The role of tetraspanin CD63 in antigen presentation via MHC class II

Interactions between MHC class II (MHC II)-positive APCs and CD4(+) T cells are central to adaptive immune responses. Using an Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell line (LCL) as MHC II-positive APCs and CD4(+) T-cell clones specific for two endogenously expressed EBV antigens, we found that shRNA knockdown of the tetraspanin protein CD63 in LCL cells consistently led to increased CD4(+) T-cell recognition. This effect was not due to enhanced antigen processing nor to changes in MHC II expression since CD63 knockdown did not influence the amount or dimerization of MHC II in LCL cells. We therefore investigated the possible involvement of exosomes, small MHC II- and tetraspanin-abundant vesicles which are secreted by LCL cells and which we found could themselves activate the CD4(+) T-cell clones in an MHC II-dependent manner. While equal loadings of exosomes purified from the control and CD63(low) LCLs stimulated T cells to a comparable degree, we found that exosome production significantly increased following CD63-knockdown, suggesting that this may underlie the greater T-cell stimulatory capacity of the CD63(low) LCLs. Taken together, our data reveal a new insight into the mechanisms by which tetraspanins are involved in the regulation of MHC II-dependent T-cell stimulation.     

Analysis of the immune response against tetanus toxoid: enumeration of specific T helper cells by the Elispot assay

Tetanus toxoid (TT) is an antigen known to induce strong T cell specific immune responses in humans after vaccination. Here we have used the Elispot assay to assess the number of TT-specific Interferon-gamma (IFN-gamma) secreting T cells present in individuals and monitored the number of TT specific T cells present in the donors for more than two years. In each of the 22 healthy volunteers tested, TT-specific T cells could be detected. Six out of 7 repetitively tested donors showed a remarkably constant number of TT-specific IFN-gamma secreting T cells over several months, whereas one donor demonstrated a transient increase during a flu-like infection. Three healthy donors received TT booster-immunizations and showed significant increases in the number of TT-specific IFN-gamma secreting T cells which reached peak levels by 4 weeks after vaccination. Depletion of either CD4+ T cells, CD8+ T cells or CD16+/CD56+ T cells by immunomagnetic separation demonstrated that TT-specific IFN-gamma secretion is mediated exclusively by CD4+ T cells. In addition, HLA class-I and -II blocking studies showed that IFN-gamma production is performed by HLA class-II restricted cells. Our data show that the Elispot assay can be reliably used to assess the number of TT-specific CD4+ IFN-gamma producing cells (i.e. probably T helper cells) and therefore maybe also useful for the assessment of reactions to other helper antigens.     

Prevention of T cell activation by interference of internalized intravenous immunoglobulin (IVIg) with MHC II-dependent native antigen presentation

Activation of self-reactive CD4⁺ T cells plays a central role in the initiation and maintenance of autoimmune diseases. We recently reported that intravenous immunoglobulin (IVIg) inhibits the MHC II-restricted CD4⁺ T cell activation induced by the presentation of immune complexes. Because native antigens can also play a role in the induction of several autoimmune diseases, we determined whether IVIg could also affect CD4⁺ T cell activation following presentation of native antigens by APCs. Here we report that IVIg significantly reduces the activation of CD4⁺ T cells by native ovalbumin. The inhibitory effect is FcγR-independent and occurs following internalization of IVIg inside APCs, where it interferes with the intracellular events leading to MHC II-dependent antigen presentation. The effect of IVIg on native antigen presentation could therefore contribute to dampen the autoimmune reaction by reducing CD4⁺ T cell activation and the subsequent inflammatory response induced by these cells.     

