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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

NK cell activity in response to K562 target cells as well as intracellular NFAT2 expression is retained in KTx patients compared to healthy individuals.(A) PBMC of healthy donors (n = 4, grey bar) or of KTx patients (n = 4, white bar) were incubated for 18h with K562 target cells and activation was quantified by IFN-γ ELISpot and multiplex analyses of supernatants for perforin and granzyme A/B. For statistical analyses, spots were normalized to 10.000 NK cells per well and mean values ± standard deviations are depicted, compared by Kruskal-Wallis test followed by Dunn’s Multiple Comparison test. (B) PBMC of healthy donors (n = 6) were pre-incubated with immunosuppressive drugs (5 μM) or DMSO solvent for 20 min and either left unstimulated (shaded bars) or P/I stimulated for additional 6h or 24h, respectively (grey bars, left and middle graph). KTx recipient-derived PBMC (n = 4) were stimulated identically, cells were stained intracellular for total NFAT2 and analyzed by flow cytometry. The right plot shows total NFAT2 in healthy donors compared to KTx patients after 24h stimulation. Mean values and standard deviations are displayed compared by two-sided One-way-ANOVA test (* = p≤0.05, ** = p≤0.01, *** = p≤0.001, only significant values are shown).
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pone.0132484.g003: NK cell activity in response to K562 target cells as well as intracellular NFAT2 expression is retained in KTx patients compared to healthy individuals.(A) PBMC of healthy donors (n = 4, grey bar) or of KTx patients (n = 4, white bar) were incubated for 18h with K562 target cells and activation was quantified by IFN-γ ELISpot and multiplex analyses of supernatants for perforin and granzyme A/B. For statistical analyses, spots were normalized to 10.000 NK cells per well and mean values ± standard deviations are depicted, compared by Kruskal-Wallis test followed by Dunn’s Multiple Comparison test. (B) PBMC of healthy donors (n = 6) were pre-incubated with immunosuppressive drugs (5 μM) or DMSO solvent for 20 min and either left unstimulated (shaded bars) or P/I stimulated for additional 6h or 24h, respectively (grey bars, left and middle graph). KTx recipient-derived PBMC (n = 4) were stimulated identically, cells were stained intracellular for total NFAT2 and analyzed by flow cytometry. The right plot shows total NFAT2 in healthy donors compared to KTx patients after 24h stimulation. Mean values and standard deviations are displayed compared by two-sided One-way-ANOVA test (* = p≤0.05, ** = p≤0.01, *** = p≤0.001, only significant values are shown).

Mentions: Since the NK cell repertoire of KTx patients under immunosuppression showed remarkable alterations, we investigated if this particular phenotype was associated with impaired NK cell function. Upon stimulation with P/I for 6h or 24h, CD16 expression on CD56dim NK cells was down-regulated at the same level in patients as in healthy donors and equivalent amounts of IFN-γ were produced (Fig 2C, S4D Fig). To test whether NK cells from KTx patients remained responsive to stimulation with HLA class I-negative K562 target cells, IFN-γ ELISpot assays were performed. In order to control for individual proportions of NK cells in healthy donors and KTx recipients, IFN-γ spots were normalized to 10,000 NK cells (Fig 3A). No significant differences in IFN-γ-secreting NK cells in response to K562 cells were observed between healthy individuals and KTx recipients. The supernatants harvested from these K562 stimulations were used to quantify perforin and granzyme A/B secretion and, again, no significant differences were measured between patients and healthy donors. Thus, NK cells of KTx recipients seem to retain their capacity to respond to strong, non-specific stimulation such as P/I as well as to specific stimulation by K562.


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)

NK cell activity in response to K562 target cells as well as intracellular NFAT2 expression is retained in KTx patients compared to healthy individuals.(A) PBMC of healthy donors (n = 4, grey bar) or of KTx patients (n = 4, white bar) were incubated for 18h with K562 target cells and activation was quantified by IFN-γ ELISpot and multiplex analyses of supernatants for perforin and granzyme A/B. For statistical analyses, spots were normalized to 10.000 NK cells per well and mean values ± standard deviations are depicted, compared by Kruskal-Wallis test followed by Dunn’s Multiple Comparison test. (B) PBMC of healthy donors (n = 6) were pre-incubated with immunosuppressive drugs (5 μM) or DMSO solvent for 20 min and either left unstimulated (shaded bars) or P/I stimulated for additional 6h or 24h, respectively (grey bars, left and middle graph). KTx recipient-derived PBMC (n = 4) were stimulated identically, cells were stained intracellular for total NFAT2 and analyzed by flow cytometry. The right plot shows total NFAT2 in healthy donors compared to KTx patients after 24h stimulation. Mean values and standard deviations are displayed compared by two-sided One-way-ANOVA test (* = p≤0.05, ** = p≤0.01, *** = p≤0.001, only significant values are shown).
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pone.0132484.g003: NK cell activity in response to K562 target cells as well as intracellular NFAT2 expression is retained in KTx patients compared to healthy individuals.(A) PBMC of healthy donors (n = 4, grey bar) or of KTx patients (n = 4, white bar) were incubated for 18h with K562 target cells and activation was quantified by IFN-γ ELISpot and multiplex analyses of supernatants for perforin and granzyme A/B. For statistical analyses, spots were normalized to 10.000 NK cells per well and mean values ± standard deviations are depicted, compared by Kruskal-Wallis test followed by Dunn’s Multiple Comparison test. (B) PBMC of healthy donors (n = 6) were pre-incubated with immunosuppressive drugs (5 μM) or DMSO solvent for 20 min and either left unstimulated (shaded bars) or P/I stimulated for additional 6h or 24h, respectively (grey bars, left and middle graph). KTx recipient-derived PBMC (n = 4) were stimulated identically, cells were stained intracellular for total NFAT2 and analyzed by flow cytometry. The right plot shows total NFAT2 in healthy donors compared to KTx patients after 24h stimulation. Mean values and standard deviations are displayed compared by two-sided One-way-ANOVA test (* = p≤0.05, ** = p≤0.01, *** = p≤0.001, only significant values are shown).
Mentions: Since the NK cell repertoire of KTx patients under immunosuppression showed remarkable alterations, we investigated if this particular phenotype was associated with impaired NK cell function. Upon stimulation with P/I for 6h or 24h, CD16 expression on CD56dim NK cells was down-regulated at the same level in patients as in healthy donors and equivalent amounts of IFN-γ were produced (Fig 2C, S4D Fig). To test whether NK cells from KTx patients remained responsive to stimulation with HLA class I-negative K562 target cells, IFN-γ ELISpot assays were performed. In order to control for individual proportions of NK cells in healthy donors and KTx recipients, IFN-γ spots were normalized to 10,000 NK cells (Fig 3A). No significant differences in IFN-γ-secreting NK cells in response to K562 cells were observed between healthy individuals and KTx recipients. The supernatants harvested from these K562 stimulations were used to quantify perforin and granzyme A/B secretion and, again, no significant differences were measured between patients and healthy donors. Thus, NK cells of KTx recipients seem to retain their capacity to respond to strong, non-specific stimulation such as P/I as well as to specific stimulation by K562.

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