Decoding the Immune Microenvironment of Primary Chronic Myelomonocytic Leukemia and Secondary Chronic Myelomonocytic Leukemia Due to DLBCL with CD19 CAR-T Failure By Single-Cell RNA-Seq

Purpose: Chimeric antigen receptor T (CAR-T) cell therapy has demonstrated promising efficacy in clinical trials for relapsed/refractory diffuse large B cell lymphoma (DLBCL). However, the potential mechanisms underlying the development of secondary chronic myelomonocytic leukemia (CMML) due to DLBCL with CD19 CAR-T failure has not been comprehensively investigated and the post-treatment microenvironmental changes remain unclear. Therefore, in this study, the changes of transcription regulation of CMML were deeply discussed by single cell sequencing, so as to better understand the biological and molecular mechanism of CMML.

Methods: Firstly, we compared the bone marrow morphology of primary CMML patients with secondary CMML patients due to DLBCL with CD19 CAR-T failure by Giemsa staining. Then we performed scRNA-seq on CD45+ cells of primary CMML and secondary CMML. The data of health control comes from public database. Finally, we performed scRNA-seq on the CD45+ cells of CMML patients before and after the treatment of Azacitidine combined with Venacla, using CellRanger pipeline and CellChat for detailed analysis.

Results: We found that the immunophenotype of primary and secondary CMML was heterogeneous. Comparing them with CD45+ single cell data of healthy controls, the results showed that the ratio of T/NK cells in patients with secondary CMML was the lowest, and the ratio of monocytes/macrophages was the highest. The results of granulocyte monocyte population analysis confirmed that there was a great difference between the distribution of cell subsets of CMML and the other two, and the expression level of the gene encoding protein, which is known to be important to KEGG, was significantly higher. In the T/NK cell population of secondary CMML, the percentages of CD4 naive T cells, CD8 naive T cells, NK cells, Tregs and proliferating T cells increased, while the percentages of CD8 cytotoxic T cells, γδT cells and NKT cells decreased. The analysis of intercellular interaction found that the increase of monocytes/macrophages in patients with secondary CMML was related to inflammatory cytokines, chemokines and mediated immunosuppression. The data of single cell before and after the treatment of secondary CMML Azacitidine combined with Venacla indicated that after treatment, the percentage of T/NK cells increased, the percentage of monocytes/macrophages and granulocytes decreased, the number of intercellular interactions increased, and the interaction intensity decreased.

Conclusions: Here, we report the immune microenvironment of primary CMML and both pretreatment and post treatment of secondary CMML due to DLBCL with CD19 CAR-T failure by single-cell RNA-seq. Our results revealed a significant increase proportion of monocytes/macrophages with several pathogenic microbial infection and decrease ratio of T/NK cells in secondary CMML before treatment. Significantly, monocytes/macrophages demonstrated enhanced inflammatory cytokines, chemokines, and immunosuppression state, which may inhibit the cytotoxicity of T/NK cells. By contrast, the cytotoxicity of T/NK cells were enhanced in secondary CMML after treatment. These findings suggest that monocytes/macrophages from secondary CMML may inhibit the cytotoxicity of T/NK cells, providing a rationale for targeting macrophages to counteract CMML treatment.

No relevant conflicts of interest to declare.