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Background Levels of circulating CMV Ag-specific lymphocytes determine CMV reactivation risk in immunocompromised individuals. Methods Frequencies of T cells producing cytokines after stimulation by CMV Ag were measured in hematopoietic stem-cell donors using flow cytometry. Results In seropositive individuals (n = 75) the mean number of CD8[sup +] (CD8[sup bright], CD8[sup dim]) and CD4[sup +] cells producing IFN-γ was respectively 3.1% (12.6/μL) and 0.38% (3.2/μL), over 10-fold higher than in seronegative subjects (n = 22). CMV stimulation induced tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in both CD4[sup +] and CD8[sup +] cells (usually together), with a shift from memory- to effector-cell phenotype, while only a small proportion of CD4[sup +] cells produced IL-4. Although the normal range was wide, neither age, sex nor HLA type affected the frequency. Discussion These quantitative studies and the recognition of CD4[sup +] cells as potential effectors of CMV immunity are of relevance for immunotherapeutic approaches to prevent CMV disease after stem-cell transplantation. [ABSTRACT FROM AUTHOR]

Despite a high response rate in chimeric antigen receptor (CAR) T cell therapy for acute lymphocytic leukaemia (ALL) 1-3 , approximately 50% of patients relapse within the first year 4-6 , representing an urgent question to address in the next stage of cellular immunotherapy. Here, to investigate the molecular determinants of ultralong CAR T cell persistence, we obtained a single-cell multi-omics atlas from 695,819 pre-infusion CAR T cells at the basal level or after CAR-specific stimulation from 82 paediatric patients with ALL enrolled in the first two CAR T ALL clinical trials and 6 healthy donors. We identified that elevated type 2 functionality in CAR T infusion products is significantly associated with patients maintaining a median B cell aplasia duration of 8.4 years. Analysis of ligand-receptor interactions revealed that type 2 cells regulate a dysfunctional subset to maintain whole-population homeostasis, and the addition of IL-4 during antigen-specific activation alleviates CAR T cell dysfunction while enhancing fitness at both transcriptomic and epigenomic levels. Serial proteomic profiling of sera after treatment revealed a higher level of circulating type 2 cytokines in 5-year or 8-year relapse-free responders. In a leukaemic mouse model, type 2 high CAR T cell products demonstrated superior expansion and antitumour activity, particularly after leukaemia rechallenge. Restoring antitumour efficacy in type 2 low CAR T cells was attainable by enhancing their type 2 functionality, either through incorporating IL-4 into the manufacturing process or by priming manufactured CAR T products with IL-4 before infusion. Our findings provide insights into the mediators of durable CAR T therapy response and suggest potential therapeutic strategies to sustain long-term remission by boosting type 2 functionality in CAR T cells., (© 2024. The Author(s).)

Current cancer immunotherapy predominately focuses on eliciting type 1 immune responses fighting cancer; however, long-term complete remission remains uncommon 1,2 . A pivotal question arises as to whether type 2 immunity can be orchestrated alongside type 1-centric immunotherapy to achieve enduring response against cancer 3,4 . Here we show that an interleukin-4 fusion protein (Fc-IL-4), a typical type 2 cytokine, directly acts on CD8 + T cells and enriches functional terminally exhausted CD8 + T (CD8 + T TE ) cells in the tumour. Consequently, Fc-IL-4 enhances antitumour efficacy of type 1 immunity-centric adoptive T cell transfer or immune checkpoint blockade therapies and induces durable remission across several syngeneic and xenograft tumour models. Mechanistically, we discovered that Fc-IL-4 signals through both signal transducer and activator of transcription 6 (STAT6) and mammalian target of rapamycin (mTOR) pathways, augmenting the glycolytic metabolism and the nicotinamide adenine dinucleotide (NAD) concentration of CD8 + T TE cells in a lactate dehydrogenase A-dependent manner. The metabolic modulation mediated by Fc-IL-4 is indispensable for reinvigorating intratumoural CD8 + T TE cells. These findings underscore Fc-IL-4 as a potent type 2 cytokine-based immunotherapy that synergizes effectively with type 1 immunity to elicit long-lasting responses against cancer. Our study not only sheds light on the synergy between these two types of immune responses, but also unveils an innovative strategy for advancing next-generation cancer immunotherapy by integrating type 2 immune factors., (© 2024. The Author(s).)

Cholangiocarcinoma (CCA) is a rare and aggressive malignancy originating from the bile ducts, with poor prognosis and limited treatment options. Traditional therapies, such as surgery, chemotherapy, and radiation, have shown limited efficacy, especially in advanced cases. Recent advancements in immunotherapy, particularly T cell-based therapies like chimeric antigen receptor T (CAR T) cells, tumor-infiltrating lymphocytes (TILs), and T cell receptor (TCR)-based therapies, have opened new avenues for improving outcomes in CCA. This review provides a comprehensive overview of the current state of T cell therapies for CCA, focusing on CAR T cell therapy. It highlights key challenges, including the complex tumor microenvironment and immune evasion mechanisms, and the progress made in preclinical and clinical trials. The review also discusses ongoing clinical trials targeting specific CCA antigens, such as MUC1, EGFR, and CD133, and the evolving role of precision immunotherapy in enhancing treatment outcomes. Despite significant progress, further research is needed to optimize these therapies for solid tumors like CCA. By summarizing the most recent clinical results and future directions, this review underscores the promising potential of T cell therapies in revolutionizing CCA treatment.

