Context-Specific Function of S6K2 in Th Cell Differentiation.

The mammalian target of rapamycin (mTOR) is essential for Th cell proliferation and effector differentiation, making the mTOR signaling network an attractive immunomodulatory target for autoimmune-related diseases. Although direct targeting of mTOR complex-1 (mTORC1) with rapamycin can provide clinical benefit, targeting downstream enzymes has the potential to offer more selective immunosuppression. In this study, we evaluated p70 ribosomal protein S6 Kinase 2 (S6K2), a downstream effector of mTORC1, for its role in T cell function and autoimmunity. S6K2 is a direct substrate of mTORC1, with a potential role in Th17 differentiation suggested by biochemical studies. Using a genetic approach with S6K2 knockout mice, we found that S6K2 loss reduces Th17 skewing and increases regulatory T cell differentiation in vitro when cultured in RPMI 1640 media. However, S6K2 was dispensable for Th17 differentiation in IMDM. In an in vivo experimental autoimmune encephalomyelitis model in which rapamycin suppresses disease, S6K2 knockout mice did not exhibit differences in clinical score or Th17 differentiation. These results suggest that S6K2 is dispensable for Th17-driven autoimmunity and highlight how distinct experimental conditions can produce significantly different results in T cell differentiation.

Deficiency of IL-17A, but not the prototypical Th17 transcription factor RORγt, decreases murine spontaneous intestinal tumorigenesis.

While inflammation has been associated with the development and progression of colorectal cancer, the exact role of the inflammatory Th17 pathway remains unclear. In this study, we aimed to determine the relative importance of IL-17A and the master regulator of the Th17 pathway, the transcription factor RORγt, in the sporadic intestinal neoplasia of APC(MIN/+) mice and in human colorectal cancer. We show that levels of IL-17A are increased in human colon cancer as compared to adjacent uninvolved colon. Similarly, naïve helper T cells from colorectal cancer patients are more inducible into the Th17 pathway. Furthermore, IL-17A, IL-21, IL-22, and IL-23 are all demonstrated to be directly mitogenic to human colorectal cancer cell lines. Nevertheless, deficiency of IL-17A but not RORγt is associated with decreased spontaneous intestinal tumorigenesis in the APC(MIN/+) mouse model, despite the fact that helper T cells from RORγt-deficient APC(MIN/+) mice do not secrete IL-17A when subjected to Th17-polarizing conditions and that Il17a expression is decreased in the intestine of RORγt-deficient APC(MIN/+) mice. Differential expression of Th17-associated cytokines between IL-17A-deficient and RORγt-deficient APC(MIN/+) mice may explain the difference in adenoma development.

Elevated IL-17 produced by TH17 cells promotes myeloma cell growth and inhibits immune function in multiple myeloma.

Elevated cytokines in bone marrow (BM) micro-environment (interleukin-6 [IL-6], transforming growth factor-beta [TGF-beta], and IL-1beta) may play an important role in observed immune dysfunction in multiple myeloma (MM). As IL-6 and TGF-beta are important for the generation of T-helper 17 (T(H)17) cells, we evaluated and observed a significantly elevated baseline and induced frequency of T(h)17 cells in peripheral blood mononuclear cells (PBMCs) and BM mononuclear cells (BMMCs) from MM patients compared with healthy donors. We observed significant increase in levels of serum IL-17, IL-21, IL-22, and IL-23 in blood and BM in MM compared with healthy donors. We also observed that myeloma PBMCs after T(H)17 polarization significantly induced IL-1alpha, IL-13, IL-17, and IL-23 production compared with healthy donor PBMCs. We next observed that IL-17 promotes myeloma cell growth and colony formation via IL-17 receptor, adhesion to bone marrow stromal cells (BMSCs) as well as increased growth in vivo in murine xenograft model of human MM. Additionally, we have observed that combination of IL-17 and IL-22 significantly inhibited the production of T(H)1-mediated cytokines, including interferon-gamma (IFN-gamma), by healthy donor PBMCs. In conclusion, IL-17-producing T(h)17 cells play an important role in MM pathobiology and may be an important therapeutic target for anti-MM activity and to improve immune function.

Autoimmunity linked protein phosphatase PTPN22 as a target for cancer immunotherapy.

BACKGROUND: Cancer immunotherapy has evolved from interferon-alpha (IFNα) and interleukin-2 in the 1980s to CTLA-4 and PD-1/PD-L1 checkpoint inhibitors (CPIs), the latter highlighting the importance of enhancing T-cell functions. While the search for novel immunomodulatory pathways continues, combination therapies augmenting multiple pathways can also increase efficacy. The association of autoimmune-related adverse events with clinical efficacy following CPI treatment has been inferred and suggests that breaking tolerance thresholds associated with autoimmunity may affect host immune responses for effective cancer immunotherapy. RESULTS: Here, we show that loss of autoimmune associated PTPN22, a key desensitization node for multiple signaling pathways, including IFNα receptor (IFNAR) and T-cell receptor, can augment tumor responses. Implantation of syngeneic tumors in Ptpn22-/- mice led to expansion and activation of peripheral and intratumoral T cells and, in turn, spontaneous tumor regression as well as enhanced responses in combination with anti-PD-L1 treatment. Using genetically modified mice expressing a catalytically inactive PTPN22 or the autoimmunity-associated human single-nucleotide polymorphism variant, augmentation of antitumor immunity was dependent on PTPN22 phosphatase activity and partially on its adaptor functions. Further, antitumor responses were dependent on both CD4+ and CD8+T cells and, in part, IFNAR function. Finally, we demonstrate that the autoimmune susceptibility Ptpn22(C1858T) variant is associated with lower risk of developing non-melanoma skin cancers, improved overall survival and increased risk for development of hyperthyroidism or hypothyroidism following atezolizumab (anti-PD-L1) treatment. CONCLUSIONS: Together, these data suggest that inhibition of PTPN22 phosphatase activity may provide an effective therapeutic option for cancer immunotherapy and that exploring genetic variants that shift immune tolerance thresholds may serve as a paradigm for finding new cancer immunotherapy targets.

