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Mechanisms of Alloantigen-Specific Regulation After Donor Specific Transfusion and Costimulatory Blockade in YFP-Foxp3/TdTomRed-Ebi3 Marker Mice.

Y. Tomita,1 W. Bracamonte-Baran,1 D. Vignali,2 W. Burlingham.1

1Department of Surgery, Division of Transplantation, University of Wisconsin-Madison, Madison, WI
2Department of Immunology, University of Pittsburgh, Pittsburgh.

Meeting: 2016 American Transplant Congress

Abstract number: 89

Keywords: Allorecognition, T cells, Tolerance, Transforming growth factor-beta (TGF-b)

Session Information

Date: Sunday, June 12, 2016

Session Name: Concurrent Session: Allograft Tolerance 1: Animal Models

Session Time: 4:30pm-6:00pm

 Presentation Time: 5:42pm-5:54pm

Location: Room 306

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Background: In mice, donor specific transfusion (DST) plus anti-CD40L costimulatory blockade (MR-1) treatment is a standard and successful protocol to induced donor-specific transplant tolerance. Hypothesis: The internal Ebi3 (iEbi3)-producing, Foxp3+ Tregs are the main source of surface Ebi3 (sEbi3) acquired by non-producing, “bystander” CD4 T cells after DST plus MR-1. Methods: CBA (H2k) spleen cells were injected i.v. on d.0 into a two types of double reporter transgenic mice (C57BL/6; H2b background): 1) ones which expressed YFP under the Foxp3 and TdTomRed under the Ebi3 promoter, and 2) ones in which both reporters were present, but Ebi3 was knocked out, such that no protein was expressed in Foxp3+ T cells [Foxp3Cre x Ebi3loxp]. MR-1 was injected i.p. into the mice of 125mg dose on d.0, 2, and 4. Mice were sacrificed on d.14 and 35 spleen and lymph nodes were harvested. In order to investigate mechanisms of alloantigen-specific regulation induced by IL35 positive Tregs, we used a novel flow-cytometry assay for sEbi3 expression by Treg cells. Results: In lymph node cells cultured overnight, we observed increased iEbi3 (p<0.05) paralleled by a slight but significant increase inFoxp3+CD25+ Tregs (p<0.01) in cultures pulsed with CBA, but not B6 or DBA/2 (3rd party) soluble antigen. The sEbi3+and surface TGFβ+ CD4 T cells were detected in much greater population in 24 hr culture with allo-specific CBA-Ag on d.35 (p<0.001). By Image Stream population microscopy analysis, the sEbi3 appeared to be secreted as exosomes by the Tregs and captured by bystander CD4 non Treg cells. In the FoxpCre x Ebi3loxp mice, freshly harvested spleen and lymph node cells on d.35 showed a 10-fold lower level of sEbi3+ population than the level in the normal double reporter mice. Interestingly, sEbi3 expression in the presence of CBA-Ag with 24-hour cultured in vitro was also eliminated by Foxp3-targeted deletion of Ebi3 gene expression. Conclusions: These results suggest that bystander memory CD4 T cells, which may amplify the regulatory response, can acquire Ebi3 from exosomes released from allo-specific Tregs developed during the DST plus MR-1 treatment Ebi3 deletion in Foxp3 Tregs eliminated the sEbi3 response, indicating that Tregs are the primary source of IL35 in allo-specific regulation.

CITATION INFORMATION: Tomita Y, Bracamonte-Baran W, Vignali D, Burlingham W. Mechanisms of Alloantigen-Specific Regulation After Donor Specific Transfusion and Costimulatory Blockade in YFP-Foxp3/TdTomRed-Ebi3 Marker Mice. Am J Transplant. 2016;16 (suppl 3).

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To cite this abstract in AMA style:

Tomita Y, Bracamonte-Baran W, Vignali D, Burlingham W. Mechanisms of Alloantigen-Specific Regulation After Donor Specific Transfusion and Costimulatory Blockade in YFP-Foxp3/TdTomRed-Ebi3 Marker Mice. [abstract]. Am J Transplant. 2016; 16 (suppl 3). https://atcmeetingabstracts.com/abstract/mechanisms-of-alloantigen-specific-regulation-after-donor-specific-transfusion-and-costimulatory-blockade-in-yfp-foxp3tdtomred-ebi3-marker-mice/. Accessed March 7, 2021.

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