Diabetogenic CD8 T Lymphocytes Cell Death Promotes Priming of Β in Situ

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In Situ β Cell Death Promotes Priming of Diabetogenic CD8 T Lymphocytes Yiqun Zhang, Bronwyn O'Brien, Jacqueline Trudeau, Rusung Tan, Pere Santamaria and Jan P. Dutz This information is current as of September 26, 2021. J Immunol 2002; 168:1466-1472; ; doi: 10.4049/jimmunol.168.3.1466 http://www.jimmunol.org/content/168/3/1466 Downloaded from

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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. In Situ ␤ Cell Death Promotes Priming of Diabetogenic CD8 T Lymphocytes1

Yiqun Zhang,* Bronwyn O’Brien,‡ Jacqueline Trudeau,†‡ Rusung Tan,† Pere Santamaria,§ Jan P. Dutz2*

CTLs are important mediators of pancreatic ␤ cell destruction in the nonobese diabetic mouse model of type 1 diabetes. Cross- presentation of Ag is one means of priming CTLs. The death of Ag-bearing cells has been implicated in facilitating this mode of priming. The role of ␤ cell death in facilitating the onset of spontaneous autoimmune diabetes is unknown. Here, we used an adoptive transfer system to determine the time course of islet-derived Ag presentation to naive ␤ cell-speciﬁc CD8 T cells in nonobese diabetic mice and to test the hypothesis that ␤ cell death enhances the presentation of ␤ cell autoantigen. We have determined that ␤ cell death enhances autoantigen presentation. Priming of diabetogenic CD8 T cells in the pancreatic lymph

nodes was negligible before 4 wk, progressively increased until 8 wk of age, and was not influenced by gender. Administration of Downloaded from multiple low doses of the ␤ cell toxin streptozotocin augmented in situ ␤ cell apoptosis and accelerated the onset and magnitude of autoantigen presentation to naive CD8 T cells. Increasing doses of streptozotocin resulted in both increased pancreatic ␤ cell death and significantly enhanced T cell priming. These results indicate that in situ ␤ cell death facilitates autoantigen-specific CD8 T cell priming and can contribute to both the initiation and the ongoing amplification of an autoimmune response. The Journal of Immunology, 2002, 168: 1466–1472. http://www.jimmunol.org/ ype 1 diabetes mellitus (DM)3 is an autoimmune disorder transfer of naive CD4 T cells specific for a pancreatic ␤ cell Ag, in which the pancreatic ␤ cells are destroyed by the cel- it has been demonstrated that these cells can be primed in the T lular immune system (1, 2). The nonobese diabetic pancreatic lymph nodes as early as, but not before, 14 days of age (NOD) mouse is a useful and well-studied animal model for hu- (14). Similarly, CD8 T cells responding to transgenically overex- man type 1 DM. Adoptive T cell transfer studies using T cells from pressed Ags in the pancreas cannot be primed in mice of 5Ð10 days prediabetic NOD mice have demonstrated that transfer of diabetes of age (14, 15). The timing and mechanism by which CD8 T cells into immunodeficient mice requires both CD4 and CD8 T cells are primed to pancreatic ␤ cell Ag in spontaneous autoimmunity (3Ð6). CD8 T cells are known to participate in pancreatic ␤ cell remains to be determined. Cross-presentation of Ag, where tissue- destruction (7Ð9). The importance of CD8 T cells in diabetogen- specific Ag is processed by APCs and presented to T cells via class by guest on September 26, 2021 esis is underscored by the fact that neither DM nor insulitis de- I MHC, is one means of priming CD8 T cells (16). Cross-presen- velop in NOD mice deficient in MHC class I expression and CD8 tation of cellular Ags expressed by tumors (17), viruses (18), and T cells (10, 11). Recent data suggest that CD8 T cells play a piv- self (19), as well as of soluble Ags, has been described (20). Cell otal role in the earliest initiative phases of autoimmune pancreatic death, specifically by apoptosis, can provide an excellent source of ␤ cell destruction (12, 13). cellular Ag for cross-presentation (21, 22). Cell death, by either The activation of naive (resting and previously unactivated) T apoptosis (23) or necrosis (24), can also provide cell-derived sig- cells requires activation of the TCR and the simultaneous delivery nals that activate APCs and promote immune responses. There- of a costimulatory signal by a specialized APC . Using adoptive fore, we hypothesized that ␤ cell death could promote the initiation and/or progression of spontaneous autoimmune diabetes. The aims of the present study were first to delineate the timing Departments of *Medicine and †Pathology and Laboratory Medicine, British Colum- bia Research Institute of Children and Women’s Health, University of British Co- of CD8 T cell priming to pancreatic ␤ cell-associated Ag in NOD lumbia, Vancouver, British Columbia, Canada; ‡Department of Kinesiology, Simon mice and, second, to determine whether ␤ cell death can promote Fraser University, Burnaby, British Columbia, Canada; and ¤Department of Micro- biology and Infectious Diseases, Faculty of Medicine, University of Calgary, Calgary, the priming of CD8 T cells to a diabetogenic self-Ag. We used an Alberta, Canada adoptive transfer system using CD8 T cells derived from a mouse Received for publication September 19, 2001. Accepted for publication November line that transgenically expresses a TCR of a diabetogenic T cell 13, 2001. clone (8). This clone uses a TCR rearrangement expressed by a The costs of publication of this article were defrayed in part by the payment of page preponderance of CD8 T cells found in early insulitic lesions of charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. NOD mice (13). The results of our investigations show that priming of diabetogenic CD8 T cells occurs in the pancreatic lymph 1 This work was supported by a grant from the Canadian Institutes of Health Re- search/Juvenile Diabetes Research Foundation International Program II (to J.P.D.), nodes and is undetectable before 3Ð4 wk of age and then progres- and grants from the Canadian Institutes of Health Research and Canadian Diabetes sively increases with age to reach maximal levels at 8 wk of age. Association (to P.S.). J.P.D. is a Junior Scholar of the Arthritis Society. P.S. is a Senior Scholar for the Alberta Heritage Foundation for Medical Research. All authors An increased incidence of apoptosis in the pancreas after the ad- are members of the Beta Cell Apoptosis Network. ministration of low doses of the ␤ cell toxin streptozotocin (STZ) 2 Address correspondence and reprint requests to Dr. Jan P. Dutz at the current ad- augmented CD8 T cell priming and allowed it to occur at ages dress: The Skin Care Center, 835 West Tenth Avenue, Vancouver, British Columbia when it does not occur spontaneously. These results suggest that ␤ V5Z 4E2, Canada. E-mail address: [email protected] cell death precedes the priming of T cells in autoimmune diabetes 3 Abbreviations used in this paper: DM, diabetes mellitus; NOD, nonobese diabetic; ␤ STZ, streptozotocin; CM, complete medium; DC, dendritic cell; PARP, poly(A)DP and implicate in situ cell death in the facilitation of autoantigen- ribose polymerase. specific CD8 T cell priming in autoimmunity.

