HLA-restricted responses to M and N determinants in the primed lymphocyte test.

We have studied the role of MNSs determinants in the primed lymphocyte test (PLT). The data demonstrate that incompatibility associated with the M or N antigens causes HLA-restricted proliferative responses in PLT. Responses to the M or N determinants required the presence of the same HLA-A antigen(s) on both the stimulator and the responder cells. No effects of S or s incompatibility were observed in this test. This is the first report of lymphocyte proliferative responses to "minor" alloantigens that require corecognition of the MHC determinants. These observations suggest possible new biological functions of these blood group antigens.

Role of the ABO, Secretor, and Lewis determinants in the primed lymphocyte test.

The antigenic determinants of the combined ABO, Lewis, and Secretor genes have been detected on the surface of lymphocytes by the lymphocytotoxicity test. We have studied the role of these determinants in the primed lymphocyte test (PLT), and the data demonstrate that Lewis incompatibility causes proliferative responses in PLT. On the other hand, no effects of ABO and Secretor incompatibilities were observed in this test. The frequency of the alloantigen-reactive cells (ARC) responding to Lewis and HLA-DR antigens in PLT was estimated by the limiting dilution analysis. The frequency of ARC to allogeneic Lewis-negative donors, who are positive for the sensitizing HLA-DR antigens ranged between 1:58 and 1:97. The incidence of ARC to Lewis-positive allogeneic donors who did not carry the sensitizing HLA-DR specificity was 1:94 to 1:142. These results demonstrate the presence of lymphocyte clones that are able to respond to antigens of the Lewis system. This study suggests that non-HLA antigens belonging to the Lewis system can cause stimulation of lymphocytes in the PLT test.

In vitro cell-mediated cytotoxicity to the male specific (H-Y) antigen in man.

We have studied the role of HLA antigens in restricting specificity of the cytotoxic lymphocytes (CTL). CTL's developed between female responder/male stimulator combinations, were tested for H-Y antigen killing in cell-mediated lympholysis. Two CTL's demonstrated HLA-restricted H-Y cytotoxicity. In both instances, the responders are married parous females and both are positive for HLA-A2. These CTL's lysed target cells from donors who are either positive for the sensitizing HLA antigen or who are HLA-A2-positive males. On the other hand, one CTL where the HLA-A2-positive responder is not a parous female did not show HLA-restricted H-Y cytotoxicity. Also, CTL's where responders do not carry HLA-A2 showed no H-Y cytotoxicity. The data suggest that pregnancy(ies) is sufficient in itself to induce HLA-restricted H-Y cytotoxicity and that it can be recalled by in vitro stimulation with lymphocytes from an unrelated male donor. Also, in these studies HLA-restricted H-Y cytotoxicity was obtained only with targets that shared HLA-A2 with the effectors.

Limiting dilution analysis of alloantigen-reactive cells which respond to allogeneic lymphocytes in human MLC and PLT.

We have determined the frequency of the alloantigen-reactive cells (ARC) in human MLC and PLT by the limiting dilution analysis. In PLT, the frequency of the ARC to the original sensitizing donor ranged between 1:32 to 1:62, an increase of six to nine-fold after priming in MLC. The MLC primed populations were also enriched (three to five fold) for the ARC responding to the PL-positive allogeneic donors. The incidence of the ARC was 1:62 to 1:118 with donors positive for the sensitizing HLA-DRw antigen and 1:77 to 1:144 with donors negative for the specific HLA-DRw determinant. The results from experiments utilizing pooled stimulating cells from the original and allogeneic donors suggest that same subpopulation of cells responds to the sensitizing HLA-DRw determinant, whether it is presented by the specific stimulator or by a third-party allogeneic donor. On the other hand, different subpopulations of alloreactive cells respond to different alloantigens. In MLC experiments between HLA-identical siblings, the incidence of the ARC ranged between 1:995 to 1:1673. The responses of the ARC to non-HLA antigens were observed under conditions where responder cells were limiting. Also, the responses of the limiting numbers of responding cells were inhibited by mitomycin-treated autologous lymphocytes. Nonresponse in MLC combinations with higher responder cell numbers was not due to small numbers of stimulating cells.

Typing for MLC determinants using lymphocytes from cousin-marriage offspring as typing cells.

A first cousin marriage family with three children was studied. One sibling (AW) was homozygous for W32.W10. When used as stimulator in one way MLC, AW elicited a very low response in family members possessing this haplotype. Serologically identical sibling and father combination showed bidirectional non-stimulation in one way MLC. Therefore, AW is assumed to be homozygous not only for SD antigens, but also for LD determinants which we call LD W10a. Among the 43 unrelated individuals tested with AW, an association between LD W10a and W10 antigen of the second HL-A locus was observed. Of 12 W10 positive individuals tested, five carry this allele as compared to one out of 31 W10 negative individuals. The data suggest a strong linkage disequilibrium between the FOUR locus determinant W10 and LD determinant LD W10a.

Inhibition of the mixed leucocyte culture reaction by plasma from renal transplatn recipients.

Plasma samples from 28 renal allograft recipients were studied for the presence of alloantibodies blocking MLC response. MLC blocking antibodies were found in plasma samples obtained from 19 recipients, six with rejected allograft and 13 with long-term surviving transplant. In many instances, these antibodies were found in the absence of detectable lymphocytotoxic antibodies. The MLC blocking activity resides in IgG fractions of plasma samples and was decreased only after absorption with lymphocytes, no effect on blocking was observed by absorption with platelets and fibroblasts.