Immunity against cutaneous herpes simplex virus infection : host defense mechanisms mediated by IFN-γ and active protection induced by DNA vaccination

The goal of this dissertation is to understand the immune responses generated against cutaneous herpes simplex virus (HSV) infection, with an emphasis on the role of gamma-interferon (IFN-γ) in mediating protection against HSV. Further, novel approaches to achieve optimal immune responses by DNA vaccination in the murine HSV model is explored. A T-cell mediated inflammatory response dominated by BFN-γ appears to play the essential role in viral clearance during cutaneous HSV infection. In accordance with this hypothesis, this dissertation shows that following infection, IFN-γ gene knock-out (GKO) mice are more susceptible to the development of skin lesions than are wild-type mice. However, following HSV immunization, GKO mice become solidly immune to viral challenge. In addition, transferring T cells from immune GKO mice to nude mice recipients renders them resistant to zosteriform lesions. These results indicate that compensatory mechanisms are available to the body to effect immunity against viral infections. To test the principle that genetically engineered epitopes in a plasmid DNA can efficiently induce specific immunity, a study on HSV-specific immune responses induced by vaccinating mice with a plasmid DNA (pcMini) encoding cytotoxic T lymphocyte (CTL), helper T and B cell epitopes from HSV is described in this dissertation. Following immunizations with pcMini, mice developed epitope-specific CTLs comparable to the responses induced by live HSV. Antibody, lymphoproliferative responses, and T cell cytokine release were also detected. The protection provided by minigene vaccination was significant; however, not as efficient as live virus immunization. The DNA minigene approach may prove useful to define and induce immune responses against minimal antigenic determinants. To address the question of whether immune responses to a vector-encoded antigen can be enhanced by the activation of antigen-presenting cells in the local environment, the effects of co-inoculation of an HSV glycoprotein (gB)-expressing plasmid with a plasmid encoding mouse granulocyte-macrophage colony-stimulatory factor (GM-CSF) on modulation of gB-specific immune responses are evaluated in this dissertation. The results show that co-administration of GM-CSF DNA provided increased resistance to HSV infection and this protective response could be related to the enhanced immune induction of CD4-t- T cells and B cells responses

Identification and structural characterization of a mutant KRAS‐G12V specific TCR restricted by HLA‐A3

Mutations in KRAS are some of the most common across multiple cancer types and are thus attractive targets for therapy. Recent studies demonstrated that mutant KRAS generates immunogenic neoantigens that are targetable by adoptive T‐cell therapy in metastatic diseases. To expand mutant KRAS‐specific immunotherapies, it is critical to identify additional HLA‐I allotypes that can present KRAS neoantigens and their cognate T‐cell receptors (TCR). Here, we identified a murine TCR specific to a KRAS‐G12V neoantigen (7VVVGAVGVGK16) using a vaccination approach with transgenic mice expressing HLA‐A*03:01 (HLA‐A3). This TCR demonstrated exquisite specificity for mutant G12V and not WT KRAS peptides. To investigate the molecular basis for neoantigen recognition by this TCR, we determined its structure in complex with HLA‐A3(G12V). G12V‐TCR CDR3β and CDR1β formed a hydrophobic pocket to interact with p6 Val of the G12V but not the WT KRAS peptide. To improve the tumor sensitivity of this TCR, we designed rational substitutions to improve TCR:HLA‐A3 contacts. Two substitutions exhibited modest improvements in TCR binding avidity to HLA‐A3 (G12V) but did not sufficiently improve T‐cell sensitivity for further clinical development. Our study provides mechanistic insight into how TCRs detect neoantigens and reveals the challenges in targeting KRAS‐G12V mutations

Removal of homeostatic cytokine sinks by lymphodepletion enhances the efficacy of adoptively transferred tumor-specific CD8(+) T cells

Depletion of immune elements before adoptive cell transfer (ACT) can dramatically improve the antitumor efficacy of transferred CD8(+) T cells, but the specific mechanisms that contribute to this enhanced immunity remain poorly defined. Elimination of CD4(+)CD25(+) regulatory T (T reg) cells has been proposed as a key mechanism by which lymphodepletion augments ACT-based immunotherapy. We found that even in the genetic absence of T reg cells, a nonmyeloablative regimen substantially augmented CD8(+) T cell reactivity to self-tissue and tumor. Surprisingly, enhanced antitumor efficacy and autoimmunity was caused by increased function rather than increased numbers of tumor-reactive T cells, as would be expected by homeostatic mechanisms. The γ (C) cytokines IL-7 and IL-15 were required for augmenting T cell functionality and antitumor activity. Removal of γ (C) cytokine–responsive endogenous cells using antibody or genetic means resulted in the enhanced antitumor responses similar to those seen after nonmyeloablative conditioning. These data indicate that lymphodepletion removes endogenous cellular elements that act as sinks for cytokines that are capable of augmenting the activity of self/tumor-reactive CD8(+) T cells. Thus, the restricted availability of homeostatic cytokines can be a contributing factor to peripheral tolerance, as well as a limiting resource for the effectiveness of tumor-specific T cells

miR-155 augments CD8(+) T-cell antitumor activity in lymphoreplete hosts by enhancing responsiveness to homeostatic gamma(c) cytokines

Lymphodepleting regimens are used before adoptive immunotherapy to augment the antitumor efficacy of transferred T cells by removing endogenous homeostatic "cytokine sinks." These conditioning modalities, however, are often associated with severe toxicities. We found that microRNA-155 (miR-155) enabled tumor-specific CD8(+) T cells to mediate profound antitumor responses in lymphoreplete hosts that were not potentiated by immune-ablation. miR-155 enhanced T-cell responsiveness to limited amounts of homeostatic gamma c cytokines, resulting in delayed cellular contraction and sustained cytokine production. miR-155 restrained the expression of the inositol 5-phosphatase Ship1, an inhibitor of the serine-threonine protein kinase Akt, and multiple negative regulators of signal transducer and activator of transcription 5 (Stat5), including suppressor of cytokine signaling 1 (Socs1) and the protein tyrosine phosphatase Ptpn2. Expression of constitutively active Stat5a recapitulated the survival advantages conferred by miR-155, whereas constitutive Akt activation promoted sustained effector functions. Our results indicate that overexpression of miR-155 in tumor-specific T cells can be used to increase the effectiveness of adoptive immunotherapies in a cell-intrinsic manner without the need for life-threatening, lymphodepleting maneuvers.112922Ysciescopu

BACH2 regulates CD8(+) T cell differentiation by controlling access of AP-1 factors to enhancers.

T cell antigen receptor (TCR) signaling drives distinct responses depending on the differentiation state and context of CD8(+) T cells. We hypothesized that access of signal-dependent transcription factors (TFs) to enhancers is dynamically regulated to shape transcriptional responses to TCR signaling. We found that the TF BACH2 restrains terminal differentiation to enable generation of long-lived memory cells and protective immunity after viral infection. BACH2 was recruited to enhancers, where it limited expression of TCR-driven genes by attenuating the availability of activator protein-1 (AP-1) sites to Jun family signal-dependent TFs. In naive cells, this prevented TCR-driven induction of genes associated with terminal differentiation. Upon effector differentiation, reduced expression of BACH2 and its phosphorylation enabled unrestrained induction of TCR-driven effector programs