CD39SP exhibits a proinflammatory TH phenotype and stimulates B-cell proliferation. Spleens from ICPi- (red) and PBS-treated (blue) young (aged 2-3 months) HOD⁺OTII⁺ mice were collected 14 days after treatment and CD4 enriched. (A) Frequency of T cells expressing purinergic signaling molecules CD39 and CD73. (B) Representative flow plots of expression of CD39 and CD73 on total CD4⁺Vα2⁺Vβ5⁺ OTII T cells (black) and CD4⁺ FoxP3⁺CD25⁺Vα2⁺Vβ5⁺ Tregs (red) are shown. (C) Frequency of CD39⁺ CD4⁺ T cells expressing FoxP3. (D) Representative flow plots of expression of CD39 and CD73 on CD4⁺Vα2⁺Vβ5⁺ OTII T cells (black) and CD49b⁺Lag3⁺IL10⁺ Tr1 Tregs (red). These data show that CD39⁺ do not express Treg markers and will be defined phenotypically as CD4⁺CD39⁺CD73^–FoxP3^–CD25^– and referred to as CD39SP. (E-H) The frequencies of CD39SP that stained positive for (E) p-S6 and p-Akt, markers of mTOR complexes 1 and 2 activation, (F) HIF1α hypoxia transcription factor, (G) transcription factors c-MAF and T-bet, and (H) proinflammatory cytokines IFN-γ and IL-17 were determined. (I) CD39SP were sorted from ICPi- (red) and PBS-treated (blue) animals and cocultured with B cells at 1:2 ratio, previously labeled with CellTrace-FR and stimulated with HEL or HEL + OVA. CellTrace-FR dilution as measure of B-cell proliferation was assessed 3 days after stimulation. CD4⁺ T cells enriched from OTII mice (green) were used as a positive control. Data presented in panels A-H are cumulative from 4 to 5 independent experiments with 3 to 4 mice per group. Data shown in panel I are cumulative of 2 independent experiments with 4 to 5 mice per group. Statistical significance was determined by 2-way ANOVA with Sidak posttest and Mann-Whitney test: ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. HIF1α, hypoxia inducible factor 1 subunit α; mTORC, mammalian target of rapamycin complex.