Immune mechanisms involved in solid organ transplantation

Abstract

Transplant rejection is an immunologically mediated phenomenon. Both T cells and circulating antibodies are induced against allografts and xeno-grafts. Antibodies produced are responsible for hyperacute rejection. T cells are mainly responsible for chronic rejection of most of the other tissues. The most important transplantation antigens, which cause rapid rejection of the allograft are found on cell membranes and are encoded by genes in the major histocompatibility complex (MHC) which is known as HLA in humans and H-2 in mice. HLA helps in discriminating between self and non-self. The approaches to enhance graft survival are gaining acceptance and wide use in human tissue and organ transplantation. The knowledge of molecular immunology, better understanding of the cellular and molecular mechanisms that underline the immunological response to transplanted organ led to the discovery of new immunosuppressive agents, such as tacrolimus, rapamycin, interleukin-2 receptor monoclonal antibodies, and mycophenolate mofetil. All these drugs show selective mechanisms for T and B cell alloimmune responses. Presently combinations of various drugs are on trial and the results show that rejection rate has been reduced tremendously. However, vigorous and prolonged immuno-suppression results in infections and malignancies. If immune-tolerance can be developed then side effects of immunosuppression can be reduced. The new generation drugs like FTY 20, antisence oligonucleotides are in the process of development. The trend is to develop agents, which are capable of blocking the co- stimulatory pathway of allorecognition which can result in tolerance.

Introduction

The clinical application of our knowledge of the immune barriers to transplantation has advanced allo-organ replacement therapy to the level of routine practice. The success of an organ transplant is the function of several variables. However, the major determinant of acceptance or non-acceptance (rejection) of an otherwise technically perfect graft is the magnitude of the immunologically mediated responses against graft. Transplant rejection can be mediated by antibodies, T lymphocytes or both can manifest itself in different ways; hyper acute rejection (during the early post transplant period), acute rejection (may occur at any time) and chronic rejection (a slowly developing process causing a progressive decline in graft function).

The delineation and application of recent discoveries in cell co-stimulatory events, antigen presentation and differential T lymphocyte signalling are opening pathways towards the development of tolerogenic protocols for clinical transplantation. The genetic differences between recipient and donor elicit immune response that could be prevented by genetic compatibility, which is determined on the basis of human leukocyte antigens (HLA). These antigens play an important role in immune discrimination between self and non-self (foreign) and effectively promote detection and eradication of foreign molecules.
Major histocompatibility complex (MHC) or HLA molecules are encoded by two highly polymorphic gene families located in a 3,600-kb region of chromosome 6p6p21.3).

HLA molecules are polymorphic in nature, these membrane bound glycoproteins, bind to the processed antigenic peptides and present them to T cells. The HLA class I A, B and C molecules are composed of an MHC -encoded heavy chain (MW45kD) non-covalently associated with a non-polymorphic polypeptide, b₂ - microglobulin (MW12kD), which is encoded on chromosome 15. These class I antigens are expressed on all nucleated cells (except fetal trophoblast cells) and platelets and function to present peptides of largely endogenous (viral) origin to CD8+ T cells, which mainly function as the cytotoxic cells. The bound peptides are highly circumscribed in length, usually 8-9 amino acids, and are held in a peptide -binding groove. X-ray crystallography has shown that this groove has allele - specific conformation¹. The polymorphic residues that distinguish between the different alleles of a particular HLA class I locus are found, mainly within peptide binding groove².

In contrast to class I molecules, HLA class II molecules, comprising three main subclasses - DR, DQ and DP - are found on a more restricted range of cell types, including B cells, activated T cells the monocyte/ macrophage lineage and are also interferon - gamma inducible. An expressed class II molecule consists of a a chain (MW 31 - 34 kD) encoded by an A gene, noncovalently associated with a b chain (MW 26 - 29 kD), encoded by a b gene. Each DR, DQ or DP subregion consists of at least one expressed a and one expressed b gene. Both a and b genes may be polymorphic, but most polymorphism resides in the b genes. These are now known to be (2 DRA, 126 DRB, 12 DQA, 22 DQB, 6 DPA and 56 different expressed DPB alleles) excluding silent substitutions¹. Both a and b chains combine to form a peptide - binding groove shown by X-ray crystallography to be very similar to the class I groove³. However, class II molecules present peptides of largely exogenous origin to CD4+ T cells of largely ‘helper’ phenotype. These bound peptides are generally longer and more variable in length than peptides which bind to class I molecules (i.e. 14- 21 amino acids), due to the more open ends of the peptide –binding groove. Both classes of HLA molecule function to present self-antigens in the thymus and so induce tolerance, while foreign antigens are presented in the context of self-HLA molecules in the periphery, invoking an immune response.

