Basics of the transplantation immunity.
Immunology of reproduction. Immune forms of infertility.
Autoimmune responses are directed against self antigens Responses to alloantigens and transplant rejection Self-tolerance and its loss
We have learned in preceding chapters that the adaptive immune response is a critical component of host defense against infection and therefore essential for normal health. Unfortunately, adaptive immune responses are also sometimes elicited by antigens not associated with infectious agents, and this may cause serious disease. These responses are essentially identical to adaptive immune responses to infectious agents; only the antigens differ. Later we saw how responses to certain environmental antigens cause allergic diseases and other hypersensitivity reactions. In this chapter we will examine responses to two particularly important categories of antigen: responses to self tissue antigens, called autoimmunity, which can lead to autoimmune diseases characterized by tissue damage; and responses to transplanted organs that lead to graft rejection. We will examine these disease processes and the mechanisms that lead to the undesirable adaptive immune responses that are their root cause.
Autoimmune responses are directed against self antigens.
Autoimmune disease occurs when a specific adaptive immune response is mounted against self antigens. The normal consequence of an adaptive immune response against a foreign antigen is the clearance of the antigen from the body. Virus-infected cells, for example, are destroyed by cytotoxic T cells, whereas soluble antigens are cleared by formation of immune complexes of antibody and antigen, which are taken up by cells of the mononuclear phagocytic system such as macrophages. When an adaptive immune response develops against self antigens, however, it is usually impossible for immune effector mechanisms to eliminate the antigen completely, and so a sustained response occurs. The consequence is that the effector pathways of immunity cause chronic inflammatory injury to tissues, which may prove lethal. The mechanisms of tissue damage in autoimmune diseases are essentially the same as those that operate in protective immunity and in hypersensitivity diseases. Some common autoimmune diseases are listed.
Autoimmune diseases classified by the mechanism of tissue damage.
Autoimmune diseases can be grouped in the same way as hypersensitivity reactions, according to the type of immune response and the mechanism by which it damages tissues. The immunopathological mechanisms are as illustrated for the hypersensitivity reactions, with the exception of the type I IgE-mediated responses, which are not a known cause of autoimmune disease. Some additional autoimmune diseases in which the antigen is a cellsurface receptor, and the pathology is due to altered signaling, are listed later.
Several immunopathogenic mechanisms operate in parallel to cause many autoimmune diseases. This is illustrated in the case of rheumatoid arthritis, which appears in more than one category of immunopathological mechanism.
There are four types of hypersensitivity reaction mediated by immunological mechanisms that cause tissue damage.
Types I III are antibody-mediated and are distinguished by the different types of antigens recognized and the different classes of antibody involved. Type I responses are mediated by IgE, which induces mast-cell activation, whereas types II and III are mediated by IgG, which can engage Fc-receptor and complement-mediated effector mechanisms to varying degrees, depending on the subclass of IgG and the nature of the antigen involved. Type II responses are directed against cell-surface or matrix antigens, whereas type III responses are directed against soluble antigens, and the tissue damage involved is caused by responses triggered by immune complexes. Type IV hypersensitivity reactions are T cell-mediated and can be subdivided into three groups. In the first group, tissue damage is caused by the activation of macro-phages by TH1 cells, which results in an inflammatory response. In the second, damage is caused by the activation by TH2 cells of inflammatory responses in which eosinophils predominate; in the third, damage is caused directly by cytotoxic T cells (CTL).
Adaptive immune responses are initiated by the activation of antigen-specific T cells, and it is believed that autoimmunity is initiated in the same way. T-cell responses to self antigens can inflict tissue damage either directly or indirectly. Cytotoxic T-cell responses and inappropriate activation of macrophages by TH1 cells can cause extensive tissue damage, whereas inappropriate T-cell help to self-reactive B cells can initiate harmful autoantibody responses. Autoimmune responses are a natural consequence of the open repertoires of both Bcell and T-cell receptors, which allow them to recognize any pathogen. Although these repertoires are purged of most receptors that bind with high affinity to self antigens encountered during development, they still include receptors of lower affinity reactive to some self antigens. It is not known what triggers autoimmunity, but both environmental and genetic factors, especially MHC genotype, are clearly
important. Transient autoimmune responses are common, but it is only when they are sustained and cause lasting tissue damage that they attract medical attention. In this section, we will examine the nature of autoimmune responses and how autoimmunity leads to tissue damage. In the last section of this chapter, we will examine the mechanisms by which selftolerance is lost and autoimmune responses are initiated.
Specific adaptive immune responses to self antigens can cause autoimmune disease.
Early in the study of immunity it was realized that the powerful effector mechanisms used in host defense could, if turned against the host, cause severe tissue damage; Ehrlich termed this horror autotoxicus. Healthy individuals do not mount sustained adaptive immune responses to their own antigens and, although transient responses to damaged self tissues occur, these rarely cause additional tissue damage. But although selftolerance is the general rule, sustained immune responses to self tissues occur in some individuals, and these autoimmune responses cause the severe tissue damage that Ehrlich predicted. In certain genetically susceptible strains of experimental animals, autoimmune disease can be induced artificially by injection of ‘self’ tissues from a genetically identical animal mixed with strong adjuvants containing bacteria . This shows that autoimmunity can be provoked by inducing a specific, adaptive immune response to self antigens and forms the basis for our understanding of how autoimmune disease arises. In humans, autoimmunity usually arises spontaneously; that is, we do not know what events initiate the immune response to self that leads to the autoimmune disease. There is evidence, as we will learn in the last part of this chapter, that some autoimmune disorders, such as rheumatic fever, may be triggered by infectious agents. There is, however, also evidence, particularly from animal models of autoimmunity, that many autoimmune disorders occur through internal dysregulation of the immune system without the participation of infectious agents.
Autoimmune diseases can be classified into clusters that are typically either organ-specific or systemic. The classification of disease is an uncertain science, especially in the absence of a precise understanding of causative mechanisms. This is well illustrated by the difficulty in classifying the autoimmune diseases. It is useful to distinguish two major patterns of autoimmune disease, the diseases in which the expression of autoimmunity is restricted to specific organs of the body, known as ‘organ-specific’ autoimmune diseases, and those in which many tissues of the body are affected, the ‘systemic’ autoimmune diseases. Examples of organspecific autoimmune diseases are
Hashimoto’s thyroiditis and Graves’ disease, each predominantly affecting the thyroid gland, and type I insulin-dependent diabetes mellitus (IDDM), which affects the pancreatic islets. Examples of systemic autoimmune disease are systemic lupus erythematosus (SLE) and primary Sjögren’s syndrome, in which tissues as diverse as the skin, kidneys, and brain may all be affected. The autoantigens recognized in these two categories of disease are themselves respectively organ-specific and systemic. Thus, Graves’ disease is characterized by the production of antibodies to the thyroid-stimulating hormone (TSH) receptor in the thyroid gland; Hashimoto’s thyroiditis by antibodies to thyroid peroxidase; and type I diabetes by anti-insulin antibodies. By contrast, SLE is characterized by the presence of antibodies to antigens that are ubiquitous and abundant in every cell of the body, such as anti-chromatin antibodies and antibodies to proteins of the pre-mRNA splicing machinery the spliceosome complex within the cell. It is likely that the organ-specific and systemic autoimmune diseases have somewhat different etiologies, which provides a biological basis for their division into two broad categories. Evidence for the validity of this classification also comes from observations that different autoimmune diseases cluster within individuals and within families. The organ-specific autoimmune diseases frequently occur together in many combinations; for example, autoimmune thyroid disease and the autoimmune depigmenting disease vitiligo are often found in the same person. Similarly, SLE and primary Sjögren’s syndrome can coexist within a single individual or among different members of a family.
These clusters of autoimmune diseases provide the most useful classification into different subtypes, each of which may turn out to have a distinct mechanism. A working classification of autoimmune diseases based on clustering is given in. It can be seen that the strict separation of diseases into ‘organ-specific’ and ‘systemic’ categories breaks down to some extent. Not all autoimmune diseases can be usefully classified in this manner. Autoimmune hemolytic anemia, for example, sometimes occurs as a solitary entity and could be classified as an organ-specific disease. In other circumstances it may occur in conjunction with SLE as part of a systemic autoimmune disease.
Some common autoimmune diseases classified according to their ‘organ-specific’ or ‘systemic’ nature.
Diseases that tend to occur in clusters are grouped in single boxes. Clustering is defined as more than one disease affecting a single patient or different members of a family. Not all autoimmune diseases can be classified according to this scheme. For example, autoimmune hemolytic anemia may occur in isolation or in association with systemic lupus erythematosus (SLE). Although anyone can, in principle, develop an autoimmune disease, it seems that some individuals are more at risk than others of developing particular diseases. We will first consider those factors that contribute to susceptibility.
Susceptibility to autoimmune disease is controlled by environmental and genetic factors, especially MHC genes.
The best evidence in humans for susceptibility genes for autoimmunity comes from family studies, especially studies of twins. A semiquantitative technique for measuring what proportion of the susceptibility to a particular disease arises from genetic factors is to compare the incidence of disease in monozygotic and dizygotic twins. If a disease shows a high concordance in all twins, it could be caused by shared genetic or environmental factors. This is because both monozygotic and dizygotic twins tend to be brought up in shared environmental conditions. If the high concordance is restricted to monozygotic rather than dizygotic twins, however, then genetic factors are likely to be more important than environmental factors. Studies with twins have been undertaken for several human diseases in which autoimmunity is important, including type I IDDM, rheumatoid arthritis, multiple sclerosis, and SLE. In each case, around 20% of pairs of monozygotic twins show disease concordance, compared with fewer than 5% of dizygotic twins. A similar technique is to compare the frequency of a disease such as diabetes in the siblings of patients who have diabetes with the frequency of that disease in the general population. The ratio of these two frequencies gives a measure of the heritability of the disease, although shared environmental factors within families could also be at least partly responsible for an increased frequency. Results from both twin and family studies show an important role for both inherited and environmental factors in the induction of autoimmune disease. In addition to this evidence from humans, certain inbred mouse strains have an almost uniform susceptibility to particular spontaneous or experimentally induced autoimmune diseases, whereas other strains do not. These findings have led to an extensive search for genes that determine susceptibility to autoimmune disease. So far, susceptibility to autoimmune disease has been most consistently associated with MHC genotype. Human autoimmune diseases that show associations with HLA type are shown. For most of these diseases, susceptibility is linked most strongly with MHC class II alleles, but in some cases there are strong associations with particular MHC class I alleles.
Associations of HLA serotype and sex with susceptibility to autoimmune disease.
The ‘relative risk’ for an HLA allele in an autoimmune disease is calculated by comparing the observed number of patients carrying the HLA allele with the number that would be expected, given the prevalence of the HLA allele in the general population. For type I insulin-dependent diabetes mellitus (IDDM), the association is in fact with the HLA-DQ gene, which is tightly linked to the DR genes but is not detectable by serotyping. Some diseases show a significant bias in the sex ratio; this is taken to imply that sex hormones are involved in pathogenesis. Consistent with this, the difference in the sex ratio in these diseases is greatest between the menarche and the menopause, when levels of such hormones are highest. The association of MHC genotype with disease is assessed initially by comparing the frequency of different alleles in patients with their frequency in the normal population. For IDDM, this approach originally demonstrated an association with HLA-DR3 and HLA-DR4 alleles identified by serotyping. Such studies also showed that the MHC class II allele HLA-DR2 has a dominant protective effect; individuals carrying HLA-DR2, even in association with one of the susceptibility alleles, rarely develop diabetes. Another way of determining whether MHC genes are important in autoimmune disease is to study the families of affected patients; it has been shown that two siblings affected with the same autoimmune disease are far more likely than expected to share the same MHC haplotypes.
Population studies show association of susceptibility to IDDM with HLA genotype.
