T Cells Demonstrate Enhanced Specificity for CD19⁺ Malignancies When Stimulated with IL-21.

T cells genetically modified to express CD19-specific chimeric antigen receptors (CARs) are being evaluated in early-phase clinical trials in patients with B-lineage malignancies. Therapeutic success is predicted by ability of infused CAR⁺ T cells to both persist and kill in an antigen-dependent fashion. The first of these dual goals can be achieved by altering the CAR molecule to provide T-cell survival signals through a chimeric CD28 endodomain (designated CD19RCD28). We now report that altering the culturing microenvironment with IL-21 improves antigen-dependent cytolysis of T cells when propagated on CD19⁺ artificial antigen presenting cells (aAPC) derived from K562. To test whether IL-21 acts in conjunction with CD28 signaling to support acquisition of redirected effector functions we electro-transferred quiescent T cells from peripheral blood with Sleeping Beauty system DNA plasmids to introduce CD19RCD28 CAR transposon. Selective outgrowth of CAR⁺ T cells was achieved on CD19⁺ aAPC that provide co-stimulation with the addition of exogenous IL-2 and/or IL-21. When IL-21 was present there was preferential numeric expansion of CD19-specific CD8⁺ T cells which lysed and produced IFN-g in response to CD19 (Figure). Furthermore, the CD8⁺CAR⁺ T cells displayed a central memory (CM) cell surface phenotype characterized as CD62L⁺ and CD28⁺. In contrast, genetically modified T cells propagated with exogenous IL-2 resulted in predominately CD19-specific CM CD4⁺ T cells. Thus, cytokines can be used to tailor the CD8/CD4 ratio of CAR⁺ T cells derived from peripheral blood. These data demonstrate that the dual goals of persistence and lysis can be achieved by altering CAR and the cytokine milieu and have implications for infusing CAR⁺ T cells in next-generation immunotherapy trials.

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