bluuebunny/crossref_metadata_2025 huggingface dataset:
Goblet cells regulate expansion of colonic iNKT cells in CD1d-dependent manner
Authors: John Vini , Barrios Bibiana , Udayan Sreeram , Floyd Alexandria , Schill Ellen M , McDonald Keely G , Blumberg Richard S , Newberry Rodney D
Published: January 2023

Invariant natural killer T (iNKT) cells are innate-like T cells that secrete a wide array of cytokines and immune mediators. These cells recognize self or microbial ligands presented by cells expressing CD1d, and can contribute to host protection or/and pathogenesis during intestinal inflammation. The colonic iNKT cell population is established in early life under the influence of the microbiota and current understanding is that this iNKT cells are not manipulable in later life. Previous studies have identified that goblet cell associated passages (GAPs) play a role in luminal antigen delivery and the induction and maintenance of peripherally induced T regulatory cells in the steady state. Colonic GAPs are largely absent in adult mice due to goblet cell (GC) microbial sensing, which inhibits GAP formation. We hypothesized, that when present, colonic GAPs may deliver glycolipids to stimulate colonic iNKT cells. We found that the glycolipids can be delivered through GAPs and that inducing colonic GAPs in adult mice using pharmacological inhibitors or transgenic mouse models resulted in significant iNKT cell expansion. Further deletion of CD1d on GCs inhibited iNKT cell expansion suggesting a role for colonic GCs in presenting glycolipids to iNKT cells. Single cell RNA sequencing of sorted colonic iNKT cells showed significantly expanded iNKT2 and iNKT1 subsets after colonic GAP induction. Furthermore, the iNKT cells expanding after opening colonic GAPs were protective in DSS-induced colitis. Our findings suggest that the GAP function and CD1d expression by GCs plays a role in modulating colonic iNKT cell subsets in adulthood and can be protective in some colitis models.
The Novel Roles of Dusp6 in Gut Barrier Modulation and Microbiome Shaping in a Mouse Colitis Model
Authors: Kao Cheng-Yuan , Chang Cherng-Shyang , Liao Yi-Chu , Huang Chih-Ting , Lin Chiao-Mei , Tsai Yi-Ting , Lin I-Jung , Ruan Jhen-Wei , Liao Yu-Chieh
Published: January 2023

Gut microbiota dysbiosis and barrier integrity are important contributors to overall health and many diseases. Our study demonstrated that dual-specificity phosphatase 6 (Dusp6)-depletion is a novel modulator that enhances baseline gut barrier integrity. We found that Dusp6-knockout mice were more resistant to dextran sulfate sodium (DSS)-induced colitis and had enhanced intrinsic colonic tight-junctions and elongated microvilli before exposure to DSS. Our FMT in germ-free mice and co-housing experiments found that the Dusp6-knockout-derived gut microbiota contribute substantially to colitis resistance in mice. Further gut microbiome analysis showed that Dusp6-knockout mice harbored fewer pathobiont facultative anaerobes and more obligate anaerobes than wild-type mice after DSS treatment. Through multi-omics analysis, we found that epithelial barrier integrity and hypoxic and oxygen homeostasis regulation related pathways were more enriched in DUSP6-depleted Caco-2 cells and with a cultivation-based approach, we reported 11 new mouse gut microbiome members. Finally using microbe-phenotype triangulation and mono-colonization of germ-free mice, we found that the newly discovered Duncaniella species contributed to the colitis protection. In sum, identifying functioning gut microbiota members that shape host physiology and disease susceptibility will be vital to the advance of microbiota-based therapeutics.

This work was supported by the following grants: IM-108-PP-04 from NHRI and 106-2628-B-400-001-MY3, 106-2923-B-400-001-MY3, 108-2321-B-400-011-, 109-2327-B-400-001-, 109-2320-B-400-008-MY3, 110-2327-B-400-005- from MOST.
Th17-driven immunity to oral candidiasis is dependent on the microbiome and can be triggered by mono-colonization with segmented filamentous bacteria.
Authors: Aggor Felix E Y , Zhou Chunsheng , Abbott Darryl , Musgrove Javonn , Bruno Vincent , Hand Timothy W , Gaffen Sarah L
Published: January 2023

The opportunistic infection oropharyngeal candidiasis (OPC; oral thrush) is a common oral infection among the immunocompromised. The oral cavity is a major entry portal to the body and oral health impacts several aspects of host immunity. While much is now known about microbiome dependent Th17 induction and protection against intestinal candidiasis and other Th17 driven responses, our understanding of the role of the microbiome in oral Th17 induction and oral antifungal immunity is rather rudimentary. We demonstrate a direct role for the microbiome in triggering protection against OPC. While wildtype (WT) mice raised under specific pathogen free (SPF) conditions are resistant to OPC, WT germ free (GF) mice succumbed to infection. Reconstitution of WT-GF mice with segmented filamentous bacteria (SFB) was sufficient to restore protection against OPC with associated increase in Th17 cells, neutrophils, and antimicrobial responses in the oral mucosa. Thus, SFB mono-colonization in WT-GF mice triggers oral mucosal Th17 induction to protect against oral thrush and reveals a role of the microbiome in promoting oral mucosal immunity.