Stromal CCR6 drives tumor growth in a murine transplantable colon cancer through recruitment of tumor-promoting macrophages.

Interactions between the inflammatory chemokine CCL20 and its receptor CCR6 have been implicated in promoting colon cancer; however, the mechanisms behind this effect are poorly understood. We have previously demonstrated that deficiency of CCR6 is associated with decreased tumor macrophage accumulation in a model of sporadic intestinal tumorigenesis. In this study, we aimed to determine the role of stromal CCR6 expression in a murine syngeneic transplantable colon cancer model. We show that deficiency of host CCR6 is associated with decreased growth of syngeneic CCR6-expressing colon cancers. Colon cancers adoptively transplanted into CCR6-deficient mice have decreased tumor-associated macrophages without alterations in the number of monocytes in blood or bone marrow. CCL20, the unique ligand for CCR6, promotes migration of monocytes in vitro and promotes accumulation of macrophages in vivo. Depletion of tumor-associated macrophages decreases the growth of tumors in the transplantable tumor model. Macrophages infiltrating the colon cancers in this model secrete the inflammatory mediators CCL2, IL-1α, IL-6 and TNFα. Ccl2, Il1α and Il6 are consequently downregulated in tumors from CCR6-deficient mice. CCL2, IL-1α and IL-6 also promote proliferation of colon cancer cells, linking the decreased macrophage migration into tumors mediated by CCL20-CCR6 interactions to the delay in tumor growth in CCR6-deficient hosts. The relevance of these findings in human colon cancer is demonstrated through correlation of CCR6 expression with that of the macrophage marker CD163 as well as that of CCL2, IL1α and TNFα. Our findings support the exploration of targeting the CCL20-CCR6 pathway for the treatment of colon cancer.

CCR6, the sole receptor for the chemokine CCL20, promotes spontaneous intestinal tumorigenesis.

Interactions between the inflammatory chemokine CCL20 and its receptor CCR6 have been associated with colorectal cancer growth and metastasis, however, a causal role for CCL20 signaling through CCR6 in promoting intestinal carcinogenesis has not been demonstrated in vivo. In this study, we aimed to determine the role of CCL20-CCR6 interactions in spontaneous intestinal tumorigenesis. CCR6-deficient mice were crossed with mice heterozygous for a mutation in the adenomatous polyposis coli (APC) gene (APCMIN/+ mice) to generate APCMIN/+ mice with CCR6 knocked out (CCR6KO-APCMIN/+ mice). CCR6KO-APCMIN/+ mice had diminished spontaneous intestinal tumorigenesis. CCR6KO-APCMIN/+ also had normal sized spleens as compared to the enlarged spleens found in APCMIN/+ mice. Decreased macrophage infiltration into intestinal adenomas and non-tumor epithelium was observed in CCR6KO-APCMIN/+ as compared to APCMIN/+ mice. CCL20 signaling through CCR6 caused increased production of CCL20 by colorectal cancer cell lines. Furthermore, CCL20 had a direct mitogenic effect on colorectal cancer cells. Thus, interactions between CCL20 and CCR6 promote intestinal carcinogenesis. Our results suggest that the intestinal tumorigenesis driven by CCL20-CCR6 interactions may be driven by macrophage recruitment into the intestine as well as proliferation of neoplastic epithelial cells. This interaction could be targeted for the treatment or prevention of malignancy.

Modulation of T Cell Metabolism and Function through Calcium Signaling.

As a vital second messenger in the activation of lymphocytes, the divalent cation Ca(2+) plays numerous roles in adaptive immune responses. Importantly, Ca(2+) signaling is essential for T cell activation, tolerance of self-antigens, and homeostasis. Supporting the essential role of Ca(2+) signaling in T cell biology, the Ca(2+) regulated protein phosphatase calcineurin is a key target of pharmacologic inhibition for preventing allograft rejection and for autoimmune therapy. Recent studies have highlighted the unique role of Stim1 and Orai1/2 proteins in the regulation of store-operated/calcium release activated calcium (CRAC) channels in the context of T cells. While Ca(2+) is known to modulate T cell activation via effects on calcineurin and its target, nuclear factor of activated T cells (NFAT), this second messenger also regulates other pathways, including protein kinase C, calmodulin kinases, and cytoskeletal proteins. Ca(2+) also modulates the unique metabolic changes that occur during in distinct T cell stages and subsets. Herein, we discuss the means by which Ca(2+) mobilization modulates cellular metabolism following T cell receptor ligation. Further, we highlight the crosstalk between mitochondrial metabolism, reactive oxygen species (ROS) generation, and CRAC channel activity. As a target of mitochondrial ROS and Ca(2+) regulation, we describe the involvement of the serine/threonine kinase DRAK2 in the context of these processes. Given the important roles for Ca(2+) dependent signaling and cellular metabolism in adaptive immune responses, the crosstalk between these pathways is likely to be important for the regulation of T cell activation, tolerance, and homeostasis.