Tonic signaling and its effects on lymphopoiesis of CAR-armed hematopoietic stem and progenitor cells

Long-term survival of adoptively transferred chimeric antigen receptor (CAR) T cells is often limited. Transplantation of hematopoietic stem cells (HSCs) transduced to express CARs could help to overcome this problem as CAR-armed HSCs can continuously deliver CAR+ multi-cell lineages (e.g. T cells, NK cells). In dependence on the CAR construct a variable extent of tonic signaling in CAR T cells was reported, thus, effects of CAR-mediated tonic signaling on the hematopoiesis of CAR-armed HSCs is unclear. To assess effects of tonic signaling two CAR constructs were established and analyzed: (i) A signaling CAR inducing a solid antigen-independent tonic signaling termed CAR-28/, and (ii) a non-stimulating control CAR construct lacking intracellular signaling domains termed CAR-Stop. Bone marrow (BM) cells from immunocompetent mice were isolated, purified for HSC-containing Lin-cKit+ (LK) cells or the LK Sca-1+ subpopulation (Lin Sca 1+cKit+, LSK) and transduced with both CAR constructs. Subsequently, modified BM cells were transferred into irradiated mice where they successfully engrafted and differentiated into hematopoietic progenitors. HSCs expressing the CAR-Stop sustained normal hematopoiesis. In contrast, expression of the CAR-28/ led to elimination of mature CAR+ T and B cells suggesting that the CAR-mediated tonic signaling mimics autorecognition via the newly recombined immune receptors in the developing lymphocytes.