A balanced immune response is required to mediate clearance of a virus infection without immune-mediated disease. CD4 and CD8 T cells are capable of both exerting antiviral effector functions and regulating the immune response. The regulatory T cell (Treg) subset of CD4 T cells helps to modulate immune activation and inflammation. During respiratory syncytial virus (RSV) infection in mice, conventional CD4 T-cell-mediated cytokine production has been shown to contribute to immune-mediated pathology. I demonstrate that Tregs are critical to control immunopathology during RSV infection. This was demonstrated through diphtheria toxin (DT)-mediated Treg elimination in a mouse strain expressing the DT receptor (DTR) under the control of the Foxp3 promoter. However, these mice were unable to maintain extended Treg depletion limiting the effectiveness of this model. In addition, DT-treated wild-type (WT) mice were found to be a necessary control for adverse DT-induced disease. In humans, I have shown that activated Tregs are reduced in the peripheral blood of RSV-infected infants compared to controls. RSV-infected infants also exhibited an increased proinflammatory cytokine response in nasal aspirates. However, the alarmin cytokine IL-33, which has been shown to mediate Treg homeostasis, was the only cytokine that exhibited reduced protein levels in RSV-infected infants compared to controls. Thus, severe RSV infection in infants may be due to lack of proper Treg-mediated immune regulation. Similar to RSV, regulation of the T cell response during chronic viral infection with lymphocytic choriomeningitis virus (LCMV) is vital to prevent immune-mediated pathology. During LCMV and human chronic viral infections, CD4 and CD8 T cells exhibit T cell exhaustion where they lose the ability to exert effector functions. However, a functional CD4 and CD8 T cell response is required for viral clearance. During human chronic viral infection, an association between increased CD4 and CD8 T cell function and enhanced viral control has been identified that can be influenced by genetic factors. I aimed to identify the contribution of the host genetic factors that contribute to enhanced CD8 T cell function and viral control using the LCMV model. I found that increasing the major histocompatibility complex (MHC) diversity resulted in enhanced viral control in both a C57BL and BALB genetic background. Thus, induction of a broader T cell response was associated with enhanced viral control. However, mice expressing a heterozygous MHC on the C57BL background also exhibited mortality following chronic viral infection. Both CD4 and CD8 T cells were shown to contribute to this mortality and exhibited reduced T cell exhaustion during LCMV infection in these mice. Heterozygous MHC expression on the C57BL mouse background was also associated with an increased T helper (Th)-1 skewed CD4 T cell response compared to mice on the BALB background. Furthermore, CD4 T-cell-mediated IFN-γ production contributed to both CD8 T cell effector activity and mortality during chronic LCMV infection. Thus, both T cell epitope diversity and host genetics contribute to LCMV-induced mortality. Collectively, my data highlight both the need for effective immune-meditated viral control and regulation of T-cell-mediated pathology during both acute and chronic viral infections.

Mycobacterium tuberculosis infection is one of the leading causes of mortality worldwide. One third of the population is estimated to be infected, however only 5-10% of those individuals can transmit the disease. While T cell immunity initially limits mycobacterium growth, it is unclear why T cell immunity fails to sterilize the infection and prevent subsequent recrudescence. One hypothesis is T cell exhaustion is mediating the failure of T cell immunity late during infection. Here we show the development of T cell exhaustion during chronic infection, and that the inhibitory receptor T cell-immunoglobulin and mucin domain containing 3 (TIM3) mediates the development of T cell exhaustion. TIM3 accumulates on the surface of T cells throughout the course of infection and there is a subsequent decrease in effector cytokine production, such as IL-2, TNFα, and IFNγ. Furthermore, antibody blockade of TIM3 restores T cell function and improves bacterial control. Our results show that TIM3 is mediating T cell exhaustion during chronic TB infection and leading to suboptimal bacterial control.

