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The class A scavenger receptors (SR-A) MARCO and SR-AI/II are expressed on lung macrophages (MΦs) and dendritic cells (DCs) and function in innate defenses against inhaled pathogens and particles. Increased expression of SR-As in the lungs of mice in an OVA-asthma model suggested an additional role in modulating responses to an inhaled allergen. After OVA sensitization and aerosol challenge, SR-AI/II and MARCO-deficient mice exhibited greater eosinophilic airway inflammation and airway hyperresponsiveness compared with wild-type mice. A role for simple SR-A-mediated Ag clearance (“scavenging”) by lung MΦs was excluded by the observation of a comparable uptake of fluorescent OVA by wild-type and SR-A-deficient lung MΦs and DCs. In contrast, airway instillation of fluorescent Ag revealed a significantly higher traffic of labeled DCs to thoracic lymph nodes in SR-A-deficient mice than in controls. The increased migration of SR-A-deficient DCs was accompanied by the enhanced proliferation in thoracic lymph nodes of adoptively transferred OVA-specific T cells after airway OVA challenge. The data identify a novel role for SR-As expressed on lung DCs in the down-regulation of specific immune responses to aeroallergens by the reduction of DC migration from the site of Ag uptake to the draining lymph nodes.

Androgen modulation of immunity to tumor antigens have implications on designing novel prostate cancer immunotherapy regimens. However, the mechanisms governing androgen action are largely unknown. Herein we compared mouse castrate and non-castrate CD4 T cell transcriptomes and revealed major perturbations in gene regulation. Ingenuity pathway analysis identified dysregulations in key pathways, including Th cell differentiation, TCR signaling, Interferon signaling, and p38 MAPK signaling. Interestingly, nearly half of the genes that constitute the T cell differentiation pathway were affected. These genes include key transcription factors such as STAT1, Tbet, Foxp3, ROR-γ, costimulatory molecules CD40L and ICOS, and T helper cytokines such as IFN-γ, IL-17, TNF-α, and IL- 2. Transcription factors driving Th1 and Th17 differentiation as well as Th1 and Th17 cytokines and costimulatory receptors were upregulated. More CD4 T cells from castrated mice produced IFN-γ than did non-castrated ones. Conversely, exposure of activated CD4 T cells to IL-12 in the presence of androgens inhibited IFN-γ production and reduced Th1 differentiation genes like T-bet and IL-12-Rβ. This was concurrent with inhibition of STAT4 phosphorylation and upregulation of Ptpn22 and Ptpn1, both of which exhibit the ability to dephosphorylate STAT proteins. This suggests androgens’ negative effects on Th1 differentiation might be partly induced through Ptpn22- and Ptpn1-dependent dephosphorylation of STAT4.

Our immune system plays an important role in preventing cancer. However, tumor-derived immuno-suppression hinders effective anti-tumor immunity. T cell dysfunction or exhaustion in tumor-bearing hosts is one such mechanism. Numerous studies have identified PD-1 and Tim-3 as markers of exhausted T cells, and antibodies blocking PD-1 and Tim-3 have been shown to partially restore T cell function. We have found that both Tim-3 and PD-1 are expressed on CD8+ tumor-infiltrating lymphocytes (TILs) in an animal model of B cell lymphoma and that these cells represent the predominant fraction of T cells infiltrating tumors. We find that iNKT stimulation using the ligand, alpha-galactosylceramide, combined with TLR9 stimulation using the ligand, CpG, can down-regulate Tim-3 and PD-1 expression in TILs, restore TIL function as measured by proliferation and production of TNF and IFNg in response to tumor, and induce long-lasting therapeutic immune responses in mice with large advanced (>1cm2) tumors.

iNKT cells have considerable therapeutic promise in cancer, but so far this has not been fully realized. At issue has been delivering iNKT ligand and tumor Ag to the same APC for optimal processing and presentation. Also, although tumor-infiltrating APCs express CD1d in both prostate cancer (PCa) models and patients, tumor induced tolerance and suppression leads to defective iNKT activation. Whole-cell tumor vaccines represent a promising treatment approach and prevent tumor progression in multiple models of cancer. Here, we use an optimized approach consisting of alpha-galactosylceramide (aGalCer) and the GM-CSF-based PCa vaccine, ‘GVax’ combined with IL-12. This approach was capable of breaking tumor-induced tolerance and causing dramatic and substantial tumor regression and survival in large advanced prostate tumors in TRAMP mice. Remarkably, 10/12 vaccinated >9 month old mice rejected tumors, 'breaking tolerance', even at this late stage of tumor growth. The vaccine induced strong immune responses capable of reversing INKT defects in tumor-bearing mice as well as in patient iNKT in vitro. Both IL-12 and aGalCer were crucial for this response. Vaccinated mice had higher levels and activation of tumor-specific CD8 T, CD4 T and iNKT cells, and greater memory CD8 T cell expansion capable of producing the effector cytokines IFN-γ and TNF-α in response to tumor. These studies shed light on how whole cell vaccines targeting iNKTs can be utilized in PCa immunotherapy.

