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STAT4 plays a critical role in the generation of both innate and adaptive immune responses. In the absence of STAT4, Th1 responses, critical for resistance to fungal disease, do not occur. Infection with the dimorphic fungus, Coccidioides, is a major cause of community-acquired pneumonia in the endemic regions of Arizona and California. In some people and often for unknown reasons, coccidioidal infection results in hematogenous dissemination and progressive disease rather than the typical self-limited pneumonia. Members of three generations in a family developed disseminated coccidioidomycosis, prompting genetic investigation. All affected family members had a single heterozygous base change in STAT4, c.1877A>G, causing substitution of glycine for glutamate at AA626 (STAT4E626G/+). A knockin mouse, heterozygous for the substitution, developed more severe experimental coccidioidomycosis than did wild-type mice. Stat4E626G/+ T cells were deficient in production of IFN-γ after anti-CD3/CD28 stimulation. Spleen cells from Stat4E626G mice showed defective responses to IL-12/IL-18 stimulation in vitro. In vivo, early postinfection, mutant Stat4E626G/+ mice failed to produce IFN-γ and related cytokines in the lung and to accumulate activated adaptive immune cells in mediastinal lymph nodes. Therefore, defective early induction of IFN-γ and adaptive responses by STAT4 prevents normal control of coccidioidomycosis in both mice and humans.

Neisseria musculi, isolated from the oral cavity of wild-caught mice, does not colonize most inbred mouse strains. N. musculi does weakly (50%) colonize C57BL/6J (B6) mice but readily colonizes CAST/EiJ (CAST) mice. In this study, we examined whether differences in the CAST and B6 host response could elucidate mechanisms governing N. musculi colonization. In vivo stimulation of B6 or CAST splenocytes with wild type (WT) Neisseria or Escherichia coli LPS showed that CAST mice had a blunted inflammatory response, producing significantly lower levels of IL-6 than B6 mice. The use of specific genetic knockouts highlighted a need for an intact innate immune system to prevent colonization. B6–RAG-1−/− mice were colonized at a similar rate as WT B6 mice, whereas B6-MyD88−/− and TLR4−/− mice were readily colonized like CAST (100%) mice. Sequence analysis revealed a unique point mutation in TLR4 in CAST mice. However, crosses to TLR4−/− mice and analysis of recombinant inbred Collaborative Cross mice showed that TLR4 from CAST mice was not sufficient to allow Neisseria colonization. In vitro stimulation of B6 bone marrow–derived macrophages or splenocytes with WT Neisseria yielded low levels of IL-6 compared with LPS stimulation. Surprisingly, UV-inactivated Neisseria induced high levels of IL-6, suggesting suppression of IL-6 production is an active bacterial process. Consistent with a critical role for IL-6 in preventing colonization, mice deficient for the IL-6 receptor were efficiently colonized, indicating host IL-6 production plays a critical role in determining host colonization susceptibility.

Coccidioidomycosis is a common fungal respiratory disease caused by the fungus Coccidioides spp. fungi in Arizona and the central valley of California. Disseminated Coccidioidomycosis (DCM) occurs in 8% of identified cases, causing significant morbidity and mortality. DCM occurred in 3 generations of a family in Tucson, Arizona who all shared a heterozygous missense mutation (p.E626G) in STAT4. Transfection of STAT4 E626G in WT human cells showed dampened responses to STAT4-dependent cytokine gene induction, suggesting the mutation is a dominant negative. To understand the function of E626G mutation, we generated the homologous mutation in Stat4 in B6 mice. Mice carrying E626G were grossly normal with normal numbers of innate and adaptive immune cells in primary and secondary lymphoid organs. To examine the effect of E626G on susceptibility to Coccidioides infection, we infected mice with C. posadasii strain 1038 intranasally. After infection E626G heterozygous mice all died by 50 days post infection (dpi), median time to death 45.5 days; range 30–50 dpi. 87% of WT B6 controls were alive when the experiment ended at a predetermined 70 dpi, resulting in a median time to death of >70 days. Mediastinal lymph nodes in E626G heterozygous mice had reduced numbers of B cells and activated (CD62Llow) T cells compared to B6 controls on 14 dpi. Cells from infected lungs from E626G heterozygotes produced less IFN-gamma after infection on 28, 35 and 42 dpi. Interferon stimulated cytokines (IP-10, MIG, MIP-2) were also significantly reduced compared to WT controls. Ongoing work includes examining the cells requiring STAT4 signaling and IFN-gamma production as well as the mechanism of reduced accumulation of immune cells in the draining lymph nodes.

