Effector Cytokine Production Research Articles

Trogocytosis-Mediated Intracellular Signaling in CD4+ T Cells Drives TH2-Associated Effector Cytokine Production and Differentiation.

CD4+ T cells have been observed to acquire APC-derived membrane and membrane-associated molecules through trogocytosis in diverse immune settings. Despite this, the consequences of trogocytosis on the recipient T cell remain largely unknown. We previously reported that trogocytosed molecules on CD4+ T cells engage their respective surface receptors, leading to sustained TCR signaling and survival after APC removal. Using peptide-pulsed bone marrow-derived dendritic cells and transfected murine fibroblasts expressing antigenic MHC:peptide complexes as APC, we show that trogocytosis-positive CD4+ T cells display effector cytokines and transcription factor expression consistent with a TH2 phenotype. In vitro-polarized TH2 cells were found to be more efficient at performing trogocytosis than TH1 or nonpolarized CD4+ cells, whereas subsequent trogocytosis-mediated signaling induced TH2 differentiation in polarized TH1 and nonpolarized cells. Trogocytosis-positive CD4+ T cells generated in vivo also display a TH2 phenotype in both TCR-transgenic and wild-type models. These findings suggest that trogocytosis-mediated signaling impacts CD4+ T cell differentiation and effector cytokine production and may play a role in augmenting or shaping a TH2-dominant immune response.

Helios deletion in CD4+T cells induces pTregs that inhibit memory recall responses

Abstract The transcription factor Helios is required for the maintenance of the suppressive phenotype of Foxp3+ regulatory T cells (Tregs) as evidenced by the spontaneous autoimmune activation that develops in mice with a Foxp3− specific Helios deficiency. Helios is also upregulated in a subset of activated CD4+Foxp3−T conventional (Tconv) cells, but its role in Tconv cells remains unknown. Mice with a CD4-Cre driven Helios deletion do not exhibit autoimmunity despite the defect in the suppressor function of their Treg population, pointing to a requisite function of Helios in the activation of Tconv cells in response to antigen. We examined the capacity of Helios-deficient, naïve Tconv cells to respond to their cognate antigen in vivo and found that Helios was not required for proliferation, differentiation into helper subsets, or the production of effector cytokines. Unexpectedly, we observed that Helios deficiency increased the susceptibility of naïve T cells to differentiate into pTregs under inflammatory conditions. These pTregs were then subsequently able to suppress the reactivation of memory T cells in an antigen-specific manner upon challenge with antigen. These findings suggest that increased pTreg differentiation in the periphery in mice with a CD4-Cre driven Helios deletion may prevent the development of autoimmunity normally associated with Helios-deficient Tregs.

Selective inhibition of gelatinases modulates cellular energetics in CD4+ T-cells

Abstract The gelatinases, MMP-2 and MMP-9, are proteolytic enzymes with diverse physiological roles. They are linked with various aspects of the immune response, such as leukocyte trafficking and cytokine activation. However, their dysregulation is associated with various pathologies such as metastasis in cancer progression and blood-brain barrier disruption in multiple sclerosis. Much of what is known about the gelatinases is affiliated with their activities in the extracellular milieu. However, recent studies have suggested that they contribute to normal intracellular function of various immune cell types, including T-cells. CD4+ T-cells play a key role in linking the innate and adaptive arms of the immune response, as well as determining the character of the immune response. Our findings show, for the first time, that intracellular gelatinase activity can modulate T-cell bioenergetics, affecting both oxidative phosphorylation and glycolytic pathways and resulting in a mitigated CD4+ T-cell response to stimulation. Upon stimulation, CD4+ T-cells lacking gelatinase activity demonstrate a reduced capacity to function as determined by the expression of early activation markers, proliferation, and the production of effector cytokines. In addition, RNA-seq analysis of control and gelatinase inhibited CD4+ T-cells highlight key proteins associated with T-cell receptor signaling, cytokine production, and metabolism. These results suggest that gelatinases can be immunomodulatory agents in CD4+ T-cell mediated diseases, such as multiple sclerosis, and thus have therapeutic potential.

