Cell Surface CD1d Research Articles

Effect of CD4(+) T cell surface CD1d molecules on progression of multiple sclerosis in mouse experimental autoimmune encephalomyelitis model

Objective: To investigate the effect of CD1d molecules on the surface of CD4(+) T cells on the progression of experimental autoimmune encephalomyelitis (experimental, allergic, encephalomyelitis, EAE) mouse models. Methods: EAE model of C57BL/6 mice was established, Splenic cells were isolated at different stages of the progression of the disease.The proportion of CD1d(+) cells on the surface of activated and non activated CD4(+) T cells was detected by flow cytometry. Results: The proportion of CD1d(+) cells in the control group (normal group and complete Freund's adjuvant (CFA) group), in the peak and recovery period of disease in the EAE group were compared.The proportion of CD1d(+) cells in the control group was (8.98 ±0.36)%, and the proportion of CD1d(+) cells in the peak and recovery period of disease in the EAE group were respectively (2.14±0.15)% and (13.80±0.84)%.The differences were statistically significant difference (P<0.05). Conclusion: The trend of the proportion of CD4(+) T cells expressing CD1d molecules during the course of EAE pathogenesis is verified, which lays a foundation for further study on the interaction between CD4(+) T cells and NKT cells in the progression of EAE models.

Human Invariant Natural Killer T Cells Respond to Antigen-Presenting Cells Exposed to Lipids from Olea europaea Pollen

Background: Allergic sensitization might be influenced by the lipids present in allergens, which can be recognized by natural killer T (NKT) cells on antigen-presenting cells (APCs). The aim of this study was to analyze the effect of olive pollen lipids in human APCs, including monocytes as well as monocyte-derived macrophages (Mϕ) and dendritic cells (DCs). Methods: Lipids were extracted from olive (Olea europaea) pollen grains. Invariant (i)NKT cells, monocytes, Mϕ, and DCs were obtained from buffy coats of healthy blood donors, and their cell phenotype was determined by flow cytometry. iNKT cytotoxicity was measured using a lactate dehydrogenase assay. Gene expression of CD1A and CD1D was performed by RT-PCR, and the production of IL-6, IL-10, IL-12, and TNF-α cytokines by monocytes, Mϕ, and DCs was measured by ELISA. Results: Our results showed that monocytes and monocyte-derived Mϕ treated with olive pollen lipids strongly activate iNKT cells. We observed several phenotypic modifications in the APCs upon exposure to pollen-derived lipids. Both Mϕ and monocytes treated with olive pollen lipids showed an increase in CD1D gene expression, whereas upregulation of cell surface CD1d protein occurred only in Mϕ. Furthermore, DCs differentiated in the presence of human serum enhance their surface CD1d expression when exposed to olive pollen lipids. Finally, olive pollen lipids were able to stimulate the production of IL-6 but downregulated the production of lipopolysaccharide- induced IL-10 by Mϕ. Conclusions: Olive pollen lipids alter the phenotype of monocytes, Mϕ, and DCs, resulting in the activation of NKT cells, which have the potential to influence allergic immune responses.

The Rho-ROCK pathway as a new pathological mechanism of innate immune subversion in chronic myeloid leukaemia.

