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The MICA-129 dimorphism affects NKG2D signaling and outcome of hematopoietic stem cell transplantation.

Isernhagen A, Malzahn D, Viktorova E, Elsner L, Monecke S, von Bonin F, Kilisch M, Wermuth JM, Walther N, Balavarca Y, Stahl-Hennig C, Engelke M, Walter L, Bickeböller H, Kube D, Wulf G, Dressel R - EMBO Mol Med (2015)

Bottom Line: A single nucleotide polymorphism causes a valine to methionine exchange at position 129.Functionally, the MICA-129Met isoform was characterized by stronger NKG2D signaling, triggering more NK-cell cytotoxicity and interferon-γ release, and faster co-stimulation of CD8(+) T cells.The MICA-129Met variant also induced a faster and stronger down-regulation of NKG2D on NK and CD8(+) T cells than the MICA-129Val isoform.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany.

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Related in: MedlinePlus

Down-regulation of NKG2D by the MICA-129Met and the MICA-129Val isoforms on CD8+ T cells and impairment of subsequent co-stimulation via NKG2DNKG2D expression on purified CD8+ T cells exposed to L-MICA-129Met (n = 19) or L-MICA-129Val clones (n = 19) for 0, 4, and 24 h was analyzed by flow cytometry. CD8+ T cells (2.5 × 105) were co-cultured with 5 × 104 target cells and analyzed for NKG2D expression after gating on CD3+CD8+ T cells. The means and SD of the MFI of NKG2D (left panel) and of the percentage of NKG2D+CD8+ T cells (right panel) are displayed. Differences between the groups were analyzed by repeated measures ANOVA, and the P-values are indicated.Purified CD8+ T cells were cultured on plate-bound anti-NKG2D (1 μg/ml) or isotype control (mIgG1) for 24 h before the NKG2D expression was measured by flow cytometry. Means and SD of MFI (upper panel) and percentage of NKG2D+ cells (lower panel) are shown (n = 6). Differences between the groups were analyzed by t-tests, and the P-values are indicated.These CD8+ T cells were subsequently CFSE-stained and cultured on plates coated with anti-CD3 (0.005 μg/ml) in combination with anti-CD28 (0.5 μg/ml) as a positive control or anti-NKG2D (0.5 μg/ml). Proliferation was measured after 60 h by flow cytometry. Untreated CFSE-stained CD8+ T cells are included for comparison. The percentage of proliferating cells and the MFI for CFSE are indicated.The MFI of CFSE in unstimulated CD8+ T cells (control) was set to 100% in individual experiments (n = 6), and the relative decrease due to proliferation was calculated. Means + SD are shown. Significant differences (Wilcoxon test) between CD8+ T cells pre-exposed to anti-NKG2D and isotype control (mIgG1) were found in these experiments at anti-CD3 concentrations of 0.01 and 0.005 μg/ml.
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fig07: Down-regulation of NKG2D by the MICA-129Met and the MICA-129Val isoforms on CD8+ T cells and impairment of subsequent co-stimulation via NKG2DNKG2D expression on purified CD8+ T cells exposed to L-MICA-129Met (n = 19) or L-MICA-129Val clones (n = 19) for 0, 4, and 24 h was analyzed by flow cytometry. CD8+ T cells (2.5 × 105) were co-cultured with 5 × 104 target cells and analyzed for NKG2D expression after gating on CD3+CD8+ T cells. The means and SD of the MFI of NKG2D (left panel) and of the percentage of NKG2D+CD8+ T cells (right panel) are displayed. Differences between the groups were analyzed by repeated measures ANOVA, and the P-values are indicated.Purified CD8+ T cells were cultured on plate-bound anti-NKG2D (1 μg/ml) or isotype control (mIgG1) for 24 h before the NKG2D expression was measured by flow cytometry. Means and SD of MFI (upper panel) and percentage of NKG2D+ cells (lower panel) are shown (n = 6). Differences between the groups were analyzed by t-tests, and the P-values are indicated.These CD8+ T cells were subsequently CFSE-stained and cultured on plates coated with anti-CD3 (0.005 μg/ml) in combination with anti-CD28 (0.5 μg/ml) as a positive control or anti-NKG2D (0.5 μg/ml). Proliferation was measured after 60 h by flow cytometry. Untreated CFSE-stained CD8+ T cells are included for comparison. The percentage of proliferating cells and the MFI for CFSE are indicated.The MFI of CFSE in unstimulated CD8+ T cells (control) was set to 100% in individual experiments (n = 6), and the relative decrease due to proliferation was calculated. Means + SD are shown. Significant differences (Wilcoxon test) between CD8+ T cells pre-exposed to anti-NKG2D and isotype control (mIgG1) were found in these experiments at anti-CD3 concentrations of 0.01 and 0.005 μg/ml.

