CD1d and invariant NKT cells at the human maternal-fetal interface

Invariant CD1d-restricted natural killer T (iNKT) cells comprise a small, but significant, immunoregulatory T cell subset. Here, the presence of these cells and their CD1d ligand at the human maternal-fetal interface was investigated. Immunohistochemical staining of human decidua revealed the expression of CD1d on both villous and extravillous trophoblasts, the fetal cells that invade the maternal decidua. Decidual iNKT cells comprised 0.48% of the decidual CD3+ T cell population, a frequency 10 times greater than that seen in peripheral blood. Interestingly, decidual CD4+ iNKT cells exhibited a striking Th1-like bias (IFN-gamma production), whereas peripheral blood CD4+ iNKT clones exhibited a Th2-like bias (IL-4 production). Moreover, compared to their peripheral blood counterparts, decidual iNKT clones were strongly polarized toward granulocyte/macrophage colony-stimulating factor production. The demonstration of CD1d expression on fetal trophoblasts together with the differential pattern of cytokine expression by decidual iNKT cells suggests that maternal iNKT cell interactions with CD1d expressed on invading fetal cells may play an immunoregulatory role at the maternal-fetal interface.     

CD1d-unrestricted human NKT cells release chemokines upon Fas engagement

Attempts at inducing allograft immune privilege by enforced Fas ligand expression have shown accelerated rejection mediated by neutrophils. While it has been proposed that Fas ligand was directly chemotactic toward neutrophils, several lines of evidence argue for an indirect recruitment mechanism. This question was addressed by using in vitro migration assays that used highly purified human leukocyte subsets. Granulocytes did not migrate in response to Fas engagement and required the presence of T cells expressing several natural killer (NK) cell markers. These rare CD8 memory T cells expressed T and NK cell markers and were not restricted to CD1d, showing that they are distinct from conventional natural killer T (NKT) cells. These cells were able to kill both NK-sensitive and -insensitive targets and secreted several CC and CXC chemokines active toward granulocytes, monocytes, and NK cells upon Fas engagement. Chemotactic factor release depended on caspase activity, in the absence of NKT cell apoptosis. The ability of CD1d-unrestricted NKT cells to recruit innate immune system cells might play a role in cancer cell eradication and contribute to inflammatory diseases.     

Requirement for CD1d expression by B cells to stimulate NKT cell-enhanced antibody production

Activation of natural killer-like T (NKT) cells with the CD1d ligand alpha-galactosylceramide enhances T-dependent humoral immune responses against coadministered T-dependent Ag. At present, there is little information on the mechanisms involved other than a dependence on CD1d expression by antigen-presenting cells and/or development of the NKT subset. We therefore tested the hypothesis that direct presentation of alpha-GC by B cells was required for NKT-enhanced Ab responses against T-dependent Ag. We reconstituted B cell-deficient microMT mice with B cells from C57Bl/6 donors or CD1d(-/-) donors before immunization with NP-KLH alone or NP-KLH mixed with alpha-GC. We made the surprising observation that B-cell expression of CD1d is absolutely required for the NKT-enhanced Ab response. Our data show that the mechanism by which NKT cells enhance humoral immune responses involves interaction with CD1d-expressing B cells.     

Lipid binding orientation within CD1d affects recognition of Borrelia burgorferi antigens by NKT cells

Invariant natural killer T cells (iNKT cells) respond to CD1d-presented glycolipids from Borrelia burgdorferi, the causative agent of Lyme disease. Although mouse and human iNKT cells respond to different antigens based on subtle differences in their fatty acids, the mechanism by which fatty acid structure determines antigenic potency is not well understood. Here we show that the mouse and human CD1d present glycolipids having different fatty acids, based in part upon a difference at a single amino acid position that is involved in positioning the sugar epitope. CD1d also can bind nonantigenic lipids, however, but unexpectedly, mouse CD1d orients the two aliphatic chains of a nonantigenic lipid rotated 180°, causing a dramatic repositioning of the exposed sugar. Therefore, our data reveal the biochemical basis for the high degree of antigenic specificity of iNKT cells for certain fatty acids, and they suggest how microbes could alter fatty acid biosynthesis as an immune evasion mechanism.     

