bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inducing/expanding Tregs in vivo to suppress autoimmunity using a new bispecific therapeutic containing IL-2 and TGFβ
Authors: Jackson Nicholas M , Hoft Stella , Ford Eric , Yakobian Natalie , Girgis Natasha , DiPaolo Richard 
Published: August 2023

Abstract
The objective of this study was to test a new injectable therapeutic, CUE-401, that is designed to induce Foxp3 expressing Tregs by signaling through both IL-2 and TGFβ receptors. Unlike IL-2 therapies that activate and expand existing Tregs, this biotherapeutic was designed to both induce new subsets of iTregs from naïve autoreactive CD4+ T cells and to activate and expand existing Tregs. Experiments were performed to determine the biological activities of CUE-401 both in vitro and in vivo after administering to mice with active and ongoing autoimmunity. The in vitro results showed that stimulating naïve CD4+ T cells in the presence of CUE-401 was effective at inducing Foxp3 expressing iTregs that were phenotypically and functionally indistinguishable from iTreg induced with recombinant IL-2 and TGFβ. When injected into mice, CUE-401 was effective at increasing Foxp3+Tregs. Unlike mice treated with IL-2 complexes, CUE-401 did not induce proliferation of autoreactive T cells. Single cell RNA sequencing revealed that CUE-401 induced a subset of Tregs that shared many transcripts known to be expressed by TGFβ-induced iTregs. Finally, CUE-401 induced iTregs were effective at suppressing organ specific autoimmunity. These studies identify a new injectable therapeutic, and new approach to induce and expand Tregs in vivo to suppress autoimmunity and other inflammatory diseases.
Investigating the interactions between regulatory T cells and cancer-associated fibroblasts and their role in lung cancer progression
Authors: Ringham Olivia Rose , Loffredo Lucas F , Arpaia Nicholas 
Published: August 2023

Abstract
The tumor microenvironment (TME) is a highly complex multicellular system and has been shown to play a fundamental role in dictating tumorigenesis. High expression of regulatory surface molecules, cytokines, and chemokines in fibroblast niches within lung tumor stroma, paired with their colocalization with tumor infiltrating lymphocytes (TILs), suggests that cancer-associated fibroblast (CAF) niches may alter the functional response of TILs and influence the anti-tumor immune response. Here, utilizing a transcriptomic approach, we have uncovered a distinct CAF population, which we term “iCAFs,” that is enriched for immunomodulatory gene signatures, T cell ligands, and chemoattractants. This population expresses a unique set of cell surface molecules that permit their identification and isolation from lung tumor tissue. Given the demonstrated importance of Tregs in suppressing anti-tumor immunity, we chose to evaluate how Tregs may be interacting with iCAFs within the TME of lung adenocarcinoma. We uncovered significant colocalization patterns of Foxp3+ Tregs with iCAF—a pattern which was found to be unique for Tregs when compared to conventional CD4+Foxp3− T cells. Moreover, the Treg to conventional CD4+ T cell ratio is significantly decreased in tumor tissue relative to normal adjacent tissue and healthy controls. Our analyses demonstrate that Tregs preferentially associate with iCAF populations within the tumor, leading us to hypothesize that interactions between Tregs and iCAFs may, in turn, influence Treg trafficking, phenotype and function within the TME.
T32AI106711
Sensory Nerves inhibit the development of protective anti-melanoma immune response and formation of tertiary lymphoid structures
Authors: Vats Kavita , Kruglov Oleg , Sahoo Bikram , Zhang Jiying , Sonam Vishal , Chandran Uma , Shurin Galina V , Skum Pavel , Shurin Michael R , Strokus Walter J , Bunimovich Yuri M L 
Published: August 2023

