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. 2010 Jun 29;5(6):e11354.
doi: 10.1371/journal.pone.0011354.

Protection Against Bronchiolitis Obliterans Syndrome Is Associated With Allograft CCR7+ CD45RA- T Regulatory Cells

Free PMC article

Protection Against Bronchiolitis Obliterans Syndrome Is Associated With Allograft CCR7+ CD45RA- T Regulatory Cells

Aric L Gregson et al. PLoS One. .
Free PMC article


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(-), and total Treg frequency did not correlate to BOS outcome. The majority of Treg were CCR4(+) and CD103(-) 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(+)CD45RA(-) 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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Figure 1
Figure 1. Representative gating strategy for identification of CD3+CD4+CD25hiFoxp3+ lymphocytes and subsets.
Gated cells (selected cells) are purple, while non-gated cells (discarded cells) are blue or black. The gating involves sequential boolean filters and the sequence of filters is demonstrated here moving left to right and then top to bottom. Purple cell populations are selected in each scatterplot and this population is then used as the parent population in the subsequent scatterplot. (top row) First, singlets are identified in a forward scatter-area vs. forward scatter-height plot and gated on prior to logicle transformation of the data. Live/CD4+ cells are then identified in a CD4 vs. UV-Blue plot. CD3+ cells are then identified in a CD4 vs. CD3 plot. (bottom row) Lymphocytes within this population are identified in a side scatter-area vs. forward scatter-area plot. Treg are identified in a Foxp3 vs. CD25 plot. Determination of gates for CD25, CD45RA, Foxp3 and CCR7 utilized fluorescence minus-one (FMO) tubes.
Figure 2
Figure 2. Flow chart description of the number of samples and outcomes from each group.
Forty-seven lung transplant recipients were in the study. Thirteen went on to develop BOS. Seventy BALF samples were collected and ten had insufficient cells to allow detection of Treg, thus 60 samples were used for total Treg analysis. Of the 60 samples, 27 were dropped from analysis for the Treg subsets due to inadequate number of Treg as explained in the Methods.
Figure 3
Figure 3. Boxplots of T regulatory lymphocytes and BOS outcomes.
(left) A boxplot of all 60 evaluable samples for total Treg. This plot demonstrates that there are no differences in total Treg frequencies between those recipients who eventually develop BOS and those that do not (p = 0.92). (middle) A boxplot of all 33 evaluable samples for Treg subsets. This plot demonstrates that there are no differences in CCR4+ Treg frequencies between those recipients who eventually develop BOS and those that do not (p = 0.12). (right) A boxplot of all 33 evaluable samples for Treg subsets. The CCR7+ subset is associated with protection against BOS (p = 0.04).
Figure 4
Figure 4. Plots of the log Cox Relative Hazard ratio for BOS against percent T regulatory lymphocyte subsets.
The log of the Cox Relative Hazard (y-axis) decreases, i.e., less likely to develop BOS, as the percent of Treg, Treg subset or CCL21 protein level (x-axis) increases. The steeper the curve, as indicated by the blue line, the more protective the association with each Treg subset. The most significant effect is seen with the CCR7+ Treg subset. Confidence intervals are given by the gray bands. (top left) A plot for total BALF Treg, which demonstrates a non-significant protective effect. (top right) A plot of percent CCR4+ Treg, which demonstrates no significant protective effect. (bottom left) A plot of percent CCR7+ Treg, which demonstrates a significant protective effect. (botom right) A plot of BALF CCL21 protein level (pg) as determined by ELISA, which demonstrates a significant protective effect.
Figure 5
Figure 5. Density plots of chemokine receptor expression by Treg.
Representative density plots of CCR4 and CCR7 expression by BALF Treg. The parent populations for determination of chemokine receptor positivity were singlet, live, CD3+CD4+CD25hiFoxp3+ lymphocytes as shown in Figure 1. (left) Demonstrates the generally high level of CCR4 expression by Treg. The CCR4 gate was determined such that <0.1% of the cells were positive in the CCR4 FMO tube (see text for detail). (right) Demonstrates the variability of CCR7 expression by Treg. The CCR7 gate was determined such that <0.3% of the cells were positive in the CCR7 FMO tube.
Figure 6
Figure 6. Histopathology demonstrating CCL21 production in healthy lung allograft.
Representative immunohistochemistry for (a) control antibody and (b) CCR7 ligand CCL21 on healthy lung allograft biopsy tissue. CCL21 protein expression is found in bronchial epithelial cells (red arrows) and alveolar macrophages (green arrows). Panels were photographed at 200× and 400× magnification.
Figure 7
Figure 7. ROC curve for CCR7+ Treg and BOS.
The ROC curve is based upon a logistic regression model for CCR7+ Treg predicting BOS outcome, which excludes samples collected after the diagnosis of BOS. The p-value for the global null hypothesis is 0.04 and the area under the curve (AUC) is 0.75; the model has good predictive value.

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