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Inflammatory Mediators Modulate Alloreactive T Cell Susceptibility to Immune-Regulation in Reconstructive Transplantation
Saami Khalifian, B.A.1, Yawah Nicholson, B.S.2, Cordelia Ziraldo, Ph.D.2, Ravi Starzl, Ph.D.3, Stefan Schneeberger, M.D.1, Angus Thomson, Ph.D., D.Sc.2, Gerald Brandacher, M.D.1, Yoram Vodovotz, Ph.D.2, Giorgio Raimondi, Ph.D.1.
1Johns Hopkins University School of Medicine, Dept. of Plastic & Reconstructive Surgery, Baltimore, MD, USA, 2University of Pittsburgh Medical Center, Starzl Transplantation Institute, Pittsburgh, PA, USA, 3Language Technologies Institute, Carnegie Mellon University, Pittsburgh, PA, USA.
PURPOSE: Tolerance induction to allografts in reconstructive transplantation remains an elusive goal, partly due to limitations in understanding the interplay between inflammatory mediators and activation/regulation of T lymphocytes. We believe specific inflammatory mediators contribute to the activation of alloreactive T cells via previously unappreciated mechanisms: i), direct costimulation, ii) simultaneous reduction of Treg suppressive activity, thereby limiting the effectiveness of tolerogenic regimens. We aimed to identify these inflammatory mediators that diminish T cell regulation and identify strategies to blunt their effect.
METHODS: In vitro assessment of inflammatory mediators and Treg activity on T cell activation and proliferation was determined via CFSE-suppression assay. The expression of 22 cytokines in the supernatant of maturing dendritic cells was quantified using Luminex. Bioinformatics analysis was used to determine which cytokines most significantly contributed to modulation of Treg suppression, and resultant cytokines were then tested in the CFSE-suppression assay. The immunomodulatory activity of specific cytokines in vivo was tested in a mouse model of inflammatory bowel disease (outcome measure=weight loss). Specifically, Rag-knockout mice were adoptively transferred with T cells (+/- Tregs), followed by injections of anti-IL-6R mAb or IL-1R antagonist (IL-1RA). To further dissect the role of IL-1 family members (e.g. IL-18), the experiment was repeated with T and Treg from MyD88 knockout mice. Production/accumulation of inflammatory cytokines in various tissues was also investigated after orthotopic hindlimb transplantation (OHTx) using Luminex at various timepoints.
RESULTS: Bioinformatics identified IL-6 and IL-1α/β as potential modulators of Tcell/Treg activity. In vitro analysis confirmed that all three cytokines directly promoted T cell proliferation; however, IL-1α/β also directly inhibited Treg suppressive activity. In vivo analysis in Rag-/- mice confirmed that blockade of IL-6R after adoptive transfer of Tcells/Treg resulted in significantly delayed weight loss in this model (not observed without Treg injection), suggesting that blockade of IL-6 signaling sufficiently slowed activation of T cells permitting Treg to effectively control disease development. Unexpectedly, blockade of IL-1R did not delay disease progression, likely due to redundant function of IL-1 with IL-18. Therefore, the experiment was repeated using T and Treg from MyD88-/- mice (IL-1 and IL-18 use MyD88 for intracellular signaling) and only when MyD88 was absent from both injected populations (Tcells and Tregs) was there a significant delay in weight loss, corroborating our in vitro observations that IL-1α/β (and now IL-18) have a counter-regulatory effect on Tregs/Tcells. IL-6, IL-1α/β, and IL-18 were all confirmed to accumulate in various tissues after OHTx--further evidence of their role in the rejection response.
CONCLUSION: Specific inflammatory cytokines promote activation of alloreactive T cells and reduce T cell susceptibility to suppression. We have identified the synergistic activities of IL-6, IL-1α/β, and IL-18, which must be taken into consideration when designing novel therapeutic modalities. Furthermore, our VCA model provides clear evidence that these cytokines play an important role in promoting rejection. Targeting these inflammatory mediators should translate into improved immunological outcomes post-VCA, and elucidation of the intracellular mechanisms used by these cytokines to prevent T cell suppression will aid in identifying targets that promote immuneregulation.
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