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We designed a primer for the PCR directed against a highly conserved sequence of the TCR V beta gene. The V beta-universal primer, in combination with a constant region-specific primer, enabled us to amplify TCR beta cDNA of allo-HLA class-II-reactive T-cell clones by PCR without prior knowledge of their V beta sequences. The amplified TCR cDNA was purified by agarose gel electrophoresis and subjected to direct sequencing. In nine of ten T-cell clones analyzed, direct TCR sequencing gave readable sequence ladders, including two-thirds of V beta, junctional, and J beta regions. One T-cell clone gave an unreadable mixed-profile sequence ladder, indicating that this clone expressed more than one major TCR beta transcript. Even in this case, however, it was possible to determine two different TCR beta sequences separately using sequence primers specific to one of the 13 J beta segments deduced from the mixed ladder. Thus, direct sequencing utilizing the single V beta-universal primer enabled a simple, rapid, and reliable sequence determination of TCR beta cDNA of all T-cell clones analyzed.

Alloreactive human T lymphocyte clones were found to lose antigen-stimulated proliferative capacity and their ability to secrete interleukin 2 (IL 2) after a critical period in tissue culture. Instead, they gained the previously absent capacity to suppress lymphoproliferative (LP) responses in the presence or absence of exogenous IL 2. Such “ex-PLT” suppressive clones continued to grow perfectly well, retaining IL 2 and filler cell dependency, apparently normal karyotypes, and their OKT4+, OKT8− phenotypes. At least two suppressive mechanisms were demonstrated: (1) the “induction” of suppressive effectors in normal peripheral lymphocytes, and (2) a direct suppressive activity on lymphocyte proliferation shown by their ability to inhibit restimulation of cloned lymphocytes lacking suppressor cell precursors. The consistent “differentiation” from IL 2-secreting “helper” status to nonspecific suppressive status may represent a novel immunoregulatory phase in the long-term differentiation of normal human T cells.

Suppressor cells (SC) that nonspecifically inhibited lymphoproliferative (LP) responses were found after culturing peripheral blood mononuclear cells: (1) with suppressor T-cell clones, (2) in mixed lymphocyte cultures (MLC), and (3) with recombinant interleukin 2 (IL-2), but were not found after culture in medium alone. A monoclonal anti-IL-2 receptor (R) antibody (MoAb), TU69, which blocked LP responses of IL 2-dependent T-cell lines, also blocked SC induction by T-cell clones, but completely failed to inhibit SC generation in MLC or with IL-2. This suggests that the IL-2R epitope defined by TU69 was not involved in SC induction in the latter systems. MoAb against HLA-DQ (TU 22), -DR (TU34, SG 157), -DP (B7/21), or DR and DP (TU43, 58), all of which were able to block stimulation of appropriately specific clones, did not block SC induction in any of the three systems studied. In contrast, the broadly reactive moAb TU39, which binds at least DR and DP but also has additional reactivity for determinants tentatively designated “DY,” blocked SC induction by T-cell clones and in MLC. Finally, an anti-HLA class I MoAb, W6/32.HL, greatly decreased SC generation in MLC, but not with rIL-2 or T-cell clones. Thus, the induction of nonspecific SC was dissected into three pathways involving: (1) class I and TU 39-defined but not DR, DQ, or DP determinants (in MLC) which was independent of the IL-2R epitope bound by TU69; (2) only TU 39-defined determinants (with T-cell clones), which were IL 2R dependent; and, (3) neither class I, class II nor TU 39-defined determinants (induction by rIL-2), which was also TU69 + IL-2R independent.

SB phenotyping was undertaken on 96 HLA-D homozygous typing cells (HTCs) and 129 normal unselected heterozygous donors in the German population, using Interleukin-2-propogated primed lymphocyte typing (PLT) reagents. The results showed that the SB antigens in the normal population behave as a system of alleles at a single locus in Hardy-Weinberg equilibrium ( p ∼ 0.20). Estimated gene ffrequencies in the German population appeared to be significantly different ( p

Human T lymphocyte clones with specific proliferative response to influenza A virus were derived by limiting dilution from peripheral blood lymphocytes (PBL) after in vitro stimulation with autologous irradiated, virus-infected PBL. Four OKT3+4+8− lymphocyte clones (TLC) that showed HLA-restricted antigen-specific proliferative responses were used for a detailed analysis of the restriction elements for antigen presentation. None of the clones showed alloreactivity and all required the presence on the antigen-presenting cell of HLA class II antigens of one or other haplotype of the donor. Restriction elements for two clones were correlated with Dw1 rather than DR1, and for two others with Dw6 rather than DRw6. These latter clones showed differential recognition of HLA-Dw6 subtypes as defined tentatively by homozygous typing cells, without relationship to putative serological “splits” of DRw6. One of the Dw6-restricted clones was specific for a Dw6.1 (now Dw18) “subtype”, confirmed by family segregation analysis, the other for a broad Dw6 (Dw18 and Dw19) specificity. Studies with a panel of monoclonal antibodies against monomorphic determinants of HLA class II antigens revealed heterogenous patterns of blocking activity, distinguishing between clones of different restriction specificity. Inhibition patterns were partly as predictable from the known activity of the monoclonal antibody in alloantigeneic PLT systems. These results provide evidence that certain structures that function as restriction elements for antigen presentation also carry alloantigeneic determinants.

