Under the Human Keratin 14 Promoter Expressed Autoimmune Disease to an Antigen a Spontaneous CD8 T Cell-Dependent

A Spontaneous CD8 T Cell-Dependent Autoimmune Disease to an Antigen Expressed Under the Human Keratin 14 Promoter

This information is current as Maureen A. McGargill, Dita Mayerova, Heather E. Stefanski, of September 26, 2021. Brent Koehn, Evan A. Parke, Stephen C. Jameson, Angela Panoskaltsis-Mortari and Kristin A. Hogquist J Immunol 2002; 169:2141-2147; ; doi: 10.4049/jimmunol.169.4.2141

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

A Spontaneous CD8 T Cell-Dependent Autoimmune Disease to an Antigen Expressed Under the Human Keratin 14 Promoter1

Maureen A. McGargill,*‡ Dita Mayerova,*‡ Heather E. Stefanski,*‡ Brent Koehn,*‡ Evan A. Parke,*‡ Stephen C. Jameson,*‡ Angela Panoskaltsis-Mortari,†‡ and Kristin A. Hogquist2*‡

Using a previously described human keratin 14 (K14) promoter, we created mice expressing a peptide Ag (OVAp) in epithelial cells of the skin, tongue, esophagus, and thymus. Double transgenic mice that also express a TCR specific for this Ag (OT-I) showed evidence for Ag-driven receptor editing in the thymus. Surprisingly, such mice exhibited a severe autoimmune disease. In this work we describe the features of this disease and demonstrate that it is dependent on CD8 T cells. Consistent with the Ag expression pattern dictated by the human K14 promoter, an inflammatory infiltrate was observed in skin and esophagus and Downloaded from around bile ducts of the liver. We also observed a high level of TNF-␣ in the serum. Given that Ag expression in the thymus induced development of T cells with dual TCR reactivity, and that dual-reactive cells have been suggested to have autoimmune potential, we tested whether they were a causal factor in the disease observed here. We found that OT-I/K14-OVAp animals on a recombinase-activating gene-deficient background still suffered from disease. In addition, OT-I animals expressing OVA broadly in all tissues under a different promoter did not experience disease, despite having a similar number of dual-specific T cells. Thus, in this model it would appear that dual-reactive T cells do not underlie autoimmune pathology. Finally, we extended these http://www.jimmunol.org/ observations to a second transgenic system involving 2C TCR-transgenic animals expressing the SIY peptide Ag with the hK14 promoter. We discuss the potential relationship between autoimmunity and self-Ags that are expressed in stratified epithelium. The Journal of Immunology, 2002, 169: 2141–2147.

