Terminal Exhaustion Research Articles

Class I HDAC inhibitors enhance antitumor efficacy and persistence of CAR-T cells by activation of the Wnt pathway

Epigenetic modification shapes differentiation trajectory and regulates the exhaustion state of chimeric antigen receptor T (CAR-T) cells. Limited efficacy induced by terminal exhaustion closely ties with intrinsic transcriptional regulation. However, the comprehensive regulatory mechanisms remain largely elusive. Here, we identify class I histone deacetylase inhibitors (HDACi) as boosters of CAR-T cell function by high-throughput screening of chromatin-modifying drugs, in which M344 and chidamide enhance memory maintenance and resistance to exhaustion of CAR-T cells that induce sustained antitumor efficacy both invitro and invivo. Mechanistically, HDACi decrease HDAC1 expression and enhance H3K27ac activity. Multi-omics analyses from RNA-seq, ATAC-seq, and H3K27ac CUT&Tag-seq show that HDACi upregulate expression of TCF4, LEF1, and CTNNB1, which subsequently activate the canonical Wnt/β-catenin pathway. Collectively, our findings elucidate the functional roles of class I HDACi in enhancing CAR-T cell function, which provides the basis and therapeutic targets for synergic combination of CAR-T cell therapy and HDACi treatment.

Abstract 4075: Targeting attenuated IL-18 to PD-1: A next generation checkpoint inhibitor with enhanced anti-tumor activity

Abstract PD-1 antagonists have shown strong clinical proof of concept, but the development of next-generation versions that can target tumors that weakly respond and/or acquire drug resistance is a critical medical need. Lymphocytes, including T cells and NK cells, upregulate PD-1 and other immunoregulatory receptors in response to neoantigens. Such immunoregulation is important for maintaining the persistence of immune responses in a controllable manner so that immune pathology does not overwhelm the host. In addition to microbial proteins, neoantigens are often expressed by tumor cells. These 'tumor antigens' are derived from mutated genes or proteins not presented during T cell education. As such, many established tumors contain areas of chronic inflammation, including a subset of PD-1+ T cells that can be 'de-immuno-regulated' by anti-PD-1 antagonist antibodies, thus enhancing anti-tumor activity. IL-18 is a cytokine that delivers a 'danger' signal to NK cells and T cell subsets that express the IL-18 receptor complex (RC) and increases their capacity to kill, induces proliferation, diminishes terminal exhaustion, and sustains survival. Importantly, IL-18RC is expressed on progenitor exhausted T cells, the population of T cells in solid tumors that is most active and responds to anti-PD-1, but not terminal exhausted T cells. The latter have been shown to be non-responsive to PD-1 antagonism. Thus, we engineered an anti-PD-1-IL-18 antibody to enhance anti-tumor immune responses. Such a therapeutic requires two key characteristics: resistance to inhibition by IL-18BP, IL-18's natural antagonist, and PD-1-dependent cis-restricted binding to the IL18RC on PD-1+ cells to reduce trans-based systemic toxicity. We have engineered an IL-18BP-resistant mutant of IL-18 and generated variants with different degrees of attenuation for PD-1-dependent cis targeting. Attenuation was achieved by sterically hindering the capacity of IL-18 to bind the IL-18RC while maintaining cis activity without a requirement for proteolytic activation. We identified a variant of IL-18 that is 10,000-fold attenuated and, when fused to an anti-PD-1 antibody, mediated (1) PD-1-dependent cis activity in vitro equivalent to ~5x that of anti-PD-1 alone and (2) tumor shrinkage in mouse tumor models, including an ~80% frequency of complete responses. Due to the high degree of IL-18 attenuation, we were able to dose mice without toxicity to levels at which full PD-1 antagonism is maintained. To our knowledge, this anti-PD-1-IL-18 variant is the first PD-1 targeted cytokine capable of activating PD-1+ T cells via the cytokine’s receptor and fully antagonizing PD-1 as a single agent. Thus, targeting a highly attenuated variant of IL-18 that retains strong cis activity to PD-1 holds promise as a clinical candidate for the treatment of cancer indications that respond weakly to anti-PD-1 and/or are associated with acquired resistance. Citation Format: Xueyuan Zhou, Jeffrey Takimoto, Sunny Yoon, Nikhita Khanna, Brian Rabinovich. Targeting attenuated IL-18 to PD-1: A next generation checkpoint inhibitor with enhanced anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4075.

Abstract 1359: Rewiring CD8+T cell responses to PD-1 immune checkpoint blockade in PDAC by preventing terminal exhaustion via the inhibitory receptor PSGL-1

