Type II NKT-T_FH cells against Gaucher lipids regulate B-cell immunity and inflammation

Natural killer T (NKT) cells are distinct innate lymphocytes that recognize lipid/glycolipid antigens in the context of the major histocompatibility complex (MHC)-like molecule CD1d.¹  NKT cells are currently classified into 2 major subsets: type I or invariant NKT (iNKT) cells that express a semi-invariant T-cell receptor (TCR) and recognize the prototypic antigen α-galactosylceramide (α-GalCer), and type II or diverse NKT cells that use diverse TCR α and β chains and do not recognize α-GalCer (reviewed in Godfrey et al² ). The widely studied type I NKT cells are more prevalent than type II NKT cells in mice as compared with humans, whereas type II NKT cells comprise the dominant subset of human CD1d-restricted T cells.³  Recent studies have begun to implicate a distinct regulatory role for type II NKT cells (or the type I/type II NKT balance) in several settings including autoimmunity, inflammation, obesity, and protection against tumors and pathogens.^4-15 

Sulfatide was the first antigen recognized as a target for murine type II NKT cells, and sulfatide-reactive T cells are currently the best-studied subset of murine type II NKT cells.^4,6  Studies with murine transgenic or sulfatide-reactive NKT cells have suggested that these cells have a diverse but oligoclonal TCR repertoire and distinct genomic profile and mode of TCR binding compared with type I NKT cells.^16-19  The spectrum of putative murine type II NKT ligands has now widened, and some of these ligands can be recognized by both type I and type II NKT cells.^20-27  Importantly, there are some species-specific differences in ligand recognition between human and murine NKT cells.^23,28  Understanding the diversity and functional properties of human type II NKT cells against defined lipids is therefore of great interest in view of their potential immunoregulatory role in several disease states.^4,5 

Dysregulation of glucosphingolipids (GSLs) has been demonstrated in several metabolic disorders, including Gaucher disease (GD) and obesity.^29,30  GD is an inborn error of metabolism due to deficiency of the lysosomal enzyme glucocerebrosidase (acid-β-glucosidase [GBA]).^30,31  GBA deficiency leads to progressive lysosomal storage of β-glucosylceramide (β-GlcCer; GL1) and its deacylated product, glucosylsphingosine (Lyso-GL1; LGL1), most conspicuously in the mononuclear phagocytes.^32,33  Elevated levels of these lipids can also be detected in circulation, leading to modest elevation in GL1 and a marked increase in LGL1 levels.³⁴  Analysis of fatty acid acyl compositions of spleen from GD patients reveals that β-glucosylceramide 22:0 (βGL1-22) and GL1-24:0 are the most abundant β-GlcCer species.^35,36  The accumulation of lipids in GD patients is associated with a chronic progressive inflammatory state with an increase in inflammatory cytokines, activation of macrophages, and high incidence of B-cell activation, manifest as polyclonal and monoclonal gammopathy.^32,37-40  Interestingly, chronic inflammation has been observed in glucocerebrosidase-deficient mice with minimal substrate accumulation lacking classically engorged macrophages,³⁷  suggesting involvement of immune cells other than just macrophages in stimulating inflammation and B-cell activation. Here, we have analyzed the host response to GD lipids to gain insights into mechanisms underlying lipid-associated inflammation.

Six- to 9-week-old mice on a C57BL/6 background were used. CD1d^−/− mice⁴¹  and Jα 18^−/− on a C57BL/6 background were kindly provided by Dr Peter Cresswell (Yale University, New Haven, CT). The generation of conditional GBA knockout mice has been previously described.⁴²  All mice were bred and maintained in compliance with Yale University’s institutional animal care guidelines. Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated from buffy coats purchased from New York Blood Center or from patients with GD, following informed consents approved by the institutional review board in accordance with the Declaration of Helsinki.

