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. 2015 Apr 14;180(2):329–340. doi: 10.1111/cei.12576

Follicular T helper cells and humoral reactivity in kidney transplant patients

G N de Graav *,✉, M Dieterich *, D A Hesselink *, K Boer *, M C Clahsen-van Groningen †, R Kraaijeveld *, N H R Litjens *, R Bouamar ‡, J Vanderlocht §, M Tilanus §, I Houba §, A Boonstra , D L Roelen **, F H J Claas **, M G H Betjes *, W Weimar *, C C Baan *
PMCID: PMC4408167  PMID: 25557528

Abstract

Memory B cells play a pivotal role in alloreactivity in kidney transplantation. Follicular T helper (Tfh) cells play an important role in the differentiation of B cells into immunoglobulin-producing plasmablasts [through interleukin (IL)-21]. It is unclear to what extent this T cell subset regulates humoral alloreactivity in kidney transplant patients, therefore we investigated the absolute numbers and function of peripheral Tfh cells (CD4POSCXCR5POS T cells) in patients before and after transplantation. In addition, we studied their relationship with the presence of donor-specific anti-human leucocyte antigen (HLA) antibodies (DSA), and the presence of Tfh cells in rejection biopsies. After transplantation peripheral Tfh cell numbers remained stable, while their IL-21-producing capacity decreased under immunosuppression. When isolated after transplantation, peripheral Tfh cells still had the capacity to induce B cell differentiation and immunoglobulin production, which could be inhibited by an IL-21-receptor-antagonist. After transplantation the quantity of Tfh cells was the highest in patients with pre-existent DSA. In kidney biopsies taken during rejection, Tfh cells co-localized with B cells and immunoglobulins in follicular-like structures. Our data on Tfh cells in kidney transplantation demonstrate that Tfh cells may mediate humoral alloreactivity, which is also seen in the immunosuppressed milieu.

Keywords: donor-specific antibodies, follicular T helper cell, humoral reactivity, kidney transplantation

Introduction

Memory B cells are important in alloreactivity in kidney transplantation. During the last decade it has become clear that acute T cell-mediated rejection biopsies contain numerous B cells as well as conventional T cells 1. A significant proportion of acute rejections can therefore be classified as a mixed, rather than a pure, T cell-mediated rejection 2,3. Hence, B cells are also involved in acute T cell-mediated rejection next to chronic antibody-mediated rejection, which involves donor-specific anti-human leucocyte antigen (HLA) antibodies (DSA) produced by B cells that differentiated into plasmablasts or plasma cells 4. The latter are formed after the differentiation of donor-specific memory B cells. Follicular T helper (Tfh) cells are of importance for the survival of memory B cells and the differentiation of B cells into immunoglobulin-producing plasmablasts or plasma cells 5. These Tfh cells are a heterogeneous population of CD4POS T helper cells, which are active in secondary lymphoid organs 6 and may be present in tertiary lymphoid structures formed in kidney allografts 7. Tfh cells also produce interleukin (IL)-21, which is an important cytokine for B cell stimulation and differentiation 5,8,9. The transcription factor for the formation of Tfh cells, B cell lymphoma 6 (Bcl-6), suppresses the transcription factors required for formation of other T helper subsets 5,9,10. Chemokine receptor 5 (CXCR5) allows migration of Tfh cells towards the germinal centre 11–13. Peripheral CD4POSCXCR5POS T cells are the counterparts of Tfh cells in blood and express low levels of inducible T cell co-stimulator (ICOS) and programmed death 1 (PD1), and lack Bcl-6 5,14.

Recent studies have revealed the pivotal role of Tfh cells in B cell-mediated immune responses in autoimmunity 15–17 and chronic viral infections 18–23 in humans. In autoimmunity, frequencies of Tfh cells are correlated positively with the peripheral blood levels of disease-specific autoantibodies 16,17 and with disease activity 15. Accordingly, the percentage of circulating Tfh cells decreased in patients with autoimmune thyroiditis after treatment 17. IL-21 is an important disease mediator in these autoimmune disorders 15–17. Also, in chronic viral infections such as hepatitis B and HIV, Tfh cells are expanded in peripheral blood 21–23 or in lymph nodes 18,19, and they can function as markers for the response to anti-viral treatment.

