Cross-presentation Of Exogenous Antigens Research Articles

Redundancy renders the glycoprotein 96 receptor scavenger receptor A dispensable for cross priming in vivo

CD8(+) T cells (T(CD8+)) differentiate into effector cells following recognition of specific peptide-major histocompatibility complex (MHC) class I complexes (pMHC-I) on the surface of professional APCs (pAPCs), such as dendritic cells. Antigenic pMHC-I can be generated from two spatially distinct sources. The direct presentation pathway involves generation of peptide from protein substrate synthesized within the cell that is presenting the pMHC-I. Alternatively, the cross presentation pathway involves presentation of antigen that is not synthesized within the presenting cell, but is derived from exogenous proteins synthesized within other donor cells. The mechanisms by which cross presentation of exogenous antigens occur in vivo remain controversial. The C-type lectin scavenger receptor A (SR-A) has been implicated in a number of potential cross presentation pathways, including the presentation of peptide bound to heat shock proteins, such as glycoprotein 96 (gp96), and the transfer of pMHC-I from a donor cell to the pAPC. We demonstrate here that initiation of T(CD8+) responses is normal in mice lacking SR-A, and that the redundancy of ligand binding exhibited by the SR family is likely to be an important mechanism that ensures cross presentation in vivo. These observations emphasize the requirement to target multiple receptors and antigen-processing pathways during the rational design of vaccines aimed at eliciting protective T(CD8+).

The effect of poly( d,l-lactide- co-glycolide) microparticles with polyelectrolyte self-assembled multilayer surfaces on the cross-presentation of exogenous antigens

A surface-engineered particulate delivery system for exogenous antigens was developed in this study. Poly( d,l-lactide- co-glycolide) (PLGA) microparticles containing ovalbumin (OVA) or fluorescein isothiocyanate-conjugated bovine serum albumin (FITC–BSA) were fabricated by the double emulsion and solvent evaporation method. Encapsulation of the PLGA microparticles was performed by physisorption of multilayers of oppositely charged polyelectrolytes, including polyethylenimine (PEI) and dextran sulfate. Surface charges of the particles after layer-by-layer (LbL) adsorption were determined by the zeta potential measurements. The uptake of these particles by the J774A.1 murine macrophages was examined by fluorescence microscopy. Generation of reactive oxygen species (ROS) in J774A.1 cells was determined by flow cytometry using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and hydroethidine (HE). Antigen presentation assays were performed in B3Z cells, a hybridoma of OVA-specific CD8 + T cells. Results obtained in this study demonstrated an effective ingestion of the PLGA microparticles and enhanced production of ROS in J774A.1 murine macrophages. Treatment of murine bone marrow-derived dendritic cells (BMDCs) with polyelectrolyte-encapsulated PLGA microparticles resulted in an in vitro activation of B3Z cells, demonstrating the feasibility of induction of adaptive immunity for class I major histocompatibility complex (MHC) by surface engineering of microparticulate vaccine delivery.

Maturation pathways of dendritic cells determine TAP1 and TAP2 levels and cross‐presenting function

Cross‐presentation of exogenous antigens in the HLA class I pathway is key to the antigen presenting function of mature tumor‐loaded dendritic cells (DC). Conditions of DC maturation have been shown to influence the ability of DCs to induce T cell effector functions. However, it remains unknown if different pathways of DC maturation are associated with modulation in the ability of DCs to cross‐present tumor‐associated antigens. We compared DC matured with three clinically relevant cytokine protocols: IL‐1β, TNF‐α, IL‐6 (DC‐0), DC‐0+PGE‐2, or IFN‐γ, IFN‐α, TNF‐α, and IL1‐β (DC‐1). We found that these DC vary in the ability to cross‐present tumor antigens to CTL, with DC‐1 protocol being significantly more effective than the other protocols. This level of TA cross presentation was correlated with the level of expression of the antigen processing machinery (APM) components, TAP1 and TAP2, and to a lesser extent, tapasin, indicating that these components could be used to standardize DC preparations for optimal function. However, upregulation of TAP1/2 was not sufficient to explain the strongest cross‐presentation ability induced by DC‐1 cells, since IFN‐γ did not generate DC as effective at cross‐presentation as the full DC‐1 cytokine cocktail. These data indicate that DC maturation pathways modulate APM component expression and that DC vary in the ability to cross‐present TA to HLA class I‐restricted CTL.

