Type II NKT-TFH Cells Against Gaucher Lipids Regulate B Cell Immunity and Inflammation
Authors: Nair Shiny , Boddupalli Chandra Sekhar , Verma Rakesh , Liu Jun , Ruhua Yang , Pastores Gregory M , Mistry Pramod , Dhodapkar Madhav V. 
Published: October 2019

Abstract
Gaucher disease (GD) is a lysosomal storage disorder due to an inherited deficiency of acid β-glucosidase (GBA) that leads to the accumulation of two key sphingolipids, glucosylceramide (βGL1) and glucosylsphingosine (LGL1) in lysosomes of mononuclear phagocytes. Pathophysiology of classic GD is complex and includes chronic inflammation with an increased risk of B cell malignancies, pointing to immune dysregulation beyond the accumulation of lipid-loaded macrophages. Although an involvement of accumulated lipids has been implicated, the mechanisms linking sphingolipid accumulation with the observed clinical phenotypes are poorly understood.
Natural Killer T (NKT) cells are a specialized lineage of T cells that recognize lipid/glycolipid antigens presented by MHC-like molecule CD1d. NKT cells are currently classified into two major subsets- type I or invariant NKT (iNKT) cells that express a conserved TCR and recognize α-galactosylceramide (α-GalCer) and type II or diverse NKT (dNKT) cells that utilize diverse TCR and do not recognize α-GalCer. Type II NKT cells comprise a major subset in humans and are increasingly implicated in immune regulation of diverse disease states. However in contrast to type I NKT cells, information on the functional properties of human type II NKT cells and their dysregulation in the context of disease is extremely limited.
We utilized human and murine CD1d-tetramers loaded with GD-associated lipids (βGL1 and LGL1) to characterize the genomic and functional properties of T cells against these lipids. Analysis of human PBMCs revealed that βGL1-22/LGL1-tetramer+ cells are a distinct subset of CD1d-restricted T cells with a naïve phenotype and diverse Vβ receptor usage. Gene expression profiling as well as multiplex cytokine analysis demonstrated a distinct genomic and cytokine profile of these cells compared to classical type I NKT cells. Using both CD1d blocking experiments and CD1d-expressing C1R cells we could show that βGL1-22 and LGL1 could activate respective lipid-specific T cells in a CD1d dependent manner. Akin to human PBMCs, murine βGL1-22/LGL1 was shown to be a cognate antigen of type II NKT cells by performing double staining with α-GalCer and βGL1-22/LGL1-loaded-CD1d tetramers and using CD1d-/- and Jα18-/- mice. Transcriptional profile of βGL1-22 and LGL1-specific T cells revealed increased expression of genes associated with T-follicular-helper (TFH) phenotype. Therefore, we analyzed whether βGL1-22 or LGL1-specific type II NKT cells acquire TFH phenotype (CXCR5hi PD1hi ICOShi BCL6+IL-21+) upon immunization with βGL1-22 or LGL1. Separate groups of WT (C57BL/6), CD1d-/- and Jα18-/- mice were immunized with vehicle, α-GalCer, βGL1-22 or LGL1, after 7 days splenocytes and serum from each mice strain was assessed for upregulation of TFH markers and germinal center (GC) B cells by flow cytometry and antibodies by ELISA respectively. In contrast to type-I NKT cells, βGL1-22 and LGL1-specific NKT cells constitutively expressed TFH phenotype. The magnitude of TFH response was dictated by the amount of antigen and directly correlated with induction of GC B cells, hypergammaglobulinemia and production of anti-lipid antibodies. Complementarily, in vitro co culture of human βGL1-22 and LGL1-specific type II NKT cells with purified autologous B cells induced TFH markers in lipid specific T cells and led to expansion of plasmablasts and Ig secretion. In order to study changes in βGL1-22/LGL1-specific T cells in the setting of a disease wherein these ligands are altered, we analyzed patients and mouse models with GD. Compared to wild type control mice, GD mice exhibited >20-fold increase of LGL1-specific T cells. Concomitant to increase in LGL1-specific T cells in GD mice, notable up regulation of TFH markers and induction of anti-lipid antibodies was also observed. Similar to GD mice, sphingolipids accumulation in GD patients results in an increase of LGL1-specific type II NKT cells which correlated with biomarkers of disease severity and show reduction upon clinical improvement with enzyme replacement therapy.
These data reveal and characterize a new subset of human and murine type II NKT-TFH cells against lipids dysregulated in Gaucher disease that regulate B cell immunity and inflammation and provide a mechanistic link between lipid storage and inflammation.

Disclosures
Pastores: Genzyme: Honoraria, Research Funding; Shire: Honoraria, Research Funding. Mistry:Genzyme, a Sanofi Company: Consultancy, Honoraria, Travel reimbursement, Research grants Other. Dhodapkar:Celgene: Research Funding.