Chimeric antigen receptor (CAR) T-cell therapy has achieved significant success in achieving durable and potentially curative responses in patients with hematological malignancies. CARs are tailored fusion proteins that direct T cells to a specific antigen on tumor cells thereby eliciting a targeted immune response. The approval of several CD19-targeted CAR T-cell therapies has resulted in a notable surge in clinical trials involving CAR T cell therapies for hematological malignancies. Despite advancements in understanding response mechanisms, resistance patterns, and adverse events associated with CAR T-cell therapy, the translation of these insights into robust clinical efficacy has shown modest outcomes in both clinical trials and real-world scenarios. Therefore, the assessment of CAR T-cell functionality through rigorous preclinical studies plays a pivotal role in refining therapeutic strategies for clinical applications. This review provides an overview of the various in vitro and animal models used to assess the functionality of CAR T-cells. We discuss the findings from preclinical research involving approved CAR T-cell products, along with the implications derived from recent preclinical studies aiming to optimize the functionality of CAR T-cells. The review underscores the importance of robust preclinical evaluations and the need for models that accurately replicate human disease to bridge the gap between preclinical success and clinical efficacy., Competing Interests: Declaration of competing interest Dr. Melenhorst has patents related to Biomarkers and manufacturing of chimeric antigen receptor-engineered T cells. The remaining authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Ltd.)

Autologous chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of lymphoid malignancies, leading to the approval of CD19-CAR T cells for B-cell lymphomas and acute leukaemia, and more recently, B-cell maturation antigen-CAR T cells for multiple myeloma. The long-term follow-up of patients treated in the early clinical trials demonstrates the possibility for long-term remission, suggesting a cure. This is associated with a low incidence of significant long-term side effects and a rapid improvement in the quality of life for responders. In contrast, other types of immunotherapies require prolonged treatments or carry the risk of long-term side effects impairing the quality of life. Despite impressive results, some patients still experience treatment failure or ultimately relapse, underscoring the imperative to improve CAR T-cell therapies and gain a better understanding of their determinants of efficacy to maximize positive outcomes. While the next-generation of CAR T cells will undoubtingly be more potent, there are already opportunities for optimization when utilizing the currently available CAR T cells. This review article aims to summarize the current evidence from clinical, translational and fundamental research, providing clinicians with insights to enhance their understanding and use of CAR T cells., (© 2024 The Author(s). British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Abstract: Autologous T-cell-based therapies, such as chimeric antigen receptor (CAR) T-cell therapy, exhibit low success rates in chronic lymphocytic leukemia (CLL) and correlate with a dysfunctional T-cell phenotype observed in patients. Despite various proposed mechanisms of T-cell dysfunction in CLL, the specific CLL-derived factors responsible remain unidentified. This study aimed to investigate the mechanisms through which CLL cells suppress CAR T-cell activation and function. We found that CLL-derived T cells get activated, albeit in a delayed fashion, and specifically that restimulation of CAR T cells in the presence of CLL cells causes impaired cytokine production and reduced proliferation. Notably, coculture of T cells with CD40-activated CLL cells did not lead to T-cell dysfunction, and this required direct cell contact between the CD40-stimulated CLL cells and T cells. Inhibition of kinases involved in the CD40 signaling cascade revealed that the Spare Respiratory Capacity (SRC) kinase inhibitor dasatinib prevented rescue of T-cell function independent of CD40-mediated increased levels of costimulatory and adhesion ligands on CLL cells. Transcriptome profiling of CD40-stimulated CLL cells with or without dasatinib identified widespread differential gene expression. Selecting for surface receptor genes revealed CD40-mediated downregulation of the Sialic acid-binding Ig-like lectin 10 (Siglec-10) ligands CD24 and CD52, which was prevented by dasatinib, suggesting a role for these ligands in functional T-cell suppression in CLL. Indeed, blocking CD24 and/or CD52 markedly reduced CAR T-cell dysfunction upon coculture with resting CLL cells. These results demonstrated that T cells derived from CLL patients can be reinvigorated by manipulating CLL-T-cell interactions. Targeting CD24- and CD52-mediated CLL-T-cell interaction could be a promising therapeutic strategy to enhance T-cell function in CLL., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: Despite recent advances in immunotherapy, a substantial population of late-stage melanoma patients still fail to achieve sustained clinical benefit. Lack of translational preclinical models continues to be a major challenge in the field of immunotherapy; thus, more optimized translational models could strongly influence clinical trial development. To address this unmet need, we designed a preclinical model reflecting the heterogeneity in melanoma patients' clinical responses that can be used to evaluate novel immunotherapies and synergistic combinatorial treatment strategies. Using our all-autologous humanized melanoma mouse model, we examined the efficacy of a novel engineered interleukin 2 (IL-2)-based cytokine variant immunotherapy., Methods: To study immune responses and antitumor efficacy for human melanoma tumors, we developed an all-autologous humanized melanoma mouse model using clinically annotated, matched patient tumor cells and peripheral blood mononuclear cells (PBMCs). After inoculating immunodeficient NSG mice with patient tumors and an adoptive cell transfer of autologous PBMCs, mice were treated with anti-PD-1, a novel investigational engineered IL-2-based cytokine (nemvaleukin), or recombinant human IL-2 (rhIL-2). The pharmacodynamic effects and antitumor efficacy of these treatments were then evaluated. We used tumor cells and autologous PBMCs from patients with varying immunotherapy responses to both model the diversity of immunotherapy efficacy observed in the clinical setting and to recapitulate the heterogeneous nature of melanoma., Results: Our model exhibited long-term survival of engrafted human PBMCs without developing graft-versus-host disease. Administration of an anti-PD-1 or nemvaleukin elicited antitumor responses in our model that were patient-specific and were found to parallel clinical responsiveness to checkpoint inhibitors. An evaluation of nemvaleukin-treated mice demonstrated increased tumor-infiltrating CD4 + and CD8 + T cells, preferential expansion of non-regulatory T cell subsets in the spleen, and significant delays in tumor growth compared with vehicle-treated controls or mice treated with rhIL-2., Conclusions: Our model reproduces differential effects of immunotherapy in melanoma patients, capturing the inherent heterogeneity in clinical responses. Taken together, these data demonstrate our model's translatability for novel immunotherapies in melanoma patients. The data are also supportive for the continued clinical investigation of nemvaleukin as a novel immunotherapeutic for the treatment of melanoma., Competing Interests: Competing interests: JL, RW and HL hold stock in Alkermes. Research performed at the Cleveland Clinic in the laboratory of BRG was funded by Alkermes., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