olerance reflects the ability of the immune system to dis- ceptor, and CD5, to reduce their sensitivity and avoid deletion tinguish between healthy and infected tissue. For T cells, (5Ð7). T cells can also avoid deletion in the thymus by secondary T an important mechanism to establish tolerance occurs TCR gene rearrangement, which occurs during allelic inclusion (8) during development in the thymus. In this study, developing T and receptor editing (9). When the secondary gene rearrangement cells that express a self-Ag-reactive TCR are induced to die via event occurs on the same allele as the primary, the primary TCR by guest on September 26, 2021 apoptosis, thereby eliminating dangerous T cell clones before they chain is replaced. When it occurs on the alternate allele it results complete development (1). At least two mechanisms exist to en- in the synthesis of two TCR chains. In this later case, the second sure that a wide variety of self-Ags are presented to T cells in the chain can compete with the first during pairing and assembly and thymus. First, bone marrow-derived APCs can cross-present ex- thereby alter the T cell reactivity. T cells with two productive ogenous Ags that are not synthesized within thymic APC (2). Sec- rearrangements of the same TCR chain are referred to as dual- ond, medullary epithelial cells are apparently specialized for pro- reactive, even though one specificity may predominate. miscuous expression of otherwise tissue-specific gene products Although receptor editing is viewed as a tolerance mechanism, (3). Peptides from these are presented to developing T cells and dual-reactive T cells have been shown to have autoimmune poten- induce immune tolerance. Nonetheless, in both humans and mice, tial. In one model, dual-reactive CD4ϩ T cells were observed in autoreactive T cells escape deletion in the thymus and circulate mice that expressed a TCR transgene specific for an influenza through the body. Their autoaggressive potential after maturation hemagglutinin along with the hemagglutinin Ag (10). The Ag was in the thymus is further controlled by peripheral tolerance mech- expressed in hemopoietic cells with the Ig ␬ promoter and in the anisms (4). pancreas with the rat insulin promoter. Although the majority of There are numerous ways in which T cells with autoreactive hemagglutinin-specific T cells were deleted in the thymus, CD4ϩ receptors could escape clonal deletion in the thymus. T cells can T cells that expressed the transgenic TCR were detected in the modulate the level of surface molecules, such as the TCR, core- peripheral lymphoid organs and the pancreas. While these T cells were anergic when tested in vitro, 25% of the mice developed mild diabetes. Adoptive transfer experiments demonstrated that it was † ‡ Departments of *Laboratory Medicine and Pathology and Pediatrics and Center for the CD4ϩ T cells that expressed low levels of the transgenic Immunology, University of Minnesota, Minneapolis, MN 55455 ␣-chain along with an endogenously rearranged ␣-chain that in- Received for publication March 4, 2002. Accepted for publication June 11, 2002. duced diabetes. In another system, constitutive expression of two The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance transgenic TCRs allowed T cells to escape deletion in the thymus with 18 U.S.C. Section 1734 solely to indicate this fact. (11). These cells were tolerant in the periphery but could be acti- 1 This work was funded by National Institutes of Health Grants PO1 AI 35296 and vated in vitro through either TCR. These experiments suggest that RO1 AI 39560 and the University of Minnesota Graduate School. dual-reactive T cells may play a dominant role in induction of 2 Address correspondence and reprint requests to Dr. Kristin A. Hogquist, De- spontaneous autoimmune disease (12). partment of Laboratory Medicine and Pathology, University of Minnesota, Box 334, 420 Delaware Street SE, Minneapolis, MN 55455. E-mail address: We previously reported a TCR-transgenic strain where expres- [email protected] sion of self-Ag in the thymus resulted in receptor editing. These

Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00 2142 SPONTANEOUS CD8 T CELL AUTOIMMUNE DISEASE mice expressed the OT-I TCR, specific for a peptide epitope from injected i.p. every fourth day from birth to wk 1 (50 ␮g), from wk 1 to wk OVA in the context of the Kb MHC molecule. When the OVA 3 (100 ␮g), and from wk 3 to wk 6 (150 ␮g). All mice were weighed and peptide Ag was expressed in the thymus under the human keratin 14 monitored for signs of disease every other day. Mice remaining at the end 3 ϩ ϩ of wk 6 were sacrificed. Serum was taken for analysis as described above. (K14) promoter, only a modest reduction in thymic CD4 CD8 Lymph node and spleen were analyzed for T cell depletion by flow cy- double positive cells was observed. TCR␣ gene rearrangement was tometry. Depletion Ͼ95% was observed in all animals. highly elevated in these precursors, and the predominant T cell population that matured expressed both the transgenic receptor and Results an endogenous TCR␣ chain. Surprisingly, OT-I/K14-OVAp ani- We studied TCR-transgenic mice that expressed the antigenic pep- mals exhibited a lethal disease between 2 and 6 wk of age. In this tide ligand in the thymus, skin, and esophagus (9). The transgenic report we describe the features of this disease and provide evi- TCR was the OT-I TCR that recognizes a peptide from chicken dence that it is a CD8-mediated autoimmune disease with signif- OVA (OVAp) in the context of the MHC class I molecule, Kb. The icant manifestation in the skin. Because of the large dual-reactive antigenic peptide, OVAp, was also expressed as a transgene under T cell population in these mice, and because such cells have pre- the control of a human keratinocyte-specific promoter, K14. This viously been reported to have autoimmune potential, we tested the promoter directs expression in epithelial cells of the thymus as hypothesis that dual-reactive T cells play a dominant role in spon- well as the skin, esophagus, and tongue (17). We previously re- taneous disease induction in this model. ported that in double transgenic (OT-I/K14-OVAp) mice the pres- ence of the antigenic peptide in the thymus did not delete the OT-I Materials and Methods transgenic T cells. On the contrary, extensive endogenous TCR␣ Mice gene rearrangement occurred (9). This receptor editing was re- Downloaded from C57BL/6 (B6) and RAG1null.B6 mice were obtained from The Jackson quired for efficient development of mature T cells. Thus, a signif- Laboratory (Bar Harbor, ME). OT-I mice express a transgenic receptor icant number of CD4 and CD8 T cells were present in periphery of b specific for the OVA257Ð264 peptide (OVAp) in the context of the H-2K OT-I/K14-OVAp mice (Table I). These cells expressed the trans- (13). 2C mice (14) express an alloreactive receptor that also has reactivity ␤ ␤ ␣ b genic TCR chain (V 5) at a high level and the transgenic -chain to a synthetic peptide (SIYRYYGL), in the context of H-2K (15). K14- (V␣2) at a reduced level (9), implying the usage of an endogenous OVAp and K14-SIYp transgenic constructs were generated using a mul-

