Marjolein Dieterich

Follicular T helper cells and humoral reactivity in kidney transplant patients

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.

Down-regulation of surface CD28 under belatacept treatment: An escape mechanism for antigen-reactive T-cells

Background The co-stimulatory inhibitor of the CD28-CD80/86-pathway, belatacept, allows calcineurin-inhibitor-free immunosuppression in kidney transplantation. However, aggressive T-cell mediated allogeneic responses have been observed in belatacept-treated patients, which could be explained by effector-memory T-cells that lack membrane expression of CD28, i.e. CD28-negative (CD28NULL) T-cells. CD28-positive (CD28POS) T-cells that down regulate their surface CD28 after allogeneic stimulation could also pose a threat against the renal graft. The aim of this study was to investigate this potential escape mechanism for CD28POS T-cells under belatacept treatment. Materials & Methods PBMCs, isolated T-cell memory subsets and isolated CD28POS T-cells were obtained from end-stage renal disease (ESRD) patients and co-cultured with allo-antigen in the presence of belatacept to mimic allogeneic reactions in kidney-transplant patients under belatacept treatment. As a control, IgG was used in the absence of belatacept. Results Despite high in vitro belatacept concentrations, a residual T-cell growth of ±30% was observed compared to the IgG control after allogeneic stimulation. Of the alloreactive T-cells, the majority expressed an effector-memory phenotype. This predominance for effector-memory T-cells within the proliferated cells was even larger when a higher dose of belatacept was added. Contrary to isolated naïve and central-memory T cells, isolated effector-memory T cells could not be inhibited by belatacept in differentiation or allogeneic IFNγ production. The proportion of CD28-positive T cells was lower within the proliferated T cell population, but was still substantial. A fair number of the isolated initially CD28POS T-cells differentiated into CD28NULL T-cells, which made them not targetable by belatacept. These induced CD28NULL T-cells were not anergic as they produced high amounts of IFNγ upon allogeneic stimulation. The majority of the proliferated isolated originally CD28POS T-cells, however, still expressed CD28 and also expressed IFNγ. Conclusion This study provides evidence that, apart from CD28NULL T-cells, also CD28POS, mostly effector-memory T-cells can mediate allogeneic responses despite belatacept treatment.

A Randomized Controlled Clinical Trial Comparing Belatacept With Tacrolimus After De Novo Kidney Transplantation

Background Belatacept, an inhibitor of the CD28-CD80/86 costimulatory pathway, allows for calcineurin-inhibitor free immunosuppressive therapy in kidney transplantation but is associated with a higher acute rejection risk than ciclosporin. Thus far, no biomarker for belatacept-resistant rejection has been validated. In this randomized-controlled trial, acute rejection rate was compared between belatacept- and tacrolimus-treated patients and immunological biomarkers for acute rejection were investigated. Methods Forty kidney transplant recipients were 1:1 randomized to belatacept or tacrolimus combined with basiliximab, mycophenolate mofetil, and prednisolone. The 1-year incidence of biopsy-proven acute rejection was monitored. Potential biomarkers, namely, CD8+CD28−, CD4+CD57+PD1−, and CD8+CD28++ end-stage terminally differentiated memory T cells were measured pretransplantation and posttransplantation and correlated to rejection. Pharmacodynamic monitoring of belatacept was performed by measuring free CD86 on monocytes. Results The rejection incidence was higher in belatacept-treated than tacrolimus-treated patients: 55% versus 10% (P = 0.006). All 3 graft losses, due to rejection, occurred in the belatacept group. Although 4 of 5 belatacept-treated patients with greater than 35 cells CD8+CD28++ end-stage terminally differentiated memory T cells/&mgr;L rejected, median pretransplant values of the biomarkers did not differ between belatacept-treated rejectors and nonrejectors. In univariable Cox regressions, the studied cell subsets were not associated with rejection-risk. CD86 molecules on circulating monocytes in belatacept-treated patients were saturated at all timepoints. Conclusions Belatacept-based immunosuppressive therapy resulted in higher and more severe acute rejection compared with tacrolimus-based therapy. This trial did not identify cellular biomarkers predictive of rejection. In addition, the CD28-CD80/86 costimulatory pathway appeared to be sufficiently blocked by belatacept and did not predict rejection.

