bluuebunny/crossref_metadata_2025_split huggingface dataset:
An experimental method to delineate responses from pre-existing memory and induced naive T cells in neoepitope candidate screening
Authors: Chihab Leila Y , Burel Julie G , Koşaloğlu-Yalçin Zeynep , Peters Bjoern 
Published: August 2023

Abstract
Identifying tumor-specific T cells is critical to the development of T-cell based immunotherapies. As a result, computational tools have been developed to predict the neoepitopes present within a tumor. A common experimental approach used to screen neoepitope candidates is the long-term in vitro expansion of patient peripheral blood mononuclear cells (PBMC) with candidate peptides-followed by a fluorospot to detect antigen-specific responses. Although this approach allows for high-throughput screening of candidates, it cannot distinguish between responses from pre-existing memory cells versus from naïve cells that were primed in vitro.
We have developed an experimental method to fulfill this need. We designed a negative control peptide pool of 15-mers with no sequential similarity to any known antigens. Through the use of this pool with depletion of naïve T cells from PBMC, we show in 12 healthy donors that this megapool successfully quantifies signal that is a result of naïve cell proliferation. Importantly, we applied this approach to screen pools of neoepitopes in this cohort of donors. We found that the majority of the neoepitope-specific responses detected were derived from induced naïve cells whereas only a maximum of 3 donors had pre-existing memory to any given neoepitope pool. This work underscores the applicability and relevance of our method in characterizing fluorospot signals and provides the foundation for others to study the role of these various T cell responses in therapeutic outcomes.
This material is based upon work supported by the National Science Foundation Graduate Research Fellowship Program under Grant no. 2038238. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.
CD38-cADPR axis mediated chronic Ca2+ signaling promotes the differentiation of TCF1lo terminal exhausted CD8+ T cells in tumor
Authors: Kar Anwesha , Ghosh Puspendu , Chatterjee Shilpak 
Published: August 2023

Abstract
Preponderance of terminally exhausted CD8+T cells (Texterm) at the tumor site is the major impediment for successful immunotherapy of cancer. Despite the recent understanding in epigenetic, transcriptomic and metabolic features of Texterm, the cellular pathway underpinning the differentiation of Texterm remains elusive. Upon assessing intratumoral CD8+ T cells from both clinical samples and pre-clinical mouse models, we observed that CD8+ T cells co-expressing CD38 and PD1 displayed the characteristic features of Texterm. Single cell RNA sequencing analysis from both murine tumor infiltrating CD8+ T cells and in vitro chronically stimulated human CD8+ T cells revealed that clusters co-expressing Cd38 and Pdcd1 diplayed the transcriptomic signature of Texterm. Most importantly, clusters expressing Cd38 and Tcf7 were found to be mutually exclusive. Further studies to decipher the role of CD38 showed that both pharmacological and genetic knockdown of CD38 maintained TCF1 expression in chronically exhausted CD8+ T cells and significantly improved their ability to control tumor growth upon adoptive transfer in B16–F10 melanoma bearing mice. Mechanistic studies revealed that CD38 expression on Textermled to chronic elevation of intracellular Ca2+ levels through cADPR-RyR2 axis, which activated Akt signalling. Activation of Akt restrained the nuclear localization of Ezh2, which was found to be pivotal in maintaining TCF1 expression in Texterm and inhibition of which in CD8+ T cells resulted in poor anti-tumor response. Thus, our data suggests that CD38 could be a drugable target to improve the immunotherapeutic efficacy of CD8+ T cells in cancer.
Supported by grants from IA/I/19/1/504277/WTDBT_/DBT-Wellcome Trust India Alliance/India CRG/2019/001334 SERB Extramural Fund CSIR Intramural Fund
DUSP2 functions as a negative regulator of cytotoxic T cell responses in cancer immunotherapy
Authors: Dellacecca Emilia Rae , Barham Whitney , Li Ying , Liu Xin , Gicobi Joanina K , Yan Yiyi , Hirdler Jacob B , Dong Haidong 
Published: August 2023