Abstract
Peripheral nervous system (PNS) is an important component of the tumor microenvironment (TME). It has been established that autonomic innervation plays an important role in cancer. However, the effect of the afferent (sensory) neurons on tumor progression remains unclear. Utilizing surgical and chemical skin sensory denervation methods, we showed that afferent neurons supported the growth of melanoma tumors in vivo and demonstrated that sensory innervation limited the activation of effective antitumor immune responses. Precisely, sensory ablation enhanced leukocyte recruitment into tumors, with decreased presence of lymphoid and myeloid immunosuppressive cells and increased activation of T-effector cells within the TME. Denervation further increased T-cell clonality and expanded the B-cell repertoire in the TME. Furthermore, Depletion of CD8+ lymphocytes prevented denervation-restricted tumor growth. Importantly, denervated tumors contained higher densities of mature high endothelial venules (HEV) associated with the formation of classic tertiary lymphoid structures (TLS), which associate with a more favorable melanoma prognosis. Transcriptional profiling of human melanomas confirmed our findings in murine melanoma models. Specifically, we observed that gene signatures of inflammation and the content of neuron-associated transcripts inversely correlated in human primary cutaneous melanomas, with the latter representing a negative prognostic marker of patient overall survival.. Our results suggest that tumor-associated sensory neurons negatively regulate the development of protective antitumor immune responses within the TME, thereby providing a new therapeutic target for melanoma treatment.
PSGL-1 deficiency eradicates orthotopic pancreatic ductal adenocarcinoma tumors in combination with anti-PD-1 therapy and enhances T cell anti-tumor immunity
Authors: Roy Sreeja , Hope Jennifer L , Zhang Yijuan , Palete Ashley B , Faso Hannah A , Otero Dennis C , Maganthi Swetha , Commisso Cosimo , Bradley Linda M 
Published: August 2023

Abstract

Background
Pancreatic ductal adenocarcinoma (PDAC) is highly refractory to all current immune checkpoint blockade (ICB) therapies and display limited CD8+ T cell infiltration. We previously showed that deficiency of P-selectin glycoprotein 1 (PSGL-1) promotes CD8+ T cell responses that clear chronic lymphocytic choriomeningitis virus (LCMV) Cl13 virus infection and dramatically inhibits the growth of anti-PD-1 blockade resistant melanoma. Here we evaluated if PSGL-1 regulates CD8+ T cell responses to PDAC tumors.

Results
PSGL-1−/− mice reproducibly showed T cell dependent, significant reduction of KPC.4662 primary PDAC tumors and reduced metastasis to lungs compared to control mice. Furthermore, anti-PD-1 treatment of PSGL-1−/− mice completely obliterated tumor growth as opposed to control mice. Histology of primary tumors revealed marked increase of immune infiltration (CD45+ cells), including CD3+ T cells in PSGL-1−/− mice. Flow cytometry and immunofluorescence staining confirmed a significant increase in tumor-infiltrating CD8+ T cells in PDAC tumors from PSGL-1−/− mice. Importantly, PSGL-1−/− CD8+ T cells were phenotypically and functionally less exhausted.

Conclusion
PSGL-1 deficiency significantly reduced growth of primary orthotopic PDAC tumors and decreased metastases and this effect was enhanced in combination with anti-PD-1 ICB treatment. This protective effect was associated with increased immune cell infiltration in the tumors and notably, increased CD8+ T cell effector responses. PSGL-1 therefore represents a novel target for promoting immune responses to PDAC tumors and responses to PD-1 blockade.

NCI Cancer Center Support Grant P30 CA030199
The Toll-like receptor 4 pathway modulates response to anti-PD-1 therapy
Authors: Whiteside Sarah K , Beresford-Jones Benjamin S , Sadar Puspendu , Kuo Paula , Xia Wangmingyu , Suyama Satoshi , Evans Alexander C , Imianowski Charlotte J , Conti Alberto G , Wesolowski Alexander J , Soderholm Amelia , Tourlomousis Panagiotis , Okkenhaug Klaus , Bryant Clare E , Pedicord Virginia , Roychoudhuri Rahul 
Published: August 2023

Abstract
Immune checkpoint inhibitors (ICI), such as anti-PD-1, have revolutionized cancer treatment. The use of antibiotics can lead to abnormal gut microbiome composition resulting in primary resistance to ICI. However, the mechanism by which this occurs is poorly understood. Metagenomic analyses of melanoma patient stool samples prior to therapy revealed an association between clinical responses to anti-PD-1 and functional differences in gut bacteria, including Gram-negative bacteria capable of lipopolysaccharide biosynthesis. By modeling anti-PD-1 therapy in mice, we found an antibiotic that binds lipopolysaccharide inhibited therapeutic efficacy. Subsequently, the inhibition of TLR4 signaling resulted in a loss of anti-PD-1 efficacy. Oral supplementation with lipopolysaccharide augmented anti-PD-1 immunology responses. These results reveal a TLR4-dependent role for gut-derived lipopolysaccharide in modulating anti-PD-1 responses.
Mitochondria targeted metabolic inhibitors promote lymphocyte infiltration into murine pancreatic tumors
Authors: Poimenidou Maria , AbuEid Mahmoud , Drouillard Donovan , McAllister Donna , Dwinell Michael B 
Published: August 2023

