Antibiotic Transport in Bacteria

Abstract

Survival or destruction of intramacrophage pathogen Leishmania depends in part on modulation of their host cell phagosome, capabilities of the infected macrophages to present parasite antigen to the host's immune system. Macrophages house these parasites as amastigotes in the acidic phagolysosomal compartment. Leishmania phagolysosome is the potential site for processing and presentation of its antigen as well as being the target site for chemotherapy in leishmaniasis. It is thought that the parasites are killed from macrophage activation by lymphokines secreted from either helper T1 cells or CD8⁺ T cells. Characterization of both the host and parasite molecules in the compartment in the context of biogenesis of Leishmania-phagolysosome and processing of the parasite antigen by this compartment are discussed. Trafficking of different drugs and new agents through this compartment and their role in chemotherapy and necessity of developing new drug carrier are also stressed.     

Trypanosoma cruzi-infected macrophages are defective in major histocompatibility complex class II antigen presentation

Trypanosoma cruzi, the intracellular protozoan parasite that causes Chagas' disease, interferes with the host immune response to establish a persistent infection. In this report, we demonstrate that macrophages infected with T. cruzi are unable to effectively present antigens to CD4 T cells. The interference is due to defective antigen-presenting cell (APC) function, as antigen-independent stimulation of the T cell in the presence of infected macrophages is not affected. The defect is distal to antigen processing and is not due to decreased major histocompatibility complex (MHC) class II expression, decreased viability, defective peptide loading in the infected macrophages, nor absence of CD28 co-stimulation. There was a role for gp39:CD40 co-stimulation during antigen presentation to the T cells we studied, but the expression of CD40 on T. cruzi-infected macrophages was not decreased. Antigen-specific adhesion between macrophages and T cells was reduced by infection. Equivalent levels of the adhesion molecules lymphocyte function-associated antigen-1, intercellular adhesion molecule-1, vascular cell adhesion molecule-1 or very late antigen-4 are found on infected and uninfected APC, suggesting that reduced expression of these adhesion molecules was not responsible for the defect in antigen-specific adhesion. The defective T cell:macrophage adhesion may be due to the reduced expression of other adhesion molecules or other changes in the cell induced by infection. Interfering with MHC class II antigen presentation in infected macrophages may help T. cruzi to blunt the immune response by the host.     

Antigens targeted to the Leishmania phagolysosome are processed for CD4+ T cell recognition

Processing of antigen for recognition by class II-restricted CD4⁺ T cells occurs within acidic compartments of the antigen-presenting cell. The exact nature of this compartment has yet to be precisely defined, however, but may vary depending upon the cell type studied and the antigen used. The acidic compartments of macrophages are also responsible for the degradation of ingested micro-organisms and play host to others which are adapted to an intracellular existance. To determine whether the phagolysosome (PL) formed in activated macrophages after ingestion of Leishmania parasites is also a site for entry of antigen into the class II presentation pathway, we have used the approach of genetic transformation. Hence, Leishmania were transfected with the genes for the protein antigens ovalbumin (OVA) and β-galactosidase (β-gal) and after infection were able to deliver these antigens specifically into the PL. Delivery of antigen to this site resulted in the ability of infected macrophages to present these antigens to antigen-specific CD4⁺ T cells. After taking into account the absolute levels of antigen uptake by macrophages, a 4-h processing period for OVA delivered by this or a soluble route led to equivalent levels of T cell activation. Unlike macrophages pulsed with soluble OVA, those with PL-targeted OVA still retained the ability to stimulate T cells after a 24-h processing period. This enhanced lifespan of antigen in macrophages corresponded to the kinetics of degradation of the parasite, suggesting slow release of antigen into the processing pathway. β-gal presentation from the PL was tenfold less efficient under the same conditions. In addition to providing the first information on antigen processing in a protozoan PL, these studies highlight the usefulness of genetically transformed parasites for these types of studies.     

