Fig 2: Cytokine staining of CD45.2+ CD8+ T cells of adoptive transfer modelPurified OT-1 CD8+ cells are adoptively transferred to CD45.1 mice and then the recipient mice are infected with LmOVA (Figure 5). 7 Days later, collect the splenocytes and restimulate them with PMA/Ionomycin and stain the cytokine.(A) FACS gating strategy of cytokine producing CD45.2+ CD8+ T cells.(B) Production and number of IFN-γ+ or GzmB+ CD45.2+CD8+ T cells from mice treated with PBS (-) (with PMA/Ionomycin) or infected with LmOVA with or without PMA/Ionomycin as indicated. All data are the mean ± s.e.m.; n = 5 independent wells per experiment. Two-tailed Student’s t test; ∗∗∗∗p < 0.0001.

Fig 3: Tissue-resident cells mediate the increase in muscle fiber size in the A3R−/− TA muscle.A Representative scatter plots of CD45.1 and CD45.2 stained blood-derived leukocytes from bone marrow-transplanted mice. B Mean and median CSA values and fiber size repartition of 22-day regenerated TA muscles isolated from wild-type mice transplanted with wild-type or A3R−/− bone marrow. C Mean and median CSA values and fiber size repartition of 22-day regenerated TA muscles isolated from wild-type or A3R−/− mice transplanted with wild-type bone marrow. D Number of muscle-infiltrating CD45+ cells isolated from the regenerating TA muscles of bone marrow-transplanted mice on day 2 post-CTX-injury. E Number of muscle-infiltrating CD45+ cells on day 2 post-CTX-injury in wild-type TA muscles co-injected either with MRS1191, an A3R antagonist (2 mg/kg), or IB- MECA, an A3R agonist (100 μg/kg). F Expression of adenosine receptors in muscle-derived CD31+ cells at various times following CTX-induced injury determined by RT-qPCR. Mean and individual values are shown, and statistical significance was determined by two-tailed Student’s t-test and ANOVA. *p < 0.05, **p < 0.01 (n = 6 in panels (B and C), n = 4 in panels (D, E and F)).

Fig 4: Increased leukocyte infiltration and altered macrophage polarization in regenerating A3R−/− TA muscles.A Alterations in the number of CD45+ leukocytes per injured muscle and B percentage of anti-F4/80 antibody stained MΦs within the CD45+ leukocyte population in A3R+/+ and A3R−/− TA muscles during the first 4 days of regeneration following CTX-induced injury (n = at least 4 samples). C MCP-1 mRNA expression levels in wild-type or A3R null TA muscles determined by RT-qPCR following CTX-induced injury (n = 4). D Representative scatter plots of CD206/Ly6C-stained and E MHCIIhigh muscle‐derived F4/80+ cells determined at the indicated days following CTX‐induced injury in the TA muscles of A3R+/+ and A3R−/− mice. F Percentages of Ly6C+, CD206+, and MHCIIhigh cells within the muscle‐derived F4/80+ population determined at the indicated days following CTX‐induced injury in the TA muscles of A3R+/+ and A3R−/− mice (n = 4). Data are expressed as mean and individual values, and statistical significance was determined by two-tailed Student’s t-test. *p < 0.05, **p < 0.01 (n = 4).

Fig 5: Loss of A3R impacts the gene expression of the muscle-derived CD45+ cells.A Single-cell analysis showing the expression of various adenosine receptors in CD45+ cells isolated from TA muscles of wild-type mice at day 4 post-CTX injury. 1. Resolution-related MΦs, 2. Growth factor-expressing MΦs, 3. Pro-inflammatory MΦs, 4. Antigen-presenting MΦs. 5. Neutrophils, 6. CD8a+ Clec9a+dendritic cells, 7. Fibroblasts, 8. Fibroblast-like cells, 9. Myeloid CD8a- dendritic cells, 10. Endothelial cells, 11. T cells, NK-T cells, 12. Lymphoid CD8a+ dendritic cells. B mRNA expressions of various adenosine receptors and inflammatory markers in muscle-derived CD45+ cells isolated at various time points post-CTX injury detected by RT-qPCR. Data are expressed as mean and individual values and statistical significance was determined by two-tailed Student’s t-test and ANOVA. *p < 0.05, **p < 0.01 (n = 4).