CD4+ CD45RA+ and CD4+ CD45RO+ T cells differ in their TCR-associated signaling responses.

Peripheral CD4+ T cells can be divided into two different functional populations based on the expression of distinct isoforms of the surface molecule CD45. We have investigated the differences in the proximal signaling induced by anti-CD3 monoclonal antibody in purified populations of "naive" CD45RA+ and "memory" CD45RO+ human CD4+ T cells. Expression of cell surface CD3, CD4 and CD28 was comparable between RA+ and RO+ cells. However, TCR-directed stimulation in the form of anti-CD3 produced markedly different patterns of intracellular signaling. Greater inositol triphosphate generation occurred in naive cells and the rise in intracellular free calcium was also substantially greater in naive than in memory cells. Cells with the naive phenotype were considerably more active in TCR-dependent tyrosine phosphorylation, both at an overall level and specifically in terms of TCR-zeta and ZAP-70 phosphorylation. Despite these differences in phosphorylation, the amounts of TCR-zeta, ZAP-70 and Ick were equivalent between the two subsets. These findings suggest that the TCR-dependent signaling is differentially regulated in naive and memory CD4+ T cells. This may be due to differences in the way that the two isoforms of the CD45 phosphatase regulate the activity of proximal kinases in the TCR signaling pathway, and could be an important means by which the unique functions of differentiated T cell populations are maintained.

Cross-linking of the CAMPATH-1 antigen (CD52) triggers activation of normal human T lymphocytes.

The CAMPATH-1 (CD52) antigen is a 21-28 kDa glycopeptide which is highly expressed on lymphocytes and macrophages and is coupled to the membrane by a glycosylphosphatidylinositol (GPI) anchoring structure. The function of this molecule is unknown. However, it is an extremely good target for complement-mediated attack and antibody-mediated cellular cytotoxicity. The humanized CAMPATH-1H antibody, which is directed against CD52, is very efficient at mediating lymphocyte depletion in vivo, and is currently being used in clinical trials for lymphoid malignancy and rheumatoid arthritis. It is therefore important to examine the functional effects of this antibody on different lymphocyte sub-populations. Because several other GPI-linked molecules expressed on the surface of T lymphocytes are capable of signal transduction resulting in cell proliferation, we have investigated whether the CAMPATH-1 antigen can also mediate these effects. In the presence of phorbol esters and cross-linking anti-Ig antibodies, mAbs specific for CD52 induced proliferation and lymphokine production in highly purified resting CD4+ and CD8+ T lymphocytes. The rat IgG2c YTH 361.10 anti-CD52 antibody, however, was able to activate resting CD4+ and CD8+ T cells directly without cross-linking or phorbol myristate acetate in the absence of Fc-bearing cells. Anti-CD52 antibodies also augmented the anti-CD3 mediated proliferative response of CD4+ and CD8+ T cells when the two antibodies were co-immobilized onto the same surface or cross-linked in solution by the same second antibody. Both CD4+ CD45RA and CD4+ CD45RO T cells were stimulated to proliferate by anti-CD52 antibodies in the presence of appropriate co-stimulatory factors. Anti-CD52 mAbs did not, however, synergize with anti-CD2 or CD28 mAb to induce CD4+ T cell proliferation. The activation of CD4+ T cells by anti-CD52 antibodies was inhibited by cyclosporin A, suggesting a role for the calcineurin-dependent signal transduction pathways. Although CD52 could transduce a signal in T cells, anti-CD52 antibodies did not inhibit antigen-specific or polyclonal T cell responses, suggesting this molecule does not play an essential co-stimulatory role in normal T cell activation.

Expression of hybrid malaria antigens in insect cells and their engineering for correct folding and secretion.

Hybrid proteins containing selected regions of the major surface antigens of the sporozoite and merozoite stages of Plasmodium falciparum were expressed in insect cells using baculovirus vectors. A recombinant protein containing the signal peptide from the precursor to the major merozoite surface antigens (PMMSA) fused to a fragment from the carboxy (C) terminus of the same gene was recognized by monoclonal antibodies specific for reduction-sensitive conformational epitopes within the C-terminal fragment, suggesting that correct disulphide cross-linking of cysteine residues within this region had occurred. Addition of 26 copies of the tetrapeptide repeat from the circumsporozoite protein (CSP) resulted in a protein recognized by anti-CSP antiserum as well as the conformation specific monoclonal antibodies. Deletion of the C-terminal putative anchor sequence from both proteins resulted in secretion of protein in a fully soluble form antigenically indistinguishable from the anchor containing products. Correct conformation was not observed when the proteins were expressed as polyhedrin fusions without the signal peptide. These data indicate that the PMMSA signal peptide is recognized in insect cells and that correct assembly of disulphide cross-links is dependent upon targeting the protein to the endoplasmic reticulum.