Anne P. Bouvy

A discriminating messenger RNA signature for bipolar disorder formed by an aberrant expression of inflammatory genes in monocytes.

CONTEXT Mood disturbances are associated with an activated inflammatory response system. OBJECTIVE To identify a discriminating and coherent expression pattern of proinflammatory genes in monocytes of patients with bipolar disorder. DESIGN A quantitative polymerase chain reaction (Q-PCR) case-control gene expression study on purified monocytes of bipolar patients, the offspring of bipolar patients, and healthy control participants after having selected 22 discriminating inflammatory genes using whole genome analyses. SETTING Academic research setting in The Netherlands. PATIENTS Forty-two bipolar patients with 25 healthy controls, 54 offspring of a bipolar parent (13 had a mood disorder and 3 developed one during follow-up), and 70 healthy children underwent Q-PCR. MAIN OUTCOME MEASURE Inflammatory gene expression levels in monocytes. RESULTS We detected in the monocytes of bipolar patients a coherent mutually correlating set (signature) of 19 aberrantly expressed (P < .01) messenger RNAs of inflammatory (PDE4B, IL1B, IL6, TNF, TNFAIP3, PTGS2, and PTX3), trafficking (CCL2, CCL7, CCL20, CXCL2, CCR2, and CDC42), survival (BCL2A1 and EMP1), and mitogen-activated protein kinase pathway (MAPK6, DUSP2, NAB2, and ATF3) genes. Twenty-three of 42 bipolar patients (55%) had a positive signature test result vs 7 of 38 healthy controls (18%) (positive test result: positivity for PDE4B, ie, a messenger RNA 1 SD higher than the mean level found in healthy controls, plus 25% of the other genes with similar positive findings). Positive signature test results were also present in 11 of 13 offspring with a mood disorder (85%), 3 of 3 offspring developing a mood disorder (100%), and 17 of 38 euthymic offspring (45%) vs 13 of 70 healthy children (19%). Lithium carbonate and antipsychotic treatment downregulated the gene expression of most inflammatory genes. CONCLUSIONS The monocytes of a large proportion of bipolar patients and offspring of bipolar parents showed an inflammatory gene expression signature. This coherent set of genes opens new avenues for biomarker development with possibilities for disease prediction in individuals genetically at risk and for the subclassification of bipolar patients who could possibly benefit from anti-inflammatory treatment.

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 impact of induction therapy on the homeostasis and function of regulatory T cells in kidney transplant patients

BACKGROUND To evaluate the influence of induction therapy on Tregs we investigated their origin, kinetics and function in kidney transplant patients after treatment with T-cell depleting rabbit antithymocyte globulin (rATG) or IL-2 receptor antagonist basiliximab. METHODS Flow cytometry was used to study thymopoiesis by CD31+ naïve Tregs, homeostatic proliferation by Ki-67+ Tregs and Treg origin by the expression of Helios (nTreg-marker). FACSsorted Tregs were analysed for the demethylation status of the Treg-specific demethylated region (TSDR) of the FoxP3 gene, and Treg-suppressive function. RESULTS Differential effects of rATG and basiliximab induction therapies were measured on the repopulation kinetics of Tregs. While decreased absolute numbers of Tregs were found in both study arms, increased percentages of Tregs were found in rATG treated patients and decreased percentages in basiliximab treated patients. In both groups, Treg repopulation was the result of homeostatic proliferation and not of thymopoiesis. At 1 month after rATG and 6 months after basiliximab therapy, high percentages of Ki-67+ Treg were measured, which in the rATG group, was accompanied by low percentages of Ki-67+Helios+ Treg, and by cells with a demethylated TSDR in the FoxP3 gene. After both rATG and basiliximab therapy, repopulated Tregs inhibited proliferation of allo-antigen activated T effector cells (Teff). CONCLUSIONS In kidney transplant patients, repopulation of Treg after rATG and basiliximab therapy is the result of homeostatic proliferation and not of thymopoiesis. These repopulated Treg were functional after both induction strategies; however only after rATG therapy, were increased proportions of Helios(-) methylated FoxP3 Treg found.

Kinetics of homeostatic proliferation and thymopoiesis after rATG induction therapy in kidney transplant patients.

