IL-6 Production by B-CLL Cells Plays a Critical Role in the Inhibition of T Cell Activation and Proliferation and Promotes a Th2 Response in Normal T Lymphocytes.

Abstract

Immune dysfunction is a hallmark of B-cell chronic lymphocytic leukemia (B-CLL) which occurs through loss of normal cell function as the malignant clone expands, as a result of therapy or because of immunoregulatory properties of the tumor itself. It has previously been shown that B-CLL cells are poor stimulators of the allogeneic mixed lymphocyte reaction (MLR) and we first determined whether this is due to lack of stimulatory activity or active immunosuppression by examining the effect of B-CLL contact and tumor supernatant (TSN) on a 3rd party MLR. Incorporation of B-CLL cells in a 5 day MLR inhibited 3H proliferation by responders in 2/10 cases, whereas TSN inhibited in all 10 cases. Studies in which normal T cells were stimulated by CD3/28 beads for 72 hours in the absence or presence of TSN showed a reduction in cell cycle entry measured by PI and FITC staining with 26+/−4.6% and 13.7+/−4.3% of cells in S +G2M in the absence and presence of TSN respectively (p<0.0001). Studies performed using CFSE labelled normal T cells showed that TSN reduced the number of T cells undergoing one or more cell divisions from a mean of 81.8+/−1.65% to 58.2+/−4.4% (p=0.0072). It is known that T cells in B-CLL have an acquired defect in CD40L expression, which has been ascribed to downregulation by CD40 present on tumor cells. Our experiments confirm that this defect is reversible since purification of B-CLL T cells restores activation induced CD40L upregulation to normal. We further demonstrate that B-CLL TSN from all 17 patients tested inhibits CD40L upregulation by normal T cells in response to PMA and ionomycin or CD3/28 beads (to a mean of 51%+/−5.6%, p<0.0001, of those activated in the absence of TSN) and a parallel inhibition of IL-2 secretion (correlation with CD40L inhibition: p=0.006, r2 = 0.54). In addition to the effects of TSN on T proliferation and activation, B-CLL TSN also induced Th2 polarisation of normal T cells. When activated using CD3/28 beads in control medium, normal T cells show an increase in IL-2, γ-interferon and TNF-α secretion consistent with the expected Th1 response. When incubated in TSN however, 10 and 1000 fold increases in IL-4 and IL6 release were observed respectively consistent with a shift to a Th2 response. B-CLL cells are known to secrete a number of cytokines and in order to determine which might be responsible for the observed effects a number were assayed either by enzyme-linked or cytokine bead array assay. The effects of TSN were not due to TGF-β , IL-10 or soluble CD40 and depletion of soluble CD25 using bead conjugated anti-CD25 had no effect on the immunosuppressive activity. High levels of IL-6 were detected in TSN from all cases studied (n=5). When normal T cell were activated in TSN, a 100 fold further increase in IL-6 level was observed suggesting that this cytokine may be responsible for at least some of the observed effects of TSN. Antibody neutralization of the IL-6 in TSN demonstrated an increase in both Th1 cytokine production and CD40L expression. Furthermore, addition of recombinant IL-6 to T cells activated in media inhibited CD40L upregulation. In summary, B-CLL cells secrete factor(s) which inhibit T cell activation and proliferation and promote Th2 polarisation. These factors might contribute to the disease phenotype by impairing T cell responses to infection, predisposing to autoimmunity and promoting the growth of the malignant clone through the action of IL-6.