Tonic Signaling and Its Effects on Lymphopoiesis of CAR-Armed Hematopoietic Stem and Progenitor Cells

Abstract

Long-term survival of adoptively transferred chimeric Ag 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+ multicell 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 the effects of tonic signaling, two CAR constructs were established and analyzed 1) a signaling CAR inducing a solid Ag-independent tonic signaling termed CAR-28/ζ and 2) a nonstimulating control CAR construct lacking intracellular signaling domains termed CAR-Stop. Bone marrow cells from immunocompetent mice were isolated, purified for HSC-containing Lin−cKit+ cells or the Lin−cKit+ Sca-1+ subpopulation (Lin−Sca-1+cKit+), and transduced with both CAR constructs. Subsequently, modified bone marrow cells were transferred into irradiated mice, in which they successfully engrafted and differentiated into hematopoietic progenitors. HSCs expressing the CAR-Stop sustained normal hematopoiesis. In contrast, expression of the CAR-28/ζ 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.

Versatile chimeric antigen receptor platform for controllable and combinatorial T cell therapy

“…Analysis of gene expression was performed using quantitative PCR analysis as described, with slight modifications. 41,42 Briefly, triplets of 1*10 4 PC3 cells were seeded and cultivated with RevCAR T cells in the presence or absence of RevTMs. After incubation for 24 h, co-transduced RevCAR T cells were sorted for EGFP + /mCherry + expression under exclusion of PI + cells.…”

“…Sorting and analysis of gene expression profile were performed as described earlier. 41 The n-fold change of gene expression between incubation with COS RevTM + SIG RevTM versus w/o RevTM was calculated using the ΔΔC P method.…”

Optimization of the proliferation and persistency of CAR T cells derived from human induced pluripotent stem cells

“…During their ex vivo expansion, CAR tonically signals to induce the exhaustion phenotype 12 . Antigen-independent tonic signalling is reported to affect the lymphopoiesis of CAR-engineered haematopoietic stem cells (HSCs) and promote NK cell-like development 13 , 14 . In contrast, T cells derived from CD19 CAR-engineered iPSCs show phenotypic similarities to γδ T cells 5 .…”

“…1c ). Recent investigations reported that 1928z transduction into HSCs impaired T-cell differentiation capability 13 , 14 and promoted NK-like cell development by suppressing the transcription factor BCL11B, which is indispensable for T-lineage development of lymphoid progenitors during early phases of ex vivo T-cell generation. It could be a possible reason also for T-cell differentiation from 1928z CAR-transduced iPSC 5 .…”

Generation and clinical potential of functional T lymphocytes from gene-edited pluripotent stem cells

“…T-iPSCs with RAG2 knockout and non-T-iPSCs with transduced TCR can also help avoid unpredictable TCR generation [ 89 ]. It is notable that CAR, as an artificial fusion molecule, may disturb the normal pattern of T lymphopoiesis [ 123 ] and this can be overcome by constructing conditional expression cassettes at the stem cell level. Additionally, eliminating the expression of GM-CSF in CAR-T cells mitigates neurotoxicity and cytokine release syndrome (CRS) [ 124 – 126 ].…”