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Histone deacetylases (HDAC) are therapeutic targets in multiple cancers. ACY241, an HDAC6 selective inhibitor, has shown anti-multiple myeloma (MM) activity in combination with immunomodulatory drugs and proteasome inhibitors. Here we show ACY241 significantly reduces the frequency of CD138+ MM cells, CD4+CD25+FoxP3+ regulatory T cells, and HLA-DRLow/-CD11b+CD33+ myeloid-derived suppressor cells; and decreases expression of PD1/PD-L1 on CD8+ T cells and of immune checkpoints in bone marrow cells from myeloma patients. ACY241 increased B7 (CD80, CD86) and MHC (Class I, Class II) expression on tumor and dendritic cells. We further evaluated the effect of ACY241 on antigen-specific cytotoxic T lymphocytes (CTL) generated with heteroclitic XBP1unspliced184–192 (YISPWILAV) and XBP1spliced367–375 (YLFPQLISV) peptides. ACY241 induces co-stimulatory (CD28, 41BB, CD40L, OX40) and activation (CD38) molecule expression in a dose- and time-dependent manner, and anti-tumor activities, evidenced by increased perforin/CD107a expression, IFN-γ/IL-2/TNF-α production, and antigen-specific central memory CTL. These effects of ACY241 on antigen-specific memory T cells were associated with activation of downstream AKT/mTOR/p65 pathways and upregulation of transcription regulators including Bcl-6, Eomes, HIF-1 and T-bet. These studies therefore demonstrate mechanisms whereby ACY241 augments immune response, providing the rationale for its use, alone and in combination, to restore host anti-tumor immunity and improve patient outcome.

The cancer therapeutic cetuximab blocks EGFR signaling on tumor cells. Pozzi et al . now show that this antibody, when combined with chemotherapy, can also kill colorectal cancer cells by triggering immunogenic cell death. Cetuximab is a monoclonal antibody that is effective in the treatment of metastatic colorectal cancer (mCRC). Cetuximab blocks epidermal growth factor receptor (EGFR)-ligand interaction and inhibits downstream RAS–ERK activation. However, only some activating mutations in RAS affect cetuximab efficacy, and it is not clear what else mediates treatment success. Here we hypothesized that cetuximab induces immunogenic cell death (ICD) that activates a potent antitumor response. We found that cetuximab, in combination with chemotherapy, fostered ICD in CRC cells, which we measured via the endoplasmic reticulum (ER) stress response and an increase in phagocytosis by dendritic cells. ICD induction depended on the mutational status of the EGFR signaling pathway and on the inhibition of the splicing of X-box binding protein 1 ( XBP1 ), an unfolded protein response (UPR) mediator. We confirmed the enhanced immunogenicity elicited by cetuximab in a mouse model of human EGFR-expressing CRC. Overall, we demonstrate a new, immune-related mechanism of action of cetuximab that may help to tailor personalized medicine.

CARM1 is often overexpressed in human cancers including in ovarian cancer. However, therapeutic approaches based on CARM1 expression remain to be an unmet need. Cancer cells exploit adaptive responses such as the endoplasmic reticulum (ER) stress response for their survival through activating pathways such as the IRE1α/XBP1s pathway. Here, we report that CARM1-expressing ovarian cancer cells are selectively sensitive to inhibition of the IRE1α/XBP1s pathway. CARM1 regulates XBP1s target gene expression and directly interacts with XBP1s during ER stress response. Inhibition of the IRE1α/XBP1s pathway was effective against ovarian cancer in a CARM1-dependent manner both in vitro and in vivo in orthotopic and patient-derived xenograft models. In addition, IRE1α inhibitor B-I09 synergizes with immune checkpoint blockade anti-PD1 antibody in an immunocompetent CARM1-expressing ovarian cancer model. Our data show that pharmacological inhibition of the IRE1α/XBP1s pathway alone or in combination with immune checkpoint blockade represents a therapeutic strategy for CARM1-expressing cancers. The unfolded protein response (UPR) promotes cell survival in cancers with hyperactive ER stress response. Here the authors show that CARM1, an arginine methyltransferase, controls the IRE1α/XBP1 pathway of the UPR and the inhibition of this pathway can inhibit growth in CARM1 expressing ovarian cancers.

