Exosomes bearing HLA-DR1 molecules need dendritic cells to efficiently stimulate specific T cells

Exosomes are small vesicles (60-100 nm) secreted by various cell types upon the fusion of endosomal compartments with the plasma membrane. Exosomes from antigen-presenting cells (APC), such as B lymphocytes and dendritic cells (DC), bear MHC class II molecules. In addition, the injection of DC-derived exosomes was reported to elicit potent T cell responses in vivo. Here, we analyzed the activation of specific T cells by MHC class II-bearing exosomes in vitro. The rat mast cell line, RBL-2H3, was engineered to express human class II molecules uniformly loaded with an antigenic peptide [HLA-DR1-hemagglutinin (HA)]. These cells secreted exosomes bearing DR1 class II molecules upon stimulation by a calcium ionophore or IgE receptor cross-linking. Exosomes bearing DR1-HA(306-318) complexes activated HA/DR1-specific T cells only weakly, whereas the cross-linking of such exosomes to latex beads increased stimulation of specific T cells. By contrast, the incubation of free exosomes with DC resulted in the highly efficient stimulation of specific T cells. Thus, exosomes bearing MHC class II complexes must be taken up by professional APC for efficient T cell activation.     

A monoclonal antibody specific for a cytochrome c T cell stimulatory peptide inhibits T cell responses and affects the way the peptide associates with antigen-presenting cells.

A monoclonal antibody (mAb) specific for the 93-104 segment of pigeon cytochrome c (cyt) was shown to block interleukin 2 production and proliferation by pigeon cyt-specific T cells in response to the pigeon cyt 81-104 peptide using either the LK35.2 B cell hybridoma or normal splenocytes as antigen-presenting cells (APC). The mAb inhibited the response to soluble peptide antigen presented by metabolically inactive paraformaldehyde-fixed APC but not the response to APC that were pre-pulsed with Ag. These results suggest that the mAb blocked the formation of peptide-major histocompatibility complex (MHC) class II molecule complexes at the cell surface but did not displace the peptide once bound to the MHC class II molecule. As determined by direct binding experiments using labeled peptide, the major means of free peptide association with live APC was fluid-phase endocytosis. No free peptide associated directly with the MHC class II molecule at the cell surface near 0 degrees C since APC pulsed with peptide on ice did not activate cyt-specific T cells. The mAb enhanced the association of the radiolabeled peptide with APC at 4 degrees C apparently by binding of the peptide-mAb complex to Fc receptors. By stripping molecules from the LK35.2 cell surface using a nonspecific protease it was shown that the peptide-mAb complexes were not internalized either at 4 degrees C or 37 degrees C. Since the mAb was found to stably bind the peptide at pH levels below that of endosomes (pH 5.5-6.2) even if the peptide-mAb complexes were taken up by fluid-phase endocytosis, it is likely that the peptide would not be able to associate with MHC class II molecules inside the APC. This mAb appears to inhibit T cell activation by blocking the formation of peptide-MHC class II molecule complexes at the cell surface and by interfering with uptake of the peptide into endosomes. Therefore, it is different from other antibodies that have been reported to block T cell receptor recognition of preformed peptide/MHC class II molecule complexes.     

Peptide-induced proliferation and lymphokine production in human T cells in the absence of antigen-presenting cells: role of T-cell activation state and costimulatory signals.

The role of T-lymphocytes as antigen-presenting cells (APCs) for other T cells was investigated. Activated rabies-virus-specific human T-cell clones were shown to present peptide to class II major histocompatibility complex (MHC)-restricted T cells of a different fine specificity, resulting in lymphokine production and cell proliferation. Furthermore, purified and activated antigen-specific T cells could produce lymphokines and proliferate as a result of the addition of antigenic peptide in the absence of APC. The functional response of T cells to peptide in the absence of APC was amplified by the addition of phorbol ester (PMA) and was inhibited with antibodies specific to class II MHC or to the CD2 molecule. Experiments performed in single-cell suspension cultures using semisolid medium prepared with 1% agar demonstrate that T-cell proliferative and lymphokine responses to peptide both in the presence and absence of APC require the interaction of T-cell antigen receptor (TCR) molecules with class II MHC-peptide complexes on different cell surfaces (cell-cell contact). On the other hand, peptide self-presentation, which occurs by the binding of TCR with class II MHC-peptide complexes on the same cell surface (at the single-cell level), resulted in T-cell activation (i.e., high expression of surface CD2, CD25, and HLA-DR molecules), without proliferation or lymphokine secretion, a pattern observed in the induction of T-cell anergy by antigen. The results are discussed in terms of the role of class II MHC molecules on activated T-lymphocytes, which enable these cells to function as "professional APC" in the development of T-cell regulatory networks.     

