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In this issue, report a mechanism by which host cells avert an autoimmune response to self-nucleic acids. They show that the nuclease Trex1 prevents the accumulation of DNA derived from endogenous retroelements that, if left unchecked, trigger elevated production of type I interferons leading to autoimmunity. [Copyright &y& Elsevier]

CD19-directed immunotherapies are clinically effective for treating B cell malignancies but also cause a high incidence of neurotoxicity. A subset of patients treated with chimeric antigen receptor (CAR) T cells or bispecific T cell engager (BiTE) antibodies display severe neurotoxicity, including fatal cerebral edema associated with T cell infiltration into the brain. Here, we report that mural cells, which surround the endothelium and are critical for blood-brain-barrier integrity, express CD19. We identify CD19 expression in brain mural cells using single-cell RNA sequencing data and confirm perivascular staining at the protein level. CD19 expression in the brain begins early in development alongside the emergence of mural cell lineages and persists throughout adulthood across brain regions. Mouse mural cells demonstrate lower levels of Cd19 expression, suggesting limitations in preclinical animal models of neurotoxicity. These data suggest an on-target mechanism for neurotoxicity in CD19-directed therapies and highlight the utility of human single-cell atlases for designing immunotherapies. • Single-cell RNA-seq reveals CD19 expression in human brain mural cells • Mural cells line blood vessels and maintain blood-brain barrier integrity • Brain mural cell CD19 expression is present across brain regions and human age • Targeting CD19+ mural cells may contribute to neurotoxicity of CAR-T therapy Single-cell RNA sequencing analysis shows that CD19, primarily considered as a B cell-specific surface antigen, is expressed in human brain mural cells that are critical for blood-brain-barrier integrity, suggesting that this cell population may contribute to the neurotoxicity of CD19-directed immunotherapy including CAR-T. [ABSTRACT FROM AUTHOR]

Pre-existing anti-human leukocyte antigen (HLA) allo-antibodies constitute a major barrier to transplantation. Current desensitization approaches fail due to ineffective depletion of allo-specific memory B cells (Bmems) and long-lived plasma cells (LLPCs). We evaluate the efficacy of chimeric antigen receptor (CAR) T cells targeting CD19 and B cell maturation antigen (BCMA) to eliminate allo-antibodies in a skin pre-sensitized murine model of islet allo-transplantation. We find that treatment of allo-sensitized hosts with CAR T cells targeting Bmems and LLPCs eliminates donor-specific allo-antibodies (DSAs) and mitigates hyperacute rejection of subsequent islet allografts. We then assess the clinical efficacy of the CAR T therapy for desensitization in patients with multiple myeloma (MM) with pre-existing HLA allo-antibodies who were treated with the combination of CART-BCMA and CART-19 (ClinicalTrials.gov: NCT03549442) and observe clinically meaningful allo-antibody reduction. These findings provide logical rationale for clinical evaluation of CAR T-based immunotherapy in highly sensitized candidates to promote successful transplantation., Competing Interests: Declaration of interests J.A.F. is an inventor on patents in the field of T cell therapy for cancer, from which he has received royalties. J.A.F. is also a member of the scientific advisory boards of Cartography Bio. and Shennon Biotechnologies, Inc. M.C.M. is an inventor on patents pending or granted related to CAR T cell technology that are assigned to the University of Pennsylvania and licensed to Novartis Ag, from which he receives royalties; these include patents on the CAR T cell therapies described in this study (PCT/US2011/064191, PCT/US2018/061239, PCT/US2018/063255). M.C.M. is also a founder, stockholder, and co-chair of the scientific advisory boards for Cabaletta Bio and Verismo