CD1d-restricted invariant natural killer T (iNKT) cells are believed to play a key role in cancer immune surveillance, and are functionally deficient in patients with chronic myeloid leukaemia (CML). Herein, we have hypothesized that this defect might originate from BCR-ABL-dependent dysfunctions in myeloid dendritic cells (mDCs). Indeed, flow cytometry and confocal microscopy revealed that cell surface expression of CD1d was downregulated in CML mDCs, relative to healthy donor (HD) controls. The decreased cell surface display of CD1d could not be ascribed to defective mDC differentiation, as attested by normal expression of HLA-DR and the CD86 maturation marker. On the other hand, reduced membrane expression was not associated with decreased intracytoplasmic levels of CD1d or its mRNA transcripts, consistent with intracellular retention. In vitro treatment of CML mDCs with the Rho-associated protein kinase (ROCK) inhibitor Y-27632 partially restored both cell surface CD1d expression and CD1d-mediated antigen presentation, whereas it had no effect on HD mDCs. An inhibitor of BCR-ABL tyrosine kinase (TK), imatinib mesylate (IM), had no such activity. Similar recovery of CD1d expression occurred with fasudil, another ROCK inhibitor that is commonly used in clinical trials. Our data support the conclusion that BCR-ABL-dependent ROCK, but not TK, is involved in CD1d downregulation. We propose that ROCK, which is most likely activated by the DH/PH domain of BCR-ABL, mediates iNKT-cell immune subversion in CML patients by downregulating CD1d expression on CML mDCs. Our study reveals the ROCK-mDC axis as a new potential target to restore immune surveillance in patients with CML, offering new therapeutic perspectives for CML treatment. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The DH/PH domain of BCR-ABL mediates INKT cell immunosubversion in chronic myeloid leukemia by downregulating cell-surface CD1D expression in myeloid dendritic cells (TUM10P.1038)

Abstract Chronic myeloid leukemia (CML) is associated with a rearrangement between the Abelson (ABL) and the BCR genes, generating a chimeric fusion gene. The constitutive tyrosine-kinase (TK) activity linked to ABL is sufficient to induce malignant transformation while BCR contains a DH/PH domain ensuring activation of the Rho/ROCK pathway that is implicated in the control of mobility of leukemia cells. iNKT cells constitute a distinct population of T cells that recognize glycolipids as α-GC presented by CD1d, and are believed to play a key role in cancer immunosurveillance. CML in chronic phase (CP) is associated with functional deficiencies of iNKT cells. Here, we hypothesized that the anergic status of iNKT cells during CML-CP results from BCR-ABL-dependent dysfunctions in APC. Accordingly, CML mDC downregulated their cell surface expression of CD1d, as compared to mDC from healthy individuals. Since Rho/ROCK pathway is a negative regulator of CD1d expression, we surmised that its enhancement by the DH/PH domain of BCR-ABL is responsible for the impaired cell-surface CD1d expression in CML mDC. In favor of this hypothesis, we showed that treatment of PBMC by Rho/ROCK inhibitors restores in vitro cell-surface expression levels of CD1d on CML-CP mDC as well as expansion of CM-CP iNKT cells in response to α-GC. Altogether, these results indicate that the DH/PH domain of BCR-ABL mediates iNKT-cell immunosubversion in CML patients by downregulating cell-surface CD1d expression in mDC.

Induction of Regulatory B-Cells by Mesenchymal Stem Cells is Affected by SDF-1α-CXCR7

Background/Aims: Mesenchymal stem cells (MSCs) possess immunomodulatory properties on a diverse array of immune cell lineages, including regulatory T and B cells (Tregs and Bregs, respectively). However, their specific effects and mechanisms underlying induction of Bregs remain unclear. The immune regulatory function of MSCs is exerted through both cell-cell contact and the release of soluble factors. The main objective of this study was to examine the role of the SDF-1-CXCR4/CXCR7 axis in the secretory action of MSCs, and potential effects on the immunoregulatory function of these cells. Methods: MSCs were isolated from mouse bone marrow and characterized according to their multilineage differentiation potential and their surface antigen expression. CD19⁺ B cells purified from mice splenocytes were co-cultured with MSCs at various ratios in the presence of LPS and αCD40. After 4 days, intracellular IL-10 production and cell surface CD1d and CD5 expression by CD19⁺ B cells were determined using flow cytometry, and the secretion of IL-10, IL-6, IgM, and IgG were assessed with ELISA. MSCs were treated with different concentrations of stromal derived factor-1α (SDF-1α) stimuli or transiently overexpressed with CXCR7. and their cell viability and immune regulatory effects of MSCs on Bregs were assessed. Results: MSCs induced IL-10-producing regulatory B cells and primarily stimulated the CD1d⁺CD5⁺B cell subset of IL-10⁺Breg cells to express IL-10. IL-10, IL-6, and IgM secretion were additionally induced by MSCs. The CXCR7 pathway was required for MSC viability and the production of paracrine factors under SDF-1α culture condition. Low concentrations of SDF-1α promoted the immunomodulatory effect of MSCs, leading to a further increase in IL-10-producing regulatory B cells and IL-10 secretion. In contrast, high concentrations of SDF-1α inhibited MSCs induction of IL-10⁺Breg cells. Notably, CXCR7 overexpression in MSCs reversed the inhibitory effect of high concentrations of SDF-1α and promoted the immunomodulatory effect of these cells. Conclusion: MSCs induce IL-10⁺Breg cells, which contribute to the generation of an immunosuppressive environment. SDF-1α and its receptor, CXCR7 play important roles in the immunomodulatory function of MSCs by regulating their paracrine actions.

