bluuebunny/crossref_metadata_2025_split huggingface dataset:
Enriching for tumor-reactive CD8 TIL (AGX148) leads to effective tumor clearance in a patient-derived xenograph model
Authors: Thalhofer Colin J , Ballinger Elaine A , Montler Ryan D , Morris Nicholas P , Rios Joshua F , Moudgil Tarsem , Weinberg Andrew D 
Published: January 2023

Abstract
Adoptive Cell Therapy (ACT) with Tumor Infiltrating Lymphocytes (TIL) can induce durable clinical responses in a subset of patients with melanoma; however, the efficacy of standard bulk TIL therapy can be limited. We hypothesize that the therapeutic efficacy of TIL ACT is dependent on the frequency and functionality of tumor-reactive T cells when infused back into patients. Our approach enhances the activity of the ACT product by enriching for the tumor-reactive CD8 TIL prior to ex vivo expansion by sorting on cells that co-express CD103 and CD39. The CD103/CD39 double positive (DP) CD8 TIL can be found in most human solid tumors but at varying frequencies in any given patient. Surgically resected human tumor samples were provided through a collaboration with the EACRI. Tumor samples were flow sorted and expanded ex vivo and patient-matched autologous tumor lines were generated. Tumors were then cocultured with the expanded autologous ACT product (AGX148) in vitro or used in xenograph mouse experiments. The AGX148 TIL products are effective at recognizing and killing autologous tumor cells, in vitro. Upon adoptive transfer into patient-derived tumor-bearing huIL-2 NOG mice, the transferred human T cells increased in frequency, migrated to the tumor, became activated in the tumor and recognized/destroyed established tumors. Interestingly there was little to no in vivo activity of CD8 TIL that did not express these two markers. We have generated a potent therapeutic ACT TIL product (AGX148) through the selective isolation and expansion of tumor-reactive T cells. This TIL product effectively induces durable curative responses in hosts harboring autologous tumors providing rationale for clinical investigation.
Targeting the high-affinity IL-2R with high-dose mIL-2/CD25 induces effective antitumor responses
Authors: Laporte Kathryn M , Hernandez Rosmely , Malek Thomas R 
Published: January 2023

Abstract
Much interest remains to harness the T cell activating properties of IL-2 to promote antitumor immunity. Current efforts largely utilize approaches directed toward the intermediate-affinity IL-2R to broadly stimulate memory-phenotypic CD8+ T and NK cells while minimizing off-target Tregs. However, this approach does not effectively engage tumor-reactive CD4+ and CD8+ T effector cells that express the high-affinity IL-2R. Recently, we developed a fusion protein where mouse IL-2 was linked to CD25 (mIL-2/CD25 or FP) with selectivity toward the high-affinity IL-2R. At a low dose, the FP is Treg-selective to limit autoimmunity, but at a high dose with a tumor vaccine it expands tumor-reactive T cells to develop antitumor immunity. Here, we assessed the efficacy of high-dose (HD) FP as a cancer monotherapy or with checkpoint blockade. HD FP supports antitumor responses in mice bearing CT26, MC38, or B16.F10 tumors. Although HD FP led to increases in Tregs in immune tissues, Tregs were at a lower frequency in the tumor microenvironment (TME), leading to a high CD8+ T cell:Treg ratio. Further, adding anti-PD-1 to HD FP treatment was more effective in controlling tumors in CT26-bearing mice. Surprisingly, at the doses tested, HD FP supported more robust antitumor responses in each model than IL-2/S4B6 anti-IL-2 complexes (IL2/IC), directed to the intermediate-affinity IL-2R. In the CT26 TME, the number of tumor antigen-specific CD8+ T cells trended upward with HD FP over IL2/IC. These studies indicate that targeting the high-affinity IL-2R on activated tumor-specific T cells with HD FP alone or with PD-1 blockade supports antitumor responses and suggests that this strategy may be more effective than targeting the intermediate-affinity IL-2R.
Supported by Bristol Myers Squibb grant support
Targeting immunosuppression for enhanced focused ultrasound efficacy in triple negative breast cancer.
Authors: Petricca Lydia E , Witter Alexandra R , Sheybani Natasha D , Thim Eric A , Bullock Timothy N , Price Richard J 
Published: January 2023

