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. 2010 Jul;16(7):907-14.
doi: 10.1016/j.bbmt.2010.02.026. Epub 2010 Mar 17.

Frequency of CD4(+)CD25(hi)FOXP3(+) Regulatory T Cells Has Diagnostic and Prognostic Value as a Biomarker for Acute Graft-Versus-Host-Disease

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Frequency of CD4(+)CD25(hi)FOXP3(+) Regulatory T Cells Has Diagnostic and Prognostic Value as a Biomarker for Acute Graft-Versus-Host-Disease

John M Magenau et al. Biol Blood Marrow Transplant. .
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The relationship between regulatory T cells (Tregs) and acute graft-versus-host disease (aGVHD) in clinical allogeneic bone marrow transplantation (BMT) recipients is not well established. We conducted a prospective analysis of peripheral blood Tregs as determined by the frequency of CD4(+)CD25(hi)FOXP3(+) lymphocytes in 215 BMT patients. Autologous BMT patients (N = 90) and allogeneic BMT patients without GVHD (N = 65) had similar Treg frequencies, whereas allogeneic patients with GVHD (N = 60) had Treg frequencies that were 40% less than those without GVHD. Treg frequencies decreased linearly with increasing grades of GVHD at onset, and correlated with eventual maximum grade of GVHD (P < .001). In addition, frequency of Tregs at onset of GVHD predicted the response to GVHD treatment (P = .003). Patients with Treg frequencies less than the median had higher nonrelapse mortality (NRM) than patients with Tregs greater than the median, but experienced equivalent relapse mortality, resulting in an inferior survival at 2 years (38% versus 63%, P = .03). Treg frequency may therefore have important prognostic value as a biomarker of aGVHD.

Conflict of interest statement

Conflict-of-interest disclosure: The authors declare no competing financial interests.


Figure 1
Figure 1. Phenotypic characterization of Tregs
Representative flow data from a patient following allogeneic transplantation. Fresh whole blood was stained with CD4-PerCP-Cy5.5 and CD25-PE antibodies followed by intracellular staining for FOXP3-APC. Treg frequency was determined using a Canto II flow cytometer (BD Biosciences) to identify CD4+CD25hiFOXP3+ triple positive cells within the total lymphocyte population. Treg frequency was performed in triplicate for each sample. (A) The total lymphocyte population (R1) was identified by light scatter after backgating on the CD4+ population. On right are histograms of FOXP3+ expression (red) gated on CD4+CD25+, CD4+CD25++, CD4+CD25+++ (CD4+CD25hi) lymphocyte subsets. Analysis was restricted to the CD4+CD25hi lymphocyte subset (R2) which was identified in R1. The frequency of CD4+CD25hiFOXP3+ lymphocytes was calculated by multiplying the percentage of CD4+CD25hi cells by the percentage of FOXP3+ cells within CD4+CD25hi.
Figure 2
Figure 2. Regulatory T cells (Tregs) at GVHD onset
Fresh blood samples from autologous and allogeneic transplant patients (N=215) were acquired within 24hrs of acute GVHD onset or at equivalent timepoints following transplantation. Mean (A) Treg frequencies (B) Absolute Treg numbers and (C) Treg / Tconv frequencies for autologous BMT patients, allogeneic BMT patients with no GVHD, and allogeneic BMT patients with GVHD. Error bars represent the SEM.
Figure 3
Figure 3. Treg frequencies prior to GVHD onset
(A) Paired analysis in GVHD patients (N = 14) comparing Treg frequencies at GVHD onset to prior timepoints (3 to 14 days). (B) Treg frequencies from matched controls with no GVHD (N = 15) over an equivalent 3-14 day interval. Bars denote mean values.
Figure 4
Figure 4. Treg frequencies and GVHD severity
Fresh blood samples from allogeneic transplant patients with GVHD (N = 60) were acquired within 24hrs of acute GVHD onset and analyzed according to GVHD severity. (A) Mean Treg frequencies, (B) absolute Treg numbers by grade of GVHD at onset, and (C) mean Treg frequencies by eventual maximum overall GVHD grade. The median interval between GVHD onset and eventual maximum overall GVHD grade was 14 days. Error bars represent the SEM.
Figure 5
Figure 5. Treg frequencies are correlated with clinical outcomes
The median T reg frequency was 0.5% in allogeneic BMT patients at time of GVHD onset (N = 60). (A) non-relapse mortality, (B) relapse mortality and (C) overall survival in patients with GVHD divided according to the median Treg frequency (high Treg ≥ 0.5% (N=30) or low Treg < 0.5 (N=30).
Figure 6
Figure 6. Treg frequencies at 4 weeks according to treatment response
Mean (±SEM) Treg frequencies were measured on paired samples at onset and 4 weeks after treatment in 25 patients with complete response or near complete response (CR) and 15 non-responders (NR).

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