Abstract
Only a small cohort of patients show durable responses to immune checkpoint inhibitor (ICI) therapy and the mechanisms regulating CD8 T cell responses to ICIs are yet to be elucidated. To that end, we explored T cell intrinsic factors that may be attributed to low ICI therapy response rates. We have identified Dual Specificity Phosphatase 2 (DUSP2) as a potential negative regulator of ICI response within CD8 T cells. We demonstrated that DUSP2 expression in human CD8 T cells is transient, peaking at two-hours post-T cell stimulation with rapid waning thereafter. Downregulation of DUSP2, via siRNA transfection, increased cytotoxicity of human CD8 T cells activated for 48-hours prior to inclusion in a four-hour tumor killing co-culture assay, despite the return of DUSP2 expression to baseline levels at this time-point. Additionally, DUSP2 overexpression, via mRNA transfection, decreased release of effector molecules (Granzyme B and Interferon-gamma) from human CD8 T cells at 24-hours post-activation when compared to control mRNA. When we utilized RNA-sequencing to analyze and compare differentially expressed genes between responders and non-responders of ICI therapy, we found DUSP2 expression in CX3CR1 positive CD8 T cells to be consistently decreased in responders but not in non-responders. Since CX3CR1 positive CD8 T cells identify a highly resilient and cytotoxic subset in response to ICI therapy, we hypothesize that DUSP2 negatively regulates ICI-responsive effector CD8 T cells. Thus, uncovering the role of DUSP2 in CD8 T cells may provide a target to improve the efficacy of ICIs in patients with limited therapeutic alternatives.
Mayo Clinic Department of Immunology Pilot Award Mayo Clinic Center for Individualized Medicine
Targeting epigenetic crosstalk that restrains the responses of exhausted T cells to immune checkpoint blockade therapy
Authors: Yousif Amir , Saadey Abbey , Castillo Chelsea , Saini Ankita , Webb Amy , Oltz Eugene M , Ghoneim Hazem 
Published: August 2023

Abstract
Epigenetic scarring of exhausted CD8 T (TEX) cells during chronic virus infections or cancer remains a major cell-intrinsic barrier to T cell immunotherapies, including immune checkpoint blockade (ICB). For successful epigenetic reprogramming of TEX cells, it is crucial to identify and target the molecular mechanisms underlying terminal exhaustion. Previous work revealed that de novo DNA methylation enforces silencing of T cell function and restrains their responses to ICB therapy. Yet, it remains largely unknown whether post-translational histone modifications crosstalk with de novo DNA methylation during the progression to terminal exhaustion. To better understand the interplay between these epigenetic mechanisms in chronically stimulated CD8 T cells, we employed our novel in vitro model of human T cell dysfunction, as well as preclinical models of T cell exhaustion including chronic LCMV infection. We found a significant relationship between dynamic histone changes and de novo DNA methylation in TEX cells. Importantly, targeting a key histone-modifying enzyme in dysfunctional human T cells improved their effector function and cytotoxicity in vitro. In addition, during chronic LCMV infection, combined treatment by a selective inhibitor and anti-PD-L1 significantly enhanced the ICB responses of stem-like and cytolytic subsets of TEX cells. We further explored the impact of targeting histone modifications on DNA methylation programming in CD8 T cells. These findings provide important mechanistic insights for developing novel therapeutic approaches to epigenetically reprogram TEX cells and enhance T cell immunotherapies.
Supported by the Ohio State University College of Medicine and the OSU Comprehensive Cancer Center.
Low avidity CD8+ T cells are less exhausted than their high avidity counterparts and provide superior anti-tumor immunity
Authors: Singhaviranon Summit , Dempsey Joseph , Hagymasi Adam , Mandoiu Ion , Srivastava Pramod K 
Published: August 2023

