Background
The risk of acute rejection after kidney transplantation has been decreased with the development of immunosuppressant and transplant technique, while chronic renal allograft dysfunction (CAD) is still the main threat for long-term allograft survival rates. Antibody-mediated injury or rejection is the leading cause of late kidney allograft dysfunction [
1,
2]. Donor-specific antibodies (DSA) could identify patients at high risk for kidney allograft loss [
3,
4]. Avoiding the influence of humoral immune factors on allograft function could decline the risk of CAD.
The production of high affinity antibody in germinal center (GC) requires the help of T follicular helper (Tfh) cells [
5]. Tfh cells in lymph node highly express C-X-C chemokine receptor 5 (CXCR5), programmed death 1 (PD-1) and inducible co-stimulator (ICOS) [
6]. Tfh cells migrate into germinal centers via gradients of C-X-C chemokine ligand 3 (CXCL13) and initiate B cells to undergo proliferation, differentiation and somatic hypermutation [
6]. Tfh differentiation relies on expression of B cell lymphoma 6 (Bcl-6), which promote ICOS and PD-1 expression [
6]. It has been demonstrated that signal transducer and activator of transcription 3 (STAT3) is required for the differentiation of Tfh cells through the induction of Bcl-6 [
7‐
10]. Bcl-6 within Tfh cells is negatively regulated by signal transducer and activator of transcription 5 (STAT5), which inhibit Tfh differentiation through increasing the expression of B lymphocyte-induced maturation protein 1 (Blimp-1) [
11,
12].
Tfh cells could also migrate to the circulation as circulating Tfh (cTfh) cells. These circulating cells express lower amounts of the Tfh markers ICOS, CXCR5, PD-1 and Bcl-6 than their germinal center counterparts and respond to CXCL13 chemokine gradients, moving back to a secondary lymphoid organ germinal center, where they may be involved in germinal center formation [
6]. Whether serum CXCL13 level is associated with cTfh cells in kidney transplantation recipients is not clear.
A recently described T follicular regulatory (Tfr) cells has revealed a new means by which the GC reaction is controlled [
13]. Tfr cells express high levels of CXCR5, which directs them to the GC to inhibit the interaction of Tfh and B cells. Tfr cells differ from Tfh cells by expressing foxkhead box P3 (FoxP3) and Blimp-1 [
13]. The dynamic proportions of Tfh and Tfr cells precede the increase in GC-B cells and antibody production [
13]. Whether the circulating Tfr (cTfr) cells or the cTfh to cTfr ratio is associated with CAD is not clear.
It is well-known that transforming growth factor beta (TGF-β) plays a critical role in immune regulation, particularly in generation, function and stabilization of regulatory T cells (Tregs) [
14]. TGF-β could also regulate the development of Tfh and Tfr cells. Schmitt et al. found that TGFβ could promote human Tfh cells differentiation through STAT3/STAT4-mediated signal pathway [
8]. TGF-β neutralization could partially weaken the inhibitory effect of Tfr cells on the proliferation and differentiation of Tfh cells and B cells [
15]. TGF-β could also contribute to the development of Tfr cells through promoting the generation and activation of Treg cells [
16]. Therefore, it is needed to identify that whether the role of serum TGF-β in kidney transplant recipients favoring immune regulation or immune reactivation.
It is not clear that whether the cTfh and cTfr cells, the cTfh to cTfr ratio, the expression of STAT3/STAT5 on CD4+CXCR5+ cells, serum CXCL13 and TGF-β are correlated with CAD in kidney transplant recipients. The aim of this study was to identity the possible association between these immune parameters and CAD, and further probe into the underlying mechanism of CAD.
Methods
Patients
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/1.73m
2 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.
Immunosuppressive regimen
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.
Flow cytometry
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.
Bio-plex
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.
Laboratory assays
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/1.73m
2) = 186 × Scr (mg/dl)
-1.154 × age
-0.203 × (0.742 if female) × 1.233.
Statistics analysis
Statistical analysis and graphics were performed using SPSS 21.0 (SPSSInc, Chicago, IL, US) and GraphPad Prism version 5.01 (GraphPad, Inc., La Jolla, CA). The Mann-Whitney U-test was utilized to identify differences in phenotype between different groups. Logistic regression was performed to assess the independent associations of immune-associated parameters, other clinical variables with CAD. Linear regression was used to assess the independent associations of immune-associated parameters, other clinical variables with eGFR. Chi-square test and Mann-Whitney U-test were used to compare the percentage of recipients with CAD between groups classified based on cTfh to cTfr ratio. Spearman correlation analysis was performed to assess the association between CXCL13 or TGF-β and the phenotype of cTfh or cTfr cells. A two-sided p-value ≤0.05 was considered significant.
Discussion
In the present study, we found that the frequency of CXCR5+ on CD4+ cells and cTfr cells were decreased in CAD group than stable group. The frequency of cTfh cells had the same trend. The cTfh to cTfr ratio in CAD group was higher than that in stable group. Serum CXCL13 in CAD group was higher than that in stable group. Serum CXCL13 was negatively associated with the frequency of cTfh cells. No association between serum CXCL13 and cTfr cells was observed. Serum TGF-β in CAD group was lower than that in stable group. No association between serum TGF-β and cTfh, cTfr, or CXCR5+STAT3+ on CD4+cells was observed. The cTfh to cTfr ratio was an independent risk factor to renal function and CAD after multiple regression analysis. After stratified analysis based on the cTfh to cTfr ratio, the percentage of recipients with CAD in Group 4 was significantly higher than that in Group 1 and Group 2. The cTfh to cTfr ratio was also significantly higher in BPR group or DSA group compared to stable group.