IFN-g-Stimulated Mesenchymal Stromal Cells Acquire the Ability To Cross-Present Exogenous Soluble Antigens and Lead to an Effective Immune Response.

Antigen cross-presentation is the mechanism by which exogenous antigens can be presented by major histocompatibility complex (MHC) class I molecules to CD8+ T cells. This process is efficiently performed by professional antigen presenting cells (APC) such as dendritic cells and macrophages. Recently, we and others have shown that IFN-g enables the upregulation of the expression of MHC class I & II molecules by marrow-derived Mesenchymal Stromal Cells (MSCs) and that MHC II-mediated antigen presentation can lead to cell-mediated protective immunity to xenoantigens [Stagg et al., Blood March 2006]. These findings led us to investigate whether MSCs also possess the ability to cross-present antigens via MHC class I. Using antigen presentation assays performed on murine MSC in the presence of MHC class I-restricted ovalbumin (OVA)-specific CD8+ T hybridoma cells or purified primary CD8+ T lymphocytes from OT1 transgenic mice, we demonstrated that MSC can robustly and effectively cross-present exogenous antigens via MHC class I molecules upon IFN-g pre-treatment in a manner comparable to professional APCs like macrophages. Use of transporter associated with antigen processing (TAP-1)-deficient mice and proteasome inhibitors suggested a MHC class I machinery-dependent pathway. Cross-presentation by IFN-g-activated MSC was also observed to be suppressed by TGF-b and regulated by cell density. In vivo, IFN-g-treated, OVA-pulsed MSCs administered to normal C57Bl/6 mice led to an effective OVA-specific, T cell cytotoxic immune response, leading to the rejection of OVA-expressing EG7 lymphoma cells. In conclusion, our findings suggest that cross-presentation properties of MSC could play a role in their effectiveness as conditional APCs in vivo and this property may be exploited as a therapeutic cell-based immune biopharmaceutical for treatment of cancer or infectious disease.

Neonatal and adult microglia cross‐present exogenous antigens

Some observations have suggested that cells from the central nervous system (CNS) could present exogenous antigens on major histocompatibility complex (MHC) class I molecules to CD8(+) T cells (a process called cross-presentation). Microglia are the major myeloid immunocompetent cells of the CNS. When activated, following the injury of the nervous parenchyma, they become fully competent antigen-presenting cells (APC) that prime CD4(+) T lymphocytes. We therefore tested the cross-presentation capacity of murine microglia. We report that a microglial cell line (C8-B4), neonatal microglia, and interestingly adult microglia cross-present soluble exogenous antigen (ovalbumin) to a OVA-specific CD8(+) T-cell hybridoma and cross-prime OVA-specific naive OT-1 CD8(+) T cells. In both these cases, C8-B4 and neonatal microglia cross-present OVA as well as peritoneal macrophages. Although cross-presentation by adult microglia is less efficient, it is increased by GM-CSF and CpG oligodeoxynucleotide (ODN) stimulation. Using microglial cells either exposed to an inhibitor of proteasome, lactacystin, or purified from TAP(-/-) mice, we demonstrate that the microglia cross-present antigen in proteasome- and TAP-dependant pathways, respectively. Last, microglia purified from adult mice injected intracerebrally with OVA efficiently stimulate OVA-specific CD8(+) T cells, thereby showing that microglia take up and process exogenous antigen into MHC class I in vivo. This first demonstration of the cross-presentation property of microglia offers novel therapeutic approaches to modulate CD8 T-cell responses in the brain.