Non-classical HLA and Non-HLA Genes in the HLA Class I / II Regions

The application of molecular techniques like cloning, sequencing and gene mapping have also revealed a number of additional HLA and non- HLA genes in the class I / II regions. In the class I region, there are known to be 17 ‘nonclassical’ genes or gene fragments, although only 3 of these - HLA - E, F and G - are known to be transcribed^4-7. Little is yet known of the possible function of HLA - E and F, more is known of HLA - G, which is closely homologous to other class I gene sequences and was thought to show little polymorphism, although this may not be so⁸. HLA - G is primarily although not exclusively expressed on fetal cytotrophoblast cells. These are derived from fetal cells in contact with maternal cells and lack expression of classical class I genes. In consequence, it is thought that the HLA - G gene product may function as a fetal antigen presenting / recognition molecule and hence in the absence of classical, highly polymorphic class I molecules, may permit maternal tolerance of the placenta⁹.

A series of gene mapping studies carried out independently in different laboratories have revealed a series of novel genes in the class II region, located between the DQ and DP subregions¹⁰. Gene sequencing, deletion mutant and transfection studies have now demonstrated a role for many of these genes in pathways of antigen processing and presentation. While HLA class I and II molecules are synthesized and assembled in the endoplasmic reticulum and peptide binding to class I molecules also occur here, it has been a conundrum as to how these peptides are generated from proteins present in the cytosol and transported into the endoplasmic reticulum. Proteasome complex of at least 16 polypeptides (each of MW 15 - 30 kD), catalyses the degradation of the vast majority of cell proteins and generate most peptides presented by class I molecules.

Two subunits of the proteasome are encoded by two genes located between DQ and DP - LMP2 and LMP7 (LMP = low molecular weight proteins). Deletion of these LMP2/ LMP7 genes alters the nature of the peptides generated by the proteasome, so that they no longer have optimal characteristics for class I binding. Two additional genes, TAP1 and TAP2 (TAP = transporter associated with antigen processing) are also located in the DQ - DP interval. These genes encode separate chains of a trans-endoplasmic reticulum membrane heterodimer which functions as a peptide pump, transporting peptides generated by the proteasome into the endoplasmic reticulum. The TAP genes show some polymorphism and this may influence the nature of the peptides transported, TAP transporter molecule preselects peptides according to sequence and length in a manner compatible with subsequent presentation by class I molecules.

The DQ- DP region is still richer in what were once termed RING or “really interesting new genes”, as defined by John Trowsdale’s group. Two further genes - DMA and DMB - map to this region and have sequences intermediate between those of classical class I and II genes, but may encode a class II - like heterodimer with a modified (more rigid) peptide - binding groove. Recent transfection experiments in mutant B lymphoblastoid cell transfection experiments in mutant B lymphoblastoid cell lines suggest that HLA - DM is expressed and appears to function at an intracellular site to promote peptide binding to classical class II molecules.

Peptide binding to class II molecules in the endoplasmic reticulum is prevented by co-assembly of the a and b chains, with a third chain, the so-called invariant chain (Ii, MW 35 kD, encoded by a gene on chromosome 5). The Ii chain also acts as an `address label’ and directs the class II - like complex to an intracellular endosomal compartment. The HLA-DM may act as a `sink’ for the removal of Ii chain- derived `CLIP’ (class II- associated invariant chain peptides) peptides in this compartment, so freeing classical class II molecules for peptide binding.
Taken together, these collected discoveries have overthrown earlier concepts of the MHC class I and II regions as solely containing genes encoding for molecules which present antigenic peptides to T cells. Rather, the current view is a genetic region encoding many different types of molecules collectively involved in pathways of antigen processing and presentation to helper and cytotoxic T cells. These gene products may have a role in immunologically mediated immune rejections.