No studies have been conducted which define the role of Tfh cells in B cell activation and humoral alloreactivity in immunosuppressed kidney transplant patients 24. In CD4-reconstituted rodents, Tfh cells induce long-lasting immunoglobulin (Ig)G alloantibody responses after heart transplantation 25, which are potentially dangerous for the graft. These cells also mediated humoral responses towards the kidney allograft in an immunosuppressed non-human primate model 26. In addition, Tfh cells from intestinal transplant patients maintain germinal centres in isolated lymphoid follicles in the transplanted intestines and subsequently ensure IgA synthesis to control commensal microflora, despite the immunosuppressed milieu 27. As B cells play a pivotal role in both cellular and humoral alloreactivity, the interaction between Tfh cells and B cells is an important target for immunosuppression. We hypothesize that in immunosuppressed kidney transplant patients Tfh cells can still stimulate B cells, which leads to their differentiation into immunoglobulin-producing plasmablasts and plasma cells. The frequency and function of peripheral Tfh cells were therefore examined before and after kidney transplantation, as well as their relationship to the presence of donor-specific antibodies (DSA). In addition, we stained acute rejection biopsies to examine the presence of intragraft Tfh cells, B cells and immunoglobulins.

Material and methods

Study population in which peripheral Tfh cells were studied

Thirty consecutive renal transplant recipients were included and followed for 1 year post-transplantation. Sixteen age- and gender-matched controls (healthy volunteers) were also included. Patients receiving a kidney transplant from a living donor all participated in a randomized controlled clinical trial with the primary aim of studying the efficacy of a genotype-based approach to tacrolimus dosing (Dutch trial registry number NTR 2226; http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=2226). All patients received induction therapy with basiliximab [Simulect®; Novartis, Basel, Switzerland; 20 mg intravenously (i.v.) on days 0 and 4], tacrolimus (Prograf®; Astellas Pharma, Tokyo, Japan; aiming for predose concentrations of 10–15 ng/ml in weeks 1–2, 8–12 ng/ml in weeks 3–4 and 5–10 ng/ml thereafter), mycophenolate mofetil (Cellcept®; Roche, Basel, Switzerland; starting dose of 1 g twice a day, aiming for predose concentrations of 1·5–3·0 mg/l) and glucocorticoids. Prednisolone was tapered to 5 mg at month 3 and withdrawn at months 4–5. For inclusion in the study, as well as the current substudy, written informed consent was required from the patient. The study was approved by the Medical Ethical Committee of the Erasmus MC (MEC number 2010-080, EudraCT 2010–018917-30). Rejections were defined as biopsy-proven acute rejection (BPAR) according to the Banff classification 2009 28.

Numbers of peripheral Tfh cells and cytokine production capacity

Peripheral Tfh cells were defined as CD3POSCD4POSCXCR5POS lymphocytes and measured in fresh whole blood samples obtained 1 day before and 3 months after transplantation. In these cells the intracellular IL-21 production capacity was determined ex vivo after 4 h of stimulation with phorbol myristate acetate (PMA) 0·5 μg/ml and ionomycin 10 μg/ml (Sigma-Aldrich, St Louis, MO, USA) at 37°C. The following monoclonal antibodies (mAbs) were used: CD3 AmCyan; CD4 Pacific Blue; CXCR5 AF647; IL-21 phycoerythrin (PE); and the IL-21 isotope mouse IgG1-PE (all from BD Biosciences, San Jose, CA, USA). To measure absolute numbers of CD3 and CD4, BD multi-test 6-colour® was used in BD TruCount Tubes® (BD Biosciences). Absolute numbers of the subsets were calculated using the percentages of these subsets within the total CD3 and CD4 populations.

Co-culture experiments of peripheral Tfh cells and memory B cells

To determine the function of peripheral Tfh cells, co-culture experiments with memory B cells were conducted. First, peripheral Tfh cells, i.e. CD3POSCD4POSCXCR5POS T cells and memory B cells, i.e. CD19POSCD27POS cells, were isolated by sorting with BD-FACSAria II SORP™ (purities ≥ 95%) from defrosted peripheral blood mononuclear cells (PBMCs). These PBMCs were obtained from patients 1 day before and 3 months after transplantation (thus isolated from an immunosuppressed milieu). As a control, PBMCs of healthy volunteers were used; mAbs were used as described above, including viability staining solution 7-aminoactinomycin (7-AAD) peridinin chlorophyll (PerCP) (BD Biosciences). Secondly, memory B cells were co-cultured for 7 days with CD4POSCXCR5POS Tfh cells in the presence of the superantigen Staphylococcus aureus antigen B (SEB; Sigma-Aldrich). Subsequently, differentiation of B cells into plasmablasts after 7 days was determined with flow cytometry. Plasmablasts were defined as CD3NULLCD4NULLCD19POSCD20NULLCD27POSCD38HIGH cells 14,29 using the following mAbs: CD3 AmCyan (BD Biosciences); CD4 Pacific Blue [Becton Dickinson (BD), Frankin Lakes, NJ, USA]; CD19 fluorescein isothiocyanate (FITC) (BD); CD20 PerCP (BD); CD27 PE-cyanin-7 (Cy7) (eBioscience, San Diego, CA, USA); and CD38 PE (eBioscience). Finally, after 7 days, IgM and IgG production were measured with a sandwich enzyme-linked immunosorbent assay (ELISA) on the supernatants of the co-cultures.

