Cholera intoxication of human enteroids reveals interplay between decoy and functional glycoconjugate ligands.

Prior research on cholera toxin (CT) binding and intoxication has relied on human colonic cancer derived epithelial cells. While these transformed cell lines have been beneficial, they neither derive from small intestine where intoxication occurs, nor represent the diversity of small intestinal epithelial cells (SI-ECs) and variation in glycoconjugate expression among individuals. Here, we used human enteroids, derived from jejunal biopsies of multipledonors to study CT binding and intoxication of human non-transformed SI-ECs. We modulated surface expression of glycosphingolipids, glycoproteins and specific glycans to distinguish the role of each glycan/glycoconjugate. Cholera-toxin-subunit-B (CTB) mutants were generated to decipher the preference of each glycoconjugate to different binding sites and the correlation between CT binding and intoxication. Human enteroids contain trace amounts of GM1, but other glycosphingolipids may be contributing to CT intoxication. We discovered that inhibition of either fucosylation or O-glycosylation sensitize enteroids to CT-intoxication. This can either be a consequence of the removal of fucosylated "decoy-like-ligands" binding to CTB's non-canonical site and/or increase in the availability of Gal/GalNAc-terminating glycoconjugates binding to the canonical site. Furthermore, simultaneous inhibition of fucosylation and O-glycosylation increased the availability of additional Gal/GalNAc-terminating glycoconjugates but counteracted the sensitization in CT intoxication caused by inhibiting O-glycosylation because of reduction in fucose. This implies a dual role of fucose as a functional glycan and a decoy, the interplay of which influences CT binding and intoxication. Finally, while the results were similar for enteroids from different donors, they were not identical, pointing to a role for human genetic variation in determining sensitivity to CT.

Fucosylated glycoproteins and fucosylated glycolipids play opposing roles in cholera intoxication.

Cholera toxin (CT) is the etiological agent of cholera. Here we report that multiple classes of fucosylated glycoconjugates function in CT binding and intoxication of intestinal epithelial cells. In Colo205 cells, knockout of B3GNT5, the enzyme required for synthesis of lacto- and neolacto-series glycosphingolipids (GSLs), reduces CT binding but sensitizes cells to intoxication. Overexpressing B3GNT5 to generate more fucosylated GSLs confers protection against intoxication, indicating that fucosylated GSLs act as decoy receptors for CT. Knockout (KO) of B3GALT5 causes increased production of fucosylated O-linked and N-linked glycoproteins, and leads to increased CT binding and intoxication. Knockout of B3GNT5 in B3GALT5 KO cells eliminates production of fucosylated GSLs but increases intoxication, identifying fucosylated glycoproteins as functional receptors for CT. These findings provide insight into molecular determinants regulating CT sensitivity of host cells.

Low-grade intestinal inflammation two decades after pelvic radiotherapy.

BACKGROUND: Radiotherapy is effective in the treatment of cancer but also causes damage to non-cancerous tissue. Pelvic radiotherapy may produce chronic and debilitating bowel symptoms, yet the underlying pathophysiology is still undefined. Most notably, although pelvic radiotherapy causes an acute intestinal inflammation there is no consensus on whether the late-phase pathophysiology contains an inflammatory component or not. To address this knowledge gap, we examined the potential presence of a chronic inflammation in mucosal biopsies from irradiated pelvic cancer survivors. METHODS: We biopsied 24 cancer survivors two to 20 years after pelvic radiotherapy, and four non-irradiated controls. Using tandem mass tag (TMT) mass spectrometry and mRNA sequencing (mRNA-seq), we charted proteomic and transcriptomic profiles of the mucosal tissue previously exposed to a high or a low/no dose of radiation. Changes in the immune cell populations were determined with flow cytometry. The integrity of the protective mucus layers were determined by permeability analysis and 16S rRNA bacterial detection. FINDINGS: 942 proteins were differentially expressed in mucosa previously exposed to a high radiation dose compared to a low radiation dose. The data suggested a chronic low-grade inflammation with neutrophil activity, which was confirmed by mRNA-seq and flow cytometry and further supported by findings of a weakened mucus barrier with bacterial infiltration. INTERPRETATION: Our results challenge the idea that pelvic radiotherapy causes an acute intestinal inflammation that either heals or turns fibrotic without progression to chronic inflammation. This provides a rationale for exploring novel strategies to mitigate chronic bowel symptoms in pelvic cancer survivors. FUNDING: This study was supported by the King Gustav V Jubilee Clinic Cancer Foundation (CB), The Adlerbertska Research Foundation (CB), The Swedish Cancer Society (GS), The Swedish State under the ALF agreement (GS and CB), Mary von Sydow's foundation (MA and VP).

