*Purpose: Despite a growing body of work on type 1 interferon (IFNAR1) signaling, there is a lack of consensus on the immunological properties of type I interferons and most notably their modulation of T-cell responses. In the context of viral infections, IFNAR1 signaling was reported as essential to T-cell-mediated antiviral immunity. Recently, however, we found a non-redundant role of IFNAR1 signaling in driving the expansion of regulatory T cells (T_Regs) in patients with worsened progression of multiple myeloma. Here, we report how dose- and time-dependent IFNAR1 signaling results in a spectrum of immunomodulatory effects and altered T-cell responses.

*Methods: In vitro. Splenic CD4+CD25- helper T cells (TH) were isolated and cultured with α-CD3/CD28, +/-IL2, and +/-IFN-β, at varying concentrations and time-points. Wild type C57BL/6 (B6) cells were stimulated for 72 hrs, and analyzed by flow cytometry. In vivo. B6.Rag1-/- mice were transplanted with BALB/c skin and injected with CD3+CD25- B6 T cells and treated five days daily with IFN-ß. Seven days post adoptive transfer, draining lymph node T cells were analyzed by flow cytometry. Statistics. Independent samples two-tailed Student’s t-tests were used.

*Results: In in vitro CD4 TReg induction assays, we observed that IFNAR1 signaling differentially affected the TGF-β-mediated conversion of CD4 T cells to TRegs, notably depending on the strength of interleukin (IL-)2 signaling. Intriguingly, the TReg conversion was suppressed in the absence of IL-2 signaling, while increasing dosages of IL-2 normalized, and ultimately enhanced TReg conversion (P<0.001). The dependence of type 1 interferons on IL-2 is in line with previous findings that the effects of IFNAR1 signaling are enhanced in competitive, cytokine-rich microenvironments. Additionally, we observed an essential role for the chronology of IFNAR1 signaling in determining the T-cell fate. Delayed IFNAR1/IL-2 signaling promoted pro-inflammatory T cell proliferation, compared to synchronous TCR/CD28 and IFNAR1/IL-2 signaling, and more importantly attenuated TReg conversion only in the type I interferon-treated condition (P<0.0001). To harness the regulatory arm of the immune system, we pursued synchronous type I interferon treatment in an in vivo skin transplant model (10,000 U/day IFN-β vs. PBS sham, n=5/group). Seven days post the adoptive transfer of mismatched B6 CD3+ cells, we found a five-fold increase in draining lymph node (DLN) TRegs (P<0.01), along with a 24.8% and 42.6% decrease in DLN effector CD44+CD62L- CD4+ (P<0.05) and CD8+ cells (P<0.05) respectively.

*Conclusions: In all, we describe the importance of dosage and timing to the possible immunomodulatory effects of IFNAR1 signaling, and the therapeutic potential of these agents in mitigating alloimmunity.

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