STING-induced regulatory B cells compromise NK function in cancer immunity.

An immunosuppressive tumour microenvironment is a major obstacle in the control of pancreatic and other solid cancers1-3. Agonists of the stimulator of interferon genes (STING) protein trigger inflammatory innate immune responses to potentially overcome tumour immunosuppression4. Although these agonists hold promise as potential cancer therapies5, tumour resistance to STING monotherapy has emerged in clinical trials and the mechanism(s) is unclear5-7. Here we show that the administration of five distinct STING agonists, including cGAMP, results in an expansion of human and mouse interleukin (IL)-35+ regulatory B cells in pancreatic cancer. Mechanistically, cGAMP drives expression of IL-35 by B cells in an IRF3-dependent but type I interferon-independent manner. In several preclinical cancer models, the loss of STING signalling in B cells increases tumour control. Furthermore, anti-IL-35 blockade or genetic ablation of IL-35 in B cells also reduces tumour growth. Unexpectedly, the STING-IL-35 axis in B cells reduces proliferation of natural killer (NK) cells and attenuates the NK-driven anti-tumour response. These findings reveal an intrinsic barrier to systemic STING agonist monotherapy and provide a combinatorial strategy to overcome immunosuppression in tumours.

Regulatory B cells in cancer.

Tumorigenesis proceeds through discrete steps where acquisition of genetic lesions and changes in the surrounding microenvironment combine to drive unrestricted neoplastic proliferation and metastasis. The ability of tumor-infiltrating immune cells to promote tumor growth via the provision of signals that enable tumor cell survival and proliferation as well as contribute to immune suppression is an active area of research. Recent efforts have provided us with mechanistic insights into how B cells can positively and negatively regulate immune responses. Negative regulation of immune responses in cancer can be mediated by regulatory B cells and is often a result of increased production of cytokines that can directly and indirectly affect anti-tumor immune function and cancer cell growth. Signals that lead to the expansion of regulatory B cells and the spectrum of their functional roles are not well understood and are the subject of active research by many groups. Here, we elaborate broadly on the history of regulatory B cells in cancer and summarize recent studies that have established genetic models for the study of regulatory B cell function and their potential for therapeutic intervention in the setting of solid cancers.

Pancreatic cancer-associated inflammation drives dynamic regulation of p35 and Ebi3.

B cells are important modulators of immune responses both in autoimmunity and cancer. We have previously shown that B regulatory (Breg) cells promote pancreatic cancer via production of IL35, a heterodimeric cytokine comprised of the subunits p35 (Il12a) and Ebi3. However, it is not known how production of IL35 is regulated in vivo in the context of cancer-associated inflammation. To begin addressing this question, we have generated a knock-in mouse model, Il12aGFP, where an IRES-emGFP gene was inserted within the 3' UTR of the Il12a locus. EmGFP signal in B cells from the Il12aGFP mice correlated with expression of p35 mRNA and protein. Using this model, we observed that in addition to Bregs, expression of GFP (p35) is upregulated in several other B cell subtypes in response to cancer. We assessed the expression of the other IL35 subunit, Ebi3, using a published tdTomato reporter model. We determined that Ebi3 expression was more tightly regulated in vivo and in vitro, suggesting that stimuli affecting Ebi3 upregulation are more likely to result in production of full IL35 heterodimer. We were also able to detect GFP and Tomato signal in myeloid & T cell lineages suggesting that these reporter models could also be used for tracking IL12-, IL27- and IL35-producing cells. Furthermore, using primary B cells isolated from reporter mice, we identified BCR, CD40 and TLR pathways as potential drivers of IL35 expression. These findings highlight the importance of pancreatic cancer-associated inflammatory processes as drivers of cytokine expression and provide a tool to dissect both disease-associated regulation of IL12- and IL35-competent lineage cells as well as establish assays for pharmacological targeting of individual subunits of heterodimeric IL12 family cytokines.

MAR binding protein SMAR1 favors IL-10 mediated regulatory T cell function in acute colitis.

