Increased circulating Tfh to Tfr ratio in chronic renal allograft dysfunction: a pilot study

The present study is a cross-sectional pilot study. Eighty-two kidney transplant recipients receiving living donor kidney in West China Hospital of Sichuan University were enrolled in this study from May 2016 to May 2017. All of the recipients with infection or autoimmune diseases at the time of analysis were excluded from this study. All of the heparin-anticoagulated whole blood from these patients were performed flow cytometry. Excluded the number of acquired target cell subsets less than 100 cells, 67 recipients were eventually included in the following analysis. Chronic allograft dysfunction was defined as estimated glomerular filtration rate (eGFR) < 60 ml/min/1.73m² after 3 months of transplantation [17, 18]. Within the 67 recipients, 34 recipients suffered from CAD (defined as CAD group) and 33 had stable renal function (defined as stable group). Among the 34 recipients with CAD, 21 recipients had undergone biopsy. According to Banff-2015 [19], 13 recipients were defined as biopsy-proven rejection (BPR) with 11 antibody-mediated rejection (ABMR) and 2 T cell-mediated rejection (TCMR), 9 recipients were defined as non-rejection (4 interstitial fibrosis tubular atrophy, 3 transplant glomerulonephropathy, 1 BK virus nephropathy, 1 recurrent glomerulonephropathy). Only 6 of all recipients with CAD got DSA detection with the results of 5 positive and 1 negative. Fifty recipients of all got panel reactive antibodies (PRA) detection with the results of 35 positive and 15 negative. BPR, non-rejection, DSA, PRA were used for sub-group analysis.

All of 67 patients received basiliximab as prophylactic therapy. Forty-eight recipients received tacrolimus-based triple immunosuppressant regimen (tacrolimus + mycophenolate mofetil + prednisone); 12 recipients received sirolimus-based triple immunosuppressant regimen (sirolimus + mycophenolate mofetil + prednisone); 2 recipients received cyclosporine A-based triple immunosuppressant regimen (cyclosporine A + mycophenolate mofetil + prednisone); 5 recipients received the combined tacrolimus-minimized and sirolimus immunosuppressant regimen (tacrolimus + sirolimus + mycophenolate mofetil + prednisone). Tacrolimus dose was administered at 1.0–1.5 mg bid. The tacrolimus-minimized regimen was 0.5 mg bid. The dose of sirolimus was 1.0 mg bid. Cyclosporine A was administered at 50–75 mg bid. Mycophenolate mofetil (MMF) was administered at 750 mg bid. The maintenance dose of prednisone was 5 mg or 10 mg qd.

To determine the percentage of T cell subsets, heparin-anticoagulated whole blood were collected and stained with CD3-PerCP (BD Bioscience, New Jersey, US), CD4-FITC (BD Bioscience, New Jersey, US), CXCR5-APC (Biolegand, California, US), PD-1-PE (eBioscience, California, US), ICOS-PE (eBioscience, California, US) and CD25-APC (BD Bioscience, New Jersey, US). After fixed and permeabilized, samples were stained with FoxP3-PE (BD Bioscience, New Jersey, US), p-STAT3-PE (BD Bioscience, New Jersey, US), p-STAT5-PE (BD Bioscience, New Jersey, US) and p-STAT4-PE (BD Bioscience, New Jersey, US). After stimulation with phorbol 12-myristate 13-acetate (PMA) (50 ng/ml) (Sigma-Aldrich, US), ionomycin (1 μg/ml) (Sigma-Aldrich, US), and Golgi stop (BD Bioscience, New Jersey, US) for 5 h, the fixation and permeablication were performed. Then samples were stained with IL-21-PE (BD Bioscience, New Jersey, US). Samples were measured with FACS Canto II (BD Biosciences, New Jersey, US). Gating strategy used for the analysis of all immune parameters was shown in Additional file 1.

Serum samples were collected and stored at − 80 °C freezer until analysis. Human Premixed Multi-Analyte Kit was purchased from R&D Systems (Minneapolis, Minnesota, USA). Serum CXCL13 and TGF-β were measured by Bio-Plex® suspension array system (Bio-Rad Laboratories Inc., California, USA). All samples were measured in duplicate. Four serum samples were excluded from this analysis as the volume were not enough for analysis. Two CXCL13 detection results were also excluded as they were reported with warning after bio-plex analysis. Eventually, 61 results of CXCL13 and 63 results of TGF-β were included in the following analysis.

Serum creatinine (Scr) was measured by picric acid method (Roche Diagnostics, Mannheim, Germany). The eGFR was calculated using the Modification of Diet in Renal Disease formula which was adjusted to Chinese [20]: eGFR (ml/min/1.73m²) = 186 × Scr (mg/dl)^-1.154 × age^-0.203 × (0.742 if female) × 1.233.