Abstract
Immunologic rejection of triple-negative breast cancer (TNBC) is rare. Focused Ultrasound (FUS) promotes anti-tumor immunity by inducing tissue destruction and inflammation. We hypothesized that thermally-ablative FUS (tFUS) would increase the immune response to TNBC. However, TNBC is commonly accompanied by the expansion of immunosuppressive myeloid cells. By implanting a metastasizing TNBC cell line, 4T1, into BALB/c mice, we showed that a combinatorial therapy of tFUS and Gemcitabine (GEM), a myeloablative chemotherapy, was able to control primary tumor growth and reduce mortality. This response is dependent on T cell-mediated immunity. While this treatment remarkably resulted in some cures, the effects were not durable in every case; suggesting immunosuppression may be re-established. We hypothesize GEM is synergizing with FUS to immunologically control tumor growth by either depleting myeloid derived suppressor cells (MDSCs) or inducing tumor cell death. To discriminate between these possibilities, we will use a Ly6G-specific 1A8 antibody to deplete the MDSC population. In parallel, we are interrogating the ability of GEM to induce immunogenic cell death in this model. Current studies are focused on understanding alterations and the role of CD4 and CD8 T cells in tumor control after FUS+GEM therapy. Finally, RNAseq analysis has been performed on treated samples to develop hypotheses to explain the effectiveness of dual therapy. Notably, similar therapeutic approaches are now ongoing as a clinical trial at our institution. We expect our studies to reveal the mechanistic basis in which FUS and GEM are eliciting an immunological response resulting in primary tumor growth control and an overall survival advantage.
Supported by grants from NIH (R01 164985 101 GB10846 41017)
A novel linked TCR: MyD88 receptor improves anti-tumor responses while maintaining antigen specificity
Authors: Magno Joey C , Guo Jitao , Davila Eduardo 
Published: January 2023

Abstract
The treatment of malignant tumors utilizing engineered T cell receptor (TCR) therapies in the clinic have demonstrated encouraging therapeutic potential in a subset of patients. Limitations to T cell-based therapies include down-regulation of tumor-antigen presentation by the tumor cells, poor TCR activation, and the immunosuppressive effects of the tumor microenvironment (TME). Attempts to ameliorate these issues by utilizing T cells engineered with tumor-reactive TCRs with increased affinity showed enhanced responses. However, affinity-modified TCRs are more promiscuous and can lead to off-target toxicity. We and others have demonstrated that activating MyD88 signaling (a key adapter protein in toll-like receptor signaling) in T cells enhances effector function and expansion while simultaneously reducing their propensity to enter an exhausted state. In these studies, we designed novel receptors in which the death and intermediate domains of MyD88 are fused to the TCR. T cells engineered to express this novel TCR:MyD88 receptor showed improved responses in a tumor-antigen dependent manner, as demonstrated by increased proliferation and expression of effector molecules, without reducing the specificity of the TCR. In mouse models, TCR:MyD88 T cells conferred improved anti-tumor responses against tumors expressing suboptimal levels of target antigen which was associated with increased T cell infiltration, reduced T cell exhaustion, and an altered TME. These results demonstrate the ability of TCR:MyD88 to improve current therapies without sacrificing the specificity of the TCR which we believe can be utilized to improve the outcome of patients suffering from advanced cancer while reducing toxicities.
Supported by -US Department of Veteran Affairs -Leukemia Lymphoma Society -University of Colorado -Shoots Medical Center -NCI (R01CA207913)
Targeting Autophagy with Carbon-monoxide Reprograms Anti-Tumor T Cells with Robust Immunometabolic Phenotype
Authors: Chakraborty Paramita , Parikh Rasesh Y , Choi Seungho , Mehrotra Meenal , Maldonado Eduardo N , Wang Hongjun , Diehl J Alan , Gangaraju Vamsi K , Mehrotra Shikhar 
Published: January 2023

Abstract
Mitochondria and Endoplasmic reticulum (ER) share structural and functional networks and activates well-orchestrated signaling processes to shape a cell’s fate and function. While persistent ER stress (ERS) response leads to mitochondrial collapse, moderate ERS condition promotes mitochondrial function. Strategies to boost anti-tumor T cell function by targeting ER-Mitochondria cross talk have not been exploited yet. Thus, we used carbon monoxide (CO), a short-lived gaseous molecule, to test if engaging moderate ERS conditions can improve T cells mitochondrial function and anti-tumor function. Using melanoma antigen specific T cells, we identify that CO induced transient activation of ERS sensor ‘protein kinase R-like endoplasmic reticulum kinase (PERK)’ dramatically increase anti-tumor T cell function. Furthermore, CO-induced PERK activation temporarily halts protein translation and induces protective autophagy (that includes mitophagy). To get further insight, we used LC3-GFP to differentiate between the cells that prepare themselves to undergo active autophagy (LC3-GFPpos) and those that fail to enter into the process (LC3-GFPneg). We found that LC3-GFPpos T cells show robust anti-tumor potential whereas, LC3-GFPneg cells show T regulatory-like phenotype, harbor dysfunctional mitochondria, and accumulate abnormal metabolite content. These anomalous ratios of metabolites render the cells with a hypermethylated state and distinct epigenetic profile limiting their anti-tumor activity. Overall, the study highlights that ERS activated autophagy pathways modify mitochondrial function and epigenetically reprogram the T cells towards a superior anti-tumor phenotype to achieve robust tumor control.
IFNγ from IL-12 virotherapy stably controls tumors independent of T cell cytotoxicity and IFNγ sensing by tumor cells
Authors: Walsh Michael , Ali Lestat , Stump Courtney , Lenehan Patrick , Dougan Michael , Knipe David , Dougan Stephanie 
Published: January 2023