During the course of infection with chronic pathogens such as Hepatitis C virus (HCV), Hepatitis B virus (HBV) and HIV, virus-specific CD8^+ T cells differentiate into heterogeneous dysfunctional subpopulations. Advances in multi-parameter flow cytometry have allowed these subpopulations to be further classified into classes of exhausted T cells, primarily by their expression of multiple inhibitory receptors. However, the exact phenotype of CD8^+ T cells during exhaustion is an area of great interest as many inhibitory receptors are also expressed on functional CD8^+ T cells. Discovering novel and specific markers of T cell exhaustion is fundamental in developing strategies to restore CD8^+ T cell function in chronic viral infections. Recently, genome wide expression profiles have identified broad molecular phenotypes in exhausted T cells that could not have been discovered by flow cytometry alone. I show how similar genomic approaches identify and further characterise the ectonucleotidase CD39 as a novel marker of CD8^+ T cell exhaustion in chronic viral infection. I show that CD39 is highly expressed in HCV and HIV-specific CD8^+ T cells and that CD39^+ CD8^+ T cells are enriched with gene signatures of exhaustion. CD39 is highly co-expressed with multiple inhibitory receptors including PD-1, enzymatically active on CD8^+ T cells in HCV infection and positively correlated with viral load in both HCV and HIV. I also demonstrate the discovery of a novel CD39^High population of cells in the mouse model of chronic Lymphocytic Choriomenigitis Virus (LCMV) infection, which express the highest degrees of PD-1, LAG3 and 2B4 in the CD39^+ fraction. Thus, CD39 is a novel and specific marker of severe CD8^+ T cell exhaustion in human and animal models of chronic viral infection.

No description available.

Most Adoptive Cell Therapies (ACT), including CAR T cell therapies, suffer failure because of the severe side effects due to loss-of-control of the therapeutic cells once they are inside the patient’s body, suggesting that novel strategies must be developed for a better in vivo control of these engineered cells. In the meantime, CAR T cell therapies targeting solid tumors have not experienced the remarkable success achieved with hematopoietic cancers, mainly due to continuous tumor antigen exposure and a suppressive tumor microenvironment. Here we designed a private passageway fusion receptor, which is composed of a ligand binding domain and a glycosylphosphatidylinositol (GPI) anchoring domain, to be expressed and localized to the surface of CAR T cells independently to the classical CAR T construct. These ligand binding domains preserve high binding affinity towards their cognate ligands and are only expressed on the CAR T cells that have been transduced. Therefore, cytotoxic drugs or immunosuppressants linked to the corresponding targeting ligands are shown to be specifically delivered to these fusion receptor positive CAR T cells for lowering the activity of the over-activated CAR T cells. On the other hand, we discovered that a potent TLR7 agonist is able to enhance the lysis effect of the exhausted CAR T cells in a co-culture model. Serial releasable and non-releasable targeted TLR7 agonists were prepared and tested. Based on these data, we suggest that our secret passageway fusion receptor platform provides a better control of the activity of CAR T cells using the corresponding targeting ligand-payload conjugates in a dose dependent manner and function as a doorway for the delivery of instructions to CAR T cells for versatile purposes.

The hallmark of HIV/AIDS is a gradual depletion of CD4 T cells. Despite effective control by antiretroviral therapy (ART), a significant subgroup of people living with HIV (PLHIV) fails to achieve complete immune reconstitution, deemed as immune non-responders (INRs). The mechanisms underlying incomplete CD4 T cell recovery in PLHIV remain unclear. In this study, CD4 T cells from PLHIV were phenotyped and functionally characterized, focusing on their mitochondrial functions. The results show that while total CD4 T cells are diminished, cycling cells are expanded in PLHIV, especially in INRs. HIV-INR CD4 T cells are more activated, displaying exhausted and senescent phenotypes with compromised mitochondrial functions. Transcriptional profiling and flow cytometry analysis showed remarkable repression of mitochondrial transcription factor A (mtTFA) in CD4 T cells from PLHIV, leading to abnormal mitochondrial and T cell homeostasis. These results demonstrate a sequential cellular paradigm of T cell over-activation, proliferation, exhaustion, senescence, apoptosis, and depletion, which correlates with compromised mitochondrial functions. Therefore, reconstituting the mtTFA pathway may provide an adjunctive immunological approach to revitalizing CD4 T cells in ART-treated PLHIV, especially in INRs.

