The neonatal FcR-mediated presentation of immune-complexed antigen is associated with endosomal and phagosomal pH and antigen stability in macrophages and dendritic cells

The FcγRs found on macrophages (Ms) and dendritic cells (DCs) efficiently facilitate the presentation or cross-presentation of immune-complexed Ags to T cells. We found that the MHC class I-related neonatal FcR for IgG (FcRn) in both Ms and DCs failed to have a strong effect on the cross-presentation of immune complex (IC) OVA Ag to CD8(+) T cells. Interestingly, endosomal FcRn enhanced the presentation of the monomeric OVA-IC to CD4(+) T cells robustly, whereas FcRn in phagosomes exerted distinctive effects on Ag presentation between Ms and DCs. The presentation of phagocytosed OVA-ICs to CD4(+) T cells was considerably enhanced on wild-type versus FcRn-deficient Ms, but was not affected in FcRn-deficient DCs. This functional discrepancy was associated with the dependence of IgG-FcRn binding in an acidic pH. Following phagocytosis, the phagosomal pH dropped rapidly to <6.5 in Ms but remained in the neutral range in DCs. This disparity in pH determined the rate of degradation of phagocytosed ICs. Thus, our findings reveal that FcRn expression has a different effect on Ag processing and presentation of ICs to CD4(+) T cells in the endosomal versus phagosomal compartments of Ms versus DCs.     

CD36 is differentially expressed by CD8+ splenic dendritic cells but is not required for cross-presentation in vivo

Cross-presentation allows the processing of Ags from donor cells into the MHC class I presentation pathway of dendritic cells (DCs). This is important for the generation of cytotoxic T cell immunity and for induction of self tolerance. Apoptotic cells are reported to be efficient targets for cross-presentation, and in vitro studies using human DCs have implicated CD36 in their capture. In support of a role for CD36 in cross-presentation, we show that this molecule is differentially expressed by CD8(+) splenic DCs, which previously have been identified as responsible for cross-presentation in the mouse. Three different cross-presentation models were examined for their dependence on CD36. These included cross-priming to OVA-coated spleen cells and cross-tolerance to OVA transgenically expressed in the pancreatic islet beta cells under constitutive conditions or during beta cell destruction. In these models, CD36 knockout DCs were equivalent to wild-type DCs in their capacity to cross-present either foreign or self Ags, indicating that CD36 is not essential for cross-presentation of cellular Ags in vivo.     

Influenza A infection enhances cross-priming of CD8+ T cells to cell-associated antigens in a TLR7- and type I IFN-dependent fashion

The initiation of antitumor immunity relies on dendritic cells (DCs) to cross-present cell-associated tumor Ag to CD8(+) T cells (T(CD8+)) due to a lack of costimulatory molecules on tumor cells. Innate danger signals have been demonstrated to enhance cross-priming of T(CD8+) to soluble as well as virally encoded Ags; however, their effect on enhancing T(CD8+) cross-priming to cell genome-encoded Ags remains unknown. Furthermore, influenza A virus (IAV) has not been shown to enhance antitumor immunity. Using influenza-infected allogeneic cell lines, we show in this study that T(CD8+) responses to cell-associated Ags can be dramatically enhanced due to enhanced T(CD8+) expansion. This enhanced cross-priming in part involves TLR7- but not TLR3-mediated sensing of IAV and is entirely dependent on MyD88 and IFN signaling pathways. We also showed that the inflammasome-induced IL-1 and IFN-γ did not play a role in enhancing cross-priming in our system. We further demonstrated in our ex vivo system that CD8(+) DCs are the only APCs able to prime TCR-transgenic T(CD8+). Importantly, plasmacytoid DCs and CD8(-) DCs were both able to enhance such priming when provided in coculture. These observations suggest that IAV infection of tumor cells may facilitate improved cross-presentation of tumor Ags and may be used to augment clinical vaccine efficacy.     

The acquisition of antigen cross-presentation function by newly formed dendritic cells

The development of Ag-presenting functions by murine dendritic cells (DCs) of the CD8(+) DC lineage was studied using a Flt-3 ligand stimulated bone-marrow culture system. Although newly formed DCs of this lineage are capable of Ag uptake and efficient presentation to T cells on MHC class II, they initially lack the ability to cross-present exogenous Ags on MHC class I. Cross-presentation capacity is acquired as a subsequent maturation step, promoted by cytokines such as GM-CSF. The development of cross-presentation capacity by the DCs in these cultures may be monitored by the parallel development of DC surface expression of CD103. However, the expression of CD103 and cross-presentation capacity are not always linked; therefore, CD103 is not an essential part of the cross-presentation machinery. These results explain the considerable variability in CD103 expression by CD8(+) DCs as well as the findings that not all DCs of this lineage are capable of cross-presentation.     

