Memory Cell Phenotype Research Articles

Broad applicability of the Goldspire™ platform for the treatment of solid tumors

Goldspire™ is a personalized immunotherapy platform that combines whole tumor-derived cells with antisense oligonucleotide (IMV-001) against Insulin-Like Growth Factor-1 Receptor (IGF-1R) in biodiffusion chambers (BDCs; 0.1 μm pore). BDCs are exposed to 5–6 Gy and implanted at abdominal sites for ∼48 h to deliver an antigenic payload and immunostimulatory factors to train the immune system. Lead product IGV-001 was evaluated in newly diagnosed glioblastoma (ndGBM) patients in Phase 1a and 1b trials (NCT02507583). A Phase 2b study (NCT04485949) recently completed enrollment.Preventative treatment with tumor-specific products manufactured with Goldspire limited tumor progression and extended overall survival in mice challenged with bladder, pancreatic, ovarian, colorectal, or renal carcinomas. The benefit of this immunotherapy was enhanced with anti-PD-1; combination treatment was superior to either monotherapy in orthotopic GBM and melanoma models. Lastly, Goldspire elicited immune T cell activation and memory phenotypes against patient-derived endometrial tumor-derived products in co-cultures with matching immune cells.

Rapamycin Prevents Expansion of Costimulation Blockade-resistant CD8+ Alloreactive Memory Cells following Depletional Induction in Renal Transplant Recipients.

Alemtuzumab induction with belatacept/rapamycin-based maintenance immunotherapy (ABR) prevents kidney allograft rejection and specifically limits early costimulation blockade-resistant rejection (CoBRR). To evaluate the mechanisms by which this regimen alters CoBRR, we characterized the phenotype and functional response of preexisting memory cells to allogeneic endothelial cells using intracellular cytokine staining and flow cytometry. IL-7-induced lymphocyte proliferation in the presence or absence of rapamycin was assessed to characterize the phenotype of proliferating cells. Lymphocytes from 40 recipients who underwent transplant using the ABR regimen were studied longitudinally. The rapid immunoresponses of preexisting alloreactive cells to allogeneic endothelial cells were predominantly CD8+TNF-α+/IFN-γ+ cells. These cells were effector memory (TEM) and terminally differentiated effector memory cells lacking CD28 expression, and most were CD57+PD1-. Neither rapamycin nor belatacept directly inhibited these cells. IL-7, a cytokine induced during lymphopenia postdepletion, provoked dramatic CD8+ TEM cell proliferation and a low level of CD8+CD57+PD1- cell expansion invitro. The IL-7 stimulation induced CD8+ cell mTOR phosphorylation, and rapamycin treatment markedly inhibited IL-7-induced TEM and CD57+PD1- cell expansion. This effect was evident in patients receiving the ABR in that the repopulation of CD8+CD57+PD1- TEM cells was substantially suppressed for at least 36 mo after transplant. These findings help define one mechanism by which a costimulation blockade/rapamycin-based therapy following alemtuzumab induction minimizes CoBRR, namely that in the presence of rapamycin, costimulation-resistant alloreactive cells are disproportionately ineffective at repopulating following post-transplant T cell depletion.

Human CD4+ memory phenotype T cells use mitochondrial metabolism to generate sensitive IFN-γ responses

The transition of naive T lymphocytes into antigenically activated effector cells is associated with a metabolic shift from oxidative phosphorylation to aerobic glycolysis. This shift facilitates production of the key anti-tumor cytokine interferon (IFN)-γ; however, an associated loss of mitochondrial efficiency in effector Tcells ultimately limits anti-tumor immunity. Memory phenotype (MP) Tcells are a newly recognized subset that arises through homeostatic activation signals following hematopoietic transplantation. We show here that human CD4+ MP cell differentiation is associated with increased glycolytic and oxidative metabolic activity, but MP cells retain less compromised mitochondria compared to effector CD4+ Tcells, and their IFN-γ response is less dependent on glucose and more reliant on glutamine. MP cells also produced IFN-γ more efficiently in response to weak Tcell receptor (TCR) agonism than effectors and mediated stronger responses to transformed B cells. MP cells may thus be particularly well suited to carry out sustained immunosurveillance against neoplastic cells.

PD-1 downregulation enhances CAR-T cell antitumor efficiency by preserving a cell memory phenotype and reducing exhaustion

BackgroundDespite the encouraging outcome of chimeric antigen receptor T cell (CAR-T) targeting B cell maturation antigen (BCMA) in managing relapsed or refractory multiple myeloma (RRMM) patients, the therapeutic side effects...

