The Jak Inhibitor CP-690,550 Preserves the Function of CD4+CD25brightFoxP3+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+CD25brightFoxP3+CD127-/low T cells (Treg) and CD4+CD25neg 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+CD25bright Treg (increased by 71%, mean) than for CD4+CD25neg 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+CD25brightTreg (% inhibition; 51%), while almost completely blocked in Teff (%inhibition; 84%, p = 0.03). The IC50 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+CD25bright 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+CD25bright regulatory T cells

End-stage renal failure and regulatory activities of CD4(+)CD25(bright+)FoxP3(+) T-cells

Background. The defensive immune system in patients with end-stage renal failure is impaired at multiple levels. This state of immune incompetence is associated with continuous activation of the immune system. An additional explanation for this state of activation may be the disturbed function of CD4(+)CD25(bright+)FoxP3(+) regulatory T-cells. Methods. The phenotype and function of peripheral regulatory T-cells from patients with end-stage renal failure (N = 80) and healthy controls (N = 17) was studied by flow cytometry, RT-PCR and mixed lymphocyte reaction. Patients were on haemodialysis (N = 40), peritoneal dialysis (N = 26) or not treated with dialysis yet (N = 14). The latter group had a glomerular filtration rate of < 20 ml/min/ 1.73 m(2). Results. The basal IL-2 mRNA level was high in patient-PBMC (P = 0.0002 versus healthy controls). The absolute number of CD4(+)CD25(bright+) T-cells was low in patients (P < 0.05 versus healthy controls). Furthermore, proliferation of patient-PBMC upon allogeneic stimulation was impaired (P < 0.0001 versus healthy controls). The regulatory function of CD4(+)CD25(bright+) T-cells was determined in the setting of direct allorecognition. First, the effect of depletion of CD25(bright+) cells from patient-PBMC on proliferation was low. Second, co-culture of CD25(bright+) cells with CD25(neg/dim) cells (1:10 ratio) showed impaired regulatory function (P < 0.001 versus healthy controls), which was especially pronounced in patients on dialysis. The FOXP3 mRNA level was also low upon stimulation (P = 0.0002 versus healthy controls). Conclusions. In line with previous studies, we observed an overactivated but functionally compromised immune system in patients with end-stage renal failure. It now appears that in this setting, regulation by CD4(+)CD25(bright+)FoxP3(+) T-cells is also impaired

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 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 effect of rabbit anti-thymocyte globulin induction therapy on regulatory T cells in kidney transplant patients

Background. Prevention of alloreactivity by rabbit anti-thymocyte globulins (rATG) may not only result from immunodepletion but also from the induction of T cells that control allogeneic immune responses. In the present prospective and controlled study, we investigated the effect of rATG on the frequency, function and phenotype of peripheral immunoregulatory CD4(+) T cells in kidney transplant (KTx) patients. Methods. After transplantation, 16 patients received ATG-induction therapy and triple therapy consisting of tacrolimus, MMF and steroids. The control group (n = 18) received triple therapy only. By flow cytometry, T cells were analysed for CD25, FoxP3, CD127, CD45RO and CCR7. To study their suppressive capacities, CD25(bright) T cells were co-cultured with CD25(-/dim) effector T cells (Teff) in mixed lymphocyte reactions (MLR), stimulated with donor and third party (3P) antigens. Results. Pre-transplant levels of FoxP3(+)CD127(-/low) T cells were 6% of CD4(+) T cells. One week post-ATG treatment, no measurable numbers of regulatory T cells were present (P < 0.01). After 4 weeks, the cell numbers of CD4(+)FoxP3(+)CD127(-/low) T cells slowly reappeared and thereafter remained low (P < 0.01). At 14 weeks, a significant shift towards the CD45RO(+)CCR7(+) (central memory) phenotype within CD4(+)FoxP3(+) T cells was observed (P < 0.01). At 26 weeks, the proliferative alloresponses of the PBMC and CD25(-/dim) Teff profoundly decreased compared to pre-transplant (P = 0.01 and P = 0.02 respectively), while the regulatory capacity of the CD25(bright) T cells, of which 90% consisted of FoxP3(+)CD127(-/low) T cells, remained unaffected. The CD25(bright) T cells suppressed the anti-donor (94%) and 3P responses (93%). Conclusion. Our findings show that rATG therapy does not spare peripheral immunoregulatory T cells in vivo, but after regeneration preserves their suppressive activity

