Els Blokland

Mismatches of Minor Histocompatibility Antigens between HLA-Identical Donors and Recipients and the Development of Graft-Versus-Host Disease after Bone Marrow Transplantation

BACKGROUND Graft-versus-host disease (GVHD) can be a major complication of allogeneic bone marrow transplantation even when the donor and recipient are siblings and share identical major histocompatibility antigens. The explanation may be a mismatch of minor histocompatibility antigens. We previously characterized five minor histocompatibility antigens, HA-1, 2, 3, 4, and 5, that are recognized by T cells in association with the major histocompatibility antigens HLA-A1 an A2. METHODS We collected peripheral-blood leukocytes from 148 bone marrow recipients and their sibling donors, who were genotypically HLA identical. Fifty pairs were positive for HLA-A1, 117 were positive for HLA-A2, and 19 were positive for both. The pairs were typed with cytotoxic-T-cell clones specific for minor histocompatibility antigens HA-1, 2, 3, 4, and 5. RESULTS Mismatches of HA-3 were equally distributed among recipients in whom GVHD developed and those in whom it did not. By contrast, a mismatch of only HA-1 was significantly correlated with GVHD of grade II or higher (odds ratio, infinity; P = 0.02) in adults. One or more mismatches of HA-1, 2, 4, and 5 were also significantly associated with GVHD (odds ratio, infinity; P = 0.006) in adults. These associations were not observed in children. CONCLUSIONS A mismatch of minor histocompatibility antigen HA-1 can cause GVHD in adult recipients of allogeneic bone marrow from HLA-identical donors. Prospective HA-1 typing may improve donor selection and identify recipients who are at high risk for GVHD.

The HLA-A*0201-Restricted H-Y Antigen Contains a Posttranslationally Modified Cysteine That Significantly Affects T Cell Recognition

A peptide recognized by two cytotoxic T cell clones specific for the human minor histocompatibility antigen H-Y and restricted by HLA-A*0201 was identified. This peptide originates from SMCY, as do two other H-Y epitopes, supporting the importance of this protein as a major source of H-Y determinants in mice and humans. In naturally processed peptides, T cells only recognize posttranslationally altered forms of this peptide that have undergone modification of a cysteine residue in the seventh position. One of these modifications involves attachment of a second cysteine residue via a disulfide bond. This modification has profound effects on T cell recognition and also occurs in other class I MHC-associated peptides, supporting its general importance as an immunological determinant.

Immunogenetics of human minor histocompatibility antigens : their polymorphism and immunodominance

Minor Histocompatibility (mH) antigens are polymorphic endogenously synthesized products that can be recognized by alloreactive T cells in the context of major histocompatibility complex molecules. In transplant situations where tissue donor and recipient are matched for HLA, mH antigens may trigger strong cellular immune responses. To gain insight into the polymorphism of mH antigens we studied their frequencies in the healthy population. Five HLA class I restricted mH antigens recognized by distinct cytotoxic T-cell (CTL) clones were used in the population genetic analysis consisting of a panel (N=100) of HLA typed target cells. Three mH antigens showed phenotype frequencies of 69% or higher, this contrasted the frequencies of two other mH antigens with 16 and 7% respectively. To gain insight into the “functional” polymorphism of the T-cell response to mH antigens, we analyzed the specificity of CTL clones within individuals. Three out of five individuals investigated shared a CTL response to one single HLA-A2 restricted mH antigen. These results indicate limited allelic polymorphism for some mH antigens in the healthy population and are suggestive of the existence of immunodominant human mH antigens.

The Hematopoietic System-specific Minor Histocompatibility Antigen HA-1 Shows Aberrant Expression in Epithelial Cancer Cells

Allogeneic stem cell transplantation (SCT) can induce curative graft-versus-tumor reactions in patients with hematological malignancies and solid tumors. The graft-versus-tumor reaction after human histocompatibility leukocyte antigen (HLA)-identical SCT is mediated by alloimmune donor T cells specific for polymorphic minor histocompatibility antigens (mHags). Among these, the mHag HA-1 was found to be restricted to the hematopoietic system. Here, we report on the HA-1 ribonucleic acid expression by microdissected carcinoma tissues and by single disseminated tumor cells isolated from patients with various epithelial tumors. The HA-1 peptide is molecularly defined, as it forms an immunogenic peptide ligand with HLA-A2 on the cell membrane of carcinoma cell lines. HA-1–specific cytotoxic T cells lyse epithelial tumor cell lines in vitro, whereas normal epithelial cells are not recognized. Thus, HA-1–specific immunotherapy combined with HLA-identical allogeneic SCT may now be feasible for patients with HA-1+ carcinomas.

