T cell clones isolated from class II MHC-disparate MLR combinations, and specific for I-Ak and I-Ek molecules, respectively, are shown to induce acute lethal graft-vs-host disease in unirradiated recipients. Cytolytic and noncytolytic clones are equally efficient in this respect. The lethal disease is dependent on recognition of the stimulatory class II molecules in the host. The clones home to lungs and liver, and become activated in these organs as demonstrated by an in vivo thymidine incorporation assay. After activation, a severe vascular leak syndrome develops causing death of the recipients within 5 d after the injection of 5 x 10(6) to 10(7) cloned cells. The disease develops without the participation of secondary host-derived inflammatory mechanisms, such as mast cell degranulation, complement activation, and the release of prostaglandins, oxygen radicals, or proteolytic enzymes. The results raise the possibility that Th cells can directly influence vascular permeability, and control, thereby, the acute inflammatory reaction of blood vessels.

The specificity of binding of stimulator-derived H-2 antigens by mixed lymphocyte culture (MLC)-activated T blasts was investigated under conditions of antigen excess. We have shown that the detectable proportion of alloantigen-binding blasts from primary MLC is a function of antigen concentration, and can represent up to more than 90 percent of total blasts, when the antigen is presented in the appropriate form (on mitomycin-treated viable stimulator cells, or membrane vesicles prepared from lipopolysaccharide blasts), and at nonlimiting concentration. Thus stimulator alloantigen-binding directly parallels the proliferative response and is not restricted to a subpopulation of T blasts. However, the marked dependence of the binding on antigen concentration indicates that cells with a wide range of receptor affinities for the stimulating determinants are involved. In view of this possibility, the specificity of binding by these cells was studied. We have demonstrated that stimulator K, I, and D region products are bound by nonoverlapping subpopulations of blasts, the sum of which may represent 93 percent of total blasts. Thus, specific distinction by these cells between different H-2 region products is not affected by the putative heterogeneity in terms of receptor affinities. However, specificity with respect to unrelated H-2 haplotypes is strictly dependent on antigen concentration. A preferential binding of stimulator membrane vesicles occurs at limiting concentrations; whereas the majority of blasts bind stimulator and third- party vesicles equally well at high vesicle concentrations. The binding of both vesicle types is specific in that it can be inhibited with the relevant anti-H-2 sera. Furthermore, stimulator and third-party vesicles seem to compete for binding sites on the same cells, as shown by cold antigen inhibition. From these results, we propose that there is an imperfect distinction between stimulator and third-party H-2 antigens by the majority of primary MLC blasts. In contrast, highly selected long-term MLC blasts do not bind third-party H-2 antigens at any concentration, and seem to have high affinity for the stimulating antigens. We conclude that large numbers of clones with low-affinity (cross- reactive) receptors are generated in primary MLC, most of which become eliminated during long-term selection. This implies that the frequency of cells strictly specific for nonshared stimulating determinants must be minute.

The proliferative T cell response of inbred mouse strains to the random copolymer poly(Glu50Tyr50) (GT) was found to fall into two categories. Some strains responded only marginally (delta cpm values less than 10,000 and stimulation indices less than 3), whereas other strains mounted a substantial response (delta cpm 10,000 to 80,000, SI 3 to 30). The response is controlled by the A alpha and A beta loci of the major histocompatibility complex (MHC), as well as by genes not linked to the MHC. Because the response is selectively inhibited by monoclonal antibodies specific for the A alpha A beta molecule, we assume that its control by A loci is manifested as an A-restriction of the participating T (Ly-1high, Ly-2-) cells. It is of interest that the responsiveness is recessive in F1 hybrids of responder and nonresponder strains that are H-2-identical, but differ at their genetic background. Nonresponsiveness of these F1 mice is caused neither by a defect of antigen presentation, nor the result of immune suppression on priming or at the effector phase of the response. It is most likely the consequence of clonal deletion during the establishment of self-tolerance.

Strain combinations generating cytotoxic T lymphocytes (CTL) specific for a single class I (K or D) or class II (A or E) MHC molecule were set up. The responder cells were separated into Ly subsets (Ly-1+2-, Ly-1-2+, and Ly-1+2+) on day 5 of culture by using lytic or non-lytic selection techniques and monoclonal Ly-specific antibodies. The separated subsets were restimulated on day 8 and tested for secondary CTL activity on day 12. Class II-specific secondary CTL could be generated from all three subsets, whereas class I-specific CTL developed only in the Ly-1+2+ and Ly-1-2+ subsets. The Ly-1+2+ cells underwent a phenotypic shift to Ly-1-2+ by day 12, whereas CTL generated from the Ly-1+2- and Ly-1-2+ subsets retained their phenotype up to the secondary effector stage. The cells separated according to their Ly phenotypes on day 5 were the progeny of unprimed progenitors expressing the same Ly phenotypes. Unprimed Ly-1+2+ cells gave rise to CTL in the absence of the other subsets, while unprimed Ly-1+2- and Ly-1-2+ cells required the help of Ly-1+2+ cells (or soluble factors) during priming to become non-lytic CTL precursors by day 5, and cytolytic cells after restimulation. The Ly-1+2- subset could generate class II-specific secondary CTL only in the absence of the other two subsets. Apparently, alloantigen-primed Ly-1+2+ and Ly-1-2+ cells suppressed the development of cytolytic activity in the Ly-1+2- subset. The combined data provide a comprehensive pathway of CTL differentiation from T cell subsets.

Inoculation of 10(8) unirradiated, minor H antigen-incompatible spleen cells into recipients leads to a failure of the induction of cytolytic T lymphocytes (CTL) specific for these antigens. In contrast, a strong CTL response against minor H antigens is obtained when the inoculated cells are irradiated or treated with Thy-1-, Lyt-1- or Lyt-2-specific antibody and complement. Thus the failure of CTL induction is probably due to suppression mediated by radiosensitive, Lyt-1+2+ T cells in the immunizing inoculum. We demonstrate here that the inoculated cells must share class I MHC loci with the recipients for the suppression to occur. Thus, the interaction between the suppressor T (Ts) cells and their targets (presumably the CTL precursors) is restricted by class I molecules. A disparity at class II loci between the inoculated cells and the recipients overrides the class I-restricted suppression, possibly through a positive allogeneic effect. The simplest interpretation of the class I restriction of Ts cell-target cell interaction is that the CTL precursors recognize minor H antigens in the context of class I molecules on the surface of the Ts cells themselves.