A type I interferon footprint in pre-operative biopsies is an independent biomarker that in combination with CD8+ T cell quantification can improve the prediction of response to neoadjuvant treatment of rectal adenocarcinoma.

Tailored treatment for patients with rectal cancer requires clinically available markers to predict their response to neoadjuvant treatment. The quantity of tumor-infiltrating lymphocytes (TILs) in pre-operative tumor biopsies has been suggested to predict a favorable response, but opposing results exist. A biopsy-adapted Immunoscore (ISB) based on TILs has recently emerged as a promising predictor of tumor regression and prognosis in (colo)rectal cancer. We aimed to refine the ISB for prediction of response using multiplex immunofluorescence (mIF) on pre-operative rectal cancer biopsies. We combined the distribution and density of conventional T cell subsets and γδT cells with a type I Interferon (IFN)-driven response assessed using Myxovirus resistance protein A (MxA) expression. We found that pathological complete response (pCR) following neoadjuvant treatment was associated with type I IFN. Stratification of patients according to the density of CD8+ in the entire tumor tissue and MxA+ cells in tumor stroma, where equal weight was assigned to both parameters, resulted in improved predictive quality compared to the ISB. This novel stratification approach using these two independent parameters in pre-operative biopsies could potentially aid in identifying patients with a good chance of achieving a pCR following neoadjuvant treatment.

Clonal relationships of memory B cell subsets in autoimmune mice.

Immunological memory protects our body from re-infection and it is composed of a cellular and a humoral arm. The B-cell branch with its memory B cells (MBCs), plasma cells and antibodies, formed either in a germinal centre (GC) -dependent or -independent manner, ensure that we can rapidly mount a recall immune response. Previous work in immunised wildtype (WT) mice have identified several subsets of MBCs whereas less is known under autoimmune conditions. Here, we have investigated the heterogeneity of the MBC compartment in autoimmune mouse models and examined the clonal relationships between MBC subsets and GC B cells in one of the models. We demonstrate the presence of at least four different MBC subsets based on their differential expression pattern of CD73, CD80 and PD-L2 in surrogate light chain-deficient (SLC-/-), MRL+/+ and MRLlpr/lpr mice, where most of the MBCs express IgM. Likewise, four MBC subsets could be identified in WT immunised mice. In SLC-/- mice, high-throughput sequencing of Ig heavy chains demonstrates that the two CD73-positive subsets are generally more mutated. Lineage tree analyses on expanded clones show overlaps between all MBC subsets and GC B cells primarily in the IgM sequences. Moreover, each of the three IgM MBC subsets could be found both as ancestor and progeny to GC B cells. This was also observed in the IgG sequences except for the CD73-negative subset. Thus, our findings demonstrate that several MBC subsets are present in autoimmune and WT mice. In SLC-/- mice, these MBC subsets are clonally related to each other and to GC B cells. Our results also indicate that different MBC subsets can seed the GC reaction.