To determine the role of IL-21 in the interaction of Tfh cells with memory B cells, 5 μg/ml of IL-21-receptor blocking antibody (IL-21-R-Fc; R&D Systems, Minneapolis, MN, USA) was added to the co-cultures of four patients (pretransplantation samples) and three healthy volunteers, according to the concentration used for blocking immunoglobulin production by 50–75% in cells of healthy controls 14, thus sufficiently blocking the IL-21-receptor without completely inhibiting plasmablast formation and immunoglobulin production. An isotype-matched control (IgG1-Fc; R&D Systems) was used. Thereafter, differentiation into plasmablasts and immunoglobulin production were measured after 7 days.

Donor-specific anti-HLA antibodies (DSA)

DSA, including C1q-binding capacity, were determined in thawed heparin plasma samples before and 3, 6 and 12 months after kidney transplantation. For all patients, the complement-dependent cytotoxicity (CDC) cross-match before transplantation was negative for both current and historic sera. DSA presence before transplantation was considered as pre-existent DSA, and DSA developing after transplantation as de-novo DSA. Plasma samples were centrifuged for 10 min at 14 170 rpm. Thirty μl of the plasma was incubated with 100 μl/ml Adsorb Out microbeads (One Lambda®, Canoga Park, CA, USA) to minimize false positive staining. Subsequently, 20 μl plasma was incubated for 30 min with 2 μl single antigen beads mix from LABScreen (One Lamda) single antigen class I and class II kits. After protocol washing procedures, plasma samples were incubated with 1 μl goat anti-human IgG-PE per well (One Lambda). Microbeads were analysed with a Luminex Labscan™ 100 (One Lambda) analyser using both Luminex 100IS and HLA Fusion version 3·0 software. All samples fulfilled the quality criteria for the control beads reactivity.

Immunohistochemistry

Kidney biopsies, diagnosed as type I acute rejection (three of type 1A and two of type 1B), were paraffin-embedded, formalin-fixed and cut into 4-μm sections. Immunohistochemistry was performed by routine diagnostics on the Benchmark Ultra Stainer (Ventana, Basel, Switzerland), using the following mAbs: CD3 (1:150 dilution; Dako, Glostrup, Denmark) was used to detect pan-T cells; CD4 (undiluted; Ventana, Tucson, AZ, USA) for T helper cells; CD8 (1:50; Dako) for cytotoxic T cells; CD20 (1:400 dilution; Dako) for B cells; C4d (1:60 dilution; Biomedica Gruppa, Vienna, Austria) for complement factor C4d; Bcl-6 (1:15 dilution; Novocastra/Leica, Solms, Germany) for the transcription factor of Tfh cells; IgM (1:80 dilution; Biogenex, Fremont, CA, USA) and IgG (1:200 dilution, Dako) for immunoglobulin production.

Incubation with antibodies was carried out for 30 min and anti-rabbit or anti-mouse amplifiers were used. As positive control for Bcl-6, tonsillar sections obtained from a paediatric tonsillectomy were used.

To identify intragraft Tfh cells, sections were double-labelled with CD3 (polyclonal rabbit; Dako; 1:200) and Bcl-6 (mouse monoclonal; Novocastra; 1:50). After incubation overnight at 4°C with the primary antibody Bcl-6, sections were subsequently incubated with a biotinylated horse anti-mouse antibody (1:500) and Dyelight®594 (red) conjugated with streptavidin (Vector Laboratories, Burlingame, CA, USA; 1:500) to visualize the Bcl-6 expression. Next, sections were incubated for 1 h with the CD3 antibody and subsequently with the Dyelight®488 (green) goat anti-rabbit antibody (Vector Laboratories; 1:200).

Statistical analyses

Differences between measurements before and after transplantation were analysed using the Wilcoxon signed-rank test. Using the Mann–Whitney U-test, the differences were analysed between transplant patients and healthy controls and between patients with and without pre-existent DSA. For comparing dichotomous outcomes, we used Fisher's exact test.

IBM spss version 20 (Armonk, NY, USA) was used for statistical analysis. P-values with a two-sided α of 0·05 were considered statistically significant. When not specified otherwise, medians (+ range_ are presented.

Results

Study population

Table 1 depicts the baseline characteristics of the patients from whom we studied the peripheral Tfh cells. Two patients discontinued tacrolimus within 3 months of transplantation. In one case this was necessitated by tacrolimus-induced thrombotic micro-angiopathy, and in the other case because of severe acute nephrotoxicity. One patient underwent a complicated transplantation with small bowel perforation and subsequently did not receive mycophenolate mofetil (MMF). The remaining 27 patients were all on a tacrolimus/MMF/prednisolone regimen for the first 3 months after transplantation. Eleven patients (36·7%) suffered from rejection within 12 months of transplantation. Median time to rejection was 99 days, ranging from 7 to 261 days. Six patients had an early BPAR, i.e. within the first 3 months of transplantation. One-year dead-censored graft survival was 100% and 1-year patient survival was 96·7%. One patient died from gastric adenocarcinoma 9 months after transplantation.