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, poorly immunogenic cancer with increasing incidence and a poor 5-year survival rate of 12%. PDAC tumors are refractory to all currently available treatments, including immune checkpoint blockade (ICB) therapies, and demonstrate limited infiltration of cytotoxic CD8+ T cells that kill tumor cells. There is therefore an urgent, unmet need for novel therapeutic options. Analysis of single-cell RNA-sequencing data of patient PDAC revealed high expression of the novel immune checkpoint, PSGL-1, on infiltrating CD8+ T cells. We hypothesize that PSGL-1-mediated immune suppression is a critical barrier to effective anti-tumor immunity in PDAC. To test this, we utilized a preclinical model of PDAC where orthotopic injection of the KPC.4662 tumor cell line into pancreata recapitulates patient tumor responses with limited T cell infiltrates and uncontrolled tumor growth in C57BL/6 (wildtype) control mice and compared to PSGL-1KO (C57BL/6 background) mice. At endpoint, tumor burden in PSGL-1KO mice was reduced by >50% compared to controls, with 45% of PSGL-1KO animals exhibiting >70% reduction. Total immune infiltration (CD45+) was 2-fold greater in tumors from PSGL-1KO mice, with a significant increase in infiltrating CD3+ T cells. Detailed analyses revealed that infiltrating CD8+ T cells from PSGL-1KO mice were not only more abundant, but also less differentiated towards terminal exhaustion with sustained TCT-1 expression compared to controls. Moreover, PSGL-1KO mice showed protection against metastatic disease. Since our data support a critical role for T cell driven anti-tumor immunity to PDAC, we transiently depleted CD4+ T cells, CD8+ T cells, or both just prior to tumor cell implantation. While depletion of CD8+ T cells did not impede tumor control by PSGL-1KO mice, depletion of all T cells or only CD4+ T cells resulted in the loss of PDAC tumor growth inhibition, demonstrating a key role of CD4+ cells in the response. Given our previous studies demonstrating an intrinsic role for PSGL-1 in development of terminal T cell exhaustion in melanoma, including loss of TCF-1+ CD8+ T cells, we hypothesized that PSGL-1-deficiency would promote responsiveness to PD-1 ICB, which is ineffective as a monotherapy in PDAC patients and mouse models. To test this prediction, PD-1 ICB was therapeutically administered to control and PSGL-1KO mice. While treatment with anti-PD-1 had no effect on the tumor growth in the control animals, PD-1 ICB resulted in complete tumor ablation in 85% of PSGL-1KO animals, demonstrating a profound synergistic effect of PD-1 inhibition in the absence of PSGL-1. From these studies, we conclude that PSGL-1 is a critical inhibitor of T cell anti-tumor responses in PDAC. PSGL-1 therefore represents a novel ICB target with high translational potential for promoting immune responses to PDAC tumors. Citation Format: Jennifer L. Hope, Yijuan Zhang, Hannah A. Hetrick, Gabriele Romano, Sreeja Roy, Michelle Lin, Ashley B. Palete, Swetha Maganti, Dennis C. Otero, Cosimo Commisso, Linda M. Bradley. Rewiring CD8+T cell responses to PD-1 immune checkpoint blockade in PDAC by preventing terminal exhaustion via the inhibitory receptor PSGL-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1359.

Abstract 6326: Targeting PRDM1 to enhance CAR T cell efficacy against diffuse large B cell lymphoma (DLBCL)

Abstract CAR T cell therapy has shown remarkable success in treating patients with relapsed or refractory (r/r) DLBCL, but a majority (>60%) will experience disease progression. A key failure mechanism is the rapid terminal differentiation and exhaustion of CAR T cells, driven by persistent antigen exposure in vitro during manufacturing and in vivo in the DLBCL environment, which culminates in poor persistence and short-term effector functions. Recent studies have identified the transcriptional repressor PR/SET domain 1 (PRDM1) as a key regulator that drives differentiation of T cells toward these unwanted fates. Further, single cell transcriptomic profiling of r/r DLBCL biopsies has revealed PRDM1 enrichment in a cluster of exhausted-like CD8+ CAR T cells. These observations indicate that targeting PRDM1 could prevent or delay terminal differentiation/exhaustion in CAR T cells, thereby enhancing their persistence for lasting anti-tumor activity. Using CRISPR/Cas9 targeting, we have demonstrated that PRDM1-deficient CAR T cells exhibit enhanced proliferative capacity, memory characteristics, and resilience against exhaustion upon repeated stimulation. Moreover, genetic ablation of PRDM1 also promoted the in vivo expansion and persistence of CAR T cells, which led to significantly improved survival of lymphoma-bearing mice. Mechanistically, loss of PRDM1 leads to de-repression of PPARGC1A (PGC1α), a master regulator of mitochondrial biogenesis and antioxidant activity. This confers elevated tolerance to oxidative damage in CAR T cells lacking PRDM1, which showed increased capacity to buffer oxidative stress induced by mitochondrial uncouplers and constant stimulation. These findings suggest that PRDM1 loss may improve CAR T cell fitness through PGC1α upregulation and mitochondrial activation. From a translational perspective, we assessed the potential synergistic effects of PGC1α agonist, bezafibrate, with CAR T cells. Dual therapy with bezafibrate and CAR T cells mediated more robust expansion and anti-tumor immunity of CAR T cells without causing discernible adverse events. Importantly, bezafibrate alone had no direct tumoricidal effects, which indicates that bezafibrate suppressed tumor growth through modulating CAR T cell functions. Moving forward, we will uncover the previously uncharted role of PRDM1 in orchestrating the mitochondrial and metabolic activity of CAR T cells. Building upon a correlation between PRDM1 expression and CAR T cell dysfunction, we aim to pioneer the use of PRDM1 and/or its target genes as novel biomarkers for predicting clinical responses of r/r DLBCL patients to CAR T cell therapy. Our endeavors will shed light on the long-standing conundrum of CAR T cell dysfunction and offer solutions that may be applicable for improving T cell-based therapy against cancer in general. Citation Format: Sidney Wang, Lishi Xie, Xiufen Chen, Alan Cooper, Joanna Chorazeczewski, Justin Kline. Targeting PRDM1 to enhance CAR T cell efficacy against diffuse large B cell lymphoma (DLBCL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6326.