Abstract
Pancreatic ductal adenocarcinoma is a malignant carcinoma with a 5-year survival rate of 11% characterized with poor prognosis, rising incidence, and resistance to chemo- and immune-therapies. Cancer cell evasion and suppression of anti-tumor immune responses in pancreas cancer are regulated in part by metabolic reprogramming within cancer and tumor accessory cells. We sought to determine if targeted inhibition of mitochondrial oxidative phosphorylation by mito-metformin (MMe) and newly developed variant pCF3-MMe influence the tumor immune microenvironment. Our results showed that MMe strongly inhibited tumor growth in murine models of pancreas cancer. Flow cytometry revealed a significant increase in total CD45+ leukocytes, with elevated cytolytic granzyme B+ and CD44+ CD8+ T cells. Immunodepleting CD8+ T cells, reversed the anti-tumor effects of MMe. Immunofluorescence microscopy and immunoblot analysis indicated that MMe stimulated oligomerization of mitochondrial antiviral-signaling protein (MAVS) and phosphorylation of IRF3 and IRF7. Further, levels of the IRF3/7 target chemokines CXCL9, CXCL10, and CXCL11 were increased in MMe-treated tumor cells, consistent with a role in CD8+ T cell infiltration. These data indicate OXPHOS inhibition stimulated virus-independent MAVS oligomerization, activation of downstream proinflammatory signaling, and reignition of the suppressed pancreatic tumor immune microenvironment.
Funding Provided by NIH (2211376.1-5 R01 CA226279-01.04) and Center for Immunology MCW
Investigation of γδ T cell function in a novel inducible model of lung adenocarcinoma
Authors: Horrigan Katherine J , Lenahan Sean M , Mathieu Erin E , Moquin Cameron A , Dienz Oliver , Seward David J , Boyson Jonathan E 
Published: August 2023

Abstract
γδ T cells have been demonstrated to both promote tumor growth/metastasis and to efficiently kill tumor cells. This dual role of γδ T cells has been attributed to their ability to produce either IL-17 (γδT17) or IFNγ (γδT1), the former being associated with tumor progression and the latter being associated with anti-tumor activity. γδ T cell function is influenced by multiple factors, including the recognition of stress-induced ligands, as well as cytokines and chemokines. However, the specific signals that lead to the selective activation and function of γδT17 or γδT1 subsets in different cancers remains unclear. Here, we explored these questions using a novel inducible mouse model of Kras-driven lung adenocarcinoma with or without a co-mutation in Stk11, a master regulator of metabolic pathways. Preliminary characterization of this model revealed that KrasG12D/Stk11fl/fl mice exhibited significantly greater morbidity than KrasG12D mice. While lung leukocyte numbers did not differ significantly between KrasG12D and KrasG12D/Stk11fl/fl mice, we noted a significantly increased frequency of γδT17, but not γδT1, subsets in KrasG12D/Stk11fl/fl lungs. Interestingly, the increased frequency of γδT17 was due to increased Vγ1 and Vγ6, but not Vγ4, γδT17 suggesting a selective expansion of these subsets. To examine a potential role for butyrophilins in these models, we assessed lung butyrophilin gene expression using qPCR. This analysis revealed increased expression of Btnl1, Btnl2, and Btnl9 in KrasG12D lungs mice as compared to controls. Together, these results suggest that this model has the potential to shed new light on how the interaction of the tumor microenvironment with specific γδ T cell subsets affects disease pathogenesis.
PSGL-1 deficiency re-wires T cell signaling to support development of stem cell like exhausted T cells that control PD-1 blockade resistant melanoma tumor growth
Authors: Otero Dennis C , Hope Jennifer L , Palete Ashley B , Faso Hannah A , Lin Michelle , Roy Sreeja , Wang Eric S , Chow Savio , Yip Kevin , Campos Alexandre Rosa , Bradley Linda M 
Published: August 2023

