Cell Surface Expression Of CD1d Research Articles

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.

Histone deacetylase inhibitors enhance CD1d-dependent NKT cell responses to lymphoma.

Histone deacetylases (HDACs) are a family of enzymes that influence expression of genes implicated in tumor initiation, progression, and anti-tumor responses. In addition to their canonical role in deacetylation of histones, HDACs regulate many non-canonical targets, such as Signal Transducer and Activator of Transcription 3 (STAT3). We hypothesize that tumors use epigenetic mechanisms to dysregulate CD1d-mediated antigen presentation, thereby impairing the ability of natural killer T (NKT) cells to recognize and destroy malignant cells. In this study, we pre-treated CD1d-expressing tumor cells with HDAC inhibitors (HDACi) and assessed CD1d-dependent NKT cell responses to mantle cell lymphoma (MCL). Pre-treatment with Trichostatin-A, a pan-HDACi, rapidly enhanced both CD1d- and MHC class II-mediated antigen presentation. Similarly, treatment of MCL cells with other HDACi resulted in enhanced CD1d-dependent NKT cell responses. The observed changes are due, at least in part, to an increase in both CD1D mRNA and CD1d cell surface expression. Mechanistically, we found that HDAC2 binds to the CD1D promoter. Knockdown of HDAC2 in tumor cells resulted in a significant increase in CD1d-mediated antigen presentation. In addition, treatment with HDACi inhibited STAT3 and STAT3-regulated inflammatory cytokine secretion by MCL cells. We demonstrated that MCL-secreted IL-10 inhibits CD1d-mediated antigen presentation and pre-treatment with TSA abrogates secretion of IL-10 by MCL. Taken together, our studies demonstrate the efficacy of HDACi in restoring anti-tumor responses to MCL through both cell-intrinsic and cell-extrinsic mechanisms and strongly implicate a role for HDACi in enhancing immune responses to cancer.

HDAC inhibitors enhance CD1d-dependent NKT cell responses to lymphoma

Abstract Histone deacetylases (HDACs) influence expression of genes implicated in tumor initiation, progression and anti-tumor responses. We hypothesize that tumors use epigenetic mechanisms to dysregulate CD1d-mediated antigen processing and presentation, which leads to a functional impairment in the ability of Natural killer T (NKT) cells to recognize and destroy cancerous cells. In this study, we treated CD1d-expressing tumor cells with HDAC inhibitors (HDACi) to assess CD1d-dependent NKT cell responses to B cell lymphoma. Consistent with previous studies, we found that treatment with Trichostatin A, a pan-HDACi, enhanced both CD1d and MHC class II-mediated antigen presentation. Similarly, treatment of B cell lymphomas with other HDACi, Panobinostat and Vorinostat, resulted in enhanced CD1d-dependent NKT cell responses. Mechanistically, we found that HDAC2 binds to the CD1d promoter and knockdown of HDAC2 in tumor cells enhanced their immunogenicity. The observed changes are due at least in part to a dose-dependent increase in both CD1d mRNA and CD1d cell surface expression. In addition, treatment with HDCAi resulted in a decrease in inflammatory cytokine secretion by B cell lymphomas. To examine the therapeutic potential of HDACi, we treated human peripheral blood mononuclear cells with TSA and Panobinostat. Treatment with these HDACi did not suppress T cell activation; conversely, treatment with a novel HDAC4 inhibitor MC1568 suppressed T cell responses. Taken together, our studies demonstrate the efficacy of HDACi in restoring anti-tumor responses to B cell lymphomas through both cell-intrinsic and extrinsic factors and strongly implicate a role for select HDACi in cancer immunotherapeutic strategies.

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.

