Reduced Frequency of Regulatory T Cells in Peripheral Blood Stem Cell Compared to Bone Marrow Transplantations Céline Blache, Joe-Marc Chauvin, Aude.

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Reduced Frequency of Regulatory T Cells in Peripheral Blood Stem Cell Compared to Bone Marrow Transplantations Céline Blache, Joe-Marc Chauvin, Aude Marie-Cardine, Nathalie Contentin, Pascal Pommier, Ingrid Dedreux, Stéphanie François, Serge Jacquot, Dominique Bastit, Olivier Boyer Biology of Blood and Marrow Transplantation Volume 16, Issue 3, Pages 430-434 (March 2010) DOI: 10.1016/j.bbmt.2009.10.027 Copyright © 2010 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 1 Reduced Treg frequencies in PBSC compared to BM transplants. (A) Flow cytometry analysis of a representative PBSC transplant. Cells were stained with RD1 anti-CD25 and ECD anti-CD4 (left), PE anti-FoxP3 and ECD anti-CD4 (intracellular staining, center), or RD1 anti-CD25 and FITC anti-CD127 (right). Data are gated on MNC (left and center) or CD4+T cells (right). Numbers indicate cell frequency in the depicted region or quadrant. (B) Treg frequencies in BM (n=29) and PBSC (n=55) transplants. Horizontal trait indicates median, box represents 25th and 75th percentiles, error bars depict 10th and 90th percentiles, and dots are individual values below or above the 10th and 90th percentiles, respectively. (C) Total CD3+T cell numbers in BM and PBSC transplants: (mean + SEM). (D) Box plots of the frequency of Annexin-V+ cells within CD4+CD25+CD127lo (Tregs) and CD4+CD25− (Tconv) subsets, in the blood of donors after G-CSF administration (before leukapheresis) and in the PBSC transplant. Horizontal trait indicates median, box represents 25th and 75th percentiles, and error bars depicts range of values. Significant P-values ≤.05, ≤.01, and ≤.001 are indicated by ∗, ∗∗, and ∗∗∗, respectively. Biology of Blood and Marrow Transplantation 2010 16, 430-434DOI: (10.1016/j.bbmt.2009.10.027) Copyright © 2010 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 2 Decrease of CD62L+ Tregs during collect of PBSC transplants. (A) Flow cytometry analysis of Tregs from PBSC donor blood samples (before G-CSF, n=19) or from BM (n=26) or PBSC (n=53) transplants. Histograms (mean+SEM) indicate the frequency of CD62L expression on Tregs, the proportion of CD62L+ Treg and the frequency of several phenotypic markers in CD4+CD25hi Tregs. Stainings were performed using RD1 anti-CD25, ECD anti-CD4 and 1 of the following FITC-labeled anti-CD62L, anti-CD45RO, anti-CD45RA, or anti-HLA-DR antibodies. (B) Flow cytometry analysis of CD62L expression at the surface of CD4+CD25hi Tregs. Box plots indicate the percentage of CD25hiCD62L+ cells within the CD4+T cell gate (n=11), in the blood of donors before G-CSF administration, after G-CSF administration (before leukapheresis), and in the PBSC transplant. Horizontal trait indicates median, box represents 25th and 75th percentiles, and error bars depicts range of values. Data were compared by ANOVA. Significant P-values ≤.01 and ≤.001 are indicated by ∗∗ and ∗∗∗, respectively. Biology of Blood and Marrow Transplantation 2010 16, 430-434DOI: (10.1016/j.bbmt.2009.10.027) Copyright © 2010 American Society for Blood and Marrow Transplantation Terms and Conditions