Leishmania mexicana promastigotes induce cytotoxic T lymphocytes in vivo that do not recognize infected macrophages

The question is addressed whether antigens of Leishmania, a parasite residing in the endosomal compartment of macrophages, can be presented in the context of major histocompatibility complex class I molecules. We used E. coli β-galactosidase as a model antigen which can be expressed in high levels in L. mexicana promastigotes (L. mexicana-gal). Infection of BALB/c mice with L. mexicanagal induces β-galactosidase-specific cytotoxic T cells (CTL), which can be isolated using a β-galactosidase-expressing mastocytoma line as an antigen-presenting cell. These CTL recognize epitopes of β-galactosidase in the context of H-2K^d; however, they do not recognize L. mexicanagal-infected macrophages even after killing of the intracellular amastigotes by drug treatment or macrophage activation by lymphokines, although class I-peptide interaction and the presentation of endogenously produced antigens is normal. It is concluded that parasite antigens can induce a CTL response in vivo but that these CTL cannot recognize infected macrophages because the relevant epitopes cannot gain access to class I molecules. The effect of priming in vivo may be explained by the well-known but ill-understood phenomenon of cross-priming.     

Leishmania infantum-specific T cell lines derived from asymptomatic dogs that lyse infected macrophages in a major histocompatibility complex-restricted manner

Protective immunity to leishmaniasis has been demonstrated in murine models to be mediated by T cells and the cytokines they produce. We have previously shown that resistance to experimental Leishmania infantum infection in the dog, a natural host and reservoir of the parasite, is associated with the proliferation of peripheral blood mononuclear cells (PBMC) to parasite antigen and to the production of interleukin-2 and tumour necrosis factor. In this study we show that PBMC from asymptomatic experimentally infected dogs produce interferon-γ upon parasite antigen-specific stimulation, whereas lymphocytes from symptomatic dogs do not. In addition, we report for the first time the lysis of L. infantum-infected macrophages by PBMC from asymptomatic dogs and by parasite-specific T cell lines derived from these animals. These T cell lines were generated by restimulation in vitro with parasite soluble antigen and irradiated autologous PBMC as antigen-presenting cells. We show that lysis of infected macrophages by T cell lines is major histocompatibility complex restricted. Characterization of parasite-specific cytotoxic T cell lines revealed that the responding cells are CD8⁺. However, for some animals, CD4⁺ T cells that lyse infected macrophages were also found. In contrast to asymptomatic dogs, lymphocytes from symptomatic dogs failed to proliferate and produce interferon-γ after Leishmania antigen stimulation in vitro and were not capable of lysing infected macrophages. These results suggest that both the production of interferon-γ and the destruction of the parasitized host cells by Leishmania-specific T cells play an important role in resistance to visceral leishmaniasis.     

Antigen processing mutant T2 cells present viral antigen restricted through H-2K

Cytotoxic T lymphocytes (CTL) recognize foreign antigens as short peptides presented by class I molecules of the major histocompatibility complex (MHC). T2 cells are profoundly defective in the presentation of endogenously synthesized antigens to CTL due to a deletion of MHC class II-encoded genes for transporters associated with antigen presentation (TAP1/TAP2). Surprisingly, we here demonstrate that T2 cells, after infection with Sendai virus, are readily killed by H-2K^b restricted CD8⁺ T cells. In contrast to classical class I-mediated antigen presentation, the presentation of Sendai virus antigen inT2K^b cells is brefeldin A (BFA) insensitive. The present findings may suggest the presence of an alternative pathway for MHC class I-mediated antigen presentation in T2 cells.     

Trypanosoma cruzi infection does not impair major histocompatibility complex class I presentation of antigen to cytotoxic T lymphocytes

Trypanosoma cruzi (T. cruzi), the etiological agent of Chagas' disease, lives free within the cytoplasm of infected host cells. This intracellular niche suggests that parasite antigens may be processed and presented on major histocompatibility complex (MHC) class I molecules for recognition by CD8⁺ T cells. However, the parasite persists indefinitely in the mammalian host, indicating its success at evading immune clearance. It has been shown that T. cruzi interferes with processing and presentation of antigenic peptides in the MHC class II pathway. This investigation sought to determine whether interference in MHC class I processing and presentation occurs with T. cruzi infection. Surface expression of MHC class I molecules was found to be unaffected or up-regulated by T. cruzi infection in vitro. A model system employing a β-galactosidase (β-gal)-specific murine cytotoxic T lymphocyte (CTL) line (0805B) showed: (i) in vitro infection of mouse peritoneal macrophages or J774 cells with T. cruzi did not inhibit MHC class I presentation of exogenous peptide (a nine-amino acid epitope of β-gal) to the CTL line, (ii) in vitro infection of a β-gal-expressing 3T3 cell line (LZEJ) with T. cruzi did not inhibit MHC class I presentation of the endogenous protein to the CTL line and (iii) mouse renal adenocarcinoma cells infected with T. cruzi and subsequently infected with adenovirus expressing β-gal were able to present antigen to the β-gal-specific CTL line. These findings indicate that the failure of the immune response to clear T. cruzi does not result from global interference by the parasite with MHC class I processing and presentation. Parasites engineered to express β-gal were unable to sensitize infected antigen-presenting cells in vitro to lysis by the CTL 0805B line. This was probably due to the intracellular localization of the β-gal within the parasite and its inaccessibility to the host cell cytoplasm.     

