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NK Cells of Kidney Transplant Recipients Display an Activated Phenotype that Is Influenced by Immunosuppression and Pathological Staging.

Hoffmann U, Neudörfl C, Daemen K, Keil J, Stevanovic-Meyer M, Lehner F, Haller H, Blume C, Falk CS - PLoS ONE (2015)

Bottom Line: Upon in vitro stimulation via Ca++-dependent signals, down-modulation of CD16 was associated with induction of interferon (IFN)-γ expression.However, secretion of other cytokines like IL-13, IL-17, IL-22 and IL-31 was significantly reduced compared to healthy donors.Thus, immunosuppressive treatment affects NK cell function at the level of NFAT-dependent gene expression whereby calcineurin inhibitors primarily impair cytokine secretion while mTOR inhibitors have only marginal effects.

View Article: PubMed Central - PubMed

Affiliation: Institute of Transplant Immunology, IFB-Tx, Hannover Medical School Hannover, Hannover, Germany.

ABSTRACT
To explore phenotype and function of NK cells in kidney transplant recipients, we investigated the peripheral NK cell repertoire, capacity to respond to various stimuli and impact of immunosuppressive drugs on NK cell activity in kidney transplant recipients. CD56dim NK cells of kidney transplanted patients displayed an activated phenotype characterized by significantly decreased surface expression of CD16 (p=0.0003), CD226 (p<0.0001), CD161 (p=0.0139) and simultaneously increased expression of activation markers like HLA-DR (p=0.0011) and CD25 (p=0.0015). Upon in vitro stimulation via Ca++-dependent signals, down-modulation of CD16 was associated with induction of interferon (IFN)-γ expression. CD16 modulation and secretion of NFAT-dependent cytokines such as IFN-γ, TNF-α, IL-10 and IL-31 were significantly suppressed by treatment of isolated NK cells with calcineurin inhibitors but not with mTOR inhibitors. In kidney transplant recipients, IFN-γ production was retained in response to HLA class I-negative target cells and to non-specific stimuli, respectively. However, secretion of other cytokines like IL-13, IL-17, IL-22 and IL-31 was significantly reduced compared to healthy donors. In contrast to suppression of cytokine expression at the transcriptional level, cytotoxin release, i.e. perforin, granzyme A/B, was not affected by immunosuppression in vitro and in vivo in patients as well as in healthy donors. Thus, immunosuppressive treatment affects NK cell function at the level of NFAT-dependent gene expression whereby calcineurin inhibitors primarily impair cytokine secretion while mTOR inhibitors have only marginal effects. Taken together, NK cells may serve as indicators for immunosuppression and may facilitate a personalized adjustment of immunosuppressive medication in kidney transplant recipients.

No MeSH data available.


Related in: MedlinePlus

CNI but not mTORi suppress cytokine production of PBMC and isolated NK cells of healthy donors.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).
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pone.0132484.g004: CNI but not mTORi suppress cytokine production of PBMC and isolated NK cells of healthy donors.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).

Mentions: In order to confirm our observations of intracellular IFN-γ production with other cytokines, supernatants of PBMC and isolated NK cells following 24h P/I stimulation in presence or absence of CNI, mTORi or MPA were harvested and applied to the multiplex protein technology to determine the Th1/Th2/Th17/Th22 profile. In PBMC, secretion of IFN-γ, TNF-α, IL-17A, IL-21, IL-22 and IL-31 was significantly suppressed by both CNI, while mTORi as well as MPA exerted only minor inhibitory effects without statistical significance (Fig 4A). A similar cytokine profile was detected for isolated NK cells, however, CNI-mediated suppression reached statistical significance only for TNF-α, IL-22 and IL-31. NK cells primarily produced IFN-γ, TNF-α, IL-13, IL-21 and IL-31 after P/I stimulation, lower amounts of IL-17A and IL-22 and hardly contributed to IL-4, IL-10, IL-17F production (S5A Fig). Of note, the capacity to produce the Th2 cytokine IL-31 has not been described for NK cells so far [26]. The efficacy of CNI in suppression of these cytokines argues for a rather broad spectrum of NFAT-dependent target genes across Th1/Th2/Th17 cytokine families. In contrast to cytokines, secretion of soluble IL-2Rα (sCD25) was neither suppressed by CNI nor by mTORi or MPA suggesting that cellular processes that are independent from transcriptional regulation via NFAT such as shedding of IL-2Rα or degranulation of cytotoxins, for instance, cannot be inhibited by these immunosuppressive drugs.


