Single-Cell Profiling of the Immune Response Following Siglec 15 Knockout in Leukemia Cells

Background: Siglec-15 (Sig15) has emerged as a putative immune checkpoint, highly expressed in several solid and hematological malignancies, including B cell acute lymphoblastic leukemia (B-ALL). Knockout (KO) of Siglec15 in murine B-ALL models has been shown to promote leukemia clearance in immunocompetent recipients while the disease progresses lethally in immunodeficient recipients, suggesting that Sig15 KO restores T effector function and is a promising immunotherapy target. Through our study, we aim to elucidate the impact of Sig15 expression on the immune response in B-ALL and its potential as a therapeutic target.

Methods: We performed single-cell RNA sequencing (scRNA-seq) on paired spleen (N=12) and bone marrow samples (N=12) from immunocompetent and immunodeficient (Rag1-/-) C57BL/6 mice engrafted with wild-type (WT) or Sig15 KO murine B-ALL cell lines. Following genomic alignment, QC, batch correction, and clustering, the resulting single-cell dataset comprised 262,702 cells forming 80 clusters, representing malignant cells and immune subpopulations spanning myeloid and lymphoid lineages in the bone marrow (BM) and spleen.

Results: ScRNA-seq analysis revealed a trend in increasing proportions of T cells, NK cells, and mature neutrophils in the BM of Sig15 KO mice compared to their WT B-ALL counterparts. Specifically, within the BM lymphoid compartment, a rise in naïve and cytotoxic CD8+ T cell subpopulations was observed, along with the differentiation of effector memory T cells and a shift in NK cells from an activated to resting state. This phenotypic shift to a resting state in NK cells has been previously associated with poor outcomes in B-ALL, prompting us to examine the survival association of resting versus activated NK gene signatures using the publicly available TARGET phase 2 B-ALL dataset. The survival analysis illustrated that an increased activated NK cell signature in the BM or peripheral blood was significantly associated with poor overall survival (P < 0.05), while an enrichment of resting NK cell signature was conversely associated with improved overall survival (P < 0.05). We further validated these findings with an external B-ALL single-cell dataset derived from human BM samples, demonstrating that increased resting versus activated NK cells were linked to remission relative to relapse. Within the BM myeloid compartment, Sig15 KO led to the abrogation of emergency myelopoiesis observed in the recipients of WT B-ALL, exhibiting a shift toward mature neutrophils. Furthermore, mature neutrophils in the BM of recipients of Sig15 KO B-ALL displayed an anti-tumoral N1 neutrophil phenotype compared to an N2 pro-tumoral phenotype observed in WT B-ALL mice. Notably, the enrichment of this N1 neutrophil signature in the TARGET phase 2 B-ALL cohort was associated with improved survival (P < 0.05). Comparing these findings to the immune profile of the spleen revealed similar patterns with an increase in circulating cytotoxic T cells and T follicular helper costimulation, along with a shift toward a resting NK cell state and mature N1 neutrophils. Lastly, to examine how immune subpopulations interact following Sig15 KO, we performed intercellular communication analysis. Our results showed that Sig15 KO in leukemia cells promotes a pro-inflammatory immune microenvironment with increased pro-inflammatory signaling between subpopulations, including classical monocyte-mediated IL-1 signaling to N1 neutrophils and cytotoxic T cells, as well as increased CCL6 and IL-16 signaling.

Conclusion: Our study identifies leukemia-expressed Sig15 as a pivotal regulator of the immune landscape in B-ALL, with its knockout promoting pro-inflammatory antitumoral immune responses and an association with improved patient outcomes. These insights underscore the potential of targeting Sig15 in hematologic malignancies to enhance antitumor immunity and improve patient outcomes.