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.     

TRAIL/DR5 plays a critical role in NK cell-mediated negative regulation of dendritic cell cross-priming of T cells

Dendritic cells (DCs) are critical in initiating immune responses by cross-priming of tumor Ags to T cells. Previous results showed that NK cells inhibited DC-mediated cross-presentation of tumor Ags both in vivo and in vitro. In this study, enhanced Ag presentation was observed in draining lymph nodes in TRAIL(-/-) and DR5(-/-) mice compared with that of wild-type mice. NK cells inhibit DC cross-priming of tumor Ags in vitro, but not direct presentation of endogenous Ags. NK cells lacking TRAIL, but not perforin, were not able to inhibit DC cross-priming of tumor Ags. DCs that lack expression of TRAIL receptor DR5 were less susceptible to NK cell-mediated inhibition of cross-priming, and cross-linking of DR5 receptor led to reduced generation of MHC class I-Ag peptide complexes, followed by attenuated cross-priming of CD8(+) T cells. In addition, key molecules involved in the TRAIL/DR5 pathway during DC/NK cell interactions were determined. In summary, these data indicate a novel alternative pathway for DC/NK cell interactions in antitumor immunity and may reflect homeostasis of both DCs and NK cells for regulation of CD8(+) T cell function in physiological conditions.     

Type I IFNs control antigen retention and survival of CD8α(+) dendritic cells after uptake of tumor apoptotic cells leading to cross-priming

Cross-presentation is a crucial mechanism for generating CD8 T cell responses against exogenous Ags, such as dead cell-derived Ag, and is mainly fulfilled by CD8α(+) dendritic cells (DC). Apoptotic cell death occurring in steady-state conditions is largely tolerogenic, thus hampering the onset of effector CD8 T cell responses. Type I IFNs (IFN-I) have been shown to promote cross-priming of CD8 T cells against soluble or viral Ags, partly through stimulation of DC. By using UV-irradiated OVA-expressing mouse EG7 thymoma cells, we show that IFN-I promote intracellular Ag persistence in CD8α(+) DC that have engulfed apoptotic EG7 cells, regulating intracellular pH, thus enhancing cross-presentation of apoptotic EG7-derived OVA Ag by CD8α(+) DC. Notably, IFN-I also sustain the survival of Ag-bearing CD8α(+) DC by selective upmodulation of antiapoptotic genes and stimulate the activation of cross-presenting DC. The ensemble of these effects results in the induction of CD8 T cell effector response in vitro and in vivo. Overall, our data indicate that IFN-I cross-prime CD8 T cells against apoptotic cell-derived Ag both by licensing DC and by enhancing cross-presentation.     

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.     

Characterization of pulmonary T cell response to helper-dependent adenoviral vectors following intranasal delivery

In spite of the extensive research in the field of gene therapy, host immune responses continue to be the major barrier in translating basic research to clinical practice. Helper-dependent adenoviral (HD-Ad) vectors show great potential for pulmonary gene therapy, but the knowledge of pulmonary immune responses toward these vectors is very limited. In this study, we show that HD-Ad vectors are potent stimulators of dendritic cell (DC) maturation, thus leading to stimulation of T cell proliferation with approximately 6% of naive CD4(+) T cells from pulmonary mediastinal lymph node responding to HD-Ad-treated DCs. In contrast to the belief that HD-Ad vectors are unable to prime adaptive immune response, we show for the first time, through in vivo pulmonary studies in mice, that HD-Ad vectors can prime CD4(+) and CD8(+) T cell responses in the lung at high and substantially low doses. This indicates cross-presentation of HD-Ad-derived epitopes by DCs to prime CD8(+) T cell responses. To assess the basis of pulmonary T cell response against HD-Ad vectors, we examined the response of conventional DCs (cDCs) and plasmacytoid DCs (pDCs) in the lung. In response to HD-Ad delivery, there is induction of maturation in both cDC and pDC subsets, but it is the cDCs, not pDCs, that migrate rapidly to draining lymph nodes within the first 2 days after vector delivery to prime adaptive immune response against these vectors. These findings have implications for development of strategies to prevent adaptive immune responses against gene therapy vectors.     