A powerful ICOS agonist that enhances anti-tumor immune responses restored by immune checkpoint inhibitors

Abstract The Inducible T cell Costimulator (ICOS, CD278) is a receptor in the CD28 family of B7-binding proteins expressed mostly by activated T cells. Upon binding to its ligand ICOS-L expressed on APCs,T cells are further activated by ICOS, resulting in their expansion and production of effector cytokines. Clinical data in patients treated with anti-CTLA-4 or anti-PD-1 mAbs have shown that ICOShiT cells correlated with an increased treatment response in these patients. Here, we report the design of a high avidity pentavalent form of the human ICOS-L extracellular domain fused to a short α-helical peptide from the human cartilage oligomeric matrix protein (COMP). This ICOS agonist, termed ICOS-L.COMP, spontaneously assembles into stable pentamers, binds tightly to both human and murine ICOS (SPR; Kd<10 nM) and co-stimulates (with anti CD3 mAb) the proliferation and cytokine release of both murine and human CD4+ and CD8+ T cells when added as a soluble factor (unlike ICOS-L.Fc). Soluble ICOS-L.COMP also phosphorylates Akt in activated T cells and enhances anti-tumor immune responses in tumor-bearing mice [murine MC38 or CT26 tumors] treated with either anti-murine PD-1 or anti-murine CTLA-4 mAb. Impressively, most mice treated with ICOS-L.COMP combined with an immune checkpoint inhibitor were either cured or displayed a stable tumor burden in contrast to checkpoint monotherapies (delayed tumor progression) or when ICOS-L.COMP was given as a monotherapy (comparable to no treatment). Importantly, ICOS-L.COMP lacks a Fc domain thus eliminating Fc-associated off-target adverse effects. In summary, our results suggest that ICOS-L.COMP represents a new and powerful biologic to be used in combination with existing immune checkpoint inhibitors.

Type1 IFN signaling on Tregs modulates the migration of Myeloid Derived Suppressor Cells

Abstract Multiple sclerosis is an autoimmune disorder whose severity is reduced with immune suppressive drugs. Therapeutic intervention with interferon-beta reduces disease exacerbations and delays relapses. The receptor for type 1 interferon, IFNAR, is present on virtually all cell types making it difficult to dissect the mechanisms involved. To address this issue, we induced experimental autoimmune encephalomyelitis (EAE) in mice with a conditional deletion (cKO) of IFNAR in T regulatory (Treg) cells (IFNARfl/flxFoxp3YFP-Cre). cKO mice developed severe EAE with an earlier onset than control mice. Although Treg cells from cKO mice appeared to be more activated, the activation status of and effector cytokine production by CD4+T cells in the draining lymph nodes (dLN) was similar in WT and cKO mice during the priming phase, but higher in cKO CD4+T cells in the effector phase. Interestingly, we noted a substantial reduction of myeloid derived suppressor cells (MDSCs) in the dLN of cKO mice, while generation of MDSCs in bone marrow and recruitment to spleen of WT and cKO mice were comparable. Surprisingly, we noted a decrease in the production of monocyte and granulocyte cell recruiting chemokine ligands by CD4+ and CD8+ T cells, B cells and endothelial cells in the dLN during the priming phase in the cKO mice. While modulation of Treg cell number and function by MDSCs has been documented, this study is one of the first to demonstrate that Treg cells modify the lymph node microenvironment by modulating MDSC homing and raises the possibility of a novel role for Treg cells in regulating the kinetics of MDSC recruitment during inflammatory conditions. This research was supported by the Intramural Research Program of NIAID, NIH.

Mucosal immune mediated protection of the CNS following olfactory viral infection

Abstract Barrier immunity, particularly at mucosal surfaces, provides a first line of defense against invading pathogens. Unlike classical mucosal barrier surfaces in the gut and respiratory tract, the olfactory epithelium within the nose is a specialized barrier structure containing layers of olfactory sensory neurons (OSNs) dedicated to our sense of smell. Within nasal airways, OSNs are vulnerable to infection but also directly connected to the brain, thus anatomically, the olfactory epithelium functions as a mucosal barrier to the central nervous system (CNS). Upper respiratory viral infections can damage the olfactory neuroepithelium and are associated with loss of olfaction. Neurotropic viruses, such as vesicular stomatitis virus (VSV), readily infect OSNs, yet the olfactory epithelial response to infection is unknown. VSV infection drove a massive influx of hematopoietic cells including antigen presenting cells and antigen specific CD4+ and CD8+ T cells that established residence within the olfactory epithelium after viral clearance. Upon rechallenge, neutralizing antibody completely protected the neuronal tissues of the olfactory mucosa and the CNS, however this protection appeared to be secretory IgA independent. Resident memory T cells within the olfactory mucosa rapidly produced effector cytokines and prevented heterotypic viral challenge from accessing the CNS. Olfactory VSV infection also resulted in widespread viral and immune mediated tissue damage that contributed to permanent changes in airway turbinate structure. Collectively, these studies offer insight into how the olfactory barrier remodels its neural-immune architecture to provide enhanced protection following a viral challenge.

