CD4+ CD28null T cells are not alloreactive unless stimulated by interleukin-15.

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 < .05). After allogeneic stimulation together with IL-15 and IL-21, frequency of degranulating CD107a+ CD4+ CD28null T cells increased significantly from 0.6% to 5.8% (P < .001). Granzyme B and perforin positivity remained similar, but production of interferon-γ and tumor necrosis factor-α increased by the combination of IL-15 and IL-21 (P < .001 and P < .05, respectively). Finally, CD4+ CD28null T cells did not show significant suppression. Thus, CD4+ CD28null T cells represent a population with absent alloreactivity unless IL-15 is present.

Co-inhibitory profile and cytotoxicity of CD57+ PD-1- T cells in end-stage renal disease patients.

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.

Pretransplant Numbers of CD16+ Monocytes as a Novel Biomarker to Predict Acute Rejection After Kidney Transplantation: A Pilot Study.

Acute rejection is one of the major immunological determinants of kidney graft function and survival. Early biomarkers to predict rejection are lacking. Emerging evidence reveals a crucial role for the monocyte/macrophage lineage cells in the pathogenesis of rejection. We hypothesized that higher pretransplant numbers of proinflammatory CD16+ monocytes can predict rejection. The study cohort consisted of 104 kidney transplant recipients (58 with no rejection and 46 with biopsy-proven rejection) and 33 healthy persons. Posttransplant median follow-up time was 14.7 mo (interquartile range 0.3-34 mo). Pretransplantation blood samples were analyzed by flow cytometry for monocyte immunophenotypes. Groups were compared by Cox regression models for the occurrence of acute rejection. We documented a significantly increased absolute number of pretransplant CD16+ monocytes in patients who developed biopsy-proven rejection after transplantation compared with those with no rejection (hazard ratio [HR] 1.60, 95% CI 1.28-2.00, p < 0.001) and healthy persons (HR 1.47, 95% CI 1.18-1.82, p < 0.001). In parallel, significantly fewer absolute numbers of CD16- monocytes were observed at pretransplant time points in rejectors versus nonrejectors (HR 0.74, 95% CI 0.58-0.94, p < 0,014). A higher pretransplant number of CD16+ monocytes is significantly associated with a higher risk of acute rejection after kidney transplantation.

Implementation of the Participatory Approach for Supervisors to Increase Self-Efficacy in Addressing Risk of Sick Leave of Employees: Results of a Cluster-Randomized Controlled Trial.

Purpose To study the effectiveness of a multifaceted strategy to implement the participatory approach (PA) for supervisors to increase their self-efficacy in addressing risk of sick leave of employees. Methods Supervisors from three organizations were invited to participate. Randomization was performed at department level. Supervisors (n = 61) in the intervention departments received the implementation strategy consisting of a working group meeting, supervisor training in PA application, and optional supervisor coaching. Supervisors in the control departments (n = 55) received written information on PA. The primary outcome was supervisors' self-efficacy to apply the PA, measured at baseline and 6 months' follow-up. The number of employees with whom supervisors discussed work functioning problems or (risk of) sick leave was also assessed. Effects were tested using multilevel analyses. Results The strategy did not increase self-efficacy to apply the PA. Subgroup analyses showed that self-efficacy increased for supervisors who at baseline reported to have discussed (risk of) sick leave with less than three employees during the last 6 months (B = 1.42, 95 % CI 0.34-2.50). Furthermore, the implementation strategy increased the number of employees with whom supervisors discussed work functioning problems or risk of sick leave (B = 1.26, 95 % CI 0.04-2.48). Conclusion Although the implementation strategy cannot be recommended for all supervisors, for supervisors who less frequently discuss (risk of) sick leave with employees the implementation strategy might be helpful. Trial registration NTR3733.

Employees' Perceptions of Social Norms as a Result of Implementing the Participatory Approach at Supervisor Level: Results of a Randomized Controlled Trial.

Purpose A multifaceted implementation strategy was targeted at supervisors to encourage them to apply a participatory approach (PA) in dealing with employees' work functioning problems due to health concerns. This paper assesses the effect on employees' perceived social norms regarding the use of the PA to deal with work functioning problems. Methods Three organizations participated in a cluster randomized controlled trial, with randomization at the department level. Supervisors in the PA intervention departments received the implementation strategy consisting of a working group meeting, supervisor training, and optional coaching. Supervisors in the control departments received written information about the PA only. In two of the organizations, employees were invited to complete surveys at baseline and at 6-month follow-up. The primary outcome was perceived social norms regarding the use of the PA to deal with work functioning problems. Secondary measures included attitudes and self-efficacy, and intention regarding joint problem solving, and sick leave data. Effects were analyzed using multilevel analyses to account for nesting of cases. Results At baseline, 273 employees participated in the survey, with follow-up analyses of 174 employees. There were no statistically significant group effects on employee outcome measures. The intervention group showed a larger reduction in mean sick days (from 4.6 to 2.4 days) versus the control group (from 3.8 to 3.6 days), but this difference did not reach statistical significance (p > .05). Conclusion The multifaceted strategy to implement the participatory approach for supervisors did not show effects on outcomes at the employee level. To gain significant effects at the employee level, may require that an implementation strategy not only targets management and supervisors, but also employees themselves. TRIAL REGISTRATION: NTR3733.

