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

The work reported here involves studies of suppressor T cells (T[sub=s]C) and their suppressor factor (SF) which specifically suppress the in vitro generation of cells cytotoxic for a syngeneic tumor, P815, in DBA/2 mice. This work can be divided into three sections: a) the immunogenetic properties and requirements of this T[sub=s]C and SF, b) the Lyt phenotype of the T[sub=s]C as well as that of the cells involved in the cytotoxic response to the syngeneic tumor, c) the properties of syngeneic and allogeneic antisera raised to the P815 specific SF. a) P815-antigen specific T[sub=s]C and suppressive extracts obtained from the thymuses of DBA/2 mice bearing small syngeneic P815 tumors, were compared for their immunogenetic properties and requirements. It was shown that pretreatment of T[sub=s]C populations with anti-la[sub=d] antiserum plus rabbit complement removed the suppressive activity. Similarly, absorption of the SF with anti-la[sup=d] antiserum removed the suppressive properties of the material. It was found that the T[sub=s]C and SF were capable of specifically suppressing the anti-P815 response of B6D2F₁ radiation chimeras possessing lymphoid cells of the H-2[sup=b] or H-2[sup=t2] haplotype equally as well as they could suppress the response of H-2[sup=d] bearing cells. This indicates that the T[sub=s]C and SF are not H-2 restricted with respect to K or D markers on responder cells in this system. b) T[sub=s]C were also identified in the spleens of DBA/2 mice injected intraperitoneally with membrane extracts of the P815 tumor. The Lyt phenotypes of various effector cells was determined. DBA/2 allogeneic killer cells were identified as Lyt-1⁺2⁺, whereas the syngeneic effector cells were found to be predominantly Lyt-1⁺2⁺. The suppressor cell population lost its ability to suppress the in vitro cytotoxic anti-P815 response after treatment with anti-Lyt-1 serum plus complement but not after treatment with anti-Lyt-2 serum, indicating that an Lyt-1⁺2⁻ cell is essential in this suppression. c) P815 tumor-specific SF was partially purified by passage of suppressive spleen extracts through an immunoadsorbent containing P815 membrane components. Antisera raised in syngeneic DBA/2 and allogeneic, C57BL/6, mice were tested. It was found that these antisera, but not their controls were capable of absorbing out the SF. The antisera were also capable, in the presence of complement, of eliminating T[sub=s]C from suppressive spleen cell populations. However, the antisera were not capable of eliminating syngeneic tumor specific in vitro generated killer cells, indicating that the receptor molecules on suppressor and effector cells in this system are distinct from each other. Only the antisera "raised in syngeneic DBA/2 mice had any observable effect on P815 tumor growth in vivo. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Thesis (Ph. D.)--University of Wisconsin--Madison, 1983. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