The fate of leukemia-specific CD8+ T cell clones is dictated by antigen specificity and the mechanism of cognate antigen presentation

Abstract We previously demonstrated that high-affinity leukemia-specific CD8+ T cells are deleted in leukemia-bearing hosts in a manner that depends on cross-presentation of the antigen by splenic Batf3-lineage dendritic cells. Here, we generated a T cell receptor transgenic mouse strain (Tg101) that expresses TCR-α and -β chains from a CD8+ T cell clone specific for a native H-2Kb-restricted antigen on C1498 leukemia cells. In naïve mice, Tg101 T cells demonstrate no evidence of self-reactivity, arguing that the Tg101 antigen is leukemia-specific. In leukemia-bearing mice, high-affinity 2C CD8+ T cells specific for the H-2Kb-restricted model antigen, SIYRYYGL, encounter antigen rapidly (day 3–4) via its cross-presentation by splenic CD8a+ DCs, proliferate briefly, and are largely deleted by day 12. Conversely, antigen encounter by Tg101 CD8+ T cells in mice with leukemia is delayed (day 8–10), and primarily occurs in the liver - a prominent site of leukemia progression. Subsequently, Tg101 T cells expand, upregulate expression of co-inhibitory receptors, including PD-1, LAG-3, TIM-3 and TIGIT, and produce effector cytokines poorly, consistent with the acquisition of a dysfunctional state. Surprisingly, in vivo antigen encounter by Tg101 T cells occurs independently of CD11c+ cells, indicating that the Tg101 antigen is poorly cross-presented. Rather, the antigen is directly presented to Tg101 T cells by C1498 cells. Collectively, our data reveal that CD8+ T cell tolerance is effectively generated in leukemia-bearing animals. However, the nature of the leukemia antigen and the context in which it is presented are critical factors in dictating the underlying mechanism of tolerance that ensues. A search for the Tg101 antigen is ongoing.

The tumor necrosis factor superfamily member RANKL suppresses effector cytokine production in group 3 innate lymphoid cells

Abstract While signals that activate group 3 innate lymphoid cells (ILC3s) have been described, the factors that negatively regulate these cells are less well understood. Here we found that the tumor necrosis factor (TNF) superfamily member receptor activator of nuclear factor kB ligand (RANKL) suppressed ILC3 activity in the intestine. Deletion of RANKL in ILC3s and T cells increased C-C motif chemokine receptor 6 (CCR6)+ ILC3 abundance and enhanced production of interleukin-17A (IL-17A) and IL-22 in response to IL-23 and during infection with the enteric murine pathogen Citrobacter rodentium. Additionally, CCR6+ ILC3s produced higher amounts of the master transcriptional regulator RORγt at steady state in the absence of RANKL. RANKL-mediated suppression was independent of T cells, and instead occurred via interactions between CCR6+ ILC3s that expressed both RANKL and its receptor, RANK. Thus, RANK-RANKL interactions between ILC3s regulate ILC3 abundance and activation, suggesting that cell clustering may control ILC3 activity.

STING-associated lung disease in mice relies on T cells but not type I interferon

Monogenic interferonopathies are thought to be mediated by type I interferon. For example, a gain-of-function mutation in stimulator of interferon genes (STING; N153S) upregulates type I interferon-stimulated genes and causes perivascular inflammatory lung disease in mice. The equivalent mutation in human subjects also causes lung disease, which is thought to require signaling through the cyclic GMP-AMP synthase (cGAS)-STING pathway and subsequent activation of interferon regulatory factors (IRFs) 3 and 7, type I interferon, and interferon-stimulated genes. We set out to define the roles of cGAS, IRF3, IRF7, the type I interferon receptor (IFN-α and IFN-β receptor subunit 1 [IFNAR1]), T cells, and B cells in spontaneous lung disease in STING N153S mice. STING N153S mice were crossed to animals lacking cGAS, IRF3/IRF7, IFNAR1, adaptive immunity, αβ T cells, and mature B cells. Mice were evaluated for spontaneous lung disease. Additionally, bone marrow chimeric mice were assessed for lung disease severity and survival. Lung disease in STING N153S mice developed independently of cGAS, IRF3/IRF7, and IFNAR1. Bone marrow transplantation revealed that certain features of STING N153S-associated disease are intrinsic to the hematopoietic compartment. Recombination-activating gene 1 (Rag1)-/- STING N153S mice that lack adaptive immunity had no lung disease, and T-cell receptor β chain (Tcrb)-/- STING N153S animals only had mild disease. STING N153S led to a reduction in percentages and numbers of naive and regulatory T cells, as well as an increased frequency of cytokine-producing effector T cells. Spontaneous lung disease in STING N153S mice develops independently of type I interferon signaling and cGAS. STING N153S relies primarily on T cells to promote lung disease in mice.