Mentions: NKG2D was strongly down-regulated on CD8+ T cells co-cultured with L-MICA-129Met but hardly after co-culture with L-MICA-129Val clones (FigEV5). The proportion of NKG2D+CD3+CD8+ cells decreased by 35.8%-points more when exposed to L-MICA-129Met clones compared to those encountering the L-MICA-129Val clones (P = 7.8 × 10−8 two-way ANOVA adjusted for MICA expression intensity on different clones), and the MFI of NKG2D decreased by 12.1 units more (P = 2.6 × 10−5; Fig7A). Notably, on CD8+ T cells, the MFI of NKG2D decreased clearly more when exposed to clones with higher MICA-129Met expression intensity (P = 0.016, two-way ANCOVA), whereas CD8 expression was not altered indicating the specificity of the effect (Appendix Fig S13). The down-regulation of NGK2D on CD8+ T cells was functionally relevant. Exposure of CD8+ T cells to anti-NKG2D for 24 h reduced NKG2D expression (Fig7B) and impaired their capability to proliferate subsequently in response to NKG2D-mediated co-stimulation (Fig7C and D and Appendix Fig S14). The strong down-regulation of NKG2D on CD8+ T cells by MICA-129Met variants appears to be important for the association of this polymorphism with the outcome of HSCT.


The MICA-129 dimorphism affects NKG2D signaling and outcome of hematopoietic stem cell transplantation.

Isernhagen A, Malzahn D, Viktorova E, Elsner L, Monecke S, von Bonin F, Kilisch M, Wermuth JM, Walther N, Balavarca Y, Stahl-Hennig C, Engelke M, Walter L, Bickeböller H, Kube D, Wulf G, Dressel R - EMBO Mol Med (2015)