Impact of thymidine phosphorylase and CD163 expression on prognosis in stage II colorectal cancer

BackgroundTumor-associated macrophages (TAM) are known to facilitate colorectal cancer (CRC) growth. High macrophage infiltration in thymidine phosphorylase (TYMP) expressing CRC may correspond to poor prognosis. The prognostic impact of the expression CD163, a receptor associated with TAM, and TYMP in stroma, respectively, tumor tissue is not yet established. The aim of this study was to identify the potential associations between TYMP and CD163 expression levels and relapse-free survival (RFS) of patients with stage II CRC, and if microdissection is of importance.MethodsStage II CRC patients, radically resected with relapse (n = 104), were matched to patients with a 5-year relapse-free follow-up (n = 206). Gene expression of TYMP and CD163 was analyzed in snap-frozen tumor tissues and in microdissected formalin-fixed tumor tissues separated into tumor epithelium and stroma.ResultsTYMP expression was high in poorly differentiated tumors, right-sided CRC, and tumors with high microsatellite instability CD163-expressing macrophages near tumor epithelial cells had high expression in poorly differentiated and T4 tumors. High TYMP expression in tumor epithelial cells was in the multivariate analyses associated with shorter relapse-free survival (hazard ratio 1.66; 95% confidence interval: 1.09–2.56; p < 0.05).ConclusionsTYMP expression in tumor epithelial cells was associated with RFS and emphasizes the need for tissue microdissection. Additional studies are needed to establish whether TYMP and CD163 could add clinically relevant information to identify high-risk stage II patients that could benefit from adjuvant chemotherapy. (AU)

A Fraction of CD8+ T Cells from Colorectal Liver Metastases Preferentially Repopulate Autologous Patient-Derived Xenograft Tumors as Tissue-Resident Memory T Cells.

The diversity of T cells in the human liver may reflect the composition of TILs in CRLM. Our ex vivo characterization of CRLM vs. adjacent liver tissue detected CD103+CD39+CD8+ TRM cells predominantly in CRLM, which prompted further assessments. These TRM cells responded to cognate antigens in vitro. As functional activities of autologous TILs are central to the implementation of personalized cancer treatments, we applied a patient-derived xenograft (PDX) model to monitor TILs' capacity to control CRLM-derived tumors in vivo. We established PDX mice with CRLMs from two patients, and in vitro expansion of their respective TILs resulted in opposing CD4+ vs. CD8+ TIL ratios. These CRLMs also displayed mutated KRAS, which enabled trametinib-mediated inhibition of MEK. Regardless of the TIL subset ratio, persistent or transient control of CRLM-derived tumors of limited size by the transferred TILs was observed only after trametinib treatment. Of note, a portion of transferred TILs was observed as CD103+CD8+ TRM cells that strictly accumulated within the autologous CRLM-derived tumor rather than in the spleen or blood. Thus, the predominance of CD103+CD39+CD8+ TRM cells in CRLM relative to the adjacent liver and the propensity of CD103+CD8+ TRM cells to repopulate the autologous tumor may identify these TILs as strategic targets for therapies against advanced CRC.

Impact of thymidine phosphorylase and CD163 expression on prognosis in stage II colorectal cancer.

BACKGROUND: Tumor-associated macrophages (TAM) are known to facilitate colorectal cancer (CRC) growth. High macrophage infiltration in thymidine phosphorylase (TYMP) expressing CRC may correspond to poor prognosis. The prognostic impact of the expression CD163, a receptor associated with TAM, and TYMP in stroma, respectively, tumor tissue is not yet established. The aim of this study was to identify the potential associations between TYMP and CD163 expression levels and relapse-free survival (RFS) of patients with stage II CRC, and if microdissection is of importance. METHODS: Stage II CRC patients, radically resected with relapse (n = 104), were matched to patients with a 5-year relapse-free follow-up (n = 206). Gene expression of TYMP and CD163 was analyzed in snap-frozen tumor tissues and in microdissected formalin-fixed tumor tissues separated into tumor epithelium and stroma. RESULTS: TYMP expression was high in poorly differentiated tumors, right-sided CRC, and tumors with high microsatellite instability CD163-expressing macrophages near tumor epithelial cells had high expression in poorly differentiated and T4 tumors. High TYMP expression in tumor epithelial cells was in the multivariate analyses associated with shorter relapse-free survival (hazard ratio 1.66; 95% confidence interval: 1.09-2.56; p < 0.05). CONCLUSIONS: TYMP expression in tumor epithelial cells was associated with RFS and emphasizes the need for tissue microdissection. Additional studies are needed to establish whether TYMP and CD163 could add clinically relevant information to identify high-risk stage II patients that could benefit from adjuvant chemotherapy.

