HIV and Antiretroviral Therapy Influence Tumor Microenvironment Architecture, Subclonal Evolution, and Immune Evasion Strategies in Diffuse Large B-Cell Lymphoma

The tumor immune microenvironment (TIME) and tumor heterogeneity shape prognosis and therapeutic response in Diffuse Large B-cell Lymphoma (DLBCL), but HIV-associated (HIV+) DLBCL remains poorly understood. To characterize the TIME and tumor-intrinsic features of DLBCL across a spectrum of HIV-associated immune dysregulation, we applied imaging mass cytometry (IMC), spatial transcriptomics (ST), and whole exome sequencing to 61 pre-treatment DLBCL biopsies, stratified by HIV status and antiretroviral therapy (ART) exposure. By IMC, a tumor-immune hot neighborhood associated with inferior survival in HIV+ DLBCL (HR=3.16, p=0.013). By ST, HIV+/ART-experienced tumors demonstrated greater macrophage infiltration (p=0.02) and enrichment for M2 macrophage markers (CD163, MRC1, MARCO) and PDCD1:CD274 coexpression. Unsupervised clustering identified 17 shared subclonal niches, with PRC1/BMI1 dysregulation in HIV+ tumors and PRC2/EZH2 enrichment in HIV-negative (HIV-) tumors. Pseudotime positioned HIV+/ART-naïve, HIV+/ART-experienced, and HIV- tumors at progressively later transcriptional states. CD47 increased over pseudotime in HIV+/ART-experienced tumors but decreased in HIV-, while CD58 loss occurred in HIV- tumors only. In an exploratory paired exome subset, high-mutational-burden tumors transitioned from proliferative to immune-engaged states, while low-burden tumors accumulated extracellular matrix and myeloid signatures suggesting fibrotic microenvironments. The data define distinct immune architectures in DLBCL across a spectrum of HIV-associated immune dysregulation. HIV and ART exposure appear to shape the DLBCL TIME in a manner that may reflect the degree of immune reconstitution during lymphomagenesis. The immunosuppressive, checkpoint-enriched microenvironment of ART-experienced tumors supports further investigation of immune checkpoint blockade and macrophage-targeting strategies in a population historically excluded from such trials.