We have reported the existence of biochemical and conformational differences in the ␣␤ T cell receptor (TCR) complex between CD4 ؉ and CD8 ؉ CD3␥-deficient (␥ ؊ ) mature T cells. In the present study, we have furthered our understanding and extended the observations to primary T lymphocytes from normal (␥ ؉ ) individuals. Surface TCR⅐CD3 components from CD4 ؉ ␥ ؊ T cells, other than CD3␥, were detectable and similar in size to CD4 ؉ ␥ ؉ controls. Their native TCR⅐CD3 complex was also similar to CD4 ؉ ␥ ؉ controls, except for an ␣␤(␦⑀) 2 2 instead of an ␣␤␥⑀␦⑀ 2 stoichiometry. In contrast, the surface TCR␣, TCR␤, and CD3␦ chains of CD8 ؉ ␥ ؊ T cells did not possess their usual sizes. Using confocal immunofluorescence, TCR␣ was hardly detectable in CD8 ؉ ␥ ؊ T cells. Blue native gels (BN-PAGE) demonstrated the existence of a heterogeneous population of TCR⅐CD3 in these cells. Using primary peripheral blood T lymphocytes from normal (␥ ؉ ) donors, we performed a broad epitopic scan. In contrast to all other TCR⅐CD3-specific monoclonal antibodies, RW2-8C8 stained CD8 ؉ better than it did CD4 ؉ T cells, and the difference was dependent on glycosylation of the TCR⅐CD3 complex but independent of T cell activation or differentiation. RW2-8C8 staining of CD8 ؉ T cells was shown to be more dependent on lipid raft integrity than that of CD4 ؉ T cells. Finally, immunoprecipitation studies on purified primary CD4 ؉ and CD8 ؉ T cells revealed the existence of TCR glycosylation differences between the two. Collectively, these results are consistent with the existence of conformational or topological lineage-specific differences in the TCR⅐CD3 from CD4 ؉ and CD8 ؉ wild type T cells. The differences may be relevant for cis interactions during antigen recognition and signal transduction.␣␤ T lymphocytes recognize peptide-major histocompatibility complex ligands by means of a multimeric protein complex termed the ␣␤ T cell receptor (TCR) 1 CD3 complex (TCR⅐CD3). This structure is composed of a variable ␣␤ TCR dimer that binds antigens and three invariant dimers (CD3␥⑀, ␦⑀, and ) that are in charge of TCR⅐CD3 complex transport, stabilization, and signal transduction (1). The minimum stoichiometry, therefore, is believed to be ␣␤␥⑀␦⑀ 2 .Mature CD4 ϩ and CD8 ϩ ␣␤ T cells differ sharply in their major histocompatibility complex ligands, but their TCR⅐CD3 complex is believed to be qualitatively identical. The reduced ␣␤ TCR⅐CD3 staining levels observed in CD8 ϩ T cells, relative to CD4 ϩ T cells, were therefore reported as quantitative under this assumption (2). Unexpectedly, peripheral blood ␣␤ TCR⅐CD3 expression was shown to be more impaired in CD8 ϩ than in CD4 ϩ cells when CD3␥ (3, 4) or CD3␦ (5) was absent. These observations were followed by the description of conformational and biochemical differences in the TCR⅐CD3 complex between CD8 ϩ and CD4 ϩ CD3␥ deficient (␥ Ϫ ) T lymphocytes (6). Biosynthetic studies showed that CD8 ϩ but not CD4 ϩ ␥ Ϫ T cells lacked normal TCR␣. Instead, the CD4 ϩ ␥ Ϫ T cells contained a small ␣␤ hetero...

