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Human CD3γ, but not CD3δ, haploinsufficiency differentially impairs γδ versus αβ surface TCR expression.

Muñoz-Ruiz M, Pérez-Flores V, Garcillán B, Guardo AC, Mazariegos MS, Takada H, Allende LM, Kilic SS, Sanal O, Roifman CM, López-Granados E, Recio MJ, Martínez-Naves E, Fernández-Malavé E, Regueiro JR - BMC Immunol. (2013)

Bottom Line: To test this hypothesis, we compared the surface TCR expression of primary αβ and γδ T cells from healthy donors carrying a single or leaky mutation in CD3G (γ+/-) or CD3D (δ+/-, δ+/leaky) with that of normal controls.Although the partial reduction in the intracellular availability of CD3γ or CD3δ proteins was comparable as a consequence of the mutations, surface TCR expression measured with anti-CD3ε antibodies was significantly more decreased in γδ than in αβ T lymphocytes in CD3γ+/- individuals, whereas CD3δ+/- and CD3δ+/leaky donors showed a similar decrease of surface TCR in both T cell lineages.A modified version of the prevailing TCR assembly model is proposed to accommodate these new data.

View Article: PubMed Central - HTML - PubMed

Affiliation: Inmunología, Facultad de Medicina, Universidad Complutense, Madrid, 28040, Spain.

ABSTRACT

Background: The T cell antigen receptors (TCR) of αβ and γδ T lymphocytes are believed to assemble in a similar fashion in humans. Firstly, αβ or γδ TCR chains incorporate a CD3δε dimer, then a CD3γε dimer and finally a ζζ homodimer, resulting in TCR complexes with the same CD3 dimer stoichiometry. Partial reduction in the expression of the highly homologous CD3γ and CD3δ proteins would thus be expected to have a similar impact in the assembly and surface expression of both TCR isotypes. To test this hypothesis, we compared the surface TCR expression of primary αβ and γδ T cells from healthy donors carrying a single or leaky mutation in CD3G (γ+/-) or CD3D (δ+/-, δ+/leaky) with that of normal controls.

Results: Although the partial reduction in the intracellular availability of CD3γ or CD3δ proteins was comparable as a consequence of the mutations, surface TCR expression measured with anti-CD3ε antibodies was significantly more decreased in γδ than in αβ T lymphocytes in CD3γ+/- individuals, whereas CD3δ+/- and CD3δ+/leaky donors showed a similar decrease of surface TCR in both T cell lineages. Therefore, surface γδ TCR expression was more dependent on available CD3γ than surface αβ TCR expression.

Conclusions: The results support the existence of differential structural constraints in the two human TCR isotypes regarding the incorporation of CD3γε and CD3δε dimers, as revealed by their discordant surface expression behaviour when confronted with reduced amounts of CD3γ, but not of the homologous CD3δ chain. A modified version of the prevailing TCR assembly model is proposed to accommodate these new data.

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Reduced surface αβ and γδ TCR expression in CD3γ+/−, CD3δ+/− and CD3δ+/leaky individuals using UCHT-1. αβ T cells were defined as CD8bright and CD4+ in 9 γ +/−, 4 δ+/−, 2 δ+/leaky and 7 +/+ donors. Similar results were obtained with other anti-CD3 mAb (SK7, S4.1, F101.01, data not shown). (A) CD3 MFI ratios (X100) ± SEM relative to controls are shown for the indicated T cell lineages and genotypes. Asterisks in bars indicate significant differences as compared with controls, other comparisons as indicated. p as in Figure 1A. (B) Representative peripheral blood CD3 reactivity patterns of γ+/−, δ+/− or δ+/leaky T cells (dashed lines) as compared with +/+ controls (solid lines). The vertical line in each panel indicates the upper limit of background fluorescence using isotype-matched irrelevant mAb. (C) Intracellular stainings of γ+/− and δ+/leaky T cells (dashed lines) as compared with +/+ controls (solid lines). Vertical lines as in Figure 2B. The numbers in each histogram indicate MFI ratios (x100) relative to controls. (D) Comparative titration of CD3 (UCHT1) binding (MFI) to γ+/− peripheral blood T cells (n = 1) versus +/+ controls (n = 2, mean ± SD). The percentage of bound cells was determined in parallel to establish the endpoint dilution (1:16.000). The arrow indicates the working dilution in all other experiments. Similar results were obtained using other antibodies (F101.01 or BMA031).
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Figure 2: Reduced surface αβ and γδ TCR expression in CD3γ+/−, CD3δ+/− and CD3δ+/leaky individuals using UCHT-1. αβ T cells were defined as CD8bright and CD4+ in 9 γ +/−, 4 δ+/−, 2 δ+/leaky and 7 +/+ donors. Similar results were obtained with other anti-CD3 mAb (SK7, S4.1, F101.01, data not shown). (A) CD3 MFI ratios (X100) ± SEM relative to controls are shown for the indicated T cell lineages and genotypes. Asterisks in bars indicate significant differences as compared with controls, other comparisons as indicated. p as in Figure 1A. (B) Representative peripheral blood CD3 reactivity patterns of γ+/−, δ+/− or δ+/leaky T cells (dashed lines) as compared with +/+ controls (solid lines). The vertical line in each panel indicates the upper limit of background fluorescence using isotype-matched irrelevant mAb. (C) Intracellular stainings of γ+/− and δ+/leaky T cells (dashed lines) as compared with +/+ controls (solid lines). Vertical lines as in Figure 2B. The numbers in each histogram indicate MFI ratios (x100) relative to controls. (D) Comparative titration of CD3 (UCHT1) binding (MFI) to γ+/− peripheral blood T cells (n = 1) versus +/+ controls (n = 2, mean ± SD). The percentage of bound cells was determined in parallel to establish the endpoint dilution (1:16.000). The arrow indicates the working dilution in all other experiments. Similar results were obtained using other antibodies (F101.01 or BMA031).

