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.

There are two major mechanisms reported to prevent the autoreactivity of islet-specific CD8 ؉ T cells: ignorance and tolerance. When ignorance is operative, naïve autoreactive CD8 ؉ T cells ignore islet antigens and recirculate without causing damage, unless activated by an external stimulus. In the case of tolerance, CD8 ؉ T cells are deleted. Which factor(s) contributes to each particular outcome was previously unknown. Here, we demonstrate that the concentration of self antigen determines which mechanism operates. When ovalbumin (OVA) was expressed at a relatively low concentration in the pancreatic islets of transgenic mice, there was no detectable cross-presentation, and the CD8 ؉ T cell compartment remained ignorant of OVA. In mice expressing higher doses of OVA, cross-presentation was detectable and led to peripheral deletion of OVA-specific CD8 ؉ T cells. When crosspresentation was prevented by reconstituting the bone marrow compartment with cells incapable of presenting OVA, deletional tolerance was converted to ignorance. Thus, the immune system uses two strategies to avoid CD8 ؉ T cell-mediated autoimmunity: for high dose antigens, it deletes autoreactive T cells, whereas for lower dose antigens, it relies on ignorance.antigen presentation ͉ transgenic mice ͉ ovalbumin ͉ apoptosis ͉ autoimmunity

An antigen administered orally can induce immunological tolerance to a subsequent challenge with the same antigen. Evidence has been provided for the efficacy of this approach in the treatment of human autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. However, oral administration of autoantigen in mice was found to induce a cytotoxic T lymphocyte response that could lead to the onset of autoimmune diabetes. Thus, feeding autoantigen can cause autoimmunity, which suggests that caution should be used when applying this approach to the treatment of human autoimmune diseases.

We have previously reported that feeding OVA to C57BL/6 mice can lead to a weak CTL response that is dependent on CD4+ T cell help and is capable of causing autoimmunity. In this study, we investigated the basis of the class I and class II-restricted Ag presentation required for such CTL induction. Two days after feeding OVA, Ag-specific CD4+ and CD8+ T cells were seen to proliferate in the Peyer’s patches and mesenteric lymph nodes. Little proliferation was evident in other lymphoid tissues, except at high Ags doses, in which case some dividing CD4+ T cells were observed in the spleen and peripheral lymph nodes. Using chimeric mice, the APC responsible for presenting orally derived Ags was shown to be derived from the bone marrow. Examination of the Ag dose required to activate either CD4+ or CD8+ T cells indicated that a single dose of 6 mg OVA was the minimum dose that consistently stimulated either T cell subset. These data indicate that oral Ags can be transported from the gut into the gut-associated lymphoid tissue, where they are captured by a bone marrow-derived APC and presented to both CD4+ and CD8+ T cells.

IntroductionSeveral years ago, we began studies to elucidate the nature of tolerance towards auto-antigens expressed in the pancreas. Early studies showed that peripheral self antigens were ignored by CD8 T cells 1,2 and we have published a report consistent with this view. 3 In our case, we had used the histocompatibility antigen H-2K b (K b ) as the model autoantigen. This antigen is recognised in its native state, but not after processing by APC. Thus, our studies did not take into account the effect of auto-antigen presentation on cells such as dendritic cells. To address this issue, we embarked on a series of new studies using ovalbumin (OVA) as a model self antigen. This approach was inspired by the ever-increasing information on the immune response to OVA and particularly by the then recent elucidation of class I-restricted epitopes of this abundant soluble protein. 4 We set about generating mice expressing OVA in their pancreatic β cells, under the control of the rat insulin promoter (RIP). These were called RIPmOVA mice, with the 'mOVA' referring to membrane-bound OVA. 5 We also developed class I-and class II-restricted T cell receptor transgenic mice (OT-I and OT-II mice, respectively) to use as tools for detecting antigen presentation and its consequences. 6,7 In our first paper examining the site of OVA presentation in RIP-mOVA mice, we used three colour flow cytometry to reveal an increase in the proportion of autoantigen-specific transgenic T cells in those lymph nodes draining OVA-expressing tissues. We also used the rather difficult technique of bromodeoxyuridine (BrdU) incorporation to show that a large proportion of these T cells were proliferating. While such studies yielded some very interesting information, they were rather crude, required complex flow cytometry and divulged only a limited picture of the process of auto-antigen presentation. After completing the first set of experiments in this model, we became aware of a study by Hodgkin, Lee and Lyons, 8 which used a technique for measuring cell proliferation developed by Weston, Lyons and Parish. 9,10 This technique involved labelling of T cells with the fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) and enabled us to track cells in vivo for their homing and subsequent proliferation in response to antigen. While our use of CFSE began some years after its initial description, 9,10 we were fortunate to still be relatively early among the first wave of investigators to use this labelling technique. 11 At the time, two points were clear: first, that CFSE provided a great advance over alternative techniques for the visualization of T cell activation and proliferation in vivo and second, that there would be numerous applications for this technique. In this review, we would like to describe a variety of experiments to illustrate how we have used CFSE to understand various aspects of in vivo antigen presentation. Site of antigen presentationWe have used CFSE for identifying the anatomical site of presentation of various f...

Oral administration of antigen is known to induce a state of specific immunological unresponsiveness to a subsequent challenge with the same antigen. Based on this, oral delivery of autoantigens has been applied as a possible strategy for the treatment of human autoimmune diseases like rheumatoid arthritis and multiple sclerosis. The precise mechanisms involved in the induction of oral tolerance are yet to be clearly defined. In an attempt to address this issue, we have generated several lines of transgenic mice using ovalbumin (OVA) as the model antigen. Our studies have shown that a cytotoxic T lymphocyte response could be induced by oral administration of antigen and that this could lead to the onset of autoimmune disease. These findings suggest caution should be applied when oral administration of antigen is used to treat human autoimmune diseases.