Supported by R37-DE022550
A novel, synthetic TLR7/8 agonist demonstrates monotherapy efficacy and synergy with anti-PD-1 in syngeneic mouse tumor models
Authors: Beyer Caleb A , Lussier Danielle , Jackson Konner , Whitacre Margaret , Ward Janine , Schoener Roman , Bazin Helene G , Burkhart David , Miller Shannon M
Published: January 2023

Cancer is the second leading cause of death in the United States, with more than half a million Americans succumbing to this disease every year. To date, immunotherapies such as anti-PD-1 and anti-CTLA-4 have shown some advancement toward treating certain cancer types, but efficacy is typically reserved for a small subset of cancer patients. Recent clinical trials have shown that melanoma and renal cell carcinoma patients had some of the highest rates of response to checkpoint treatment, but even then, the rate of response was only 20% and 22%, respectively. Furthermore, immunotherapies can lead to severe adverse reactions due to their ability to activate non-specific immune responses. Inimmune has developed and tested a novel TLR7/8 agonist as an immunotherapy for cancer. In vivo mouse studies demonstrated that IV treatment with our novel, synthetic TLR7/8 agonist in combination with anti-PD-1 resulted in increased efficacy in eliminating tumors or slowing tumor growth over anti-PD-1 treatment alone in B16F10 and MC38 tumor models in C57BL/6 mice. Furthermore, lead formulations of our novel, synthetic TLR7/8 agonist demonstrated efficacy as a monotherapy in BALB/c mice inoculated with a highly tumorigenic Lewis Lung Carcinoma (LLC) cell line. Taken together, our data indicate that this novel, synthetic TLR7/8 agonist was tolerated systemically, functioned as a monotherapy, and synergized well with anti-PD-1 treatment in various mouse syngeneic tumor models. As we move this compound to Phase 1 clinical trials, these data suggest that our TLR7/8 agonist can be dosed safely as a monotherapy or in combination with checkpoint therapy and may drive efficacious anti-tumor responses in humans.
Complement downregulation promotes an inflammatory signature that renders colorectal cancer susceptible to immunotherapy
Authors: Guglietta Silvia , Weber Lukas M , Fosso Bruno , Hardiman Gary , Olcina Monica M , Marzano Marinella , Robinson Mark D , Krieg Carsten
Published: January 2023

The role of inflammation in colorectal cancer (CRC) development and response to therapy is highly debated. While inflammation is a known driver of CRC, inflammatory immune infiltrates are a positive prognostic factor in CRC as inflammation can enhance response to immune checkpoint blockade (ICB) therapy. Yet, the majority of CRC are poorly infiltrated and even in presence of an inflammatory infiltrate half of these cancers become refractory to ICB. Therefore, there is a need to identify mechanisms to establish therapy supportive inflammation. We have previously reported that certain patients with CRC down-regulate the receptor for the complement anaphylatoxin C3a (C3aR). The complement system is a first line of defense against pathogens and a central component of the immune response. Emerging evidence suggests that C3aR may play a role in intestinal homeostasis. However, to date, it is unknown how C3aR affects CRC development and response to therapy with ICB.

Using our novel mouse model of CRC (APCMin/+/C3aR−/−) we showed that loss of C3aR results in enhanced immune infiltration in typically “cold” tumors and changes in the intestinal microbiota. Similarly, we found that down-regulation of the complement C3aR in patients correlates with changes in specific tumor-infiltrating immune cells. Notably, when treated with a-PD1, APCMin/+/C3aR−/− mice showed a significant reduction of tumors, suggesting that the loss of C3aR unleashes in the colon a microbiota-mediated immune response that can be exploited for therapeutic purposes.

Our findings reveal that C3aR may act as a previously unrecognized checkpoint to enhance antitumor immunity in CRC and that C3aR inhibition may be used to overcome ICB resistance in a larger group of CRC patients.

This project was supported in part by NIH P20 GM120475 (core facility) at the MUSC Digestive Disease Research Core Center.
CD28: A roadblock to designing long-lasting CARs?
Authors: Honikel Mackenzie M , Holling G Aaron , Burchett Rebecca , Bramson Jonathan , Lee Kelvin P , Olejniczak Scott H
Published: January 2023

Multiple myeloma (MM) remains an incurable hematological malignancy by conventional chemotherapeutic approaches. Chimeric antigen receptor T (CAR-T) cells targeting B cell maturation antigen (BCMA) on MM have shown remarkable efficacy in relapsed/refractory patients, leading to their recent FDA approval. However, many MM patients fail to achieve durable remissions leading to relapse in the months following treatment, highlighting the need to better understand mechanisms of CAR-T cell failure. Recent studies demonstrate that reducing CD28 co-stimulatory signals delivered by CARs promote CAR-T cell persistence and reduce exhaustion. Whether endogenous CD28 co-stimulation contributes to in vivo CAR-T cell transience or dysfunction is unknown. Our preliminary data indicate that genetic deletion of the endogenous CD28 receptor does not impair CAR-T cell cytotoxicity but promotes in vivo persistence while targeted mutation of CD28 reduces exhaustion marker expression. These findings suggest that targeting endogenous CD28 on CAR-T cells with CTLA4-Ig (Abatacept) may represent a viable clinical strategy to increase the durability of a potent CAR-T cell design. Importantly, in the context of MM, we have previously shown that CD28 is expressed on and delivers pro-survival signals to MM, contributing to therapy resistance. Moreover, given the abundance of CD28 ligands, CD80 and CD86, within the bone marrow, we hypothesize that CD28 signaling reduces the efficacy of BCMA CAR-T therapy by providing a survival signal to MM cells while simultaneously limiting the durability of CAR-T cells. The combination therapy of BCMA CAR-T cells and Abatacept is expected to enhance the efficacy of adoptive CAR-T cell therapy for MM patients.

