Activated CD4+ T Cells and Highly Differentiated Alloreactive CD4+ T Cells Distinguish Operationally Tolerant Liver Transplantation Recipients

Abstract

Spontaneous operational tolerance to the allograft develops in a proportion of liver transplantation (LT) recipients weaned off immunosuppressive (IS) drugs. Several studies have investigated whether peripheral blood circulating T cells could play a role in the development or identify operational tolerance, but never characterized alloreactive T cells in detail due to the lack of a marker for these T cells. In this study, we comprehensively investigated phenotypic and functional characteristics of alloreactive circulating T cell subsets in tolerant LT recipients (n = 15) using multiparameter flow cytometry and compared these with LT recipients on IS drugs (n = 23) and healthy individuals (n = 16). Activation-induced CD137 was used as a marker for alloreactive T cells upon allogenic stimulation. We found that central and effector memory CD4+ T cells were hyporesponsive against donor and third-party splenocyte stimulation in tolerant LT recipients, whereas an overall hyperresponsiveness was observed in alloreactive terminally differentiated effector memory CD4+ T cells. In addition, elevated percentages of circulating activated T helper cells were observed in these recipients. Lastly, tolerant and control LT recipients did not differ in donor-specific antibody formation. In conclusion, a combination of circulating hyperresponsive highly differentiated alloreactive CD4+ T cells and circulating activated T helper cells could discriminate tolerant recipients from a larger group of LT recipients.

FIG. 1

Higher relative numbers of circulating activated T helper cells in tolerant LT recipients. Percentages of (A) CD4⁺ and CD8⁺ T cells, (B) CD4⁺FoxP3⁺CD25⁺ T cells and gating strategy, (C) aTh, aTreg, and rTreg defined by gating strategy with FoxP3 and CD45RA, and (D) IFNγ‐ or IL17‐positive cells in PMA/IONO‐stimulated aTh are presented. HC, n = 13; CTRL, n = 20; and TOL, n = 13. Statistical analyses were performed with 1‐way ANOVA or Kruskal‐Wallis and posttests. *P < 0.05 and **P < 0.01.

FIG. 2

CMV‐positive serostatus is associated with a higher Vδ1:Vδ2 γδT cell ratio in all LT recipients. Percentages and ratios of Vδ1 and Vδ2 γδT cells of (A) entire groups and (B) in CMV+ and CMV– individuals sorted by serostatus at the end of follow‐up are presented. ^~These individuals are CMV seronegative but were transplanted with a CMV+ donor. HC, n = 13; CTRL, n = 20; and TOL, n = 13. Statistical analyses were performed with 1‐way ANOVA, Kruskal‐Wallis or Friedman, and posttests. *P < 0.05 and **P < 0.01.

FIG. 3

Alloreactive memory CD4⁺ T cells are more differentiated in tolerant LT recipients. (A) Representative dot plots indicating CD137 expression in CD4⁺ and CD8⁺ T cells cultured in the absence or presence of allogenic splenocytes. Ratios of CD137 expression in T cells stimulated by allogeneic splenocytes (S) for HC, donor splenocytes (D), and third‐party splenocytes (T) against unstimulated T cells (S/–, D/–, or T/–) and/or donor against third party (D/T) are presented in B‐D and F. Ratios are presented for (B) CD137‐expressing CD4⁺ and CD8⁺ T cells; (C) CD137‐expressing aTh, allTreg, and allTh in CD4⁺ T cells; (D) CD137‐expressing IFNγ‐producing CD4⁺ (left) and CD8⁺ (right) T cells; and (F) CD137‐expressing T cell subsets naïve, CM, EM, and EMRA in CD4⁺ T cells. A solid line represents a ratio of 1. In (E) percentages of IFNγ‐positive cells in PMA/IONO‐stimulated CD4⁺ or CD8⁺ CD137⁺ T cells are presented. Panel B/C: HC, n = 13; CTRL, n = 19; TOL, n = 12; Panel D: HC, n = 8; CTRL, n = 13; TOL, n = 8; Panel F: HC, n = 8; CTRL, n = 12; TOL, n = 9. Statistical analyses were performed with 1‐way ANOVA, Kruskal‐Wallis or Friedman, and posttests. *P < 0.05, **P < 0.01, and ***P < 0.001.

FIG. 4

A tolerance profile discriminating tolerant LT recipients could be established. (A) A heat map with hierarchical clustering analysis is depicted for all LT recipients in whom all significantly different markers between TOL and CTRL were measured in this study. To avoid a selection bias, the LT recipients in whom not all significantly different markers were measured were not included. Analysis was performed with the public Galaxy server, version 3.0.1, R gplots package (R Foundation, Vienna, Austria) with the Euclidean distance method and the complete hierarchical clustering method. Data from each recipient were scaled with a z score according to total data of TOL and CTRL for that marker (color key). (B) Principal component analysis of the significantly different markers between TOL and CTRL is depicted. Rotated component matrix analysis was performed using Varimax with Kaiser normalization. CTRL, n = 12; TOL, n = 9.

FIG. 5

Findings in a small group of prospectively IS drug‐weaned group of LT recipients. Percentages of (A) FoxP3⁺CD25⁺ in CD4⁺ T cells; (B) aTh defined by FoxP3 and CD45RA expression; and (C) percentages and ratios of Vδ1 and Vδ2 γδT cells before, during, and after IS drug weaning are presented. Ratios of T cell responses against donor (D) and third‐party (T) splenocytes against unstimulated T cells (D/– or T/–) measured by flow cytometric determination of activation‐induced CD137 are presented in D and E. Ratios are depicted for (D) CD4⁺ and CD8⁺ CD137⁺ T cells and (E) CD137‐expressing aTh, allTreg, and allTh in CD4⁺ T cells.

FIG. 6

No significant differences are present in the development of DSAs in tolerant and control LT recipients. Development of DSA formation (A) individually and (B) for the entire group with specific subtypes in TOL before, during, and after IS drug weaning and in CTRL at matching time points are depicted. (C) The cumulative MFI for each DSA+ LT recipient is presented for TOL and CTRL. Statistical analyses were performed with 2‐sided Fisher’s exact test or Pearson chi‐square test. Before versus after weaning: a = 0.06, b, c = 0.13.