Background Lymphocyte-depleting therapy is associated with long-lasting effects on repopulated T cells and subsequent increased rates of infections and malignancies. The mechanisms of T-cell repopulation and their posttransplantation kinetics are not fully understood. Methods We studied thymopoiesis by CD31+ naïve T cells (recent thymic emigrants) and homeostatic proliferation by Ki-67+ T cells in rabbit antithymocyte globulin (rATG)–treated patients the first 6 months after transplantation. Patients receiving basiliximab or no induction therapy served as controls. Results At 6 months after transplantation, T-cell numbers were lower than before transplantation in rATG-treated patients, whereas T-cell numbers remained stable in both control groups. In this time period, thymopoiesis was similar between the three treatment groups; CD8+ T cells showed the highest percentage of recent thymic emigrants. At month 1, percentages of Ki-67+ naïve and memory CD4+ and CD8+ T cells were the highest in rATG-treated patients, but these percentages declined in the months thereafter. When CD31 was used to distinguish between cytokine- and antigen-driven proliferation in naïve T cells, we found evidence for cytokine-dependent proliferation. Cytokine-dependent proliferation was also shown by in vivo increased percentages of phosphorylated STAT5 and high expression levels of the interleukin-7 receptor-&agr; and interleukin-15 receptor-&agr; by T cells. Conclusion These findings demonstrate that, in the first month after rATG therapy, cytokine-induced homeostatic proliferation is involved in T-cell repopulation of both naïve and memory T cells. At later time points, the contribution of homeostatic proliferation diminished, which explains the observed incomplete T-cell recovery.

Phospho-specific flow cytometry for pharmacodynamic monitoring of immunosuppressive therapy in transplantation

Organ transplant recipients frequently suffer from toxicity or from lack of efficacy of immunosuppressive drugs, which can be attributed to individual variations in drug sensitivity. This problem can be resolved by applying pharmacodynamic monitoring that focuses on measuring the biological effects of drugs. Here we discuss the new technique called phospho-specific flow cytometry to monitor the activity of intracellular immune signaling pathways at the single-cell level in whole blood samples. Through this tool the efficacy of immunosuppressive medication can be assessed, novel targets can be identified, and differences in drug sensitivity between cells and patients can be clarified.

Alemtuzumab as Antirejection Therapy: T Cell Repopulation and Cytokine Responsiveness.

Background Alemtuzumab induction therapy in kidney transplant patients results in T cell depletion followed by slow immune reconstitution of memory T cells with reduced immune functions. The kinetics and functional characteristics of T cell reconstitution when alemtuzumab is given during immune activation, ie, as antirejection therapy, are unknown. Methods Patients (n = 12) with glucocorticoid-resistant or severe vascular kidney transplant rejection were treated with alemtuzumab. Flow cytometric analysis was performed on whole blood to measure cell division by the marker Ki-67, and cytokine responsiveness by IL-2–mediated and IL-7–mediated phosphorylation of signal transducer and activator of transcription 5 of T cells before and during the first year after rejection therapy. Results At 1 year after alemtuzumab antirejection therapy, the total T cell population recovered to baseline level. Repopulation of CD4+ and CD8+ T cells was associated with increased percentages of Ki-67+ proliferating T cells (P < 0.05). In addition, both populations showed a phenotypic shift toward relatively more memory T cells (P < 0.01). At the functional level, IL-7 reactivity of CD4+ memory T cells was diminished, reflected by a decreased capacity to phosphorylate signal transducer and activator of transcription 5 during the first 6 months after alemtuzumab treatment (P < 0.05), whereas reactivity to IL-2 was preserved. CD8+ T cells were affected in terms of both IL-2 and IL-7 responses (both P < 0.05). After reconstitution, relatively more regulatory T cells were present, and a relatively high proportion of Ki-67+ T cells was observed. Conclusions Preliminary data from this small series suggest that alemtuzumab antirejection therapy induces homeostatic proliferation of memory and regulatory T cells with diminished responsiveness to the homeostatic cytokine IL-7. IL-2 responsiveness was affected in repopulated CD8+ T cells.

