Comprehensive phenotyping of regulatory T cells after liver transplantation

Abstract

Regulatory T cells (Tregs) play an important role in controlling alloreactivity after solid organ transplantation, but they may also impair antiviral immunity. We hypothesized that the Treg frequency and the Treg phenotype are altered in hepatitis C virus (HCV)-infected recipients of liver transplantation (LT) with possible prognostic implications. Tregs from 141 individuals, including healthy individuals, LT recipients with or without persistent HCV infections, and nontransplant patients with chronic HCV, were studied. A comprehensive phenotypic analysis was performed with multicolor flow cytometry, which included standard Treg markers [CD4(+), CD25(hi), CD127(-), and FoxP3(+) in addition to HLA DR, CCR7, CD45RA, CD62L, CD49d, CD39, ICOS and LAP-TGFβ stainings. Healthy individuals and LT patients displayed similar Treg frequencies and largely comparable Treg phenotypes, which were stable over time after transplantation. In contrast, Tregs with a CD45RA(-) CCR7(-) effector phenotype were enriched in LT recipients with chronic HCV versus HCV-negative transplant patients. HCV infection, rather than LT, altered the expression of functional markers on Tregs. A principal component analysis revealed distinct Treg phenotypes in HCV-infected LT recipients with rejection and patients with recurrent graft HCV. In conclusion, Treg phenotypes are altered in HCV-infected LT patients. An investigation of Tregs may possibly help to distinguish recurrent HCV from graft rejection. Further functional studies are needed to define the role of Tregs in determining the balance between antiviral and allogenic immunity.

Figure 1

Phenotypical characterization of Tregs. (A) Representative gating strategy for the characterization of Tregs. Lymphocytes were gated on the basis of their FSC‐A/SSC‐A location (not shown). Afterwards, CD4⁺ and CD25^hi were gated. For CD4⁺CD25^hi cells, 3 different Treg gating strategies were applied (CD127^–, FoxP3⁺ or CD127^– and FoxP3⁺). (B) Comparison of surface and intracellular definitions of Tregs. CD4⁺CD25^hiCD127^– Tregs were correlated with CD4⁺CD25^hiFoxP3⁺ Tregs, and CD4⁺CD25^hiFoxP3⁺ was correlated for 2 different fluorochromes. When the data followed a normal distribution, a Pearson correlation was applied; if not, a Spearman correlation was used. 102, 80, and 80 pairs, respectively, were tested after they were stained for the combinations listed in Table 4.

Figure 2

Comparison of Tregs in LT patients (83 patients and 121 samples) and healthy individuals (34 individuals). Percentages of Tregs stained with (A) surface markers as CD4⁺CD25^hiCD127^– and (B) intracellular markers as CD4⁺CD25^hiFoxP3⁺ are compared for samples from LT patients and healthy individuals. On Tregs (gated as CD4⁺CD25^hiCD127^–), (C) different subpopulations according to a CCR7/CD45RA classification and (D) CD45RA⁺CD62L⁺ naive Tregs are compared between the 2 groups. On Tregs (gated as CD4⁺CD25^hiFoxP3⁺), (E) HLA‐DR⁺ and (F) CD49d^– percentages in LT patients and healthy controls are shown. An unpaired t test was performed. Scatter plots show means and SDs.

Figure 3

Comparison of Tregs in HCV LT patients (35 patients and 51 samples) and non‐HCV LT patients (24 patients and 31 samples). Percentages of Tregs stained with (A) surface markers as CD4⁺CD25^hiCD127^– and (B) intracellular markers as CD4⁺CD25^hiFoxP3⁺ are compared for samples from HCV LT patients and non‐HCV LT patients. On Tregs (gated as CD4⁺CD25^hiCD127^–), (C) different subpopulations according to a CCR7/CD45RA classification and (D) CD45RA⁺CD62L⁺ naive Tregs are compared between the 2 groups. On Tregs (gated as CD4⁺CD25^hiFoxP3⁺), (E) HLA‐DR⁺ and (F) CD49d^– percentages in HCV LT patients and non‐HCV LT patients are shown. An unpaired t test was performed. Statistically significant differences are marked with asterisks (P < 0.05). Scatter plots show means and SDs.

