Daclizumab expands CD56 bright NK cells by inducing their proliferation in vivo

Multiple Sclerosis (MS) is a chronic, debilitating, inflammatory disease of the central nervous system (CNS). MS affects young adults and occurs in twice as many females as males. Although the cause of MS is unknown, there seem to be definite genetic links as well as environmental components (Noseworthy et al, 2000). MS is characterized by lesions in the brain and spinal cord that pathologically range from inflammation to progressive demyelination of axons. Although there is no doubt that the disease is immune-mediated, the exact population of immune cells that are pathogenic as well as the specific antigen being targeted are unknown. While there is no cure for MS, there are some current treatments, such as corticosteroids, interferons, and glatiramer acetate, which help reduce inflammation. However, these treatments have potentially serious side effects and more effective, better tolerated therapies are needed (Keegan & Noseworthy, 2002).

Daclizumab is one of many experimental therapies in MS currently being studied. It is a humanized monoclonal antibody against the α subunit of the IL-2 receptor (IL-2R). IL-2 cytokine is a T cell growth factor that promotes T cell proliferation and differentiation, but is also responsible for their activation-induced cell death. IL-2R is composed of three subunits: α (CD25), β (CD122), γ (CD132). IL-2 signals through the CD122 and CD132 chains-however, with the presence of CD25, there is a 10-fold increase in the binding affinity of IL-2 (Malek, 2008). Daclizumab blocks CD25, which significantly decreases the binding affinity of IL-2, shutting down the IL-2 signaling pathways of T cells (Muraro & Bielekova, 2007).

IL-2 also regulates the development and functional activity of natural killer (NK) cells. NK cells are cytotoxic cells of the innate immune system and play an important role in anti-viral immunity and in defense against tumor cells. In humans, NK cells express differing amounts of CD56 on their surface and can be divided into two subsets based on the density of the CD56 surface protein on the cell membrane: CD56^bright and CD56^dim cells (Lima et al, 2001). CD56^bright NK cells have a higher expression of CD122, possess more surface adhesion molecules, and produce immunoregulatory cytokines (Farag & Caliguiri, 2006). CD56^bright NK cells are believed to have an immunoregulatory function because CD56^bright NK cells were shown to be able to kill autologous activated T cells, thus participating in the termination of the adaptive immune response and promoting immune tolerance (Bielekova et al, 2006). Following Daclizumab treatment, there is a vast expansion of these CD56^bright NK cells and this expansion induced by Daclizumab in MS patients correlates with the inhibitory effect of Daclizumab on brain inflammatory activity (Bielekova et al, 2006). By understanding the mechanism of how Daclizumab therapy leads to expansion of CD56^bright NK cells, we can develop more targeted, ideally oral therapies for expansion of immunoregulatory CD56^bright NK cells in MS and potentially other autoimmune diseases.

My project is part of a collaborative effort to dissect the mechanism for an in vivo expansion of CD56^bright NK cells during Daclizumab therapy. Based on preliminary in vitro experiments we hypothesized that CD56^bright NK cells are expanded during Daclizumab therapy because they are not dependent on CD25 for IL-2 signaling (due to their very high expression of CD122) and therefore can receive IL-2 signals during Daclizumab therapy. In contrast, T cells are dependent on CD25 for IL-2 signaling and therefore, the Daclizumab-mediated blockade of CD25 will result in diminished consumption of IL-2 by T cells, making more IL-2 available for CD56^bright NK cells. We wanted to test this hypothesis in several ways:

1. CD25 expressing FoxP3+ T regulatory cells (T-regs) are dependent on IL-2 for their in vivo proliferation and survival (Wuest et al, 2008). Therefore we wanted to assess numbers and in vivo proliferation of T-regs before and during Daclizumab therapy.
2. CD56^bright NK cells proliferate in vitro when exposed to IL-2 (Bielekova et al, 2006). Therefore we wanted to assess in vivo proliferation of CD56^bright NK cells before and during Daclizumab therapy.
3. We also studied signaling and function of T cells and NK cells before and during Daclizumab therapy, but those studies were performed by different scientists in the laboratory.

Peripheral blood mononuclear cells (PBMCs) from 26 MS patients from two separate clinical trials (Daclizumab monotherapy or IFN-β/Daclizumab combination therapy) were used for an intracellular cytokine stain for FoxP3 and Ki67. FoxP3 is a T-reg marker and Ki67 is a proliferation marker, both of which bind to the chromatin in the cell nucleus. Three separate time points were taken: baseline, month 3 of treatment and month 8 of treatment to examine the time course effects of Daclizumab treatment. The PBMCs were stained for surface proteins: CD4, CD8, CD3, CD127 and CD25. CD127 is the α subunit of the IL-7 receptor; IL-7 is also important for T cell growth and proliferation. By staining for CD127, we may be able to see if T cells upregulate CD127 to compensate for the blocked CD25 and inability to utilize IL-2. The cells were then permeabilized/ fixed and a human immunoglobulin solution was added to help block nonspecific binding. The intracellular antibodies FoxP3 and Ki67 were then added for 1 hour incubation in the refrigerator. Cells were then washed with a permeabilization wash buffer and resuspended in FACS buffer. The cells were then analyzed using the flow cytometer. All stainings were performed in duplicates and isotype controls were subtracted from sample wells. Average and standard deviation was then calculated per each time point for each study subject. Group differences between pre-treatment baseline and therapy time-points were then assessed by statistical methods, specifically by repeated measure analysis of variance.