Epigenetic modulation of CD1d-mediated antigen presentation by B cell lymphomas (TUM10P.1054)

Abstract Histone deacetylases (HDACs) are a family of enzymes that regulate diverse cellular events such as gene expression, cell proliferation, and immune pathways through deacetylation of their protein targets. We hypothesize that tumors use epigenetic mechanisms to dysregulate CD1d-mediated antigen processing and presentation, which leads to a functional impairment in the ability of natural killer T (NKT) cells to recognize and destroy cancerous cells. We examined CD1d-mediated antigen presentation to NKT cells following treatment with HDAC inhibitors (HDACi). Consistent with previous studies, we found that preventing deacetylation by treatment with Trichostatin A, a pan-HDACi, enhanced both CD1d and MHC class II-mediated antigen presentation. Similarly, treatment of B cell lymphomas with Panobinostat resulted in increased CD1d-dependent NKT cell responses, due at least in part to a dose-dependent increase in CD1d-cell surface expression. In addition, treatment with HDCAi results in a decrease in STAT3 expression as well as IL-10 secretion by B cell lymphomas. Overall, our studies demonstrate the efficacy of HDACi in restoring anti-tumor responses to B cell lymphomas through both cell-intrinsic and extrinsic factors. Collectively, these results suggest that HDACi may work to enhance the immune response and increases the immunogenicity of the tumor itself.

Herpes Simplex Virus 1 US3 Phosphorylates Cellular KIF3A To Downregulate CD1d Expression.

Herpes simplex virus 1 (HSV-1) causes one of the most prevalent herpesviral infections in humans and is the leading etiological agent of viral encephalitis and eye infections. Our understanding of how HSV-1 interacts with the host at the cellular and organismal levels is still limited. We and others previously reported that, upon infection, HSV-1 rapidly and efficiently downregulates CD1d cell surface expression and suppresses the function of NKT cells, a group of innate T cells with critical immunoregulatory function. The viral protein kinase US3 plays a major role in this immune evasion mechanism, and its kinase activity is required for this function. In this study, we investigated the cellular substrate(s) phosphorylated by US3 and how it mediates US3 suppression of CD1d recycling. We identified the type II kinesin motor protein KIF3A as a critical kinesin factor in the cell surface expression of CD1d. Interestingly, KIF3A is phosphorylated by US3 both in vitro and in infected cells. Mass spectrometry analysis of purified KIF3A showed that it is phosphorylated predominantly at serine 687 by US3. Ablation of this phosphorylation abolished US3-mediated downregulation of CD1d expression, suggesting that phosphorylation of KIF3A is the primary mechanism of HSV-1 suppression of CD1d expression by US3 protein. Understanding of the precise mechanism of viral modulation of CD1d expression will help to develop more efficient vaccines in the future to boost host NKT cell-mediated immune responses against herpesviruses. Herpes simplex virus 1 (HSV-1) is among the most common human pathogens. Little is known regarding the exact mechanism by which this virus evades the human immune system, particularly the innate immune system. We previously reported that HSV-1 employs its protein kinase US3 to modulate the expression of the key antigen-presenting molecule CD1d to evade the antiviral function of NKT cells. Here we identified the key cellular motor protein KIF3A as a cellular substrate phosphorylated by US3, and this phosphorylation event mediates US3-induced immune evasion.

Increase in natural killer T (NKT) Cell activation in hypoxic environments via the induction of HIF1alpha (162.16)

Abstract Tumors often experience hypoxic microenvironments due to poor vascular access in the early stage of neoplastic growth and continue to experience oxygen deficits in late stage progression despite the increase in neovascularization to the primary tumor or metastasis. This persistent hypoxic state selects for tumors cells that are able to compensate for low oxygen levels by increasing use of the glycolytic cycle under aerobic conditions (The Warburg Effect), suppressing apoptosis and inducing decreased genomic stability. These effects have been shown to be mediated, at least in part, by the transcription factor hypoxia inducible factor (HIF)-1alpha. Natural Killer T (NKT) cells are a subset of T cells that recognize a wide range of lipid antigens presented by the MHC Class I-like CD1d molecule. In this study, we sought to investigate the effects of hypoxia induction on CD1d-mediated NKT cell responses. Pharmacological agents were used to induce HIF-1a in LCD1d, and then we assessed their ability to present endogenous antigen to NKT cells. It was found that the induction of HIF-1a resulted in a significant increase in CD1d-mediated NKT cell activation, as assessed by IL-2, GM-CSF, and IL-13 production. The increase in NKT function was not due to with an increase in cell surface expression of CD1d. These results suggest that hypoxia in tumor cells may alter the repertoire of endogenous lipids presented in the context of CD1d which permits their recognition by NKT cells.