CD14⁺ monocytes were separated from PBMCs with CD14 magnetic beads (Miltenyi Biotec) using the manufacturer’s protocol. MNCs from thymus, spleen, and liver were isolated following a protocol described earlier.⁴³ 

Data were acquired with the LSRII system (BD Biosciences) and FACSCalibur (BD Biosciences) and analyzed with FlowJo (Tree Star). Doublets were excluded with FSC1-FSC-H linearity. Mouse antibodies (clones) were as follows: anti-CD3e(145-2C11), anti-CD4(RM4-5), anti-CD8(53-6.7), anti-CD19(1D3), anti-B220(RA3-6B2), anti-F4/80(45-4801), anti-IgM(eB121-15F9), anti-IgD(11-26c), anti-CD279/PD1(J43), anti-CD185/CXCR5(SPRCL5), anti-MHC II(M5/114.15.2) (from eBioscience), anti-BCl-6(K112-91), anti-foxp3(MF23), anti-ICOS(15F9), and anti-IL-21(FFA21). The human antibodies anti-CD3e (SK7), anti-CD4(RPA-T4), anti-CD8(SK1), anti-CD45 RA(H100), anti-CD45RO(UCHL1), anti-CD56(MY31), anti-IFN-γ(B27), isotype control (MOPC-21), anti-CD19(SJ25C1), anti-CD27 (M-T271), and anti-IL-21(3A3-N2.1) were from BD Pharmingen; anti-TCR Vα24(C15) and anti-TCR Vβ11 (C21) were from Beckman Coulter; and anti-CD62L(FN50), anti-PLZF(Mags.21F7), anti- CD279/PD1(J105), anti-CD185/CXCR5(MU5UBEE), anti-CD38(HB7), anti-FAS(15A7), and anti-GL7(GL7) were from eBioscience.

NKT ligand α-galactosyl-ceramide (α-GalCer) was kindly provided by Kirin Breweries (Tokyo, Japan). β-D-GlcCer d_18:1-C24:1(15Z),C12:0 was from Avanti Polar Lipids. β-D-GlcCer d_18:1-C22:0 (thin-layer chromatography purity >98%) and lysoglucocerebroside (thin-layer chromatography purity >98%) were from Matreya. Mouse and human empty CD1d tetramer were obtained from the National Institutes of Health (NIH) tetramer facility (Atlanta, GA). A molar loading ratio (lipid to CD1d) of 200:1 was used for tetramerization studies for both mouse and human tetramers as used in other studies.^21,25  The unloaded multimer control was prepared by mixing CD1d tetramer with the carrier only.

For stimulation with anti-CD3/CD28 beads (Miltenyi Biotec), sorted tetramer-positive cells were stimulated with anti-CD3/CD28 beads in a 96-well U-bottom plate. For some experiments, sorted βGL1-22/LGL1/α-GalCer tetramer-positive cells were cultured with CD1d-expressing cell line (C1rd cells; kindly provided by Dr Mark Exley, Boston) or mock-transfected control cells at a T-cell: antigen-presenting cell (APC) ratio of 1: 0, as described previously,^22,44  with or without lipid pulsing.

Genomic DNA was isolated from CD1d tetramer flow-sorted βGL1-22/LGL1–specific T cells or type I NKT cells, and the diversity of TCR was profiled using high-throughput sequencing of rearranged TCR β loci from genomic DNA by Adaptive Biotechnologies (Seattle, WA) as previously described.^45,46 

C57BL/6, CD1d^−/−, or Jα18^−/− mice were injected intraperitoneally with phosphate-buffered saline, α-GalCer (4 μg/mouse), or βGL1-22 or LGL1 (200 μg/mouse) each week for 6 weeks in a volume of 100 μL. The mice were sacrificed 7 days after the last injection.

RNA from CD1d tetramer-sorted β-GL122/LGL1–specific T cells or iNKT cells was amplified, labeled, and hybridized on the Affymetrix Human Genome U133 Plus 2.0 microarray chips. The data were analyzed with GeneSpring GX 12.5 (Agilent Technologies) and Partek Genomice Suite (6.6) software, as described previously.^47,48 

The Student t test was used to compare data between 2 groups. Significance was set at P < .05. Correlation coefficient between 2 variables was calculated with the Pearson correlation test.

