An overview of T follicular cells in transplantation: spotlight on their clinical significance.

Introduction: For late stage organ failure patients, transplantation is the best option to increase life expectancy with a superior quality of life. Unfortunately, after transplantation many patients are at risk of cellular and antibody-mediated rejection (ABMR). The latter is initiated by donor specific antibodies (DSA) which depend on the actions of B cells, T follicular helper (Tfh) cells and T follicular regulatory (Tfr) cells that are present in the germinal center of lymphoid organs.Areas covered: In this overview paper, we discuss the biology and function of Tfh and Tfr cells in lymphoid tissues, transplanted organs and their circulating counterparts. We report on their relevance to alloimmunity and on the effects of immunosuppressive drugs on these immunocompetent cell populations.Expert opinion: Growing knowledge about the actions of Tfh and Tfr allows for a better understanding of the immunological mechanisms of ABMR after organ transplantation. This understanding feeds the hypothesis that immunosuppressive drugs targeting the actions of Tfh cells have huge therapeutic potential. This new concept in the treatment of the humoral rejection response will improve graft and patient survival after organ transplantation.

Inhibition of T Helper Cell Differentiation by Tacrolimus or Sirolimus Results in Reduced B-Cell Activation: Effects on T Follicular Helper Cells.

The effect of immunosuppressive drugs on the generation of T follicular helper (Tfh) cells, specialized in supporting B-cell differentiation, is largely unknown. We examined whether the calcineurin inhibitor tacrolimus (TAC) and the mammalian target of rapamycin (mtor) inhibitor sirolimus (SRL) inhibit Tfh cell differentiation, and affect subsequent B-cell functions. Isolated naive T cells were polarized into Tfh-like cells in the presence of TAC or SRL. To demonstrate their functionality, we co-cultured these cells with isolated B cells in the presence of alloantigen and studied the activation and differentiation of these B cells. Tfh-like cells were defined as CD4+CXCR5+ T cells, expressing immunoinhibitory programmed death protein 1 (pd1) and inducible T-cell costimulator (icos). We found that TAC and SRL significantly inhibited Tfh-like cell differentiation. Therapeutic concentrations of TAC and SRL reduced the percentage of pd1+ and icos+ Tfh cells compared to controls. In addition, T cells grown in the presence of TAC or SRL expressed less IL-21 and provided less B-cell help. TAC and SRL both inhibited Tfh-dependent alloantigen-activated B-cell proliferation and differentiation into plasma cells and transitional B cells. In conclusion, TAC and SRL inhibited the differentiation of naive T cells into functional Tfh-like cells, a finding that can be extrapolated to immunosuppressive regimens in transplant patients.

Pediatric Mesenchymal Stem Cells Exhibit Immunomodulatory Properties Toward Allogeneic T and B Cells Under Inflammatory Conditions.

Mesenchymal stem cells from pediatric patients (pMSCs) are an attractive cell source in regenerative medicine, due to their higher proliferation rates and better differentiation abilities compared to adult MSCs (aMSCs). We have previously characterized the immunomodulatory abilities of pMSCs on T cells under co-culture. It has also been reported that aMSCs can inhibit B cell proliferation and maturation under inflammatory conditions. In this study, we therefore aimed to clarify the immunomodulatory effect of pMSCs toward T and B cells in an inflammatory microenvironment. Bone marrow derived pMSCs were primed to simulate inflammatory conditions by exposure with 50 ng/mL of IFN-γ for 3 days. To analyze the interaction between pMSCs and T cells, CD3/CD28 stimulated peripheral blood mononuclear cells (PBMCs) were co-cultured with primed or unprimed pMSCs. To investigate B cell responses, quiescent B cells obtained from spleens by CD43 negative selection were stimulated with anti-IgM, anti-CD40, IL-2, and co-cultured with either IFN-γ primed or unprimed pMSC. pMSC phenotype, B and T cell proliferation, and B cell functionality were analyzed. Gene expression of indoleamine 2,3-dioxygenease (IDO), as well as the expression of HLA-ABC, HLA-DR and the co-stimulatory molecules CD80 and CD86 was upregulated on pMSCs upon IFN-γ priming. IFN-γ did not alter the immunomodulatory abilities of pMSCs upon CD4+ nor CD8+ stimulated T cells compared to unprimed pMSCs. IFN-γ primed pMSCs but not unprimed pMSCs strongly inhibited naïve (CD19+CD27-), memory (CD19+CD27+), and total B cell proliferation. Antibody-producing plasmablast (CD19+CD27highCD38high) formation and IgG production were also significantly inhibited by IFN-γ primed pMSCs compared to unprimed pMSCs. Collectively, these results show that pMSCs have immunomodulatory effects upon the adaptive immune response which can be potentiated by inflammatory stimuli. This knowledge is useful in regenerative medicine and allogeneic transplantation applications toward tailoring pMSCs function to best modulate the immune response for a successful implant engraftment and avoidance of a strong immune reaction.