␣ ␣ http://www.jimmunol.org/ tistep PCR procedure as previously described (16). Act-mOVA mice were TCR chains. Interestingly, the level of V 2 on CD8 T cells from obtained from B. Ehst and M. Jenkins (University of Minnesota, Minne- the spleen or lymph nodes of OT/K14-OVAp mice was consis- apolis, MN). These mice express a transmembrane form of OVA under tently higher than the level of V␣2 on mature CD8 thymocytes in ␤ control of the -actin promoter and CMV immediate-early enhancer. OVA the same animals (Fig. 1A). In addition, the cell surface phenotype could be detected on the surface of all white blood cells by flow cytometry with an OVA-specific Ab (Sigma-Aldrich, St. Louis, MO) and in all tissues of CD8 T cells from OT-I/K14-OVAp animals differed from con- high low ϩ by immunohistochemistry (data not shown). All four major thymic APCs trol OT-I animals, being CD44 , CD62L , and CD69 (9). (cortical and medullary epithelial cells, dendritic cells, and macrophages) Together these features suggest that peripheral CD8 T cells in from Act-mOVA mice were able to stimulate OT-I T cells (data not OT-I/K14-OVAp mice were activated. shown). All mice were treated in accordance with federal guidelines ap- proved by the University of Minnesota Institutional Animal Care and Use Double transgenic mice develop an acute disease by guest on September 26, 2021 Committee. OT-I/K14-OVAp double transgenic animals experienced a lethal Flow cytometric analysis of lymphoid organs disease. The outward signs included hair loss, skin lesions, weight Thymus, lymph nodes, and spleen were harvested from the indicated mice loss, and a hunched appearance (Fig. 2). Disease was not observed between 2 and 8 wk of age and stained with Abs to CD4 (L3T4, RMA-5), in OT-I or K14-OVAp single transgenic littermates (Fig. 3A and ␤ CD8 (53-6.7), CD69 (H1.2F3), Thy 1.1 (HIS51), and TCR (H57-597), all data not shown). Onset occurred at an average age of 2Ð4wkand obtained from BD PharMingen (San Diego, CA). V␤5 (MR9.1)- and V␣2 (B20.1.1)-specific Abs were used to detect the OT-I transgene, although it was incompletely penetrant, with 20% of animals showing no should be noted that these Abs also recognize endogenous V␤5 and V␣2 signs of disease (Fig. 3A). Animals with outward signs of the dis- TCR chains. A clonotype-specific Ab (1B2) was used to detect the 2C ease showed, on average, higher levels of TNF-␣ in the serum ␤ receptor along with an Ab to V 8 (F23.1). Data were collected using a (Fig. 4). In addition, the level of TNF-␣ in the serum was elevated FACSCalibur (BD Biosciences, San Jose, CA) and analyzed with FlowJo software (TreeStar, San Carlos, CA). in a few of the double transgenic mice that were not acutely ill. However, there were no major differences in cellular composition Histology or T cell surface phenotype of lymph node and spleen cells from Tissues from OT-I/K14-OVAp or OT-I control animals were arranged in acutely sick double transgenics and age-matched littermates that aluminum foil cups, snap-frozen in liquid nitrogen, and stored at Ϫ80¡C. showed no signs of disease (data not shown). Cryosections were cut 5 mm thick, mounted onto glass slides, fixed for 5 min in acetone, and stained with H&E. Table I. T lymphocyte cellularity in various strains of micea Serum cytokine measurement