Table 1.

Baseline characteristics and outcomes

Study group (n = 30)
Recipient age in years (median, range) 52 (19–75)
Donor age in years (median, range) 49 (21–86)
Recipient gender (% male) 67%
HLA-A mismatches (mean ± s.d.) 0·9 (± 0·8)
HLA-B mismatches (mean ± s.d.) 1·1 (± 0·8)
HLA-DR mismatches (mean ± s.d.) 1·0 (± 0·6)
Cause of end-stage renal disease
• Hypertensive nephropathy 23·3% (7)
• Glomerulonephritis 16·7% (5)
• Polycystic kidney disease 13·3% (4)
• Focal segmental glomerulosclerosis 10% (3)
• Diabetic nephropathy 6·7% (2)
• Congenital dysplasia 6·7% (2)
• Miscellaneous 23·3% (7)
Previous kidney-transplantation* 20% (6)
• Third kidney transplantation 6·7% (2)
• Second kidney transplantation 13·3% (4)
Renal replacement therapy before transplantation 53·3% (16)
• Haemodialysis 26·7% (8)
• Peritoneal dialysis 26·7% (8)
Rejection within 12 months of transplantation 36·7% (11)
• Type I T cell-mediated rejection 54·5% (6)
• Type II T cell-mediated rejection 27·3% (3)
• Pure humoral (C4d+) rejection 0% (0)
• Mixed rejection 18·2% (2)
Time to rejection in days (median, range) 99 (7–261)
1-year death-censored graft survival 100%
1-year patient survival 96·7%
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Patient number is depicted between brackets unless otherwise specified.

*

Two patients received two previous grafts: one received a graft from a post-mortem donor and from a living donor; the other received both previous grafts from a post-mortem donor. Four patients received one previous graft, all from a living donor. HLA = human leucocyte antigen; s.d. = standard deviation.

Numbers of peripheral Tfh cells remain stable after kidney transplantation, while their IL-21 production capacity decreases

We studied the number of peripheral Tfh cells before and 3 months after kidney transplantation. Absolute numbers of CD4POSCXCR5POS T cells were lower in patients before and after transplantation compared to the healthy controls (P < 0·01), but remained stable after transplantation (Fig. 1a,b).

Fig 1.

Fig 1

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Numbers of peripheral (CD4POSCXCR5 POS) follicular T helper (Tfh) cells remained equal after transplantation, while interleukin (IL)-21-production capacity decreased after transplantation. (a) CD4POSCXCR5POS were gated from CD3-positive cells within the lymphocytes, which were defined by forward- and side-scatter. A typical example of CXCR5-expression within CD4-positive T cells is shown for a patient before and 3 months after kidney transplantation. (b) Absolute numbers of CD4POSCXCR5POS T cells were compared before and 3 months after transplantation (n = 30), and between patients and healthy controls (n = 16). (c) The proportion of IL-21-producing cells is shown upon 4 h phorbol myristate acetate (PMA)/ionomycin stimulation in typical examples for CD4POSCXCR5POS T cells and CD4POSCXCR5NULL T cells (total measured n = 30). An unstimulated (i.e. negative) control for CD4POSCXCR5POS T cells is depicted. (d) Absolute numbers of IL-21-producing CD4POSCXCR5POS T cells upon 4 h PMA/ionomycin stimulation are depicted before and after transplantation (n = 30), and in healthy controls (n = 16).

To confirm that IL-21 is produced by CD4POSCXCR5POS T cells, their IL-21 production capacity upon PMA/ionomycin stimulation was compared with that of CD4POSCXCR5NULL T cells. A representative example of the IL-21 production by CD4POSCXCR5POS and CD4POSCXCR5NULL T cells is illustrated (Fig. 1c). A higher percentage of CD4POSCXCR5POS T cells produced IL-21 than CD4POSCXCR5NULL T cells [6·9% (1·5–12·6%) versus 3·2% (0·4–8·5%), P < 0·0001].

The numbers of IL-21-producing Tfh cells after stimulation were comparable between healthy controls and patients before transplantation (Fig. 1d). After transplantation, a decrease in IL-21 production capacity of CD4POSCXCR5POS T cells was observed, P < 0·05. (Fig. 1d). Patients (n = 6) with a BPAR within 3 months after transplantation, which consequently received high doses of pulse steroids, had similar numbers of CD4POSCXCR5POS T cells. However, a lower IL-21 production capacity was observed compared to patients who did not reject within the first 3 months after transplantation (n = 24), P < 0·05 (Supporting information, Fig. S1).

