Alterations in CD4+ T-cell Subsets in Living Donor Liver Transplantation Associated With Graft Rejection

Abstract

Background and objectives: Regulatory T-cells (Tregs) play a key role in immune homeostasis after organ transplantation. However, the role of CD4⁺ T cell subsets in early acute rejection is still not well understood. Therefore, our aim was to determine changes in CD4⁺ T-cell subsets in living donor liver transplantation (LDLT).

Methods: LDLT patients were assessed for T-cell subsets, Tregs frequencies and their functionality by flow-cytometry at peri- and post-transplant in the span of 1 year.

Results: 33 patients were followed up and 11 (33%) patients have developed early acute cellular rejection (ACR). At peri-transplant time point, MFI of Foxp3⁺ Tregs was significantly increased compared to HC (P = 0.04). However, CD4⁺CD25⁺Foxp3⁺/CD127^- Tregs numbers and IL-10, IL-17 and TGF-β secreting functional Tregs were significantly decreased at 3 months compared to peri-transplant (P = 0.003). But in patients with rejection, CD4⁺CD25⁺FOXP3⁺ and CD4⁺CD25⁺CD127^- Tregs were significantly decreased at day 3 compared to no rejection group (P = 0.048). Patients with rejection also showed significantly decreased numbers of IL-17 and TGF-β secreting CD4⁺CD25⁺FOXP3⁺ Tregs at peri-transplant time (P = 0.04, P = 0.03) compared to no rejection. Further, rejection group showed decreased terminally differentiated effector memory (TEMRA) at peri-transplant and day 7 (P = 0.048 and P = 0.01). Additionally, CD4⁺ central memory (CM) was decreased at peri-transplant (P = 0.05), 1 month (P = 0.04), and 3 to 6 month (P = 0.02).

Interpretation and conclusion: Tregs frequencies were significantly decreased in peri-TX in rejection patients. Further, decreased frequencies of CD4⁺ TEMRA and CD4⁺ CM at day 7 and 1 month were associated with rejection.

Keywords: effector and memory T cells; living donor liver transplantation; rejection; tregs.

Figure 1

Changes in percentage frequency of CD4⁺T-cells and their subsets post-Tx. (A) Gating strategy showing lymphocytes gating on FSC/SSC dot plot followed by CD4⁺T-cells and its subsets based on CD45RA⁺, CCR7⁺. Dot plot representing % frequency of (B) CD4⁺T-cells, (C–F) and its subsets i.e central memory (CM) cells, naïve T-cells, TEMRA (T effector memory cells) and effector memory (EM) cells. Colored circle: pre-Tx data, colored square: 1 days, colored triangle: 3 days, colored opposite triangle: 7 days, colored rhombus: 1 month, open circle: 3 months, open square: 6 month, and open triangle: 1-year data. P values < 0.05 were considered significant.

Figure 2

Phenotypical characterization of T regulatory cells (Tregs) (A) Representative gating strategy used for the characterization of Tregs. Lymphocytes were gated on FSC-A/SSC-A dot plot, after that FOXP3⁺, FOXP3⁺CD127^- and CD127^- cell population was gated on CD4⁺CD25⁺T-cells and showed as contour plots. Dot plots showing percentage frequency and Mean fluorescence intensity (MFI) of (B) CD4⁺CD25⁺FOXP3⁺ Tregs in healthy controls (HC) and end stage liver disease (ESLD) containing all etiologies together and separately at pre-Tx. Dot plot graphs showing percentage frequency and MFI of (C–D) CD4⁺CD25⁺FOXP3⁺, CD4⁺CD25⁺CD127^- Tregs and CD4⁺CD25⁺HLADR⁺ Tregs at various time points in end stage liver disease (ESLD) patients. (F) % frequency of CD4⁺CD25⁺CD45RA⁺CD62L⁺ at various time points checked in all etiologies. P values < 0.05 were considered significant.

Figure 3

Expression of cytokines for suppressive activity of Tregulatory cells (Tregs). (A) Gating strategy showing counter plots used to gate all cytokines like IL-10, IL-17 and TGF-β on CD4⁺CD25⁺FOXP3⁺. (B–D) Dot plots showing MFI of IL-10, IL-17 and TGF-β expressing CD4⁺CD25⁺ Tregs in all the time points. (E–G) Dot plots showing mean flourscence intensity (MFI) of IL-10, IL-17 and TGF-β expressing CD4⁺CD25⁺ FOXP3⁺ Tregs in all the time points.

Figure 4

Transplant rejection and its linkage to T effector memory cells (TEMRA) population in CD4⁺T-cells (A–D) Percent frequency of TEMRA in rejection vs no rejection at pre-Tx, 1-day, 3-day and 7-day.

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