Article Figures & Data
Figures
- FIGURE 1.
Time course of chemokine receptor expression in allogeneic and syngeneic cultures. PMBC were purified from two unrelated donors and cultured under syngeneic or allogeneic conditions. Immediately after preparation of cultures (day 0) or at the time points indicated cells were labeled with anti-CD4 and either anti-CCR5 (A, C, and D) or anti-CCR6 (B, E, and F), and analyzed by flow cytometry. A and B, The percentage of double-positive cells was calculated as a proportion of the total CD4+ cells and plotted as a function of time for allogeneic (solid line) and syngeneic (dashed line) cultures. Values are the mean ± SEM (n = 4). A statistically significant difference between allogeneic and syngeneic samples at a given time point is indicated by an asterisk. C–F, Representative density plots comparing the expression of CCR5 and CCR6 in syngeneic (C and E) and allogeneic (D and F) cultures on day 9.
- FIGURE 2.
Coregulation of chemokine receptors and memory/activation markers on CD4+ T cells. PMBC were purified from two unrelated donors and mixed to generate an allogeneic MLR. On day 9 of culture, cells were labeled with anti-CD4 and either anti-CCR5 (A–C) or anti-CCR6 (D–F) and anti-CD45RA (A and D), anti-CD45RO (B and E), or anti-CD25 (C and F). The cells were then analyzed by flow cytometry. CD4+ T cells were electronically gated, and staining for the remaining markers was displayed on bivariate density plots. Data shown are representative of at least four experiments.
- FIGURE 3.
CCR5 and CCR6 define distinct subpopulations of CD4+ T cells following allogeneic activation. PMBC were purified from two unrelated donors and mixed to generate an allogeneic MLR. On day 9 of culture cells were labeled with anti-CD4, anti-CCR5, and anti-CCR6 and analyzed by flow cytometry. CD4+, CD4normal, or CD4high T cells were electronically gated as indicated, and staining for CCR5 and CCR6 is displayed on bivariate density plots. Data shown are representative of four experiments.
- FIGURE 4.
Up-regulation of CCR5 and CCR6 mRNA in allogeneic MLR. PMBC were purified from two unrelated donors and cultured under syngeneic or allogeneic conditions for 9 days. RNA was extracted from cell pellets, reverse transcribed, and used in PCR reactions with CCR5-, CCR6-, or GAPDH-specific primers. A representative agarose gel is shown in A, while pooled data from three experiments are shown in B, presented as the ratio of band intensity relative to GAPDH. ∗, Statistical significance at p < 0.05.
- FIGURE 5.
Detection of intracellular chemokine receptor protein. PMBC were purified from two unrelated donors and cultured under syngeneic or allogeneic conditions for 9 days. A and B, Cells were collected from syngeneic (A) or allogeneic (B) cultures, fixed in paraformaldehyde, then either permeabilized or left untreated. Permeabilized and nonpermeabilized cells were labeled with anti-CD4 and either anti-CCR5 or anti-CCR6 and analyzed by flow cytometry, and the percentage of double-positive cells was calculated as a proportion of total CD4+ cells. Data are presented as the mean ± SEM (n = 4–8). C, Cells were spun onto microscope slides, fixed, permeabilized, stained with anti-CCR5 or anti-CCR6 as indicated, and subjected to fluorescent microscopic analysis. Images shown are representative of at least four experiments. ∗, Statistical significance at p < 0.05.
- FIGURE 6.
Division and time dependence of chemokine receptor regulation. PMBC were purified from two unrelated donors, mixed to generate an allogeneic MLR, then labeled with CFSE and cultured for 6 or 9 days. Following culture, cells were labeled with anti-CD4 and either anti-CCR5 or anti-CCR6 and analyzed by flow cytometry. On day 6 the intensity of CFSE fluorescence was used to designate cells as undivided or as having undergone the indicated number of cell divisions. On day 9 it was not possible to accurately discriminate the number of cell division cycles; accordingly, all divided cells were considered as a single population. Data represent the percentage of CD4+ T cells expressing CCR5 (A), CCR6 (B), CXCR3 (C), or CXCR4 (D) within each population (mean ± SEM; n = 4–6).
- FIGURE 7.
Chemotactic responsiveness of cells from MLR cultures to MIP-1β/CCL4 and MIP-3α/CCL20. PMBC were purified from two unrelated donors and cultured under syngeneic or allogeneic conditions. On day 9 of culture cells were collected and subject to Transwell chemotaxis assays using MIP-1β/CCL4, MIP-3α/CCL20, or diluent alone in the lower chamber. A, The absolute number of migrated cells was determined by duplicate hemocytometer counts, and the migration index was calculated, as described in Materials and Methods. B, The percent migration was determined fluorometrically, as described in Materials and Methods, and the migrated cells were labeled with anti-CD4 and subjected to flow cytometric analysis to determine the percentage of CD4+ T cells migrated. Values are the mean ± SEM (n = 4–6). ∗, Statistical significance at p < 0.05.
Tables
- Table I.
Coregulation of chemokine receptors and memory/activation markers on CD4+ T cellsa
Percentage Coexpressing CD45RA CD45RO CD25 CCR5 14.6 ± 2.8 91.3 ± 5.2 90.3 ± 1.1 CCR6 19.1 ± 4.8 93.7 ± 3.2 80.1 ± 1.6 -
a PMBC were purified from two unrelated donors and mixed to generate an allogeneic MLR. On day 9 of culture, cells were labeled with anti-CD4 and the other Abs as indicated and analyzed by flow cytometry. CD4+ T cells were electronically gated, and the percentage of cells double-positive for the chemokine receptor and the memory/activation marker was determined as a proportion of total chemokine receptor-positive cells (mean ± SEM; n = 4).
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