Phagosomes acquire nascent and recycling class II MHC molecules but primarily use nascent molecules in phagocytic antigen processing

Phagosomes contain class II MHC (MHC-II) and form peptide:MHC-II complexes, but the source of phagosomal MHC-II molecules is uncertain. Phagosomes may acquire nascent MHC-II or preexisting, recycling MHC-II that may be internalized from the plasma membrane. Brefeldin A (BFA) was used to deplete nascent MHC-II in murine macrophages to determine the relative contributions of nascent and recycling MHC-II molecules to phagocytic Ag processing. In addition, biotinylation of cell-surface proteins was used to assess the transport of MHC-II from the cell surface to phagosomes. BFA inhibited macrophage processing of latex bead-conjugated Ag for presentation to T cells, suggesting that nascent MHC-II molecules are important in phagocytic Ag processing. Furthermore, detection of specific peptide:MHC-II complexes in isolated phagosomes confirmed that BFA decreased formation of peptide:MHC-II complexes within phagosomes. Both flow organellometry and Western blot analysis of purified phagosomes showed that about two-thirds of phagosomal MHC-II was nascent (depleted by 3 h prior treatment with BFA) and primarily derived from intracellular sites. About one-third of phagosomal MHC-II was preexisting and primarily derived from the plasma membrane. BFA had little effect on phagosomal H2-DM or the degradation of bead-associated Ag. Thus, inhibition of phagocytic Ag processing by BFA correlated with depletion of nascent MHC-II in phagosomes and occurred despite the persistent delivery of plasma membrane-derived recycling MHC-II molecules and other Ag-processing components to phagosomes. These observations suggest that phagosomal Ag processing depends primarily on nascent MHC-II molecules delivered from intracellular sites, e.g., endocytic compartments.     

Co-dominant restriction by a mixed-haplotype I-A molecule (alpha k beta b) for the lysozyme peptide 52-61 in H-2k x H-2b F1 mice

Helper (CD4+) T lymphocytes recognize protein Ag as peptides associated to MHC class II molecules. The polymorphism of class II alpha- and beta-chains has a major influence on the nature of the peptides presented to CD4+ T lymphocytes. For instance, T cell responses in H-2k and H-2b mice are directed at different epitopes of the hen egg lysozyme (HEL) molecule. The current studies were undertaken with the aim of defining the role of mixed haplotype I-A (alpha k beta b and alpha b beta k) molecules in T cell responses to HEL in (H-2k x H-2b)F1 mice, as well as the nature of the immunogenic peptides of HEL recognized in the context of I-A alpha k beta b and I-A alpha b beta k. A series of HEL-reactive T cell lines and hybridomas derived from MHC class II heterozygous (C57BL/6 x C3H F1) mice were established. Their responsiveness to HEL and synthetic HEL peptides was analyzed with the use of L cells transfected with either I-A alpha k beta b or I-A alpha b beta k as APC. Out of 28 clonal T cell hybridomas tested, 13 (46%) only responded to HEL presented by I-A alpha k beta b, 11 (40%) by I-A alpha b beta k (and to a minor extent I-A alpha k beta k), only 4 (14%) were primarily restricted by I-Ak, and none by I-Ab. All the I-A alpha k beta b-restricted T cell hybridomas responded to the HEL peptide 46-61 and to its shorter fragment 52-61, even at concentrations as low as 0.3 nM. As this determinant has been previously defined as immunodominant for I-Ak but not for I-Ab mice, these results suggest a role for the I-A alpha k chain in the selection and immunodominance of HEL 52-61 in H-2k mice. The fine specificity of I-A alpha k beta b-restricted T cell hybridomas for a series of different HEL peptides around the sequence 52 to 61 suggests that peptide 52-61 binds to I-A alpha k beta b with higher affinity than to I-A alpha k beta k. The peptides recognized in the context of I-A alpha b beta k and I-A alpha k beta k were not identified.     