Down-regulation of MHC class II molecules and inability to up-regulate class I molecules in murine macrophages after infection with Toxoplasma gondii

Toxoplasma gondii is able to invade phagocytic cells of the monocyte-macrophage lineage and replicates within a parasitophorous vacuole. Since macrophages may activate specific T lymphocytes by presenting pathogen-derived antigens in association with molecules of the MHC, we investigated the in vitro expression of host cell molecules involved in antigen processing and presentation before and during infection of murine bone marrow-derived macrophages (BMM) with T. gondii. Fifty-one hours after addition of T. gondii tachyzoites at different parasite-to-host ratios, up-regulation of total MHC class II molecules by interferon-gamma (IFN-γ) was dose-dependently abrogated in up to 50% of macrophages compared with uninfected control cultures. Quantitative analyses by flow cytometry revealed that the IFN-γ-induced surface expression of class II antigens as well as the IFN-γ-induced up-regulation of class I molecules was significantly decreased in T. gondii-infected macrophage cultures compared with uninfected controls. However, the constitutive expression of MHC class I antigens was not altered after parasitic infection, and infected BMM remained clearly positive for these molecules. After infection of macrophages preactivated with IFN-γ for 48 h, T. gondii also actively down-regulated an already established expression of MHC class II molecules. Furthermore, kinetic analysis revealed that the reduction in intracellular and plasma membrane-bound class II molecules started ≈ 20 h after infection. While MHC class II antigens were most prominently reduced in parasite-positive host cells, culture supernatant from T. gondii-infected BMM cultures also significantly inhibited expression of these molecules in uninfected macrophages. However, down-regulation of MHC class II molecules was not mediated by an increased production of prostaglandin E₂, IL-10, transforming growth factor-beta or nitric oxide by infected BMM compared with uninfected controls. Our data indicate that intracellular T. gondii interferes with the MHC class I and class II antigen presentation pathway of murine macrophages and this may be an important strategy for evasion from the host's immune response and for intracellular survival of the parasite.     

Expression of class II,but not class I,major histocompatibility complex molecules is required for granuloma formation in infection with Schistosoma mansoni

Previous studies have suggested that granulomatous inflammation in schistosomiasis is mediated by CD4⁺ T helper lymphocytes sensitized to parasite egg antigens. However, CD8⁺ T cells have also frequently been associated with the immune response to schistosome eggs. To examine more precisely the role of CD4⁺ and CD8⁺ T cells in the pathology of the schistosomal infection, we used mice with targeted mutations in major histocompatibility complex (MHC) class II or class I molecules. These mutations lead, respectively, to the virtual absence of CD4⁺ and CD8⁺ T cells. The results clearly show that schistosome-infected MHC class II mutant mice failed to form granulomas around parasite eggs. In contrast, infected MHC class I mutant mice displayed characteristic granulomatous lesions that were comparable to those in wild-type control mice. Moreover, lymphoid cells from MHC class II mutant mice were unable to react to egg antigens with either proliferative or cytokine [interferon-gamma, interleukin (IL)-4, IL-10] responses; nor were they able to present egg antigens to specifically sensitized CD4⁺ T helper cells from infected syngeneic control mice. By comparison, cells from MHC class I mutant mice exercised all these functions in a manner comparable with those from wild-type controls. These observations clearly demonstrate that schistosomal egg granulomas are mediated by MHC class II-restricted CD4⁺ T helper cells. They also suggest that CD8⁺ T cells do not become sensitized to egg antigens and play little role, if any, in the pathogenesis of schistosomiasis.     

