Gluten dependent activation of CD4+ T cells by MHC class II-expressing epithelium.

BACKGROUND AND AIMS: Intestinal epithelial cell (IEC) damage is a hallmark of celiac disease (CeD); however, its role in gluten-dependent T-cell activation is unknown. We investigated IEC-gluten-T cell interactions in organoid monolayers expressing human MHC class II (HLA-DQ2.5), which facilitates gluten antigen recognition by CD4+ T cells in CeD. METHODS: Epithelial MHC class II (MHCII) was determined in active and treated CeD, and in non-immunized and gluten-immunized DR3-DQ2.5 transgenic mice, lacking mouse MHCII molecules. Organoid monolayers from DR3-DQ2.5 mice were treated with or without IFN-γ, and MHCII expression was evaluated by flow cytometry. Organoid monolayers and CD4+ T cell co-cultures were incubated with gluten, pre-digested, or not by elastase-producing Pseudomonas aeruginosa or its lasB mutant. T cell function was assessed based on proliferation, expression of activation markers, and cytokine release in the co-culture supernatants. RESULTS: Active CeD patients and gluten-immunized DR3-DQ2.5 mice demonstrated epithelial MHCII expression. Organoid monolayers derived from gluten-immunized DR3-DQ2.5 mice expressed MHCII, which was upregulated by IFN-γ. In organoid monolayer-T cell co-cultures, gluten increased the proliferation of CD4+ T cells, expression of T cell activation markers, and the release of IL-2, IFN-γ, and IL-15 in co-culture supernatants. Gluten metabolized by P. aeruginosa, but not the lasB mutant, enhanced CD4+ T cell proliferation and activation. CONCLUSIONS: Gluten antigens are efficiently presented by MHCII-expressing IECs, resulting in the activation of gluten-specific CD4+ T cells, which is enhanced by gluten pre-digestion with microbial elastase. Therapeutics directed at IECs may offer a novel approach for modulating both adaptive and innate immunity in CeD patients.

Constitutive expression of interleukin-27 diminishes proinflammatory cytokine production without impairing effector function of engineered T cells.

Immunomodulatory cytokines can alter the tumor microenvironment and promote tumor eradication. Interleukin (IL)-27 is a pleiotropic cytokine that has potential to augment anti-tumor immunity while also facilitating anti-myeloma activity. We engineered human T cells to express a recombinant single-chain (sc)IL-27 and a synthetic antigen receptor targeting the myeloma antigen, B-cell maturation antigen, and evaluated the anti-tumor function of T cells bearing scIL-27 in vitro and in vivo. We discovered that T cells bearing scIL-27 sustained anti-tumor immunity and cytotoxicity yet manifested a profound reduction in pro-inflammatory cytokines granulocyte-macrophage colony-stimulating factor and tumor necrosis factor alpha. IL-27-expressing T cells therefore present a potential avenue to avert treatment-related toxicities commonly associated with engineered T-cell therapy due to the reduced pro-inflammatory cytokine profile.

A positive feedback loop involving nuclear factor IB and calpain 1 suppresses glioblastoma cell migration.

Glioblastoma (GBM) is a brain tumor that remains largely incurable because of its highly-infiltrative properties. Nuclear factor I (NFI)-type transcription factors regulate genes associated with GBM cell migration and infiltration. We have previously shown that NFI activity depends on the NFI phosphorylation state and that calcineurin phosphatase dephosphorylates and activates NFI. Calcineurin is cleaved and activated by calpain proteases whose activity is, in turn, regulated by an endogenous inhibitor, calpastatin (CAST). The CAST gene is a target of NFI in GBM cells, with differentially phosphorylated NFIs regulating the levels of CAST transcript variants. Here, we uncovered an NFIB-calpain 1-positive feedback loop mediated through CAST and calcineurin. In NFI-hyperphosphorylated GBM cells, NFIB expression decreased the CAST-to-calpain 1 ratio in the cytoplasm. This reduced ratio increased autolysis and activity of cytoplasmic calpain 1. Conversely, in NFI-hypophosphorylated cells, NFIB expression induced differential subcellular compartmentalization of CAST and calpain 1, with CAST localizing primarily to the cytoplasm and calpain 1 to the nucleus. Overall, this altered compartmentalization increased nuclear calpain 1 activity. We also show that nuclear calpain 1, by cleaving and activating calcineurin, induces NFIB dephosphorylation. Of note, knockdown of calpain 1, NFIB, or both increased GBM cell migration and up-regulated the pro-migratory factors fatty acid-binding protein 7 (FABP7) and Ras homolog family member A (RHOA). In summary, our findings reveal bidirectional cross-talk between NFIB and calpain 1 in GBM cells. A physiological consequence of this positive feedback loop appears to be decreased GBM cell migration.

Effects of nuclear factor I phosphorylation on calpastatin (CAST) gene variant expression and subcellular distribution in malignant glioma cells.