All chimeric antigen receptor (CAR) T-cell products currently approved by the FDA are autologous, which poses several challenges for widespread use. In this issue, Degagné and colleagues present their preclinical research on creating off-the-shelf CAR T cells for multiple myeloma. They utilized the CRISPR/Cas12a genome editing platform and gene knock-in techniques to eliminate alloreactivity and decrease susceptibility to natural killer (NK)-cell elimination. This work has led to an ongoing phase I trial of off-the-shelf CAR T cells for multiple myeloma treatment. See related article by Degagné et al., p. 462 (2)., (©2024 American Association for Cancer Research.)

Competing Interests: Competing interests: No, there are no competing interests.

Multiple clinical studies have treated mesothelin (MSLN)-positive solid tumors by administering MSLN-directed chimeric antigen receptor (CAR) T cells. Although these products are generally safe, efficacy is limited. Therefore, we generated and characterized a potent, fully human anti-MSLN CAR. In a phase 1 dose-escalation study of patients with solid tumors, we observed two cases of severe pulmonary toxicity following intravenous infusion of this product in the high-dose cohort (1-3 × 10 8 T cells per m 2 ). Both patients demonstrated progressive hypoxemia within 48 h of infusion with clinical and laboratory findings consistent with cytokine release syndrome. One patient ultimately progressed to grade 5 respiratory failure. An autopsy revealed acute lung injury, extensive T cell infiltration, and accumulation of CAR T cells in the lungs. RNA and protein detection techniques confirmed low levels of MSLN expression by benign pulmonary epithelial cells in affected lung and lung samples obtained from other inflammatory or fibrotic conditions, indicating that pulmonary pneumocyte and not pleural expression of mesothelin may lead to dose-limiting toxicity. We suggest patient enrollment criteria and dosing regimens of MSLN-directed therapies consider the possibility of dynamic expression of mesothelin in benign lung with a special concern for patients with underlying inflammatory or fibrotic conditions., Competing Interests: Declaration of interests C.H.J., R.M.Y., and M.M.D. are inventors on patents and/or patent applications licensed to Novartis Institutes of Biomedical Research and receive license revenue from such licenses. J.L.B., B.E., K.M., and L.Z. are holders of stock options and patents with Novartis Institutes for Biomedical Research. R.M.Y. and M.M.D. are inventors on patents and/or patent applications licensed to Tmunity Therapeutics and receive license revenue from such licenses. C.H.J. and A.C. are scientific cofounders of Tmunity Therapeutics. C.H.J. is a scientific cofounder of Capstan Therapeutics and is a member of the scientific advisory boards of AC Immune, Alaunos, BluesphereBio, Cabaletta, Carisma, Cartography, Cellares, Celldex, Decheng, Poseida, Verismo, and WIRB-Copernicus. B.E. and R.J.G. completed work on this study before becoming employees at Miltenyi Biotec and AstraZeneca, respectively. R.J.G. holds or may hold AstraZeneca stock. N.C.S. holds equity in Fate Therapeutics and Pfizer. J.A.F. has received grants and personal fees from Cartography Bio., grants from Tmunity Therapeutics, and personal fees from Retro Bio and Shennon Bio outside the submitted work. Additionally, J.A.F. holds patents related to CAR T cells for cancer that are licensed and associated with royalties. S.F.L. is an inventor on patents in the areas of CAR T and biomarkers at Penn that were assigned to Novartis; received research funding from Novartis, Tmunity, and Cabaletta; and consults for Kite/Gilead. M.M.D. is a consultant for Tmunity Therapeutics and is a Consultant and Member on the Scientific Advisory Board of Cellares Corporation., (Copyright © 2023 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Chimeric antigen receptor (CAR) T-cell therapies have transformed the treatment landscape of blood cancers. These engineered receptors which endow T cells with antibody-like target cell recognition combined with the typical T cell target cell lysis abilities. Introduced into the clinic in the 2010s, CAR T-cells have shown efficacy in chronic B lymphocytic leukemia (CLL), but a majority of patients do not achieve sustained remission. Here we discuss the current treatment landscape in CLL using small molecules and allogeneic stem cell transplantation, the niche CAR T-cells filled in this context, and what we have learned from biomarker and mechanistic studies. Several product parameters and improvements are introduced as examples of how the bedside-to-bench is translated into improved CAR T-cells for CLL. We hope to convey to our readers the crucial role translational medicine plays in transforming the treatment outcomes for patients with CLL and how this line of research is an essential component of modern medicine., (Copyright © 2023 Elsevier Inc. All rights reserved.)

In chronic lymphocytic leukemia (CLL) patients who achieve a complete remission (CR) to anti-CD19 chimeric antigen receptor T cells (CART-19), remissions are remarkably durable. Preclinical data suggesting synergy between CART-19 and the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib prompted us to conduct a prospective single-center phase 2 trial in which we added autologous anti-CD19 humanized binding domain T cells (huCART-19) to ibrutinib in patients with CLL not in CR despite ≥6 months of ibrutinib. The primary endpoints were safety, feasibility, and achievement of a CR within 3 months. Of 20 enrolled patients, 19 received huCART-19. The median follow-up for all infused patients was 41 months (range, 0.25-58 months). Eighteen patients developed cytokine release syndrome (CRS; grade 1-2 in 15 of 18 subjects), and 5 developed neurotoxicity (grade 1-2 in 4 patients, grade 4 in 1 patient). While the 3-month CR rate among International Working Group on CLL (iwCLL)-evaluable patients was 44% (90% confidence interval [CI], 23-67%), at 12 months, 72% of patients tested had no measurable residual disease (MRD). The estimated overall and progression-free survival at 48 months were 84% and 70%, respectively. Of 15 patients with undetectable MRD at 3 or 6 months, 13 remain in ongoing CR at the last follow-up. In patients with CLL not achieving a CR despite ≥6 months of ibrutinib, adding huCART-19 mediated a high rate of deep and durable remissions. ClinicalTrials.gov number, NCT02640209., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

