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High levels of IL1β can result in chronic inflammation, which in turn can promote tumor growth and metastasis. Inhibition of IL1β could therefore be a promising therapeutic option in the treatment of cancer. Here, the effects of IL1β blockade induced by the mAbs canakinumab and gevokizumab were evaluated alone or in combination with docetaxel, anti–programmed cell death protein 1 (anti–PD-1), anti-VEGFα, and anti-TGFβ treatment in syngeneic and humanized mouse models of cancers of different origin. Canakinumab and gevokizumab did not show notable efficacy as single-agent therapies; however, IL1β blockade enhanced the effectiveness of docetaxel and anti–PD-1. Accompanying these effects, blockade of IL1β alone or in combination induced significant remodeling of the tumor microenvironment (TME), with decreased numbers of immune suppressive cells and increased tumor infiltration by dendritic cells (DC) and effector T cells. Further investigation revealed that cancer-associated fibroblasts (CAF) were the cell type most affected by treatment with canakinumab or gevokizumab in terms of change in gene expression. IL1β inhibition drove phenotypic changes in CAF populations, particularly those with the ability to influence immune cell recruitment. These results suggest that the observed remodeling of the TME following IL1β blockade may stem from changes in CAF populations. Overall, the results presented here support the potential use of IL1β inhibition in cancer treatment. Further exploration in ongoing clinical studies will help identify the best combination partners for different cancer types, cancer stages, and lines of treatment.

Supplementary tables S1-S5 and figures S1-S7

High levels of IL1β can result in chronic inflammation, which in turn can promote tumor growth and metastasis. Inhibition of IL1β could therefore be a promising therapeutic option in the treatment of cancer. Here, the effects of IL1β blockade induced by the mAbs canakinumab and gevokizumab were evaluated alone or in combination with docetaxel, anti–programmed cell death protein 1 (anti–PD-1), anti-VEGFα, and anti-TGFβ treatment in syngeneic and humanized mouse models of cancers of different origin. Canakinumab and gevokizumab did not show notable efficacy as single-agent therapies; however, IL1β blockade enhanced the effectiveness of docetaxel and anti–PD-1. Accompanying these effects, blockade of IL1β alone or in combination induced significant remodeling of the tumor microenvironment (TME), with decreased numbers of immune suppressive cells and increased tumor infiltration by dendritic cells (DC) and effector T cells. Further investigation revealed that cancer-associated fibroblasts (CAF) were the cell type most affected by treatment with canakinumab or gevokizumab in terms of change in gene expression. IL1β inhibition drove phenotypic changes in CAF populations, particularly those with the ability to influence immune cell recruitment. These results suggest that the observed remodeling of the TME following IL1β blockade may stem from changes in CAF populations. Overall, the results presented here support the potential use of IL1β inhibition in cancer treatment. Further exploration in ongoing clinical studies will help identify the best combination partners for different cancer types, cancer stages, and lines of treatment.

Immunosuppressive tumor microenvironments (TMEs) reduce the effectiveness of immune responses in cancer. Mesenchymal stromal cells (MSCs), precursors to cancer-associated fibroblasts (CAFs), promote tumor progression by enhancing immune cell suppression in colorectal cancer (CRC). Hyper-sialylation of glycans promotes immune evasion in cancer through binding of sialic acids to their receptors, Siglecs, expressed on immune cells, which results in inhibition of effector functions. The role of sialylation in shaping MSC/CAF immunosuppression in the TME is not well characterized. In this study, we show that tumor-conditioned stromal cells have increased sialyltransferase expression, α2,3/6-linked sialic acid, and Siglec ligands. Tumor-conditioned stromal cells and CAFs induce exhausted immunomodulatory CD8+ PD1+ and CD8+ Siglec-7+/Siglec-9+ T cell phenotypes. In vivo, targeting stromal cell sialylation reverses stromal cell-mediated immunosuppression, as shown by infiltration of CD25 and granzyme B-expressing CD8+ T cells in the tumor and draining lymph node. Targeting stromal cell sialylation may overcome immunosuppression in the CRC TME.

Despite the increasing interest in targeting stromal elements of the tumor microenvironment, we still face tremendous challenges in developing adequate therapeutics to modify the tumor stromal landscape. A major obstacle to this is our poor understanding of the phenotypic and functional heterogeneity of stromal cells in tumors. Herein, we perform an unbiased interrogation of tumor mesenchymal cells, delineating the co-existence of distinct subsets of cancer-associated fibroblasts (CAFs) in the microenvironment of murine carcinomas, each endowed with unique phenotypic features and functions. Furthermore, our study shows that neutralization of TGFβ in vivo leads to remodeling of CAF dynamics, greatly reducing the frequency and activity of the myofibroblast subset, while promoting the formation of a fibroblast population characterized by strong response to interferon and heightened immunomodulatory properties. These changes correlate with the development of productive anti-tumor immunity and greater efficacy of PD1 immunotherapy. Along with providing the scientific rationale for the evaluation of TGFβ and PD1 co-blockade in the clinical setting, this study also supports the concept of plasticity of the stromal cell landscape in tumors, laying the foundation for future investigations aimed at defining pathways and molecules to program CAF composition for cancer therapy., Understanding the tumor microenviroment is important before it can be exploited therapeutically. Here, the authors use single cell sequencing to study stromal cells in mouse tumors and identify a subset of interferon-licensed cancer associated fibroblasts that appear after anti-TGFβ treatment.