This work is supported by grants from NIH (R03 CA256122, R01 AI155499).
Fluorescence tagging to monitor CD8 T cell recirculation from the tumor to the tumor-draining lymph node: the impact of focal radiation therapy on recirculation
Authors: Gough Michael J , Blair Tiffany , Dowdell Alexa K , Bambina Shelly , Kramer Gwen , Piening Brian D , Crittenden Marka R
Published: January 2023

T cells are continuously moving through tissues and recirculating back into the peripheral blood via lymph nodes. In cancer, tumor-specific T cells are enriched in the tumor environment and their presence is associated with improved outcome in patients and in preclinical models. T cells are known to contribute to tumor control following radiotherapy despite evidence indicating that radiation can be directly cytotoxic to lymphocytes. The impact of radiation therapy on recirculation of T cells through the tumor to the tumor draining lymph node (TDLN) and their trafficking to distant sites is unclear. By photoconverting tumor infiltrating cells using focal UV in Kaede mice we can use fluorescence tagging to directly identify T cells in the TDLN that originated in the tumor. We demonstrate that radiation therapy significantly decreases CD8 T cell trafficking from the tumor to the TDLN. Using single cell RNASeq and flow cytometry, we demonstrate that radiation therapy is locally cytotoxic to proliferating effector CD8 T cells within the tumor environment. This results in the loss of this effector population in the TDLN following treatment. Using flow cytometry and direct blockade of T cell entry into the LN, we characterize the CD8 T cells in the TDLN that proliferate following radiation therapy. Our data demonstrate that radiation therapy is locally cytotoxic and restricts CD8 T cell recirculation to the TDLN. Since tumor control following radiation therapy is partially dependent on CD8 T cell responses, these data force us to re-evaluate both the type and location of T cell populations that contribute to tumor control following radiation therapy.

Supported by grants from the NIH (R01CA182311, R01CA244142, R01CA208644)
Induction of Inflammatory Macrophages in Solid Tumors by All-trans Retinoic Acid Augments Radiation Efficacy
Authors: Liang Hua Laura , Rao Enyu , Hou Yuzhu , Wang Jiaai , Huang Xiaona , Yu Xianbin , Wang Liangliang , He Chuan , Vokes Everett , Weichselbaum Ralph
Published: January 2023

Radiotherapy is an important anticancer treatment modality that activates innate and adaptive immune responses. Local RT induces an influx of myeloid cells (mostly myeloid-derived suppressor cells MDSCs), which suppress T-cell function, in the tumor microenvironment (TME). Alleviating therapy-induced immunosuppression would address a significant barrier to the efficacy of current cancer immunotherapeutic approaches. When all-trans retinoic acid (RA) was administered with radiation, we observed superior antitumor responses compared with ionizing radiation (IR) alone or RA alone. The effects of combination treatment were accompanied by a marked increase of tumor necrosis factor–α– (TNFα) and inducible nitric oxide synthase (iNOS)–producing inflammatory macrophages in local and distal nonirradiated tumors. Inflammatory macrophages (Inf-MAC) are essential for the therapeutic efficacy of combination treatment by inducing effector T cell infiltration and enhancing the effector T cell to regulatory T cell ratio in local and distal tumors. T cells and T cell–derived interferon-γ are crucial for increasing inflammatory macrophage levels in IR- and RA-treated tumors. The synergistic positive feedback loop of inflammatory macrophages and adaptive immunity is required for the antitumor efficacy of IR + RA combination treatment. Our findings provide a translational and relatively nontoxic strategy for enhancing the local and systemic antitumor effects of IR. Single cell RNAseq of immune infiltrates revealed unique transcriptional changes delineating the differentiation of Inf-Mac in the TME which may lead to potential specific therapeutic targets.
Preoperative exercise therapy attenuates liver metastases following surgical stress by inducing Kupffer cells-mediated anti-tumor immunity
Authors: Zhang Hongji , Cheng Xiang , Deng Meihong , Tsung Allan , Huang Hai
Published: January 2023

Introduction

Resection of colorectal cancer hepatic metastases improves overall disease-free survival. Unfortunately, not all patients have a successful surgical outcome. Pre-operative exercise therapy (PEx) have been demonstrated to be beneficial in the prevention of post-operative complications. We hypothesize that PEx initially reverts pro-tumorigenic inflammatory responses following surgical stress and maintains an anti-tumor immune microenvironment through modulating Kupffer cells (KCs) with anti-tumor immunity.

Methods

8W C57BL/6 mice were randomly divided into PEx and sedentary (Sed) groups, mice with PEx run on a motorized treadmill at a speed of 12.5 m/min for 60 min/day, 5 days/W for 4 weeks. 105 MC38 cells were injected directly through portal vein and surgical stress was subjected to partial hepatic ischemia and reperfusion model. Both hepatic and tumor CD45+ cells from PEx or Sed mice 3 W after IR were submitted for single-cell RNA sequencing (scRNA-seq).

Results

4 weeks of PEx significantly reduces metastasis in the liver along 3 weeks after IR, compared with Sed controls. For the scRNA-seq, 11 cell lineages were identified and annotated according to the transcriptomic profile of feature genes expression. Surprisingly, discontinued 4-week-PEx still led to a significant transcriptomic shift in the KCs. 3 clusters from the entire KCs population were unique in PEx mice. In contrast, 2 Clusters were derived from Sed mice. PEx-relevant KCs are enriched in gene expression related to the anti-tumor phenotype, whereas Sed-relevant KCs are enriched in gene expression related to pro-tumor or immunosuppressive phenotype, which suggests that PEx modulates transcriptomic changes in KCs towards an anti-tumor phenotype.

