Effect of everolimus-based drug regimens on CMV-specific T-cell functionality after renal transplantation: 12-month ATHENA subcohort-study results

Post-transplant cytomegalovirus (CMV) infections and increased viral replication are associated with CMV-specific T-cell anergy. In the ATHENA-study, de-novo everolimus (EVR) with reduced-exposure tacrolimus (TAC) or cyclosporine (CyA) showed significant benefit in preventing CMV infections in renal transplant recipients as compared to standard TAC + mycophenolic acid (MPA). However, immunomodulatory mechanisms for this effect remain largely unknown. Ninety patients from the ATHENA-study completing the 12-month visit on-treatment (EVR + TAC n = 28; EVR + CyA n = 19; MPA + TAC n = 43) were included in a posthoc analysis. Total lymphocyte subpopulations were quantified. CMV-specific CD4 T cells were determined after stimulation with CMV-antigen, and cytokine-profiles and various T-cell anergy markers were analyzed using flow cytometry. While 25.6% of MPA + TAC-treated patients had CMV-infections, no such events were reported in EVR-treated patients. Absolute numbers of lymphocyte subpopulations were comparable between arms, whereas the percentage of regulatory T cells was significantly higher with EVR + CyA versus MPA + TAC (p = 0.019). Despite similar percentages of CMV-specific T cells, their median expression of CTLA-4 and PD-1 was lower with EVR + TAC (p < 0.05 for both) or EVR + CyA (p = 0.045 for CTLA-4) compared with MPA + TAC. Moreover, mean percentages of multifunctional CMV-specific T cells were higher with EVR + TAC (27.2%) and EVR + CyA (29.4%) than with MPA + TAC (19.0%). In conclusion, EVR-treated patients retained CMV-specific T-cell functionality, which may contribute to enhanced protection against CMV infections

Donor NK and T Cells in the Periphery of Lung Transplant Recipients Contain High Frequencies of Killer Cell Immunoglobulin-Like Receptor-Positive Subsets

Introduction For end-stage lung diseases, double lung transplantation (DLTx) is the ultimate curative treatment option. However, acute and chronic rejection and chronic dysfunction are major limitations in thoracic transplantation medicine. Thus, a better understanding of the contribution of immune responses early after DLTx is urgently needed. Passenger cells, derived from donor lungs and migrating into the recipient periphery, are comprised primarily by NK and T cells. Here, we aimed at characterizing the expression of killer cell immunoglobulin-like receptors (KIR) on donor and recipient NK and T cells in recipient blood after DLTx. Furthermore, we investigated the functional status and capacity of donor vs . recipient NK cells. Methods Peripheral blood samples of 51 DLTx recipients were analyzed pre Tx and at T0, T24 and 3wk post Tx for the presence of HLA-mismatched donor NK and T cells, their KIR repertoire as well as activation status using flow cytometry. Results Within the first 3 weeks after DLTx, donor NK and T cells were detected in all patients with a peak at T0. An increase of the KIR2DL/S1-positive subset was found within the donor NK cell repertoire. Moreover, donor NK cells showed significantly higher frequencies of KIR2DL/S1-positive cells (p<0.01) 3wk post DLTx compared to recipient NK cells. This effect was also observed in donor KIR + T cells 3wk after DLTx with higher proportions of KIR2DL/S1 (p<0.05) and KIR3DL/S1 (p<0.01) positive T cells. Higher activation levels of donor NK and T cells (p<0.001) were detected compared to recipient cells via CD25 expression as well as a higher degranulation capacity upon activation by K562 target cells. Conclusion Higher frequencies of donor NK and T cells expressing KIR compared to recipient NK and T cells argue for their origin in the lung as a part of a highly specialized immunocompetent compartment. Despite KIR expression, higher activation levels of donor NK and T cells in the periphery of recipients suggest their pre-activation during the ex situ phase. Taken together, donor NK and T cells are likely to have a regulatory effect in the balance between tolerance and rejection and, hence, graft survival after DLTx

The Streptococcal Exotoxin Streptolysin O Activates Mast Cells To Produce Tumor Necrosis Factor Alpha by p38 Mitogen-Activated Protein Kinase- and Protein Kinase C-Dependent Pathways