Background: Hypersialylation (excessive sialoglycans) on tumor cells has been known to be associated with poorer cancer outcomes for more than 40 years. Sialoglycans are immune suppressive and promote tumor immune evasion by binding to sialoglycan receptors (e.g. Siglecs) expressed on immune cells. However, redundant immune cell expression among Siglecs has posed a challenge for receptor-targeting therapeutic approaches. E-602 is a first-in-class fusion protein of engineered human sialidase (Neu2) and a human IgG1 Fc region. The dimeric sialidase moieties of E-602 circumvent the redundancy of this biology by directly cleaving terminal sialic acid residues from sialoglycans on immune and tumor cells. In preclinical studies, E-602 enhanced immune function by augmenting antigen-specific priming and activation of T cells and restoring function of exhausted-like T cells, and E-602 demonstrated antitumor activity as monotherapy in multiple mouse tumor models. Methods: A Phase 1/2 first-in-human dose escalation study is evaluating the safety, pharmacokinetics (PK), pharmacodynamics (PD), and antitumor activity of E-602 in patients with advanced cancers. Eligible patients with select advanced solid tumors were treated with E-602 IV once weekly at dose levels between 1 and 30 mg/kg. Circulating immune cells were analyzed for changes in sialylation and immunophenotyping by flow cytometry. Changes in circulating cytokines were measured by immunoassays. Results: As of January 6, 2023, 32 patients were treated with at least one dose of E-602. The most common tumors treated were colorectal (n=18) and pancreatic (n=6). Doses up to 30 mg/kg were tolerated with no dose-limiting toxicities. The most frequent adverse event was infusion-related reactions which were primarily grade 1-2 and clinically manageable. PK was linear across the evaluated dose range with an estimated T1/2 of 9-24 hours. Dose dependent PD observations included (1) desialylation of peripheral CD8+ T, CD4+ T, and NK cells, remaining detectable in some patients at 7 days post-dose; (2) increases in the immune activation marker, CD69, on peripheral CD8+ T, CD4+ T, and NK cells; and (3) increases in the IFNγ-dependent chemokine CXCL10, TNF-α, and MIP1-β. Thus far, response-evaluable patients have had stable or progressive disease. Additional safety, PK, PD, and clinical outcomes for other patients treated as part of backfill at multiple dose levels will be reported at the meeting. Conclusion: E-602 is tolerated at doses up to 30 mg/kg. Consistent with preclinical findings, dose-dependent desialylation and immune system activation were observed. Based on the observed tolerability and PD effects, the Phase 2 portion of the study to evaluate clinical activity of E-602 monotherapy in patients with checkpoint-inhibitor resistant NSCLC and melanoma will proceed. Citation Format: Jason J. Luke, Melissa Johnson, Anthony Tolcher, Christopher T. Chen, Tong Dai, Brendan D. Curti, Anthony El-Khoueiry, Mario Sznol, Brian S. Henick, Christine Horak, Pushpa Jayaraman, Christopher B. Cole, Dawn Wilson, Lizhi Cao, Li Peng, David Feltquate, Deanne Lathers, Manish R. Sharma. GLIMMER-01: initial results from a phase 1 dose escalation trial of a first-in-class bi-sialidase (E-602) in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT034.

Objectives Sabatolimab is a humanized monoclonal antibody (hIgG4, S228P) directed against human T-cell immunoglobulin domain and mucin domain-3 (TIM-3). Herein, we describe the development and characterization of sabatolimab. Methods Sabatolimab was tested for binding to its target TIM-3 and blocking properties. The functional effects of sabatolimab were tested in T-cell killing and myeloid cell cytokine assays. Antibody-mediated cell phagocytosis (ADCP) by sabatolimab was also assessed. Results Sabatolimab was shown to (i) enhance T-cell killing and inflammatory cytokine production by dendritic cells (DCs); (ii) facilitate the phagocytic uptake of TIM-3-expressing target cells; and (iii) block the interaction between TIM-3 and its ligands PtdSer/galectin-9. Conclusion Taken together, our results support both direct anti-leukemic effects and immune-mediated modulation by sabatolimab, reinforcing the notion that sabatolimab represents a novel immunotherapy with immuno-myeloid activity, holding promise for the treatment of myeloid cell neoplasms.