T cells Exhibit Reduced Signal Transducer and Activator of Transcription 5 Phosphorylation and Upregulated Coinhibitory Molecule Expression after Kidney Transplantation

Background T-cell depletion therapy is associated with diminished interleukin (IL)-7/IL-15-dependent homeostatic proliferation resulting in incomplete T-cell repopulation. Furthermore, it is associated with impaired T-cell functions. We hypothesized that this is the result of impaired cytokine responsiveness of T cells, through affected signal transducer and activator of transcription (STAT)5 phosphorylation and upregulation of coinhibitory molecules. Materials and Methods Patients were treated with T cell–depleting rabbit antithymocyte globulin (rATG) (6 mg/kg, n = 17) or nondepleting, anti-CD25 antibody (basiliximab, 2 × 40 mg, n = 25) induction therapy, in combination with tacrolimus, mycophenolate mofetil, and steroids. Before and the first year after transplantation, IL-7 and IL-2 induced STAT5 phosphorylation, and the expression of the coinhibitory molecules programmed cell death protein 1 (PD-1), T cell immunoglobulin mucin-3 (TIM-3), lymphocyte activation gene-3 (LAG-3), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), cluster of differentiation (CD) 160, and CD244 was measured by flow cytometry. Results The first year after rATG, CD4+, and CD8+ T cells were affected in their IL-7–dependent phosphorylation of STAT5 (pSTAT5) which was most outspoken in the CD8+ memory population. The capacity of CD4+ and CD8+ T cells to pSTAT5 in response to IL-2 decreased after both rATG and basiliximab therapy. After kidney transplantation, the percentage of TIM-3+, PD-1+, and CD160+CD4+ T cells and the percentage of CD160+ and CD244+CD8+ T cells increased, with no differences in expression between rATG- and basiliximab-treated patients. The decrease in pSTAT5 capacity CD8+ T cells and the increase in coinhibitory molecules were correlated. Conclusions We show that memory T cells in kidney transplant patients, in particular after rATG treatment, have decreased cytokine responsiveness by impaired phosphorylation of STAT5 and have increased expression of coinhibitory molecules, processes which were correlated in CD8+ T cells.

Efficacy of Rabbit Anti-Thymocyte Globulin Therapy for Severe Acute Rejection in Kidney Transplant Patients using Calcineurin Inhibitor and Mycophenolate Mofetil based Immunosuppressive Therapy

Background T-cell depleting antibody therapy is the treatment of choice for glucocorticoid-resistant kidney allograft rejection and is used as first-line therapy in severe T-cell mediated rejection (TCMR). Most studies investigating the effectiveness of this treatment were conducted when cyclosporine and azathioprine combinatiwere therapy. However, there is little evidence for its effectiveness in combination with current standard maintenance immunosuppressive therapy consisting of tacrolimus (TAC), mycophenolate mofetil (MMF), and corticosteroids (CS) with or without induction therapy. Here, the long-term outcome of rATG therapy for AR in patients using the current standard immunosuppressive therapy (i.e. tacrolimus and mycophenolate mofetil), is described. Methods Between 2002 to 2012, we identified 108 patients, treated with rabbit anti-thymocyte globulin (rATG) for biopsy-proven severe (Banff TCMR grade IIA or more) or glucocorticoid-resistant acute rejection; 78 patients had TAC/MMF and/or CS therapy at time of rejection. Patients were treated with high dose methylprednisolone, followed by rATG when kidney function showed further deterioration or a biopsy showed TCMR grade II. Results Overall survival after rATG was comparable to overall survival of all kidney transplant patients (p = 0.10). Allograft survival 5 years after rATG was 55.6%. Serum creatinine 1 year after rATG was 179 &mgr;mol/L (interquartile range (IQR) 136-234 &mgr;mol/L) and was comparable to baseline serum creatinine (p = 0.22). Early AR showed better allograft survival than late AR (p = 0.0007). Remarkably, allograft survival was worse in patients with acute T cell mediated rejection (aTCMR) grade I versus aTCMR grade II+III (p = 0.03). This was associated with a longer interval between pulse glucocorticoids and rATG (31 days (IQR 23-92) versus 8 days (IQR 3-15; p < 0.05). After rATG (mean follow-up 1948 days) 212 infections requiring hospitalization, 12 solid tumors and 2 lymphomas occurred. Conclusion rATG is an effective anti-rejection therapy in patients using current standard immunosuppressive therapy, even in patients with poor allograft function. Treatment with rATG for AR in this study seems not to be associated with increased mortality compared to patients without rATG treatment. The timing of rATG therapy is important. Early recognition of severe and /or glucocorticoid-resistant AR followed by aggressive treatment leads to better allograft function and allograft survival. When this window of opportunity is used, the benefits may outweigh the risks of adverse events.

