Different composition of the human and the mouse gammadelta T cell receptor explains different phenotypes of CD3gamma and CD3delta immunodeficiencies

Abstract

The gammadelta T cell receptor for antigen (TCR) comprises the clonotypic TCRgammadelta, the CD3 (CD3gammaepsilon and/or CD3deltaepsilon), and the zetazeta dimers. gammadelta T cells do not develop in CD3gamma-deficient mice, whereas human patients lacking CD3gamma have abundant peripheral blood gammadelta T cells expressing high gammadelta TCR levels. In an attempt to identify the molecular basis for these discordant phenotypes, we determined the stoichiometries of mouse and human gammadelta TCRs using blue native polyacrylamide gel electrophoresis and anti-TCR-specific antibodies. The gammadelta TCR isolated in digitonin from primary and cultured human gammadelta T cells includes CD3delta, with a TCRgammadeltaCD3epsilon(2)deltagammazeta(2) stoichiometry. In CD3gamma-deficient patients, this may allow substitution of CD3gamma by the CD3delta chain and thereby support gammadelta T cell development. In contrast, the mouse gammadelta TCR does not incorporate CD3delta and has a TCRgammadeltaCD3epsilon(2)gamma(2)zeta(2) stoichiometry. CD3gamma-deficient mice exhibit a block in gammadelta T cell development. A human, but not a mouse, CD3delta transgene rescues gammadelta T cell development in mice lacking both mouse CD3delta and CD3gamma chains. This suggests important structural and/or functional differences between human and mouse CD3delta chains during gammadelta T cell development. Collectively, our results indicate that the different gammadelta T cell phenotypes between CD3gamma-deficient humans and mice can be explained by differences in their gammadelta TCR composition.

Figure 1.

CD3γ-deficient patients show abundant peripheral blood γδ T cells with high levels of γδ TCR. (A) Presence of γδ T cells in hCD3γ deficiency. Peripheral blood cell counts from four CD3γ-deficient individuals are plotted as a function of age in comparison with the normal distribution (dashed line). Three were homozygous for a p.K69X mutation (triangles), and one was compound heterozygous for p.M1V and p.N28V/H29X (squares). CD3δ-deficient patients (circles) are included for comparison. Filled symbols identify SCID patients, who died before 1 yr of age. (B) CD3 expression is higher on CD3γ-deficient γδ than αβ T cells. Flow cytometry histograms of anti-CD3 (SK7)–stained CD3γ-deficient T cells (dashed lines) are compared with healthy controls (continuous lines) either in αβ (CD8 and CD4; top and middle) or γδ (double negative; bottom) T cells. Numbers indicate TCR expression (mean fluorescence intensity) on cells from CD3γ-deficient patients as a percentage of that on cells from healthy donors. The vertical dashed line indicates the background fluorescence using an irrelevant antibody. (C) Quantification of the CD3 expression on the indicated cell types from CD3γ-deficient patients as a percentage of that on the same cell types from healthy donors (percentage of CD3 expression). Data are expressed as the percent mean fluorescence intensity ± SEM from three different patients using the anti-CD3 antibodies SK7 (left) or UCHT1 (right). Similar results were obtained using other anti-CD3 antibodies, as well as other gating criteria (not depicted). *, P < 0.05 compared with γδ T cells.

Figure 2.

The human γδ TCR includes CD3δ. (A) Human γδ T cell clones incorporate CD3δ into the γδ TCR. Anti-ζ immunopurified TCRs from Jurkat, αβ (αβB6 and αβPA), and γδ (γδ19 and γδ46) T cell clones were separated on nonreducing SDS-PAGE and analyzed via Western blotting using anti-CD3δ and anti-ζ antibodies. In the control (C), which was loaded on another gel, anti-ζ antibodies and protein G–coupled sepharose were incubated in lysis buffer alone. (B) αβ TCR– and γδ TCR–associated CD3δ chains are differentially glycosylated. Anti-ζ immunopurified TCRs from αβ (αβB6 and αβPA) and γδ (γδ19) T cell clones, as well as the Jurkat variant Jγ9δ2, were left untreated (−) or subject to N-glycosidase F treatment (+) and analyzed as in A. Glycosylated (CD3δ) and deglycosylated (dg-CD3δ) CD3δ chains are indicated by arrowheads. (C) The γδ TCR on primary human γδ T cells contains CD3δ. TCRs from Jurkat and human PBMCs were immunopurified using anti-TCRβ and anti-TCRγδ antibodies and subjected to deglycosylation and analysis as in B.