The HLA genotypes (determined by serotyping) of diabetic patients (bottom panel) are not representative of those found in the population (top panel). Almost all diabetic patients express HLA-DR3 and/or HLA-DR4, and HLADR3/ DR4 heterozygosity is greatly overrepresented in diabetics compared with controls. These alleles are linked tightly to HLA-DQ alleles that confer susceptibility to IDDM. By contrast, HLA-DR2 protects against the development of IDDM and is found only extremely rarely in diabetic patients. The small letter x represents any allele other than DR2, DR3, or DR4
Family studies show strong linkage of susceptibility to IDDM with HLA genotype.
In families in which two or more siblings have IDDM, it is possible to compare the HLA
genotypes of affected siblings. Affected siblings share two HLA haplotypes much more frequently than would be expected if the HLA genotype did not influence disease susceptibility. As HLA genotyping has become more exact through the sequencing of HLA alleles, disease associations that were originally discovered through HLA serotyping using antibodies have been defined more precisely. For example, the association between IDDM and the DR3 and DR4 alleles is now known to be due to their tight genetic linkage to DQβ alleles that confer susceptibility to disease. Indeed, disease susceptibility is most closely associated with polymorphisms at a particular position in the DQβ amino acid sequence. The most abundant DQβ amino acid sequence has an aspartic acid at position 57 that is able to form a salt bridge across the end of the peptidebinding cleft of the DQ molecule. By contrast, the diabetic patients in Caucasoid populations mostly have valine, serine, or alanine at that position and thus make DQ molecules that lack this salt bridge. The nonobese diabetic (NOD) strain of mice, which develops spontaneous diabetes, also has a serine at that position in the homologous MHC class II molecule, known as I-Ag7.
Amino acid changes in the sequence of an MHC class II protein correlate with susceptibility to and protection from diabetes.
The HLA-DQβ1 chain contains an aspartic acid (Asp) at position
The genes that have been associated with the development of systemic lupus erythematosus provide important clues to the etiology of the disease.
The major serological abnormality in SLE is the presence of autoantibodies to ubiquitous and abundant intracellular antigens, such as chromatin. How is tolerance broken to such all-pervasive self antigens? A number of genes have been implicated in the etiology of SLE in humans and mice. These can be classified into three categories on the basis of their physiological function. The first comprises genes whose products are active in the body’s mechanisms for disposing of dead and dying cells, which could provide a source of autoantigens. Genetic knockout in mice of four genes in this category has produced animal models of SLE. One of these genes codes for the complement protein C1q, which, together with other complement proteins, is involved in the effective clearance of immune complexes and apoptotic cells. A second gene in this category encodes serum amyloid P component, which binds chromatin and may mask it from the immune system. Its deletion results in the development of antibodies against chromatin and development of glomerulonephritis caused by deposition of immune complexes of these antibodies in the kidney. Third, deletion of DNase I, an enzyme that digests extracellular chromatin, results in the development of anti-chromatin antibodies and glomerulonephritis. Fourth, a similar phenotype has been seen in mice in which the secretory portion of the immunoglobulin μ chain is deleted, and which thus lack secreted IgM, which may have an important role in the clearance of effete cells. However, the majority of cases of spontaneous SLE are likely to be influenced by far more complex genetic factors than these single-gene defects.
Amino acid changes in the sequence of an MHC class II protein correlate with susceptibility to and protection from diabetes.
The HLA-DQβ1 chain contains an aspartic acid (Asp) at position
for SLE includes those encoding proteins that regulate the thresholds for tolerance and activation of T and B lymphocytes, such as Fas, Fas ligand, the signaling molecule SHP-1, the B-cell inhibitory receptor CD22, FcγRIIB, and the cell-cycle inhibitor p21. The third category of genes encode proteins that could modify the expression of SLE in individual organs by their involvement in immune complex-mediated inflammation. Examples are the polymorphic genes for FcγRIIa and FcγRIII, where the variant proteins are thought to differ in their ability to bind immune complexes and are associated with the presence of nephritis in SLE. A further very important factor in disease susceptibility to SLE is the hormonal status of the patient. Indeed, many autoimmune diseases show a strong sex bias. Where a bias towards disease in one sex is observed in experimental animals, castration or the administration of estrogen to males usually normalizes disease incidence between the two sexes. Furthermore, many autoimmune diseases that are more common in females show peak incidence in the years of active child bearing, when production of the female sex hormones estrogen and progesterone is at its greatest. A thorough understanding of how these genetic and hormonal factors contribute to disease susceptibility might allow us to prevent the autoimmune response.
Responses to alloantigens and transplant rejection.
The transplantation of tissues to replace diseased organs is now an important medical therapy. In most cases, adaptive immune responses to the grafted tissues are the major impediment to successful transplantation. Rejection is caused by immune responses to alloantigens on the graft, which are proteins that vary from individual to individual within a species, and are thus perceived as foreign by the recipient. In blood transfusion, which was the earliest and is still the most common tissue transplant, blood must be matched for ABO and Rh blood group antigens to avoid the rapid destruction of mismatched red blood cells by antibodies. Because there are only four major ABO types and two Rh blood types, this is relatively easy. When tissues containing nucleated cells are transplanted, however, T-cell responses to the highly polymorphic MHC molecules almost always trigger a response against the grafted organ. Matching the MHC type of donor and recipient increases the success rate of grafts, but perfect matching is possible only when donor and recipient are related and, in these cases, genetic differences at other loci still trigger rejection. In this section, we will examine the immune response to tissue grafts, and ask why such responses do not reject the one foreign tissue graft that is tolerated routinely the mammalian fetus.
Graft rejection is an immunological response mediated primarily by T cells.
The basic rules of tissue grafting were first elucidated by skin transplantation between inbred strains of mice. Skin can be grafted with 100% success between different sites on the same animal or person (an autograft), or between genetically identical animals or people (a syngeneic graft). However, when skin is grafted between unrelated or allogeneic individuals (an allograft), the graft is initially accepted but is then rejected about 10-13 days after grafting. This response is called a first-set rejection and is quite consistent. It depends on a T-cell response in the recipient, because skin grafted onto nude mice, which lack T cells, is not rejected. The ability to reject skin can be restored to nude mice by the adoptive transfer of normal T cells.
Skin graft rejection is the result of a T cell-mediated anti-graft response.
Grafts that are syngeneic are permanently accepted (first panels) but grafts differing at the MHC are rejected around 10-13 days after grafting (first-set rejection, second panels). When a mouse is grafted for a second time with skin from the same donor, it rejects the second graft faster (third panels). This is called a second-set rejection and the accelerated response is MHC-specific; skin from a second donor of the same MHC type is rejected equally fast, whereas skin from an MHC-different donor is rejected in a first-set pattern (not shown). Naive mice that are given T cells from a sensitized donor behave as if they had already been grafted (final panels). When a recipient that has previously rejected a graft is regrafted with skin from the same donor, the second graft is rejected more rapidly (6-8 days) in a second-set rejection. Skin from a third-party donor grafted onto the same recipient at the same time does not show this faster response but follows a first-set rejection course. The rapid course of second-set rejection can be transferred to normal or irradiated recipients by transferring T cells from the initial recipient, showing that graft rejection is caused by a specific immunological reaction. Immune responses are a major barrier to effective tissue transplantation, destroying grafted tissue by an adaptive immune response to its foreign proteins. These responses can be mediated by CD8 T cells, by CD4 T cells, or by both. Antibodies can also contribute to second-set rejection of tissue grafts.
Matching donor and recipient at the MHC improves the outcome of transplantation.
When donor and recipient differ at the MHC, the immune response, which is known as an alloreactive response as it is directed against antigens (alloantigens) that differ between members of the same species, is directed at the nonself allogeneic MHC molecule or molecules present on the graft. In most tissues, these will be predominantly MHC class I antigens. Once a recipient has rejected a graft of a particular MHC type, any further graft bearing the same nonself MHC molecule will be rapidly rejected in a second-set response. As we learned in Chapter 5, the frequency of T cells specific for any nonself MHC molecule is relatively high, making differences at MHC loci the most potent trigger of the rejection of initial grafts; indeed, the major histocompatibility complex was originally so named because of its central role in graft rejection. Once it became clear that recognition of nonself MHC molecules is a major determinant of graft rejection, a considerable amount of effort was put into MHC matching between recipient and donor. Although HLA matching significantly improves the success rate of clinical organ transplantation, it does not in itself prevent rejection reactions. There are two main reasons for this. First, HLA typing is imprecise, owing to the polymorphism and complexity of the human MHC; unrelated individuals who type as HLA-identical with antibodies against MHC proteins rarely have identical MHC genotypes. This should not be a problem with HLA-identical siblings: because siblings inherit their MHC genes as a haplotype, one sibling in four should be truly HLA-identical. Nevertheless, grafts between HLA-identical siblings are invariably rejected, albeit more slowly, unless donor and recipient are identical twins. This rejection is the result of differences between minor histocompatibility antigens, which is the second reason for the failure of HLA matching to prevent rejection reactions. These minor histocompatibility antigens will be discussed in the next section. Thus, unless donor and recipient are identical twins, all graft recipients must be given immunosuppressive drugs to prevent rejection. Indeed, the current success of clinical transplantation of solid organs is more the result of advances in immunosuppressive therapy, than of improved tissue matching. The limited supply of cadaveric organs, coupled with the urgency of identifying a recipient once a donor organ becomes available, means that accurate matching of tissue types is achieved only rarely.
In MHC-identical grafts, rejection is caused by peptides from other alloantigens bound to graft MHC molecules.
When donor and recipient are identical at the MHC but differ at other genetic loci, graft rejection is not so rapid The polymorphic antigens responsible for the rejection of MHC-identical grafts are therefore termed minor histocompatibility antigens or minor H antigens. Responses to single minor H antigens are much less potent than responses to MHC differences because the frequency of responding T cells is much lower. However, most inbred mouse strains that are identical at the MHC differ at multiple minor H antigen loci, so grafts between them are still uniformly and relatively rapidly rejected. The cells that respond to minor H antigens are generally CD8 T cells, implying that most minor H antigens are complexes of donor peptides and MHC class I molecules. However, peptides bound to MHC class II molecules can also participate in the response to MHC-identical grafts.
Even complete matching at the MHC does not ensure graft survival.
Although syngeneic grafts are not rejected (left panels), MHC-identical grafts from donors that differ at other loci (minor H antigen loci) are rejected (right panels), albeit more slowly than MHC-disparate grafts (center panels). Minor H antigens are now known to be peptides derived from polymorphic proteins that are presented by the MHC molecules on the graft. MHC class I molecules bind and present a selection of peptides derived from proteins made in the cell, and if polymorphisms in these proteins mean that different peptides are produced in different members of a species, these can be recognized as minor H antigens. One set of proteins that induce minor H responses is encoded on the male-specific Y chromosome. Responses induced by these proteins are known collectively as H-Y. As these Y chromosome-specific genes are not expressed in females, female anti-male minor H responses occur; however, male anti-female responses are not seen, because both males and females express X-chromosome genes. One H-Y antigen has been identified in mice and humans as peptides from a protein encoded by the Y-chromosome gene Smcy. An X-chromosome homologue of Smcy, called Smcx, does not contain these peptide sequences, which are therefore expressed uniquely in males. The nature of the majority of minor H antigens, encoded by autosomal genes, is unknown, but one, HA-2, has been identified as a peptide derived from myosin.
Minor H antigens are peptides derived from polymorphic cellular proteins bound to MHC class I molecules.
Self proteins are routinely digested by proteasomes within the cell’s cytosol, and peptides derived from them are delivered to the endoplasmic reticulum, where they can bind to MHC class I molecules and be delivered to the cell surface. If a polymorphic protein differs between the graft donor (shown in red on the left) and the recipient (shown in blue on the right), it can give rise to an antigenic peptide (red on the donor cell) that can be recognized by the recipient’s T cells as nonself and elicit an immune response. Such antigens are the minor H antigens. The response to minor H antigens is in most ways analogous to the response to viral infection. However, an antiviral response eliminates only infected cells, whereas all cells in the graft express minor H antigens, and thus the entire graft is destroyed in the response against these antigens. Thus, even though MHC genotype might be matched exactly, polymorphism in any other protein could elicit potent T-cell responses that would destroy the entire graft. It is no wonder that successful transplantation requires the use of powerful immunosuppressive drugs.