Materials and Methods achieved with 3,3Ј-diaminobenzidine tetrahydrochloride as the substrate. Mice and cell lines To provide a negative control for the TUNEL, TdT was omitted from the labeling mix. The ␤ cell origin of the apoptosis was verified by double NOD mice were obtained from Taconic Farms (Germantown, NY) and staining for insulin using guinea pig anti-porcine insulin Ab (DAKO, Mis- bred in a specific pathogen-free environment. The 8.3-NOD mice, express- sissauga, Ontario, Canada), a biotinylated anti-guinea pig IgG as secondary ing the rearranged TCR genes of the diabetogenic CTL clone NY8.3, have Ab (Vector Laboratories, Burlington, Ontario, Canada), and the chromogen been previously described (8) and were bred at the University of Calgary New Fuschin (DAKO). Cells with brown nuclei (TUNEL positive) and (Calgary, Alberta, Canada). C57BL/6 mice were obtained from The Jack- pink cytoplasm (insulin positive) were identified as apoptotic ␤ cells. Ap- son Laboratory (Bar Harbor, ME). All animal experiments were approved optosis was quantified in all ␤ cells present in stained sections from each by the Animal Care Committee of the University of British Columbia. EL4 animal, each section being separated from the next by at least 40 ␮m. thymoma and E.G7-OVA cells (EL4 cells transfected to express OVA) Approximately 1000 ␤ cells were counted for each animal by a single (20) were obtained from the American Type Culture Collection (Manassas, observer, blinded to the treatment status of the animal. The number of VA) and cultured in complete medium (CM) consisting of RMPI 1640 apoptotic ␤ cells present was expressed as a percentage of the total number medium (Invitrogen, Burlington, Ontario, Canada) supplemented with of ␤ cells observed. penicillin (100 U/ml), streptomycin (100 ␮g/ml), 5 ϫ 10Ϫ5 M 2-ME, and 10% FCS. Assessment of diabetes development Adoptive transfers and cytofluorometric analysis Blood glucose was measured in tail vein blood using Elite glucostrips and glucometer (Bayer, Etobicoke, Canada). Animals were considered to be Naive 8.3 CD8 T cells were isolated from 4- to 5-wk-old 8.3-NOD mice. overtly diabetic when two consecutive blood glucose measurements ex- Single-cell suspensions were prepared from peripheral lymph nodes and ceeded 14 mmol/L. spleen, and CD8 T cells were purified using immunomagnetic cell sepa-