Background TIM-3 is expressed on leukemic stem cells (LSCs) and blasts in AML,1 2 and TIM-3 expression on MDS blasts correlates with disease progression.3 Functional evidence for TIM-3 in AML was established with an anti-TIM-3 antibody which inhibited engraftment and development of human AML in immuno-deficient murine hosts.1 TIM-3 promotes an autocrine stimulatory loop via the TIM-3/Galectin-9 interaction, supporting LSC self-renewal.4 In addition to its cell-autonomous role on LSCs/blasts, TIM-3 also has a critical role in immune system regulation, in adaptive (CD4+ and CD8+ T effector cells, regulatory T cells) and innate (macrophages, dendritic cells, NK cells) immune responses.5 MBG453 is a high-affinity, humanized anti-TIM-3 IgG4 antibody (Ab) (stabilized hinge, S228P), which blocks the binding of TIM-3 to phosphatidylserine (PtdSer). Recent results from a multi-center, open label phase Ib dose-escalation study (NCT03066648) in patients with high-risk MDS and no prior hypomethylating agent therapy evaluating MBG453 in combination with decitabine demonstrated encouraging preliminary efficacy with an overall response rate of 58%,6 and MBG453 combined with azacitidine also showed encouraging response rates.7 Preclinical experiments were undertaken to define the mechanism of action of the hypomethylating agent and anti-TIM-3 combination. Methods THP-1 cells (a human monocytic AML cell line) were pre-treated with decitabine and co-cultured with anti-CD3 activated healthy human donor peripheral blood mononuclear cells (PBMCs) in an Incucyte-based assay to measure cell killing. The ability of MBG453 to mediate antibody-dependent cellular phagocytosis (ADCP) was measured by determining the phagocytic uptake of an engineered TIM-3-overexpressing Raji cell line in the presence of MBG453 by phorbol 12-myristate 13-acetate (PMA)-activated THP-1 cells. Patient-derived AML xenograft studies were undertaken in immune-deficient murine hosts to evaluate the combination of decitabine and MBG453. Results MBG453 was determined to partially block the TIM-3/Galectin-9 interaction in a plate-based MSD (Meso Scale Discovery) assay, supported by a crystal structure of human TIM-3.8 Pre-treatment of THP-1 cells with decitabine enhanced sensitivity to immune-mediated killing in the presence of MBG453. MBG453 was determined to mediate modest ADCP, relative to controls. MBG453 did not enhance the anti-leukemic activity of decitabine in patient-derived xenograft studies in immuno-deficient hosts. Conclusions Taken together, these results support both direct anti-leukemic effects and immune-mediated modulation by MBG453. Further studies are ongoing to determine: (1) whether MBG453 can mediate physiologically relevant ADCP of TIM-3-expressing leukemic cells; and (2) the potential of MBG453 to impact the autocrine feedback loop of TIM-3/Galectin-9. Ethics Approval The human tissue used in these studies was under the Novartis Institutes of BioMedical Research Ethics Board IRB, Approval Number 201252867. References Kikushige Y, Shima T, Takayanagi S, et al. TIM-3 is a promising target to selectively kill acute myeloid leukemia stem cells. Cell Stem Cell 2010;7(6):708–717. Jan M, Chao MP, Cha AC, et al. Prospective separation of normal and leukemic stem cells based on differential expression of TIM3, a human acute myeloid leukemia stem cell marker. Proc Natl Acad Sci USA 2011; 108(12): 5009–5014. Asayama T, Tamura H, Ishibashi M, et al. Functional expression of Tim-3 on blasts and clinical impact of its ligand galectin-9 in myelodysplastic syndromes. Oncotarget 2017;8(51): 88904–88917. Kikushige Y, Miyamoto T, Yuda J, et al. A TIM-3/Gal-9 autocrine stimulatory loop drives self-renewal of human myeloid leukemia stem cells and leukemic progression. Cell Stem Cell 2015; 17(3):341–352. Acharya N, Sabatos-Peyton C, Anderson AC. Tim-3 finds its place in the cancer immunotherapy landscape. J Immunother Cancer 2020; 8(1):e000911. Borate U, Esteve J, Porkka K, et al. Phase Ib Study of the Anti-TIM-3 Antibody MBG453 in combination with decitabine in patients with high-risk myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Blood 2019;134 (Supplement_1):570. Borate U, Esteve J, Porkka K, et al. Abstract S185: Anti-TIM-3 antibody MBG453 in combination with hypomethylating agents (HMAs) in patients (pts) with high-risk myelodysplastic syndrome (HR-MDS) and acute myeloid leukemia (AML): a Phase 1 study. EHA 2020. Sabatos-Peyton C. MBG453: A high affinity, ligand-blocking anti-TIM-3 monoclonal Ab. AACR 2016.

The differentiation of effector CD8+ T cells is a dynamically regulated process that varies during different infections and is influenced by the inflammatory milieu of the host. In this study, we define three signals regulating CD8+ T cell responses during tuberculosis by focusing on cytokines known to affect disease outcome: IL-12, type I IFN, and IL-27. Using mixed bone marrow chimeras, we compared wild-type and cytokine receptor knockout CD8+ T cells within the same mouse following aerosol infection with Mycobacterium tuberculosis. Four weeks postinfection, IL-12, type 1 IFN, and IL-27 were all required for efficient CD8+ T cell expansion in the lungs. We next determined if these cytokines directly promote CD8+ T cell priming or are required only for expansion in the lungs. Using retrogenic CD8+ T cells specific for the M. tuberculosis Ag TB10.4 (EsxH), we observed that IL-12 is the dominant cytokine driving both CD8+ T cell priming in the lymph node and expansion in the lungs; however, type I IFN and IL-27 have nonredundant roles supporting pulmonary CD8+ T cell expansion. Thus, IL-12 is a major signal promoting priming in the lymph node, but a multitude of inflammatory signals converge in the lung to promote continued expansion. Furthermore, these cytokines regulate the differentiation and function of CD8+ T cells during tuberculosis. These data demonstrate distinct and overlapping roles for each of the cytokines examined and underscore the complexity of CD8+ T cell regulation during tuberculosis.

Introduction: TPI is a hallmark of cancer and plays a pivotal role in tumor initiation, development, progression, and invasiveness. High levels of tumor-promoting inflammatory cytokines e.g. IL-1β and IL-6 are associated with advanced malignancies and reduced survival. Selective IL-1β inhibitors like canakinumab (CAN) and gevokizumab (GEV) aim to target TPI within the tumor microenvironment (TME) by reducing tumor-associated immune suppression. In phase III CANTOS study, CAN significantly reduced lung cancer (LC) incidences and LC related mortalities in atherosclerosis patients, providing the first clinical evidence for the role of IL-1β inhibition in LC. We have utilized preclinical models to gain further insights into the mechanistic role of CAN and GEV in cancer development and progression. Methods: Preclinical efficacy of anti-human IL-1β antibodies, CAN and GEV was assessed using humanized mouse models of non-small cell lung cancer (NSCLC, H358), triple-negative breast cancer (TNBC, MDA-MB-231) and colorectal cancer (CRC, SW480) either as single agents or in combination with other immunomodulatory agents including PD-1 inhibitor pembrolizumab (PEM) and anti-VEGF therapy. A murine surrogate IL-1β blocking antibody clone 01BSUR was used to evaluate efficacy in murine syngeneic models of breast (4T1) and lung (LL-2) cancer either as single agent or with PD-1 inhibitor and docetaxel (DOC). Tumor infiltrating lymphocytes (TIL) characterization was carried out by flow cytometry and IHC in syngeneic and humanized models, respectively. Results: The IL-1β inhibitors showed anti-tumor activity in both murine syngeneic and humanized models as single agents or with other agents. In the H358 NSCLC mouse model, CAN + PEM significantly inhibited tumor growth and was associated with >50% regression vs control. Decreased tumor growth was also observed with GEV treatment in the SW480 CRC mouse model, both alone and with anti-VEGF therapy. In GEV+anti-VEGF arm, a decrease in tolerogenic DC-10 immune populations and an increase in CD45+ immune cells and CD68+ myeloid cells were also observed. In the MDA-MB-231 TNBC mouse model, CAN single-agent showed a decrease in tumor growth vs control. In murine syngeneic 4T1 and LL-2 models, anti-IL-1β alone and anti-IL-1β + anti-PD-1 resulted in increased presence of CD8+ T cells and reduction of immunosuppressive myeloid-derived suppressor cells, neutrophils, and regulatory T cells in the tumor, suggesting that the TME may be shifting to a less suppressive phenotype after IL-1β blockade. Similar changes were observed with combination of anti-IL-1β with DOC and anti-VEGF in murine syngeneic models. Conclusions: These data provide key insights into the biology behind IL-1β blockade currently being pursued in clinical trials with CAN and GEV. Additional preclinical studies to investigate mechanism of action of these agents and other novel combinations are planned. Citation Format: Pushpa Jayaraman, Vanessa Rodrik-Outmezguine, John Millholland, Neil O'Brien, Connie Wong, Rohan Diwanji, Muchun Wang, Elizabeth Choi, Ronald Linnartz, Kristine Rose, Marc Pelletier, Alexander Savchenko, Tong Luo, Valerie Rezek, Scott Kitchen, Jason Baum, Catherine Sabatos-Peyton, Anne-Marie Martin, Glenn Dranoff. Targeting tumor-promoting inflammation (TPI) via the IL-1βpathway for cancer immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5640.