Abstract
The interaction of T cell receptors (TCRs) on T cells with MHC I-peptide complexes on cancer cells elicits the cytotoxic activity of CD8+ T cells. Here we analyze how the strength of this interaction, or T cell avidity, shapes the exhaustion of CD8+ TILs and dictates anti-tumor immunity. We developed a novel tetramer decay assay to isolate T cells based on their TCR avidities. We used this method to study low and high avidity CD8+ TILs responding to the tumor neoantigen PDPRMUT of the murine sarcoma Meth A. Single cell sequencing reveals that high avidity CD8+ TILs are more terminally exhausted (51.41% vs 27.21%) and less effector-like (28.39% vs. 60.0%) than low avidity CD8+ TILs. We used flow cytometry to analyze PDPRMUT-specific CD8+ TILs with low or high avidity from 28 day-old Meth A tumors. There were significantly more TIM3+PD-1+ cells (Paired t-test; P &lt; 0.0001) and TOX+PD-1+ cells (Paired t-test; P &lt; 0.05) in the high avidity CD8+ TILs than the low avidity CD8+ TILs. We adoptively transferred 1,000 PDPRMUT-specific low or high avidity CD8+ T cells, or control CD8+ T cells into tumor-bearing mice 10 days after tumor challenge. Low avidity T cells significantly improved the survival of mice (Mantel-Cox; P = 0.0044), while high avidity t cells did not do so (Mantel-Cox; P = 0.7081). These results establish a novel correlation between avidity, exhaustion and T cell-mediated tumor control in vivo.
Temporal requirements for TGF-β and IL-7 in generating resident memory T cell responses to melanoma
Authors: Ramirez Delaney E , Hawkes Aaron R , Searles Tyler G , Wang Chen-Yu , Nemeth Alexander N , Pioli Patricia A , Turk Mary Jo 
Published: August 2023

Abstract
Resident memory CD8+ (TRM) cells persist in tissues and are critical mediators of the host anti-tumor response. We previously demonstrated that protective, melanoma/melanocyte antigen-specific CD8+ TRM cells are generated in skin and B16 tumor-draining lymph nodes (LNs) of mice following CD4+ T cell depletion and tumor excision surgery. Whereas TGF-β and IL-7 have previously been shown to play important roles in generating viral Ag-specific TRM cells, a role for these cytokines in generating TRM responses against tumor antigens had not been studied. We employed antibody-mediated blockade of TGF-β (mAb clone 1D11.16.8) or IL-7 receptor (IL-7R; mAb clone A7R34) during primary tumor growth or after tumor excision to define a temporal role for these cytokines in generating the TRM response. Sentinel gp100-specific pmel CD8+ T cells were used to monitor priming and memory generation. TGF-β blockade during tumor growth did not impair primary responses but significantly reduced CD103+ CD69+ TRM formation in skin and draining LNs one month later. TGF-β signaling was only required during priming, as delayed blockade after tumor excision had no effect. Conversely, IL-7R blockade during priming did not impair TRM generation in skin or LN. However, its blockade after tumor excision significantly impaired TRM formation in both locations. These studies demonstrate temporally distinct requirements for TGF-β and IL-7 in promoting TRM responses against tumor antigens.
Supported by grants from the NIH (R01CA22502, T32-AI007363), The Knights of the York Cross of Honour and O. Ross McIntyre, M.D. Endowment to MJT
PD-1 in balancing expansion of effector T cells and attrition of memory T cell during tumor-reactive T cell responses
Authors: Mao Zhiming , Hirdler Jacob , Harrington Susan , Liu Xin , Gicobi Joanina , Dellacecca Emilia , Hsu Michelle A , Zhang Wenjing , Park Sean , Dong Haidong 
Published: August 2023

Abstract
Though anti-PD-1/PD-L1 immune checkpoint blockade therapy has demonstrated robust anti-tumor immunity in human patients with advanced cancers such as melanoma, only a fraction of patients achieved such durable anti-tumor responses. Among all the non-responders, some patients responded initially but developed resistance to the therapy in the later treatments. This reflects an urgent need to understand the role of PD-1 signals in CD8+ T cells at priming and at effector stages during tumor development for the benefit of immunotherapy. The controversy of rapid tumor progression in PD-1 conditional knockout in regulatory T cells and tumor regression in PD-1 targeted deletion in regulatory T cells indicated germline PD-1 knockout mice might not be sophisticated enough to study the role of PD-1 in CD8+ T cells during tumor development. Thus, we produced a CD8 T cell-specific PD-1 knockout mouse model (CD8 E8ICre-PD-1fl/fl, also called CD8-PD-1 cKO mice) in which PD-1 is deleted in CD8+ T cells in the peripheral tissues. When CD8-PD-1 cKO mice were challenged with B16-OVA mouse melanoma cells, we found a delayed tumor growth and prolonged survival of CD8-PD-1 cKO compared to control mice. However, after 20 days of tumor challenge, the delayed tumor growth rebound in CD8-PD-1 cKO mice. In an OVA antigen immunization model, we found increased effector cells and tissue resident cells in the spleen of CD8-PD-1 cKO mice compared with control mice on day 7 after immunization. Taken together, our results suggest that PD-1 may restrain the expansion of effector T cells that contribute to the early phase of tumor control, but the augmented effector T cell response may cause an attrition of memory T cell pool that will be needed for long term protection.
R01CA256927
Thymoquinone treatment enhances T cell-mediated anti-tumor response
Authors: Pandey Rachana , Sirbu Cristian , Hankins Gerald , Fandy Tamer , Du Wei 
Published: August 2023

