Single-Cell Spatial Transcriptomics Identifies Lymphoid Aggregates with Location-Specific Alterations of Adaptive Immune Cells in Human MDS Bone Marrow

Myelodysplastic syndromes (MDS) represent a heterogenous group of myeloid neoplasms characterized by ineffective hematopoiesis, dysplastic morphology, and risk of transformation to acute leukemia. While neoplastic cell-intrinsic mechanisms driving MDS pathogenesis have been intensely investigated, the spatial architecture and in situ immune microenvironment in human MDS has not been studied at single-cell resolution. We hypothesized that the MDS bone marrow microenvironment is characterized by altered hematopoietic and non-hematopoietic cell distributions and transcriptional profiles including the composition and location of adaptive immune subsets. We developed an approach for high-definition spatial transcriptomic analysis of fixed and EDTA-decalcified human marrow core biopsies, enabling interrogation of hundreds of genes simultaneously in the context of their cellular location. We designed a marrow and immune focused 433 gene panel for the Xenium platform (10x Genomics) and applied this to 7 decalcified core marrow biopsies from patients with early-stage untreated MDS, and 3 control marrows, yielding in situ spatial data for 750,688 cells. In parallel, we performed 32-color spectral flow cytometry along with 5' single-cell CITE- and T cell receptor- (TCR) sequencing (N=31 MDS, N=9 controls) capturing 521,853 cells and 145,176 TCRs. Single-cell spatial transcriptomics identified >20 distinct cell types including populations that are poorly captured by analyses of single-cell suspensions from bone marrow aspirates: megakaryocytes, osteoblasts, endothelial cells, mesenchymal stromal cells, vascular smooth muscle cells, and adipocytes. Nearest neighbor analyses revealed reduced cell type diversity in MDS compared to controls, particularly surrounding osteoblasts (p=1.7x10-2). Within the immune compartment, we captured B, T, and natural killer (NK) cell types for further stratification into FOXP3+ T regulatory cells, naïve and memory CD4 or CD8 T cells, along with numerous phenotypic and functional markers. Spatial data were integrated with single-cell proteomic, RNA, and TCR sequencing in the same individuals to further elucidate the adaptive immune composition and T cell repertoire of the MDS marrow. In MDS samples, B cells were reduced in frequency whereas T cells were the most abundant lymphocyte cell type. Although MDS T cell subset composition was similar to control marrows, the spatial distribution of immune cells in MDS was distinct. Spatial analysis identified aggregates of T, B, and plasmacytoid dendritic cells (pDC) reminiscent of tertiary lymphoid structures in the marrow (which we defined as clusters of B, T, NK and pDCs with > 10 T cells). Immune aggregates were larger in MDS (>75 cells/aggregate, range 83-390) compared to controls (no aggregates >72 cells) with increased proportions of T cells (p=1.2x10-3) and frequencies of CD4 and CD8 T cell subsets. TCF7, a transcription factor linked to T cell stemness, and CXCR4, a key marker of bone marrow homing, were increased in CD4 (p=7.0x10-4 and p=1.5x10-5 respectively) and CD8 (p=1.6x10-2 and p=8.9x10-7 respectively) T cells in MDS immune aggregates. These findings suggest the emergence of immunologically active niches within the MDS marrow and would not have been captured via single-cell RNA-seq alone. Outside of immune aggregates, we found increased memory CD8 T cells (p=1.4x10-2) in MDS. In contrast, control marrows showed no difference in T cell subset frequency between cells inside or outside of immune aggregates. CITE/TCR-seq revealed clonal T cell expansions in MDS within GZMB+ CD8s, distinct from controls (where clonal expansions were found in GZMK+ CD8s), supporting altered CD8 T cell immunity in MDS. Our integrative analyses, including single-cell spatial transcriptomic profiling, revealed active remodeling of the adaptive immune system in early stage MDS. The finding of large immune aggregates in the MDS marrow with more T cells bearing signatures of active cell recruitment and growth, combined with increased clonally expanded memory CD8 T cells outside immune aggregates, point toward localized changes in the adaptive immune response in early stage MDS. Spatial analyses revealed location-specific T cell differences within the MDS marrow, underscoring the importance of combined phenotypic and spatial approaches to elucidate disease pathophysiology in MDS.