We describe here the properties of mAb GK1.5, which recognizes a cell surface molecule designated L3T4; the determinant on L3T4 recognized by mAb GK1.5 is designated L3T4a. We present evidence here that: i) the expression of L3T4a by murine T cell clones correlates primarily with class II MHC antigen-reactivity; ii) mAb GK1.5 blocks all class II MHC antigen-specific functions (cytolysis, proliferation, release of lymphokines) by murine class II MHC antigen-reactive T cell clones, although there appears to be clonal heterogeneity in the degree to which these functions are blocked by mAb GK1.5; iii) mAb GK1.5 blocks class II MHC antigen-specific release of IL-2 from cloned T cell hybridomas by blocking class II MHC antigen-specific binding; and iv) L3T4 is very similar to the human Leu3/T4 antigen. The properties of mAb GK1.5 (complement fixation, reactivity with all mouse strains tested, profound blocking of all class II MHC antigen-specific functions by murine T cells, usefulness for FACS analyses, and usefulness for immuno-precipitation/SDS-PAGE analyses) make it suitable for investigating both the role of class II MHC antigen-reactive T cells in various immunological phenomena and the mechanistic basis, at the molecular level, of class II MHC antigen-reactivity by murine T cells.

The characterization of the antigen-recognizing structure on the various classes of T cells has been a difficult problem for immunologists. Three aspects of this problem are particularly controversial: first, whether the genes that code for the variable portion of immunoglobulin also code for the antigen-recognizing portion of the receptor on all classes of T cells (1-4); second, whether T cell receptors specific for allo-major histocompatibility complex (MHC) 1 products are unique or are included within the population of receptors for conventional antigens (5-10); and third, how T cells, particularly those of the cytotoxic and helper classes, are able to demonstrate an apparent dual specificity for both a conventional antigen and a product of the MHC such that they function only when confronted with both the appropriate antigen and MHC product (11-16). Various models have been proposed to explain this phenomenon. Some have proposed that antigen and MHC are recognized independently via two receptors on the T cell (17-23). Others have proposed various forms of dependent recognition, involving, for example, a single receptor on the T cell, and/or a physical interaction between antigen and MHC products in antigenpresenting cells (24)(25)(26). A number of experimental results have been used by various investigators to argue for one or the other of these models, but there is little evidence that distinguishes the various possibilities.A direct attack on these questions has been hampered by the lack of suitable sources of clonal T cell lines analogous to the B cell myelomas. Recently, two techniques have offered promise in this regard. The first is the establishment of long-term cultures of normal antigen-specific T cells through the use of repeated in vitro antigen stimulation and/or the growth factor, interleukin 2 (IL-2; formerly, T cell growth factor) (27-30). The second is the production of immortal T cell hybrids between normal antigenspecific T cells and T cell tumor lines (31-36). In the present study, we combined these two methods to produce inducible, antigen-specific, H-2-restricted T cell hy-*

An antibody-secreting B cell hybridoma, KJ1-26.1, has been prepared from mice immunized with the T cell hybridoma DO-11.10, which recognizes chicken ovalbumin in association with I-Ad (cOVA/I-Ad). KJ1-26.1 blocks I-restricted antigen recognition by DO-11.10 and a subclone of this T cell hybridoma, DO-11.10.24, which has the same specificity for cOVA/I-Ad as its parent. KJ1-26.1 does not block I-restricted antigen recognition by any other T cell hybridoma tested, including a number of T cell hybridomas closely related to DO-11.10, with similar, but not identical, specificities for antigen/I. Moreover, KJ1-26.1 binds to DO-11.10 and DO-11.10.24, but not to any other T cell hybridomas tested, including three subclones of DO-11.10 that have lost the ability to recognize cOVA/I-Ad. Thus, in every regard KJ1-26.1 appears to be binding to all or part of the receptors for antigen/I on the T cell hybridoma DO-11.10. KJ1-26.1 appears to bind to approximately 15,000 molecules/cell on the surface of DO-11.10. The antibody precipitates an 80,000 dimer from the cells, which on reduction migrates as 40-44,000 monomers. The receptor(s) for antigen/I on DO-11.10 therefore includes molecules with these properties.

We examined the ability of a set of cloned chicken ovalbumin (cOVA)-specific, Id-restricted, T cell hybridomas to produce interleukin-2 in response to cOVA presented by the Ia+ B cell lymphoma line, A20-2J. Although viable A20-2J cells presented native, denatured, and fragmented cOVA more or less equally well, A20-2J cells that were glutaraldehyde-fixed could present only enzymatically or chemically fragmented cOVA. These results suggest that antigen fragmentation may be both necessary and sufficient to define accessory cell processing of soluble antigens so that they may be recognized in association with I-region molecules by T cells.