Abstract 82: Single-cell cytokine secretion kinetics probed by Time-lapse flow (TM) cytometry

Abstract Cytokine secretion from immune cells is a central element of the immune response and a major determinant of immunotherapy efficacy. Several methods, including bulk in-solution assays and flow cytometry, have been used to measure cytokine secretion. Population-level analyses have shown that different cytokines can have different rates of secretion, depending upon their differentiation state or the stimulus itself. However, no current method can interrogate cytokine secretion from individual cells over time. Here, we describe a novel flow cytometry-based assay for measuring single-cell cytokine secretion kinetics. Our approach, called “Time-lapse flowTM” uses semiconductor probes called laser particles (LPs) to optically barcode individual cells, providing a means to track single cells across a series of measurements over time. We applied this approach to measure T-cell cytokine secretion kinetics using stimulated primary human T cells. Isolated T cells from cryopreserved human PBMCs were barcoded with LPs and stained with a bispecific antibody that binds to the cell surface on one end and “catches” secreted cytokines (IFNy and IL-2) on the other end. This permits analysis of cytokines from live cells. Next, T cells underwent three sequential stimulations over the course of 3 hours with PMA/Ionomycin. After the first stimulation, cells were stained with a surface marker panel that defined T cell memory phenotypes (CD3, CD4, CD8, CD45RA, CCR7, CD57, CD27, and CD95) and “detection” antibodies that recognized IFNy and IL-2 bound to the cell surface. The detection antibodies were re-added after each stimulation to quantify the accumulating cytokine on the cell surface. In total, kinetic profiles from over 100,000 CD3+ cells were measured. We discovered massive heterogeneity of responding cells, identifying at least three distinct groups of cells defined by their initial time of cytokine secretion (early, middle, and late responders) and varying intensities of cytokine accumulation over time. Cells peaking in cytokine expression at the middle time points were mainly memory types with higher intensity, while later responders skewered towards naïve cells with less cytokine. However, IFNy variation wasn’t solely due to these markers, as diverse kinetics occurred across all subsets. For IL-2, we found that most responses initiated later than those of IFNy, and there was a reduced heterogeneity of both intensity and kinetics at these time points. These results were consistent across multiple donors and replicates (n=3). Laser particle barcoding enables measurement of single-cell function over time. This novel time-lapse data allows interrogation of functional heterogeneity of immune cells at scale, enabling discovery of new relationships between T cell responses, diseases, and immunotherapies. Citation Format: Marissa Fahlberg, Sarah Forward, Emane Rose Assita, Trevor Brown, Pratip Chattopadhyay, Sheldon Kwok. Single-cell cytokine secretion kinetics probed by Time-lapse flow (TM) cytometry [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 82.

Abstract 17: Metabolic screening of T cells guides the enrichment of stem cell memory phenotype

Abstract The efficacy of T cell-mediated immunotherapy is improved by the proliferation of T cells with a stem cell memory phenotype (Tscm). These cells are capable of long-term persistence, self-renewal, multipotency, and robust engagement with cancer cells. This study focuses on refining T cell activation protocols to bolster the presence of Tscm within the therapeutic arsenal against cancer. Through a series of cell health and metabolic assays, we traced the activation-induced metabolic reprogramming that results from Tscm enrichment. Ex vivo activation was characterized by a rapid 2.5-3 fold increase in ATP production and a 2 fold shift in intracellular reducing potential, paralleling the metabolic demands of Tscm in the cancer microenvironment. Progression of activation intensified the metabolic flux through catabolic and anabolic pathways, with significant fluctuations in metabolites such as glucose, lactate, glutamine, glutamate, branched chain amino acids, and pyruvate. These metabolic alterations were accompanied by a 2 fold increase in the prevalence of Tscm cells, and demonstrated a dependency on media composition. Our findings reveal that metabolic profiling during T cell activation provides early and actionable insights into the enrichment of Tscm, and may serve as a strategic guideline for the enhancement of cancer immunotherapies. Citation Format: Anthony Lauer, Natasha Karassina, Kayla Sylvester, Jolanta Vidugiriene. Metabolic screening of T cells guides the enrichment of stem cell memory phenotype [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 17.

Endogenous Signaling Molecule Activating (ESMA) CARs: A Novel CAR Design Showing a Favorable Risk to Potency Ratio for the Treatment of Triple Negative Breast Cancer.

As chimeric antigen receptor (CAR) T cell therapy continues to gain attention as a valuable treatment option against different cancers, strategies to improve its potency and decrease the side effects associated with this therapy have become increasingly relevant. Herein, we report an alternative CAR design that incorporates transmembrane domains with the ability to recruit endogenous signaling molecules, eliminating the need for stimulatory signals within the CAR structure. These endogenous signaling molecule activating (ESMA) CARs triggered robust cytotoxic activity and proliferation of the T cells when directed against the triple-negative breast cancer (TNBC) cell line MDA-MB-231 while exhibiting reduced cytokine secretion and exhaustion marker expression compared to their cognate standard second generation CARs. In a NOD SCID Gamma (NSG) MDA-MB-231 xenograft mouse model, the lead candidate maintained longitudinal therapeutic efficacy and an enhanced T cell memory phenotype. Profound tumor infiltration by activated T cells repressed tumor growth, further manifesting the proliferative capacity of the ESMA CAR T cell therapy. Consequently, ESMA CAR T cells entail promising features for improved clinical outcome as a solid tumor treatment option.

Excess generation and activation of naturally arising memory-phenotype CD4+ T lymphocytes are inhibited by regulatory T cells in steady state.