Memory NK Cell Features Exploitable in Anticancer Immunotherapy.

Besides their innate ability to rapidly produce effector cytokines and kill virus-infected or transformed cells, natural killer (NK) cells display a strong capability to adapt to environmental modifications and to differentiate into long-lived, hyperfunctional populations, dubbed memory or memory-like NK cells. Despite significant progress in the field of NK cell-based immunotherapies, some factors including their short life span and the occurrence of a tumor-dependent functional exhaustion have limited their clinical efficacy so that strategies aimed at overcoming these limitations represent one of the main current challenges in the field. In this scenario, the exploitation of NK cell memory may have a considerable potential. This article summarizes recent evidence in the literature on the peculiar features that render memory NK cells an attractive tool for antitumor immunotherapy, including their long-term survival and in vivo persistence, the resistance to tumor-dependent immunosuppressive microenvironment, the amplified functional responses to IgG-opsonized tumor cells, and in vitro expansion capability. Along with highlighting these issues, we speculate that memory NK cell-based adoptive immunotherapy settings would greatly take advantage from the combination with tumor-targeting therapeutic antibodies (mAbs), as a strategy to fully unleash their clinical efficacy.

Abstract A030: Identification of α-fetoprotein-specific T-cell receptors for hepatocellular carcinoma immunotherapy

Abstract Hepatocellular carcinoma (HCC) is the major form of liver cancer for which there is no effective therapy. Genetic modification with T-cell receptors (TCR) specific for HCC-associated antigens, such as α-fetoprotein (AFP), can potentially redirect human T-cells to specifically recognize and kill HCC tumor cells to achieve antitumor effects. In this study, by using lentivector and peptide immunization, we identified a population of CD8-T-cells in HLA-A2 transgenic AAD mice that recognized AFP158 epitope on human HCC cells. Adoptive transfer of the AFP158-specific mouse CD8-T-cells eradicated HepG2 tumor xenografts as large as 2cm in diameter in immunocompromised NSG mice. We then established T-cell hybridoma clones from the AFP158-specific mouse CD8-T-cells and identified three sets of paired TCR genes out of 5 hybridomas. Expression of the murine TCR genes redirected primary human T-cells to bind HLA-A2/AFP158 tetramer. The TCR gene-engineered human T-cells (TCR-T) also specifically recognized HLA-A2+AFP+ HepG2 HCC tumor cells and produced effector cytokines. Importantly, the TCR-T-cells could specifically kill HLA-A2+AFP+ HepG2 tumor cells without significant toxicity to normal primary hepatocytes in vitro. Adoptive transfer of the AFP-specific human TCR-T-cells could eradicate HepG2 tumors in NSG mice. Conclusion: We have identified novel AFP-specific murine TCR genes that can redirect human T-cells to specifically recognize and kill HCC tumor cells, and those AFP158-specific TCRs have a great potential to engineer a patient’s autologous T-cells to treat HCC tumors. Citation Format: Yukai He, Wei Zhu, Yibing Peng, Lan Wang, Yuan Hong, Juan Wu, Esteban Celis. Identification of α-fetoprotein-specific T-cell receptors for hepatocellular carcinoma immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A030.

Abstract A196: Systematic identification of markers and drug targets on exhausted CD8+ T-cells

Abstract CD8+ T-cells are responsible for detection and killing of hosT-cells expressing non-self proteins. Upon antigen persistence, as in cancer or chronic viral infection, antigen-specific CD8+ T-cells lose their effector function, a phenomenon known as T-cell exhaustion. This process is concomitant with increased surface expression of negative regulatory proteins such as PD-1 and CTLA-4. Blockade of these molecules has become a widespread and successful clinical strategy for the treatment of malignancies. Unfortunately, not all tumor types are responsive to checkpoint molecule blockade, nor do all patients with susceptible tumor types exhibit clinical responses. These failures highlight the need for identification of additional negative regulators on exhausted, tumor-specific CD8+ T-cells. Here, we perform a systematic approach to identify surface proteins expressed on exhausted murine and human CD8+ T-cells. Using RNA-sequencing data from antigen-specific CD8+ T-cells from the mouse lymphocytic choriomeningitis virus (LCMV) infection model as well as data from human tumor-infiltrating CD8+ T-cells, we show high cross-species concordance of genes encoding membrane proteins in T-cell exhaustion. These expression profiles not only include many known checkpoint molecules such as PD-1, but also several proteins that negatively regulate T-cell receptor signaling but are largely unexplored in the process of CD8+ T-cell exhaustion. We validate these results performing by flow cytometry on both mouse and human CD8+ T-cells, including on T-cells obtained from resected human tumor tissue. To substantiate the importance of these markers, we further explore the function of one identified surface protein, CD101, in the murine LCMV infection model. We demonstrate that CD101 is induced on antigen-specific of CD8+ T-cells weeks after infection, in contrast with the early expression of canonical exhaustion markers such as PD-1. We show that antigen-specific CD101+ cells arise from CD101- precursors and that this differentiation process results in a loss of effector function, cytokine production, and proliferative capacity with simultaneous large transcriptomic changes. Together, these results demonstrate that exhaustion-associated surface protein expression patterns are largely conserved between humans and mice and provide a comprehensive catalog of phenotypic markers and potential drug targets for CD8+ T-cell-directed immunotherapy. Citation Format: Will H. Hudson, Julia L. Gensheimer, Haydn T. Kissick, Rafi Ahmed. Systematic identification of markers and drug targets on exhausted CD8+ T-cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A196.

