Abstract B050: Spatial Investigation of CD4+ T-cell priming during leukemia initiation

Abstract Relapse is common in B-cell acute lymphoblastic leukemia (B-ALL) due to the persistence of measurable residual disease (MRD). MRD can be mediated by dysfunctional immune responses, enabling leukemic cells to evade immune clearance. The expansion of PD1+/TIM3+/Foxp3- CD4+ T-cells is a particularly strong dysfunctional immune correlate and is associated with an 8-10-fold higher risk of relapse. We previously showed that PD1+/TIM3+/Foxp3- CD4+ T-cells are predominantly comprised of Type-1 regulatory cells (Tr1s), which secrete IL-10 and suppress bystander T-cells. This ultimately inhibits anti-tumor immune responses. Here, we examine the immune microenvironment in a mouse model of B-ALL to elucidate which cell types prime leukemia-specific CD4+ T-cells towards Tr1 states. Other groups have demonstrated that hematopoietic stem and progenitor cells can act as antigen-presenting cells by presenting neoantigen-derived peptides in an MHCII context to peptide-specific CD4+ T-cells. This ultimately polarized CD4 T-cells towards Tr1 fates. We hypothesized that mature leukemia cells could co-opt this mechanism to directly prime neoantigen-specific CD4 T-cells towards a Tr1-like fate. We injected a leukemic-specific fixed-TCR CD4+ T-cell (HV1) into B6 recipients, followed 24 hours later by injection of a BCR-ABL+ ALL cell line (LM138). We examined the leukemic microenvironment at day 5 (n=3), day 7 (n=4), and day 9 (n=4) by flow cytometry and using the multiparameter spatial proteomics platform, Cellscape. By day 9, 49% of HV1s were TIM3+ and IL10+ in the bone marrow (± 36.07), suggesting that Tr1 transcriptional programming was active. We then examined the spatial neighborhoods of HV1s in the bone marrow at day 7. After segmentation and thresholding in Qupath, cellular neighborhoods were examined in CytoMAP, and direct cell interactions within 20 µm were quantified using Spatstat in R. We found that 86% of LM138 cells were MHCII- at this time point. 28% of naïve HV1s (CD44-) were in direct contact with LM138s, and only 9% of naïve HV1s were in direct contact with an MHCII+ leukemia cell. When we examined the 50 nearest neighbors of naïve HV1s, we found that two populations, CD4+ T cells and F4/80+ cells, were enriched. We further subsetted F4/80+ cells by MHCII positivity and found that MHCII+F4/80+ macrophages were enriched in the neighborhoods of naïve HV1s, compared to a random spatial distribution (1.49 ± 0.63). Our findings suggest that neoantigen-specific naïve CD4 T-cells in the bone marrow interact predominantly with MHCII+ macrophages at early priming timepoints, while direct contact with LM138s is infrequent. This suggests that macrophages may play an indispensable role in initiating Tr1 generation in the bone marrow during leukemia initiation. Citation Format: Kyra Boorsma Bergerud, Enoc Granados Centeno, Michael Farrar, Sean Tracy. Spatial Investigation of CD4+ T-cell priming during leukemia initiation abstract. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr B050.