Conventional CD4+ T lymphocytes consist of naïve, foreign antigen-specific memory, and self-antigen-driven memory-phenotype (MP) cell compartments at homeostasis. We recently showed that MP cells tonically proliferate in response to self-antigens and differentiate into the T-bet+ subset in steady state. How excess proliferation and differentiation of MP cells are inhibited remains unclear. Given immunosuppressive function of regulatory T cells (Tregs), it is possible that they are also involved in inhibition of spontaneous MP cell activation. Here we show using Foxp3-diphtheria toxin receptor-transgenic mice that both MP and naïve CD4+ T cells spontaneously proliferate and differentiate into Th1 cells upon acute Treg depletion. At an early time point post Treg depletion, MP as compared to naïve CD4+ T cells are preferentially activated while at a later stage, the response is dominated by activated cells originated from the naïve pool. Moreover, we argue that MP cell proliferation is driven by TCR and CD28 signaling whereas Th1 differentiation mediated by IL-2. Furthermore, our data indicate that such activation of MP and naïve CD4+ T lymphocytes contribute to development of multi-organ inflammation at early and later time points, respectively, after Treg ablation. Together our findings reveal that Tregs tonically inhibit early, spontaneous proliferation and Th1 differentiation of MP CD4+ T lymphocytes as well as late activation of naïve cells, thereby contributing to maintenance of T cell homeostasis.

4-1bb Enrichment Enhances Adoptive T Cell Therapy for Hematological Malignancies Using a Novel Approach for Ex Vivo Immune Cell Priming with DC/Tumor Fusion Vaccine

Introduction: While CAR T cells have transformed outcomes for patients with hematologic malignancies, application to patients with acute myeloid leukemia (AML) is limited by lack of surface antigens that differentiate between malignant and normal hematopoietic precursors. We have developed a personalized vaccine created by fusion of patient-derived tumor with autologous dendritic cells (DCs). The vaccine presents an array of tumor antigens stimulating polyclonal immunity. In a phase II clinical trial of patients with AML in first remission, vaccination with DC/AML fusions resulted in expansion of leukemia specific T cells associated with durable disease remission. The ex vivo generation of vaccine educated T cells provides a powerful substrate of effector cells for adoptive immunotherapy that are selective and capture tumor heterogeneity. This novel adoptive cell therapy enhances T-cell potency with potential to mediate disease regression. Enrichment of vaccine-educated T cells for activated antigen-specific effector cells via agonistic 4-1bb antibody-based selection further enhances for activation and memory cell phenotype; superior T cell efficacy was observed in AML and MM models. Methods: DC/tumor fusion vaccines were generated from C57BL/6J mice DCs and syngeneic C1498 mCh/luc + AML cells or C57BL/KaLwRij and syngeneic 5TGM1 mCh/luc + MM cells, respectively. T cells isolated from spleen were co-cultured with autologous irradiated DC/tumor fusions in presence of IL-2/7/15. Vaccine-educated T cells underwent selection with agonistic 4-1bb (3H3) followed by expansion with anti-CD3/CD28 activation beads. T cells were phenotyped for markers of activation (CD25/CD69), immune checkpoint (PD1/LAG3/TIM3), and memory (CD44+CD62L-) and for enrichment (anti-rat H&L). Cytotoxicity was evaluated on luminescent assay. Mice engrafted with C1498 mCh/luc + received the T cell therapy 7 days later. Disease progression and survival was monitored for 100 days by BLI. Results: In the C1498 model, vaccine-educated T cells demonstrated evidence of immune activation (CD25+CD69+) and memory (CD44+CD62L-) phenotype compared to unstimulated naïve T-cell controls (TN) (4-fold, p=0.1143; 1.3-fold, p=0.0195, respectively). Vaccine-educated T cells selected based on 4-1bb expression showed 9-fold higher expression of activation markers (p=0.0034) while memory markers were 4.4-fold higher compared to TN (p=0.0274). Selection enriched for 4-1bb + vaccine-educated T cells resulting in enhanced antigen-specific recognition as measured by induction of IFNg expression. Tumor specificity and activation was maintained following CD3/CD28-mediated expansion. The 4-1bb positive vaccine-educated T cells showed 11-fold enhanced cytotoxicity compared to TN at 10:1 E:T (P=0.0005), while vaccine-educated T cells showed 4-fold increase (P=0.0226). Phenotypic and functional analysis support 4 days as the optimal duration of time for T-cell vaccine education. Cell fold-expansion supports 3-5 days as the optimal duration for anti-CD3/CD28 expansion. In vivo, 60% of mice treated with 4-1bb + vaccine-educated cells were alive at 60 days vs 20% treated with unselected vaccine-educated cells. In the 5TGM1 model, activation, memory, immune checkpoint phenotype and cytotoxicity were increased in vaccine-educated T cells selected for 4-1bb expression. Conclusion: Vaccine-educated T cells represent a unique platform for adoptive immunotherapy for AML further enhanced by selection for 4-1bb expression by agonistic antibody. T-cell stimulation by fusion vaccine and subsequent enrichment by agonistic 4-1bb selection enhances cytotoxicity, activation and memory cell phenotype. Thus, ex vivo vaccine stimulation and 4-1bb selection presents a novel approach for cell-based immunotherapy for AML.

