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Antibodies are hallmarks of most effective vaccines. For successful T-dependent antibody responses, conventional dendritic cells (cDC) have been largely attributed the role of priming T cells. By contrast, follicular dendritic cells and macrophages have been seen as responsible for B cell activation, due to their strategic location within secondary lymphoid tissues and capacity to present native antigen to B cells. This review summarizes the mounting evidence that cDC can also present native antigen to B cells. cDC2 have been the main subset linked to humoral responses, based largely on their favorable location, capacity to prime CD4 + T cells, and ability to present native antigen to B cells. However, studies using strategies to deliver antigen to receptors on cDC1, reveal this subset can also contribute to naïve B cell activation, as well as T cell priming. cDC1 location within lymphoid tissues reveals their juxtaposition to B cell follicles, with ready access to B cells for presentation of native antigen. These findings support the view that both cDC1 and cDC2 are capable of initiating humoral responses provided antigen is captured by relevant surface receptors attuned to this process. Such understanding is fundamental for the development of innovative humoral vaccination approaches., (© 2021 Wiley-VCH GmbH.)

B cells are efficiently activated by antigens presented on cell membranes, which provide opportunity for receptor cross-linking and antigen capture. The two main cell types implicated in native antigen presentation to B cells are follicular dendritic cells (FDC), which reside in B cell follicles, and CD169 + macrophages, which line the antigen-exposed surfaces of these follicles in both the lymph nodes and the spleen. There is mounting evidence, however, that conventional dendritic cells (cDC) can also participate in native antigen presentation to B cells. This underappreciated role, largely hidden by the simultaneous need for cDC to participate in T cells priming, appears to be primarily mediated by the type 2 subset of cDC (cDC2), but may also be a function of cDC1. Better understanding of how cDC participate in B cell priming is likely to improve our capacity to develop effective humoral vaccines., (Crown Copyright © 2019. Published by Elsevier Ltd. All rights reserved.)

CD4 + T cell responses are crucial for the control of many intracellular pathogens, yet the requirements for their induction are not fully understood. To better understand the role that various dendritic cell (DC) subtypes play in CD4 + T cell priming, we compared in vivo T cell responses to skin inoculation of mice with infectious or UV-inactivated HSV type 1. Localized infection elicited a Th1 response that was primed in skin-draining lymph nodes involving Ag presentation by migratory dermal and lymph node-resident DC. However, expansion and Th1 differentiation was impaired in response to UV-inactivated virus (UV-HSV), and this defect correlated with a restriction of Ag presentation to migratory CD103 - dermal DC. A similar differentiation defect was seen in infected mice lacking CD8α + and CD103 + classical type 1 DC (cDC1). Finally, Th1 differentiation after UV-HSV inoculation was rescued by targeted Ag delivery to CD8α + and CD103 + cDC1 using an anti-Clec9A Ab construct. This suggests that Ag presentation by cDC1 is crucial for optimal Th1 immunity to HSV type 1 infection and potentially other pathogens of the skin., (Copyright © 2019 by The American Association of Immunologists, Inc.)

We describe an MHC class II (I-A b )-restricted TCR transgenic mouse line that produces CD4 + T cells specific for Plasmodium species. This line, termed PbT-II, was derived from a CD4 + T cell hybridoma generated to blood-stage Plasmodium berghei ANKA (PbA). PbT-II cells responded to all Plasmodium species and stages tested so far, including rodent (PbA, P. berghei NK65, Plasmodium chabaudi AS, and Plasmodium yoelii 17XNL) and human ( Plasmodium falciparum ) blood-stage parasites as well as irradiated PbA sporozoites. PbT-II cells can provide help for generation of Ab to P. chabaudi infection and can control this otherwise lethal infection in CD40L-deficient mice. PbT-II cells can also provide help for development of CD8 + T cell-mediated experimental cerebral malaria (ECM) during PbA infection. Using PbT-II CD4 + T cells and the previously described PbT-I CD8 + T cells, we determined the dendritic cell (DC) subsets responsible for immunity to PbA blood-stage infection. CD8 + DC (a subset of XCR1 + DC) were the major APC responsible for activation of both T cell subsets, although other DC also contributed to CD4 + T cell responses. Depletion of CD8 + DC at the beginning of infection prevented ECM development and impaired both Th1 and follicular Th cell responses; in contrast, late depletion did not affect ECM. This study describes a novel and versatile tool for examining CD4 + T cell immunity during malaria and provides evidence that CD4 + T cell help, acting via CD40L signaling, can promote immunity or pathology to blood-stage malaria largely through Ag presentation by CD8 + DC., (Copyright © 2017 by The American Association of Immunologists, Inc.)