Sustained cross‐presentation capacity of murine splenic dendritic cell subsets in vivo

An exclusive feature of dendritic cells (DCs) is their ability to cross‐present exogenous antigens in MHC class I molecules. We analyzed the fate of protein antigen in antigen presenting cell (APC) subsets after uptake of naturally formed antigen‐antibody complexes in vivo. We observed that murine splenic DC subsets were able to present antigen in vivo for at least a week. After ex vivo isolation of four APC subsets, the presence of antigen in the storage compartments was visualized by confocal microscopy. Although all APC subsets stored antigen for many days, their ability and kinetics in antigen presentation was remarkably different. CD8α⁺ DCs showed sustained MHC class I‐peptide specific CD8⁺ T‐cell activation for more than 4 days. CD8α^? DCs also presented antigenic peptides in MHC class I but presentation decreased after 48 h. In contrast, only the CD8α^? DCs were able to present antigen in MHC class II to specific CD4⁺ T cells. Plasmacytoid DCs and macrophages were unable to activate any of the two T‐cell types despite detectable antigen uptake. These results indicate that naturally occurring DC subsets have functional antigen storage capacity for prolonged T‐cell activation and have distinct roles in antigen presentation to specific T cells in vivo.     

Antigen processing and CD24 expression determine antigen presentation by splenic CD4+ and CD8+ dendritic cells

To examine heterogeneity in dendritic cell (DC) antigen presentation function, murine splenic DCs were separated into CD4⁺ and CD8⁺ populations and assessed for the ability to process and present particulate antigen to CD4⁺ and CD8⁺ T cells. CD4⁺ and CD8⁺ DCs both processed exogenous particulate antigen, but CD8⁺ DCs were much more efficient than CD4⁺ DCs for both major histocompatibility complex (MHC) class II antigen presentation and MHC class I cross-presentation. While antigen processing efficiency contributed to the superior antigen presentation function of CD8⁺ DCs, our studies also revealed an important contribution of CD24. CD8⁺ DCs were also more efficient than CD4⁺ DCs in inducing naïve T cells to acquire certain effector T-cell functions, for example generation of cytotoxic CD8⁺ T cells and interferon (IFN)-γ-producing CD4⁺ T cells. In summary, CD8⁺ DCs are particularly potent antigen-presenting cells that express critical costimulators and efficiently process exogenous antigen for presentation by both MHC class I and II molecules.     

Norovirus antagonism of B-cell antigen presentation results in impaired control of acute infection

Human noroviruses are a leading cause of gastroenteritis, and so, vaccine development is desperately needed. Elucidating viral mechanisms of immune antagonism can provide key insight into designing effective immunization platforms. We recently revealed that B cells are targets of norovirus infection. Because noroviruses can regulate antigen presentation by infected macrophages and B cells can function as antigen-presenting cells, we tested whether noroviruses regulate B-cell-mediated antigen presentation and the biological consequence of such regulation. Indeed, murine noroviruses could prevent B-cell expression of antigen presentation molecules and this directly correlated with impaired control of acute infection. In addition to B cells, acute control required MHC class I molecules, CD8⁺ T cells, and granzymes, supporting a model whereby B cells act as antigen presenting cells to activate cytotoxic CD8⁺ T cells. This immune pathway was active prior to the induction of antiviral antibody responses. As in macrophages, the minor structural protein VP2 regulated B-cell antigen presentation in a virus-specific manner. Commensal bacteria were not required for the activation of this pathway and ultimately only B cells were required for the clearance of viral infection. These findings provide new insight into the role of B cells in stimulating antiviral CD8⁺ T-cell responses.     

MHC presentation via autophagy and how viruses escape from it

T cells detect infected and transformed cells via antigen presentation by major histocompatibility complex (MHC) molecules on the cell surface. For T cell stimulation, these MHC molecules present fragments of proteins that are expressed or taken up by the cell. These fragments are generated by distinct proteolytic mechanisms for presentation on MHC class I molecules to cytotoxic CD8⁺ and on MHC class II molecules to helper CD4⁺ T cells. Proteasomes are primarily involved in MHC class I ligand and lysosomes, in MHC class II ligand generation. Autophagy delivers cytoplasmic material to lysosomes and, therefore, contributes to cytoplasmic antigen presentation by MHC class II molecules. In addition, it has been recently realized that this process also supports extracellular antigen processing for MHC class II presentation and cross-presentation on MHC class I molecules. Although the exact mechanisms for the regulation of these antigen processing pathways by autophagy are still unknown, recent studies, summarized in this review, suggest that they contribute to immune responses against infections and to maintain tolerance. Moreover, they are targeted by viruses for immune escape and could maybe be harnessed for immunotherapy.     