CRISPR Activation Screen to Optimize Chimeric Antigen Receptor (CAR) T Cell Immunophenotype

Background Adoptive cellular therapies (ACT) offer potential cures for a wide range of human diseases. Immune cells genetically engineered with chimeric antigen receptors (CAR) have a clear role in B cell malignancies, and broader applications to other malignancies are under investigation. Significant obstacles to the long-term durability and potency of these therapies remain. As CAR T-cells remain persistently activated and exposed to the tumor microenvironment, they may become functionally impaired. This exhaustion can result in reduced production of cytokines, cytotoxic molecules, and other factors necessary for effective tumor cell killing. While CAR T-cell exhaustion remains a challenge, ongoing research and clinical trials aim to further understand the underlying mechanisms and develop strategies to overcome this limitation. Clinical studies have demonstrated that sustained in vivo persistence and function of CAR T-cells is correlated with infusion product phenotype. Persistence is associated with a less differentiated, memory-like phenotype. Naive (T N), Central Memory (T CM) and Stem Cell Memory (T SCM) are related to good proliferative potential and durability. CRISPR technologies offer powerful, functional genomic screens that permit the inference and modification of gene regulatory networks to optimize CAR T-cell persistence and function. Genome-wide CRISPR knock out screens have identified the CD2-CD58 axis as critical to T-cell potency and the knock-out of the mediator kinase module as beneficial to potency. Here we report on in vitro CRISPR activation screening of human CAR T-cells for gene expression that enhances T cell phenotypes associated with durable function. Method We designed 9,983 single guide RNA (sgRNA) to target the transcription start sites (TSS) of all known human transcription factors as well as a select set of genes with critical functions in T-cells. The vast majority of genes was targeted with 5 different sgRNA. To this we added a pool of 1,000 non-targeting sgRNA. We lentivirally transduced the sgRNA in tandem to a second-generation anti-CD20 CAR into primary T cells from healthy human donors. In a second lentiviral transduction we integrated catalytically dead Cas9 (dCas9) fused to the strong transcription activator, VP64 and in tandem to a puromycin resistance gene. After puromycin selection, we cultured the CRISPR activated CAR T-cells either alone or together with Raji or K562 cells for nine days before immunophenotypically sorting the CAR T-cells into CD8 + Naive, CD8 + Transitional Memory, CD8 + Central Memory, CD8 + Stem Cell Memory, CD8 + Transitional Effector, CD8 + Effector Memory, CD4 + Naive and CD4 + Memory T-cell populations. We extracted DNA from the sorted cells and prepared sequencing libraries targeting the sgRNA. We carried out two-way ANOVA analysis on the experimental conditions (trivial, Raji or K562 co-culture versus addition of CD2/CD28 activation beads) and T-cell subset to determine which genes targeted by CRISPRa resulted in favorable phenotypes. Result Across the different culture conditions, sgRNA activating IL23A and KLF4 were enriched in Transitional Memory versus Stem Cell Memory T-cells. Across the different CAR T-cell immunophenotypes, sgRNA targeting the TGFß (including KLF10), TNFα (including NFATC4) and FOXO signaling pathways were enriched in CAR T-cells cultured alone compared to CAR T-cells activated with CD2/CD28. Conclusion In this study we have investigated genes whose activation results in CAR T-cell immunophenotypes that are associated with more potent and durable CAR T-cell therapies. Our experiment suggests that the activation of genes, such as IL23 and KLF4, enriches for a Transitional Memory over Stem Cell Memory phenotype in a manner that is largely independent of environment (culture conditions). The activation of other genes, such as KLF10 and NFATC4, results in enrichment of CAR T-cells across all T-cell subsets, but in a manner that is dependent on environment. Gene expression requires coordinated control of gene-proximal and -distal cis-regulatory elements (CREs), whose state varies as cells differentiate or respond to environmental signals. Abrogating or adding CRE may result in more durable and potent adoptive cellular therapies independent of environment.

TMIC-50. SIGLEC-15 IS A NEW MYELOID CHECKPOINT IN GBM, WHOSE SUPPRESSION LEADS TO EXTREMELY DURABLE RESPONSES WITH ONCOLYTIC THERAPY

Abstract Zika virus (ZIKV) significantly increases survival in multiple syngeneic glioma mouse models; like other oncolytic therapies, it generates a potent, anti-tumor CD8+ T-cell response. Post-ZIKV, we observe a 32-fold increase in myeloid cells in the tumor microenvironment. Siglec-15 is a newly identified myeloid immune checkpoint in cancer, which suppresses T-cell activation; an antibody targeting Siglec-15 is currently in clinical trial for lung cancer. Using immunostaining, for the first time, we report Siglec-15 is expressed on myeloid cells in human GBM specimens. In syngeneic murine models, anti-Siglec-15 alone does not improve survival. However, in combination with ZIKV, anti-Siglec-15 significantly improves survival in the CT2A, SB28, and GL261 models. For example, in the CT2A model, the combination increased long-term survivors (at least 90 days) to 60%, compared to 20% with ZIKV alone and 0% with no treatment. To further confirm the role of Siglec-15, we used Siglec-15 knockout (KO) mice and observed 70% long-term survivor rate in Siglec-15 KO mice treated with ZIKV, compared to 40% in wild-type mice (B/6) with ZIKV. ZIKV treatment in Siglec-15 KO mice led to activation of CD8 T-cells and increased expression of cytotoxic molecules granzyme B and perforin, indicating enhanced anti-tumor activity. Additionally, we detected high levels IFN-gamma and TNF-alpha, further supporting the anti-tumor response. We further improved efficacy by targeting PD-1. This combination resulted in 90% long-term survivors. Moreover, in a model for recurrence, upon contralateral rechallenge with CT2A in long-term survivors, we observed long-term memory T-cell responses, with increased numbers of CD8+ T-cells displaying effector and resident memory cell phenotypes, compared to age-matched controls. We obtained similar results with immune-resistant SB28 model. This work suggests that oncolytic therapy combined with blocking myeloid checkpoint Siglec-15, using an agent already in humans, and blocking CD8+ T cell checkpoint with anti-PD1, is worthy of study in humans.