Melanoma cell necrosis facilitates transfer of specific sets of antigens onto MHC class II molecules of dendritic cells

Vaccine strategies that target dendritic cells (DC) in order to elicit immunity against tumors are the subject of intense research. For the induction and maintenance of anti-tumor immunity, CD4+ helper T cells are often required, which need to see appropriate MHC class II-peptide complexes on DC. So far, it remained widely unclear what type of tumor cells can feed the MHC class II processing pathway of DC with what type of antigens. Here, we report that peptide loading onto MHC class II molecules of myeloid DC is facilitated by melanoma cells undergoing necrotic rather than apoptotic cell death. Importantly, the set of MHC class II-associated peptides induced by necrotic tumor cells differed from those found upon engagement of apoptotic tumor cells. This may be due to the fact that only necrotic cells liberated heat shock proteins, which bind tumor-derived peptides and thereby may promote processing by DC. The potential of DC to activate T cells was kinetically controlled through their antigen receptivity: CD4+ T cells were easily stimulated upon encountering antigen early in DC maturation, whereas antigen capture at later maturation stages favored activation of CD8+ T cells. These findings may aid in designing future vaccination strategies and in identifying novel tumor-specific helper T cell antigens.     

Effective antigen presentation to helper T cells by human eosinophils

Although eosinophils are inflammatory cells, there is increasing attention on their immunomodulatory roles. For example, murine eosinophils can present antigen to CD4⁺ T helper (Th) cells, but it remains unclear whether human eosinophils also have this ability. This study determined whether human eosinophils present a range of antigens, including allergens, to activate Th cells, and characterized their expression of MHC class II and co‐stimulatory molecules required for effective presentation. Human peripheral blood eosinophils purified from non‐allergic donors were pulsed with the antigens house dust mite extract (HDM), Timothy Grass extract (TG) or Mycobacterium tuberculosis purified protein derivative (PPD), before co‐culture with autologous CD4⁺ Th cells. Proliferative and cytokine responses were measured, with eosinophil expression of HLA‐DR/DP/DQ and the co‐stimulatory molecules CD40, CD80 and CD86 determined by flow cytometry. Eosinophils pulsed with HDM, TG or PPD drove Th cell proliferation, with the response strength dependent on antigen concentration. The cytokine responses varied with donor and antigen, and were not biased towards any particular Th subset, often including combinations of pro‐ and anti‐inflammatory cytokines. Eosinophils up‐regulated surface expression of HLA‐DR/DP/DQ, CD80, CD86 and CD40 in culture, increases that were sustained over 5 days when incubated with antigens, including HDM, or the major allergens it contains, Der p I or Der p II. Human eosinophils can, therefore, act as effective antigen‐presenting cells to stimulate varied Th cell responses against a panel of antigens including HDM, TG or PPD, an ability that may help to determine the development of allergic disease.     

Longitudinal analysis of T cells responding to tetanus toxoid in healthy subjects as well as in pediatric patients after bone marrow transplantation: the identification of identical TCR-CDR3 regions in time suggests long-term stability of at least part of the antigen-specific TCR repertoire

To understand the nature of long-term Th immune responses, we investigated in the present study the TCRBV gene repertoire of CD4(+) T cells specific for the recall antigen tetanus toxoid (TT) in recipients of an allogeneic bone marrow transplantation (allo-BMT) at several time points after transplantation and in their BM donors. We observed that the TCR repertoire of TT-specific CD4(+) Th cells was heterogeneous, and differed between allo-BMT recipients and their respective donors. Some individuals, however, used similar TCR-complementarity-determining region (CDR) 3 motifs that could reflect recognition of and selection by similar promiscuous epitopes of TT. Longitudinal analysis of this TT-specific T cell response revealed that T cells with completely identical TCR were present at several time points after the first analysis in allo-BMT recipients, most probably reflecting long-term stability of at least part of the antigen-specific TCR repertoire. Similar stability of the TT-specific TCR repertoire in time was also noted in the allo-BMT donors. These observations reveal that within a given individual the dominant antigen-specific T cell clones persist in time in an otherwise diverse TT-specific CD4(+) T cell immune response.     