Mentions: We have previously observed in γ−/− individuals that CD3 expression levels are overestimated when T cells are defined using antibodies against TCR-associated epitopes [7], such as BMA031 (for TCRαβ) or Immu510 (for TCRγδ). To avoid a similar bias in haploinsufficient donors, TCR-independent electronic gates were first defined in order to identify αβ or γδ T cell subsets (Figure 1B). The results indicated that CD3+ cells within CD4+ or CD8bright lymphocytes were >98% αβ T cells, whereas CD3+ double negative (DN) lymphocytes were 78 ± 6% γδ T cells. Accordingly, αβ and γδ T cells were gated as CD4+/CD8bright and DN cells, respectively, for further analyses. Using several CD3-specific antibodies, analysis of surface TCR expression consistently showed reduced antibody binding in γ+/− and δ+/− T lymphocytes as compared to normal controls (50-90% as judged by their relative mean fluorescence intensity, Figure 2A, B). These results were confirmed in family members of two newly reported patients with a leaky mutation in CD3D (termed δ+/leaky) [8]. Consistent with their relatively higher CD3δ content as compared to δ+/− donors, δ+/leaky donors showed a milder, but nevertheless clear decrease in surface TCR expression (Figure 2A, B). In order to establish if these results were associated with reduced availability of each CD3 chain, we measured intracellular CD3γ (iCD3γ) or CD3δ (iCD3δ) by flow cytometry in haploinsufficient γ+/− and δ+/leaky donors. The results showed that this was indeed the case (Figure 2C), confirming previous reports of decreased CD3γ protein in haploinsufficient donors [3].


Human CD3γ, but not CD3δ, haploinsufficiency differentially impairs γδ versus αβ surface TCR expression.

Muñoz-Ruiz M, Pérez-Flores V, Garcillán B, Guardo AC, Mazariegos MS, Takada H, Allende LM, Kilic SS, Sanal O, Roifman CM, López-Granados E, Recio MJ, Martínez-Naves E, Fernández-Malavé E, Regueiro JR - BMC Immunol. (2013)

