Expression of a nonpolymorphic MHC class I-like molecule, CD1D, by human intestinal epithelial cells.

The human CD1 locus encodes three nonpolymorphic MHC class I-like cell surface glycoproteins, CD1a-c, which are expressed primarily by immature thymocytes. A mAb and antipeptide antiserum were utilized to determine the tissue distribution of a fourth CD1 molecule, CD1d. Within the lymphoid lineage, CD1d was expressed on B cells but not on thymocytes. Immunoperoxidase staining of fresh frozen intestinal tissues demonstrated that the majority of intestinal epithelial cells, with the exception of cells at the base of some crypts, expressed CD1d. The CD1d staining was observed in the cytoplasm and along the basolateral membranes of the epithelial cells. The intestinal epithelial cell expression of CD1d was confirmed by immunoblotting with a CD1d antipeptide antiserum. Further immunoperoxidase studies indicated that CD1d, unlike murine CD1, was also expressed by nonlymphoid tissues outside of the gastrointestinal tract. The expression of CD1d outside the lymphoid and myeloid lineages clearly distinguishes this molecule from CD1a-c and suggests that it may serve a distinct function. The prominent expression of CD1d by intestinal epithelial cells suggests that this molecule may be an important ligand for T lymphocytes within the gut-associated lymphoid tissue.

Oligoclonal expansion and CD1 recognition by human intestinal intraepithelial lymphocytes.

A human intestinal intraepithelial lymphocyte (IEL) T cell line was established from jejunum to characterize the structure and function of the alpha beta T cell antigen receptors (TCRs) expressed by this population. Single-sided polymerase chain reaction (PCR) amplification cloning and quantitative PCR amplification of the TCR chains from the cell line and from fresh IELs demonstrated that IELs were oligoclonal. The IEL T cell line exhibited CD1-specific cytotoxicity and a dominant IEL T cell clone was CD1c-specific. Thus, human jejunal intraepithelial lymphocytes are oligoclonal and recognize members of the CD1 gene family.

Genetic reconstitution of the T cell receptor (TcR) alpha/beta heterodimer restores the association of CD3 zeta 2 with the TcR/CD3 complex.

The cell surface expression of the T cell receptor (TcR)/CD3 complex and, consequently, the functional competence of the cell is partly dependent on CD3 zeta. In its absence, a pentameric complex (TcR alpha/beta/CD3 gamma delta epsilon) is formed which is inefficiently transported to the cell surface. Reconstitution of CD3 zeta by transfection, in turn, restores the cell surface expression and function of the complex. Through the use of transfection experiments, we here provide direct evidence that the association of CD3 zeta 2 with the TcR/CD3 complex is dependent on the presence of both the TcR alpha and beta polypeptide chains. Despite wild-type levels of the CD3 zeta protein in a TcR alpha-negative mutant human T cell line, a complex was formed intracellularly which lacked CD3 zeta 2 and consisted of beta gamma delta epsilon and beta 2 gamma delta epsilon. Upon transfection of the mutant with a TcR alpha cDNA, a TcR/CD3 complex which contained CD3 zeta 2 was observed intracellularly. In contrast to the partial subcomplex on the cell surface of the untransfected cell line, the TcR/CD3 complex on the transfectant was functional as demonstrated by its ability to mobilize intracellular calcium after stimulation with a mitogenic CD3 epsilon-specific monoclonal antibody. Transient transfection studies performed in COS cell fibroblasts indicated that CD3 zeta 2 was not interacting with the TcR alpha protein alone, implying that a conformation provided by either the TcR alpha/beta heterodimer or the TcR alpha/beta/CD3 gamma delta epsilon complex was necessary for the association of CD3 zeta 2. Transfection studies performed in a TcR alpha/beta-negative murine T-T hybridoma confirmed the requirement of both the TcR alpha and beta proteins in CD3 zeta 2 binding. We conclude that the TcR alpha and beta chains harbor polypeptide sequences essential for the association of CD3 zeta 2 with the TcR/CD3 complex.

Assembly and function of the T cell antigen receptor. Requirement of either the lysine or arginine residues in the transmembrane region of the alpha chain.

The T cell receptor (TCR) for antigen consists, on the majority of peripheral lymphocytes, of an immunoglobulin-like, disulfide-linked heterodimeric glycoprotein: the alpha and beta chain. These proteins are noncovalently linked to at least four nonvariant proteins which comprise the CD3 complex: CD3 gamma, delta, epsilon, and zeta. Whereas the TCR alpha and beta proteins have positively charged residues in the transmembrane region, all the CD3 proteins have similarly placed negatively charged amino acid residues. It has been suggested that these basic and acidic amino acid residues may play an important role in TCR.CD3 complex assembly and/or function. In this paper, the structural and functional role of the lysine and arginine residues of the TCR alpha chain was addressed using oligonucleotide mediated site directed mutagenesis. The Arg256 and Lys261 residues of the TCR alpha cDNA of the HPB-ALL cell line were mutated to either Gly256 and/or Ile261. The altered cDNAs were transfected into a TCR alpha negative recipient mutant cell line of REX, clone 20A. Metabolic labeling of the T cell transfectants showed that mutation of either the Arg256 or Lys261 amino acid residues had no effect on the ability of the TCR alpha chain to form either a heterodimer with the TCR beta chain or a complex with the CD3 gamma, delta, and epsilon proteins. Consequently, the Arg256 to Gly256 and Lys261 to Ile261 mutations did not prevent the formation of a mature, functional TCR.CD3 complex on the cell surface as determined by immunofluorescence, cell surface radioiodination, and the ability of the transfectants to mobilize intracellular calcium after stimulation with a mitogenic anti-CD3 epsilon monoclonal antibody. In contrast, a mutant cDNA in which both the Arg256 and Lys261 residues were mutated to Gly256 and Ile261, respectively, failed to reconstitute the cell surface expression of the TCR.CD3 complex and, consequently, the ability to respond to mitogenic stimuli. In the absence of both the Arg256 and Lys261 residues, TCR alpha beta heterodimer formation was not observed. Cotransfection studies in COS cells showed that the failure of assembly of a heterodimer was likely due to an inability of the mutated TCR alpha chain to form a subcomplex with either the CD3 gamma, delta, epsilon, or zeta proteins.(ABSTRACT TRUNCATED AT 400 WORDS)