Benzofuran sulfonates and small self-lipid antigens activate type II NKT cells via CD1d

Natural killer T (NKT) cells detect lipids presented by CD1d. Most studies focus on type I NKT cells that express semi-invariant αβ T cell receptors (TCR) and recognize α-galactosylceramides. However, CD1d also presents structurally distinct lipids to NKT cells expressing diverse TCRs (type II NKT cells), but our knowledge of the antigens for type II NKT cells is limited. An early study identified a nonlipidic NKT cell agonist, phenyl pentamethyldihydrobenzofuransulfonate (PPBF), which is notable for its similarity to common sulfa drugs, but its mechanism of NKT cell activation remained unknown. Here, we demonstrate that a range of pentamethylbenzofuransulfonates (PBFs), including PPBF, activate polyclonal type II NKT cells from human donors. Whereas these sulfa drug–like molecules might have acted pharmacologically on cells, here we demonstrate direct contact between TCRs and PBF-treated CD1d complexes. Further, PBF-treated CD1d tetramers identified type II NKT cell populations expressing αβTCRs and γδTCRs, including those with variable and joining region gene usage (TRAV12-1–TRAJ6) that was conserved across donors. By trapping a CD1d–type II NKT TCR complex for direct mass-spectrometric analysis, we detected molecules that allow the binding of CD1d to TCRs, finding that both selected PBF family members and short-chain sphingomyelin lipids are present in these complexes. Furthermore, the combination of PPBF and short-chain sphingomyelin enhances CD1d tetramer staining of PPBF-reactive T cell lines over either molecule alone. This study demonstrates that nonlipidic small molecules, which resemble sulfa drugs implicated in systemic hypersensitivity and drug allergy reactions, are targeted by a polyclonal population of type II NKT cells in a CD1d-restricted manner.

Natural killer T (NKT) cells are defined as T cells that are restricted to the lipid antigen-presenting molecule, CD1d. The most extensively studied are type I NKT cells, which typically express an invariant T cell receptor (TCR)-α chain consisting of TRAV10–TRAJ18 in humans (TRAV11–TRAJ18 in mice) paired with a constrained repertoire of TCR-β chains, enriched for TRBV25 in humans (TRBV13, 29, 1 in mice) (reviewed in ref. 1). Type I NKT cells are defined by their strong responses to α-galactosylceramide (α-GalCer) and structurally related hexosylceramides presented by CD1d, while in contrast, type II NKT cells are defined as CD1d-restricted T cells that express diverse TCRs and do not recognize α-GalCer (reviewed in refs. 1 and 2). Very little is known about the chemical identity of antigens for type II NKT cells; however, some studies suggest that these cells are abundant in humans (3–5), and, by virtue of their greater TCR diversity, they can interact with a broader range of antigens compared to type I NKT cells (2, 6–14).In 2004, a nonlipidic molecule, phenyl pentamethyldihydrobenzofuransulfonate (PPBF), was described that stimulated a human TRAV10⁻ (type II) NKT cell line (clone ABd) in a CD1d-dependent manner (15). These observations were notable because PPBF resembles various sulfonamide drugs: furosemide (diuretic), sulfasalazine (disease-modifying antirheumatic), and celecoxib (anti-inflammatory) as well as “sulfa” antibiotics such as sulfonamide, sulfapyridine, sulfamethoxazole, sulfadiazine, and sulfadoxine (16). These drugs can cause systemic delayed-type hypersensitivity reactions, which are thought to be mediated by T cells (17–20). Most of our limited understanding of drug hypersensitivity reactions comes from work focusing on Human Leukocyte Antigen (HLA)-restricted conventional T cells, which has led to the proposal of four main mechanisms for small-drug immune activity as reviewed in ref. 21: 1) hapten/prohapten formation, whereby the drug reacts with self-Ags to generate a neo-product that undergoes processing and presentation to T cells; 2) noncovalent/labile pharmacological interaction with immune receptors on the cell surface; 3) superantigen mediating direct linkage of TCRs and Ag-presenting molecules; and 4) anchor site occupation by small molecules in Ag-presenting molecules inducing an altered self-Ag repertoire (22). Whether the CD1d-NKT cell axis is implicated in drug hypersensitivity remains unclear (23). Whereas most antigens in the CD1d system are lipids that use their alkyl chains to bind to CD1d, PPBF is a polycyclic small molecule and so would have to act through an atypical display mechanism. As previous attempts to stain the ABd clone with CD1d-PPBF tetramers were unsuccessful, the atypical drug-like structure of PPBF raised the possibility of direct pharmacological action on T cells rather than the presentation of CD1d-PPBF complexes to TCRs. However, the mechanism of PPBF-mediated type II NKT cell activation remains undefined (15).Here, using TCR-transduced cell lines, CD1d tetramers treated with PPBF, and new analogs in the pentamethylbenzofuransulfonate (PBF) family, we discovered that several molecules stimulate polyclonal NKT cells. Using CD1d tetramers treated with a newly identified and more potent analog of PPBF, we identified populations of type II NKT cells that comprise a polyclonal repertoire of both αβ and γδ T cells, including those with conserved TCR sequences. This enigmatic nature of T cell responses to PBF molecules was resolved using TCR trap technology (24, 25). Mass-spectrometric analysis of all molecules present in CD1d-PBF-TCR complexes indicates that CD1d binds PBFs and small self-lipids that promote CD1d-TCR binding. These data support a model of type II NKT cell recognition of small sulfa drug–like compounds in association with CD1d and flag a possible mechanism in which such cells may be involved in sulfa drug hypersensitivity.     