CD19-redirected chimeric antigen receptor (CAR) T cells have shown remarkable activity against B-cell cancers. While second-generation CARs induce complete remission in >80% of patients with acute lymphoblastic leukemia, similar monotherapy induces long-term remissions in only 26% of patients with chronic lymphocytic leukemia (CLL). This disparity is attributed to cell-intrinsic effector defects in autologous CLL-derived T cells. However, the mechanisms by which leukemic cells impact CAR T-cell potency are poorly understood. Herein we describe an in vitro assay that recapitulates endogenous CLL-mediated T-cell defects in healthy donor CAR T cells. Contact with CLL cells insufficiently activates, but does not irreversibly impair, CAR T-cell function. This state is rescuable by strong antigenic stimulation or IL2, and is not driven by immune suppression. Rather, this activation defect is attributable to low levels of costimulatory molecules on CLL cells, and exogenous costimulation enhanced CAR T-cell activation. We next assessed the stimulatory phenotype of CLL cells derived from different niches within the same patient. Lymph node (LN)-derived CLL cells had a strong costimulatory phenotype and promoted better CAR T-cell degranulation and cytokine production than matched peripheral blood CLL cells. Finally, in vitro CD40L-activated CLL cells acquired a costimulatory phenotype similar to the LN-derived tumor and stimulated improved CAR T-cell proliferation, cytokine production, and cytotoxicity. Together, these data identify insufficient activation as a driver of poor CAR T-cell responses in CLL. The costimulatory phenotype of CLL cells drives differential CAR T-cell responses, and can be augmented by improving costimulatory signaling., Significance: CLL cells insufficiently activate CAR T cells, driven by low levels of costimulatory molecules on the tumor. LN-derived CLL cells are more costimulatory and mediate enhanced CAR T-cell killing. This costimulatory phenotype can be modeled via CD40 L activation, and the activated tumor promotes stronger CAR T-cell responses., Competing Interests: M.A. Collins reports grants from Parker Institute for Cancer Immunotherapy during the conduct of the study. A.P. Kater reports grants and other from Abbvie, AstraZeneca, BMS; and other from Janssen and LAVA outside the submitted work; in addition, A.P. Kater has a patent to Janssen pending and a patent to LAVA pending. C. Sun reports grants from Genmab outside the submitted work. A. Wiestner reports grants from Pharmacyclics, Acerta, Merck, Nurix, Verastem, and Genmab outside the submitted work. J.J. Melenhorst reports grants from Parker Institute for Cancer Immunotherapy during the conduct of the study; personal fees from IASO Biotherapeutics, Poseida Therapeutics, and Kite Pharma outside the submitted work; in addition, J.J. Melenhorst has a patent to Methods for improving the efficacy and expansion of immune cells issued, a patent to Biomarkers predictive of therapeutic responsiveness to chimeric antigen: issued, a patent to Methods of making chimeric antigen receptor - expressing cells issued, a patent to Methods for improving the efficacy and expansion of chimeric antigen receptor: issued, a patent to CAR T-cell therapies with enhanced efficacy issued, and a patent to Biomarkers predictive of cytokine release syndrome issued. No disclosures were reported by the other authors., (© 2022 The Authors; Published by the American Association for Cancer Research.)

Purpose: To study the biology and identify markers of severe cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) in children after chimeric antigen receptor T-cell (CAR T) treatment., Experimental Design: We used comprehensive proteomic profiling to measure over 1,400 serum proteins at multiple serial timepoints in a cohort of patients with B-cell acute lymphoblastic leukemia treated with the CD19-targeted CAR T CTL019 on two clinical trials., Results: We identified fms-like tyrosine kinase 3 (FLT3) and mast cell immunoglobulin-like receptor 1 (MILR1) as preinfusion predictive biomarkers of severe CRS. We demonstrated that CRS is an IFNγ-driven process with a protein signature overlapping with hemophagocytic lymphohistiocytosis (HLH). We identified IL18 as a potentially targetable cytokine associated with the development of ICANS., Conclusions: We identified preinfusion biomarkers that can be used to predict severe CRS with a sensitivity, specificity, and accuracy superior to the current gold standard of disease burden. We demonstrated the fundamental role of the IFNγ pathway in driving CRS, suggesting CRS and carHLH are overlapping rather than distinct phenomena, an observation with important treatment implications. We identified IL18 as a possible targetable cytokine in ICANS, providing rationale for IL18 blocking therapies to be translated into clinical trials in ICANS., (©2022 American Association for Cancer Research.)

Chimeric antigen receptor (CAR) T cell therapies targeting CD19 and CD22 have been successful for treating B cell cancers, but CAR T cells targeting non-B cell cancers remain unsuccessful. We propose that rather than being strictly a side effect of therapy, the large number of CAR interactions with normal B cells may be a key contributor to clinical CAR T cell responses.

A notable number of acute lymphoblastic leukemia (ALL) patients develop CD19-positive relapse within 1 year after receiving chimeric antigen receptor (CAR) T cell therapy. It remains unclear if the long-term response is associated with the characteristics of CAR T cells in infusion products, hindering the identification of biomarkers to predict therapeutic outcomes. Here, we present 101,326 single-cell transcriptomes and surface protein landscape from the infusion products of 12 ALL patients. We observed substantial heterogeneity in the antigen-specific activation states, among which a deficiency of T helper 2 function was associated with CD19-positive relapse compared with durable responders (remission, >54 months). Proteomic data revealed that the frequency of early memory T cells, rather than activation or coinhibitory signatures, could distinguish the relapse. These findings were corroborated by independent functional profiling of 49 patients, and an integrative model was developed to predict the response. Our data unveil the molecular mechanisms that may inform strategies to boost specific T cell function to maintain long-term remission.

