Jurjen H.L. Velthuis

Differences in Baseline Lymphocyte Counts and Autoreactivity Are Associated With Differences in Outcome of Islet Cell Transplantation in Type 1 Diabetic Patients

OBJECTIVE The metabolic outcome of islet cell transplants in type 1 diabetic patients is variable. This retrospective analysis examines whether differences in recipient characteristics at the time of transplantation are correlated with inadequate graft function. RESEARCH DESIGN AND METHODS Thirty nonuremic C-peptide–negative type 1 diabetic patients had received an intraportal islet cell graft of comparable size under an ATG-tacrolimus–mycophenolate mofetil regimen. Baseline patient characteristics were compared with outcome parameters during the first 6 posttransplant months (i.e., plasma C-peptide, glycemic variability, and gain of insulin independence). Correlations in univariate analysis were further examined in a multivariate model. RESULTS Patients that did not become insulin independent exhibited significantly higher counts of B-cells as well as a T-cell autoreactivity against insulinoma-associated protein 2 (IA2) and/or GAD. In one of them, a liver biopsy during posttransplant year 2 showed B-cell accumulations near insulin-positive β-cell aggregates. Higher baseline total lymphocytes and T-cell autoreactivity were also correlated with lower plasma C-peptide levels and higher glycemic variability. CONCLUSIONS Higher total and B-cell counts and presence of T-cell autoreactivity at baseline are independently associated with lower graft function in type 1 diabetic patients receiving intraportal islet cells under ATG-tacrolimus–mycophenolate mofetil therapy. Prospective studies are needed to assess whether control of these characteristics can help increase the function of islet cell grafts during the first year posttransplantation.

CD4+ CD25bright+ Regulatory T Cells Can Mediate Donor Nonreactivity in Long‐Term Immunosuppressed Kidney Allograft Patients

CD4+ CD25bright+ FoxP3+ T cells are potent regulators of T‐cell reactivity, but their possible involvement in donor‐specific nonresponsiveness after clinical kidney transplantation remains to be elucidated. We assessed the proliferative donor‐reactivity in 33 kidney allograft recipients who were maintained on a combination of proliferation inhibitors (mycophenolate mofetil (MMF) or Azathioprine (Aza)) and prednisone, long (>5 years) after transplantation. Of the 33 patients, 8 still exhibited donor‐reactivity, whereas 25 were classified as donor nonreactive patients. Within these 25 donor nonreactive patients, we assessed the involvement of CD4+ CD25bright+ regulatory T cells both by depleting them from the responder population as well as by reconstituting them to the CD25−/dim effector population. The absence of proliferation in these 25 patients, was abolished in 7 (28%) recipients upon depletion of the CD4+ CD25bright+ T cells. Reconstitution of these cells suppressed the donor‐reactivity in a dose‐dependent manner. Adding‐back CD4+ CD25bright+ T cells inhibited the anti‐third party response in all recipients, indicating that functional CD4+ CD25bright+ T cells circulate despite more then 5 years of immunosuppressive treatment.

Intragraft FOXP3 mRNA expression reflects antidonor immune reactivity in cardiac allograft patients

Background. Regulatory FOXP3+ T cells control immune responses of effector T cells. However, whether these cells regulate antidonor responses in the graft of cardiac allograft patients is unknown. Therefore, we analyzed the gene expression profiles of regulatory and effector T-cell markers during immunological quiescence and acute rejection. Methods. Quantitative real-time polymerase chain reaction was used to analyze mRNA expression levels in time-zero specimens (n=24) and endomyocardial biopsies (EMB; n=72) of cardiac allograft patients who remained free from rejection (nonrejectors; n=12) and patients with at least one histologically proven acute rejection episode (rejectors; International Society for Heart and Lung Transplantation [ISHLT] rejection grade >2; n=12). Results. For all analyzed regulatory and effector T-cell markers, mRNA expression levels were increased in biopsies taken after heart transplantation compared with those in time-zero specimens. Posttransplantation, the FOXP3 mRNA levels were higher in EMB assigned to a higher ISHLT rejection grade than the biopsies with grade 0: the highest mRNA levels were detected in the rejection biopsies (rejection grade >2; P=0.003). In addition, the mRNA levels of CD25, glucocorticoid-induced TNF receptor family-related gene, cytotoxic T lymphocyte-associated antigen 4, interleukin-2, and granzyme B were also significantly higher in rejecting EMB than in nonrejecting EMB (rejection grade ≤2). This increase in expression levels in relation to the histological rejection grade was only observed in patients who developed an acute rejection episode; the mRNA levels of nonrejectors remained stable irrespective of ISHLT rejection grade. Conclusions. These observations suggest that, after clinical heart transplantation, FOXP3+ T cells do not prevent acute rejection, but rather are a response to antidonor effector T-cell activity.

Monotherapy rapamycin allows an increase of CD4+ CD25bright+ FoxP3+ T cells in renal recipients

CD4+ CD25bright+ FoxP3+ regulatory T cells (Tregs) may control donor‐specific allogeneic responses in kidney transplant recipients. Recent evidence demonstrated that three phenotypical Treg‐subsets, naive (CCR7+CD45RO−), central‐memory (CCR7+CD45RO+) and effector‐memory (CCR7−CD45RO+), are essential for the development and function of antigen‐specific suppression in the lymphoid and peripheral tissues. Also, it has been appreciated that Tregs are affected by immunosuppressive agents. In clinical practice, however, the effect of a single drug remains to be determined. Therefore, we analyzed the effect of several immunosuppressive agents on the number, phenotype and function of peripheral Tregs from 46 stable kidney transplant recipients. These patients were converted to monotherapy with tacrolimus (n = 15), rapamycin (n = 17) or mycophenolate mofetil (n = 14). Blood was obtained at inclusion and 6 months thereafter. The number of Tregs increased significantly in patients on monotherapy with rapamycin (P < 0.001), which was caused by increased numbers of Tregs with a central‐memory and an effector‐memory phenotype (both P < 0.05). At 6 months after conversion, however, the suppressive function of Tregs did not significantly change in co‐cultures stimulated with donor‐Ag. Therefore, monotherapy with rapamycin allows the signals that are needed to increase the number of functional Tregs with a memory phenotype, thereby enhancing the potential capacity to regulate donor‐specific responses in the lymphoid and the peripheral tissues.

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+CD25bright+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+CD25bright+ T cells of 79 patients before, 3, 6, and 12 months after kidney transplantation. The immune regulatory capacities of CD4+CD25bright+ T cells were assessed by their depletion from peripheral blood mononuclear cells and in co-culture with CD25neg/dim responder T-cells in the mixed lymphocyte reactions. Results. In the first year after transplantation, the number and proportion of CD4+CD25bright+ T cells significantly decreased (P<0.05 and P<0.001, respectively). In the mixed lymphocyte reactions, we observed donor-specific hyporesponsiveness 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 CD25brigth+ 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 CD25neg/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+CD25bright+ regulatory T-cell function is generated in fully immunosuppressed renal recipients in the first year after transplantation.

Functional CD25bright+ alloresponsive T cells in fully immunosuppressed renal allograft recipients

Abstract:  Background:  Evidence from animal studies indicate a crucial role for CD25bright+ regulatory T cells in transplantation tolerance.