Abstract A215: Thymocyte selection-associated HMG box protein TOX is a master regulator of tumor-specific T-cell dysfunction

Abstract Tumor-specific CD8 T-cells in cancers enter a state of dysfunction characterized by the expression of inhibitory receptors and failure to produce effector cytokines and cytotoxic molecules. Here we identify the nuclear factor, Thymocyte selection-associated HMG box protein, TOX, as a master regulator of tumor-specific T-cell dysfunction. TOX is uniquely expressed in dysfunctional CD8 T-cells from mouse and human tumors but absent in functional T-cells. TOX expression is driven by continuous TCR stimulation and NFAT activity. Forced expression of TOX in functional effector T-cells was sufficient to induce a transcriptional program of dysfunction through the concerted expression of genes encoding numerous inhibitory receptors and dysfunction-associated transcription factors. Notably, TOX-deficient tumor-infiltrating T-cells did not upregulate inhibitory receptors such as PD1, LAG3, CD38, or CD39 and maintained high TCF1 expression. Surprisingly, despite their normal, “non-exhausted” phenotype, TOX-deficient T-cells failed to make effector cytokines, suggesting that loss of effector function in tumor-specific T-cells is uncoupled from inhibitory receptor expression. Furthermore, TOX-deficient T-cells failed to persist in tumors, ultimately undergoing activation-induced cell death. We propose that the TOX-induced transcriptional program of hyporesponsiveness is a physiologic negative feedback mechanism that prevents overstimulation; thus TOX is absolutely required for T-cell survival in the setting of chronic antigen stimulation as in cancers. Citation Format: Andrew C. Scott, Steven Camara, Peter Lauer, Alexandra Synder, Dmitriy Zamarin, Tyler Walther, Olivier Levy, Michael Glickman, Jonathan Kaye, Mary Philip, Andrea Schietinger. Thymocyte selection-associated HMG box protein TOX is a master regulator of tumor-specific T-cell dysfunction [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A215.

Abstract B146: The tumor necrosis factor superfamily member RANKL suppresses effector cytokine production in group 3 innate lymphoid cells

Abstract While signals that activate group 3 innate lymphoid cells (ILC3s) have been described, the factors that negatively regulate these cells are less well understood. Here we found that the tumor necrosis factor (TNF) superfamily member receptor activator of nuclear factor κB ligand (RANKL) suppressed ILC3 activity in the intestine. Deletion of RANKL in ILC3s and T-cells increased C-C motif chemokine receptor 6 (CCR6)+ ILC3 abundance and enhanced production of interleukin-17A (IL-17A) and IL-22 in response to IL-23 and during infection with the enteric murine pathogen Citrobacter rodentium. Additionally, CCR6+ ILC3s produced higher amounts of the master transcriptional regulator RORγt at steady state in the absence of RANKL. RANKL-mediated suppression was independent of T-cells, and instead occurred via interactions between CCR6+ ILC3s that expressed both RANKL and its receptor, RANK. Thus, RANK-RANKL interactions between ILC3s regulate ILC3 abundance and activation, suggesting that cell clustering may control ILC3 activity. Citation Format: Jennifer Kaoru Bando, Susan Gilfillan, Christina Song, Keely McDonald, Stanley C-C. Huang, Rodney D. Newberry, Yasuhiro Kobayashi, David S.J. Allan, James R. Carlyle, Marina Cella, Marco Colonna. The tumor necrosis factor superfamily member RANKL suppresses effector cytokine production in group 3 innate lymphoid cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B146.

Innate Lymphoid Cells: A Link between the Nervous System and Microbiota in Intestinal Networks.