There are two ways of presenting alloantigens on the transplant to the recipient’s T lymphocytes.
We saw that alloreactive effector T cells that bind directly to allogeneic MHC class I molecules in mismatched organ grafts are an important cause of graft rejection; this is called direct allorecognition. Before they can cause rejection, naive alloreactive T cells must be activated by antigenpresenting cells that both bear the allogeneic MHC molecules and have co-stimulatory activity. Organ grafts carry with them antigen-presenting cells of donor origin, known as passenger leukocytes, and these are an important stimulus to alloreactivity. This route for sensitization of the recipient to a graft seems to involve donor antigen-presenting cells leaving the graft and migrating via the lymph to regional lymph nodes. Here they can activate those host T cells that bear the corresponding T-cell receptors. The activated alloreactive effector T cells are then carried back to the graft, which they attack directly. Indeed, if the grafted tissue is depleted of antigen-presenting cells by treatment with antibodies or by prolonged incubation, rejection occurs only after a much longer time. Also, if the site of grafting lacks lymphatic drainage, no response to the graft results.
Alloantigens in grafted organs are recognized in two different ways. Direct recognition of a grafted organ (red in upper panel) is mediated by T cells whose receptors have specificity for the allogeneic MHC class I or class II molecule in combination with peptide. These alloreactive T cells are stimulated by donor antigen-presenting cells (APC), which express both the allogeneic MHC molecule and co-stimulatory activity (bottom left panel). Indirect recognition of the graft (bottom right panel) is mediated by T cells whose receptors are specific for allogeneic peptides that are derived from the grafted organ. Proteins from the graft are processed by the recipient’s antigen-presenting cells and are therefore presented by self (recipient) MHC class I or class II molecules.
The initiation of graft rejectioormally involves migration of donor antigen-presenting cells from the graft to local lymph nodes.
The example of a skin graft is illustrated here, in which Langerhans’ cells are the antigen-presenting cells. They display peptides from the graft on their surface. After traveling to a lymph node, these antigen-presenting cells encounter recirculating naive T cells specific for graft antigens, and stimulate these T cells to divide. The resulting activated effector T cells migrate via the thoracic duct to the blood and home to the grafted tissue, which they rapidly destroy. Destruction is highly specific for donorderived cells, suggesting that it is mediated by direct cytotoxicity and not by nonspecific inflammatory processes. A second mechanism of allograft recognition leading to graft rejection is the uptake of allogeneic proteins by the recipient’s own antigen-presenting cells and their presentation to T cells by self MHC molecules The recognition of allogeneic proteins presented in this way is known as indirect allorecognition, in contrast to the direct recognition by T cells of allogeneic MHC class I and class II molecules expressed on the graft itself. Among the graft-derived peptides presented by the recipient’s antigen-presenting cells are the minor H antigens and also peptides from the foreign MHC molecules themselves, which are a major source of the polymorphic peptides recognized by the recipient’s T cells. The relative contributions of direct and indirect allorecognition in graft rejection are not known. Direct allorecognition is thought to be largely responsible for acute rejection, especially when MHC mismatches mean that the frequency of directly alloreactive recipient T cells is high. Furthermore, a direct cytotoxic T-cell attack on graft cells can be carried out only by T cells that recognize the graft MHC molecules directly. Nonetheless, T cells with indirect allospecificity can contribute to graft rejection by activating macrophages, which cause tissue injury and fibrosis, and are likely to be important in the development of an alloantibody response to a graft.
Antibodies reacting with endothelium cause hyperacute graft rejection.
Antibody responses are also an important potential cause of graft rejection. Preexisting alloantibodies to blood group antigens and polymorphic MHC antigens can cause rapid rejection of transplanted organs in a complement-dependent reaction that can occur within minutes of transplantation. This type of reaction is known as hyperacute graft rejection. Most grafts that are transplanted routinely in clinical medicine are vascularized organ grafts linked directly to the recipient’s circulation. In some cases, the recipient might already have circulating antibodies against donor graft antigens, produced in response to a previous transplant or a blood transfusion. Such antibodies can cause very rapid rejection of vascularized grafts because they react with antigens on the vascular endothelial cells of the graft and initiate the complement and blood clotting cascades. The vessels of the graft become blocked, causing its immediate death. Such grafts become engorged and purple-colored from hemorrhaged blood, which becomes deoxygenated. This problem can be avoided by cross-matching donor and recipient. Cross-matching involves determining whether the recipient has antibodies that react with the white blood cells of the donor. If antibodies of this type are found, they are a serious contraindication to transplantation, as they lead to near-certain hyperacute rejection.
Preexisting antibody against donor graft antigens can cause hyperacute graft rejection.
In some cases, recipients already have antibodies to donor antigens, which are often blood group antigens. When the donor organ is grafted into such recipients, these antibodies bind to vascular endothelium in the graft, initiating the complement and clotting cascades. Blood vessels in the graft become obstructed by clots and leak, causing hemorrhage of blood into the graft. This becomes engorged and turns purple from the presence of deoxygenated blood. A very similar problem prevents the routine use of animal organs xenografts in transplantation. If xenogeneic grafts could be used, it would circumvent the major limitation in organ replacement therapy, namely the severe shortage of donor organs. Pigs have been suggested as a potential source of organs for xenografting as they are a similar size to humans and are easily farmed. Most humans and other primates have antibodies that react with endothelial cell antigens of other mammalian species, including pigs. When pig xenografts are placed in humans, these antibodies trigger hyper-acute rejection by binding to the endothelial cells of the graft and initiating the complement and clotting cascades. The problem of hyperacute rejection is exacerbated in xenografts because complement-regulatory proteins such as CD59, DAF (CD55), and MCP (CD46) work less efficiently across a species barrier; the complement-regulatory proteins of the xenogeneic endothelial cells cannot protect them from attack by human complement. A recent step toward xenotransplantation has been the development of transgenic pigs expressing human DAF. Preliminary experiments have shown prolonged survival of organs transplanted from these pigs into recipient cynomolgus monkeys, under cover of heavy immunosuppression. However, hyperacute rejection is only the first barrier faced by a xenotransplanted organ. The T lymphocyte-mediated graft rejection mechanisms might be extremely difficult to overcome with present immunosuppressive regimes.
The converse of graft rejection is graft-versus-host disease.
Allogeneic bone marrow transplantation is a successful therapy for some tumors derived from bone marrow precursors, such as certain leukemias and lymphomas. It may also be successful in the treatment of some primary immunodeficiency diseases and inherited bone marrow diseases, such as the severe forms of thalassemia. In leukemia therapy, the recipient’s bone marrow, the source of the leukemia, must first be destroyed by aggressive cytotoxic chemotherapy. One of the major complications of allogeneic bone marrow transplantation is graft-versus-host disease (GVHD), in which mature donor T cells that contaminate the allogeneic bone marrow recognize the tissues of the recipient as foreign, causing a severe inflammatory disease characterized by rashes, diarrhea, and pneumonitis. Graft-versus-host disease occurs not only when there is a mismatch of a major MHC class I or class II antigen but also in the context of disparities between minor H antigens. Graft-versus-host disease is a common complication in recipients of bone marrow transplants from HLA-identical siblings, who typically differ from each other in many polymorphic proteins encoded by genes unlinked to the MHC. The presence of alloreactive T cells can easily be demonstrated experimentally by the mixed lymphocyte reaction (MLR), in which lymphocytes from a potential donor are mixed with irradiated lymphocytes from the potential recipient. If the donor lymphocytes contain alloreactive T cells, these will respond by cell division The MLR is sometimes used in the selection of donors for bone marrow transplants, when the lowest possible alloreactive response is essential. However, the limitation of the MLR in selection of bone marrow donors is that the test does not accurately quantitate alloreactive T cells. A more accurate test is a version of the limiting-dilution assay, which precisely counts the frequency of alloreactive T cells.
The mixed lymphocyte reaction (MLR) can be used to detect histoincompatibility.
Lymphocytes from the two individuals who are to be tested for compatibility are isolated from peripheral blood. The cells from one person (yellow), which will also contain antigen-presenting cells, are either irradiated or treated with mitomycin C; they will act as stimulator cells but cannot now respond by DNA synthesis and cell division to antigenic stimulation by the other person’s cells. The cells from the two individuals are then mixed (top panel). If the unirradiated lymphocytes (the responders, blue) contain alloreactive T cells, these will be stimulated to proliferate and differentiate to effector cells. Between 3 and 7 days after mixing, the culture is assessed for T-cell proliferation (bottom left panel), which is mainly the result of CD4 T cells recognizing differences in MHC class II molecules, and for the generation of activated cytotoxic T cells (bottom right panel), which respond to differences in MHC class I molecules. Although graft-versus-host disease is usually harmful to the recipient of a bone marrow transplant, there can be some beneficial effects. Some of the therapeutic effect of bone marrow transplantation for leukemia can be due to a graft-versus-leukemia effect, in which the allogeneic bone marrow recognizes minor H antigens or tumorspecific antigens expressed by the leukemic cells, leading the donor cells to kill the leukemic cells. One such minor H antigen, HB-1, is a B-cell lineage marker that is expressed by acute lymphoblastic leukemia cells, which are B-lineage cells, and by B lymphocytes transformed with Epstein-Barr virus (EBV). One of the treatment options for suppressing the development of graft-versus-host disease is the elimination of mature T cells from the donor bone marrow in vitro before transplantation, thereby removing alloreactive T cells. Those T cells that subsequently mature from the donor marrow in vivo in the recipient are tolerant to the recipient’s antigens. Although the elimination of graft-versus-host disease has benefits for the patient, there is an increase in the risk of leukemic relapse, which provides strong evidence in support of the graft-versus-leukemia effect.
Chronic organ rejection is caused by inflammatory vascular injury to the graft.
The success of modern immunosuppression means that approximately 85% of cadaveric kidney grafts are still functioning a year after transplantation. However, there has beeo improvement in rates of long-term graft survival: the half-life for functional survival of renal allografts remains about 8 years. The major cause of late organ failure is chronic rejection, characterized by concentric arteriosclerosis of graft blood vessels, accompanied by glomerular and tubular fibrosis and atrophy. Mechanisms that contribute to chronic rejection can be divided into those due to alloreactivity and those due to other pathways, and into early and late events after transplantation. Alloreactivity may occur days and weeks after transplantation and cause acute graft rejection. Alloreactive responses may also occur months to years after transplantation, and be associated with clinically hard-to-detect gradual loss of graft function. Other important causes of chronic graft rejection include ischemia-reperfusion injury, which occurs at the time of grafting but may have late adverse effects on the grafted organ, and later-developing adverse factors such as chronic cyclosporin toxicity or cytomegalovirus infection. Infiltration of the graft vessels and tissues by macrophages, followed by scarring, are prominent histological features of late graft rejection. A model of injury has been developed in which alloreactive T cells infiltrating the graft secrete cytokines that stimulate the expression of endothelial adhesion molecules and also secrete chemokines such as RANTES, which attracts monocytes that mature into macrophages in the graft. A second phase of chronic inflammation then supervenes, dominated by macrophage products including interleukin (IL)-1, TNF-α and the chemokine MCP, which leads to further macrophage recruitment. These mediators conspire to cause chronic inflammation and scarring, which eventually leads to irreversible organ failure. Animal models of chronic rejection also show that alloreactive IgG antibodies may induce accelerated atherosclerosis in transplanted solid organs. \
Properties of selected chemokines.
Chemokines fall mainly into two related but distinct groups: the CC chemokines, which in humans are mostly encoded in one region of chromosome 4, have two adjacent cysteine residues in their amino-terminal region; CXC chemokines, the genes for which are mainly found in a cluster on chromosome 17, have an amino acid residue between the equivalent two cysteines. These chemokines can be divided further by the presence or absence of an amino acid triplet (ELR; glutamic acid leucine arginine) preceding the first of these invariant cysteines. All the chemokines that attract neutrophils have this motif, while most of the other CXC chemokines, including the chemokines reacting with CXCR3, 4, and 5 lack it. A C chemokine with only one cysteine at this location, and fractalkine, a CX3C chemokine, are encoded elsewhere in the genome. Each chemokine interacts with one or more receptors, and affects one or more types of cell.
