In the clinic, chimeric antigen receptor-modified T (CAR T) cell therapy is frequently associated with life-threatening cytokine-release syndrome (CRS) and neurotoxicity. Understanding the nature of these pathologies and developing treatments for them are hampered by the lack of appropriate animal models. Herein, we describe a mouse model recapitulating key features of CRS and neurotoxicity. In humanized mice with high leukemia burden, CAR T cell-mediated clearance of cancer triggered high fever and elevated IL-6 levels, which are hallmarks of CRS. Human monocytes were the major source of IL-1 and IL-6 during CRS. Accordingly, the syndrome was prevented by monocyte depletion or by blocking IL-6 receptor with tocilizumab. Nonetheless, tocilizumab failed to protect mice from delayed lethal neurotoxicity, characterized by meningeal inflammation. Instead, the IL-1 receptor antagonist anakinra abolished both CRS and neurotoxicity, resulting in substantially extended leukemia-free survival. These findings offer a therapeutic strategy to tackle neurotoxicity and open new avenues to safer CAR T cell therapies.

In allogeneic bone marrow transplantation (allo-BMT), donor lymphocytes play a central therapeutic role in both graft-versus-leukemia (GvL) and immune reconstitution. However, their use is limited by the risk of severe graft-versus-host disease (GvHD). Eight patients who relapsed or developed Epstein-Barr virus-induced lymphoma after T cell-depleted BMT were then treated with donor lymphocytes transduced with the herpes simplex virus thymidine kinase (HSV-TK) suicide gene. The transduced lymphocytes survived for up to 12 months, resulting in antitumor activity in five patients. Three patients developed GvHD, which could be effectively controlled by ganciclovir-induced elimination of the transduced cells. These data show that genetic manipulation of donor lymphocytes may increase the efficacy and safety of allo-BMT and expand its application to a larger number of patients.

Targeted genome editing by artificial nucleases has brought the goal of site-specific transgene integration and gene correction within the reach of gene therapy. However, its application to long-term repopulating Hematopoietic Stem Cells (HSCs) has remained elusive. Here we show that poor permissiveness to gene transfer and limited proficiency of the homology directed DNA repair pathway constrain gene targeting in human HSCs. By tailoring delivery platforms and culture conditions we overcame these barriers and provide stringent evidence of targeted integration in human HSCs by long-term multilineage repopulation of transplanted mice. We demonstrate the therapeutic potential of our strategy by targeting a corrective cDNA into the IL2RG gene of HSCs from healthy donors and a subject with X-linked Severe Combined Immunodeficiency (SCID-X1). Gene edited HSCs sustained normal hematopoiesis and gave rise to functional lymphoid cells that possess a selective growth advantage over those carrying disruptive IL2RG mutations. These results open new avenues for treating SCID-X1 and other diseases.

Key Points• Identification of the biologic requirements for memory stem T cell (T SCM ) generation and expansion from naive precursors ex vivo.• Differentiation, expansion, and genetic manipulation of human T SCM for cancer adoptive cellular therapy. Long-living memory stem T cells (T SCM) IntroductionAdaptive immunity is a potent and flexible system able to combat microbes and cancer cells. 1,2 In the presence of infections or cancer, antigen-specific lymphocytes expand and differentiate into effectors devoted to rapidly clearing the pathogen and memory cells able to persist long-term to patrol the entire organism for recurrence and minimal residual disease. 3,4 However, the mechanism and hierarchical differentiation path underlying the generation of memory precursors and terminal effector cells remain to be fully elucidated. 5 This process has been proposed to involve a self-renewing, stem cell-like memory T-cell subset capable of differentiating into effectors on antigen reencounter. 6,7 This T-cell subset, referred to as memory stem T cells (T SCM ), and initially described in mice, 8,9 begins to be unveiled in humans. 10 T SCM potential biodistribution and long-term persistence represent appealing features to overcome the current limitations of cancer adoptive immune-gene therapy. [11][12][13] At present, clinical-grade protocols able to obtain or preserve T SCM functional and phenotypic characteristics remain to be defined. We previously showed that costimulation of unselected T cells and culture with ␥-chain cytokines allow the preferential generation of gene-modified T cells with a functional central memory (T CM ) phenotype, superior to effector/effector memory (T EM ) counterparts for expansion potential and antitumor activity. 14,15 Compared with T CM and T EM lymphocytes, naive T cells (T N ) are endowed with the highest developmental plasticity and are unique in the ability to generate daughter cells with potential to enter the entire spectrum of immunologic memory, including T SCM . We thus hypothesized that, starting from naive precursors, we could differentiate and genetically engineer human T SCM . We report that IL-7 and IL-15 support the generation of postmitotic costimulated CD8 ϩ T cells with molecular and functional features of T SCM cells. These cells-defined by the expression of CD45RA, CD45R0, CD62L, CCR7, IL-7R␣, and CD95-can be identified among healthy subjects, are selectively enriched in hematopoietic stem cell transplant (HSCT) recipients, and reveal a phenotypic and functional profile distinct from that of T CM and T EM cells for extensive expansion capacity and ability Submitted May 22, 2012; accepted October 25, 2012. Prepublished online as Blood First Edition paper, November 15, 2012; DOI 10.1182 DOI 10. /blood-2012 There is an Inside Blood commentary on this article in this issue.The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this arti...