Antigen-Presenting B Cells Program the Efferent Lymph T Helper Cell Response.

B cells interact with T follicular helper (Tfh) cells in germinal centers (GCs) to generate high-affinity antibodies. Much less is known about how cognate T-B-cell interactions influence Th cells that enter circulation and peripheral tissues. Therefore, we generated mice lacking MHC-II expressing B cells and, by thoracic duct cannulation, analyzed Th cells in the efferent lymph at defined intervals post-immunization. Focusing on gut-draining mesenteric lymph nodes (MLNs), we show that antigen-specific α4ß7+ gut-homing effector Th cells enter the circulation prior to CXCR5+PD-1+ Tfh-like cells. B cells appear to have no or limited impact on the early generation and egress of gut-homing Th cells but are critical for the subsequent appearance of Tfh-like cells that peak in the lymph before GCs have developed. At this stage, antigen-presenting B cells also reduce the proportion of α4ß7+ Th cells in the MLN and efferent lymph. Furthermore, cognate B-cell interaction drives a broad transcriptional program in Th cells, including IL-4 that is confined to the Tfh cell lineage. The IL-4-producing Tfh-like cells originate from Bcl6+ precursors in the LNs and have gut-homing capacity. Hence, B cells program the efferent lymph Th cell response within a limited window of time after antigenic challenge.

Interleukin-22 regulates B3GNT7 expression to induce fucosylation of glycoproteins in intestinal epithelial cells.

Interleukin (IL)-22 is a cytokine that plays a critical role in intestinal epithelial homeostasis. Its downstream functions are mediated through interaction with the heterodimeric IL-22 receptor and subsequent activation of signal transducer and activator of transcription 3 (STAT3). IL-22 signaling can induce transcription of genes necessary for intestinal epithelial cell proliferation, tissue regeneration, tight junction fortification, and antimicrobial production. Recent studies have also implicated IL-22 signaling in the regulation of intestinal epithelial fucosylation in mice. However, whether IL-22 regulates intestinal fucosylation in human intestinal epithelial cells and the molecular mechanisms that govern this process are unknown. Here, in experiments performed in human cell lines and human-derived enteroids, we show that IL-22 signaling regulates expression of the B3GNT7 transcript, which encodes a ß1-3-N-acetylglucosaminyltransferase that can participate in the synthesis of poly-N-acetyllactosamine (polyLacNAc) chains. Additionally, we find that IL-22 signaling regulates levels of the α1-3-fucosylated Lewis X (Lex) blood group antigen, and that this glycan epitope is primarily displayed on O-glycosylated intestinal epithelial glycoproteins. Moreover, we show that increased expression of B3GNT7 alone is sufficient to promote increased display of Lex-decorated carbohydrate glycan structures primarily on O-glycosylated intestinal epithelial glycoproteins. Together, these data identify B3GNT7 as an intermediary in IL-22-dependent induction of fucosylation of glycoproteins and uncover a novel role for B3GNT7 in intestinal glycosylation.

Antigen Presenting Cells from Tumor and Colon of Colorectal Cancer Patients Are Distinct in Activation and Functional Status, but Comparably Responsive to Activated T Cells.

