Browsing by Author "Yang, Yiping"

Macrophages are terminally differentiated cells of the hematopoietic system that are involved in various important cellular processes, including protection against pathogenic infections, inflammation and tissue homeostasis. Additionally, macrophages coinhabit with malignant cells in various stages of tumor development and can be polarized to two distinctive phenotypes – the pro-inflammatory M1 phenotype and the anti-inflammatory M2 phenotype. These M2 polarized tumor-associated macrophages (TAMs) are known to suppress immune responses and promote neoplastic progression. We sought to gain insights into the regulation of TAM functions and mechanisms by which TAMs exert immunosuppressive changes within the tumor microenvironment (TME).

The hedgehog (Hh) signaling pathway is one of the most important signaling pathways in cancer. Its aberrant activation within cancer cells can lead to increased proliferation and metastasis. Hh ligands, including sonic hedgehog (Shh), desert hedgehog (Dhh) and indian hedgehog (Ihh) can also be secreted from tumor cells to modify the TME in order to create a more favorable environment to foster tumorigenesis. However, little is known about how Hh ligands change the TME to promote tumor progression and what cell type(s) are involved in this process. Using a murine model, we found initial evidence that hedgehog signaling is active in TAMs and deletion of the pathway leads to suppressed tumor growth.

In the first aim of this dissertation, we first sough to investigate whether Hh signaling plays a role in TAM functions. Specifically, we asked whether Hh activation could promote TAM M2 polarization. Using several murine tumor models, we confirmed that Hh signaling in myeloid cells is critical for promoting the immunosuppressive M2 phenotype in TAMs, resulting in accelerated tumor growth. We also found that tumor cells secrete Shh and that tumor derived Shh drives M2 polarization. Furthermore, Hh-induced M2 polarization in TAMs can suppress CD8+ T cell recruitment to the TME through the inhibition of C-X-C motif chemokine ligand 9 (CXCL9) and CXCL10 production by TAMs. Lastly, we demonstrated that Krüppel-like factor 4 (Klf4) mediates Hh-dependent TAM M2-polarization and the immunosuppressive function. Collectively, these findings highlight a critical role for tumor derived Shh in promoting TAM M2 polarization and inhibiting CD8+ T cell infiltration in tumor.

In the second aim of this study, we further elucidated whether Hh-induced M2 TAMs can directly subvert CD8+ tumor-infiltrating lymphocyte (TIL) cytotoxic functions. Specifically, we queried whether expression of programmed death ligand 1 (PD-L1) on TAMs, promoted by Hh signaling activation, play a role in suppressing CD8+ TILs. Using several murine models including a novel myeloid-specific Pdl1 knock-out mouse, we showed the tumor-derived Shh ligand promoted PD-L1 expression on TAMs, resulting in decreased interferon- (IFN-) and granzyme B (GzmB) production in CD8+ TILs. Furthermore, TAM-derived PD-L1 was found to be the major source of PDL1-programmed death 1 (PD1)-axis-mediated CD8+ TIL suppression. Lastly, we demonstrated signal transducer and activator of transcription 3 (Stat3) mediates Hh-dependent PD-L1 upregulation in TAMs. Together, these findings demonstrated that hedgehog signaling plays an important role in suppressing CD8+ TIL effector functions through PD-L1-expressing TAMs.

Collectively, this work sought to dissect the role of hedgehog signaling in TAMs and immunosuppression within the TME in order to identify novel cancer therapeutic strategies.

Natural killer (NK) cells are lymphocytes of the innate immune response with well-demonstrated activities against viral infections and tumors. Because of these abilities, we sought to glean insights into the mechanisms of NK cell activation so that they may be applied toward the design of new therapies.

NK cells are particularly critical for the control of poxviral infections. Vaccinia virus (VV) is the most-studied member of the poxviral family. It is robustly immunogenic and functions as the live vaccine responsible for the successful elimination of smallpox. VV infection provides a useful model for studying NK cell activation: NK cells play an important role in its clearance and the virus efficiently activates NK cells and recruits them to the site of infection. We had previously used this model to identify Toll-like receptor (TLR)-dependent and -independent mechanisms of NK cell activation to VV. One method of TLR-independent activation to VV requires the activation receptor NKG2D, which recognizes host ligands expressed upon viral infection by accessory cells such as dendritic cells (DCs) and macrophages.