Modulation of proinflammatory responses to Pneumocystis carinii f. sp. muris in neonatal mice by granulocyte-macrophage colony-stimulating factor and IL-4: role of APCs

Clearance of Pneumocystis carinii f. sp. muris (PC) organisms from the lungs of neonatal mice is delayed due to failure of initiation of inflammation over the first 3 wk after infection. The ability of neonatal lung CD11c(+) dendritic cells (DCs) to induce Ag-specific T cell proliferative responses was significantly reduced compared with adult lung DCs. However, neonatal bone marrow-derived DCs were as competent at presenting PC Ag as were adult bone marrow-derived DCs. Because GM-CSF mRNA expression and activity were significantly reduced in neonatal lungs compared with adults, we treated neonates with exogenous GM-CSF and IL-4 and found enhanced clearance of PC compared with untreated neonates. This was associated with increased lung TNF-alpha, IL-12p35, and IL-18 mRNA expression, indicating enhanced innate immune responses. Cytokine-treated mice had marked expansion of CD11c(+) DCs with up-regulated MHC-II in the lungs. Moreover, increased numbers of activated CD4(+)CD44(high)CD62L(low) cells in the lungs and draining lymph nodes suggested improved Ag presentation by the APCs. Together these data indicate that neonatal lungs lack maturation factors for efficient cellular functioning, including APC maturation.     

Enhanced tumor-specific long-term immunity of hemagglutinating [correction of hemaggluttinating] virus of Japan-mediated dendritic cell-tumor fused cell vaccination by coadministration with CpG oligodeoxynucleotides

Immunization with dendritic cells (DCs) using various Ag-loading approaches has shown promising results in tumor-specific immunotherapy and immunoprevention. Fused cells (FCs) that are generated from DCs and tumor cells are one of effective cancer vaccines because both known and unknown tumor Ags are presented on the FCs and recognized by T cells. In this study, we attempted to augment antitumor immunity by the combination of DC-tumor FC vaccination with immunostimulatory oligodeoxynucleotides containing CpG motif (CpG ODN). Murine DCs were fused with syngeneic tumor cells ex vivo using inactivated hemagglutinating virus of Japan (Sendai virus). Mice were intradermally (i.d.) immunized with FCs and/or CpG ODN. Coadministration of CpG ODN enhanced the phenotypical maturation of FCs and unfused DCs, and the production of Th1 cytokines, such as IFN-gamma and IL-12, leading to the induction of tumor-specific CTLs without falling into T cell anergy. In addition, immunization with FCs + CpG ODN provided significant protection against lethal s.c. tumor challenge and spontaneous lung metastasis compared with that with either FCs or CpG ODN alone. Furthermore, among mice that rejected tumor challenge, the mice immunized with FCs + CpG ODN, but not the mice immunized with FCs or CpG ODN alone, completely rejected tumor rechallenge, indicating that CpG ODN provided long-term maintenance of tumor-specific immunity induced by FCs. Thus, the combination of DC-tumor FCs and CpG ODN is an effective and feasible cancer vaccine to prevent the generation and recurrence of cancers.     

APC stimulated by CpG oligodeoxynucleotide enhance activation of MHC class I-restricted T cells

Oligonucleotides containing unmethylated CpG motifs (cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG ODN)) are potent immunostimulatory agents capable of enhancing the Ag-specific Th1 response when used as immune adjuvants. We evaluated the cellular mechanisms responsible for this effect. Development of a CTL response was enhanced when mice were immunized with peptide-pulsed dendritic cells (DCs) treated with CpG ODN. However, in vitro, CpG ODN had no direct effect on highly purified T cells. In vitro, CpG ODN treatment of peptide- or protein-pulsed DCs enhanced the ability of the DCs to activate class I-restricted T cells. The presence of helper T cells enhanced this effect, indicating that treatment with CpG ODN does not obviate the role of T cell help. The enhanced ability of CpG ODN-treated DCs to activate T cells was present but blunted when DCs derived from IL-12 knockout mice were used. Fixation of Ag-pulsed, CpG ODN-treated DCs limited their ability to activate T cells. In contrast, fixation had little effect on DC activation of T cells when DCs were not exposed to CpG ODN. This indicates that production of soluble factors by DCs stimulated with CpG ODN plays a particularly important role in their ability to activate class I-restricted T cells. We conclude that CpG ODN enhances the development of a cellular immune response by stimulating APCs such as DCs, to produce IL-12 and other soluble factors.     