A marked reduction in priming of cytotoxic CD8+ T cells mediated by stress-induced glucocorticoids involves multiple deficiencies in cross-presentation by dendritic cells

Protracted psychological stress elevates circulating glucocorticoids, which can suppress CD8(+) T cell-mediated immunity, but the mechanisms are incompletely understood. Dendritic cells (DCs), required for initiating CTL responses, are vulnerable to stress/corticosterone, which can contribute to diminished CTL responses. Cross-priming of CD8(+) T cells by DCs is required for initiating CTL responses against many intracellular pathogens that do not infect DCs. We examined the effects of stress/corticosterone on MHC class I (MHC I) cross-presentation and priming and show that stress/corticosterone-exposed DCs have a reduced ability to cross-present OVA and activate MHC I-OVA(257-264)-specific T cells. Using a murine model of psychological stress and OVA-loaded β(2)-microglobulin knockout "donor" cells that cannot present Ag, DCs from stressed mice induced markedly less Ag-specific CTL proliferation in a glucocorticoid receptor-dependent manner, and endogenous in vivo T cell cytolytic activity generated by cross-presented Ag was greatly diminished. These deficits in cross-presentation/priming were not due to altered Ag donation, Ag uptake (phagocytosis, receptor-mediated endocytosis, or fluid-phase uptake), or costimulatory molecule expression by DCs. However, proteasome activity in corticosterone-treated DCs or splenic DCs from stressed mice was partially suppressed, which limits formation of antigenic peptide-MHC I complexes. In addition, the lymphoid tissue-resident CD11b(-)CD24(+)CD8α(+) DC subset, which carries out cross-presentation/priming, was preferentially depleted in stressed mice. At the same time, CD11b(-)CD24(+)CD8α(-) DC precursors were increased, suggesting a block in development of CD8α(+) DCs. Therefore, glucocorticoid-induced changes in both the cellular composition of the immune system and intracellular protein degradation contribute to impaired CTL priming in stressed mice.     

The relative efficiency of acquisition of MHC:peptide complexes and cross-presentation depends on dendritic cell type

Intercellular exchange of MHC molecules has been reported between many cells, including professional and nonprofessional APCs. This phenomenon may contribute to T cell immunity to pathogens. In this study, we addressed whether the transfer of MHC class I:peptide complexes between cells plays a role in T cell responses and compare this to conventional cross-presentation. We observed that dsRNA-matured bone marrow-derived dendritic cells (BMDCs) acquired peptide:MHC complexes from other BMDCs either pulsed with OVA(257-264) peptide, soluble OVA, or infected with a recombinant adenovirus expressing OVA. In addition, BMDCs were capable of acquiring MHC:peptide complexes from epithelial cells. Spleen-derived CD8alpha(+) and CD8alpha(-) dendritic cells (DCs) also acquired MHC:peptide complexes from BMDCs pulsed with OVA(257-264) peptide. However, the efficiency of acquisition by these ex vivo derived DCs is much lower than acquisition by BMDC. In all cases, the acquired MHC:peptide complexes were functional in that they induced Ag-specific CD8(+) T cell proliferation. The efficiency of MHC transfer was compared with cross-presentation for splenic CD8alpha(+) and CD8alpha(-) as well as BMDCs. CD8alpha(+) DCs were more efficient at inducing T cell proliferation when they acquired Ag via cross-presentation, the opposite was observed for BMDCs and splenic CD8alpha(-) DCs. We conclude from these observations that the relative efficiency of MHC transfer vs cross-presentation differs markedly between different DC subsets.     

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.     

