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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

CD16 down-regulation is associated with IFN-γ induction following stimulation of NK cells from healthy individuals and KTx patients.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.
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pone.0132484.g002: CD16 down-regulation is associated with IFN-γ induction following stimulation of NK cells from healthy individuals and KTx patients.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.

Mentions: In order to investigate the molecular mechanism of decreased CD16 expression in CD56dim NK cells of KTx patients, PBMC of healthy donors were stimulated in vitro in the presence or absence of immunosuppressive drugs, i.e. CsA, Tac, Sir, Ever or MPA. After 6h stimulation with PMA/ionomycin (P/I), CD16 was significantly down-regulated and, thus, the majority of CD56dim NK cells became CD16-negative (Fig 2A and 2B). Incubation with inhibitors alone had no effect on CD16 expression. Intracellular staining revealed that IFN-γ induction was associated with loss of CD16 expression (panel 1) since virtually no IFN-γ/CD16-double-positive NK cells were detected and remaining CD16-positive NK cells did not produce IFN-γ. In the presence of CsA or Tac, CD16 down-regulation was impaired while mTORi and MPA had minor effects. Upon CNI treatment, IFN-γ production was blocked almost completely, whereas treatment with mTORi or MPA could not significantly inhibit IFN-γ production. While the inhibitory effect of CNI on IFN-γ production was still continued after 24h, a block of CD16 down-regulation could no longer be observed (Fig 2, S4A Fig). These in vitro findings indicated that both IFN-γ production which depends on transcriptional activity via NFAT signaling and CD16 down-regulation were both suppressed by CNI but not by mTORi and MPA.


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)

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

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getmorefigures.php?uid=PMC4492590&req=5

pone.0132484.g002: CD16 down-regulation is associated with IFN-γ induction following stimulation of NK cells from healthy individuals and KTx patients.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.
Mentions: In order to investigate the molecular mechanism of decreased CD16 expression in CD56dim NK cells of KTx patients, PBMC of healthy donors were stimulated in vitro in the presence or absence of immunosuppressive drugs, i.e. CsA, Tac, Sir, Ever or MPA. After 6h stimulation with PMA/ionomycin (P/I), CD16 was significantly down-regulated and, thus, the majority of CD56dim NK cells became CD16-negative (Fig 2A and 2B). Incubation with inhibitors alone had no effect on CD16 expression. Intracellular staining revealed that IFN-γ induction was associated with loss of CD16 expression (panel 1) since virtually no IFN-γ/CD16-double-positive NK cells were detected and remaining CD16-positive NK cells did not produce IFN-γ. In the presence of CsA or Tac, CD16 down-regulation was impaired while mTORi and MPA had minor effects. Upon CNI treatment, IFN-γ production was blocked almost completely, whereas treatment with mTORi or MPA could not significantly inhibit IFN-γ production. While the inhibitory effect of CNI on IFN-γ production was still continued after 24h, a block of CD16 down-regulation could no longer be observed (Fig 2, S4A Fig). These in vitro findings indicated that both IFN-γ production which depends on transcriptional activity via NFAT signaling and CD16 down-regulation were both suppressed by CNI but not by mTORi and MPA.

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