Therapeutics. A.L.G. has received grants from Novartis, grants from National Institutes of Health, and grants from the Leukemia & Lymphoma Society while this study was conducted, grants and personal fees from Janssen, personal fees from GlaxoSmithKline, personal fees from Legend Biotech, grants from CRISPR Therapeutics, grants from Tmunity Therapeutics, personal fees from Amgen, grants from the Leukemia & Lymphoma Society, and grants from National Institutes of Health outside the submitted work; in addition, A.L.G. has a patent for US15/757,123 pending and licensed to Novartis, a patent for US16/764,459 pending, and a patent for US16/768,260 pending and stock ownership in Cabaletta Bio. V.G.B. is an inventor on patents pending or granted related to CAR T cell technology that are assigned to the University of Pennsylvania and licensed to Cabaletta Bio and Tmunity, from which he receives royalties., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Several studies have shown the influence of commensal microbes on T cell function, specifically in the setting of checkpoint immunotherapy for cancer. In this study, we investigated how vancomycin-induced gut microbiota dysbiosis affects chimeric antigen receptor (CAR) T immunotherapy using multiple preclinical models as well as clinical correlates. In two murine tumor models, hematopoietic CD19 + -A20 lymphoma and CD19 + -B16 melanoma, mice receiving vancomycin in combination with CD19-directed CAR T cell (CART-19) therapy displayed increased tumor control and tumor-associated antigens (TAAs) cross-presentation compared with CART-19 alone. Fecal microbiota transplant from human healthy donors to pre-conditioned mice recapitulated the results obtained in naive gut microbiota mice. Last, B cell acute lymphoblastic leukemia patients treated with CART-19 and exposed to oral vancomycin showed higher CART-19 peak expansion compared with unexposed patients. These results substantiate the role of the gut microbiota on CAR T cell therapy and suggest that modulation of the gut microbiota using vancomycin may improve outcomes after CAR T cell therapy across tumor types., Competing Interests: Declaration of interests M.R.: BMS, BAYER, GSK, consultancy; Novartis, patents and royalties; AbClon, consultancy, research funding; Tmunity, patents and royalties; viTToria Biotherapeutics, research funding. N.F.: Sana Biotechnology, consultancy; Novartis, research funding; Kite Pharma, consultancy; Syndax Pharmaceuticals, consultancy. C.H.J.: Tmunity, DeCART, BluesphereBio, Carisma, Cellares, Celldex, Cabaletta, Poseida, Verismo, and Ziopharm, current equity holder in publicly traded company; AC Immune, DeCART, BluesphereBio, Carisma, Cellares, Celldex, Cabaletta, Poseida, Verismo, Ziopharm, consultancy; Novartis, patents and royalties. D.L.P.: American Society for Transplantation and Cellular Therapy, honoraria; ASH and DeCart, membership on the Board of Directors or advisory committee; Genentech, current employment, current equity holder in publicly traded company; Incyte and Janssen, Kite/Gilead, and National Marrow Donor Program, membership on an entity’s board of directors or advisory committee; Novartis, membership on an entity’s board of directors or advisory committee, patents and royalties, and research funding; Unity, patents and royalties; and Wiley and Sons Publishing, honoraria. S.J.S.: TG Therapeutics, research funding; Incyte, research funding; Adaptive Biotechnologies, research funding; Pharmacyclics, research funding; Merck, research funding; Genentech/Roche, consultancy, research funding; Tessa Therapeutics, consultancy; Loxo Oncology, consultancy; Juno Therapeutics, consultancy, research funding; BeiGene, consultancy; Alimera Sciences, consultancy; Acerta Pharma/AstraZeneca, consultancy; Novartis, consultancy, honoraria, patents and royalties, research funding; AbbVie, consultancy, research funding; Nordic Nanovector, consultancy; Celgene, consultancy, honoraria, research funding., (Copyright © 2023. Published by Elsevier Inc.)

