Aberrant CD200/CD200R1 expression and function in systemic lupus erythematosus contributes to abnormal T-cell responsiveness and dendritic cell activity

Introduction

CD200 is a type I transmembrane glycoprotein that can regulate the activation threshold of inflammatory immune responses, polarize cytokine production, and maintain immune homeostasis. We therefore evaluated the functional status of CD200/CD200 receptor 1 (CD200R1) interactions in subjects with systemic lupus erythematosus (SLE).

Methods

Serum CD200 level was detected by ELISA. The expression of CD200/CD200R1 by CD4⁺ T cells and dendritic cells (DCs) was examined by flow cytometry, and then compared between SLE patients and healthy controls. Peripheral blood mononuclear cells were stained with carboxyfluorescein diacetate succinimidyl ester and annexin V/propidium iodide for evaluation of the effect of CD200 on cell proliferation and apoptosis. In addition, the effect of CD200 on DC function was determined by transwell migration assay as well as by measurement of binding and phagocytosis of apoptotic cells.

Results

In SLE patients, the number of CD200⁺ cells and the level of soluble CD200 were significantly higher than in healthy controls, whereas the expression of CD200R1 by CD4⁺ T cells and DCs was decreased. Furthermore, the increased CD200 expression by early apoptotic cells contributed to their diminished binding and phagocytosis by DCs in SLE. Importantly, the engagement of CD200 receptor on CD4⁺ T cells with CD200-Fc fusion protein in vitro reduced the differentiation of T-helper type 17 cells and reversed the defective induction of CD4⁺CD25^highFoxP3⁺ T cells by transforming growth factor beta in SLE patients. Conversely, blockade of CD200-CD200R1 interaction with anti-CD200R1 antibody promoted CD4⁺ T-cell proliferation.

Conclusion

CD200 and CD200R1 expression and function are abnormal in SLE and may contribute to the immunologic abnormalities in SLE.     

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.     

Conserved and heterogeneous lipid antigen specificities of CD1d-restricted NKT cell receptors

CD1d-restricted NKT cells use structurally conserved TCRs and recognize both self and foreign glycolipids, but the TCR features that determine these Ag specificities remain unclear. We investigated the TCR structures and lipid Ag recognition properties of five novel Valpha24-negative and 13 canonical Valpha24-positive/Vbeta11-positive human NKT cell clones generated using alpha-galactosylceramide (alpha-GalCer)-loaded CD1d tetramers. The Valpha24-negative clones expressed Vbeta11 paired with Valpha10, Valpha2, or Valpha3. Strikingly, their Valpha-chains had highly conserved rearrangements to Jalpha18, resulting in CDR3alpha loop sequences that are nearly identical to those of canonical TCRs. Valpha24-positive and Valpha24-negative clones responded similarly to alpha-GalCer and a closely related bacterial analog, suggesting that conservation of the CDR3alpha loop is sufficient for recognition of alpha-GalCer despite CDR1alpha and CDR2alpha sequence variation. Unlike Valpha24-positive clones, the Valpha24-negative clones responded poorly to a glucose-linked glycolipid (alpha-glucosylceramide), which correlated with their lack of a conserved CDR1alpha amino acid motif, suggesting that fine specificity for alpha-linked glycosphingolipids is influenced by Valpha-encoded TCR regions. Valpha24-negative clones showed no response to isoglobotrihexosylceramide, indicating that recognition of this mammalian lipid is not required for selection of Jalpha18-positive TCRs that can recognize alpha-GalCer. One alpha-GalCer-reactive, Valpha24-positive clone differed from the others in responding specifically to mammalian phospholipids, demonstrating that semi-invariant NKT TCRs have a capacity for private Ag specificities that are likely conferred by individual TCR beta-chain rearrangements. These results highlight the variation in Ag recognition among CD1d-restricted TCRs and suggest that TCR alpha-chain elements contribute to alpha-linked glycosphingolipid specificity, whereas TCR beta-chains can confer heterogeneous additional reactivities.     