Allograft Rejection

Allograft rejection remains the single largest impediment to the success in the field of transplantation. Graft rejection is different from other immune responses as two different sets of antigen presenting cells are involved, one from the donor and other from the recipient. Exact mechanism by which allograft rejection can occur is still not fully understood because of the complex immune mechanisms involved in the graft rejection. Rejection episodes lead to adverse immune response and affect the allograft survival. The immune response following an allograft is primarily against major histocompatibility complex (MHC) molecules of the donor from which recipients differ. As many as 8-10% of the normal adult T cell repertoire is capable of recognizing and responding to the foreign MHC molecules. This response is not to the hosts benefit but occurs due to cross reactivity of some of host T cells whose TCR were selected to recognize MHC plus foreign peptide during thymic education and recognize foreign MHC antigens in context of self MHC and get activated.

T cells recognize major histocompatability complex antigens in transplantation by two-pathway i.e. direct pathway and indirect pathway¹¹. There are three evidences in support of the direct recognition pathway in allograft rejection. (i) Stimulation is very high in primary allogenic mixed lymphocyte culture (MLR), (ii) The depletion of donor APCs can some time prolong the allograft survival, (iii) Donor MHC are more important than minor antigens in causing graft rejection. Hornick et al¹² have shown in cardiac transplant rejection that two populations of T cells with direct allospecificity are activated after recognition of intact MHC alloantigens displayed at the surface of donor passenger leukocytes, carried along within the graft. The direct recognition pathway involves T cells that recognize intact allogenic MHC/peptide complexes on the surface of donor target cells. This form of recognition does not require processing and presentation by host (APCs) and is therefore, not MHC restricted. Because the frequency of T cells that are able to recognize alloantigen directly is very high, even in non-immunized responders, it is believed that this process reflects T cell recognition of allogenic MHC/peptide complex via molecular mimicry with other antigenic structures. Although the majority of T cells infiltrating the graft during early acute rejection exhibit direct recognition ability, it is unlikely that these cells can mediate late or chronic rejection because their stimulation requires the presence in the graft of passenger APC of donor for a long time further, the absence of costimulatory molecules on the surface of graft endothelial and parenchymal cells renders such putative targets more likely to induce anergy rather than to stimulate recipient’s T lymphocytes¹³.

In contrast to the direct recognition pathway, T cells that react against peptides derived from the processing of allogenic MHC and proteins mediate indirect alloimmune responses through host APCs¹⁴. Peptides resulting from the proteolysis of allogenic MHC molecules bind to MHC- class II antigens of host APC and trigger T cell alloimmune responses. This form of alloreactivity is restricted by host HLA-DR antigens and is carried out by an oligoclonal population of T cells, which are capable of recognising the dominant epitope of the allogenic MHC molecule. Because the stimulatory peptide can be generated continuously from soluble MHC alloantigens released from the graft and processed by host APC, the direct pathway may be responsible both for initiation and perpetuation of allograft rejection.
The exact mechanism by which T cells destroy the graft is not yet clear. Both CD4 and CD8 subclass of effector cells probably destroy graft cells by classical cytotoxic T cell mechanisms. Another important consequence of T cell activation is the release of other lymphokines, especially interferon (IFN- g). IFN-g induces increased expression of HLA-A, -B and DR on graft tissues thus potentially making the graft more vulnerable to effector mechanisms. IFN-g also activates monocytes to mediate a destructive delayed hypersensitivity response against the graft.

In addition to IL-2 and IFN-g released from activated T cells, IL-4 and IL-5 play a role in directing B cell for the production of antibodies. Antibody-mediated damage may then take place directly through complement activation or recruitment of antibody dependent cell mediated cytotoxic (ADCC) effector cells. Most of the cells that arrive in the graft early after transplantation are lymphocytes, which migrate out of the capillary beds, after 7 days a remarkably heterogenous collection of cell types appears. Those of the lymphocytic series predominate over the monocytes/macrophages although few polymorphonuclear neutrophils are also present.

Mechanisms involved in allograft rejection

Immunological mechanisms involved in rejection could be (i) cell mediated (ii) antibody mediated (iii) delayed type hypersensitivity (DTH) and (iv) natural killer cell.