The in-vitro function of peripheral Tfh cells is conserved in patients after transplantation

To determine the functionality of peripheral (CD4POSCXCR5POS) Tfh cells, we performed co-culture experiments of pure, isolated Tfh cells and memory B cells from patients from whom materials were available before and after transplantation (n = 11). For gating strategies see Fig. 2a–c. After transplantation, less Tfh cell-dependent B cell differentiation into plasmablasts was observed than before transplantation (Fig. 2d, P < 0·05). Nevertheless, this did not lead to altered IgM and IgG production levels: among samples obtained before and 3 months after transplantation, comparable levels of IgM and IgG were measured in the co-cultures with CD4POSCXCR5POS T cells (Fig. 2e,2f). Tfh cell-dependent IgM production was lower in co-cultures of patient-derived cells than in co-cultures of healthy control-derived cells (Fig. 2e, P < 0·01). This difference was not observed for Tfh cell-dependent IgG production (Fig. 2f).

Fig 2.

Fig 2

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After kidney transplantation CD4POSCXCR5POS T cells were still capable of stimulating memory B cells to differentiate into immunoglobulin-producing plasmablasts. This process was interleukin (IL)-21-dependent. (a) A typical example of the fluorescence activated cell sorting (FACS) strategy to obtain CD4POSCXCR5POS T cells is depicted. Cells were gated from viable [i.e. 7-aminoactinomycin D (7-AAD)-negative] lymphocytes, which were defined by forward- and side-scatter. (b) A typical example of the FACS strategy to obtain memory B cells is depicted. Cells were gated from viable (i.e. 7-AAD-negative) cells, which were defined by forward- and side-scatter. (c) A typical example of the FACS measurement of differentiation of memory B cells into plasmabast is depicted (after 7-day co-culture with CD4POSCXCR5POS T cells). (d–f) CD4POSCXCR5POS T cells were obtained before and after transplantation (n = 11). Their capacity to stimulate memory B cell differentiation into plasmablasts (d), and to stimulate IgM (e) and immunoglobulin (Ig)G production (f) is compared. Also the stimulation capacity of healthy controls' CD4POSCXCR5POS T cells is depicted (n = 8). (g–i) The percentage of memory B cells differentiated into plasmablasts (g), immunoglobulin (Ig)M production (h) and IgG production (i) are given on day 7 in the presence or absence of an anti-IL-21R-antagonist (5 μg/ml IL-21R-Fc). Dashed lines represent the healthy controls (n = 3) and solid lines the patients (n = 4). *P < 0·05; **P < 0·01; ***P < 0·001; y-axes for immunoglobulin production are scaled log-linearly. 3 M = 3 months; KT = kidney transplantation; TFH cells = follicular T helper cells.

Plasmablast formation and immunoglobulin production regulated by peripheral Tfh cells are dependent on IL-21

The importance of IL-21 in the functional interaction between Tfh cells and B cells was established by adding an anti-IL-21-R antibody to co-cultures containing CD4POSCXCR5POS T cells and memory B cells from healthy controls or patients before transplantation. Differentiation of memory B cells into plasmablasts was inhibited by 48·4% (ranging from 11·3 to 89·9%, P < 0·05, Fig. 2g). Subsequently, both IgM and IgG production were inhibited by 89·2% (27·4–97·5%), P < 0·05 (Fig. 2h) and 80·4% (3·6–99·8%), P < 0·05 (Fig. 2i).

After transplantation, numbers of peripheral Tfh cells are higher in patients with pre-existent DSA than in patients without pre-existent DSA

To study whether Tfh cells may induce the B cell differentiation in vivo, we determined the relation between peripheral Tfh (CD4POSCXCR5POS) cell numbers and the presence of DSA. For this purpose the absolute numbers of CD4POSCXCR5POS T cells, before and after transplantation, were compared between patients with pre-existent DSA and patients without pre-existent DSA (Fig. 3). To cover the different definitions for DSA positivity, different mean fluorescence intensity (MFI) cut-offs were used to determine DSA positivity or negativity. No differences were found at MFI cut-offs ≥1000, ≥2000 or ≥3000 (data not shown). When cut-offs for the MFI of ≥4000 or ≥5000 were used, patients with pre-existent DSA had more peripheral Tfh cells after transplantation than patients without pre-existent DSA (Fig. 3a,b, P < 0·05 and P < 0·001). See Table 2 for baseline characteristics in patients with and without pre-existent DSA at an MFI cut-off ≥4000. All pre-existent DSA (MFI ≥ 4000) were initially C1q-negative and were persistent after transplantation. In two patients the pre-existent DSA converted into C1q-positive, i.e. complement-binding DSA, after transplantation. In the other patients the DSA remained C1q-negative after transplantation. Five patients developed de-novo DSA after transplantation. In three of these patients DSA de novo were C1q-positive, two of whom suffered from an acute rejection within the first year after transplantation. Despite the limited numbers of patients with de-novo DSA, we compared them with patients without de-novo DSA and found no differences among the numbers of peripheral Tfh cells (data not shown).