Direct proteasome-independent cross-presentation of viral antigen by plasmacytoid dendritic cells on major histocompatibility complex class I

Although plasmacytoid dendritic cells (pDCs) respond to virus replication in a nonspecific way by producing large amounts of type I interferon, a rapid, direct function for pDCs in activating antiviral lymphocytes is less apparent. Here we show that pDCs were able to rapidly initiate antigen-specific antiviral CD8+ T cell responses. After being exposed to virus, pDCs efficiently and rapidly internalized exogenous viral antigens and then presented those antigens on major histocompatibility complex (MHC) class I to CD8+ T cells. Processing of exogenous antigen occurred in endocytic organelles and did not require transit of antigen to the cytosol. Intracellular stores of MHC class I partially localized together with the transferrin receptor and internalized transferrin in endosomes, which suggested that such recycling endosomes are sites for loading peptide onto MHC class I or for peptide transit. Our data demonstrate that pDCs use 'ready-made' stores of MHC class I to rapidly present exogenous antigen to CD8+ T cells.     

Hypersensitivities following allergen antigen recognition by unconventional T cells

Conventional T cells recognise protein-derived antigens in the context of major histocompatibility complex (MHC) class Ia and class II molecules and provide anti-microbial and anti-tumour immunity. Conventional T cells have also been implicated in type IV (also termed delayed-type or T cell–mediated) hypersensitivity reactions in response to protein-derived allergen antigens. In addition to conventional T cells, subsets of unconventional T cells exist, which recognise non-protein antigens in the context of monomorphic MHC class I-like molecules. These include T cells that are restricted to the cluster of differentiation 1 (CD1) family members, known as CD1-restricted T cells, and mucosal-associated invariant T cells (MAIT cells) that are restricted to the MHC-related protein 1 (MR1). Compared with conventional T cells, much less is known about the immune functions of unconventional T cells and their role in hypersensitivities. Here, we review allergen antigen presentation by MHC-I-like molecules, their recognition by unconventional T cells, and the potential role of unconventional T cells in hypersensitivities. We also speculate on possible scenarios of allergen antigen presentation by MHC-I-like molecules to unconventional T cells, the hallmarks of such responses, and the expected frequencies of hypersensitivities within the human population.     

T cell tolerance and activation to a transgene-encoded tumor antigen

Much has been learned in recent years concerning the nature of tumor antigens recognized by T cells. To apply this knowledge clinically, the nature of the host response to individual and multiple tumor antigens has to be characterized. This will help to define the efficacy of immune surveillance and the immune status of the host following exposure to tumor antigens expressed on pre-neoplastic tissue. To approach these questions, we have developed a transgenic mouse which expresses the tumor-specific antigen P91A. The single amino acid substitution in P91A results in the expression of a new MHC class I (H-2L^d)-binding peptide. In transgenic tissue, the H-2L^d/P91A complex is expressed in isolation from other tumor-associated antigens, allowing definition of the immune response to a single defined tumor antigen, a situation closely analogous to events during tumorigenesis. We show that CD8⁺ T cell immune surveillance of P91A is ineffective without the introduction of a helper determinant operating through stimulation of CD4⁺ T cells. Recognition of the isolated P91A tumor antigen on normal tissue by CD8⁺ T cells is a tolerogenic process. Induction of T cell tolerance suggests tumor antigen-T cell interactions occurring during tumorigenesis may elicit T cell tolerance and hence confound some immunotherapeutic approaches.     

Antigen unfolding and disulfide reduction in antigen presenting cells

CD4+ helper T cells recognize short peptides stably associated with class II MHC molecules displayed on the surface of antigen presenting cells. Very little is known about the sequence of events that lead to the generation of these peptides from protein antigens. It is likely that native proteins must partially unfold before they are cleaved by endopeptidases or bind to MHC proteins. For many antigens, the rate-limiting step in unfolding may involve reduction of disulfide bonds. Evidence that disulfide reduction occurs in endocytic compartments is reviewed and potential mechanisms for the reduction of antigen disulfide bonds are proposed.     