Hsp70 promotes antigen-presenting cell function and converts T-cell tolerance to autoimmunity in vivo

Pathogens or pathogen-associated molecular patterns can signal to cells of the innate immune system and trigger effective adaptive immunity. However, relatively little is known about how the innate immune system detects tissue injury or necrosis. Evidence suggests that the release of heat-shock proteins (HSPs) may provide adjuvant-like signals, but the ability of HSPs to promote activation or tolerance in vivo has not been addressed. In this study we show that Hsp70 promotes dendritic cell (DC) function and, together with antigen, triggers autoimmune disease in vivo.     

Toll-like receptor expression and function in human dendritic cell subsets: implications for dendritic cell-based anti-cancer immunotherapy

Dendritic cells (DCs) are central players of the immune response. To date, DC-based immunotherapy is explored worldwide in clinical vaccination trials with cancer patients, predominantly with ex vivo-cultured monocyte-derived DCs (moDCs). However, the extensive culture period and compounds required to differentiate them into DCs may negatively affect their immunological potential. Therefore, it is attractive to consider alternative DC sources, such as blood DCs. Two major types of naturally occurring DCs circulate in peripheral blood, myeloid DCs (mDCs) and plasmacytoid (pDCs). These DC subsets express different surface molecules and are suggested to have distinct functions. Besides scavenging pathogens and presenting antigens, DCs secrete cytokines, all of which is vital for both the acquired and the innate immune system. These immunological functions relate to Toll-like receptors (TLRs) expressed by DCs. TLRs recognize pathogen-derived products and subsequently provoke DC maturation, antigen presentation and cytokine secretion. However, not every TLR is expressed on each DC subset nor causes the same effects when activated. Considering the large amount of clinical trials using DC-based immunotherapy for cancer patients and the decisive role of TLRs in DC maturation, this review summarizes TLR expression in different DC subsets in relation to their function. Emphasis will be given to the therapeutic potential of TLR-matured DC subsets for DC-based immunotherapy.     

Observation of antigen-dependent CD8+ T-cell/ dendritic cell interactions in vivo

In order to track hematopoetic cells of all lineages unambiguously at all stages of development, we have developed C57BL/6 mice that express a transgene coding for green fluorescent protein (GFP) under control of the human ubiquitin C promoter. These mice, called UBI-GFP/BL6, express GFP in all tissues examined, with high levels of GFP expression observed in hematopoetic cells. UBI-GFP/BL6 mice are unique in that B cells, T cells, and dendritic cells have distinct levels of GFP fluorescence. In cell transfer experiments, leukocytes from UBI-GFP/BL6 mice are readily identified by FACS or fluorescence microscopy. We demonstrate that transplanted UBI-GFP/BL6 dendritic cells are easily identified in secondary lymphoid tissues. Direct interactions between individual dendritic cells and multiple na?ve CD8+ T cells are observed in lymph nodes within 12 h of cell transfer and require loading of the dendritic cells with the appropriate peptide antigen. Dendritic cells undergo specific morphologic changes following interactions with antigen-specific T cells.     

CD1d-restricted NKT cells: an interstrain comparison

CD1d-restricted Valpha14-Jalpha281 invariant alphabetaTCR(+) (NKT) cells are well defined in the C57BL/6 mouse strain, but they remain poorly characterized in non-NK1.1-expressing strains. Surrogate markers for NKT cells such as alphabetaTCR(+)CD4(-)CD8(-) and DX5(+)CD3(+) have been used in many studies, although their effectiveness in defining this lineage remains to be verified. Here, we compare NKT cells among C57BL/6, NK1.1-congenic BALB/c, and NK1.1-congenic nonobese diabetic mice. NKT cells were identified and compared using a range of approaches: NK1.1 expression, surrogate phenotypes used in previous studies, labeling with CD1d/alpha-galactosylceramide tetramers, and cytokine production. Our results demonstrate that NKT cells and their CD4/CD8-defined subsets are present in all three strains, and confirm that nonobese diabetic mice have a numerical and functional deficiency in these cells. We also highlight the hazards of using surrogate phenotypes, none of which accurately identify NKT cells, and one in particular (DX5(+)CD3(+)) actually excludes these cells. Finally, our results support the concept that NK1.1 expression may not be an ideal marker for CD1d-restricted NKT cells, many of which are NK1.1-negative, especially within the CD4(+) subset and particularly in NK1.1-congenic BALB/c mice.     

