Cytotoxic T lymphocytes (CTLs) which carry the CD8 antigen recognize antigens that are presented on target cells by the class I major histocompatibility complex. CTLs are responsible for the killing of antigen-bearing target cells, such as virus-infected cells. Although CTL effectors can act alone when killing target cells, their differentiation from naive CD8-positive T cells is often dependent on 'help' from CD4-positive helper T (TH) cells. Furthermore, for effective CTL priming, this help must be provided in a cognate manner, such that both the TH cell and the CTL recognize antigen on the same antigen-presenting cell. One explanation for this requirement is that TH cells are needed to convert the antigen-presenting cell into a cell that is fully competent to prime CTL. Here we show that signalling through CD40 on the antigen-presenting cells can replace the requirement for TH cells, indicating that T-cell 'help', at least for generation of CTLs by cross-priming, is mediated by signalling through CD40 on the antigen-presenting cell.

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 β 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.

SummaryOvalbumin (OVA)-specific CD8 + T cells from the T cell receptor-transgenic line OT-I (OT-I cells) were injected into unirradiated transgenic 1KIP-mOVA mice, which express a membranebound form of OVA (mOVA) in the pancreatic islet [3 cells and the renal proximal tubular cells. OT-I cells accumulated in the draining lymph nodes (LN) of the kidneys and pancreas and in no other LN. They displayed an activated phenotype and a proportion entered cell cycle. Unilateral nephrectomy 7-13 d before inoculation of OT-I ceils into 1KIP-mOVA mice allowed the injected T cells to home only to the regional LN of the remaining kidney (and pancreas), but when the operation was performed 4 h before injecting the T ceils, homing to the LN of the excised kidney was evident. When the bone marrow of RIP-mOVA mice was replaced with one of a major histocompatibility haplotype incapable of presenting OVA to OT-I cells, no homing or activation was detectable. Therefore, OT-I cells were activated by OVA presented by short-lived antigen-presenting cells of bone marrow origin present in the draining LN of OVA-expressing tissue. These results provide the first evidence that tissue-associated "self" antigens can be presented in the context of class I via an exogenous processing pathway. This offers a constitutive mechanism whereby T cells can be primed to antigens that are present in nonlymphoid tissues, which are not normally surveyed by recirculating 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.

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We previously described a mechanism for the maintenance of peripheral self-tolerance. This involves the cross-presentation of tissue-associated antigens by a bone marrow–derived cell type that stimulates the proliferation and ultimate deletion of self-reactive CD8 T cells. This process has been referred to as cross-tolerance. Here, we characterize the elusive cell type responsible for inducing cross-tolerance as a CD8α+ dendritic cell (DC). To achieve this aim, transgenic mice were generated expressing yellow fluorescent protein (YFP) linked to CTL epitopes for ovalbumin and glycoprotein B (gB) of herpes simplex virus under the rat insulin promoter (RIP). Although tracking of YFP was inconclusive, the use of a highly sensitive gB-specific hybridoma that produced β-galactosidase on encounter with antigen, enabled detection of antigen presentation by cells isolated from the pancreatic lymph node. This showed that a CD11c+CD8α+ cell was responsible for cross-tolerance, the same DC subset as previously implicated in cross-priming. These data indicate that CD8α+ DCs play a critical role in both tolerance and immunity to cell-associated antigens, providing a potential mechanism by which cytotoxic T lymphocyte can be immunized to viral antigens while maintaining tolerance to self.

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.

Self-antigens expressed in extrathymic tissues such as the pancreas can be transported to draining lymph nodes and presented in a class I–restricted manner by bone marrow-derived antigen-presenting cells. Such cross-presentation of self-antigens leads to CD8+ T cell tolerance induction via deletion. In this report, we investigate the influence of CD4+ T cell help on this process. Small numbers of autoreactive OVA-specific CD8+ T cells were unable to cause diabetes when adoptively transferred into mice expressing ovalbumin in the pancreatic β cells. Coinjection of OVA-specific CD4+ helper T cells, however, led to diabetes in a large proportion of mice (68%), suggesting that provision of help favored induction of autoimmunity. Analysis of the fate of CD8+ T cells indicated that CD4+ T cell help impaired their deletion. These data indicate that control of such help is critical for the maintenance of CD8+ T cell tolerance induced by cross-presentation.