Protection against Bronchiolitis Obliterans Syndrome Is Associated with Allograft CCR7 + CD45RA 2 T Regulatory Cells Aric L. Gregson 1 *, Aki Hoji 2¤ , Vyacheslav Palchevskiy 3 , Scott Hu 3 , S. Samuel Weigt 3 , Eileen Liao 4 , Ariss Derhovanessian 3 , Rajeev Saggar 3 , Sophie Song 5 , Robert Elashoff 4 , Otto O. Yang 1,2,6 , John A. Belperio 3 1 Division of Infectious Diseases, Department of Medicine, University of California Los Angeles, Los Angeles, California, United States of America, 2 UCLA AIDS Institute, University of California Los Angeles, Los Angeles, California, United States of America, 3 Division of Pulmonary and Critical Care, Department of Medicine, University of California Los Angeles, Los Angeles, California, United States of America, 4 Department of Biomathematics/Biostatistics, University of California Los Angeles, Los Angeles, California, United States of America, 5 Department of Pathology and Laboratory Medicine, University of California Los Angeles, Los Angeles, California, United States of America, 6 Department of Microbiology, Immunology, and Molecular Genetics, University of California Los Angeles, Los Angeles California, United States of America Abstract Bronchiolitis obliterans syndrome (BOS) is the major obstacle to long-term survival after lung transplantation, yet markers for early detection and intervention are currently lacking. Given the role of regulatory T cells (Treg) in modulation of immunity, we hypothesized that frequencies of Treg in bronchoalveolar lavage fluid (BALF) after lung transplantation would predict subsequent development of BOS. Seventy BALF specimens obtained from 47 lung transplant recipients were analyzed for Treg lymphocyte subsets by flow cytometry, in parallel with ELISA measurements of chemokines. Allograft biopsy tissue was stained for chemokines of interest. Treg were essentially all CD45RA 2 , and total Treg frequency did not correlate to BOS outcome. The majority of Treg were CCR4 + and CD103 2 and neither of these subsets correlated to risk for BOS. In contrast, higher percentages of CCR7 + Treg correlated to reduced risk of BOS. Additionally, the CCR7 ligand CCL21 correlated with CCR7 + Treg frequency and inversely with BOS. Higher frequencies of CCR7 + CD3 + CD4 + CD25 hi Foxp3 + C- D45RA 2 lymphocytes in lung allografts is associated with protection against subsequent development of BOS, suggesting that this subset of putative Treg may down-modulate alloimmunity. CCL21 may be pivotal for the recruitment of this distinct subset to the lung allograft and thereby decrease the risk for chronic rejection. Citation: Gregson AL, Hoji A, Palchevskiy V, Hu S, Weigt SS, et al. (2010) Protection against Bronchiolitis Obliterans Syndrome Is Associated with Allograft CCR7 + CD45RA 2 T Regulatory Cells. PLoS ONE 5(6): e11354. doi:10.1371/journal.pone.0011354 Editor: Carol Feghali-Bostwick, University of Pittsburgh, United States of America Received April 16, 2010; Accepted June 8, 2010; Published June 29, 2010 Copyright: ß 2010 Gregson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work was supported by National Institutes of Health grants HL080206 and HL086491 to JAB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: a.gregson@ucla.edu ¤ Current address: Hillman Cancer Center, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America Introduction Lung transplantation is a therapeutic option for end-stage lung disorders, but is complicated by allograft rejection with an incidence and severity that is among the highest of solid organ transplants [1]. Long-term survival is largely dependent upon recipients remaining free of bronchiolitis obliterans syndrome (BOS). BOS is a chronic alloimmune mediated, fibro-obliterative syndrome characterized by progressive airflow obstruction and graft dysfunction [2–5]. BOS affects over 60% of lung transplant recipients within five years after transplantation [1,6,7] and imparts a 3-year mortality of . 50% [1]. Over the last 20 years nearly 10,000 lung transplants have been performed in the USA, suggesting that over 6,000 individuals have developed and 3,000 died from BOS; a tremendous human and financial burden [8]. Despite BOS being a major obstacle to long- term survival post-lung transplantation, there is presently no effective means of early detection, prevention, or treatment [9]. The regulatory T lymphocyte (Treg, CD3 + CD4 + CD25 hi - Foxp3 + ) is recognized as a cell integral to protection against autoimmunity and allograft rejection via the down-regulation of cellular immunity [10–17]. Treg are believed to suppress the activity of alloreactive, effector CD4 + and CD8 + T cells, and thereby contribute to allograft survival [18–21]. To our knowl- edge, no studies have examined the frequency of bronchoalveolar lavage fluid (BALF) Treg subsets or the chemokines responsible for their recruitment and accumulation in the lung allograft. We recently found no difference in BALF Treg (CD4 + CD25 hi - Foxp3 + ) frequencies and ratios to effector T cells (CD8 + CD38 + ) in lung transplant recipients with or without rejection [22], although some recipients with increased Treg during rejection did not go on to develop BOS (unpublished data). We therefore hypothesized that allograft Treg may be protective against BOS. Herein we describe our characterization of Treg subsets and chemokine protein expression in BALF from a larger cohort of lung transplant recipients with known BOS outcomes. Materials and Methods Study Design and Patient Population Forty-seven participants underwent routine screening bron- choscopy with transbronchial biopsy and were recruited non- PLoS ONE | www.plosone.org 1 June 2010 | Volume 5 | Issue 6 | e11354