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Mature T cells segregate phenotypically into one of two classes: those that express the surface glycoprotein CD4, and those that express the glycoprotein CD8. The CD4 molecule is expressed primarily on helper T cells whereas CD8 is found on cytotoxic and suppressor cells. A more stringent association exists, however, between these T-cell subsets and the major histocompatibility complex (MHC) gene products recognized by their T-cell receptors (TCRs). CD8+ lymphocytes interact with targets expressing class I MHC gene products, whereas CD4+ cells interact with class II MHC-bearing targets. To explain this association, it has been proposed that these 'accessory' molecules bind to monomorphic regions of the MHC proteins on the target cell, CD4 to class II and CD8 to class I products. This binding could hold the T cell and its target together, thus improving the probability of the formation of the trimolecular antigen: MHC: TCR complex. Because the TCR on CD4+ cells binds antigen in association with class II MHC, it has been difficult to design experiments to detect the association of CD4 with a class II molecule. To address this issue, we devised a xenogeneic system in which human CD4 complementary DNA was transfected into the murine CD4-, CD8- T-cell hybridoma 3DT-52.5.8, the TCR of which recognizes the murine class I molecule H-2Dd. The murine H-2Dd-bearing target cell line, P815, was cotransfected with human class II HLA-DR alpha, beta and invariant chain cDNAs. Co-culture of the parental T-cell and P815 lines, or of one parental and one transfected line resulted in a low baseline response. In contrast, a substantial increase in response was observed when CD4+ 3DT-52.5.8 cells were co-cultured with HLA-DR+ P815 cells. This result strongly indicates that CD4:HLA-DR binding occurs in this system and that this interaction augments T-cell activation.

During development in the thymus, invariant natural killer T (iNKT) cells commit to one of three major functionally different subsets, iNKT1, iNKT2, and iNKT17. Here, we show that T cell antigen receptor (TCR) signal strength governs the development of iNKT cell subsets, with strong signaling promoting iNKT2 and iNKT17 development. Altering TCR diversity or signaling diminishes iNKT2 and iNKT17 cell subset development in a cell-intrinsic manner. Decreased TCR signaling affects the persistence of Egr2 expression and the upregulation of PLZF. By genome-wide comparison of chromatin accessibility, we identify a subset of iNKT2-specific regulatory elements containing NFAT and Egr binding motifs that is less accessible in iNKT2 cells that develop from reduced TCR signaling. These data suggest that variable TCR signaling modulates regulatory element activity at NFAT and Egr binding sites exerting a determinative influence on the dynamics of gene enhancer accessibility and the developmental fate of iNKT cells.

We have examined the role of the murine homologue of Leu-3 T4, L3T4, in recognition of antigen in association with products of the major histocompatibility complex (Ag/MHC) by murine T cell hybridomas. A series of ovalbumin (OVA)/I-Ad-specific T cell hybridomas were ranked in their sensitivity to Ag/I by measuring their ability to respond to low doses of OVA, or their sensitivity to inhibition by anti-I-Ad antibodies. T cell hybridomas with low apparent avidity for OVA/I-Ad, i.e. that did not respond well to low concentrations of OVA and were easily inhibited by anti-I-Ad, were also easily inhibited by anti-L3T4 antibodies. The reverse was true for T cell hybridomas with apparent high avidity for Ag/MHC. We found that the presence of low doses of anti-L3T4 antibodies caused T cell hybridomas to respond less well to low doses of Ag, and to be more easily inhibited by anti-I-Ad antibodies. These results suggested that the role of the L3T4 molecule is to increase the overall avidity of the reaction between T cells and Ag-presenting cells. In support of this idea was the discovery of several L3T4- subclones of one of our L3T4+ T cell hybridomas, D0.11.10. The L3T4- subclones had the same amount of receptor for OVA/I-Ad as their L3T4+ parent, as detected by an anti-receptor monoclonal antibody. The L3T4- subclones, however, responded less well to low doses of OVA, and were more easily inhibited by anti-I-Ad antibodies than their L3T4/ parent. These results showed that the L3T4 molecule was not required for surface expression of, or functional activity of, the T cell receptor for Ag/MHC. The L3T4 molecule did, however, increase the sensitivity with which the T cell reacted with Ag/MHC on Ag-presenting cells.