Bcl6 is required for the development of mouse CD4+ and CD8α+ dendritic cells

Th2-type inflammation spontaneously shown in Bcl6-knockout (KO) mice is mainly caused by bone marrow (BM)-derived nonlymphoid cells. However, the function of dendritic cells (DCs) in Bcl6-KO mice has not been reported. We show in this article that the numbers of CD4(+) conventional DCs (cDCs) and CD8α(+) cDCs, but not of plasmacytoid DCs, were markedly reduced in the spleen of Bcl6-KO mice. Generation of cDCs from DC progenitors in BM cells was perturbed in the spleen of irradiated wild-type (WT) mice transferred with Bcl6-KO BM cells, indicating an intrinsic effect of Bcl6 in cDC precursors. Although cDC precursors were developed in a Bcl6-KO BM culture with Fms-like tyrosine kinase 3 ligand, the cDC precursors were more apoptotic than WT ones. Also p53, one of the molecular targets of Bcl6, was overexpressed in the precursors. The addition of a p53 inhibitor to Bcl6-KO BM culture protected apoptosis, suggesting that Bcl6 is required by cDC precursors for survival by controlling p53 expression. Furthermore, large numbers of T1/ST2(+) Th2 cells were naturally developed in the spleen of Bcl6-KO mice. Th2 skewing was accelerated in the culture of WT CD4 T cells stimulated with Ags and LPS-activated Bcl6-KO BM-derived DCs, which produced more IL-6 and less IL-12 than did WT DCs; the addition of anti-IL-6 Abs to the culture partially abrogated the Th2 skewing. These results suggest that Bcl6 is required in cDC precursors for survival and in activated DCs for modulating the cytokine profile.     

Human cytomegalovirus pp65- and immediate early 1 antigen-specific HLA class I-restricted cytotoxic T cell responses induced by cross-presentation of viral antigens

Dendritic cells (DCs) play a pivotal role in the development of anti-viral CD8(+) CTL responses. This is straightforward if they are directly infected with virus, but is less clear in response to viruses that cannot productively infect DCS: Human CMV (HCMV) shows strain-specific cell tropism: fibroblast (Fb)-adapted laboratory strains (AD169) and recent clinical isolates do not infect DCs, whereas endothelial cell-adapted strains (TB40/E) result in productive lytic DC infection. However, we show here that uninfected DCs induce CD8(+) T cell cytotoxicity and IFN-gamma production against HCMV pp65 and immediate early 1 Ags following in vitro coculture with HCMV-AD169-infected Fbs, regardless of the HLA type of these FBS: CD8(+) T cell stimulation was inhibited by pretreatment of DCs with cytochalasin B or brefeldin A, indicating a phagosome/endosome to cytosol pathway. HCMV-infected Fbs were not apoptotic as measured by annexin V binding, and induction of apoptosis of infected Fbs in vitro did not augment CTL induction by DCs, suggesting a mechanism other than apoptosis in the initiation of cross-presentation. Furthermore, HCMV-infected Fbs provided a maturation signal for immature DCs during coculture, as evidenced by increased CD83 and HLA class II expression. Cross-presentation of HCMV Ags by host DCs enables these professional APCs to bypass some of the evasion mechanisms HCMV has developed to avoid T cell recognition. It may also serve to explain the presence of immediate early 1 Ag-specific CTLs in the face of pp65-induced inhibition of Ag presentation at the level of the infected cell.     

Hematopoietic cells are required to initiate a Chlamydia trachomatis-specific CD8+ T cell response

Chlamydia trachomatis is a global human pathogen causing diseases ranging from blinding trachoma to pelvic inflammatory disease. To explore how innate and adaptive immune responses cooperate to protect against systemic infection with C. trachomatis L2, we investigated the role of macrophages (Mphi) and dendritic cells (DCs) in the stimulation of C. trachomatis-specific CD8(+) T cells. We found that C. trachomatis infection of Mphi and DCs is far less productive than infection of nonprofessional APCs, the typical targets of infection. However, despite the limited replication of C. trachomatis within Mphi and DCs, infected Mphi and DCs process and present C. trachomatis CD8(+) T cell Ag in a proteasome-dependent manner. These findings suggest that although C. trachomatis is a vacuolar pathogen, some Ags expressed in infected Mphi and DCs are processed in the host cell cytosol for presentation to CD8(+) T cells. We also show that even though C. trachomatis replicates efficiently within nonprofessional APCs both in vitro and in vivo, Ag presentation by hematopoietic cells is essential for initial stimulation of C. trachomatis-specific CD8(+) T cells. However, when DCs infected with C. trachomatis ex vivo were adoptively transferred into naive mice, they failed to prime C. trachomatis-specific CD8(+) T cells. We propose a model for priming C. trachomatis-specific CD8(+) T cells whereby DCs acquire C. trachomatis Ag by engulfing productively infected nonprofessional APCs and then present the Ag to T cells via a mechanism of cross-presentation.     