(i) T Cell mediated Rejection

Thymus derived T cells have an essential role in acute allograft rejection. If the host is naturally or experimentally deprived of T cells (eg. nude mice, SCID mice, thymectomized mice) it is unable to reject allograft in the first set. If the passive transfer of T cells into athymic mice is done vigorous graft rejection will take place. In clinical transplantation, the role of T cells has been confirmed by the dramatic effects of anti-T cell antibodies, including monoclonal anti-CD3 antibody (OKT3), antithymocyte globulin and antilymphocyte globulin, the effectiveness of which is often limited by the side effects of non-specific immunosuppression. Treatment of rhesus monkey by CD3 immunotoxin just before transplantation resulted in long-term graft acceptance in more than 50% of the monkeys. The allografts differ from host at class I and class II loci. Both CD8+ and CD4+ T cells are activated by recognition of alloantigens of the grafts; the CD8+ T cells recognize foreign MHC class I molecules, which are expressed by all the cells in the graft. The differentiation of cytotoxic T lymphocytes (CTLs) is largely dependent on CD4+ T helper cells being stimulated by allogenic class II molecules present on antigen presenting cells (APCs) in the allograft. Several lines of evidences suggest that the CD4 subset and its lymphokine products are the principle mediators of rejection¹⁵ various studies on mice and rat model convincingly suggest that CD4 cells mediate rejection however, to delineate mechanisms of different lymphokines is difficult¹⁶.

There are evidences, which suggest that some CD8+ T cells can also provide sufficient help to allow cytotoxic T lymphocytes to differentiate independent of CD4+ T cells. However, these CD8+ T cells appear to depend upon the same professional APCs, as those required by conventional CD8+T cell. The most important APCs stimulating an antigraft response may be dendritic cells residing in the interstitium of the graft. Dendritic cells are now regarded as critical instigators and regulators of immune reactivity, which play a key role in both the direct and indirect pathways of allorecognition. Molecular signalling between dendritic cells and Th cells directs the differentiation of naive (Tho) cells into either Th1 or Th2 cells. Specific cytokines such as IL –10 and other factors can inhibit IL-12. Experimental dendritic cell targeted approaches to the therapy of organ allograft rejection include administration of co-stimulation of blocking agents together with donor dendritic cells or administration of inhibitors of NF – KB and genetic engineering of the dendritic cells to express tolerance promoting molecules.

(ii) Antibody Mediated Rejection

The role of antibody in hyperacute rejection has been clearly established¹⁷. A direct correlation is seen between positive pretransplant crossmatch which detects anti-MHC class I antibodies and the development of hyperacute rejection¹⁸. Anti-graft antibodies can be eluted from donor kidneys after hyperacute rejection. The passive transfer of antigraft antibodies in experimental models can provoke hyperacute rejection. It is likely that antibodies also play a role in other types of rejection; however, their mechanisms remain incompletely understood and also controversial especially in chronic rejection¹⁹. The scanty cellular infiltrate in most cases of chronic rejection is antibody mediated rejection. However, direct evidence for antibody-mediated damage in chronic dysfunction is inconclusive. The antibodies causing hyperacute rejection may be preformed²⁰ or they may develop under the influence of immunosuppressive drugs, which could modulate their rate of production. Antibodies can bind to the graft, making the detection of soluble antigraft antibody difficult. Thus the role of antibody in the pathogenesis of chronic dysfunction remains undetermined.

Role of cytokines in allograft rejection

Cytokines are soluble mediators secreted by one cell that acts on another cell or organ; the term is generally reserved for protein mediators. Naïve T cells could be converted into either Th1 or Th2 type cells. Th1 produces high levels of interferon (IFN–g) and TNF - a, both
IFN-g and TNF–b which may promote cell mediated cytotoxicity and delayed type hypersensitivity reactions, Th2 cells produce high levels of IL –4, IL-5, IL-10 and IL-13 which promote humoral response. Both Th1 and Th2 responses counter regulate one another. Th2 cytokines may evoke allograft rejection by recruitment of alternate effector mechanisms. Hence the exact mechanisms underlying this phenomenon in general is not yet defined and both Th1 and Th2 clones can reject skin grafts. Th1 to Th2 immune deviation can induce islet allograft tolerance across multiple minor histocompatibility antigen barriers. Apoptosis may promote the development of immunoregulaory T cells and facilitate active immunosuppression²¹. Apoptosis is tightly controlled and regulated via several mechanisms including Fas / Fas ligand interactions, the effect of cytokines such as tumour necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF – beta) and the influence of pro and anti apoptotic mitochondria associated proteins of the B cell lymphoma – 2 Bcl-2) family.