Abstract 3862: Identification, profiling and clonotypic tracking of tumor-reactive T cells derived from bronchoalveolar lavage in lung cancer patients

Abstract Immunotherapeutic strategies for disinhibiting T cell responses have revolutionized lung cancer treatment. However, the identification of tumor-reactive T cells and the acquisition of adequate tissue for molecular and immune profiling remains a challenge that hinders our ability to adequately characterize the mechanisms of immunotherapy response versus resistance. To address this gap, we are investigating the use of bronchoalveolar lavage (BAL) as an alternative to blood or tumor biopsy samples to identify tumor reactive T cells. We collected cells from a prospective bronchoscopic biorepository of patients undergoing lung cancer evaluation that were subjected to bilateral BAL, phlebotomy, and tissue sampling. We then employed flow cytometry and single-cell multi-omic analyses to identify, characterize and clonotypically track tumor-reactive T cells obtained from BAL, blood and tumor samples from lung cancer patients. Analysis of BAL samples revealed a significant enrichment of terminally exhausted CD8+CD39+CD103+ T cells in BAL from tumor-bearing lungs compared to non-tumor bearing lung BAL and blood PBMCs in 14 patients. We then used single-cell sequencing-based multi-omics to deeply profile bilateral BAL cells, blood PBMCs and tumor tissues from 5 of these patients. By tracking T cell receptor (TCR) sequences across patient-matched samples, we found BAL derived T cell clonotypes that expressed markers of terminal exhaustion, such as CD39 and CXCL13 in the tumor, which strongly implicates them as being tumor reactive and relatively abundant in BAL. Further validation through functional assays is being used to confirm the tumor reactivity of these clones. These data demonstrate the feasibility of capturing tumor-specific T cells during bronchoscopy, providing promising opportunities for patient outcome characterization and exploration of novel therapies. This relatively non-invasive and practical test during routine bronchoscopy offers a cost-effective and safe alternative to identifying tumor-reactive T cells from surgical specimens or biopsies. Citation Format: Heeju Ryu, Wei Sun, Michelle A. Wurscher, Viswam S. Nair, Evan W. Newell. Identification, profiling and clonotypic tracking of tumor-reactive T cells derived from bronchoalveolar lavage in lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3862.

Abstract A076: T-cell exhaustion is an independent predictive biomarker of clinical outcome in high grade serous ovarian cancer regardless of homologous recombination deficiency status

Abstract Epithelial ovarian cancer is the most lethal gynecologic malignancy, with 5-year survival rates of 46%. High grade serous carcinoma (HGSC) is the most frequent subtype of epithelial ovarian cancer. Approximately 50% of cases are homologous recombination deficient (HRD). Whereas maintenance treatment by PARP inhibitors has significantly improved the prognosis of patients with HRD tumors, clinical outcome in this malignancy is still poor. Despite the immunogenic nature of HGSC, in which T-cell infiltration correlates to improved clinical outcome following surgery and chemotherapy, the disease exhibits low response rates to immune checkpoint blockade (ICB) monotherapy in advanced line settings. We have previously identified epithelial malignancies including HGSC, CD4 and CD8 populations of tumor-infiltrating and antigen-specific T cells that display features of terminal exhaustion. These populations were key players in the response to anti-PD-1 in patients with epithelial tumors. Here, we set out to characterize tumor-infiltrating T cells in a cohort of 80 HGSC patients treated at our institution from whom we collected tumor samples prior to therapy in which HRD status was assessed. We used multiplex immunofluorescence of FFPE tumor samples to assess infiltration by CD4 and CD8 T cells and multiparametric flow cytometry of frozen viable cell suspensions to assess CD4 and CD8 T-cell exhaustion. The link between biological parameters and clinical outcome was assessed by univariable and multivariable analyses. Assessment of the T-cell infiltrate by immunofluorescence showed no difference in the absolute numbers, per tumor or stroma mm2, of CD8 or CD4 T cells between HRD and homologous recombination proficient (HRP) tumors. Instead, analysis of exhausted CD8 and CD4 T cells showed an increased frequency of terminally exhausted PD-1HighTIM-3+ CD8 T cells in HRD tumors. This population, which expresses the ectonucleotidase CD39, was significantly correlated to the presence of T regulatory cells (Treg), which agreed with previous studies showing comigration of effector and regulatory T cells in ovarian cancer. In addition, terminally exhausted CD4 T cells, expressing the immune checkpoints PD-1 and TIM-3 as well as CD39, were enriched in HRD tumors. Among HRD tumors, no difference was observed between the exhaustion profile in tumors presenting deleterious BRCA1/2 mutations compared to those with other mutations in the homologous recombination pathway. Univariable analyses showed exhaustion parameters to be significant predictors of progression-free survival (PFS). The frequencies of exhausted CD4 and CD8 T cells stood out as predictors of PFS in multivariable analyses. Altogether, these results provide the first evidence of a link between T-cell exhaustion and HRD status and reveal T-cell exhaustion as an independent predictor of disease outcome. They also underline the major role played by the antitumor immune response in HGSC and warrant the development of combination immunotherapies despite the lack of response to ICB monotherapy. Citation Format: Anna Salvioni, Mathilde Del, Marie Michelas, Pierre Vuattoux, Clara-Maria Scarlata, Carlos Martinez-Gomez, Nathalie Van Acker, François-Xavier Frenois, Guillaume Bataillon, Jean-Pierre Delord, Alejandra Martinez, Maha Ayyoub. T-cell exhaustion is an independent predictive biomarker of clinical outcome in high grade serous ovarian cancer regardless of homologous recombination deficiency status [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A076.