In order to detect human CD1d-restricted T cells specific for the GSLs βGL1-22 and its deacylated product, LGL1 (Figure 1A), βGL1-22– and LGL1-loaded human CD1d tetramers were used to stain freshly isolated human PBMCs from healthy blood donors. βGL1-22/LGL1–loaded CD1d tetramer-positive cells consisted predominantly of Vα24⁻Vβ11⁻CD3ε⁺ T cells (Figure 1B), whereas α-GalCer–loaded tetramer-positive cells were primarily Vα24⁺Vβ11⁺ T cells. These reagents specifically stained only CD3ε⁺ T cells, and there was no staining of CD3ε⁻ cells (data not shown). Frequency of βGL1-22–specific T cells was higher than LGL1-specific T cells in PBMCs from healthy donors (Figure 1C). βGL1-22/LGL1 tetramer-positive T cells were predominantly CD4⁺ or CD8⁺ T cells, as opposed to α-GalCer tetramer-positive T cells, which consisted mainly of CD4⁺ and double-negative (DN) cells (Figure 1D). βGL1-22/LGL1 tetramer-positive cells displayed naive phenotype (CD45RA^hi, CD62L^hi, CD69^neg/lo) and had low expression of natural killer (NK)-cell markers like CD56 and CD161. In contrast, α-GalCer tetramer-positive T cells revealed an effector/memory phenotype (CD45RO^hi, CD62L^lo, CD69^hi) and displayed classical NK-cell markers like CD56 and CD161 (Figure 1E). Both βGL1-22 and LGL1 tetramer-positive T cells were found to express NKT-associated transcription factor PLZF⁴⁹  similar to type I NKT cells, but at lower levels (Figure 1F). To gain additional insight into the overall spectrum of TCR use by βGL1-22/LGL1 tetramer-positive T cells, we analyzed Vβ chain usage of sorted βGL1-22/LGL1–specific T cells by TCR sequencing, which revealed diverse Vβ receptor usage among βGL1-22/LGL1–reactive T cells. Together, these data demonstrate that βGL1-22– and LGL1-specific CD1d-restricted human T cells can be detected in human PBMCs, use diverse TCRs, express PLZF, and are phenotypically distinct from α-GalCer–reactive type I NKT cells.

In order to further validate the ligand-specificity of tetramer-positive cells, we analyzed the capacity of tetramer-positive cells to respond to specific lipids in culture. Freshly isolated human PBMCs were cultured with α-GalCer, βGL1-22, or LGL1, and interferon-γ (IFN-γ) production in response to the glycolipids was measured by intracellular staining. We found that βGL1-22/LGL1 was able to stimulate IFN-γ production, and this effect was completely blocked by mAb specific to CD1d (Figure 2A-B). Compared with type I NKT cells, βGL1-22/LGL1 type II NKT cells secreted lower levels of IFN-γ (see MFI in Figure 2B), suggesting a lower stimulatory capacity of these ligands. Coculture of purified tetramer-positive cells with CD1d-expressing C1R cells confirmed that βGL1-22/LGL1 tetramer-positive T-cell responses to βGL1-22/LGL1 pulsed APCs were CD1d dependent, as the CD1d-negative parental cell line pulsed with either βGL1-22 or LGL1 did not stimulate cytokine secretion (Figure 2C). In order to further confirm antigen specificity, βGL1-22/LGL1–specific CD1d-restricted T cells were expanded in culture using respective lipid-loaded human monocyte–derived dendritic cells (DCs). βGL1-22/LGL1–loaded DCs led to expansion of lipid-specific T cells without concurrent expansion of invariant TCR (iTCR)-positive (Vα24⁺Vβ11⁺) cells (including in tetramer-negative fraction), unlike α-GalCer–loaded DCs, which led to clear expansion of Vα24⁺Vβ11⁺ T cells (Figure 2D). The diverse TCR-V-β repertoire of expanded T cells was also confirmed with the use of a flow cytometry–based V-β repertoire assay (supplemental Figure 1, available on the Blood Web site). Together, these data demonstrate that βGL1-22/LGL1–loaded CD1d tetramers detect T cells that respond functionally to the specific lipid in a CD1d-dependent manner.

The ability to detect and isolate βGL1-22/LGL1–specific human type II NKT cells allowed us to compare the global gene expression profiles of these cells with type I NKT cells using microarray analysis. Principal component analysis revealed that the gene expression profile signature for βGL1-22 and LGL1-specific T cells both before and after activation with anti-CD3/CD28 beads is distinct from that of type I NKT cells (Figure 2E and supplemental Table 1). Selected genes that are differentially expressed by βGL1-22/LGL1–specific type II NKT (relative to type I NKT) are shown in Figure 2F. Metacore analysis revealed that the top differentially expressed pathways involved DAP12 and Th17 pathways (supplemental Table 2). To further confirm the differences observed with respect to cytokines, we compared the cytokine secretion of sorted βGL1-22/LGL1 tetramer-positive cells with type I NKT cells in response to anti-CD3 stimulation. Compared with type I NKT cells, βGL1-22– and LGL1-specific T cells secreted higher levels of interleukin-5 (IL-5), IL-6, IL-10, IL-13, IL-17, granulocyte macrophage colony-stimulating factor, macrophage-derived chemokine, and macrophage inflammatory protein 1β (Figure 2G). This response was CD1d dependent, as it was not observed in cocultures with CD1d-deficient APCs (Figure 2H), corroborating the antigen specificity of βGL1-22/LGL1–specific type II NKT cells. Together, these data demonstrate that the human type II NKT cells identified here have a distinct genomic and cytokine profile compared with type I NKT cells, alluding to their potential for distinct roles in regulating human immune response.