Figure 3.

The human γδ TCR has a stoichiometry of TCRγδCD3ε₂γδζ₂. (A) The digitonin-solubilized γδ TCR is the same size as the αβ TCR. TCRs from Jurkat, Peer, and the γδ T cell clones γδ19 and γδ46 were purified, separated by BN-PAGE, and analyzed via Western blotting using the anti-ζ antibody. (B) Digitonin-extracted TCRs from γδ T cell clone γδ19 were incubated with the indicated amounts of antibodies against hCD3ε (UCHT1), hCD3γ (HMT3.2), hCD3δ (APA1/2), ζ (G3), and hTCRγδ (5A6.E9), separated by BN-PAGE and analyzed as in A. The number of shifts correlates with the number of antibody binding sites in the TCR complex, as indicated by arrowheads. The marker protein is ferritin in its 24-meric (f1, 440 kD) and 48-meric (f2) forms.

Figure 4.

The mouse γδ TCR has a stoichiometry of TCRγδCD3ε₂γ₂ζ₂. (A) The γδ TCR on primary mouse γδ T cells does not contain CD3δ. TCRs from splenocytes from wild-type (Bl/6) and TCRβ^−/−γ1δ6tg mice were immunopurified with an anti-ζ antiserum. They were left untreated or were deglycosylated, separated via SDS-PAGE and analyzed by Western blotting as in Fig. 2 (B and C). (B) The mouse γδ TCR has two CD3εγ dimers. Splenocytes from wild-type (Bl/6) and TCRβ^−/−γ1δ6tg mice were lysed in digitonin, and purified TCRs were incubated with the indicated amounts of antibodies against CD3ε (145-2C11) or CD3γ (17A2), separated by BN-PAGE, and analyzed by Western blotting as in Fig. 3. Lanes 1, 4, 6, and 8 show TCRs alone. The number of shifts correlates with the number of antibody binding sites in the TCR complex, as indicated by arrowheads. The marker protein is ferritin in its 24-meric (f1, 440 kD) and 48-meric (f2) forms.

Figure 5.

hCD3δ can substitute both mCD3γ and mCD3δ in γδ T cell development. Thymocytes and splenocytes from wild-type (A), CD3γ^−/−(B), CD3γδ^−/−mCD3δtg (C), and CD3γδ^−/−hCD3δtg (D) mice were surface stained with anti-CD3 (145-2C11) and anti-TCRγδ (GL3) antibodies and analyzed by flow cytometry. Percentages of cells in the marked regions and the total number of γδ T cells (in millions) in the thymi are shown within and above the dot plots, respectively. (E) CD3 expression is higher on CD3γ-deficient γδ than αβ T cells. Flow cytometry histograms of anti-CD3 (2C11)–stained CD3γδ^−/−hCD3δtg T cells (dashed lines) are compared with wild-type mice (continuous lines) either in αβ (top) or γδ (bottom) T cells from the thymus (left) or spleen (right). (F) Quantification of the CD3 expression on αβ or γδ T cells from CD3γδ^−/−hCD3δtg mice as a percentage of that on the same cell types from wild-type mice (percentage of CD3 expression). The CD3 high population was used for the αβ TCR in thymocytes. Data are expressed as the percent mean fluorescence intensity ± SEM from two independent experiments. *, P < 0.05 compared with γδ T cells. mio, millions.