Unique Abnormalities of CD4+ and CD8+ Central Memory Cells Associated with Chronic Graft-versus-Host Disease Improve after Extracorporeal Photopheresis  Kouhei Yamashita, Mitchell E. Horwitz, Akua Kwatemaa, Effie Nomicos, Kathleen Castro, Robert Sokolic, Susan F. Foster, Mary Garofalo, Uimook Choi, Mark Ryherd, Margaret R. Brown, Susan F. Leitman, Alan S. Wayne, Daniel H. Fowler, Michael R. Bishop, Richard W. Childs, A. John Barrett, Steven Z. Pavletic, Harry L. Malech  Biology of Blood and Marrow Transplantation  Volume 12, Issue 1, Pages 22-30 (January 2006) DOI: 10.1016/j.bbmt.2005.11.004 Copyright © 2006 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 1 A, Flow cytometry dot plot analyses of peripheral blood CD4+ T cells assessing surface expression of CD62L (allophycocyanin chromoflor) on vertical axis and CCR7 (CyChrome chromoflor) on horizontal axis. Left, Typical pattern seen in healthy individuals. Middle and right panels, Patterns seen in two patients with cGVHD with extreme change noted in about 10% (right) and more moderate change typical of majority where loss of expression of CCR7 is more profound than loss of CD62L expression (middle). Vertical line in each graph was set just above staining by isotype CyChrome control antibody, whereas horizontal line was set just below population of cells in healthy donors with high expression of CD62L. Numbers in each quadrant indicate percent of events (cells) falling into that quadrant. B, Scatter plot (vertical logarithmic scale) of mathematic transformation of data from flow cytometry generated dot plots identical to those shown in A, where ratio of CCR7+CD62Lhigh/CCR7−CD62Llow CD4+ T cells (percent in upper right quadrant divided by percent in lower left quadrant) in 52 healthy individuals (solid square, healthy), 21 patients postallogeneic transplantation without cGVHD (▴, no GVHD), and 37 patients postallogeneic transplantation with symptomatic cGVHD (solid circle, cGVHD). Each dot represents one patient. Biology of Blood and Marrow Transplantation 2006 12, 22-30DOI: (10.1016/j.bbmt.2005.11.004) Copyright © 2006 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 2 Scatter plot showing data from flow cytometry analysis of subtypes of peripheral blood CD8+ T cells. Each filled symbol represents value for analysis of healthy individual whereas each open symbol represents value for analysis of patient with symptomatic cGVHD. Indicated are comparisons of populations of CD8+ T cells that are defined as naive (CD45RA+CCR7+), effector memory (Tem = CD45RA−CCR7−), RA+ effector memory (Temra = CD45RA+CCR7−) and central memory (Tcm = CD45RA−CCR7+). Each dot represents one patient. Biology of Blood and Marrow Transplantation 2006 12, 22-30DOI: (10.1016/j.bbmt.2005.11.004) Copyright © 2006 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 3 Plots combine data for peripheral blood CD4+ and CD8+ T-cell subpopulations, respectively, as shown from scatter plots in Figure 1B (vertical axis) and Figure 2 (horizontal axis) for subset of healthy donors (left) and patients with symptomatic cGVHD (right) for which both sets of data are available. Regular symmetric ellipse was drawn and placed to tightly include all values for healthy donors and then reproduced over data similarly plotted for patients with cGVHD. Lines were produced by best-fit algorithm of regression analysis as indicated in formulae in each graph. Biology of Blood and Marrow Transplantation 2006 12, 22-30DOI: (10.1016/j.bbmt.2005.11.004) Copyright © 2006 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 4 Paired measurements of ratio of CCR7+CD62Lhigh/CCR7−CD62Llow CD4+ T cells (percent in upper right quadrant divided by percent in lower left quadrant) (A) and percent of CD8+ CD45RO+T-cell that are also CCR7+ (i.e., percent of central memory CD8+ T cells) (B) from each of 7 patients before (blue) and after 6 months of ECP treatments (red). Biology of Blood and Marrow Transplantation 2006 12, 22-30DOI: (10.1016/j.bbmt.2005.11.004) Copyright © 2006 American Society for Blood and Marrow Transplantation Terms and Conditions

Figure 5 Proposed scheme indicating differentiation pathways for T cells from healthy individuals, patients with cGVHD, and patients with cGVHD after response to ECP treatment. Arrows from left to right indicate direction of differentiation. Size of circles suggests relative size of that population. Naive, effector memory (Tem), RA+ effector memory (Temra), and central memory (Tcm) cells are indicated. Biology of Blood and Marrow Transplantation 2006 12, 22-30DOI: (10.1016/j.bbmt.2005.11.004) Copyright © 2006 American Society for Blood and Marrow Transplantation Terms and Conditions