Serum was tested in duplicate with TNF-␣ or IL-1␤ ELISA kits (R&D Total Systems, Minneapolis, MN) according to the manufacturer’s protocol. A Strain CD8 CD4 CD4 ϩ CD8 standard curve was generated with known amounts of cytokine provided in the kit, and the values in the serum were extrapolated from this. B6 6.7 Ϯ 1.1 9.7 Ϯ 1.8 16.4 OT-I 14.6 Ϯ 4.9 1.95 Ϯ .77 16.6 Ab treatment OT-I/K14-OVAp 1.28 Ϯ .38 4.19 Ϯ 1.9 5.5 Ϯ Ϯ ␣ ␤ OT-I/Act-mOVA 4.1 4.7 4.3 5.7 8.4 Mice were injected i.p. with depleting Abs to CD8 (2.43), CD8 (H35- OT-I/RAGnull 7.30 Ϯ 5.57 0.12 Ϯ .08 7.4 17), or rat Ig beginning at day 1 after birth and continuing every other day OT-I/K14-OVAp RAGnull 0.33 Ϯ .27 0.06 Ϯ .05 0.4 until 6 wk of age. An initial dose of 50 ␮g was increased to 100 ␮g after Ϯ Ϯ ␮ ␣ 2C 2.8 2.1 0.8 0.5 3.2 2 wk of age and to 150 g after 4 wk. A neutralizing Ab to TNF- (TN3- 2C/K14-SIY 2.1 Ϯ 1.1 4.4 Ϯ 5.0 6.5 19.12; a gift of K. Sheehan, Washington University, St. Louis, MO) was a The average number of CD4 or CD8 T cells (ϫ106) from spleens of various transgenic mice is indicated, along with the SD. The data are from 8Ð16 adult animals 3 Abbreviations used in this paper: K14, human keratin 14; RAG, recombinase-acti- per group for B6 or OT-I strains. The data are from six 2-wk-old 2C or 2C/K14-SIY vating gene; LC, Langerhans cell. mice. The Journal of Immunology 2143

FIGURE 2. Disease in OT-I/K14-OVAp mice. Photograph was taken at 4 wk of age. The OT-I/K14-OVAp mouse is on the left next to an OT-I control littermate. Downloaded from

mononuclear cell infiltrate. Transgenes driven by the hK14 promoter were shown to be expressed in a manner similar to endogenous K14, specifically in basal cells of stratified epithelium in

skin, esophagus, and tongue (17Ð21). Therefore, the pattern of in- http://www.jimmunol.org/ filtration reproducibly observed in skin and esophagus is consistent with the predicted Ag expression pattern in these animals. We also observed an inflammatory infiltrate in the liver (Fig. 5). The infiltrate was not observed in the parenchyma of the liver or around hepatic venules, but surrounded the bile duct. Although transgene expression was not detected in total liver RNA by PCR (9), it was detected in RNA from bile duct (data not shown), perhaps explain- ing the unique pattern of infiltration in this organ. Nonetheless, bile duct was not obliterated and function was not impaired, at least as by guest on September 26, 2021