To investigate the role of Aire in thymic selection, we examined the cellular requirements for generation of ovalbumin (OVA)-specific CD4 and CD8 T cells in mice expressing OVA under the control of the rat insulin promoter. Aire deficiency reduced the number of mature single-positive OVA-specific CD4(+) or CD8(+) T cells in the thymus, independent of OVA expression. Importantly, it also contributed in 2 ways to OVA-dependent negative selection depending on the T-cell type. Aire-dependent negative selection of OVA-specific CD8 T cells correlated with Aire-regulated expression of OVA. By contrast, for OVA-specific CD4 T cells, Aire affected tolerance induction by a mechanism that operated independent of the level of OVA expression, controlling access of antigen presenting cells to medullary thymic epithelial cell (mTEC)-expressed OVA. This study supports the view that one mechanism by which Aire controls thymic negative selection is by regulating the indirect presentation of mTEC-derived antigens by thymic dendritic cells. It also indicates that mTECs can mediate tolerance by direct presentation of Aire-regulated antigens to both CD4 and CD8 T cells.

Three surface molecules of mouse CD8(+) dendritic cells (DCs), also found on the equivalent human DC subpopulation, were compared as targets for Ab-mediated delivery of Ags, a developing strategy for vaccination. For the production of cytotoxic T cells, DEC-205 and Clec9A, but not Clec12A, were effective targets, although only in the presence of adjuvants. For Ab production, however, Clec9A excelled as a target, even in the absence of adjuvant. Potent humoral immunity was a result of the highly specific expression of Clec9A on DCs, which allowed longer residence of targeting Abs in the bloodstream, prolonged DC Ag presentation, and extended CD4 T cell proliferation, all of which drove highly efficient development of follicular helper T cells. Because Clec9A shows a similar expression pattern on human DCs, it has particular promise as a target for vaccines of human application.

Despite extensive evidence that Plasmodium species are capable of stimulating the immune system, the association of malaria with a higher incidence of other infectious diseases and reduced responses to vaccination against unrelated pathogens suggests the existence of immune suppression. Recently, we provided evidence that blood-stage Plasmodium berghei infection leads to suppression of MHC class I-restricted immunity to third party (non-malarial) antigens as a consequence of systemic DC activation. This earlier study did not, however, determine whether reactivity was also impaired to MHC class II-restricted third party antigens or to Plasmodium antigens themselves. Here, we show that while P. berghei-expressed antigens were presented early in infection, there was a rapid decline in presentation within 4 days, paralleling impairment in MHC class I- and II-restricted presentation of third party antigens. This provides important evidence that P. berghei not only causes immunosuppression to subsequently encountered third party antigens, but also rapidly limits the capacity to generate effective parasite-specific immunity.

The importance of conventional dendritic cells (cDCs) in the processing and presentation of antigen is well established, but the contribution of plasmacytoid dendritic cells (pDCs) to these processes, and hence to T cell immunity, remains unclear. Here we showed that unlike cDCs, pDCs continued to synthesize major histocompatibility complex (MHC) class II molecules and the MHC class II ubiquitin ligase MARCH1 long after activation. Sustained MHC class II-peptide complex formation, ubiquitination and turnover rendered pDCs inefficient in the presentation of exogenous antigens but enabled pDCs to continuously present endogenous viral antigens in their activated state. As the antigen-presenting abilities of cDCs and pDCs are fundamentally distinct, these two cell types may activate largely nonoverlapping repertoires of CD4(+) T cells.

