Conformational and Biochemical Differences in the TCR·CD3 Complex of CD8+ Versus CD4+ Mature Lymphocytes Revealed in the Absence of CD3γ

Abstract

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...

Biochemical Differences in the αβ T Cell Receptor·CD3 Surface Complex between CD8+ and CD4+ Human Mature T Lymphocytes

“…5 and 7, respectively), which were regulated by activation and differentiation for certain epitopes, likely due to lipid raft-dependent arrangements or glycosylation. These results confirm our previous biosynthetic and phenotypic studies in CD3␥-deficient T cells (6) and extend the observations to primary T lymphocytes from normal individuals. Biosynthetic studies showed that CD8 ϩ but not CD4 ϩ CD3␥-deficient T cells contained a small ␣␤ heterodimer composed of abnormally glycosylated TCR␤ and an abnormally small CD3-associated chain that was not recognized by TCR␣-specific antibodies.…”

“…However, as shown above and in Ref. 6, the analysis of ␥ Ϫ TCR⅐CD3 complexes suggested the existence of hitherto unrecognized structural differences between both cell lineages. These results prompted us to search for qualitative ␣␤ TCR⅐CD3 complex differences in normal CD4 ϩ and CD8 ϩ primary T lymphocytes.…”

“…1A, right). This finding correlated with complete endo-␤-N-acetylglucosaminidase H resistance (6). Therefore, we next analyzed the CD3␦ from CD4 ϩ ␥ Ϫ cells.…”

“…Endo-␤-N-acetylglucosaminidase H and N-glycosidase F (Roche Applied Science) treatments were done as described (6,7). Finally, samples were resuspended in Laemmli sample buffer and boiled for 5 min before a short spin at 12,000 ϫ g. SDS-PAGE was performed on 10% polyacrylamide gels and analyzed by autoradiography.…”

“…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␣.…”