ATRA Upregulates Cell Surface CD1D on Myeloma Cells and Sensitizes Them to iNKT Cell-Mediated Lysis

Invariant natural killer T (iNKT) cells recognize lipid antigens in the context of CD1D and have been shown to effectively kill CD1D-expressing multiple myeloma (MM) cells. It has been reported that iNKT cells in MM patients are deficient and dysfunctional and that CD1D is variably expressed among MM patients and is completely lost in MM cell lines. Recent clinical study suggests that targeting iNKT cells may prevent progression of asymptomatic MM (AMM) to active disease (Richter at al. Blood 2013). All-Trans Retinoic Acid (ATRA) has been used for the treatment of MM but its modes of action have not been completely elucidated. The aims of this study were to shed light on the mechanism of action of ATRA and to identify ATRA target genes in MM. ATRA at clinically relevant concentrations (1-2 μM, 4 days) had minimal growth inhibitory effect on the majority of MM cell lines (10±13% and 14±16%, respectively; n=10). Gene expression profile (GEP) of MM cell lines and primary cases (n=13) treated with ATRA (1 μM, 2 days) revealed upregulation of CD1D (381±204 vs. 5692±1675 mean±SEM GEP signal in CONT and ATRA groups, respectively; p<0.006). Flow cytometry analysis for cell surface CD1D in 21 MM cases treated in vitro with ATRA revealed significant CD1D induction in 11 cases, intermediate induction in 2 cases and no induction in 8 cases. Clinically, MM cells from patients who received ATRA as part of their salvage therapy also had induced CD1D gene expression compared to levels prior to ATRA therapy. Analysis of abnormal plasma cells based on a single 8-color markers flow cytometry showed higher CD1D coefficient of variation (CV) in AMM cases molecularly classified as high-risk (n=5) than low-risk cases (n=9, p<0.003), while mean florescence intensity (MFI) was insignificantly lower in high-risk AMM cases. In vitro ATRA treatment of BM cells from high-risk AMM patient resulted in increased CD1D MFI by 2 folds and reduced CV in abnormal plasma cell restricted population. To test the functional consequences of CD1D upregulation in MM cells, iNKT cells were purified from healthy donors’ peripheral blood mononuclear cells using anti-Vα24Jα18 immunomagnetic beads and expanded in vitro via stimulation with irradiated autologous α-GalCer-loaded mature dendritic cells (DCs) in the presence of IL-2. ATRA treated (1 μM, 2 days) compared to vehicle treated MM cells (n=4) were more sensitive to iNKT cell-mediated lysis in 5 hour 51chromium-release assays or bioluminescence viability assays (e.g. 21% vs. 75% lysis of H929 cells, p<0.0001, effector: target ratio 2.5:1). ATRA-induced MM cell lysis by iNKT cells was markedly blocked by CD1D neutralizing antibody (1-40 μg/ml) indicating that the enhanced cell lysis was mediated through CD1D. Further GEP analysis identified RARRES3 (retinoic acid receptor responder protein 3) as an additional top ATRA target gene in MM cells. RARRES3 gene expression was commonly but variably induced by ATRA whereas MM cells expressing high baseline RARRES3 were resistant to ATRA-induced CD1D gene and protein expression. Expression levels of CD1D and RARRES3 were inversely correlated in MM cells from newly diagnosed patients (n=449, r=-0.4, p<0.0001). Among molecularly classified groups, the MF subtype, known to be associated with poor survival, had the lowest CD1D/highest RARRES3 expression, whereas the favorable CD2 subtype had a reversed pattern. We conclude that CD1D and RARRES3 are ATRA target genes in MM cells and that stimulation of cell surface CD1D expression on MM cells enhances sensitivity to iNKT cell-mediated lysis. Disclosuresvan Rhee:Senesco: PI Other.