Inflammation in cancer can both suppress tumor growth and promote tumorigenesis. In the tumor microenvironment (TME), inflammation characterized by inflammatory cells and other mediators including cytokines affects tumor development and progression and can have an impact on response to therapy. The inflammatory cytokine IL-1b is upregulated in several conditions including rheumatoid arthritis, chronic obstructive pulmonary disease, inflammatory bowel disease, as well as in different cancers including breast, gastric, pancreatic, and lung cancers. IL-1b contributes to the generation and accumulation of myeloid-derived suppressor cells (MDSCs). In the tumor microenvironment, MDSCs consist of a mixture of immature macrophages, immature dendritic cells and neutrophils, which can suppress the function of antitumor T cells. In addition, IL-1b is produced via activation of the inflammasome, which has been shown to promote the infiltration of MDSCs and tumor-associated macrophages (TAMs) into the TME. Downstream of IL-1b production are other inflammatory cytokines such as IL-6, IL-8, and IL-17, which can also suppress the immune system. Recently published data show that blocking IL-1b in patients without cancer can lead to a decrease in fatal cancer and more specifically lung cancer incidences and fatalities (Ridker et al., 2017). While the study was not designed to study the anticancer effects of canakinumab (Ilaris), this finding has led to increased interest in understanding the role of IL-1b and IL-1b blockade in cancer development and progression. Emerging data aim to elucidate the role of IL-1b blockade on tumor growth and progression, as well as characterize changes to tumor-infiltrating lymphocyte (TIL) populations and their function after treatment with an anti-IL-1b antibody. Reference: Ridker PM et al. Effect of interleukin-1b inhibition with canakinumab on incident lung cancer in patients with atherosclerosis: exploratory results from a randomized, double-blind, placebo-controlled trial. Lancet 2017;390:1833-42. Citation Format: Reshma Singh, Rohan Diwanji, Pushpa Jayaraman, Derek Chiang, Catherine Sabatos-Peyton, Glenn Dranoff. Anti IL-1b as a cancer immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A76.

Despite the introduction almost a century ago of Mycobacterium bovis BCG (BCG), an attenuated form of M. bovis that is used as a vaccine against Mycobacterium tuberculosis, tuberculosis remains a global health threat and kills more than 1.5 million people each year. This is mostly because BCG fails to prevent pulmonary disease – the contagious form of tuberculosis. Although there have been significant advances in understanding how the immune system responds to infection, the qualities that define protective immunity against M. tuberculosis remain poorly characterized. The ability to predict who will maintain control over the infection and who will succumb to clinical disease would revolutionize our approach to surveillance, control, and treatment. Here we review the current understanding of pulmonary T cell responses following M. tuberculosis infection. While infection elicits a strong immune response that contains infection, M. tuberculosis evades eradication. Traditionally, its intracellular lifestyle and alteration of macrophage function are viewed as the dominant mechanisms of evasion. Now we appreciate that chronic inflammation leads to T cell dysfunction. While this may arise as the host balances the goals of bacterial sterilization and avoidance of tissue damage, it is becoming clear that T cell dysfunction impairs host resistance. Defining the mechanisms that lead to T cell dysfunction is crucial as memory T cell responses are likely to be subject to the same subject to the same pressures. Thus, success of T cell based vaccines is predicated on memory T cells avoiding exhaustion while at the same time not promoting overt tissue damage.