Rabbit Antithymocyte Globulin Impairs the Capacity for Homeostatic Proliferation of T Cells in Kidney Transplant Patients: 1113

Introduction: Short term rabbit antithymocyte globulin (rATG) induction therapy leads to long-lasting depletion of T cells. Numbers of T cells return to baseline levels approximately one year after kidney transplantation. The biological mechanism of this phenomenon is not elucidated yet. Here we studied the hypothesis that it could be explained by an effect on the homeostatic proliferation of T cells. Material and methods: The phenotype and homeostatic proliferation capacity of T cells from renal transplant recipients (n=14) treated with rATG induction therapy (3x2mg/kg/day) in combination with tacrolimus, mycophenolate mofetil (MMF) and steroids were investigated in the first year after transplantation by whole blood phospho-specific flow cytometry. Patients (n=23) treated with the non-depleting basiliximab induction therapy (day 0; 4; 20 mg) served as a control group. Results: After a significant decrease in the absolute number of both CD4 and CD8 T cells one week after rATG therapy (p< 0.0001), T cells begin to repopulate. However, while CD8 T cells reach baseline levels approximately 3 months after induction therapy, CD4 T cells do not reach 40% of their baseline levels 12 months after transplantation. Functional analysis of the repopulated CD4 and CD8 T cells revealed an impaired Interleukin 7 (IL-7) induced Signal Transducer and Activator of Transcription 5 (STAT5) phosphorylation capacity of repopulated T cells. CD4 memory T cells, including central memory (CD45RO+CCR7+) and effector memory (CD45RO+CCR7-) subpopulations, showed a decrease in the proportion of IL-7 induced phosphorylation of STAT5 (p< 0.04 vs pre-transplantation). After rATG induction therapy, also in the CD8 memory T cells an impaired IL-7 induced STAT5 phosphorylation capacity was found that even decreases further during follow-up the first year after transplantation (p< 0.022 vs pre-transplantation). This decreased STAT5 phosphorylation capacity included the central memory, effector memory and EMRA (CD45RO-CCR7-) subpopulations. CD4 and CD8 T cells of basiliximab treated patients did not show impaired IL-7 induced STAT5 phosphorylation responses. Conclusion: CD4 and CD8 T cells showed a long-lasting impaired IL-7 induced phosphorylation capacity of STAT5 after rATG induction therapy which can explain the absence of full immune reconstitution approximately one year after rATG induction therapy. 1566

Reconstitution of T Cells after rATG Induction Therapy in Kidney Transplant Patients Is the Result of Homeostatic Proliferation and not of Thymopoiesis: 1120

Introduction: Rabbit antithymocyte globulin (rATG) induction therapy is followed by peripheral immune reconstitution. To define the biology of this phenomenon we studied two key mechanisms involved in the reconstitution of the peripheral CD4 and CD8 T cell pool: thymopoiesis and homeostatic proliferation driven by STAT5 (Signal Transducer and Activator of Transcription-5) activating cytokines. Material and methods: Adult patients were treated with rATG (3 x 2mg/ kg/day, n=8) or non-depleting anti-CD25 antibody basiliximab (day 0, 4; 20 mg, n=8) induction therapy in combination with tacrolimus, MMF and steroids. Flow cytometric analyses were performed to study Ki67, a molecular marker of proliferation, in combination with CD31, the marker of recent thymic emigrants (RTEs) in naïve T cells (CD45RO-CCR7+). Ki67 expression was also analyzed in memory T cell subsets; central memory (CD45RO+CCR7+), effector memory (CD45RO+CCR7-) and EMRA T cells (CD45RO-CCR7-). Results: In our rATG treated patient population we found no evidence of increased thymopoiesis. At one month after transplantation the percentage of RTEs (Ki67-CD31+ thymicnaïve T cells) was comparable between rATG and basiliximab treated patients. Homeostatic proliferation of the Ki67+CD31central naïve T cells, responding upon self-antigens was not increased in rATG treated patients. In contrast, the Ki67+CD31+ central naïve T cell subset, in which proliferation depends on homeostatic cytokines and Ki67+ memory T cell subsets were proportionally higher in the rATG patient group compared to the basiliximab group (p< 0.01 for all subsets). These results were similar for both CD4 and CD8 T cell subsets. Our findings were supported by whole blood STAT5 analysis that demonstrated elevated levels of phosphorylated STAT5 in unstimulated peripheral naïve and memory T cells of the rATG group 1 month after depletion but not in the basiliximab group (p< 0.04). Conclusion: Our results show that rATG has no effect on thymic output. Homeostatic proliferation and not thymopoiesis is the main mechanism by which the peripheral CD4 and CD8 T cell pool reconstitutes. 1123