Malignant glioma (MG) is the most lethal primary brain tumor. In addition to having inherent resistance to radiation treatment and chemotherapy, MG cells are highly infiltrative, rendering focal therapies ineffective. Genes involved in MG cell migration and glial cell differentiation are up-regulated by hypophosphorylated nuclear factor I (NFI), which is dephosphorylated by the phosphatase calcineurin in MG cells. Calcineurin is cleaved and thereby activated by calpain proteases, which are, in turn, inhibited by calpastatin (CAST). Here, we show that the CAST gene is a target of NFI and has NFI-binding sites in its intron 3 region. We also found that NFI-mediated regulation of CAST depends on NFI's phosphorylation state. We noted that occupation of CAST intron 3 by hypophosphorylated NFI results in increased activation of an alternative promoter. This activation resulted in higher levels of CAST transcript variants, leading to increased levels of CAST protein that lacks the N-terminal XL domain. CAST was primarily present in the cytoplasm of NFI-hypophosphorylated MG cells, with a predominantly perinuclear immunostaining pattern. NFI knockdown in NFI-hypophosphorylated MG cells increased CAST levels at the plasma membrane. These results suggest that NFI plays an integral role in the regulation of CAST variants and CAST subcellular distribution. Along with the previous findings indicating that NFI activity is regulated by calcineurin, these results provide a foundation for further investigations into the possibility of regulatory cross-talk between NFI and the CAST/calpain/calcineurin signaling pathway in MG cells.

Endogenous CD28 drives CAR T cell responses in multiple myeloma.

Recent FDA approvals of chimeric antigen receptor (CAR) T cell therapy for multiple myeloma (MM) have reshaped the therapeutic landscape for this incurable cancer. In pivotal clinical trials B cell maturation antigen (BCMA) targeted, 4-1BB co-stimulated (BBζ) CAR T cells dramatically outperformed standard-of-care chemotherapy, yet most patients experienced MM relapse within two years of therapy, underscoring the need to improve CAR T cell efficacy in MM. We set out to determine if inhibition of MM bone marrow microenvironment (BME) survival signaling could increase sensitivity to CAR T cells. In contrast to expectations, blocking the CD28 MM survival signal with abatacept (CTLA4-Ig) accelerated disease relapse following CAR T therapy in preclinical models, potentially due to blocking CD28 signaling in CAR T cells. Knockout studies confirmed that endogenous CD28 expressed on BBζ CAR T cells drove in vivo anti-MM activity. Mechanistically, CD28 reprogrammed mitochondrial metabolism to maintain redox balance and CAR T cell proliferation in the MM BME. Transient CD28 inhibition with abatacept restrained rapid BBζ CAR T cell expansion and limited inflammatory cytokines in the MM BME without significantly affecting long-term survival of treated mice. Overall, data directly demonstrate a need for CD28 signaling for sustained in vivo function of CAR T cells and indicate that transient CD28 blockade could reduce cytokine release and associated toxicities.

Pyroptosis activates conventional type I dendritic cells to mediate the priming of highly functional anticancer T cells.

BACKGROUND: Initiation of antitumor immunity is reliant on the stimulation of dendritic cells (DCs) to present tumor antigens to naïve T cells and generate effector T cells that can kill cancer cells. Induction of immunogenic cell death after certain types of cytotoxic anticancer therapies can stimulate T cell-mediated immunity. However, cytotoxic therapies simultaneously activate multiple types of cellular stress and programmed cell death; hence, it remains unknown what types of cancer cell death confer superior antitumor immunity. METHODS: Murine cancer cells were engineered to activate apoptotic or pyroptotic cell death after Dox-induced expression of procell death proteins. Cell-free supernatants were collected to measure secreted danger signals, cytokines, and chemokines. Tumors were formed by transplanting engineered tumor cells to specifically activate apoptosis or pyroptosis in established tumors and the magnitude of immune response measured by flow cytometry. Tumor growth was measured using calipers to estimate end point tumor volumes for Kaplan-Meier survival analysis. RESULTS: We demonstrated that, unlike apoptosis, pyroptosis induces an immunostimulatory secretome signature. In established tumors pyroptosis preferentially activated CD103+ and XCR1+ type I conventional DCs (cDC1) along with a higher magnitude and functionality of tumor-specific CD8+ T cells and reduced number of regulatory T cells within the tumor. Depletion of cDC1 or CD4+ and CD8+ T cells ablated the antitumor response leaving mice susceptible to a tumor rechallenge. CONCLUSION: Our study highlights that distinct types of cell death yield varying immunotherapeutic effect and selective activation of pyroptosis can be used to potentiate multiple aspects of the anticancer immunity cycle.

A rational relationship: Oncolytic virus vaccines as functional partners for adoptive T cell therapy.

Tumours employ a variety of immune-evasion and suppression mechanisms to impair development of functional tumor-specific T cells and subvert T cell-mediated immunity in the tumour microenvironment. Adoptive T cell therapy (ACT) aims to overcome these barriers and overwhelm tumor defenses with a bolus of T cells that were selectively expanded ex vivo. Although this strategy has been effective in liquid tumors and melanomas, many tumors appear to be resistant to ACT. Several factors are thought to play into this resistance, including poor engraftment and persistence of transferred cells, tumour cell heterogeneity and antigen loss, poor immune cell recruitment and infiltration into the tumour, and susceptibility to local immunosuppression in the tumor microenvironment. Oncolytic viruses (OV) have been identified as powerful stimulators of the anti-tumour immune response. As such, OVs are inherently well-positioned to act in synergy with ACT to bolster the anti-tumour T cell response. Further, OV vaccines, wherein tumour-associated antigens are encoded into the viral backbone, have proven to be remarkable in boosting antigen-specific T cell response. Pre-clinical studies have revealed remarkable therapeutic outcomes when OV vaccines are paired with ACT. In this scenario, OV vaccines are thought to function in a "push and pull" manner, where push refers to expanding T cells in the periphery and pull refers to recruiting those cells into the tumour that has been rendered amenable to T cell attack by the actions of the OV. In this review, we discuss barriers that limit eradication of tumors by T cells, highlight attributes of OVs that break down these barriers and present strategies for rational combinations of ACT with OV vaccines.