Supported by National Institutes of Health R01-CA214865-01 and R01-GM95566-06 to AT, National Institutes of Health R01-AI152044 to MD, and National Institutes of Health R01-GM137203 and Joseph A. Patrick Research Fellowship in Transplantation to HH.
β-adrenergic signaling modulates the development and activity of erythroid suppressor cells
Authors: Chawla Arshmeet K , Nevin James , Moussa Marmar , Geyer Rory , Mandoiu Ion , Srivastava Pramod K
Published: January 2023

The neuroimmune response to stress has been linked to enhanced tumor proliferation, angiogenesis, and metastasis. In particular, signaling through β-adrenergic receptors has been demonstrated to promote a highly immunosuppressive tumor microenvironment by favoring the development and recruitment of suppressive immune cells, including regulatory T cells and myeloid-derived suppressive cells, that inhibit the cytotoxic CD8+ T cell response and enhance tumor progression. Interestingly, the complexity and scope of cancer immunosuppression extends beyond classical immune cells. Immunosuppressive erythroid progenitors, deemed erythroid suppressor cells (ESCs), have been shown to be prominent players in suppressing CD8+ T cell-mediated tumor responses; however, little is known about how these cells develop and are regulated. Thus, we examined if β-adrenergic signaling affects the development of ESCs and their role in tumor immunity. In this study, we show that β-adrenergic signaling promotes the accumulation of CD71+ Ter119+ erythroid progenitors consistent with the phenotypic description of ESCs in tumor-bearing mice. In characterizing the phenotype of these cells by flow cytometry and single-cell RNA sequencing, we elucidate unique features that endow these cells with immunosuppressive activity and illustrate their sensitivity to regulation by the sympathetic nervous system. We further demonstrate these erythroid precursors exhibit suppressive effects on the CD8 response. These findings elucidate a previously unappreciated axis of neuroimmune coordination in cancer and are pivotal to understanding crucial elements of tumor progression that will help shape the course of targeted immunotherapies in the future.
TNF-α blockade improves immunotherapy efficacy by altering the tumor microenvironment and enhancing tumor-specific T cell function in pancreatic ductal adenocarcinoma
Authors: Burrack Adam L , Schmiechen Zoe , Miller Ebony , Stromnes Ingunn
Published: January 2023

Pancreatic ductal adenocarcinoma (PDA) is a particularly lethal malignancy with a 5-year survival rate of 9%. A recent phase 1 clinical trial suggests CD40 agonist has antitumor activity in some patients. We developed an orthotopic PDA mouse model to track tumor specific CD8 T cells, identify critical antitumor mechanisms, and determine pathways of immunotherapy resistance. Here, we exploit this model to uncover a novel combination immunotherapy that includes CD40 agonist, PD-L1 blockade and TNF-α neutralization (e.g., 4PT). Interfering with TNF-α significantly improves overall mouse survival and cure rate compared to CD40+PDL1 only (4P). Critically, 4PT enhanced the generation of tumor-specific long-lived effector and central memory T cells. TNF-α neutralization significantly reduced T cell exhaustion, as indicated by reduced Lag-3 and increased IFN-γ production by intratumoral tetramer+ CD8 T cells. Additionally, 4PT increased CD4+Foxp3− T cell frequency and decreased CD4+Foxp3+ T cells as compared to 4P-treated mice, consistent with enhanced antitumor CD4 T cell reactivity in the absence of chronic TNF-α signaling. Lastly, abrogating Tnfr1 significantly reduced splenic and intratumoral Ly6G+ granulocytes following 4P. Thus, disrupting TNF-α via genetic deletion or monoclonal antibodies alters the tumor microenvironment to promote highly functional tumor-specific CD8 T cells. We conclude that perturbation of TNF-α-mediated chronic inflammation is an appealing approach to enhance immunotherapy efficacy for pancreatic cancer patient treatment.

Supported by NIH 1R01CA249393-01A1
Resistance to Anti-PD-1 Therapy is mediated via the Microbiota-Th17-Prostoglandin E2 Axis in the LSLKrasG12D Lung Cancer Model
Authors: Li Qingsheng , Goggin Kevin Edward , Seo Jamie , Egilmez Nejat
Published: January 2023

We previously demonstrated that anti-PD-1 antibody mediated activation of type 17 T-cells undermined checkpoint inhibitor therapy in the LSLKrasG12D murine lung cancer model. In this study we confirm that the Th17 subset is the primary driver of resistance to anti-PD-1; demonstrate that the ontogeny of dysplasia-associated Th17 cells is driven by microbiota-conditioned macrophages; and identify IL-17-COX-2/Prostoglandin E2 (PGE2) axis as the mediator of CD8+ cytotoxic T-lymphocyte (CTL) de-sensitization to checkpoint inhibitor therapy. Specifically, anti-PD-1 treatment of CD4+ T-cell RORc-deficient LSLKrasG12D mice resulted in a 50% increase in CTL cytotoxicity and a 2.2-fold reduction in tumor burden confirming the critical role of Th17 cells in resistance to therapy. Lung-specific depletion of microbiota reduced Th17 cell prevalence and tumor burden by ~2 and ~1.5 fold, respectively, establishing a link between microbiota and the Th17-driven tumorigenesis. Importantly, interstitial macrophages from microbiota sufficient, but not from microbiota-deficient, mice polarized naïve CD4+ T-cells towards a Th17 phenotype, highlighting their role in bridging microbiota and lung Th17 immunity. Neutralization of IL-17 diminished COX-2/PGE2 levels, whereas inhibition of COX-2 rescued CTL activity and restored tumor suppression in anti-PD-1 antibody-treated mice, revealing the molecular basis of IL-17-driven anti-PD-1 resistance. The clinical implications of these findings are discussed.

