Effect of mitochondrial transfer from bone marrow stroma cells to anti-tumor T cells on T cell differentiation and function

Mitochondrialloss and dysfunctiondrive T cell exhaustion, representing major barriers to successful T cell-based immunotherapies. We found that bone marrow stromal cells (BMSCs) transfer stromal cell mitochondria into CD8+ T cells. CD8+ T cells with donated mitochondria displayed enhanced mitochondrial respiration and spare respiratory capacity. When transferred into tumor-bearing hosts, these supercharged T cells expanded more robustly, infiltrated the tumor more efficiently, and exhibited fewer signs of exhaustion compared to T cells that did not take up mitochondria. As a result, mitochondria-boosted CD8+ T cells mediated superior antitumor responses, prolonging animal survival. Overall design: We co-cultured activated CD8+ T cells from 3 donor mice with BMSCs in which mitochondria are stably labeled with dsRed. After co-culture, we sorted dsRed+ (MitoPositive) T cells that had taken up mitochondria from BMSCs and dsRed-negative (MitoNegative) T cells. Sorted T cells were either processed for immediate scRNA-Seq ("d0") or transferred into B16KVP tumor-bearing B6 mice and re-isolated by flow cytometry on d7. For multiplexing of d0 samples, biological replicates were marked using hashtags 1,3,5 and 2,4,6 for MitoPositive and MitoNegative cells, respectively. Transferred T cells were isolated from spleen (#1 and #2) and tumor (#3 and #4 for MitoPositive and MitoNegative T cells, respectively) and pooled by donor after mulitplexing.