Murine βGL1-22 and LGL1 tetramer-positive T cells are composed of both CD4⁺ (∼60% and 62%) and CD8⁺ T cells (∼35.1% and 29.6%), respectively, in contrast to α-GalCer tetramer-positive type I NKT cells, which are predominantly composed of CD4⁺ T cells (∼82%) (Figure 3A). βGL1-22 tetramer-positive and LGL1 tetramer-positive cells constitute ∼0.9% and 0.06% of lymphocytes in spleen and ∼4% and 1.5% of lymphocytes in liver, respectively, whereas α-GalCer tetramer-positive type I NKTs represent ∼2% of lymphocytes in spleen and ∼30% of lymphocytes in liver (Figure 3B). Frequency of βGL1-22–specific T cells was higher than LGL1-specific T cells in both splenocytes and liver MNCs (Figure 3B). To determine whether murine βGL1-22/LGL1–reactive T cells are a type II NKT population similar to human PBMCs, splenic as well as liver MNCs were simultaneously stained with α-GalCer and βGL1-22/LGL1–loaded CD1d tetramers conjugated to different fluorochromes. Both βGL1-22– and LGL1-specific T cells had a nonoverlapping profile with type I NKT cells (Figure 3C). This observation is further validated by the detection of βGL1-22/LGL1 tetramer-positive, but not α-GalCer tetramer-positive, cells in Jα18^−/− mice, which completely lack type I NKT cells. Both βGL1-22– and LGL1-specific T cells were deficient in splenocytes from CD1d^−/− mice, consistent with their CD1d-dependent nature (Figure 3D).

Transcriptional profiling data of βGL1-22– and LGL1-specific T cells revealed expression of genes associated with T_FH phenotype. Therefore, we examined if βGL1-22– or LGL1-specific type II NKT cells acquire the typical CXCR5^hi PD-1^hi ICOS^hi Bcl-6^hi T_FH phenotype upon immunization with βGL1-22 or LGL1. Phenotypic analysis revealed that βGL1-22– and LGL1-specific T cells showed constitutive expression of T_FH phenotype (defined as CXCR5^hi PD-1^hi ICOS ^hi Bcl-6^hi foxp3^neg IL-21⁺) in wild-type (WT) (Figure 4A) as well as Jα18^−/− mice (Figure 4B). Interestingly, α-GalCer CD1d tetramer-positive T cells showed T_FH phenotype in only 3% of iNKT cells at steady-state level (Figure 4A). Injection of βGL1-22 or LGL1 increased the frequency of antigen-specific T cells in vivo in the spleen of WT as well as Jα18^−/− mice (Figure 4C and supplemental Figures 2 and 3) exhibiting a sustained upregulation of T_FH markers CXCR5^hi PD-1^hi (Figure 4D), ICOS (supplemental Figure 4), and IL-21 (supplemental Figure 5). Contrary to the findings at steady-state level, T_FH phenotype was also induced in type I NKT cells following activation by α-GalCer in a Jα18-dependent manner, as expected (Figure 4D). Injection of the lipids in CD1d^−/− mice did not result in the induction of either lipid-specific T cells or T_FH markers (data not shown). Injection of lipids did not lead to changes in CD8⁺ T cells (data not shown). Notably, injection of βGL1-22 in WT mice led to reduction in α-GalCer CD1d tetramer-positive cells (Figure 4C) concurrent with activation of ICOS and IL-21 expression (supplemental Figures 4 and 5), underscoring a possible crosstalk between βGL1-22–specific type II NKT cells and type I NKT cells.^5,7,50  Together, these data show that murine βGL1-22– and LGL1-specific type II murine NKT cells respond to ligand-specific stimulation in vivo and constitutively express T_FH phenotype, in contrast to type I NKT cells, wherein T_FH phenotype requires stimulation by α-GalCer.