Antigen presentation by T cells: T cell receptor ligation promotes antigen acquisition from professional antigen-presenting cells

The purpose of this study was to determine whether the clonotypic specificity of the T cell receptor influences the specificity of T cell-mediated antigen presentation. We have previously shown that myelin basic protein (MBP)-specific Lewis rat GP2.E5/R1 (R1) T cells cultured with antigen, irradiated syngeneic splenocytes (IrrSPL) and tolerogenic monoclonal antibody become highly effective antigen-presenting cells (APC). In the current studies, we investigated the transfer of specific (MBP) and unrelated (conalbumin) antigens from antigen-pulsed SPL to R1 T cells. R1 T cells cultured with IrrSPL that were pulsed simultaneously with both MBP and conalbumin acquired and presented both antigens to the appropriate T cell responders in a secondary assay. These results suggested a physical transfer of major histocompatibility complex (MHC)/peptide complexes from professional APC to R1 T cells. Transfer of conalbumin from professional APC to R1 T cells required specific recognition of MBP and was optimal when both conalbumin and MBP were presented on the same group of professional APC. Antigens transfer did not occur when allogeneic SPL were used as APC. The anti-I-A mAb OX6 inhibited antigen transfer but only when added during the initiation of culture. OX6 also inhibited antigen acquisition by R1-trans, a variant of the R1 T cell line which constitutively synthesizes high levels of I-A, from MBP-pulsed IrrSPL but blockade of I-A did not inhibit antigen acquisition when soluble MBP was added directly to the culture. Despite constitutive synthesis of I-A, R1-trans T cells did not acquire guinea pig MBP from pulsed allogeneic APC. These studies demonstrate that although T cells of a particular specificity can present unrelated antigens, the cognate interaction of the T cell antigen receptor with the appropriate antigen/self-MHC complex strongly promotes acquisition of these complexes from professional APC.     

Probing the activation requirements for naive CD8+ T cells with Drosophila cell transfectants as antigen presenting cells

Summary: Activation of T cells involves multiple receptor-ligand interactions between T cells and antigen presenting cells (APC), At least two signals are required for T-cell activation: Signal 1 results from recognition of MHC/peptide complexes on the APC by cell surface T-cell receptors (TCR). whereas Signal 2 is induced by the interactions of co-stimulatory molecules on APC with their complementary receptors on T cells. This review focuses on our attempts to understand these various signals in a model system involving the 2C TCR. The structural basis of Signal 1 was investigated by determining the crystal structure of 2C TCR alone and in complex with MHC/peptide. Analysis of these structures has provided some basic rules for how TCR and MHC/peptide interact; however, the critical question of how this interaction transduces Signal I to T cells remains unclear. The effects of Signal 1 and Signal 2 on T-cell activation were examined with naive T cells from the 2C TCR transgenic mice, defined peptides as antigen and transfected Drosophila cells as APC. The results suggest that, except under extreme conditions, Signal I alone is unable to activate naive CD8 T cells despite the induction of marked TCR downregulation. Either B7 or intercellular adhesion molecule (ICAM)-l can provide the second signal for CD8 T-cell activation. However, especially at low MHC/peptide densities, optimal activation and differentiation of CD8 T cells required interaction with both B7 and [CAM-1 on the same APC. Thus, the data suggest that at least two qualitatively different co-stimulation signals are required for full activation of CD8 T cells under physiological conditions.     