Abstract
Oil from Nigella sativa seeds (“black seed oil”) has been used for health care and medical purposes for over 2000 years. The major active compound thymoquinone (TQ) has been shown to have immune regulatory effects in various diseases. Tumor microenvironment is critical for tumor development. Immune cells, particularly T cells, are central players to kill tumor cells and affect tumor progression and metastasis. The impact of TQ on human T cell-mediated anti-tumor response was investigated. Using human PBMCs, we activated T cells with anti-CD3/CD28 in vitro, and treated cells with TQ. First, we tested the impact of TQ on T cell activation, proliferation and apoptosis; second, the anti-tumor effect of T cells after treatment with TQ; last, we determined alterations in T cells gene expression at the RNA level after TQ treatment. TQ treatment increased activation and proliferation of T cells at days 2–4 and more activation-induced cell death at day 3. After 7–11 days, TQ-treated cells showed less survival than cells treated with control medium. At low TQ concentration (10uM), treatment induced more CD3+ T cell proliferation; while at 20uM and 50uM TQ showed more cytotoxic effect to T cells, and more cell death. When co-cultured with multiple types of cancer cells, TQ-treated T cells exhibited significantly enhanced anti-tumor activity, indicated by more tumor cell apoptosis and less cell expansion. Gene expression study showed upregulation of NFAT signaling pathway, including 10+ fold up-regulation of IL-2, which might explain the enhanced anti-tumor effect after TQ treatment. In summary, TQ treatment can enhance human T cell-mediated anti-tumor effect in vitro. This immunoregulatory function could benefit patients with different cancers.
Supported by grants from NIH/ NIGMS (2U54GM104942-02, 5U54GM104942-03).
Loss of OGT enhances type I interferon-dependent antitumor immunity
Authors: Wen Haitao , Chen Jianwen 
Published: August 2023

Abstract
The O-GlcNAc transferase (OGT) is an essential enzyme that mediates protein O-GlcNAcylation, a unique form of posttranslational modification of many nuclear and cytosolic proteins. Recent studies observed increased OGT and O-GlcNAcylation level in a broad range of human cancer tissues compared to adjacent normal tissues, indicating a universal effect of OGT in promoting tumorigenesis. Here, we show that OGT is essential for tumor growth in immunocompetent hosts via repressing cyclic GMP-AMP synthase (cGAS)-dependent DNA sensing pathway. We found that deletion of OGT (Ogt−/−) caused a markedly reduction in tumor growth in both syngeneic tumor models and genetic colorectal cancer (CRC) model induced by mutation of the Apc gene (Apcmin). Pharmacological inhibition or genetic deletion of OGT induced a robust genomic instability (GIN), leading to cGAS-dependent production of type I interferon (IFN-I) and IFN-stimulated genes (ISGs). As a result, deletion of Cgas from Ogt−/− MC38 CRC cells restored tumor growth, which correlated with impaired CD8+ T cell antitumor immunity. Mechanistically, we found that OGT-dependent cleavage of host cell factor C1 (HCF-1) is required for the avoidance of GIN and IFN-I production. In sum, our results identify OGT-mediated genomic stability as an important tumor cell-intrinsic mechanism to repress antitumor immunity.
Supported by the National Institutes of Health R01GM120496 (H.W.)
Enforced expression of c-Myc and T58A-mutant c-Myc in adult thymic epithelial cell subsets
Authors: Swanbery Nathan J 
Published: August 2023

