Faculty Bibliography

With the advent of checkpoint blockade therapies, immunotherapy is now a critical modality for the treatment of some cancers. While some patients respond well to checkpoint blockade, many do not, necessitating the need for other forms of therapy. Vaccination against malignancy has been a long sought goal of science. For cancers holding a microbial etiology, vaccination has been highly effective in reducing the incidence of disease. However, vaccination against established malignancy has been largely disappointing. In this review, we discuss efforts to develop diverse vaccine modalities in the treatment of cancer with a particular focus on melanoma. Recent work has suggested that vaccines targeting patient-specific tumor mutations may be more relevant than those targeting unmutated proteins. Nonetheless, tumor cells utilize many strategies to evade host immunity. It is likely that the full potential of cancer vaccination will only be realized when vaccines are combined with other therapies targeting tumor immunoevasive mechanisms. By modulating inhibitory molecules, regulatory immune cells, and the metabolic resources and demands of T cells, scientists and clinicians can ensure vaccine-stimulated T cells are fully functional within the immunosuppressive tumor microevironment.

Sphingosine Kinase-2 (Sphk2) is responsible for the production of the bioactive lipid Sphingosine-1 Phosphate, a key regulator of tissue repair. Here we address the in vivo significance of Sphingosine Kinase -2 in renal inflammation/fibrosis in response to unilateral ureteral obstruction using both genetic and pharmacological strategies. Obstructed kidneys of Sphk2-/- mice showed reduced renal damage and diminished levels of the renal injury markers TGFβ1 and αSMA when compared to wild type controls. We found a consistently significant increase in anti-inflammatory (M2) macrophages in obstructed Sphk2-/- kidneys by flow cytometry and a decrease in mRNA levels of the inflammatory cytokines, MCP1, TNFα, CXCL1 and ILβ1, suggesting an anti-inflammatory bias in the absence of Sphk2. Indeed, metabolic profiling showed that the pro-inflammatory glycolytic pathway is largely inactive in Sphk2-/- bone marrow-derived macrophages. Furthermore, treatment with the M2-promoting cytokines IL-4 or IL-13 demonstrated that macrophages lacking Sphk2 polarized more efficiently to the M2 phenotype than wild type cells. Bone marrow transplant studies indicated that expression of Sphk2-/- on either the hematopoietic or parenchymal cells did not fully rescue the pro-healing phenotype, confirming that both infiltrating M2-macrophages and the kidney microenvironment contribute to the damaging Sphk2 effects. Importantly, obstructed kidneys from mice treated with an Sphk2 inhibitor recapitulated findings in the genetic model. These results demonstrate that reducing Sphk2 activity by genetic or pharmacological manipulation markedly decreases inflammatory and fibrotic responses to obstruction, resulting in diminished renal injury and supporting Sphk2 as a novel driver of the pro-inflammatory macrophage phenotype.

The spleen is an important site for generating protective immune responses against pathogens. After infection, immune cells undergo rapid reorganization to initiate and maintain localized inflammatory responses; however, the mechanisms governing this spatial and temporal cellular reorganization remain unclear. We show that the strategic position of splenic marginal zone CD169⁺ macrophages is vital for rapid initiation of antibacterial responses. In addition to controlling initial bacterial growth, CD169⁺ macrophages orchestrate a second phase of innate protection by mediating the transport of bacteria to splenic T cell zones. This compartmentalization of bacteria within the spleen was essential for driving the reorganization of innate immune cells into hierarchical clusters and for local interferon-γ production near sites of bacterial replication foci. Our results show that both phases of the antimicrobial innate immune response were dependent on CD169⁺ macrophages, and, in their absence, the series of events needed for pathogen clearance and subsequent survival of the host was disrupted. Our study provides insight into how lymphoid organ structure and function are related at a fundamental level.

Memory CD8⁺ T cells were originally thought to exist as two populations (effector and central memory). In recent years, a third population called resident memory T cells has been discovered and further to this these populations are being divided into different subtypes. Understanding the function and developmental pathways of memory CD8⁺ T cells is key to developing effective therapies against cancer and infectious diseases. Here we have reviewed what is currently known about all three subsets of memory CD8⁺ T populations and as to how each population was originally discovered and the developmental pathways of each subpopulation. Each memory population appears to play a distinct role in adaptive immune responses but we are still a long way from understanding how the populations are generated and what roles they play in protection against invading pathogens and if they contribute to the pathogenesis of inflammatory diseases.

Mucosal antigen-specific CD4 T-cell responses to intestinal pathogens remain incompletely understood. Here we examined the CD4 T-cell response after oral infection with an internalin A 'murinized' Listeria monocytogenes (Lm). Oral Lm infection induced a robust endogenous listeriolysin O (LLO)-specific CD4 T-cell response with distinct phenotypic and functional characteristics in the intestine. Circulating LLO-specific CD4 T cells transiently expressed the 'gut-homing' integrin α₄β₇ and accumulated in the intestinal lamina propria and epithelium where they were maintained independent of interleukin (IL)-15. The majority of intestinal LLO-specific CD4 T cells were CD27^- Ly6C^- and CD69⁺ CD103^- while the lymphoid LLO-specific CD4 T cells were heterogeneous based on CD27 and Ly6C expression and predominately CD69^-. LLO-specific effector CD4 T cells transitioned into a long-lived memory population that phenotypically resembled their parent effectors and displayed hallmarks of residency. In addition, intestinal effector and memory CD4 T cells showed a predominant polyfunctional Th1 profile producing IFNγ, TNFα, and IL-2 at high levels with minimal but detectable levels of IL-17A. Depletion of CD4 T cells in immunized mice led to elevated bacterial burden after challenge infection highlighting a critical role for memory CD4 T cells in controlling intestinal intracellular pathogens.