Supported by grant from DOD grant (to N.E., OGMB190737) and in part through the NIH/NIGMS P20-GM135004 (1P20GM135005-01)
NASAL NANO-VACCINE PREVENTS PRIMARY BREAST TUMOR FROM MAKING THE LUNG ITS NEW HOME
Authors: Donkor Michael Ahenkora , Jones Harlan
Published: January 2023

Despite medical advances in the diagnosis and treatment of cancer, metastatic breast cancers remain a leading cause of death in the U.S. Increasingly, novel immune-based treatments which harness the patient’s immune system have promise for improving survivorship. Such therapies take advantage of the immune system’s natural defense mechanisms to halt progression of breast cancer. This is through the early activation of innate immune cells such as natural killer cells and the subsequent activation of the adaptive immune responses such as T and B lymphocytes. Researchers have taken advantage of these immune mechanisms of tumor defense as a complementary approach to current radio-chemo treatments, which have shown to be limited by adverse off-target effects on patients. Ongoing research in our laboratory is focused on using nanotechnology to develop immune-based vaccines to target local immune protection against metastatic lung disease. Because the lung is naturally tolerogenic, making it easy for disseminated tumor cells to grow, the expectation is that boosting immune responses at the lungs before seeding tumors from primary organs would mitigate metastasis and reduce mortality risks. Using an experimental murine breast cancer model of metastasis, we examined the effect of intranasal vaccination to induce local and systemic adaptive immune responses. We hypothesized that an intranasal vaccine protocol would increase antigen-specific adaptive responses across the respiratory tract. Our results demonstrated that intranasal vaccination provides protection against secondary lung metastasis due to increased accumulation functional CD4+ and CD8+ T cells in the lungs as shown by flow cytometry and ELISA techniques.

Supported by National Institute of Cancer Research of the Health under Award 1 P20 CA233355-01
P2RX7 enhances tumor control by CD8+ T cells in adoptive cell therapy
Authors: Wanhainen Kelsey , Peng Changwei , O’flanagan Stephen , Burbach Brandon , da Silva Henrique Borges , Jameson Stephen
Published: January 2023

Expression of the purinergic receptor P2RX7 by CD8+ T cells promotes generation of memory populations following acute infections and is critical for responses to chronic infection. Despite these findings, recent studies have indicated that P2RX7 may limit the efficacy of anti-tumor responses. We show that P2RX7 is beneficial for optimal melanoma control in a mouse CD8+ T cell adoptive transfer model. Tumor specific P2rx7−/− CD8+ T cells exhibited impaired mitochondrial maintenance and function, but do not display signs of overt exhaustion or poor survival early in the anti-tumor response. However, as tumor burden increases the frequency P2RX7 deficient CD8+ T cells progressively declines relative to WT CD8+ T cells within the tumor. This was associated with worsening mitochondrial function and increased expression of some markers of T cell exhaustion among P2rx7−/− CD8+ T cells. Extending these studies, we found that transient in vitro stimulation of P2RX7 using the ATP analog BzATP led to enhanced B16 tumor control by CD8+ T cells. The improved ability of BzATP stimulated CD8+ T cells to prolong survival of tumor bearing mice was accompanied by enhanced mitochondrial function of tumor specific CD8+ T cells. These findings are in keeping with the concept that extracellular ATP (eATP) sensing by P2RX7 on CD8+ T cells is required for their ability to efficiently eliminate tumors by promoting mitochondrial fitness and indicates the potential for P2RX7 stimulation as a novel therapeutic treatment to enhance tumor immunotherapy.

Supported by grants F30 award from the NIH (1F30CA250321-01)
Tumor-induced, TCR-independent upregulation of A2AR expression in effector CD8+ T cells
Authors: Zhou Maggie , da Silva Henrique Borges
Published: January 2023

The tumor microenvironment (TME) is characterized by high levels of extracellular ATP (eATP) and adenosine, which are sensed by purinergic receptors. CD8+ T cells express two important receptors that bind these ligands: the extracellular receptor P2RX7 binds eATP and is generally immunostimulatory, while the adenosine receptor A2AR promotes immunosuppression. However, the role of the TME itself in the T cell expression of purinergic receptors is not clear. We used in vitro co-cultures of tumor cells and CD8+ T cells, and specifically found that A2AR is significantly upregulated after activation, in a tumor cell number-correlated manner. These tumor-induced changes in A2AR expression are independent of TCR:MHC contact, which was evidenced by co-cultures using transwells to physically separate tumor cells and T cells, as well as distinct combination of transgenic T cells and tumors. In vivo murine solid tumor models confirmed that tumor-infiltrating CD8+ T cells upregulate A2AR. As previously found by our group, P2RX7 expression is limited to a small percentage of tumor-infiltrating CD8+ T cells. Interestingly, we identified that these few P2RX7-expressing tumor-infiltrating CD8+ T cells are enriched for high A2AR expression – suggesting that co-expression of these two receptors is favored in T cells exposed to tumors. Overall, our findings suggest that TME exposure – likely through TCR-independent factors – directly promote the expression of A2AR in CD8+ T cells. We are currently investigating the nature of the tumor-derived factor(s) promoting this upregulation, as well as the possible role of A2AR/P2RX7 co-expressing tumor-infiltrating CD8+ T cells as effector antitumor cells with longer intratumoral lifespan.

Supported by grants from NIH (R00 AI139381, NIAID).
CD8 T cell responses to conserved DNAJB1-PRKACA fusion neoantigens in fibrolamellar carcinoma
Authors: Kirk Allison M , Chou Ching-Heng , Awad Walid , Crawford Jeremy Chase , Allen E Kaitlynn , Zhang Xiaoyu , Zamora Anthony E , Strome Scott E , Thomas Paul G
Published: January 2023