Glycolipid activation of invariant T cell receptor+ NK T cells is sufficient to induce airway hyperreactivity independent of conventional CD4+ T cells

Asthma is an inflammatory lung disease, in which conventional CD4+ T cells producing IL-4/IL-13 appear to play an obligatory pathogenic role. Here we show, in a mouse model of asthma, that activation of pulmonary IL-4/IL-13 producing invariant TCR+ CD1d-restricted natural killer T (NKT) cells is sufficient for the development of airway hyperreactivity (AHR), a cardinal feature of asthma, in the absence of conventional CD4+ T cells and adaptive immunity. Respiratory administration of glycolipid antigens that specifically activate NKT cells (alpha-GalactosylCeramide and a Sphingomonas bacterial glycolipid) rapidly induced AHR and inflammation typically associated with protein allergen administration. Na?ve MHC class II-deficient mice, which lack conventional CD4+ T but have NKT cells, showed exaggerated baseline AHR and, when challenged with alpha-GalactosylCeramide, demonstrated even greater AHR. These studies demonstrate an expanded role for NKT cells, in which NKT cells not only produce cytokines that influence adaptive immunity but also function as critical effector cells that can induce AHR. These results suggest that NKT cells responding to glycolipid antigens, as well as conventional CD4+ T cells responding to peptide antigens, may be synergistic in the induction of AHR, although in some cases, each may independently induce AHR.     

非酒精性脂肪性肝病患者外周血恒定自然杀伤T细胞和CD4+/CD8+T细胞活化差异研究*

目的 分析不同非酒精性脂肪性肝病(NAFLD)患者外周血恒定自然杀伤T细胞(iNKT)、CD4⁺和CD8⁺ T细胞活化标记物(CD69、CD25、HLA-DR和NKG2D)的表达差异。方法 2020年1月～2022年7月我院诊治的NAFLD患者64例和同期健康体检者50例,对NAFLD患者行肝穿刺活检,使用流式细胞仪检测外周血iNKT、CD4⁺和CD8⁺T细胞CD69、CD25、HLA-DR和NKG2D表达。结果 在64例NAFLD患者中,经组织病理学检查,诊断NAFL 37例和NASH 27例;健康对照者、NAFL和NASH患者健康对照者、NAFL和NASH患者外周血CD69⁺iNKT细胞百分比分别为(10.1±1.7)%、(6.1±1.3)%和(26.7±3.6)%(P<0.05),CD25⁺iNKT细胞百分比分别为(83.0±5.9)%、(94.1±8.0)%和(90.8±7.5)%(P<0.05),HLA-DR⁺iNKT细胞百分比分别为(15.3±1.7)%、(15.8±2.0)%和(22.3±2.0)%(P>0.05),NKG2D⁺iNKT细胞百分比分别为(44.5±3.5)%、(59.7±4.0)%和(71.3±6.0)%(P<0.05);外周血CD69⁺CD4⁺ T细胞百分比分别为(0.7±0.2)%、(0.4±0.1)%和(0.5±0.1)%(P>0.05),CD25⁺CD4⁺ T细胞百分比分别为(1.4±0.6)%、(3.0±1.3)%和(1.5±0.7)%(P>0.05),HLA-DR⁺CD4⁺ T细胞百分比分别为(2.7±0.7)%、(4.1±1.0)%和(3.9±1.0)%(P<0.05),NKG2D⁺CD4⁺ T细胞百分比分别为(1.6±0.5)%、(0.6±0.2)%和(0.9±0.2)%(P<0.05);外周血CD69⁺CD8⁺ T细胞百分比分别为(2.0±0.4)%、(1.6±0.3)%和(2.1±0.6)%(P>0.05),CD25⁺CD8⁺ T细胞百分比分别为(1.3±0.3)%、(1.1±0.2)%和(1.0±0.2)%(P>0.05),HLA-DR⁺CD8⁺ T细胞百分比分别为(5.0±0.7)%、(6.5±1.0)%和(9.6±1.4)%(P<0.05),NKG2D⁺CD8⁺ T细胞百分比分别为(0.6±0.1)%、(0.5±0.1)%和(0.9±0.2)%(P<0.05)。结论 本研究发现NAFL与NASH患者可能存在外周血iNKT细胞、CD4⁺和CD8⁺ T细胞活化的免疫表型差异,显示NASH患者CD69⁺iNK T细胞百分比增高,可能对诊断有帮助,值得进一步研究。     