A variety of organs are transplanted routinely in clinical medicine.
Although the immune response makes organ transplantation difficult, there are few alternative therapies for organ failure. Three major advances have made it possible to use organ transplantation routinely in the clinic. First, the technical skill to carry out organ
replacement surgery has been mastered by many people. Second, networks of transplantation centers have been organized to ensure that the few healthy organs that are available are HLA-typed and so matched with the most suitable recipient. Third, the use of powerful immunosuppressive drugs, especially cyclosporin A and FK-506, known as tacrolimus, to inhibit T-cell activation, has increased graft survival rates dramatically. The different organs that are transplanted in the clinic are listed Some of these operations are performed routinely with a very high success rate. By far the most frequently transplanted solid organ is the kidney, the organ first successfully transplanted between identical twins in the 1950s. Transplantation of the cornea is even more frequent; this tissue is a special case, as it is not vascularized, and corneal grafts between unrelated people are usually successful even without immunosuppression.
Tissues commonly transplanted in clinical medicine.
All grafts except corneal and some bone marrow grafts require long-term immunosuppression. The number of organ grafts performed in the United States in 1999 is shown. Figures in brackets are for the organ alone, while the total figure includes combination transplants, e.g., heart and lungs. *The 5-year survival values are an average; closer matching between donor and recipient generally gives better survival. Data courtesy of United Network for Organ Sharing. Data for 2005 courtesy of National Eye Institute. Data for 1998 courtesy of International Bone Marrow Transplant Registry. There are, however, many problems other than graft rejection associated with organ
transplantation. First, donor organs are difficult to obtain; this is especially a problem when the organ involved is a vital one, such as the heart or liver. Second, the disease that destroyed the patient’s organ might also destroy the graft. Third, the immunosuppression required to prevent graft rejection increases the risk of cancer and infection. Finally, the procedure is very costly. All of these problems need to be addressed before clinical transplantation can become commonplace. The problems most amenable to scientific solution are the development of more effective means of immunosuppression, the induction of graft-specific tolerance, and the development of xenografts as a practical solution to organ availability.
The fetus is an allograft that is tolerated repeatedly.
All of the transplants discussed so far are artefacts of modern medical technology. However, one tissue that is repeatedly grafted and repeatedly tolerated is the mammalian fetus. The fetus carries paternal MHC and minor H antigens that differ from those of the mother, and yet a mother can successfully bear many children expressing the same nonself MHC proteins derived from the father. The mysterious lack of rejection of the fetus has puzzled generations of reproductive immunologists and no comprehensive explanation has yet emerged. One problem is that acceptance of the fetal allograft is so much the norm that it is difficult to study the mechanism that prevents rejection; if the mechanism for rejecting the fetus is rarely activated, how can one analyze the mechanisms that control it?
The fetus is an allograft that is not rejected.
Although the fetus carries MHC molecules derived from the father, and other foreign antigens, it is not rejected. Even when the mother bears several children to the same father, no sign of immunological rejection is seen. Various hypotheses have been advanced to account for the tolerance shown to the fetus. It has been proposed that the fetus is simply not recognized as foreign. There is evidence against this hypothesis, as women who have borne several children usually make antibodies directed against the father’s MHC proteins; indeed, this is the best source of antibodies for human MHC typing. However, the placenta, which is a fetus-derived tissue, seems to sequester the fetus away from the mother’s T cells. The outer layer of the placenta, the interface between fetal and maternal tissues, is the trophoblast. This does not express classical MHC class I and class II proteins, making it resistant to recognition and attack by maternal T cells. Tissues lacking class I expression are, however, vulnerable to attack by NK cells. The trophoblast might be protected from attack by NK cells by expression of a nonclassical and minimally polymorphic HLA class I molecule HLA-G. This protein has been shown to bind to the two major inhibitory NK receptors, KIR1 and KIR2, and to inhibit NK killing. The placenta may also sequester the fetus from the mother’s T cells by an active mechanism of nutrient depletion. The enzyme indoleamine 2,3-dioxygenase (IDO) is expressed at a high level by cells at the maternalfetal interface. This enzyme catabolizes, and thereby depletes, the essential amino acid tryptophan at this site. T cells starved of tryptophan show reduced responsiveness. Inhibition of IDO in pregnant mice, using the inhibitor 1-methyltryptophan, causes rapid rejection of allogeneic but not syngeneic fetuses. This supports the hypothesis that maternal T cells, alloreactive to paternal MHC proteins, may be held in check in the placenta by tryptophan depletion. It is likely that fetal tolerance is a multifactorial process. The trophoblast does not act as an absolute barrier between mother and fetus, and fetal blood cells can cross the placenta and be detected in the maternal circulation, albeit in very low numbers. There is direct evidence from experiments in mice for specific T-cell tolerance against paternal MHC alloantigens. Pregnant female mice whose T cells bear a transgenic receptor specific for a paternal alloantigen showed reduced expression of this T-cell receptor during pregnancy. These same mice lost the ability to control the growth of an experimental tumor bearing the same paternal MHC alloantigen. After pregnancy, tumor growth was controlled and the level of the T-cell receptor increased. This experiment demonstrates that the maternal immune system must have been exposed to paternal MHC alloantigens, and that the immune response to these antigens was temporarily suppressed. Yet another factor that might contribute to maternal tolerance of the fetus is the secretion of cytokines at the maternal-fetal interface. Both uterine epithelium and trophoblast secrete cytokines, including transforming growth factor (TGF)-β, IL-4, and IL-10. This cytokine pattern tends to suppress TH1 responses. Induction or injection of cytokines such as interferon (IFN)-γ and IL-12, which promote TH1 responses in experimental animals, promote fetal resorption, the equivalent of spontaneous abortion in humans. The fetus is thus tolerated for two main reasons: it occupies a site protected by a nonimmunogenic tissue barrier, and it promotes a local immunosuppressive response in the mother. We will see later that several sites in the body have these characteristics and allow prolonged acceptance of foreign tissue grafts. They are usually called immunologically privileged sites.
Summary.
Clinical transplantation is now an everyday reality, its success built on MHC matching, immunosuppressive drugs, and technical skill. However, even accurate MHC matching does not prevent graft rejection; other genetic differences between host and donor can result in allogeneic proteins whose peptides are presented as minor H antigens by MHC molecules on the grafted tissue, and responses to these can lead to rejection. As we lack the ability to specifically suppress the response to the graft without compromising host defense, most transplants require generalized immunosuppression of the recipient. This can be significantly toxic and increases the risk of cancer and infection. The fetus is a natural allograft that must be accepted it almost always is or the species will not survive. Tolerance to the fetus might hold the key to inducing specific tolerance to grafted tissues, or it might be a special case not applicable to organ replacement therapy.
Self-tolerance and its loss.
Tolerance to self is acquired by clonal deletion or inactivation of developing lymphocytes. Tolerance to antigens expressed by grafted tissues can be induced artificially, but it is very difficult to establish once a full repertoire of functional B and T lymphocytes has been produced, which occurs during fetal life in humans and around the time of birth in mice. We have already discussed the two important mechanisms of self-tolerance clonal deletion by ubiquitous self antigens and clonal inactivation by tissue-specific antigens presented in the absence of co-stimulatory signals. These processes were first discovered by studying tolerance to nonself, where the absence of tolerance could be studied in the form of graft rejection. In this section, we will consider tolerance to self and tolerance to nonself as two aspects of the same basic mechanisms. These mechanisms consist of direct induction of tolerance in the periphery, either by deletion or by anergy. There is also a state referred to as immunological ignorance, in which T cells or B cells coexist with antigen without being affected by it. Finally, there are mechanisms of tolerance that involve T-cell-T-cell interactions, known variously as immune deviation or immune suppression. In an attempt to understand the related phenomena of autoimmunity and graft rejection, we also examine instances where tolerance to self is lost.
Many autoantigens are not so abundantly expressed that they induce clonal deletion or anergy but are not so rare as to escape recognition entirely.
We saw in Chapter 7 that clonal deletion removes immature T cells that recognize ubiquitous self antigens and in Chapter 8 that antigens expressed abundantly in the periphery induce anergy or clonal deletion in lymphocytes that encounter them on tissue cells. Most self proteins are expressed at levels that are too low to serve as targets for T-cell recognition and thus cannot serve as autoantigens. It is likely that very few self proteins contain peptides that are presented by a given MHC molecule at a level sufficiently high to be recognized by effector T cells but too low to induce tolerance. T cells able to recognize these rare antigens will be present in the individual but will not normally be activated. This is because their receptors only bind self peptides with very low affinity, or because they are exposed to levels of self peptide that are too low to deliver any signal to the T cell. Such T cells are said to be in a state of immunological ignorance. This state has been demonstrated experimentally using transgenic animals in which ovalbumin was expressed at high or very low concentrations in the pancreas. Lymphocytes reactive to ovalbumin were transferred to these animals. The lymphocytes transferred to animals expressing high levels of ovalbumin proliferated in response to ovalbumin presented by antigen-presenting cells and then died. In contrast, the lymphocytes transferred to animals expressing very low levels of pancreatic ovalbumin did not divide but persisted and could be stimulated normally when exposed to high levels of ovalbumin in vitro.
T cells are ignorant of very low levels of autoantigen .
Transgenic mice were developed that expressed ovalbumin in the pancreas at high or very low levels. CD8 lymphocytes specific for ovalbumin were injected into these mice. After 3 days, the regional lymph nodes draining the pancreas were isolated and the amount of proliferation of the ovalbumin-specific CD8 T cells was measured. In the mice expressing high levels of ovalbumin, these T cells were proliferating, in contrast to mice expressing low levels of ovalbumin, in which no proliferation was observed. After 4 weeks, the spleens were obtained, the ovalbumin-specific T cells were enumerated and their spontaneous proliferation and proliferation in response to ovalbumin in vitro was assessed. At this time no ovalbumin-specific T cells were recovered from the spleens of the mice expressing high levels of ovalbumin, and there was no proliferative response to ovalbumin in vitro. Thus in these mice the ovalbumin-specific T cells divided early, after encounter with ovalbumin in the periphery, and then died, illustrating the phenomenon of peripheral tolerance by deletion. In contrast, in the mice expressing very low levels of ovalbumin, the ovalbumin-specific T cells persisted without proliferating in the periphery, were recovered from the spleen at 4 weeks, and responded normally to ovalbumin presented in vitro. This silent persistence in the presence of low levels of autoantigen illustrates the phenomenon of T-cell ignorance. Miscarriages can be Prevented An unexpected miscarriage can shatter dreams. Two or more can be devastating. But now there is hope, and a solution. One in every 200 couples are too genetically similar to
achieve successful pregnancy. And usually, they don’t know it. That’s why early detection is vital. Without intervention, the painful pattern of miscarriage occurs again and again.
Reproductive Immunology
One in two hundred couples will experience two or more consecutive miscarriages. There are five reasons for miscarriage which have been identified:
Some women have multiple reasons for miscarriages. At Reproductive Immunology Associates we evaluate patients for immune related miscarriages. Your obstetrician will test for most other causes of pregnancy loss. The Immune System Advances in immunology, the study of the body’s defense systems, enable us to understand how during pregnancy, the mother’s immune system is altered so that the fetus is not rejected by her body and allows the fetus to grow. The immune system is comprised of white blood cells, also known as leukocytes, which make a variety of antibodies . Some of the antibodies protect us and others are harmful to our bodies.
When the immune system is the cause of miscarriage, the chances of mother having a successful pregnancy without treatment after three miscarriages is 30%, after four miscarriages 25%, and after 5 miscarriages 5%. Antiphospholipid Antibodies Phospholipid molecules are normal components of all cell membranes. Some also have glue like properties and allow cells to fuse (as you will see later). Antibodies to phospholipid molecules can, therefore, cause problems. Specifically, they can damage the inside of the blood vessel wall. This allows blood cells to stick to the site of the injury and cause blood clots.