NK Cells of Kidney Transplant Recipients Display an Activated Phenotype that Is Influenced by Immunosuppression and Pathological Staging.

Hoffmann U, Neudörfl C, Daemen K, Keil J, Stevanovic-Meyer M, Lehner F, Haller H, Blume C, Falk CS - PLoS ONE (2015)

CNI but not mTORi suppress cytokine production of PBMC and isolated NK cells of healthy donors.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).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492590&req=5

pone.0132484.g004: CNI but not mTORi suppress cytokine production of PBMC and isolated NK cells of healthy donors.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).
Mentions: In order to confirm our observations of intracellular IFN-γ production with other cytokines, supernatants of PBMC and isolated NK cells following 24h P/I stimulation in presence or absence of CNI, mTORi or MPA were harvested and applied to the multiplex protein technology to determine the Th1/Th2/Th17/Th22 profile. In PBMC, secretion of IFN-γ, TNF-α, IL-17A, IL-21, IL-22 and IL-31 was significantly suppressed by both CNI, while mTORi as well as MPA exerted only minor inhibitory effects without statistical significance (Fig 4A). A similar cytokine profile was detected for isolated NK cells, however, CNI-mediated suppression reached statistical significance only for TNF-α, IL-22 and IL-31. NK cells primarily produced IFN-γ, TNF-α, IL-13, IL-21 and IL-31 after P/I stimulation, lower amounts of IL-17A and IL-22 and hardly contributed to IL-4, IL-10, IL-17F production (S5A Fig). Of note, the capacity to produce the Th2 cytokine IL-31 has not been described for NK cells so far [26]. The efficacy of CNI in suppression of these cytokines argues for a rather broad spectrum of NFAT-dependent target genes across Th1/Th2/Th17 cytokine families. In contrast to cytokines, secretion of soluble IL-2Rα (sCD25) was neither suppressed by CNI nor by mTORi or MPA suggesting that cellular processes that are independent from transcriptional regulation via NFAT such as shedding of IL-2Rα or degranulation of cytotoxins, for instance, cannot be inhibited by these immunosuppressive drugs.

Bottom Line: Upon in vitro stimulation via Ca++-dependent signals, down-modulation of CD16 was associated with induction of interferon (IFN)-γ expression.However, secretion of other cytokines like IL-13, IL-17, IL-22 and IL-31 was significantly reduced compared to healthy donors.Thus, immunosuppressive treatment affects NK cell function at the level of NFAT-dependent gene expression whereby calcineurin inhibitors primarily impair cytokine secretion while mTOR inhibitors have only marginal effects.

View Article: PubMed Central - PubMed

Affiliation: Institute of Transplant Immunology, IFB-Tx, Hannover Medical School Hannover, Hannover, Germany.

ABSTRACT
To explore phenotype and function of NK cells in kidney transplant recipients, we investigated the peripheral NK cell repertoire, capacity to respond to various stimuli and impact of immunosuppressive drugs on NK cell activity in kidney transplant recipients. CD56dim NK cells of kidney transplanted patients displayed an activated phenotype characterized by significantly decreased surface expression of CD16 (p=0.0003), CD226 (p<0.0001), CD161 (p=0.0139) and simultaneously increased expression of activation markers like HLA-DR (p=0.0011) and CD25 (p=0.0015). Upon in vitro stimulation via Ca++-dependent signals, down-modulation of CD16 was associated with induction of interferon (IFN)-γ expression. CD16 modulation and secretion of NFAT-dependent cytokines such as IFN-γ, TNF-α, IL-10 and IL-31 were significantly suppressed by treatment of isolated NK cells with calcineurin inhibitors but not with mTOR inhibitors. In kidney transplant recipients, IFN-γ production was retained in response to HLA class I-negative target cells and to non-specific stimuli, respectively. However, secretion of other cytokines like IL-13, IL-17, IL-22 and IL-31 was significantly reduced compared to healthy donors. In contrast to suppression of cytokine expression at the transcriptional level, cytotoxin release, i.e. perforin, granzyme A/B, was not affected by immunosuppression in vitro and in vivo in patients as well as in healthy donors. Thus, immunosuppressive treatment affects NK cell function at the level of NFAT-dependent gene expression whereby calcineurin inhibitors primarily impair cytokine secretion while mTOR inhibitors have only marginal effects. Taken together, NK cells may serve as indicators for immunosuppression and may facilitate a personalized adjustment of immunosuppressive medication in kidney transplant recipients.

No MeSH data available.


Related in: MedlinePlus