Background microRNA-23b (miR-23b) is a multiple functional miRNA. We hypothesize that miR-23b plays a role in the pathogenesis of sepsis. Our study investigated the effect of miR-23b on sepsis-induced immunosuppression. Methods Mice were treated with miR-23b inhibitors by tail vein injection 2 days after cecal ligation puncture (CLP)-induced sepsis. Apoptosis in spleens and apoptotic signals were investigated, and survival was monitored. T-cell immunoreactivities were examined during late sepsis. Nuclear factor B (NF-B)-inducing kinase (NIK), tumor necrosis factor (TNF)-receptor associated factor 1 (TRAF1), and X-linked inhibitor of apoptosis protein (XIAP), the putative targets of miR-23b, were identified by a dual-luciferase reporter assay. Results miR-23b expression is upregulated and sustained during sepsis. The activation of the TLR4/TLR9/p38 MAPK/STAT3 signal pathway contributes to the production of miR-23b in CLP-induced sepsis. miR-23b inhibitor decreased the number of spleen cells positive by terminal deoxynucleotidyl transferase dUTP nick-end labeling and improved survival. miR-23b inhibitor restored the immunoreactivity by alleviating the development of T-cell exhaustion and producing smaller amounts of immunosuppressive interleukin 10 and interleukin 4 during late sepsis. We demonstrated that miR-23b mediated immunosuppression during late sepsis by inhibiting the noncanonical NF-B signal and promoting the proapoptotic signal pathway by targeting NIK, TRAF1, and XIAP. Conclusions Inhibition of miR-23b reduces late-sepsis-induced immunosuppression and improves survival. miR-23b might be a target for immunosuppression.

abstract: Osteosarcoma is the most common bone cancer in children and adolescents. Patients with metastatic osteosarcoma are typically refractory to treatment. Numerous lines of evidence suggest that cytotoxic T-lymphocytes (CTL) limit the development of metastatic osteosarcoma. I have investigated the role of Programmed Death Receptor-1 (PD-1) in limiting the efficacy of immune mediated control of metastatic osteosarcoma. I show that human metastatic, but not primary, osteosarcoma tumors express the ligand for PD-1 (PD-L1) and that tumor infiltrating CTL express PD-1, suggesting this pathway may limit CTL control of metastatic osteosarcoma in patients. PD-L1 is also expressed on the K7M2 osteosarcoma tumor cell line that establishes metastases in mice, and PD-1 is expressed on tumor infiltrating CTL during disease progression. Blockade of PD-1/PD-L1 interactions dramatically improves the function of osteosarcoma-reactive CTL in vitro and in vivo, and results in decreased tumor burden and increased survival in the K7M2 mouse model of metastatic osteosarcoma. My results suggest that blockade of PD-1/PD-L1 interactions in patients with metastatic osteosarcoma should be pursued as a therapeutic strategy. However, PD-1/PD-L1 blockade treated mice still succumb to disease due to selection of PD-L1 mAb resistant tumor cells via up-regulation of other co-inhibitory T cell receptors. Combinational α-CTLA-4 and α-PD-L1 blockade treated mice were able to completely eradicate metastatic osteosarcoma, and generate immunity to disease. These results suggest that blockade of PD-1/PD-L1 interactions in patients with metastatic osteosarcoma, although improves survival, may lead to tumor resistance, requiring combinational immunotherapies to combat and eradicate disease. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2015