An Acute Cellular Rejection With Detrimental Outcome Occurring Under Belatacept-Based Immunosuppressive Therapy: An Immunological Analysis

Background Belatacept has been associated with an increased acute rejection rate after kidney transplantation. This case report sheds light on the possible immunological mechanisms underlying this phenomenon by analyzing the immunological mechanisms in patient serum, peripheral blood mononuclear cells, rejected kidney tissue, and graft infiltrating cells. Methods A 61-year-old woman treated with belatacept, who received her first kidney transplant from her husband was admitted with an acute, vascular rejection 56 days after transplantation which necessitated a transplantectomy. Histology and immunohistochemistry were performed on biopsy and explant tissue. CD86 expression on peripheral monocytes was assessed. Using Ficoll density methods, peripheral blood, and graft infiltrating lymphocytes were isolated and phenotyped. Results The explant showed a vascular rejection (Banff ACR grade III) and a perivascular infiltrate mostly consisting of T cells. No evidence for antibody-mediated rejection was found. In contrast to the peripheral blood monocytes, CD86 was still expressed by part of the mononuclear cells in the explant. Isolated graft cells were mostly CCR7−CD45RO+ effector memory CD4+ and CD8+ T cells (60-70%). CD28-positive as CD28-negative T cells were present in the explant, showing a great IFN-&ggr; production capacity and expressing granzyme B. Conclusions We postulate that this glucocorticoid-resistant cellular rejection occurring under belatacept was predominantly mediated by cytotoxic memory T cells, which are less susceptible to costimulatory blockade by belatacept, or resulted from incomplete CD80/86 blockade at the tissue level.

IL-21 Receptor Antagonist Inhibits Differentiation of B Cells toward Plasmablasts upon Alloantigen Stimulation

Interaction between T follicular helper (Tfh) cells and B cells is complex and involves various pathways, including the production of IL-21 by the Tfh cells. Secretion of IL-21 results in B cell differentiation toward immunoglobulin-producing plasmablasts. In patients after kidney transplantation, the formation of alloantibodies produced by donor antigen-activated B cells are a major cause of organ failure. In this allogeneic response, the role of IL-21-producing Tfh cells that regulate B cell differentiation is unknown. Here, we tested, in an alloantigen-driven setting, whether Tfh cell help signals control B cell differentiation with its dependency on IL-21. Pre-transplantation patient PBMCs were sorted into pure CD4posCXCR5pos Tfh cells and CD19posCD27pos memory B cells and stimulated with donor antigen in the presence or absence of an IL-21 receptor (IL-21R) antagonist (αIL-21R). Donor antigen stimulation initiated expression of the activation markers inducible co-stimulator (ICOS) and programmed death 1 (PD-1) on Tfh cells and a shift toward a mixed Tfh2 and Tfh17 phenotype. The memory B cells underwent class switch recombination and differentiated toward IgM- and IgG-producing plasmablasts. In the presence of αIL-21R, a dose-dependent inhibition of STAT3 phosphorylation was measured in both T and B cells. Blockade of the IL-21R did not have an effect on PD-1 and ICOS expression on Tfh cells but significantly inhibited B cell differentiation. The proportion of plasmablasts decreased by 78% in the presence of αIL-21R. Moreover, secreted IgM and IgG2 levels were significantly lower in the presence of αIL-21R. In conclusion, our results demonstrate that IL-21 produced by alloantigen-activated Tfh cells controls B cell differentiation toward antibody producing plasmablasts. The IL-21R might, therefore, be a useful target in organ transplantation to prevent antigen-driven immune responses leading to graft failure.

Interleukin-17-producing CD4 + cells home to the graft early after human heart transplantation

BACKGROUND Interleukin-17 (IL-17) is regarded as a major effector cytokine with pro-inflammatory actions. It has pleiotropic and environment-specific functions by promoting adaptive cytotoxic T-lymphocyte responses during inflammation. Therefore, it is tempting to speculate that IL-17 plays a major role in inflammatory responses in transplant recipients. We questioned whether IL-17 is expressed in the transplanted heart during acute rejection (AR), or during immunologic quiescence, and which graft-infiltrating lymphocytes produce IL-17. In addition, we analyzed donor-specific IL-17-producing cells in peripheral blood cells in comparable periods after transplantation. METHODS Endomyocardial biopsies from heart transplant recipients with early or late AR or in an immunologic quiescence period were analyzed for the presence of IL-17 mRNA. In addition, the capacity of graft-infiltrating lymphocytes (GILs) to produce IL-17 was analyzed. Moreover, we determined the frequency of donor-reactive IL-17-producing peripheral blood mononuclear cells (PBMCs) using an Elispot assay. RESULTS Twenty-one percent (14 of 67) of the biopsies assessed were positive for IL-17 mRNA. Thirteen of 41 biopsies were observed in the early period (≤3 months) after transplantation. One (of 26) of the late biopsies expressed IL-17 (p = 0.006). Specifically, IL-17 was expressed during early AR (57%, or 8 of 14), whereas biopsies from late AR (0 of 5) did not express IL-17 mRNA (p = 0.02). During AR, IL-17 is derived from IL-17-producing CD4(+)CD161(+), and not CD8(+), GILs. In contrast to the graft findings, we detected circulating donor-reactive IL-17-producing cells mostly during immunologic quiescence. CONCLUSIONS Particularly early after heart transplantation, IL-17-producing CD4(+) T cells home to the graft, which contributes to the AR process.

