Expansion Of Effector Research Articles

Mucosal Single-Cell Profiling of Crohn’s-Like Disease of the Pouch Reveals Unique Pathogenesis and Therapeutic Targets

The pathophysiology of Crohn's-like disease of the pouch (CDP) in patients with a history of ulcerative colitis (UC) is unknown. We examined mucosal cells from patients with and without CDP using single-cell analyses. Endoscopic samples were collected from pouch body and prepouch ileum (pouch/ileum) of 50 patients with an ileal pouch-anal anastomosis. Single-cell RNA sequencing was performed on pouch/ileal tissues of patients with normal pouch/ileum and CDP. Mass cytometry was performed on mucosal immune cells from patients with UC with normal pouch/ileum, CDP, pouchitis, and those with familial adenomatous polyposis after pouch formation. Findings were independently validated using immunohistochemistry. The cell populations/states in the pouch body differed from those in the prepouch ileum, likely secondary to increased microbial burden. Compared with the familial adenomatous polyposis pouch, the UC pouch was enriched in colitogenic immune cells even without inflammation. CDP was characterized by increases in T helper 17 cells, inflammatory fibroblasts, inflammatory monocytes, TREM1+ monocytes, clonal expansion of effector T cells, and overexpression of T helper 17 cells-inducing cytokine genes such as IL23, IL1B, and IL6 by mononuclear phagocytes. Ligand-receptor analysis further revealed a stromal-mononuclear phagocytes-lymphocyte circuit in CDP. Integrated analysis showed that up-regulated immune mediators in CDP were similar to those in CD and pouchitis, but not UC. Additionally, CDP pouch/ileum exhibited heightened endoplasmic reticulum stress across all major cell compartments. CDP likely represents a distinct entity of inflammatory bowel disease with heightened endoplasmic reticulum stress in both immune and nonimmune cells, which may become a novel diagnostic biomarker and therapeutic target for CDP.

A randomized phase II trial of atezolizumab with or without tiragolumab before and after definitive chemoradiation for unresectable stage III non-small cell lung cancer (NSCLC; AFT-57).

TPS8123 Background: A minority of patients with unresectable stage III non-small cell lung cancer (NSCLC) may be cured by chemoradiotherapy (CRT). Adjuvant checkpoint inhibition through PD-L1 blockade after CRT improved survival compared to placebo in the PACIFIC trial (median progression-free survival (mPFS) 16.9 vs 5.6 months (mo), median overall survival (mOS) 47.5 vs 29.1 mo), but overall survival at 5 years remains below 50% (Spigel D, et al J Clin Oncol 2022, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9015199/ . Only patients completing CRT without progression and with good performance status were eligible for this approach. Neoadjuvant checkpoint inhibition would allow more patients the opportunity to benefit from immunotherapy and may attenuate tumor-related immunosuppression via depletion of regulatory T cells and clonal expansion of effector T cells, thereby improving tumor immunogenicity. Neoadjuvant atezolizumab had favorable outcomes compared to historic PACIFIC data in the phase II single arm trial, AFT-16 (mPFS 30 mo and mOS not reached). Whether combination immunotherapy therapy before CRT will improve outcomes in this setting is the question posed in the AFT-57 trial. Methods: This phase II randomized Alliance Foundation Trials study (AFT-57) will evaluate safety and efficacy of atezolizumab with or without the anti-TIGIT monoclonal antibody, tiragolumab, before and after definitive CRT with concurrent atezolizumab. The primary endpoint is PFS in the two experimental arms with a goal to select the most promising regimen for further study. AFT-57 will randomly assign 158 patients with stage III NSCLC (AJCC v8), Eastern Cooperative Oncology Group performance status 0-1, no active autoimmune disease and no underlying organ dysfunction to 2 cycles of neoadjuvant atezolizumab (1200 mg intravenously [IV] every 21 days) with or without tiragolumab (600 mg IV). Non-progressing patients will receive carboplatin and paclitaxel weekly with atezolizumab 1200 mg IV every 21 days concurrent with 60 Gray involved field thoracic radiation in 30 daily fractions (Monday - Friday) followed by atezolizumab with or without tiragolumab on the same schedule as the neoadjuvant doses, with a plan to complete one year of therapy. A pilot cohort of 20 patients for whom tiragolumab will be combined with atezolizumab during chemoradiotherapy is planned toward the end of accrual. Correlative science includes tissue and blood-based immune-related biomarkers at baseline and during therapy. This trial utilizes the Alliance Participant Engagement Portal, a patient-facing site designed to provide information and resources to Alliance clinical trial participants. The trial was activated on 12/7/23 with the first patient screened 1/19/24. Clinical trial information: NCT05798663 .