Neisseria musculi, isolated from the oral cavity of wild caught mice, weakly (50%) colonizes C57BL/6J (B6) mice but readily colonizes CAST/EiJ (CAST) mice. In vitro stimulation of B6 or CAST splenocytes with WT Neisseria or E. coli LPS showed that CAST mice had a blunted inflammatory response producing significantly lower levels of IL-6 than B6 mice. The use of specific genetic knockouts highlighted a need for an intact innate immune system to prevent colonization. B6-RAG-1−/− were colonized at a similar rate to WT B6 while B6-MyD88−/− and TLR4−/− mice were readily colonized like CAST (100%). In vitro stimulation of B6 BMDM or splenocytes with WT Neisseria yielded only low levels of IL-6. A series of Neisseria mutants in the Type IV Pilus (TFP), a key component of bacterial-host interaction, failed to colonize susceptible CAST mice and showed increased levels of IL-6 compared to WT Neisseria stimulation, indicating that the TFP plays a role in down regulating host IL-6 production. Surprisingly, UV-inactivated Neisseria induced high levels of IL-6 regardless of TFP status supporting that this is an active bacterial process and the presence of TFP alone was unable to down regulate IL-6 production. Consistent with a critical role for IL-6 is preventing colonization, mice deficient for the IL-6 receptor were colonized at a rate of 100% indicating host IL-6 signaling plays a critical role in determining host colonization susceptibility.

CD25+CD4+ T regulatory (Treg) cells regulate peripheral self tolerance and possess the ability to suppress antitumor responses, which may in part explain the poor clinical response of cancer patients undergoing active immunization protocols. We have previously shown that in vitro incubation of human PBMC with LMB-2, a CD25-directed immunotoxin, significantly reduced CD25+FOXP3+CD4+ Treg cells without impairing the function of the remaining lymphocytes. In the current study, eight patients with metastatic melanoma were treated with LMB-2 followed by MART-1 and gp100-specific peptide vaccination. LMB-2 administration resulted in a preferential, transient reduction in mean circulating CD25+CD4+ T cell number, from 83 ± 16 cells/μl to a nadir of 17 ± 5 cells/μl, a 79.1% reduction. FOXP3 analysis revealed a less robust depletion with mean FOXP3+CD4+ Treg cell number decreasing from 74 ± 15 cells/μl to 36 ± 8 cells/μl, a 51.4% reduction. FOXP3+CD4+ Treg cells that survived LMB-2-mediated cytotoxicity expressed little or no CD25. Similar to the peripheral blood, immunohistochemical analysis showed a 68.9% mean reduction in FOXP3+CD4+ Treg cell frequency in evaluable lesions. Despite inducing a reduction in Treg cell numbers in vivo, LMB-2 therapy did not augment the immune response to cancer vaccination and no patient experienced an objective response or autoimmunity. These data demonstrate the capacity of a CD25-directed immunotoxin to selectively mediate a transient partial reduction in circulating and tumor-infiltrating Treg cells in vivo, and suggest that more comprehensive Treg cell elimination may be required to bolster antitumor responses in patients with metastatic melanoma.

Cell-based antitumor immunity is driven by CD8+ cytotoxic T cells bearing TCR that recognize specific tumor-associated peptides bound to class I MHC molecules. Of several cellular proteins involved in T cell:target-cell interaction, the TCR determines specificity of binding; however, the relative amount of its contribution to cellular avidity remains unknown. To study the relationship between TCR affinity and cellular avidity, with the intent of identifying optimal TCR for gene therapy, we derived 24 MART-1:27-35 (MART-1) melanoma Ag-reactive tumor-infiltrating lymphocyte (TIL) clones from the tumors of five patients. These MART-1-reactive clones displayed a wide variety of cellular avidities. α and β TCR genes were isolated from these clones, and TCR RNA was electroporated into the same non-MART-1-reactive allogeneic donor PBMC and TIL. TCR recipient cells gained the ability to recognize both MART-1 peptide and MART-1-expressing tumors in vitro, with avidities that closely corresponded to the original TCR clones (p = 0.018–0.0003). Clone DMF5, from a TIL infusion that mediated tumor regression clinically, showed the highest avidity against MART-1 expressing tumors in vitro, both endogenously in the TIL clone, and after RNA electroporation into donor T cells. Thus, we demonstrated that the TCR appeared to be the core determinant of MART-1 Ag-specific cellular avidity in these activated T cells and that nonreactive PBMC or TIL could be made tumor-reactive with a specific and predetermined avidity. We propose that inducing expression of this highly avid TCR in patient PBMC has the potential to induce tumor regression, as an “off-the-shelf” reagent for allogeneic melanoma patient gene therapy.

