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Proinflammatory, cytotoxic CD4+ CD28null T cells can be substantially expanded in patients with end-stage renal disease. These cells have been associated with the risk for rejection, but their alloreactive potential is unknown. CD4+ CD28null T cells were stimulated with HLA-mismatched antigen presenting cells in the absence/presence of exogenous cytokines. Alloreactive potential was evaluated based on proliferation, degranulation, cytotoxicity, and cytokine production. Further, their suppressive capacity was assessed by measuring inhibition of proliferating alloreactive CD28+ T cells. CD4+ CD28null T cells contained alloreactive (CD137+ ) T cells but did not proliferate in response to allogeneic stimulation, unless interleukin (IL)-15 was added. However, they could proliferate on stimulation with cytomegalovirus antigen without exogenous cytokines. IL-15 increased the frequency of proliferating alloreactive CD4+ CD28null T cells to 30.5% without inducing CD28 expression (P

Summary Blockade of the CD80/86-CD28 pathway by belatacept after kidney transplantation is associated with an increased risk of rejection compared with standard, calcineurin inhibitor (CNI)-based therapy. CD28− T cells, which express CD57, are not susceptible to belatacept treatment. High numbers of CD4+CD57+programmed death 1 (PD-1)− T cells pretransplantation have been associated with a higher chance of rejection, although conflicting data have been reported. To investigate the working mechanism behind this possible higher chance of rejection, we studied the expression of co-inhibitory molecules (CD223, CD244 and PD-1), proliferative capacity and cytotoxic potential of fluorescence activated cell sorted (FACS) CD4+CD57+PD-1− and CD8+CD57+PD-1− T cells, and their CD57− control populations, after alloantigen stimulation. The effect of belatacept on the cytotoxic capacity of pretransplantation peripheral blood mononuclear cells from 20 patients who received belatacept post-transplantation was also tested. Expression of co-inhibitory molecule CD223 increased by approximately 10-fold after allogeneic stimulation in all four T cell subsets. Proliferation and up-regulation of CD244 and PD-1 was observed for CD4+CD57−PD-1− T cells after allogeneic stimulation, but no up-regulation of these markers occurred on CD8+ T cells or CD4+CD57+PD-1− T cells. However, CD4+CD57+PD-1− T cells and, to a lesser extent, CD8+CD57+PD-1− T cells displayed higher cytotoxicity as indicated by granzyme B expression. Belatacept inhibited the cytotoxic potential of CD4+CD57+PD-1− T cells (median of inhibition 31%, P < 0·01) and CD8+CD57+PD-1− T cells (median of inhibition 10%, P < 0·05). In conclusion, alloantigen-activated CD4+CD57+PD-1− T cells exhibited a less proliferative but more cytotoxic profile than their CD57− counterparts. Their cytotoxic capacity can be inhibited partly by belatacept and was not associated with development of rejection after kidney transplantation.

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+CD25brightCD127− 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 (

Summary 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.

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

Summary The novel immunosuppressant sotrastaurin is a selective inhibitor of protein kinase C isoforms that are critical in signalling pathways downstream of the T cell receptor. Sotrastaurin inhibits nuclear factor (NF)-κB, which directly promotes the transcription of forkhead box protein 3 (FoxP3), the key regulator for the development and function of regulatory T cells (Tregs). Our center participated in a randomized trial comparing sotrastaurin (n = 14) and the calcineurin inhibitor Neoral (n = 7) in renal transplant recipients. We conducted ex vivo mixed lymphocyte reaction (MLR) and flow cytometry studies on these patient samples, as well as in vitro studies on samples of blood bank volunteers (n = 38). Treg numbers remained stable after transplantation and correlated with higher trough levels of sotrastaurin (r = 0·68, P = 0·03). A dose-dependent effect of sotrastaurin on alloresponsiveness was observed: the half maximal inhibitory concentration (IC50) to inhibit alloactivated T cell proliferation was 45 ng/ml (90 nM). In contrast, Treg function was not affected by sotrastaurin: in the presence of in vitro-added sotrastaurin (50 ng/ml) Tregs suppressed the proliferation of alloactivated T effector cells at a 1:5 ratio by 35 versus 47% in the absence of the drug (P = 0·33). Signal transducer and activator of transcription 5 (STAT)-5 phosphorylation in Tregs remained intact after incubation with sotrastaurin. This potent Treg function was also found in cells of patients treated with sotrastaurin: Tregs inhibited the anti-donor response in MLR by 67% at month 6, which was comparable to pretransplantation (82%). Sotrastaurin is a potent inhibitor of alloreactivity in vitro, while it did not affect Treg function in patients after kidney transplantation.