Fibrolamellar carcinoma (FLC) is an often-lethal liver malignancy of adolescents and young adults. No systemic therapies are currently approved for FLC, so novel treatments are needed to improve patient outcomes. A promising therapeutic vulnerability in FLC is a highly-conserved gene fusion between DNAJB1 and PRKACA. The resultant fusion protein is identical in more than 90% of patients, making it a potentially ideal neoantigen target for T cell-based immunotherapy. The goal of our study was to test if DNAJB1-PRKACA neoantigens could elicit T cell responses and serve as targets for immunotherapy. Spatial transcriptomic analyses of FLC tumor tissue indicated that CD8 T cells could infiltrate FLC tumors, but expressed exhaustion markers such as TOX. Nevertheless, we expanded T cells from an FLC patient tumor ex vivo and detected T cells that produced IFNγ and TNFα in response to stimulation with a fusion neoantigen. We then used functional response after peptide stimulation or peptide-MHC tetramer staining to identify fusion-specific T cell receptors (TCRs) in both FLC patients and healthy donors. We expressed these TCRs in primary human T cells and found that cells expressing fusion-specific TCRs bound to their cognate tetramer and produced IFNγ, TNFα, and IL-2 in response to stimulation with their cognate peptide. TCR-expressing cells also specifically killed target cells presenting their cognate fusion peptide in vitro. Ongoing experiments will test if cells expressing these TCRs can control growth of fusion-expressing tumors in vivo. Together, these studies have defined the first reported fusion-specific T cell response in an FLC patient, as well as fusion-specific TCRs that hold promise for development in adoptive T cell therapies.

Supported by grants from NIH (R01 AI136514, F31 CA254423) and The Mark Foundation for Cancer Research (Aspire Award)
PIK3IP1/TrIP Immune Regulation on CD8+ T Cells Restricts Anti-Tumor Immunity
Authors: Murter Benjamin M , Banerjee Hridesh , Szymczak-Workman Andrea , Kane Lawrence P
Published: January 2023

The signaling pathways involving phosphoinositide-3-kinases (PI3Ks) are highly conserved and tightly regulated to influence the activation, proliferation, and survival of all cell types. Our lab has recently shown protein TrIP (Transmembrane Inhibitor of PI3K, Pik3ip1) has a distinctly high expression on T cells and is capable of downregulating PI3K signaling in these cells, acting as a negative regulator of T cell immune responses. These studies revealed that CD4+ T cells lacking TrIP expression exhibit a more Th1 inflammatory phenotype compared to WT T cells, both in vivo and in vitro. These data have led us to propose that TrIP restricts the inflammatory activity of T cells more generally, including CD8+ T cells, and that targeting/knockout of this regulator may promote anti-tumor immunity.

Using a conditional TrIP knockout mouse model developed in our lab, we have performed syngeneic tumor challenges in CD8+ T cell-specific TrIP knockout mice (TrIPfl/fl E8icre) which show that TrIP knockout mice are resistant to growth of syngeneic tumors. In addition to this increased resistance, tumors harvested from our knockout mice contain twice as many infiltrating T cells compared to their WT counterparts. Of those, CD8+ T cells appeared to be the main drivers of this increased T cell infiltration as their frequency was double that of the CD4+ population. In addition, these TrIP KO CD8+ T cells have a significantly more inflammatory phenotype detailed through RNAseq analysis.

In conclusion, TrIP plays a significant role in the antitumor immune activity of CD8+ T cells. The TrIP KO mice are resistant to tumor challenge and display an increase in TIL T cell infiltrate. These data describes TrIP as an interesting potential target for immunotherapy.

Supported by grants from the NIH 5R01GM136148-02 and 5T32CA082084-20
Cancer vaccines used in combination with TCR-like antibody blockade of the Qa-1b/Qdm complex, the ligand for the immune checkpoint NKG2A, induce anti-tumor immunity
Authors: Ghaffari Soroush
Published: January 2023

Antagonizing antibodies against immune checkpoint inhibitory molecules (ICI) has gained an excellent reputation in immuno-oncology. Anti CTLA-4 and anti-PD-1 are among the first cancer checkpoint immunotherapies that got FDA approval to treat certain malignancies. However, not everyone can benefit from them due to the insufficient expression of the checkpoint marker on the surface of the tumor cells.

CD94/NKG2A receptor is another inhibitory marker known to be expressed by natural killer cells and sub-population of CD8+ T cells. Specific peptides loaded by Qa-1b (mouse) or HLAE (human) on the surface of tumor cells, immune infiltrating lymphocytes, and myeloid cells are NKG2A ligand and are known to have a significant role in modulating anti-tumor effector cell responses.

Our group previously developed a single-domain T-cell receptor-like antibody (EXX-1) with specificity for the Qa-1b/Qdm peptide complex, the ligand for the murine NKG2A/CD94 receptor. We have shown that treating tumor-bearing mice with EXX-1 as a single agent blocking antibody can create a modest delay in mice tumor growth and overall survival time.

The presented study focused on utilizing EXX-1efficacy as a checkpoint blocking antibody in combination with a cancer vaccine. Our findings indicate that cancer vaccine effectiveness can be markedly enhanced when combined with EXX-1 antibody, leading to tumor-free survival in 35% of the mice compared to control groups. Taken together, our results indicate targeting the NKG2A axis through blocking Qa-1b/Qdm peptide complex is an effective means to treat cancer. Also, using HLAE/Qa-1b blocking antibody can improve the efficacy of the cancer vaccines and lead to better clinical outcomes when combined.
T cell receptor revision as a novel mechanism of action of immune checkpoint blockade
Authors: Brice David , Thomas Paul G , McGargill Maureen A
Published: January 2023

Background

Immune checkpoint blockade (ICB) is a promising therapy prescribed to combat tumors. However, acquired resistance to ICB highlights the need for a better understanding of ICB mechanisms. Analysis of T cell receptor (TCR) sequences in individuals before and after ICB showed that a distinct TCR repertoire was observed following ICB, suggesting that T cells with novel specificities contribute to tumor clearance. While the source of these T cells with distinct TCRs is not clear, ICB has been found to lead T cells infiltrating both mouse and human tumors to express components of TCR revision. Therefore, one potential mechanism by which ICB leads to anti-tumor immunity is by inducing TCR revision.