Renal transplant patients monitored by the Cell-Mediated-Lympholysis assay : evaluation of its clinical value

Donor-specific cytotoxic T cell activity was measured over a period of 5 years after transplantation using the cell-mediated lympholysis (CML) test in 124 recipients of unrelated kidney allografts who received conventional immunosuppressive therapy consisting of azathioprine and prednisone. Since patients with a functioning transplant frequently display donor-specific CML non-responsiveness in vitro, we addressed the question of whether the CML status has a predictive value regarding the graft prognosis at any time interval until 5 years posttransplantation. From log-rank type analyses we conclude that the estimated relative risk calculated over the whole follow-up period of a CML-responder in the category of transplant rejectors is 1.25 with 95% confidence bounds between 0.94 and 1.65. Measurements of CML responder status during follow-up seem to have only limited prognostic value, although the relative risk is borderline significant when the analysis is restricted to the period between 2 weeks and 6 months posttransplantation.

Month-related variability in immunological test results; implications for immunological follow-up studies.

This longitudinal study was originally designed to detect changes in the in vitro immune response of healthy subjects as a result of a psychological intervention. In this study a significant proportion, about 70%, of the immunological variability in the test results was accounted for by the differences in immunological response levels of the subjects. Apart from this between‐subject‐effect, a significant proportion of the variability in test results was related to the month of data sampling. The month‐effect was computed in such a way that the between‐subject variation was taken into account. This resulted in a more accurate estimation of the month‐effect. Even after correction for the intervention, i.e. the defence of the PhD thesis, the effect of month of data sampling remains significant for mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, percentage of CD4 and CD8 cells, and for the response to the mitogens phytohaemagglutinin, pokeweed mitogen and concanavalin A as well as the results for the mixed lymphocyte culture for one pool out of three. In contrast, no significant month‐effect was observed for the whole blood cell counts, for the differential white blood cell counts as determined by monoclonal antibody staining for cell surface markers CD3, GD16, TAG and OKM1, nor for the immunoglobulin IgM and IgG serum levels. Likewise the cell‐mediated lympholysis activities measured against three pools of stimulator cells remained unaltered. We discuss the implications for future immunological follow‐up studies of the observation that a significant proportion of the variability in immunological test results is related to differences between subjects and to the month of data sampling.

A genetic analysis of human minor histocompatibility antigens demonstrates Mendelian segregation independent of HLA

An analysis of the genetic traits of human minor histocompatibility (mH) antigens is, unlike with inbred mice, rather complicated. Moreover, the fact that mH antigens are recognized in the context of MHC molecules creates an additional complication for reliable segregation analysis. To gain insight into the mode of inheritance of the mH antigens, we relied upon a series of HLA-A2-restricted cytotoxic T-cell (CTL) clones specific for four mH antigens. To perform segregation analysis independent of HLA-A2 gene: we transfected HLA-A2-negative cells with the HLA-A2 gene: this results in the cell surface expression of the HLA-A2 gene product and, if present, mH antigen recognition. The mode of inheritance of the HLA-A2-restricted mH antigens HA-1, -2, -4, and -5 was analysed in 25 families whoese members either naturally expressed positive. Analysis of distribution of the mH antigens in the parent population among the mating types, together with their inheritance patterns in the families, demonstrated that the four mH antigens behaved as Mendelian traits, whereby each can be considered a product of a gene with two alleles, one expressing and one not expressing the detected specificity. We also showed that the loci encoding the HA-1 and HA-2 antigens are not closely linked to HLa (lod scores Z (0 = 0.05) <−4.0). Some indication was obtained that the HA-4- and HA-5-encoding loci may be losely linked to HLA. While we are aware of the limited results of this nonetheless comprehensive study, we feel the similarity in immunogenetic traits between human and mouse mH antigens is at least striking.