Introduction: Inflammation is a hallmark of cancer wherein diverse immune cells exert either pro- or antitumor properties and affect therapeutic resistance (Ritter B et al, JEM 2019). High concentrations of tumor-promoting inflammatory cytokines such as IL-1β (Litmanovich A et al, Oncol Ther 2018) and IL-6 (Altundag O et al, J Clin Oncol 2005) correlate with advanced malignancies and are associated with reduced survival. A recent phase III clinical trial (CANTOS) demonstrated that canakinumab, a selective IL-1β inhibitor, could significantly reduce lung cancer incidences and lung cancer related mortalities as a secondary endpoint in atherosclerosis patients, providing the first clinical evidence for the role of IL-1β inhibition in lung cancer. Canakinumab and other selective IL-1β inhibitors such as gevokizumab aim to target tumor promoting inflammation (TPI) within the tumor microenvironment (TME) by reducing tumor-associated immune suppression. In an effort to gain further insights into the clinical mechanistic role of canakinumab and gevokizumab in cancer development and progression, we have utilized preclinical models to understand the immunological events following IL-1β blockade. Methods: Preclinical efficacy of anti-human IL-1β antibodies, canakinumab and gevokizumab, was assessed in humanized mouse models of NSCLC (H358) and CRC (SW480) respectively. Anti-mouse IL-1β blocking antibody was used to evaluate efficacy in murine syngeneic models of breast (4T1) and lung (LL-2) cancer either as a single agent or in combination with PD-1 inhibitor and docetaxel. Tumor infiltrating lymphocytes (TIL) characterization was carried out either by flow cytometry or by IHC in syngeneic and humanized models. Results: Using both murine syngeneic and humanized mouse models, we demonstrate that IL-1β inhibition remodels the TME and slows tumor growth either alone or in combination with other agents. In the humanized H358 NSCLC mouse model, the combination of canakinumab and the anti-PD-1 monoclonal antibody, pembrolizumab, significantly slowed tumor growth by more than 50% vs control antibodies. Furthermore, canakinumab alone and in combination resulted in increased CD8 and CD3 positive TIL infiltration within the tumor. Decreased tumor growth was also observed with another IL-1β inhibitor, gevokizumab, in a humanized SW480 CRC mouse model, both alone and in combination with anti-VEGF therapy. In syngeneic 4T1 and LL-2 models, anti-IL-1β alone and in combination with anti-PD-1 resulted in increased infiltration of CD8+ cells along with the reduction of immunosuppressive myeloid derived suppressor cells (MDSCs), neutrophils and regulatory T cells (Tregs) into the tumor, suggesting that the TME may be shifting to a less suppressive phenotype after IL-1β blockade. Similar changes to the tumor microenvironment were observed with the combination of anti-IL-1β with docetaxel. Additional combinations are being explored in other tumor models reflecting different indications. Conclusions: Our results highlight the role for IL-1β in tumor immunomodulation and that the pathophysiological role of the IL-1β pathway in innate immunity might have important consequences on T cell function and checkpoint blockade in cancer. These results support the ongoing clinical evaluation of IL-1β inhibitors such as canakinumab and gevokizumab and in combination with other therapeutic agents across several cancers. Citation Format: Pushpa Jayaraman, John Millholland, Neil O’Brien, Connie Wong, Rohan Diwanji, Muchun Wang, Elizabeth Choi, Ronald Linnartz, Kristine Rose, Vanessa Rodrik-Outmezguine, Marc Pelletier, Alexander Savchenko, Tong Luo, Valerie Rezek, Scott Kitchen, Jason Baum, Catherine Sabatos-Peyton, Anne-Marie Martin, Glenn Dranoff . Targeting IL-1β pathway for cancer immunotherapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C103. doi:10.1158/1535-7163.TARG-19-C103

In vivo control of Mycobacterium tuberculosis reflects the balance between host immunity and bacterial evasion strategies. Effector Th1 cells that mediate protective immunity by depriving the bacterium of its intracellular niche are regulated to prevent overexuberant inflammation. One key immunoregulatory molecule is Tim3. Although Tim3 is generally recognized to downregulate Th1 responses, we recently described that its interaction with Galectin-9 expressed by M. tuberculosis–infected macrophages stimulates IL-1β secretion, which is essential for survival in the mouse model. Why IL-1β is required for host resistance to M. tuberculosis infection is unknown. In this article, we show that IL-1β directly kills M. tuberculosis in murine and human macrophages and does so through the recruitment of other antimicrobial effector molecules. IL-1β directly augments TNF signaling in macrophages through the upregulation of TNF secretion and TNFR1 cell surface expression, and results in activation of caspase-3. Thus, IL-1β and downstream TNF production lead to caspase-dependent restriction of intracellular M. tuberculosis growth.

IL-21 is produced predominantly by activated CD4(+) T cells and has pleiotropic effects on immunity via the IL-21 receptor (IL-21R), a member of the common gamma chain (gamma(c)) cytokine receptor family. We show that IL-21 signaling plays a crucial role in T cell responses during Mycobacterium tuberculosis infection by augmenting CD8(+) T cell priming, promoting T cell accumulation in the lungs, and enhancing T cell cytokine production. In the absence of IL-21 signaling, more CD4(+) and CD8(+) T cells in chronically infected mice express the T cell inhibitory molecules PD-1 and TIM-3. We correlate these immune alterations with increased susceptibility of IL-21R(-/-) mice, which have increased lung bacterial burden and earlier mortality compared to WT mice. Finally, to causally link the immune defects with host susceptibility, we use an adoptive transfer model to show that IL-21R(-/-) T cells transfer less protection than WT T cells. These results prove that IL-21 signaling has an intrinsic role in promoting the protective capacity of T cells. Thus, the net effect of IL-21 signaling is to enhance host resistance to M. tuberculosis. These data position IL-21 as a candidate biomarker of resistance to tuberculosis., This work was supported by National Institutes of Health Grants R21 AI100766, R01 AI106725, and P01 AI073748.

T cell Ig and mucin domain 3 (Tim3) is an inhibitory molecule involved in immune tolerance, autoimmune responses, and antiviral immune evasion. However, we recently demonstrated that Tim3 and Galectin-9 (Gal9) interaction induces a program of macrophage activation that results in killing of Mycobacterium tuberculosis in the mouse model of infection. In this study, we sought to determine whether the Tim3–Gal9 pathway plays a similar role in human pulmonary TB. We identified that pulmonary TB patients have reduced expression of Tim3 on CD14+ monocytes in vivo. By blocking Tim3 and Gal9 interaction in vitro, we show that these molecules contribute to the control of intracellular bacterial replication in human macrophages. The antimicrobial effect was partially dependent on the production of IL-1β. Our results establish that Tim3–Gal9 interaction activates human M. tuberculosis –infected macrophages and leads to the control of bacterial growth through the production of the proinflammatory cytokine IL-1β. Data presented in this study suggest that one of the potential pathways activated by Tim3/Gal9 is the secretion of IL-1β, which plays a crucial role in antimicrobial immunity by modulating innate inflammatory networks.