Treg cells are not only crucial for controlling immune responses to autoantigens but also prevent those directed towards commensal pathogens. Control of effector immune responses by Treg cells depend on their capacity to accumulate at inflammatory site and accordingly accommodate to inflammatory environment. Till date, the factors associated with maintaining these aspects of Treg phenotype is not understood properly. Here we have shown that a known nuclear matrix binding protein SMAR1 is selectively expressed more in colonic Treg cells and is required for their ability to accumulate at inflammatory site and to sustain high levels of Foxp3 and IL-10 expression during acute colitis. Elimination of anti-inflammatory subsets revealed a protective role for IL-10 producing Treg cells in SMAR1(-/-) mice. Moreover, a combined action of Foxp3 and SMAR1 restricts effector cytokine production and enhance the production of IL-10 by colonic Treg cells that controls acute colitis. This data highlights a critical role of SMAR1 in maintaining Treg physiology during inflammatory disorders.

Regulation of T cell lineage commitment by SMAR1 during inflammatory & autoimmune diseases.

BACKGROUND & OBJECTIVES: CD4 + T cells are involved in abnormal inflammatory responses causing adverse effects to the body. Th17 cells play a major role in immune disorders and the exact mechanism by which CD4 + T cells regulate its effector Th1 and Th17 phenotype at chromatin level is not clearly understood. This study was aimed to understand the role of matrix associated region (MAR) binding protein SMAR1 (scaffold/matrix attachment region binding protein 1) in T cell differentiation during inflammatory and autoimmune condition using SMAR1 transgenic mice as model. METHODS: Wild type (C57BL/6J) and SMAR1 transgenic mice were used for isolation of T cells and further identification of different T cell lineages, along with histological analysis. Further, we studied autoimmune and inflammatory diseases using chemically induced and T cell transfer model of colitis and rheumatoid arthritis to better understand the role of SMAR1 in immune responses. RESULTS: SMAR1 transgenic mice were resistant to dextran sodium sulphate (DSS) induced colitis with decreased expression of Th1 and Th17 specific cytokines. Overexpression of SMAR1 repressed Th17 response by negatively regulating RORγt and IL-17 expression. Downregulation of SMAR1 upregulated signal transducer and activator of transcription 3 (pSTAT3) and IL-17 expression that caused generation of more proinflammatory Th1 and Th17 cells leading to inflammation and disease. INTERPRETATION & CONCLUSIONS: Our results show an important role of SMAR1 in regulating CD4 + T cell differentiation during inflammatory disorders via regulation of both Th1 and Th17 signaling pathways. This study reveals a critical role of SMAR1 in maintaining the proinflammatory immune responses by repressing Th1 and Th17 cell function and it gives the novel insight into immune regulatory mechanisms.

Tumor promoting roles of IL-10, TGF-ß, IL-4, and IL-35: Its implications in cancer immunotherapy.

Cytokines play a critical role in regulating host immune response toward cancer and determining the overall fate of tumorigenesis. The tumor microenvironment is dominated mainly by immune-suppressive cytokines that control effector antitumor immunity and promote survival and the proliferation of cancer cells, which ultimately leads to enhanced tumor growth. In addition to tumor cells, the heterogeneous immune cells present within the tumor milieu are the significant source of immune-suppressive cytokines. These cytokines are classified into a broad range; however, in most tumor types, the interleukin-10, transforming growth factor-ß, interleukin-4, and interleukin-35 are consistently reported as immune-suppressive cytokines that help tumor growth and metastasis. The most emerging concern in cancer treatment is hijacking and restraining the activity of antitumor immune cells in the tumor niche due to a highly immune-suppressive environment. This review summarizes the role and precise functions of interleukin-10, transforming growth factor-ß, interleukin-4, and interleukin-35 in modulating tumor immune contexture and its implication in developing effective immune-therapeutic approaches. CONCISE CONCLUSION: Recent effort geared toward developing novel immune-therapeutic approaches faces significant challenges due to sustained mutations in tumor cells and a highly immune-suppressive microenvironment present within the tumor milieu. The cytokines play a crucial role in developing an immune-suppressive environment that ultimately dictates the fate of tumorigenesis. This review critically covers the novel aspects of predominant immune-suppressive cytokines such as interleukin-10, transforming growth factor-ß, interleukin-4, and interleukin-35 in dictating the fate of tumorigenesis and how targeting these cytokines can help the development of better immune-therapeutic drug regimens for the treatment of cancer.

