Gretchen N. de Graav

CD4+CXCR5+ T cells in chronic HCV infection produce less IL-21, yet are efficient at supporting B cell responses

BACKGROUND & AIMS During chronic HCV infection, T cell dependent virus-specific antibodies are produced. However, the role of B-T cell interaction in chronic HCV is largely unknown. CD4(+)CXCR5(+) T follicular helper (TFH)-cells activate B cells and are important for clearance of various chronic viral infections. We investigated the function of TFH cells and B cells in liver and in peripheral blood of chronic HCV patients. METHODS T cells from chronic HCV patients and healthy individuals were analysed for expression of CXCR5, PD-1, ICOS, and IL-21 and IFN-γ production by flow cytometry. CD19(+) B cell subpopulations were identified on the basis of CD27 and IgD expression. In order to assess the frequency and function of T cells and B cells in liver follicles, immunohistochemistry was performed for CD3, CXCR5, Bcl6, IL-21, CD20, IgD, IgM, and IgG. RESULTS The frequency of IL-21-producing CXCR5(+)CD4(+) T cells in blood was lower in HCV patients compared to healthy individuals (p=0.002), which was reflected by lower serum IL-21 levels (p<0.001). Nonetheless, CXCR5(+)CD4(+) T cells from HCV patients and healthy individuals were equally capable to stimulate CD19(+)CD27(+) memory B cells into IgG and IgM-producing plasmablasts. Importantly, human intrahepatic TFH cells and their related function were identified by immunohistochemistry on liver biopsies for CD3, Bcl6, and CD20 within portal areas and follicles. CONCLUSIONS The specific localization of TFH cells and IgG and IgD/IgM-producing B cells suggests a functional B-T cell environment in liver follicles during HCV infection. The decreased frequency of IL-21-producing CXCR5(+)CD4(+) T cells and lower serum IL-21 levels in chronic HCV patients did not lead to an altered TFH-B cell interaction.

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.

THERAPEUTIC DRUG MONITORING OF BELATACEPT IN KIDNEY TRANSPLANTATION

Abstract: Belatacept is a novel immunosuppressive drug that inhibits the co-stimulatory signal required for T-cell activation and has been approved for the prevention of acute rejection after kidney transplantation. In this article, the need for and possibility of therapeutic drug monitoring (TDM) of belatacept is reviewed. Clinical studies have defined the upper limit of the therapeutic window, but the lower limit is unknown. The pharmacokinetics and pharmacodynamics of belatacept display only limited interpatient variability but no data are available on the intrapatient variability of these parameters. Several assays to measure serum belatacept concentrations and its in vitro immunologic effects have been developed, but these are not commercially available and require validation. Importantly, pharmacodynamic assays have not been correlated with clinical outcomes (both efficacy and safety) and have only used surrogate laboratory readouts. TDM is likely to become feasible in the near future if these assays are developed further. However, because its pharmacokinetics and pharmacodynamics seem to vary little between individual patients, it may not be necessary to perform TDM for this drug. There could be a role for such an approach if one seeks to lower the belatacept doses further in an attempt to minimize adverse events. A future, prospective concentration-ranging study that defines the lower end of the belatacept therapeutic window should, however, be conducted first to provide the rationale for performing TDM of this novel immunosuppressant.

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.

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.

Renal Allograft Transcription Analysis Reveals Similar Signature of Acute T Cell Mediated Rejection in Patients Treated with Tacrolimus or Belatacept

Introduction Histopathological examination of kidney allograft biopsies is the gold standard for diagnosing transplant pathology. However, limited reproducibility and differential diagnostic dilemma’s remain a problem. Identification of biomarkers of acute kidney allograft rejection (AR) can potentially lead to improved diagnostics. Here, we analyzed the expression of 209 genes in biopsies of kidney transplant patients with AR with the NanoString® nCounter® analysis system. With this novel technique, only low quantities of RNA from formalin fixed paraffin embedded (FFPE) biopsies are required and no amplification is needed. Therefore, residual material used for histopathological diagnosis can be analyzed. The objectives of this study were: i) to examine the gene expression profile in biopsies of patients with acute T cell-mediated rejection (aTCMR) versus patients without aTCMR and ii) to compare the gene expression profiles in patients with aTCMR treated with tacrolimus versus patients treated with belatacept (an inhibitor of the CD28-CD80/86 co-stimulatory pathway). Materials and Methods Biopsies from 21 kidney transplant were studied. Seven biopsies from patients with aTCMR (Banff 1b-3, without C4d) treated with tacrolimus as maintenance immunosuppressive therapy, 9 biopsies from patients with aTCMR treated with belatacept, and 5 negative controls (for-cause biopsies without histomorphological changes) were included. Patients were matched for age, days after transplantation and Banff 2015 category. RNA was extracted from FFPE biopsies and gene expression was analyzed using the NanoString® nCounter® analysis system. Gene expression was identified by scaled estimates (JMP, Fit Model). P values were corrected for false discovery (JMP, addin) Results and Discussion A distinct pattern was seen in biopsies with aTCMR compared to biopsies without rejection. Comparison of aTCMR and controls (Banff, no rejection) identified 60 genes with higher expression (FDRPV <0.05 to 2E-6). The most significant were T cell associated genes, CD3, CD8, and CD4 (p < 10E-5), and interferon (p = 2x10E-3) inducible genes (CXCL9, CCL5, TBX21 p< 10E-3), plus effector genes (GNLY, ITGAX p<10E-3). This overall pattern is that of aTCMR. Interestingly, pairwise estimates showed no significant differences between belatacept or tacrolimus treated subjects with aTCMR. Conclusion Gene expression analysis on FFPE biopsies with the novel technique NanoString® nCounter® analysis system can distinguish kidney transplant biopsies showing aTCMR from those of without aTCMR. Interestingly, we found no differences in gene expression profiles in renal allograft biopsies showing aTCMR in subjects receiving tacrolimus or belatacept-based immunosuppressive regimens. The limitations of this study may relate to the small sample size.

Analysis of NFATc1 amplification in T cells for pharmacodynamic monitoring of tacrolimus in kidney transplant recipients

Background Therapeutic drug monitoring (TDM) of tacrolimus, based on blood concentrations, shows an imperfect correlation with the occurrence of rejection. Here, we tested whether measuring NFATc1 amplification, a member of the calcineurin pathway, is suitable for TDM of tacrolimus. Materials and methods NFATc1 amplification was monitored in T cells of kidney transplant recipients who received either tacrolimus- (n = 11) or belatacept-based (n = 10) therapy. Individual drug effects on NFATc1 amplification were studied in vitro, after spiking blood samples of healthy volunteers with either tacrolimus, belatacept or mycophenolate mofetil. Results At day 30 after transplantation, in tacrolimus-treated patients, NFATc1 amplification was inhibited in CD4+ T cells expressing the co-stimulation receptor CD28 (mean inhibition 37%; p = 0.01) and in CD8+CD28+ T cells (29% inhibition; p = 0.02), while this was not observed in CD8+CD28- T cells or belatacept-treated patients. Tacrolimus pre-dose concentrations of these patients correlated inversely with NFATc1 amplification in CD28+ T cells (rs = -0.46; p < 0.01). In vitro experiments revealed that 50 ng/ml tacrolimus affected NFATc1 amplification by 58% (mean; p = 0.02). Conclusion In conclusion, measuring NFATc1 amplification is a direct tool for monitoring biological effects of tacrolimus on T cells in whole blood samples of kidney transplant recipients. This technique has potential that requires further development before it can be applied in daily practice.