Alternate class I MHC antigen processing is inhibited by Toll-like receptor signaling pathogen-associated molecular patterns: Mycobacterium tuberculosis 19-kDa lipoprotein,CpG DNA,and lipopolysaccharide

Pathogen-associated molecular patterns (PAMPs) signal through Toll-like receptors (TLRs) to activate immune responses, but prolonged exposure to PAMPs from Mycobacterium tuberculosis (MTB) and other pathogens inhibits class II MHC (MHC-II) expression and Ag processing, which may allow MTB to evade CD4(+) T cell immunity. Alternate class I MHC (MHC-I) processing allows macrophages to present Ags from MTB and other bacteria to CD8(+) T cells, but the effect of PAMPs on this processing pathway is unknown. In our studies, MTB and TLR-signaling PAMPs, MTB 19-kDa lipoprotein, CpG DNA, and LPS, inhibited alternate MHC-I processing of latex-conjugated Ag by IFN-gamma-activated macrophages. Inhibition was dependent on TLR-2 for MTB 19-kDa lipoprotein (but not whole MTB or the other PAMPs); inhibition was dependent on myeloid differentiation factor 88 for MTB and all of the individual PAMPs. Inhibition of MHC-II and alternate MHC-I processing was delayed, appearing after 16 h of PAMP exposure, as would occur in chronically infected macrophages. Despite inhibition of alternate MHC-I Ag processing, there was no inhibition of MHC-I expression, MHC-I-restricted presentation of exogenous peptide or conventional MHC-I processing of cytosolic Ag. MTB 19-kDa lipoprotein and other PAMPs inhibited phagosome maturation and phagosome Ag degradation in a myeloid differentiation factor 88-dependent manner; this may limit availability of peptides to bind MHC-I. By inhibiting both MHC-II and alternate MHC-I Ag processing, pathogens that establish prolonged infection of macrophages (>16 h), e.g., MTB, may immunologically silence macrophages and evade surveillance by both CD4(+) and CD8(+) T cells, promoting chronic infection.     

Bacterial heat shock proteins enhance class II MHC antigen processing and presentation of chaperoned peptides to CD4+ T cells

APCs process heat shock protein (HSP):peptide complexes to present HSP-chaperoned peptides on class I MHC molecules, but the ability of HSPs to contribute chaperoned peptides for class II MHC (MHC-II) Ag processing and presentation is unclear. Our studies revealed that exogenous bacterial HSPs (Escherichia coli DnaK and Mycobacterium tuberculosis HSP70) delivered an extended OVA peptide for processing and MHC-II presentation, as detected by T hybridoma cells. Bacterial HSPs enhanced MHC-II presentation only if peptide was complexed to the HSP, suggesting that the key HSP function was enhanced delivery or processing of chaperoned peptide Ag rather than generalized enhancement of APC function. HSP-enhanced processing was intact in MyD88 knockout cells, which lack most TLR signaling, further suggesting the effect was not due to TLR-induced induction of accessory molecules. Bacterial HSPs enhanced uptake of peptide, which may contribute to increased MHC-II presentation. In addition, HSPs enhanced binding of peptide to MHC-II molecules at pH 5.0 (the pH of vacuolar compartments), but not at pH 7.4, indicating another mechanism for enhancement of MHC-II Ag processing. Bacterial HSPs are a potential source of microbial peptide Ags during phagocytic processing of bacteria during infection and could potentially be incorporated in vaccines to enhance presentation of peptides to CD4+ T cells.     

