The MICA-129 dimorphism affects NKG2D signaling and outcome of hematopoietic stem cell transplantation.
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.
Affiliation: Institute of Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany.
- Graft vs Host Disease/mortality*/pathology*
- Hematopoietic Stem Cell Transplantation*
- Histocompatibility Antigens Class I/genetics*
- Mutant Proteins/genetics*
- NK Cell Lectin-Like Receptor Subfamily K/metabolism*
- Signal Transduction*
- Aged, 80 and over
- CD8-Positive T-Lymphocytes/immunology
- Child, Preschool
- Cytotoxicity, Immunologic
- Killer Cells, Natural/immunology
- Middle Aged
- Mutation, Missense
- Survival Analysis
- Treatment Outcome
- Young Adult
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fig02: NKG2D binding to the MICA-129Met and MICA-129Val isoform and triggering of phosphorylation of SRC family kinasesThe linear regression of MICA expression intensity and binding of a recombinant NKG2D-Fc fusion protein both determined as MFI by flow cytometry is displayed for L-MICA-129Met (n = 79, left panel) and L-MICA-129Val clones (n = 81, right panel). The coefficients of determination (R2), the regression coefficients (reg. coeff.), and the P-values for Pearson correlation are indicated.Purified IL-2-stimulated (100 U/ml for 4 days) NK cells (106) were stimulated with immobilized MICA-129Met-mIgG2a-Fc or MICA-129Val-mIgG2a-Fc or OVA-mIgG2a-Fc fusion proteins (10 μg/ml) for 3, 10, or 30 min. The protein lysates of these cells were separated by SDS–PAGE, and the blot was probed subsequently with an anti-phospho-Tyr mAb, an anti-phospho-SRC family (Tyr419) kinases Ab, and an anti-β-actin mAb as a loading control. The arrow points toward phosphorylated SRC family kinases.Blots obtained from three independent experiments were analyzed by densitometry, and the means plus SD of the ratio between phospho-SRC family kinase and β-actin signals is displayed. The difference between NK cells stimulated for 10 min by MICA-129Met-Fc or MICA-129Val-Fc proteins was assessed by t-test.Purified IL-2-stimulated NK cells (100 U/ml for 4 days, 106) were incubated with the SRC kinase inhibitor PP2 (25 μM), the vehicle DMSO, or medium only (Ø) for 30 min before being added to immobilized MICA-129Met-Fc, MICA-129Val-Fc, or OVA-Fc fusion proteins (10 μg/ml) for 10 min. The protein lysates of these cells were separated by SDS–PAGE, and the blot was probed subsequently with an anti-phospho-Tyr mAb and an anti-β-actin mAb as a loading control. The blot is representative for two independent experiments.In parallel, degranulation of the NK cells was measured by anti-CD107a staining in flow cytometry. The difference between DMSO- and PP2-treated cells with respect to CD107a+ cells and the MFI of CD107a is indicated in the histograms. The results are representative for two independent experiments.
We generated two sets of tools to analyze the functional effects of the MICA-129Val/Met dimorphism. First, we stably transfected L cells, which like all mouse cells do not possess a MICA gene, with expression constructs encoding a MICA-129Met or MICA-129Val variant. The MICA-129Met variant was the MICA*00701 allele, which has a methionine at amino acid position 129. In the MICA-129Val construct, the amino acid position 129 was changed to valine. The resulting L-MICA-129Met and L-MICA-129Val cells, in contrast to vector-only transfected L-con cells, expressed MICA and bound a human NKG2D-Fc protein (Appendix Fig S1A). A broad range of MICA expression intensities was observed on different clones, but on average, these intensities were similar for both variants (Appendix Fig S1B). Analysis of the ratio of MICA expression and NKG2D binding revealed clearly a higher avidity of the MICA-129Met than MICA-129Val variant for NKG2D (Appendix Fig S1C and D) in accord with previous results (Steinle et al, 2001). Notably, binding of NKG2D to the MICA-129Met isoform was more dependent on the intensity of MICA expression on individual clones (coefficient of determination R2 = 0.62) than to the MICA-129Val isoform (R2 = 0.39). The slope (regression coefficient) of NKG2D binding with increasing MICA expression intensity was steeper for the MICA-129Met (0.23; Fig2A, left panel) than for the MICA-129Val variant (0.08; Fig2A, right panel).