Abstract
Although some work has been done to understand the equilibrium phase of cancer immunoediting, it is still common for immunotherapies to generate stable disease or for patients to relapse after being seemingly tumor-free. We have repurposed the non-lytic herpes simplex virus (HSV)-1 d106S vaccine vector for local delivery of IL-12. Our replication-defective d106S virus releases a large burst of IL-12 locally within the tumor environment, synergizing with a type I IFN response induced by the virus. We have shown that d106S-IL12 induces regression and long-term stable immune equilibrium in murine B16 melanoma. Intriguingly, individual depletion of CD8+ T cells, CD4+ T cells, or NK cells does not affect stable tumor control. However, triple depletion of these cell types leads to tumor outgrowth suggesting these cells have redundant functions. Indeed, IFNγ, likely secreted by these lymphocytes, is essential for controlling tumors in response to d106S-IL12 treatment. The inability of tumors to respond to IFNγ and present antigen to CD8+ T cells through MHC-I often renders tumors resistant to checkpoint blockade and other immunotherapies reliant on direct T cell cytotoxicity. Surprisingly, B16 tumors defective in IFNγ-sensing or MHC-I presentation are still stably maintained in response to IL-12 virotherapy. This suggests that while IFNγ secretion by lymphocytes is necessary, IFNγ and the lymphocytes themselves are not required to act directly on cancer cells to stably control tumor growth. Single-cell RNA-sequencing of immune cells infiltrating the tumor indicates that IFNγ is primarily acting on myeloid cells. Thus, myeloid cells activated by IFNγ may be a method to control growth of checkpoint blockade-resistant tumors.
Supported by grants from NIH (P01-AI098681) and Melanoma Research Alliance
Molecular regulation of CD8 T cell activity and heterogeneity in pancreatic cancer
Authors: Green William D , Mullins Genevieve Nicole , Green Jarred Michael , Baldwin Albert , Milner J Justin 
Published: January 2023

Abstract
T cell dysfunction or ‘exhaustion’ is a hallmark of solid malignancies. Despite the critical importance of CD8 T lymphocytes in controlling tumor growth and immunotherapy efficacy, the mechanisms governing T cell exhaustion remain unclear. Here, we focused on resolving the heterogeneity and molecular programming of exhausted CD8 T cell states in the context of pancreatic cancer. Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a discouraging 5-year survival rate of approximately 8%. PDAC tumors commonly present as immunologically ‘cold’, devoid of protective T cell populations, and are typically resistant to promising immunotherapies such as immune checkpoint blockade or adoptive cell therapy. Therefore, understanding the unique microenvironmental and CD8 T cell-intrinsic signals governing T cell exhaustion and accumulation in PDAC tumors may reveal crucial insight for engineering CD8 T cells for optimal immunotherapy efficacy. Here, we developed preclinical PDAC models for profiling tumor-specific murine CD8 T cell responses. Through application of spectral flow cytometry and multimodal single-cell sequencing approaches, we have uncovered impressively diverse antigen-specific CD8 T cell states in PDAC tumors. Select CD8 T cell populations identified within the PDAC tumor microenvironment included canonical populations of terminally-exhausted and progenitor-exhausted CD8 T cells as well as unexpected and uncharacterized CD8 T cell populations. Accordingly, we also have identified critical transcription factors controlling key features of CD8 T cell heterogeneity and persistence in PDAC tumors. These findings may ultimately provide insight for tailoring CD8 T cell fate and function in PDAC.
Supported by 2022 AAI Intersect Fellowship for Computational Scientists and Immunologists
Tumors bearing defective transcription elongation are immune hot but resistant to immune checkpoint inhibitors
Authors: Modur Vishnu , Guo Fukun 
Published: January 2023