It is important to keep in mind that only IL4 and IL10 confer tolerance but also the patient donor cytokine genotype may have differential effect on renal transplantation outcome. Effect of cytokine genotype on allograft outcome has to be seen keeping in mind the DR background of the donor and the recipient. As cytokines play an important role in allograft rejection and tolerance, hence there are various drugs being used, which inhibit selectively different steps of the cell, and cytokine mediated immune cascade. Both the calcineurin inhibitors cyclosporine and tacrolimus for instance, block IL-2 production by activated T cells. Rapamycin also inhibits signal transduction through selected cytokine receptors. Mycophenolate modetil blocks the salvage pathway of purine metabolism upon which lymphocytes are particularly dependent. Treatment with this drug causes 50% reduction in acute rejection. Monoclonals targeted against IL-2 receptors have recently been approved to treat acute rejections.

Role of Chemokines and their receptors

Chemokines are 8 – 11 kDa molecules. The criteria used for their classification is based on the relative positions of their amino – terminal cysteins. In CC chemokines, cysteins are adjacent, in C x C chemokines cysteins are separated by one residue, in Cx 3C cysteins are separated by more than 3 residues. So far more than 40 chemokines and over 18 chemokine receptors have been reported. However, the knowledge about exact role of chemokines in transplant biology is fragmentary.
As now anti-chemokines and anti-chemokine receptor mAbs and gene knokout animals are available the role of the chemokines is explainable to some extent. In renal transplant polyclonal anti -b chemokines antibodies i.e. MCP –a and MCP – 2 have been used and it has been shown that the expression of RANTES or MIP – 1 a which is not significantly related to developing rejections, since MCP –1 and MIP -bB are ligands for CCR2 and CCR5 respectively, these data are consistant, firstly, with the fact that CCR5 mRNA although not present in normal kidneys is localized to infiltrating mononuclear cells in rejected human renal allografts. There are some studies which demonstrates that MCP1, MIP – IB and IL-8 proteins were increased in renal allograft rejection as well as during acute tubular necrosis, cyclosporin toxicity and pyelonephritis^22,23,24. Hence these chemokines can be used as indicators of renal tubular injury because of different causes. Rejection is associated with Cx CR4 chemokines, however, the exact machenism is not known. Various studies on lung, liver, cardiac allografts suggests that clinical applications of chemokines^25-27 and mAbs and cDNA arrys for monitoring graft rejection is likely to yield new diagnostic and prognostic application in transplantation.

Role of HLA matching in transplantation

It has been established that the graft immunogenicity plays a key role during the allograft rejection, which is determined by HLA antigen.
Although much debated, it is clear from the data summarised in national as well as international transplantation registries that HLA matched renal grafts out perform less matched allografts^28,29 Not only graft survival but also patient survival is improved with HLA matching. Both broad specificities (routine serology) and split antigens should be matched. For this purpose molecular typing, (SSP, SSOP and DNA sequencing) have been suggested. Further innovation in DNA sequencing and chip-based approaches should enhance our ability to identify HLA specificities and to match recipients and donor organs better. This strategy, in conjunction with identification of the recipients immune responder status, perhaps by genotyping for cytokines and other immuno-regulatory molecules, has the significant potential of placing well matched grafts in the high responders (to alloantigens) and using the poorly matched grafts in the low responders.

Considerable evidence has been obtained that matching for HLA reduces allograft rejection thereby promoting survival of kidney and heart transplant patients. Although the HLA system comprises multiple class I and especially class II genes, most matching strategies consider only HLA-A, HLA-B and HLA-DR. Because of the co dominant expression of HLA genes, the degree of compatibility ranges from 0 – 6 matches (two for each locus). Survival statistics from kidney and heart transplant registries have shown the best survival rates of cadaver transplants with 6 matches followed by 5 matches, etc. Although it makes sense to find a perfect match for each transplant patient, the reality of practice dictates the selection of less well matched donors and the urgency status of the patient. However, recent experience has shown that transplanted kidneys with “permissible” HLA mismatches have excellent graft survival.