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.

Differential T Cell Signaling Pathway Activation by Tacrolimus and Belatacept after Kidney Transplantation: Post Hoc Analysis of a Randomised-Controlled Trial.

Pharmacokinetic immunosuppressive drug monitoring poorly correlates with clinical outcomes after solid organ transplantation. A promising method for pharmacodynamic monitoring of tacrolimus (TAC) in T cell subsets of transplant recipients might be the measurement of (phosphorylated) p38MAPK, ERK1/2 and Akt (activated downstream of the T cell receptor) by phospho-specific flow cytometry. Here, blood samples from n = 40 kidney transplant recipients (treated with either TAC-based or belatacept (BELA)-based immunosuppressive drug therapy) were monitored before and throughout the first year after transplantation. After transplantation and in unstimulated samples, p-p38MAPK and p-Akt were inhibited in CD8+ T cells and p-ERK in CD4+ T cells but only in patients who received TAC-based therapy. After activation with PMA/ionomycin, p-p38MAPK and p-AKT were significantly inhibited in CD4+ and CD8+ T cells when TAC was given, compared to pre-transplantation. Eleven BELA-treated patients had a biopsy-proven acute rejection, which was associated with higher p-ERK levels in both CD4+ and CD8+ T cells compared to patients without rejection. In conclusion, phospho-specific flow cytometry is a promising tool to pharmacodynamically monitor TAC-based therapy. In contrast to TAC-based therapy, BELA-based immunosuppression does not inhibit key T cell activation pathways which may contribute to the high rejection incidence among BELA-treated transplant recipients.

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.

Pharmacodynamic Monitoring of Tacrolimus-Based Immunosuppression in CD14+ Monocytes After Kidney Transplantation.

BACKGROUND: Monocytes significantly contribute to ischemia-reperfusion injury and allograft rejection after kidney transplantation. However, the knowledge about the effects of immunosuppressive drugs on monocyte activation is limited. Conventional pharmacokinetic methods for immunosuppressive drug monitoring are not cell type-specific. In this study, phosphorylation of 3 signaling proteins was measured to determine the pharmacodynamic effects of immunosuppression on monocyte activation in kidney transplant patients. METHODS: Blood samples from 20 kidney transplant recipients were monitored before and during the first year after transplantation. All patients received induction therapy with basiliximab, followed by tacrolimus (TAC), mycophenolate mofetil, and prednisolone maintenance therapy. TAC whole-blood predose concentrations were determined using an antibody-conjugated magnetic immunoassay. Samples were stimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycin, and phosphorylation of p38MAPK, ERK, and Akt in CD14 monocytes was quantified by phospho-specific flow cytometry. RESULTS: Phosphorylation of p38MAPK and Akt in monocytes of immunosuppressed recipients was lower after 360 days compared with before transplantation in the unstimulated samples [mean reduction in median fluorescence intensity 36%; range -28% to 77% for p-p38MAPK and 20%; range -22% to 53% for p-Akt; P < 0.05]. P-ERK was only decreased at day 4 after transplantation (mean inhibition 23%; range -52% to 73%; P < 0.05). At day 4, when the highest whole-blood predose TAC concentrations were measured, p-p38MAPK and p-Akt, but not p-ERK, correlated inversely with TAC (rs = -0.65; P = 0.01 and rs = -0.58; P = 0.03, respectively). CONCLUSIONS: Immunosuppressive drug combination therapy partially inhibits monocyte activation pathways after kidney transplantation. This inhibition can be determined by phospho-specific flow cytometry, which enables the assessment of the pharmacodynamic effects of immunosuppressive drugs in a cell type-specific manner.

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, CD8CD28, CD4CD57PD1, and CD8CD28 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 CD8CD28 end-stage terminally differentiated memory T cells/µL 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.

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-1ß and phagocytosis by monocytes were not affected by tacrolimus concentrations, whereas MPA did inhibit IL-1ß 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.

Natural regulatory T cells from patients with end-stage renal disease can be used for large-scale generation of highly suppressive alloantigen-specific Tregs.