Innate lymphoid cells (ILCs) are a novel family of innate immune cells that act as key coordinators of intestinal mucosal surface immune defense and are essential for maintaining intestinal homeostasis and barrier integrity by responding to locally produced effector cytokines or direct recognition of exogenous or endogenous danger patterns. ILCs are also involved in the pathogenesis of inflammatory bowel disease (IBD). Many studies have demonstrated the occurrence of crosstalk between ILCs and intestinal microbiota, and ILCs have recently been shown to be connected to the enteric nervous system (ENS). Thus, ILCs may act as a key link between the nervous system and microbiota in intestinal networks. In this review, we briefly summarize the role of the ILCs in the intestinal tract (particularly in the context of IBD) and discuss the relationship between ILCs and the microbiota/ENS.

CCR9 Expressing T Helper and T Follicular Helper Cells Exhibit Site-Specific Identities During Inflammatory Disease.

CD4+ T helper (Th) cells that express the gut homing chemokine receptor CCR9 are increased in the peripheral blood of patients with inflammatory bowel disease and Sjögren's syndrome and in the inflamed lesions of autoimmune diseases that affect the accessory organs of the digestive system. However, despite the important role of the GIT in both immunity and autoimmunity, the nature of CCR9-expressing cells in GIT lymphoid organs and their role in chronic inflammatory diseases remains unknown. In this study, we analyzed the characteristics of CCR9+ Th and T follicular helper (Tfh) cells in GIT associated lymphoid tissues in health, chronic inflammation and autoimmunity. Our findings reveal an association between the transcriptome and phenotype of CCR9+ Th in the pancreas and CCR9+ Tfh cells from GIT-associated lymphoid tissues. GIT CCR9+ Tfh cells exhibited characteristics, including a Th17-like transcriptome and production of effector cytokines, which indicated a microenvironment-specific signature. Both CCR9+ Tfh cells and CCR9+ Th cells from GIT-associated lymphoid tissues migrated to the pancreas. The expression of CCR9 was important for migration of both subsets to the pancreas, but Tfh cells that accumulated in the pancreas had downmodulated expression of CXCR5. Taken together, the findings provide evidence that CCR9+ Tfh cells and Th cells from the GIT exhibit plasticity and can accumulate in distal accessory organs of the digestive system where they may participate in autoimmunity.

The Potential for Cancer Immunotherapy in Targeting Surgery-Induced Natural Killer Cell Dysfunction.

Natural Killer (NK) cells are granular lymphocytes of the innate immune system that are able to recognize and kill tumor cells without undergoing clonal selection. Discovered over 40 years ago, they have since been recognized to possess both cytotoxic and cytokine-producing effector functions. Following trauma, NK cells are suppressed and their effector functions are impaired. This is especially important for cancer patients undergoing the removal of solid tumors, as surgery has shown to contribute to the development of metastasis and cancer recurrence postoperatively. We have recently shown that NK cells are critical mediators in the formation of metastasis after surgery. While research into the mechanism(s) responsible for NK cell dysfunction is ongoing, knowledge of these mechanisms will pave the way for perioperative therapeutics with the potential to improve cancer outcomes by reversing NK cell dysfunction. This review will discuss mechanisms of suppression in the postoperative environment, including hypercoagulability, suppressive soluble factors, the expansion of suppressive cell populations, and how this affects NK cell biology, including modulation of cell surface receptors, the potential for anergy, and immunosuppressive NK cell functions. This review will also outline potential immunotherapies to reverse postoperative NK dysfunction, with the goal of preventing surgery-induced metastasis.

Notch Activation Rescues Exhaustion in CISH-Deleted Human iPSC-Derived Natural Killer Cells to Promote In Vivo Persistence and Enhance Anti-Tumor Activity