Abstract
Tumors infiltrated with abundant T lymphocytes are generally thought to respond to immune checkpoint inhibitors (ICI). However, a significant proportion of immune hot tumors are resistant to ICI. The immune nature of such tumors is poorly understood. We recently reported a tumor type that is defective in transcription elongation (TEdef) and irresponsive to ICI in several clinical cohorts. Here, we show that TEdef tumors are unexpectedly infiltrated with cytotoxic T cells (CTLs) in patients and in animal models. Mechanistically, T cell infiltration is facilitated by cytoplasmic DNA/double strand RNA-nucleic acid sensing-TBK1-ccl5/cxcl/9/10 signaling cascade. However, single-cell RNA sequencing combined with flow cytometry reveals that CTLs in TEdef tumors are exhausted. Furthermore, immune suppressive regulatory T cells are enriched in TEdef tumors. ICI treatment of TEdef tumors in mice and human patients fails to further induce T cell infiltration. This study reveals a mechanism underlying inert inflammation and ICI resistance of TEdef tumors. We suggest that TEdef, alongside the surrogate markers of tumor mutational burden and CTLs, should be assessed in immunotherapy-candidate patients.
Supported by 5R01CA234038-03
Suppression of local IFN-I by commensal microbiota-derived butyrate impairs antitumor effects of ionizing radiation
Authors: Yang Kaiting , Fu Yang-Xin , Weichselbaum Ralph R 
Published: January 2023

Abstract
The antitumor effects of ionizing radiation (IR) are mediated in part through activation of innate and adaptive immunity. Here, we report that gut microbiota influences tumor control following IR. Vancomycin decreased the abundance of butyrate-producing gut bacteria, accompanied with reduction of butyric acid in circulation and tumor tissues, and enhanced antitumor responses to IR. Oral administration of Lachnospiraceae, a family of vancomycin-sensitive bacteria, associated with increased systemic and intratumoral butyric acid levels and impaired the efficacy of IR in germ-free (GF) mice. Local butyrate inhibited STING-activated type I IFN expression in dendritic cells (DCs) through blockade of TBK 1 and IRF3 phosphorylation, which abrogated IR-induced tumor-specific cytotoxic T cell immune responses without directly protecting tumor cells from radiation. Our findings demonstrate that the selective targeting of butyrate-producing microbiota may provide a novel therapeutic option to enhance tumor radiation sensitivity.
This work was supported by a grant from the Ludwig Foundation to R.R. Weichselbaum, and National Institutes of Health/National Cancer Institute Provocative Questions grants R21 CA227528 and R21 CA231273-01 (R.R. Weichselbaum).
Metabolic reprogramming of antitumor CD8+ T cells by modulation of GSH-Gpx4 axis
Authors: Chen Siqi , Fan Jie , Xie Ping , Ahn Jihae , Fernandez Michelle , Chandel Navdeep , Zhang Bin 
Published: January 2023

Abstract
The CD8+ T cells within tumor microenvironment are the most essential component in tumor immunity. Activation of CD8+ T cells is coupled to various metabolic reprogramming. Glutathione (GSH) as the key role in oxidative metabolismto buffer increased reactive oxygen species (ROS) in activated T cells, is important for T cells effector functions in the context of inflammation. The function and mechanism by GSH metabolism in regulating anti-tumor immunity of CD8+ T cells remain unknown. Here, we show that GSH is dependent on glutathione peroxidase 4 (Gpx4) to maintain CD8+ T cell activity, and A2AR signaling pathway interacts with the GSH-Gpx4 axis to reprogram the metabolism and survival of functional CD8+ T cells. Interestingly, A2AR signaling blockade increases effector function of antitumor CD8+ T cells, but facilitates the assumption of intracellular GSH, leading to rapid ferroptosis. Notably, combination treatment with a potent ferroptosis inhibitor liproxstatin-1 (Lip-1) and A2AR antagonists elicits a synergistic antitumor efficacy and enhanced mitochondrial functionality of antitumor CD8+ T cells in multiple mouse tumor models. Finally, we generate a gene expression signature for GSH metabolism in tumor-infiltrating CD8+ T cells positively correlating with favorable clinical outcomes. Our work demonstrate a critical role of GSH metabolism in modulating antitumor CD8+ T cell survival and functionality, pointing to new strategies of targeting these cells for cancer immunotherapy.
CD8 T cells licensed with immune checkpoint blockade kill murine tumors lacking MHC-I
Authors: Lerner Emily C , Tomaszewski William , D’Anniballe Vincent , Perera Jonathan , Cui Xiuyu , Wilkinson Daniel S , Waibl-Polania Jessica , Gunn Michael , Fecci Peter E , Woroniecka Karolina 
Published: January 2023