The proportions of both cTfh and cTfr cells in recipients with CAD were lower than that in recipients with stable renal function. Tfh and Tfr cells share a lot of common differentiation signal pathway. Bcl-6 is a key transcription factor for the differentiation of Tfh and Tfr cells [
6,
13]. Tfh and Tfr cells express CXCR5 and migrate into GC under the gradient of CXCL13 [
6,
13]. Interleukin-2 (IL-2) could inhibit the differentiation of Tfh and Tfr cells through STAT5-Blimp-1 signal pathway [
21‐
23]. A recent study demonstrated that RNA-binding protein (Roquin) could inhibit the differentiation from Naïve T cells to Tfh cells, while inhibit the conversion of Treg to Tfr cells through inhibiting protein kinase B signal pathway [
24]. Several studies have demonstrated that STAT3 was indispensable for Tfh and Tfr cell differentiation by inducing the expression of Bcl-6 during immunization or infection [
9,
10,
25,
26]. Therefore, it is probably that the increased number of Tfh cells would be accompanied with the increase of Tfr cells in kidney transplant recipients.
We found that serum CXCL13 in CAD group was significantly higher than that in stable group and high expression of serum CXCL13 is negatively associated with the frequency of cTfh cells. Havenar-Daughton et al. demonstrated in mouse, macaques model and HIV-infected human that plasma CXCL13 levels correlated with GC activity in draining lymph nodes [
27]. Mabuka et al. found that early serum CXCL13 but not B cell frequency could predict the later emergence of detectable HIV neutralizing antibodies [
28]. cTfh cells could migrate into GC via CXCL13 gradient, which might contribute to the lower frequency of cTfh cells in CAD group, initiate the GC formation, and promote the humoral immune response. Dedeoglu et al. recently demonstrated that the frequency of CD4
+ T cells within CD3
+ T cells in lymph node from end-stage renal disease (ESRD) patients was significantly higher than that in peripheral blood [
29]. Tfh or Tfr cells, as a main subset of CD4
+ T cells within GC, their frequency in GC was also probably higher than that in circulation, particularly under the chemotaxis of high level of CXCL13.
The cTfh to cTfr ratio was an independent risk factor to renal function and CAD after multiple regression analysis. The proportion of Tfh and Tfr cells is dramatically changed. In skin-draining lymph nodes without antigen stimulation, Tfr cells constitute approximately 50% of all CD4
+CXCR5
+ cells. Seven days after stimulation, Tfr cells comprised only approximately 20% of the CD4
+CXCR5
+ population [
13]. When there was influenza infection, Tfr cells was approximately 5–8% of CD4
+CXCR5
+ cells [
13]. Constant allograft antigen stimulation would make the imbalance between Tfh cells to Tfr cells, dysregulation of humoral immunity, and eventually lead to allograft rejection.
After stratified analysis based on the cTfh to cTfr ratio, the percentage of recipients with CAD in Group 4 was significantly higher than that in Group 1 and Group 2. The Tfh to Tfr ratio has been demonstrated to act as a biomarker of humoral immunity. Fan et al. recently demonstrated in a simian immunodeficiency virus-infected (SIV) rhesus macaques model that the Tfh to Tfr ratio in peripheral lymphatic tissues is critical for regulating autoreactive antibody production in chronic SIV infection [
30]. Xu et al. found that the cTfh to cTfr ratio was associated with disease activity in systemic lupus erythematosus [
25]. The cTfh to cTfr ratio indicated ectopic lymphoid structure formation in minor salivary gland, strongly associated with B cell, CD4
+ T cell, and PD-1
+ICOS
+ T cell infiltration in minor salivary gland and allowed discrimination between Sjogren’s syndrome patients and healthy donors [
31]. Chen et al. showed that the frequency of cTfr cells and the number of Tfr cells in renal graft tissues in ABMR group were significantly lower than that of non-ABMR group, although no cTfh to cTfr ratio was analyzed [
15]. In the present study, we found that the cTfh to cTfr ratio was significantly higher in recipients with ABMR or DSA than recipients with stable renal function. The cTfh to cTfr ratio is a potential biomarker for kidney transplant recipients with CAD, ABMR and the production of DSA and might identify recipients at the risk of allograft failure.
No association between serum TGF-β level and cTfh cells nor CXCR5
+STAT3
+ on CD4
+ T cells was observed. Serum TGF-β has a trend to be positively associated with cTfr cells. As it has been reported, TGF-β could not only contribute the Tfh differentiation but also Tfr production [
8,
15,
16]. Tfh and Tfr cells are activated in germinal canter, which might explain why serum TGF-β did not significantly affect the frequency of cTfh and cTfr cells in kidney transplant recipients.
This study has some limitations. Only the percentage of each cell subset was detected, but not the cell function. The analysis of donor-specific immune cell would be better than the cell phenotyping for organ transplantation. In our previous study [
32], we already found that donor-specific IL-21 producing cells at 6 months after kidney transplantation could predict rejection within 5 years, while cTfh or cTfr cells was not found to be associated with rejection. In the present study, cTfr cells and cTfh to cTfr ratio correlated with rejection, but not IL-21. Donor-specific IL-21 producing cells might be more sensitive in rejection prediction than cTfh and cTfr. However, with the prolonged transplant duration time, the expression of cTfh and cTfr might be changed. The different degree of CXCR5
+ cells migrating to GC and allograft might lead to different outcomes. Considering the detection difficulty of donor-specific IL-21 producing cells, the cTfh to cTfr ratio might be more potential as a biomarker of CAD in kidney transplant recipients.
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