FIGURE 1. T cell phenotype in lymph nodes of various transgenic strains. A, Lymph node cells from various adult transgenic mice were stained for CD4, CD8, V␣2, and CD69. Histograms show the level of V␣2 or CD69 on the CD8-positive cells. For comparison, the level of V␣2on CD8 single positives in the thymus of the double transgenic is also included. B, Lymph nodes from OT-I RAGnull and the OT-I/K14-OVAp RAGnull mice were stained as above. These cells were also stained for Thy 1 due to a high number of cells in the OT-I/K14-OVAp RAGnull lymph nodes that were not T cells. C, Spleens from 2C or 2C/K14-SIY mice at 2 wk of age were harvested and stained as in A.

Histology of tissue sections revealed an extensive infiltrate of mononuclear cells directly beneath the epidermis and also around the hair follicles (Fig. 5). This infiltrate consisted of CD4 T cells, CD8 T cells, and macrophages, but not B cells (data not shown). Hyperkeratosis and sloughing were observed in some areas of the skin. In the esophagus (Fig. 5) and tongue (data not shown), inflammation was again localized to the epithelial layer. Double FIGURE 3. Percentage of survival of double transgenic mice. Mice transgenic animals showed occasional incidence of diarrhea and were monitored every other day for signs of disease until 12 wk of age. The intestinal necrosis. However, this was not a consistent feature of age at which the mice died or were euthanized due to severe disease was this disease and in most animals the epithelium of the stomach, recorded and plotted as the percentage of mice that were alive at each time small intestine, and colon appeared intact, with no significant point. The number of mice in each group is indicated in parentheses. 2144 SPONTANEOUS CD8 T CELL AUTOIMMUNE DISEASE

esophagus, and liver, the composition of the inﬁltrate was similar, being comprised of both CD4 and CD8 T cells and macrophages but not B cells (data not shown). Other organs that were analyzed and appeared normal included heart, lung, pancreas, and kidney (data not shown).

Disease was dependent on CD8␣␤ T cells The activated phenotype of the CD8 T cells and the fact that there was a mononuclear cell inﬁltrate found in areas of Ag expression suggested that the disease in the OT-I/K14-OVAp mice might be due to the autoaggressive CD8 T cells. To test this, CD8 T cells were depleted with administration of Abs to CD8␣ or CD8␤ from birth to 6 wk of age. As a control, littermates were injected with rat Ig, and the mice were monitored for signs of disease. All mice injected with CD8 Abs survived, whereas only 3 of the 10 mice injected with the control Ab survived (Table II). The percentage of survival in mice injected with the control Ab was similar to un-

treated OT-I/K14-OVAp mice. Thus, CD8 T cells are required for Downloaded from disease induction. It had previously been shown that T cells expressing CD8␣␣ homodimers could mature extrathymically in TCR-transgenic mice (22, 23). FACS analysis of the spleen and lymph nodes from OT-I/K14-OVAp mice indicated that there was only a very small

increase in CD8␣␣ cells, but that these cells also had an activated http://www.jimmunol.org/ phenotype (data not shown). To test whether it was these cells or FIGURE 4. High levels of TNF in the serum of the double transgenic conventional CD8␣␤ heterodimer expressing T cells of thymic mice. Mice were euthanized between 1 and 12 wk of age. A, Serum TNF-␣ origin that induced disease, we treated mice with a CD8␤ Ab. was measured using an ELISA. The SDs were 8, 43, 151, and 53 for OT-I, Similar to the CD8␣ depletion, all of the mice injected with the OT-I/K14-OVAp, OT-I/K14-OVAp acutely ill, and OT-I/K14-OVAp CD8␤ Ab survived, indicating that the CD8␣␣ T cells alone did null RAG acutely ill, respectively. The p values obtained from a two-tailed not cause disease. Mice depleted with either CD8␣ or CD8␤ Ab t test, assuming unequal variance in comparison to OT-I, were 0.26, 0.01, did not display elevated levels of TNF in the serum (data not and 0.03 for OT-I/K14-OVAp, OT-I/K14-OVAp acutely ill, and OT-I/ shown). Because of the correlation between elevated levels of K14-OVAp RAGnull acutely ill, respectively. B, Serum from nine acutely ␣ ␣ by guest on September 26, 2021 ill animals was tested for both IL-1␤ and TNF-␣. Lines though the axis and TNF- and disease, we also tested whether TNF- was required abscissa indicate the assay sensitivity threshold. for disease. Six OT-I/K14-OVAp mice were injected with a neutralizing Ab to TNF-␣ (24), beginning at day 1 after birth and continuing every 4 days until 6 wk of age. Surprisingly, ﬁve of the reﬂected by the normal levels of bilirubin and ␥-glutamyl six mice suffered from acute disease and died by 6 wk of age transpeptidase present in the serum (data not shown). In skin, (Table II). This indicated that, while TNF-␣ levels were high in