Dendritic cells (DC) are a heterogeneous cell population that bridge the innate and adaptive immune systems. CD8alpha DC play a prominent, and sometimes exclusive, role in driving amplification of CD8(+) T cells during a viral infection. Whether this reliance on a single subset of DC also applies for CD4(+) T cell activation is unknown. We used a direct ex vivo antigen presentation assay to probe the capacity of flow cytometrically purified DC populations to drive amplification of CD4(+) and CD8(+) T cells following infection with influenza virus by different routes. This study examined the contributions of non-CD8alpha DC populations in the amplification of CD8(+) and CD4(+) T cells in cutaneous and systemic influenza viral infections. We confirmed that in vivo, effective immune responses for CD8(+) T cells are dominated by presentation of antigen by CD8alpha DC but can involve non-CD8alpha DC. In contrast, CD4(+) T cell responses relied more heavily on the contributions of dermal DC migrating from peripheral lymphoid tissues following cutaneous infection, and CD4 DC in the spleen after systemic infection. CD4(+) T cell priming by DC subsets that is dependent upon the route of administration raises the possibility that vaccination approaches could be tailored to prime helper T cell immunity.

The entry of exogenous antigen into the MHC class I-restricted cross-presentation pathway can contribute to CD8(+) T cell tolerance and immunity, in particular to peripheral self-antigens or selected viruses and bacteria showing restricted tissue tropism. Dendritic cells are the key cross-presenting cells and, as such, they are thought to carry specialized machinery dedicated to this purpose. Two recent papers describe intracellular components tailored to the dendritic cell cross-presentation pathway.

The phenotypic and functional studies carried out in recent years on dendritic cells (DC) purified from spleen and lymph nodes has revealed the existence of heterogeneous populations with distinct life cycles, migratory properties and antigen presenting functions. A major subdivision can be made between "tissue derived" DC that migrate to the lymph nodes from peripheral tissues, both in the steady state and in the course of infections, and "blood-derived" DC, which reside in the spleen and lymph nodes throughout their life cycle. These two groups of DC can be subdivided into smaller subsets. The tissue-derived and the blood-derived DC also show fundamental differences in maturational status and antigen presenting capabilities. In this review, we summarize the roles played by the different DC types in the steady state and during pathogen infections, relating those roles to maintenance of peripheral tolerance and the induction of immunity. We point out the caveats of assuming that the DC that collect antigens are the ones involved in their presentation, emphasizing the phenomenon of antigen transfer as an important component of the immune response.

CD8alpha(+) dendritic cells (DCs) have been shown to be the principal DC subset involved in priming MHC class I-restricted CTL immunity to a variety of cytolytic viruses, including HSV type 1, influenza, and vaccinia virus. Whether priming of CTLs by CD8alpha(+) DCs is limited to cytolytic viruses, which may provide dead cellular material for this DC subset, or whether these DCs selectively present intracellular Ags, is unknown. To address this question, we examined Ag presentation to a noncytolytic virus, lymphocytic choriomeningitis virus, and to an intracellular bacterium, Listeria monocytogenes. We show that regardless of the type of intracellular infection, CD8alpha(+) DCs are the principal DC subset that initiate CD8(+) T cell immunity.

It has been reported that MHC class I-restricted Ag presentation persists for only a short period following infection with certain pathogens, declining in parallel with the emergence of specific CTL activity. We have examined this issue in the case of murine infection with HSV-1. We found that the period of Ag presentation capable of priming naive CD8(+) T cells is comparatively prolonged, persisting for at least 7 days after infection, and continuing despite the appearance of localized CTL activity. Ag presentation was abbreviated to 3 or 4 days postinfection by surgical excision of the inoculation site early after infection. This intervention attenuated the size of the primary CTL response, implying that prolonged presentation is necessary to drive maximal CTL expansion. Combined, these data show that, in some types of infection, CTL priming can extend well beyond the first 24-48 h after primary inoculation.

During lung infection with virus, airway-derived dendritic cells (DC) have been thought to be the dominant cell type involved in acquisition, transport, and direct antigen presentation for cytotoxic T lymphocyte priming. Contrary to this view, we have found that both an airway-derived CD8alpha(-)CD11b(-) DC subset and distinct CD8alpha(+) lymph node resident DC can present class I-restricted antigens after lung infection with influenza virus or herpes simplex virus 1. Presentation by a nonairway-derived DC population argues that cytotoxic T lymphocyte priming may involve interplay between different DC subsets, not all of which originate within the site of infection.