Fig 3.

Fig 3

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After kidney transplantation, the numbers of CD4POSCXCR5POS T cells were higher in patients with pre-existent donor-specific anti-human leucocyte antigen (HLA) antibodies (DSA) than in patients without pre-existent DSA. (a) The study population (n = 23) is divided into patients with pre-existent (=pre-transplantation) DSA (n = 15) and patients without pre-existent DSA (n = 8). In this analysis a mean fluorescence intensity (MFI) of 4000 or higher was considered as positive DSA. Numbers of CD4POSCXCR5POS T cells were compared between patients with and without DSA, before and after transplantation. (b) The study population (n = 23) is divided into patients with pre-existent (= pre-transplantation) DSA (n = 17) and patients without pre-existent DSA (n = 6). In this analysis an MFI of 5000 or higher was considered as positive DSA. Numbers of CD4POSCXCR5POS T cells were compared between patients with and without DSA, before and after transplantation. (c) The study population (n = 23) is divided into patients with pre-existent (= pre-transplantation) DSA (n = 15) and patients without pre-existent DSA (n = 8). In this analysis an MFI of 4000 or higher was considered as positive DSA. Numbers of interleukin (IL)-21+CD4POSCXCR5POS T cells were compared between patients with and without DSA, before and after transplantation. (d) The study population (n = 23) is divided into in patients with pre-existent (=pre-transplantation) DSA (n = 17) and patients without pre-existent DSA (n = 6). In this analysis an MFI of 5000 or higher was considered as positive DSA. Numbers of IL-21+CD4POSCXCR5POS T cells were compared between patients with and without DSA, before and after transplantation. Black lines represent the median; the upper and lower border of the boxes represent the 25th and 75th percentiles; the error lines represent the minimal and maximal value within 1·5 quartile distances of the box; values above 1·5 quartile distances of the box are considered outliers and are represented by a dot.

Table 2.

Baseline characteristics of patients with pre-existent donor-specific anti-human leucocyte antigen (HLA)-antibodies (DSA) and without pre-existent DSA when a cut-off of a mean fluorescence intensity (MFI) ≥ 4000 is used

No pre-existent DSA (n = 16) Pre-existent DSA (n = 7) P-value (two-sided)
Recipient age in years (median, range) 58 (19–75) 51 (22–58) 0·02
Donor age in years (median, range) 55 (26–86) 48 (25–54) 0·08
Recipient gender (% male) 69% (11) 43% (3) 0·36
HLA-A mismatches (mean ± s.d.) 1·1 (± 0·7) 0·9 (± 0·9) 0·56
HLA-B mismatches (mean ± s.d.) 1·3 (± 0·8) 1·1 (± 0·9) 0·86
HLA-DR mismatches (mean ± s.d.) 1·1 (± 0·6) 1·1 (± 0·4) 0·97
Cause of end-stage renal disease 0·02
• Hypertensive nephropathy 31·2% (5) 14·3% (1)
• Glomerulonephritis 0% (0) 28·6% (2)
• Polycystic kidney disease 18·8% (3) 0% (0)
• Focal segmental glomerulosclerosis 0·0% (0) 28·6% (2)
• Diabetic nephropathy 12·5% (2) 0% (0)
• Congenital dysplasia 12·5% (2) 0% (0)
• Miscellaneous 25% (4) 28·6% (2)
Previous kidney transplantation* 0% (0) 43% (3) 0·02
• Third kidney transplantation – 14·3% (1)
• Second kidney transplantation – 28·6% (2)
Replacement therapy before transplantation 43·8% (7) 71·4% (5) 0·37
• Haemodialysis 31·2% (5) 14·3% (1)
• Peritoneal dialysis 12·5% (2) 57·1% (4)
Rejection within 12 months of transplantation 31·3% (5) 42·9% (3) 0·47
Time to rejection in days (median, range) 78 (7–261) 129 (8–216) 0·71
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Patient number is depicted between brackets unless otherwise specified.

*

Two patients received two previous grafts: one received a graft from a post-mortem donor and from a living donor; the other received both previous grafts from a post-mortem donor. Four patients received one previous graft, all from a living donor. s.d. = standard deviation.

Tfh cells are present in follicular-like structures in acute T cell-mediated rejection kidney biopsies

Acute T cell-mediated rejection biopsies (n = 5) were stained to determine the presence of Tfh cells (representative example in Fig. 4). Semi-quantitative analyses of the immunohistochemistry are depicted in Table 3. Follicular-like structures containing CD4POS T cells and CD20POS B cells were observed. Simultaneous staining of CD3 and Bcl-6 revealed Bcl-6-expressing T cells on the T–B cell border of the follicular-like structures, suggesting that these cells interact physically at the graft site to regulate B cell function (Fig. 4f–g). Confirming this, the immunohistochemistry data showed that these biopsies were also positive for IgM and IgG (Fig. 4i,j). All biopsies were C4d-negative (data not shown).

