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Reduced frequency of FOXP3+ CD4+CD25+ regulatory T cells in patients with chronic graft-versus-host disease

Emmanuel Zorn, Haesook T. Kim, Stephanie J. Lee, Blair H. Floyd, Despina Litsa, Sankari Arumugarajah, Roberto Bellucci, Edwin P. Alyea, Joseph H. Antin, Robert J. Soiffer and Jerome Ritz

Article Figures & Data

Figures

  • Figure 1.

    Decreased frequency of circulating CD4+CD25+ T cells in patients with cGVHD. Percent CD4+CD25+ in PBLs was measured by flow cytometry in patient and donor samples. Values represent total positive cells including CD4+ CD25+dim and CD25+high cells. Box plots define the values for median, range, 25th, and 75th percentiles. One outlier with a value of 37.5% in the group of patients with cGVHD is not shown in the figure. P values were calculated using the Wilcoxon rank-sum test; cGVHD versus no cGVHD, P = .007; cGVHD versus healthy donors, P = .29; no cGVHD versus healthy donors, P < .001.

  • Figure 2.

    Decreased expression levels of FOXP3 in patients with cGVHD. (A) Expression levels of the Treg-specific transcription factor FOXP3 was assessed by quantitative PCR in patient and donor samples, normalized, and reported as a function of total lymphocytes (AU, arbitrary units). Box plots define the values for median, range, 25th, and 75th percentiles. P values were calculated using the Wilcoxon rank-sum test; cGVHD versus no cGVHD, P = .009; cGVHD versus healthy donors, P = .01; no cGVHD versus healthy donors, P = .34. (B) Correlation between CD4+CD25+ phenotypic values and FOXP3 expression levels in patients with cGVHD was calculated using a rank-based Spearman test (P < .001; rs = 0.60). (C) Patients with cGVHD were subdivided according to whether they previously developed aGVHD. Box plots define the values for median, range, 25th, and 75th percentiles. P values were calculated using the Wilcoxon rank-sum test; aGVHD and cGVHD versus no aGVHD and cGVHD, P = .29; aGVHD and cGVHD versus no aGVHD and no cGVHD, P < .001; no aGVHD and cGVHD versus no aGVHD and no cGVHD, P = .005.

  • Figure 3.

    Correlation between CD3+CD8+ and CD4+CD25+ T cells in active cGVHD. (A) Percent CD3+CD8+ in PBLs was measured by flow cytometry in patient and donor samples. Lines represent median values. (B) Percent CD4+CD25+ and CD3+CD8+ T cells in total PBLs are shown for patients with cGVHD. Correlation between the 2 variables was calculated using a rank-based Spearman test (rs = -0.81, P < .001).

  • Figure 4.

    Percent CD4+CD25+ in PBLs correlates with Treg suppressive function in cGVHD. (A) CTL reactivity against a pool of class I-restricted common viral peptides was assessed by IFNγ ELISPOT assays before and after CD25+ T-cell depletion using 15 samples collected from patients with cGVHD. Depletion of CD25+ cells was confirmed by flow cytometry for each sample. (B) Percent inhibition was measured for 15 patients with cGVHD and values are plotted together with percent CD4+CD25+. One sample generated a negative value and was interpreted as null. Correlation between the 2 variables was calculated using a rank-based Spearman test (rs = 0.59, P = .02).

  • Figure 5.

    Patient Tregs express normal levels of suppression. CD4+CD25+ Tregs were immunopurified from PBMCs and assessed for their capacity to suppress the proliferation of autologous CD4+CD25- cells at a responder/Treg ratio of 1:1. Percent inhibition of responder cell proliferation due to the presence of Tregs was measured using 17 samples collected from 5 cGVHD patients, 7 patients without cGVHD, and 5 healthy individuals. The solid lines represent the median values.

  • Figure 6.

    Allo-transplant patients have impaired thymus function. Thymic activity was assessed in patient and donor samples using the TREC assay and represented as TREC copy number per 105 CD3+ T cells. Box plots define the values for median, range, 25th, and 75th percentiles. One outlier with a value of 1292 in the group of no-cGVHD patients is not shown in the figure. P values were calculated using the Wilcoxon rank-sum test; cGVHD versus no cGVHD, P = .13; cGVHD versus healthy donors, P < .001; no cGVHD versus healthy donors, P = .02.

Tables

  • Table 1.

    Patient characteristics

    cGVHDNo cGVHDP
    Number 30 27 NA
    Median age, y 44 36 .22
    Sex .43
    Male 17 12
    Female 13 15
    Diagnosis NA
    Chronic myeloid leukemia 11 5
    Acute lymphoblastic leukemia 3 1
    Acute myeloid leukemia 8 7
    Myelodysplastic syndrome 7 5
    Multiple myeloma 1 0
    Non-Hodgkin lymphoma 0 5
    Hodgkin lymphoma 0 3
    Aplastic anemia 0 1
    Conditioning regimen .09
    Myeloablative 29 22
    Nonmyeloablative 1 5
    Stem cell source <.001
    Bone marrow 26 10
    Peripheral blood 4 17
    Donor .06
    Related 13 19
    Unrelated 17 8
    HLA .09
    Matched 25 25
    Mismatched 5 2
    Sex match, patient/donor NA
    M/M 7 6
    M/F 10 6
    F/M 4 4
    F/F 9 11
    Acute GVHD grade II to IV .01
    No 14 22
    Yes 16 5
    Median time after transplantation
    (range), mo 24 (7-124) 19 (5-127) .08
    Immunosuppressive drugs 29 12 <.001
    cGVHD grade NA
    Mild 23 NA
    Moderate 6 NA
    Severe 1 NA
    • NA indicates not applicable.

  • Table 2.

    CD4+ CD25+ Tregs in peripheral blood

    CD4+CD25+/PBL ratioscGVHDNo cGVHDHealthy donors
    Less than or equal to 3%, no. (%) 15/30 (50.0) 1/27 (3.7) 2/26 (7.7)
    Greater than 3%, no. (%) 15/30 (50.0) 26/27 (96.3) 24/26 (92.3)
    • cGVHD versus no cGVHD: P<.001; cGVHD versus healthy donors: P<.001; no cGVHD versus healthy donors: P = .61.

  • Table 3.

    Phenotypic and molecular Treg markers in patients without cGVHD

    Previous occurrence of cGVHDNoYes (resolved)P
    %CD4+CD25+/PBLs, median (range) 8.3 (2.6-12.4) 8.7 (4.0-16.6) .44
    FOXP3 expression, AU, median (range) 17548 (268.5-48 327) 14872 (4 916-51 029) .94
    • AU indicates arbitrary units.