• Applying MyD88 signaling in CD8+ T cells to enhance anti-tumor immunity

      Kaczanowska, Sabina; Davila, Eduardo, Ph.D.; 0000-0003-0191-6371 (2016)
      T cell immunotherapy is a promising strategy for the treatment of patients with advanced cancers. Despite promising results in patients with hematological malignancies, response rates to solid tumors remain low. One of the biggest challenges is effectively activating and sustaining anti-tumor T cell responses in the context of an immunosuppressive tumor environment. Several approaches are currently being explored to improve the efficacy of adoptive T cell transfer, including the activation of co-stimulatory signaling pathways in T cells. Toll-like receptor (TLR) engagement on T cells is a potent co-stimulatory signal that increases anti-tumor activity by enhancing T cell proliferation, effector function, and T cell survival. We developed and tested two genetic engineering strategies to exploit the co-stimulatory effects of TLR signaling in CD8+ T cells. First, CD8+ T cells were modified to express and secrete the TLR5 ligand (TLR5L), flagellin, as a means to deliver this immune adjuvant to the tumor for enhanced anti-tumor activity. TLR5L-secreting T cells exhibited improved proliferation, cytokine secretion, and anti-tumor activity in both xenogeneic and syngeneic models of melanoma. The anti-tumor activity of TLR5L-secreting T cells was associated with decreased numbers of phenotypically exhausted T cells and fewer myeloid-derived suppressor cells. Second, we designed and characterized a synthetic fusion protein composed of the T cell co-receptor CD8α and the TLR adaptor protein MyD88, termed CD8α:MyD88. The expression of CD8α:MyD88 on T cells increased T cell responses to low concentrations of tumor antigens as well as augmented the expression of effector molecules and co-stimulatory proteins. These effects were antigen-dependent and accompanied by elevated levels of TLR signaling-related proteins. The enhanced anti-tumor activity of CD8α:MyD88-expressing T cells in tumor-bearing mice was associated with a unique tumor cytokine/chemokine signature, improved T cell infiltration, elevated levels of antigen presentation, and fewer macrophages with an immunosuppressive phenotype. The use of T cells as vehicle to deliver TLR5L to the tumor site and the co-stimulation of T cells through a synthetic CD8α:MyD88 receptor represent two novel and versatile approaches for modulating the tumor microenvironment to enhance anti-tumor immunity.
    • Differential FOXO1 Localization in SLE and Healthy Human Lymphocyte Subsets

      Hritzo, Molly K.; Golding, Amit; 0000-0001-9614-732X (2018)
      Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease characterized by elevated levels of circulating autoantibodies and multi-organ damage. Although SLE is a highly heterogeneous disease, one factor unifies it: lymphocyte hyperactivity driving immunopathogenesis. This involves CD4 helper T cells potentiating autoreactive B cells to produce pathogenic autoantibodies. In healthy individuals, lymphocyte activation is a closely regulated kinetic process controlled by key transcription factors (TF) signaling downstream of the T cell (TCR) and B cell receptor (BCR). Forkhead box O1 (FOXO1) is one such TF that integrates activation and differentiation signals in human lymphocytes. When active, it remains in the nucleus, but upon Akt phosphorylation downstream of TCR or BCR signaling, FOXO1 is inactivated and shuttles to the cytoplasm, linking FOXO1 localization to function. In SLE, both T and B cells are hyperactive, and respond more quickly and strongly to antigen, producing a disproportionate inflammatory response. Thus, we hypothesize that SLE lymphocytes will have altered FOXO1 localization, reflecting altered lymphocyte activation. To address this hypothesis, we first developed a method of examining dynamic native FOXO1 localization in human peripheral lymphocyte subsets using imaging flow cytometry (IFC). IFC combines the quantitative power of flow cytometry with the qualitative images of microscopy and can be performed with many fewer cells than are needed for the more traditional methods. We demonstrated that we can visualize native FOXO1 and detect significant kinetic differences in localization within user-defined subsets of HuT102 cells, a human CD4 T cell line with baseline nuclear FOXO1, as well as primary peripheral human T and B cells. We then used IFC to compare FOXO1 localization in SLE and healthy donor lymphocytes. Interestingly, we found that most T and B cell subsets have nuclear FOXO1 localization in both health and SLE. However, FOXO1 is significantly more cytoplasmic in SLE double negative (DN) atypical memory B cells. Based on our findings, we propose a model by which these DN B cells are highly active in disease flares and may serve as a death-resistant reservoir of autoreactive cells. Future experiments will be aimed at elucidating at how these cells persist in the periphery.
    • Dissecting the protective memory CD4 T cell response to influenza virus infection