FIGURE 5. Histopathology in OT-I/K14-OVAp mice. Tissues were taken from healthy OT-I control (lower panels) or diseased OT-I/K14-OVAp mice. Shown are representative H&E-stained tissue sections from liver (left panels), esophagus (middle panels), and skin (right panels). In liver, note inflammation around the bile duct (B) but not hepatic venules (V). In esophagus, note the extensive infiltrate beneath the basal cell layer (bracket). “L” indicates the lumen .(sloughing, and follicular infiltrate (arrow ,(ء) of the esophagus. In skin, note the hyperkeratosis The Journal of Immunology 2145

Table II. Disease was dependent on CD8␣␤ T cellsa ulation on peripheral cells might be a result of T cell activation to the K14-OVAp Ag. Indeed, the CD69ϩ CD8 T cells seen in OT- Number of Mice That I/K14-OVAp are predominantly among this higher V␣2-express- Ab Died Before 6 wk ing subset (data not shown). These results suggest that disease may CD8␣ 0/6 be associated with Ags expressed under control of the hK14 pro- CD8␤ 0/5 moter rather than with receptor editing as a tolerance mechanism. TNF-␣ 5/6 To test whether Ag expression by K14 results in disease in other Rat Ig 7/10 TCR-transgenic models, we studied the 2C TCR-transgenic strain. a Neonatal animals were given 50 ␮g of Ab i.p. every other day (for CD8 deple- The 2C TCR recognizes the peptide mimetope SIYRYGGL when tion) or every fourth day (for TNF neutralization) until 6 wk of age. presented by H-2Kb (15). We created transgenic mice expressing this peptide under the control of the human K14 promoter. In the thymus of 2C/K14-SIY mice there was a profound reduction in the acutely sick mice, this cytokine was not the sole mediator of dis- number of CD4ϩCD8ϩ precursor cells and no signiﬁcant receptor ␤ ease. In this regard, we noted that IL-1 was elevated in seven of editing at the TCR␣ locus, in contrast to that observed in OT-I nine serum samples from acutely ill mice (Fig. 4B); therefore, it is mice (D. Mayerova and K. A. Hogquist, manuscript in prepara- ␤ ␣ possible that IL-1 could mediate inﬂammation in TNF- tion). The small number of CD8 T cells in the thymus, as well as neutralized mice. those in the spleen, expressed both the transgenic ␣- and ␤-chains of the 2C receptor (Fig. 1). Despite the small number of CD8 T

