Myeloid subclustering resolved 19 distinct populations (Figure 3D), including dendritic cells (DCs), monocytes, neutrophils, and various macrophage subsets (Figure S3C, Table S4). Rapamycin altered macrophage composition, increasing Il1b^hi inflammatory macrophages while reducing Nlrp3^hi and Ccr2^hi subsets, and decreasing Trem2^hi macrophages, whereas foam-like macrophages were slightly increased. In line with our flow cytometry data, neutrophil clusters were consistently reduced, while dendritic cells and mast cells were not affected. Although subset-specific variation was observed, inflammatory gene signatures (Table S5) within Nlrp3^hi and Mox macrophages were overall reduced in rapamycin-treated mice (Figure 3E).

B cell and plasma cell subclustering identified 10 different subsets (Figure 3F, Figure S3D, E, Table S6). While most B2-like (cluster 0 and 1; Fcer2a, Cr2), activated (cluster 3; Myc^hi Egr3^hi), and memory (cluster 4; Bach22⁺, Cd83⁺) B cell populations were relatively unchanged, rapamycin reduced resting B cells (cluster 5; ribosomal genes) and B1/Breg-like cells (cluster 2; S100a6, Ebi3, Cd9). Strikingly, cluster 6, enriched for proliferating germinal center (GC) B cell markers (Mef2b, Aicda, Fas, Mki67), suggesting these cells are derived from GCs, were nearly absent following rapamycin treatment, accompanied by a reduction in plasma cells (cluster 9; Jchain, Prdm1, Sdc1, Xbp1). Consistently, expression of plasma cell-and immunoglobulin-related genes (e.g., Jchain, Sdc1, Iglc1, and Ighg2c) was reduced (Figure 3G), indicating suppression of antigen-driven humoral responses.

In-depth analysis of conventional T cells revealed 14 subpopulations (Figure 3H, Table S7), broadly separating into CD4⁺ and CD8⁺ T populations (Figure S3F, G). Rapamycin drastically reduced effector memory CD4⁺ (cluster 6; Cd44^hi, Sell⁻, Jun^low) and CD8⁺ (clusters 0 and 7; Gzmk⁺, Tox^hi, and cluster 3; Gzma^hi, Gzmb^hi) T cell subsets, while increasing naïve (clusters 1 and 4; Cd44^low, Sell⁺, Jun⁺) and central memory (cluster 5: Cd44⁺, Sell⁺) subsets. Proliferating (cluster 11; Mki67, Top2a, Hells) and IFN-induced CD8⁺ T cells (cluster 13; Ifit1, Ifit3, Ifi204) remained largely unaffected, whereas apoptotic/dying CD8⁺ T cells (cluster 8; mitochondrial genes, Malat1) were nearly absent after rapamycin treatment. Importantly, Tregs (cluster 9; Foxp3, Tnfrsf4, Ctla4) were preserved and showed elevated expression of Foxp3 and Tgfb1 (Figure 3I), together with enrichment of IL-10 and CTLA-4 signaling pathways (Figure 3J), indicating enhanced immunoregulatory activity.