Azithromycin Induces the Expansion of Regulatory T Cells and Diminishes Alloreactive T Cell Proliferation in Vitro and in Vivo

Background: Donor T cell activation in response to alloantigens presented by host antigen presenting cells (APC) is a critical step in the pathophysiology of Graft Versus Host Disease. The macrolide Azithromycin (Azi) is studied in lung transplant and allogeneic hematopoietic cell transplant (HCT) recipients to decrease graft rejection and progression of lung injury, respectively. We now tested whether Azi plays a role in the modulation of adaptive immune responses by determining its effects on alloantigen-dependent T cell activation.

Methods:

In vitro: Mixed lymphocyte reactions (MLR) using C57BL/6 (H2b) T cells as responders and B6D2F1 (H2bd) or BALB/c (H2d) splenocytes as stimulators, as well as TCR dependent, alloantigen-independent CD3/CD28 or lectin-type stimulation (Concavalin) assays of C57BL/6 Tcells were performed in the absence or presence of Azi at varying concentrations 5µg/ml, 10µg/ml, 20µg/ml, 50µg/ml. T cell proliferation was assessed by 3HT-uptake. T cells were phenotyped and supernatant was tested for cytokine expression.

In vivo: B6D2F1 were pretreated for 2 weeks with Azi at a dose of 3mg/ml drinking water or received untreated water as control. Then, animals were conditioned with 12Gy TBI and transplanted with 4X10E6 bone marrow cells and 6X10E6 splenocytes from allogeneic C57BL/6 donors. Recipients continued to receive Azi-supplemented or untreated water, and on day+7, splenic T cell expansion and T cell phenotyping were done.

Results:

Alloreactive T cell proliferation in vitro showed an Azi dose-dependent decline at 96 hours along with reductions in TNF, IFNg, IL-2, IL-6, L-10, IL-17. Interestingly, a significant relative expansion of CD4+FOXP3+ T cells (Tregs) within the CD4+ T cell fraction was observed with increasing Azi-levels from 2.5±0.1% (0µg/ml) to 5.0±0.2% (50µg/ml). No differences in T cell proliferation were seen when T cell stimulation occurred APC-independent either via Concavalin A or via CD3/CD28 except for slight suppression at Azi 50 µg/ml.

In vivo, when compared to controls, pretreatment with Azi resulted in decreased splenic expansion of both CD4+ T cells (2.1X10E6±0.2 vs. 4.3X10E6±0.6, p<0.01) and CD8+ T cells (4.9X10E6±0.5 vs. 6.4X10E6±0.4, p<0.05) along with a slight and non-significant increase of Tregs (1.5± 0.3% vs 1.1± 0.1%).

Conclusion: Azithromycin suppresses alloreactive T cell response in vitro and in vivo, paralleled by the induction of Tregs, supporting its use in lung transplantation and allogeneic HCT