Granzyme F: Exhaustion Marker and Modulator of Chimeric Antigen Receptor T Cell-Mediated Cytotoxicity.

Granzymes are a family of proteases used by CD8 T cells to mediate cytotoxicity and other less-defined activities. The substrate and mechanism of action of many granzymes are unknown, although they diverge among the family members. In this study, we show that mouse CD8+ tumor-infiltrating lymphocytes (TILs) express a unique array of granzymes relative to CD8 T cells outside the tumor microenvironment in multiple tumor models. Granzyme F was one of the most highly upregulated genes in TILs and was exclusively detected in PD1/TIM3 double-positive CD8 TILs. To determine the function of granzyme F and to improve the cytotoxic response to leukemia, we constructed chimeric Ag receptor T cells to overexpress a single granzyme, granzyme F or the better-characterized granzyme A or B. Using these doubly recombinant T cells, we demonstrated that granzyme F expression improved T cell-mediated cytotoxicity against target leukemia cells and induced a form of cell death other than chimeric Ag receptor T cells expressing only endogenous granzymes or exogenous granzyme A or B. However, increasing expression of granzyme F also had a detrimental impact on the viability of the host T cells, decreasing their persistence in circulation invivo. These results suggest a unique role for granzyme F as a marker of terminally differentiated CD8 T cells with increased cytotoxicity, but also increased self-directed cytotoxicity, suggesting a potential mechanism for the end of the terminal exhaustion pathway.

Extraislet expression of islet antigen boosts T cell exhaustion to partially prevent autoimmune diabetes.

Persistent antigen exposure results in the differentiation of functionally impaired, also termed exhausted, T cells which are maintained by a distinct population of precursors of exhausted T (TPEX) cells. T cell exhaustion is well studied in the context of chronic viral infections and cancer, but it is unclear whether and how antigen-driven T cell exhaustion controls progression of autoimmune diabetes and whether this process can be harnessed to prevent diabetes. Using nonobese diabetic (NOD) mice, we show that some CD8+ T cells specific for the islet antigen, islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) displayed terminal exhaustion characteristics within pancreatic islets but were maintained in the TPEX cell state in peripheral lymphoid organs (PLO). More IGRP-specific T cells resided in the PLO than in islets. To examine the impact of extraislet antigen exposure on T cell exhaustion in diabetes, we generated transgenic NOD mice with inducible IGRP expression in peripheral antigen-presenting cells. Antigen exposure in the extraislet environment induced severely exhausted IGRP-specific T cells with reduced ability to produce interferon (IFN)γ, which protected these mice from diabetes. Our data demonstrate that T cell exhaustion induced by delivery of antigen can be harnessed to prevent autoimmune diabetes.

Integrated multi-omics profiling to dissect the spatiotemporal evolution of metastatic hepatocellular carcinoma.

556 Background: Comprehensive molecular analyses of metastatic hepatocellular carcinoma (HCC) are lacking. Methods: Here, we generated genomic, transcriptomic, and T cell repertoire data coupled with digital pathology and digital spatial profiling of 257 primary, 28 relapsed, and 176 metastatic regions from 182 HCC patients. Results: Primary tumors rich in hypoxia signatures tended to give rise to polyclonal dissemination, which was associated with a poor clinical outcome. Genomic divergence between primary and metastatic HCC was high, and early dissemination was prevalent. The remarkable neoantigen intratumor heterogeneity observed in metastases was associated with decreased T cell reactivity, which may have resulted from disruptions to neoantigen presentation. We identified macroevolutionary somatic copy number alterations as highly selected events driving metastasis. Furthermore, we found that subclonal Wnt pathway mutations were not selected during spreading, whereas subclones without Wnt mutations showed a strong selective advantage for metastasis and were characterized by a reactive microenvironment rich in activated fibroblasts favoring an invasive tumor cell phenotype. Finally, metastases without Wnt mutations exhibited higher enrichment of immunosuppressive B cells that mediated terminal exhaustion of CD8+ T cells via HLA-E – NKG2A checkpoint axis. Conclusions: Collectively, our results provide a multi-dimensional dissection of the complex evolutionary process of metastasis.

Mitochondrial isocitrate dehydrogenase impedes CAR T cell function by restraining antioxidant metabolism and histone acetylation

The efficacy of chimeric antigen receptor (CAR) Tcell therapy is hampered by relapse in hematologic malignancies and by hyporesponsiveness in solid tumors. Long-lived memory CAR Tcells are critical for improving tumor clearance and long-term protection. However, during rapid exvivo expansion or invivo tumor eradication, metabolic shifts and inhibitory signals lead to terminal differentiation and exhaustion of CAR Tcells. Through a mitochondria-related compound screening, we find that the FDA-approved isocitrate dehydrogenase 2 (IDH2) inhibitor enasidenib enhances memory CAR Tcell formation and sustains anti-leukemic cytotoxicity invivo. Mechanistically, IDH2 impedes metabolic fitness of CAR Tcells by restraining glucose utilization via the pentose phosphate pathway, which alleviates oxidative stress, particularly in nutrient-restricted conditions. In addition, IDH2 limits cytosolic acetyl-CoA levels to prevent histone acetylation that promotes memory cell formation. In combination with pharmacological IDH2 inhibition, CAR Tcell therapy is demonstrated to have superior efficacy in a pre-clinical model.