B-cell linker protein expression contributes to controlling allergic and autoimmune diseases by mediating IL-10 production in regulatory B cells

Regulatory B cells that exhibit the cell-surface CD1d(hi)CD5(+) phenotype and produce IL-10 are termed B10 cells. Although B10 cells exert potent suppressive functions in patients with various allergic and autoimmunity disorders, the precise signaling mechanisms required for B10 cell functions remain unknown. B-cell linker protein (BLNK) is an essential component of the B-cell antigen receptor signaling pathway and is required for optimal B-cell development. We sought to elucidate the signaling pathways that are responsible for IL-10 production in B10 cells and in vivo mechanisms of how impaired B10 cell functions influence allergic and autoimmune responses. For in vitro assays, splenic CD1d(hi)CD5(+) B cells from BLNK-deficient (BLNK(-/-)) mice were analyzed for intracellular signaling pathways and cytokine production. Contact hypersensitivity (CHS) and experimental autoimmune encephalomyelitis were examined by using BLNK(-/-) mice. Although the CD1d(hi)CD5(+) B-cell population was present in BLNK(-/-) mice, IL-10 production was impaired both in vitro and in vivo. BLNK(-/-) mice had exaggerated CHS and experimental autoimmune encephalomyelitis responses, which were normalized by adoptive transfer of splenic CD1d(hi)CD5(+) B cells from wild-type mice. In mice with CHS, BLNK(-/-) mice exhibited decreased B-cell and regulatory T-cell percentages and increased CD8(+) T-cell percentages in the skin and lymph nodes. In vitro BLNK was required for LPS-induced signal transducer and activator of transcription 3 phosphorylation in CD1d(hi)CD5(+) B cells. Finally, secreted IL-10 leads to autocrine expansion of IL-10-producing B cells. BLNK serves as a critical signaling component for B10 cell function by mediating IL-10 production.

Requirement for Invariant Chain in Macrophages for Mycobacterium tuberculosis Replication and CD1d Antigen Presentation

Mycobacterium tuberculosis is an intracellular bacterium that persists in phagosomes of myeloid cells. M. tuberculosis-encoded factors support pathogen survival and reduce fusion of phagosomes with bactericidal lysosomal compartments. It is, however, not entirely understood if host factors that mediate endosomal fusion affect M. tuberculosis intracellular localization and survival. Neither is it known if endosomal fusion influences induction of host immune reactivity by M. tuberculosis-infected cells. Lysosomal degradation of M. tuberculosis appears to be pivotal for making available lipid substrates for assembly into lipid-CD1d complexes to allow activation of CD1d-restricted invariant natural killer T (iNKT) cells. To clarify the role for endosomal fusion in M. tuberculosis survival and induction of host CD1d-mediated immune defense, we focused our studies on the invariant chain (Ii). Ii regulates endosome docking and fusion and thereby controls endosomal transport. Through direct binding, Ii also directs intracellular transport of the class II major histocompatibility complex and CD1d. Our findings demonstrate that upon infection of Ii-knockout (Ii(-/-)) macrophages, M. tuberculosis is initially retained in early endosomal antigen 1-positive lysosomal-associated membrane protein 1-negative phagosomes, which results in slightly impaired pathogen replication. The absence of Ii did not affect the ability of uninfected and infected macrophages to produce nitric oxide, tumor necrosis factor alpha, or interleukin-12. However, induction of cell surface CD1d was impaired in infected Ii(-/-) macrophages, and CD1d-restricted iNKT cells were unable to suppress bacterial replication when they were cocultured with M. tuberculosis-infected Ii(-/-) macrophages. Thus, while the host factor Ii is not essential for the formation of the M. tuberculosis-containing vacuole, its presence is crucial for iNKT cell recognition of infected macrophages.