Disease was not dependent on dual-reactive T cells Downloaded from cells maturing in the thymus, 2-wk-old mice displayed a significant Because it was previously shown that T cells with two TCRs could number of CD8 T cells in the spleen (Table I). These cells were induce autoimmune disease, and because the majority of CD8 T apparently able to initiate disease, because these mice suffered cells in these animals express two receptors, we wanted to test the from a similar lethal disease to the OT-I/K14-OVAp strain (Fig. role of dual-reactive T cells in disease induction in this model. To 3B). Altogether, these data suggest that the K14 promoter results in do this, we crossed the OT-I/K14-OVAp mice to the recombinase- an Ag expression pattern or level that is difficult to achieve com- null activating gene (RAG)1 background. In the absence of RAG1 plete immunological tolerance to. The breakdown of tolerance in http://www.jimmunol.org/ protein, rearrangement of endogenous TCRs cannot occur, thereby this case does not correlate with the extent of clonal deletion or eliminating dual-reactive T cells. As shown previously, this dra- receptor editing in the thymus and may reflect a breakdown in an matically limited the development of CD8 T cells in the thymus as-yet-unidentified peripheral tolerance process. (9). Nevertheless, some T cells were present in the periphery of the OT-I/K14-OVAp RAGnull mice (Table I), and these cells expressed the OT-I TCR and CD8, albeit at levels slightly lower than controls (Fig. 1B). Fig. 3 shows that these mice also suffered from Discussion We report in this work the observation of spontaneous autoim- acute disease, indicating that dual reactivity is not required. How- ever, the disease in the OT-I/K14-OVAp RAGnull mice was de- mune disease in OT-I TCR-transgenic animals when the Ag is by guest on September 26, 2021 layed in the time of onset compared with the double transgenics on expressed under control of the human K14 promoter. This is a a wild-type background (Fig. 3A). The symptoms of the disease lethal disease, although the precise cause of death was not deter- were similar to wild-type OT-I/K14-OVAp mice including hair mined. An inflammatory infiltrate was observed surrounding the loss, skin lesions, and weight loss. There were also elevated levels bile ducts of the liver, presumably related to transgene expression of TNF-␣ in the serum of the OT-I/K14-OVAp RAGnull mice, but in the bile duct. Nonetheless, liver and bile duct function were ␣ the levels on average were not as high as the OT-I/K14-OVAp apparently normal. The high levels of TNF- observed in the se- mice on a wild-type background (Fig. 4). These data show that T rum of these mice might account for the lethality, because aspects ␣ cell dual reactivity is not required for cells to escape deletion in the of this disease are similar to those seen in mice expressing TNF- ␣ thymus or to induce autoimmune disease in this model. as a transgene (20). Nonetheless, serum levels of TNF- varied, ␣ Not only are dual-reactive T cells dispensable for disease, but and a TNF- neutralization treatment did not prevent lethal dis- ␣ ␤ data from a second Ag transgenic we studied suggests that dual ease. IL-1 and IL-1 were also elevated in double transgenics ␣ receptor-expressing T cells are not sufficient to induce disease ei- (Fig. 4), and in the absence of TNF- these cytokines might con- ther. In this case we obtained transgenic mice where an actin pro- tribute to disease. Thus, disease initiation does not require TNF-␣, moter was used to drive OVA expression at a high level in all and the end stages of the disease process could involve multiple tissues (Act-mOVA). Interestingly, a thymic phenotype consistent factors including TNF-␣. CD8 T cells were required for the dis- with receptor editing was observed in this mouse, as in OT-I/K14- ease, because treatment with Ab to either CD8␣ or CD8␤ pre- OVAp mice (D. Mayerova and K. A. Hogquist, manuscript in vented lethality and no disease characteristics were observed in preparation). The thymus and spleen showed a large number of treated animals. CD4 and CD8 T cells with low-level expression of the transgenic T cells with dual TCR reactivity were previously shown to have ␣-chain (Fig. 1A and Table I). The transgenic ␤-chain was not autoimmune potential (10, 11). Because the OT-I/K14-OVAp an- decreased, indicating endogenous TCR␣ chain usage. Nonetheless, imals showed a high number of dual-reactive CD8 T cells, we these mice were completely healthy (Fig. 3B), again suggesting considered whether this was a factor in disease induction. OT-I/ that autoimmune disease in OT-I/K14-OVAp is not a reflection of K14-OVAp animals on a RAGnull background still experienced receptor editing. In Fig. 1A, note that CD8 T cells from both thy- disease, although the average age of onset was delayed by ϳ4 wk. mus and lymph node of OT-I/Act-mOVA mice expressed a very This experiment formally proved that dual-reactive T cells are not low level of the transgenic ␣-chain and had not up-regulated required for disease. However, the delayed onset of disease in CD69. However, peripheral CD8 T cells from OT-I/K14-OVAp RAGnull animals might suggest that dual-reactive T cells can ac- always expressed a higher level of V␣2 than CD8 thymocytes celerate or exacerbate disease. However, such a conclusion would from the same mice, although this level was not as high as CD8 T be premature, because RAGnull animals also have a B cell defi- cells expressing only the transgenic ␣- and ␤-chains. This up-reg- ciency, severe lymphopenia, and a greatly reduced total number of 2146 SPONTANEOUS CD8 T CELL AUTOIMMUNE DISEASE