Cross-presentation of cell-bound Ags from established, solid tumors to CD8 cells is efficient and likely to have a role in determining host response to tumor. A number of investigators have predicted that when tumor Ags are derived from apoptotic cells either no response, due to Ag "sequestration," or CD8 cross-tolerance would ensue. Because the crucial issue of whether this happens in vivo has never been addressed, we induced apoptosis of established hemagglutinin (HA)-transfected AB1 tumors in BALB/c mice using the apoptosis-inducing reagent gemcitabine. This shrank the tumor by approximately 80%. This induction of apoptosis increased cross-presentation of HA to CD8 cells yet neither gross deletion nor functional tolerance of HA-specific CD8 cells were observed, based on tetramer analysis, proliferation of specific CD8 T cells, and in vivo CTL activity. Interestingly, apoptosis primed the host for a strong antitumor response to a second, virus-generated HA-specific signal in that administration of an HA-expressing virus after gemcitabine administration markedly decreased tumor growth compared with viral administration without gemcitabine. Thus tumor cell apoptosis in vivo neither sequesters tumor Ags nor cross-tolerizes tumor-specific CD8 cells. This observation has fundamental consequences for the development of tumor immunotherapy protocols and for understanding T cell reactivity to tumors and the in vivo immune responses to apoptotic cells.

The immune response to cutaneous herpes simplex virus type 1 (HSV-1) infection begins with remarkable rapidity. Activation of specific cytotoxic T lymphocytes (CTL) begins within hours of infection, even though the response within the draining lymph nodes peaks nearly 5 days later. HSV gene products are classified into three main groups, alpha, beta, and gamma, based on their kinetics and requirements for expression. In C57BL/6 mice, the immunodominant epitope from HSV is derived from glycoprotein B (gB(498-505)). While gB is considered a gamma or "late" gene product, previous reports have indicated that some level of gene expression may occur soon after infection. Using brefeldin A as a specific inhibitor of viral antigen presentation to major histocompatibility complex class I-restricted CTL, we have formally addressed the timing of gB peptide expression in an immunologically relevant manner following infection. Presentation of gB peptide detected by T-cell activation was first observed within 2 h of infection. Comparison with another viral epitope expressed early during infection, HSV-1 ribonucleotide reductase, demonstrated that gB is presented with the same kinetics as this classical early-gene product. Moreover, this rapidity of gB expression was further illustrated via rapid priming of naïve transgenic CD8(+) T cells in vivo after HSV-1 infection of mice. These results establish that gB is expressed rapidly following HSV-1 infection, at levels capable of effectively stimulating CD8(+) T cells.

Dendritic cells (DC) are considered nature's adjuvants. They are potent stimulators of naive T cells and key inducers of primary immune responses. In recent times it has become clear that they can also play a central role in the development of T cell tolerance. Further complicating our understanding of DC function is the realization that DC can no longer be viewed as a homogeneous cell type. Rather, they exist as a complex mixture of strikingly different cell populations. The mechanisms that drive the conflicting immunological outcomes of tolerance and immunity have been the subject of intense scrutiny in recent years, most recently in terms of how the various DC subsets are involved in these events. Here we review recent experiments that provide insights into how DC subsets control the outcome of T cell activation and in so doing select between immunity and tolerance induction.

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.

Peripheral antigens can be captured by APC and cross-presented to naive CD8(+) T cells. Notably, cross-presentation of pancreatic antigen is not seen in neonatal mice, although presentation of antigen expressed by the kidney is still intact. In this report, we examined why pancreatic antigens are not cross-presented in neonatal mice. First, we established that antigen expression was not limiting, as neonatal islets expressed as much antigen per cell as adult islets, and vastly more than neonatal renal cells. Next, we analyzed the APC subsets present in the lymph node draining the neonatal pancreas. No obvious population was absent. Finally, we examined whether cross-presentation occurred during inflammation. This showed that inflammation caused by CTL attack of islet tissue facilitated cross-presentation of antigens in neonatal mice. These data indicate that constitutive cross-presentation of islet antigens is inactive during neonatal life, but that under inflammatory conditions this antigen presentation pathway becomes available.