Intracellular processing and presentation of T cell epitopes,expressed by recombinant Escherichia coli and Salmonella typhimurium,to human T cells

Vaccines based on recombinant attenuated bacteria represent a potentially safe and effective immunization strategy. A carrier system was developed to analyze in vitro whether foreign T cell epitopes, inserted in the outer membrane protein PhoE of Escherichia coli and expressed by recombinant bacteria, are efficiently processed and presented via human leukocyte antigen (HLA) class I and II molecules by bacterial infected human macrophages. A well-defined HLA-B27-restricted cytotoxic T cell (CTL) epitope and an HLA-DR53 restricted T helper (T_h) epitope of the fusion protein of measles virus were genetically inserted in a surface-exposed region of PhoE, and the chimeric proteins were expressed in E. coli and Salmonella typhimurium. Macrophages infected with both recombinant bacteria presented the T_h epitope to the specific CD4⁺ T cell clone, but failed to present the CTL epitope to the specific CD8⁺ T cell clone. Presentation of the T_h epitope by the infected macrophages was inhibited by cytochalasin D, indicating that phagocytic processing of intact bacteria within infected macrophages was essential for antigen presentation via HLA class II. Presentation of the T_h epitope to the CD4⁺ T cell clone by infected macrophages was blocked by brefeldin A and cycloheximide, indicating the requirement of nascent HLA class II molecules for presentation. The efficiency of macrophages to process and present the inserted T_h epitope was similar for both the recombinant E. coli and S. typhimurium strains.     

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

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

Is Secretion of Tumour‐specific Antigen Important for Cancer Eradication by CD4+ T Cells? – Implications for Cancer Immunotherapy by Adoptive T Cell Transfer

The potential for cancer immunotherapy by adoptive transfer of CD4⁺ T cells is gaining increased attention. Most cancer cells lack major histocompatibility complex (MHC) class II molecules and cannot present tumour‐specific antigens (TSA) directly to CD4⁺ T cells. We have reported that tumour‐specific CD4⁺ T cells collaborate with macrophages and dendritic cells. These professional antigen‐presenting cells endocytose and process TSA to display antigenic peptides on their MHC class II molecules for indirect cancer cell recognition by CD4⁺ T cells. We hypothesized that this critical step may depend on secretion of TSA by cancer cells. This was investigated in a mouse model for myeloma immunosurveillance mediated by CD4⁺ T cells. From this study, several conclusions could be drawn. First, TSA secretion facilitates cancer immunosurveillance. Second, TSA secretion results in stronger activation of naïve tumour‐specific CD4⁺ T cells in lymph nodes. Third, TSA concentration within the tumour extracellular matrix must reach a certain threshold to allow successful cancer immunosurveillance. Fourth, treatment by local injection of purified TSA enhances immunity against cancer cells that do not secrete TSA. Fifth, secretion of TSA by at least some cancer cells within a tumour favours antitumour immunity. Therefore, we propose that CD4⁺ T cells that recognize secreted TSA may be superior for immunotherapy by T cell transfer, because the local extracellular antigen concentration will be higher for secreted TSA. Thus, it is anticipated that secreted TSA will be more readily detected in vivo by transferred CD4⁺ T cells, resulting in more efficient tumour eradication.     

LAMP-2-deficient human B cells exhibit altered MHC class II presentation of exogenous antigens

Major histocompatibility complex (MHC) class II molecules present antigenic peptides derived from engulfed exogenous proteins to CD4⁺ T cells. Exogenous antigens are processed in mature endosomes and lysosomes where acidic proteases reside and peptide‐binding to class II alleles is favoured. Hence, maintenance of the microenvironment within these organelles is probably central to efficient MHC class II‐mediated antigen presentation. Lysosome‐associated membrane proteins such as LAMP‐2 reside in mature endosomes and lysosomes, yet their role in exogenous antigen presentation pathways remains untested. In this study, human B cells lacking LAMP‐2 were examined for changes in MHC class II‐restricted antigen presentation. MHC class II presentation of exogenous antigen and peptides to CD4⁺ T cells was impaired in the LAMP‐2‐deficient B cells. Peptide‐binding to MHC class II on LAMP‐2‐deficient B cells was reduced at physiological pH compared with wild‐type cells. However, peptide‐binding and class II‐restricted antigen presentation were restored by incubation of LAMP‐2‐negative B cells at acidic pH, suggesting that efficient loading of exogenous epitopes by MHC class II molecules is dependent upon LAMP‐2 expression in B cells. Interestingly, class II presentation of an epitope derived from an endogenous transmembrane protein was detected using LAMP‐2‐deficient B cells. Consequently, LAMP‐2 may control the repertoire of peptides displayed by MHC class II molecules on B cells and influence the balance between endogenous and exogenous antigen presentation.     