Blood monocyte subsets differentially give rise to CD103+ and CD103- pulmonary dendritic cell populations

There are two major myeloid pulmonary dendritic cell (DC) populations: CD103+ DCs and CD11bhigh DCs. In this study, we investigated in detail the origins of both myeloid DC pools using multiple experimental approaches. We show that, in resting lung, Ly-6ChighCCR2high monocytes repopulated CD103+ DCs using a CCR2-dependent mechanism, and these DCs preferentially retained residual CCR2 in the lung, whereas, conversely, Ly-6ClowCCR2low monocytes repopulated CD11bhigh DCs. CX3CR1 was required to generate normal numbers of pulmonary CD11bhigh DCs, possibly because Ly-6Clow monocytes in the circulation, which normally express high levels of CX3CR1, failed to express bcl-2 and may have diminished survival in the circulation in the absence of CX3CR1. Overall, these data demonstrate that the two circulating subsets of monocytes give rise to distinct tissue DC populations.     

Monocyte-derived CD1a+ and CD1a- dendritic cell subsets differ in their cytokine production profiles, susceptibilities to transfection, and capacities to direct Th cell differentiation

We describe a phenotypically and functionally novel monocyte-derived dendritic cell (DC) subset, designated mDC2, that lacks IL-12 synthesis, produces high levels of IL-10, and directs differentiation of Th0/Th2 cells. Like conventional monocyte-derived DC, designated mDC1, mDC2 expressed high levels of CD11c, CD40, CD80, CD86, and MHC class II molecules. However, in contrast to mDC1, mDC2 lacked expression of CD1a, suggesting an association between cytokine production profile and CD1a expression in DC. mDC2 could be matured into CD83+ DC cells in the presence of anti-CD40 mAbs and LPS plus IFN-gamma, but they remained CD1a- and lacked IL-12 production even upon maturation. The lack of IL-12 and CD1a expression by mDC2 did not affect their APC capacity, because mDC2 stimulated MLR to a similar degree as mDC1. However, while mDC1 strongly favored Th1 differentiation, mDC2 directed differentiation of Th0/Th2 cells when cocultured with purified human peripheral blood T cells, further indicating functional differences between mDC1 and mDC2. Interestingly, the transfection efficiency of mDC2 with plasmid DNA vectors was significantly higher than that of mDC1, and therefore mDC2 may provide improved means to manipulate Ag-specific T cell responses after transfection ex vivo. Taken together, these data indicate that peripheral blood monocytes have the capacity to differentiate into DC subsets with different cytokine production profiles, which is associated with altered capacity to direct Th cell differentiation.     

CD1d-restricted NKT cells contribute to the age-associated decline of T cell immunity

NKT cells are known to regulate effector T cell immunity during tolerance, autoimmunity, and antitumor immunity. Whether age-related changes in NKT cell number or function occur remains unclear. Here, we investigated whether young vs aged (3 vs 22 mo old) mice had different numbers of CD1d-restricted NKT cells and whether activation of NKT cells by CD1d in vivo contributed to age-related suppression of T cell immunity. Flow cytometric analyses of spleen and LN cells revealed a 2- to 3-fold increase in the number of CD1d tetramer-positive NKT cells in aged mice. To determine whether NKT cells from aged mice differentially regulated T cell immunity, we first examined whether depletion of NK/NKT cells affected the proliferative capacity of splenic T cells. Compared with those from young mice, intact T cell preparations from aged mice had impaired proliferative responses whereas NK/NKT-depleted preparations did not. To examine the specific contribution of NKT cells to age-related T cell dysfunction, Ag-specific delayed-type hypersensitivity and T cell proliferation were examined in young vs aged mice given anti-CD1d mAb systemically. Compared with young mice, aged mice given control IgG exhibited impaired Ag-specific delayed-type hypersensitivity and T cell proliferation, which could be significantly prevented by systemic anti-CD1d mAb treatment. The age-related impairments in T cell immunity correlated with an increase in the production of the immunosuppressive cytokine IL-10 by splenocytes that was likewise prevented by anti-CD1d mAb treatment. Together, our results suggest that CD1d activation of NKT cells contributes to suppression of effector T cell immunity in aged mice.     