Injection with βGL1-22 or LGL1 in mice led to the induction of FAS⁺GL-7⁺ splenic germinal center (GC) B cells in WT and Jα18^−/− mice, but not in CD1d^−/− mice (Figure 4E and 4F). Increase in T_FH and GC B cells was also associated with an increase in total immunoglobulin G1 (IgG1), IgG2b, and IgM in WT and Jα18^−/− mice injected with respective lipids, but not in CD1d^−/− mice, confirming CD1d-restricted help provided by βGL1-22– and LGL1-specific type II NKT cells to B cells (Figure 4G). In order to evaluate the effect of these lipids on the induction of lipid-specific antibodies, we analyzed the sera from these mice for lipid-specific antibodies by enzyme-linked immunosorbent assay (ELISA). Injection of βGL1-22 and LGL1 led to the induction of corresponding lipid-specific antibodies in WT and Jα18^−/− mice, but not in CD1d-deficient mice (Figure 4H). Injection of α-GalCer also led to some increase in βGL1-22-specific antibodies, albeit less than following the injection of specific ligands. Together, these data indicate that lipid-mediated activation of βGL1-22/LGL1–specific type II NKT cells is associated with the induction of GC B cells and lipid-specific antibodies in vivo in a CD1d-dependent manner.

Coculture of human βGL1-22 and LGL1-specific type II NKT cells and autologous B cells induced expansion of CD27^hiCD38^hi plasmablasts in the presence of respective lipids from cocultured B cells (Figure 5A-B). B cells pulsed with vehicle or cultured with tetrameter-negative cells did not show any significant effect on the frequency of CD27^hiCD38^hi cells (Figure 5B). Coculture of βGL1-22 and LGL1-specific type II NKT cells with autologous B cells induced greater immunoglobulin secretion relative to B cells cultured alone or with non-NKT cells (Figure 5C). Parallel to B-cell differentiation into plasmablasts in the coculture, βGL1-22– and LGL1-specific type II NKT cells were found to express T_FH markers (CXCR5^hiPD1^hi ICOS^hi Bcl-6^hi, IL-21⁺) (Figure 5D). Interestingly α-GalCer tetramer-positive T cells did lead to some induction of plasmablasts and immunoglobulin, albeit less than with type II NKT cells (Figure 5A,C), despite not having T_FH phenotype (Figure 5D) in these cultures. Together, these results demonstrate that human βGL1-22– and LGL1-specific type II NKT cells can provide efficient help to B cells.

In order to test if these NKT cells are altered in vivo in the context of a disease state associated with alteration of corresponding lipids, we analyzed mouse models and patients with GD. Mistry et al recently developed a murine model that faithfully recapitulates the clinical phenotype of type I human GD.⁴²  Therefore, we analyzed T cells from the spleen and lymph nodes of these mice for the presence of βGL1-22– and LGL1-reactive T cells using CD1d tetramers (Figure 6A). Compared with WT control mice, GBA^−/− mice exhibited a >20-fold increase of LGL1-specific T cells (Figure 6B). In contrast, compared with the WT control mice, the proportion of type I NKT cells was strikingly reduced in GBA^−/− mice (Figure 6B). Therefore, lysosomal GSL accumulation in GBA^−/− mice was associated with an altered balance of type I vs type II NKT cells. Concomitant to increase in LGL1-specific T cells in GBA^−/− mice, upregulation of T_FH markers in these cells was also observed (Figure 6C).