Methods

Wildtype C57BL/6, as well as TCR revision-deficient TCRa+/−.Rag2fl/fl.CD4cre (TCRa+/−.Rag2CD4) mice received subcutaneous injections of either 4.5 × 105 B16F10 or MC38F10 cells. Mice then received either PBS or a combination of 250 μg each of anti-CTLA-4, anti-PD-1, and anti-PDL-1 three times per week starting Day 7 after tumor cell injection. Tumor size and mouse survival were monitored, and baseline splenic and thymic immune cell numbers were measured via flow cytometry.

Results

No differences in T or B cell subsets were detected at baseline between the mouse strains. ICB significantly reduced tumor growth and mortality in the wildtype, but not the TCRa+/−.Rag2CD4, mice for both tumor models tested.

Conclusions

TCR revision capabilities seem to be important in ICB efficacy. Further investigation using these mouse models will help explore the possibility of future targeting of TCR revision pathways to increase ICB efficacy.
Inflammation suppresses elevated TCF1 and FOXO1 expression in vaccine-elicited T cells
Authors: Ivanova Daria Leonidovna , Kilgore Augustus , Klarquist Jared , Thompson Scott , Kedl Ross
Published: January 2023

T cells that express TCF1 display self-renewing capacity and are essential for the long-term T cell response during chronic infections and cancer. Due to their increased responsiveness to checkpoint blockade, TCF1+ T cells are recognized as promising targets for immunotherapies. We found that, in contrast to T cells responding to infections (Tinf), the majority of adjuvanted subunit vaccine-elicited T cells (Tvac) rapidly acquired a memory phenotype (CD127hi) and were TCF1hi. This suggests that Tvacs could be programmed by TCF1 to commit to a memory cell fate. We found that Tvac peak and memory responses were more compromised in the absence of TCF1 than those of Tinf. However, TCF1 KO Tvacs still developed memory, suggesting that additional factors were involved. FOXO1 is important for T cell memory after infections. We found that Tvacs expressed more FOXO1 than Tinfs. In contrast to Tinf, FOXO1 was required for both the peak and memory Tvac responses. Moreover, even though FOXO1 drives TCF1 in Tinfs, Tvacs expressed TCF1 independently of FOXO1. To explore the cause for such high expression of TCF1 and FOXO1 in Tvacs, we next examined the role of inflammatory mediators in their regulation. Tvac TCF1 and FOXO1 inversely correlated with adjuvant-elicited pro-inflammatory signals. Moreover, addition of pro-inflammatory TLR9 ligand CpG reduced Tvac TCF1, FOXO1 and memory. In contrast to prior reports, anti-IL-12 failed to reverse the effect of CpG on Tvacs. Anti-IFNγ, however, restored the Tvac memory phenotype even as CpG drove additional inflammatory mediators. Our data highlight the differences in the transcriptional regulation of vaccine- and infection-elicited T cells and provide insights into the mechanisms regulating TCF1 and FOXO1.

Supported by grants from NIH (T32 AI074491, R01 AI066121)
Cell-specific regulation of antigen-specific germinal center formation and antibody responses by thymic stromal lymphopoietin
Authors: Domeier Phillip P , Ziegler Steven F
Published: January 2023

Germinal centers (GCs) are dynamic immune structures that expand upon T-dependent immune challenge to generate high-affinity antibody-producing plasma cells and long-lived memory. GCs require stringent regulation to optimize GC-derived antibody responses. Here, we identified thymic stromal lymphopoietin (TSLP), as a novel regulator of GCs. After immunization with NP-OVA in Alum, TSLP receptor-deficient (TSLPR−/−) mice fail to form antigen-specific GCs and antibodies. TSLPR expression is elevated on T follicular helper cells (Tfh) and GC B cells during inflammation, so we asked how cell-specific loss of TSLPR signaling could disrupt overall GC activity. When TSLPR was deleted in CD4 T cells with a tamoxifen-inducible Cre (CD4CreERT2/+ TSLPRFLOX/FLOX), we observed a similar reduction in T follicular helper cells (Tfh), GC formation, and antibody responses after immunization when compared to CD4CreERT2/+ and TSLPRFLOX/FLOX controls. Conversely, when TSLPR signaling was lost on mature B cells (CD21Cre/+ TSLPRFLOX/FLOX) and GC B cells (AIDCre/+ TSLPRFLOX/FLOX), we observed an increase in antigen-specific GC B cells and antigen-specific memory B cells. TSLPR signaling regulates IRF4 expression in dendritic cells, and we observed a similar reduction in IRF4 expression in Tfh and GC B cells from TSLPR−/− mice compared to TSLPR+/+ controls. Collectively, these findings suggest that TSLPR signaling promotes IRF4 expression in GC B cells and Tfh cells, resulting in distinct roles in the GC. TSLPR and IRF4 signaling in T cells is required for the establishment and maintenance of antigen-specific Tfh cells, whereas TSLPR/IRF4 signaling in B cells inhibits antigen-specific GC B cell retention and the establishment of B cell memory.

Supported by a grant from the NIH (F32 AI154787)
Inhibition of perforin enhances T cell and humoral responses after immunization
Authors: Krishnamurthy Durga , Cevik Hilal , Ali Ayad , Canaday Laura , Mukhopadhyay Eshana , Seibel William , Risma Kimberly , Jordan Michael B , Waggoner Stephen N
Published: January 2023

Cytotoxic lymphocytes such as natural killer (NK) cells release perforin to form pores on plasma membrane of target cells to allow delivery of granzymes that induce apoptosis. We previously showed that NK cells exert a perforin-dependent suppression of follicular helper CD4 T cell and germinal center B cell responses following infection and immunization. Consistent with this activity, genetic ablation of either perforin or NK cells non-additively enhanced the quality and magnitude of humoral immune responses. Therefore, we hypothesize that transient inhibition of perforin represents an innovative means to enhance vaccine efficacy to prevent viral infections such as HIV. Intraperitoneal injection of a selective inhibitor of perforin (35mg/kg) to mice reduced killing activity of NK cells by >50%. A single injection of this inhibitor to mice during alum adjuvanted NP-KLH immunization resulted in a greater than two-fold enhancement of follicular helper CD4 T cell, germinal center B cell, and antigen-specific antibody responses. In summary, transient inhibition of pore-forming protein perforin can block NK-cell immunosuppression to enhance vaccine-elicited T and B cell responses.