In vivo,dendritic cells can cross-present virus-like particles using an endosome-to-cytosol pathway

Recombinant parvovirus-like particles (PPV-VLPs) are particulate exogenous Ags that induce strong CTL response in the absence of adjuvant. In the present report to decipher the mechanisms responsible for CTL activation by such exogenous Ag, we analyzed ex vivo and in vitro the mechanisms of capture and processing of PPV-VLPs by dendritic cells (DCs). In vivo, PPV-VLPs are very efficiently captured by CD8alpha- and CD8alpha+ DCs and then localize in late endosomes of DCs. Macropinocytosis and lipid rafts participate in PPV-VLPs capture. Processing of PPV-VLPs does not depend upon recycling of MHC class I molecules, but requires vacuolar acidification as well as proteasome activity, TAP translocation, and neosynthesis of MHC class I molecules. This study therefore shows that in vivo DCs can cross-present PPV-VLPs using an endosome-to-cytosol processing pathway.     

Recombinant Escherichia coli express a defined, cytoplasmic epitope that is efficiently processed in macrophage phagolysosomes for class II MHC presentation to T lymphocytes.

Although the processing of soluble Ag for presentation to T cells has been extensively studied in vitro, similar studies of phagocytic Ag processing have been limited. We have developed an in vitro model system to study the ability of macrophages to process recombinant Escherichia coli strain HB101 with cytoplasmic or surface expression of the well characterized T cell epitope of hen egg lysozyme (HEL) 52-61. This epitope was expressed within full length HEL or within a fusion protein containing the HEL epitope. Phagocytosis of E. coli with cytoplasmic expression of HEL or the HEL fusion protein resulted in strong presentation of HEL(52-61) to T cells. Surface-conjugated HEL was processed with even greater efficiency. Processing required viable macrophages, was inhibited by cytochalasin D, and was achieved within 20 min of bacterial contact with the macrophages. Within this time span, phagosomes containing bacteria fused with lysosomes, and the bacteria were extensively degraded. Uptake of as few as four bacteria per macrophage produced an Ag-specific T cell response. We conclude that bacterial compartmentalization of the antigenic epitope (cytoplasmic vs surface) had some effect on its processing, but that phagocytic Ag processing organelles contain extensive capacity to degrade internalized bacteria and liberate intracellular Ag epitopes for recycling and presentation, consistent with a central role for phagolysosomes. Thus, future recombinant bacterial vaccines may be effectively designed with T cell epitopes expressed either on the surface or within the bacterial cytoplasm.     

The endothelial cell-produced antiangiogenic cytokine vascular endothelial growth inhibitor induces dendritic cell maturation

Angiogenesis is an essential component of chronic inflammation that is linked to carcinogenesis. In this study, we report that human vascular endothelial growth inhibitor (VEGI, TNF superfamily 15), an endothelial cell-produced antiangiogenic cytokine, induces mouse dendritic cell (DC) maturation, a critical event in inflammation-initiated immunity. VEGI-stimulated bone marrow-derived immature DCs display early activation of maturation signaling molecules NF-kappaB, STAT3, p38, and JNK, and cytoskeleton reorganization and dendrite formation. The activation signals are partially inhibited by using a neutralizing Ab against death domain-containing receptor-3 (DR3) or a truncated form of DR3 consisting of the extracellular domain, indicating an involvement of DR3 in the transmission of VEGI activity. A VEGI isoform, TL1A, does not induce similar activities under otherwise identical experimental conditions. Additionally, the cells reveal significantly enhanced expression of mature DC-specific marker CD83, secondary lymphoid tissue-directing chemokine receptor CCR7, the MHC class-II protein (MHC-II), and costimulatory molecules CD40, CD80, and CD86. Functionally, the cells exhibit decreased Ag endocytosis, increased cell surface distribution of MHC-II, and increased secretion of IL-12 and TNF. Moreover, VEGI-stimulated DCs are able to facilitate the differentiation of CD4+ naive T cells in cocultures. These findings suggest that the anticancer activity of VEGI arises from coupling the inhibition of endothelial cell growth with the promotion of the adaptive immune mechanisms through the stimulation of DC maturation.     