HY Immune Tolerance Is Common in Women without Male Offspring

Background Sex difference is an established risk factor for hematopoietic stem cell transplantation (HSCT)-related complications like graft versus host disease (GVHD). CD8pos cytotoxic T cells specific for Y chromosome-encoded minor Histocompatibility antigens (HY) play an important role therein. Prior to HSC donation, female donors may encounter HY antigens through fetomaternal or transmaternal cell flow, potentially leading to the induction of HY-specific cytotoxic or regulatory immune responses. Whether HY priming occurs independent of parity, and whether HY priming is dependent on the presence of male microchimerism, is as yet unknown. Methods We investigated the presence of HY-specific regulatory T cells (Treg) and male microchimerism in 45 healthy women with a fully documented pregnancy and family history. HY peptide-induced linked suppression, a commonly reported functional feature of CD4pos and CD8pos Treg, was measured by trans vivo Delayed Type Hypersensitivity testing. As source of HY antigens, male microchimerism was analyzed by real-time PCR and defined by the presence of male DNA in at least one purified leukocyte cell type. Results HLA class I or class II restricted HY-specific Treg were detected in 26/42 (62%) women eligible for analysis. The prevalence of HY-specific Treg was significantly higher in women who had never given birth to sons than in women with male offspring (p = 0.004). Male microchimerism could be detected in 24 out of 45 (53%) women but did not correlate with the presence of HY specific Treg. Conclusions HY-specific Treg in women with male offspring have been described previously. Here we show for the first time that, in fact, HY specific Treg are more common in nulliparous women and in parous women with female offspring. Their presence is independent of the presence of male microchimerism. Whether HY-specific Treg presence in female stem cell grafts might decrease the GVHD incidence in male HSCT recipients needs to be investigated.

Transmaternal cell flow leads to antigen-experienced cord blood

Umbilical cord blood (UCB) is used for HSCT. It is known that UCB can comprise Ag-specific T cells. Here we question whether solely transmaternal cell flow may immunize UCB. Twenty-three female UCB samples were collected from healthy mothers and analyzed for minor histocompatibility Ag HY-specific responses. Forty-two of 104 tetramer(pos) T-cell clones, isolated from 16 of 17 UCB samples, showed male-specific lysis in vitro. Male microchimerism was present in 6 of 12 UCB samples analyzed. In conclusion, female UCB comprises HY-specific cytotoxic T cells. The immunization is presumably caused by transmaternal cell flow of male microchimerism present in the mother. The presence of immune cells in UCB that are not directed against maternal foreign Ags is remarkable and may explain the reported clinical observation of improved HSCT outcome with younger sibling donors.

The recognition of abnormal sex chromosome constitution by HLA-restricted anti-H-Y cytotoxic T cells and antibody

Using the cell-mediated lympholysis (CML) technique, we studied lymphocytes of six individuals with discrepancies between the karyotypic and phenotypic sex. Two sets of cytotoxic T cells (CTLs) obtained from two multitransfused female aplastic anemia patients were used as typing reagents. These cells were previously shown to kill allogeneic target cells from HLA-A2- or B7-positive male donors. An antiserum obtained from one of the patients likewise killed HLA-A2 male lymphocytes. The six patients studied were selected for the required antigens. Positive reactions were obtained with lymphocytes from a 46, XY woman with pure gonadal dysgenesis and a 45, XO male. Target cells of the mother of the latter patient were also lysed. One individual with a 45, XO/46, X, del(Y)? karyotype was weakly positive, while three 46, XX males were completely negative. The reactivity of the HLA-A2-restricted H-Y-specific antibody showed the same discriminatory patterns. The results obtained by theHLA-restricted CTLs as well as by the antiserum did not correlate with the presence of testes as is the case in a different test system for the serologically detectable male (SDM) antigen in man. On the other hand, there was a correlation with the presence of cytologically detectable Y-chromosome material in five of the six individuals studied. The HLA-restricted CTLs and the antibody might recognize the classical transplantation antigen H-Y.