The Effect of Tacrolimus and Mycophenolic Acid on CD14+ Monocyte Activation and Function

Monocytes and macrophages play key roles in many disease states, including cellular and humoral rejection after solid organ transplantation (SOT). To suppress alloimmunity after SOT, immunosuppressive drug therapy is necessary. However, little is known about the effects of the immunosuppressive drugs tacrolimus and mycophenolic acid (MPA) on monocyte activation and function. Here, the effect of these immunosuppressants on monocytes was investigated by measuring phosphorylation of three intracellular signaling proteins which all have a major role in monocyte function: p38MAPK, ERK and Akt. In addition, biological functions downstream of these signaling pathways were studied, including cytokine production, phagocytosis and differentiation into macrophages. To this end, blood samples from healthy volunteers were spiked with diverse concentrations of tacrolimus and MPA. Tacrolimus (200 ng/ml) inhibited phosphorylation of p38MAPK by 30% (mean) in CD14+ monocytes which was significantly less than in activated CD3+ T cells (max 60%; p < 0.05). This immunosuppressive agent also partly inhibited p-AKT (14%). MPA, at a therapeutic concentration showed the strongest effect on p-AKT (27% inhibition). p-ERK was inhibited with a maximum of 15% after spiking with either tacrolimus or MPA. The production of IL-1β and phagocytosis by monocytes were not affected by tacrolimus concentrations, whereas MPA did inhibit IL-1β production by 50%. Monocyte/macrophage polarization was shifted to an M2-like phenotype in the presence of tacrolimus, while MPA increased the expression of M2 surface markers, including CD163 and CD200R, on M1 macrophages. These results show that tacrolimus and MPA do not strongly affect monocyte function, apart from a change in macrophage polarization, to a clinically relevant degree.

Belatacept Does Not Inhibit Follicular T Cell-Dependent B-Cell Differentiation in Kidney Transplantation

Humoral alloreactivity has been recognized as a common cause of kidney transplant dysfunction. B-cell activation, differentiation, and antibody production are dependent on IL-21+CXCR5+follicular T-helper (Tfh) cells. Here, we studied whether belatacept, an inhibitor of the costimulatory CD28-CD80/86-pathway, interrupts the crosstalk between Tfh- and B-cells more efficiently than the calcineurin inhibitor tacrolimus. The suppressive effects of belatacept and tacrolimus on donor antigen-driven Tfh–B-cell interaction were functionally studied in peripheral blood mononuclear cells from 40 kidney transplant patients randomized to a belatacept- or tacrolimus-based immunosuppressive regimen. No significant differences in uncultured cells or donor antigen-stimulated cells were found between belatacept- and tacrolimus-treated patients in the CXCR5+Tfh cell generation and activation (upregulation of PD-1). Belatacept and tacrolimus in vitro minimally inhibited Tfh-cell generation (by ~6–7%) and partially prevented Tfh-cell activation (by ~30–50%). The proportion of IL-21+-activated Tfh-cells was partially decreased by in vitro addition of belatacept or tacrolimus (by ~60%). Baseline expressions and proportions of activated CD86+ B-cells, plasmablasts, and transitional B-cells after donor antigen stimulation did not differ between belatacept- and tacrolimus-treated patients. Donor antigen-driven CD86 upregulation on memory B-cells was not fully prevented by adding belatacept in vitro (~35%), even in supratherapeutic doses. In contrast to tacrolimus, belatacept failed to inhibit donor antigen-driven plasmablast formation (~50% inhibition vs. no inhibition, respectively, p < 0.0001). In summary, donor antigen-driven Tfh-B-cell crosstalk is similar in cells obtained from belatacept- and tacrolimus-treated patients. Belatacept is, however, less potent in vitro than tacrolimus in inhibiting Tfh-cell-dependent plasmablast formation.

