Many antigen-specific T cells die after exposure to antigen in animals. These cells also die if they are isolated from animals shortly after activation and cultured. Various cytokines were tested for their ability to interfere with this in vitro death. Surprisingly, tumor necrosis factor ␣ and other inf lammatory cytokines did not prevent the in vitro death of activated T cells, even though these cytokines do prevent activated T cell death in animals. Therefore, the inf lammatory cytokines probably act on T cells in vivo via an intermediary factor.

Tumor-associated macrophages (TAMs) express programmed cell death ligand 1 (PD-L1) and contribute to the immune-suppressive tumor microenvironment. Although the role of the PD-L1 and PD-1 interaction to regulate T-cell suppression is established, less is known about PD-L1 signaling in macrophages and how these signals may affect the function of TAMs. We used and models to investigate PD-L1 signaling in macrophages and the effects of PD-L1 antibody treatment on TAM responses. Treatment of mouse and human macrophages with PD-L1 antibodies increased spontaneous macrophage proliferation, survival, and activation (costimulatory molecule expression, cytokine production). Similar changes were observed in macrophages incubated with soluble CD80 and soluble PD-1, and in PD-L1 macrophages. Macrophage treatment with PD-L1 antibodies upregulated mTOR pathway activity, and RNAseq analysis revealed upregulation of multiple macrophage inflammatory pathways. treatment with PD-L1 antibody resulted in increased tumor infiltration with activated macrophages. In tumor-bearing RAG mice, upregulated costimulatory molecule expression by TAMs and reduced tumor growth were observed. Combined PD-1/ PD-L1 antibody treatment of animals with established B16 melanomas cured half of the treated mice, whereas treatment with single antibodies had little therapeutic effect. These findings indicate that PD-L1 delivers a constitutive negative signal to macrophages, resulting in an immune-suppressive cell phenotype. Treatment with PD-L1 antibodies reverses this phenotype and triggers macrophage-mediated antitumor activity, suggesting a distinct effect of PD-L1, but not PD-1, antibody treatment. .

Francisella tularensis is an obligate intracellular bacterium that induces severe, acute, often fatal disease when acquired by the respiratory route. Despite the seriousness of this pathogen, very little is understood about its interaction with key target cells in the airways and lungs (alveolar macrophages and airway dendritic cells (DC)) after inhalation. In this study we demonstrate replication of F. tularensis in primary DC. Early after infection, F. tularensis induced increased expression of MHC class II and CD86 on DC, but not macrophages. This was followed by depletion of DC from the airways and lungs. Despite logarithmic replication and phenotypic maturation of DC, F. tularensis failed to induce production of several key proinflammatory cytokines, including TNF-α and IL-6, from DC. However, F. tularensis infection did elicit production of the potent immunosuppressive cytokine, TGF-β. Furthermore, F. tularensis actively suppressed the ability of DC to secrete cytokines in response to specific TLR agonists. Finally, we also found that infection of DC and macrophages in the lungs appears to actually increase the severity of pulmonary infection with F. tularensis. For example, depletion of airway DC and alveolar macrophages before infection resulted in significantly prolonged survival times. Together, these data suggest F. tularensis is able to selectively uncouple Ag-presenting functions from proinflammatory cytokine secretion by critical APCs in the lungs, which may serve to create a relatively immunosuppressive environment favorable to replication and dissemination of the organism.

Complexing TLR9 agonists such as plasmid DNA to cationic liposomes markedly potentiates their ability to activate innate immunity. We therefore reasoned that liposomes complexed with DNA or other TLR agonists could be used as effective vaccine adjuvants. To test this hypothesis, the vaccine adjuvant effects of liposomes complexed to TLR agonists were assessed in mice. We found that liposomes complexed to nucleic acids (liposome-Ag-nucleic acid complexes; LANAC) were particularly effective adjuvants for eliciting CD4+ and CD8+ T cell responses against peptide and protein Ags. Notably, LANAC containing TLR3 or TLR9 agonists effectively cross-primed CD8+ T cell responses against even low doses of protein Ags, and this effect was independent of CD4+ T cell help. Ag-specific CD8+ T cells elicited by LANAC adjuvants were functionally active and persisted for long periods of time in tissues. In a therapeutic tumor vaccine model, immunization with the melanoma peptide trp2 and LANAC adjuvant controlled the growth of established B16 melanoma tumors. In a prophylactic vaccine model, immunization with the Mycobacterium tuberculosis protein ESAT-6 with LANAC adjuvant elicited significant protective immunity against aerosol challenge with virulent M. tuberculosis. These results suggest that certain TLR agonists can be combined with cationic liposomes to produce uniquely effective vaccine adjuvants capable of eliciting strong T cell responses against protein and peptide Ags.

One approach to enhancing the T cell response to tumors is vaccination with mimotopes, mimics of tumor epitopes. While mimotopes can stimulate proliferation of T cells that recognize tumor-associated antigens (TAAs), this expansion does not always correlate with control of tumor growth. We hypothesized that vaccination with mimotopes of optimal affinity in this interaction will improve antitumor immunity. Using a combinatorial peptide library and a cytotoxic T lymphocyte clone that recognizes a TAA, we identified a panel of mimotopes that, when complexed with MHC, bound the TAA-specific TCR with a range of affinities. As expected, in vitro assays showed that the affinity of the TCR-peptide-MHC (TCR-pMHC) interaction correlated with activity of the T cell clone. However, only vaccination with mimotopes in the intermediate-affinity range elicited functional T cells and provided protection against tumor growth in vivo. Vaccination with mimotopes with the highest-affinity TCR-pMHC interactions elicited TAA-specific T cells to the tumor, but did not control tumor growth at any of the peptide concentrations tested. Further analysis of these T cells showed functional defects in response to the TAA. Thus, stimulation of an antitumor response by mimotopes may be optimal with peptides that increase but do not maximize the affinity of the TCR-pMHC interaction.