Streptolysin O (SLO), a major virulence factor of pyogenic streptococci, binds to cholesterol in the membranes of eukaryotic cells and oligomerizes to form large transmembrane pores. While high toxin doses are rapidly cytocidal, low doses are tolerated because a limited number of lesions can be resealed. Here, we report that at sublethal doses, SLO activates primary murine bone marrow-derived mast cells to degranulate and to rapidly induce or enhance the production of several cytokine mRNAs, including tumor necrosis factor alpha (TNF-α). Mast cell-derived TNF-α plays an important protective role in murine models of acute inflammation, and the production of this cytokine was analyzed in more detail. Release of biologically active TNF-α peaked ∼4 h after stimulation with SLO. Production of TNF-α was blunted upon depletion of protein kinase C by pretreatment of the cells with phorbol-12 myristate-13 acetate. Transient permeabilization of mast cells with SLO also led to the activation of the stress-activated protein kinases p38 mitogen-activated protein (MAP) kinase and c-jun N-terminal kinase (JNK), and inhibition of p38 MAP kinase markedly reduced production of TNF-α. In contrast, secretion of preformed granule constituents triggered by membrane permeabilization was not dependent on p38 MAP kinase or on protein kinase C. Thus, transcriptional activation of mast cells following transient permeabilization might contribute to host defense against infections via the beneficial effects of TNF-α. However, hyperstimulation of mast cells might also lead to overproduction of TNF-α, which would then promote the development of toxic streptococcal syndromes

Cytokine response of PBMC derived from KTx patients is partially impaired compared to healthy individuals.

<p>PBMC of KTx patients (n = 4, white bars) were stimulated for 24h with P/I or left untreated as described, supernatants were collected, analyzed for cytokine production and compared to P/I stimulated PBMCs of healthy donors (n = 6, grey bars). Data are represented as mean values compared by two-sided One-way ANOVA test with Tukey’s post test (* = p≤0.05, ** = p≤0.01, *** = p≤0.001, only significant values are shown).</p

CD16 down-regulation is associated with IFN-γ induction following stimulation of NK cells from healthy individuals and KTx patients.

<p>PBMC of healthy donors (n = 6) or KTx-patients (n = 4) were pre-incubated with 5 μM inhibitor or equal concentrations of DMSO solvent, stimulated with P/I for 6 or 24 h, respectively and stained for surface CD3, CD56, CD16 and intracellular IFN-γ. (A) FACS dot plot analysis of gated CD3^-CD56⁺ NK cells of one representative healthy donor is shown. CD16^-IFN- γ⁺ subset used for statistical evaluation was labeled as 1. (B) Corresponding statistics of NK cells of 6 healthy individuals regarding IFN-γ-positive subsets in combination with CD16 after 6h stimulation are shown as mean values ± standard deviation compared by Kruskal-Wallis test followed by Dunn’s Multiple Comparison test (* = p<0.05, ** = p<0.01, *** = p<0.001, only significant values are shown). (C) Corresponding statistics of NK cell subsets of healthy donors (n = 6) in comparison with KTx patients (n = 4) after 6h stimulation are displayed.</p

Surface expression of CD16, CD226 and CD161 is significantly reduced in KTx patients, while CD25, CD69 and HLA-DR surface expression is increased.

<p>Phenotypic characterization of peripheral NK cells from healthy individuals (n = 11, circles) and KTx patients (n = 29, triangles) was performed by flow cytometry. (A) CD16, CD226 (DNAM-1), CD161, HLA-DR, CD25 and CD69 expression was determined on CD56^dim NK cells, and compared between healthy donors (HD) and KTx patients (left plots). Displayed are mean values using unpaired Student’s t test (* = p≤0.05, ** = p≤0.01 and *** = p≤0.001, only significant values are shown). The impact of immunosuppression (right plots) was determined by grouping patients according to their immunosuppressive regimen: CsA, Tac or combination of Tac and Sir (T/S). Displayed are mean values, D'Agostino & Pearson omnibus normality test was performed to determine Gaussian distribution, subsequently either One-way-ANOVA or Kruskal-Wallis test were used to determine statistical significance. (B) Patients were grouped according to the histopathology of their biopsies (Banff classification): unsuspicious, borderline, T cell-mediated (TCMR) or antibody-mediated (AMR) rejection. (C) The impact of time after Tx was determined by grouping patients according to the time interval after Tx: ≤3, 6 or ≥ 9 months. Data are shown as scatter plots and display mean values. Asterisks indicate p-values * = p≤0.05, ** = p≤0.01 and *** = p≤0.001, only significant values are shown.</p

CNI but not mTORi suppress cytokine production of PBMC and isolated NK cells of healthy donors.

<p>PBMC of healthy donors (n = 6) were pre-incubated for 20 min with 5 μM inhibitor or DMSO solvent, stimulated with P/I for 24h, supernatants were collected and analyzed for cytokine secretion. Mean values ± standard deviation are shown. To determine statistical significance, Kruskal-Wallis test with Dunn’s post test comparing the different inhibitor treatments to DMSO control was performed. NK cells were negatively MACS-isolated from healthy donor PBMC and stimulated as described. To determine statistical significance, One-Way-ANOVA with Dunnett’s multiple comparison test was performed (* = p≤0.05, ** = p≤0.01, *** = p≤0.001, only significant values are shown).</p