PGE2 inhibits TIL expansion by disrupting IL-2 signalling and mitochondrial function

Expansion of antigen-experienced CD8+ T cells is critical for the success of tumour-infiltrating lymphocyte (TIL)-adoptive cell therapy (ACT) in patients with cancer1. Interleukin-2 (IL-2) acts as a key regulator of CD8+ cytotoxic T lymphocyte functions by promoting expansion and cytotoxic capability2,3. Therefore, it is essential to comprehend mechanistic barriers to IL-2 sensing in the tumour microenvironment to implement strategies to reinvigorate IL-2 responsiveness and T cell antitumour responses. Here we report that prostaglandin E2 (PGE2), a known negative regulator of immune response in the tumour microenvironment4,5, is present at high concentrations in tumour tissue from patients and leads to impaired IL-2 sensing in human CD8+ TILs via the PGE2 receptors EP2 and EP4. Mechanistically, PGE2 inhibits IL-2 sensing in TILs by downregulating the IL-2Rγc chain, resulting in defective assembly of IL-2Rβ–IL2Rγc membrane dimers. This results in impaired IL-2–mTOR adaptation and PGC1α transcriptional repression, causing oxidative stress and ferroptotic cell death in tumour-reactive TILs. Inhibition of PGE2 signalling to EP2 and EP4 during TIL expansion for ACT resulted in increased IL-2 sensing, leading to enhanced proliferation of tumour-reactive TILs and enhanced tumour control once the cells were transferred in vivo. Our study reveals fundamental features that underlie impairment of human TILs mediated by PGE2 in the tumour microenvironment. These findings have therapeutic implications for cancer immunotherapy and cell therapy, and enable the development of targeted strategies to enhance IL-2 sensing and amplify the IL-2 response in TILs, thereby promoting the expansion of effector T cells with enhanced therapeutic potential.

PGE2 limits effector expansion of tumour-infiltrating stem-like CD8+ T cells

Cancer-specific TCF1+ stem-like CD8+ T cells can drive protective anticancer immunity through expansion and effector cell differentiation1–4; however, this response is dysfunctional in tumours. Current cancer immunotherapies2,5–9 can promote anticancer responses through TCF1+ stem-like CD8+ T cells in some but not all patients. This variation points towards currently ill-defined mechanisms that limit TCF1+CD8+ T cell-mediated anticancer immunity. Here we demonstrate that tumour-derived prostaglandin E2 (PGE2) restricts the proliferative expansion and effector differentiation of TCF1+CD8+ T cells within tumours, which promotes cancer immune escape. PGE2 does not affect the priming of TCF1+CD8+ T cells in draining lymph nodes. PGE2 acts through EP2 and EP4 (EP2/EP4) receptor signalling in CD8+ T cells to limit the intratumoural generation of early and late effector T cell populations that originate from TCF1+ tumour-infiltrating CD8+ T lymphocytes (TILs). Ablation of EP2/EP4 signalling in cancer-specific CD8+ T cells rescues their expansion and effector differentiation within tumours and leads to tumour elimination in multiple mouse cancer models. Mechanistically, suppression of the interleukin-2 (IL-2) signalling pathway underlies the PGE2-mediated inhibition of TCF1+ TIL responses. Altogether, we uncover a key mechanism that restricts the IL-2 responsiveness of TCF1+ TILs and prevents anticancer T cell responses that originate from these cells. This study identifies the PGE2–EP2/EP4 axis as a molecular target to restore IL-2 responsiveness in anticancer TILs to achieve cancer immune control.