Figure 4

Comparison of Tregs in HCV LT patients (35 patients and 51 samples) and HCV CHR patients (24 patients). Percentages of Tregs stained with (A) surface markers as CD4⁺CD25^hiCD127^– and (B) intracellular markers as CD4⁺CD25^hiFoxP3⁺ are compared for samples from HCV LT patients and HCV CHR patients. On Tregs (gated as CD4⁺CD25^hiCD127^–), (C) different subpopulations according to a CCR7/CD45RA classification and (D) CD45RA⁺CD62L⁺ naive Tregs are compared between the 2 groups. On Tregs (gated as CD4⁺CD25^hiFoxP3⁺), (E) HLA‐DR⁺ and (F) CD49d^– percentages in HCV LT patients and HCV CHR patients are shown. The groups were compared with (A,D) the Mann‐Whitney test and (B,C,E,F) an unpaired t test. Statistically significant differences are marked as follows: **P < 0.01 and ***P < 0.001.

Figure 5

Comparison of functional associated markers on Tregs from healthy patients, HCV LT patients, non‐HCV LT patients, and HCV CHR patients. Tregs were defined as CD4⁺CD25^hiFoxP3⁺ cells. Percentages of (A) positive CD39⁺ Tregs, (B) positive ICOS⁺ Tregs, and (C) positive LAP‐TGFβ⁺ Tregs were analyzed. The scatter plots show means and SDs. (D) Multiparameter analysis of Tregs. ICOS⁺, LAP‐TGFβ⁺, and CD39⁺ Tregs were grouped, and percentages of Tregs expressing no markers (0 parameters), 1 marker, 2 markers, or all 3 markers are shown. The groups included healthy individuals (n = 34), HCV LT patients (n = 35), non‐HCV LT patients (n = 24), and HCV CHR patients (n = 24).

Figure 6

PCA plots generated with Treg subpopulations and surface markers in order to find characteristics varying between the different patient groups. (A) No major differences in the quality of the Tregs were observed between LT patients and healthy controls. (B) Samples from HCV LT patients showed partial clustering on the PCA plot and could be slightly distinguished from those from non‐HCV LT patients on the basis of the variations in the frequencies of Tregs (CD127^low), Tregs with a CD45RA^–CCR7^– effector phenotype, and CD45RA⁺CCR7⁺ Tregs, which were statistically significant. (C) Points representing HCV LT patients clustered together, and they could be separated from HCV CHR patients. Naive Tregs (CD62L⁺CD45RA⁺), CD49d^– Tregs, Tregs with a CD45RA^–CCR7^– effector phenotype, CD45RA⁺CCR7⁺ Tregs, LAP‐TGFβ⁺ Tregs, and memory Tregs varied significantly between the 2 groups.

Figure 7

Comparison of Tregs in HCV LT patients with rejection or graft HCV. (A) Samples from HCV LT patients with rejection clustered together and could be clearly differentiated from the samples of those who suffered graft HCV on the basis of differences in CD62L⁺CD45RA⁺ Tregs, HLA‐DR⁺ Tregs, and CD39^–ICOS⁺LAP‐TGFβ^– Tregs. (B) The percentage of HLA‐DR⁺ Tregs was reduced in HCV LT individuals suffering rejection versus those with graft HCV. (C) CD62L⁺CD45RA⁺ Tregs showed an increase in HCV LT patients with rejection versus patients with graft HCV. (D) The difference in CD39^–ICOS⁺LAP‐TGFβ^– Tregs between the 2 patient groups was not statistically significant. Statistically significant differences are marked by * P < 0.05, ** P < 0.01.