It is technically challenging to generate large doses of regulatory T cells (Tregs) engineered to express a chimeric antigen receptor (CAR) in non-human primates (NHP). Here, we have optimized the manufacturing of CAR Tregs by stringent sorting of Tregs, stimulation by artificial antigen-presenting cells, transduction by simian tropic lentiviral vectors, and antigen-specific expansion. The result of this method is highly suppressive CAR Tregs for use in a pre-clinical, large animal model of transplant tolerance. For complete details on the use and execution of this protocol, please refer to Ellis et al. (2022)., Competing Interests: J.L.R. and G.I.E. have submitted patents related to use of the Bw6 CAR and the aAPCs described in this manuscript. J.L.R. is a founder and equity holder of Tmunity Therapeutics., (© 2022 The Author(s).)

Adoptive transfer of chimeric antigen receptor regulatory T cells (CAR Tregs) is a promising way to prevent allograft loss without the morbidity associated with current therapies. Non-human primates (NHPs) are a clinically relevant model to develop transplant regimens, but manufacturing and engraftment of NHP CAR Tregs have not been demonstrated yet. Here, we describe a culture system that massively expands CAR Tregs specific for the Bw6 alloantigen. In vitro, these Tregs suppress in an antigen-specific manner without pro-inflammatory cytokine secretion or cytotoxicity. In vivo, Bw6-specific CAR Tregs preferentially traffic to and persist in bone marrow for at least 1 month. Following transplant of allogeneic Bw6 + islets and autologous CAR Tregs into the bone marrow of diabetic recipients, CAR Tregs traffic to the site of islet transplantation and maintain a phenotype of suppressive Tregs. Our results establish a framework for the optimization of CAR Treg therapy in NHP disease models., Competing Interests: Declaration of interests J.L.R. and G.I.E. have submitted patents related to use of the HLA-A2 and Bw6 CARs described in this manuscript. J.L.R. is an equity holder of and receives sponsored research funding from Tmunity Therapeutics. The remaining authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Vaccine-mediated immunity often relies on the generation of protective antibodies and memory B cells, which commonly stem from germinal center (GC) reactions. An in-depth comparison of the GC responses elicited by SARS-CoV-2 mRNA vaccines in healthy and immunocompromised individuals has not yet been performed due to the challenge of directly probing human lymph nodes. Herein, through a fine-needle aspiration-based approach, we profiled the immune responses to SARS-CoV-2 mRNA vaccines in lymph nodes of healthy individuals and kidney transplant recipients (KTXs). We found that, unlike healthy subjects, KTXs presented deeply blunted SARS-CoV-2-specific GC B cell responses coupled with severely hindered T follicular helper cell, SARS-CoV-2 receptor binding domain-specific memory B cell, and neutralizing antibody responses. KTXs also displayed reduced SARS-CoV-2-specific CD4 and CD8 T cell frequencies. Broadly, these data indicate impaired GC-derived immunity in immunocompromised individuals and suggest a GC origin for certain humoral and memory B cell responses following mRNA vaccination., Competing Interests: Declaration of interests E.J.W. is consulting or is an advisor for Merck, Elstar, Janssen, Related Sciences, Synthekine, and Surface Oncology. E.J.W. is a founder of Surface Oncology and Arsenal Biosciences. E.J.W. is an inventor on a patent (US Patent number 10,370,446) submitted by Emory University that covers the use of PD-1 blockade to treat infections and cancer. S.E.H. has received consultancy fee from Sanofi Pasteur, Lumen, Novavax, and Merck for work unrelated to this report. A.S. is a consultant for Gritstone, Flow Pharma, Arcturus, Immunoscape, CellCarta, Oxford Immunotech, and Avalia., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Prior to adoptive transfer, CAR T cells are activated, lentivirally infected with CAR transgenes, and expanded over 9 to 11 days. An unintended consequence of this process is the progressive differentiation of CAR T cells over time in culture. Differentiated T cells engraft poorly, which limits their ability to persist and provide sustained tumor control in hematologic as well as solid tumors. Solid tumors include other barriers to CAR T cell therapies, including immune