Thyroid epithelial cells frequently express one or more members of the rearranged during transfection/papillary thyroid carcinoma (RET/PTC) fusion oncogene family during early stages of cancer, and fusion gene transcripts have been found in inflammatory conditions of the thyroid such as the autoimmune disease, Hashimoto’s thyroiditis. Because these oncogenes encode chimeric proteins, novel RET/PTC epitopes may be targets of antitumor immune responses. We have been interested in the RET/PTC3 (RP3) fusion protein because this family member is more frequently expressed in radiation-induced and childhood papillary carcinomas than other members of the fusion oncogene family. We hypothesized that the activated kinase of c-RET, in the form of RP3, when expressed in patients with thyroid disease, presents an unusual altered self target for T cell recognition. Interestingly, we find that immunization with mouse RP3 protein can induce a strongly immunogenic response to RP3, although this response is not directed against the peptide comprising the unique fusion region. Rather, the responses are specific for the carboxyl-terminal portion of RP3 that is derived from the self protein c-RET. Furthermore, transplantation of RP3-expressing thyroid tumors into naive mice resulted in leukocytic infiltration, tumor rejection, and induction of RP3-specific T cells. Thus, the somatic fusion of two unrelated self proteins results in the development of a uniquely immunogenic response directed against self epitopes within RP3. These studies may better define the mechanisms controlling the initiation of thyroid-specific immune responses and provide insight into the design of novel molecules for invoking tumor-specific immunity.

Ovarian cancer causes more deaths than any other cancer of the female reproductive system. However, novel therapies, including immunotherapy are now being utilized to treat this disease. In addition, efforts have been made to identify and evaluate the subsets of cells within the tumor environment that correspond with patient overall survival. The presence of intraepithelial CD3+ T cells and CD8+ T cells have been correlated with ovarian cancer patient survival and our lab has recently identified CD137 as a biomarker for naturally-occurring tumor-reactive T cells in melanoma and ovarian cancers. However, a thorough elucidation of the immune landscape of endogenous bulk and tumor-reactive T-cell has not been conducted. In our study, we used an in-depth multi-parameter flow cytometry panel to determine the expression of multiple inhibitory molecules, Th1 cytokines, and the effector phenotype in the bulk and tumor-reactive subsets in human ovarian cancer. We observed that the CD4+ and CD8+ T cells found within the tumor environment express inhibitory receptors, very limited Th1 cytokine producing cells and a decreased expression of Granzyme B within the CD8+ subset. The tumor-reactive subset expressed inhibitory molecules, but was still able to elicit an effector phenotype and comprise Th1 cytokine producing cells. In conclusion, using multi-parameter flow cytometry enabled us to investigate the expression of inhibitory molecules and determine the effector phenotype in the bulk TIL and tumor-reactive CD137+ subsets.

Tumor polarization of macrophages represents an essential immune escape mechanism thus targeting tumor-associated macrophages (TAMs) could boost anti-tumor immune response. To model tumor-induced macrophage polarization, we developed an in vitro system of transwell co-culture with human macrophages, T cells and ovarian cancer cells (OVCAR3) where in vitro-matured macrophages and CD3/CD28 subactivated T cells were separated from tumor cells; in vitro-generated TAMs overexpressed the immunosuppressive molecule B7-H4 and inhibited T cell functions. We then hypothesized that targeting B7-H4 could block T cell inhibition mediated by B7-H4+ TAMs. We isolated anti-human B7-H4 scFvs from a novel yeast-display scFv library derived from B cells of ovarian cancer patients’ ascites. T cell proliferation and activation were restored in presence of anti-B7-H4 scFv #56 during transwell co-culture, suggesting that scFv #56 could block B7-H4 inhibitory signals mediated by TAMs. To further address whether scFv #56 activity was TCR-dependent, we set up experiments with antigen-specific T cells (MART1) and wild type or B7-H4-transduced artificial APCs (T2) loaded with MART1 peptide. T cell cytokine response was downregulated by B7-H4 expression on T2 and partially restored in presence of scFv#56. Our results indicate that competing with B7-H4 inhibitory signals on TAMs can promote T cell activity. We propose that in vivo use of anti-B7-H4 scFv could increase anti-tumor T-cell immunity.