Down-regulation of NKG2D by the MICA-129Met and the MICA-129Val isoforms on CD8+ T cells and impairment of subsequent co-stimulation via NKG2DNKG2D expression on purified CD8+ T cells exposed to L-MICA-129Met (n = 19) or L-MICA-129Val clones (n = 19) for 0, 4, and 24 h was analyzed by flow cytometry. CD8+ T cells (2.5 × 105) were co-cultured with 5 × 104 target cells and analyzed for NKG2D expression after gating on CD3+CD8+ T cells. The means and SD of the MFI of NKG2D (left panel) and of the percentage of NKG2D+CD8+ T cells (right panel) are displayed. Differences between the groups were analyzed by repeated measures ANOVA, and the P-values are indicated.Purified CD8+ T cells were cultured on plate-bound anti-NKG2D (1 μg/ml) or isotype control (mIgG1) for 24 h before the NKG2D expression was measured by flow cytometry. Means and SD of MFI (upper panel) and percentage of NKG2D+ cells (lower panel) are shown (n = 6). Differences between the groups were analyzed by t-tests, and the P-values are indicated.These CD8+ T cells were subsequently CFSE-stained and cultured on plates coated with anti-CD3 (0.005 μg/ml) in combination with anti-CD28 (0.5 μg/ml) as a positive control or anti-NKG2D (0.5 μg/ml). Proliferation was measured after 60 h by flow cytometry. Untreated CFSE-stained CD8+ T cells are included for comparison. The percentage of proliferating cells and the MFI for CFSE are indicated.The MFI of CFSE in unstimulated CD8+ T cells (control) was set to 100% in individual experiments (n = 6), and the relative decrease due to proliferation was calculated. Means + SD are shown. Significant differences (Wilcoxon test) between CD8+ T cells pre-exposed to anti-NKG2D and isotype control (mIgG1) were found in these experiments at anti-CD3 concentrations of 0.01 and 0.005 μg/ml.
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fig07: Down-regulation of NKG2D by the MICA-129Met and the MICA-129Val isoforms on CD8+ T cells and impairment of subsequent co-stimulation via NKG2DNKG2D expression on purified CD8+ T cells exposed to L-MICA-129Met (n = 19) or L-MICA-129Val clones (n = 19) for 0, 4, and 24 h was analyzed by flow cytometry. CD8+ T cells (2.5 × 105) were co-cultured with 5 × 104 target cells and analyzed for NKG2D expression after gating on CD3+CD8+ T cells. The means and SD of the MFI of NKG2D (left panel) and of the percentage of NKG2D+CD8+ T cells (right panel) are displayed. Differences between the groups were analyzed by repeated measures ANOVA, and the P-values are indicated.Purified CD8+ T cells were cultured on plate-bound anti-NKG2D (1 μg/ml) or isotype control (mIgG1) for 24 h before the NKG2D expression was measured by flow cytometry. Means and SD of MFI (upper panel) and percentage of NKG2D+ cells (lower panel) are shown (n = 6). Differences between the groups were analyzed by t-tests, and the P-values are indicated.These CD8+ T cells were subsequently CFSE-stained and cultured on plates coated with anti-CD3 (0.005 μg/ml) in combination with anti-CD28 (0.5 μg/ml) as a positive control or anti-NKG2D (0.5 μg/ml). Proliferation was measured after 60 h by flow cytometry. Untreated CFSE-stained CD8+ T cells are included for comparison. The percentage of proliferating cells and the MFI for CFSE are indicated.The MFI of CFSE in unstimulated CD8+ T cells (control) was set to 100% in individual experiments (n = 6), and the relative decrease due to proliferation was calculated. Means + SD are shown. Significant differences (Wilcoxon test) between CD8+ T cells pre-exposed to anti-NKG2D and isotype control (mIgG1) were found in these experiments at anti-CD3 concentrations of 0.01 and 0.005 μg/ml.
Mentions: NKG2D was strongly down-regulated on CD8+ T cells co-cultured with L-MICA-129Met but hardly after co-culture with L-MICA-129Val clones (FigEV5). The proportion of NKG2D+CD3+CD8+ cells decreased by 35.8%-points more when exposed to L-MICA-129Met clones compared to those encountering the L-MICA-129Val clones (P = 7.8 × 10−8 two-way ANOVA adjusted for MICA expression intensity on different clones), and the MFI of NKG2D decreased by 12.1 units more (P = 2.6 × 10−5; Fig7A). Notably, on CD8+ T cells, the MFI of NKG2D decreased clearly more when exposed to clones with higher MICA-129Met expression intensity (P = 0.016, two-way ANCOVA), whereas CD8 expression was not altered indicating the specificity of the effect (Appendix Fig S13). The down-regulation of NGK2D on CD8+ T cells was functionally relevant. Exposure of CD8+ T cells to anti-NKG2D for 24 h reduced NKG2D expression (Fig7B) and impaired their capability to proliferate subsequently in response to NKG2D-mediated co-stimulation (Fig7C and D and Appendix Fig S14). The strong down-regulation of NKG2D on CD8+ T cells by MICA-129Met variants appears to be important for the association of this polymorphism with the outcome of HSCT.

Bottom Line: A single nucleotide polymorphism causes a valine to methionine exchange at position 129.Functionally, the MICA-129Met isoform was characterized by stronger NKG2D signaling, triggering more NK-cell cytotoxicity and interferon-γ release, and faster co-stimulation of CD8(+) T cells.The MICA-129Met variant also induced a faster and stronger down-regulation of NKG2D on NK and CD8(+) T cells than the MICA-129Val isoform.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany.

Show MeSH
Related in: MedlinePlus