View Abstract

A4731 - Calcineurin-Inhibition Drives IL13 and IL5 Production in Differentiating and Memory T Cells in a Ca++-Dependent Manner
Author Block: U. Gehrmann, K. Bjorhall, J. Jirholt; AstraZeneca, RIA IMED, Mölndal, Sweden.
RATIONALE
The classical Th2 cytokines interleukin 13 and 5 play an important role in the development of allergic diseases due to their role in leukocyte recruitment and tissue remodeling. Phase II clinical studies testing the potency of neutralizing antibodies to both cytokines in asthma patients are currently ongoing with promising results. However, IL13 has also been implicated as a major driver of tissue fibrosis, a hallmark of non-Th2 chronic lung diseases such as bronchiolitis obliterans (BOS). Strikingly, 50% of lung transplant recipients develop fibrotic disease 5-years post-transplantation despite immunosuppressive treatment. Standard immunosuppression is based on Calcineurin (CNI)-inhibition and it has recently been proposed that side-effects of CNI-inhibition might contribute to the development of lung fibrosis.
METHODS
Naïve and memory T cells were isolated from blood of healthy donors and activated with aCD3/aCD28 coated Dynabeads and cytokines cocktails for naïve T cell differentiation in the presence or absence of Calcineurin inhibitors for up to 5 days. Calcium-flux and intracellular cytokine production was assessed by flow cytometry, cytokine secretion was measured in supernatants using MSD technology and qPCR was performed on isolated RNA from activated T cells using primers for IL13, IL5 and other relevant cytokines and controls.
RESULTS
We report that low-dose inhibition of Calcineurin by Cyclosporine A or related compounds strongly enhances production of IL13 from activated memory CD4 T cells. Strikingly, naïve CD4 T cells that were differentiated in vitro into effector Th1, Th2, Th17 or regulatory T cells in the presence of low dose Cyclosporine A increased production of IL5 and IL13 by up to 50-fold, both on mRNA and secreted protein level while expression of IL4, IL17 and IFNg were strongly decreased. Increased IL13 transcription was detected as early as 12hrs after TCR ligation and suggests a cell intrinsic effect of Calcineurin. Pre-treatment of T cells with Cyclosporine A increased Ca++-flux upon TCR activation and inhibition of CRAC1 channels on the cell surface blocked the Cyclosporine A based IL13-induction, suggesting that this effect was dependent on the influx of extracellular Ca++.
CONCLUSIONS
Together, these results suggest that immunosuppressive regimens based on Calcineurin inhibition might contribute to development of fibrosis in the lung and potentially other tissues due to the induction of IL13 in T cells. Understanding the signaling pathway downstream of Calcineurin inhibition will be important to understand the molecular mechanisms behind IL13-mediated fibrosis and might lead to the identification of new treatment targets for fibrotic diseases.