In the first aim of this thesis, we sought to determine how the ligands for the NKG2D activation receptor become upregulated in the context of VV infection. Specifically, we asked whether interleukin-18 (IL-18), known to play a role in the innate immune response, could boost the expression of NKG2D ligands on DCs in response to viral infection. Using an in vivo infection model with IL-18R-deficient mice, our results confirmed an important role for IL-18 in NK cell activation to VV and viral control. We then made use of an NK-DC co-culture to show that IL-18 signaling on DCs, in addition to NK cells, is necessary to achieve efficient NK cell activation to viral infection. We further demonstrated in a cell-transfer experiment that cell-extrinsic IL-18 signaling is critical for NK cell activation in vivo. DC ablation via a mouse model designed to specifically ablate CD11c+ cells showed that DCs are also required for NK cell activation to VV in vivo. We finally showed how IL-18 can act on DCs in vivo and in vitro to boost the expression of Rae-1, an NKG2D ligand. Collectively, our data uncover a novel mechanism whereby NK cells become activated by IL-18 control of NKG2D ligand expression on DCs.

In the second aim of this project, we detailed how IL-18 signaling results in the upregulation of the NKG2D ligand Rae-1. Using an in vitro macrophage model, we showed how recombinant IL-18 was sufficient to upregulate Rae-1 expression. We compared IL-18 control of Rae-1 expression to LPS, a TLR ligand that also signals through the common adaptor MyD88 to govern Rae-1 expression. Using chemical inhibitors to cell signaling molecules, we then identified the importance of MyD88 signaling through PI3K. We then revealed that glycogen synthase kinase 3 (GSK-3) can act as a negative regulator of Rae-1 expression downstream of IL-18/TLR signaling. Specifically, we have shown that during inflammatory signaling, PI3K (acting downstream of MyD88) can inhibit GSK-3 to relieve its tonic suppression of Rae-1 expression and upregulate the NKG2D ligand. Finally, we showed that PI3K and GSK-3 signaling are also important to Rae-1 expression on DCs - the accessory cell where IL-18 signals to control Rae-1 expression to boost NK cell activation against VV.

In its entirety, this work seeks to address how NK cells become activated in the context of VV infection in order to identify new ways NK cells may be harnessed therapeutically.

Vaccinia virus (VV) is the most thoroughly studied member of the Poxviridae family and the vaccine used to achieve the only successful eradication of a human disease. Over the years, it has proven itself as a useful tool for the study of antiviral immunity, vaccine development, and potentially cancer immunotherapy. VV is capable of eliciting a robust immune response; however the mechanisms by which VV accomplishes this task remain unknown. The overall goal of this thesis project is to determine how VV activates the innate immune system, and how this activation contributes to viral clearance in vivo. We determined that VV or VV-DNA activated the TLR8-MyD88 pathway in plasmacytoid dendritic cells (pDC), resulting in the production of type I interferons (IFN). We also demonstrated that TLR8-mediated production of type I IFN by pDC was crucial to efficient VV control and clearance in vivo. Moreover, we identified the polyA- and polyT-rich sequences in VV-DNA was the possible motif recognize by TLR8. Type I IFN, known for ability to establish the "antiviral state", are also critical mediators of NK cell activation. In the setting of VV infection, we demonstrated that direct action of type I IFN on NK cells, but not accessory cells such as DC, was necessary for NK cell activation in vivo. We further demonstrated that type I IFN-dependent activation of NK cells was required for optimal VV clearance in vivo. Given the importance of NK cells in anti-VV innate immunity, we next examined what role the TLR2-MyD88 pathway, critical for activation of cDC, played in the activation of NK cells. NK cells from TLR2-/- or MyD88-/- mice displayed a reduction in activation and cytolytic function, and this defect was independent of pro-inflammatory cytokine signaling. We were able to demonstrate that direct TLR2 signaling on NK cells was required for their optimal activation and function in response to VV infection. Moreover, we were able to demonstrate that TLR2-MyD88 signaling resulted in the activation of the PI3K-ERK pathway, which was necessary for NK cell cytotoxicity. In addition, we identified the NKG2D pathway as critical for efficient NK cell activation and function in response to VV infection, independent of the TLR2 pathway. Both the NKG2D and TLR2 pathways were crucial for optimal VV clearance and control in vivo. Collectively, this project illuminates the roles and mechanisms of the innate immune system in the control of VV in vivo.

The role of CD4 T cell help in primary and secondary CD8 T cell responses to infectious pathogens remains incompletely defined. The primary CD8 T response to infections was initially thought to be largely independent of CD4 T cells, but it is not clear why some primary, pathogen-specific CD8 T cell responses are CD4 T cell-dependent. Furthermore, although the generation of functional memory CD8 T cells is CD4 T cell help-dependent, it remains controversial when the "help" is needed. The goal of this thesis project is to determine requirement and mechanisms of CD4 help during the CD8 response to vaccinia viral (VV) infection.