Abstract
MHC-I downregulation is a well described mechanism of tumor immune escape. Thus, targeting tumors with low or no MHC-I expression remains a significant challenge for T cell-based immunotherapies, including immune checkpoint blockade (ICB).
We previously demonstrated that the combination of PD-1 blockade and 4-1BB agonism has marked efficacy against intracranial murine CT2A glioma in a CD8 T cell-dependent manner. Surprisingly, this combination therapy remained effective in a β2 microglobulin knockout CT2A line overexpressing TRP2 (CT2A-TRP2-β2mKO). These tumors lack MHC-I but retain the Trp2 rejection antigen. Remarkably, the persisting efficacy remained dependent on CD8 T cells, but independent of NK cells, CD4 T cells, and B cells. Furthermore, the efficacy was demonstrated to be antigen-specific in vivo, as adoptively transferred Trp2-specific T cells prolonged survival of mice bearing CT2A-TRP2-β2mKO tumors, but not those bearing CT2A-β2mKO, which lacked the TRP2 antigen.
To analyze the mechanism driving antigen-dependent killing of tumor cells lacking MHC-I, we performed a series of in vitro cytotoxicity experiments using Trp2-specific T cells in the presence or absence of Trp2 antigen-loaded bone marrow-derived macrophages (BMDM). These studies demonstrated that in the absence of BMDM, Trp2-specific T cells efficiently killed MHC-I-expressing CT2A-Trp2, but failed to kill MHC-I deficient CT2A-Trp2-B2mKO tumors. However, killing was restored in the presence of Trp2 loaded BMDMs; the latter of which possessed little to no cytotoxic effect alone. These findings suggest a novel role for myeloid populations in mediating the influence of ICB on T cell function and challenge the traditional model of T cell tumor killing.
Adoptive transfer of Trac-targeted T cell receptor engineered T cells with defective Tgfbr2 signaling promotes pancreatic cancer eradication
Authors: Rollins Meagan R , Burrack Adam L , Miller Ebony , Stromnes Ingunn 
Published: January 2023

Abstract
Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy characterized by a suppressive tumor microenvironment (TME) with elevated Tgfb. Adoptive transfer of T cell receptor (TCR) engineered T cells specific to mesothelin (Msln) effectively targets autochthonous PDA, but the TME promotes engineered T cell dysfunction and limits efficacy. To test our hypothesis that Tgfb is a key mediator of engineered T cell dysfunction in PDA, we first created a Msln-specific TCR Trac knock-in mouse model to generate a uniform source of Msln406-414:H-2Db-specific T cells. Using CRISPR/Cas9, we next knocked out Tgfbr2 in Msln-specific T cells. Both Tgfbr2WT and Tgfbr2KO engineered T cells preferentially accumulate in orthotopic KPC tumors, comprising over 70% of the total CD8+ intratumoral T cells. In combination with vaccination, engineered T cells cause a 10-fold reduction in tumor weight at day 12 post tumor implantation. Loss of Tgfbr2 in effector T cells increases Klrg1, IFNg, TNFa and decreases exhaustion markers PD1, Lag3 and Tox in engineered T cells. Further supporting our data, Tgfbr2 cKO mice receiving orthotopic KPC2a PDA implantation had reduced tumor growth compared to Tgfbr2 WT littermates. Analysis of the endogenous tumor-specific T cells using a tetramer support the role of Tgfb in promoting T cell exhaustion and tumor growth in PDA. Ongoing 2-photon studies on the extent Tgfβ impacts engineered T cell migration, interactions with dendritic cells and stromal modifications will be discussed. Overall, disruption of Tgfβ is a key mediator of both engineered and endogenous tumor-antigen-specific T cell fate by promoting potent Klrg1+ T cells at the expense of Tox+ exhausted T cells and leading to durable tumor control in PDA.
Supported by grants from NIH (R01 CA249393, R01 CA255039, T32 AI 007313), Dennis W. Watson Fellowship, American Association for Cancer Research (AACR) Pancreatic Cancer Action Network Career Development Award (17-20-25-STRO), an AACR Pancreatic Cancer Action Network Catalyst Award (19-35-STRO), an American Cancer Society Institutional Research Grant (124166-IRG-58-001-55-IRG65)