Abstract
Thymic epithelial cells (TEC) are essential for T lymphopoiesis. Cortical (c)TEC support T lineage commitment and positive selection, whereas medullary (m)TEC delete self-reactive T cells to enforce central tolerance. Both cTEC and mTEC contain several distinct subtypes that were only recently characterized, and precursor-progeny relationships among adult TEC remains incompletely understood. Prior work in our lab demonstrated that enforced expression of c-Myc broadly in TEC under a constitutive FoxN1-Cre increased thymus size and TEC numbers, while maintaining morphology and function. To elucidate which TEC subtypes require enforced c-Myc expression to drive thymic expansion in adults, we’ve developed and acquired Cre-ERT2 systems under the control of TEC-specific genes FoxN1, Aire, and Ccl21a. This allows us to target expression of c-Myc or stabilized T58A-mutant c-Myc to specific subsets of TEC in adult mice. Analysis of these systems is currently ongoing and results will be presented. We hypothesize that enforced expression of c-Myc or stabilized T58A-mutant c-Myc in specific adult TEC subsets will expand the targeted population and may eventually result in transformation. Regardless of whether our hypothesis is correct, our experiments will contribute to better understanding of c-Myc-driven thymic hyperplasia and may provide a mouse model for human thymoma.
CEACAM marks intratumoral Tregs that exhibited highly suppressive and activated phenotypes
Authors: Chung Youseung , Jeon Seung Hyuck , Jeon Minwoo , Kim A Reum , Lee Yong Joon , Kim Eui-soon , Shin Eui-Cheol , Park Su-Hyung 
Published: August 2023

Abstract
Regulatory T cells (Tregs) exert immunosuppressive functions and hamper anti-tumor immune responses in the tumor microenvironment. Understanding the heterogeneity of intratumoral Tregs, and how it changes with tumor progression, will provide clues regarding novel target molecules of Treg-directed therapies. Here we characterized the phenotypes and suppressive function of Tregs from tumor tissues of 42 patients with renal cell carcinoma using multicolor flow cytometry, RNA sequencing, and in vitro functional assay. We found that carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) was selectively expressed on intratumoral Tregs, while its expression on peripheral Tregs or other immune cells was minimal. CEACAM1+ intratumoral Tregs accumulated with tumor progression, while the CEACAM1− subset did not. Notably, we found that CEACAM1 marked intratumoral Tregs that exhibited highly suppressive and activated phenotypes with substantial clonal expansion. Depletion of CEACAM1-expressing cells from tumor-infiltrating leukocytes led to increased effector functions of exhausted T cells. Moreover, CEACAM1+ cell depletion further enhanced anti-PD-1-–mediated reinvigoration of exhausted CD8+ T cells. Collectively, our results demonstrate that CEACAM1 delineates highly suppressive subset of intratumoral Tregs, and can be a target for selective depletion of intratumoral Tregs.
Biodegradable Nanoparticles Immunotherapy for the Treatment of Carcinogen-Induced Bladder Cancer
Authors: Podojil Joseph R , Cogswell Andrew C , Chiang Ming-Yi , Neef Tobias , Yu Yanni , Murthy Tushar , Boyne Michael T , Elhofy Adam , Meeks Joshua J , Miller Stephen D 
Published: August 2023

Abstract
Patients with locally advanced and metastatic urothelial carcinoma have a low survival rate (median 15.7 months, 13.1–17.8), with a 23% response rate to monotherapy treatment with anti-PD-L1 immunotherapy. We recently reported the potent effects of i.v. infusions of immune modifying poly(lactic-co-glycolic acid) (PLGA) nanoparticles (IMPs; named ONP-302) to both decrease B16.F10, MC-38, LLC, and 4T1 tumor growth and allow for B16.F10 tumor responsiveness to anti-PD-1 treatment. We next sought to determine if ONP-302 treatment could significantly decrease the more clinically relevant BBN-carcinogen induced bladder cancer. The present data show that ONP-302 treatment significantly decreases bladder cancer stage and overall bladder weight. While minor epithelial hyperplasia remained, ONP-302 treatment inhibited widespread carcinoma in situ and muscle invasive tumor development, as compared to saline treated mice. Mechanistically, ONP-302 infusion decreased tumor growth via the activation of the cGAS/STING pathway within myeloid cells, and subsequently increased CD8+ T cell and NK cell activation via IL-15. In subsequent studies to assess the cellular mechanisms involved in ONP-302 function, we found that in vitro treatment with ONP-302 rapidly induced oxidized DNA within the cells that took up PLGA nanoparticles and oxidized DNA was present within the supernatant as well. Furthermore, co-treatment of ONP-302 with DNase inhibited the STING mediated cytokine production. These findings indicate that ONP-302 allows for tumor control via reprogramming myeloid cells and inducing a STING pathway activation, increases anti-PD-1 response rates, and significantly decreases BBN-carcinogen induced bladder cancer.