Fig 4.

Fig 4

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In a typical acute T cell-mediated rejection biopsy Bcl-6POS T cells were co-localized with CD20POS B cells and immunoglobulins in follicular-like structures. (a–d) Immunohistochemistry for CD3 (a), CD4 (b), CD8 (c) and CD20 (d) was performed to determine the location of, respectively, T cells, T helper cells, cytotoxic T cells and B cells in the same acute T cell-mediated rejection biopsy. (e) Immunohistochemistry for Bcl-6 was performed to determine the location of this transcription factor for follicular T helper cells among others in the same acute cellular renal rejection biopsy. (f,g) CD3 and Bcl-6 double staining showed the presence of the transcription factor for follicular T helper cells in T cells. (h) Positive control for double-staining procedure on a tonsil. (i,j) Immunohistochemistry of immunoglobulin (Ig)M (i) and IgG (j) to determine the presence of IgM and IgG in the same acute cellular renal rejection biopsy. Magnification of a–e: ×20; insert in e: ×40; f,g: ×63; h–f: ×20.

Table 3.

Immunofluorescence on five biopsies of kidney transplants undergoing acute T cell-mediated rejection

PA diagnosis CD3 CD4 CD20 BCL6 IgM IgG
1 TCMR I ++ ++ ++ + ++ ++
2 TCMR I ++ ++ ++ ++ Not enough material Not enough material
3 TCMR I ++ ++ ++ ++ − −
4 TCMR I ++ ++ ++ + + +++
5 TCMR I ++ ++ +++ + ++ ++
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TCRM I = T cell (cellular)-mediated rejection type I. +++, Very strongly positive; ++, strongly positive; +, positive; +/−, weakly positive; −/+, very weakly positive; −, negative. Ig = immunoglobulin; PA = Pathology.

Discussion

We have studied the involvement of Tfh cells in B cell-mediated immune responses after kidney transplantation in vitro and in vivo. Several new insights were gained: (i) despite the decreased capacity of peripheral Tfh cells to stimulate B cell differentiation after transplantation, the levels of IgM and IgG production capacity by plasmablasts in co-cultures were not affected by the immunosuppressive drugs in vivo; (ii) Tfh cell numbers post-transplantation were associated with high DSA titres before transplantation; (iii) graft infiltrating Tfh cells co-localized with B cells in follicular-like structures; and (iv) locally produced immunoglobulins were present in transplanted kidneys during rejection. Overall, the combination of in-vitro and in-vivo measurements in samples of patients before and after transplantation emphasize the importance of Tfh cells in humoral alloreactivity.

Even though the immunoglobulin production remained intact after transplantation, Tfh cell-dependent B cell differentiation into plasmablasts was reduced, which suggests that either the Tfh cells or B cells are influenced partially by immunosuppressive drugs. As IgM and IgG levels were comparable after transplantation, this indicates that at the single-cell level, differentiated B cells produced higher amounts of IgM and IgG 30,31. The sustained Tfh cell-mediated immunoglobulin production after transplantation by differentiated B cells in vitro indicates that peripheral Tfh cells are capable of supporting B cells. Meier et al. also concluded that Tfh cells are present and functional in the immunosuppressed milieu of intestinal transplant patients 27. IgM production capacity of the cells isolated from pretransplant patients was lower than in healthy controls (Fig. 2e), which may be explained by an impaired immune response caused by uraemia in end-stage renal disease patients 32, resulting in a lower antibody response towards new antigen 33. IgG production by plasma cells represents a memory response, which was not impaired in our study cohort, suggesting that memory B cell or plasmablast functions are not susceptible to the devastating effects of end-stage kidney disease. Because, in our study, the function of peripheral Tfh cells was demonstrated by the differentiation of memory B cells into IgM- and IgG-producing plasmablasts in the presence of a superantigen, further studies will need to establish whether these peripheral Tfh cells are fully capable of providing B cell help during allogeneic stimulation.