Mature CD4؉ and CD8 ؉ T lymphocytes are believed to build and express essentially identical surface ␣␤ T-cell receptor-CD3 (TCR⅐CD3) complexes. However, TCR⅐CD3 expression has been shown to be more impaired in CD8 ؉ cells than in CD4؉ cells when CD3␥ is absent in humans or mice. We have addressed this paradox by performing a detailed phenotypical and biochemical analysis of the TCR⅐CD3 complex in human CD3␥-deficient CD8؉ and CD4 ؉ T cells. The results indicated that the membrane TCR⅐CD3 complex of CD8 ؉ T lymphocytes was conformationally different from that of CD4 ؉ lymphocytes in the absence of CD3␥. In addition, CD8؉ , but not CD4 ؉ , CD3␥-deficient T lymphocytes were shown to contain abnormally glycosylated TCR␤ proteins, together with a smaller, abnormal TCR chain (probably incompletely processed TCR␣). These results suggest the existence of hitherto unrecognized biochemical differences between mature CD4؉ and CD8 ؉ T lymphocytes in the intracellular control of ␣␤TCR⅐CD3 assembly, maturation, or transport that are revealed when CD3␥ is absent. Such lineage-specific differences may be important in receptor-coreceptor interactions during antigen recognition.Mature ␣␤ T lymphocytes recognize pathogen-derived peptides on antigen-presenting cells by means of the multimeric membrane protein ensemble termed the T-cell receptor (TCR) 1 ⅐CD3 complex. This TCR⅐CD3 complex includes two clonally distributed variable chains that directly interact with antigens (TCR␣ and TCR␤) and four invariant polypeptides that regulate assembly and signal transduction (CD3␥, CD3␦, CD3⑀, and ) (1). The assembly of complete TCR⅐CD3⅐ complexes takes place in a highly ordered manner within the endoplasmic reticulum: first CD3 chains, then TCR chains, and finally chains. Further conformational maturation, including carbohydrate processing, occurs in the Golgi apparatus before exportation of mature complexes to the T cell surface. The biochemical machinery involved in the assembly, processing, and exportation of TCR⅐CD3 complexes is assumed to be shared by all ␣␤ T-lineage cells. Thus, CD4ϩ and CD8 ϩ are believed to build biochemically and conformationally identical antigen receptors, although differences in the numbers that reach or remain at the cell surface have been noted (2). Therefore, the lack of any CD3 chain would be expected to affect to a similar extent the assembly and exportation of TCR⅐CD3 complexes by mature CD4 ϩ and CD8 ϩ T cells. However, this was not the case in several murine and human CD3 deficiencies (reviewed in Ref.3). In particular, it has been consistently shown that in the absence of CD3␥ or CD3␦, TCR⅐CD3 expression (or conformation) is more impaired in mature peripheral CD8 ϩ cells than in their CD4 ϩ counterparts, both in human and in murine deficiencies (3-7). Three other observations suggested the existence of CD8 ϩ cell-specific defects in human CD3␥ deficiency: first, the proband died after a viral infection (a cytolytic T-celldependent function) despite normal antibody responses (helper T-cell-dependent) (8...

The contribution of CD3γ to the surface expression, internalization, and intracellular trafficking of the TCR/CD3 complex (TCR) has not been completely defined. However, CD3γ is believed to be crucial for constitutive as well as for phorbol ester-induced internalization. We have explored TCR dynamics in resting and stimulated mature T lymphocytes derived from two unrelated human congenital CD3γ-deficient (γ−) individuals. In contrast to γ− mutants of the human T cell line Jurkat, which were selected for their lack of membrane TCR and are therefore constitutively surface TCR negative, these natural γ− T cells constitutively expressed surface TCR, mainly through biosynthesis of new chains other than CD3γ. However, surface (but not intracellular) TCR expression in these cells was less than wild-type cells, and normal surface expression was clearly CD3γ-dependent, as it was restored by retroviral transduction of CD3γ. The reduced surface TCR expression was likely caused by an impaired assembly or membrane transport step during recycling, whereas constitutive internalization and degradation were apparently normal. Ab binding to the mutant TCR, but not phorbol ester treatment, caused its down-modulation from the cell surface, albeit at a slower rate than in normal controls. Kinetic confocal analysis indicated that early ligand-induced endocytosis was impaired. After its complete down-modulation, TCR re-expression was also delayed. The results suggest that CD3γ contributes to, but is not absolutely required for, the regulation of TCR trafficking in resting and Ag-stimulated mature T lymphocytes. The results also indicate that TCR internalization is regulated differently in each case.

CD3 proteins may have redundant as well as specific contributions to the intracellular propagation and final effector responses of TCR-mediated signals at different checkpoints during T cell differentiation. We report here on the participation of CD3 gamma in the activation and effector function of human mature T lymphocytes at the antigen recognition checkpoint. Following TCR-CD3 engagement of human CD3 gamma-deficient T cell lines, and despite their lower TCR-CD3 surface levels compared to normal controls, mature T cell responses such as protein tyrosine phosphorylation and the regulation of expression of several cell surface molecules, including the TCR-CD3 itself, were either normal or only slightly affected. In contrast, other physiological responses like the specific adhesion and concomitant cell polarization on ICAM-1-coated dishes were selectively defective, and activation-induced cell death was increased. Our data indicate that CD3 gamma contributes essential specialized signaling functions to certain mature T cell responses. Failure to generate appropriate interactions may abort cytoskeleton reorganization and initiate an apoptotic response.

Major histocompatibility complex class II (MHCII) deficiency is a primary immunodeficiency resulting from defects in one of four different MHCII-specific transcription factors-CIITA, RFX5, RFXAP, and RFXANK. Despite this genetic heterogeneity, the phenotypical manifestations are homogeneous. It is frequently difficult to establish a definitive diagnosis of the disease on the basis of clinical and immunological criteria. Moreover, the phenotypical homogeneity precludes unambiguous identification of the regulatory gene that is affected. Identification of the four genes mutated in the disease has now allowed us to develop a rapid and straightforward diagnostic test for new MHCII-deficiency patients. This test is based on direct correction of the genetic defect by transduction of cells from patients with lentiviral vectors encoding CIITA, RFXANK, RFX5, or RFXAP. We have validated this approach by defining the molecular defects in two new patients. The RFXANK vector restored MHCII expression in a T cell line from one patient. The RFXAP vector corrected primary cells (PBL) from a second patient. Molecular analysis confirmed the presence of homozygous mutations in the RFXANK and RFXAP genes, respectively. Direct genetic correction represents a valuable tool for the diagnosis and classification of new MHCII-deficiency patients.