Antiphospholipid antibodies can also cause blood vessels to constrict, causing decreased blood flow throughout the circulatory system.
The combination of blood clots and constricted blood vessels may impair blood supply to the fetus and placenta resulting in complete fetal demise or growth retardation.
Some phospholipid molecules have adhesion properties i.e. glue like, and allow cells to fuse. The formation of the normal placenta involves the fusion of small cells called cytotrophoblasts into giant cells known as syncytiotrophoblasts. The syncytiotrophoblasts play a key role in the regulation of nutrients going to the baby.
Antibodies to phospholipid molecules can, therefore, interfere with the development of the placenta. With each pregnancy loss, there is a 10% chance that the mother will develop an antibody to a phospholipid molecule. Most women with antiphospholipid antibodies are not sick. However, some have underlying autoimmune tendencies and should be appropriately evaluated. Women with underlying autoimmune diseases may have antiphospholipid antibodies even before they ever become pregnant.
Treatment for Antiphospholipid Antibodies
Antiphospholipid antibodies are treated with low dose (baby) aspirin and a blood thinner called Heparin. Heparin is a very large molecule and is unable to cross the placenta. Aspirin is able to cross the placenta but the dose used is so small that the fetus is unaffected. The effectiveness of treatment is much greater when the medication, if indicated, is started prior to conception and continued throughout the pregnancy. All medication, if indicated should be discussed with one’s physician. Antinuclear Antibodies The nucleus is the ” brain ” of the cell. It contains the information that regulates the function of the cell. Some people have antibodies to different nuclear components. What causes these antibodies to be made is currently under investigation but there appears to be a genetic susceptibility which may be reflected by the HLA tissue type (refer back to the section on blocking antibodies). The disease that we typically associate with antinuclear antibodies is Systemic Lupus Erythematosus (SLE). The miscarriage rate in SLE patients is much higher than that of the general population. Although most women who suffer recurrent miscarriages do not have clinical signs of SLE, many exhibit autoimmune phenomena which is similar to that seen in SLE patients. The placentas in these women are inflamed and weakened.
As the body is dynamic, antibody levels may change over time. This is illustrated in the figure above. Most people have no antinuclear antibodies all the time (A,B). Many women who miscarry have borderline (C,D,E) or abnormal levels of antinuclear antibodies (F,G). Patients who develop new autoantibodies like antinuclear and antiphospholipid antibodies during pregnancy have a more guarded prognosis.
Treatment of Antinuclear Antibodies
Women with ANA are treated with prednisone, a corticosteroid, which suppresses the inflammatory process and stabilizes the cell. Prednisone does not pass through the placenta easily and is also broken down by enzymes in the placenta so that the fetus is exposed to only trace amounts. Additionally, the body produces the equivalent of 8 mg per day of this corticosteroid. When indicated, Prednisone should be started prior to conception. Antithyroid Antibodies In 1990, Stagnaro-Green demonstrated in a prospective analysis that thyroid antibodies were markers for “at-risk” pregnancies. The two antibodies studied, anti-thyroid peroxidase and anti-thyroglobulin antibodies, are collectively referred to as anti-thyroid antibodies (ATA). Many reports have since corroborated the markedly increased prevalence of ATA in women who experience reproductive failure, especially first trimester miscarriages. Pratt, et. al., showed that 67% of women with recurrent first trimester losses had ATA, compared to 17% of controls. None of the participants in either group had clinical manifestations of thyroid disease. Although there is a highly positive correlation between the presence of ATA and fetal loss, no definitive pathophysiology has been identified. Several hypotheses have been proposed to explain this phenomenon. One hypothesis states that these patients have very mild hypothyroidism. Studies to date fail to indicate low thyroid hormone levels in those who miscarried. Proponents suggest that serum hormone levels do not necessarily reflect thyroid dysfunction. Another opinion is that ATA are markers for predisposition to autoimmune disease, and that the latter is what actually causes the miscarriage. Notable is that ATA is present in up to 45% of patients with systemic lupus erythematosus (SLE). In another study, 70.8% of patients with recurrent spontaneous abortion (RSA) had various autoantibodies, leading the authors to conclude that some patients with unexplained infertility and RSA suffer from polyclonal B-cell activation. Antithyroid antibodies appear to be markers for abnormal T-lymphocyte function. Significant increases in the endometrial T-cell population and the cytokine interferon gamma have been observed in infertile women with ATA. It can be presumed that infertile patients who demonstrate ATA can be classified as having the reproductive autoimmune failure syndrome (RAFS). Patients with RAFS should have immune evaluations that include blocking antibodies, ANA and APA panels, NK cell number and activity, DQ alpha genotyping, and gene mutations leading to inherited thrombophilias.
Treatment for Antithyroid Antibodies
In IVF patients, antithyroid antibodies (ATAs) are treated with intravenous immune globulin (IVIg) before the IVF transfer. There is no specific treatment for ATA in patients with recurrent miscarriage unless it is associated with other abnormalities. Immunophenotypes: Natural Killer Cells and Cytotoxic B-Cells The immune system is composed of more than 30 types of white blood cells including neutrophils, monocytes and lymphocytes. Lymphocytes, particularly B-cells (antibody producers), T-cells (helper and suppressor) and killer (NK) cells have been the focus of intense research interest to the discipline of reproductive immunology. Immunophenotype refers to the relative amounts of T, B and NK cells in the bloodstream. The immunophenotype assay involves labeling a sample of blood with fluorescent dyes directed to specific markers for each type of lymphocyte: CD4 for T-helper cells, and CD56 for NK cells. The specimen is then placed into a cell flow cytometer in which the cells, via laminar flow fluidics, pass in single file across an argon laser that excites the dyes and causes them to fluoresce. Intensity of the fluorescence is measured by electronic tubes and digitized, allowing a computer to calculate the relative percentages of different lymphocyte subsets. There is a special class of NK cells (CD3-, CD16-, CD56+) in the placenta that promotes cell growth, secretes growth molecules for the placenta and down regulates the mother’s immune response locally at the maternal/placenta interface. Opposing is another group of NK cells (CD3-, CD16+, CD56+), when activated by the cytokine IL-2, are cytotoxic to placental trophoblast. The same cells secrete tumor necrosis factor (TNF) which can destroy the placenta. Women with CD16+, CD56+ NK cells in excess of 20% are at risk for miscarriage despite optimal immune treatment (paternal leukocyte immunization, prednisone, aspirin and heparin). In a subset of women who have had multiple failed IVFs, it is believed that TNF is secreted in amounts that inhibit implantation and early formation of the placenta resulting in an IVF cycle which does not produce a clinical pregnancy.
Treatment for Immunophenotypes
Women who have an elevation of NK cells are candidates for immunologlobulin G infusion (IVIg). The dosage of IVIg is 400 mg/kg/day for three consecutive days, monthly, until the NK cells become normal or until the 28th week of pregnancy. In some studies, autoantibodies to phospholipid and nuclear epitopes were demonstrably lower after IVIg. Some researchers have used Enbrel, a TNF alpha inhibitor, instead of, or in addition to IVIg. Presently, there is not enough data to assess the true efficacy of Enbrel therapy. Success A recent study reports an 80% success rate in women treated with IVIg who either had a history of miscarriage despite optimal immunotherapy (paternal leukocyte immunization, aspirin, heparin and prednisone), or had a history of IUGR. DQ Alpha Genotyping DQ Alpha genotyping refers to a specific kind of HLA (tissue) typing done at the DNA level. The Class II HLA, found on the surface of white blood cells (WBC), include HLA-DR and HLA-DQ. B-cell is a type of WBC that manufactures antibodies. Each tissue type is made up of an “A” or alpha part and a “B” or beta part. Researchers have discovered that mothers who are HLA-DQ alpha (DQA1) and/or DQ beta (DQB1) compatible with their fetuses tend to have a high rate of miscarriage before eight weeks of pregnancy. Others studies have shown that mother’s who are DQ alpha compatible with their fetuses can develop an exacerbation of autoimmune processes, such as rheumatoid arthritis and antiphospholipid antibody syndrome, during the early portions of their pregnancy. Once the association is discovered, more aggressive treatments can be tailored to a patient’s situation to prevent the autoimmune process from causing another miscarriage. Inherited Thrombophilias The Inherited Thrombophilias comprise a group of genetic disorders of the blood clotting pathways, leading to abnormal blood clot formation (thrombi). A common route involves resistance to a natural anticoagulant called activated protein C (APC). These diseases have been shown in several studies to cause vascular complications that lead to miscarriage, intrauterine fetal death, pre-eclampsia (toxemia of pregnancy), and the HELLP syndrome which is a severe form of pre-eclampsia characterized by hemolysis (blood cells breaking up), elevated levels of liver enzymes, and thrombocytopenia (a low platelet count). Women who carry the genes for Inherited Thrombophilias are more likely (2 to 14 times) to have a clotting problem leading to a miscarriage, compared with the normal population. The three major gene mutations that lead to Inherited Thrombophilias are: Factor V Leiden mutation. Factor II (Prothrombin) G20210 gene mutation. Methylene-tetrahydrofolate reductase (MTHFR) mutation, leading to hyperhomocytseinemia. The most common cause of APC resistance arises from the point (one DNA based-pair) mutation at the cleavage site of factor V, called factor V Leiden. It is the most common of the Inherited Thrombophilias, with a prevalence of 10% in the Caucasian population. The mutation has been discovered in 60% of patients who have clot formation during pregnancy, and is also a major cause of blood clots associated with oral contraceptive use. The Prothrombin (factor II) gene mutation has been shown to occur in 7.8% of women who experienced fetal loss due to a clotting disorder. Factor II is one of the major factors in the human clotting pathway. Homocysteine is normally present in low levels in the bloodstream. It is derived from dietary methionine, an amino acid. A gene mutation for the enzyme methylene-tetrahydrofolate reductase (MTHFR), will lead to build up of homocysteine in the bloodstream. This condition, called hyperhomocytseinemia, results in blood clot formation and hardening of the arteries, even in childhood. Nutritional lack of vitamins B6, B12 and folic acid aggravate the problem. Women who have the homozygous form of the MTHFR gene mutation (both of her alleles having the mutation) are more than a two-fold increased risk for a miscarriage.
Treatment of Inherited Thrombophilias
A combination of low-dose aspirin plus low molecular weight heparin injections is used to treat the inherited thrombophilias. The therapy starts before pregnancy occurs, and continued four to six weeks after birth. Folic acid supplementation is given to patients with the MTHFR gene mutation. Blocking Antibodies Early in pregnancy, the mother’s immune system receives signals from the tiny fetus. Many of the signals are hormonal, but others come directly from genetic messages that the father has contributed. Some of the messages involve the tissue type, also known as the human leukocyte antigens (HLA) and the white blood cell (leukocyte) type. HLA are expressed on white blood cells. They are unique to each individual and allow the body to identify anything foreign to it such as infections, cancers, transplanted organs and fetuses. One half of the fetus’s HLA type is contributed by mother and the other half by father. When a woman becomes pregnant, her body’s immune system usually recognizes the father’s HLA as different from her own, and the white blood cells in her uterus produce protective, blocking antibodies. These antibodies coat the baby’s cells and protect the fetus from mother’s killer cells. If father’s HLA is too similar to mother’s, her cells may not recognize differences that are vital to the production of blocking antibodies.
Women who have successful pregnancies and have no history of miscarriages normally, have high levels of blocking antibodies even in the nonpregnant state vs women who miscarry and whose levels tend to be low even when pregnant. Through HLA tissue typing we can identify couples who look too much “alike”. In addition we can measure the ability of a couple’s cells to respond to each other i.e. level of blocking antibodies, using sophisticated equipment which combine computers and lasers (cell flow cytometry).