Clinical Experience with Cranial Nerve Impairment in the CARTITUDE-1, CARTITUDE-2 Cohorts A, B, and C, and Cartitude-4 Studies of Ciltacabtagene Autoleucel (Cilta-cel)

Introduction: Cilta-cel is a dual-binding, BCMA-targeting CAR-T cell therapy that has shown high rates of deep and durable response in patients (pts) with relapsed/refractory multiple myeloma (RRMM), and significantly prolonged PFS vs SOC in pts with lenalidomide-refractory MM after 1-3 prior lines of therapy (LOT; HR, 0.26) in the phase 3 CARTITUDE-4 trial. CAR-T therapies are associated with AEs related to immune activation, including neurologic toxicities (Gonzalez Castro. Neuro-Oncol Pract. 2020). Here, we describe the clinical experience with presentation and management of cranial neuropathy (CNP) in pts treated with cilta-cel in the CARTITUDE-1, CARTITUDE-2 Cohorts A, B, and C, and CARTITUDE-4 studies. Methods: CARTITUDE-1 and CARTITUDE-2 Cohort C pts had ≥3 prior LOT, incl PIs, IMiDs, and anti-CD38 mAbs, or were double-refractory to PI and IMiD; CARTITUDE-2 Cohort C pts also had non-cellular anti-BCMA therapy. CARTITUDE-2 Cohort B pts relapsed within 12 months of initial therapy. CARTITUDE-2 Cohort A and CARTITUDE-4 pts had 1-3 prior LOT, incl PI and IMiD, and were lenalidomide-refractory. After apheresis, pts received bridging therapy, then 1 cilta-cel infusion (target dose 0.75×10 6 CAR+ viable T cells/kg) 5-7 days (d) after lymphodepletion. Pts presenting with signs/symptoms of cranial nerve impairment commonly underwent a diagnostic workup that included CSF analysis and brain MRI at investigator discretion. Correlative data were available for pts in CARTITUDE-4: cilta-cel levels and T cell memory phenotypes in peripheral blood were assessed by flow cytometry, and serum cytokines were measured using multiplex sandwich immunoassays on the Meso Scale Discovery platform. Results: Of the 332 pts infused with cilta-cel as study treatment in CARTITUDE-1, CARTITUDE-2 (Cohorts A, B, and C), and CARTITUDE-4, 21 (6.3%) developed CNP (TABLE); most cases were grade (gr) 2 (n=3 gr 3). 6 pts had CNP that affected both sides; most unilateral impairments were left-sided. Median time to onset was 22 d (range 17-101; 81% had onset on d 22 +/- 5). Cranial nerve (CN) VII was involved in all pts; 2 pts had additional CNs involved (1 CN III [gr 3], 2 CN V [gr 3]). Twelve pts had concurrent neurologic symptoms/neurotoxicities (headache, n=7; n=1 each for dysgeusia, parosmia, restlessness, peripheral sensory neuropathy, polyneuropathy, amnesia, aphasia, agitation, depressed level of consciousness). Clinical characteristics of pts with or without CNP were comparable. In the 21 pts with CNP, median age was 64 years; 81% were male; at baseline, 1 pt had high disease burden (BM 95% plasma cells), 4 had plasmacytomas (1 bone-based); 1 pt had ISS stage III. Most pts responded to bridging therapy. Six pts had an infection after cilta-cel infusion and prior to CNP onset (bacterial, n=5; CMV, n=2; both, n=1). CRS rate was comparable between pts with vs without CNP. 90% of pts with CNP had preceding CRS (all gr 1/2; median onset, d 7; median duration, 3 d); 13 received tocilizumab for CRS. One pt had preceding gr 2 ICANS; no pts had movement/neurocognitive TEAEs (MNT) at any time. CSF analysis and brain MRI were performed in 14 and 17 pts, respectively. No evidence of infectious or malignant etiology was identified in any of these cases; MRI showed facial nerve enhancement in 7 pts, in whom there were no other significant findings. Most CNP cases were treated with corticosteroids for median 13 d. CNP resolved in 19/21 pts within median 66 d, including the 3 pts with gr 3 CNP. In CARTITUDE-4, pts with CNP had significantly higher levels of CAR+ T cell expansion (C max) and greater exposure to CAR+ T cells (AUC 0-CNP onset) than those without CNP (FIGURE). Pts with CNP had a trend toward higher peak concentration and exposure level (AUC 0-CNP onset) of IL-6, IL-10, and IL-2Rα, but not in IFNγ. The differentiation pattern of memory T cells from apheresis to the time of peak CAR+ T expansion (T max) was comparable in pts with vs without CNP; CAR+ T cells at T max were dominant with central memory T cells in both groups. Conclusions:Ptstreated with CAR-T cells, including cilta-cel, may experience CNP.The pathogenesis of the events in these 3 studies was unknown/idiopathic, but it is important to rule out etiology of infection or MM progression. Most cases were low-grade and resolved with a limited course of corticosteroids. CNP in CARTITUDE-4 was associated with higher exposure to CAR-T cells before onset, but no predictive clinical factors have been established.