Tissue-Resident Memory T Cells of Donor Origin are Short-Lived in Renal Allografts after Transplantation

Introduction Tissue-resident memory T (TRM) cells provide protective immunity to infection by rapidly responding to antigen in non-lymphoid tissues. These non-migrating memory T cells are characterized by surface expression of CD69 and CD103. In transplanted kidneys the existence, origin and properties of TRM cells are unclear. In this study, we used the unique tissue resource of transplant nephrectomies to determine whether TRM cells reside in rejected kidney allografts and whether these cells are of donor or recipient origin. Materials and Methods Thirteen transplant nephrectomy specimens were studied. These grafts failed because of acute (n=4) or chronic (n=9) rejection and were removed after a mean time of 6.7 years (range: 8 days – 26 years). Half of the explanted renal allograft (cortex and medulla) was processed into a single cell suspension. Isolated cells were stained and analyzed by flow cytometry to determine their phenotype. The origin of the cells was measured by mAb directed against HLA epitopes of the donor or acceptor. Results Functional CD3+ T cells were isolated from all explanted kidney allografts as 57.8 ± 16.5% (mean ± SD) of the cells had the capacity to produce IFN&ggr;; 16.1 ± 6.8% produced IL-2; 1.8 ± 1.2% IL-17, and 4.6 ± 5.2% IL-4 after PMA/ionomycin stimulation. The isolated T cells consisted of 43.2 ± 19.1% CD4+ T cells and 45.3 ± 20.6% CD8+ T cells.Of the CD8+ T cells, 27.9 ± 15.5% expressed CD69 and CD103, reflecting CD8+ TRM cells. The majority of these TRM cells did not express CD28 (61.6 ± 18.2%), indicating a phenotype associated with highly-reactive effector functions. The isolated CD4+ T cells also included a population of TRM cells, though this fraction was relatively small (1.9 ± 2.2%). We confirmed that CD69+CD103+ TRM cells were exclusively present in the renal allografts and not in the circulation of healthy controls (p=0.002). No differences in proportions of TRM cells were found between acute and chronically rejecting kidney allografts. High proportions of donor CD4+ and CD8+ T cells were present in the renal allografts removed within the first month after transplantation (6.8 ± 5.7% CD4+ T cells; 9.8 ± 9.2% CD8+ T cells) compared to low proportions in the renal allografts removed after one month (0.4 ± 0.3% CD4+ T cells; 0.3 ± 0.3% CD8+ T cells). Remarkably, within the CD8+ TRM cells the ratio between donor versus recipient cells was 3.6 times higher compared to this ratio within the total CD8+ T cells. Conclusion Our results demonstrate that both donor and patient CD4+ and CD8+ TRM cells reside in the rejecting transplanted kidney. Over time, the donor TRM cells disappear from the allograft.

Single-Cell Analysis of NFATc1 Amplification in T Cells for Pharmacodynamic Monitoring of Tacrolimus

Introduction Therapeutic drug monitoring (TDM) of the calcineurin inhibitor tacrolimus (TAC) is based on blood concentrations that show an imperfect correlation with the occurrence of acute rejection. A pharmacodynamic method that reflects the direct inhibitory effects of TAC may therefore be preferable over traditional pharmacokinetic TDM. Here, we tested whether measuring the amplification of NFATc1, a member of the calcineurin pathway, is suitable for TDM of TAC. Materials and Methods NFATc1 amplification was monitored in T cells of kidney transplant recipients who received either TAC- (n = 11) or a belatacept (BELA)-based (n = 10) immunosuppressive therapy. Heparinized blood samples were collected at days 0 (pre-transplantation), 4, 30, 90, 180 and 360 after transplantation and stimulated with PMA/ionomycin. In addition, individual drug effects on NFATc1 amplification were studied in vitro, after spiking blood samples of healthy volunteers with either TAC, BELA or mycophenolate mofetil. Results and Discussion In TAC- treated patients, at day 30 after transplantation, NFATc1 amplification was significantly inhibited in CD4+ T cells expressing the co-stimulation receptor CD28 (mean inhibition 37%; p = 0.01) and in CD8+CD28+ T cells (mean inhibition 29%; p = 0.02), while this was not observed in CD8+CD28- T cells or in BELA-treated patients. The TAC pre-dose concentrations of these patients correlated inversely with NFATc1 amplification in CD28+ T cells (rs = -0.46; p < 0.01). The in vitro study revealed a dose dependent effect of TAC on NFATc1 amplification in all three tested T cell subsets (mean inhibition 58% at 50 ng/ml TAC; p = 0.02), while belatacept and mycophenolate mofetil did not show an effect. Only one patient under TAC-based therapy suffered from a rejection and, as a consequence, no conclusions could be drawn on the association between NFATc1 amplification and rejection risk. Conclusion In conclusion, measuring NFATc1 amplification is a promising tool for monitoring the biological effects of TAC on T cell subsets directly, which might be useful for further transplantation diagnostics.