Localized cutaneous herpes simplex virus type 1 (HSV-1) infection leads to arming and initial expansion of cytotoxic T lymphocytes (CTLs) in the draining popliteal lymph nodes (PLNs) followed by migration and further proliferation in the spleen. To accurately characterize the sequence of events involved in the activation and generation of anti-HSV CTLs, we used T cell receptor (TCR) transgenic mice specific for the immunodominant epitope from HSV glycoprotein B (gB(498-505)). We describe the detection of the initiation of antigen presentation in the draining lymph nodes by 4-6 h after infection with HSV-1. Analysis of CD69 up-regulation revealed activation of gB-specific CD8(+) T cells by 6-8 h after infection. Furthermore, we show that T cell proliferation begins no sooner than 24 h after activation and is marked by the concurrent appearance of CTL activity in the PLNs. These events are not dependent on the presence of virus in the draining lymph nodes, and suggest a requirement for recruitment of professional antigen-presenting cells to the site of T cell activation. Consequently, we have defined the initiation of the CD8(+) T cell-mediated response to cutaneous HSV-1 infection, demonstrating that the immune response to localized viral infection depends only on the appearance of cells presenting virus-derived antigen and commences with remarkable swiftness.

T lymphocytes recognize antigen presented on the surface of antigen-presenting cells byMHC class I and class II molecules. Classically, MHC class I molecules present self- or pathogen-derived antigens that are synthesized within the cell, whereas exogenous antigens derived via endocytic uptake are loaded onto MHC class II molecules for presentation to CD4+ T cells. It is becoming increasingly clear that some dendritic cells are also specialized to process exogenous antigens into the MHC class I pathway for presentation to CD8+ T cells. This process is known as cross-presentation. It provides a mechanism that can drive dendritic cells to generate either tolerance to self-antigens or immunity to pathogens. The cells responsible for, and mechanisms underlying, this decision between tolerance and immunity via cross-presentation has become the focus of intense study to determine how various dendritic cell subsets effect the different outcomes.

T lymphocytes recognize peptide antigens presented by class I and class II molecules encoded by the major histocompatibility complex (MHC). Classical antigen-presentation studies showed that MHC class I molecules present peptides derived from proteins synthesized within the cell, whereas MHC class II molecules present exogenous proteins captured from the environment. Emerging evidence indicates, however, that dendritic cells have a specialized capacity to process exogenous antigens into the MHC class I pathway. This function, known as cross-presentation, provides the immune system with an important mechanism for generating immunity to viruses and tolerance to self.

To better understand the antigenic requirements for cross-presentation, we compared the in vivo efficiency of presentation of cell-associated vs soluble OVA with the OT-I (CD8) and OT-II (CD4) TCR transgenic lines. Cross-presentation of cell-associated OVA was very efficient, requiring as little as 21 ng of OVA to activate OT-II cells and 100-fold less to activate OT-I cells. In contrast, soluble OVA was presented inefficiently, requiring at least 10,000 ng OVA for activation of either T cell subset. Thus, cell-associated OVA was presented 500-fold more efficiently than soluble OVA to CD4 T cells and 50,000-fold more efficiently to CD8 T cells. These data, which represent the first quantitative in vivo analysis of cross-presentation, show that cell-associated OVA is very efficiently presented via the class I pathway.

Mouse spleen contains three distinct mature dendritic cell (DC) populations (CD4(+)8(-), CD4(-)8(-), and CD4(-)8(+)) which retain a capacity to take up particulate and soluble AGS: Although the three splenic DC subtypes showed similar uptake of injected soluble OVA, they differed markedly in their capacity to present this Ag and activate proliferation in OVA-specific CD4 or CD8 T cells. For class II MHC-restricted presentation to CD4 T cells, the CD8(-) DC subtypes were more efficient, but for class I MHC-restricted presentation to CD8 T cells, the CD8(+) DC subtype was far more effective. This differential persisted when the DC were activated with LPS. The CD8(+) DC are therefore specialized for in vivo cross-presentation of exogenous soluble Ags into the class I MHC presentation pathway.

This review examines the role of cross-presentation in tolerance and immunity. We discuss (a) the antigenic requirements for cross-presentation, (b) the phenotype of the antigen presenting cell (APC), (c) the cellular interactions and molecular signals involved in cross-priming, and (d) the factors that direct the immune system toward tolerance or immunity. A large part of this review is dedicated to summarizing our current knowledge of the cross-presenting APC.

