PRUEBA DE HIPÓTESIS

MAb's have been used to identify several cell surface antigens which have been proposed to distinguish naive from memory T cells. Two early candidates were the antigens Ta1 and Leu-8. LeuB is now known to be the human homologue of the murine lymph node homing receptor molecule, MEL-14 (Camerini, etal., 1989). However, the subsets defined by presence or absence of Leu8 are not completely functionally discrete (reviewed in Beverley, 1989). Tai identified an antigen of 105kd (CDw26) expressed on activated I cells, I cell lines and clones. The T cell proliferative response to recall antigens such as tetanus toxoid and mumps virus resides entirely in the Tai+ subset of T cells (Fox, etal., 1984; Hafler, etal., 1986). Despite this, the expression of Tai does not correlate with levels of other markers such as LFA-3 which have also been used to separate memory and naive subsets (Sanders et al., 1988a). Latterly, two T cell antigens have emerged as front runners for separating naive and memory cells: CD44 and the isoforms of CD45.

The expression of the antigen pgp-1 (Trowbridge, etal., 1982), now known as CD44, has been extensively studied in the mouse and appears to define a functional division within CD4 and CD8 T cells. The human homologue has also been described (Isacke, etal., 1986). In the mouse, this 95 kd antigen was shown to be present on most bone marrow cells, some T cells, but few thymocytes, as well as on many non-lymphoid cells such as lung, brain and liver (Trowbridge etal., 1982). Interestingly, most early foetal thymocytes express CD44 and the percentage decreases with increasing foetal age (Lesley, etal., 1985). This almost certainly represents the altered ratio of double negative cells to other thymocyte populations during ontogeny. Two colour flow cytometry of thymocyte subsets has supported this view (Lynch & Ceredig, 1988). Depletion of CD44+ bone marrow cells abolished thymic repopulation and reduced the number of spleen colonies in irradiated mice suggesting that prothymocytes are CD44+ (Lesley etal., 1985).

The earliest suggestion that CD44 might define memory cells arose from studies on murine CD8+ T cells (Budd et al., 1987b). In these, it was shown that following immunisation with H-Y antigen virtually all the H-Y specific CTL precursors (CTLp) were in the CD44+ subset of CD8+ spleen cells. Furthermore, following alloimmunization the frequency of allospecific CTLp resistant to inhibition by anti-CD8 antibody was markedly enriched within the CD44+ subset suggesting an enrichment for CTLp bearing high avidity antigen receptors. Using a definition of memory T cells as increased precursor frequency together with higher avidity for the recall antigen it appeared that CD44+CD8+ cells might represent the memory subset. These data were extended by determining the effects of thymectomy and in vitro stimulation (Budd, et al., 1987a). Very few mature thymocytes express CD44 and thymectomy leads to a virtual absence of CD44- cells suggesting that peripheral CD44- T cells are generated from the thymus. Studies in vivo and in vitro showed that the subsequent acquisition of surface CD44 was found to be a stable differentiation event occurring concomitantly with primary antigenic stimulation (Budd etal., 1987a). These observations were extended to CD4+ T cells by showing that the minor CD44+ subset of CD4+ cells also contains the antigen specific T helper cells after immunization with either KLH or SWM (Butterfield, etal., 1989). In keeping with the concept of CD44 being a marker of memory cells, others demonstrated that CD44hi cells accumulate with age in mice (Lerner, etal., 1989).

However, although the functional data are compatible with the notion that expression of CD44 might correlate with a memory phenotype there are problems. In particular, in the mouse, expression of CD44 varies between strains, e.g. in BALB/c 41% of thymocytes express CD44 compared to only 3-9% in CBA/CaJ, C57BL/6J and AKR/J mice (Husmann, etal., 1988). Similarly, in specific pathogen free (SPF) BALB/c mice essentially all peripheral T cells are CD44+ compared with 25% of CD8+ cells and 15% of CD4+ cells in C57BL/6 mice (Lynch & Ceredig, 1988). The basis of this strain difference is not known and it is not clear that such variation in expression occurs among humans. However, a marker that is strain dependent is limited in its usefulness in defining functional T cell subsets.

Cloning of the human and murine CD44 molecules (Goldstein, et al., 1989; Stamenkovic, et al., 1989; Zhou, et al., 1989) revealed that the highly

conserved NH2 domain of CD44 is homologous to link protein and the core

protein of large proteoglycans both of which can bind the g lyco sa m in o g lyca n , hyaluronate. H yaluronate, like o th e r glycosaminoglycans, participates in the assembly of the extracellular matrix (ECM) and is thought to play an important role in embryogenesis, wound healing and inflammation as well as being associated with tumour growth (reviewed in Sy, eta l., 1991). Subsequently it has been shown that hyaluronate can function as a cell adhesion molecule and that CD44 is the principal cell surface receptor for hyaluronate (Aruffo etal., 1990; Miyake et al., 1990). Two isoforms of CD44 have now been described, the 80-90kd equivalent to the molecule described in the preceeding section and a 150kd species which does not bind hyaluronate and which is expressed on a subpopulation of epithelial cells (Stamenkovic et al., 1989). Recent data have demonstrated that transfection of the 90kd isoform, but not the 150 kd isoform, into human B cell lymphoma cells greatly enhanced both local tumor formation and metastatic proclivity of the lymphoma cells (Sy et al., 1991). Thus, the 90kd isoform of CD44 is an adhesion molecule and expression may well alter the traffic of the T cells which express it.