LAT Down-Regulation in T Lymphocytes from B-Cell Chronic Lymphocytic Leukemia: A Possible Mechanism for T Cell Incompetence.

Patients with chronic lymphocytic leukaemia (CLL) show, in addition to the clonal expansion of the malignant B cells, several abnormalities in the non-malignant T cells population. These include T cell lymphocytosis, an increase of CD8+ T cells which results in a low CD4/CD8 ratio and a defective proliferative response to mitogenic stimuli. A down-regulation of TCR-related CD3 zeta protein in T cells of CLL has been reported suggesting that T cell immune deficiency may result from an impairment of cell activation machinery. In addition recent data show a decreased level of the costimolatory CD28 molecules and a concomitant augment of the downregulatory CTLA-4 molecules suggesting that T cells may be in a partial state of anergy which may account for their failure to display a full activation response following TCR engagement. Despite the evidence of a compromission in theT cell signal machinery, little is known about possible defects in other molecules involved in the cell signal transduction pathway. In order to investigate the expression of the proteins involved in the early TCR signalling events CD3+ T cells of 7 healthy donors and of 21 B-CLL patients were purified by negative selection using magnetic beads (purity >95%) and lysed in 1% Triton X-100. Subsequently, equal amounts of protein for each sample were analyzed by Western Blotting and the following molecules were evaluated: CD3 epsilon, p56Lck (Lck) and linker for activation of T cells (LAT). Overall results showed that CD3 epsilon in T cells of B-CLL was expressed in an amount comparable to normal healthy donors in 15/21 (71%) cases while was reduced in 6/21 (29%), Lck was reduced in 3/21 (14%) cases and interestingly LAT was reduced in 15/21 (71%) cases. Our data show that, apart from CD3 zeta, other molecules involved in the early activation events, are reduced in T cells of B-CLL. CD3 epsilon was reduced in a third of the cases tested. This subunit, along with CD3 zeta, plays a crucial role in the early TCR signalling events, its reduction therefore may contribute to explain the hyporesponsiveness showd by T lymphocytes. Interestingly we observed a significant reduction in the expression of LAT molecule. LAT is an adaptor molecule which, even if lacking in enzymatic activity partecipates to cell signalling acting as a plasma membrane scaffold. This molecule following TCR engagement is phosphorilated by ZAP-70 kinase and in the active form promotes the recruitment at the plasma membrane of molecules with SH2 domains and the assembly of numerous signalling complexes. LAT reduced expression in T cell is of particular interest in the light of recent data, obtained in mice, showing that anergic T cells are defective in LAT activation. To our best knowledge so far there are no available biochemical data about abnormalities in the expression degree of trasduction molecules in human anergic T cells, so our data may represent a preliminary contribution in understanding a possible molecular mechanism at the basis of the anergic condition displayed by T cell compartment of B-CLL.