Large B-cell lymphoma microenvironment archetype profiles (LymphoMAPs). [macrophage]

Large B-cell lymphomas (LBCL) are clinically and biologically heterogeneous lymphoid malignancies with complex microenvironments that are central to disease etiology. Here we have employed single-nucleus multiome profiling of 232 tumor and control biopsies to characterize diverse cell types and subsets that are present in LBCL tumors, effectively capturing the lymphoid, myeloid, and non-hematopoietic cell compartments. Cell subsets co-occurred in stereotypical Lymphoma Microenvironment Archetype Profiles (LymphoMAPs) defined by; (i) a sparsity of T-cells and high frequencies of cancer-associated fibroblasts and tumor-associated macrophages [FMAC]; (ii) lymph node architectural cell types with naïve and memory T-cells [LN]; or (iii) activated macrophages and exhausted CD8 T-cells [TEX]. Divergent patterns of cell-cell communication underpinned the transcriptional phenotypes of archetype-defining cell subsets resulting in exclusion, support or suppression of T-cells, respectively. Consistent with this, LymphoMAPs were associated with significantly different clinical outcomes following CD19 CAR T-cell therapy. Peripheral blood-derived monocytes were isolated from cryopreserved peripheral blood mononuclear cells obtained from 3 different healthy donors (Stemcell) using pan-monocyte isolation kit (Miltenyi) with autoMACS Neo cell separator (Miltenyi) according to the manufacturer’s instructions. Cells were cultured in ImmunoCult SF Macrophage medium (Stemcell) containing 50 ng/mL of recombinant human M-CSF (Stemcell) in temperature-responsive polymer-treated UpCell plates (Thermo Fisher) for 4 days, followed by either supplementation with 50 ng/mL of recombinant human IFN-gamma (Stemcell) or replacement with supernatant of exhausted T-cells (Tex sup) for an additional 2 days. For Tex sup production, a serial killing assay was conducted using healthy donor-derived CAR T cells and HT cell line, starting at a 1:1 ratio in IMDM (Gibco) supplemented with 10% FBS (Gibco). HT cells were added every 2 days along with medium exchange. The supernatant was collected on day 10 and then cryopreserved until use. Tex sup and ImmunoCult SF macrophage medium were mixed at a 1:1 ratio and supplemented with 50 ng/mL rhM-CSF. RNA was extracted using RNeasy micro kit (QIAGEN) according to the manufacturer’s protocol after washing the cells with PBS, followed by library preparation for RNAseq.