Treatment for Blocking Antibodies
Two treatments have been offered for low blocking antibody levels: paternal white cell immunizations and IVIg. Immunizing the mother with concentrates of the father’s white blood cells amplifies the HLA signal. Approximately 50% of patients have a discernible increase in the blocking antibody level after 2 treatments. The other 50% require additional white cell immunizations. To determine if additional preparations will be required, the blocking antibody level should be measured 3 to 4 weeks after the second and all subsequent immunizations. When blocking levels are elevated, prior to conception, the rate of successful pregnancy is nearly 80%. Table 2: Success rate with varying treatment modalities in women with history of RSA
The risk associated with white blood cell immunization is the possible transmission of infectious agents that the father’s blood may be harboring. This can be avoided by testing his blood for any significant infections. Very uncommonly, there can be a local skin infection caused by bacteria on mother’s own skin. This is easily treated. IVIg is an alternative to white cell preparations. The doses vary between 10 and
· Blocking Antibody level (by flow cytometry)
· T cell IgG
· B cell IgG
· Antiphospholipid Antibody Panel
· Anticardiolipin antibodies — IgG, IgM, IgA
· Antiphosphoglycerol antibodies — IgG, IgM, IgA
· Antiphosphoserine antibodies — IgG, IgM, IgA
· Antiphosphoethanolamine antibodies — IgG, IgM, IgA
· Antiphosphatidic acid antibodies — IgG, IgM, IgA
· Antiphosphoinositol antibodies — IgG, IgM, IgA
· Activated partial thromboplastin time (APTT)
· Lupus anticoagulant (LA)
· VDRL
· Antinuclear Antibody Panel
· Double stranded DNA
· SSA
· SSB
· RNP
· SM
· Antihistone Antibody
· HLA Tissue Typing
· ABC
· DR,DQ
· DQA1 DNA fingerprinting
· DQB1 DNA fingerprinting
· Chromosome analysis
· Immunophenotype
· Natural Killer Cell Activation Assay
· Natural Killer Cell Activation/IVIg Assay
· Intracellular Tumor Necrosis Factor (TNF) Alpha Assay
· Quantitative Immunoglobulin
· Factor V Leiden Gene Mutation
· Factor II (Prothrombin) Gene Mutation
Methylene Tetrahydrofolate Reductase (MTHFR) Gene MutationMiscarriages and Immunotherapy It is important that patients who have experienced immune-mediated reproductive failure follow any medication recommendations made, unless they have had an allergic reaction, serious side effect or medical problem that precludes the use of the drug(s): Aspirin Heparin Prednisone Immunoglobulin G Infusion Enbrel Paternal Leukocyte Immunization Some patients are uncomfortable about taking any medication during pregnancy. Rest assured that all medication and doses have been thoughtfully selected to minimize any drug related side effects. As your pregnancy progresses your blood test results may change, justifying a modification in your medication regime. If changes are indicated, they will be communicated to you and initiated immediately. Aspirin Aspirin is an anti-inflammatory and antiplatelet agent. Should you require low dose aspirin, the recommendation is 80 mg per day, which is equivalent to a baby aspirin. Aspirin, like all medication, can cause allergic reactions. Manifestations of aspirin allergy may include dermatitis, rhinitis, bronchospasm and even anaphylaxis. People who have a history of asthma and nasal polyps are at increased risk for allergic reactions. Side effects of low dose aspirin are infrequent but can include nausea, reflux esophagitis, abdominal discomfort, anorexia and gastrointestinal and urinary tract bleeding. Use of high dose aspirin during pregnancy may affect maternal and neonatal blood clotting mechanisms, leading to an increased risk of bleed. High dose aspirin may also impair maternal kidney function and has been causally related to increased perinatal mortality, intrauterine growth retardation and congenital defects. Aspirin at low doses has not been associated with these risks. Aspirin is excreted into breast milk in low concentration ranging from 1.1 to 10 mcg/ml. Adverse effects of platelet function in the nursing infants have not been reported, but are a potential risk. If you choose to breast feed your baby you should not take aspirin at that time. Routine laboratory testing while on aspirin should include complete blood count, chemistries, APTT and antiphospholipid antibody panel. Heparin Heparin, an anticoagulant, is a purified preparation derived from animal tissue. It is delivered as a subcutaneous (sq) injection and a typical dose would be 5000 IU twice a day. Low molecular weight heparins like Lovenox and Fragmin are more purified forms of heparin. The typical dose for Lovenox is 40 mg sq once a day. The dosing for Fragmin is 5000 IU sq once a day. Their advantage is that they only have to be administered once a day. Also, fewer side effects have been reported. Low molecular weight heparin preparations are the medication of choice for patients who suffer Inherited Thrombophilias. Both drugs are significantly more expensive than standard heparin. When heparin is recommended, it should be started 14 days before IVF transfer or 5 days before a natural cycle. Duration of treatment depends upon the clinical history. Patients with miscarriage use medication through week 34. Those who only have failed IVF and + APA should continue at least through week 12. Patients suffering from hereditary thrombophilia continue with medication six weeks post partum. Allergic reactions may include chills, fever, dermatitis, asthma and anaphylactic shock. Before a therapeutic dose is administered, a trial of 1000 IU would be prudent. Fortunately, allergy to heparin is rare. Because of heparin’s blood “thinning” property the user is more susceptible to bleeding (skin, nose, gastrointestinal tract, bladder, etc.). Almost all patients experience some bruising at the site where heparin is injected. Long term heparin therapy has been associated with osteoporosis and spontaneous fractures in patients who have received in excess of 15,000 units per day for more than six months. One study of 117 patients on long term heparin (up to 15 years) report no spontaneous fracture when subjects received less that 10,000 units per day.
Although our protocol utilizes low dose heparinization, supplementation (dietary) with calcium is recommended. Exercise and sun tanning may also prevent osteoporosis. Please note that the study group consisted of women who had severe maternal disease necessitating high dose anticoagulant therapy. Although heparin is the preferred anticoagulant during pregnancy it is not risk free. Heparin is not excreted in breast milk. Relative contraindications to the use of heparin are active bleeding, hemophilia, thrombocytopenia or other blood dyscrasia, endocarditis or tuberculosis. Caution should be exercised when there is underlying hypertension and liver kidney disease. Routine platelet, hematocrit, APTT and antiphospholipid antibody panel monitoring during heparinization is recommended. Heparin Treatment See Instructions for Heparin Injections Prednisone Prednisone is a corticosteroid. If indicated, prednisone should be started prior to conception; 30 days before an IVF cycle or 5 days before ovation in a natural cycle. Drug dosage may be adjusted depending upon follow-up blood tests. Duration of treatment may continue well into the second trimester. Allergy to prednisone is rare, as the human body manufactures a similar compound. In fact prednisone is used to treat moderate to life threatening allergies. Possible adverse reaction(s) to moderate and high doses of prednisone include fluid and electrolyte imbalance; metabolic disturbances e.g. hyperglycemia or gestational diabetes and osteoporosis; susceptibility to infection; peptic ulcer; behavioral changes e.g. nervousness, insomnia, irritability and mood swings; myopathy; and cataracts. Prednisone should be used with caution in people with hypertension, congestive heart failure, diabetes mellitus, osteoporosis, ulcerative colitis, ocular herpes and others (please consult with doctor if you have any chronic illness). Osteoporosis can be retarded with calcium supplementation and exercise. Prednisone should be discontinued if pregnancy is achieved, but should be resumed five days prior to ovation of the next cycle. Rapid withdrawal of prednisone may cause fatigue, myalgias, arthralgias, dizziness, hypotension, hypoglycemia and dyspnea.If you experience these symptoms, please contact your doctor. There are a number of studies that review the use of prednisone during pregnancy and effects on the fetus. The fetus appears to be protected by at least three mechanisms: 1) enzymes in the placenta degrade the drug to an inactive form, 2) prednisone in maternal circulation is bound to a large protein making it harder to cross the placenta and 3) fetal liver is not able to activate prednisone until the end of the second trimester. Trace amounts of prednisone have been measured in breast milk.
Although these quantities are of doubtful clinical significance, your baby’s pediatrician should be notified. Laboratory studies to monitor while on prednisone include complete blood count, chemistries, electrolytes and antinuclear antibody panel. Immunoglobulin G Infusion Immunoglobulin G is a preparation of human derived antibodies. In some patients, conventional immunotherapy with aspirin, heparin and paternal white cell immunization may have to be supplemented with this medication. Patients at risk for developing intruauterine growth retardation, oligohydramnios, toxemia, or severe side effects of steroids, or have preexisting maternal disease are prime candidates. Immunoglobulin G is contradindicated in patients who are known to have had anaphylactic or severe systemic reaction to human immune globulin. Patients with IgA deficiency should not receive this product. Side effects to immunoglobulin G include fever, chills, headache, nausea, malaise and back pain. Mild erythema following infiltration at the site of infusion has been reported. Laboratory tests that need to be followed while on this treatment are quantitative immunoglobulins, immunophenotype, natural killer cells (NK) activation studies and intrecellular tumor necrosis factor alpha (TNFa) assay and blocking antibody levels. Enbrel Enbrel (etanercept) is an inhibitor of tumor necrosis factor (TNF) and works by binding directly to it. TNF alpha is a chemical (cytokine) that is secreted by NK and some other cells. This cytokine can lead to destruction of the placenta. Enbrel is administered as a subcutaneous injection, 25 mg twice per week. It is usually started one month prior to the cycle of planned conception and stopped when there is ultrasonic evidence of a fetal heartbeat. Some of the more common side effects are injection site reactions (swelling and itching), headache, nausea, dizziness, infections and rash. Toxicity studies performed in pregnant rats and rabbits have revealed no evidence of harm to the fetus, even at dose equivalent to 60 times the normal human dose. No controlled studies have yet been performed in pregnant women. Paternal Leukocyte Immunization Paternal leukocyte immunization (PLI), a purified preparation of husband’s white blood cell, is administered intradermally. Because this is a blood product, the recipient (wife) risks acquiring infectious diseases that donor (husband) may harbor. Rh sensitization is also possible; however, extensive steps are taken to prevent this. Most women will experience redness and itching at the site of immunization. Please notify your doctor if you have any unexpected or serious reaction. Maternal antipaternal leukocyte antibodies (blocking antibodies), by flow cytometry, should be followed to monitor efficacy of treatment. IVF and Immunotherapy Society for Assisted Reproductive Technology (SART) data has shown that the per-cycle success rate for in-vitro fertilization (IVF) has remained at 24%. Although there has been an increase in success when the cause is male factor infertility and intra-cytoplasmic sperm injection (ICSI) is implemented, the rate for women whose etiology is female organic pelvic disease has not changed. The reproductive autoimmune failure syndrome (RAFS), first described in 1988, is the association of pregnancy wastage, infertility and endometriosis with circulating autoantibodies. Patients with RAFS have polyclonal B-cell activation; that is, their antibody producing cells including those that manufacture autoantibodies are very active. The pathophysiologic mechanisms that cause in-vitro fertilization failures are complex. Antiphospholipid antibodies (APA) play a central role in this process. Phospholipids are adhesion molecules — they help cells stick to each other. At a very basic level, they help the fetus “stick” to the uterus. Antiphospholipid antibodies interfere with this process, so that the transferred fetus has difficultly implanting, i.e., attaching to the uterus. Furthermore, APA cause problems with uterine and placental blood flow, making the uterus unhealthy for successful implantation. Antinuclear antibodies (ANA) cause inflammation in various tissues, including the uterus. This inflammatory process prevents the uterus from being able to host a proper implantation. CD56+CD16+ natural killer cells (NK) cells normally kill cancer cells before they grow into large tumors. These cells may misinterpret the implanting fetus as a cancer and kill it too. It is believed that antithyroid antibodies are markers for polyclonal B-cell activation and do not have a direct effect on implantation or the fetus. Any patient who has antithyroid antibodies should be carefully evaluated for APA, ANA and increased NK cell number and/or activity.