In vivo, the peripheral Tfh cells were also linked to humoral reactivity. The numbers of peripheral Tfh cells were associated with the presence of pretransplantation DSA measured by Luminex (Fig. 3a–d). When cut-offs at MFIs ≥4000 or ≥5000 were used, identifying DSA with a low prevalence for false positivity 34, patients with pre-existent DSA had more peripheral Tfh cells after transplantation than patients without pre-existent DSA. This can be explained by the fact that peripheral follicular T cells, including other CXCR5POS T cells, are memory T cells, which are less susceptible for the given immunosuppressive medication 5,35–37. As patients with pre-existent DSA were mainly patients who had had previous kidney transplants (Table 2), it is expected that HLA-specific memory T cells are present in these patients' circulation. Another explanation could be that pre-existent DSA indicates the presence of antigen-specific memory B cells, which can present donor antigen to naive T cells. These naive T cells consequently differentiate into antigen-specific Tfh cells and other subtypes of matured T cells 5. Finally, the higher numbers of peripheral Tfh cells in patients with pre-existent DSA might be related to an underlying autoimmune disease. Two patients in the pre-existent DSA group suffered from glomerulonephritis before transplantation, which could have led to higher numbers of circulating Tfh cells 38. The remaining five patients, however, did not have an autoimmune disease and still had higher numbers of circulating Tfh cells. Larger patient cohorts are needed to determine other confounders for the numbers of circulating Tfh cells.

All pre-existent DSA were non-complement binding, i.e. C1q-negative. The clinical relevance of non-complement binding C1q-negative DSA is under debate, and conflicting results are reported. C1q-negative DSA are often dismissed as clinically irrelevant 39,40; however, patients with C1q-negative DSA have a more severe Banff score during rejection 4, as well as a worse graft survival compared to patients without DSA 4,41. Hence, C1q-negative DSA cannot be labelled automatically as clinically irrelevant. C1q-positive DSA are known to predict graft loss 4,41. Despite these ongoing discussions we observed an association between peripheral Tfh cells and DSA, which suggests the involvement of Tfh cells in anti-donor B cell mediated alloreactivity in kidney transplant patients. Interference of the Tfh–B cell interaction may be an approach to block B cell differentiation into immunoglobulin-producing plasmablasts and plasma cells. One method of interfering in this interaction is the blockade of the IL-21 pathway, as demonstrated in our in-vitro studies (Fig. 2g–i). The immunosuppressed regimen in this cohort probably partially interferes with this interaction 29,30. IL-21 production capacity by peripheral Tfh cells as measured in whole blood was decreased after transplantation. However, the presence of circulating DSA and the support by peripheral Tfh cells to humoral reactivity in vitro indicate that the interference by the current calcineurin-based immunosuppressive regimen is only partially effective.

In addition to pure humoral alloreactivity, Tfh cells are involved in B cell alloreactivity classified as T cell-mediated rejection 2,3. The presence of Bcl-6 expressing Tfh cells was demonstrated in C4d-negative acute T cell-mediated rejection biopsies of kidney grafts. These findings strengthen our observations that Tfh cells contribute actively to the anti-donor response and that rejection is the result of T and B cell interactions, even when the rejection is not classified as mixed or antibody-mediated 3,28. As well as their effector functions, B cells still could exhibit their well-known antigen-presenting function during rejection. The Bcl-6 expressing T cells were, however, located on the T–B cell border, which is the same localization pattern of Tfh cells in lymph nodes to provide B cell help 5. Furthermore, the presence of IgM and IgG in these follicular-like structures reflects humoral alloreactivity and thus effector function of differentiated B cells. Tfh cells may be the link between cellular and humoral reactivity in acute rejection in kidney transplantation. Whether these Tfh cells are always present in transplanted kidneys, such as their presence in stable allografts from intestinal transplant patients 27, is as yet unknown. In contrast to Tfh cell function in the intestines, the B cell supporting function of Tfh cells in transplanted kidneys is not desirable.

In conclusion, our blood and biopsy data on Tfh cells in kidney transplantation demonstrate that Tfh cells may mediate humoral alloreactivity and also in the immunosuppressed milieu of tacrolimus combined with MMF and steroids.

Acknowledgments

The authors thank D. Lindenbergh-Kortleve for the technical assistance with the double labelling experiments and S. F. H. de Witte for the grammatical corrections. Part of this study was supported by a Mozaiek grant awarded to Mrs Bouamar by the Dutch Scientific Organization (NWO). Studies by Mr A. Boonstra are supported by the Virgo consortium, funded by the Dutch government project number FES0908.

Disclosure

The authors have no conflicts of interest to declare in relation to this paper.

Supporting Information

Additional Supporting information may be found in the online version of this article at the publisher's web-site:

Fig. S1. Absolute numbers of CD4POSCXCR5POS T lymphocytes (a) and interleukin (IL)-21+CD4POSCXCR5POS T lymphocytes (b) in patients who rejected within the first 3 months after transplantation (R < M3) and patients who did not (NR). KT = kidney transplantation.

cei0180-0329-sd1.tif (802.2KB, tif)

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Supplementary Materials

Fig. S1. Absolute numbers of CD4POSCXCR5POS T lymphocytes (a) and interleukin (IL)-21+CD4POSCXCR5POS T lymphocytes (b) in patients who rejected within the first 3 months after transplantation (R < M3) and patients who did not (NR). KT = kidney transplantation.

cei0180-0329-sd1.tif (802.2KB, tif)

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