Group 3 innate lymphoid cells (ILC3s) are key players in intestinal homeostasis. ER stress is linked to inflammatory bowel disease (IBD). Here, we used cell culture, mouse models, and human specimens to determine whether ER stress in ILC3s affects IBD pathophysiology. We show that mouse intestinal ILC3s exhibited a 24-hour rhythmic expression pattern of the master ER stress response regulator inositol-requiring kinase 1α/X-box–binding protein 1 (IRE1α/XBP1). Proinflammatory cytokine IL-23 selectively stimulated IRE1α/XBP1 in mouse ILC3s through mitochondrial ROS (mtROS). IRE1α/XBP1 was activated in ILC3s from mice exposed to experimental colitis and in inflamed human IBD specimens. Mice with Ire1α deletion in ILC3s ( Ire1α ΔRorc ) showed reduced expression of the ER stress response and cytokine genes including Il22 in ILC3s and were highly vulnerable to infections and colitis. Administration of IL-22 counteracted their colitis susceptibility. In human ILC3s, IRE1 inhibitors suppressed cytokine production, which was upregulated by an IRE1 activator. Moreover, the frequencies of intestinal XBP1s + ILC3s in patients with Crohn’s disease before administration of ustekinumab, an anti-IL-12/IL-23 antibody, positively correlated with the response to treatment. We demonstrate that a noncanonical mtROS-IRE1α/XBP1 pathway augmented cytokine production by ILC3s and identify XBP1s + ILC3s as a potential biomarker for predicting the response to anti–IL-23 therapies in IBD.

Type 3 innate lymphoid cells (ILC3s) are key regulators of intestinal homeostasis and epithelial barrier integrity. In this issue of the JCI , Cao and colleagues found that a sensor of endoplasmic reticulum (ER) stress, the inositol-requiring kinase 1α/X-box–binding protein 1 (IRE1α/XBP1) pathway, fine-tuned the functions of ILC3s. Activation of IRE1α and XBP1 in ILC3s limited intestinal inflammation in mice and correlated with the efficacy of ustekinumab, an IL-12/IL-23 blocker, in patients with Crohn’s disease. These results advance our understanding in the use of ILCs as biomarkers not only to predict disease outcomes but also to indicate the response to biologicals in patients with inflammatory bowel disease.

Macrophages are among the most abundant immune cells in colorectal cancer (CRC). Re-educating tumor-associated macrophages (TAMs) to switch from protumoral to anti-tumoral activity is an attractive treatment strategy that warrants further investigation. However, little is known about the key pathway that is activated in TAMs. In this study, infitrating CD206 + TAMs in CRC were sorted and subjected to RNA-seq analysis. Differentially expressed genes were found to be enriched in unfolded protein response/endoplasmic reticulum stress response processes, and XBP1 splicing/activation was specifically observed in TAMs. XBP1 activation in TAMs promoted the growth and metastasis of CRC. Ablation of XBP1 inhibited the expression of the pro-tumor cytokine signature of TAMs, including IL-6, VEGFA, and IL-4. Simultaneously, XBP1 depletion could directly inhibit the expression of SIRPα and THBS1, thereby blocking “don’t eat me” recognition signals and enhancing phagocytosis. Therapeutic XBP1 gene editing using AAV2-sgXBP1 enhanced the anti-tumor activity. Together, XBP1 activation in TAMs drives CRC progression by elevating pro-tumor cytokine expression and secretion, as well as inhibiting macrophage phagocytosis. Targeting XBP1 signaling in TAMs may be a potential strategy for CRC therapy.

In vitro systems based on human peripheral blood mononuclear cells (PBMCs) can bridge the gap between preclinical and clinical vaccine evaluation but have so far mainly been exploited to assess vaccine effects on antigen-presenting cells and T cells. Our study aimed to assess whether B cells present in PBMCs also respond to vaccines and reflect the effects of different vaccine formulations and adjuvants. We stimulated PBMCs with whole inactivated virus (WIV) or split virus (SIV) H5N1 influenza vaccine, with or without the addition of the adjuvant cytosine phosphoguanine (CpG) ODN 2395, and collected the cells and supernatants at different timepoints. B cell subsets were measured by flow cytometry, immunoglobulin (IgG) levels by ELISA, B cell-related genes by qPCR, and cytokine levels by intracellular staining. B cells differentiated more readily to plasmablasts and plasma cells and produced more IgG when PBMC cultures were stimulated with WIV than when stimulated with SIV. In line, PRDM1, XBP1, and AICDA, genes associated with the differentiation of B cells to antibody-secreting cells, were expressed at higher levels in WIV- than in SIV-stimulated PBMCs. The combination of WIV and CpG consistently induced the highest levels of antibody-secreting cell differentiation, IgG production, and B-cells secreting IL-6 and IL-10. Taken together, B cells in human PBMC cultures show distinct responses to different types of vaccines and vaccine/CpG combinations. This underlines the suitability of unfractionated PBMCs for evaluating vaccine effects on different types of human immune cells before running costly clinical trials. •In long-term cultures of human PBMCs, B cell responses to different types of influenza vaccines reflect the in vivo properties of the vaccines.•B cells respond to vaccines in vitro by changes in transcription factor expression and B cell subset ratios, and increases in ASCs and total IgG.•In contrast to split virus influenza vaccine, whole inactivated virus vaccine and the adjuvant CpG ODN 2395 activate B cells in a polyclonal manner.•Human PBMCs, in the absence of added cytokines, are suitable to inform about the immune-stimulating properties of vaccines.