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

Regulation of the immune response is mediated, in part, by the action of suppressor T cells (Ts). One intriguing aspect of these cells is the description of T cell suppressor factor (TsF): a soluble analog of the cell that shares many of its properties, such as the ability to bind free antigen (Ag) and suppress an Ag-specific immune response. The exact molecular nature of TsF and the relationship of TsF to Ts are unknown. The immune response to the small, bacterial protein, ferredoxin (Fd), was used as a model system to study TsF. A Fd-specific suppressor cell network has been described in mice that are genetically nonresponsive to this Ag. Previously, a soluble mediator, known as Fd11F, was found in the culture supernatant (SN) of the Ts hybridoma, Fd11. Fd11F possessed both Ag-binding activity and the ability to suppress the anti-Fd Ab response in mice. The TsF-specific monoclonal antibody, B16G, was used for both the recovery of Fd11F-enriched material from SN and its detection by the enzyme-linked immunosorbent assay. ' Further immunochemical, biological, and biochemical characterization of Fd11F was done with emphasis on describing the Ag-binding properties of Fd11F. It was found that Fd11F bound to solid- and liquid-phase Fd, and demonstrated preferential binding to the carrier determinant of the Ag. A spleen cell culture assay was devised which showed that Fd11F suppressed Ab production in a concentration-dependent manner. Additional experiments suggested that the suppressive effect was Ag-specific. The identification of the Ag-binding molecule was attempted by the fractionation of Fd11F-enriched material using high performance gel filtration or preparative SDS-PAGE (run under non-reducing conditions). Using SDS-PAGE, a unique, single polypeptide of about 30k relative molecular mass (Mr) was identified as the Ag-binding moiety of Fd11F. The possible relationship of this moiety to other identified materials is discussed. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The absence of invariant Natural Killer T cells (iNKT cells) in mice infected with Influenza A virus (flu) has previously been shown to augment the expansion of Myeloid-derived suppressor cells (MDSCs), a bone marrow derived population that powerfully suppress the development of viral and tumor immune responses. Moreover, iNKT cell adoptive transfer into flu-infected mice has been shown to abolish the expansion and flu-induced suppressive activity of the MDSCs in a CD1d- and CD40-dependent manner. However, the mechanisms by which this relatively small subset of T cells influence myelopoiesis and MDSC differentiation remain largely unknown. In this manuscript we firstly better define the MDSCs found in flu-infection as IL-10-secreting neutrophils that can suppress T cell proliferation. We then go further to show that the flu-induced ability to suppress T cells is acquired as early as the level of the Granulocyte-Macrophage Progenitors (GMPs) in the bone marrow and that iNKT cells can not only abrogate the suppressive activity of the IL-10-secreting neutrophils in the periphery but also that of the GMPs by a direct CD1d-dependent GCSF-mediated crosstalk. MDSC expansion has previously been shown to be associated with the expression of the myeloid-related protein S100A9, and the mechanism of action of granulocytic-MDSCs shown to be ARG1-dependent. We built upon both these findings to show that iNKT cells influence the expansion and function of the MDSCs in part by regulating S100A9 and ARG1 expression. Following this we then showed for the first time that the acute phase protein Serum Amyloid A (SAA), shown to increase during flu-infection, has a dual reciprocal role: having the ability to up-regulate S100A9 and ARG1 in myeloid cells and differentiate IL-10-secreting suppressive neutrophils, while simultaneously facilitating the ability of the MDSCs to crosstalk with iNKT cells in a CD1d-dependent GCSF-mediated manner to abrogate the SAA-induced suppressive activity. All together the data highlights the complexity of the immune response and the role iNKT cells play in influencing the differentiation of MDSCs during demand-driven myelopoiesis. More importantly however, it further affirms that research into harnessing the immunomodulatory capacity of iNKT cells remains an exciting prospect in bolstering future vaccination strategies and should continue to be pursued.

The tumour microenvironment consists of an immunosuppressive niche created by the complex interactions between cancer cells and surrounding stromal cells. A critical component of this environment are myeloid-derived suppressor cells (MDSCs), a heterogeneous group of immature myeloid cells arrested at different stages of differentiation and expanded in response to a variety of tumour factors. MDSCs exert diverse effects in modulating the interactions between immune effector cells and malignant cells. An increased presence of MDSCs is associated with tumour progression, poorer outcomes, and decreased effectiveness of immunotherapeutic strategies. In this project, we sought to quantify and characterise MDSC populations in patients with Acute Myeloid Leukaemia (AML) and delineate the mechanisms underlying their expansion. We have demonstrated that immune suppressive MDSCs are expanded in the peripheral blood and bone marrow of patients with AML. Furthermore, AML cells secrete extra-cellular vesicles (EVs) that skew the tumour microenvironment from antigen-presentation to a tumour tolerogenic environment, through the expansion of MDSCs. We then demonstrated that MDSC expansion is dependent on tumour and EV expression of the oncoproteins MUC1 and c-Myc. Furthermore, we determined that MUC1 signalling promotes c-MYC expression in a microRNA (miRNA) dependent mechanism. This observation lead us to elucidate the critical role of MUC1 in suppressing microRNA-genesis in AML, via the down-regulation of the DICER protein, a key component of miRNA processing machinery. Finally, exploiting this critical pathway, we showed that MDSCs can be targeted by MUC1 inhibition or by the use of a novel hypomethylating agent SGI-110.