Plasmacytoid dendritic cell‐derived secretome ameliorates neuropathic corneal pain

Aims/Purpose: Prior work from our group has demonstrated a role for plasmacytoid dendritic cells (pDCs) in corneal nerve homeostasis. Herein, we sought to determine if pDC‐derived secretome could be used for the treatment of neuropathic corneal pain.Methods: The BDCA2‐DTR transgenic mice, in which pDCs can be selectively depleted result in subsequent nerve degeneration, were utilized for ex vivo electrophysiology studies. Animals were treated with pDC secretome drops, beginning 1 day after initial depletion. Electrophysiologic recordings were taken at day 3 post‐depletion analysed. A ciliary nerve ligation model of neuropathic corneal pain (NCP), was used to assess the therapeutic potential of pDC secretome. Spontaneous and evoked pain responses were assessed by palpebral opening measurements and hyperosmolar saline, respectively.Results: There was a significant reduction in cooling threshold following pDC depletion, which was rescued by pDC secretome treatment (26.85 ± 0.65°C vs. 29.52 ± 0.42°C; p < 0.05), restoring the cooling threshold to control levels (29.21 ± 0.51). Furthermore, nerve terminal exhaustion was reduced following pDC depletion and rescued by pDC secretome treatment (0.55 ± 0.08 vs. 1.11 ± 0.10; p < 0.01). Spontaneous pain responses were worse in the vehicle‐treated compared to pDC secretome‐treated group (0.67 ± 0.03 vs. 0.85 ± 0.02; p < 0.001). Similarly, evoked pain responses were worse in the vehicle‐treated compared to pDC secretome‐treated group (30.25 ± 1.50 wipes/30 sec vs. 15.38 ± 1.29; p < 0.0001).Conclusions: Herein, we demonstrate that pDC secretome restores normal functioning of corneal cold receptors. Importantly, in a translational model of NCP, pDC secretome treatment improved both spontaneous and evoked pain responses. Thus, pDC secretome is a viable therapeutic modality for further development and may hold promise in treatment of NCP and other corneal nerve related diseases.Reference1. Jamali A, Kenyon B, Ortiz G, Abou‐Slaybi A, Sendra VG, Harris DL, Hamrah P. Plasmacytoid Dendritic Cells in the Eye. Prog Retin Eye Res 2021; 80: 100877.2. Jamali A, Hu K, Sendra VG, Blanco T, Qazi Y, Zheng L, Turhan A, Hamrah P. Characterization of Resident Corneal Plasmacytoid Dendritic Cells and Their Pivotal Role in Herpes Simplex Keratitis. Cell Rep 2020; 32: 108099.

Recently activated CD4 T cells in tuberculosis express OX40 as a target for host-directed immunotherapy

After Mycobacterium tuberculosis (Mtb) infection, many effector T cells traffic to the lungs, but few become activated. Here we use an antigen receptor reporter mouse (Nur77-GFP) to identify recently activated CD4 T cells in the lungs. These Nur77-GFPHI cells contain expanded TCR clonotypes, have elevated expression of co-stimulatory genes such as Tnfrsf4/OX40, and are functionally more protective than Nur77-GFPLO cells. By contrast, Nur77-GFPLO cells express markers of terminal exhaustion and cytotoxicity, and the trafficking receptor S1pr5, associated with vascular localization. A short course of immunotherapy targeting OX40+ cells transiently expands CD4 T cell numbers and shifts their phenotype towards parenchymal protective cells. Moreover, OX40 agonist immunotherapy decreases the lung bacterial burden and extends host survival, offering an additive benefit to antibiotics. CD4 T cells from the cerebrospinal fluid of humans with HIV-associated tuberculous meningitis commonly express surface OX40 protein, while CD8 T cells do not. Our data thus propose OX40 as a marker of recently activated CD4 T cells at the infection site and a potential target for immunotherapy in tuberculosis.

Integrated multi-omics profiling to dissect the spatiotemporal evolution of metastatic hepatocellular carcinoma

Comprehensive molecular analyses of metastatic hepatocellular carcinoma (HCC) are lacking. Here, we generate multi-omic profiling of 257 primary and 176 metastatic regions from 182 HCC patients. Primary tumors rich in hypoxia signatures facilitated polyclonal dissemination. Genomic divergence between primary and metastatic HCC is high, and early dissemination is prevalent. The remarkable neoantigen intratumor heterogeneity observed in metastases is associated with decreased Tcell reactivity, resulting from disruptions to neoantigen presentation. We identify somatic copy number alterations as highly selected events driving metastasis. Subclones without Wnt mutations show a stronger selective advantage for metastasis than those with Wnt mutations and are characterized by a microenvironment rich in activated fibroblasts favoring a pro-metastatic phenotype. Finally, metastases without Wnt mutations exhibit higher enrichment of immunosuppressive B cells that mediate terminal exhaustion of CD8+ Tcells via HLA-E:CD94-NKG2A checkpoint axis. Collectively, our results provide a multi-dimensional dissection of the complex evolutionary process of metastasis.