Cell-contact-dependent activation of CD4+ T cells by adhesion molecules on synovial fibroblasts

Objective: To determine how cell–cell contact with synovial fibroblasts (SF) influence on the proliferation and cytokine production of CD4^+?T cells.

Methods: Naïve CD4^+?T cells were cultured with SF from rheumatoid arthritis patients, stimulated by anti-CD3/28 antibody, and CD4^+?T cell proliferation and IFN-γ/IL-17 production were analyzed. To study the role of adhesion molecules, cell contact was blocked by transwell plate or anti-intracellular adhesion molecule-1 (ICAM-1)/vascular cell adhesion molecule-1(VCAM-1) antibody. To study the direct role of adhesion molecules for CD4^+?T cells, CD161^+?or CD161^- naïve CD4^+?T cells were stimulated on plastic plates coated by recombinant ICAM-1 or VCAM-1, and the source of IFN-γ/IL-17 were analyzed.

Results: SF enhanced naïve CD4^+?T cell proliferation and IFN-γ/IL-17 production in cell-contact and in part ICAM-1-/VCAM-1-dependent manner. Plate-coated ICAM-1 and VCAM-1 enhanced naïve CD4^+?T cell proliferation and IFN-γ production, while VCAM-1 efficiently promoting IL-17 production. CD161^+?naïve T cells upregulating LFA-1 and VLA-4 were the major source of IFN-γ/IL-17 upon interaction with ICAM-1/VCAM-1.

Conclusion: CD4^+?T cells rapidly expand and secrete IFN-γ/IL-17 upon cell-contact with SF via adhesion molecules. Interfering with ICAM-1-/VCAM-1 may be beneficial for inhibiting RA synovitis.     

Roles of CD1d-restricted NKT cells in the intestine

Natural killer T (NKT) cells are a subset of lymphocytes that express cell surface molecules of both conventional T cells and natural killer cells and share the features of both innate and adaptive immune cells. NKT cells have been proposed to make both protective and pathogenic contributions to inflammatory bowel diseases (IBD). On the one hand, recent studies have shown that these cells are involved in the maintenance of mucosal homeostasis. On the other, NKT cells were shown to play a pathogenic role in human ulcerative colitis. Similar contrasting data have been generated in murine models of IBD. Whether the apparent differences in NKT response patterns depend on variations in NKT antigens and/or on the presence of specific subsets of mucosal NKT cells remains to be elucidated. In this article we review the current literature on intestinal NKT cells and their roles in IBD pathogenesis. Specifically, the nomenclature, NKT antigens, and immune mechanisms of NKT cells within the intestinal mucosa are discussed.     

Polymorphisms in CD1d affect antigen presentation and the activation of CD1d-restricted T cells

CD1 proteins constitute a distinct lineage of antigen-presenting molecules specialized for the presentation of lipid antigens to T cells. In contrast to the extensive sequence polymorphism characteristic of classical MHC molecules, CD1 proteins exhibit limited sequence diversity. Here, we describe the identification and characterization of CD1d alleles in wild-derived mouse strains. We demonstrate that polymorphisms in CD1d affect the presentation of endogenous and exogenous ligands to CD1d-restricted T cells, including type I (Vα14i) and type II (non-Vα14i) natural killer T (NKT) cells. Using congenic mice, we found CD1d polymorphisms affect the thymic selection of type I NKT cells and induce allogeneic T cell responses. Collectively, results from these studies demonstrate a role for polymorphisms in influencing the development and function of CD1d-restricted T cells.     