Although mouse models of CRC treatments have demonstrated robust immune activation, it remains unclear to what extent CRC patients' APCs and TILs interact to fuel or quench treatment-induced immune responses. Our ex vivo characterization of tumor and adjacent colon cell suspensions suggest that contrasting environments in these tissues promoted inversed expression of T cell co-stimulatory CD80, and co-inhibitory programmed death (PD)-ligand1 (PD-L1) on intratumoral vs. colonic APCs. While putative tumor-specific CD103+CD39+CD8+ TILs expressed lower CD69 (early activation marker) and higher PD-1 (extended activation/exhaustion marker) than colonic counterparts, the latter had instead higher CD69 and lower PD-1 levels. Functional comparisons showed that intratumoral APCs were inferior to colonic APCs regarding protein uptake and upregulation of CD80 and PD-L1 after protein degradation. Our attempt to model CRC treatment-induced T cell activation in vitro showed less interferon (IFN)-γ production by TILs than colonic T cells. In this model, we also measured APCs' CD80 and PD-L1 expression in response to activated co-residing T cells. These markers were comparable in the two tissues, despite higher IFN- γ exposure for colonic APCs. Thus, APCs within distinct intratumoral and colonic milieus showed different activation and functional status, but were similarly responsive to signals from induced T cell activation.

The Microenvironment of Small Intestinal Neuroendocrine Tumours Contains Lymphocytes Capable of Recognition and Activation after Expansion.

Traditionally, immune evasion and immunotherapy have been studied in cancers with a high mutational load such as melanoma or lung cancer. In contrast, small intestinal neuroendocrine tumours (SINETs) present a low frequency of somatic mutations and are described as genetically stable tumours, rendering immunotherapies largely unchartered waters for SINET patients. SINETs frequently metastasise to the regional lymph nodes and liver at the time of diagnosis, and no curative treatments are currently available for patients with disseminated disease. Here, we characterised the immune landscape of SINET and demonstrated that tumour-infiltrating lymphocytes (TILs) can be expanded and activated during autologous tumour challenge. The composition of lymphocyte subsets was determined by immunophenotyping of the SINET microenvironment in one hepatic and six lymph node metastases. TILs from these metastases were successfully grown out, enabling immunophenotyping and assessment of PD-1 expression. Expansion of the TILs and exposure to autologous tumour cells in vitro resulted in increased T lymphocyte degranulation. This study provides insights into the largely unknown SINET immune landscape and reveals the anti-tumour reactivity of TILs, which might merit adoptive T cell transfer as a feasible treatment option for patients with SINET.

Biomarkers and cell-based models to predict the outcome of neoadjuvant therapy for rectal cancer patients.

Rectal cancer constitutes approximately one-third of all colorectal cancers and contributes to considerable mortality globally. In contrast to colon cancer, the standard treatment for localized rectal cancer often involves neoadjuvant chemoradiotherapy. Tumour response rates to treatment show substantial inter-patient heterogeneity, indicating a need for treatment stratification. Consequently researchers have attempted to establish new means for predicting tumour response in order to assist in treatment decisions. In this review we have summarized published findings regarding potential biomarkers to predict neoadjuvant treatment response for rectal cancer tumours. In addition, we describe cell-based models that can be utilized both for treatment prediction and for studying the complex mechanisms involved.

Cryopreservation of Whole Tumor Biopsies from Rectal Cancer Patients Enable Phenotypic and In Vitro Functional Evaluation of Tumor-Infiltrating T Cells.