Balance between immunoregulatory B cells and plasma cells drives pancreatic tumor immunity.

Plasma cell responses are associated with anti-tumor immunity and favorable response to immunotherapy. B cells can amplify anti-tumor immune responses through antibody production; yet B cells in patients and tumor-bearing mice often fail to support this effector function. We identify dysregulated transcriptional program in B cells that disrupts differentiation of naive B cells into anti-tumor plasma cells. The signaling network contributing to this dysfunction is driven by interleukin (IL) 35 stimulation of a STAT3-PAX5 complex that upregulates the transcriptional regulator BCL6 in naive B cells. Transient inhibition of BCL6 in tumor-educated naive B cells is sufficient to reverse the dysfunction in B cell differentiation, stimulating the intra-tumoral accumulation of plasma cells and effector T cells and rendering pancreatic tumors sensitive to anti-programmed cell death protein 1 (PD-1) blockade. Our findings argue that B cell effector dysfunction in cancer can be due to an active systemic suppression program that can be targeted to synergize with T cell-directed immunotherapy.

IL-12 Family Cytokines in Cancer and Immunotherapy.

The IL-12 family cytokines are a group of unique heterodimeric cytokines that include IL-12, IL-23, IL-27, IL-35 and, most recently, IL-39. Recent studies have solidified the importance of IL-12 cytokines in shaping innate and adaptive immune responses in cancer and identified multipronged roles for distinct IL-12 family members, ranging from effector to regulatory immune functions. These cytokines could serve as promising candidates for the development of immunomodulatory therapeutic approaches. Overall, IL-12 can be considered an effector cytokine and has been found to engage anti-tumor immunity by activating the effector Th1 response, which is required for the activation of cytotoxic T and NK cells and tumor clearance. IL-23 and IL-27 play dual roles in tumor immunity, as they can both activate effector immune responses and promote tumor growth by favoring immune suppression. IL-35 is a potent regulatory cytokine and plays a largely pro-tumorigenic role by inhibiting effector T cells. In this review, we summarize the recent findings on IL-12 family cytokines in the control of tumor growth with an emphasis primarily on immune regulation. We underscore the clinical implications for the use of these cytokines either in the setting of monotherapy or in combination with other conventional therapies for the more effective treatment of malignancies.

IL-35 Detection in B Cells at the mRNA and Protein Level.

Emerging research suggests that IL-35-producing regulatory B cells accumulate in patients and mouse models of pancreatic cancer, one of the most lethal cancers, characterized by late diagnosis, high mortality, and morbidity. Identification of IL-35-producing B cells can be challenging due to the heterodimeric nature of IL-35 and diversity of cell surface markers that define regulatory B-cell subsets across spectrum of diseases. In this chapter, we describe the methods for the isolation of splenic and tumor-infiltrating murine regulatory B cells and subsequent detection of IL-35 by RT-qPCR and intracellular staining, as well as detection of circulating IL-35 by ELISA. We also describe methods for the detection of IL-35-producing human B cells by flow cytometry, RT-qPCR, and immunofluorescence in the context of pancreatic cancer. This chapter should facilitate the study of regulatory IL-35+ B cells in cancer, autoimmunity, and inflammation.

B Cell Receptor Signaling and Protein Kinase D2 Support Regulatory B Cell Function in Pancreatic Cancer.

B cells can act as potent suppressors of anti-tumor T cell immunity, presenting a mechanism of resistance to immunotherapy. In pancreatic ductal adenocarcinoma, B cells can display a T cell-suppressive or regulatory phenotype centered on the expression of the cytokine Interleukin 35 (IL-35). While B cell-mediated immunosuppression presents a barrier to anti-tumorigenic T cell function, it is not clear how regulatory B cell function could be targeted, and the signals that promote this suppressive phenotype in B cells are not well understood. Here we use a novel IL-35 reporter model to understand which signaling pathways are important for immunosuppressive properties in B cells. In vitro analysis of IL-35 reporter B cells revealed a synergy between the BCR and TLR4 signaling pathways is sufficient to induce IL-35 expression. However, in vivo, B cell receptor activation, as opposed to MyD88 signaling in B cells, is central to B cell-mediated suppression and promotion of pancreatic cancer growth. Further analysis identified protein kinase D2 (PKD2) as being a key downstream regulator of IL-35 expression in B cells. Regulatory B cells with an inactivating mutation in PKD2 failed to produce IL-35 or fully suppress effector T cell function in vitro. Furthermore, inhibition of PKD in B cells decreased tumor growth and promoted effector T cell function upon adoptive transfer into B cell-deficient mice. Collectively, these data provide insight into how regulatory B cell function is promoted in pancreatic cancer and identify potential therapeutic targets to restrain this function.