The first aim of this project was to determine when CD4 T cell help is required during the CD8 response to VV infection. Using both CD4-deficient mice and mice with transient depletion of CD4 T cells, we demonstrated that CD4 T cell help was not needed for the activation and effector differentiation of CD8 T cells during the primary response to VV infection. However, the activated CD8 T cells showed poor survival without CD4 T cell help, leading to a reduction in clonal expansion and a diminished, but stable CD8 memory pool. In addition, we observed that CD4 T cell help provided during both the primary and secondary responses was required for the survival of memory CD8 T cells during recall expansion. Our study indicates that CD4 T cells play a crucial role in multiple stages of CD8 T cell response to VV infection and may help to design effective vaccine strategies.

Given that CD4 T cell help is critical for the survival of activated CD8 T cells during both the primary and memory recall responses, it is still unclear how CD4 T cell help promotes CD8 T cell survival. The second aim of this project was to determine the mechanism of CD4 help for the survival of activated CD8 cells. We first showed that CD4 help in vitro was mediated by IL-21, a cytokine produced predominantly by activated CD4 T cells. We then demonstrated direct action of IL-21 on CD8 T cells was critical for the VV-specific CD8 T cell response in vivo. This intrinsic IL-21 signaling was essential for the survival of activated CD8 T cells and the generation of long-lived memory cells. We further revealed that IL-21 promoted CD8 T cell survival in a mechanism dependent on activation of the STAT1 and STAT3 pathways and subsequent upregulation of the pro-survival molecules Bcl-2 and Bcl-xL. Collectively, these results identify a critical role for CD4-derived IL-21 signaling in CD8 T cell responses to acute VV infection in vivo and may help design effective vaccine strategies in situations where CD4 cells are not fully functional.

Immune responses against viral infections are mediated through a complex process by diverse populations of cells, that can be harnessed for tumor immunotherapies and vaccinations. Vaccinia virus (VV) is the most studied member of the poxvirus family and is responsible for the successful elimination of smallpox worldwide. It is unique among well-studied viruses in that it replicates solely in the host cytoplasm and is able to elicit one of the longest lasting immunity in recorded human history. Its success in vaccination has led to the development of adjuvants with VV epitopes and recombinant VV vectors for other infectious diseases and cancer immunotherapy. However, the mechanism behind how VV elicits such a strong immune response from the immune system remains insufficiently understood.

Previous studies have shown that although activation of NK cells is critical for the initial control of VV infection, efficient activation of CD8+ T cell response is required for the eradication of VV infection. It has also been demonstrated that gammadelta T cells play an important part in the immune response against VV infection. However, both processes remain relatively undefined. What promotes CD8+ T cell activation and subsequent generation of CD8+ memory T cells in response to VV infection is still not very well dissected, and the mechanisms that govern gammadelta T cells response to VV are relatively unknown. This thesis examines these questions through three main aims: 1) influence of gammadelta T cells on CD8+ T cell activation, 2) gammadelta T cell direct cytotoxicity against VV infection, and 3) mechanisms that govern CD8+ memory T cell formation. The overall goal of this thesis is to understand the mechanisms behind gammadelta T and CD8+ T cells responses against VV infection.

We found that gammadelta T cells play an important role in promoting CD8+ T cell response to VV infection. We showed that gammadelta T cells serve not only as antigen presenting cells to CD8+ T cell activation, but also as mediators of other signals of CD8+ T cell response in vivo. We further demonstrated that cell-intrinsic MyD88 signaling in gammadelta T cells is required for activation of CD8+ T cells. Contrary to conventional expectations, we found that NKG2D expression in both NK and CD8+ T cells only have partial effect on the elimination of VV post-infection. Instead, we found that NKG2D is an important activator of gammadelta T cell cytotoxicity for VV clearance. Lastly, we demonstrated that Notch1, but not Notch2, deficiency increases the formation of CD8+ memory T cells, through modulating the expression of TCF1/Tcf7. We discovered that cleaved Notch1 intracellular domain binds upstream of Tcf7 and controls the expression of Tcf7 for CD8+ memory T cell formation.

These results demonstrated a critical role for gammadelta T cells in viral clearance and the regulation of adaptive T cell response, with insights into the formation of CD8+ memory T cells. Collectively, this dissertation seeks to better understand how gammadelta T and CD8+ T cells respond to VV infection, with the hopes of shedding additional light on the design of more effective vaccine strategies based on the precise manipulation of immune cell populations for infectious diseases and cancer immunotherapy.

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