Background/Purpose: Regulatory T cells (Treg) are necessary for immune homeostasis but are functionally deficient in inflammatory/autoimmune diseases such as rheumatoid arthritis (RA). On the other hand, polymorphonuclear neutrophils (PMN) are known for their proinflammatory properties and are highly recruited at inflammation sites such as RA joints. However, recent evidences show that PMN exert also immunoregulatory functions. We hypothesized that Treg/PMN interact and that this interaction is dysregulated in RA. We aimed at deciphering the phenotype and activity of both cells upon in vitro Treg/PMN co-cultures and characterized the mechanisms involved. Finally, we analyzed the fate of Treg/PMN interaction in RA patients.

Methods: Splenic mouse Treg and bone marrow PMN (C57BL/6 mice) as well as peripheral blood Treg and PMN of healthy donors and RA patients were freshly purified by magnetic sorting. Co-cultures were unstimulated or exposed to anti-CD3/anti-CD28 antibodies and/or LPS. CD4⁺FoxP3⁺ Treg (mouse and human), Ly6G⁺ (mouse) and CD66b⁺ (human) PMN were identified by flow cytometry. Cell activation was further studied by flow cytometry using antibodies against CD25, CTLA-4 (Treg) and CD11b, CD39, PD-L1 (PMN). Treg maintenance was evaluated as the frequency of FoxP3 expression among CD4⁺ cells, and cell proliferation by CFSE staining followed by flow cytometry analysis. In some case, co-cultures were performed using transwell or Tofacitinib (a JAK inhibitor). Cytokine levels were quantified in culture supernatants by ELISA. Collagen-induced arthritis (CIA) was induced by immunization with type II collagen in complete Freund’s adjuvant.

Results: Upon co-culture in vitro with PMN, Treg proliferated, showed increased maintenance of FoxP3 expression and up-regulated CTLA-4 as well as CD25 compared to Treg cultured alone, only when both Treg and PMN were stimulated. Reciprocally, upon co-culture with Treg, PMN were activated and expressed regulatory molecules, as evidenced by CD11b, CD39 and PD-L1 up-regulation, as compared to PMN cultured alone, when both cell types were stimulated. The co-culture led to higher secretion of MIP-2/IL-8, IL-6 and IL-17. All these effects were abrogated when co-cultures were done using transwell or when blocking JAK-STAT signalling. Most importantly, PMN sustain Treg suppressive effect on effector (CD4⁺FoxP3^–) T cells (inhibition of cell proliferation). Similar results were observed in co-cultures of PMN/Treg isolated from mice (both naïve and CIA mice) and healthy donors. However, and importantly, we found out that PMN from RA patients were unable to increase CTLA-4 expression on Treg in co-culture, CTLA-4 being involved in Treg suppressive activity.

Conclusion: Our results reveal the existence of a new link between Treg and PMN, leading to an activation of both cell types and a more suppressive phenotype in healthy conditions. Although cell contacts are clearly required, soluble mediators are also involved and probably as a second signal. This is the first demonstration of an effect of PMN on Treg phenotype. In RA patients, we evidenced a defect in this Treg/PMN cross-talk. This Treg/PMN interaction might unveil new therapeutic targets against RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/polymorphonuclear-neutrophils-and-regulatory-t-lymphocytes-treg-cooperate-to-sustain-treg-activity-but-this-interaction-is-altered-in-rheumatoid-arthritis-patients/