CD1d, a Sentinel Molecule Bridging Innate and Adaptive Immunity, Is Downregulated by the Human Papillomavirus (HPV) E5 Protein: a Possible Mechanism for Immune Evasion by HPV

CD1d and CD1d-restricted natural killer T (NKT) cells serve as a natural bridge between innate and adaptive immune responses to microbes. CD1d downregulation is utilized by a variety of microbes to evade immune detection. We demonstrate here that CD1d is downregulated in human papillomavirus (HPV)-positive cells in vivo and in vitro. CD1d immunoreactivity was strong in HPV-negative normal cervical epithelium but absent in HPV16-positive CIN1 and HPV6-positive condyloma lesions. We used two cell lines for in vitro assay; one was stably CD1d-transfected cells established from an HPV-negative cervical cancer cell line, C33A (C33A/CD1d), and the other was normal human vaginal keratinocyte bearing endogenous CD1d (Vag). Flow cytometry revealed that cell surface CD1d was downregulated in both C33A/CD1d and Vag cells stably transfected with HPV6 E5 and HPV16 E5. Although the steady-state levels of CD1d protein decreased in both E5-expressing cell lines compared to empty retrovirus-infected cells, CD1d mRNA levels were not affected. Confocal microscopy demonstrated that residual CD1d was not trafficked to the E5-expressing cell surface but colocalized with E5 near the endoplasmic reticulum (ER). In the ER, E5 interacted with calnexin, an ER chaperone known to mediate folding of CD1d. CD1d protein levels were rescued by the proteasome inhibitor, MG132, indicating a role for proteasome-mediated degradation in HPV-associated CD1d downregulation. Taken together, our data suggest that E5 targets CD1d to the cytosolic proteolytic pathway by inhibiting calnexin-related CD1d trafficking. Finally, CD1d-mediated production of interleukin-12 from the C33A/CD1d cells was abrogated in both E5-expressing cell lines. Decreased CD1d expression in the presence of HPV E5 may help HPV-infected cells evade protective immunological surveillance.

Uncoupling between CD1d upregulation induced by retinoic acid and conduritol-B-epoxide and iNKT cell responsiveness

Gaucher disease (GD) is associated with upregulation of CD1d and MHC-class II expression by monocytes. While the physiological impact of CD1d upregulation remains uncertain, it has been proposed that MHC-class II upregulation is associated with inflammation. Hereby, we show that the decrease in MHC-class II expression seen in GD patients under therapy correlates positively with chitotriosidase activity, a marker of inflamed macrophages. We also show that retinoic acid (RA) and the β-glucocerebrosidase inhibitor conduritol-B-epoxide (CBE) lead to upregulation of CD1d expression by THP-1 cells, which correlated with an increase in mRNA expression. In vitro co-culture experiments showed that RA treated THP-1 cells were more stimulatory for CD4 + than for CD8 + T cells, as determined by CFSE loss, in comparison to untreated THP-1 cells. Interestingly, even though addition of exogenous isoglobotrihexosylceramide (iGb3), a physiological CD1d ligand, augmented the percentage of dividing CD4 + T cells, we could not detect a significant expansion of CD4 +Vα24 + invariant Natural Killer T (iNKT) cells. In contrast, addition of α-galactosylceramide (α-GC) induced expansion of Vα24 + iNKT cells as determined by using α-GC-loaded human CD1d dimers. These results strengthen the existence of a cross-talk between monocyte lipid accumulation, inflammation and changes in cell surface CD1d and MHC-class II in monocytes, which may result in inappropriate recognition events by immune cells and perpetuate chronic inflammation.

