B-lineage acute lymphoblastic leukaemia disrupts a LepR+ bone marrow stromal niche which is critical for CAR-T cell persistence Free

B-lineage acute lymphoblastic leukaemia (B-ALL) is an aggressive haematological malignancy with high treatment failure rates. Whilst outcomes have improved through the use of genetically modified, chimeric antigen receptor (CAR)-T cells, most responding patients will relapse within two years. Poor persistence of CAR-T cells is implicated in most relapses. ALL is associated with marked disruption to the population architecture of the bone marrow (BM), including loss of stromal niches critical for hematopoietic and progenitor cells. We hypothesised that such disruption would also affect BM mesenchymal stromal cells (MSC) which express T cell-supporting cytokines, and that this would contribute to the failure of CAR-T cells to persist. In particular, we addressed the role of MSCs identified by the expression of the leptin receptor (LepR) ('LepR⁺ MSCs') which express IL-15 and IL-7. To address this hypothesis, we first performed confocal imaging of the BM of 12-week old, immunocompetent Lepr fate reporter mice (tdTomato^fl/wt; Lepr^cre/wt) infused with the murine pre-B cell leukaemia, E2A:PBX1. Analysis of 3-dimensional, segmented images demonstrated marked disruption of the LepR⁺ MSC population in the presence of B-ALL, including changes in overall network architecture and abnormal cell morphology. To investigate how these changes in distribution and morphology related to cellular state, we performed single cell mRNA sequencing (10X platform) of stromal and haematopoietic fractions from the BM of 12-week old, healthy or B-ALL-bearing, B6 mice +/- treatment with anti-CD19 CAR-T cells with cyclophosphamide pre-conditioning. In steady state, Il7 was expressed almost exclusively by LepR⁺ MSCs in the bone marrow, whilst Il15 was expressed by both haematopoietic and stromal cells (in the latter, principally by LepR⁺ MSCs). In the presence of B-ALL, we observed substantial re-modelling of the LepR⁺ MSC compartment, with the emergence of additional LepR⁺ MSC subsets, characterised by signatures in keeping with response to inflammation but also a profound depletion of both Il7 and Il15. After 15 days, treatment ofB-ALL-bearing mice with CAR-T had only a limited effect upon correcting the inflammatory remodelling of the LepR⁺ MSC stromal compartment. To functionally investigate the role of LepR⁺ MSC-derived IL-7 and IL-15 in supporting endogenous and CAR-T cell persistence, we applied the Cre-LoxP system to conditionally deplete IL-7 or IL-15 from LepR⁺ MSCs ('KO') and compare T cell numbers/frequency/phenotype with those from littermate controls. Using flow cytometry of BM, spleen and lymph node samples, we found that conditional depletion of IL-15 from LepR⁺ MSCs had no effect upon T cell numbers/frequency/phenotype in either steady state or after infusion of anti-CD19 CAR-T cells. Conditional depletion of IL-7 also had no effect on T cell numbers/frequency/phenotype in steady state. However, in the setting perturbed by CAR-T cell infusion, we found a significant decrease in absolute CAR-T cell numbers (median 69% decrease in BM CAR T cell number, p<0.05) at 8 weeks after infusion. In addition, absolute endogenous (non-CAR) T cell numbers were also reduced at this time point (median 30% decrease in BM T cell number, p<0.01). T cells in the BM expressed higher levels of IL-7 receptor alpha (IL-7Rα) in LepR⁺Il7 KO mice versus controls, consistent with a lack of access and down-regulation to functional ligand in the former. Similarly, activated T cells from healthy donors, co-incubated ex vivo with LepR⁺ MSCs isolated from B-ALL-bearing mice, also demonstrated higher IL-7Rα expression than controls. In summary, using a combination of confocal imaging, single cell sequencing, in vitro testing and functional in vivo testing, we demonstrate that B-ALL induces major structural and functional disruption of the BM LepR⁺ MSC population leading to loss of IL-7 which is required to support CAR-T cell persistence in vivo. This study therefore identifies B-ALL-induced changes in LepR⁺ MSCs as a novel target to improve the efficacy and persistence of CAR-T cells.