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Ex-vivo generation of regulatory T-cells expressing transcription factor foxp3 and their application in cornea transplantation

Creator:

Maenz, Martin;

Subject Keywords: Immunology; Cornea; Transplantation; Immune-modulation; Regulatory T-cells;
Region:
Description:

This project investigated the therapeutic potential of allo-antigen specific regulatory
T-cells in the context of penetrating keratoplasty. Two methods of ex-vivo generation
of T regulatory cells have been explored in this study. Using recombinant retroviruses
gene transfer of the transcription factor foxp3 was achieved into allo-antigen specific
primary rat T-cells. However, expansion of foxp3 gene modified cells necessary for
in-vitro and in-vivo studies, was found to be too cumbersome. The second approach
exploited a non-depleting anti-CD4 antibody. Allo-primed mixed lymphocyte cultures
were treated with low-dose anti-CD4 antibody resulting in an outgrowth of foxp3
positive T-cells. CFSE labelled responder T-cells targeted with anti-CD4 treatment
indeed confirmed preferential growth of regulatory T-lymphocytes. Tregs generated
by this procedure were studied by flowcytometry and found to express high levels of
CD25, Ox-40 and ICOS, confirming their suppressor T-cell phenotype. To study Treg
effects on the outcome of allo-graft survival, a full allogeneic cornea transplant model
was established. Two different strain combinations were tested. First, a high
responder LEW-DA model was set-up but was found to be too fragile and unreliable
to serve as a preclinical model. A second novel strain combination was tested and
discovered to be of low-responder characteristics and remarkably robust and highly
suitable for in-vivo applications. The rejection process of the BN-PVG model was
comprehensively studied using flowcytometric analysis of draining lymph nodes.
Moreover, a gentle digestion protocol was established to isolate viable graft
infiltration lymphocytes. Applying multi-parameter FACS six distinct cell populations
were observed among which were CD8+ cytotoxic T-cells, CD4+ T-cells, CD3- CD8+/-
CD161high NK, CD3+ CD8+ CD161dull NK-T-cells, CD161dull large granular
lymphocytes and MHC-2 positive cells. Additionally a serum analysis found evidence
for IgM, IgG1 and IgG2a allo-antibodies in rejection animals. Finally ex-vivo
generated Tregs were tested towards their ability to prevent allo-graft rejection. CD3
sorted regulatory T-cell preparations with direct allo-antigen specificity did not
prevent or delay rejection of allogeneic corneal grafts. The implications of this finding
for the clinical application of adoptive Treg therapies have been discussed in detail.

Suggested citation:

Maenz, Martin; . () Ex-vivo generation of regulatory T-cells expressing transcription factor foxp3 and their application in cornea transplantation [Online]. Available from: http://publichealthwell.ie/node/642774 [Accessed: 15th October 2019].

  

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