Spatial and mechanistic separation of cross-presentation and endogenous antigen presentation

Antiviral or antitumor immunity requires activation of cytotoxic CD8+ T cells by dendritic cells, which present viral or tumor antigens on major histocompatibility complex (MHC) class I molecules. The intracellular mechanisms facilitating MHC class I-restricted presentation of extracellular antigens ('cross-presentation') are unclear. Here we demonstrate that cross-presentation of soluble antigen occurred in an early endosomal compartment distinct from the endoplasmic reticulum where endogenous antigen is loaded onto MHC class I. Efficient cross-presentation required endotoxin-induced, Toll-like receptor 4- and signaling molecule MyD88-dependent relocation of the transporter associated with antigen processing, essential for loading of MHC class I, to early endosomes. Transport of cross-presented antigen from endosomes to the cell surface was inhibited by primaquine, which blocks endosomal trafficking. Thus, cross-presentation is spatially and mechanistically separated from endogenous MHC class I-restricted antigen presentation and is biased toward antigens containing microbial molecular patterns.     

Polymorphonuclear neutrophils as accessory cells for T-cell activation: major histocompatibility complex class II restricted antigen-dependent induction of T-cell proliferation

Polymophonuclear cells (PMN) of healthy donors do not express major histocompatibility complex (MHC) class II antigens or the T-cell costimulatory molecules CD80 or CD86. Expression of these receptors, however, is seen in patients with chronic inflammatory diseases. We now report that, by culturing PMN of healthy donors with autologous serum, interferon-gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF), de novo synthesis of MHC class II, CD80 and CD86 could be induced. MHC class II-positive PMN acquired the capacity to present staphylococcus enterotoxin to peripheral T cells, apparent as induction of interleukin-2 (IL-2) synthesis and proliferation of the T cells. Moreover, the PMN also processed tetanus toxoid (TT) and induced proliferation of TT-specific T cells in a MHC class II-restricted manner. Taken together, these data indicate that PMN can be activated to function as accessory cells for T-cell activation.     

MHC class II-dependent activation of CD4+ T cell hybridomas by human mast cells through superantigen presentation.

Human mast cells (MC) were examined for expression of MHC class II antigens and for their ability to activate CD4+ T cell hybridomas through presentation of superantigen (SAg). HMC-1, a leukemic immature MC line expressing class II Ags, was shown to efficiently present the staphylococcal enterotoxin B (SEB) SAg to responding T cell hybridoma on treatment with interferon-gamma (IFN-gamma), which up-regulated class II molecules. The study was then extended to human normal MC. Almost pure (>99%) cord blood-derived MC (CBMC) were shown to express class II Ags (HLA-DR and HLA-DQ) and CD80, which were up-regulated by IFN-gamma treatment and, to a lesser extent, by interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). CBMC directly activated CD4+ T cell hybridomas through presentation of SEB and TSST1 SAgs. The production of IL-2 required a cell-to-cell contact between T cells and CBMC and it was inhibited by anti-class II antibodies. Furthermore, an additional pretreatment of CBMC by IFN-gamma or GM-CSF or IL-4 had no effect on their presenting efficiency. This previously unknown function of human MC, i.e., MHC class II-dependent activation of CD4+ T cells, may be critical in subsequent cellular activation events because colocalization of mast and T cells is frequently observed at sites of antigen entry.     

Human tumor antigens: implications for cancer vaccine development

The adoptive transfer of tumor-infiltrating lymphocytes along with interleukin 2 into autologous patients resulted in the objective regression of tumor in about 30% of patients with melanoma, indicating that these T cells play a role in tumor rejection. To understand the molecular basis of the T cell-cancer cell interaction we and others started to search for tumor antigens expressed on cancer cells recognized by T cells. This led to the identification of several major histocompatibility complex (MHC) class I restricted tumor antigens. These tumor antigens have been classified into several categories: tissue-specific differentiation antigens, tumor-specific shared antigens, and tumor-specific unique antigens. Because CD4+ T cells play a central role in orchestrating the host immune response against cancer, infectious diseases, and autoimmune diseases, a novel genetic approach has recently been developed to identify these MHC class II restricted tumor antigens. The identification of both MHC class I and II restricted tumor antigens provides new opportunities for the development of therapeutic strategies against cancer. This review summarizes the current status of tumor antigens and their potential applications to cancer treatment.     