Mycobacterium tuberculosis is an intracellular bacterium that persists in phagosomes of myeloid cells. M. tuberculosis -encoded factors support pathogen survival and reduce fusion of phagosomes with bactericidal lysosomal compartments. It is, however, not entirely understood if host factors that mediate endosomal fusion affect M. tuberculosis intracellular localization and survival. Neither is it known if endosomal fusion influences induction of host immune reactivity by M. tuberculosis -infected cells. Lysosomal degradation of M. tuberculosis appears to be pivotal for making available lipid substrates for assembly into lipid-CD1d complexes to allow activation of CD1d-restricted invariant natural killer T (iNKT) cells. To clarify the role for endosomal fusion in M. tuberculosis survival and induction of host CD1d-mediated immune defense, we focused our studies on the invariant chain (Ii). Ii regulates endosome docking and fusion and thereby controls endosomal transport. Through direct binding, Ii also directs intracellular transport of the class II major histocompatibility complex and CD1d. Our findings demonstrate that upon infection of Ii-knockout (Ii −/− ) macrophages, M. tuberculosis is initially retained in early endosomal antigen 1-positive lysosomal-associated membrane protein 1-negative phagosomes, which results in slightly impaired pathogen replication. The absence of Ii did not affect the ability of uninfected and infected macrophages to produce nitric oxide, tumor necrosis factor alpha, or interleukin-12. However, induction of cell surface CD1d was impaired in infected Ii −/− macrophages, and CD1d-restricted iNKT cells were unable to suppress bacterial replication when they were cocultured with M. tuberculosis -infected Ii −/− macrophages. Thus, while the host factor Ii is not essential for the formation of the M. tuberculosis -containing vacuole, its presence is crucial for iNKT cell recognition of infected macrophages.

The interaction between Tim3 on Th1 cells and galectin-9 on Mycobacterium tuberculosis–infected macrophages restricts the bacterial growth by stimulating caspase-1–dependent IL-1β secretion., T cell immunoglobulin and mucin domain 3 (Tim3) is a negative regulatory molecule that inhibits effector TH1-type responses. Such inhibitory signals prevent unintended tissue inflammation, but can be detrimental if they lead to premature T cell exhaustion. Although the role of Tim3 in autoimmunity has been extensively studied, whether Tim3 regulates antimicrobial immunity has not been explored. Here, we show that Tim3 expressed on TH1 cells interacts with its ligand, galectin-9 (Gal9), which is expressed by Mycobacterium tuberculosis–infected macrophages to restrict intracellular bacterial growth. Tim3–Gal9 interaction leads to macrophage activation and stimulates bactericidal activity by inducing caspase-1–dependent IL-1β secretion. We propose that the TH1 cell surface molecule Tim3 has evolved to inhibit growth of intracellular pathogens via its ligand Gal9, which in turn inhibits expansion of effector TH1 cells to prevent further tissue inflammation.

We assessed differences in the character and specificity of autologous neutralizing antibodies (ANAbs) against individual viral variants of the quasispecies in a cohort of drug-naïve subjects with long-term controlled human immunodeficiency virus type 1 (HIV-1) infection and moderate levels of broad heterologous neutralizing antibodies (HNAb). Functional plasma virus showed continuous env evolution despite a short time frame and low levels of viral replication. Neutralization-sensitive variants dominated in subjects with intermittent viral blips, while neutralization-resistant variants predominated in elite controllers. By sequence analysis of this panel of autologous variants with various sensitivities to neutralization, we identified more than 30 residues in envelope proteins (Env) associated with resistance or sensitivity to ANAbs. The appearance of new sensitive variants is consistent with a model of continuous selection and turnover. Strong ANAb responses directed against autologous Env variants are present in long-term chronically infected individuals, suggesting a role for these responses in contributing to the durable control of HIV replication.

To model human immunodeficiency virus (HIV) perinatal transmission, we studied infection of simian-human immunodeficiency virus (SHIV) SF162P3 in 10 pregnant Macaca nemestrina females and their offspring. Four of nine infants born to and suckled by these dams had evidence of infection, a transmission rate of 44.4% (95% confidence interval, 13.7% to 78.8%). We quantified transplacentally acquired and de novo Env-specific immunoglobulin G (IgG), IgM, and neutralizing antibodies in newborns. Transmission of escape variants was confirmed. In utero infection ( n = 1) resulted in high viremia, depletion of peripheral CD4 + T cells, and rapid evolution of env in blood and tissues. Peripartum or postpartum SHIV infection ( n = 3) resulted in postacute viral control that was undetectable by very sensitive multiplex PCR, despite increasing antibodies. Seropositive infants with highly controlled viremia had homogeneous peripheral blood env sequences, and their tissues had