T cell stimulation in the absence of exogenous antigen: a T cell signal is induced by both MHC-dependent and -independent mechanisms

Mature T cells residing in peripheral lymphoid organs have frequent contact with antigen presenting cells (APC). Such contact may be required for T cell survival, but the degree to which signals in mature T cells are induced by TCR recognition of self peptide/MHC complexes is unclear. We have used induction of the early growth response gene 1 (Egr1) as an indicator of signal transduction in 3.L2 (I-Ek-restricted) T cells interacting with APC in the absence of exogenous antigen. The data show that Egr1 can be induced in 3.L2 T cells by TCR recognition of self peptides presented by I-Ek. However, a more transient induction of Egr1 can be induced in 3.L2 T cells interacting with dendritic cells derived from class II/beta2m double-deficient mice. Egr1 induction after T cell-APC contact was also observed in a freshly isolated polyclonal CD4 T cell population. The data suggest that self peptide/MHC recognition by the TCR induces a signal in T cells and that dendritic cells can also induce a more transient T cell signal by an MHC-independent mechanism.     

Selection of aberrant class II restricted CD8+ T cells in NOD mice expressing a glutamic acid decarboxylase (GAD)65-specific T cell receptor transgene

We previously described the generation of non-obese diabetic (NOD) mice expressing a transgenic T cell receptor (TCR) specific for peptide epitope 286-300 of the diabetes related self antigen, glutamic acid decarboxylase (GAD)65 in the context of I-A(g7) class II MHC, that are paradoxically protected from diabetes. In this report, we examine the atypical CD8+ cells in these mice. Unlike typical class II restricted TCR transgenic mice, GAD286 mice have normal numbers of CD8+ cells, half of which express high levels of the transgenic TCR. These MHC mismatched CD8+ cells persist in the periphery and proliferate to GAD286-300 peptide in vitro and in vivo in a class II restricted fashion. Interestingly, the CD8+ tetramer(-) T cells that are expressing endogenous TCR can delay diabetes induction in a transfer model, as we previously showed for CD4+ tetramer+ T cells in these mice. The MHC mismatched CD8+ cells appear to be positively selected in an atypical fashion, in that they do not upregulate CD69 or reexpress CD44, and they escape negative selection. We find that production of these CD8+ cells is not dependent on NOD thymus or high affinity of the TCR, but is dependent on the atypical TCR transgenic thymic environment.     

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.     

Cytosolic targeting of hen egg lysozyme gives rise to a short-lived protein presented by class I but not class II major histocompatibility complex molecules

A way to study the role of intracellular trafficking of an antigen in its presentation to T cells is to target the antigen to various cell compartments of the antigen-presenting cells (APC) and compare the nature of the complexes associating major histocompatibility complex (MHC) molecules and antigenic peptides, expressed on the cell surface. MHC class I+ and MHC class II+ mouse L fibroblasts secreting hen egg lysozyme (HELs cells) or expressing HEL in their cytosol (HELc cells) were obtained after transfection with HEL cDNA and signal sequence-deleted HEL cDNA, respectively. HEL was evidenced in both HELs- and HELc-transfected cells and the former type of transfectant secreted a large amount of HEL. However, HEL produced in the cytosol exhibited a short half-life of less than 5 min. HEL-derived peptides could not be shown biochemically either in HELc- nor in HELs-transfected cells. We then studied the capacity of these cells to present HEL to HEL-specific class I- and class II-restricted T cells. Both cell types could be recognized by the HEL-specific MHC class I-restricted CTL clones. In contrast, MHC class II-HEL peptide complexes, recognized by HEL-specific helper T cell hybridomas, could be detected on MHC class II+ HELs- but not HELc-transfected cells. In vivo experiments showed, however, that HELc-transfected cells could provide host APC with HELc-derived peptides able to associate with MHC class II molecules. This was inferred from the capacity of MHC class II-HELc-transfected cells, unable by themselves to elicit any anti-HEL antibody response, to prime syngeneic and allogeneic mice against HEL. The priming was revealed by the induction of an antibody response after a boost with an amount of HEL unable itself to elicit an antibody response.     

