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...

High mobility group box 1 (HMGB1) is an abundant chromatin protein that acts as a cytokine when released in the extracellular milieu by necrotic and inflammatory cells. Here, we show that extracellular HMGB1 and its receptor for advanced glycation end products (RAGE) induce both migration and proliferation of vessel-associated stem cells (mesoangioblasts), and thus may play a role in muscle tissue regeneration. In vitro, HMGB1 induces migration and proliferation of both adult and embryonic mesoangioblasts, and disrupts the barrier function of endothelial monolayers. In living mice, mesoangioblasts injected into the femoral artery migrate close to HMGB1-loaded heparin-Sepharose beads implanted in healthy muscle, but are unresponsive to control beads. Interestingly, α-sarcoglycan null dystrophic muscle contains elevated levels of HMGB1; however, mesoangioblasts migrate into dystrophic muscle even if their RAGE receptor is disabled. This implies that the HMGB1–RAGE interaction is sufficient, but not necessary, for mesoangioblast homing; a different pathway might coexist. Although the role of endogenous HMGB1 in the reconstruction of dystrophic muscle remains to be clarified, injected HMGB1 may be used to promote tissue regeneration.

Although lymphoid dendritic cells (DC) are thought to play an essential role in T cell activation, the initial physical interaction between antigen-bearing DC and antigen-specific T cells has never been directly observed in vivo under conditions where the specificity of the responding T cells for the relevant antigen could be unambiguously assessed. We used confocal microscopy to track the in vivo location of fluorescent dye-labeled DC and naive TCR transgenic CD4+ T cells specific for an OVA peptide–I-Ad complex after adoptive transfer into syngeneic recipients. DC that were not exposed to the OVA peptide, homed to the paracortical regions of the lymph nodes but did not interact with the OVA peptide-specific T cells. In contrast, the OVA peptide-specific T cells formed large clusters around paracortical DC that were pulsed in vitro with the OVA peptide before injection. Interactions were also observed between paracortical DC of the recipient and OVA peptide-specific T cells after administration of intact OVA. Injection of OVA peptide-pulsed DC caused the specific T cells to produce IL-2 in vivo, proliferate, and differentiate into effector cells capable of causing a delayed-type hypersensitivity reaction. Surprisingly, by 48 h after injection, OVA peptide-pulsed, but not unpulsed DC disappeared from the lymph nodes of mice that contained the transferred TCR transgenic population. These results demonstrate that antigen-bearing DC directly interact with naive antigen-specific T cells within the T cell–rich regions of lymph nodes. This interaction results in T cell activation and disappearance of the DC.

T cells activated by antigen receptor stimulation in the absence of accessory cell-derived costimulatory signals lose the capacity to synthesize the growth factor interleukin-2 (IL-2), a state called clonal anergy. An analysis of CD3- and CD28-induced signal transduction revealed reduced ERK and JNK enzyme activities in murine anergic T cells. The amounts of ERK and JNK proteins were unchanged, and the kinases could be fully activated in the presence of phorbol 12-myristate 13-acetate. Dephosphorylation of the calcineurin substrate NFATp (preexisting nuclear factor of activated T cells) also remained inducible. These results suggest that a specific block in the activation of ERK and JNK contributes to defective IL-2 production in clonal anergy.

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