This report examines the use of 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) to determine the site, duration and cell type responsible for antigen presentation in vivo. Evidence that CFSE-labelled T cells can be used to determine where various types of antigens are presented, including auto-antigens, oral antigens and cell-associated foreign antigens, is provided. Using this technique, the length of time antigen is presented after acquisition by APC was measured. Finally, CFSE labelling was used to identify the origin of the APC responsible for different forms of antigen presentation.

This report investigates the response of CD8(+) T cells to antigens presented by B cells. When C57BL/6 mice were injected with syngeneic B cells coated with the Kb-restricted ovalbumin (OVA) determinant OVA257-264, OVA-specific cytotoxic T lymphocyte (CTL) tolerance was observed. To investigate the mechanism of tolerance induction, in vitro-activated CD8(+) T cells from the Kb-restricted, OVA-specific T cell receptor transgenic line OT-I (OT-I cells) were cultured for 15 h with antigen-bearing B cells, and their survival was determined. Antigen recognition led to the killing of the B cells and, surprisingly, to the death of a large proportion of the OT-I CTLs. T cell death involved Fas (CD95), since OT-I cells deficient in CD95 molecules showed preferential survival after recognition of antigen on B cells. To investigate the tolerance mechanism in vivo, naive OT-I T cells were adoptively transferred into normal mice, and these mice were coinjected with antigen-bearing B cells. In this case, OT-I cells proliferated transiently and were then lost from the secondary lymphoid compartment. These data provide the first demonstration that B cells can directly tolerize CD8(+) T cells, and suggest that this occurs via CD95-mediated, activation-induced deletion.

There is now convincing evidence that CD8+ T cells can be activated by professional antigen-presenting cells which present antigens derived from non-lymphoid tissues in association with MHC class I molecules in the draining lymph nodes. This mechanism, referred to as cross-presentation, enables the immune system to respond to those microorganisms that infect only non-lymphoid tissues. Consistent with this view, cross-presentation was found to focus on antigens expressed in high concentrations and those released from dying cells, which can be expected to result from viral infections. Recent evidence, however, demonstrates that high dose self antigens can be cross-presented constitutively, resulting in the activation of autoreactive CD8+ T cells. This does not lead to auto immunity under physiologic conditions, but to CD95-mediated deletion of the T cells. Cross-presentation can thus engage a well-defined pathway of antigen-induced T-cell death and purge the immune system of autoreactive CD8+ T cells. Low dose self antigens are not cross-presented and are consequently ignored. The immune system therefore uses two strategies to avoid CD8+ T-cell-mediated autoimmunity in the periphery: deletion of autoreactive CD8+ T cells responding to high dose self antigens and ignorance of self antigens expressed at low concentrations.

Recently, we demonstrated that major histocompatibility complex class I-restricted cross-presentation of exogenous self-antigens can induce peripheral T cell tolerance by deletion of autoreactive CD8+ T cells. In these studies, naive ovalbumin (OVA)-specific CD8+ T cells from the transgenic line OT-I were injected into transgenic mice expressing membrane-bound OVA (mOVA) under the control of the rat insulin promoter (RIP) in pancreatic islets, kidney proximal tubules, and the thymus. Cross-presentation of tissue-derived OVA in the renal and pancreatic lymph nodes resulted in activation, proliferation, and then the deletion of OT-I cells. In this report, we investigated the molecular mechanisms underlying this form of T cell deletion. OT-I mice were crossed to tumor necrosis factor receptor 2 (TNFR2) knockout mice and to CD95 (Fas, Apo-1) deficient mutant lpr mice. Wild-type and TNFR2-deficient OT-I cells were activated and then deleted when transferred into RIP-mOVA mice, whereas CD95-deficient OT-I cells were not susceptible to deletion by cross-presentation. Furthermore, cross-presentation led to upregulation of the CD95 molecule on the surface of wild-type OT-I cells in vivo, consistent with the idea that this is linked to rendering autoreactive T cells susceptible to CD95-mediated signaling. This study represents the first evidence that CD95 is involved in the deletion of autoreactive CD8+ T cells in the whole animal.