The epidermal growth factor receptor variant III (EGFRvIII) has been investigated as a therapeutic target for chimeric antigen receptor (CAR) T cell therapy in glioblastoma. Earlier research demonstrated that phenotypic and genotypic characteristics in T cells and CAR T product predicted therapeutic success in hematologic malignancies, to date no determinants for clinical response in solid tumors have been identified. We analyzed apheresis and infusion products from the first-in-human trial of EGFRvIII-directed CAR T for recurrent glioblastoma (NCT02209376) by flow cytometry. Clinical response was quantified via engraftment in peripheral circulation and progression-free survival (PFS), as determined by the time from CAR T infusion to first radiographic evidence of progression. The CD4 + CAR T cell population in patient infusion products demonstrated PD1 expression which positively correlated with AUC engraftment and PFS. On immune checkpoint inhibitor analysis, CTLA-4, TIM3, and LAG3 did not exhibit significant associations with engraftment or PFS. The frequencies of PD1 + GZMB + and PD1 + HLA-DR + CAR T cells in the CD4 + infusion products were directly proportional to AUC and PFS. No significant associations were observed within the apheresis products. In summary, PD1 in CAR T infusion products predicted peripheral engraftment and PFS in recurrent glioblastoma., Competing Interests: DO’R receives laboratory support from Tmunity Therapeutics. DO’R and ZB are on patents relating to CAR T cell therapy for GBM. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tang, Tian, Yoder, Xu, Kulikovskaya, Gupta, Melenhorst, Lacey, O’Rourke and Binder.)

Objective: Heterogeneity has hampered sepsis trials, and sub-phenotyping may assist with enrichment strategies. However, biomarker-based strategies are difficult to operationalize. Four sub-phenotypes defined by distinct temperature trajectories in the first 72 h have been reported in adult sepsis. Given the distinct epidemiology of pediatric sepsis, the existence and relevance of temperature trajectory-defined sub-phenotypes in children is unknown. We aimed to classify septic children into de novo sub-phenotypes derived from temperature trajectories in the first 72 h, and compare cytokine, immune function, and immunometabolic markers across subgroups., Methods: This was a secondary analysis of a prospective cohort of 191 critically ill septic children recruited from a single academic pediatric intensive care unit. We performed group-based trajectory modeling using temperatures over the first 72 h of sepsis to identify latent profiles. We then used mixed effects regression to determine if temperature trajectory-defined sub-phenotypes were associated with cytokine levels, immune function, and mitochondrial respiration., Results: We identified four temperature trajectory-defined sub-phenotypes: hypothermic, normothermic, hyperthermic fast-resolvers, and hyperthermic slow-resolvers. Hypothermic patients were less often previously healthy and exhibited lower levels of pro- and anti-inflammatory cytokines and chemokines. Hospital mortality did not differ between hypothermic children (17%) and other sub-phenotypes (3-11%; P = 0.26)., Conclusions: Critically ill septic children can be categorized into temperature trajectory-defined sub-phenotypes that parallel adult sepsis. Hypothermic children exhibit a blunted cytokine and chemokine profile. Group-based trajectory modeling has utility for identifying subtypes of clinical syndromes by incorporating readily available longitudinal data, rather than relying on inputs from a single timepoint., Competing Interests: The authors report no conflicts of interest., (Copyright © 2022 by the Shock Society.)

Along with the clinical success of immuno-oncology drugs and cellular therapies, T-cell biology has attracted considerable attention in the immunology community. Long-term immunity, traditionally analyzed in the context of infection, is increasingly studied in cancer. Many signaling pathways, transcription factors, and metabolic regulators have been shown to participate in the formation of memory T cells. There is increasing evidence that the signal transducer and activator of transcription-3 (STAT3) signaling pathway is crucial for the formation of long-term T-cell immunity capable of efficient recall responses. In this review, we summarize what is currently known about STAT3 role in the context of memory T-cell formation and antitumor immunity.

The adoptive transfer of T lymphocytes reprogrammed to target tumour cells has demonstrated potential for treatment of various cancers 1-7 . However, little is known about the long-term potential and clonal stability of the infused cells. Here we studied long-lasting CD19-redirected chimeric antigen receptor (CAR) T cells in two patients with chronic lymphocytic leukaemia 1-4 who achieved a complete remission in 2010. CAR T cells remained detectable more than ten years after infusion, with sustained remission in both patients. Notably, a highly activated CD4 + population emerged in both patients, dominating the CAR T cell population at the later time points. This transition was reflected in the stabilization of the clonal make-up of CAR T cells with a repertoire dominated by a small number of clones. Single-cell profiling demonstrated that these long-persisting CD4 + CAR T cells exhibited cytotoxic characteristics along with ongoing functional activation and proliferation. In addition, longitudinal profiling revealed a population of gamma delta CAR T cells that prominently expanded in one patient concomitant with CD8 + CAR T cells during the initial response phase. Our identification and characterization of these unexpected CAR T cell populations provide novel insight into the CAR T cell characteristics associated with anti-cancer response and long-term remission in leukaemia., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Purpose: B-cell maturation antigen (BCMA)-targeted chimeric antigen receptor (CAR) T cells (CART-BCMA) are a promising treatment for relapsed/refractory multiple myeloma (r/rMM). We evaluated the safety and feasibility of bridging radiation (RT) in subjects treated on a phase I trial of CART-BCMA., Experimental Design: Twenty-five r/rMM subjects were treated in three cohorts with two doses of CART-BCMA cells ± cyclophosphamide. We retrospectively analyzed toxicity, response, and CART manufacturing data based on RT receipt., Results: Thirteen subjects received no RT <1 year before CART infusion (Group A). Eight subjects received RT <1 year before CART infusion (Group B) with median time from RT to apheresis of 114 days (range 40-301). Four subjects received bridging-RT (Group C) with a median dose of 22 Gy and time from RT to infusion of 25 days (range 18-35). Group C had qualitatively lower rates of grade 4 (G4) hematologic toxicities (25%) versus A (61.5%) and B (62.5%). G3-4 neurotoxicity occurred in 7.7%, 25%, and 25% in Group A, B, and C, respectively. G3-4 cytokine release syndrome was observed in 38.5%, 25%, and 25% in Group A, B, and C, respectively. Partial response or better was observed in 54%, 38%, and 50% of Group A, B, and C, respectively. RT administered <1 year ( P = 0.002) and <100 days ( P = 0.069) before apheresis was associated with lower in vitro proliferation during manufacturing; however, in vivo CART-BCMA expansion appeared similar across groups., Conclusions: Bridging-RT appeared safe and feasible with CART-BCMA therapy in our r/rMM patients, though larger future studies are needed to draw definitive conclusions., (©2021 American Association for Cancer Research.)

