InVivoMAb anti-human/monkey CD5

Lymphoid tissue in formalin fixed and snap frozen human fetal ileum has been studied using immunohistochemistry. At 11 weeks gestation clusters of cells expressing CD4 (leu-3a positive) are present in fetal ileum but these do not express CD3 (UCHT1 negative) and are probably macrophages. Aggregates of lymphoid tissue are apparent from 14 weeks gestation which contain T cells of helper/inducer and suppressor/cytotoxic phenotype. Both B and T cells are present at 16 weeks but with no cellular zonation. By 19 weeks, distinct follicles of B cells are present surrounded by T cells of helper/inducer and suppressor/cytotoxic phenotype. Follicular dendritic cells are also present within the B cell areas. The B cells at this age express surface IgM and IgD, C3b- and C3d-receptors. They also express the antigen CD5 which has been shown by others to be present on some fetal B cells but which is almost exclusively associated with T cells in the adult. HLA-D region antigens are present on apparently all of the cells within the fetal lymphoid follicles. The antigen on activated B cells, CD23 (recognized by MHM6), was present on some cells scattered within the B cell follicle. This is indicative of antigen independent B cell proliferation.

Recognition by scavenger receptor cysteine-rich domains on membrane proteins regulates innate and adaptive immune responses. Two receptors expressed primarily on T cells, CD5 and CD6, are linked genetically and are structurally similar, both containing three scavenger receptor cysteine-rich domains in their extracellular regions. A specific cell surface interaction for CD5 has been difficult to define at the molecular level because of the susceptibility of CD5 protein to denaturation. By using soluble CD5 purified at neutral pH to preserve biological activity, we show that CD5 mediates species-specific homophilic interactions. CD5 domain 1 only is involved in the interaction. CD5 mAbs that have functional effects in humans, rats, and mice block homophilic binding. Ag-specific responses by mouse T cells in vitro were increased when engagement of human CD5 domain 1 was inhibited by mutation or by IgG or Fab fragment from a CD5 mAb. This showed that homophilic binding results in productive engagement. Enhancement of polyclonal immune responses of rat lymph node cells by a Fab fragment from a CD5 mAb shown to block homophilic interactions provided evidence that the extracellular region of CD5 regulates inhibition in normal cells. These biochemical and in vitro functional assays provide evidence that the extracellular region of CD5 regulates immunity through species-specific homophilic interactions.

In this work we report that CD5, a T cell accessory activation antigen and receptor for the B cell surface protein CD72, is associated with the T cell antigen receptor (TcR)/CD3 complex in human T lymphocytes. In vitro phosphorylation of either CD3 or CD5 immunoprecipitates prepared from CD3-stimulated Jurkat and peripheral blood T cells in the presence of the detergent polyoxyethelene 10 oleyl ether (Brij96) showed, unexpectedly, an identical pattern of five phosphopolypeptides of 70, 59, 56, 21 and 18 kDa, respectively. Peptide mapping of the five bands demonstrated that the same protein kinase substrates co-precipitated with both CD3 and CD5 and that the majority of the protein phosphorylation occurred on tyrosine residues. These data suggested that the TcR/CD3 complex and and the CD5 antigen might be associated in T cells. Evidence to support this hypothesis was obtained from analysis of immunoprecipitates prepared from surface-iodinated T cells. Bands characteristic of the TcR and CD3 antigens were identified in CD5 immunoprecipitates and conversely, CD5 was identified in CD3 immunoprecipitates. Conformation that CD3 and CD5 co-precipitated in the presence of Brij96 was obtained by Western blotting. Quantitative immunodepletion demonstrated that between 10%-20% of cell surface CD5 was associated with the TcR/CD3 complex in Brij96 detergent lysates of human T cells and, furthermore, that this association was independent of T cell activation. The association of these two receptors provides a possible physical basis for the accessory role of the CD5 antigen in T cell activation.

T lymphocytes and a subpopulation of B lymphocytes express the CD5 coreceptor. Its functional importance is evident from the multiple levels and developmental stages of the regulation of its expression. We here report the discovery of a novel regulatory exon upstream of the noncoding region of the CD5 gene in humans. This alternate exon 1 is designated E1B (with the conventional exon 1 renamed E1A) and was shown to regulate the expression of CD5. E1B-containing transcripts existed exclusively in B lymphocytes and encoded a protein that was truncated and retained intracellularly. As a consequence, the amount of E1A-containing transcripts was down-regulated and the membrane CD5 expression was diminished in the presence of E1B-containing transcripts. High levels of E1A transcripts were found in chronic lymphocytic leukemia, and there were no E1A transcripts in 697 pre-B cells, which have no membrane CD5. Introduction of E1B into Jurkat cells reduced their membrane expression of CD5, and sequence analysis revealed that the E1B motif is a defective human endogenous retrovirus. A balance between the 2 alternative exons 1 might be central to the regulation of membrane CD5 in human B cells, and, through CD5-associated SH2-containing phosphatase 1, to the modulation of B-cell antigen receptor-transduced signals.