Cytokine-inducible SH2-containing protein (CIS) is a critical negative regulator of IL-15 signaling in natural killer (NK) cells and is encoded by CISH gene in human. Recent studies in a murine model demonstrate that CIS is a potent inhibitory checkpoint in NK cell-mediated tumor immunity. However, it is unclear if CIS similarly regulates human NK cell-mediated anti-tumor activity. Unlike CTLA-4 and PD1 that are expressed at the cell surface and can be blocked by antibody-mediated therapy, CIS is expressed intracellularly. Therefore, we deleted CISH gene in human induced pluripotent stem cells (iPSCS) using CRISPR/Cas9 technology and characterized the CISH-knockout iPSC lines. Our study demonstrates that CISH does not regulate maintenance of undifferentiated human pluripotent stem cells. We then derived NK cells from these gene-modified iPSCs using a two-stage in vitro hematopoietic-lymphoid differentiation protocol developed by our group. Initial hematopoietic differentiation of the iPSCs was unaffected by CISH deletion. However, deletion of CISH in iPSCs markedly delayed the second stage of in vitro NK cell differentiation. Specifically, whereas NK cell differentiation was typically fully complete with >90% NK cells after 4 weeks using WT iPSCs, the CISH-/- iPSC- cells only produced ~10% CD45+CD56+ NK cells at 4 weeks, though by 5 weeks these cultures were >80% NK cells. The CISH-/- iPSC-derived NK cells demonstrated typical NK surface maker expression, including CD94, CD16, NKG2D, NKp44, NKp46, FasL, and KIRs. Initial studies demonstrated that CISH-/- human iPSC-derived NK cells had significantly reduced ability to expand in vitro with evidence of NK cell exhaustion, including increased TIM-3 expression, decreased IFN-γ production and decreased cytotoxicity. RNA-seq analysis also confirmed that expression of exhaustion-related genes, including TIM3, CTLA4, and 2B4, were all significantly increased in CISH-/- NK cells. Interestingly, mass cytometric (CyTOF) analysis of CISH-/- NK cells with a panel of 36 phenotypic and functional NK cell markers identified an exhausted sub-population (increased expression of exhaustion marker TIM3 and inhibitory receptors such as ILT2 and Siglec7, as well as decreased proliferation marker Ki67). Importantly, this exhaustion could be rescued by co-culturing CISH-/- NK cells with Notch ligand-expressing OP9-DL4 stromal cells, leading to production of fully functionally mature NK cells. The CISH-/- NK cells stimulated by Notch maintained better expansion and improved cytotoxic function with low concentrations of either IL15 or IL2 compared to WT-iPSC-derived NK cells. CISH-/- NK cells demonstrate increased cytotoxic activity against leukemia cell lines K562 and MOLM-13 cells in vitro. Moreover, single-cell cytokine response analysis of CISH-/- NK cells after Notch stimulation showed >10 fold enrichment of polyfunctional cell subsets with effector cytokine production (Granzyme B, IFN-γ, MIP-1α, Perforin, TNF-α) compared with WT NK cells or NK cells from peripheral blood. In a MOLM-13 xenograft model, CISH-/- NK cells displayed significantly increased persistence in peripheral blood in comparison with WT NK cells (CISH-/- NK cells 6.81 ± 0.85 % vs WT NK cells 2.05 ± 0.25 N=5). More importantly, CISH-/- NK cells show significantly better control of tumor progression in the MOLM-13 xenograft model compared with WT NK cells. Together, these studies demonstrate CISH plays a key role to regulate NK cell activation-induced exhaustion and that Notch activation prevents this exhaustion to enable production of functionally hyperactive NK cells. DisclosuresGuan:Vivace: Equity Ownership. Malmberg:Fate Therapeutics Inc.: Consultancy, Research Funding. Kaufman:Fate Therapeutics: Consultancy, Research Funding.

Ex Vivo Evidence for the Combination of Immune Checkpoint Inhibition with TGF-β Blockade to Enhance Anti-Tumor T Cell Responses in Multiple Myeloma