Neutrophil involvement in cross-priming CD8+ T cell responses to bacterial antigens

Substantial CD8(+) T cell responses are generated after infection of mice with recombinant Listeria monocytogenes strains expressing a model epitope (lymphocytic choriomeningitis virus NP(118-126)) in secreted and nonsecreted forms. L. monocytogenes gains access to the cytosol of infected cells, where secreted Ags can be accessed by the endogenous MHC class I presentation pathway. However, the route of presentation of the nonsecreted Ag in vivo remains undefined. In this study we show that neutrophil-enriched peritoneal exudate cells from L. monocytogenes-infected mice can serve as substrates for in vitro cross-presentation of both nonsecreted and secreted Ag by dendritic cells as well as for in vivo cross-priming of CD8(+) T cells. In addition, specific neutrophil depletion in vivo by low dose treatment with either of two Ly6G-specific mAb substantially decreased the relative CD8(+) T cell response against the nonsecreted, but not the secreted, Ag compared with control Ab-treated mice. Thus, neutrophils not only provide rapid innate defense against infection, but also contribute to shaping the specificity and breadth of the CD8(+) T cell response. In addition, cross-presentation of bacterial Ags from neutrophils may explain how CD8(+) T cell responses are generated against Ags from extracellular bacterial pathogens.     

No advantage of cell-penetrating peptides over receptor-specific antibodies in targeting antigen to human dendritic cells for cross-presentation

Induction of CTL responses by dendritic cell (DC)-based vaccines requires efficient DC-loading strategies for class I Ags. Coupling Ags to cell-penetrating peptides (CPPs) or receptor-specific Abs improves Ag loading of DCs. In contrast to CPPs, receptor-specific Abs deliver conjugated Ags to DCs with high specificity, which is advantageous for in vivo strategies. It has, however, been speculated that CPPs facilitate uptake and endosomal escape of conjugated Ags, which would potently enhance cross-presentation. In this study, we directly compare the in vitro targeting efficiency of a humanized D1 Ab directed against the human DC surface receptor DC-SIGN hD1 to that of three CPPs. The three CPPs colocalized within endosomes when targeted to human monocyte-derived DCs simultaneously, whereas hD1 was present in a different set of endosomes. However, within 75 min after uptake CPPs and hD1 colocalized extensively within the lysosomal compartment. Ab-mediated targeting of class I-restricted peptides to DC-SIGN enhanced cross-presentation of the peptides, while only one of the CPPs enhanced peptide presentation. This CPP and hD1 enhanced cross-presentation with equal efficiencies. Thus, we found no evidence of CPP specifically favoring the delivery of conjugated Ag to the DC class I presentation pathway. Given the specificity with which Abs recognize their targets, this favors the use of DC receptor-specific Abs for in vivo vaccination strategies.     

Rab7b, a novel lysosome-associated small GTPase, is involved in monocytic differentiation of human acute promyelocytic leukemia cells

Rab7 is a small Rab GTPase that regulates vesicular traffic from early to late endosomal stages of the endocytic pathway. Here we report the cloning and characterization of a novel Rab7-like GTPase, which shares highest homology with Rab7 and thus is designated as Rab7b. Northern blot analysis shows that Rab7b mRNA is expressed in human heart, placenta, lung, skeletal muscle, and peripheral blood leukocyte. RT-PCR or Western blot analysis of Rab7b expression shows that Rab7b is selectively expressed in monocytes, monocyte-derived immature dendritic cells (DCs), and promyeloid or monocytic leukemia cell lines. In the peripheral blood, Rab7b is specifically detected in CD14(+) cells, but not in CD4(+), CD8(+), CD19(+) or CD56(+) cells. When immature DCs are matured with lipopolysaccharide (LPS), Rab7b expression is gradually downregulated, while Rab7b is upregulated when monocytes are activated by LPS treatments. In acute promyelocytic leukemia (APL) HL-60 and NB4 cell lines, Rab7b expression is upregulated after phorbol myristate acetate (PMA)-induced monocytic differentiation. By immunofluorescence confocal microscopy, we demonstrate that Rab7b is associated with lysosomal organelles. Our data suggest that Rab7b is a lysosome-localized monocytic cell-specific small GTPase, and is involved in PMA-induced APL cell differentiation and possibly in regulation of monocyte functions.