The Efficacy of Rabbit Anti-Thymocyte Globulin for Acute Kidney Transplant Rejection in Patients Using Calcineurin Inhibitor and Mycophenolate Mofetil-Based Immunosuppressive Therapy.

BACKGROUND T cell depleting antibody therapy with rabbit anti-thymocyte globulin (rATG) is the treatment of choice for glucocorticoid-resistant acute kidney allograft rejection (AR) and is used as first-line therapy in severe AR. Almost all studies investigating the effectiveness of rATG for this indication were conducted at the time when cyclosporine A and azathioprine were the standard of care. Here, the long-term outcome of rATG for AR in patients using the current standard immunosuppressive therapy (i.e., tacrolimus and mycophenolate mofetil) is described. MATERIAL AND METHODS Between 2002 to 2012, 108 patients were treated with rATG for AR. Data on kidney function in the year following rATG and long-term outcomes were collected. RESULTS Overall survival after rATG was comparable to overall survival of all kidney transplantation patients (P=0.10). Serum creatinine 1 year after rATG was 179 µmol/L (interquartile range (IQR) 136-234 µmol/L) and was comparable to baseline serum creatinine (P=0.22). Early AR showed better allograft survival than late AR (P=0.0007). In addition, 1 year after AR, serum creatinine was lower in early AR (157 mol/L; IQR 131-203) compared to late AR (216 mol/L; IQR 165-269; P<0.05). The Banff grade of rejection, kidney function at the moment of rejection, and reason for rATG (severe or glucocorticoid resistant AR) did not influence the allograft survival. CONCLUSIONS Treatment of AR with rATG is effective in patients using current standard immunosuppressive therapy, even in patients with poor allograft function. Early identification of AR followed by T cell depleting treatment leads to better allograft outcomes.

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.

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-α and interleukin-15 receptor-α 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.

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.