Kinetics and Cellular Site of Glycolipid Loading Control the Outcome of Natural Killer T Cell Activation

SummaryCD1d-restricted natural killer T cells (NKT cells) possess a wide range of effector and regulatory activities that are related to their ability to secrete both T helper 1 (Th1) cell- and Th2 cell-type cytokines. We analyzed presentation of NKT cell activating α galactosylceramide (αGalCer) analogs that give predominantly Th2 cell-type cytokine responses to determine how ligand structure controls the outcome of NKT cell activation. Using a monoclonal antibody specific for αGalCer-CD1d complexes to visualize and quantitate glycolipid presentation, we found that Th2 cell-type cytokine-biasing ligands were characterized by rapid and direct loading of cell-surface CD1d proteins. Complexes formed by association of these Th2 cell-type cytokine-biasing αGalCer analogs with CD1d showed a distinctive exclusion from ganglioside-enriched, detergent-resistant plasma membrane microdomains of antigen-presenting cells. These findings help to explain how subtle alterations in glycolipid ligand structure can control the balance of proinflammatory and anti-inflammatory activities of NKT cells.

Vesicular Stomatitis Virus Matrix Protein Impairs CD1d-Mediated Antigen Presentation through Activation of the p38 MAPK Pathway

Natural killer T (NKT) cells are unique T lymphocytes that recognize CD1d-bound lipid antigens and play an important role in both innate and acquired immune responses against infectious diseases and tumors. We have already shown that a vesicular stomatitis virus (VSV) infection results in the rapid inhibition of murine CD1d-mediated antigen presentation to NKT cells. In the present study, it was found that the VSV matrix (VSV-M) protein is an important element in this decrease in antigen presentation postinfection. The VSV-M protein altered the intracellular distribution of murine CD1d molecules, resulting in qualitative (but not quantitative) changes in cell surface CD1d expression. The M protein was distributed throughout the infected cell, and it was found to activate the mitogen-activated protein kinase (MAPK) p38 very early postinfection. Infection of CD1d(+) cells with a temperature-sensitive VSV-M mutant at the nonpermissive temperature both substantially reversed the inhibition of antigen presentation by CD1d and delayed the activation of p38. Thus, the VSV-M protein plays an important role in permitting the virus to evade important components of the innate immune response by regulating specific MAPK pathways.

Phage display‐derived recombinant antibodies with TCR‐like specificity against α‐galactosylceramide and its analogues in complex with human CD1d molecules

The glycolipid alpha-galactosylceramide (alpha-GalCer) is a potent activator of invariant natural killer T (iNKT) cells and has been shown to be an effective agent against cancer, infections and autoimmune diseases. The effectiveness of alpha-GalCer and its alkyl chain analogues depends on efficient loading and presentation by the antigen-presenting molecule CD1d. To monitor the ability of CD1d to present the glycolipids, we have used a phage display strategy to generate recombinant antibodies with T cell receptor-like (TCRL) specificity against the human CD1d (hCD1d)-alpha-GalCer complex. These Fab fragments were able to detect specifically hCD1d-alpha-GalCer complexes in cell-free systems such as surface plasmon resonance and ELISA, as well as on the surface of hCD1d(+) antigen-presenting cells (APC) by flow cytometry and immunofluorescence microscopy, the latter of which could also detect intracellular complexes. We show that our TCRL antibodies can stain dendritic cells from CD11c-hCD1d-transgenic mice administered in vivo with alpha-GalCer and its analogues. Furthermore, the antibody was also able to detect the presentation by hCD1d molecules of analogues of alpha-GalCer with the same polar head structure. Using this reagent, we were able to confirm directly that the alpha-GalCer analogue C20:2 preferentially loads onto cell surface CD1d rapidly without the need for internalization, while the loading of alpha-GalCer is improved with longer incubation times on professional APC. This reagent will be essential for assessing the loading and presenting capabilities of hCD1d of alpha-GalCer and its analogues.

Role of Type I and Type II NKT cells in the host’s innate antitumor immune response to a B-cell lymphoma (50.26)