Apoptosis and systemic autoimmunity: the dendritic cell connection

Much effort has been devoted in recent years to the events linking recognition and disposal of apoptotic cells to sustained immunity towards the antigens they contain. Programmed death via apoptosis indeed provides most of the raw material the immune system exploits to establish self tolerance, i.e. to learn how to distinguish between self constituents and foreign antigens, belonging to invading pathogens. In parallel, events occurring during cell death may enable a restricted array of molecules endowed with diverse structure, function and intracellular distribution to satisfy the requirement to evoke and maintain autoimmune responses. Dendritic cells (DCs), the most potent antigen presenting cells, appear to play a crucial role. Here we will discuss some of the constrains regulating the access of dying cells' antigens to DCs, as well as censorship mechanisms that prevent their maturation and the full explication of their antigen presenting function.     

The complexity of the CD4 T-cell responses: old and new T-cell subsets

The T-cell compartment of the immune system reacts to an enormous variety of antigens, including self antigens, due to its a wide repertoire of T-cell clones. Self-reactive T cells undergo a negative selection process resulting in apoptosis of T cells with high affinity for self-peptides. Self-reactive T cells escaped to negative selection are then controlled by natural T regulatory (Treg) cells. Regulation also controls excessive effector T-cell responses. Three types of effector T cells are recognized: T helper 1 (Th1) cells, which protect against intracellular bacteria; Th2 cells, which play a role against parasites; Th17 cells, which would face extracellular bacteria, but also are involved in autoimmunity. Effector T-cell polarization is determined by the complex interaction of antigen-presenting cells with naive T cells and involves a multitude of factors, including the dominant cytokine environment, costimulatory molecules, type and load of antigen presented and signaling cascades. The decision for the immune response to go in a certain direction is based not onto one signal alone, rather onto many different elements acting synergistically, antagonistically and through feedback loops leading to activation of Th1, Th2, or Th17 responses. Both Th1 and Th2 can be suppressed by adaptive Treg cells through contact-dependent mechanisms and/or cytokines.     

Functional specialization of islet dendritic cell subsets

Dendritic cells (DC) play important roles in both tolerance and immunity to β cells in type 1 diabetes. How and why DC can have diverse and opposing functions in islets remains elusive. To answer these questions, islet DC subsets and their specialized functions were characterized. Under both homeostatic and inflammatory conditions, there were two main tissue-resident DC subsets in islets, defined as CD11b(lo/-)CD103(+)CX3CR1(-) (CD103(+) DC), the majority of which were derived from fms-like tyrosine kinase 3-dependent pre-DC, and CD11b(+)CD103(-)CX3CR1(+) (CD11b(+) DC), the majority of which were derived from monocytes. CD103(+) DC were the major migratory DC and cross-presented islet-derived Ag in the pancreatic draining lymph node, although this DC subset displayed limited phagocytic activity. CD11b(+) DC were numerically the predominant subset (60-80%) but poorly migrated to the draining lymph node. Although CD11b(+) DC had greater phagocytic activity, they poorly presented Ag to T cells. CD11b(+) DC increased in numbers and percentage during T cell-mediated insulitis, suggesting that this subset might be involved in the pathogenesis of diabetes. These data elucidate the phenotype and function of homeostatic and inflammatory islet DC, suggesting differential roles in islet immunity.     

Distinctive lack of CD48 expression in subsets of human dendritic cells tunes NK cell activation