The transmembrane isoform of mucin 1 (MUC1/TM) is a well recognized tumor antigen, contributing to tumorigenesis and immune evasion. While MUC1/TM has been correlated with malignancy, it appears that a secreted splice variant of MUC1 (MUC1/sec) has antitumor properties and prevents tumor development. It was discovered that MUC1/sec expressing tumor cells (DA-3/sec) have a significant reduction in expression of urokinase plasminogen activator (uPA) relative to the parental tumor line, and tumor cells expressing MUC1/TM (DA-3/TM). The serine protease uPA, has been found to be involved in growth promoting signaling, angiogenesis, and induction of matrix remodeling leading to metastasis. Furthermore, the tumor suppressive and interferon responsive Stat1 transcription factor is dramatically upregulated in DA-3/sec cells. In addition, treatment of various murine and human cell lines with conditioned media containing MUC1/sec results in up-regulation of Stat1. DA-3/sec tumor cells are also sensitized to the anti-proliferative effects of IFN-g. Furthermore, transfection of the Stat1 gene into DA-3 tumor cells leads to a downregulation of uPA, and delays tumor progression. Since myeloid-derived suppressor cells (MDSC) play a critical role in tumor-induced immunosuppression, we investigated their recruitment by DA-3/sec and DA-3/TM cells. DA-3/sec tumor cells recruit dramatically lower levels of MDSC, relative to DA-3/TM cells. Since MUC1/sec down-regulates tumor expression of uPA, its potential role in MDSC recruitment was investigated. Tumor-derived uPA is capable of recruiting MDSC, and correlates with tumor development. In addition to diminishing recruitment of MDSC, the effect of MUC1/sec on MDSC suppressive mechanisms was investigated. MUC1/sec, or its unique immunoenhancing peptide (IEP), is capable of blocking expression of arginase 1 and production of reactive oxygen species (ROS) in MDSC, implicated in the suppression of T cells. These findings demonstrate a new mechanism of MDSC recruitment, and provide evidence that MUC1/sec has antitumor properties affecting both tumor cells and MDSC. Furthermore, it was discovered that MDSC home to the liver in addition to the tumor, bone marrow, blood, and spleen of tumor bearers, as previously described. The liver is thus an organ where MDSC accumulate and can contribute to immunosuppression directly and indirectly, via interactions with a variety of immune cells.

Myeloid-derived suppressor cells (MDSCs), characterized by the co-expression of CD11b and Gr1, are a heterogeneous population of immature myeloid cells that exhibit strong suppressive functions against T cell responses. In inflammatory conditions like IBD, there is an increase in MDSCs but this is not sufficient to improve intestinal inflammation in IBD. Herein, we investigated the expansion of MDSCs in TNBS-induced acute colitis and whether the adoptive transfer of in vitro generated MDSCs ameliorated intestinal inflammation. We found that CD11b+Gr1+ MDSCs were significantly increased in experimental colitis. Further, this increase correlated to some extent with the severity of the disease. As per our protocol, MDSCs were generated from bone marrow cells co-cultured with hepatic stellate cells (HSCs), an essential cell type to obtain functional MDSCs in vitro. Adoptive transfer of HSC-induced MDSCs improved body weight loss and significantly downregulated inflammatory cytokines TNF, IFN-γ, and IL-17 in colonic tissue. Our results indicate MDSCs are immunoregulatory players in intestinal inflammation and that the adoptive transfer of in vitro generated MDSCs may provide a novel therapeutic approach for inflammatory bowel disease.