Efferocytosis induces macrophages to produce IL-4 and activate invariant NKT cells to suppress inflammation (117.17)

Abstract Efferocytosis of apoptotic neutrophils by macrophages is fundamental to the resolution of inflammation. In this study we demonstrate using a mouse sterile peritonitis model, a circuit requiring the activation of NKT cells by efferocytosing macrophages to resolve inflammation. Following ingestion of apoptotic neutrophils, macrophages produced IL-4 and IL-13, which in an autocrine manner, resulted in cells with an M2 phenotype. Efferocytosing macrophages displayed increased cell surface expression of CD1d and activated invariant NKT (iNKT) cells that produced IL-4 during the later phase of inflammation. Peritoneal inflammation was exaggerated in mice lacking IL-4 or NKT cells, suggesting both are required to suppress inflammation. Furthermore, sterile peritoneal inflammation was also enhanced and prolonged in NADPH oxidase-deficient mice with X-linked chronic granulomatous disease (X-CGD). We found that X-CGD exudate macrophages expressed reduced surface CD1d and were impaired in activating iNKT cells, which contributed to the persistent inflammation in these mice. Therefore, efferocytosis-induced IL-4 production and activation of iNKT cells by macrophages are immunomodulatory events in an innate immune circuit required to resolve sterile inflammation.

Human CD1d molecules are resistant to human cytomegalovirus US2- and US11-mediated degradation

Natural killer T (NKT) cells may play a crucial role in controlling viral infection by bridging the innate and adaptive immune systems. These cells are activated by lipids presented by CD1d molecules, which are structurally homologous to major histocompatibility complex class I (MHC-I) molecules. Although human cytomegalovirus (HCMV) can avoid T cell recognition by down-regulating MHC-I-mediated antigen presentation, it remains unknown whether it can also interfere with CD1d-mediated lipid presentation. Here, we show that CD1d is resistant to rapid degradation induced by the HCMV gene products US2 and US11, which cause dislocation of MHC-I molecules from the endoplasmic reticulum (ER) to the cytosol for destruction by proteasomes. The resistance of CD1d to US11 is mainly due to the short cytosolic tail of CD1d; a hybrid CD1d protein, whose cytosolic tail was replaced with that of HLA-A2.1, was efficiently degraded by US11. Finally, we found that HCMV infection did not significantly influence the cell surface expression of CD1d. Thus, these results suggest that antigen presentation by CD1d is largely unaffected by the multiple immune-modulating functions of HCMV.

NKT cell mediated responses to mantle cell lymphoma (48.35)

Abstract Mantle cell lymphoma (MCL) is an aggressive subtype of Non-Hodgkin’s lymphoma that is characterized by the abnormal accumulation of CD20+CD5+ B cells in the lymph nodes, spleen, gastrointestinal tract, bone marrow and blood. Although treatment with combination chemotherapy can be effective, most patients relapse, and the outcome for MCL remains poor. Natural killer T (NKT) cells are important in regulating immune responses to tumors. The observation that lymphoma patients have a reduction in both NKT cell number and function suggests that the anti-tumor effects mediated by these cells may be compromised. In these studies, we have investigated NKT cell activation in response to MCL. We found that CD1d cell surface expression was highly variable in a panel of well-characterized MCL cell lines. Importantly, cell surface expression of CD1d did not directly correlate with the ability of the MCL lines to activate primary NKT cells. Circulating NKT cells in the peripheral blood of MCL patients were not stimulated by autologous B cells, even in the presence of the potent antigen, α-GalCer. However, when NKT cells from healthy donors were co-cultured with purified B cells from MCL patients, the NKT cells specifically recognized the malignant B cells and produced IFN-γ, but not inflammatory or regulatory cytokines. These data suggest that B cell lymphomas may condition autologous NKT cells not to recognize them in order to evade NKT cell mediated anti-tumor responses.

