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Mice were immunized i.p. with soluble or heat-denatured protein antigens [ovalbumin, beta-galactosidase, or recombinant E7 protein of human papilloma virus type 16 (HBV)]. Heat-denatured (100 degrees C) preparations of these proteins were able to induce cytotoxic T lymphocytes (CTL) that recognize cells expressing the respective genes, whereas native protein was either inefficient or required up to 30-fold higher doses. If the heat-treated proteins were separated into aggregated and soluble fractions by ultracentrifugation, only the aggregated fractions were able to induce specific CTL; this is probably because of the easier access to one of the major histocompatibility complex class I loading pathways for exogenous antigen. Addition of the adjuvant aluminium hydroxide (alum) to aggregated proteins abolished their ability to induce CTL; thus, a condition leading to a strong antibody response appeared to inhibit CTL induction. Interestingly, immunization with heat-denatured ovalbumin plus alum increased the IgM/IgG1 ratio compared to immunization with native ovalbumin and alum. Immunization of B6 mice transgenic for an HLA-A2/H-2K(b) hybrid gene with heat-denatured, recombinant HPV 16-E7 protein induced D(b)-restricted CTL specific for the peptide 49-57 of E7, indicating that this epitope is immunodominant over any A2-restricted E7 epitope in these mice. A whole influenza virus preparation heated to 100 degrees C or even autoclaved was still able to induce virus-specific CTL and BALB/c spleen cells heated to 100 degrees C could still cross-prime minor H-specific CTL in B6 mice, although with lower efficiency than fresh spleen cells. Thus, aggregated proteins can be considered as components for future vaccines.

SIT (SHP2-interacting transmembrane adaptor protein) is a recently identified transmembrane adaptor protein, which is expressed in lymphocytes. Its structural properties, in particular the presence of five potential tyrosine phosphorylation sites, suggest involvement of SIT in TCR-mediated recruitment of SH2 domain-containing intracellular signaling molecules to the plasma membrane. Indeed, it has recently been demonstrated that SIT inducibly interacts with the SH2-containing protein tyrosine phosphatase 2 (SHP2) via an immunoreceptor tyrosine-based inhibition motif (ITIM). Moreover, SIT is capable to inhibit TCR-mediated signals proximal of activation of protein kinase C. However, inhibition of T cell activation by SIT occurs independently of SHP2 binding. The present study was performed to further characterize the molecular interaction between SIT and intracellular effector molecules and to identify the protein(s) mediating its inhibitory function. We demonstrate that SIT not only interacts with SHP2 but also with the adaptor protein Grb2 via two consensus YxN motifs. However, mutation of both Grb2-binding sites also does not influence the inhibitory function of SIT. In contrast, mutation of the tyrosine-based signaling motif Y(168) ASV completely abrogates the ability of SIT to inhibit T cell activation. Co-precipitation experiments revealed that the tyrosine kinase p50(csk) could represent the negative regulatory effector molecule that binds to this motif.

The previously demonstrated role of H-2 gene products in nonimmune cell interactions was further analyzed. As model of such interactions served the erythropoietic performance (in terms of 59Fe uptake) of bone marrow cell (BMC) inocula in the cellular environment of the spleen of massively irradiated hosts. When the performance in syngeneic hosts is taken as standard, the performance of cells from the same pool in H-2 disparate (congenic) hosts is subnormal. The inhibition was also observed in two congenic strain combinations where the H-2 disparity did not involve the I region. The capacity of BMC to distinguish H-2 -identical and H-2 -disparate hosts could be greatly reduced or virtually abolished by enzymatic digestion of certain cell surface carbohydrates. An essential effect of neuraminidase was further increased by emulsin. When the digested cells were left for 6 h in an enzyme-free medium, their capacity to dinstinguish H-2 disparities recovered. A pretreatment with papain, which cleaves a large fragment of the H-2 protein carrying both the antigenic site and the carbohydrate chain, had an effect comparable with but no greater than the sugar-removing enzymes. The enzymatic effect was largely due to the impairment of the performance of BMC in H-2 -identical hosts rather than to an improvement in H-2 -disparate ones. The neuraminidase- and emulsin-sensitive groupings thus seem to be essential for the cells to recognize “self” rather than “non-self” and to interact with H-2 -identical cells in physiological processes. A hypothesis is proposed that in H-2 glycoproteins, the antigenically and physiologically active sites are distinct, being located in the protein and carbohydrate parts of the molecules, respectively. It is further suggested that the normal function of the H-2 products might consist in providing the cell surface with structures which are complementary to similar structures on other cells; the carbohydrate moiety seems to be essential for this type of physiological interaction in contrast to the key and lock-type of complementarity in immune interactions which is based on protein structures.

Two monoclonal antibodies, MEM-15 and MEM-18, were prepared which recognize a monocyte 53-kDa antigen. A soluble form of this antigen was found in most normal sera and in urine of some nephrotic patients. Milligram amounts of this glycoprotein were isolated by immunoaffinity chromatography from urine and used for quantitative amino acid and carbohydrate analysis and N-terminal amino acid sequencing. The reactivity of the isolated antigen with several previously described monoclonal antibodies indicates that it is the antigen previously called MY-4 or MY-23. A significant homology exists between the sequenced N-terminal portion of the antigen and sequences found in the kinase-related transforming protein src and in the precursor of epidermal growth factor.