The requirements for activation of autocrine proliferation in human helper T cell clones (Th-TCC) by allogeneic cells were examined in monoclonal antibody (MoAb) blocking studies. Stimulation was not blocked by CD4, CD5, CD6, CD7, or CD45 MoAbs, despite high levels of expression of these antigens on the TCC. Only CD2 and CD11a (LFA-1) MoAbs blocked activation, the latter only when peripheral blood mononuclear cells (PBMCs) and not B-lymphoblastoid cell line (B-LCL) cells were used at stimulators. Responses to interleukin 2 (IL 2) were only minimally blocked by any of the MoAbs. All TCC were CD3 + and expressed the α/β chain T cell receptor (TCR) as detected by moAb WT31. Accordingly, CD3 and WT31 MoAbs consistently blocked stimulation by B-LCL, and in addition one anti-DR5 TCC and one anti-DQw3 TCC were blocked by MoAb 42/1C1, which is directed to an idiotypic determinant of the HPB-ALL leukemic line TCR. Only these two TCC reacted with moAb 42/1C1 in flow cytometry. These observations suggest that CD2- but not LFA-1-mediated interactions, as well as TCR and stimulating antigen binding, are absolutely necessary to activate Th-TCC.

Specificity analysis of two unusual alloreactive T-cell clones (TCC) is presented. In repeated in vitro sensitizations of various donors against irradiated HLA-A2, B44, DRw11, DRw52, DQw3, Dw5 homozygous stimulating cells, 202 autonomously proliferating TCC were isolated. Two of these, from different donors, responded against a majority of DRw11 homozygotes, but not against DRw11+ individuals from within or without the family of the original priming cells. Kinetic and stimulator cell titration experiments showed that the reason for this was not to be found in gene-dose effects. One out of 14 non-DRw11+ homozygotes was found that stimulated both TCC: this donor was DRw8+, Dw8+. Stimulation of the TCC was blocked by some anti-DR, and by multi-locus, monoclonal antibodies, but not by those specific for DQ or DP products. Thus, the existence of unique DR-associated stimulating determinants carried only by homozygous cells is demonstrated in man. Physiologically, this might imply that the actual restriction repertoire of HLA-DR homozygotes is not reduced to one half that of heterozygotes as expected.

Alloantigenic determinants causing secondary lymphoproliferative responses of primed T cells were investigated by blocking stimulation with monoclonal antibodies TU22, 34, 35, 36, 37, 39, 43, and 58 binding differentially to HLA-DR and SB or MB associated molecules. In particular, the use of cloned and functionally defined T cell responders greatly facilitated the assignment of stimulator-level inhibition caused by these antibodies. Thus, a novel way of functional "mapping" for stimulatory epitopes for sets of clones with restimulation specificities associated with HLA-D, SB, MB, or hitherto unidentified class II determinants is presented here. This considerably helps to elucidate the distinct immunoregulatory roles of the three major class II alloantigen systems thus far defined.

Lymphocytes alloactivated by Dw3 homozygous typing cells were cloned by the method of limiting dilution and cultured for prolonged periods using T-cell growth factor and irradiated pooled leukocytes (as feeder cells). Restimulation specificity of two clones functioning as primed lymphocyte typing reagents was investigated in panel and family studies. Cells from one of the clones (12-2) were always specifically stimulated by HLA-Dw3 antigens shared between the original priming cells and the stimulating panel cells. In an informative family KOH, however, cells from this clone seemed to detect a split in the Dw3 cluster. Cells from the other clone (12-8) failed to respond to Dw3 antigens as expressed by the original priming cells or by panel stimulating cells; rather, specificity of restimulation seemed to be associated with the expression of Dw4 antigens. Family segregation analysis did not support this conclusion however, since stimulating products segregated with one of the three Dw3-bearing haplotypes and with none of three Dw4-bearing haplotypes. This suggested that 12-8 cells may be responsive to antigens different from those detected by homozygous typing cells.

From a total of 37 different priming combinations between donors matched for HLA-A,B, and/or Dw/DR, but mismatched for SB, antigens, T cell clones strongly restimulated with concordance for SB specificities were isolated from only two. Most of the alloproliferative (PLT) clones obtained were restimulated by determinants not correlated with any currently known HLA product. Nonetheless, their stimulation was inhibited by a monoclonal antibody TU 39, which preferentially blocks stimulation by SB-, rather than by Dw/DR-associated determinants. Despite having an OKT4+, OKT-, Leu8- phenotype, and secreting Interleukin-2 after contact with stimulatory cells, these clones strongly suppressed proliferative responses of cloned PLT reagents as well as unprimed lymphocytes in mixed leukocyte cultures. They may thus represent a novel type of immunoregulatory T cell, stimulated by SB-related antigens, which despite their "helper/inducer" phenotype are able directly to suppress lymphoproliferative responses.