Regulatory CD56(bright) natural killer cells mediate immunomodulatory effects of IL-2Ralpha-targeted therapy (daclizumab) in multiple sclerosis

Abstract

Administration of daclizumab, a humanized mAb directed against the IL-2Ralpha chain, strongly reduces brain inflammation in multiple sclerosis patients. Here we show that daclizumab treatment leads to only a mild functional blockade of CD4(+) T cells, the major candidate in multiple sclerosis pathogenesis. Instead, daclizumab therapy was associated with a gradual decline in circulating CD4(+) and CD8(+) T cells and significant expansion of CD56(bright) natural killer (NK) cells in vivo, and this effect correlated highly with the treatment response. In vitro studies showed that NK cells inhibited T cell survival in activated peripheral blood mononuclear cell cultures by a contact-dependent mechanism. Positive correlations between expansion of CD56(bright) NK cells and contraction of CD4(+) and CD8(+) T cell numbers in individual patients in vivo provides supporting evidence for NK cell-mediated negative immunoregulation of activated T cells during daclizumab therapy. Our data support the existence of an immunoregulatory pathway wherein activated CD56(bright) NK cells inhibit T cell survival. This immunoregulation has potential importance for the treatment of autoimmune diseases and transplant rejection and toward modification of tumor immunity.

Fig. 1.

Correlation between expansion of CD56^bright NK cells and inhibition of brain inflammatory activity during the daclizumab trial. Percentages of CD56^bright NK cells were averaged from two combination therapy samples (months 0–5.5; IFN-β plus daclizumab, open circles) and two monotherapy samples (months 6.5–15.5; daclizumab, target circles) for each patient (n = 12) and correlated with the average number of contrast-enhancing lesions on brain MRI during 5.5 combination therapy months and 12 monotherapy months.

Fig. 2.

Inhibitory effect of daclizumab on T cell survival in in vitro cultures appears to be mediated by NK cells and requires NK–T cell contact. (A) PBMC or NK-depleted PBMC (by CD56 microbeads) from the same samples were stained with CFSE, polyclonally activated (plate-bound CD3/CD28) for 72 h in the presence/absence of daclizumab, washed and reseeded in T cell media enriched for IL-7 and IL-15 (with or without daclizumab), and followed for long-term survival. Corresponding flow cytometry profiles of equivalent proportions of cultures (CFSE proliferation together with intracellular cytokine staining for IL-2) at day 8 after stimulation are depicted from a representative patient from samples before and during daclizumab therapy. (B and C) Similar experimental design to A except that the NK cells that were depleted from PBMC were added into TW cultures at a 1:10 NK:T cell ratio so that T cells and NK cells were not in contact. (B Left) Rate of T cell proliferation by CFSE dilution at day 6 (no. of mitoses per 100 gated cells). (B Right) Thymidine incorporation from the same cultures at same time point (cpm). (C) T cell survival at day 18 of culture was assessed by two independent investigators counting the live cells in culture by light microscopy using trypan blue exclusion and by calculating the final number of T cells based on the proportions of CD3⁺ T cell in the cultures analyzed by flow cytometry. For each of the three panels, T cells in the PBMC are compared with T cells in NK-depleted PBMC, and depleted NK cells were placed in TW. Each plot represents representative experiment from two to five patients.

Fig. 3.

NK cells are cytotoxic toward activated autologous T cells. (A) NK-mediated cytotoxicity against autologous T cells from a healthy donor. Both T cells and NK cells were purified by negative magnetic bead selection to >95% purity and tested in a 4-h chromium-release cytotoxicity assay immediately (Left) or after the T cells were polyclonal activated for 24 h (Right), and NK cells were cultured with 10 units/ml IL-2. Open circles indicate the effect of daclizumab (10 μg/ml) in the culture and cytotoxicity assay. (B) NK-mediated cytotoxicity against autologous activated T cells from a daclizumab-treated MS patient. T cells were purified by negative selection and activated (by PMA/ionomycin) from frozen samples of a MS patient from pretreatment baseline period and during daclizumab therapy and stained overnight with chromium. The next morning the NK cells were purified from the same frozen samples by negative selection and used fresh as effector cells. Left compares cytotoxicity of NK cells isolated from baseline samples, and Right shows the activity of NK populations from therapy samples against the same targets. (C) Cytotoxicity of purified CD56^dim and CD56^bright NK cells from MS patients undergoing daclizumab therapy on autologous T cells. T cells were left unstimulated or were stimulated with PMA/ionomycin for 12 h. Daclizumab (10 μg/ml) was added to the assays as indicated. Inhibition of NK subset cytotoxicity by anti-CD16 Ab (1 μg/ml) is depicted in red. Each plot corresponds to a representative experiment from two to five subjects.