Burkholderia pseudomallei, the etiologic agent of melioidosis, is a Gram negative bacterium designated as a Tier 1 threat. This bacterium is known to be endemic in Southeast Asia and Northern Australia and can infect humans and animals by several routes. Inhalational melioidosis has been associated with monsoonal rains in endemic areas and is also a significant concern in the biodefense community. There are currently no effective vaccines for B. pseudomallei and antibiotic treatment can be hampered by non-specific symptomology and also the high rate of naturally occurring antibiotic resistant strains. Well-characterized animal models will be essential when selecting novel medical countermeasures for evaluation prior to human clinical trials. Here, we further characterize differences between the responses of BALB/c and C57BL/6 mice when challenged with low doses of a low-passage and well-defined stock of B. pseudomallei K96243 via either intraperitoneal or aerosol routes of exposure. Before challenge, mice were implanted with a transponder to collect body temperature readings, and daily body weights were also recorded. Mice were euthanized on select days for pathological analyses and determination of the bacterial burden in selected tissues (blood, lungs, liver, and spleen). Additionally, spleen homogenate and sera samples were analyzed to better characterize the host immune response after infection with aerosolized bacteria. These clinical, pathological, and immunological data highlighted and confirmed important similarities and differences between these murine models and exposure routes.

Mouse models have been essential to generate supporting data for the research of infectious diseases. Burkholderia pseudomallei, the etiological agent of melioidosis, has been studied using mouse models to investigate pathogenesis and efficacy of novel medical countermeasures to include both vaccines and therapeutics. Previous characterization of mouse models of melioidosis have demonstrated that BALB/c mice present with an acute infection, whereas C57BL/6 mice have shown a tendency to be more resistant to infection and may model chronic disease. In this study, either BALB/c or C57BL/6 mice were exposed to aerosolized human clinical isolates of B. pseudomallei. The bacterial strains included HBPUB10134a (virulent isolate from Thailand), MSHR5855 (virulent isolate from Australia), and 1106a (relatively attenuated isolate from Thailand). The LD50 values were calculated and serial sample collections were performed in order to examine the bacterial burdens in tissues, histopathological features of disease, and the immune response mounted by the mice after exposure to aerosolized B. pseudomallei. These data will be important when utilizing these models for testing novel medical countermeasures. Additionally, by comparing highly virulent strains with attenuated isolates, we hope to better understand the complex disease pathogenesis associated with this bacterium.

One obstacle in eliciting potent anti-tumor immune responses is the induction of tolerance to tumor antigens. TCRlo mice bearing a TCR transgene specific for the melanoma antigen Tyrosinase-related protein-2 (TRP-2, Dct) harbor T cells that maintain tumor antigen responsiveness but lack the ability to control melanoma outgrowth. We used this model to determine whether higher avidity T cells could control tumor growth without becoming tolerized. As part of the current study, we developed a second TRP-2-specific TCR transgenic mouse line (TCRhi) that bears higher avidity T cells and spontaneously developed autoimmune depigmentation. In contrast to TCRlo T cells, which were ignorant of tumor-derived antigen, TCRhi T cells initially delayed subcutaneous B16 melanoma tumor growth. However, persistence in the tumor microenvironment resulted in reduced IFN-γ production and CD107a (Lamp1) mobilization, hallmarks of T cell tolerization. IFN-γ expression by TCRhi T cells was critical for up-regulation of MHC-I on tumor cells and control of tumor growth. Blockade of PD-1 signals prevented T cell tolerization and restored tumor immunity. Depletion of tumor-associated dendritic cells (TADCs) reduced tolerization of TCRhi T cells and enhanced their antitumor activity. In addition, TADCs tolerized TCRhi T cells but not TCRlo T cells in vitro. Our findings demonstrate that T cell avidity is a critical determinant of not only tumor control but also susceptibility to tolerization in the tumor microenvironment. For this reason, care should be exercised when considering T cell avidity in designing cancer immunotherapeutics.

Maintaining antitumor immunity remains a persistent impediment to cancer immunotherapy. We and others have previously reported that high-avidity CD8+ T cells are more susceptible to tolerance induction in the tumor microenvironment. In the present study, we used a novel model where T cells derived from two independent TCR transgenic mouse lines recognize the same melanoma antigenic epitope but differ in their avidity. We tested whether providing CD4+ T cell help would improve T cell responsiveness as a function of effector T cell avidity. Interestingly, delivery of CD4+ T cell help during in vitro priming of CD8+ T cells improved cytokine secretion and lytic capacity of high-avidity T cells, but not low-avidity T cells. Consistent with this observation, copriming with CD4+ T cells improved antitumor immunity mediated by higher avidity, melanoma-specific CD8+ T cells, but not T cells with similar specificity but lower avidity. Enhanced tumor immunity was associated with improved CD8+ T cell expansion and reduced tolerization, and it was dependent on presentation of both CD4+ and CD8+ T cell epitopes by the same dendritic cell population. Our findings demonstrate that CD4+ T cell help preferentially augments high-avidity CD8+ T cells and provide important insight for understanding the requirements to elicit and maintain durable tumor immunity.