Immunosuppressive milieu of multiple myeloma is associated with various cellular and non-cellular factors that foster immune escape leading to tumor progression. While immune checkpoint inhibitors have achieved significant clinical success in many types of solid tumors, recent clinical trials of immune checkpoint blockade performed in patients with multiple myeloma failed to demonstrate significant anti-tumor efficacy. To enhance the clinical efficacy of immune checkpoint blockade in multiple myeloma, elaborate characterization of tumor antigen-specific T cells is an essential prerequisite. In the present study, we investigated the immunophenotypic and functional characteristics of tumor antigen-specific T cells in patients with multiple myeloma. In addition, using direct ex vivo experimental techniques, we tried to examine how to manipulate the immunosuppressive microenvironment to maximize anti-myeloma responses of the tumor-specific T cells.We first tried to define and characterize CD8+ T cell population capable of specifically recognizing and reacting to myeloma cells in bone marrow of myeloma patients using MHC multimer technique. In selected patients, we could successfully define CD8+ T cell population specifically recognizing the HLA-A*0201-restricted epitopes (either “SLLMWITQC” or “LLLGIGILV”), included in myeloma tumor antigens NY-ESO-1 and HM1.24. The vast majority of myeloma-antigen specific CD8+ T cells expressed high level of PD-1 and also co-expressed other types T cell inhibitory receptors. More strikingly, PD-1+ myeloma-specific CD8+ T cells had a distinct pattern of transcriptional factor expression, high level of Eomes and low level of T-bet (EomeshiT-betlo), indicating that they were profoundly exhausted functionally and that a simple blockade of PD-1/PD-L1 binding might not be enough to reinvigorate their anti-myeloma activity.Consistent with the immunophenotypes of myeloma-specific CD8+ T cells, malignant plasma cells in bone marrow of myeloma patients, defined as CD14-CD19-CD138+CS1+CD56hi, also expressed PD-L1 abundantly, compared to normal plasma cells, suggesting that PD-1/PD-L1 axis plays a major role in making myeloma-recognizing T cells unresponsive to TCR stimulation. Interestingly, in addition to tumor cells, various types of immune cells comprising myeloma microenvironment also highly express PD-L1. Indeed, in response to ex vivo TCR stimulation with anti-CD3, CD8+ T cells from myeloma bone marrow showed lower proliferation and reduced production of anti-tumor effector cytokines (INF-γ and TNF-α), compared to marrow-infiltrating CD8+ T cells of diffuse large B cell lymphoma and Hodgkin lymphoma patients with extensive bone marrow involvement. However, even in the presence of anti-PD-1, myeloma-specific responses of marrow-infiltrating CD8+ T cells was only modestly improved in terms of proliferation and cytokine production.Next, we investigated whether blocking TGF-β signaling in combination with PD-1/PD-L1 axis blockade could restore the function of marrow-infiltrating CD8+ T cells of myeloma patients, since TGF-β produced by clonal plasma cells and bone marrow stromal cells is critical in immunosuppressive microenvironment of multiple myeloma. To this end, we found that combination of TGF-β signaling blockade (either anti-TGF-β1 or Galunisertib, a small molecule inhibitor of TGF-β receptor I) with anti-PD-1 significantly increased the frequencies of IFN-γ- and/or TNF-α-producing CD8+ T cells in response to ex vivo TCR stimulation, compared to a single PD-1 or a single TGF-β blockade. Likewise, myeloma antigen-specific proliferation of CD8+ T cells was significantly enhanced with addition of TGF-β signaling blockade.Taken together, although PD-1/PD-L1 axis acts as a major component of immunosuppressive milieu in multiple myeloma, the efficacy of PD-1 blockades in multiple myeloma might be hampered by complicated microenvironment consisting of T cell-intrinsic and -extrinsic factors. Our results provide an ex vivo evidence of incorporating TGF- β signaling blockade to immune checkpoint inhibition to enhance anti-tumor T cell responses in multiple myeloma DisclosuresNo relevant conflicts of interest to declare.

Human Double-Negative Regulatory T Cells Modulate Effector Functions of Conventional T Cells By Selectively Blocking mTOR Signaling

Regulatory T cells play an important role in the maintenance of immune tolerance to self-antigens and are involved in modulating immune responses to promote resolution of inflammation. The population of TCRαβ+ CD4-/CD8- (double-negative, DN) T cells has attracted growing attention as a result of their potent immune regulatory function. In murine models, DN T cells were able to prevent rejection of allogeneic and xenogeneic organ grafts by effectively suppressing reactive T cells. In addition, DN T cells possess the capacity to resolve various inflammatory conditions, including graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation. Notably, first clinical studies in patients after stem cell transplantation demonstrated an inverse correlation between the frequency of circulating DN T cells and the severity of acute GvHD, suggesting a therapeutic potential of human DN T cells. To gain a better understanding of the molecular mechanism of suppression, we investigated whether human DN T cells modulate distinct signaling processes in conventional T cells. We found that DN T cells selectively block mTOR signaling but not activation of mitogen-activated protein kinases. Enforced activation of the mTOR pathway by a chemical activator rendered conventional T cells unsusceptible to DN T cell-mediated suppression, confirming the critical function of mTOR signaling. Given that mTOR is a major regulator of cellular metabolism, we further determined the impact of DN T cells on the metabolic framework of conventional T cells. Of interest, DN T cells diminished upregulation of the glycolytic machinery and glucose uptake in conventional T cells, whereas fatty acid uptake was not modified. Next, we investigated the fate and function of effector cells after DN T cell co-culture. Of importance, DN T cells suppressed proliferation but also altered expression of differentiation markers, transcription factors and homing receptors. Further analyses demonstrated that CD4+ T cells failed to produce effector cytokines IL-17 and IFN-γ after coculture with DN T cells, whereas IL-2 secretion was amplified. The selective modification was induced by a direct cell-cell contact dependent mechanism between CD4 and DN T cells and not as a consequence of competition for nutrients or growth factors. Together, our findings expand the understanding of DN T cell functionality and support that human DN T cells represent an interesting opportunity to limit and modulate T-cell reactivity. DisclosuresNo relevant conflicts of interest to declare.