Presentation of soluble antigens to CD8+ T cells by CpG oligodeoxynucleotide-primed human naive B cells

Naive B lymphocytes are generally thought to be poor APCs, and there is limited knowledge of their role in activation of CD8(+) T cells. In this article, we demonstrate that class I MHC Ag presentation by human naive B cells is enhanced by TLR9 agonists. Purified naive B cells were cultured with or without a TLR9 agonist (CpG oligodeoxynucleotide [ODN] 2006) for 2 d and then assessed for phenotype, endocytic activity, and their ability to induce CD8(+) T cell responses to soluble Ags. CpG ODN enhanced expression of class I MHC and the costimulatory molecule CD86 and increased endocytic activity as determined by uptake of dextran beads. Pretreatment of naive B cells with CpG ODN also enabled presentation of tetanus toxoid to CD8(+) T cells, resulting in CD8(+) T cell cytokine production and granzyme B secretion and proliferation. Likewise, CpG-activated naive B cells showed enhanced ability to cross-present CMV Ag to autologous CD8(+) T cells, resulting in proliferation of CMV-specific CD8(+) T cells. Although resting naive B cells are poor APCs, they can be activated by TLR9 agonists to serve as potent APCs for class I MHC-restricted T cell responses. This novel activity of naive B cells could be exploited for vaccine design.     

Distinct MHC Class II Molecules Are Associated on the Dendritic Cell Surface in Cholesterol-dependent Membrane Microdomains

Very small amounts of MHC class II-peptide complexes expressed on the surface of antigen-presenting cells (APCs) are capable of stimulating antigen-specific CD4 T cells. There is intense interest to elucidate the molecular mechanisms by which these small amounts of MHC-II can cluster, cross-link T cell receptors, and promote T cell proliferation. We now demonstrate that a significant fraction of the total pool of MHC-II molecules on the surface of dendritic cells is physically associated in macromolecular aggregates. These MHC-II/MHC-II interactions have been probed by co-immunoprecipitation analysis of the MHC-II I-A molecule with the related I-E molecule. These molecular associations are maintained in gentle detergents but are disrupted in harsh detergents such as Triton X-100. MHC-II I-A/I-E interactions are disrupted when plasma membrane cholesterol is extracted using methyl β-cyclodextrin, suggesting that lipid raft microdomains are important mediators of these MHC-II interactions. Although it has been proposed that tetraspanin proteins regulate molecular clustering, aggregation, and co-immunoprecipitation in APCs, genetic deletion of the tetraspanin family members CD9 or CD81 had no effect on MHC-II I-A/I-E binding. These data demonstrate that the presence of distinct forms of MHC-II with plasma membrane lipid rafts is required for MHC-II aggregation in APCs and provides a molecular mechanism allowing dendritic cells expressing small amounts of MHC-II-peptide complexes to cross-link and stimulate CD4 T cells.     

A subset of Toll-like receptor ligands induces cross-presentation by bone marrow-derived dendritic cells

Dendritic cells (DCs) are capable of cross-presenting exogenous Ag to CD8(+) CTLs. Detection of microbial products by Toll-like receptors (TLRs) leads to activation of DCs and subsequent orchestration of an adaptive immune response. We hypothesized that microbial TLR ligands could activate DCs to cross-present Ag to CTLs. Using DCs and CTLs in an in vitro cross-presentation system, we show that a subset of microbial TLR ligands, namely ligands of TLR3 (poly(inosinic-cytidylic) acid) and TLR9 (immunostimulatory CpG DNA), induces cross-presentation. In contrast to presentation of Ag to CD4(+) T cells by immature DCs, TLR-induced cross-presentation is mediated by mature DCs, is independent of endosomal acidification, and relies on cytosolic Ag processing machinery.     

CD13 regulates dendritic cell cross-presentation and T cell responses by inhibiting receptor-mediated antigen uptake