Abstract CT112: An off-the-shelf multivalent vaccine containing cancer’s dark matter, DPV-001, combined with PD-1 +/- GITR in head & neck cancer: safety, efficacy, and immunodynamics from the phase 1 GITRVax trial

Abstract Background: Immune responses to shared cancer antigens have correlated with durable clinical responses. DPV-001 is a novel microvesicle vaccine that contains >300 proteins for genes commonly overexpressed by cancer and is enriched for short-lived proteins (SLiPs), and defective ribosomal products (DRiPs), including non-canonical peptides, representing potential shared non-mutated alternative cancer neoantigens (dark matter). Preclinical studies identified increased therapeutic efficacy when this vaccine strategy was combined with anti-PD-1 and anti-GITR, leading to this clinical trial for patients with advanced or metastatic HNSCC. Methods: Following safety run-in, eligible pts were randomly assigned 1:1 to receive DPV-001 +/- GITR agonist mAb (INCAGN-1949; q2wks). All received sequenced PD-1 mAb (retifanlimab; q4wks) starting D15. Safety was evaluated as the primary endpoint and efficacy was evaluated as a secondary endpoint. Tumor biopsies were taken pre-treatment, Wk 2 and 8, and assessed by CITE-seq, scRNA-seq, BCR-seq, and TCR-seq. Multiplex immunofluoresence was performed on biopsies. Blood samples were taken pre-treatment and at multiple timepoints and analyzed by flow cytometry and seromics. Results: 18 pts received backbone therapy of DPV-001 + sequenced PD-1; of these, 9 pts also received GITR starting D1 (Arm 2). G3 irAE's included: hypothyroidism & mucositis (Arm 1, same pt); pneumonitis and adrenal insufficiency (Arm 2, separate pts). No G4 toxicity was observed. Combining both arms, the response rate (RR) for PD-1 naïve pts was 55% (5/9), and 33% (3/9) for PD-1 experienced pts. Two CR's have been observed in Arm 2. Durability of response (DOR) and progression-free survival (PFS) will be reported at the meeting. DPV-001 induced increased Ki67+ CD4 EM (p<0.042) by D15, prior to first dose of PD-1. Addition of GITR at D1 further increased Ki67+ CD4 (p=0.008) & CD8 EM cells (P=0.0006) by D15. Activated or proliferating CD4 and CD8 EM were significantly increased in both arms by D30 (p<0.025). Across above specified analyses, pts receiving GITR had the greatest increase. Evaluable pts had significantly (all p<0.032) increased numbers of TIL expressing IFN-γ and/or GZMB, LAG-3, and TIM3 in on-treatment biopsies. TCR, BCR, and seromic analyses, as well as studies to identify targets of anti-cancer immunity, are ongoing. Conclusions: This 18 pt trial of DPV-001 and sequenced PD-1 +/- GITR shows a promising RR and evidence of increased activation and expansion of effector T cells in PBL and tumor. Upregulation of LAG-3 and TIM3 by T cells that infiltrate the tumor and have increased in number, provide a rationale for including inhibitors for both in this treatment strategy. Current efforts include evaluating whether immune responses target shared non-canonical alternative neoantigens, or Dark Matter, contained in DPV-001, and whether synchronized antibody and cellular response is evidenced. Acknowledgements: Incyte, Providence Portland Medical Foundation, Steve and Cindy Harder, Trial Registry: NCT04470024, Ethics Approval: PSJH IRB# 2020000480 Citation Format: Rom S. Leidner, Christopher Paustian, Shawn M. Jensen, Tarsem Moudgil, Yoshinobu Koguchi, Noah Simons, Venkatesh Rajamanickam, Ryan Meng, Marcus A. Couey, Quyen Vu, Matthew H. Taylor, Anne Long, Abigail J. Staeck, Lakhvir Kaur, Tijana Jovanovic, Brady Bernard, Tanisha Christie, Noriko Iwamoto, Yuriko Minegishi, Koji Ueda, William Redmond, Lessli M. Rushforth, Takashi Shimada, Patrick Rethwisch, Ashish Patel, Richard B. Bell, Carlo B. Bifulco, Traci L. Hilton, Hong-Ming Hu, Brian Piening, Walter J. Urba, Bernard A. Fox. An off-the-shelf multivalent vaccine containing cancer’s dark matter, DPV-001, combined with PD-1 +/- GITR in head & neck cancer: safety, efficacy, and immunodynamics from the phase 1 GITRVax trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT112.