Abstract Natural killer T (NKT) cells are a T cell subpopulation known to possess immunoregulatory functions and recognize CD1d molecules. The majority of NKT cells express an invariant TCR [alpha] chain rearrangement and are called Type I NKT cells; all other NKT cells are Type II. In the current study, we have analyzed the roles for NKT cell subsets in the host’s innate antitumor response against the murine NS0 B-cell lymphoma model in vivo. As the NS0 cell line lacks detectable cell surface CD1d expression, murine CD1d1 cDNA or empty vector was transfected into NS0 cells to generate CD1d+ and control cell lines. In tumor-bearing mice, we found that Type I NKT cells conferred protection in a CD1d-dependent manner, whereas Type II NKT cells exhibited inhibitory activity. Pro- and anti-inflammatory cytokines secreted by splenocytes from tumor-bearing mice were predictive of tumor progression. Myeloid suppressor cells (CD11b+Gr1+) were present in larger numbers at the tumor site and in the spleen of tumor-bearing Type I NKT-deficient mice, suggesting a possible inhibition of antitumor immunosurveillance mediated by these cells. Therefore, there are distinct roles for NKT cell subsets in response to a B-cell lymphoma in vivo, pointing to potential novel targets to be exploited in the immunotherapy of blood cancers. This study was supported by NIH grants RO1 CA89029 and AI46455 to RRB.[alpha]

Interplay of Cytokines and Microbial Signals in Regulation of CD1d Expression and NKT Cell Activation

In this study we show that like MHC class I and class II molecules, cell surface CD1d expression on APC is regulated and affects T cell activation under physiological conditions. Although IFN-gamma alone is sufficient for optimum expression of MHC, CD1d requires two signals, one provided by IFN-gamma and a second mediated by microbial products or by the proinflammatory cytokine TNF. IFN-gamma-dependent CD1d up-regulation occurs on macrophages following infection with live bacteria or exposure to microbial products in vitro and in vivo. APC expressing higher CD1d levels more efficiently activate NKT cell hybridomas and primary NKT cells independently of whether the CD1d-restricted TCR recognizes foreign or self-lipid Ags. Our findings support a model in which CD1d induction regulates NKT cell activation.

BCR targeting of biotin-α-galactosylceramide leads to enhanced presentation on CD1d and requires transport of BCR to CD1d-containing endocytic compartments

CD1d is a non-polymorphic MHC class I-related protein that binds and presents glycolipid antigens to T cell antigen receptors expressed by NK-like T (NKT) cells. CD1d-dependent immune responses play critical roles in infectious disease, autoimmunity, allergy and cancer. We tested the hypothesis that B cell antigen receptor (BCR) targeting of a biotin-modified version of the CD1d-binding antigen alpha-galactosylceramide (biotin-alpha-GalCer) results in enhanced murine CD1d-mediated presentation as compared with presentation of non-targeted biotin-alpha-GalCer. Presentation of BCR-targeted antigen to NKT cells was enhanced 100- to 1000-fold compared with non-targeted antigen. CD1d presentation of BCR-targeted antigen was observed after 4 h treatment, consistent with a requirement for endosomal trafficking. Furthermore, unlike non-targeted antigen, BCR-targeted antigen was not loaded directly onto cell-surface CD1d. Blocking BCR signaling with the Syk tyrosine kinase inhibitor piceatannol inhibited presentation of BCR-targeted antigen but not non-targeted antigen. Piceatannol blocked transport of the BCR to CD1d-containing endosomes, showing that intersection of BCR-targeted antigen with endosomes is required for enhanced mCD1d antigen presentation. Our data suggest that the BCR facilitates capture of low quantities of mCD1d antigens to enhance CD1d-dependent immune responses.

Regulation of CD1d expression and function by a herpesvirus infection

Little is known about the role of CD1d-restricted T cells in antiviral immune responses. Here we show that the lytic replication cycle of the Kaposi sarcoma-associated herpesvirus (KSHV) promotes downregulation of cell-surface CD1d. This is caused by expression of the 2 modulator of immune recognition (MIR) proteins of the virus, each of which promotes the loss of surface CD1d expression following transfection into uninfected cells. Inhibition of CD1d surface expression is due to ubiquitination of the CD1d alpha-chain on a unique lysine residue in its cytoplasmic tail, which triggers endocytosis. Unlike MIR-mediated MHC class I downregulation, however, CD1d downregulation does not appear to include accelerated lysosomal degradation. MIR2-induced downregulation of CD1d results in reduced activation of CD1d-restricted T cells in vitro. KSHV modulation of CD1d expression represents a strategy for viral evasion of innate host immune responses and implicates CD1d-restricted T cells as regulators of this viral infection.