The Cross‐priming Pathway: A Portrait of an Intricate Immune System

Antigen presentation by professional antigen-presenting cells (pAPCs) to cytotoxic CD8(+) T cells can occur via two processing routes - the direct and cross-presentation pathways. Cross-presentation of exogenous antigens in the context of major histocompatibility complex (MHC) class I molecules has recently attracted a lot of research interest because it may prove crucial for vaccine development. This alternative pathway has been implicated in priming CD8(+) T-cell responses to pathogens as well as tumours in vivo (cross-priming). In cross-presentation, the internalized antigens can be processed through diverse intracellular routes. As many unresolved questions regarding the molecular basis that controls the cross-priming process still exist, it is essential to explore the various elements involved therein, to better elucidate this pathway. In this review, we summarize current data that explore how the source and nature of antigens could affect their cross-presentation. Moreover, we will discuss and outline how recent advances regarding pAPCs' properties have increased our appreciation of the complex nature of the cross-priming pathway in vivo. In conclusion, we contemplate how the direct and cross-presentation pathways can function to allow the immune system to deal efficiently with diverse pathogens.

A Role for the Endoplasmic Reticulum Protein Retrotranslocation Machinery during Crosspresentation by Dendritic Cells

Crosspresentation of exogenous antigens (Ags) to CD8(+) T cells by dendritic cells generally requires their entry into the cytosol. Here we show that both soluble and phagocytosed extracellular Ags accessed the cytosol via molecular components required for endoplasmic reticulum (ER)-associated degradation (ERAD). Exogenous Pseudomonas aeruginosa Exotoxin A, which inhibits protein translocation from the ER to the cytosol, abrogated crosspresentation. Exotoxin A also prevented the transporter associated with antigen processing (TAP) inhibitor, ICP47, from entering the cytosol and blocking TAP-mediated peptide transport. In an in vitro model of retrotranslocation, the AAA ATPase p97, an enzyme critical for ERAD, was the only cytosolic cofactor required for protein export from isolated phagosomes. Functional p97 was also required for crosspresentation but not conventional presentation. Thus, crosspresentation appears to result from an adaptation of the retrotranslocation mechanisms involved in the degradation of misfolded ER proteins.

A Novel Pathway of Antigen Presentation by Dendritic and Endothelial Cells: Implications for Allorecognition and Infectious Diseases

Dendritic cells (DCs) are the major antigen presenting cells capable of stimulating T cell responses following either organ transplantation or a viral infection. In the context of allorecognition, T cells can be activated following presentation of alloantigens by donor DCs (direct), as well as by recipient DCs presenting processed donor major histocompatibility complex (MHC) as peptides (indirect). We have recently described another mechanism by which alloreactive T cells are activated. Recipient DCs can acquire donor MHC through cell-to-cell contact and this acquired MHC can stimulate a T cell response (the semidirect pathway). Similarly, during a viral infection, DCs are capable of stimulating T cells directly, as occurs when infected DCs present processed viral antigens, or indirectly by a process known as cross-presentation. Although cross-presentation of exogenous antigen is an important mechanism for controlling infectious diseases, it is possible that peptide:MHC acquisition (the semidirect pathway) may also play a part in immunity against pathogens. In this review, we discuss the possible contributions of the semidirect pathway/MHC transfer in infectious disease.

