The suppressor circuit and it's components have been studied extensively in this laboratory and others for the past ten years. This laboratory has produced factor-secreting hybridoma cells which are analogous to first-order T suppressor cells directed against the tumor P815 . A monoclonal antibody has been raised which recognizes a common portion of suppressor cells and factors. These tools are used in this study. It had been seen that when 20 µg A10F (factor secreted by A10, the Ts1 analogue) was injected into a mouse concurrently with P815, the suppression of the mouse's immune response was boosted. This led to increased tumor growth and accelerated death. However, when A10F was injected ten days prior to the mouse receiving P815, the opposite effect was seen. Mice had smaller tumors and longer survival times. This was not the contrasuppressive effect described by other laboratories, as the effect seen was not merely an abrogation of suppression, but rather enhancement of the immune response. The specificity and dose response of the effect was examined. This immune enhancement effect was not specific within the context of syngeneic tumor systems. It was found that the same effects were seen when P815 was replaced with L1210 or M-1, both also being H-2d tumors. In fact, L1210 was more sensitive to A10F than P815. There was some level of specificity to the enhancement effect. When A10F was replaced with Fd11F, a suppressor factor raised to ferredoxin, no effect was seen. Conversely, A10F did not produce the same effects as Fd11F in the ferredoxin system. Suppressor deletion therapy was used in both of these systems to confirm that suppressor cells were responsible for the effects seen. Dose response studies showed that the enhancement effect was dose dependent. Doses of A10F below 20 µg did not produce enhancement in the P815 system. Enhancement was seen with lower doses of A10F in the L1210 system, but the effect did decrease at the lower doses. A model is proposed for the data presented. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

A large amount of effort has gone into the elucidation of the mechanism of suppression of the immune system. This level of immunoregulation has been demonstrated to be mediated by both antigen-nonspecific and antigen-specific protein factors elicited by leukocytes. In this work, two different modes of immunosuppression were investigated. First, an attempt was made to purifiy an antigen-nonspecific protein factor, SIF (Suppressor Inducer Factor) secreted by the Natural Suppressor cell line M1-A5. M1-A5 culture supernatants were subjected to ion exchange chromatogrphy (IEC) and fast protein liquid chromatography (FPLC). Bioactivity of eluted fractions was determined by the plaque forming cell assay and followed through the purification. Reducing SDS-PAGE of selected fractions suggested that bands with Mr's of >110 KD and/or 55 KD were mediating the suppressive activity. In addition, an assay was developed to further investigate the mode of action of SIF. Second, the binding of two antisera raised to components associated with murine antigen-specific suppression was studied using human peripheral leucocytes and several human tumour cell lines. Anti-p80 and anti-p30 binding was found to be variable (within a range) and to involve two populations of human mononuclear cells. Subsequently, it was found that all CD3⁺ (T cells), CD19⁺ (B cells) and neutophils expressed both the p80 and p30 determinants. Four human leukemic cell lines were found to express varying levels of the p80 and p30 determinants. Cell lysates from each of the cell lines were subjected to Western blot analyses using anti-p30. The results showed that anti-p30 binds to a major band of 42 KD and minor bands of 60 and 80 KD in all lysates. In addition, a 25 KD band was observed in RAJI and CEM-CM3 lysates only. Thus, it appears that HuT 78 cells sythesize but are unable to express the p30-containing, 42 KD molecule on the cell surface. No firm conclusions can be made with respect to the biochemical nature or mechanisism of action of either of the two suppressor factors studied in this work. Research into the mechanisms of suppression of the immune system is complex and multi-faceted, and it seems that for now, there will remain a gap in our overall understanding of immune regulation. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The work reported in this thesis involved the purification, biochemical analysis and sequencing of a novel suppressor factor secreted by a T cell hybridoma, A10. The factor, A10F, isolated from spent medium of A10 cells, was found to consist of two forms with molecular weights at 140 - 160 and 80 kD as suggested by NH₂-terminal sequencing, Western blotting and tryptic peptide mapping experiments. Both forms of A10F were found to be capable of suppressing the in vitro generation of cytotoxic T lymphocyte (CTL) specific for P815 cells by syngeneic (DBA/2) splenocytes. In vitro ³⁵S methionine labeling experiments clearly demonstrated that the 80 kD protein was a secretory product of the A10 cells. The protein, which was specific to the monoclonal antibody (B16G), was absent in the control NS1 and BW5147 cells. Biochemical analysis indicated that the 80 kD molecule, was either a degradation product or a monomer of the 140 - 160 kD molecule. Further degradation products such as the 32 kD molecules were also found. This peptide, however, did not seem to cause substantial suppression in the in vitro CTL assay. When the 140 - 160, 80 and 32 kD proteins were sequenced at the NH₂ terminus, both 140 - 160 and 80 kD proteins were found to possess the same NH₂ terminus sequence. The 32 kD protein, on the other hand, was found to have an NH₂-terminus quite different from that of the 80 kD protein. These findings suggested that the 32 kD fragment was probably located at the distal end of the 140 - 160 kD molecule. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

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