Lowering the affinity between antigen and the B cell receptor can enhance antigen presentation

The B cell receptor (BCR) enables antigen-specific B cells to bind, internalize and target antigens for processing into small peptide fragments. These epitopes are then expressed on the plasma membrane in association with MHC class II molecules for recognition by CD4+ T cells. The affinity of the interaction between the BCR and antigen plays an important part in determining T cell epitope generation. In this report we provide evidence that the efficiency of antigen presentation by specific B cells does not need to be directly proportional to antigen/BCR affinity. We show that increased presentation can result from lowering the affinity of the antigen/BCR interaction. This finding suggests a novel mechanism by which B cells can recruit T cell help and obtain survival signals. Activation of these cells may have consequences for the generation of the B cell repertoire.     

Microglial cells in human brain have phenotypic characteristics related to possible function as dendritic antigen presenting cells

The resting human microglia have previously been shown to be cells of dendritic morphology expressing class II MHC antigens and macrophage specific antigens by immunocytochemical techniques. To examine the relationship between the microglia and the family of dendritic antigen presenting cells (APC), normal white matter from eight normal adults with no neurological disease at autopsy was examined by immunocytochemical techniques to localize antibodies to leukocyte common antigen (LCA), HLA-DR, CD1 (T6), CD4 (T4), and glial fibrillary acidic protein. In addition, enzyme histochemical staining for ATPase, non-specific esterase (NSE), and acid phosphatase (ACP) was performed. The normal microglia are ATPase +ve, NSE -ve, ACP -ve, HLA-DR +ve, LCA +ve, CD1 (T6) +ve and weakly CD4 (T4) +ve. This specialized phenotype closely resembles that of Langerhans cells and suggests that microglia are not simply quiescent phagocytes, but may have a primary role as microenvironmentally specialized APC. The finding of weak anti-CD4 (T4) immunoreactivity supports suggestions for a central role for this cell in infection of the central nervous system by human immunodeficiency virus type 1.     

Antitumour activity mediated by CD4+ cytotoxic T lymphocytes against MHC class II-negative mouse hepatocellular carcinoma induced by dendritic cell vaccine and interleukin-12

When BALA/c mice with BNL hepatocellular carcinoma (HCC) were treated with dendritic cells fused with BNL cells (DC/BNL) and recombinant murine interleukin (IL)-12, tumour development was significantly suppressed, whereas treatment with either DC/BNL or IL-12 alone did not show a tumour-suppressive effect. Antitumour activity induced by DC/BNL + IL-12 was abrogated by depletion of CD4+ T cells, but not by depletion of CD8+ T cells or natural killer cells. Splenic CD4+ T cells and CD8+ T cells from DC/BNL-treated mice showed cytotoxic activity against BNL cells after 3 days of incubation with DC/BNL, although BNL cells do not express major histocompatibility complex (MHC) class II molecules even after treatment with interferon (INF)-gamma. Furthermore, CD4+ T cells killed syngeneic-irrelevant CT26 cells and even allogeneic Hepa1-6 cells. This cytotoxicity was blocked by concanamycin A, but not by an anti-Fas ligand (FasL) monoclonal antibody, indicating that cytotoxic activity was mediated by perforin. Immunofluorescence microscopy demonstrated that abundant CD4+ T cells and MHC class II-positive macrophages, but not CD8(+) T cells, had infiltrated tumour tissue in mice treated with DC/BNL + IL-12. Flow cytometric analysis of tumour-infiltrating cells in mice treated with DC/BNL + IL-12 showed increases in CD4+ T cells and MHC class II+ CD11b+ cells but not in CD8+ T cells or MHC class I+ CD11b+ cells. Our results suggest that, in BNL-bearing mice treated with DC/BNL + IL-12, tumour macrophages activated by INF-gamma produced by IL-12-stimulated T cells might present BNL tumour antigens and activate DC/BNL-primed CD4+ cytotoxic T lymphocytes (CTLs) in a MHC class II-dependent manner, leading to perforin-mediated bystander killing of neighbouring MHC class II-negative tumour cells.     