Comment on "An Injectable Hydrogel to Modulate T Cells for Cancer Immunotherapy".

Recent clinical successes of immune checkpoint blockade (ICB) therapies represents a milestone as a novel anti-tumor strategy beyond surgery, radiotherapy, chemotherapy, and targeted therapy in cancer therapy. T cells, especially CD8+ T cells, play crucial roles in anti-tumor immune responses. However, most T cells in the tumor microenvironment express high inhibitory receptors, such as PD-1, TIM-3, and LAG-3, and decreased T cell response in response to stimuli. Applying ICB therapies, such as anti-PD-1, promotes T cell activation and increases cytotoxic T lymphocyte (CTL) response, leading to the enhanced anti-tumor immune response in patients with malignancy. Therefore, studies aimed to define novel targets that can restrain T cell terminal exhaustion are urgently required to provide new strategies for patients resistant to immunotherapy. The previously published study by Zhang etal. (An Injectable Hydrogel to Modulate T Cells for Cancer Immunotherapy, https://doi.org/10.1002/smll.202202663) introduces a new type of injectable hydrogel that can regulate the function of T cells, thereby improving their effectiveness in cancer immunotherapy. However, it remains to be discussed for its conclusion, as the flow cell assay of this article may not be proper.

Deletion of the protein tyrosine phosphatase PTPN22 for adoptive T cell therapy facilitates CTL effector function but promotes T cell exhaustion

BackgroundAdoptive cell therapy (ACT) is a promising strategy for treating cancer, yet it faces several challenges such as lack of long-term protection due to T cell exhaustion induced by chronic...

Fructose sweetens the adipocyte-T cell alliance against tumors

Dietary fructose is implicated in tumorigenesis, but whether dietary fructose regulates antitumor immunity remains elusive. In this issue of Cell Metabolism, Zhang etal. show that dietary fructose promotes adipocyte-derived leptin production, which attenuates terminal exhaustion programming and boosts the effector function of CD8+ Tcells for improved tumor control.

Abstract PR01: Regulation of exhausted CD8+ T cell differentiation by IKZF transcription factors

Abstract CD8+ T cells are indispensable for pathogen and tumor clearance, although can induce immunopathology if left unrestricted. T cell exhaustion, progressively programmed in the context of persistent antigen exposure, ensures partial control while mitigating immunopathological risk. However, exhaustion is characterized by curtailed proliferative capacity, cytokine production and cytotoxic functions, which tumors and chronic pathogens exploit to persist. As such, transiently disrupting exhaustion has emerged as a therapeutic strategy for treating cancer. Exhausted cells transition through a range of differentiation states, from stem-like progenitors that mediate response to checkpoint blockade, through to terminal effector or exhausted cells. Understanding the molecular pathways moderating the exhausted states and promoting terminal exhaustion is essential for augmenting cancer immunotherapy. We identify IKZF transcription factors, Ikaros (IKZF1), Helios (IKZF2) and Aiolos (IKZF3), as key mediators of T cell exhaustion, using gene knock-out mice and a CRISPR-mediated genetic knock-out system in both chronic infection and tumor models. IKZF1 and IKZF3 are critical regulators of exhausted subset expansion, retention, phenotype and cytokine function. Strikingly, dual IKZF1 and IKZF3 ablation boosts the response of anti-tumor and anti-viral exhausted T cells, potentially via the formation of atypical exhausted T cell populations and the enrichment of functional exhausted subsets. Cumulatively, our data highlight a novel role for IKZF transcription factors in biasing exhausted T cell differentiation and provides potentially important clinical implications, directing future cancer immunotherapies targeting T cell exhaustion. Citation Format: Sinead M Reading, Isabelle Munoz, Maria Nogueira de Menezes, Nicole Y.L Saw, Krutika Ambani, Antonio Ahn, Simone Nussing, Sara Roth, Shienny Sampurno, Kelly M Ramsbottom, Joseph A Trapani, Kim L Good-Jacobson, Ricky W Johnstone, Shom Goel, Paul A Beavis, Ian A Parish. Regulation of exhausted CD8+ T cell differentiation by IKZF transcription factors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr PR01.

Runx2 Overexpression Enhances Potency and Counteracts Exhaustion in CD8+ CAR T Cells