To extend the findings from GD mice to patients, we examined the presence of βGL1-22– and LGL1-specific T cells as well as type I NKT cells in PBMCs from GD patients (n = 20) and compared the findings to healthy donors. Clinical features of the type 1 GD patient cohort are shown in supplemental Table 3. In contrast to healthy donors, the majority of βGL1-22– and LGL1-specific T cells had a CD45RO⁺ memory and T_FH phenotype, consistent with ligand-mediated activation in vivo (supplemental Figures 6 and 7). The proportion of circulating LGL1-specific T cells was significantly increased in GD patients (percent mean ± SEM: 0.16 ± 0.06 vs 1.263 ± 0.25, P < .001), whereas the proportion of type I NKT cells was not significantly different (Figure 7A). The percentage of LGL1-specific, but not βGL1-22–specific, T cells or type 1 NKT cells correlated with the serum level of chitotriosidase, an established biomarker of clinical disease activity in GD (R = 0.70; P < .001) (Figure 7B). The increase in LGL1-specific T cells was most noticeable in untreated patients (n = 5), and the levels of these T cells declined after enzyme-replacement therapy (Figure 7C). Analysis of cytokine profile in the serum of GD patients revealed an increase in several cytokines (IL-17, IL-6, macrophage inflammatory protein 1β, fms-related tyrosine kinase 3 ligand, IL-8, and soluble IL-2 receptor) (Figure 7D), which correlated with the cytokine profile of LGL1-reactive T cells. Together, these data show that as with GD mice, GSL accumulation in GD patients is associated with an altered balance of type I and II NKT cells and, importantly, that disease severity in the GD patients correlates with circulating LGL1-specific NKT cells.

Polyclonal type II NKT cells were initially discovered in the context of MHCII^−/− mice and are currently identified as CD1d-restricted T cells lacking iTCR.^51,52  In contrast to type I NKT cells, information on functional properties of human type II NKT cells and their dysregulation in the context of human disease is extremely limited. We focused our studies on 2 distinct GSLs, βGL1-22 and LGL1, which are known to be dysregulated in several human metabolic disorders. We show that in contrast to type I NKT cells,⁵³  βGL1-22– and LGL1-specific type II NKT cells constitutively express T_FH phenotype. We also show that these cells have a distinct cytokine profile and provide robust help to B cells. This study therefore identifies a novel pathway mediated by type II NKT-T_FH cells for B-cell immunity and inflammation.

β-Glucosylceramide with acyl chains (12:0 and 24:1) was initially identified as a ligand for type I NKT cells.²¹  However, recent studies using sulfatide-reactive type II NKT cell hybridoma XV19 and Jα18-deficient IL-4 reporter mice report recognition of β-GlcCer (12:0 and 24:1) by murine type II NKT cells.^24,26  In contrast to initial findings from Brennan et al, our data pointed to β-glucosylceramide as a ligand for type II as opposed to type I NKT cells. Recent studies have attributed Brennan’s initial findings to a contaminating minor lipid species, consistent with endogenous α-linked glucosylceramide.^54-56  These new studies therefore provide further support to our conclusion that β-GlcCer is primarily a type II ligand. Interestingly, in a small minority of donors/experiments (supplemental Figure 8), we did observe an increase in human iNKT cells following stimulation with β-GlcCer. This was not specific for individual lipid preparations but rather individual donors and tracked with higher baseline iNKT cells. In light of new studies, underlying reasons may include contaminating lipids or variations in endogenous α-anomeric lipid antigens in humans, which need further study.

In contrast to βGL1-22, LGL1-specific T cells were found to belong exclusively to the polyclonal human type II NKT subset. Phenotypic characterization, TCR sequencing, and transcriptional and cytokine profiling provided additional confirmation that human βGL1-22/LGL1–specific T cells are distinct from type I NKT cells. Notably, these cells express the NKT-lineage factor PLZF, albeit at lower level than in type I NKT cells. In addition to its well-documented role in type I NKT cells, PLZF was recently implicated in the differentiation of type II NKT cells as well.^26,57  Crosstalk between type I and II NKT cells is increasingly implicated in the regulation of immunity to pathogens, tumors, and autoimmunity.^5,7,50,58  The mechanism underlying the crosstalk between NKT subsets requires further study and may be more prominent with specific ligands. For example, in our studies, injection of βGL1-22 (but not LGL1) led to reduction in type I NKT cells in vivo. Similar findings have been made in other studies wherein activation of type II NKT cells led to decline in type I NKT cells in vivo.⁵⁰  Although human T cells specific for both βGL1-22 and LGL1 can be detected using lipid-loaded CD1d tetramers, the broad pattern of staining that we observed suggests a range of binding affinities. Further studies are needed to better understand the biological significance of this variation. This may be particularly important in the context of TCR usage, as it is possible that there may be some oligoclonality, particularly with higher-affinity type II NKT cells. Oligoclonality has also been observed with sulfatide-reactive type II NKT cells.¹⁶ 