This study eventually enrolled in a total of 67 candidates. Within the 67 kidney transplant recipients, 34 recipients suffered chronic allograft dysfunction and 33 maintained stable renal function. The demographic and clinical characteristics in the present study were described in Table 1. There were no significant differences in age, gender, pre-PRA level, HLA mismatch and transplant duration time between CAD group and stable group. Sixty-two recipients received tacrolimus, sirolimus or cyclosporine A based immunosuppressive regiment combined with prednisone and mycophenolate mofetil. Five recipients received combined-use of calcineurin inhibitor (CNI) and sirolimus in CAD group. The use of immunosuppressant was statistically different between CAD group and stable group (P = 0.045). The eGFR level was significantly different between CAD group and stable group (median value: 33.2 vs 74.1 ml/min/1.73m², respectively, P < 0.001).

The frequency of CXCR5⁺ on CD4⁺ cells was significantly lower in CAD group compared to stable group (17.3% vs 22.2%, P = 0.035). The frequency of cTfh cells (CXCR5⁺Foxp3⁻ on CD4⁺) had a lower trend in CAD group compared to stable group (16.8% vs 21.2%, P = 0.058, Figs. 1a, 2a). The frequency of cTfr (CXCR5⁺Foxp3⁺ on CD4⁺) cells in CAD group was observed significantly lower than that in stable group (0.31% vs 0.68%, P = 0.002, Figs. 1b, 2b). The cTfh to cTfr ratio was significantly higher in CAD group compared to stable group (55.4 vs 25.3, P = 0.013, Fig. 2c). Tregs (CD25⁺Foxp3⁺ on CD4⁺) showed the same trend as cTfr cells (1.03% vs 1.66%, P = 0.009, Fig. 2d).

The differences of the frequency of CXCR5⁺PD-1⁺ on CD4⁺ cells or PD-1 expression on CD4⁺CXCR5⁺ cells were not significant between CAD group and stable group (4.9% vs 5.3%, P = 0.607, Fig. 2e; 29.3% vs 29.1%, P = 0.259, Fig. 2f, respectively). Neither as the frequency of CXCR5⁺ICOS⁺ on CD4⁺ cells or ICOS expression on CD4⁺CXCR5⁺ cells between CAD group and stable group (0.66% vs 0.86%, P = 0.135, Fig. 2g; 2.6% vs 3.6%, P = 0.158, Fig. 2h, respectively).

There was significantly lower frequency of CXCR5⁺STAT3⁺ on CD4⁺ or CXCR5⁺STAT5⁺ on CD4⁺ cells in CAD group than patients with stable renal function (10.1% vs 17.2%, P < 0.0001, Fig. 2i; 12.3% vs 18.5%, P = 0.0002, Fig. 2j). The STAT3 expression on CD4⁺CXCR5⁺ cells in CAD group was also significantly lower than that in stable group (70.3% vs 86.4%, P = 0.003, Fig. 2k), while the expression of STAT5 on CD4⁺CXCR5⁺ cells had no significant differences between CAD group and stable group (84.3% vs 88.1%, P = 0.151, Fig. 2l).

However, the frequency of CXCR5⁺STAT4⁺ on CD4⁺ and the expression of STAT4 on CD4⁺CXCR5⁺ cells had no significant differences between CAD group and stable group (0.29% vs 0.40%, P = 0.159, Fig. 2m; 2.0% vs 1.9%, P = 0.855, Fig. 2n, respectively). Neither as the frequency of CXCR5⁺IL-21⁺ on CD4⁺ cells or IL-21 expression on CD4⁺CXCR5⁺ cells between CAD group and stable group (1.11% vs 1.00%, P = 0.624, Fig. 2o; 6.3% vs 7.5%, P = 0.734, Fig. 2p, respectively).

Serum CXCL13 median level were 21.9 (15.2–29.6) ng/ml in patients with stable renal function and 30.4 (18.7–86.9) ng/ml in patients with CAD. Serum CXCL13 was significantly higher in CAD group when compared to stable group (P = 0.025, Fig. 2q). The median level of serum TGF-β were 976 (704–1235) pg/ml in patients with stable renal function and 716 (572–1014) pg/ml in patients with CAD. It was significantly lower in CAD group compared to stable group (P = 0.035, Fig. 2r).

