Membrane trafficking of CD1c on activated T cells.

We investigated the regulation of and the intracellular trafficking involved in the membrane expression of CD1c antigen on activated mature T cells. Membrane expression of this glycoprotein was highly regulated and dependent on the activation state of the cells. The presence of the CD1c antigen on activated peripheral blood mononuclear cells (PBMCs) was confirmed by flow cytometry, reverse transcriptase-PCR (RT-PCR), and immunoperoxidase staining. The RT-PCR analysis of the alpha3- and 3'-untranslated regions of CD1C showed that phytohemagglutinin (PHA) activation induced expression of transcripts that encode the three isoforms (soluble, membrane, and cytoplasmic/soluble). Immunocytochemical studies showed a specific association of CD1c with the cell membrane and a cytoplasmic, perinuclear distribution. Although flow-cytometric staining confirmed the intracellular presence of CD1c, membrane expression on PHA blast cells was not detected. We found that membrane detection of CD1c antigen was temperature dependent. Cell surface binding of the anti-CD1c monoclonal antibody (mAb) was consistently negative at 4 and 37 degrees C but was detected at room temperature (18-22 degrees C). At physiologic temperatures, activated PBMCs showed intracellular accumulation of the anti-CD1c mAbs, indicating that CD1c cycled between cell surface and intracellular compartments. The CD1c exocytosis pathway was sensitive to Brefeldin A, cytochalasin B, and chloroquine.

Activation-induced expression of CD1d antigen on mature T cells.

In the present study, we investigated the expression of human CD1d antigen on activated mature T cells. Expression of this glycoprotein was found to be highly regulated and dependent on PHA stimulation. Flow cytometry studies using the NOR3.2 antibody, which recognized CD1d under denaturing conditions, showed a clear increase in its expression after PHA stimulation. Expression of this molecule after PHA activation was confirmed by analysis of its corresponding transcript by RT-PCR. A single band representing mRNA for CD1d membrane isoform was observed in activated PBMC as well as in ER3 CD1D-transfected and MOLT-4, pre-T cell lines, which were used as controls. Western blot analysis revealed an activation-dependent increase in CD1d protein expression when PBMC and enriched T cells were activated for different time periods. Activation-dependent expression of CD1d antigen was also confirmed in allogenic-activated T cells, suggesting that this event could have biological significance. Finally, immunocytochemical studies showed the presence of this protein at the plasma membrane accompanied by a cytoplasmic and perinuclear distribution. Results presented herein provide the first experimental evidence showing that CD1d antigen is present on circulating, activated T lymphocytes, suggesting that its expression is dependent on the activation state of the cells. Elucidation of the molecular mechanisms implicated in the activation-dependent expression of this nonclassical antigen will provide new insights into the understanding of antigen presentation and immune regulation.

Antibodies recognizing CD24 LAP epitope on human T cells enhance CD28 and IL-2 T cell proliferation.

Membrane expression of the CD24 molecule on activated T lymphocytes is not elucidated fully. We previously described the intracellular and cell-surface expression of the CD24 sialic acid-dependent epitope(s) on phytohemagglutinin-activated peripheral blood mononuclear cells. However, the CD24 core protein was not detected previously on human T cells. This study reinvestigated the expression and role of CD24 in T cell subsets. We analyzed binding of anti-CD24 monoclonal antibodies (mAbs) to sialic and leucine-alanine-proline (LAP) epitopes in resting and activated, normal T lymphocytes. CD24 LAP and CD24 sialic epitopes were detected on activated CD4- and CD8-positive cells. Although expression of CD24 sialic epitopes remained stably expressed in interleukin (IL)-2-dependent cultures, T cell expression of the LAP epitope was transient. Anti-LAP antibodies strongly enhanced the response of T cells to a combination of anti-CD3/CD28 mAbs and enhanced proliferative response induced by recombinant IL-2. We found similarities in the tissue distribution and function of the human CD24 LAP molecule and the murine, heat-stable antigen, which suggests that CD24 might function as a signaling molecule on human T cells.