National Institute of Heath (NIH) (R01-AI148080)
A Novel Solution to Better Understand the Link Between T Cell Metabolism and Function
Authors: Walls Jessica , Kam Yoonseok , Romero Natalia
Published: January 2023

In recent years, metabolism has emerged as a key driver of immune cell function in both healthy and disease settings. Indeed, it has been shown that metabolic reprogramming is a determinant of T cell phenotype and fate. Moreover, reprogramming of the metabolic pathways of T cells can be used as a strategy to improve the antitumor efficacy of adoptive T cell therapies. The Agilent Seahorse XF T cell Metabolic Profiling (XF TCMP) kit is a new solution that, combined with the Seahorse XF analyzer, allows for the simultaneous measurement of basal metabolic demand, metabolic poise and respiratory capacity in T cells. These parameters have been linked with optimal function and improved persistence of T cell therapies. The assay uses an improved uncoupler (BAM15) for more robust measurements of T cell bioenergetic capacity with less optimization than other uncouplers used. The XF TCMP kit was used to evaluate the metabolic phenotype of human T cells expanded in different nutrient conditions. T cells expanded in different cell culture media resulted in populations with different metabolic profiles. These metabolic profiles correlated with different functional markers, supporting the link between metabolism and function. The XF TCMP kit was also used to assess metabolic poise and bioenergetic capacity under nutrient-restriction, allowing us to better understand fuel requirements after expansion. Together, the data show that the XF TCMP kit provides a complete metabolic assessment of T cells in real-time. The assay can be used to develop improved anti-tumor T cell therapies by evaluating strategies that improve the persistence of infused cells or assess how T cells sustain metabolic fitness in nutrient restricted environments to maintain function.
Gene editing at position 71 in DRB1*04:01 blocks collagen sensitization and avoids allorecognition.
Authors: Jha Vibha , Coulombe Marilyne , Rosloniec Edward F , Matsuda Jennifer , Freed Brian M , Roark Christina L
Published: January 2023

DRB1*04:01 is strongly associated with susceptibility to rheumatoid arthritis (RA). Our previous work demonstrated that changing lysine (K) at position 71 in DRB1*04:01 to glutamic acid (E), an amino acid found in resistant alleles, abrogated binding of the immunodominant collagen258–272 peptide. The purpose of this study was to determine if the K71E mutation in DRB1*04:01 would prevent sensitization to collagen and be tolerated by the host immune system in mice. DRB1*04:01 and DRB1*04:01K71E transgenic mice were created on the murine MHC class II knockout background (B6.129S2-H2dlAb1-Ea/J). DRB1*04:01 and DRB1*04:01K71E mice were immunized twice with bovine type II collagen in complete Freund’s adjuvant on days 0 and 21. On day 56, lymph node cells were harvested and stimulated in vitro with collagen258–272 peptide. Introduction of the K71E mutation in DRB1*04:01 resulted in a >90% reduction in proliferation as compared to CD4+ cells from collagen-immunized DRB1*04:01 mice. Next, skin grafts were transplanted from DRB1*04:01K71E mice onto DRB1*04:01 recipients to test for rejection. The skin grafts from DRB1*04:01K71E mice were accepted by the recipient DRB1*04:01 mice with no signs of rejection out to day 70. Our results demonstrate that introducing the K71E mutation in DRB1*04:01 blocks collagen sensitization and avoids allorecognition suggesting that this mutation could safely halt progression of RA by blocking autoimmune T cell responses to collagen.

Supported by IOS-2103367
Novel immunological tools to investigate CD4+ T cell activation in the canine species
Authors: Lang Haeree Park , Dileepan Thamotharampillai , Jenkins Marc K , Friedenberg Steven
Published: January 2023

Opportunities to identify T cell epitopes in the dog have been thwarted by a lack of immunological tools specific for the species. As a result, antigen-specific triggers in diseases that affect dogs like autoimmune disorders and allergies remain unknown. Our objective was to develop immunological tools such as: IFN-γ enzyme-linked immunosorbent spot (ELISpot) and activation induced marker (AIM) assays, and an MHC class II tetramer to help overcome these hurdles. We collected splenocytes from dogs that underwent a routine splenectomy. We used these splenocytes to develop: an ELISpot assay detecting canine IFN-γ production by stimulated T cells, an AIM assay to assess early activation by CD40L expression on CD4+CD5+ canine splenocytes using a cross-reactive anti-human monoclonal antibody, and finally, we produced the first-ever canine MHC II tetramer specific for a common canine DR allele. We confirmed the efficacy of these tools in the presence of positive and negative controls, Staphylococcus aureus enterotoxin B (SEB) and dimethyl sulfoxide (DMSO), respectively. SEB induces a robust canine IFN-γ ELISpot readout quantifiable in canine splenocytes. Similarly, we discovered that canine CD40L expression peaks at 3 hours post-stimulation with SEB. Finally, we confirmed the development of the first canine pMHCII tetramer through size discriminatory SDS-PAGE analysis and peptide exchange assays. Our novel canine-specific reagents will help identify and characterize antigen-specific T cells and their cognate antigens driving canine MHC class II-restricted disease, thus guiding research towards the advancement of antigen-specific immunotherapies.

Supported by a grant from the NIH (K01 OD027058)