Natural occurring regulatory T cells (nTregs) have the potential to offer a targeted approach of immunosuppression and are the cell type of interest for inducing tolerance in kidney transplantation. End-stage renal disease (ESRD) profoundly affects the composition and function of circulating T cells but little is known with respect to how nTreg potential is affected. To address this, nTregs of patients with ESRD (on dialysis or not) and healthy individuals were isolated, expanded using allogeneic mature monocyte-derived dendritic cells followed by anti-CD3/anti-CD28-coated beads and the different nTregs were extensively characterized by the demethylation status of the Treg-specific demethylated region within FOXP3 and expression of typical nTreg markers. Additionally, the suppressive capacity as well as cytokine producing cells were analyzed for allogeneic mature monocyte-derived dendritic cell-expanded nTregs. Compared to age- and gender-matched healthy individuals, similar frequencies of nTregs were present within the circulation of patients with ESRD either on dialysis or not. The isolated nTregs could be equally well or even better expanded using allogeneic mature monocyte-derived dendritic cells and extensive characterization did not reveal significant differences. The demethylation status of the Treg-specific demethylated region was maintained or even further promoted as was the expression of markers characteristic for nTregs. Moreover, suppressive capacity and the cytokine profile of allogeneic mature monocyte-derived dendritic cell-expanded nTregs was similar to that of healthy individuals. Thus, circulating nTregs of patients with ESRD can effectively be expanded to stable allo antigen-specific nTregs with potential clinical applicability.

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.

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-γ 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.

Allogeneic Mature Human Dendritic Cells Generate Superior Alloreactive Regulatory T Cells in the Presence of IL-15.

Expansion of Ag-specific naturally occurring regulatory T cells (nTregs) is required to obtain sufficient numbers of cells for cellular immunotherapy. In this study, different allogeneic stimuli were studied for their capacity to generate functional alloantigen-specific nTregs. A highly enriched nTreg fraction (CD4(+)CD25(bright)CD127(-) T cells) was alloantigen-specific expanded using HLA-mismatched immature, mature monocyte-derived dendritic cells (moDCs), or PBMCs. The allogeneic mature moDC-expanded nTregs were fully characterized by analysis of the demethylation status within the Treg-specific demethylation region of the FOXP3 gene and the expression of both protein and mRNA of FOXP3, HELIOS, CTLA4, and cytokines. In addition, the Ag-specific suppressive capacity of these expanded nTregs was tested. Allogeneic mature moDCs and skin-derived DCs were superior in inducing nTreg expansion compared with immature moDCs or PBMCs in an HLA-DR- and CD80/CD86-dependent way. Remarkably, the presence of exogenous IL-15 without IL-2 could facilitate optimal mature moDC-induced nTreg expansion. Allogeneic mature moDC-expanded nTregs were at low ratios (<1:320), potent suppressors of alloantigen-induced proliferation without significant suppression of completely HLA-mismatched, Ag-induced proliferation. Mature moDC-expanded nTregs were highly demethylated at the Treg-specific demethylation region within the FOXP3 gene and highly expressed of FOXP3, HELIOS, and CTLA4. A minority of the expanded nTregs produced IL-10, IL-2, IFN-γ, and TNF-α, but few IL-17-producing nTregs were found. Next-generation sequencing of mRNA of moDC-expanded nTregs revealed a strong induction of Treg-associated mRNAs. Human allogeneic mature moDCs are highly efficient stimulator cells, in the presence of exogenous IL-15, for expansion of stable alloantigen-specific nTregs with superior suppressive function.

Genetic polymorphisms in ABCB1 influence the pharmacodynamics of tacrolimus.

INTRODUCTION: Tacrolimus has a large interindividual pharmacokinetic variability, and quantification of its effect is difficult. It is a substrate of ABCB1, an efflux pump expressed more on CD8 T cells than on CD4 T cells. The ABCB1 3435C>T single-nucleotide polymorphism (SNP) has been associated with interindividual differences in ABCB1 activity and may influence drug efficacy. Here the influence of this SNP on the biological effect of tacrolimus was studied. METHODS: Rhodamine (Rh123) efflux was used to study ABCB1 activity, with or without the addition of the ABCB1 inhibitor verapamil. Intracellular interleukin (IL) 2 production in T cells was used to measure the pharmacodynamic effect of tacrolimus after phorbol-12-myristate-13-acetate/ionomycin stimulation of whole blood. In addition, the ABCB1 genotype of 36 tacrolimus-treated renal transplant patients was related to ABCB1 activity and tacrolimus efficacy. RESULTS: The mean Rh123 efflux was higher in CD8 T cells compared with CD4 T cells: 40% versus 19% of cells, respectively (P < 0.001). Verapamil almost completely blocked Rh123 efflux (to 1.8% of CD4 T cells and 0.5% of CD8 T cells), whereas tacrolimus did not change Rh123 efflux. Tacrolimus 10 ng/mL reduced the production of IL-2 in CD4 and CD8 T cells by 28.9% and 45.4% (P < 0.05). Tacrolimus-mediated inhibition of IL-2 was enhanced by verapamil (P < 0.05). This effect on tacrolimus pharmacodynamics was associated with ABCB1 3435C>T SNP in renal transplant patients: verapamil reduced the percentage of IL-2-producing CD4 and CD8 T cells by 14% and 22% in patients with the CC genotype (P < 0.05) but not in patients with the TT genotype. Moreover, the ratio of tacrolimus C0 over the percent of IL-2-producing CD8 T cells in CC genotype patients was significantly higher compared with TT genotype patients (P < 0.05), showing a smaller pharmacodynamic effect in CC genotype patients. CONCLUSION: The ABCB1 3435C>T SNP influences ABCB1 activity of T cells and the pharmacodynamic effect of tacrolimus in kidney transplant patients.