The test for the cancer stem cell (CSC) hypothesis is to find a target expressed on all, and only CSCs in a patient tumor, then eliminate all cells with that target that eliminates the cancer. That test has not yet been achieved, but CSC diagnostics and targets found on CSCs and some other cells have resulted in a few clinically relevant therapies. However, it has become apparent that eliminating the subset of tumor cells characterized by self-renewal properties is essential for long-term tumor control. CSCs are able to regenerate and initiate tumor growth, recapitulating the heterogeneity present in the tumor before treatment. As great progress has been made in identifying and elucidating the biology of CSCs as well as their interactions with the tumor microenvironment, the time seems ripe for novel therapeutic strategies that target CSCs to find clinical applicability. On May 19-21, 2021, researchers in cancer stem cells met virtually for the Keystone eSymposium "Cancer Stem Cells: Advances in Biology and Clinical Translation" to discuss recent advances in the understanding of CSCs as well as clinical efforts to target these populations., (© 2021 New York Academy of Sciences.)

The current standard of care in hematological malignancies has brought considerable clinical benefits to patients. However, important bottlenecks still limit optimal achievements following a current medical practice. The genetic complexity of the diseases and the heterogeneity of tumor clones cause difficulty in ensuring long-term efficacy of conventional treatments for most hematological disorders. Consequently, new treatment strategies are necessary to improve clinical outcomes. Chimeric antigen receptor T-cell (CAR T) immunotherapy opens a new path for targeted therapy of hematological malignancies. In this review, through a representative case study, we summarize the current experience of CAR T-cell therapy, the management of common side effects, the causative mechanisms of therapy resistance, and new strategies to improve the efficacy of CAR T-cell therapy., (© 2021. Higher Education Press.)

As a rapidly emerging treatment in the oncology field, adoptive transfer of autologous, genetically modified chimeric antigen receptor (CAR) T cells has shown striking efficacy and is curative in certain relapsed/refractory patients with hematologic malignancy. This treatment modality of using a "living drug" offers many tantalizing and novel therapeutic strategies for cancer patients whose remaining treatment options may have otherwise been limited. Despite the early success of CAR T cells in hematologic malignancies, many barriers remain for widespread adoption. General barriers include cellular manufacturing limitations, baseline quality of the T cells, adverse events post-infusion such as cytokine release syndrome (CRS) and neurotoxicity, and host rejection of non-human CARs. Additionally, each hematologic disease presents unique mechanisms of relapse which have to be addressed in future clinical trials if we are to augment the efficacy of CAR T treatment. In this review, we will describe current barriers to hindering efficacy of CAR T-cell treatment for hematologic malignancies in a disease-specific manner and review recent innovations aimed at enhancing the potency and applicability of CAR T cells, with the overall goal of building a framework to begin incorporating this form of therapy into the standard medical management of blood cancers., (© 2020. Japanese Society of Hematology.)

The fifth annual Blood and Marrow Transplant Clinical Trials Network Myeloma Intergroup Workshop on Immune Profiling and Minimal Residual Disease Testing in Multiple Myeloma was conducted as one of the American Society of Hematology Annual Meeting Scientific Workshops on Thursday December 3, 2020. This workshop focused on four main topics: (1) integrating minimal residual disease into clinical trial design and practice; (2) the molecular and immunobiology of disease evolution and progression in myeloma; (3) adaptation of next-generation sequencing, next-generation flow cytometry, and cytometry by time of flight techniques; and (4) chimeric antigen receptor T-cell and other cellular therapies for myeloma. In this report, we provide a summary of the workshop presentations and discuss future directions in the field., (Copyright © 2021 The American Society for Transplantation and Cellular Therapy. All rights reserved.)

Chimeric antigen receptor (CAR) T cells have induced remarkable antitumor responses in B cell malignancies. Some patients do not respond because of T cell deficiencies that hamper the expansion, persistence, and effector function of these cells. We used longitudinal immune profiling to identify phenotypic and pharmacodynamic changes in CD19-directed CAR T cells in patients with chronic lymphocytic leukemia (CLL). CAR expression maintenance was also investigated because this can affect response durability. CAR T cell failure was accompanied by preexisting T cell-intrinsic defects or dysfunction acquired after infusion. In a small subset of patients, CAR silencing was observed coincident with leukemia relapse. Using a small molecule inhibitor, we demonstrated that the bromodomain and extra-terminal (BET) family of chromatin adapters plays a role in downregulating CAR expression. BET protein blockade also ameliorated CAR T cell exhaustion as manifested by inhibitory receptor reduction, enhanced metabolic fitness, increased proliferative capacity, and enriched transcriptomic signatures of T cell reinvigoration. BET inhibition decreased levels of the TET2 methylcytosine dioxygenase, and forced expression of the TET2 catalytic domain eliminated the potency-enhancing effects of BET protein targeting in CAR T cells, providing a mechanism linking BET proteins and T cell dysfunction. Thus, modulating BET epigenetic readers may improve the efficacy of cell-based immunotherapies.