Dendritic cells, but not macrophages or B cells, activate major histocompatibility complex class II-restricted CD4+ T cells upon immune-complex uptake in vivo

Professional antigen-presenting cells (APC) are able to process and present exogenous antigen leading to the activation of T cells. Antigen-immunoglobulin (Ig)G complexes (IC) are much more efficiently processed and presented than soluble antigen. Dendritic cells (DC) are known for their ability to take up and process immune complex (IC) via FcgammaR, and they have been shown to play a crucial role in IC-processing onto major histocompatibility complex (MHC) class I as they contain a specialized cross-presenting transport system required for MHC class I antigen-processing. However, the MHC class II-antigen-processing pathway is distinct. Therefore various other professional APC, like macrophages and B cells, all displaying FcgammaR, are thought to present IC-delivered antigen in MHC class II. Nonetheless, the relative contribution of these APC in IC-facilitated antigen-presentation for MHC class II in vivo is not known. Here we show that, in mice, both macrophages and DC, but not B cells, efficiently capture IC. However, only DC, but not macrophages, efficiently activate antigen-specific MHC class II restricted CD4(+) T cells. These results indicate that mainly DC and not other professional APC, despite expressing FcgammaR and MHC class II, contribute significantly to IC-facilitated T cell activation in vivo under steady-state conditions.     

MHC Class II-Dependent Peptide Antigen Versus Superantigen Presentation to T Cells

T lymphocytes expressing the CD4 coreceptor can be activated by two classes of major histocompatibility complex (MHC) class II-bound ligands. The elaboration of a conventional T-cell mediated immune response involves recognition of an antigenic peptide bound to the MHC class II molecules by a T-cell receptor (TCR) specific to that particular antigen. Conversely, superantigens (SAgs) also bind to MHC class II molecules and activate T cells, leading to a completely different functional outcome; indeed, SAg-responsive T cells die through apoptosis following stimulation. Superantigens are proteins that are secreted by various bacteria. They interact with the TCR using molecular determinants that are distinct from the residues involved in the recognition of nominal antigenic peptides. Despite the similarities between the recognition of the two classes of ligands by the TCR, considerable structural difference is observed. Here, we discuss the current knowledge on the presentation of SAgs to T cells and compare the different aspects of the SAg response with the recognition of antigenic peptide/MHC complexes.     

Differential glycosylation of MHC class II molecules on gastric epithelial cells: implications in local immune responses

Class II major histocompatibility complex (MHC) expression is a hallmark of antigen presenting cells (APC). Human gastric epithelial cells (GEC) express class II MHC and this expression increases during infection with Helicobacter pylori as does the number of CD4 T cells found adjacent or in between epithelial cells. These observations suggested that human GEC act as APCs. To characterize and compare class II MHC complexes with those present in conventional APC, immunoprecipitated class II MHC from GEC and B cells, as prototypic APC, were separated by two-dimensional electrophoresis. Although the composition of class II MHC from both cell phenotypes was similar, their electrophoretic mobility differed. Methodical elimination of carbohydrates, either enzymatically with endoglycosidase-H or blocking with tunicamycin, revealed that the deviations were due to differences in glycosylation in both cell phenotypes. When deglycosylated class II MHC alpha chains, beta chains, and the invariant chain from both cell phenotypes were mixed and run in the same gel, the core proteins had identical migration patterns. Because differences in glycosylation of class II MHC proteins may affect peptide selection and/or recognition by T cells, the noted differences in glycosylation of class II MHC expressed by GEC could be important in considering their potential role as APC locally.     

Human CD8+ T cell clone regulates autologous CD4+ myelin basic protein specific T cells.

Normal human CD8+ T cell clones were co-isolated from the same culture wells as CD4+ T effector cell clones specific for myelin basic protein (MBP). Microcultures from which the CD8+ clones were isolated initially proliferated weakly to whole MBP and to an MBP peptide spanning residues 90-170. This pattern of response was similar to strongly proliferating wells that yielded CD4+ T cell clones specific for the 90-170 peptide. After repeated stimulation, however, no response to MBP or MBP 90-170 was detected, even though the number of cells increased after stimulation. Phenotyping and TCR analyses revealed the presence of two CD8+, CD4-, IL-2R+ T cell isolates that expressed a single V beta gene (V beta 17) that differed from the CD4+ isolates that uniformly expressed V beta 14. One of these CD8+ clones (C9) inhibited the antigen-driven proliferation of an autologous MBP 90-170 reactive clone but not an autologous clone specific for Herpes simplex virus (HSV), without affecting MHC non-restricted mitogen responses of the same clones. Moreover, C9 did not inhibit heterologous CD4+ T cell clones specific for MBP 1-38 or 90-170. A culture supernatant of the CD8+ clone showed the same pattern but lower levels of inhibition. C9 had mild cytolytic activity when incubated at high ratios with an autologous MBP-specific CD4+ clone. Lysis was blocked completely by anti-MHC class I antibodies, but not by anti-MHC II antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antigen-independent acquisition of MHC class II molecules by human T lymphocytes