TILs comprise functionally distinct conventional and unconventional T cell subsets and their role in responses to CRC treatments is poorly understood. We explored recovery of viable TILs from cryopreserved tumor biopsies of (chemo)-radiated patients with rectal cancer to establish a platform for retrospective TIL analyses of frozen tumors from pre-selected study cohorts. Frequencies of TIL subsets and their capacity to mount IFN-γ responses in cell suspensions of fresh vs. cryopreserved portions of the same tumor biopsies were determined for platform validation. The percentages and proportions of CD4+ TILs and CD8+ cytotoxic T lymphocytes (CTLs) among total TILs were not affected by cryopreservation. While recovery of unconventional γδ T cells and mucosal-associated invariant T cells (MAIT cells) was stable after cryopreservation, the regulatory T cells (Tregs) were reduced, but in sufficient yields for quantification. IFN-γ production by in vitro-stimulated CD4+ TILs, CTLs, γδ T cells, and MAIT cells were proportionally similar in fresh and cryopreserved tumor portions, albeit the latter displayed lower levels. Thus, the proposed platform intended for TIL analyses on cryopreserved tumor biobank biopsies holds promises for studies linking the quantity and quality of TIL subsets with specific clinical outcome after CRC treatment.

Targeting Multiple Binding Sites on Cholera Toxin B with Glycomimetic Polymers Promotes the Formation of Protein-Polymer Aggregates.

The canonical binding site on the B subunit of cholera toxin (CTB) binds to GM1 gangliosides on host cells. However, the recently discovered noncanonical binding site on CTB with affinity for fucosylated molecules has raised the possibility that both sites can be involved in initiating intoxication. Previously, we showed that blocking CTB binding to human and murine small intestine epithelial cells can be increased by simultaneously targeting both binding sites with multivalent norbornene-based glycopolymers [ACS Infect. Dis. 2020, 6, 5, 1192-1203]. However, the mechanistic origin of the increased blocking efficacy was unclear. Herein, we observed that mixing CTB pentamers and glycopolymers that display fucose and galactose sugars results in the formation of large aggregates, which further inhibits binding of CTB to human granulocytes. Dynamic light scattering analysis, small-angle X-ray scattering analysis, transmission electron microscopy, and turbidimetric assays revealed that the facial directionality of CTB promotes interchain cross-linking, which in turn leads to self-assembly of protein-polymer networks. This cross-linking-induced self-assembly occurs only when the glycopolymer system contains both galactose and fucose. In an assay of the glycopolymer's ability to block CTB binding to human granulocytes, we observed a direct correlation between IC50 and self-assembly size. The aggregation mechanism of inhibition proposed herein has potential utility for the development of low-cost macromolecular clinical therapeutics for cholera that do not have exotic architectures and do not require complex synthetic sequences.

Fucose-Galactose Polymers Inhibit Cholera Toxin Binding to Fucosylated Structures and Galactose-Dependent Intoxication of Human Enteroids.

A promising strategy to limit cholera severity involves blockers mimicking the canonical cholera toxin ligand (CT) ganglioside GM1. However, to date the efficacies of most of these blockers have been evaluated in noncellular systems that lack ligands other than GM1. Importantly, the CT B subunit (CTB) has a noncanonical site that binds fucosylated structures, which in contrast to GM1 are highly expressed in the human intestine. Here we evaluate the capacity of norbornene polymers displaying galactose and/or fucose to block CTB binding to immobilized protein-linked glycan structures and also to primary human and murine small intestine epithelial cells (SI ECs). We show that the binding of CTB to human SI ECs is largely dependent on the noncanonical binding site, and interference with the canonical site has a limited effect while the opposite is observed with murine SI ECs. The galactose-fucose polymer blocks binding to fucosylated glycans but not to GM1. However, the preincubation of CT with the galactose-fucose polymer only partially blocks toxic effects on cultured human enteroid cells, while preincubation with GM1 completely blocks CT-mediated secretion. Our results support a model whereby the binding of fucose to the noncanonical site places CT in close proximity to scarcely expressed galactose receptors such as GM1 to enable binding via the canonical site leading to CT internalization and intoxication. Our finding also highlights the importance of complementing CTB binding studies with functional intoxication studies when assessing the efficacy inhibitors of CT.