Chimeric antigen receptor (CAR) T-cell therapies have evolved from a research tool to a paradigm-shifting therapy with impressive responses in B cell malignancies. This review summarizes the current state of the CAR T-cell field, focusing on CD19- and B cell maturation antigen-directed CAR T-cells, the most developed of the CAR T-cell therapies. We discuss the many challenges to CAR-T therapeutic success and innovations in CAR design and T-cell engineering aimed at extending this therapeutic platform beyond hematologic malignancies.

Background: Autologous T cells engineered to express a chimeric antigen receptor (CAR) specific for CD19 molecule have transformed the therapeutic landscape in patients with highly refractory leukemia and lymphoma, and the use of donor-generated allogeneic CAR T is paving the way for further breakthroughs in the treatment of cancer. However, it remains unknown how the intrinsic heterogeneities of these engineered cells mediate therapeutic efficacy and whether allogeneic products match the effectiveness of autologous therapies., Methods: Using single-cell mRNA sequencing in conjunction with CITE-seq, we performed multiomics characterization of CAR T cells generated from healthy donor and patients with acute lymphoblastic leukemia. CAR T cells used in this study were manufactured at the University of Pennsylvania through lentiviral transduction with a CD19-4-1BB-CD3ζ construct. Besides the baseline condition, we engineered NIH-3T3 cells with human CD19 or mesothelin expression to conduct ex vivo antigen-specific or non-antigen stimulation of CAR T cells through 6-hour coculture at a 1:1 ratio., Results: We delineated the global cellular and molecular CAR T landscape and identified that transcriptional CAR tonic signaling was regulated by a mixture of early activation, exhaustion signatures, and cytotoxic activities. On CD19 stimulation, we illuminated the disparities of CAR T cells derived from different origins and found that donor CAR T had more pronounced activation level in correlation with the upregulation of major histocompatibility complex class II genes compared with patient CAR T cells. This finding was independently validated in additional datasets from literature. Furthermore, GM-CSF( CSF2 ) expression was found to be associated with functional gene productions, but it induced little impact on the CAR T activation., Conclusions: Through integrated multiomics profiling and unbiased canonical pathway analyses, our results unveil heterogeneities in the transcriptional, phenotypic, functional, and metabolic profiles of donor and patient CAR T cells, providing mechanistic basis for ameliorating clinical outcomes and developing next-generation 'off- the-shelf' allogeneic products., Competing Interests: Competing interests: RF is a cofounder of IsoPlexis, Singleron Biotechnologies and AtlasXomics and a member of their scientific advisory boards with financial interests, which could affect or have the perception of affecting the author’s objectivity. The interests of RF were reviewed and managed by Yale University Provost’s Office in accordance with the University’s conflict of interest policies., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Competing Interests: Dr. Melenhorst conflicts of interest disclosure: JJM receives research funding from IASO Biotherapeutics and Kite Pharma, a Gilead company. He furthermore served as speaker for Novartis and Johnson & Johnson and consults for Simcere of America, Shanghai Unicar Therapy, Janssen Research & Development, LLC, Poseida, Allogene, and IASO Biotherapeutics, and is on the Medical and Scientific Advisory Board of IASO Biotherapeutics. Lastly, JJM holds patents related to CAR T cell manufacturing and biomarkers.

Abstract: Chimeric antigen receptor T therapy has heralded a new era in the treatment of acute lymphoblastic leukemia (ALL) and other hematologic malignancies. In this autologous immunotherapy, patient-derived T cells are genetically engineered and then infused back to kill the leukemia cells. The observed response rates in ALL are a testament to the success of this therapy. However, there have been instances where the patients either did not respond or relapsed after initial response. Emergence of resistance due to antigen loss and T-cell exhaustion has been observed. This poses a challenge in making this therapy successful for every ALL patient and warrants deeper understanding of emergence of resistance and potential approaches to overcome them. Here we discuss current perspectives and advances in this area., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article. S.A.G. reports research support from Novartis, Servier, CRISPR/Vertex, and Kite/Gillead, and SAB/SSC membership and/or consulting from Novartis, CRISPR/Vertex, Allogene, CBMG, Adaptimmune, TCR2, Juno, Jazz, Cellectis, Roche, GSK, Cabaletta, and Janssen/J&J. J.J.M. is supported by the NIH grant R01 CA241762., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Purpose of Review: High-dimensional flow cytometry experiments have become a method of choice for high-throughput integration and characterization of cell populations. Here, we present a summary of state-of-the-art R-based pipelines used for differential analyses of cytometry data, largely based on chimeric antigen receptor (CAR) T cell therapies. These pipelines are based on publicly available R libraries, put together in a systematic and functional fashion, therefore free of cost., Recent Findings: In recent years, existing tools tailored to analyze complex high-dimensional data such as single-cell RNA sequencing (scRNAseq) have been successfully ported to cytometry studies due to the similar nature of flow cytometry and scRNAseq platforms. Existing environments like Cytobank (Kotecha et al., 2010), FlowJo (FlowJo™ Software) and FCS Express (https://denovosoftware.com) already offer a variety of these ported tools, but they either come at a premium or are fairly complicated to manage by an inexperienced user. To mitigate these limitations, experienced cytometrists and bioinformaticians usually incorporate these functions into an RShiny (https://shiny.rstudio.com) application that ultimately offers a user-friendly, intuitive environment that can be used to analyze flow cytometry data. Computational tools and Shiny-based tools are the perfect answer to the ever-growing dimensionality and complexity of flow cytometry data, by offering a dynamic, yet user-friendly exploratory space, tailored to bridge the space between the lab experimental world and the computational, machine learning space.