Mettl3-dependent m6A modification is essential for effector differentiation and memory formation of CD8+ T cells

Efficient immune responses rely on the proper differentiation of CD8+ T cells into effector and memory cells. Here, we show a critical requirement of N6-Methyladenosine (m6A) methyltransferase Mettl3 during CD8+ T cell responses upon acute viral infection. Conditional deletion of Mettl3 in CD8+ T cells impairs effector expansion and terminal differentiation in an m6A-dependent manner, subsequently affecting memory formation and the secondary response of CD8+ T cells. Our combined RNA-seq and m6A-miCLIP-seq analyses reveal that Mettl3 deficiency broadly impacts the expression of cell cycle and transcriptional regulators. Remarkably, Mettl3 binds to the Tbx21 transcript and stabilizes it, promoting effector differentiation of CD8+ T cells. Moreover, ectopic expression of T-bet partially restores the defects in CD8+ T cell differentiation in the absence of Mettl3. Thus, our study highlights the role of Mettl3 in regulating multiple target genes in an m6A-dependent manner and underscores the importance of m6A modification during CD8+ T cell response.

Quantitative DNA Repair Biomarkers and Immune Profiling for Temozolomide and Olaparib in Metastatic Colorectal Cancer.

O6-methylguanine DNA methyltransferase (MGMT)-silenced tumors reveal sensitivity to temozolomide (TMZ), which may be enhanced by PARP inhibitors. Approximately 40% of colorectal cancer has MGMT silencing and we aimed to measure antitumoral and immunomodulatory effects from TMZ and olaparib in colorectal cancer. Patients with advanced colorectal cancer were screened for MGMT promoter hypermethylation using methylation-specific PCR of archival tumor. Eligible patients received TMZ 75 mg/m2 days 1-7 with olaparib 150 mg twice daily every 21 days. Pretreatment tumor biopsies were collected for whole-exome sequencing (WES), and multiplex quantitative immunofluorescence (QIF) of MGMT protein expression and immune markers. MGMT promoter hypermethylation was detected in 18/51 (35%) patients, 9 received study treatment with no objective responses, 5/9 had stable disease (SD) and 4/9 had progressive disease as best response. Three patients had clinical benefit: carcinoembryonic antigen reduction, radiographic tumor regression, and prolonged SD. MGMT expression by multiplex QIF revealed prominent tumor MGMT protein from 6/9 patients without benefit, while MGMT protein was lower in 3/9 with benefit. Moreover, benefitting patients had higher baseline CD8+ tumor-infiltrating lymphocytes. WES revealed 8/9 patients with MAP kinase variants (7 KRAS and 1 ERBB2). Flow cytometry identified peripheral expansion of effector T cells. Our results indicate discordance between MGMT promoter hypermethylation and MGMT protein expression. Antitumor activity seen in patients with low MGMT protein expression, supports MGMT protein as a predictor of alkylator sensitivity. Increased CD8+ TILs and peripheral activated T cells, suggest a role for immunostimulatory combinations. TMZ and PARP inhibitors synergize in vitro and in vivo in tumors with MGMT silencing. Up to 40% of colorectal cancer is MGMT promoter hypermethylated, and we investigated whether TMZ and olaparib are effective in this population. We also measured MGMT by QIF and observed efficacy only in patients with low MGMT, suggesting quantitative MGMT biomarkers more accurately predict benefit to alkylator combinations.

Targeting monocytic‐myeloid suppressor cells through CSF1R‐blockade enhances CD19‐CAR T‐cell response in DLBCL

