BACKGROUND:The memory response to LCMV in mice persists for months to years with only a small decrease in the number of epitope specific CD8 T cells. This long persistence is associated with resistance to lethal LCMV disease. In contrast to studies focused on the number and surface phenotype of the memory cells, relatively little attention has been paid to the diversity of TCR usage in these cells. CD8+ T cell responses with only a few clones of identical specificity are believed to be relatively ineffective, presumably due to the relative ease of virus escape. Thus, a broad polyclonal response is associated with an effective anti-viral CD8+ T cell response.RESULTS:In this paper we show that the primary CD8+ T cell response to the LCMV gp33-41 epitope is extremely diverse. Over time while the response remains robust in terms of the number of gp33-tetramer+ T cells, the diversity of the response becomes less so. Strikingly, by 26months after infection the response is dominated by a small number TCRbeta sequences. In addition, it is of note the gp33 specific CD8+ T cells sorted by high and low tetramer binding populations 15 and 22months after infection. High and low tetramer binding cells had equivalent diversity and were dominated by a small number of clones regardless of the time tested. A similar restricted distribution was seen in NP396 specific CD8+ T cells 26months after infection. The identical TCRVbeta sequences were found in both the tetramerhi and tetramerlo binding populations. Finally, we saw no evidence of public clones in the gp33-specific response. No CDR3 sequences were found in more than one mouse.CONCLUSIONS:These data show that following LCMV infection the CD8+ gp33-specific CD8 T cell response becomes highly restricted with enormous narrowing of the diversity. This narrowing of the repertoire could contribute to the progressively ineffective immune response seen in aging.

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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

<p>As a major component of the adaptive immune system, CD4+ T cells play a vital role in host defense and immune tolerance. The potency and accuracy of CD4+ T cell-mediated protection lie in their ability to differentiate into distinct subsets that could carry out unique duties. In this dissertation, we dissected the roles and interplays between two emerging mechanisms, miRNAs and epigenetic processes, in regulating CD4+ T cell-mediated responses. Using both gain- and loss-of-function genetic tools, we demonstrated that a miRNA cluster, miR-17-92, is critical to promote Th1 responses and suppress inducible Treg differentiation. Mechanistically, we found that through targeting Pten, miR-17-92 promotes PI3K activation. Strong TCR-PI3K activation leads to the accumulation of DNMT1, elevated CpG methylation in the foxp3 promoter, and suppression of foxp3 transcription. Furthermore, we demonstrated that an epigenetic regulator, methyl CpG binding protein 2 (MeCP2), is critical to sustain Foxp3 expression in Tregs, and to support Th1 and Th17 differentiation in conventional CD4+ T cells (Tcons). In Tregs, MeCP2 directly binds to the CNS2 region of foxp3 locus to promote its local histone H3 acetylation; while in Tcons, MeCP2 enhances the locus accessibility and transcription of miR-124, which negatively controls SOCS5 translation to support STAT1, STAT3 activation and Th1, Th17 differentiation. Overall, miRNAs and epigenetic processes may crosstalk to control CD4+ T cell differentiation and function.</p> / Dissertation

The immune system recognizes and eliminates an enormous array of pathogens due to the diverse antigen receptor repertoire of T and B lymphocytes. However, the development of lymphocytes bearing receptors with unique specificities requires the generation of programmed double strand breaks (DSB) coupled with bursts of proliferation, rendering lymphocytes susceptible to mutations and oncogenic transformation. Thus, mechanisms responsible for monitoring global genomic integrity, such as those coordinated by the ATM (ataxia-telangiectasia mutated) kinase, must be activated during lymphocyte development to limit the oncogenic potential of antigen receptor locus recombination. I show that ATM deficiency compromises TCRα recombination and the post-mitotic survival of T-cell receptor αβ (TCRαβ+) CD4+CD8+ (DP) thymocytes, providing a molecular and developmental basis for the immunodeficiency characteristic of ATM loss. Moreover, I show that in early thymocyte progenitors undergoing TCRβ recombination, ATM loss leads to cell cycle defects and developmental arrest, likely facilitating the acquisition of mutations that contribute to leukemogenesis. Using ATM deficiency as a murine model of T cell precursor acute lymphoblastic leukemia (T-ALL), I demonstrate that IL-7 signaling, a critical survival and proliferation signal during early stages of normal thymocyte development, is also required for leukemic maintenance. Moreover, we show for the first time that in normal and leukemic thymocyte precursors, interleukin 7 receptor (IL-7R) expression and function are controlled by Notch signaling, a key determinant of T cell fate. Collectively, these findings provide insight into the mechanisms by which ATM promotes normal lymphocyte development and protects from neoplastic transformation, while establishing the groundwork for assessing the molecular events that lead to the initiation and stepwise progression of T cell leukemogenesis.