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Name of Student:
Terry-Ann Curran
Research Supervisor: Dr. Aziz Ghahary
Title of Presentation: Novel finding of antigen specific immunomodulation in an IDO generated
microenvironment
Abstract
The immune system’s cytotoxic response to foreign antigens is important in relation to defence
against harmful pathogens. However, in the context of skin transplantation, immune activation
culminates ultimately in graft rejection. Immune suppression can be prescribed to prolong
graft survival, but at the expense of generalised immune deficiency and organotoxicity.
Another avenue to prolong skin graft survival is through upregulation of regulatory T cells
(Treg), the body’s natural tolerance cells. This can be achieved through the activity of the
intracellular enzyme indoleamine 2,3dioxygenase (IDO); The IDO generated
microenvironment is supportive of Treg cell proliferation and survival but toxic to CD8+ and
CD4+CD25- T cells. Treg cells recognise and are activated by specific antigens presented to
them on MCH II by antigen presenting cells. Fibroblasts are non-professional antigen
presenting cells that express specific MCH class II. Although not ubiquitously expressed, they
can be induced to express IDO on stimulation with interferon-gamma (IFN-γ). We
hypothesized that Treg cells upregulated through an allogeneic fibroblast-IFN-γ co-culture
may induce tolerance to specific allo-antigens.
Methods: Splenocytes from BalbC mice were co-cultured with C57BL/6 fibroblasts which
had been pre-stimulated with IFN-γ, for 3 days. Flow cytometry was used to determine
CD4+CD25+Foxp3 cell upregulation and expression of the surface receptor CTLA-4, to
establish functionality. CD4+CD25+ were additionally isolated using MACS and a mixed
lymphocyte reaction using BalbC CD8+ cells stained with CFSE and antigen presenting
C57BL/6 or FVB strain cells. Suppression of CD8+ cells was then determined.
Results: A three-fold increase in CD4+CD25+Foxp3 cell was seen in the IFN-γ treated
fibroblast group compared to monoculture of splenocytes and two-fold increase compared to
control fibroblasts co-culture. CTLA-4 expression was also significantly increased in the IFNγ group compared to control. Greater suppression was evidenced in the IFN-γ Treg cells in a
C57BL/6 APC- CD8+ proliferation assay with 32% non-proliferating CD8+ cells compared to
14% CD8+ cells in the control Treg cells. Antigen specificity of the IFN-γ treated fibroblast
Treg was shown by the presence of only 5% non-proliferating CD8+ cells in the FVB APC
culture compared to 32% in the C57BL/6 culture.
Conclusion: In conclusion this is a novel method of generating antigen specific tolerance in
vitro. Through this method toxic immunosuppression and non-specific Treg tolerance is
avoided. We are currently conducting our in vivo trials and looking at local Treg delivery as an
alternative to systemic Treg injection.