The goal of our study was to investigate in detail effects of daclizumab therapy on ILC subtypes, the mechanisms behind these effects, as well as to explore potential role of ILC subsets in the MS disease process. We observed that although untreated MS patients have significantly elevated levels of pro-inflammatory ILCs, defined as c-kit+
lineage negative cells, which are known to contain LTi cells, daclizumab therapy restores ILC numbers to levels comparable to those observed in healthy subjects. Furthermore, daclizumab therapy skews the phenotype of ILCs away from the LTi lineage and toward the NK cell lineage. We were able to reproduce these in vivo
effects by adding IL-2 (or IL-15) to in vitro
differentiation assays of CD34+
HPC or c-kit+
ILCs: In both instances, addition of IL-2 drove differentiation of ILCs away from the LTi lineage and toward the NK cell lineage, in agreement with published animal studies (18
). We conclude that CD25 blockade alters differentiation of ILCs by paradoxically enhancing IL-2 signaling on common precursor(s) that can differentiate to either LTi cells or CD56bright
NK cells, depending on environmental cues. By blocking high affinity IL-2 signaling on T cells, daclizumab increases the in vivo
availability of IL-2 for cells that are capable of signaling via the intermediate affinity IL-2R (11
), such as CD56bright
NK cells (7
) and subtypes of ILCs.
The crucial question is whether the phenotypic switch of ILCs from LTi to NK cell lineage induced by daclizumab inhibits aberrant adaptive immune responses associated with autoimmunity. Utilizing an open-label cross-over trial methodology, we previously demonstrated that daclizumab, either in combination with IFN-β (3
) or as a monotherapy (8
) significantly inhibits formation of brain inflammatory lesions in MS and stabilizes neurological disability. These findings have now been fully reproduced in an independent double-blind, placebo-controlled clinical trial (4
Although the current study is the first direct evidence linking adult LTi cells with autoimmunity in humans, by facilitating OX40 and CD30 signaling on T cells, LTi cells may play an important role in promoting the development of CD4 T cell memory (28
) and production of CD4 T cell-dependent high-affinity Ab (36
). Both of these advanced functions of adaptive immunity are linked to secondary lymphoid tissues or organized lymphoid aggregates (such as “ectopic” or “tertiary” lymphoid follicles) that have been observed in many autoimmune diseases (26
), including MS (42
). Indeed, the hallmark of MS is intrathecal production of IgG, measured clinically as IgG index, with characteristics of an antigen-driven affinity maturation process (44
). Formation of high affinity IgG against protein antigens is dependent on CD4 T cell help and occurs only in germinal centers of lymphoid organs. Persistent presence of oligoclonal bands (expanded IgG clonotypes detected only in the CSF but not in the serum), presence of all stages of B cell differentiation in the CSF of MS patients (46
), and the recent discovery of CXCL13-rich tertiary lymphoid follicles in the meninges of some MS patients (42
) collectively imply that the intrathecal production of IgG in MS is facilitated and/or sustained within meningeal lymphoid aggregates.
Hence, we searched for in vivo
evidence that would link our observation of daclizumab-driven inhibition of LTi cells to meningeal inflammation in MS. There is an abundance of literature linking ectopic lymphoid follicles in a variety of human autoimmune diseases with CXCL13 expression in the affected tissue (39
) and elegant animal studies linked CXCL13 mechanistically to lymphoid neogenesis (40
). We observed that CSF CXCL13 levels decreased by 50.4% after 6.5 months of daclizumab therapy. Furthermore, this was associated with 13.5% decrease in the intrathecal production of IgG, measured as IgG index. This effect on IgG production was specific for the intrathecal compartment, as we have previously reported that daclizumab therapy has no effect on systemic production of immunoglobulins (3
). To our knowledge, daclizumab is the first immunomodulatory drug that decreases levels of intrathecally produced IgG in MS, and it is doing so without depleting or limiting access of immune cells to the intrathecal compartment (13
). Furthermore, because meningeal lymphoid follicles have been associated with neuronal loss in the underlying brain tissue (43
), inhibition of meningeal inflammation would be expected to slow down accelerated development of gray matter atrophy observed in MS. Indeed, our pilot data comparing 27 MS patients on long-term daclizumab therapy (total of 982 MRI scans) to a matched cohort of 44 MS patients treated with first line FDA-approved therapies (mainly interferons) indicate that daclizumab inhibits atrophy of brain structures that have direct contact with meninges/CSF by 38–60% in comparison to standard treatments (48
). Although these encouraging observations need to be reproduced in large controlled studies, our data suggest that inhibition of LTi cells may be a highly efficacious therapeutic modality for MS.
There are limitations of the current study: Although our in vivo observations and in vitro mechanistic studies reinforce the evidence for a developmental link between LTi cells and NK cells through regulation of a common precursor, we have not performed detailed investigation and single cell cloning experiments that would define the precise phenotype of this precursor. Similarly, because the visualization of meningeal lymphoid follicles or even direct quantification of diffuse meningeal inflammation is currently not possible in living human subjects, our study provides only an indirect, albeit plausible, link between LTi cells and intrathecal inflammation in MS. Clearly, follow-up studies evaluating ILCs in autoimmune diseases amenable to biopsies of the targeted tissue, or clinical trials of novel therapies that target specifically pro-inflammatory ILCs will be necessary to establish a definite pathogenic role of LTi cells in human autoimmunity. We hope the current study will spark the necessary interest in these innate lymphoid effectors, which will lead to a better understanding of their role in human health and disease. Such understanding is the necessary prerequisite for development of targeted therapies.