Langerhans cells cross-present antigen derived from skin

Dendritic cells (DC) efficiently cross-present exogenous antigen on MHC class I molecules to CD8+ T cells. However, little is known about cross-presentation by Langerhans cells (LC), the DCs of the epidermis. Therefore, we investigated this issue in detail. Isolated murine LCs were able to cross-present soluble ovalbumin protein on MHC-class I molecules to antigen-specific CD8+ T cells, albeit less potently than the CD8+ DC subsets from spleen. Furthermore, LCs cross-presented cell-associated ovalbumin peptide and protein expressed by neighboring keratinocytes. Use of transporter associated with antigen processing (TAP-1)-deficient mice suggested a TAP-dependent pathway. Similar observations were made with migratory LC. Antigen expressed in the epidermis was ingested by LCs during migration from the epidermis and presented to antigen-specific T cells in vitro. Cross-presentation of ovalbumin protein by LCs induced IFN-gamma production and cytotoxicity in antigen-specific CD8+ T cells. Additionally, epicutaneous application of ovalbumin protein induced in vivo proliferation of OT-I T cells in the draining lymph nodes; this was markedly enhanced when antigen was applied to inflamed, barrier-disrupted skin. Thus, LCs cross-present exogenous antigen to CD8+ T cells and induce effector functions, like cytokine production and cytotoxicity, and may thereby critically contribute in epicutaneous vaccination approaches.

Chemokine CXCL10 (IP-10) is sufficient to trigger an immune response to injected antigens in a mouse model

The induction of chemokines by interferons might represent a link between innate and adaptive immunity. Whether these induced chemokines might be useful by themselves to induce an immune response is not known. We hypothesized that the interferon-inducible chemokine CXCL10 could stimulate dendritic cells (DC) to mature and cross-present exogenous antigen to T cells, resulting in a Th1-type immune response. We found that injecting mice with CXCL10 together with ovalbumin (OVA) as a test antigen was sufficient to produce functional OVA-specific T cells in 7 of 10 mice. Further, using only CXCL10 and a peptide antigen derived from vaccinia virus, we were able to induce peptide-specific cytotoxic T cells in 4 of 4 mice tested. These cytotoxic T cells protected 9 of 10 mice from subsequent infectious challenge with vaccinia virus. Unlike traditional adjuvants, no side effects were observed in any of the injected mice. We conclude that CXCL10 co-administration with a variety of antigens may represent a unique strategy of inducing a protective T cell response to a number of pathogens that merits further study.

Very small size proteoliposomes derived from Neisseria meningitidis: An effective adjuvant for generation of CTL responses to peptide and protein antigens

The development of potent adjuvants, conditioning innate and adaptative immunity, particularly CTL responses, has become currently a hot point in the rational design of vaccines for cancer immunotherapy. We have described a new approach, in which gangliosides are incorporated into vesicles from Neisseria meningitidis to form Very Small Size Proteoliposomes (VSSP). VSSP is a good alternative to the existing adjuvants for use in whole cells vaccines since it promotes 80% tumour rejection and growing delay in the CT26 and F3II tumour models respectively. Also VSSP induces activation of CTL responses to co-injected trimmed peptides and soluble proteins. This phenomena is facilitated by the cross-presentation of exogenous antigen and do not need cooperation of CD4 T cells for primary CD8 T cells expansion.

The heat shock protein Hsp70 enhances antigen-specific proliferation of human CD4+ memory T cells

Heat shock proteins (HSP) can interact with a wide variety of peptides and the resulting HSP:peptide complexes are known to be highly immunogenic. The ability of HSP:peptide complexes to elicit CD8+ T cell responses by cross-presentation of exogenous antigen via MHC class I is well known. In contrast, their role in the activation of CD4+ T cells is less clearly defined, although several recent studies in mice and T cell lines suggest an involvement of HSP in the presentation of antigenic peptides via MHC class II. In this study we have investigated the potential of antigenic peptides from tetanus toxin and influenza hemagglutinin complexed to the human stress-inducible Hsp70 to enhance activation and proliferation of human memory CD4+ T cells. Hsp70:peptide complexes were found to amplify the proliferation of antigen-specific CD4+ T cells as confirmed by HLA-DR tetramer staining. Complex formation of the antigenic peptide with Hsp70 was absolutely required to elicit an antigen-specific amplification. This effect was most pronounced at low doses of antigen and decreasing APC/CD4+ T cell ratios. Taken together, we show the potential of Hsp70 to enhance antigen-specific CD4+ T cell proliferation and to increase the immunogenicity of presented peptides in human CD4+ T cells.