CD48 is a glycosyl phosphatidylinositol anchor protein known to be virtually expressed by all human leukocytes. Its ligand, 2B4, is a signaling lymphocyte activation molecule-related receptor involved in NK cell activation. Because dendritic cells (DCs) are strong inducers of NK cell functions, we analyzed the expression of CD48 in different human DC subsets. We observed that monocytes differentiating in DCs promptly down-regulate CD48. Similarly, DCs isolated from inflamed lymph nodes generally do not express CD48. Plasmocytoid DCs do not express CD48 either, whereas myeloid DCs harbored in blood, bone marrow, and thymus express it. In addition, we showed that CD48 expression in DCs affects NK cell functions during NK/DC cross-talk, because NK cells obtained from normal donors and from X-linked lymphoproliferative disease patients are, respectively, triggered or inhibited by DCs expressing surface CD48. Remarkably, IFN-gamma production by lymph node NK cells, in contrast to blood NK cells, can be negatively modulated by 2B4/CD48 interactions, indicating a 2B4 inhibitory pathway in lymph node NK cells. Therefore, the CD48 deficiency of DCs harbored in inflamed lymph nodes that we report in this study might be relevant to successfully activate lymph node NK cells in the early phase of the immune response. Our results show that distinct subsets of human DCs, differently from all other mononuclear hemopoietic cells, specifically do not express CD48. Moreover, the expression of CD48 depends on the anatomic location of DCs and might be related to the tissue-specific 2B4 function (activating or inhibitory) of the NK cells with which they interact.     

Regulation of antitumour immunity by CD1d-restricted NKT cells

An understanding of the complex interactions occurring between tumours and the immune system is a prerequisite for the rational design of effective cancer immunotherapies. To date, attention has focused mainly on the role the adaptive immune system plays in controlling tumourigenesis, with conventional T cells, which recognize peptide antigens presented by classical MHC molecules, coming under close scrutiny. Accumulating reports now suggest that an additional T-cell subset, known as CD1d-restricted natural killer T (NKT) cells, also plays a pivotal role in modulating antitumour responses. Found in both humans and mice, CD1d-restricted NKT cells are a highly specialized cell type that, in contrast to conventional T cells, recognize lipid/glycolipid antigens presented by the non-classical MHC molecule CD1d. Several features of NKT cells, including their ability to rapidly produce large quantities of cytokines upon primary stimulation, make them ideal targets for developing anticancer immunotherapies. This intriguing cell type is the focus of this review.     

Functional plasticity of dendritic cell subsets as mediated by CD40 versus B7 activation

Murine dendritic cells (DCs) can present Ag in an immunogenic or tolerogenic fashion, the distinction depending on either the occurrence of specialized DC subsets or the maturation or activation state of the DC. Although DC subsets may be programmed to direct either tolerance or immunity, it is not known whether appropriate environmental stimulation can result in complete flexibility of a basic program. Using splenic CD8(-) and CD8(+) DCs that mediate the respective immunogenic and tolerogenic presentation of self peptides, we show that both the in vivo and in vitro activities of either subset can be altered by ligation of specific surface receptors. Otherwise immunogenic CD8(-) DCs become tolerogenic upon B7 ligation by soluble CTLA-4, a maneuver that initiates immunosuppressive tryptophan catabolism. In contrast, CD40 ligation on tolerogenic CD8(+) DCs makes these cells capable of immunogenic presentation. Thus, environmental conditioning by T cell ligands may alter the default function of DC subsets to meet the needs of flexibility and redundancy.     

CD49d overexpression and T cell autoimmunity

D10.G4.1 (D10) cells, a murine conalbumin-reactive Th2 cell line, made to overexpress the beta(2) integrin LFA-1 by pharmacological manipulation or by transfection become autoreactive and are capable of inducing in vivo autoimmunity. However, whether this is specific to LFA-1 and whether overexpression of other T cell integrin molecules has the same effect are unknown. We examined the functional consequences of T cell CD49d (alpha(4) integrin) overexpression by transfecting murine CD49d cDNA into D10 cells. Similar to the LFA-1-transfected cells, the CD49d-overexpressing T cells are autoreactive and proliferate in response to APCs in an MHC class II-dependent manner in the absence of nominal Ag. Additionally, CD49d overexpression is associated with increased in vitro adhesion to endothelial cells and increased in vivo splenic homing. However, in contrast to LFA-1 overexpression, increased T cell CD49d expression is not associated with autoreactive cytotoxicity or the ability to induce in vivo autoimmunity. In addition to the novel observation that CD49d overexpression is sufficient to induce T cell autoreactivity, our results also support the hypothesis that the ability to induce in vivo autoimmunity is related to T cell cytotoxicity and not to T cell proliferation function in the D10 murine adoptive transfer model of autoimmunity.