The results of our experiment confirmed the hypotheses that both T-reg cell numbers would decrease and CD56^bright NK cells would expand and proliferate following Daclizumab treatment. There was a 10.8% decrease from baseline to month 3 of treatment in FoxP3+CD4 T cells (T-regs) and a 12.5% decrease from baseline to month 8 of treatment (p<0.001). There was no significant change in T-reg cell numbers between months 3 and 8 of Daclizumab treatment. Contrary to what we had hypothesized, T-regs showed no significant changes in proliferation following Daclizumab treatment, perhaps due to the small subject pool we used in this study. However, we did observe that of the T-reg cells that were proliferating, there was a 45.4% increase in CD127 expression from baseline to month 3 of treatment (p<0.05) and a 44.5% increase in CD127 expression from baseline to month 8 of treatment (p<0.05), but no significant change from month 3 to month 8. One explanation for this finding may be that because CD25 on T-reg cells is blocked by Daclizumab, the cells are unable to utilize IL-2 (which is necessary for survival) and subsequently upregulate CD127 expression in an attempt to compensate for the blocked CD25. We observed a 56.3% increase in CD56^bright NK cells from baseline to month 3 and 43.1% increase from month 3 to month 8 of treatment (p<0.05). Overall, there was a 75.1% increase of CD56^bright NK cells from baseline to month 8 (p<0.05). There was a 26.2% increase in proliferating Ki67+ CD56^bright NK cells from baseline to month 3 (p<0.05) and a 30% increase from baseline to month 8 of Daclizumab treatment (p<0.05). There were no significant changes in numbers or proliferation of CD56^dim NK cells, B cells or monocytes following Daclizumab therapy. Some other interesting results were that CD4 cells overall proliferated much less than FoxP3 expressing CD4+ T-reg cells in vivo which is not the case in vitro (Valencic et al, 2007). Future studies may explore this difference.

Our findings indicate that Daclizumab has a definite effect on populations of T-reg cells and CD56^bright NK cells following treatment. Based on our results, we could hypothesize that the combination of a low (non-toxic) dose of IL-2 with the standard regimen of Daclizumab would lead to further expansion and activation of CD56^bright NK cells, and this may be the direction of future studies.

References

Bielekova, B., Catalfamo, M., Reichert-Scrivner, S., Packer, A., Cerna, M., Waldmann, T.A., McFarland, H., Henkart, P.A.& Martin, R. Regulatory CD56^bright natural killer cells mediate immunomodulatory effects of IL-2α-targeted therapy (daclizumab) in multiple sclerosis. PNAS 2006; 103, 5941-5946.

Farag S.S. & Caliguiri, M. Human natural killer cell development and biology. Blood Reviews. 2006; 20:123-137.

Fehinger, T.A, Bluman, E.M., Porter, M.M., Mrózek, E., Cooper, M.A., VanDeusen, J.B., Frankel, S.R., Stock, W. & Caliguiri, M.A. Potential mechanisms of human natural killer cell expansion in vivo during low dose IL-2 therapy. J. Clin. Immun. 2000; 106:117-124.

Keegan, B.M. & Noseworthy, J.H. Multiple Sclerosis. Annu. Rev. Med. 2002; 53: 285-302.

Lima, M., Teixeira, M.A., Queirs, M.L., Leite, M., Santos, A.H., Justiça, B. & Orfăo, A. Immunophenotypic Characterization of Normal Blood CD56+lo Versus CD56+hi NK-Cell Subsets and Its Impact on the Understanding of their Tissue Distribution and Functional Properties. Blood Cells, Molecules, and Diseases. 2001; 27: 731-743.

Malek, T.R. The Biology of Interleukin-2. Annu. Rev. Immunol. 2008; 26:453-79.

Muraro, P. & Bielekova, B. Emerging therapies for Multiple Sclerosis. Neurotherapeutics. 2007; 4: 676-692.

Noseworthy, J.H., Lucchinetti, C., Rodriguez, M., & Weinshenker, B.G. Multiple Sclerosis. New England Journal of Medicine. 2000; 343: 938-952.

Valencic, E., Piscianz, E., Tommasini, A., & Granzotto, M. T cells stimulated in vitro have a suppressive function but do not contain only regulatory T cells. Clin. Exp. Immunol. 2007; 150: 561-566.

Wuest, T.Y., Willette-Brown, J., Durum, S. & Hurwitz, A.A. The influence of IL-2 family cytokines on activation and function of naturally occurring regulatory T cells. J. Leukocyte Biology. 2008; 1-8.