While T cell immunity initially limits Mycobacterium tuberculosis infection, why T cell immunity fails to sterilize the infection and allows recrudescence is not clear. One hypothesis is that T cell exhaustion impairs immunity and is detrimental to the outcome of M. tuberculosis infection. Here we provide functional evidence for the development T cell exhaustion during chronic TB. Second, we evaluate the role of the inhibitory receptor T cell immunoglobulin and mucin domain–containing-3 (TIM3) during chronic M. tuberculosis infection. We find that TIM3 expressing T cells accumulate during chronic infection, co-express other inhibitory receptors including PD1, produce less IL-2 and TNF but more IL-10, and are functionally exhausted. Finally, we show that TIM3 blockade restores T cell function and improves bacterial control, particularly in chronically infected susceptible mice. These data show that T cell immunity is suboptimal during chronic M. tuberculosis infection due to T cell exhaustion. Moreover, in chronically infected mice, treatment with anti-TIM3 mAb is an effective therapeutic strategy against tuberculosis., Author Summary Tuberculosis is a leading cause of morbidity and mortality across the globe. Fortunately, most people infected with M. tuberculosis mount a protective immune response and only a small fraction develops active disease. Impairment of immunity late during the course of disease can lead to bacterial recrudescence; however, why immunity fails is poorly understood. We investigated whether T cell exhaustion develops and contributes to immunological impairment during disease. Our studies provide definitive evidence that CD4+ T cells become functionally exhausted early after infection, and subsequently, CD8+ T cells also show signs of dysfunction. T cell exhaustion in both subsets was associated with the expression of multiple inhibitory receptors. Exhausted T cells expressed TIM3 plus other inhibitory receptors (e.g., PD1, TIM3, Lag-3, and 2B4), TIM3+PD1+ T cells were more likely to be poor producers of IL-2, IFNγ, and TNF and instead produce IL-10. Evaluation of gene expression by Nanostring confirmed that TIM3+PD1+ T cells in the lungs of infected mice had a transcriptional profile characteristic of exhausted T cells. Thus, this phenotype identified T cells that were truly exhausted and correlates well with previously established paradigm that co-expression of TIM3 with other inhibitory receptors such as PD1 contributes to impairment of T cell function during chronic inflammatory conditions. Most importantly, treatment of chronically infected mice with blocking antibodies specific for TIM3 led to a significant gain in bacterial control. Treatment was associated with an increase in IL-2, IFNγ, and TNF production by T cells. Based on this key result, we infer that TIM3-mediated T cell exhaustion impairs host resistance to M. tuberculosis. Thus, these data suggest that blockade of inhibitory T cell signals has the potential to be a therapeutic strategy against tuberculosis. The data in our report significantly advances our current knowledge of the biology of TIM3, the role of inhibitory T cell receptors during chronic infection, and the pathogenesis of tuberculosis.

Invariant natural killer T (iNKT) cells are activated during infection, but how they limit microbial growth is unknown in most cases. We investigated how iNKT cells suppress intracellular Mycobacterium tuberculosis (Mtb) replication. When co-cultured with infected macrophages, iNKT cell activation, as measured by CD25 upregulation and IFNγ production, was primarily driven by IL-12 and IL-18. In contrast, iNKT cell control of Mtb growth was CD1d-dependent, and did not require IL-12, IL-18, or IFNγ. This demonstrated that conventional activation markers did not correlate with iNKT cell effector function during Mtb infection. iNKT cell control of Mtb replication was also independent of TNF and cell-mediated cytotoxicity. By dissociating cytokine-driven activation and CD1d-restricted effector function, we uncovered a novel mediator of iNKT cell antimicrobial activity: GM-CSF. iNKT cells produced GM-CSF in vitro and in vivo in a CD1d-dependent manner during Mtb infection, and GM-CSF was both necessary and sufficient to control Mtb growth. Here, we have identified GM-CSF production as a novel iNKT cell antimicrobial effector function and uncovered a potential role for GM-CSF in T cell immunity against Mtb., Author Summary Mycobacterium tuberculosis (Mtb) is the cause of tuberculosis, a leading cause of sickness and death worldwide. Although much is known about CD4+ and CD8+ T cell responses to Mtb, the role of other T cell subsets is poorly understood. Invariant natural killer T (iNKT) cells are innate lymphocytes that express a semi-invariant T cell receptor and recognize lipid antigens presented by CD1d. Although iNKT cells participate in the immune response to many different pathogens, little is known about how iNKT cells directly kill microbes. We previously showed that when co-cultured with Mtb-infected macrophages, iNKT cells inhibit intracellular Mtb replication. Now, we used this model to dissociate the signals that induce iNKT cell activation markers including IFNγ production, from the signals that activate iNKT cell antimicrobial activity. This allowed us to uncover a novel antimicrobial effector function produced by iNKT cells: GM-CSF. GM-CSF is essential for immunity to Mtb, but its role has never been defined. This study is the first report to demonstrate a protective function of GM-CSF production by any T cell subset during Mtb infection. T cell production of GM-CSF should be considered as a potential mechanism of antimicrobial immunity.

Author Manuscript, Clinical trials of vaccines against Mycobacterium tuberculosis are well under way and results are starting to come in. Some of these results are not so encouraging, as exemplified by the latest Aeras-422 and MVA85A trials. Other than empirically determining whether a vaccine reduces the number of cases of active tuberculosis, which is a daunting prospect given the chronic nature of the disease, we have no way of assessing vaccine efficacy. Therefore, investigators seek to identify biomarkers that predict vaccine efficacy. Historically, focus has been on the production of interferon-γ by CD4(+) T cells, but this has not been a useful correlate of vaccine-induced protection. In this Opinion article, we discuss recent advances in our understanding of the immune control of M. tuberculosis and how this knowledge could be used for vaccine design and evaluation., The authors are supported by the following grants from the US National Institutes of Heath (NIH) and US National Institute of Allergy and Infectious Diseases (NIAID): R21AI100766, R01AI085669, R01AI098637, and R01AI106725

Tuberculosis is a pulmonary disease, caused by Mycobacterium tuberculosis (Mtb) infection, which affects approximately one third of the global population and is a major public health concern. Although T cells are essential in mediating protection against Mtb infection, they are often unable to sterilize infected tissues. One possible reason for an ineffective immune response is the onset of T cell exhaustion or dysfunction. T cell exhaustion is characterized by the progressive loss of T cell effector function accompanied by an increase in expression of inhibitory receptors. Here we show that during Mtb infection, T cells express the inhibitory molecules Tim3 and PD1, and T cells that express both Tim3 and PD1 have decreased IFNγ and TNF production. In addition, antibody blockade of Tim3 improves T cell function and decreases bacterial burden. These results indicate that T cells are functionally exhausted during chronic Mtb infection and that blockade of inhibitory receptors can re-invigorate T cell function and improve the outcome of infection. Based on a possible role for CEACAM1 in the T cell inhibitory signal mediated by Tim3, we are currently performing experiments to determine how CEACAM1 affects host defense against Mtb. Understanding the roles of inhibitory receptors, such as Tim3 and PD1, during Mtb infection is providing insight into why T cell immunity is insufficient to eradicate Mtb infection.