and metabolic checkpoints that suppress effector function and durability. Sialic acids are ubiquitous surface molecules with known immune checkpoint functions. The enzyme C. perfringens neuraminidase (CpNA) removes sialic acid residues from target cells, with good activity at physiologic conditions. In combination with galactose oxidase (GO), NA has been found to stimulate T cell mitogenesis and cytotoxicity in vitro. Here we determine whether CpNA alone and in combination with GO promotes CAR T cell antitumor efficacy. We show that CpNA restrains CAR T cell differentiation during ex vivo culture, giving rise to progeny with enhanced therapeutic potential. CAR T cells expressing CpNA have superior effector function and cytotoxicity in vitro. In a Nalm-6 xenograft model of leukemia, CAR T cells expressing CpNA show enhanced antitumor efficacy. Arming CAR T cells with CpNA also enhanced tumor control in xenograft models of glioblastoma as well as a syngeneic model of melanoma. Given our findings, we hypothesize that charge repulsion via surface glycans is a regulatory parameter influencing differentiation. As T cells engage target cells within tumors and undergo constitutive activation through their CARs, critical thresholds of negative charge may impede cell-cell interactions underlying synapse formation and cytolysis. Removing the dense pool of negative cell-surface charge with CpNA is an effective approach to limit CAR T cell differentiation and enhance overall persistence and efficacy., Competing Interests: Declaration of interests M.C.M. is an inventor on patent applications related to CAR technology and has received licensing royalties from Novartis corporation; S.G. and M.C.M. are inventors on patent applications related to methods of manufacturing CAR T cells. D.M.O., Z.B., L.J., R.T., and V.B. are inventors on patents related to CAR T cells that have been filed by the University of Pennsylvania. The other authors declare no financial or other conflicts of interest., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Metastatic medullary thyroid cancer (MTC) is a rare but often aggressive thyroid malignancy with a 5-year survival rate of less than 40% and few effective therapeutic options. Adoptive T cell immunotherapy using chimeric antigen receptor (CAR)-modified T cells (CAR Ts) is showing encouraging results in the treatment of cancer, but development is challenged by the availability of suitable target antigens. We identified glial-derived neurotrophic factor (GDNF) family receptor alpha 4 (GFRα4) as a putative antigen target for CAR-based therapy of MTC. We show that GFRα4 is highly expressed in MTC, in parafollicular cells within the thyroid from which MTC originates, and in normal thymus. We isolated two single-chain variable fragments (scFvs) targeting GFRα4 isoforms a and b by antibody phage display. CARs bearing the CD3ζ and the CD137 costimulatory domains were constructed using these GFRα4-specific scFvs. GFRα4-specific CAR Ts trigger antigen-dependent cytotoxicity and cytokine production in vitro , and they are able to eliminate tumors derived from the MTC TT cell line in an immunodeficient mouse xenograft model of MTC. These data demonstrate the feasibility of targeting GFRα4 by CAR T and support this antigen as a promising target for adoptive T cell immunotherapy and other antibody-based therapies for MTC., Competing Interests: D.L.S., V.G.B., C.R., R.S.G., and M.C.M. have filed a patent application on technology presented in this manuscript., (© 2021.)

Adoptive cellular therapy using T cells with tumor specificity derived from either natural T cell receptors (TCRs) or an artificial chimeric antigen receptor (CAR) has reached late phase clinical testing, with two CAR T cell therapies achieving regulatory approval within the United States in 2017. The effective use of these therapies depends upon an understanding of their pharmacology, which is quite divergent from traditional small molecule or biologic drugs. We review the different types of T cell therapy under clinical development, the factors affecting cellular kinetics following infusion, and the relationship between these cellular kinetics and anti-cancer activity. We also discuss the toxicity associated with T cell therapies, with an emphasis on cytokine release syndrome and neurotoxicity, and the gaps in knowledge regarding these frequent and unique adverse effects.