Isolation and characterization of the suppressor regulatory circuit controlling the immune response to the mastocytoma P815 and studies of cancer immunotherapy by suppressor modulation
Creators: name: Steele, John Kevin
Publisher: name: University of British Columbia
Year: 2010

description: This thesis is in three parts, each representing the progression of work over the 3 year period in which it was done. Initially, work centered on the B16G monoclonal antibody. This antibody had been previously raised by immunization of Balb/c mice with affinity-purified P815 tumor specific T-suppressor cell factor (TsF). Preliminary studies with B16G had indicated it could pan out T-suppressor cell (TsC) populations from DBA/2 splenocytes, resulting in an enhanced mixed lymphocyte reaction (MLR), and could slow in vivo the growth of P815 or M-l tumors in DBA/2 mice. Since the monoclonal appeared to bind to a constant determinant of DBA TsC's and TsF's, a study (Chapter I) was undertaken to identify the factor to which B16G bound. Bl6G-reactive material was found to be isolatable from the membranes of DBA/2, MRL/lpr, and CBA mice splenocytes, as well as human tonsillar lymphocytes. These purified factors were found to be specifically suppressive in the MLR, and were major histocompatibility complex restricted, that is, would not cross an H-2 (or HLA in humans) barrier. Biochemically, these TsF's (or solubilized TsC receptors) were found to have a native molecular weight of 80 kD (±10 kD) by gel filtration, and to be readily dissociatable into 45 kD and 25 kD moieties as observed by SDS-PAGE analysis. The crossreactivity of B16G between murine and human proteins was fortuitous, but B16G is not apparently reactive with TsF from all mammalian species. For example, canine splenocyte extracts purified over B16G-4B columns resulted in no yield of TsF at all. Since B16G was demonstrated to be pan-reactive with DBA/2 TsC's, it was used as a probe to isolate hybridomas producing T-suppressor factors specific for the P815 tumor system (Chapter II and III). Two hybridomas were isolated using this method; A10, which produces a TsF₁ which is id⁺, (binds to nominal P815 antigen) and specifically inhibits the response to P815 in vivo and in vitro (assayed by cytotoxic T-lymphocyte activity (CTL), and A29, which produces anti-idiotypic (id⁻) TsF₂ with similar inhibitory characteristics. Both A10 and A29 TsF have a native m.w. of 70,000, with 45 and 25 kD subunits, similar to the polyclonal TsF isolated before. Both factors bind B16G and an antiserum raised to A10, demonstrating their biochemical and serological similarity. These 2 hybridomas, therefore, represent the hybrid analogues of the Ts₁and TS₂ suppressor population, and delineate the id/αid regulatory network controlling the immune response to the P815 tumor. The final part of the thesis (Chapters IV and V) demonstrates two methods of utilizing this information to evolve several approaches to cancer therapy by suppressor modulation. Deletion of whole polyclonal suppressor populations by administration of B16G-toxin conjugates in vivo was found to frequently cause complete tumour regression, even in mice with advanced tumor growth. Mice so treated experienced long term survival, with no apparent ill effects or induction of autoimmunity induction. Similarly, immunization of DBA animals with A10 TsF prior to tumor dose was found to have a powerful protective effect against the growth of a variety of DBA/2 tumors, due to interference in the development of suppressor populations. Since B16G is demonstrably active against human suppressors, these approaches to cancer therapy might be applicable to the treatment of human neoplasms. The thesis, therefore, demonstrates the value of B16G as a tool for monitoring levels of suppressors, the profound impact that suppressors and their factors have on tumor growth, and the use of suppressor modulation therapies in treating cancer.