Adhesion molecules expression in allograft rejection

It is important to know the role of various molecules in transplantation. As after knowing various regulatory mechanisms the drugs can be designed to block the positive signals for induction of allograft tolerance, it is known that cyclosporine blocks positive signals required for T cell proliferation and apoptosis^30,31. Antigens specific lymphocyte immune response requires at least 2 stimuli from the antigen presenting cells. If the second stimulus (co-stimulation) does not occur, tolerance ensues. Two signals are: TCR-peptide - MHC recognition (specific response). T-cells ligand (CD28 “on” and / or CTLA4 “off”) - B7 - 1 or -2 binding (non specific co-stimulation provide + or -support). CD28 / B7 may not be the only co-stimulator pair. In addition other cell - cell interactions (as between ICAM - LFA) are also important³². In bone marrow transplantation exvivo manipulation (graft engineering) is being attempted more frequently using a variety of methods, including co-stimulation blockade to prevent graft-versus-host-disease by tolerising donor T cells. This technique also lends itself to solid organs transplantation, where graft tissue is not amenable to prolonged exvivo manipulation. In this case, host T cells are tolerised to alloantigens using cytokines such as IL-10 and TGF - b to induce regulatory T cells exvivo, it should be possible to induce antigen specific suppression for allo and auto antigens if known.

Recently gene therapies in clinical transplantation have a potential future. Various genes associated with chronic rejection are CAM-I, nitric oxide, oxygen free radicals, protooncogene C-myc, ribozymes etc. Recently there are efforts in targeting these molecules and use the gene therapy approach³³. Even after transplantation the damage to the allograft keep on occurring leading to chronic rejection. Smooth muscle cells are seen to proliferate in the vasculature of the transplanted organ some time resulting into transplant atherosclerosis. To prevent this various approaches have been followed for e.g. anti ICAM-I murine model have been developed to use antisense oligo nucleotides that are targeted against the messenger RNA (mRNA)^33-35. The initial proliferation of vessels in transplanted organs is another indication of chronic rejection. Sendai virus virosome has been used to deliver the endothelial cell nitric oxide synthase gene in vivo. This resulted into the reduction of proliferation by 70%. Recombinant adeno virus have been used to deliver inducible nitric oxide synthase to the sites of artherial injury invivo this has also caused the increase of local level of nitric oxide and hence prevented intimal hyperplasia^36-38. Recombinant adenoviruses encoding SOD have been used, to prevent ischaemia reperfusion injury, however, results are not very successful.

It has been reported that protoconcogene C-myc is involved in the mitogen - induced proliferation of vascular smooth muscle cells, which constitute a major pathway of atherogenesis. There are studies where antisense^39,40 or ribozymes⁴¹ have been used to inhibit the proliferation of smooth muscle cells however, the success is controversial. Ribozymes are known to target mRNA and have been used in targetting xenoantigens⁴¹, Fas ligand, perforins⁴² and also targetting inflammatory cytokines and chemokines.

Role of anti HLA antibodies in rejection

Preexisting HLA antibodies against the donor are associated with acute rejection in case of renal transplantation, which have a worse prognosis, often requiring aggressive early treatment with anti-CD3 cell antibodies⁴³. Flow cytometery is shown to be a better technique for detecting these antibodies⁴⁴.

Panel reactive antibodies

Panel reactive antibodies are not against individual HLA gene products but are expressed as percentage positivity against a panel of cells. High prevalence of PRA shows that a patient is sensitised. Highly sensitised patients are one at increased risk of early graft loss. It is recommended⁴⁵ that patients waiting for transplant should be tested for PRA, if need be erythropoietin⁴⁶ should be given as it causes reduction in the sensitisation.
HLA gene products consist of private and public determinants both of which are defined by antibodies reacting to a single epitope. Antibodies reacting to a single epitope define private and public determinants. A private determinant is unique to a single HLA gene product, where as a public determinant is shared by multiple HLA gene products.

It is therefore, possible to reduce the large number of HLA alleles to a small number of closely related groups that share common HLA derived antigenic targets; these groups are known as cross reactive groups. (CREG). It is also thought that because many of the CREG were initially defined using sera from patients who had received a previous transplant, or had multiple blood transfusions or been pregnant, the targets of these antibodies are likely to be clinically relevant. An evaluation of more than 50,000 serum samples from immunized individuals revealed that 96% of the definable antibodies reacted with 12 CREG. The amino acid residues that compose the A2 CREG epitope are located exclusively on the a1 and a2 domains and more specifically, mainly on the a helices that form the peptide-binding groove. If matching is at CREG the chances of finding matched donor increases, even beneficial effect of CREG matching have been reported⁴⁷.