Nonalcoholic fatty liver disease (NAFLD) is one of the most common causes of liver diseases in the United States and can progress to cirrhosis, end-stage liver disease and need for liver transplantation. There are limited therapies for NAFLD, in part, due to incomplete understanding of the disease pathogenesis, which involves different cell populations in the liver. Endoplasmic reticulum stress and its adaptative unfolded protein response (UPR) signaling pathway have been implicated in the progression from simple hepatic steatosis to nonalcoholic steatohepatitis (NASH). We have previously shown that mice lacking the UPR protein X-box binding protein 1 (XBP1) in the liver demonstrated enhanced liver injury and fibrosis in a high fat sugar (HFS) dietary model of NAFLD. In this study, to better understand the role of liver XBP1 in the pathobiology of NAFLD, we fed hepatocyte XBP1 deficient mice a HFS diet or chow and investigated UPR and other cell signaling pathways in hepatocytes, hepatic stellate cells and immune cells. We demonstrate that loss of XBP1 in hepatocytes increased inflammatory pathway expression and altered expression of the UPR signaling in hepatocytes and was associated with enhanced hepatic stellate cell activation after HFS feeding. We believe that a better understanding of liver cell-specific signaling in the pathogenesis of NASH may allow us to identify new therapeutic targets.

X-box binding protein 1 (XBP1), CD138 (Syndecan-1) and CS1 (SLAMF7) are highly expressed antigens in cancers including multiple myeloma (MM). Here, we identify and characterize immunogenic HLA-A24 peptides derived from these antigens for potential vaccination therapy of HLA-A24+ patients with MM. The identified immunogenic HLA-A24-specific XBP1 unspliced (UN)185-193 (I S P W I L A V L), XBP1 spliced (SP)223-231 (V Y P E G P S S L), CD138265-273 (I F A V C L V G F) and CS1240-248 (L F V L G L F L W) peptides induced antigen-specific CTL with anti-MM activity in an HLA-A24 restricted manner. Furthermore, a cocktail containing the four HLA-A24 peptides evoked MM-specific CTL with distinct phenotypic profiles (CD28, CD40L, 41BB, CD38, CD69) and anti-tumor activities, evidenced by perforin upregulation, CD107a degranulation (cytotoxicity) and Th1-type cytokines (IFN-γ/IL-2/TNF-α) production in response to HLA-A24+ MM cells. The multipeptide-specific CTL included antigen-specific memory CD8+ T cells expressing both T-cell activation (CD38, CD69) and immune checkpoints antigens (CTLA, PD-1, LAG-3, TIM-3). These results provide the framework for a multipeptide vaccination therapy to induce tumor-specific CTL in HLA-A24-positive patients with myeloma and other cancers expressing these antigens.

Acute graft- vs. -host disease (GVHD) is an important cause of morbidity and death after allogeneic hematopoietic cell transplantation (HCT). We identify a new approach to prevent GVHD that impairs monocyte-derived dendritic cell (moDC) alloactivation of T cells, yet preserves graft- vs.-leukemia (GVL). Exceeding endoplasmic reticulum (ER) capacity results in a spliced form of X-box binding protein-1 (XBP-1s). XBP-1s mediates ER stress and inflammatory responses. We demonstrate that siRNA targeting XBP-1 in moDCs abrogates their stimulation of allogeneic T cells. B-I09, an inositol-requiring enzyme-1α (IRE1α) inhibitor that prevents XBP-1 splicing, reduces human moDC migration, allo-stimulatory potency, and curtails moDC IL-1β, TGFβ, and p40 cytokines, suppressing Th1 and Th17 cell priming. B-I09-treated moDCs reduce responder T cell activation via calcium flux without interfering with regulatory T cell (Treg) function or GVL effects by cytotoxic T lymphocytes (CTL) and NK cells. In a human T cell mediated xenogeneic GVHD model, B-I09 inhibition of XBP-1s reduced target-organ damage and pathogenic Th1 and Th17 cells without impacting donor Tregs or anti-tumor CTL. DC XBP-1s inhibition provides an innovative strategy to prevent GVHD and retain GVL.