Treatment
The treatment of primary immunodeficiencies depends foremost on the nature of the abnormality. This may range from immunoglobulin replacement therapy in antibody deficiencies—in the form of intravenous immunoglobulin (IVIG) or subcutaneous immunoglobulin (SCIG)—to hematopoietic stem cell transplantation for SCID and other severe immunodeficiences. Reduction of exposure to pathogens may be recommended, and in many situations prophylactic antibiotics may be advised.
The primary immunodeficiency disorders reflect abnormalities in the development and maturation of cells of the immune system.
These defects result in an increased susceptibility to infection; recurrent pyogenic infections occur with defects of humoral immunity, and opportunistic infections with defects of cell-mediated immunity. These two broad categories of illness correspond
roughly to defects in the two principal types of immunocompetent cells, B lymphocytes and T lymphocytes. Defective development of B cells results in abnormalities in humoral immunity, whereas defects in the development of T cells cause problems with cellular immunity.
When pathogens are taken up by macrophages or dendritic cells, their antigens are degraded and presented on the cell surface to T cells, which subsequently induce the maturation of B cells through the release of cytokines. T cells also recruit other cells (macrophages, eosinophils, basophils, and mast cells) to induce an inflammatory response. The specific immune responses of T cells and antibodies (along with components of serum complement) result in resistance to infection. These responses are assisted by natural killer cells, which nonspecifically kill tumor cells and virus-infected cells.
We describe recent advances in the understanding of six of the primary immunodeficiencies: X-linked agammaglobulinemia, the hyper-IgM syndrome, common variable immunodeficiency, severe combined immunodeficiency, defects in the expression of the major histocompatibility complex (MHC), and the Wiskott–Aldrich syndrome. The underlying defects in each can be better understood by reference to Figure, which shows the development and maturation of T and B lymphocytes from the multipotent hematopoietic stem cell.
X-Linked Agammaglobulinemia
X-linked agammaglobulinemia was first described 43 years ago and remains the prototypic syndrome of a pure B-cell deficiency. The disorder has a relatively homogeneous clinical presentation — a characteristic that suggested a monogenic defect, inherited as an X- linked recessive trait. Recently, the genetic defect has been shown to result from mutations in a hitherto unknown cytoplasmic signal-transducing molecule. This discovery has broadened the description of the phenotype and led to the realization that X-linked agammaglobulinemia is more common than was previously thought. Affected boys are usually well for the first 9 to 12 months of life because they are passively protected by transplacentally acquired IgG from their mothers. Subsequently, they have recurrent pyogenic infections such as otitis media, sinusitis, conjunctivitis, pneumonia, and pyoderma. These infections are mainly due to Haemophilus influenzae and Streptococcus pneumoniae and less frequently to Staphylococcus aureus and Streptococcus pyogenes. Although readily controlled by antibiotics, these recurrent infections lead to anatomical destruction, particularly of the lungs; chronic obstructive lung disease and bronchiectasis invariably result when proper prophylactic treatment is not undertaken. Boys with X-linked agammaglobulinemia may have persistent viremia and are at risk of acquiring paralytic poliomyelitis from live-virus vaccines. They are also susceptible to an unusual and potentially fatal form of persistent enterovirus (usually echovirus) infection that is associated with chronic meningoencephalitis and a syndrome resembling dermatomyositis. Giardia lamblia infestation leads to chronic diarrhea, weight loss, protein-losing enteropathy, and steatorrhea. About 35 percent of affected boys present with arthritis of the large joints; this symptom is usually controlled by immune globulin therapy. In many cases it is probably due to mycoplasma (Ureaplasma urealyticum).
During the past decade prophylaxis with intravenous immune globulin has become the standard therapy for X-linked agammaglobulinemia. With the use of this route it has been possible to administer large doses of immune globulin so that patients can lead relatively normal lives. The optimal dose and frequency of administration must be determined for each patient. Usually, a dose of 350 to 500 mg per kilogram of body weight is given each month, but it may be preferable to divide this dose for administration every other week. Some patients require as much as 600 mg of immune globulin per kilogram to maintain adequate prophylaxis. Immune globulin is a safe biologic product. Nevertheless, the risk of transmitting hepatitis C virus is ever present, and a few unfortunate outbreaks have occurred. This problem may soon be overcome with detergent treatment of immune globulin preparations. Attempts are also being made to pump immune globulin into subcutaneous tissue.
Typically, the serum of patients with X-linked agammaglobulinemia contains less than 100 mg of IgG per deciliter and no detectable IgM or IgA. The patients are incapable of making antibodies in response to standard antigenic provocations. B cells are virtually absent from the blood. Cell-mediated immunity is relatively normal in affected male patients; they have normal numbers of circulating T cells, which can respond appropriately to specific and nonspecific mitogens. It has recently been recognized, since the identification of the genetic defect, that these laboratory findings define X-linked agammaglobulinemia too narrowly; this diagnosis should be suspected in all young boys with recurrent pyogenic infections, decreased immunoglobulin levels, or low numbers of B cells.
Standard linkage analysis was used to map the gene for X-linked agammaglobulinemia to the long arm of the X chromosome at position Xq21.2–22, and X-linked agammaglobulinemia exhibited no crossovers with a polymorphic DNA marker. A candidate signal-transducing molecule encoded by a tyrosine kinase gene called btk was identified and cloned. Missense and nonsense mutations in btk, as well as splice-site mutations, have been identified. The gene is called btk for Bruton’s, or B-cell, tyrosine kinase and is expressed early in B-cell development, but not in T cells or plasma cells. It is expressed in myeloid cells, where its role is obviously less crucial than in B cells, because granulocyte defects are rarely seen in X-linked agammaglobulinemia. Early B cells are generated in the bone marrow of affected male patients. However, these early B cells mature at an extremely low rate. The btk gene must therefore have a vital but as yet undetermined role in the maturation of B-lineage cells.
In X-linked agammaglobulinemia, mutations have been found in all parts of the btk gene.,
There is no apparent correlation between the phenotype and the different mutations, although there can be marked phenotypic differences among affected male patients in a single kindred. A point mutation has been found in btk in male CBA/N mice with a mild humoral immunodeficiency (called Xid for X-linked immunodeficiency). Recently, it has been possible to disrupt btk in mice. The severity of the phenotype in these mice depends on their genetic background. This may provide an excellent model for experimental gene therapy.
Female carriers of X-linked agammaglobulinemia, who are immunologically normal, have two populations of B-cell precursors. The
Hyper-IgM Syndrome
In 1961 two boys with a syndrome that resembled X-linked agammaglobulinemia clinically were found to have elevated serum concentrations of IgM but no IgA and very low concentrations of IgG. This new defect was presumed to be inherited as an X-linked recessive trait. Then, two female patients with the same clinical phenotype were identified.
Many patients with this syndrome have since been described, approximately 70 percent of whom have the X-linked form of the hyper-IgM syndrome. The mode of inheritance in the remaining patients is not clear-cut. The genetic defect in the X-linked form has recently been discovered and reveals new facets of collaboration between T and B lymphocytes.
Male patients with X-linked hyper-IgM syndrome have a clinical history of pyogenic infections that resembles that encountered in male patients with X-linked agammaglobulinemia. In addition, they are susceptible to opportunistic infections, particularly those due to Pneumocystis carinii. They also are prone to autoimmune diseases involving the formed elements of the blood — autoimmune hemolytic anemia, thrombocytopenic purpura, and most important of all, recurrent, often severe, and prolonged neutropenia. The neutropenia responds well to immune globulin and granulocyte–macrophage colony-stimulating factor. In these patients administration of intravenous immune globulin in the same doses as are used in X-linked agammaglobulinemia is recommended. This therapy usually results in a decrease in the serum IgM concentration. In the second decade of life there may be uncontrollable proliferation of IgM-producing plasma cells, which extensively invade the gastrointestinal tract, liver, and gallbladder. Although the proliferating cells always exhibit polyclonality, the cellular infiltrates may be so extensive as to prove fatal. Patients with X-linked hyper-IgM syndrome also have an increased risk of abdominal cancers.
The serum of patients with X-linked hyper-IgM syndrome usually contains undetectable amounts of IgA and IgE and very low concentrations of IgG (<150 mg per deciliter). However, IgM concentrations may be in the high normal range, and may even be 1000 mg per deciliter or more; IgD concentrations are also elevated. The blood contains a normal number of B lymphocytes, but they have only surface IgM and IgD. As in X-linked agammaglobulinemia, there is no germinal-center development in the lymph nodes and spleen despite the presence of normal numbers of B cells and T cells.
In an immune response IgM and IgD antibodies are produced first. As the immune response progresses, IgG antibodies appear, followed by IgA antibodies and finally by IgE antibodies. The sequential appearance of different classes of immunoglobulins is called class switching. It was possible to induce class switching to IgG and IgA in B cells from patients with X-linked hyper-IgM syndrome by incubating B cells with activated CD4+ T cells from a woman with the Sézary syndrome. Studies of class switching iormal B cells show that two signals are required for the B cell to switch from synthesizing and secreting IgM to synthesizing and secreting IgE: the binding of interleukin-4 secreted by T cells to the interleukin-4 receptor on B cells and the interaction of CD40 on the B-cell surface with the CD40 ligand, which is expressed on activated T cells. This observation led to the discovery that T cells of patients with X-linked hyper-IgM syndrome could not synthesize the CD40 ligand. The gene for X-linked hyper-IgM syndrome was mapped to Xq26 on the long arm of the X chromosome. The gene encoding the CD40 ligand was cloned and mapped to the same location. Several groups simultaneously discovered that the defect in X-linked hyper-IgM syndrome resides in mutations in the gene for the CD40 ligand. Missense as well as nonsense mutations and deletions have been found in the CD40 ligand gene in patients with X-linked hyper-IgM syndrome.
This syndrome illustrates the importance of physical contact between B cells and T cells through CD40 and its ligand in the activation of B cells and in germinal-center formation and immunoglobulin class switching. Further work is required to explain the high frequency of autoimmune disease and neutropenia, susceptibility to opportunistic infections, and the lymphoproliferative complications in X-linked hyper-IgM syndrome. Because the CD40 ligand molecule is not required for the normal development of T lymphocytes, female obligate heterozygous carriers of X-linked hyper-IgM syndrome, unlike carriers of X-linked agammaglobulinemia, have random inactivation of the X chromosome in lymphocytes. A polymorphic region at the 3′ end of the CD40 ligand gene makes prenatal diagnosis of X-linked hyper-IgM syndrome possible.
Common Variable Immunodeficiency
The term “common variable immunodeficiency” is used to designate a group of as yet undifferentiated syndromes. All are characterized by defective antibody formation. The diagnosis is based on the exclusion of other known causes of humoral immune defects. As would be expected in a heterogeneous group of undifferentiated diseases, several patterns of inheritance (autosomal recessive, autosomal dominant, and X-linked) have beeoted. Sporadic cases are, however, most common.
Among populations of European origin, common variable immunodeficiency is the most frequent of the primary specific immunodeficiency diseases.51 It affects men and women equally. The usual age at presentation is the second or third decade of life. The terms “late-onset hypogammaglobulinemia,” “adult-onset hypogammaglobulinemia,” and “acquired immunodeficiency,” which were used in the past, are no longer appropriate.
The clinical presentation of common variable immunodeficiency disease is generally that of recurrent pyogenic sinopulmonary infections. Appropriate early investigation and diagnosis are important; many cases are only identified after serious chronic obstructive lung disease and bronchiectasis have developed. A few patients with common variable immunodeficiency present with infections involving unusual organisms such as P. carinii, mycobacteria, or various fungi. Recurrent attacks of herpes simplex are common, and herpes zoster develops in about one fifth of patients. As in patients with X-linked agammaglobulinemia, some patients with common variable immunodeficiency have unusual enteroviral infections with a chronic meningoencephalitis and a syndrome resembling dermatomyositis. The patients are also highly prone to infection with enteric pathogens, such as chronic G. lamblia infection.
