Elaine Reed

INDIRECT RECOGNITION OF DONOR HLA-DR PEPTIDES IN ORGAN ALLOGRAFT REJECTION

To determine whether indirect allorecognition is involved in heart allograft rejection T cells obtained from peripheral blood and graft biopsy tissues were expanded in the presence of IL-2 and tested in limiting dilution analysis (LDA) for reactivity to synthetic peptides corresponding to the hypervariable regions of the mismatched HLA-DR antigen(s) of the donor. Serial studies of 32 patients showed that T cell reactivity to donor allopeptides was strongly associated with episodes of acute rejection. The frequency of allopeptide reactive T cells was 10-50-fold higher in the graft than in the periphery indicating that T cells activated via the indirect allorecognition pathway participate actively in acute allograft rejection. In recipients carrying a graft differing by two HLA-DR alleles the response appeared to target only one of the mismatched antigens of the donor. Indirect allorecognition was restricted by a single HLA-DR antigen of the host and directed against one immunodominant peptide of donor HLA-DR protein. However, intermolecular spreading was demonstrated in patients with multiple rejection episodes by showing that they develop allopeptide reactivity against the second HLA-DR antigen. These data imply that early treatment to suppress T cell responses through the indirect pathway of allorecognition, such as tolerance induction to the dominant donor determinant, may be required to prevent amplification and perpetuation of the rejection process.

Anti-HLA antibody ligation to HLA class I molecules expressed by endothelial cells stimulates tyrosine phosphorylation, inositol phosphate generation, and proliferation

The major threat to long-term survival of solid organ allografts is chronic rejection. Progressive narrowing and ultimate luminal occlusion of the arteries and arterioles of the transplanted organ are the hallmarks of the disease. The mechanism of chronic rejection is poorly understood, but it is suspected that the associated vascular changes are a result of anti-HLA antibody-mediated injury to the endothelium. We have postulated that anti-HLA antibodies initiate chronic rejection by binding to class I molecules on the endothelium and transducing signals that result in endothelial cell activation and proliferation. Our data demonstrate that anti-HLA class I antibodies transduce signals in endothelial cells stimulating increased tyrosine phosphorylation of intracellular proteins. Antibody binding to class I antigens also leads to the generation of inositol phosphate and endothelial cell proliferation. These results indicate that anti-HLA antibodies can deliver functionally important signals to endothelial cells, a finding that may be fundamental to an understanding of the mechanisms of chronic rejection.

The alloantibody response of pregnant women and its suppression by soluble HLA antigens and anti-idiotypic antibodies

The aim of this study was to investigate the time course of maternal allosensitization to fetal HLA antigens during normal human pregnancy and to explore mechanisms of suppression of anti-HLA alloantibodies. We found that the mother produces antibodies against some but not all of the mismatched HLA antigens of the fetus as early as the 8th week of pregnancy. These antibodies (Ab1), however, are often complexed with soluble HLA alloantigens and become detectable when immune complexes are dissociated. Soluble HLA antigens of fetal origin are present in the maternal circulation throughout the entire pregnancy beginning at 8 weeks. In some women the production of anti-anti-HLA antibodies (Ab2) became evident as early as the first trimester, while in others Ab2 was documented during the second or third trimester. Analysis of antibody specificity showed that some healthy primipara develop antibodies reactive with self HLA antigens. Although the allo- and autoantibody responses appear to be modulated by soluble HLA antigens, cyclic variations in the level of alloantibodies, as well as the mothers selective response to some, but not all, paternal HLA antigens, are best explained by the development of anti-idiotypic antibodies.

Complexes of soluble HLA antigens and anti-HLA autoantibodies in human sera: possible role in maintenance of self-tolerance.

The MHC plays an essential role in regulating the process of self-nonself discrimination. T cells recognize nonself antigens only in the context of self-MHC gene products. It is not clear, however, whether B cells are also endowed with immune receptors for self-MHC antigens. We have explored the existence of antibodies against self-MHC antigens (HLA) in the human by analyzing the specificity of anti-HLA antibodies developed by a population of 727 dialysis patients who had been monitored monthly over a period of 3-78 months. Anti-HLA autoantibodies were identified by serum screening in 119 patients. Twenty-five of these 119 patients had not been exposed to alloantigens before, indicating that the production of anti-HLA autoantibodies is not necessarily stimulated by allogeneic HLA antigens. Cross-matching of sera with autologous lymphocytes, confirmed the autoreactive nature of these anti-HLA antibodies which were of IgM or of IgG isotype in approximately equal numbers of patients. The fine specificities of anti-HLA autoantibodies was ascertained in studies which showed that antibodies can be adsorbed, and the eluted, from the membranes of target cells carrying then relevant HLA antigen. Since the antibodies characterized in our studies may be functionally active in vivo we examined the possibility that their level is controlled by anti-idiotypic antibodies or by soluble HLA antigens. We found that the titer of anti-HLA autoantibodies increased significantly if soluble HLA antigens were depleted from the serum. Our data suggest that circulating HLA antigens form immune complexes with anti-HLA autoantibodies and contribute to the maintenance of self-tolerance by inhibiting antibody binding to membrane HLA antigens. The finding that the immune repertoire includes B cells with receptors for self-MHC opens new perspectives for the study of network perturbations in autoimmune diseases.

Influence of HLA class I and class II antigens on the peripheral T-cell receptor repertoire

Using TCR-specific mAbs, we analyzed the TCR distribution in the CD4 and CD8 compartments of peripheral blood T cells from members of six families, including two which were informative for an HLA-B/DR crossover. TCR distribution in the CD4 compartment is influenced by MHC class II antigens as it was similar in the child carrying the HLA-B/DR recombination and in the HLA-DR-identical sibling. There was also a relatively small degree of difference between TCR distribution in the CD8 compartment of a child with an HLA-B/DR crossover and his HLA class-I-identical sibling. Analysis of TCR V beta gene usage in the CD4 compartment of unrelated individuals sharing an HLA-DR allele showed an increased usage of V beta 8a and V beta 12a in DR4-positive subjects. These data suggest that studies of TCR V beta gene usage in individuals with HLA-associated autoimmune diseases may provide further information about MHC-dependent biases in the T-cell repertoire.

