Early Enrichment and Subsequent Dysfunction of Marrow ILC Subsets in Monoclonal Gammopathy: Target for IMiD Therapy

Innate lymphoid cells (ILCs) are a new family of innate immune cells that lack re-arranged antigen receptors and reside predominantly in tissues. Three distinct human ILC subsets have been described based on their cytokine profile and transcriptional regulation. Murine models suggest a potential role for ILCs in immune surveillance of tumors. However, data relating to changes in ILC subsets in the tumor microenvironment in human preneoplastic states is lacking. All cases of multiple myeloma (MM) are preceded by monoclonal gammopathy of undetermined significance (MGUS). Prior studies have shown the capacity of the immune system to recognize these MGUS lesions, which correlated with a reduced risk of progression to clinical malignancy. Immune modulatory drugs (IMiDs) form a component of standard MM therapy, and act in part by inducing cereblon-mediated degradation of ikaros and aiolos. IMiD-mediated T/NK-T activation depends on antigen-mediated stimulation. However, the full spectrum of immune targets of IMiDs still remains to be characterized.

In this study, we have characterized phenotypic and functional properties of ILCs in the bone marrow of healthy humans and patients with plasma cell disorders (PCD) and evaluated changes in ILCs in MM patients undergoing therapy with pomalidomide (Pom). Compared to healthy donors, patients with PCD had an increased proportion of ILCs in the bone marrow, but not in circulation. Bone marrow of patients with PCD also had altered proportions of ILC subsets with an increase in the proportion of ILC1 and reduction in ILC2. In contrast, the proportion of circulating ILC2 subset was increased in PCD patients. The capacity of both ILC1 and ILC2 to secrete lineage-specific cytokines (IFNg and IL13, respectively) was somewhat preserved in the setting of MGUS, but significantly declined in aymptomatic multiple myeloma (AMM) patients. To further dissect profiles of NK versus ILC1 in the marrow, we sorted purified ILC1 and CD3-CD56+ NK cells from bone marrow and analyzed transcriptional profiles using single cell sequencing. NK cells and ILCs each form two distinct clusters, with ILC versus NK distinguished by LTb, IL7R, Granulysin and GranzymeB expression. ILC1 clusters (cluster1: TRAC+CCL5+GZMA+SELL-; cluster 2:SELL+CCL5-GZMA-TRAC-) were distinct from NK clusters (cluster 1:GNLY+GZMB+FGFBP2+; cluster2: XCL1+XCL2+GZMB^lo). The finding that human ILC1 express high levels of aiolos, a known target for IMiD-mediated degradation, encouraged us to directly test the impact of Pom on ILCs both in vitro and in vivo . Culture of ILCs with Pom led to a reduction in ikaros and aiolos detected within 4-hours. ILCs cultured with Pom also exhibited greater cytokine secretion upon in vitro stimulation. Circulating ILCs from Pom-treated MM patients isolated just 4 hours after first dose had reduced levels of ikaros and aiolos and increased capacity for cytokine production. Together these data indicate that Pom leads to enhanced function of ILC1 in vivo .

These data provide evidence that ILCs are among the earliest immune cell subsets enriched in the tumor microenvironment during the evolution of monoclonal gammopathies. Decline in ILC function in AMM patients suggests that dysfunction of these cells occurs early in myelomagenesis. These data also demonstrate that ILCs are a direct target of IMiDs. Pomalidomide-mediated aiolos depletion provides a novel mechanism whereby IMiDs might modulate immune microenvironment, without the need for antigen-mediated signals. Further studies are needed to identify the signals that mediate enrichment of ILCs in monoclonal gammopathies and alter their functional properties.