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Stem cells are maintained by transcriptional programs that promote self-renewal and repress differentiation. Here, we found that the transcription factor c-Myb was essential for generating and maintaining stem cells in the CD8+T cell memory compartment. Following viral infection, CD8+T cells lacking Mybunderwent terminal differentiation and generated fewer stem cell–like central memory cells than did Myb-sufficient T cells. c-Myb acted both as a transcriptional activator of Tcf7(which encodes the transcription factor Tcf1) to enhance memory development and as a repressor of Zeb2(which encodes the transcription factor Zeb2) to hinder effector differentiation. Domain-mutagenesis experiments revealed that the transactivation domain of c-Myb was necessary for restraining differentiation, whereas its negative regulatory domain was critical for cell survival. Myboverexpression enhanced CD8+T cell memory formation, polyfunctionality and recall responses that promoted curative antitumor immunity after adoptive transfer. These findings identify c-Myb as a pivotal regulator of CD8+T cell stemness and highlight its therapeutic potential.

Long-lived, self-renewing, multipotent T memory stem cells (TSCM) can trigger profound and sustained tumor regression but their rareness poses a major hurdle to their clinical application. Presently, clinically compliant procedures to generate relevant numbers of this T-cell population are undefined. Here, we provide a strategy for deriving large numbers of clinical-grade tumor-redirected TSCM starting from naive precursors. CD8+CD62L+CD45RA+ naive T cells enriched by streptamer-based serial-positive selection were activated by CD3/CD28 engagement in the presence of interleukin-7 (IL-7), IL-21, and the glycogen synthase-3β inhibitor TWS119, and genetically engineered to express a CD19-specific chimeric antigen receptor (CD19-CAR). These conditions enabled the generation of CD19-CAR–modified CD8+ TSCM that were phenotypically, functionally, and transcriptomically equivalent to their naturally occurring counterpart. Compared with CD8+ T cells generated with clinical protocols currently under investigation, CD19-CAR–modified CD8+ TSCM exhibited enhanced metabolic fitness and mediated robust, long-lasting antitumor responses against systemic acute lymphoblastic leukemia xenografts. This clinical-grade platform provides the basis for a phase 1 trial evaluating the activity of CD19-CAR–modified CD8+ TSCM in patients with B-cell malignancies refractory to prior allogeneic hematopoietic stem cell transplantation.

Long-lived, self-renewing, multipotent T memory stem cells (TSCM) can trigger profound and sustained tumor regression but their rareness poses a major hurdle to their clinical application. Presently, clinically compliant procedures to generate relevant numbers of this T-cell population are undefined. Here, we provide a strategy for deriving large numbers of clinical-grade tumor-redirected TSCMstarting from naive precursors. CD8+CD62L+CD45RA+naive T cells enriched by streptamer-based serial-positive selection were activated by CD3/CD28 engagement in the presence of interleukin-7 (IL-7), IL-21, and the glycogen synthase-3β inhibitor TWS119, and genetically engineered to express a CD19-specific chimeric antigen receptor (CD19-CAR). These conditions enabled the generation of CD19-CAR–modified CD8+TSCMthat were phenotypically, functionally, and transcriptomically equivalent to their naturally occurring counterpart. Compared with CD8+T cells generated with clinical protocols currently under investigation, CD19-CAR–modified CD8+TSCMexhibited enhanced metabolic fitness and mediated robust, long-lasting antitumor responses against systemic acute lymphoblastic leukemia xenografts. This clinical-grade platform provides the basis for a phase 1 trial evaluating the activity of CD19-CAR–modified CD8+TSCMin patients with B-cell malignancies refractory to prior allogeneic hematopoietic stem cell transplantation.