Reduced surface αβ and γδ TCR expression in CD3γ+/−, CD3δ+/− and CD3δ+/leaky individuals using UCHT-1. αβ T cells were defined as CD8bright and CD4+ in 9 γ +/−, 4 δ+/−, 2 δ+/leaky and 7 +/+ donors. Similar results were obtained with other anti-CD3 mAb (SK7, S4.1, F101.01, data not shown). (A) CD3 MFI ratios (X100) ± SEM relative to controls are shown for the indicated T cell lineages and genotypes. Asterisks in bars indicate significant differences as compared with controls, other comparisons as indicated. p as in Figure 1A. (B) Representative peripheral blood CD3 reactivity patterns of γ+/−, δ+/− or δ+/leaky T cells (dashed lines) as compared with +/+ controls (solid lines). The vertical line in each panel indicates the upper limit of background fluorescence using isotype-matched irrelevant mAb. (C) Intracellular stainings of γ+/− and δ+/leaky T cells (dashed lines) as compared with +/+ controls (solid lines). Vertical lines as in Figure 2B. The numbers in each histogram indicate MFI ratios (x100) relative to controls. (D) Comparative titration of CD3 (UCHT1) binding (MFI) to γ+/− peripheral blood T cells (n = 1) versus +/+ controls (n = 2, mean ± SD). The percentage of bound cells was determined in parallel to establish the endpoint dilution (1:16.000). The arrow indicates the working dilution in all other experiments. Similar results were obtained using other antibodies (F101.01 or BMA031).
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Figure 2: Reduced surface αβ and γδ TCR expression in CD3γ+/−, CD3δ+/− and CD3δ+/leaky individuals using UCHT-1. αβ T cells were defined as CD8bright and CD4+ in 9 γ +/−, 4 δ+/−, 2 δ+/leaky and 7 +/+ donors. Similar results were obtained with other anti-CD3 mAb (SK7, S4.1, F101.01, data not shown). (A) CD3 MFI ratios (X100) ± SEM relative to controls are shown for the indicated T cell lineages and genotypes. Asterisks in bars indicate significant differences as compared with controls, other comparisons as indicated. p as in Figure 1A. (B) Representative peripheral blood CD3 reactivity patterns of γ+/−, δ+/− or δ+/leaky T cells (dashed lines) as compared with +/+ controls (solid lines). The vertical line in each panel indicates the upper limit of background fluorescence using isotype-matched irrelevant mAb. (C) Intracellular stainings of γ+/− and δ+/leaky T cells (dashed lines) as compared with +/+ controls (solid lines). Vertical lines as in Figure 2B. The numbers in each histogram indicate MFI ratios (x100) relative to controls. (D) Comparative titration of CD3 (UCHT1) binding (MFI) to γ+/− peripheral blood T cells (n = 1) versus +/+ controls (n = 2, mean ± SD). The percentage of bound cells was determined in parallel to establish the endpoint dilution (1:16.000). The arrow indicates the working dilution in all other experiments. Similar results were obtained using other antibodies (F101.01 or BMA031).
Mentions: We have previously observed in γ−/− individuals that CD3 expression levels are overestimated when T cells are defined using antibodies against TCR-associated epitopes [7], such as BMA031 (for TCRαβ) or Immu510 (for TCRγδ). To avoid a similar bias in haploinsufficient donors, TCR-independent electronic gates were first defined in order to identify αβ or γδ T cell subsets (Figure 1B). The results indicated that CD3+ cells within CD4+ or CD8bright lymphocytes were >98% αβ T cells, whereas CD3+ double negative (DN) lymphocytes were 78 ± 6% γδ T cells. Accordingly, αβ and γδ T cells were gated as CD4+/CD8bright and DN cells, respectively, for further analyses. Using several CD3-specific antibodies, analysis of surface TCR expression consistently showed reduced antibody binding in γ+/− and δ+/− T lymphocytes as compared to normal controls (50-90% as judged by their relative mean fluorescence intensity, Figure 2A, B). These results were confirmed in family members of two newly reported patients with a leaky mutation in CD3D (termed δ+/leaky) [8]. Consistent with their relatively higher CD3δ content as compared to δ+/− donors, δ+/leaky donors showed a milder, but nevertheless clear decrease in surface TCR expression (Figure 2A, B). In order to establish if these results were associated with reduced availability of each CD3 chain, we measured intracellular CD3γ (iCD3γ) or CD3δ (iCD3δ) by flow cytometry in haploinsufficient γ+/− and δ+/leaky donors. The results showed that this was indeed the case (Figure 2C), confirming previous reports of decreased CD3γ protein in haploinsufficient donors [3].

Bottom Line: To test this hypothesis, we compared the surface TCR expression of primary αβ and γδ T cells from healthy donors carrying a single or leaky mutation in CD3G (γ+/-) or CD3D (δ+/-, δ+/leaky) with that of normal controls.Although the partial reduction in the intracellular availability of CD3γ or CD3δ proteins was comparable as a consequence of the mutations, surface TCR expression measured with anti-CD3ε antibodies was significantly more decreased in γδ than in αβ T lymphocytes in CD3γ+/- individuals, whereas CD3δ+/- and CD3δ+/leaky donors showed a similar decrease of surface TCR in both T cell lineages.A modified version of the prevailing TCR assembly model is proposed to accommodate these new data.

View Article: PubMed Central - HTML - PubMed

Affiliation: Inmunología, Facultad de Medicina, Universidad Complutense, Madrid, 28040, Spain.

ABSTRACT

Background: The T cell antigen receptors (TCR) of αβ and γδ T lymphocytes are believed to assemble in a similar fashion in humans. Firstly, αβ or γδ TCR chains incorporate a CD3δε dimer, then a CD3γε dimer and finally a ζζ homodimer, resulting in TCR complexes with the same CD3 dimer stoichiometry. Partial reduction in the expression of the highly homologous CD3γ and CD3δ proteins would thus be expected to have a similar impact in the assembly and surface expression of both TCR isotypes. To test this hypothesis, we compared the surface TCR expression of primary αβ and γδ T cells from healthy donors carrying a single or leaky mutation in CD3G (γ+/-) or CD3D (δ+/-, δ+/leaky) with that of normal controls.

Results: Although the partial reduction in the intracellular availability of CD3γ or CD3δ proteins was comparable as a consequence of the mutations, surface TCR expression measured with anti-CD3ε antibodies was significantly more decreased in γδ than in αβ T lymphocytes in CD3γ+/- individuals, whereas CD3δ+/- and CD3δ+/leaky donors showed a similar decrease of surface TCR in both T cell lineages. Therefore, surface γδ TCR expression was more dependent on available CD3γ than surface αβ TCR expression.

Conclusions: The results support the existence of differential structural constraints in the two human TCR isotypes regarding the incorporation of CD3γε and CD3δε dimers, as revealed by their discordant surface expression behaviour when confronted with reduced amounts of CD3γ, but not of the homologous CD3δ chain. A modified version of the prevailing TCR assembly model is proposed to accommodate these new data.

Show MeSH