abstract: CD8+ T-lymphocytes (CTLs) are central to the immunologic control of infections and are currently at the forefront of strategies that enhance immune based treatment of a variety of tumors. Effective T-cell based vaccines and immunotherapies fundamentally rely on the interaction of CTLs with peptide-human leukocyte antigen class I (HLA-I) complexes on the infected/malignant cell surface. However, how CTLs are able to respond to antigenic peptides with high specificity is largely unknown. Also unknown, are the different mechanisms underlying tumor immune evasion from CTL-mediated cytotoxicity. In this dissertation, I investigate the immunogenicity and dysfunction of CTLs for the development of novel T-cell therapies. Project 1 explores the biochemical hallmarks associated with HLA-I binding peptides that result in a CTL-immune response. The results reveal amino acid hydrophobicity of T-cell receptor (TCR) contact residues within immunogenic CTL-epitopes as a critical parameter for CTL-self/nonself discrimination. Project 2 develops a bioinformatic and experimental methodology for the identification of CTL-epitopes from low frequency T-cells against tumor antigens and chronic viruses. This methodology is employed in Project 3 to identify novel immunogenic CTL-epitopes from human papillomavirus (HPV)-associated head and neck cancer patients. In Project 3, I further study the mechanisms of HPV-specific T-cell dysfunction, and I demonstrate that combination inhibition of Indoleamine 2, 3-dioxygenase (IDO-1) and programmed cell death protein (PD-1) can be a potential immunotherapy against HPV+ head and neck cancers. Lastly, in Project 4, I develop a single-cell assay for high-throughput identification of antigens targeted by CTLs from whole pathogenome libraries. Thus, this dissertation contributes to fundamental T-cell immunobiology by identifying rules of T-cell immunogenicity and dysfunction, as well as to translational immunology by identifying novel CTL-epitopes, and therapeutic targets for T-cell immunotherapy. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2017

Cancer immunotherapy has gained attention in recent years for its successes in potentiating immune responses that can elicit tumor control. In particular, adoptive cell therapy (ACT), which involves the autologous/allogeneic transplant of ex vivo-cultivated tumor-specific T lymphocytes, can mediate potent tumor recognition and killing; however, durable clinical responses are often difficult to obtain in solid tumors. Solid tumors and their unique microenvironments have the capacity to evade and suppress antitumor immune responses and represent significant hurdles for effective ACT. Recently, we have discovered that chemical inhibition of histone deacetylases via MS-275 (Entinostat) during ACT can subvert tumor resistance to foster potent, broad-spectrum antitumor immunity. Overall, the work described supports the efficacy of ACT in the treatment of immunosuppressive, solid tumors; however, consistency in durable clinical outcomes can only be achieved through the concurrent therapeutic targeting of tumor resistance mechanisms. This thesis uses pre-clinical models to describe how tumor resistance to ACT can manifest, and demonstrates that concurrent MS-275 delivery drives extensive immunomodulation to promote sustained tumor clearance. This includes: 1) The polarization of tumor-infiltrating myeloid cells into cytotoxic effectors with the ability to reject immune escape variants 2) The inflammatory remodeling of the tumor microenvironment to potentiate epitope spreading against secondary tumor antigens 3) The transcriptional reprogramming of adoptively transferred T cells to overcome tumor-burden-dependent exhaustion We expect that the results will help facilitate the development of next-generation ACT platforms that will feature strategies for multi-mechanistic perturbation of tumor resistance. / Thesis / Doctor of Philosophy (PhD) / The host immune system has the ability to recognize and destroy tumor cells. Therapeutic platforms that leverage antitumor immune cells, specifically T cells, have shown potency in the elimination of cancer. In the clinic, cancer immunotherapies have demonstrated early success against hematological malignancies; however, are unreliable in the treatment of solid tumors. Solid tumors utilize intrinsic and adapted mechanisms of resistance to mitigate the effectiveness of cancer immunotherapy. This thesis pursues research questions aimed at understanding how tumors resist immunotherapy, what mechanisms are utilized, and how to overcome these obstacles. We anticipate that these results will contribute to the development and incorporation of strategies to subvert tumor resistance and potentiate T cells against solid tumors.