Introduction: Adoptive immunotherapies such as chimeric antigen receptor (CAR) T cell therapy have strongly improved the outcome of patients with diffuse large cell lymphoma (DLBCL) that are relapsed or refractory after standard chemo-immunotherapy. However, approx. 50% of patients with DLBCL are not durably responding to CAR T cell therapy. CAR T cell expansion is associated with durable responses. However, the molecular mechanisms that mediate suppression of CAR T cells leading to resistance still remain elusive. Methods: We performed bulk RNA sequencing of DLBCL patients before CAR T cell therapy. We performed gene set enrichment analysis (GSEA) using signatures derived from published genes associated with an immunosuppressive lymphoma microenvironment. To investigate CAR T cell therapy in an immunocompetent autochthonous mouse model we established murine CD19-redirected CAR-T cells that were generated using splenic T cells isolated from mice harbouring a C57BL/6N background. We treated DLBCL derived from PPMBC (PRDM1-KO, Myd88 + BCL2 overexpression, CD19: Cre) mice with this murine CD19 CAR-T cells in combination with an anti-CSF1R targeted antibody compared to controls. Results: We found increased immunosuppressive metabolic characteristics in CD19 CAR T refractory DLBCL patients indicated by increased glycolysis, hypoxia and elevated reactive oxygen species (ROS) in GSEA analysis. This metabolic signature was associated with an enriched monocytic-myeloid cell signature and a lack of expansion of effector T cells in CD19 CAR T refractory DLBCL. CSF1-CSFR1 (CD115) signaling is one major pathway that mediates the differentiation of myeloid derived cells into immunosuppressive MSCs. CD115-positive MSCs are important immune regulators in the tumor microenvironment that mediate inhibition of T cells and induce the proliferation of Tregs. We therefore hypothesize that combined treatment with a CSFR1 inhibitor will enhance CAR T cell expansion and thus improve CAR T cell response. We show that CSF1R blockade shifts the immunosuppressive lymphoma microenvironment into an proinflammatory environment in CD19 CAR T cell treated mice with DLBCL and abrogation of the expansion of lymphoma associated myelo-monocytic suppressor cells (LAMMs). This shift into an immunosupportive lymphoma microenvironment was accompanied with an increase of T cell expansion within the tumor and expansion of CD19-CAR T cells. We next evaluated whether the combination of CD19-CAR T cell therapy with CSF1R inhibition improves therapeutic outcome PPMBC DLBCL mice. Strikingly, we show that CSF1R Inhibition displays synergistic efficacy in combination with CD19 CAR T cell therapy. Conclusions: Our data strongly indicates that CSF1R inhibition improves CD19-CAR T expansion, promotes an immunosupportive microenvironment and could enhance CD19-CAR T cell therapy efficacy in patients with DLBCL. Keywords: Aggressive B-cell non-Hodgkin lymphoma, Cellular therapies, Microenvironment No conflicts of interests pertinent to the abstract.

PD-L1 checkpoint blockade promotes regulatory T cell activity that underlies therapy resistance

Despite the clinical success of immune checkpoint blockade (ICB), in certain cancer types, most patients with cancer do not respond well. Furthermore, in patients for whom ICB is initially successful, this is often short-lived because of the development of resistance to ICB. The mechanisms underlying primary or secondary ICB resistance are incompletely understood. Here, we identified preferential activation and enhanced suppressive capacity of regulatory T cells (Treg cells) in αPD-L1 therapy-resistant solid tumor-bearing mice. Treg cell depletion reversed resistance to αPD-L1 with concomitant expansion of effector T cells. Moreover, we found that tumor-infiltrating Treg cells in human patients with skin cancer, and in patients with non-small cell lung cancer, up-regulated a suppressive transcriptional gene program after ICB treatment, which correlated with lack of treatment response. αPD-1/PD-L1-induced PD-1+ Treg cell activation was also seen in peripheral blood of patients with lung cancer and mesothelioma, especially in nonresponders. Together, these data reveal that treatment with αPD-1 and αPD-L1 unleashes the immunosuppressive role of Treg cells, resulting in therapy resistance, suggesting that Treg cell targeting is an important adjunct strategy to enhance therapeutic efficacy.

Influenza virus infection modulates expression of the anti-inflammatory mucin 1 in both epithelial and immune cells

Abstract Cell-associated mucin 1(MUC1/Muc1) is a component of the mucus barrier in the respiratory tract that acts as a physical barrier and signaling molecule. Our prior work shows that MUC1 expression is increased during influenza virus (IAV) infection in human airway epithelial (HAE) cultures in vitro. Here we further detail MUC1 expression patterns and report that IAV-induced cytokines, including type I interferons (IFNs), trigger the expression of a low-molecular-weight form of MUC1 which localizes to the cytoplasm and nucleus, and is expressed in different epithelial cell types. Further, IFN-driven changes in the cellular composition of HAE toward a more secretory phenotype require MUC1, suggesting that MUC1 plays an important role in shaping the epithelial response during viral infection. Using a mouse model of IAV infection, we confirmed the upregulation of Muc1 throughout the epithelium in vivoand observed altered Muc1 levels in lung immune cells. Specifically, Muc1 was upregulated in interstitial macrophages but downregulated in antigen-presenting cells (APCs) including alveolar macrophages and dendritic cells as early as 2 days post-infection (dpi). By 5 dpi, Muc1 was significantly upregulated in various T cell subsets and their effector counterparts including CD4 +, CD8 +, and double-negative effector T cells. We also found a negative correlation between Muc1 levels in APCs and the expansion of effector T cell populations, supporting the putative immunoregulatory roles of MUC1/Muc1. Our ongoing work further explores the impact of Muc1 expression in specific cellular subsets on the immune response and IAV pathogenesis. This work was supported by the National Heart, Lung, and Blood Institute (R01HL151840, to MAS)

