Sézary syndrome (SS) is an aggressive leukemic variant of cutaneous T-cell lymphoma characterized by immune dysregulation and constitutive STAT3 activation. CD39, an ectonucleotidase encoded by the ENTPD1 gene, regulates extracellular ATP metabolism, contributes to tumor-associated immunosuppression and is upregulated in SS. However, the mechanism driving CD39 overexpression remains unclear. Given that STAT3 has been reported to regulate CD39 expression in Th17 cells, we hypothesized a functional link between CD39 and JAK/STAT3 signaling in SS.

Primary PBMCs and purified CD4+ T cells from SS patients and healthy donors (HD), along with SS cell lines (MyLa, HUT-78, HH), were used for functional and molecular analyses. Cells were stimulated with anti-CD3/CD28 or IL-2, with or without POM-1 (CD39 inhibitor), Stattic V (STAT3 inhibitor), or Ruxolitinib (JAK inhibitor). Experimental interventions included flow cytometry, Western blot, RT-qPCR, ChIP assays, ATP consumption, and PrestoBlue viability assays.

In SS cell lines, higher CD39 expression correlated with elevated STAT3 phosphorylation at Try705. Consistently, CD39⁺CD4⁺ T cells from SS patients displayed significantly higher p-STAT3 compared to CD39⁻ SS cells. ChIP analysis in HUT-78 cells demonstrated direct STAT3 binding to the ENTPD1 promoter, supporting transcriptional regulation of CD39 by STAT3. Functionally, CD39 silencing in HUT-78 cells reduced basal STAT3 phosphorylation, ATP hydrolysis ability, and cell proliferation. In primary PBMCs and CD4+ T cells, CD3/CD28 and IL-2 stimulation enhanced JAK/STAT3 signaling, whereas CD39 inhibition with POM-1 selectively decreased STAT3 phosphorylation in CD39⁺ SS cells but not in HD. Conversely, inhibition of STAT3 using Stattic V reduced both STAT3 activation and CD39 expression in SS cell lines. Ruxolitinib, a JAK inhibitor currently in phase II clinical evaluation for T cell lymphomas, preferentially reduced STAT3 phosphorylation in CD39+ cell lines (MyLa and HUT-78), while no significant effect was observed in the CD39– HH cell line. Together, our data support a bidirectional interaction between CD39 and JAK/STAT3 signaling in SS.

These results suggest a functional link between CD39-mediated adenosinergic signaling and JAK/STAT3 activation in SS. Targeting this axis may offer a novel therapeutic strategy to impair tumor survival and overcome immunosuppression in CD39+ SS patients.

This research was funded by the Research Projects of National Relevant Interest (PNRR). LL’s scholarship is funded by the China Scholarship Council.