Fratricide-Resistant Anti-CD2 Chimeric Antigen Receptor T-Cells with Endogenous CD2 Knockout Are Highly Effective Against T-Cell Neoplasms

T cell neoplasms, such as peripheral T cell lymphomas (PTCL) and acute T cell lymphoblastic leukemia (T-ALL), have some of the worst prognoses of all hematological malignancies. Poor outcomes are driven by a limited number of effective treatments, particularly in relapsed and refractory settings. While chimeric antigen receptor T cell (CAR-T) immunotherapy has been successful in other hematological diseases, its clinical use against T cell neoplasms is limited due to a lack of unique tumor-specific antigens. Additionally, CAR specificity against pan-T cell antigens causes CAR-T fratricide and insufficient manufacturability for therapeutic dosing. Early studies of CAR-T against CD5- and CD7-expressing T cell neoplasms have shown responses, however, 1) most patients ultimately relapse, and 2) in some patients, antigen negative relapse has occurred. To circumvent these obstacles, we hypothesized that targeting CD2, a pan-T cell antigen, using anti-CD2 CAR-T cells that are engineered to lack CD2, would permit effective CAR-T manufacturing against T cell neoplasms. In a retrospective analysis of clinical flow cytometry diagnostic samples, we found CD2 expression in 25 of 33 (75.8%) and 37 of 51 (72.5%) pediatric PTCL and T-ALL cases, respectively. Similarly, we found 82 of 89 (92.1%) adult PTCL cases with CD2 immunohistochemical expression, which exceeded CD7 expression (38 of 90, 42.2%) that is currently being evaluated in early phase clinical trials. We optimized early CD2 knockout (KO) during CAR-T cell manufacturing from human donor T cells (KO efficiency: 81.2% ± 2.4, n=9 donors). We next engineered a best-in-class, 4-1BB-costimulated, second-generation CD2KO anti-CD2 CAR-T product (CART2) via lentiviral transduction ( PANEL A) using seven distinct single-chain variable fragments (transduction efficiency: 60.2% ± 5.5, n=7 donors) based on cellular expansion and stressed in vivo efficacy against CD2+ T-ALL cells (Jurkat). There were no significant differences in population doublings during CART2 expansion as compared to both CD2 wild-type (CD2 WT) untransduced T cells (Mock UTD) and CD2KO untransduced T cells (CD2KO UTD) (6.20 ± 0.42 vs. 6.12 ± 0.62 vs. 6.67 ± 0.68, respectively, Day 15, n=3 donors), while CD2 WT CART2 cells failed to expand due to fratricide. CART2 cells had no significant differences in T cell memory phenotypes or T cell exhaustion markers, including PD1 and LAG3 expression, at the end of the expansion. Expanded CART2 cells eradicated primary patient-derived CD2+ Sezary cells and CD2+ T-ALL leukemic blasts as measured by in vitro cytotoxicity assays. Cytotoxicity correlated with CD8+ CART2 cells expressing IL-2 (20.4% vs. 0.2% vs. 0.35%), TNFα (28.5% vs. 0.27% vs. 0.2%), and IFNγ (11.27% vs. 0.18% vs. 0.14%) relative to Mock UTD and CD2KO UTD controls stimulated with CD2+ Jurkat cells (n=2 donors). Importantly, we also found both CD2KO UTD and CART2 cells were capable of TNFα and IFNγ release upon interaction with cytomegalovirus and influenza peptides, suggesting that T cells with CD2 perturbations are still able to mediate effector T cell responses. We further demonstrated that CART2 cells were highly effective in controlling systemic tumor burden of T-ALL patient-derived xenograft mouse (PDX) models and significantly improved median overall survival as compared to Mock UTD and CD2KO UTD controls (138 days vs. 30 days vs. 30 days, p=0.0007, Mantel-Cox) ( PANEL B). Long-term tumor control was proportional to CART2 peripheral blood persistence at Day 28 (CD2KO UTD: 186.6 ± 83.7, n=5 vs. CART2: 936.3 ± 241.7, n=5) and Day 100 (CD2KO UTD: N/A vs. CART2: 514.0 ± 441, n=3). We also found that rescue infusion of CART2 could eliminate relapsed T-ALL in PDX mice previously in a remission after initial treatment with anti-CD5 CAR-T cells, with long-term persistence of CD2-negative T cells thereafter. In conclusion, our data demonstrate that CART2 are manufacturable and highly efficacious against T-cell neoplasms. To our knowledge, this is the first report of prolonged efficacy using a CD2-targeted cellular monotherapy in human leukemia preclinical models. This product could be delivered alone or in combination with additional CARs for dual targeting, such as anti-CD5 or anti-CD7 CARs, to reduce the potential of antigen-negative escape. Further research is ongoing to study the effect of CD2 deletion in anti-CD19 CAR T cells and the possible implication for clinical translation.

Effects of Cannabidiol on the Functions of Chimeric Antigen Receptor T Cells in Hematologic Malignancies.

