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Published on: May 6, 2019
Nonsense CD247 mutations show dominant-negative features in T-cell receptor expression and function
Alejandro C Briones1, Rebeca F Megino1, Ana V Marin1
1Department of Immunology, Ophthalmology and ENT, Complutense University School of Medicine and 12 de Octubre Health Research Institute (imas12), Madrid, Spain.
Background:
The invariant TCR ζ/CD247 homodimer is crucial for TCR/CD3 expression and signaling through its 3 immunoreceptor tyrosine-based activation motifs (ITAMs). Homozygous null mutations in CD247 lead to immunodeficiency, while carriers exhibit 50% reduced surface CD3. It is unclear whether carriers of other CD247 variants show dominant-negative effects.
Objective:
We sought to analyze and model the potential impact on T-cell receptor (TCR) expression and function of heterozygous nonsense CD247 mutations found in patients with signs of immunodeficiency or autoimmunity.
Methods:
Jurkat T cells, either wild-type (WT) or CRISPR/Cas9-edited CD247-deficient (ZKO), were lentivirally transduced with WT CD247 or mutations ablating 1 (Q142X), 2 (Q101X), or 3 (Q70X) ITAMs.
Results:
Three patients from unrelated families were studied. Two heterozygous nonsense CD247 mutations were identified (p.Y152X and p.Q101X), which affected ITAM-3 and ITAM-2 and ITAM-3, respectively. Both mutations were associated with low surface CD3 expression and normal intracellular CD247 levels using a transmembrane-specific antibody, but very low intracellular CD247 levels using an ITAM-3-specific one, suggesting the presence of truncated variants in T cells. Transduction of the mutations lacking 1, 2, or 3 ITAMs into ZKO cells could not restore normal surface CD3 expression (only 60%, 22%, and 10%, respectively), whereas in WT cells, normal surface CD3 expression was reduced (to 39%, 19%, and 9% of normal levels), and both effects were dependent on ITAM number. All 6 transfectants showed reduced CD69 induction (25% to 50%), indicating that they were unable to signal downstream properly, neither isolated nor associated with WT CD247.
Conclusions:
Our results suggest that CD247 variants lacking ITAMs due to nonsense, but not null, mutations are defective for normal TCR assembly and exert a dominant-negative effect on TCR expression and signaling in vitro. This, in turn, may correlate with clinical features in vivo.
Insights
Heterozygous nonsense mutations in CD247, impacting ITAMs, impair T-cell receptor (TCR) assembly and signaling. These variants may exert dominant-negative effects on TCR expression, potentially linking to immunodeficiency.
Area of Science:
- Immunology
- Molecular Biology
Background:
- The invariant TCR ζ/CD247 homodimer is essential for TCR/CD3 expression and signaling via its 3 ITAMs.
- CD247 mutations can cause immunodeficiency, but the impact of other CD247 variants on TCR function is unclear.
Purpose of the Study:
- To analyze and model the effects of heterozygous nonsense CD247 mutations on T-cell receptor (TCR) expression and function.
- Investigate potential dominant-negative effects in patients with immunodeficiency or autoimmunity.
Main Methods:
- Jurkat T cells (WT and CD247-deficient ZKO) were engineered using CRISPR/Cas9.
- Cells were transduced with WT CD247 or variants with mutated ITAMs (Q142X, Q101X, Q70X).
Main Results:
- Two heterozygous nonsense CD247 mutations (p.Y152X, p.Q101X) were identified in patients, affecting ITAM-2 and/or ITAM-3.
- Mutated CD247 variants reduced surface CD3 expression and impaired T-cell signaling (CD69 induction).
- Expression of CD247 variants lacking ITAMs in ZKO cells failed to restore normal CD3 levels; in WT cells, it reduced CD3 expression.
Conclusions:
- CD247 variants with absent ITAMs, resulting from nonsense mutations, are defective in TCR assembly.
- These variants exert a dominant-negative effect on TCR expression and signaling in vitro.
- This in vitro effect may correlate with observed clinical symptoms in patients.
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