CpG oligodeoxynucleotides enhance FcgammaRI-mediated cross presentation by dendritic cells.

Dendritic cells (DC) can trigger naive CD8(+) T cell responses by their capacity to cross-present exogenous antigens via the major histocompatibility complex class I pathway. The myeloid class I IgG receptor, FcgammaRI (CD64), is expressed on DC, and in vivo targeting of antigens to FcgammaRI induces strong humoral and cellular immune responses. We studied the capacity of human FcgammaRI (hFcgammaRI) to facilitate DC-mediated cross presentation and T cell activation, and assessed the effect of CpG oligodeoxynucleotides on this process. We generated hFcgammaRI expressing immature DC from hFcgammaRI transgenic and immature DC from non-transgenic mice. Antigens were targeted to Fcgamma receptors as ovalbumin immune complexes, or selectively to hFcgammaRI via ovalbumin-CD64 mAb fusion proteins. Co-incubation of immature DC with CpG ODN led to markedly increased MHC class I presentation of FcgammaR-targeted antigens. When OVA was selectively targeted to hFcgammaRI, few differences were observed between Tg and NTg DC. However, upon co-incubation with CpG ODN, hFcgammaRI-triggered cross presentation was enhanced. These results document the capacity of hFcgammaRI on DC to trigger cross presentation via MHC class I upon co-culture with CpG ODN.

Cross-presentation: dendritic cells and macrophages bite off more than they can chew!

As immunologists, our knowledge of the molecular mechanisms which underlie the presentation of antigens derived from extracellular or 'exogenous' sources to CD8 cytotoxic lymphocytes (CTL) has been limited. This process, termed 'cross-presentation', has been linked to the elicitation of protective CTL responses against tumours and may be extremely important in generating immune responses against clinically relevant pathogens that do not infect tissues of haemopoietic origin. It is now known that cross-presentation of exogenous antigens on major histocompatibility complex (MHC) class I occurs through several distinct cellular pathways. In this review we outline and discuss some recent advances in our understanding of these pathways.

Heat shock-induced dendritic cell maturation is coupled by transient aggregation of ubiquitinated proteins independently of heat shock factor 1 or inducible heat shock protein 70

The transition of dendritic cells (DCs) from immature to mature states is critical for the optimal priming of the adaptive immune response. This highly regulated process is accompanied by structural and functional alterations of DCs, including rapid and dramatic redistributions of MHC class I and class II molecules to cell surfaces, nuclear translocation of NF-κB,and transient appearance of dendritic cell aggresome-like induced structures (DALIS) in the cytosol. We have previously found that DCs can be matured by a non-inflammatory stress stimulation, i.e., heat shock. In this study, we examined if thermomanipulation could induce DALIS formation. We found that heat shock of DCs, but not of other cell types, led to the appearance of aggresome-like structures that were structurally indistinguishable from DALIS induced by lipopolysaccharide (LPS). Furthermore, the induction of DALIS in DCs by heat, but not by LPS, correlated with the increased ability of DCs to cross-present exogenous antigens to MHC I. Since the canonical biochemical response to heat shock is the induction of heat shock proteins (HSP) by heat shock factors (Hsf), we studied the contribution of both HSP and Hsf in heat shock-mediated DALIS formation using gene knockout mice. We demonstrated that neither inducible HSP70 nor the major mammalian heat shock transcription factor Hsf1 was involved in the formation of DALIS. Our results highlighted the important roles of heat shock in modulating the function of DCs, and they further suggested that heat-mediated immune regulation can be uncoupled from heat shock response.

