The role of the different CD3γ domains in TCR expression and signaling.

The CD3 subunits of the T-cell antigen receptor (TCR) play a central role in regulation of surface TCR expression levels. Humans who lack CD3γ (γ-) show reduced surface TCR expression levels and abolished phorbol ester (PMA)-induced TCR down-regulation. The response to PMA is mediated by a double leucine motif in the intracellular (IC) domain of CD3γ. However, the molecular cause of the reduced TCR surface expression in γ- lymphocytes is still not known. We used retroviral vectors carrying wild type CD3γ or CD3δ or the following chimeras (EC-extracellular, TM-transmembrane and IC): δECγTMγIC (δγγ for short), γγδ, γδδ and γγ-. Expression of γγγ, γγδ, γδδ or γγ- in the γ- T cell line JGN, which lacks surface TCR, demonstrated that cell surface TCR levels in JGN were dependent on the EC domain of CD3γ and could not be replaced by the one of CD3δ. In JGN and primary γ- patient T cells, the tested chimeras confirmed that the response to PMA maps to the IC domain of CD3γ. Since protein homology explains these results better than domain structure, we conclude that CD3γ contributes conformational cues that improve surface TCR expression, likely at the assembly or membrane transport steps. In JGN cells all chimeric TCRs were signalling competent. However, an IC domain at CD3γ was required for TCR-induced IL-2 and TNF-α production and CD69 expression, indicating that a TCR without a CD3γ IC domain has altered signalling capabilities.

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

BACKGROUND: The T cell antigen receptors (TCR) of αß and γδ T lymphocytes are believed to assemble in a similar fashion in humans. Firstly, αß 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 αß and γδ T cells from healthy donors carrying a single null 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 αß 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 αß 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.

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

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.

Differential biological role of CD3 chains revealed by human immunodeficiencies.

The biological role in vivo of the homologous CD3gamma and delta invariant chains within the human TCR/CD3 complex is a matter of debate, as murine models do not recapitulate human immunodeficiencies. We have characterized, in a Turkish family, two new patients with complete CD3gamma deficiency and SCID symptoms and compared them with three CD3gamma-deficient individuals belonging to two families from Turkey and Spain. All tested patients shared similar immunological features such as a partial TCR/CD3 expression defect, mild alphabeta and gammadelta T lymphocytopenia, poor in vitro proliferative responses to Ags and mitogens at diagnosis, and very low TCR rearrangement excision circles and CD45RA(+) alphabeta T cells. However, intrafamilial and interfamilial clinical variability was observed in patients carrying the same CD3G mutations. Two reached the second or third decade in healthy conditions, whereas the other three showed lethal SCID features with enteropathy early in life. In contrast, all reported human complete CD3delta (or CD3epsilon) deficiencies are in infants with life-threatening SCID and very severe alphabeta and gammadelta T lymphocytopenia. Thus, the peripheral T lymphocyte pool was comparatively well preserved in human CD3gamma deficiencies despite poor thymus output or clinical outcome. We propose a CD3delta >> CD3gamma hierarchy for the relative impact of their absence on the signaling for T cell production in humans.