Introduction: CD19-chimeric antigen receptor (CAR) T cell therapy is a promising immunotherapy for cancer treatment that has shown remarkable clinical responses, leading to approval by the FDA for relapsed and refractory B cell hematological malignancy treatment. Cannabidiol (CBD) is a nonpsychoactive cannabinoid compound that has been utilized as a palliative treatment in cancer patients due to its immunosuppressive properties. Currently, studies on using CBD during immunotherapy have gained increasing attention. However, the possible interaction between CBD and CAR T cell therapy has not been studied. Therefore, in this study, we aimed to examine the direct effects of CBD on CD19-CAR T cell function against hematologic malignancies. Materials and Methods: The cytotoxic effect of CBD was determined by a cell proliferation reagent water-soluble tatrazolium salt (WST-1) assay. CAR T cells were generated by retroviral transduction and treated with CBD at a nontoxic dose. The effect of CBD on immune characteristics, including transgene expression, T cell subset, and memory phenotype, was analyzed by flow cytometry. Proliferation, apoptosis, and cell cycle distribution were analyzed with standard methods. The effect on cytotoxic function was evaluated using degranulation assays, and antitumor activity was evaluated using flow cytometry. Results: The half-maximum inhibitory concentration (IC50) of CBD on NALM6, Raji, and T cells ranged from 16 to 22 μM. The maximum nontoxic dose of CBD that maintained cell viability at ∼100% was 8 μM. For the generation of CD19-CAR T cells, primary T cells were activated and transduced with a retroviral vector encoding CD19-CAR. CBD did not alter the surface expression or immune characteristics, including the T cell subset and memory phenotype, of CD19-CAR T cells. However, CBD suppressed CD19-CAR T cell proliferation by inducing apoptosis, as evidenced by an increase in the proportion of cells in the Sub-G1 phase in cell cycle arrest. However, the antitumor activity and cytokine secretion of CD19-CAR T cells were not altered by exposure to CBD in this study. Conclusions: In this study, a nontoxic dose of CBD affected CD19-CAR T cell proliferation but not its immune characteristics or cytotoxic function.

FOXO1 Inhibition Generates Potent Nonactivated CAR T Cells against Solid Tumors.

Chimeric antigen receptor (CAR) T cells have shown promising results in the treatment of B-cell malignancies. Despite the successes, challenges remain. One of them directly involves the CAR T-cell manufacturing process and especially the ex vivo activation phase. While this is required to allow infection and expansion, ex vivo activation dampens the antitumor potential of CAR T cells. Optimizing the nature of the T cells harboring the CAR is a strategy to address this obstacle and has the potential to improve CAR T-cell therapy, including for solid tumors. Here, we describe a protocol to create CAR T cells without ex vivo preactivation by inhibiting the transcription factor FOXO1 (CAR TAS cells). This approach made T cells directly permissive to lentiviral infection, allowing CAR expression, with enhanced antitumor functions. FOXO1 inhibition in primary T cells (TAS cells) correlated with acquisition of a stem cell memory phenotype, high levels of granzyme B, and increased production of TNFα. TAS cells displayed enhanced proliferative and cytotoxic capacities as well as improved migratory properties. In vivo experiments showed that CAR TAS cells were more efficient at controlling solid tumor growth than classical CAR T cells. The production of CAR TAS from patients' cells confirmed the feasibility of the protocol in clinic.

The role of the CD8+ T cell compartment in ageing and neurodegenerative disorders.

CD8+ lymphocytes are adaptive immunity cells with the particular function to directly kill the target cell following antigen recognition in the context of MHC class I. In addition, CD8+ T cells may release pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), and a plethora of other cytokines and chemoattractants modulating immune and inflammatory responses. A role for CD8+ T cells has been suggested in aging and several diseases of the central nervous system (CNS), including Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, limbic encephalitis-induced temporal lobe epilepsy and Susac syndrome. Here we discuss the phenotypic and functional alterations of CD8+ T cell compartment during these conditions, highlighting similarities and differences between CNS disorders. Particularly, we describe the pathological changes in CD8+ T cell memory phenotypes emphasizing the role of senescence and exhaustion in promoting neuroinflammation and neurodegeneration. We also discuss the relevance of trafficking molecules such as selectins, mucins and integrins controlling the extravasation of CD8+ T cells into the CNS and promoting disease development. Finally, we discuss how CD8+ T cells may induce CNS tissue damage leading to neurodegeneration and suggest that targeting detrimental CD8+ T cells functions may have therapeutic effect in CNS disorders.

An immunosuppressed microenvironment distinguishes lateral ventricle-contacting glioblastomas.

Radiographic contact of glioblastoma (GBM) tumors with the lateral ventricle and adjacent stem cell niche correlates with poor patient prognosis, but the cellular basis of this difference is unclear. Here, we reveal and functionally characterize distinct immune microenvironments that predominate in subtypes of GBM distinguished by proximity to the lateral ventricle. Mass cytometry analysis of isocitrate dehydrogenase wild-type human tumors identified elevated T cell checkpoint receptor expression and greater abundance of a specific CD32+CD44+HLA-DRhi macrophage population in ventricle-contacting GBM. Multiple computational analysis approaches, phospho-specific cytometry, and focal resection of GBMs validated and extended these findings. Phospho-flow quantified cytokine-induced immune cell signaling in ventricle-contacting GBM, revealing differential signaling between GBM subtypes. Subregion analysis within a given tumor supported initial findings and revealed intratumor compartmentalization of T cell memory and exhaustion phenotypes within GBM subtypes. Collectively, these results characterize immunotherapeutically targetable features of macrophages and suppressed lymphocytes in GBMs defined by MRI-detectable lateral ventricle contact.