Role of Minor histocompatability Antigens (mi-HAgs)

Minor histocompatability antigens may play an important role in the graft rejection and are defined as cell surface antigens other than the MHC antigens. These antigens may not be universally present on all the cells and they don’t interact functionally with MHC antigens. However, the role of these antigens is not well defined in humans. Experimental data obtained from studies of congenic strains of mice suggests that polymorphism of minor HLA antigens may be similar to that of the MHC. The important difference important difference between them are that minor histocompatability antigens mi-HAgs are less potent and immunogenic and they don’t initiate the immune response independently, while, MHC antigens are more immunogenic and can trigger the antibody production against incompatible alloantigens. These mi-HAgs accounted for comparatively slower and more chronic rejections. Goulmy et al. have first reported the possible involvement of mi-HAgs in human transplantation⁴⁸.

It dealt with a clinical observation in female patient who received the bone marrow of male HLA identical sibling after ATG pre-treatment. Invitro analysis of the post transplant peripheral blood lymphocytes of the female patient showed unambiguously that there were strong cytotoxic lymphocyte (CTL) responses that were specific for the male donor HLA matched target cells. Naturally the impact of mi-HAgs on the outcome of an organ and bone marrow graft is dependent on other factors their tissue including cord endothelial cells and kidney proximal tubular epithelial cells or restricted to hematopoietic cell lineage including epidermal derived langerhance cells⁴⁹.

Linkage studies in congenic strains of mice have shown that mi-HAgs loci are scattered throughout the genome⁵⁰. Total number of mi-HAgs is not known but theoretical estimates based on breeding and transplantation studies in mice suggest that there may be several hundred minor histocompatability antigens⁵¹. So far, forty minor histoincompatibilities antigens have been found between C57BL/6 and BALB/c strains of mice. Recently, from the genetic analysis of CTLs defined HLA-A2.1 restricted mi-HAgs have been characterised and categorised into the HA-4, & HA-5 antigen⁵². These antigens can be considered as the product of a gene with allele expressing the detectable specificity and one more alleles not expressing it. The immune response to the minor histocompatability antigens is T cell mediated, predominantly by cytotoxic T lymphocytes^53-55.

Role of Tissue Specific Antigens

Tissue specific antigens are defined as an antigen system that is expressed only on one type of organ, tissue or cell. These tissue specific antigens are independent from the systemic antigens such as HLA antigens, which have a wide distribution throughout the body. In 1969, Clane et al. first described the phenomenon of differential allograft survival between organs from the same donor⁵⁶. Whereas skin and kidneys were acutely rejected and, liver allograft survival seemed to be prolonged in unrelated pigs. Several cases of multiple organ transplants have been reported in which one organ is rejected while other continues to function. One possible explanation for this observation is the affect of tissue specific antigens. Poindexter et al. have characterized a kidney specific peptide, which recognize kidney cell line but not MHC identical B-lymphoblastoid between cell line⁵⁷. These peptides are nanomer with proline and lysine residue, which are presented on the allograft kidney and may be target of CTL recognition. This may further result into acute or chronic rejections. If all relevant transplant antigens were ubiquitous graft survival should be fairly confirmed. HLA compatibility between recipient and donor may prevent sensitization to the tissue specific antigens. However, HLA identity and negative MLC does not prevent immune response to the increasingly well-studied VEL- specific antigens.

Vascular endothelial cells of transplanted organs are at the interface between the graft and the recipient’s blood containing immuno-competent cells. The VEC antigens are expressed in abundance throughout the renal vasculature. In extra renal vasculature, the VEC antigens are expressed on the endothelial cells of major abdominal vessels i.e. on both arterial and venous. VEC plays an important role in the rejection process. These are not only transplant carriers but also are an endocrine like organ capable of synthesizing clotting factors and inactivating hormones. VEC serve as antigen presenting cells and are also able to phagocytosize and are partly responsible for the normal functioning of the platelets. Antibody specific to VEC is the most commonly encounter antibody in-patients rejecting a renal allograft. Ninety six percent of the patients who experienced a chronic rejection developed anti VEC antibody^58,59. Antibody to VEC is rarely encountered in normal controls and only in low frequency in patients experiencing a benign clinical post transplantation course. Most of these patients had anti VEC antibody present in the absence of any anti HLA antibody to the donors. The VEC antigen appears to be an important immunogen in non-HLA identical combination.