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.

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.

Summary Rabbit anti-thymocyte globulins (rATG) induce CD4+CD25+forkhead box P3 (FoxP3+) regulatory T cells that control alloreactivity. In the present study, we investigated whether rATG convert T cells into functional CD4+CD25+FoxP3+CD127−/low regulatory T cells in the presence of drugs that may hamper their induction and function, i.e. calcineurin inhibitors. CD25neg T cells were stimulated with rATG or control rabbit immunoglobulin G (rIgG) in the absence and presence of tacrolimus for 24 h. Flow cytometry was performed for CD4, CD25, FoxP3 and CD127 and the function of CD25+ T cells was examined in suppression assays. MRNA expression profiles were composed to study the underlying mechanisms. After stimulation, the percentage CD4+CD25+FoxP3+CD127−/low increased (from 2% to 30%, mean, P

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 C-0 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.

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-kappa B plays a key functional role in T cell activation. Therefore, blockade of the NF-kappa B activation cascade by immunosuppressive drugs prevents immune activation. Here we studied whether TAC blocks NF-kappa B activation in peripheral human T cells. After anti-CD3/CD28-activation of T cells from healthy volunteers, NF-kappa B (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 mu g/mL, sotrastaurin 500 nM (positive control) and mycophenolic acid 10 mg/mL (negative control; n = 6). NF-kappa B transcriptional activity was measured by ELISA and intracellular TNF alpha protein, a downstream target, was measured by flow cytometry to assess the functional consequences of NF-kappa B blockade. Anti-CD3/28-activation induced NF-kappa B phosphorylation in CD3+ T cells, CD4+ T cells and CD8+ T cells by 34% (mean), 38% and 30% resp. (p

Background. Rabbit antithymocyte globulins (rATGs) are known to convert CD4(+) CD25(-) FoxP3(-) T cells from healthy individuals to CD4(+) CD25(+) FoxP3(+) 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 CD4(+) CD25(+) FoxP3(+) T 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 CD4(+) CD25(+) FoxP3(+) CD127(-/low) T cells and CD8(+) CD25(+) FoxP3(-) CD127(+) T cells became higher in the rATG-treated samples compared with the rabbit immunoglobulin-treated samples (P < 0.01). The rATG-induced CD25(+) T cells, whether CD4(+) or CD8(+) inhibited the allogeneic responses of CD25(-/dim) effector T cells as vigorously as natural CD25(+) T cells. However, the proportion of FoxP3(+) within the top 2% rATG-induced CD4(+) CD25(+) T-cells was lower than within the natural CD4(+) CD25(+) T-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 CD25(+) T-cells (all P = 0.03), whereas CTLA4 (P = 0.03), transforming growth factor-beta (P = 0.02), and ROR gamma t (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.

The Jak inhibitor CP-690,550 inhibits alloreactivity and is currently being investigated for prevention of allograft rejection after transplantation. In this study, we examined the effect of CP-690,550 on IL-2-mediated Jak/STAT5 phosphorylation by CD4(+)CD25(bright)FoxP3(+)CD127(-/low) T cells (Treg) and CD4(+)CD25(neg) effector T cells (Teff) in kidney transplant (KTx) patients. Phosphospecific flow cytometry was used to study the effect of CP-690,550 on IL-2-induced intracellular STAT5-phosphorylation. IL-2-induced phosphorylation of STAT5 (P-STAT5) in both Treg and Teff, which was significantly higher for CD4(+)CD25(bright) Treg (increased by 71%, mean) than for CD4(+)CD25(neg) Teff (increased by 42%). In the presence of 100 ng/mL CP-690,550, a clinically relevant exposure, IL-2-induced P-STAT5 was partially inhibited in CD4(+)CD25(bright)Treg (% inhibition; 51%), while almost completely blocked in Teff (%inhibition; 84%, p = 0.03). The IC(50) was 2-3 times higher for Treg (104 ng/mL) than for Teff (40 ng/mL, p = 0.02). In the presence of CP-690,550, Treg exhibited additional suppressive activities on the alloactivated proliferation of Teff (56%, mean). In addition, CD4(+)CD25(bright) Treg from KTx-patients receiving CP-690,550 vigorously suppressed the proliferation of Teff (87%, mean). Our findings show that CP-690,550 effectively inhibits Teff function but preserves the suppressive activity of CD4(+)CD25(bright) regulatory T cells.