PD-1 in balancing expansion of effector T cells and attrition of memory T cell during tumor-reactive T cell responses

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-1 fl/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

Programming of checkpoint blockade responsive exhausted T cells by rheostatic IL-2 signals during priming by dendritic cells

Abstract Stem-like progenitor exhausted CD8 T cells are critical for maintaining long-term resistance during chronic infections and cancer, and represent an important checkpoint blockade immunotherapy target for functional reinvigoration and disease control. Here, we show a fate-deterministic role of rheostatic IL-2 signals and differential DC priming in programming the development of stem-like progenitor exhausted CD8 T cells during chronic viral infection. In vivo fate-tracking studies reveal that strong IL-2 signals during priming drive terminal differentiation. In contrast, tempered IL-2 signals are associated with TCF-1 Histem-like precursors, which give rise to progenitor exhausted CD8 T cells, capable of long-term persistence and potent responsiveness to anti-PD-1 therapy in later stages of chronic viral infection. In the context of human tumors as well, single cell RNA-seq analyses of total or antigen-specific tumor infiltrating lymphocytes show an inverse relationship of IL-2 signaling signature with T cell stemness, as well as checkpoint blockade therapy outcomes in melanoma. Our studies further show that the rheostatic control of exhausted T cell fates by differential IL-2 signals is physiologically mediated through differential cell surface expression of IL-2Rα during early stages of T cell activation, which in turn is pioneered during priming by distinct dendritic cell subsets. Notably, moderation of in vivo IL-2 signals during priming promotes the development of stem-like TCF-1 Hilineage, thus supporting a unique strategy for improving clinical immunotherapy outcomes by enhancing the development of long-lived, therapy-responsive stem-like cells with vigorous effector expansion capabilities. Pediatric Cancer Research Foundation to SS, the Rachel Lynn Henley Foundation to VK, the Hopes and Smiles For Children Foundation to VK, In Concert for Cancer Foundation to VK and the National Institutes of Health (CA254168 to TP; CA225517 to PN; AI132819, AI103748 to SS; 5P30CA015704; AI154363 to VK)

BMI-1 repression of the CD8 +T cell effector program is essential for memory formation

Abstract NaïveCD8 +T cell activation results in a program of proliferation and differentiation that results in large scale changes in phenotype and function that is critical for pathogen control and establishment of immunological memory. While these changes are underpinned by structural and biochemical changes in genome regulatory elements, the mechanisms that mediate these changes are not fully elucidated. Polycomb Repressive complexes (PRC) are chromatin remodeling complexes implicated in silencing genes encoding crucial developmental regulators. We demonstrated that upregulation of PRC1 components occurs rapidly upon naïve CD8 +T cell activation and was associated with maintenance of ubiquitination at key effector transcriptional regulators. Importantly, in vitro activation of PRC1-deficient CD8 +T cells resulted in greater proliferation and more rapid acquisition of effector function compared to wildtype cells. Use of two different viral infection models demonstrated that T cell specific PRC1 deficiency resulted in a greater effector expansion and effector differentiation with establishment of dysfunctional memory T cell populations. Strikingly, even after acute infection, the resulting memory T cells exhibited phenotypic and molecular markers of T cell exhaustion and were unable to be recalled. Our data suggest that PRC1 regulation early after CD8 +T cell activation is essential for restraining early effector T cell commitment and is a key step in establishing immunological CD8 +T cell memory. Supported by grants from National Health and Medical Research Council of Australia (Ideas Grant 2020562).

Berberine governs NOTCH3/AKT signaling to enrich lung-resident memory T cells during tuberculosis.

Stimulation of naïve T cells during primary infection or vaccination drives the differentiation and expansion of effector and memory T cells that mediate immediate and long-term protection. Despite self-reliant rescue from infection, BCG vaccination, and treatment, long-term memory is rarely established against Mycobacterium tuberculosis (M.tb) resulting in recurrent tuberculosis (TB). Here, we show that berberine (BBR) enhances innate defense mechanisms against M.tb and stimulates the differentiation of Th1/Th17 specific effector memory (TEM), central memory (TCM), and tissue-resident memory (TRM) responses leading to enhanced host protection against drug-sensitive and drug-resistant TB. Through whole proteome analysis of human PBMCs derived from PPD+ healthy individuals, we identify BBR modulated NOTCH3/PTEN/AKT/FOXO1 pathway as the central mechanism of elevated TEM and TRM responses in the human CD4+ T cells. Moreover, BBR-induced glycolysis resulted in enhanced effector functions leading to superior Th1/Th17 responses in human and murine T cells. This regulation of T cell memory by BBR remarkably enhanced the BCG-induced anti-tubercular immunity and lowered the rate of TB recurrence due to relapse and re-infection. These results thus suggest tuning immunological memory as a feasible approach to augment host resistance against TB and unveil BBR as a potential adjunct immunotherapeutic and immunoprophylactic against TB.