Conversion From Calcineurin Inhibitor to Mycophenolate Mofetil-Based Immunosuppression Changes the Frequency and Phenotype of CD4(+)FOXP3(+) Regulatory T Cells

Background. CD4(+)Foxp3(+) regulatory T cells (Treg) depend on interleukin (IL)-2 for their function and survival. By interfering with the IL-2 production, calcineurin inhibitors (CNI) may negatively affect Treg. Here, we describe the effects of conversion from CNI to mycophenolate mofetil (MMF) monotherapy on renal function, and on Treg frequency and phenotype in liver transplant recipients. Methods. Patients (n=16) with renal impairment on CNI were converted to MMF and received a single dose of IL-2-receptor blocking antibody (Daclizumab). Control patients (n=8) continued CNI treatment. Results. Renal function rapidly and significantly improved after conversion. Daclizumab treatment resulted in a 75% blocking of CD25 at 1 month causing a significant reduction in the percentage of CD4(+)CD25(+) cells but not affecting the percentage of CD4(+)CD25(+)Foxp3(+) cells. Six months after conversion to MMF, the percentage of CD4+CD25+Foxp3+ cells increased significantly by 125%. FOXP3 mRNA analysis of mononuclear cells confirmed the enrichment of Foxp3 in peripheral blood. Interestingly, the CD25 expression level on CD4(+)Foxp3(+), but not CD4(+)Foxp3(-), cells significantly increased compared with preconversion. Conclusion. Conversion to MMF increases the percentage and CD25 expression of CD4(+)Foxp3(+) cells indicating that MMF therapy can overturn the repressive effect of CNI on circulating Treg levels and therefore may promote Treg-mediated suppression of alloreactivity

Generation of Donor-Specific Regulatory T-Cell Function in Kidney Transplant Patients

Background. In the search for mechanisms that can induce and maintain transplant tolerance, donor-specific CD4(+)CD25(bright) (+)Foxp3(+) regulatory T cells have been frequently mentioned. However, it remains to be demonstrated, whether these cells are generated after clinical transplantation. Methods. We prospectively analyzed the phenotype and function of peripheral regulatory CD4(+)CD25(bright+) T cells of 79 patients before, 3, 6, and 12 months after kidney transplantation. The immune regulatory capacities of CD4(+)CD25(bright+) T cells were assessed by their depletion from peripheral blood mononuclear cells and in co-culture with CD25(neg/dim) responder T-cells in the mixed lymphocyte reactions. Results. In the first year after transplantation, the number and proportion of CD4(+)CD25(bright+) T cells significantly decreased (P<0.05 and P<0.001, respectively). In the mixed lymphocyte reactions, we observed donor-specific hypo-responsiveness in the presence of significantly increased proliferation to third and fourth Party-Ag, (P<0.001 and P<0.05, respectively). Furthermore, functional analysis of CD25(bright+) cells showed that the effect of depletion of these cells from peripheral blood mononuclear cells, and their suppressive capacities in co-culture with donor-Ag stimulated CD25(neg/dim) responder T-cells (1:10 ratio) significantly improved (P<0.01 and P<0.001, respectively). Moreover, the difference between the stimulation with donor-Ag and third Party-Ag became apparent at 6 months after transplantation. Conclusions. These findings demonstrate that donor-specific CD4(+)CD25(bright+) regulatory T-cell function is generated in fully immunosuppressed renal recipients in the first year after transplantation