Expression of CD1d and Ligand-Induced Cytokine Production Are Tissue Specific in Mucosal Epithelia of the Human Lower Reproductive Tract

Mucosal epithelia of the human lower reproductive tract (vagina, cervix, and penile urethra) are exposed to sexually transmitted microbes, including Chlamydia trachomatis. The in vivo susceptibility of each tissue type to infection with C. trachomatis is quite distinct. CD1d is expressed on the surface of antigen-presenting cells, including mucosal epithelial cells, and interacts specifically with invariant NKT cells. Invariant NKT cells play a role in both innate and adaptive immune responses to microbes. Here we assessed CD1d expression in normal reproductive tissues by using immunohistochemistry. Immortalized epithelial cell lines from the human lower reproductive tract (vagina, endocervix, and penile urethra) were examined for CD1d expression and for ligand-induced cytokine production induced by CD1d cross-linking. CD1d expression in normal tissue was strong in the vagina but weak in the endocervix and penile urethra. Gamma interferon exposure induced CD1d transcription in all of the cell types studied, with the strongest induction in vaginal cells. Flow cytometry revealed cell surface expression of CD1d in vaginal and penile urethral epithelial cells but not in endocervical cells. Ligation of surface-expressed CD1d by monoclonal antibody cross-linking promoted interleukin-12 (IL-12) and IL-15, but not IL-10, production in vaginal and penile urethral cells. No induction was demonstrated in endocervical cells. CD1d-mediated cytokine production in penile urethral cells was abrogated by C. trachomatis infection. Basal deficiency in CD1d-mediated immune responsiveness may result in susceptibility to sexually transmitted agents. Decreased CD1d-mediated signaling may help C. trachomatis evade detection by innate immune cells.

Injury-Induced Suppression of Effector T Cell Immunity Requires CD1d-Positive APCs and CD1d-Restricted NKT Cells

Overwhelming infection remains the leading cause of death from serious burn injury despite recent advances in the care of burn patients and a better understanding of immune and inflammatory consequences of injury. In this study, we report a critical requirement for CD1d-restricted NKT cells and CD1d expression by APCs in the immune dysfunction that occurs early after burn injury. Using a well-established murine scald injury model with BALB/c and BALB/c CD1d knockout mice, we investigated whether peripheral T cell immunity was affected by the presence or absence of CD1d-restricted NKT cells in the early stages after injury. Using Ag-specific delayed-type hypersensitivity, T cell proliferation, and cytokine production as indices of immune responsiveness, we observed that both CD1d expression by APCs and CD1d-restricted NKT cells are required for immune suppression after injury. Via adoptive transfer of splenocytes from injured mice to uninjured recipients, we found injury-induced suppression of immunity to be Ag specific, long lasting, and critically dependent on cell surface expression of CD1d by APCs. Together, our results suggest that the defects in T cell responsiveness that occur subsequent to severe burn injury are not merely the result of global or passive suppression, but instead represent an active form of CD1d/NKT cell-dependent immunologic tolerance.

Immature Human Dendritic Cells Infected with Leishmania infantum Are Resistant to NK-Mediated Cytolysis but Are Efficiently Recognized by NKT Cells

Dendritic cells (DC) play an important role in innate and adaptive immunity, interacting with T cells, NK, and NKT cells. A critical step in the interaction of the parasitic protozoa Leishmania with their host is the evasion of both innate and adaptive immunity, producing a long-lasting chronic infection. There is growing evidence that these parasites can modify the Ag-presenting and immunoregulatory functions of DCs. The cells and mechanisms involved in innate immune response against Leishmania are still poorly understood. In this study, we investigated how Leishmania infantum infection affects DC interactions with NK and invariant NKT (iNKTs) cells in humans. We found that infected immature DCs (iDCs) do not up-regulate HLA class I molecules. Despite this, iDCs become resistant to killing mediated by autologous NK cells due to the up-regulation of HLA-E expression, which protects target cells from NK-mediated lysis through interaction with the inhibitory receptor CD94/NKG2A. Furthermore, iDCs infected with L. infantum up-regulate CD1d cell surface expression and consequently can be efficiently recognized and killed by iNKT cells that produce IFN-gamma. These data suggest that L. infantum could be able to evade NK recognition; in contrast, iNKTs may play an important role in the immune response against Leishmania.