T cell-responsive macroporous hydrogels for in situ T cell expansion and enhanced antitumor efficacy

Adoptive T cell therapy has demonstrated great promise for treating cancer and other diseases. While extensive effort has been made to improve ex vivo expansion of T cells, strategies for maintaining the proliferation and function of T cells post adoptive transfer are still lacking. Here we report an injectable T cell-responsive macroporous hydrogel that enables in situ activation and expansion of T cells. The macroporous gel is composed of a polymeric network with dispersed macropores (∼150 μm) that are large enough to home T cells. In the presence of T cells that can gradually disrupt the gel network surrounding the macropores, activation cues can be gradually released for in situ activation and expansion of T cells. This T cell-responsive macroporous gel enables expansion of effector T cells in vivo, is stable over weeks upon subcutaneous injection, and results in enhanced CD8+ T cell response and antitumor efficacy. We further show that the T cell-responsive macroporous gel could achieve comparable antitumor efficacy to conventional T cell therapy with a much lower cell dose. This injectable, T cell-responsive macroporous gel provides a platform for in vivo expansion of engineered T cells in a controlled manner, for timely and effective treatment of diseases.

Potentiating effect of reovirus on immune checkpoint inhibition in microsatellite stable colorectal cancer.

The majority of colorectal cancers (CRCs) are microsatellite stable (MSS) and resistant to immunotherapy. The current study explores the possibility of using oncolytic reovirus to sensitize MSS CRC to immune checkpoint inhibition. While reovirus reduced metabolic activity among KRAS Mut cells, microarray/computational analysis revealed microsatellite status-oriented activation of immune-response pathways. Reovirus plus anti-PD-1 treatment increased cell death among MSS cells ex vivo. Reduced tumorigenicity and proliferative index, and increased apoptosis were evident among CT26 [MSS, KRAS Mut], but not in MC38 [microsatellite unstable/MSI, KRAS Wt] syngeneic mouse models under combinatorial treatment. PD-L1-PD-1 signaling axis were differentially altered among CT26/MC38 models. Combinatorial treatment activated the innate immune system, pattern recognition receptors, and antigen presentation markers. Furthermore, we observed the reduction of immunosuppressive macrophages and expansion of effector T cell subsets, as well as reduction in T cell exhaustion. The current investigation sheds light on the immunological mechanisms of the reovirus-anti-PD-1 combination to reduce the growth of MSS CRC.

Adaptive immune responses in patients requiring revision after total knee arthroplasty.

Dissatisfaction occurs in nearly 20% of patients after total knee arthroplasty (TKA); however, there remains only limited understanding of the biologic mechanisms that may contribute to suboptimal postoperative outcomes requiring revision surgery. Expansion of effector T and B cells, could promote an abnormal healing response via local or peripheral immune system mechanisms and contribute to inferior outcomes necessitating revision TKA. In this pilot study, we hypothesized that patients suffering from complications of arthrofibrosis or instability may exhibit differences in adaptive immune function. Patients (n = 31) undergoing revision TKA for an indication of arthrofibrosis or instability were prospectively enrolled. Whole blood and synovial fluid (SF) from the operative knee were collected at time of surgery. Peripheral blood mononuclear cells were isolated and analyzed by flow cytometry. Serum and SF were assessed for immunoglobulin levels by Luminex and antiphospholipid antibodies by enzyme-linked immunoassay. No significant differences were observed in peripheral blood T/B cell populations or serum immunoglobulins levels between groups. SF analysis demonstrated significant differences between the two groups, with higher levels of immunoglobulin G1 (IgG1) (p = 0.0184), IgG3 (p = 0.0084) and antiphosphatidyl serine IgG (p = 0.034) in arthrofibrosis relative to instability patients. Increased levels of both IgG subclasses and antiphospholipid antibodies in the SF suggest that intra-articular T-B cell interactions, potentially triggered by exposure to apoptotic components generated during post-op healing, could be functioning as a source of immune complexes that fuel fibrous tissue growth in arthrofibrotic patients.