The role of endosomes and lysosomes in MHC class II functioning

B cells, dendritic cells and macrophages present peptides derived from exogenous antigens to CD4⁺ T cells. After uptake, antigens are processed into peptides, bound to major histocompatibility complex (MHC) class II molecules and transported to the plasma membrane. Here, Hans Geuze reviews the current controversy surrounding the identity of the compartments that play a role in antigen processing and peptide loading.     

Synergy between activated Leishmania major-specific CD4+ T lymphocytes and bone-marrow-derived cells in the exacerbation of murine cutaneous leishmaniasis

Mechanisms of exaberbation of murine cutaneous leishmaniasis mediated by Leishmania major-specific CD4⁺ T lymphocytes were studied. Using a limiting dilution assay for the quantification of Leishmania parasites, the infected tissues (footpad) of lethally irradiated mice were found to contain tenfold less parasites at four days of infection than the footpads of infected unirradiated animals. Injection of bone marrow cells depleted of T cells into irradiated mice at the site of infection led to an increase in parasite numbers to levels equivalent to those seen in unirradiated mice. After injection of either L. major-specific CD4⁺ T cells, previously shown to exacerbate cutaneous leishmaniasis, into the infected footpad or the intravenous (i.v.) injection of bone marrow cells depleted of T cells, the numbers of parasites in lesions of irradiated mice never reached the values found in unirradiated control mice. In contrast, the concomitant transfer of CD4⁺ T-cell populations in situ and bone marrow cells depleted of T cells intravenously led to an increase in parasite loads in irradiated mice up to levels comparable to those of the unirradiated mice. This suggested that recruitment of myelomonocytic cells at the site of the lesions plays a role in the exacerbation of murine cutaneous leishmaniasis mediated by these CD4⁺ T lymphocytes. Finally, a similar effect was observed with T cells specific for an antigen unrelated to Leishmania, provided that this antigen was added to the L. major infecting inoculum.     

Cross-presentation: underlying mechanisms and role in immune surveillance

Summary: It was originally thought that a cell's major histocompatibility complex (MHC) class I molecules presented peptides derived exclusively from proteins synthesized by the cell itself. However, in some circumstances, antigens from the extracellular environment can be presented on MHC class I molecules and stimulate CD8⁺ T‐cell immunity, a process termed cross‐presentation. Cross‐presentation was originally discovered as an obscure phenomenon in transplantation immunity. However, it is now clear that it is a major mechanism by which the immune system monitors tissues and phagocytes for the presence of foreign antigen. Cross‐presentation is the only pathway by which the immune system can detect and respond to viral infections or mutations that exclusively occur in parenchymal cells rather than in bone marrow‐derived antigen‐presenting cells (APCs). Professional APCs, such as dendritic cells, are the principal cells endowed with the capacity to cross‐present antigens. In this process, the APCs acquire proteins from other tissue cells through endocytic mechanisms, especially phagocytosis or macropinocytosis. The internalized antigen can then be processed through at least two different mechanisms. In one pathway, the antigen is transferred from the phagosome into the cytosol, where it is hydrolyzed by proteasomes into oligopeptides that are then transported by the transporter associated with antigen processing to MHC class I molecules in the endoplasmic reticulum or phagosomes. In a second pathway, the antigen is cleaved into peptides by endosomal proteases, particularly cathepsin S, and bound by class I molecules probably in the endocytic compartment itself. Depending on the nature of the antigen, one or both of these pathways can contribute to cross‐presentation in vivo. The outcome of cross‐presentation can be either tolerance or immunity. Which of these outcomes occurs is thought to depend on whether antigens are acquired by themselves alone, leading to tolerance, or with immunostimulatory signals, leading to immunity. One source of such signals is from dying cells that release immunostimulatory ‘danger’ signals that promote the generation of immunity to their cellular antigens. In addition to the critical role of cross‐presentation in normal immune physiology, this pathway has considerable potential for being exploited for developing subunit vaccines that elicit both CD4⁺ and CD8⁺ T‐cell immunity.