Impaired cell surface expression of human CD1d by the formation of an HIV-1 Nef/CD1d complex

The HIV-1 Nef protein causes a decrease in major histocompatibility complex (MHC) class I and CD4 molecule expression on the cell surface. To determine if Nef can affect components of the innate immune response, we assessed the ability of Nef to alter the cell surface expression of human CD1d. In cells co-expressing CD1d and Nef, a substantial reduction in the cell surface level of CD1d was observed, with a concomitant reduction in the activation of CD1d-restricted NKT cells. Nef had a minimal effect on the cell surface expression of a mutant CD1d molecule in which the last 6 or 10 amino acids of the cytoplasmic tail were deleted. Additionally, it was found that Nef physically interacted with wild-type (but not tail-deleted) CD1d. Therefore, one means by which HIV-1 may be able to establish a foothold in an infected individual is by directly interfering with the functional cell surface expression of CD1d.

Human immunodeficiency virus gp120 downregulates CD1d cell surface expression

CD1d is an MHC class I-like surface molecule that presents endogenous glycoplipid antigens. The effect of HIV infection on CD1d surface expression has not yet been reported. FACS analysis revealed significantly lower levels of CD1d on CD14 + monocytes from HIV-infected subjects compared to HIV-infected subjects on HAART and healthy controls. CD1d expression correlated inversely with viral load in infected individuals. CD1d surface expression on human cell lines was downregulated after infection with M-tropic HIV, T-tropic HIV, or after exposure to HIV gp120 in vitro. These data suggest that CD1d-mediated responses are altered during HIV infection and may thus contribute to the global immunodeficiency seen in these patients.

Reduction in CD1d expression on dendritic cells and macrophages by an acute virus infection

Mice were infected with lymphocytic choriomeningitis virus (LCMV) to determine if changes in CD1d expression occurred during an acute virus infection. It is interesting that a decrease in CD1d expression on splenic dendritic cells (DC) and macrophages (MPhi) was observed for at least 3 months post-LCMV infection, and vaccinia virus and vesicular stomatitis virus induced similar changes in CD1d upon infection with those viruses. The reduction of CD1d cell-surface expression on DC and MPhi was independent of interferon-gamma and interleukin-12 expression but partially recovered in transporter associated with antigen processing-1-deficient mice, suggesting that CD8+ T cells may play a role. Thus, one consequence of the induction of a cellular immune response is a change in CD1d expression, which may constitute a key element in regulating antiviral immunity.

Antigen-specific regulation of CD1 expression in humans.

During the last decade the CD1 family of cell surface glycoproteins has been implicated in the presentation of nonpeptide antigens in man. Recent findings by our group indicate that CD1 molecules also can be involved in the presentation of certain bacterial proteins. However, CD1a, b, and c (group 1 CD1 molecules) are not present at significant levels on circulating monocytes unless their expression is induced by cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF). In this study we investigated the cell surface expression of CD1 molecules following the antigenic stimulation in vivo via immunization of healthy volunteers with tetanus toxoid vaccine and in vitro cell cultures using the same antigen. Both the in vivo and in vitro studies demonstrated clear up-regulation of the surface expression of CD1a, b, and c on monocytes as a result of antigenic stimulation with tetanus toxoid, supporting the idea that CD1 molecules participate in the presentation of this protein antigen in man. In vitro, antigen-triggered expression of these molecules was mediated by GM-CSF, since neutralization of this cytokine with specific antibody totally abrogated CD1a, b, and c expression. In contrast to the group 1 CD1 molecules, CD1d was found to be constitutively expressed on the majority of circulating monocytes and B lymphocytes prior to immunization. There was no effect of antigenic stimulation with tetanus toxoid on the cell surface expression of CD1d, suggesting major differences in regulation of the expression and function of the different CD1 molecules in humans. Altogether our results point to antigen-driven up-regulation of CD1a, b, and c expression on human monocytes that is mediated by GM-CSF and no effect on CD1d expression.