T cell immunoglobulin-3 (Tim-3) has been identified as a marker of differentiated interferon-γ-producing CD4 + T helper type 1 and CD8 + T cytotoxic type 1 cells. The interaction of Tim-3 with its ligand, galectin-9 (Gal-9), induces cell death, and in vivo blockade of this interaction results in exacerbated autoimmunity and abrogation of tolerance in experimental models, establishing Tim-3 as a negative regulatory molecule. Recent studies have uncovered additional mechanisms by which Tim-3 negatively regulates T cell responses, such as promoting the development of CD8 + T cell exhaustion and inducing expansion of myeloid-derived suppressor cells. In contrast to this inhibitory effect on T cells, Tim-3–Gal-9 interaction promotes macrophage clearance of intracellular pathogens. Here, we focus on the emerging role for Tim-3 in tumor and antimicrobial immunity.

The development and maturation of Vα14 invariant (i)NKT cells in mice requires CD1d-mediated lipid antigen presentation in the thymus and the periphery. Cortical thymocytes mediate positive selection, while professional APCs are involved in thymic negative selection and in terminal maturation of iNKT cells in the periphery. CD1d requires entry in the endosomal pathway to allow antigen acquisition for assembly as lipid/CD1d complexes for display to iNKT cells. This process involves tyrosine-based sorting motifs in the CD1d cytoplasmic tail and invariant chain (Ii) that CD1d associates with in the endoplasmic reticulum. The function of Ii in iNKT cell thymic development and peripheral maturation had not been fully understood. Using mice deficient in Ii and the Ii-processing enzyme cathepsin S (catS), we addressed this question. Ii(-/-) mice but not catS(-/-) mice developed significantly fewer iNKT cells in thymus, that were less mature as measured by CD44 and NK1.1 expression. Ii(-/-) mice but not catS(-/-) mice developed fewer Vβ7(+) cells in their iNKT TCR repertoire than WT counterparts, indicative of a change in endogenous glycolipid antigen/CD1d-mediated iNKT cell selection. Finally, using a Mycobacterium tuberculosis infection model in macrophages, we show that iNKT developed in Ii(-/-) but not catS(-/-) mice have defective effector function. Our data support a role for professional APCs expressing Ii, but no role for catS in the thymic development and peripheral terminal maturation of iNKT cells.

Maternal HIV-1-specific antibodies are efficiently transferred to newborns, but their role in disease control is unknown. We administered neutralizing IgG, including the human neutralizing monoclonal IgG1b12, at levels insufficient to block infection, to six newborn macaques before oral challenge with simian-HIV strain SF162P3 (SHIV(SF162P3)). All of the macaques rapidly developed neutralizing antibodies and had significantly reduced plasma viremia for six months. These studies support the use of neutralizing antibodies in enhancing B cell responses and viral control in perinatal settings.

Despite progress in the use of antiretroviral drugs, mother-to-child transmission of HIV still remains a serious medical problem in resource-poor areas. There is a need to find the best method for drug delivery to reduce transmission, while keeping the risk of selection for drug-resistant viral variants low. Even when infection is prevented during pregnancy, the risk of acquiring infection by breast feeding remains significant and in some settings, is unavoidable. The ability of antiretroviral drugs or vaccines to limit transmission by breast milk is unknown. HIV vaccines are still in an early phase of development and have not yet been tested in newborns, in part due to concerns about potential of low immunogenicity due to transplacental transfer of maternal antibodies. Alternative strategies have been proposed to limit transmission using passive prophylaxis by human monoclonal antibody, but to insure product safety, trials have been slowed. Due to such concerns, animal models may provide an alternative for testing efficacy in human newborns. In this review, advances made using such models will be compared for mother-to-child transmission of lentivirus with that of HIV-1. In addition, some perspectives on integrating the data obtained from these models as a groundwork for future clinical work will be presented.

We developed a SHIV/macaque model of transmission from infected dams to their infants. Ten pregnant dams were infected intravenously with 100 MID(50) of macaque-titered SHIV-SF162P3 during the second trimester. Nine infants were born; the seven surviving beyond day of birth suckled for 6 months. Four of nine infants were infected (transmission rate = 44.4%), with one infection in utero, and three intrapartum and/or immediately post-birth via suckling. Varying levels of binding and neutralizing antibodies were transplacentally transferred to infants. Passive antibodies were detected in plasma on the day of birth and persisted for 5 weeks. Infants infected at or after birth controlled acute and post-acute viremia. Exposure to maternal SHIV-SF162P3 during birth and suckling in the presence of autologous maternal neutralizing antibodies may have affected transmission or pathogenesis in the infants. This transmission model can allow investigation of key parameters involved in perinatal transmission of HIV.

In vivo control of Mycobacterium tuberculosis (Mtb) reflects the balance between host-immunity and bacterial evasion strategies. Effector TH1 cells that mediate protective immunity by depriving the bacterium of its intracellular niche are regulated in many ways to prevent over-exuberant inflammation. Although Tim3 is generally recognized to down regulate TH1 responses and mediate T cell exhaustion, we recently described that its interaction with Galectin-9 (Gal9) expressed by Mtb infected macrophages (Mϕ) stimulates IL-1β secretion, which is essential for host resistance to Mtb. IL-1β signaling is both necessary for Tim3 mediated Mtb control and sufficient to directly kill Mtb in murine and human Mϕ. The molecular mechanism by which IL-1β restricts bacterial growth is not known. We report that IL-1β kills Mtb through the recruitment of other antimicrobial effector molecules. IL-1β directly augments TNF signaling through increased TNF secretion and TNFR cell surface expression. Furthermore, Tim3/Gal9 and IL-1β activates the executioner caspase-3. Thus, Tim3/Gal9, via IL-1β and downstream TNF production lead to apoptosis and restriction of intracellular Mtb growth. We propose that Tim3/Gal9 interaction acts as a bidirectional molecular rheostat that activates pathways to clear intracellular pathogens in innate immune cells while fine-tuning termination of TH1 responses to prevent immunopathology.