Abstract
We previously identified that the adhesion molecule P-selectin glycoprotein ligand-1 (PSGL-1) regulates CD8+ T cell effector functions and promotes exhaustion in responses to chronic viral infection and melanoma tumors. In this study, we investigated cellular and molecular mechanisms by which intrinsic PSGL-1 signaling regulates the differentiation of exhausted T cells (TEX). We show that PSGL-1 localizes with the TCR upon co-ligation and find that PSGL-1 associates with Zap70 and the Zap70 inhibitor, Sts-1. In the absence of PSGL-1, expression of Sts-1 is reduced allowing for greater activation of Zap70 and the downstream signaling molecules Akt and Erk1/2. In addition, PSGL-1-deficiency empowers CD8+ T cells to respond to low affinity TCR ligands. In vivo, PSGL-1 deficiency leads to growth inhibition of a PD-1-blockade resistant melanoma. Notably, PSGL-1-deficiency in T cells enables retention of effector T cell (TEFF) functions, and an increased representation of stem cell-like T cells (TSC) that express TCF-1 and low levels of TOX, which are associated with sustained effector responses and responsiveness to ICB. We now show that concomitant PSGL-1 signaling with repeated TCR stimulation of human TEFF elicits functional exhaustion. Importantly, blockade of PSGL-1 recapitulates PSGL-1 deficiency in anti-tumor as well as antiviral responses, and its distinct mechanism of T cell inhibition underscores the translational potential of targeting PSGL-1 by ICB.
Supported by grants from NIH (R01 AI106895, R21 CA249353, R21 CA216678, R03 CA252144) Melanoma Research Alliance MRA 696326, Department of Defense W81XWH-20-1-0324, Sanford Burnham Prebys NCI-Designated Cancer Center Support Grant, P30 CA030199 American Cancer Society Postdoctoral Fellowship (PF-20-113-01-LIB)
Human CAR19 CD8+ iTreg exceed CAR19 CD8 CTLs both in suppressing GVHD lethality and eliminating CD19+ Nalm-6 lymphoma cells in vivo
Authors: Larson Jemma H , Compeer Ewoud B , Torghabeh Mehrdad Hefazi , Smith Kyle D , Jin Sujeong , Dougherty Phillip , McDonald-Hyman Cameron , Houle Bailey , Dustin Michael , Hippen Keli L , Blazar Bruce R 
Published: August 2023

Abstract
Simultaneously achieving a graft versus leukemia (GVL) effect while suppressing graft versus host disease (GVHD) would facilitate the development of immunotherapies for hematopoietic stem cell transplantation. Here, we show that human peripheral blood CD8+CD25− T-cells cultured with IL-2, TGF-β and rapamycin generates CD8 induced Tregs (iTregs). CD8 iTregs expressed FoxP3, CD25 and higher CD103 and CD39, but lower Helios, CTLA-4, and TIGIT than CD4 Tregs. While in vitro suppressor function was modestly reduced with CD8 iTreg, in vivo suppression of GVHD lethality was comparable to that of CD4 Treg. In vitro, cytolytic activity with an αCD3×αCD19 bispecific engager against CD19+ Nalm-6 lymphoma cells was comparable to CD8 CTLs. We subsequently generated CD8 iTreg and CTLs expressing αCD19scFv (CAR19). Following CAR19-EGFR sort purification, CAR19 CD8 iTreg and CAR19 CTL had similar expression of perforin, IFNγ and TNFα. In a 48h Incucyte killing assay against CD19+Nalm-6 targets, using CAR19 CD8 iTreg and CAR19 CTLs generated from the same donors, no significant differences were noted; CD8 iTreg killing was perforin-dependent and granzyme-independent. No killing was observed against CD19 koNalm-6 targets. Unexpectedly, CAR19 CD8 iTreg were found to be significantly more effective at limiting tumor burden in vivo and significantly delayed tumor-related mortality in a xenogeneic Nalm-6 tumor model compared to CAR19 CTLs (p = 0.0018). Mechanistic studies are ongoing. Together, these findings highlight distinct advantages of CD8 iTreg as a concurrent immunotherapy and provide a rationale for CD8 iTreg based CAR therapies to prevent GVHD and promote GVL.
Supported by grants from NIH (R01 HL11879-07 and 2P01 CA065493-25) as well as funding from the Childrens’ Cancer Research Fund
Tissue Engineering Scaffolds Remotely Surveil Presymptomatic Allograft Rejection
Authors: Urie Russell R , Xiao Chengchuan , Hughes Elizabeth , Lombard Elizabeth , Chen Judy , Morris Aaron , Farris Diana , Goldstein Daniel , Shea Lonnie 
Published: August 2023