Dendritic cell (DC) Ag cross-presentation is generally associated with immune responses to tumors and viral Ags, and enhancement of this process is a focus of tumor vaccine design. In this study, we found that the myeloid cell surface peptidase CD13 is highly and specifically expressed on the subset of DCs responsible for cross-presentation, the CD8(+) murine splenic DCs. In vivo studies indicated that lack of CD13 significantly enhanced T cell responses to soluble OVA Ag, although development, maturation, and Ag processing and presentation of DCs are normal in CD13KO mice. In vitro studies showed that CD13 regulates receptor-mediated, dynamin-dependent endocytosis of Ags such as OVA and transferrin but not fluid-phase or phagocytic Ag uptake. CD13 and Ag are cointernalized in DCs, but CD13 did not coimmunoprecipitate with Ag receptors, suggesting that CD13 does not control internalization of specific receptors but regulates endocytosis at a more universal level. Mechanistically, we found that phosphorylation of the endocytic regulators p38MAPK and Akt was dysregulated in CD13KO DCs, and blocking of these kinases perturbed CD13-dependent endocytic uptake. Therefore, CD13 is a novel endocytic regulator that may be exploited to enhance Ag uptake and T cell activation to improve the efficacy of tumor-targeted vaccines.     

Antigen-antibody immune complexes empower dendritic cells to efficiently prime specific CD8+ CTL responses in vivo

Dendritic cells (DCs) require a maturation signal to acquire efficient CTL-priming capacity. In vitro FcgammaR-mediated internalization of Ag-Ab immune complexes (ICs) can induce maturation of DCs. In this study, we show that IC-induced DC maturation in vitro enables DCs to prime peptide-specific CD8+ CTLs in vivo, independently of CD4+ Th cells. Importantly, OVA/anti-OVA IC-treated DCs not only primed CD8+ CTLs to an exogenously loaded peptide nonrelated to OVA, but also efficiently primed CTLs against the dominant CTL epitope derived from the OVA Ag present in the ICs. Our studies show that ICs fulfill a dual role in priming of CD8+ CTL responses to exogenous Ags: enhancement of Ag uptake by DCs and activation of DCs, resulting in "license to kill." These findings indicate that the presence of specific Abs can crucially affect the induction of cytotoxic cellular responses.     

Regulation of MHC class I transport in human dendritic cells and the dendritic-like cell line KG-1

Dendritic cells (DCs) progress through distinct maturational phases; immature DCs capture Ag while mature DCs are optimized for Ag presentation. Proper control of immunity requires regulated compartmentalization of MHC class II molecules. We report that DCs also regulate MHC class I trafficking throughout maturation. Although mature human DCs express high levels of surface MHC class I, immature DCs exhibit lower surface levels while retaining MHC class I-peptide complexes in the Golgi. A cell line, KG-1, behaves similarly. We confirm the similarity of KG-1 to DCs by demonstrating its capacity to present exogenous Ags in an MHC class I-restricted fashion to CD8(+) T cell hybridomas, a phenomenon called cross-presentation. Biochemical characterization of MHC class I trafficking throughout maturation showed that, in early KG-1 dendritic-like cells, surface arrival of MHC class I-peptide complexes is delayed by their retention in the Golgi. In mature dendritic-like cells, these complexes relocate to the surface and their stability increases, concomitant with up-regulation of costimulatory molecules. Maturation induces qualitative changes in the MHC class I-associated peptide repertoire demonstrated by increased thermostability. The differential processing of MHC class I throughout maturation may prevent premature immune activation while promoting T cell responses in lymph nodes to Ags acquired at sites of inflammation.     

Mycobacterium tuberculosis LprG (Rv1411c): a novel TLR-2 ligand that inhibits human macrophage class II MHC antigen processing

MHC class II (MHC-II)-restricted CD4(+) T cells are essential for control of Mycobacterium tuberculosis infection. This report describes the identification and purification of LprG (Rv1411c) as an inhibitor of primary human macrophage MHC-II Ag processing. LprG is a 24-kDa lipoprotein found in the M. tuberculosis cell wall. Prolonged exposure (>16 h) of human macrophages to LprG resulted in marked inhibition of MHC-II Ag processing. Inhibition of MHC-II Ag processing was dependent on TLR-2. Short-term exposure (<6 h) to LprG stimulated TLR-2-dependent TNF-alpha production. Thus, LprG can exploit TLR-2 signaling to inhibit MHC-II Ag processing in human macrophages. Inhibition of MHC-II Ag processing by mycobacterial lipoproteins may allow M. tuberculosis, within infected macrophages, to avoid recognition by CD4(+) T cells.