Co-expression of master transcription factors determines CD4+ T cell plasticity and functions in auto-inflammatory diseases.

Master CD4+ T cell lineage determined transcription factors are found to be dysregulated in pathogenesis of autoimmune and inflammatory diseases. CD4+ T cells categorized into different lineages based on their functions, cell surface markers and master transcription factors those required for expression of lineage specific cytokines. T-bet, GATA3, RORγt and Foxp3 are major transcription regulators of Th1, Th2, Th17 and Treg cells respectively. Significant progress has been made in understanding expression of lineage specific master regulators that drives CD4+ T cell differentiation. It is known that each CD4+ T cell lineage express precise determined transcription factor and due to cross regulation between these factors the CD4+ T cells able to maintain thier specific phenotype. However, recent studies shows that the lineage specifying transcription factors frequently co-expressed. There is an emerging area of research revealing that the co-expression of lineage-specifying transcription factors alters the potential function and flexibility of subsets of CD4+ T cell, this in turn favors the autoimmune pathology. Here, we discuss similarities and differences between mutually co-expressed transcription factors in CD4+ T cell subsets and then recapitulates on cell type specific and dynamic balance between the lineage restricted transcription factors in determining plasticity of CD4+ T cell subsets. Furthermore, we discuss abnormal regulation of such transcription factors that establishes a pathogenic CD4+ T cell phenotype in autoimmune diseases and how this understanding will provide further insight into potential therapeutic development.

B cell-Derived IL35 Drives STAT3-Dependent CD8+ T-cell Exclusion in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignancy characterized by a paucity of tumor-proximal CD8+ T cells and resistance to immunotherapeutic interventions. Cancer-associated mechanisms that elicit CD8+ T-cell exclusion and resistance to immunotherapy are not well-known. Here, using a Kras- and p53-driven model of PDA, we describe a mechanism of action for the protumorigenic cytokine IL35 through STAT3 activation in CD8+ T cells. Distinct from its action on CD4+ T cells, IL35 signaling in gp130+CD8+ T cells activated the transcription factor STAT3, which antagonized intratumoral infiltration and effector function of CD8+ T cells via suppression of CXCR3, CCR5, and IFNγ expression. Inhibition of STAT3 signaling in tumor-educated CD8+ T cells improved PDA growth control upon adoptive transfer to tumor-bearing mice. We showed that activation of STAT3 in CD8+ T cells was driven by B cell- but not regulatory T cell-specific production of IL35. We also demonstrated that B cell-specific deletion of IL35 facilitated CD8+ T-cell activation independently of effector or regulatory CD4+ T cells and was sufficient to phenocopy therapeutic anti-IL35 blockade in overcoming resistance to anti-PD-1 immunotherapy. Finally, we identified a circulating IL35+ B-cell subset in patients with PDA and demonstrated that the presence of IL35+ cells predicted increased occurrence of phosphorylated (p)Stat3+CXCR3-CD8+ T cells in tumors and inversely correlated with a cytotoxic T-cell signature in patients. Together, these data identified B cell-mediated IL35/gp130/STAT3 signaling as an important direct link to CD8+ T-cell exclusion and immunotherapy resistance in PDA.

Antitumor Responses in the Absence of Toxicity in Solid Tumors by Targeting B7-H3 via Chimeric Antigen Receptor T Cells.