We report here that human T lymphocytes have the capacity of acquiring large amounts of MHC class II molecules from various types of antigen-presenting cells (APC) in an antigen-independent manner. The transfer of MHC class II molecules from APC to T cell required direct cell-to-cell contact and appeared to involve the interaction of numerous adhesion molecules between these cells. Depletion of cholesterol from the plasma membrane reduced the amount of MHC class II transferred onto the T cells. Most significantly, the newly acquired MHC class II molecules were capable of efficiently presenting antigen to T helper cells. These results suggest that T cells are able to interact with other T cells to regulate immune responses by presenting MHC peptide complexes that have been snatched away from nearby APC.     

Stimulation of CD2-negative T cells by staphylococcal enterotoxin superantigens.

A minor fraction of CD3+ T cells lacks expression of the CD2 antigen, which is the target for an "alternative" T cell activation pathway. CD2-CD3+ T cells can be stimulated by anti-CD3 or anti-T cell receptor (TCR) antibodies, indicating that the CD3/TCR signal transduction pathway functions in the absence of cell surface CD2. In the present study we have analyzed whether CD2-CD3+ T cells also respond to antigen stimulation. We show here that cloned CD2-negative T cells expressing the alpha/beta TCR are activated by one or several staphylococcal enterotoxin "superantigens". Activation of CD2-CD3+ T cell clones by staphylococcal enterotoxins resulted in IL-2 production and/or proliferative activity, and was dependent on the presence of HLA class II-bearing feeder cells. These data demonstrate that T cells can recognize (and respond to) antigen in the absence of a functional CD2 molecule.     

Human anti-idiotypic T cells induced by TCR peptides corresponding to a common CDR3 sequence motif in myelin basic protein-reactive T cells

T cells recognizing myelin basic protein (MBP) are potentially involved in the pathogenesis of multiple sclerosis (MS). In vivo clonal expansion of MBP-reactive T cells in MS may relate in part to dysfunction of peripheral regulatory mechanisms, including the anti-idiotypic immune network. In this study, we examined anti-idiotypic immune responses and the functional properties of anti-idiotypic T cells in patients with MS and healthy controls using TCR peptides corresponding to a CDR3 sequence motif preferentially expressed among T cells recognizing the 83-99 immunodominant peptide of MBP in some patients with MS. The study demonstrated that anti-idiotypic T cells could be induced in vitro by 8mer and 15mer peptides containing the CDR3 motif in MS patients and healthy controls respectively. The estimated precursor frequency of the anti-idiotypic T cells was slightly reduced in MS patients compared to control subjects. The obtained anti-idiotypic T cells recognizing the 15mer TCR peptide were found to express the CD4 phenotype, produce predominantly IL-10 and inhibit the proliferation of autologous T cells recognizing the immunodominant peptide of MBP. Anti-idiotypic T cells induced by the 8mer TCR peptide were predominantly CD8+ cytotoxic T cells and exhibited cytotoxic activity against autologous MBP-specific T cells expressing the CDR3 sequence. When added in primary culture, both TCR peptides had a significant inhibitory effect on the T cell responses to the immunodominant peptide of MBP. The findings suggest that anti-idiotypic immune responses can be activated by selected TCR peptides and may play an important role in the in vivo regulation of MBP-reactive T cells.     

Association of MHC class II-peptide complexes with plasma membrane lipid microdomains

Activation of CD4(+) T cells requires the interaction of multiple T-cell receptors with MHC class II-peptide complexes on the surface of antigen-presenting cells (APCs). Recent studies have shown that MHC class II complexes are clustered in APC plasma membrane microdomains, thereby providing a mechanism for localized concentration of MHC class II-peptide complexes. The integrity of one type of APC membrane microdomain, the lipid raft, is important for antigen presentation to T cells. We propose a model in which the coordinated processes of MHC class II peptide loading and intracellular trafficking enhance T-cell activation by loading specific MHC class II-peptide complexes in discrete lipid raft microdomains.     