In model 1, we assessed whether the association between immune parameters and eGFR remained independent of adjustment for age, gender, transplantation duration time, pre-PRA level, HLA mismatch and immunosuppressant. The cTfh to cTfr ratio, CXCR5⁺STAT3⁺ on CD4⁺ cells, CXCR5⁺STAT5⁺ on CD4⁺ cells, Tregs or CXCL13 was independent factor to eGFR (standardized coefficient = − 0.279, P = 0.030; standardized coefficient = 0.328, P = 0.007; standardized coefficient = 0.327, P = 0.008; standardized coefficient = 0.399, P = 0.001; standardized coefficient = − 0.380, P = 0.006, respectively). To assess whether these five parameters were independent of each other, second regression analysis was performed after including these five parameters in one multiple linear regression analysis. The cTfh to cTfr ratio was observed an independent risk factor to declined eGFR (standardized coefficient = − 0.420, P = 0.012, shown in Table 2).

In model 2, we assessed whether the association between immune parameters and CAD remained independent of adjustment for age, gender, transplantation duration time, pre-PRA level, HLA mismatch and immunosuppressant. The cTfh to cTfr ratio, CXCR5⁺STAT3⁺ on CD4⁺ cells, CXCR5⁺STAT5⁺ on CD4⁺ cells, cTfr, Tregs or CXCL13 was independent factor to eGFR (standardized coefficient = 1.019, P = 0.024; standardized coefficient = 0.868, P = 0.002; standardized coefficient = 0.327, P = 0.008; standardized coefficient = 0.250, P = 0.033; standardized coefficient = 0.344, P = 0.007; standardized coefficient = 1.038, P = 0.031, respectively). Second regression analysis was also performed after including these six parameters in one multiple linear regression analysis. Transplantation duration and cTfh to cTfr ratio were independent risk factors to CAD (OR = 1.042, 95%CI 1.007–1.078, P = 0.018; OR = 1.043, 95%CI 1.004–1.085, P = 0.031, shown in Table 2).

Based on the quartile of cTfh to cTfr ratio, the kidney transplant recipients were classified into four groups, Group 1 (ratio ≤ 16), Group 2 (16 < ratio ≤ 35), Group 3 (35 < ratio ≤ 60), Group 4 (ratio > 60). Within Group 1, Group 2, Group 3 or Group 4, the percentage of recipients with CAD was 33.3, 33.3, 64.7, 70.6%, respectively. The composition ratio of recipients with stable renal function and CAD within these four groups was significantly different (P = 0.046) by Chi-square test. Through post-hoc test by Mann-Whitney U methods, the percentage of recipients with CAD in Group 4 was significantly higher than that in Group 1 and Group 2 (P = 0.038; P = 0.030, respectively, Table 3). No significant difference of the percentage of recipients with CAD between Group 1 and Group 2, Group 1 and Group3, Group 2 and Group 3, Group 3 and Group 4 was found (P = 1.000, P = 0.081, P = 0.067, P = 0.718, respectively).

After correlation analysis, a negative association between serum CXCL13 and frequency of CXCR5⁺ on CD4⁺ cells was observed in kidney transplant recipients (spearman r = − 0.332; P = 0.008, Table 4). The frequency of cTfh cells was also negatively correlated with CXCL13 (spearman r = − 0.312; P = 0.013, Table 4). No association between serum CXCL13 and cTfr cells was observed (spearman r = − 0.108; P = 0.435, Table 4). No association between serum TGF-β and cTfh, cTfr, CXCR5⁺STAT3⁺ on CD4⁺ cells, or Tregs was observed (Table 4).

When immune parameters were compared between BPR group and stable group, the percentage of cTfr, cTfh to cTfr ratio, the expression of ICOS, STAT3, STAT5 were significantly different (Fig. 3). The differences of other immune parameters were not significant (shown in Additional file 2). The percentage of cTfr, cTfh to cTfr ratio and ICOS expression was also significantly different between DSA positive group and stable group (Fig. 4, Additional file 3). The comparisons between non-rejection group and stable group, between non-rejection group and BPR group were also done. Only the percentage of CXCR5⁺STAT3⁺ on CD4⁺ was found significantly different between non-rejection group and stable group (shown in Additional file 4). Only cTfh to cTfr ratio and ICOS expression were found significantly different between BPR group and non-rejection group (shown in Additional file 5). The analysis results between PRA positive group and PRA negative group had the same trend as comparison between CAD group and stable group (shown in Additional file 6). Patients with CAD (eGFR< 60 ml/min/1.73m²) were divided into three groups: Group 1 with eGFR from 30 to 60 ml/min/1.73m² (N = 19); Group 2 with eGFR from 15 to 30 ml/min/1.73m² (N = 12); Group 3 with eGFR less than 15 ml/min/1.73m² (N = 3). No significant differences of immune parameters were observed between these three groups (shown in Additional file 7).