Analysis of Polyfunctionality for Enhanced CAR T-Cell Therapy for Hematologic Malignancies.

Chimeric antigen receptor (CAR) T-cell therapy has emerged as a promising immunotherapeutic strategy for eradicating human cancers. Their therapeutic success and durability of clinical responses hinges, in large part, on their functional capacity, including the ability of these engineered cells to simultaneously expand and persist after infusion into patients. CD19 CAR T-cell polyfunctionality, assessing the simultaneous functions of cytokine production, proliferation, and cytotoxicity has been reported to correlate with clinical outcomes. Assay optimization is potentially limited by the heterogeneous nature of CAR T-cell infusion products and target specificity. We optimized a single-cell platform for polyfunctionality using CAR T cell products manufactured from healthy donors, engineered against a novel target, BAFF-R, and validated the protocol using CD19 CAR T cells. We observed distinct qualitative differences between BAFF-R and CD19 CAR T cells relative to the proportions of stimulatory vs. effector cytokines, based on target antigen density, and generally, CD19 CAR T cells exhibited lower indices of polyfunctionality. Finally, we applied our assay to the autologous BAFF-R CAR T-cell product generated from the first NHL patient treated on an ongoing clinical trial who had progressed after prior CD19 CAR T-cell therapy. We observed robust indicators of polyfunctionality, which correlated with successful CAR T cell expansion after infusion and achievement of durable complete remission ongoing after 18 months. The precise identification of factors determining the role of BAFF-R CAR T-cell fitness on toxicity and clinical outcome will require the application of this robust assay in the analysis of additional treated patients.

Transcriptional establishment of cell-type identity: dynamics and causal mechanisms of T-cell lineage commitment.

Precursor cell entry into the T-cell developmental pathway can be divided into two phases by the closure of T-lineage commitment. As cells decide against the last alternative options to the T-cell fate, they turn on the transcription factor Bcl11b and silence expression of a group of multipotent progenitor regulatory factors that include hematopoietic transcription factor PU.1. Functional perturbation tests show that Bcl11b is needed for commitment while PU.1 actively participates in keeping open access to alternative fates, until it is silenced; however, PU.1 and Bcl11b both contribute positively to T-cell development. Our recent work reviewed here sheds light on the transcriptional regulatory network that determines the timing and irreversibility of Bcl11b activation, the ways that Notch signaling from the thymic microenvironment restricts the action of PU.1 to prevent it from diverting cells to non-T fates, and the target genes that PU.1 still regulates under the influence of Notch signaling to contribute to T-cell generation. We argue that T-cell development depends on the sequential operation of two interlaced, but mutually antagonistic, gene regulatory networks, one initially supporting expansion before commitment and the other imposing a "terminal" differentiation process on committed cells.

Architecture of a lymphomyeloid developmental switch controlled by PU.1, Notch and Gata3.

Hematopoiesis is a classic system with which to study developmental potentials and to investigate gene regulatory networks that control choices among alternate lineages. T-cell progenitors seeding the thymus retain several lineage potentials. The transcription factor PU.1 is involved in the decision to become a T cell or a myeloid cell, and the developmental outcome of expressing PU.1 is dependent on exposure to Notch signaling. PU.1-expressing T-cell progenitors without Notch signaling often adopt a myeloid program, whereas those exposed to Notch signals remain in a T-lineage pathway. Here, we show that Notch signaling does not alter PU.1 transcriptional activity by degradation/alteration of PU.1 protein. Instead, Notch signaling protects against the downregulation of T-cell factors so that a T-cell transcriptional network is maintained. Using an early T-cell line, we describe two branches of this network. The first involves inhibition of E-proteins by PU.1 and the resulting inhibition of Notch signaling target genes. Effects of E-protein inhibition can be reversed by exposure to Notch signaling. The second network is dependent on the ability of PU.1 to inhibit important T-cell transcription factor genes such as Myb, Tcf7 and Gata3 in the absence of Notch signaling. We show that maintenance of Gata3 protein levels by Myb and Notch signaling is linked to the ability to retain T-cell identity in response to PU.1.