      Teijaro, John; Farber, Donna L. (2009)
      An important distinguishing feature of the adaptive immune response is the de-velopment of immunologic memory, which provides protective immunity against repeat pathogen encounters. T lymphocytes, including the CD4 and CD8 T cell subsets, are both known to persist as memory. Despite increasing knowledge of the mechanisms un-derlying T cell activation and function, the generation, maintenance and protective effi-cacy of T cell memory remains poorly defined. At present, there are no existing vaccine strategies to generate memory T cell mediated protective immunity. Furthermore, the qualities that define a protective memory T cell response are incompletely understood. Our laboratory has identified heterogeneous populations of influenza hemaggluti-nin (HA) specific memory CD4 T cells, with respect to phenotype, function, and migra-tion to both secondary lymphoid and peripheral tissues. However, how these diverse memory CD4 T cell subsets mediate protection to influenza virus infection remains un-explored. In order to investigate protection mediated my memory CD4 T cells, we established complementary models using TCR-transgenic and polyclonal influenza-specific T cells. Herein we deomonstrate that the presence of memory CD4 T cells in BALB/c mice resulted in enhanced viral clearance in the context of slightly increased morbidity as measured by weight loss and physical appearance. In addition, reduction in viral loads by memory CD4 T cells was not affected in the absence of B cells or CD8 T cells, and ap-peared to be IFN-γ dependent. In addition, we demonstrate that lung-derived memory preferentially migrate to and are retained within the lung tissue and facilitate rapid clear-ance of virus compared to spleen-derived memory CD4 T cells, which provided no bene-ficial protection compared to naive controls. Moreover, viral clearance in mice receiving lung memory CD4 T cells occurred in the context of reduced weight loss and physical morbidity. Finally, we demonstrate that the physiological outcome of a memory CD4 T cell-mediated secondary response to influenza can be significantly improved by targeting the CD28 pathway with the costimulation modulator CTLA4Ig. While CTLA4Ig is sup-pressive for primary immune responses to influenza, leading to increased viral loads, re-duced lung function and increased morbidity. In contrast, CTLA4Ig treatment of mem-ory CD4 T cell-mediated secondary responses to influenza is remarkably curative, result-ing in less morbidity and immunopathology, improved lung function, and enhanced re-covery. The optimized secondary response induced by CTLA4Ig treatment is character-ized by reduced lymphoid memory responses and recruitment to the lung, with intact in situ lung memory T cell responses and effector function. Our results reveal the dual na-ture of memory T-cell-mediated secondary responses and identify protective subsets, and suggest costimulation modulation as a novel strategy to optimize anti-viral immunity by limiting the memory T-cell response to its protective capacities.
    • Regulation of adaptive immune responses by the phagocyte-type NADPH oxidase in T cells and antigen presenting cells

      Shatynski, Kristen Elizabeth; Williams, Mark S., Ph.D. (2012)
      Absence of phagocyte NADPH oxidase (NOX2) activity causes chronic granulomatous disease (CGD), a primary immunodeficiency characterized by recurrent bacterial infections. In contradiction to this innate immune dysfunction, CGD patients or animal models of the disease display improved response to infectious agents such as Helicobacter pylori, influenza virus or Cryptococcus neoformans. These and other data imply an altered adaptive immune response in CGD. In addition to antigen presenting cells (APCs), NOX2 is expressed in T cells, and the goal was to determine if these differences are T cell inherent, or if NOX2-deficient APCs promote T-helper polarization. We hypothesize that NOX2 shapes both adaptive and innate immune responses in a T cell and APC-dependent fashion. In order to study T-helper polarization in vivo, wild type and NOX2-deficient (NOX2<super>(-/-)</super>) mice were immunized with OVA in CFA or Alum. Upon in vitro restimulation, lymph node cells from NOX2<super>(-/-)</super> mice had increased Th1 but decreased Th2 cytokine production. Adoptive transfer of OT-II T cells into wild type or NOX2<super>(-/-)</super> hosts followed by immunization also revealed increased IL-17 and IFN-γ, but decreased IL-4 after restimulation in vitro. NOX2<super>(-/-)</super> APCs (resident peritoneal cells and adherent splenocytes) displayed enhanced proinflammatory cytokine secretion in vitro, and stimulation of OT-II T cells with antigen-pulsed NOX2<super>(-/-)</super> APCs in vitro induced altered cytokine production, suggesting that NOX2 deficiency modifies APC function, resulting in T helper skewing. In addition to APC-induced changes, T cells from NOX2<super>(-/-)</super> mice were inherently skewed. Naïve T cells from NOX2<super>(-/-)</super>mice demonstrated Th1 skewed cytokine secretion as compared to their wild type counterparts, through increased IFN-γ, but decreased IL-4 in response to anti-CD3 and anti-CD28 stimulation. There were also selective decreases in GATA-3 and phosphorylated STAT5, with decreased Il4 gene expression, suggesting a mechanism for decreased Th2 responses. Finally, treatment with antioxidants recapitulated these selective changes in TCR-induced transcription factor activation. These findings indicate that TCR-induced reactive oxygen species generation from NOX2 activation selectively promotes STAT5 phosphorylation and downstream Th2 development in CD4+ T cells. Taken together, these findings suggest NOX2 affects cross talk between the innate and adaptive immune systems, resulting in changes in T helper differentiation.