Chimeric antigen receptor (CAR) T cells are highly effective in treating B cell malignancies, but 50% of patients eventually relapse, often due to progressive dysfunction of the remaining CAR T cell population. While several others have implicated the bZIP transcription factor (TF) family in CAR T cell exhaustion (Seo et al. Nat Immunol. 2021, Lynn et al. Nature. 2019, Zhang et al. Cancer Cell. 2022), here we reveal a novel role for the TF Runx2 in mediating resistance to exhaustion and enhancing potency of CD8+ CAR T cells (CAR8). We have previously shown that CAR8 derived from naïve or memory T cell subsets maintain functional features of the T cell population from which they were derived (DeGolier et al. ASH. 2021). We predicted that an epigenomic and transcriptomic comparison of CAR8 derived from naïve or memory cells could reveal drivers of T cell differentiation and function. To precisely control T cell antigen experience, and to study T cell persistence in the absence of xenogeneic GvHD, we chose to use a syngeneic murine model, with adoptive transfer of E2A-PBX B cell acute lymphoblastic leukemia treated by anti-murine CD19 CAR T cells (Qin et al. Blood. 2018). We used a well-characterized ovalbumin vaccination model to produce memory T cells with which to generate memory-derived CAR8, and concurrently generated naïve-derived CAR8 from naïve hosts (Ahonen et al. J Exp Med. 2004). Comparative ATACseq of naïve CD8+ T cells to memory CD8+ T cells at 28 days post-vaccination revealed differential chromatin accessibility at target loci for Runx family transcription factors. These differences were maintained upon transduction of these T cell populations with a CAR, and after reinfusion into leukemia-bearing hosts. While the Runx family contains three members, RNAseq analysis uncovered that only Runx2 transcripts were highly enriched in memory over naïve cells prior to CAR-transduction but showed that gene expression levels converged upon CAR transduction and reinfusion. Additionally, broad comparisons of chromatin accessibility and transcriptomic profiles to published datasets showed that naïve-derived CAR8 progressively became more “memory-like”, while memory-derived CAR8 became more “effector-like.” We hypothesized that establishing Runx2 expression in naïve-derived CD8+ CAR T cells (CAR8 ND) could enhance the existing “memory-like” state of these T cells and boost T cell potency and anti-leukemia response. Indeed, overexpression of Runx2 in naïve-derived CAR8 (Runx2-CAR8 ND) strongly enhanced leukemia clearance (p<0.05) in mice treated with a sub-curative 1e5 CAR+ T cell dose. While there was no difference in the PD1+ proportion (p = 0.47), indicating similar activation and/or exposure to target antigen, mice treated with Runx2-CAR8 ND exhibited a lower proportion of PD1+/CD39+ cells (p<0.001) as well as a dramatically reduced proportion of PD1+/TOX+ cells (p<0.001), suggesting that Runx2 overexpression counteracts the differentiation trajectory toward terminal exhaustion. Additionally, Runx2-CAR8 ND showed enhanced T cell proportions in the marrow at 11 days post-CAR infusion (p<0.01), with >2-fold higher levels of Runx2-CAR8 ND compared to control CAR8 ND. Finally, in mice treated with a curative 2e6 CAR+ T cell dose, Runx2-CAR8 ND demonstrated a trend toward increased persistence in the blood at 19 days post-CAR infusion, relative to control CAR8 ND (p = 0.053). In conclusion, a comparison of naïve and memory-derived CD8+ CAR T cells at the epigenetic level revealed Runx2 overexpression in naïve-derived CAR T cells as a novel strategy to enhance the anti-leukemia response. Preliminary findings suggest Runx2-CAR8 ND show enhanced T cell proportions and decreased leukemic burden, as well as favorable exhaustion phenotypes. Future studies will expand our characterization of Runx2-CAR8 ND to better understand the effects of Runx2 on CAR T cell epigenetic profile, function and persistence in the bone marrow microenvironment, as well as test the effects of Runx2 overexpression in human CAR T cells. We anticipate that Runx2 overexpression will prove to be a useful strategy in enhancing the durability of CAR-mediated leukemia remissions.

Adoptively Infused Memory-like Natural Killer Cells Impact Adaptive Immune Responses in Patients with Acute Myeloid Leukemia