Naive T cells recirculate mainly within the secondary lymphoid compartment, but once activated they can enter peripheral tissues and perform effector functions. To activate naive T cells, foreign antigens must traffic from the site of infection to the draining lymph nodes, where they can be presented by professional antigen presenting cells. For major histocompatibility complex class I-restricted presentation to CD8+ T cells, this can occur via the cross-presentation pathway. Here, we investigated the conditions allowing antigen access to this pathway. We show that the level of antigen expressed by peripheral tissues must be relatively high to facilitate cross-presentation to naive CD8+ T cells. Below this level, peripheral antigens did not stimulate by cross-presentation and were ignored by naive CD8+ T cells, although they could sensitize tissue cells for destruction by activated cytotoxic T lymphocytes (CTLs). Interestingly, CTL-mediated tissue destruction facilitated cross-presentation of low dose antigens for activation of naive CD8+ T cells. This represents the first in vivo evidence that cellular destruction can enhance access of exogenous antigens to the cross-presentation pathway. These data indicate that the cross-presentation pathway focuses on high dose antigens and those released during tissue destruction.

Upon encounter with foreign antigen, tissue-associated antigen-presenting cells (APCs) migrate to draining lymph nodes to prime specific T cells. Using the transgenic RIP-mOVA model, we recently demonstrated that self antigens derived from peripheral tissues are constitutively transported to draining lymph nodes, and can be presented in association with MHC class I molecules by a bone marrow-derived APC population. This form of class I-restricted presentation of exogenous antigen has been referred to as cross-presentation and can induce activation and proliferation of antigen-specific CD8+ T cells. In the absence of CD4+ T cell help, activation of CD8+ T cells is inefficient, and cross-presentation leads to peripheral deletion of autoreactive CD8+ T cells, acting as a mechanism to maintain self-tolerance. If CD4+ T cell help is available, CD8+ T cell responses to self antigens can be rendered immunogenic, leading to autoreactive responses. Whether autoimmunity results from such responses also depends on the tissue location of the antigen. In RIP-mOVA mice, which express the model antigen mOVA (a membrane-bound form of ovalbumin) in the pancreatic beta cells and kidney proximal tubules, OVA-specific CD8+ T cells, activated by cross-presentation, infiltrated the pancreas and caused B cell destruction. Interestingly, however, these cells did not infiltrate the kidney, suggesting that proximal tubular cells are to some extent protected from immune destruction. Analysis of the role of antigen concentration indicates that high doses were required for efficient cross-presentation, suggesting that this pathway is directed towards immune responses to high-dose antigens, such as may be present during viral infection.

In this report, we show that cross-presentation of self-antigens can lead to the peripheral deletion of autoreactive CD8(+) T cells. We had previously shown that transfer of ovalbumin (OVA)-specific CD8(+) T cells (OT-I cells) into rat insulin promoter-membrane-bound form of OVA transgenic mice, which express the model autoantigen OVA in the proximal tubular cells of the kidneys, the beta cells of the pancreas, the thymus, and the testis of male mice, led to the activation of OT-I cells in the draining lymph nodes. This was due to class I-restricted cross-presentation of exogenous OVA on a bone marrow-derived antigen presenting cell (APC) population. Here, we show that adoptively transferred or thymically derived OT-I cells activated by cross-presentation are deleted from the peripheral pool of recirculating lymphocytes. Such deletion only required antigen recognition on a bone marrow-derived population, suggesting that cells of the professional APC class may be tolerogenic under these circumstances. Our results provide a mechanism by which the immune system can induce CD8(+) T cell tolerance to autoantigens that are expressed outside the recirculation pathway of naive T cells.

Class I-restricted presentation is usually associated with cytoplasmic degradation of cellular proteins and is often considered inaccessible to exogenous antigens. Nonetheless, certain exogenous elements can gain entry into this so-called endogenous pathway by a mechanism termed cross-presentation. This is known to be effective for class I-restricted cytotoxic T lymphocyte (CTL) cross-priming directed against a variety of exogenous tumor, viral, and minor transplantation antigens. The related effect of cross-tolerance can also effectively eliminate responses to selected self components. In both cases, this presentation appears to require the active involvement of a bone marrow-derived antigen presenting cell (APC). Here, we show that CTL induction by cross-priming with cell-associated ovalbumin requires the active involvement of CD4+ helper T cells. Importantly, this CD4+ population is only effective when both the helper and CTL determinants are recognized on the same APC. Moreover, we would argue that the cognitive nature of this event suggests that the CD4+ T cell actively modifies the APC, converting it into an effective stimulator for the successful priming of the CTL precursor.