Galectin-1: a key effector of regulation mediated by CD4+CD25+ T cells

The naturally occurring population of dedicated regulatory T cells that coexpress CD4 and CD25 is known to play a key role in the maintenance of peripheral T-cell tolerance; however, their mechanism of action has remained obscure. Here we report that a member of the family of beta-galactoside-binding proteins, galectin-1, is overexpressed in regulatory T cells, and that expression is increased after activation. Most importantly, blockade of galectin-1 binding significantly reduced the inhibitory effects of human and mouse CD4+CD25+ T cells. Reduced regulatory activity was observed in CD4+CD25+ T cells obtained from galectin-1-homozygous null mutant mice. These results suggest that galectin-1 is a key effector of the regulation mediated by these cells.     

Human CD1d knock-in mouse model demonstrates potent antitumor potential of human CD1d-restricted invariant natural killer T cells

Despite a high degree of conservation, subtle but important differences exist between the CD1d antigen presentation pathways of humans and mice. These differences may account for the minimal success of natural killer T (NKT) cell-based antitumor therapies in human clinical trials, which contrast strongly with the powerful antitumor effects in conventional mouse models. To develop an accurate model for in vivo human CD1d (hCD1d) antigen presentation, we have generated a hCD1d knock-in (hCD1d-KI) mouse. In these mice, hCD1d is expressed in a native tissue distribution pattern and supports NKT cell development. Reduced numbers of invariant NKT (iNKT) cells were observed, but at an abundance comparable to that in most normal humans. These iNKT cells predominantly expressed mouse Vβ8, the homolog of human Vβ11, and phenotypically resembled human iNKT cells in their reduced expression of CD4. Importantly, iNKT cells in hCD1d knock-in mice exert a potent antitumor function in a melanoma challenge model. Our results show that replacement of mCD1d by hCD1d can select a population of functional iNKT cells closely resembling human iNKT cells. These hCD1d knock-in mice will allow more accurate in vivo modeling of human iNKT cell responses and will facilitate the preclinical assessment of iNKT cell-targeted antitumor therapies.     

Regulation of hematopoiesis in vitro and in vivo by invariant NKT cells

Invariant natural killer T cells (iNKT cells) are a small subset of immunoregulatory T cells highly conserved in humans and mice. On activation by glycolipids presented by the MHC-like molecule CD1d, iNKT cells promptly secrete T helper 1 and 2 (Th1/2) cytokines but also cytokines with hematopoietic potential such as GM-CSF. Here, we show that the myeloid clonogenic potential of human hematopoietic progenitors is increased in the presence of glycolipid-activated, GM-CSF-secreting NKT cells; conversely, short- and long-term progenitor activity is decreased in the absence of NKT cells, implying regulation of hematopoiesis in both the presence and the absence of immune activation. In accordance with these findings, iNKT-cell-deficient mice display impaired hematopoiesis characterized by peripheral-blood cytopenias, reduced marrow cellularity, lower frequency of hematopoietic stem cells (HSCs), and reduced early and late hematopoietic progenitors. We also show that CD1d is expressed on human HSCs. CD1d-expressing HSCs display short- and long-term clonogenic potential and can present the glycolipid alpha-galactosylceramide to iNKT cells. Thus, iNKT cells emerge as the first subset of regulatory T cells that are required for effective hematopoiesis in both steady-state conditions and under conditions of immune activation.     

免疫性肝损伤动物模型建立及4-1BB、CD4+CD25+T淋巴细胞表达

4-1BB是一种主要表达在活化T淋巴细胞上共刺激信号,是神经生长因了/肿瘤坏死因子受体家族成员之一。CD4^＋CD25^＋T淋巴细胞是一种免疫耐受细胞,具有免疫无能性及免疫抑制性两方面功能。本实验对免疫性肝损伤中4 1BB及CD4^＋CD25^＋T淋巴细胞的变化进行研究。[第一段]     

CD4+CD25+调节性T细胞与1型糖尿病

CD4^＋CD25^＋调节性T细胞是调节性T细胞亚群中的重要组成成分，可组成性表达许多细胞表面分子，包括CD25、细胞毒性T淋巴细胞相关抗原-4（CTLA-4）、糖皮质激素诱导肿瘤坏死因子受体（GITR）、CD122、CD103和转录因子FOXp3，并具有免疫无能性和免疫抑制性两大特性。1型糖尿病的发病与调节性T细胞的数目、功能以及调节性T细胞与效应性T细胞的比例失衡有关。多项实验表明，在体内诱生或过继调节性T细胞能预防1型糖尿病的发生和发展。