It has been suggested that one factor limiting cancer immunotherapy in clinical trials are immunological checkpoints that impede vaccine-induced anti-tumor responses. We postulate that manipulating lymphocytes and dendritic cells through the Tim-1 receptor, a membrane protein that regulates the vigor and differentiation of these cells, using an agonist monoclonal antibody could enhance cytotoxic lymphocyte (CTL) responses to prostate tumor-associated antigens. We used an approach that combines concomitant immunization against a model prostate tumor-associated antigen (TAA) SV40 Tag and treatment with an agonist anti-Tim1 antibody and assessed Tag-specific CTL responses two weeks later. Ab treatment of TRAMP mice resulted in elevated Tag-specific CTL in both spleen and prostate-draining lymph nodes, indicating that manipulating Tim1 results in circumventing immune tolerance to Tag antigen. We then tested this strategy for human epitopes derived from the prostate cancer-specific antigen ERG and SIM2 by combining anti-Tim1 antibody with an ERG- and SIM2-derived HLA-A2.1-restricted peptide vaccine in HLA-A2.1 transgenic mice and showed that CTL responses to ERG and SIM2 epitopes are elevated in the presence of anti-Tim1 Ab. Further characterization of lymphocyte differentiation post-immunization/anti-Tim1 treatment revealed that major shifts in T cell differentiation occur. Our findings suggest that targeting Tim1 could be successfully combined with prostate cancer vaccines.

The suitability of immunotherapy as a treatment for prostate cancer (PCa) has recently been demonstrated in a number of clinical trials. While these studies have provided a strong proof of principle, there is considerable room for improvement. One potential strategy is to identify new targets specific to PCa that could be immunized against and with this in mind, we have implemented an in silico-in vitro-in vivo¬ procedure for designing and assessing the suitability of MHC-I-restricted peptides as anti-PCa vaccines. In this study, we investigated peptides designed from ERG, a transcription factor expressed in around 50% of human PCa cases. We used in silico protein cleavage and peptide-binding algorithms to design HLA-A2.1 restricted peptides from the Erg protein. The binding of the in silico designed Erg peptides was then assessed in vitro and shown to bind HLA-A2.1 effectively. Additionally, when peptides were tested in vivo, they generated a strong immune response in humanized HLA-A2.1+ mice and overcame peripheral tolerance in prostate specific Erg expressing TRAMP-HLA-A2.1-ERG+ mice. Finally, it was found that a greater CD8+ T-cell recall response against human ERG+ PCa cell lines occurred in ERG immunized HLA-A2.1+ mice compared to controls. Together these data support the validity of our peptide design method and indicate that peptides derived from the overexpressed gene Erg could be used in an anti-PCa vaccine.

Metabolic pathways that drive T cell differentiation, activation and tolerance remain poorly understood. Here we used mass spectrometry to partially describe the metabolic profile of CD4 and CD8 T cells that underwent in vitro activation and differentiation in the presence or absence of prostate cancer conditioned media. T cell subsets were purified from mouse spleen or human PBMC, activated in vitro in the presence of various combinations of cytokines or prostate cancer cell line conditioned media. Metabolites were extracted using methanol, injected and analyzed using a 5500 QTRAP triple quadruple mass spectrometer coupled to a Prominence UFLC HPLC system via selected reaction monitoring (SRM) of a total of 255 endogenous water soluble metabolites for steady-state analyses of samples. Our data show that TCR/CD28-mediated activation imposes a distinct metabolic profile on mouse T cells. This profile is distinct for each cell lineage when activated cells are differentiated into either Th1, Th2, Th9, Th17 or regulatory T cell phenotypes. Likewise, metabolic profiles of activated CD4 and CD8 T cells from both mouse and human are significantly affected by the presence of prostate cancer cell line conditioned media, with major changes in the urea cycle, ammonia recycling, and protein and nucleotide biosynthesis . Together, our findings provide a basis for further development of drugs that could be used to manipulate T cells for a better outcome of prostate cancer vaccines.

The inhibitory role of regulatory T-cells (Tregs) in cancer is now well established. Furthermore, inhibition of Treg function has been shown both experimentally and clinically to improve the immune response towards a variety of cancers. Developing new and more effective strategies interfering with the function of Tregs in cancer requires a deep understanding of Treg suppressor machinery, and a thorough dissection of the molecular elements that orchestrate their differentiation from T cells. With this in mind, we sorted Tregs and non-Treg CD4 cells from the prostate-draining LN or spleens of prostate tumor-bearing male mice and their control age-matched controls. Gene expression profiling was performed and data were analyzed to identify differential gene expression and major dysregulations in biological pathways, functions and networks. These changes encompassed key pathways such as T cell differentiation, TREM1, IL-17 and IL-10 signaling, as well as many important biologic functions that include inflammation, cellular movement, immune cell trafficking and cell-to-cell interaction. In addition, the most affected network is centered on TGF-β. Interestingly, prostate cancer Tregs exhibit many markers that are distinctive of macrophage and B cell lineages. This work paves the way for a better understanding of Treg biology in the context of prostate cancer and offers new avenues for Treg manipulation toward improving the outcome of immunotherapy.