Deficient MHC class I cross-presentation of soluble antigen by murine neonatal dendritic cells

AbstractNeonates respond suboptimally to many vaccines. The reasons for this defect are unclear, but suboptimal antigen presentation by dendritic cells has been suggested as one possibility. In this report we describe an in vitro system that allows the generation of large numbers of resting murine neonatal dendritic cells facilitating their study. Using this system, we show a clear reduction in the ability of neonatal dendritic cells to present soluble ovalbumin, while the capacity to present ovalbumin peptide is intact. This suggests a specific defect in cross-presentation of exogenous antigen via the major histocompatibility complex (MHC) class I pathway. Deficient cross-presentation may contribute to the suboptimal CD8 T-cell response to vaccines in neonates. (Blood. 2004;103:4240-4242)

Early phagosomes in dendritic cells form a cellular compartment sufficient for cross presentation of exogenous antigens.

Conventionally, MHC class I-restricted antigen (Ag) processing requires the action of the multimolecular peptide-loading complex within the endoplasmic reticulum (ER). Here we show that early phagosomes from human dendritic cells (DCs) contain the peptide-loading complex, incorporating MHC class I, beta2 microglobulin, transporter associated with Ag processing (TAP), calreticulin, tapasin, and ERp57. Antigenic peptides could be translocated into purified phagosomes by TAP and loaded onto cognate class I molecules, inducing their specific dissociation from the loading complex. Endoglycosidase H-sensitive class I molecules were detected at the DC cell surface, suggesting that these molecules traffic there directly from phagosomes. Macropinocytosis also allowed internalized soluble Ags access to an ER-like compartment containing the class I loading complex. Blockade of TAP by endocytosis of a soluble derivative of human cytomegalovirus protein US6 confirmed that, although retrotranslocation into the cytosol is critical for processing, efficient association of class I molecules with peptides derived from exogenous Ags occurs within a compartment directly accessible to internalized proteins. Together, this evidence suggests that early phagosomes and pinosomes facilitate cross presentation of exogenous Ags by DCs.

The heat-shock protein receptor CD91 is up-regulated in monocytes of HIV-1–infected “true” long-term nonprogressors

AbstractA small proportion of patients with human immunodeficiency virus type 1 (HIV-1) remains asymptomatic for a long period after infection. It is thought that a vigorous immune response may contribute to long-term nonprogression, though studies are confounded by heterogeneity among patients. We studied the levels of HIV-1 receptors, costimulatory T-cell molecules, and dendritic cell (DC) numbers in 18 patients with long-term infection, CD4 counts greater than 400 cells/mm3, and HIV-1 viral loads lower than 50 copies/mL. These patients were further differentiated through the presence or absence of 2-LTR DNA circles, a possible marker for residual ongoing HIV-1 replication. A statistically significant increase in levels of CD91, the heat-shock protein (HSP) receptor, was observed in therapy-naive patients who had no evidence of ongoing viral replication (P = .01). This difference was most notable on their monocytes. High levels of CD91 may be a host factor that contributes to the maintenance of long-term nonprogression. The ability of CD91 to internalize α-defensins and to cross-present exogenous antigen to cytotoxic T lymphocytes through major histocompatibility complex (MHC) class 1 may maintain CD8+ responses in these patients.

Efficient presentation of exogenous antigen by liver endothelial cells to CD8+ T cells results in antigen-specific T-cell tolerance.

Myeloid antigen-presenting cells (APC) are known to cross-present exogenous antigen on major histocompatibility class I molecules to CD8+ T cells and thereby induce protective immunity against infecting microorganisms. Here we report that liver sinusoidal endothelial cells (LSEC) are organ-resident, non-myeloid APC capable of cross-presenting soluble exogenous antigen to CD8+ T cells. Though LSEC employ similar molecular mechanisms for cross-presentation as dendritic cells, the outcome of cross-presentation by LSEC is CD8+ T cell tolerance rather than immunity. As uptake of circulating antigens into LSEC occurs efficiently in vivo, it is likely that cross-presentation by LSEC contributes to CD8+ T cell tolerance observed in situations where soluble antigen is present in the circulation.