The high expression across multiple tumor types and restricted expression in normal tissues make B7-H3 an attractive target for immunotherapy. We generated chimeric antigen receptor (CAR) T cells targeting B7-H3 (B7-H3.CAR-Ts) and found that B7-H3.CAR-Ts controlled the growth of pancreatic ductal adenocarcinoma, ovarian cancer and neuroblastoma in vitro and in orthotopic and metastatic xenograft mouse models, which included patient-derived xenograft. We also found that 4-1BB co-stimulation promotes lower PD-1 expression in B7-H3.CAR-Ts, and superior antitumor activity when targeting tumor cells that constitutively expressed PD-L1. We took advantage of the cross-reactivity of the B7-H3.CAR with murine B7-H3, and found that B7-H3.CAR-Ts significantly controlled tumor growth in a syngeneic tumor model without evident toxicity. These findings support the clinical development of B7-H3.CAR-Ts.

IL35 Hinders Endogenous Antitumor T-cell Immunity and Responsiveness to Immunotherapy in Pancreatic Cancer.

Although successes in cancer immunotherapy have generated considerable excitement, this form of treatment has been largely ineffective in patients with pancreatic ductal adenocarcinoma (PDA). Mechanisms that contribute to the poor antitumor immune response in PDA are not well understood. Here, we demonstrated that cytokine IL35 is a major immunosuppressive driver in PDA and potentiates tumor growth via the suppression of endogenous antitumor T-cell responses. The growth of pancreatic tumors in mice deficient for IL35 was significantly reduced. An analysis of tumor-infiltrating immune cells revealed a role for IL35 in the expansion of regulatory T cells and the suppression of CD4+ effector T cells. We also detected a robust increase in both the infiltration and activation of cytotoxic CD8+ T cells, suggesting that targeting IL35 may be an effective strategy to convert PDA from an immunologically "cold" to "hot" tumor. Although PDA is typically resistant to anti-PD-1 immunotherapy, we demonstrated robust synergistic reduction in tumor growth when IL35 deficiency was combined with anti-PD-1 treatment. These findings provide new insight into the function of IL35 in the pathogenesis of pancreatic cancer and underscore the potential significance of IL35 as a therapeutic target for use in combination immunotherapy approaches in this deadly malignancy. Cancer Immunol Res; 6(9); 1014-24. ©2018 AACR.

Chromatin Remodeling Protein SMAR1 Is a Critical Regulator of T Helper Cell Differentiation and Inflammatory Diseases.

T cell differentiation from naïve T cells to specialized effector subsets of mature cells is determined by the iterative action of transcription factors. At each stage of specific T cell lineage differentiation, transcription factor interacts not only with nuclear proteins such as histone and histone modifiers but also with other factors that are bound to the chromatin and play a critical role in gene expression. In this review, we focus on one of such nuclear protein known as tumor suppressor and scaffold matrix attachment region-binding protein 1 (SMAR1) in CD4+ T cell differentiation. SMAR1 facilitates Th1 differentiation by negatively regulating T-bet expression via recruiting HDAC1-SMRT complex to its gene promoter. In contrast, regulatory T (Treg) cell functions are dependent on inhibition of Th17-specific genes mainly IL-17 and STAT3 by SMAR1. Here, we discussed a critical role of chromatin remodeling protein SMAR1 in maintaining a fine-tuned balance between effector CD4+ T cells and Treg cells by influencing the transcription factors during allergic and autoimmune inflammatory diseases.

Carbon nanospheres mediated delivery of nuclear matrix protein SMAR1 to direct experimental autoimmune encephalomyelitis in mice.

Owing to the suppression of immune responses and associated side effects, steroid based treatments for inflammatory encephalitis disease can be detrimental. Here, we demonstrate a novel carbon nanosphere (CNP) based treatment regime for encephalomyelitis in mice by exploiting the functional property of the nuclear matrix binding protein SMAR1. A truncated part of SMAR1 ie, the DNA binding domain was conjugated with hydrothermally synthesized CNPs. When administered intravenously, the conjugate suppressed experimental animal encephalomyelitis in T cell specific conditional SMAR1 knockout mice (SMAR(-/-)). Further, CNP-SMAR1 conjugate delayed the onset of the disease and reduced the demyelination significantly. There was a significant decrease in the production of IL-17 after re-stimulation with MOG. Altogether, our findings suggest a potential carbon nanomaterial based therapeutic intervention to combat Th17 mediated autoimmune diseases including experimental autoimmune encephalomyelitis.