Targeting Lymphoma Associated Myeloid-Monocytic Cells through CSF1R Blockade Enhances CAR-T Cell Response in Aggressive B Cell Lymphoma

Introduction: Chimeric antigen receptor (CAR) T cell therapy has substantially improved the outcome of patients suffering from relapsed and/or refractory (r/r) aggressive B cell lymphoma. However, around 60% of patients do not show long-term remissions after CAR-T cell therapy. Recent studies have indicated a relevant role of the lymphoma microenvironment (LME) in response and resistance to CAR-T cell therapy. However, targeting the LME in aggressive B cell lymphoma to boost CAR-T cell efficacy has not yet been sufficiently explored. We therefore aimed to unravel the immunosuppressive capacity of the LME and its myelo-monocytic cell compartment with the ultimate goal to identify potential therapeutic targets and enhance CAR-T cell response.

Methods: To elucidate hallmarks associated with an immunosuppressive LME and CAR-T cell resistance in patients with r/r B cell lymphoma, we applied multi-dimensional analyses to pre- and post-CAR-T cell-treated human lymphoma specimens (n = 41), including bulk RNA sequencing, single-cell RNA sequencing of 47,078 live cells and Imaging Mass Cytometry (IMC). To validate our findings and explore the potential of new therapeutic targets, we utilized ex vivo co-culture experiments, a fully murine CD19 CAR-T cell therapy platform in an immunocompetent, autochthonous DLBCL mouse model and performed bulk RNA sequencing and IMC of diseased spleens.

Results: In our cohort of CAR-T cell treated patients (n = 104) durable response, defined as complete remission six months after CAR-T cell therapy, resulted in prolonged progression-free and overall survival. In CAR-T cell non-durable responding lymphoma patients, we identified a prognostically relevant lymphoma-associated myelo-monocytic (LAMM) signature including genes such as CD14, CD68, MARCO, ITGAM, IL1B, IL10 and S100A9. Furthermore, non-durable response was characterized by increased hypoxia and reduced (CD8⁺) T cell infiltration. In particular, in-depth profiling using single-cell RNA sequencing and IMC revealed a distinct CSF1R⁺CD14⁺CD68⁺ LAMM cell population associated with non-durable response and poor clinical outcome in CAR-T cell-treated patients with r/r B cell lymphoma. Importantly, high LAMM and low CD8⁺ T cell infiltration prior to CAR-T cell therapy showed a reduced progression-free survival when compared to low LAMM and high CD8⁺ T cell infiltration in r/r B cell lymphoma samples. Next, in ex vivo co-culture experiments we demonstrated that CSF1R⁺ LAMM cells strongly inhibit the proliferation and the cytotoxic capacity of CAR-T cells. To elaborate on LAMM-T cell interaction at a molecular level, we performed inference analysis of cell-cell communication in our single-cell RNA sequencing dataset using CellphoneDB which revealed that LAMM cells exert their immunosuppressive function by direct interaction with T cells via prostaglandin E2 (PGE₂) and EP2/EP4 receptor signaling. Most strikingly, applying a fully autochthonous DLBCL CAR-T cell mouse model, we demonstrated that the combination of CD19 CAR-T cell therapy with CSF1R blockade switches an immunosuppressive LME into a T cell-enriched LME, which was accompanied by a follicular architecture and blood vessel normalization of diseased spleens indicated by IMC analysis. Finally, we showed that the combination of CSF1R inhibition and CD19 CAR-T cell therapy displayed synergistic treatment effects and prolonged survival with long-lasting, complete remissions.

Conclusion: Our multiomic data and preclinical models provide strong evidence that CSF1R⁺ LAMM cells contribute to CAR-T cell failure in r/r aggressive B cell lymphoma and that CSF1R inhibition synergistically improves CD19 CAR-T cell response, promotes an immunosupportive microenvironment and restores anti-lymphoma immunity. Given that CSF1R inhibitors have already been clinically evaluated and FDA-approved in other malignancies, this therapeutic combination has the potential for rapid clinical translation. Based on our findings, we propose to test the combination of CAR-T cell therapy and CSF1R inhibitors in patients with r/r aggressive B cell lymphoma within prospective clinical trials.