CD8 T cells. Such factors could also contribute to the age of dis- rine autoimmune disease model. Perhaps in the context of other ease onset. The study of TCR␣null double transgenics and devel- predisposing factors, peripheral tolerance more frequently fails to opment of an adoptive transfer system to mimic disease are being control the T cell response to self-Ags that are repeatedly or chron- used to approach this issue further. ically presented by APC from the skin but not from other tissues. The lack of correlation between the number of dual-reactive T Skin injury has been shown to lead to pathologic lesions in indi- cells and disease occurrence in two additional strains we examined viduals predisposed to the human autoimmune disease psoria- also suggested that dual reactivity was not a key factor in this sis—a phenomenon referred to as Koebner’s effect (33). model of autoimmune disease. OT-I/Act-mOVA animals had a A predisposing factor in this model is clearly the high Ag-spe- slightly higher number of dual receptor-expressing CD4 and CD8 cific T cell clone frequency. We emphasize that both K14-OVAp T cells in the spleen compared with OT-I/K14-OVAp animals and K14-SIYp single transgenic animals appear healthy and via- (Fig. 1 and Table I). However, this strain showed no signs of ble; disease was observed only after crossing K14 transgenic an- disease. The fact that OT-I/K14 and OT-I/mOVA strains had a imals to a TCR-transgenic specific for the Ag. This is consistent similar thymic tolerance phenotype but differed absolutely in dis- with CTL tolerance to a viral Ag driven by the K14 promoter, ease occurrence suggests that the immune dysregulation in OT-I/ observed in a non-TCR-transgenic system (34). Thus, an exagger- K14 may be related to how or where the Ag is presented in the ated frequency or continued onslaught of T cells may be required peripheral tissues of K14 mice. Consistent with this, we also ob- to shift the tolerance/immunity balance in this model, as in all served disease in 2C animals expressing Ag under control of the TCR-transgenic models of spontaneous disease (10, 35Ð39). In human K14 promoter. Thymic tolerance (via clonal deletion) was humans, expansion of T cell clones occurs during the immune very profound and these animals matured few CD8 T cells in thy- response (40, 41) and under conditions of lymphopenia (42, 43). Downloaded from mus, similar to 2C animals expressing Ld (14). Thus again, the However, it is not clear whether this is a predisposing factor in disease incidence did not correlate with thymic tolerance, at least human autoimmune disease (44). Because of this, murine TCR- in terms of whether clonal deletion or receptor editing was more transgenic models may not represent ideal systems to study the predominant. In other TCR-transgenic models of spontaneous au- basis of immune tolerance loss. Nonetheless, TCR transgene-based toimmune disease 1) thymic tolerance did not occur (25), 2) de- models of autoimmune disease can potentially be useful to define letion occurred but was partial (26), 3) deletion occurred and was pathogenic processes and autoimmune spreading mechanisms, and http://www.jimmunol.org/ profound (10), or 4) allelic inclusion allowed selective maturation to test treatment protocols. Future experiments will be aimed at of cells with dual reactivity (10). Thus, a breakdown of clonal determining the potential roles of skin injury in disease induction deletion does not appear to correlate with spontaneous disease in and escalation in this model. other transgenic models. 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