Implementation of the participatory approach for supervisors to prevent sick leave: a process evaluation.

PURPOSE: To perform a process evaluation of a multifaceted strategy to implement the participatory approach for supervisors to prevent sick leave in three organisations. METHODS: The implementation strategy incorporated a working group meeting with stakeholder representatives, supervisor training, and optional supervisor coaching. Context, recruitment, reach, dose delivered, dose received, fidelity, and satisfaction with the strategy were assessed at organisational and supervisor level using questionnaires and registration forms. RESULTS: At least 4 out of 6 stakeholders were represented in the working group meetings, and 11 % (n = 116) of supervisors could be reached. The working group meetings and supervisor training were delivered and received as planned and were well appreciated within all three organisations. Three supervisors made use of coaching. At 6-month follow-up, 11 out of 41 supervisors (27 %) indicated that they had applied the participatory approach at least one time. CONCLUSION: The implementation strategy was largely carried out as intended. However, reach of both supervisors and department managers should be improved. Future studies should consider targeting employees with the strategy.

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 (CD4(POS) CXCR5(POS) 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.

The protein kinase C inhibitor sotrastaurin allows regulatory T cell function.

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.

The effect of low and ultra-low dosages Thymoglobulin on peripheral T, B and NK cells in kidney transplant recipients.

INTRODUCTION: Rabbit Anti-Thymocyte Globulin (r-ATG) is a polyclonal antibody preparation, used to prevent and treat acute rejection episodes after organ transplantation. However, despite more than 40 years of clinical use, the optimal dose of r-ATG is still not defined. To find a better balance between efficacy and infectious complications, we embarked on a controlled study and monitored the effect of low and ultra-low dosages Thymoglobulin (Genzyme) on peripheral T, B, and NK cells. PATIENTS AND METHODS: Kidney transplant recipients received either 0.5 mg/kg, 1.0 mg/kg or 2.0 mg/kg on the first 3 consecutive days post-transplantation. Thus, total doses were 1.5 mg/kg, 3.0 mg/kg and 6.0 mg/kg. A total of 40 patients were enrolled, including 11 controls. All patients were treated with Prednisolon, Advagraf (Astellas) and Mycophenolate Mofetil (Roche). T (CD3+), B (CD19+) and NK (CD3-CD16+56+) cells were analyzed by flow cytometry. Baseline cell counts were compared to forty age and sex matched healthy persons. Post-transplantation cell counts of the 3 Thymoglobulin groups were compared to the 11 control patients, who received no induction therapy. RESULTS: Absolute numbers of T, B, and NK cells were comparable in all patients pre-transplantation, but T and B cells were lower than in healthy persons (p=0.007 and p=0.0003, Mann Whitney test). In the first week, T cells and NK cells were significantly lower in all Thymoglobulin groups compared to controls. B cells were not affected. One month after Thymoglobulin NK cells had returned to control numbers in all groups, while T cells had already recovered to control counts in the 1.5 mg/kg group. During follow-up, T cells in the 3.0mg/kg group also returned to control values, but at one year the patients in the 6.0 mg/kg group still had significantly lower T cells (p=0.03). Patient and graft survival, rejection and infection incidence and renal function did not differ between groups. CONCLUSION: Patients with end stage renal disease have significantly lower peripheral T and B cell counts than healthy persons. (Ultra-) low Thymoglobulin schedules deplete peripheral lymphocytes in a dose dependent way. Knowledge of the duration of this depletion contributes to finding the optimal immunosuppressive strategy for kidney transplant recipients.

The Jak inhibitor CP-690,550 preserves the function of CD4CD25FoxP3 regulatory T cells and inhibits effector T cells.

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.

The calcineurin inhibitor tacrolimus allows the induction of functional CD4CD25 regulatory T cells by rabbit anti-thymocyte globulins.

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. CD25(neg) 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 < 0.01) and was higher in the rATG samples than in control rIgG samples (2%, P < 0.01). Interestingly, FoxP3(+)T cells were also induced when tacrolimus was present in the rATG cultures. Blockade of the interleukin (IL)-2 pathway did not affect the frequency of rATG-induced FoxP3(+) T cells. The rATG tacrolimus-induced CD25(+) T cells inhibited proliferative responses of alloantigen-stimulated effector T cells as vigorously as rATG-induced and natural CD4(+)CD25(+)FoxP3(+)CD127(-/low) T cells (67% +/- 18% versus 69% +/- 16% versus 45% +/- 20%, mean +/- standard error of the mean, respectively). At the mRNA-expression level, rATG-induced CD25(+) T cells abundantly expressed IL-10, IL-27, interferon (IFN)-gamma, perforin and granzyme B in contrast to natural CD25(+) T cells (all P = 0.03), while FoxP3 was expressed at a lower level (P = 0.03). These mRNA data were confirmed in regulatory T cells from kidney transplant patients. Our findings demonstrate that tacrolimus does not negatively affect the induction, phenotype and function of CD4(+)CD25(+) T cells, suggesting that rATG may induce regulatory T cells in patients who receive tacrolimus maintenance therapy.