An important and novel aspect of these studies is the potential role of type II NKT-T_FH cells in providing help to B cells and regulating antibody responses. Several studies have demonstrated that type I NKT cells can express BCL6 upon stimulation and co-opt the T_FH pathway to provide direct or indirect help to B-cell responses against model antigens and pathogens.^53,59-65  Initial studies suggested that type I NKT-induced antibody responses were rapid but transient, although a recent study challenged this view by showing that type I NKT cells can promote prolonged antibody responses to pneumococcal capsular polysaccharides by extrafollicular B-cell responses.^53,59-61,66  Intriguingly, βGL1-22/LGL1–specific T cells were found to constitutively express T_FH phenotype (CXCR5^hi PD1^hi ICOS^hi BCL6⁺IL-21⁺) in nearly 40% to 50% of lipid-reactive CD1d tetramer-positive T cells at steady state as opposed to type I NKT cells, which differentiated into T_FH cells only after activation by α-GalCer. A specific expansion of CD1d-restricted lipid-reactive tetramer-positive cell numbers was observed following injection of either βGL1-22 or LGL1 in WT/Jα18^−/− mice, but not in CD1d^−/− mice. Differences in T_FH phenotype and B-cell help between type I and II NKT cells are particularly prominent with human NKT cells, wherein type I NKT have only a weak T_FH phenotype.⁶⁷  Therefore, type II (as opposed to type I) NKT cells may be the major contributors to NKT-mediated B-cell help in humans, particularly as the relative proportions of type I vs II NKT cells are reversed between mice and humans.³ 

In order to study changes in βGL1-22/LGL1–specific T cells in a disease setting,³⁴  we analyzed patients and mouse models with GD. It is of interest that βGL1-22/LGL1–specific T cells in GD patients have a memory phenotype consistent with their in vivo activation due to dysregulation of these lipids. Analysis of the phenotype of these cells in cord blood would also be of interest. Although a causative link between the increase in LGL1-specific T cells and clinical phenotype of GD remains to be established, the observed correlation between levels of LGL1-specific T cells and disease activity and the reduction in the level of these T cells in vivo upon clinical improvement following therapy suggest that host immune response to lipids may contribute to the pathogenesis of chronic inflammation in GD. The finding that LGL1-specific T cells (including in GD patients) are enriched for a T_FH phenotype and lead to GC B-cell activation with production of antibodies suggests potential role of these cells in providing help for B-cell activation and increased risk of B-cell malignancy, such as myeloma observed in GD.^68-70  Further studies are needed to evaluate this possibility.

As the GSL ligands studied here, particularly LGL1, are commonly altered in several inflammatory states, our findings should prompt an evaluation of human LGL1-specific type II NKT cells in several clinical settings.²⁹  One clinical setting of particular interest is obesity, wherein an increase in these GSL ligands and changes in type II NKT cells have been previously implicated, although antigenic specificity of these T cells was not examined.^14,15,29,71  Further studies of subsets of human type II NKT cells described herein may have broad implications in metabolic disorders associated with inflammation.

The authors thank Anumeha Shah for technical help with Luminex assays, Cindy Desmarais at Adaptive Biotechnologies for help with TCR sequencing, and Kavita M. Dhodapkar for discussions. They acknowledge the NIH Tetramer Core Facility (contract HHSN27201300006C) for provision of unloaded human and murine CD1d tetramers.

M.V.D. is supported in part by funds from the NIH National Cancer Institute (CA135110, CA106802, CA156689) and the Leukemia and Lymphoma Society. P.K.M. is supported by NIH National Institute of Arthritis and Musculoskeletal and Skin Diseases (65932) and Center of Excellence Grant in Clinical Translational Research from Genzyme, a Sanofi company.

Contribution: S.N. designed and performed the majority of experiments, analyzed data, and wrote the manuscript; C.S.B. performed experiments and analyzed data; R.V. analyzed data; J.L. and R.Y. performed some experiments; G.M.P. provided patient materials and analyzed data; P.K.M. provided mouse models of GD and patient materials, and helped design some of the experiments, interpret data, and write the paper; and M.V.D. developed and supervised the entire project, designed experiments, interpreted data, and wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

The current affiliation for G.M.P. is Mater Misericordiae University Hospital, Dublin, Ireland.

Correspondence: Madhav V. Dhodapkar, 333 Cedar St, WWW 211b, Yale University School of Medicine, New Haven, CT 06515; e-mail: madhav.dhodapkar@yale.edu.