Poster Presentation Australasian Society for Immunology Annual Scientific Meeting 2014

Test of transplant tolerance by in vitro assay of CD4+CD25+T cells (#201)

Catherine Robinson 1 , Bruce M Hall 1 , Karren M Plain 2 , Nirupama D Verma 3 , Rochelle Boyd 4 , Masaru Nomura 1 , Suzanne J Hodgkinson 1
  1. UNSW Medicine, Eveleigh, NSW, Australia
  2. Veterinary Science, The University of Sydney, Camden, NSW, Australia
  3. Dendritic Cell Biology and Therapeutics Program, ANZAC Research Institute, Concord Repatriation General Hospital, Concord, NSW, Australia
  4. The Australian School of Advanced Medicine, Macquarie University, Macquarie park, NSW, Australia

Aim  To compare the reactivity of tolerant and naïve CD4+, CD4+CD25+ and CD4+CD25- T cells.  Antigen (Ag) specific Treg require Ag stimulation and cytokines to survive, thus we examined which Th1 and Th2 cytokines were required. 

Methods   Cells from DA rats tolerant to fully allogeneic PVG hearts for >100 days were assayed in MLC.  Different Th1 or Th2 cytokines were added to MLC and cytokine receptor expression assayed by RT-PCR.

Results Peripheral T cells subsets were similar in naïve and tolerant: CD4+(49v42.5%), CD8+(16v15.5%), CD4+CD25+(6.1v5.6%) and CD25+Foxp3+(10.9v9.1%).   Serial dilution showed similar proliferation of both naïve and tolerant CD4+T cells to PVG and to third party Lewis. CD4+CD25-T cells proliferated less to PVG than to Lewis. Tolerant CD4+CD25+ cells did not respond to PVG, and were similar to self but proliferated to Lewis similar to naïve CD4+CD25+T cells proliferation to PVG and Lewis.  Proliferation of tolerant CD4+CD25+ T cells was enhanced to PVG but not to Lewis or self, by IL-5 or interferon-gamma (IFN-γ) but not by IL-10 or IL-13.  IL-2 and IL-4 induced non-Ag specific proliferation.  Tolerant CD4+CD25+T cells had enhanced expression of the receptor for IFN-γ (ifngr) and IL-5 (il5ra) compared to naïve CD4+CD25+T cells.  After culture with IL-5, il5ra expression was retained but not ifngr. After culture with IFN-γ, ifngr expression was retained and il5ra lost.

Conclusions Tolerant hosts have activated antigen specific CD4+CD25+Treg that express receptors for IFN-γ and IL-5 and require these cytokines to support their survival.  These in vitro assays may be developed further to diagnose transplant tolerance.