Activated Peyer's patch B cells sample antigen directly from M cells in the subepithelial dome.

The germinal center (GC) reaction in Peyer's patches (PP) requires continuous access to antigens, but how this is achieved is not known. Here we show that activated antigen-specific CCR6+CCR1+GL7- B cells make close contact with M cells in the subepithelial dome (SED). Using in situ photoactivation analysis of antigen-specific SED B cells, we find migration of cells towards the GC. Following antigen injection into ligated intestinal loops containing PPs, 40% of antigen-specific SED B cells bind antigen within 2 h, whereas unspecifc cells do not, indicating B cell-receptor involvment. Antigen-loading is not observed in M cell-deficient mice, but is unperturbed in mice depleted of classical dendritic cells (DC). Thus, we report a M cell-B cell antigen-specific transporting pathway in PP that is independent of DC. We propose that this antigen transporting pathway has a critical role in gut IgA responses, and should be taken into account when developing mucosal vaccines.

Endothelial repair is dependent on CD11c+ leukocytes to establish regrowing endothelial sheets with high cellular density.

Endothelial injury makes the vessel wall vulnerable to cardiovascular diseases. Injured endothelium regenerates by collective sheet migration, that is, the endothelial cells coordinate their motion and regrow as a sheet of cells with retained cell-cell contacts into the wounded area. Leukocytes appear to be involved in endothelial repair in vivo; however, little is known about their identity and role in the reparative sheet migration process. To address these questions, we developed a high-quality en face technique that enables visualizing of leukocytes and endothelial cells simultaneously following an endoluminal scratch wound injury of the mouse carotid artery. We discovered that regrowing endothelium forms a broad proliferative front accompanied by CD11c+ leukocytes. Functionally, the leukocytes were dispensable for the initial migratory response of the regrowing endothelial sheet, but critical for the subsequent formation and maintenance of a front zone with high cellular density. Marker expression analyses, genetic fate mapping, phagocyte targeting experiments, and mouse knock-out experiments indicate that the CD11c+  leukocytes were mononuclear phagocytes with an origin from both Ly6Chigh and Ly6Clow monocytes. In conclusion, CD11c+ mononuclear phagocytes are essential for a proper endothelial regrowth following arterial endoluminal scratch injury. Promoting the endothelial-preserving function of CD11c+  leukocytes may be a strategy to enhance endothelial repair following surgical and endovascular procedures.

Innate and adaptive stimulation of murine diverse NKT cells result in distinct cellular responses.

Natural killer T (NKT) cells recognize glycolipids presented on CD1d. They share features of adaptive T lymphocytes and innate NK cells, and mediate immunoregulatory functions via rapid production of cytokines. Invariant (iNKT) and diverse (dNKT) NKT cell subsets are defined by their TCR. The immunological role of dNKT cells, that do not express the invariant TCRα-chain used by iNKT cells, is less well explored than that of iNKT cells. Here, we investigated signals driving Toll-like receptor (TLR) ligand activation of TCR-transgenic murine dNKT cells. IFN-γ production by dNKT cells required dendritic cells (DC), cell-to-cell contact and presence of TLR ligands. TLR-stimulated DC activated dNKT cells to secrete IFN-γ in a CD1d-, CD80/86- and type I IFN-independent manner. In contrast, a requirement for IL-12p40, and a TLR ligand-selective dependence on IL-18 or IL-15 was observed. TLR ligand/DC stimulation provoked early secretion of pro-inflammatory cytokines by both CD62L+ and CD62L- dNKT cells. However, proliferation was limited. In contrast, TCR/co-receptor-mediated activation resulted in proliferation and delayed production of a broader cytokine spectrum preferentially in CD62L- dNKT cells. Thus, innate (TLR ligand/DC) and adaptive (TCR/co-receptor) stimulation of dNKT cells resulted in distinct cellular responses that may contribute differently to the formation of immune memory.