Controversial Role of the Immune Checkpoint OX40L Expression on Platelets in Breast Cancer Progression.

In conventional T cells, OX40 has been identified as a major costimulating receptor augmenting survival and clonal expansion of effector and memory T cell populations. In regulatory T cells, (Treg) OX40 signaling suppresses cellular activity and differentiation. However, clinical trials investigating OX40 agonists to enhance anti-tumor immunity, showed only limited success so far. Here we show that platelets from breast cancer patients express relevant levels of OX40L and platelet OX40L (pOX40L) inversely correlates with platelet-expressed immune checkpoint molecules GITRL (pGITRL) and TACI (pTACI). While high expression of pOX40L correlates with T and NK cell activation, elevated pOX40L levels identify patients with higher tumor grades, the occurrence of metastases, and shorter recurrence-free survival (RFS). Of note, OX40 mRNA levels in breast cancer correlate with enhanced expression of anti-apoptotic, immune-suppressive, and tumor-promoting mRNA gene signatures. Our data suggest that OX40L on platelets might play counteracting roles in cancer and anti-tumor immunity. Since pOX40L reflects disease relapse better than the routinely used predictive markers CA15-3, CEA, and LDH, it could serve as a novel biomarker for refractory disease in breast cancer.

Therapeutic potential of interleukin-2 in autoimmune diseases.

Autoimmune diseases are characterized by dysregulation and aberrant activation of cells in the immune system. Therefore, restoration of the immune balance represents a promising therapeutic target in autoimmune diseases. Interleukin-2 (IL-2) can promote the expansion and differentiation of different immune cell subsets dose-dependently. At high doses, IL-2 can promote the differentiation and expansion of effector and memory T cells, whereas at low doses, IL-2 can promote the differentiation, survival, and function of regulatory T (Treg) cells, a CD4+ T cell subset that is essential for the maintenance of immune homeostasis and immune tolerance. Therefore, IL-2 exerts immunostimulatory and immunosuppressive effects in autoimmune diseases. The immunoregulatory role of low-dose IL-2 has sparked excitement for the therapeutic exploration of modulating the IL-2-Treg axis in the context of autoimmune diseases. In this review, we discuss recent advances in the therapeutic potential of IL-2 or IL-2-derived molecules in the treatment of autoimmune diseases.

Cell-intrinsic expression of the hemichannel Pannexin-1 promotes effector and memory CD8+ T cells via distinct metabolic pathways

Abstract Tissue-derived extracellular ATP (eATP) is a “danger signal” sensed by immune cells via purinergic receptors (e.g. P2RX7). In addition, healthy cells can export eATP through transmembrane channels, such as the hemichannel Pannexin-1 (Panx1). CD8+ T cells express Panx1 constitutively throughout developmental and immune response stages. However, the biological role of Panx1 in CD8+ T cells is undefined. Here, we used genetic and pharmacological tools to address this question. T-cell specific Panx1 deletion did not affect CD8+ T cell thymic development. In contrast, CD8+ T cell responses to viral infection (LCMV) are impaired by Panx1 inhibition or deletion. This is true for effector expansion of CD8+ T cells and for the survival of memory CD8+ T cells. Using adoptive transfer of tamoxifen-conditional Panx1 knockout LCMV-specific (P14) cells into LCMV-infected mice, we showed that memory CD8+ T cell survival requires sustained Panx1 – except for small intestinal-resident cells. Mechanistically, Panx1 promotes memory CD8+ T cell survival via P2RX7, which also favors memory CD8+ T cells - as suggested by our data with Panx1/P2RX7-double knockout P14 cells. The role of Panx1 for effector CD8+ T cell expansion, however, is unlikely to be via P2RX7 since this receptor does not have a role in effector CD8+ T cells. Our transcriptional and metabolic data suggests that, while memory CD8+ T cells require Panx1 for optimal mitochondrial function and expression of pro-memory genes, effector CD8+ T cells lacking Panx1 had decreased activation of the glycolytic pathway. Collectively, these results suggest that, in response to acute virus, Panx1 induces CD8+ T cell effector expansion and memory survival through distinct metabolic pathways. Supported by grants from NIH (R00 AI139381, NIAID).