Cell Contact with CLL Cells Induces Defects in T Cell Differentiation and Effector Pathways: Impact of Silencing Specific Cytokine Production.
Authors: Holderried Tobias A.W. , Gorgun Gullu , Gribben John G. 
Published: October 2019

Abstract
Previous studies have suggested that the development and progression of B cell CLL is dependent on interactions between malignant cells and normal components of the immune system. Although the T cell count may be normal or even increased, T cell dysfunction is a feature of CLL, with abnormal CD4/CD8 ratio, impaired mitogen response, and altered expression of surface antigens in response to antigen presentation. We are evaluating the impact of tumor cells on the immune system and have reported differences in gene expression profiles in T cells in previously untreated CLL patients. Specifically, in both CD4 and CD8 T cells we identified defects in genes regulating cytoskeleton formation, intracellular transportation and control of cytokines and chemokines. Analysis of the abnormal gene expression profiles suggested that many such abnormalities are induced by signaling through surface receptors on T cells by interaction with CLL cells or the microenvironment. To detemine the mechanism, we performed ex vivo tumor cell-T cell interaction assays using patient derived serum, transwell membrane and cell contact assays using CLL and CD4 or CD8 T cells from CLL patients and B cells and CD4 or CD8 T cells from healthy donors. Since the majority of the differentially expressed genes were involved in cell cytoskeleton formation and intracellular vesicle transportation pathways, we examined the impact on those specific pathways using proteomics. In keeping with the gene expression profiles in the T cells of CLL patients, we noted significant decreased expression of NFkB p65 and GDI1 and increase in Arp2/3 in healthy CD4 T cells and decreased expression of Rho-GAP and increased Arp2/3 in healthy CD8 T cells following CLL-T cell contact compared to healthy B cell-T cell contact. In contrast there was no change after exposure to patient sera or tumor cell derived soluble factors for 48h. To further analyze whether tumor cell derived cytokines have an impact on T cells in CLL, we inhibited IL-10 and IL-4 production in CLL cells and healthy B cells using siRNA targeting IL-10 and/or IL-4. The transfection efficiency monitored by flow cytometry with fluorescence labeled non-silencing RNA and observed 60–98 % transfection efficiency and at 48 h observed 40–85% inhibition in IL-10 protein expression. After 48 h incubation of autologous and allogeneic CD4 or CD8 T cells from CLL and healthy donors with non-transfected, mock transfected or IL-10 siRNA transfected CLL or healthy B cells for 48h, T cells were isolated and there was no significant difference in expression of cytoskeletal proteins in both CD4 or CD8 T cells. Addition of anti-IL-10 neutralizing monoclonal antibody also had no effect. Although no effect was noted on cytoskeletal proteins, after incubation with CLL but not healthy B cells, silencing or neutralization of IL-10 induced changes in expression of CXCR1, CXCR2, CXCR3, CXCR4 and CCR5 in CD4 and CCR5 and CCR4 in CD8 cells from healthy donors. We conclude that cell contact with CLL cells induces changes in expression of cytoskeletal proteins in healthy T cells similar to those observed in T cells from CLL patients and this is not induced by soluble factors including IL-10. However, IL-10 and potentially other soluble factors induce changes in chemokines and chemokine receptors on T cells, suggesting multiple mechanisms impair T cell function in CLL cells. Ongoing studies are assessing ways to repair the defects identified here to enhance immune responsiveness in this disease.