Role of heat shock proteins (HSP) in transplantation

Heat shock proteins may be involved in the pathogenesis of chronic rejection. This hypothesis was tested with a rat cardiac allograft model in recipients pretreated with donor bone marrow cells. Chronic rejection was manifested in this group by obliterative arteriopathy and the epicardium and endocaridum containing lymphocytic infiltrates⁶⁰. Current experimental evidences support the concept that during cellular rejection, graft-infiltrating cells induce a stress response within the allograft which increases the expression of heat-shock proteins and triggers the recruitment and activation of hsp-dependent lymphocytes. A variety of stress proteins exhibit higher tissue levels during the different phases of allograft rejection⁶¹.

Hsp 70 does not appear to stimulate graft-infiltrating T-cells as a conventional antigen. Rather, structurally intact hsp 70 molecules seem to interact with self-APC, which then stimulate certain types of autoreactive CD4+T-cells proteins exhibit higher tissue levels during the different phases of allograft rejection⁶¹.
Hsp 70 does not appear to stimulate graft-infiltrating T-cells as a conventional antigen. Rather, structurally intact hsp 70 molecules seem to interact with self-APC, which then stimulate certain types of autoreactive CD4+T-cells to undergo proliferation. This hsp effect implies a previously unrecognized mechanism of transplant immunity and the peptide-binding properties of stress proteins might be relevant. The members of the hsp 70 family function as molecular chaperones which have a common structure consisting of a C – terminal peptide-binding domain and a N-terminal ATPase domin which influences peptide binding. For instance, 78 hsp can bind peptides of minimally 7 –8 residues and the peptide-binding region contains four major pockets that can accommodate large hydrophobic residues.

Through its peptide-binding properties, 78 hsp might participate in the transport and processing of autologous peptides presented by APC. Recent studies have demonstrated⁶³ that stress proteins such as 94 hsp (also called gp 96) and PDI, participate through their peptide-binding properties, in alternative pathways of antigen processing and presentation. Grp⁹⁴ and PDI are similar to grp 78 in that they are resident proteins in the endoplasmic reticulum and that they are upregulated during a stress response. Tissue injury leads to a denaturation of intracellular proteins and an accumulation of protein degradation products. Elevated levels of heat shock proteins that function as chaperones in protein renaturation and in other cytoprotective processes manifest the stress response to tissue injury. Conversely, the degradation of denatured protein may generate (auto) antigenic peptides that bind to heat shock proteins such as grp 78, grp 94 and PDI and such complexes might be translocated into cellular compartments involved with antigen processing and presentation. Thus, stress responses may activate hsp-dependent pathways of (auto) antigen-induced T- cell activation.
When APC are stressed by heat, chemical treatment or ultraviolet irradiation, they lose their ability to stimulate in mixed leukocyte cultures. It has been shown that animals with stressed allogenic cells often exhibit prolonged graft survivals and even transplant tolerance. Since stressed cells have increased hsp expression, it is possible that these effects might be related to hsp-mediated immunomodulation.

The involvement of hsp in the immunosuppression induced by certain HLA class I peptide has been illustrated⁶⁴ . These peptides inhibit T cell function and promote rat allograft survival and this correlates with their binding to the constitutively expressed hse 70 and the heat-inducible hsp⁷⁰. Non inhibitory peptides do not bind these hsp. The immunosuppressive effects of deoxyspergualin also involve the binding to hsc 70 and this drug interferes with antigen presentation. Therefore, certain members of the hsp 70 family have been proposed to represent a third class of immunophilins in addition to the cyclophilins and FK-binding proteins. Hence it may be concluded that stress proteins are important in transplant immunity. Their major role seems related to antigen presentation. The stress protein in transplant immunity offers perspectives of the various immune mechanisms leading to rejection, chronic dysfunction and conversely, transplant tolerance and how these processes are affected by infection and ischemia/reperfusion injury.

Post-transplant Monitoring of organ transplants

Different methodologies have been developed to monitor various organ transplants. In kidney transplant patients. The easiest way to assess renal function is by measuring serum creatinine levels. Elevations suggest rejection although cyclosporine induced nephrotoxicity may also be responsible. Histopathological examination of a renal biopsy may enable a differential diagnosis between rejection and cyclosporine toxicity. Immunostaining of renal tubular cells, a primary target of infiltrating T cells, shows increased expression of HLA class II antigens during rejection. Heart transplant patients are monitored by histopathological analysis of endomyocardial biopsies at regular intervals. These biopsies are obtained through a catheter passed into the right ventricle and the histological rejection is assessed by the degree of cellular

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Source: Indian Journal of Nephrology / July to September 2002 / Volume 12 Number 3

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