Abstract
As there is no assay to predict the risk of acute cellular allograft rejection (ACAR), clinicians rely on graft biopsy and aggressive, one-size-fits-all immunosuppression. Endomyocardial biopsy (EMB) is a flawed standard for heart transplant surveillance and diagnosing ACAR, as histological evidence of rejection inherently lags behind molecular biomarkers and suffers from variability. Noninvasive alternatives to EMB in ACAR surveillance, including gene profiling and donor-derived cell-free DNA, also measure lagging indicators of ACAR. A minimally invasive surveillance method is urgently needed to quantify early risk of rejection for minimizing biopsy and personalizing immunosuppression. We have developed porous biomaterial “scaffolds” for minimally-invasive sampling. These scaffolds amass immune cells producing biomarkers as an immunological niche.
We identified predictive biomarkers of ACAR conserved in both in murine heterotopic heart transplant and skin transplant through the remote scaffold. We identified 43 highly differentially-expressed genes as organ-agnostic biomarkers of T cell-mediated rejection. These novel biomarkers differentiate between pre-symptomatic ACAR and recipients with healthy allografts, without invasive biopsy. Scaffold cell capture technology provides a leading indicator of ACAR by monitoring tissue-specific immune responses associated with allograft rejection. This implantable scaffold enables minimally invasive histological evaluation and molecular calculation of the early risk of rejection to reduce the frequency of routine EMB and personalize immune suppression that could prolong transplant life while minimizing risk.
Supported by the Falk Medical Research Trust Catalyst Award and Transformational Award
TLR2 in γδ T cells is required for metabolic support of their IL-17A response to a commensal bacterium on the ocular surface
Authors: Xu Xiaoyan , Zhu Wenjie , Peng Zixuan , Caspi Rachel 
Published: August 2023

Abstract
The ocular surface is colonized by commensal microbiota, which tune the local mucosal immune response. We previously identified an ocular surface commensal, Corynebacterium mastitidis (C. mast), which induced IL-17A production from γδ T cells in the conjunctiva and eye-draining lymph nodes (DLN), and enhanced host resistance to fungal and bacterial pathogens. To elucidate how IL-17A producing γδ T cells (γδT17) are activated by C. mast, we associated WT and TLR2−/− mice with C. mast, and investigated the role of TLR2 in this process. This was based on the rationale that C. mast is a Gram-positive bacterium and that TLR2 expression on γδT cells increased after C. mast association. Mechanistic studies showed that TLR2 expression was required not only on DCs (for IL-1 production) but also on the γδT cells. RNA-Seq revealed that genes related to mitochondrial oxidative phosphorylation were dysregulated in TLR2−/− γδT17 cells, indicating that engagement of TLR2 may regulate metabolic processes in γδT17 cells. Seahorse assay confirmed that TLR2−/− γδ T17 cells displayed impaired oxygen consumption rates and lower ATP production. Interestingly, lysosomal activity, which plays a central role in cellular metabolism, was decreased in TLR2−/− γδ T17 cells. Enhancement of lysosomal activity by curcumin C1 restored ATP generation in TLR2−/− γδ T17 cells. We propose that intrinsic TLR2 signaling in γδ T cells is required for metabolic support of their IL-17A response.
Sympathetic regulation of skin microbiota-induced innate-like T cells
Authors: Rao Indira N , Enamorado Michel , Merrill Dean , Link Verena , Gribonika Inta , Bouladoux Nicolas , Belkaid Yasmine 
Published: August 2023

Abstract
Recent discoveries in microbiota-mediated neuroimmune crosstalk highlight how co-evolved systems collaboratively facilitate host fitness. However, in the skin – the largest and outermost protective barrier – little is known about the role of microbiota in neuro-immune regulation. We find that murine skin colonization with commensal Staphylococci induces significant accumulation of dermal IL-17A-producing Vg6+ gdT (Vg6 gdT17) cells in IL-1 and microbiota-dependent manner. Compared to sedentary Vg6 gdT17 from unassociated controls, intravital imaging shows that commensal-induced dermal Vg6 gdT17 form large highly motile aggregates. Transcriptomic analysis revealed significant differences between Vg6 gdT17 from commensal-colonized mice versus unassociated controls. Among these, the cells from colonized mice showed significant downregulation of sympathetic beta-adrenoreceptor (bAR) expression. Beta-AR signaling has recently been shown to impact conventional T cell motility in the skin. We observe that, upon systemic b2AR agonist administration, the highly dynamic commensal-induced dermal Vg6+ gdT17 clusters are rendered immobile, showing sympathetic control of gdT17 cell motility. Intriguingly, skin commensal exposure leads to systemic induction of Vg6+ gdT17 cells, implying a role for these cells beyond local surveillance. Further studies are underway to test whether, following exposure to skin commensal, sympathetic regulation promotes inter-organ gdT cell dissemination and impacts their functional potential. Overall, this work aims to uncover the mechanism(s) underlying microbiota-induced skin neuroimmune interactions in promoting organismal homeostasis.