Self antigen-driven, undifferentiated memory-phenotype CD4 T lymphocytes can induce mild and systemic inflammation by differentiating into effector and regulatory T cells

Abstract In adaptive immune responses, naïve CD4 +T lymphocytes that have T cell receptors (TCRs) reactive to challenged foreign antigens are activated to differentiate into antigen-specific effector and memory cells. We have recently reported that in steady state, peripheral naïve precursors can also recognize self antigens to spontaneously acquire a memory phenotype (MP) and that such naturally arising “MP cells” can exert innate immune function in host defense. Given the self-reactivity of MP cells, it is likely that the same T lymphocyte population also has the capacity to induce autoimmune and/or inflammatory disease. Here we show that in lymphopenic environment, self antigen-driven MP cells can evoke mild and persistent inflammation in various organs including colon, lungs, kidneys, and liver. The MP cell-driven colitis is dependent on commensal microbiota as well as cytokines IFN-γ and IL-17A while inflammation in the other organs is independent of these factors. On the other hand, we demonstrate using combined single cell RNA sequencing, TCR sequencing, and flow cytometric approaches that MP as compared to naïve CD4 +T lymphocytes more efficiently differentiate into regulatory T (Treg) cells in a TGF-β-dependent fashion, thereby partially ameliorating inflammation. We further provide evidence arguing that these Th1, Th17, and Treg cells are generated from preexisting, undifferentiated T-bet −rather than T-bet +MP subset. Altogether, our results identify self-driven T-bet −MP cells as a multi-potent CD4 +T lymphocyte population that can induce systemic and mild inflammation by differentiating into Th1 and Th17 effector as well as Treg cells. This work was supported by the Japan Society for the Promotion of Science, Astellas Foundation for Research on Metabolic Disorders, Bristol-Myers Squibb, Cell Science Research Foundation, Chemo-Sero-Therapeutic Research Institute, Daiichi Sankyo Foundation of Life Science, GlaxoSmithKline, Gonryo Foundation for the Promotion of Medical Science, G-7 Scholarship Foundation, Harmonic Ito Foundation, Ichiro Kanehara Foundation for the Promotion of Medical Sciences and Medical Care, Inamori Foundation, Intelligent Cosmos Foundation, Japan Allergy Foundation, Japan Intractable Diseases Research Foundation, Kanae Foundation for the Promotion of Medical Science, Kobayashi Foundation, Life Science Foundation of Japan, Mitsubishi Foundation, Mochida Memorial Foundation for Medical and Pharmaceutical Research, MSD Life Science Foundation, Nakajima Foundation, Nakayama Foundation for Human Science, Ohyama Health Foundation, Okinaka Memorial Institute for Medical Research, Pharmacodynamics Research Foundation, Senri Life Science Foundation, Senshin Medical Research Foundation, Sumitomo Foundation, Takeda Science Foundation, Ube Industries Foundation, Uehara Memorial Foundation, and Waksman Foundation of Japan. Akihisa Kawajiri was supported by the PhD Scholarship (Kibou Project) from Japanese Society for Immunology.

Anti-HVEM mAb therapy improves antitumoral immunity both in vitro and in vivo, in a novel transgenic mouse model expressing human HVEM and BTLA molecules challenged with HVEM expressing tumors

BackgroundTumor necrosis factor superfamily member 14 (TNFRSF14)/herpes virus entry mediator (HVEM) is the ligand for B and T lymphocyte attenuator (BTLA) and CD160-negative immune co-signaling molecules as well as viral...

CD127 and Sca1 mark self-driven memory-phenotype CD4 +T cells that differ from foreign antigen-specific memory cells and possess inflammatogenic and anti-tumor activities

Abstract Under steady-state conditions, conventional CD4 +T lymphocytes are divided into naïve (CD44 loCD62L hi) and memory (CD44 hiCD62L lo) cell compartments. While the latter population is presumed to comprise a mixture of explicit foreign antigen (Ag)-specific “authentic” memory and foreign Ag-independent memory-phenotype (MP) cells, phenotypic markers differentially expressed in these two cell types have yet to be identified. Moreover, it is unknown whether MP cells consist of distinct subsets defined by marker expression. In this study we demonstrate using combined single cell RNA sequencing and flow cytometric approaches that self-driven MP CD4 +T lymphocytes are divided into CD127 hiSca1 lo, CD127 hiSca1 hi, CD127 loSca1 hi, and CD127 loSca1 losubpopulations that are Bcl2 lowhile foreign Ag-specific memory cells are CD127 hiSca1 hiBcl2 hi. We further show that amongst the four MP subsets, CD127 hiSca1 hilymphocytes represent the most quiescent, cell division-experienced subpopulation derived from peripheral naïve precursors. Finally, we provide new evidence arguing that the CD127 hiSca1 hiMP subpopulation tonically expresses T-bet and possesses the capacity to trigger systemic inflammation including colitis, interstitial pneumonia, and nephritis as well as to exert anti-tumor activity against malignant lymphoma. Together our findings define MP CD4 +T lymphocytes as a unique, self-driven population consisting of distinct subsets that differ from conventional foreign Ag-specific memory cells in marker expression and reveal the inflammatogenic nature of the mature CD127 hiSca1 hiMP cells.