Despite many years of research, cancer vaccines have largely been ineffective in the treatment of established cancers. Many barriers to immune-mediated destruction of malignant cells exist, and these likely limit the efficacy of cancer vaccines. In this study, we sought to enhance the efficacy of a cytomegalovirus (CMV)-based vaccine targeting melanoma by combining vaccination with other forms of immunotherapy. Adoptive cell therapy in humans and in animal models has been shown to be effective for tumor regression. Thus, in this study, we assessed whether CMV-based vaccines in combination with adoptively transferred antitumor T cells could provide greater antitumor protection than either therapy alone. Our results show that adoptive cell therapy greatly enhanced the antitumor effects of CMV-based vaccines targeting the foreign model antigen, OVA, or the melanoma differentiation antigen, gp100. Combination adoptive cell therapy and vaccination induced the upregulation of the inhibitory ligands, PD-L1, and Qa-1^b, on B16 tumor cells. This expression paralleled the infiltration of tumors by vaccine-stimulated T cells which also expressed high levels of the receptors PD-1 and NKG2A/C/E, suggesting a potential mechanism of tumor immune evasion. Surprisingly, therapeutic blockade of the PD-1/PD-L1 and NKG2A/Qa-1^b axes did not delay tumor growth following vaccination, suggesting that the presence of inhibitory ligands within malignant tissue may not be an effective biomarker for successful combination therapy with CMV-based vaccines. Overall, our studies show that therapeutic CMV-based vaccines in combination with adoptive T cell transfer alone are effective for tumor rejection.

Local anatomy of lymphoid tissues during infection has emerged as a critical regulator of immunity; thus, studying the cellular choreography in the context of an intact tissue environment in situ is crucial. Following an infection, the local pathogen-specific T cell migration and the subsequent egress of effector T cells from the draining lymph nodes are important and complex biological processes. The mechanisms that regulate this complex process can now be investigated by directly visualizing T cell dynamics in vivo using intravital two-photon (2P) microscopy. In addition, static whole-mount imaging technique can provide us with a comprehensive assessment of global changes in the distribution of cellular populations within an intact tissue. Thus, in this chapter, we detail methods to visualize the migration and egress of endogenous antigen-specific CD8 T cells following viral infection using two methods-intravital 2P microscopy and multicolor whole-mount in situ tetramer staining.

The nuclear pregnane X receptor (PXR) plays a central role in regulating xenobiotic metabolism. We now report a novel role for PXR as a critical negative regulator of innate immunity after infection. Pxr(-/-) mice exhibited remarkably elevated pro-inflammatory cytokine and chemokine production following infection with Listeria monocytogenes (Lm). Despite the more robust innate immune response, Pxr(-/-) mice were highly susceptible to Lm infection. Surprisingly, disruption of the Toll-like receptor 4 (TLR4) but not TLR2 signaling restored the inflammation to normal levels and the ability to clear Lm in Pxr(-/-) mice. Mechanistically, the heightened inflammation in Pxr(-/-) mice resulted in the death of inflammatory monocytes that led to the enhanced susceptibility to Lm infection. These data demonstrated that PXR regulated pathogen-induced inflammation and host defense against Lm infection through modulating the TLR4 pathway. In summary, we discovered an apical role for PXR in regulating innate immunity. In addition, we uncovered a remarkable negative impact of the TLR4 pathway in controlling the quality of the inflammatory response and host defense against a gram-positive bacterial infection.

Memory γδ T cells are important for the clearance of Listeria monocytogenes infection in the intestinal mucosa. However, the mechanisms by which memory γδ T cells provide protection against secondary oral infection are poorly understood. Here we used a recombinant strain of L. monocytogenes that efficiently invades the intestinal epithelium to show that Vγ4(+) memory γδ T cells represent a resident memory (Trm) population in the mesenteric lymph nodes (MLNs). The γδ Trm exhibited a remarkably static pattern of migration that radically changed following secondary oral L. monocytogenes infection. The γδ Trms produced IL-17A early after rechallenge and formed organized clusters with myeloid cells surrounding L. monocytogenes replication foci only after a secondary oral infection. Antibody blocking studies showed that in addition to IL-17A, the chemokine receptor C-X-C chemokine receptor 3 (CXCR3) is also important to enable the local redistribution of γδ Trm cells and myeloid cells specifically near the sites of L. monocytogenes replication within the MLN to restrict bacterial growth and spread. Our findings support a role for γδ Trms in orchestrating protective immune responses against intestinal pathogens.