Objectives: Acute respiratory distress syndrome occurring in the setting of direct versus indirect lung injury may reflect different pathobiologies amenable to different treatment strategies. We sought to test whether a panel of plasma biomarkers differed between children with sepsis-associated direct versus indirect acute respiratory distress syndrome. We hypothesized that a biomarker profile indicative of endothelial activation would be associated with indirect acute respiratory distress syndrome., Design: Observational cohort., Setting: Academic PICU., Subjects: Patients less than 18 years old with sepsis-associated direct (pneumonia, n = 52) or indirect (extrapulmonary sepsis, n = 46) acute respiratory distress syndrome., Interventions: None., Measurements and Main Results: Of 58 biomarkers examined, 33 differed by acute respiratory distress syndrome subtype. We used classification and regression tree methodology to examine associations between clinical and biochemical markers and acute respiratory distress syndrome subtype. The classification and regression tree model using only clinical variables (age, sex, race, oncologic comorbidity, and Pediatric Risk of Mortality-III score) performed worse than the classification and regression tree model using five clinical variables and 58 biomarkers. The best classification and regression tree model used only four endothelial biomarkers, including elevated angiopoietin-2/angiopoietin-1 ratio, vascular cell-adhesion molecule, and von Willebrand factor, to identify indirect acute respiratory distress syndrome. Test characteristics were 89% (80-97%) sensitivity, 80% (69-92%) specificity, positive predictive value 84% (74-93%), and negative predictive value 86% (76-96%)., Conclusions: Indirect lung injury in children with acute respiratory distress syndrome is characterized by a biomarker profile indicative of endothelial activation, excess inflammation, and worse outcomes. A model using four biomarkers has the potential to be useful for more precisely identifying patients with acute respiratory distress syndrome whose pathobiology may respond to endothelial-targeted therapies in future trials., Competing Interests: The authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2020 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of the Society of Critical Care Medicine.)

B cells infiltrate pancreatic ductal adenocarcinoma (PDAC) and in preclinical cancer models, can suppress T cell immunosurveillance in cancer. Here, we conducted a pilot study to assess the safety and feasibility of administering lentiviral-transduced chimeric antigen receptor (CAR)-modified autologous T cells redirected against mesothelin to target tumor cells along with CART cells redirected against CD19 to deplete B cells. Both CARs contained 4-1BB and CD3ζ signaling domains. Three patients with chemotherapy-refractory PDAC received 1.5 g/m 2 cyclophosphamide prior to separate infusions of lentiviral-transduced T cells engineered to express chimeric anti-mesothelin immunoreceptor SS1 (CART-Meso, 3 × 10 7 /m 2 ) and chimeric anti-CD19 immunoreceptor (CART-19, 3 × 10 7 /m 2 ). Treatment was well tolerated without dose-limiting toxicities. Best response was stable disease (1 of 3 patients). CART-19 (compared to CART-Meso) cells showed the greatest expansion in the blood, although persistence was transient. B cells were successfully depleted in all subjects, became undetectable by 7-10 days post-infusion, and remained undetectable for at least 28 days. Together, concomitant delivery of CART-Meso and CART-19 cells in patients with PDAC is safe. CART-19 cells deplete normal B cells but at the dose tested in these 3 subjects did not improve CART-Meso cell persistence., (Copyright © 2020 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Chimeric antigen receptor (CAR) T cell therapy has come of age, offering a potentially curative option for patients who are refractory to standard anti-cancer treatments. The success of CAR T cell therapy in the setting of acute lymphoblastic leukemia and specific types of B cell lymphoma led to rapid regulatory approvals of CD19-directed CAR T cells, first in the United States and subsequently across the globe. Despite these major milestones in the field of immuno-oncology, growing experience with CAR T cells has also highlighted the major limitations of this strategy, namely challenges associated with manufacturing a bespoke patient-specific product, intrinsic immune cell defects leading to poor CAR T cell function as well as persistence, and/or tumor cell resistance resulting from loss or modulation of the targeted antigen. In addition, both on- and off-tumor immunotoxicities and the financial burden inherent in conventional cellular biomanufacturing often hamper the success of CAR T cell-based treatment approaches. Herein, we provide an overview of the opportunities and challenges related to the first form of gene transfer therapy to gain commercial approval in the United States. Ongoing advances in the areas of genetic engineering, precision genome editing, toxicity mitigation methods and cell manufacturing will improve the efficacy and safety of CAR T cells for hematologic malignancies and expand the use of this novel class of therapeutics to reach solid tumors.

Chimeric antigen receptor (CAR) T-cells directed against CD19 have drastically altered outcomes for children with relapsed and refractory acute lymphoblastic leukemia (r/r ALL). Pediatric patients with r/r ALL treated with CAR-T are at increased risk of both cytokine release syndrome (CRS) and sepsis. We sought to investigate the biologic differences between CRS and sepsis and to develop predictive models which could accurately differentiate CRS from sepsis at the time of critical illness. We identified 23 different cytokines that were significantly different between patients with sepsis and CRS. Using elastic net prediction modeling and tree classification, we identified cytokines that were able to classify subjects as having CRS or sepsis accurately. A markedly elevated interferon γ (IFNγ) or a mildly elevated IFNγ in combination with a low IL1β were associated with CRS. A normal to mildly elevated IFNγ in combination with an elevated IL1β was associated with sepsis. This combination of IFNγ and IL1β was able to categorize subjects as having CRS or sepsis with 97% accuracy. As CAR-T therapies become more common, these data provide important novel information to better manage potential associated toxicities., (© 2020 by The American Society of Hematology.)