There is an unusually high incidence of malignant lymphoreticular and gastrointestinal conditions in common variable immunodeficiency. A 50-fold increase in gastric carcinoma has been observed. Lymphoma is about 300 times more frequent in women with common variable immunodeficiency than in affected men. In contrast to patients with X-linked agammaglobulinemia, many patients with common variable immunodeficiency have diffuse lymphadenopathy and often splenomegaly. The lymph nodes and spleen show a striking reactive follicular hyperplasia. The gastrointestinal tract is also commonly involved in the process, with nodular lymphoid hyperplasia. As in celiac disease, malabsorption with weight loss, diarrhea, and associated findings such as hypoalbuminemia and vitamin deficiencies is seen. Inflammatory bowel diseases are more frequent. Patients with common variable immunodeficiency are also prone to a variety of other autoimmune disorders (e.g., pernicious anemia, hemolytic anemia, thrombocytopenia, and neutropenia). Noncaseating (sarcoid-like) granulomas occur in the skin, the gut, and other viscera.
Defective antibody formation is accompanied by decreased serum IgG concentrations and usually by decreased serum IgA and IgM concentrations. There is no convincing evidence of any intrinsic B-cell defects. Although the number of B cells may be reduced, with appropriate stimulation they can produce and secrete immunoglobulins. However, the B cells are immature. The findings in common variable immunodeficiency are consistent with insufficient in vivo stimulus for B-cell activation rather than an intrinsic failure of B cells to differentiate.
Relatives of patients with common variable immunodeficiency have an unusually high incidence of IgA deficiency and an increased incidence of autoimmune disorders, autoantibodies (including antilymphocyte antibodies), and malignant conditions. Families whose members include persons with common variable immunodeficiency and IgA deficiency often have certain fixed haplotypes in the MHC. One or more genes in the MHC may be involved in the pathogenesis of common variable immunodeficiency and IgA deficiency.
Since immature B cells in common variable immunodeficiency appear to be functionally intact, the defect might logically reside in the T-cell component of the interaction between B cells and T cells requisite for B-cell maturation. However, it is often difficult to interpret studies of T cells in common variable immunodeficiency, because of the activation of T cells that is probably a result of recurrent or chronic infections or infusions of intravenous immune globulin.
In most patients with common variable immunodeficiency, stimulation of T-cell receptors produces diminished responses and there is decreased gene transcription of cytokines such as interleukin-2, interleukin-4, interleukin-5, and interferon-. Decreased production of interleukin-2 after direct stimulation of T-cell receptorsis correlated with diminished expression of CD40 ligand and may reflect an abnormality in CD4+ T cells in common variable immunodeficiency. This abnormality of T-cell triggering can be bypassed by direct activation of signal transduction.
Thus, many patients with common variable immunodeficiency appear to have defective interactions between T cells and B cells. Defective T-cell signal transduction could contribute to the diminished humoral immunity found in these disorders in the presence of immature but otherwise potentially normal B cells. In the absence of appropriate T-cell signaling, B cells would fail not only to produce antibody, but also to proliferate and differentiate, which would result in both the decreased numbers and the arrested maturation of B cells seen frequently in common variable immunodeficiency.
Severe Combined Immunodeficiency
In contrast to X-linked agammaglobulinemia and X-linked hyper-IgM syndrome, severe combined immunodeficiency has many genetic causes even though the phenotype is fairly uniform. Usually, affected infants are ill by three months of age with persistent thrush or an extensive rash in the diaper area due to monilia. They may have intractable diarrhea or a persistent pertussis-like cough due to interstitial pneumonia caused by P. carinii.
Although growth and development may have proceeded normally for the first three months of life, growth and weight gain subsequently fall off, and failure to thrive becomes a striking feature. Sometimes these infants have a morbilliform rash shortly after birth, due to transplacental passage of maternal lymphocytes, which mount a graft-versus-host reaction. The rash becomes hyperpigmented. Death from varicella, herpes, adenovirus, or cytomegalovirus may occur very rapidly after infection. Giant-cell pneumonia has resulted from measles infection and live-measles vaccine, and progressive vaccinia has occurred after smallpox vaccination; both were uniformly fatal. A diagnosis of severe combined immunodeficiency represents a medical emergency; this immunodeficiency can be rapidly fatal if affected infants are not rendered immunocompetent by bone marrow transplantation in a timely fashion.
Infants with severe combined immunodeficiency almost invariably have profound lymphopenia (<1000 lymphocytes per cubic millimeter). The number of natural killer cells may be normal or high. In the X-linked form of severe combined immunodeficiency the number of B cells is normal or elevated, but these B cells fail to mature and function normally. CD3+ T cells, when present, may be of maternal origin. These infants are not capable of cell-mediated immunity. Lymphocytes do not respond in vitro to nonspecific mitogens such as phytohemagglutinin and concanavalin A. They also do not respond to an allogeneic stimulus or to specific antigens such as tetanus toxoid, which may have been used as an immunogen. The failure of the thymus to become a lymphoid organ is a common feature. The thymic shadow cannot be seen on a chest film. The serum immunoglobulin concentrations are all low. Rarely, an M component may be present in the serum, and in an infant this is virtually diagnostic of severe combined immunodeficiency.
Severe combined immunodeficiency is three times as common in boys as in girls, because the most common form — accounting for 50 to 60 percent of the cases — is X-linked. It has been mapped to Xq13. The genetic defect in X-linked severe combined immunodeficiency has recently been identified as a mutation of the gamma chain of the interleukin-2 receptor, which had been cloned earlier. Mutations were found in the gamma chain of the interleukin-2 receptor in a number of patients with X-linked severe combined immunodeficiency. The disorder has also been found in basset hounds, and these dogs also have a mutation in the gamma chain of the interleukin-2 receptor.
At first this finding was very surprising because it did not seem likely that such a profound immunodeficiency could result from mutations in the gamma chain of the interleukin-2 receptor. It was soon discovered that the gamma chain is a component of several interleukin receptors — namely, receptors for interleukin-4, interleukin-7, interleukin-11, and interleukin-15. Thus, the early lymphoid progenitor cells in X-linked severe combined immunodeficiency, lacking intact interleukin receptors, fail to be stimulated by all these growth factors that are vital to the normal development and differentiation of T cells and the late phases of B-cell development. The T cells, natural killer cells, and late-stage B cells of obligate female heterozygous carriers of X-linked severe combined immunodeficiency exhibit nonrandom X-chromosome inactivation; only cells with the normal X chromosome survive.
The remainder of the cases of severe combined immunodeficiency result from autosomal recessive inheritance. The most common causes of the autosomal recessive form are inherited deficiencies of the purine-degradation enzymes adenosine deaminase and nucleoside phosphorylase. Deficiencies of both these enzymes have been extensively reviewed. Many patients with adenosine deaminase deficiency have benefited from regular injections of adenosine deaminase conjugated to polyethylene glycol. Adenosine deaminase deficiency was also the underlying problem treated in the first successful gene therapy.
In 1968 the first successful bone marrow transplantation in an infant with X-linked severe combined immunodeficiency was performed. Subsequently, scores of these infants have been treated successfully with bone marrow transplantation from histoidentical related donors as well as from unrelated donors and from donors with one or more HLA mismatches whose bone marrow was depleted of T cells. These bone marrow transplantations should be performed as quickly as possible, since X-linked severe combined immunodeficiency is invariably fatal. In families with a previously affected infant, prenatal diagnosis is now possible so that preparations for transplantation can be made in advance of the birth.
In a few rare instances, cases of severe combined immunodeficiency have been reported as a result of defective interleukin-1 receptors, mutated interleukin-2 genes, failure of signal transduction in T cells, or defective T-cell–specific promoters. In these cases, the lymphocyte counts may be normal but the T cells are not functional.
Defects in the Expression of the MHC
Defects in the expression of the MHC were originally called the “bare lymphocyte syndrome.” Use of this unfortunate term should be dropped since it fails to differentiate the several distinct types of MHC class I and class II defects that have been described. The MHC class II molecules are constitutively expressed on antigen-presenting cells such as B lymphocytes, dendritic cells, and cells of monocyte or macrophage lineage and on thymic epithelial cells, where they have an important role in the maturation of CD4+ T cells. The MHC class II molecules are also expressed on activated T cells. The chief function of these molecules is to bind and present antigenic fragments to the T-cell receptor on CD4+ T cells and thereby activate them. This interaction is vital to both cell-mediated immunity and humoral immunity. In contrast, MHC class I molecules are expressed on virtually all cells. They bind and present antigenic peptides to the T-cell receptor of CD8+ T cells and activate their cytotoxic function. Class I molecules also have a vital role in the intrathymic maturation of CD8+ T cells.
A number of children, largely of North African origin, with a moderately severe immunodeficiency, were found to be unable to express MHC class II molecules. These children had severe, protracted diarrhea, frequently associated with candidiasis and cryptosporidiosis, and failure to thrive. Sclerosing cholangitis supervened in a number of them after prolonged gastrointestinal symptoms. Pneumonia, in addition to severe upper respiratory tract infections, was frequent. When given bacille Calmette–Guérin (BCG) in infancy, they survived, in contrast to infants with severe combined immunodeficiency, who invariably die of progressive bacille Calmette–Guérin infection. Graft-versus-host disease does not develop after transfusion with whole blood, whereas it is the inevitable outcome of transfusion in infants with severe combined immunodeficiency.
MHC class II deficiency is inherited as an autosomal recessive trait, but the defect does not segregate with the MHC genes, which are encoded on chromosome 6.
As expected, children with MHC class II deficiency have insufficient numbers of CD4+ T cells but not of CD8+ T cells and cannot mount delayed hypersensitivity reactions. Although the number of B cells is normal, affected children have hypogammaglobulinemia. The in vitro responses to phytohemagglutinin and other nonspecific mitogens are normal, but T cells fail to respond to specific antigens.
There are three major MHC class II molecules: HLA-DP, DQ, and DR. The coordinate expression of these molecules on the surface of B cells and macrophages is regulated in a complex way. At least three unique promoter boxes, called the Y, X, and S boxes, upstream of the MHC genes are involved in the regulation of transcription of MHC class II molecules. The regulation of this transcription is defective in MHC class II deficiency.
Complementation analysis has shown that there are several different types of MHC class II defects. B lymphocytes from patients with MHC class II deficiency were transformed with Epstein–Barr virus and maintained in culture. When transformed B cells from certain patients were fused, they corrected one another’s defect to allow the expression of MHC class II molecules. This led to the identification of so-called complementation groups of MHC class II deficiency. Only cells within one complementation group do not cross-correct; cells from any two complementation groups cross-correct.
It is well known that interferon- induces the expression of MHC class II molecules after a considerable lag period. However, interferon- fails to induce the expression of MHC class II molecules in patients with MHC class II deficiency. During the lag period that follows exposure to interferon- the cells synthesize a new protein called class II transactivator, which does not itself bind to the Y, X, or S boxes but appears to coordinate the binding of the promoters that do. This intracellular protein is defective in one of the complementation groups. The gene encoding the class II transactivator maps to chromosome
Although MHC class II deficiency is less clinically severe than the profound immunodeficiency of severe combined immunodeficiency, it is uniformly fatal in the first or second decade of life. Bone marrow transplantation has resulted in long-term survival.
Because the expression of MHC class I molecules may be decreased in MHC class II deficiency, the identification of isolated MHC class I defects has been elusive. The molecular basis of MHC class I deficiency has recently been defined in a Moroccan family, in which two affected siblings had recurrent, severe bacterial pulmonary infections starting in late childhood. In this large kindred the MHC class I deficiency segregated with the MHC genes, unlike MHC class II deficiency. From this observation it was possible to define the defect. The assembly of MHC class I molecules in the Golgi apparatus proceeds successfully when the chain of these molecules associates with beta2-microglobulin and the complex is joined by antigenic peptides transported across the Golgi membrane by a transporter protein, called TAP. The unit then moves to the cell membrane. If the assembly of the components cannot be completed, usually because no antigenic peptide is loaded onto the chain, the MHC class I complex is destroyed in the cytoplasm of the cell. TAP is encoded by two genes in the MHC, TAP1 and TAP2. The defect in the family studied results from a nonsense mutation in the TAP2 gene.113 As expected, the affected children have a deficiency of CD8+ T cells.
References.
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