Cross-presentation of antigens by dendritic cells

T lymphocytes recognize antigen presented on the surface of antigen-presenting cells byMHC class I and class II molecules. Classically, MHC class I molecules present self- or pathogen-derived antigens that are synthesized within the cell, whereas exogenous antigens derived via endocytic uptake are loaded onto MHC class II molecules for presentation to CD4+ T cells. It is becoming increasingly clear that some dendritic cells are also specialized to process exogenous antigens into the MHC class I pathway for presentation to CD8+ T cells. This process is known as cross-presentation. It provides a mechanism that can drive dendritic cells to generate either tolerance to self-antigens or immunity to pathogens. The cells responsible for, and mechanisms underlying, this decision between tolerance and immunity via cross-presentation has become the focus of intense study to determine how various dendritic cell subsets effect the different outcomes.     

不同种类乙型肝炎表面抗原诱导小鼠早期细胞免疫应答的比较

目的 比较不同表达系统来源的乙型肝炎(乙肝)表面抗原(HBsAg)免疫小鼠诱导早期脾淋巴细胞抗原特异性细胞免疫应答的特点,探讨影响乙肝疫苗保护效果的因素.方法 3种HBsAg(汉逊酵母、CHO细胞和血源)分别皮下接种不同组小鼠(BALB/c,H-2d),每只3μg,于免疫后4d分离脾单个核细胞(MNC),经细胞分选仪(MACs)分选后,获得纯度高于90％的CD4+和CD8+T细胞,应用ELISPOT测定MNC、CD4+和CD8+T细胞体外刺激后所产生的细胞因子IFN-γ、IL-2斑点数( SFC).结果 细胞分选后,汉逊抗原诱导CD8+T细胞分泌IFN-γ的水平和CD4+T细胞分泌IL-2水平均显著高于CHO抗原组(8/10、2/10,P=0.035;6/10、0/10,P=0.005).汉逊抗原组与血源抗原组CD8+T细胞经体外刺激诱导IFN-y全部阳转(10/10),但分泌水平上汉逊抗原组显著高于血源抗原组(t=2.479,P=0.035);汉逊抗原组诱导CD4+T细胞IFN-γ阳转率及分泌水平均显著高于血源抗原组(10/10、4/10,P=0.005;t=3.967,P=0.003).结论 乙肝抗原免疫小鼠4d即可诱导细胞免疫应答,且汉逊酵母抗原早期诱导抗原特异性IFN-γ、IL-2的能力显著高于CHO和血源抗原,与其临床考核母婴传播阻断HBV保护率显著优于CHO和血源乙肝疫苗相一致,为及时接种乙肝疫苗的必要性和高危新生儿选择接种乙肝疫苗的类型提供依据.     

Presentation of the candidate rheumatoid arthritis autoantigen aggrecan by antigen-specific B cells induces enhanced CD4(+) T helper type 1 subset differentiation

Effective immune responses require antigen uptake by antigen-presenting cells (APC), followed by controlled endocytic proteolysis resulting in the generation of antigen-derived peptide fragments that associate with intracellular MHC class II molecules. The resultant peptide-MHC class II complexes then move to the APC surface where they activate CD4(+) T cells. Dendritic cells (DC), macrophages and B cells act as efficient APC. In many settings, including the T helper type 1 (Th1) -dependent, proteoglycan-induced arthritis model of rheumatoid arthritis, accumulating evidence demonstrates that antigen presentation by B cells is required for optimal CD4(+) T cell activation. The reasons behind this however, remain unclear. In this study we have compared the activation of CD4(+) T cells specific for the proteoglycan aggrecan following antigen presentation by DC, macrophages and B cells. We show that aggrecan-specific B cells are equally efficient APC as DC and macrophages and use similar intracellular antigen-processing pathways. Importantly, we also show that antigen presentation by aggrecan-specific B cells to TCR transgenic CD4(+) T cells results in enhanced CD4(+) T cell interferon-γ production and Th1 effector sub-set differentiation compared with that seen with DC. We conclude that preferential CD4(+) Th1 differentiation may define the requirement for B cell APC function in both proteoglycan-induced arthritis and rheumatoid arthritis.