Caracterización del compartimento no tumoral del sistema inmunitario en pacientes con LLC-B y su relación con la expresión

Abstract

Background: B-cell chronic lymphocytic leukemia (B-CLL) is a chronic lymphoproliferative disease, are characterized by progressive accumulation of monoclonal B cells that appear mature but are to be functionally incompetent. The clinical evolution of B-CLL is very heterogeneous. The pathogenesis of this heterogeneity is poorly understood. Both the leukemic B cells and the non transformed immune cells of the patient appear to be involved in the clinical behavior of the disease. The clinical staging systems developed by Rai and Binet have been effectively used for decades to assess the diagnostic, prognostic and therapeutic requirements at the time of diagnosis. However these systems cannot identify stable or progressive forms of the patients at the early stages of the disease. There has been a persistent effort to identify other prognostic factors in B-CLL. Actually, several biologic markers have been associated with a low or high levek of clinical risk in B-CLL, there include the percentage of ZAP-70 expression by B cells. The expression of the intracellular signalling molecule ZAP-70 in leukemic B-cells has been identified in a majority of BCLL cases without IgVH mutations. ZAP-70 expression is also highly predictive of patients with more rapid disease progression and death. The aim of this study is to investigate the distribution, survival and functional state of cells from diferent immune system compartments in peripheral blood B-CLL patients classified by the expression of ZAP-70 in leukemic cells. Material and methods: Peripheral blood mononuclear cells (PBMC) from heparinised blood samples from thirty four LLC-B patients were obtained. A further requirement was all the patients were in Binet A and RAI 0 stage. The exclusion criteria were anything causing primary or acquired immunodeficiency, previous immunosuppressive or immunomodulation chemotherapy or radiotherapy, autoimmune or allergic disease. Eighteen blood samples from healthy donors with age- and sex-matched were obtained too. The B-CLL patients were grouped in two groups (ZAP-70– B-CLL and ZAP-70+B-CLL patients) according to the expression of ZAP-70 for their tumour cells. The samples were analyzed by both four and nine colors flow cytometry by study the distribution of dendritic cells, monocytes, T, B and NK cells. Apoptosis and intracellular production of cytokines by T lymphocytes were also studied. Results: Significant diferences in the distribution of DCs were observed between ZAP70+ B-CLL patients and both ZAP-70– B-CLL patients and healthy controls. On the other hand, significant differences in the absolute counts and percentages of diferent subsets of monocytes were observed between ZAP-70+ B-CLL patientes and both ZAP-70– B-CLL patients and healthy controls too. In addition the patients ZAP-70+ showed a significant redistribution of the circulating monocytes with an increase of the subpopulations CD14–CD16+high and CD14+lowCD16+high and a decrease of the CD14+hiCD16–. The disease is associated with a decrease of monocyte counts and with decrease of the expression of molecules involved in the coestimulation and cellular migration of the majority of the monocytes subsets. The ZAP-70+ B-CLL patientes showed significant increases in absolute counts and percentages of CD4+FOXP-3+ cells when compared with both ZAP-70– B-CLL patients and healthy controls. These patients presented a deep redistribution of CD4+ CD8+ T lymphocytes, with a significant increase of efector CD4+ T cells, a significant decrease of naive CD4+ T cells, a significant decrease of the naive and effector memory CD8+ T cells and a significant increase of the central memory CD8+ T cells. On the other hand the ZAP-70+ B-CLL patients presentedsignificant decrease of NK cells and significant increase of TK cells with increase of expression of cell activation surface antigens. When we analyzed apoptosis in T cells, we found that ZAP-70+ BCLL patients presented an increased spontaneus apoptosis in vitro in all the populations and subsets of T lymphocytes studied with regard to both ZAP-70– B-CLL patients and healthy controls. The studies on intracellular cytokine production showed that in the ZAP-70+ B-CLL patients there is an increase of the CD4+ T lymphocytes with Th2 pattern of cytokine production (high production of IL-4 and IL-10) with regard to the ZAP-70– B-CLL patients, being the naive and effector subsets more affected by these alterations. Conclusion: Our results have demonstrated alterations of the populations of the immune system in B-CLL patients, that are markedly different in the patients according to their degree of ZAP-70 expression on leukemic cells. These different alterations can be involved in the pathogeny of the different clinical behaviour of both groups.