Anti-idiotypic antibodies specific for HLA in heart and kidney allograft recipients

Chronic rejection is the major threat to both heart and renal allograft survival. We have explored the possibility that some patients with anti-donor HLA antibodies (Ab1) develop specific anti-idiotypic antibodies (Ab2) which suppress the production of Ab1, and subsequently, the progression of chronic rejection. analysis of Ab2 in sera obtained from Ab1 producers showed that 22% of heart and 18% of kidney recipients produced Ab2. The 4- and 5-year actuarial graft survivals in Ab2 producers were 100% and 83%, respectively, compared to 57% in patients who formed Ab1 but not Ab2 (p<0.004). Patients carrying the DR2 alleles, DRB1*1501,*1502 or*1601 were at a lower risk of producing anti-donor HLA antibodies.

In vitro studies of the effect of MAb NDA 4 linked to toxin on the proliferation of a human EBV-transformed lymphoblastoid B cell line and of gibbon MLA leukemia cell line.

The rejection of allografts is mediated by cytolytic T cells and antibody-secreting B cells. Selective ablation of these activated cells from peripheral blood lymphocytes may offer a a method of controlling allograft rejection. An immunotoxin was prepared from the monoclonal antibody (mAb) NDA 4, which recognizes a differentiation antigen (NDA 4) common to activated B and T cells. MAb NDA 4 was conjugated to the ribosome-inhibiting protein gelonin via a cleavable disulfide bond provided by a crosslinking reagent. The purified immunotoxin was evaluated for in vitro cytotoxicity on NDA 4 positive T and B cell lines. Conjugation of mAb NDA 4 to gelonin increased the in vitro cytotoxicity by a concentration factor of 1000, compared to gelonin alone. The specificity and saturability of mAb NDA 4 binding, as well as the number of antigenic sites per cell on resting versus activated T lymphocytes, were also evaluated. Resting T cells expressed 400-800 sites per cell. PHA-activated T cells and the MLA T cell leukemia expressed 10,000 to 80,000 sites per cell. Peripheral blood mononuclear cells obtained from allografted baboons in quiescence or undergoing rejection were compared for NDA 4 expression by flow cytometry. Lymphocytes obtained from baboons rejecting a heart allograft expressed NDA 4, whereas transplant recipients in quiescence showed no detectable NDA 4. These results suggest that mAb NDA 4-derived immunotoxins may be valuable for the selective depletion of activated lymphocytes while sparing the resting population.

Modulation of T-cell antigen receptor on lymphocyte membrane

A mouse monoclonal antibody, Mo. Ab. 108.45, detecting cell-surface determinants associated with the T-cell receptor for alloantigen was produced by immunizing mice with an alloreactive human T-cell clone and fusing the splenocytes with the NS1 plasmocytoma. This Mo. Ab. (1) reacts with the immunizing T-cell clone but not with autologous or allogeneic lymphocytes, lymphoblasts or monocytes; (2) stimulates the proliferation of the immunizing T cells in the absence of the alloantigen; (3) inhibits the response to the specific stimulator; and (4) precipitates a disulfied linked heterodimer composed of two distinct glycoproteins of molecular weights 40 000 and 46 000. The receptor molecule detected by Mo. Ab. 108.45 modulates on the surface of the cells, reaching the highest levels 5 days following exposure to the specific stimulator. The receptor-associated molecule detected by Mo. Ab. 108.45 was expressed by T-cell clones obtained independently in two different mixed lymphocyte cultures between the same responder and stimulator.

HLA antigens in Asian Indians: HLA-D-DR relationships

Fifty-nine Asian Indians were typed for HLA-A, B, D, and DR antigens. Peculiar to the population that we have tested was the absence of HLA-A25, B13, B14, DR1, DW1, LD13 (a DR1-associated HLA-D allele), and LD12 (a DR4-associated HLA-D allele). Certain haplotypes that exhibit high frequencies in Caucasians (such as A2-BW50, AW24-B18, B8-DR3, BW44-DR7, B18-DR5) or in Blacks (such as A29-B7) also show significant delta in Asian Indians. The HLA-D-DR associations previously described in European and North American Caucasians were also found in Asian Indians. Additionally, however, Asian Indians exhibited two new HLA-D antigens, one associated with DR5 and the other with DRw6. The genetic distance between Asian Indians and Caucasians, Blacks, or Mongoloids is of the same order of magnitude.

Major histocompatibility complex-restricted recognition of autologous chronic lymphocytic leukemia by tumor-specific T cells

From the peripheral blood of a patient with chronic lymphocytic leukemia (CLL) we generated a T-cell line and clones which recognized autologous CLL. The line comprised T-cell clones which responded to the CLL as well as to autologous Epstein-Barr virus (EBV)-transformed B cells in an HLA-DR-restricted fashion. In addition, the line comprised clones which were CLL-sepcific and showed no reactivity against EBV-transformed B cells and against autologous peripheral blood mononuclear cells obtained during remission. The proliferative response of the CLL-specific T-cell clone was inhibited by monoclonal antibodies to HLA-DR11, the major histocompatibility complex (MHC)-restrictive element. These results indicate that the MHC class-II molecule of CLL binds a tumor-specific peptide which is recognized by autologous T cells in an MHC class-II-restricted fashion. Such a peptide may serve as a target for immunotherapy.