Treg Tissue Stability Depends on Adenosine Receptor Driven Lymphatic Endothelial Cell Responses

V. Saxena¹, W. Piao¹, L. Li¹, M. W. Shirkey¹, R. Lakhan¹, S. Walden², K. L. Hippen², B. R. Blazar², J. S. Bromberg¹

¹University of Maryland School of Medicine, Baltimore, MD, ²University of Minnesota, Minneapolis, MN

Meeting: 2022 American Transplant Congress

Abstract number: 385

Keywords: Endothelial cells, Graft survival, Mice, knockout, T cells

Topic: Basic Science » Basic Science » 10 - Treg/Other Regulatory Cell/Tolerance

Session Information

Session Name: Immunoregulation and Tolerance

Session Type: Rapid Fire Oral Abstract

Date: Monday, June 6, 2022

Session Time: 5:30pm-7:00pm

Presentation Time: 6:40pm-6:50pm

Location: Hynes Room 302

*Purpose: Regulatory T cell (Treg) lymphatic migration and maintenance of transcription factor Foxp3 expression are required for suppressor function and allograft protection. Migrating Tregs were found to be ectonucleotidase CD39^hi, important for generating adenosine. We tested the hypothesis that adenosine is important for Treg migration and sustaining Foxp3 expression.

*Methods: Treg stability, migration and function were analyzed in vivo in islet allograft and footpad migration models, and in vitro in a transwell based lymphatic endothelial cell (LEC) migration assay. Tregs were analyzed by flow cytometry and immunofluorescence microscopy.

*Results: Treg sequential migration from the target tissue to the draining lymph nodes (dLN) was required for maturation and optimal execution of suppressor function. During afferent lymphatic migration, Treg LTα1β2 signals LTβR on LEC. Disruption of Treg LTα1β2-LEC LTβR interactions resulted in impaired islet allograft survival from 25d to 15d in LTβR^-/- mice (p<.03) and to 13d in mice receiving LTα^-/- Treg (p<.03). Treg were retained in the graft with poor Treg migration from tissues to the dLN. Non-migrating Tregs were converted to Foxp3^loCD25^lo exTreg. In a transwell based Treg-LEC co-culture assay, conversion to exTreg was accompanied by increased methylation at the Foxp3 locus and decreased suppressor function. The non-migrating Treg became exTreg and lost Foxp3, CD25, LTαβ, and ectonucleotidase CD39 expression. CD39 catalyzes adenosine production, and supplementation with adenosine to cultures or in footpad migration assays prevented exTreg conversion in an adenosine receptor 2A (A2AR) dependent manner. Adenosine altered LEC structure, integrity, and permeability. Since LTβR stimulates the NFκB classical pathway to secrete IL-6, blockade of NFκB or neutralization of IL-6 prevented exTreg conversion, and adenosine altered IL-6 levels. Role of adenosine was confirmed with human Tregs migrating across human LEC.

*Conclusions: Treg-LEC LTαβ-LTβR interactions are important for Treg migration, stability, and suppressor function. Disruption of the interactions results in poor migration, increased retention in grafts, loss of Foxp3 expression, and thereby the ability to protect the allograft. The exTreg have the potential to become T effector cells and cause allograft rejection. ExTreg conversion can be prevented therapeutically by inhibiting IL-6 or LTβR NFκB, or by stimulating A2AR.

To cite this abstract in AMA style:

Saxena V, Piao W, Li L, Shirkey MW, Lakhan R, Walden S, Hippen KL, Blazar BR, Bromberg JS. Treg Tissue Stability Depends on Adenosine Receptor Driven Lymphatic Endothelial Cell Responses [abstract]. Am J Transplant. 2022; 22 (suppl 3). https://atcmeetingabstracts.com/abstract/treg-tissue-stability-depends-on-adenosine-receptor-driven-lymphatic-endothelial-cell-responses/. Accessed May 16, 2025.

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