Modulation of CD4 T cell function by soluble MHC II-peptide chimeras

Peptides antigens of 8 to 24 amino acid residues in length that are derived from processing of foreign proteins by antigen presenting cells (APC), and then presented to T cells in the context of major histocompatibility complex molecules (MHC) expressed by APC, are the only physiological ligands for T cell receptor (TCR). Co-ligation of TCR and CD4 co-receptor on T cells by MHC II-peptide complexes (signal 1) leads to various T cell functions depending on the nature of TCR and CD4 co-ligation, and whether costimulatory receptors (signal 2) such as CD28, CTLA-4, CD40L are involved in this interaction. Recently, the advance of genetic engineering led to the generation of a new class of antigen-specific ligands for TCR, i.e., soluble MHC class I, and MHC class II-peptide chimeras. In principle, these chimeric molecules consist of an antigenic peptide which is covalently linked to the amino terminus of α-chain in the case of MHC I, or β-chains in the case of MHC II molecules. Conceptually, such TCR/CD4 ligands shall provide the signal I to T cells. Since soluble MHC-peptide chimeras showed remarkable regulatory effects on peptide-specific T cells in vitro and in vivo, they may represent a new generation of immunospecific T cell modulators with potential therapeutic applicability in autoimmune and infectious diseases. This review is focused on the immunomodulatory effects of soluble, MHC class II-peptide chimeras, and discuss these effects in the context of the most accepted theories on T cell regulation.     

Modulation of CD4 T cell function by soluble MHC II-peptide chimeras

Peptides antigens of 8 to 24 amino acid residues in length that are derived from processing of foreign proteins by antigen presenting cells (APC), and then presented to T cells in the context of major histocompatibility complex molecules (MHC) expressed by APC, are the only physiological ligands for T cell receptor (TCR). Co-ligation of TCR and CD4 co-receptor on T cells by MHC II-peptide complexes (signal 1) leads to various T cell functions depending on the nature of TCR and CD4 co-ligation, and whether costimulatory receptors (signal 2) such as CD28, CTLA-4, CD40L are involved in this interaction. Recently, the advance of genetic engineering led to the generation of a new class of antigen-specific ligands for TCR, i.e., soluble MHC class I-, and MHC class II-peptide chimeras. In principle, these chimeric molecules consist of an antigenic peptide which is covalently linked to the amino terminus of alpha-chain in the case of MHC I, or beta-chains in the case of MHC II molecules. Conceptually, such TCR/CD4 ligands shall provide the signal 1 to T cells. Since soluble MHC-peptide chimeras showed remarkable regulatory effects on peptide-specific T cells in vitro and in vivo, they may represent a new generation of immunospecific T cell modulators with potential therapeutic applicability in autoimmune and infectious diseases. This review is focused on the immunomodulatory effects of soluble, MHC class II-peptide chimeras, and discuss these effects in the context of the most accepted theories on T cell regulation.     

Rapid constitutive generation of a specific peptide-MHC class II complex from intact exogenous protein in immature murine dendritic cells

MHC class II molecules present peptides, derived largely from exogenous antigens, to CD4+ T cells. Complex-generation occurs mainly in the specialized late endosomal MHC class II-rich compartment (MIIC) vesicles of antigen-presenting cells (APC). Dendritic cells (DC) have been reported to store intact antigen in MIIC until the receipt of an activation signal, when they process it into peptide-MHC class II complexes. However, constitutive migration of DC from the periphery to secondary lymphoid organs has been observed, and antigen presentation by nonactivated DC is proposed to play a role in the induction of tolerance to peripheral antigens. Thus, constitutive peptide-MHC class II complex generation must also occur in DC in immunologically quiescent situations. We have used a monoclonal antibody that detects a specific peptide-MHC class II complex to directly demonstrate constitutive complex generation in immature murine DC. Protein-derived peptide-MHC class II complexes were detected by flow cytometry at the DC surface within 1 h of antigen exposure in the absence of an exogenous activation signal, and could be detected by confocal microscopy in the MIIC within 5 min of antigen exposure. This processing activity was endotoxin independent. These data provide evidence for constitutive peptide-MHC class II complex generation in immature DC, and thus support a role for this activity in the induction of peripheral tolerance.