Natural killer (NK) therapies have been challenging owing to antigen escape, restricted trafficking, and limited tumor infiltration, translating into modest clinical benefit. We have shown that memory-like (ML)-NK cells acquire an in vivo differentiated phenotype distinct from conventional NK cells and are well-tolerated without cytokine release syndrome, GVHD, or neurotoxicity. WU-NK-101 (W-NK) is an allogeneic cytokine-reprogrammed, expanded, cryopreserved ML-NK cell product derived from healthy human peripheral blood mononuclear cells, with scalable manufacturability, that is currently in clinical development in relapsed/refractory (R/R) AML (NCT# 05470140). We conducted a multi-modal analysis of the immune tumor microenvironment (TME) in 13 patients with R/R AML receiving ML-NK cells (NCT#01898793) with the aim to determine whether TME modifications induced by ML-NK cells correlate with clinical benefit. NK cells were purified from leukapheresate, and activated with IL-12, IL-15, and IL-18 for 12-16 hours. Patients were lymphodepleted with fludarabine and cyclophosphamide. NK cells were infused in escalating doses: 0.5 × 10 6/kg, 1.0 × 10 6/kg, and all NK cells from a single leukapheresis, capped at 10 × 10 6/kg. Response was defined as <5% BM blasts on d+28. BM trephines for immune gene expression profiling (nCounter IO360®) and spatial proteomics (GeoMx™ DSP; n=53 immuno-oncology [IO] targets; NanoString Technologies, Seattle) were collected at baseline (BL) and on treatment (OT). Immune infiltration, NK and T cell number from BM samples was quantified manually on immunofluorescent imaging. Oncomine™ TCR Pan-Clonality assay (Thermo Fisher) was used to profile TCRb and g repertoires by NGS. The Shannon index was used to describe TCR repertoire diversity, and clonal evenness was used as a measure of the similarity of clone sizes. BM infiltration with CD8 + effectors, enhanced glycolysis and cell cycling was higher in BM samples from responders (CR) to ML-NK cells. By contrast, OXPHOS RNA scores and gene signatures of immune senescence and terminal T-cell exhaustion were higher in non-responders (NR). We employed machine learning to construct an 8-gene predictor of response which encompassed T/NK markers, such as CD7, and IFN regulatory factors and TLRs. This score was significantly higher at baseline in CR and positively correlated with response duration. Principal component analysis (PCA) of spatially resolved IO proteins separated CR from NR, particularly in OT specimens, where T-cell markers CD3/CD8 and inhibitory receptors CTLA4/LAG3 were among the top contributing features. CD34, type I IFN signaling molecule STING1 and PARP1 were significantly higher at baseline compared with OT, with OT showing higher expression of cytotoxicity markers and inhibitory receptors (e.g., Tim-3 and CD276). This observation strongly suggests modulation of the immune TME after the infusion of ML-NK cells. Response to ML-NK cells was associated with coordinated up-regulation of T cell, NK cell and B cell protein markers. To further substantiate the co-localization of T cells and NK cells in the BM FFPE sections (Fig. A), and its correlation with clinical efficacy we annotated the 93 ROIs in our spatial proteomics dataset based on a median split of CD3 and CD56 protein barcodes. ROIs from CR were largely CD3 highCD56 high, both at BL and OT. Conversely, ROIs from BM sections of NR to ML-NK cells had a predominantly CD3 lowCD56 low phenotype. Quantitative analysis of T-cell infiltration post ML-MK dosing showed a significant association with response, 0.9 (PD) vs. 5.3 (CR)-fold-increase ( P=0.01). Interestingly, data demonstrate selected intra-BM clonal expansion as evidenced by decreased TCR diversity (mean ± SEM, 4.6±0.3 vs. 3.3±0.4, pre vs. post; P=0.013)and increased clonality (Fig. B). Thus, pre-existing T-cell responses appear to be co-opted in the AML TME of CR, suggesting the potential for durable effectiveness of ML-NK cells. Multi-modal analyses of WU-NK-101 highlight unique transcriptional and functional states, which could underpin in vivo potency and persistence. WU-NK-101 are currently being evaluated in a phase 1 study of R/R AML (#NCT05470140).

Type I Interferon Blockade Enhances Transduction Efficiency and Efficacy of Non-Activated CAR T Cells

Chimeric antigen receptor (CAR) T-cell therapies have revolutionized the treatment of advanced hematologic cancers, showing impressive response rates in pivotal trials. Despite this success, challenges in CAR T-cell adoption persist due to tumor resistance mechanisms, manufacturing failures, and limitations in CAR T-cell persistence and effector function. Strategies that promote T-cell persistence, proliferation, and cytotoxic activity are essential to improving long-term outcomes of CAR T therapy. The most common protocol for CAR T-cell manufacturing involves lentiviral vector transduction of activated T-cells followed by ex vivo culture. However, this approach may drive terminal T-cell differentiation and exhaustion, compromising therapeutic efficacy. In contrast, non-activated CAR T-cell manufacturing has shown promise in enhancing T-cell differentiation and tumor control. Yet, the low transduction efficiency of quiescent T-cells remains a challenge. In this study, we explore the role of type I interferon (IFN)-mediated innate immune responses in limiting CAR T manufacturing efficiency. We hypothesize that blocking these responses using the vaccinia virus-encoded type I interferon-binding protein can enhance transduction efficiency and promote less-differentiated CAR T-cell phenotypes, including naïve and central memory-like differentiation. Our findings demonstrate that lentiviral vector transduction triggers an innate immune response in non-activated T-cells, resulting in reduced transduction efficiency, and altered T-cell differentiation. Inhibiting the anti-viral response via anti-IFN leads to improved T-cell differentiation and exhaustion characteristics. Importantly, anti-IFN exposure supports the enrichment of naive-like T cells: a subset noted for superior clinical efficacy. Anti-IFN also reduces LAG3 expression. Collectively, these findings position anti-IFN as a novel means to enhance CAR T-cell persistence and anti-tumor function. We further investigate the influence of sustained anti-IFN exposure by designing lentiviral transfer plasmids encoding both a CAR and secreted type I interferon-binding protein. These cells showed enhanced cytolytic activity and cytokine production against target cells. Our studies in mice show promising results, suggesting that the protein secretion enhance therapeutic activity against tumors employing sustained type I interferon signaling for immune escape. Overall, targeting the type I interferon response during non-activated CAR T-cell manufacturing offers a potential solution to improve transduction efficiency and enhance CAR T-cell functionality. Given the therapeutic promise of non-activated CAR T cells, our findings have immediate translational relevance for broadening their appeal against cancer. Figure 1. Exposure to anti-IFN increases transduction efficiency and naïve-like differentiation of lentivirus-transduced non-activated T-cells A) Freshly isolated human T-cells were incubated in media with aIFN or vehicle, for one hour at room temperature. The cells were resuspended in complete media and then transduced with lentiviral vector encoding iRFP. Expression of iRFP was evaluated 96 h after transduction by flow cytometry. b, Human T-cells were isolated and prepared as above. At 96 h post-transduction, cells were stained with anti-CD45RO and anti-CCR7 antibodies and samples were evaluated by flow cytometry. Data for A and B are representative of multiple experiments with three independent donors.