Tacrolimus inhibits NF-κB activation in peripheral human T cells.

The calcineurin inhibitor, tacrolimus (TAC), inhibits the protein phosphatase activity of calcineurin, leading to suppression of the nuclear translocation of NFAT and inhibition of T cell activation. Apart from NFAT also the transcription factor NF-κB plays a key functional role in T cell activation. Therefore, blockade of the NF-κB activation cascade by immunosuppressive drugs prevents immune activation. Here we studied whether TAC blocks NF-κB activation in peripheral human T cells. After anti-CD3/CD28-activation of T cells from healthy volunteers, NF-κB (p65) phosphorylation was measured by flow cytometry in CD3+ T cells, CD4+ helper T cells and CD8+ cytotoxic T cells in the absence and presence of TAC 10 ng/mL, sotrastaurin 500 nM (positive control) and mycophenolic acid 10 µg/mL (negative control; n = 6). NF-κB transcriptional activity was measured by ELISA and intracellular TNFα protein, a downstream target, was measured by flow cytometry to assess the functional consequences of NF-κB blockade. Anti-CD3/28-activation induced NF-κB phosphorylation in CD3+ T cells, CD4+ T cells and CD8+ T cells by 34% (mean), 38% and 30% resp. (p<0.01). Sotrastaurin inhibited NF-κB activation in the respective T cell subsets by 93%, 95% and 86% (p<0.01 vs. no drug), while mycophenolic acid did not affect this activation pathway. Surprisingly, TAC also inhibited NF-κB phosphorylation, by 55% (p<0.01) in CD3+ T cells, by 56% (p<0.01) in CD4+ T cells and by 51% in CD8+ T cells (p<0.01). In addition, TAC suppressed NF-κB DNA binding capacity by 55% (p<0.05) in CD3+ T cells and TNFα protein expression was inhibited in CD3+ T cells, CD4+ T cells and CD8+ T cells by 76%, 71% and 93% resp. (p<0.01 vs. no drug), confirming impaired NF-κB signaling. This study shows the suppressive effect of TAC on NF-κB signaling in peripheral human T cell subsets, measured at three specific positions in the NF-κB activation cascade.

Characterization of rabbit antithymocyte globulins-induced CD25+ regulatory T cells from cells of patients with end-stage renal disease.

BACKGROUND.: Rabbit antithymocyte globulins (rATGs) are known to convert CD4CD25FoxP3 T cells from healthy individuals to CD4CD25FoxP3 T cells. In this study, we investigated the effect of rATG on the induction of regulatory T cells (Tregs) from blood cells of patients with end-stage renal disease who are candidates for transplantation and rATG-induction therapy. The induced Tregs were analyzed and compared with naturally occurring CD4CD25FoxP3T cells. METHODS.: The CD25 T cells of pretransplant patients (n=7) and healthy controls (n=4) were stimulated with rATG or control rabbit immunoglobulins for 24 hr. The phenotype of induced Tregs was examined by flow cytometry, and their function was studied in the conventional suppression assay. Further characterization was performed by mRNA analyses. RESULTS.: After 24 hr, the percentage of CD4CD25FoxP3CD127 T cells and CD8CD25FoxP3CD127 T cells became higher in the rATG-treated samples compared with the rabbit immunoglobulin-treated samples (P<0.01). The rATG-induced CD25T cells, whether CD4 or CD8 inhibited the allogeneic responses of CD25 effector T cells as vigorously as natural CD25T cells. However, the proportion of FoxP3 within the top 2% rATG-induced CD4CD25T-cells was lower than within the natural CD4CD25T-cells (11%+/-2% vs. 95%+/-5%, P<0.01). The mRNA-expression levels of interleukin-27, interleukin-10, interferon-gamma, perforin, and granzyme B were markedly higher compared with natural CD25T-cells (all P=0.03), whereas CTLA4 (P=0.03), transforming growth factor-beta (P=0.02), and RORgammat (P=0.04) were lower. CONCLUSION.: rATG allows the induction of Tregs from patient peripheral blood mononuclear cell in vitro. In comparison with natural Tregs, the rATG-induced Tregs are phenotypically distinct but have similar regulatory activities. rATG may beneficially contribute to the mechanisms that control alloreactivity.