Y Torimoto

Biochemical characterization of CD26 (dipeptidyl peptidase IV) : functional comparison of distinct epitopes recognized by various anti-CD26 monoclonal antibodies

In this paper, we performed further biochemical characterization of the CD26 antigen, as defined by the mAbs in anti-1F7 and anti-Ta1, in order to clarify the observed functional differences among these mAbs. For this purpose, we developed a mAb, anti-5F8, which recognizes yet another epitope on the CD26 antigen different from that recognized by anti-1F7 and anti-Ta1 and compared their respective effect on T cell activation as well as the structures recognized by these mAbs. Functionally, anti-5F8 did not exhibit a comitogenic effect on T cell activation via the CD3 and CD2 pathways. Peptide mapping studies suggested that the 110 kDa molecules precipitated by these mAbs are identical. We showed that the 110 kDa CD26 structure on human T cells is composed of a family of heterogeneous molecules, as determined by isoelectric focusing studies. In addition, we demonstrated that the CD26 antigen has a DPPIV enzyme activity and this enzyme activity is found only on the principal basic structure of CD26 but not on the additional acidic structures. Biochemical studies also revealed that these mAbs recognized distinct epitopes on the CD26 antigen. Pulse-chase studies showed the the 1F7 epitope was found on both the immature (100 kDa) and mature (110 kDa) forms of the CD26 antigen. On the other hand, the Ta1 and 5F8 epitopes were expressed mainly on the mature form of the CD26 antigen. Moreover, anti-IF7 consistently precipitated an additional 43 kDa molecule in association with the principal 110 kDa molecule. Taken together, these data suggested that the additional 43 kDa structure or the distinct epitope recognized by anti-IF7 may play a role in human T cell activation via the CD3 and CD2 pathways.

Activation of T cells through a T cell-specific epitope of CD45

The 180- and 190-kDa isoforms of CD45 are preferentially expressed on the helper inducer (memory) subset of CD4 cells. In order to generate monoclonal antibodies against the extracellular domains of these isoforms and determine whether they could regulate the function and activation of these cells, we developed a mAb, anti-4H2D, by immunizing Balb/c mice with an isogenic mouse pre-B cell line expressing the human 190-kDa CD45 isoform. Anti-4H2D reacts with approximately 60% of T cells, 70% of CD4 cells, and 60% of CD8 cells. The CD4 cell population defined by this mAb corresponds functionally and phenotypically to that defined by the CD45RO+CD29+ subset. Western blotting demonstrated that anti-4H2D reacts primarily with the 190-kDa isoform of CD45 and to a minor extent, the 205- and 180-kDa CD45 isoforms. Interestingly, this mAb reacted with only a subpopulation of mature thymocytes and peripheral T cells, despite the fact that the 190-kDa CD45 isoform, as well as CD45RO and CD29, is more widely distributed on cells of hematopoietic origin. The 4H2D epitope was neuraminidase sensitive, indicating that anti-4H2D reacts with a carbohydrate epitope which is present on only a subset of the T cells containing the 190-kDa CD45 isoform epitopes. Functional studies showed that soluble anti-4H2D augmented T cell proliferation induced by the CD2 and CD3 pathways, and treatment of T cells with this mAb up-regulated [Ca2+]i flux induced by both anti-CD2 and anti-CD3 mAbs. These results suggest that the 190-kDa CD45 isoform on human CD4 cells is heterogeneous and that the 190-kDa isoform recognized by anti-4H2D regulates the function and activation of CD4 helper T cells.

Signaling from LFA-1 contributes signal transduction through CD2 alternative pathway in T cell activation

LFA-1, a member of the integrin family of molecules, is involved in mediating cellular adhesion in all phases of the immune response, playing a role in the interaction of helper T cells as well as in killing of target cells by both cytotoxic T cells and natural killer cells. We have developed a monoclonal antibody, anti-HVS6B6, which recognizes a functionally unique epitope of the LFA-1 molecule. Although this mAb itself was not mitogenic against T cells, it induced a strong proliferative response when added to T cells with submitogenic concentrations of anti-CD2 (anti-T11(2) and anti-T11(3)) mAbs. In contrast, other anti-LFA-1 mAbs (CD11a and CD18) suppressed this anti-CD2 mAb-induced T cell proliferation. Kinetic studies showed that anti-HVS6B6 acts on an early event in CD2-mediated T cell activation. Although T11(3)-epitope expression induced by anti-T11(2) mAb was not affected by treatment of cells with anti-HVS6B6, both Ca2+ influx and phosphatidylinositol turnover induced by anti-CD2 mAbs were markedly enhanced by the pretreatment of T cells with anti-HVS6B6 mAb. These results indicate that the LFA-1 mediating signal contributes to a very early phase of signal transduction during CD2-mediated T cell activation.