Cytotoxic treatment with rabbit antiserum raised against purified glycosphingolipid "asialo GM1" was capable of eliminating natural killer (NK) activity of spleen cells from different inbred mouse strains including CBA/J, C57BL/6, BALB/c, AKR, and athymic nude mice. The anti-asialo GM1 antiserum showed little cross-reactivity with structurally related glycolipids, e.g. GM), GD 1 b and asialo GM2 in the microflocculation test. The specific reactivity of this antiserum with NK cells was confirmed by the quantitative absorption of anti-NK activity with graded amounts of asialo GM1 but not with other glycosphingolipids. The absorption of anti-brain-associated T cell antigen (anti-BAT) with asialo GM1 also effectively diminished its anti-NK activity, leaving the ability to kill T cells intact. This suggests that the antibody to asialo GM1 is responsible for the anti-NK activity contained in the anti-BAT antiserum. In contrast to the extreme sensitivity of NK cells to anti-asialo GM1, alloreactive cytotoxic T killer cells generated in the mixed lymphocyte culture were not killed by anti-asialo GM1 and complement. These results indicate that asialo GM1 is expressed on mouse NK cells in a high concentration.

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CD69, an 'activation marker' that is rapidly induced on mature T cells after stimulation through the T cell antigen receptor (TCR) was found to be expressed on approximately 10% of normal thymocytes. All of these CD69+ thymocytes express alpha beta TCR, and they include both TCRlowCD4+CD8+ and TCRhighCD4+CD8- or CD4-CD8+ thymocytes. The CD69+ cells can be further segregated into heat-stable antigen (HSA)+TCRlow, HSA+TCRhigh and HSA-TCRhigh thymocyte populations. None of CD69+ cells express the mature T cell marker Qa-2. Thus CD69+ cells present in vivo appear phenotypically to represent transitional cell populations between immature TCRlowHSA+Qa-2-double-positive cells and mature TCRhighHSA-QA-2+ single-positive cells. In addition, TCR engagement by MHC molecules is required for CD69 expression in the thymus. Taken together, the CD69+ thymocytes appear to represent the cells auditioning in positive selection process or they are the cells that have been positively selected recently. Analysis of a TCR transgenic mouse model revealed an increased number of CD69+ thymocytes in a positively selecting thymus, whereas no CD69+ transgenic TCR+ thymocytes were observed in the non-selecting thymus. Based on the results of this study, we suggest that the surface expression of CD69 serves as a useful marker to identify and trace those thymocytes that are engaged in the TCR-mediated positive selection process in the thymus.

The locus of the gene that codes for the antigen-specific suppressive T-cell factor was determined to be in a new subregion "I-J" which locates between I-B and I-C subregions in the H-2 histocompatibility complex. This was shown by two different lines of evidence: (a) The absorbing capacity for the suppressive T-cell factor of several alloantisera against restricted I subregions did not correlate with their specificity for previously known Ia molecules which are coded for by genes in I-A and I-C subregions, but was associated with the specificity for the products of genes putatively present between I-B and I-C subregions. By the occurrence of special recombinant strains, i.e. B10.A(5R), B10.A(3R), B10.S(9R), and B10.HTT, which differ with respect to the I-J subregion, we were able to produce alloantisera which distinguish I-J subregion gene products. The absorption studies using these special alloantisera directed to I-J subregion clearly indicated that the suppressive T-cell factor is a product of I-J subregion gene(s), and that the molecule is distinct from known Ia molecules expressed on splenic B cells. (b) Taking advantage of the fact that there is a strict histocompatibility requirement for the effective suppression between the donor and recipient strains of the suppressive T-cell factor, we were able to determine the required identities of the genes in the H-2 complex existing among those present between I-B and I-C. Again, utilizing the T-cell factors obtained from special recombinant strains, i.e. B10.A(4R) and B10.A(5R), we were able to locate the gene that codes for the suppressive T-cell factor reactive only with relevant haplotype strains between I-B and I-C subregions. These results are most reasonably explained by the presence of a new subregion I-J which is specialized in coding for the suppressive T-cell factor as a different molecule from previously known Ia molecules.

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Since the original discovery of T-and B-cell collaboration in the antibody response, it has been generally accepted that the helper T cell represents a single subset in the multimember family of T cells. There are, however, some controversial findings on the nature of the help with respect to its specificity, class preference, and genetic restrictions. In the antibody response to a hapten.carrier conjugate, the helper T cell is believed to recognize the carrier determinant and then to help antibody synthesis by B cells which recognize another determinant (hapten) linked to the same molecule. This type of interaction is supported by a phenomenon known as the carrier effect, in which the hapten-specific secondary antibody response is successfully elicited by the hapten coupled to the same carrier by which animals have been primarily immunized (1-4). A similar cooperative interaction between hapten-specific B cells and carrier-specific T cells is readily demonstrable in the adoptive secondary antibody response (5-8). Since in these cases hapten and carrier determinants must be present on a same single molecule, the T-B collaboration should occur upon recognition of the hapten by B cells, and recognition of the adjacent carrier determinants by T cells in a cognate form.There are certainly no denials of the presence of this type of interaction. Nevertheless, there are several examples in which the cognate interaction is not likely to occur in certain T-cell-dependent antibody responses. In fact, various antigen-nonspecific factors derived from T cells can trigger the B-cell response, in which the factors themselves do not recognize carrier determinants (9-13). Several investigators are also aware that the hapten-specific B cells can be triggered under certain circumstances in which B and T cells are independently stimulated by corresponding determinants present on two distinctly separate molecules (8,12,14,15). Hence, cognate interaction is not the only pathway for the effective T-B cell collaboration. One could ask whether the same or different helper T cells are involved in these diverse pathways of T-B cell collaboration.Such problems are now even more complicated by the growing evidence suggesting that the helper T cell may recognize not only the carrier determinants, but also the products of major histocompatibility gene complex (MHC).

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During development of CD4+ T lymphocytes in the periphery, differential expression of cytokine genes, such as those of interleukin (IL)‐2 and IL‐4, occurs in distinct T‐cell subsets. IL‐4 is a cytokine produced by T‐helper 2 (Th2) cells, and the IL‐4 receptor (IL‐4R)‐mediated signaling pathway is thought to be required for commitment to the Th2 phenotype. However, the molecular basis for development of the Th subset‐specific production of IL‐4 remains unclear. We demonstrate here that the IL‐4 promoter is functional in Th1 and B cells which do not normally form IL‐4 transcripts as well as in IL‐4‐producing T cells. Based on studies of the effect of several different upstream and downstream regions of the IL‐4 gene on IL‐4 promoter activity, a Th1‐specific IL‐4 silencer element was identified in the 3′‐untranslated region. The silencer region contained a consensus sequence for a transcriptional factor that is normally regulated by the IL‐4 R signaling pathway, STAT6. Nuclear expression of STAT6 protein, which was shown to bind to the silencer region, was observed in Th2 cells but not in Th1 cells. Deletion of the STAT6‐binding site from the silencer region and inhibition of STAT6 function resulted in the appearance of silencing function even in Th2 cells. These results provide evidence that the silencer element, and the binding of STAT6 to this element, play a permissive role in determining the commitment into Th2 phenotype.