ration kits (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s Statistical analysis Downloaded from specifications. Purified 8.3 CD8 T cells were labeled with 5 ␮mol/L CFSE (Molecular Probes, Eugene, OR) in PBS for 10 min. The labeled cells Groups were compared using one- or two-tailed Student’s t tests. (10 ϫ 106) were transferred to NOD recipients by tail vein injection (for 8-wk-old animals) or i.p. (for younger animals). Three-channel FACS anal- Results ysis was performed on a FACScalibur flow cytometer (BD Biosciences, Proliferating 8.3 CD8 T cells are detected in NOD pancreatic San Jose, CA) using Cell Quest software (BD Biosciences). Briefly, lymph nodes after adoptive transfer 200,000 cells were analyzed after staining with appropriate Abs or tetramer. Kb-OVA tetramers were synthesized as described (25). Abs to Cytotoxic T cells are known to infiltrate pancreatic islets, where http://www.jimmunol.org/ CD44, CD25, and TCR V8.1/8.2 were purchased from Cedarlane (Hornby, they participate in pancreatic ␤ cell damage (7Ð9). A significant Canada). Abs to CD8␣ (clone 53-6.7) and CD69 were purchased from BD PharMingen (San Diego, CA). The Ab to TCR V␤ 8.1/8.2 recognizes the fraction of CD8 T cells in NOD mice express highly homologous V␤-chain used by the 8.3 TCR (8). In vitro stimulation of purified 8.3 CD8 TCR ␣-chains (V␣17 and J␣42 elements, joined by the N-region T cells was conducted in the presence of 1 ␮g/ml of the indicated peptides sequence M-R-D/E) (7, 13, 27) and are cytotoxic to ␤ cells in the and CM using irradiated (3000 rad) syngeneic splenocytes as APCs. context of the H-2Kd MHC class I molecule. The 8.3-NOD mice d Immunizations and cytotoxicity assay were generated using the TCR ␣ and ␤ genes of one such H-2K - restricted ␤ cell cytotoxic CD8 T cell clone isolated from the islets C57BL/6 mice (5Ð6 wk old) were immunized in the flank by s.c. injection of an acutely diabetic NOD mouse (8). We first purified 8.3 CD8 with 100 ␮g OVA V (Sigma-Aldrich, St. Louis, MO) in PBS with or by guest on September 26, 2021 without 5 ϫ 106 EL4 thymoma cells that had been either irradiated or T cells from 8.3-NOD mice using either negative or positive se- freeze thawed for four cycles. The gamma irradiation consisted of 20,000 lection. Phenotype analysis using the activation markers CD69, rads followed by incubation for4hinserum-free medium and induced CD25, and CD44 revealed that our positive selection protocol apoptosis in 82% of cells at the time of injection, as confirmed by Annexin yielded both resting and naive CD8 T cells. By combinatorial pep- V positivity (BD PharMingen). Mice were boosted at 1 wk and sacrificed at day 14. EL4 cells were Mycoplasma negative, as tested by PCR (Amer- tide library screening, an agonist peptide (NRP-A7, KYNKA ican Type Culture Collection). For tetramer analysis, spleen cells were NAFL) for the 8.3 TCR has been identified (28). The purified cells depleted of RBCs and analyzed by fluorescence cytometry. For CTL gen- up-regulated early activation markers in response to agonist pep- 6 6 eration, 5 ϫ 10 splenocytes were stimulated with 2 ϫ 10 irradiated E.G7- tide but not to a control peptide (TUM, KYQAVTTTL) (Fig. 1; OVA cells and cultured in 10 ml CM for 5 days. Human rIL-2 (20 IU/ml) (BD PharMingen) was added on day 3. Chromium release assays were Ref. 29). Adoptive transfer of fluorochrome-labeled Ag-specificT conducted using 51Cr-labeled E.G7-OVA cells as targets. Plates were in- cells has been used as a method to track the activation of T cell in cubated for4hat37¡C. Supernatants were collected and gamma irradiation vivo and can be used to determine the location and timing of cog- was measured. Specific lysis of target cells was calculated using the following nate Ag presentation (30). Using this method, it has been deter- Ϫ Ϫ formula: [(release by CTL medium release)/(detergent release medium mined that diabetogenic CD4 T cells encounter cognate Ag in the release)] ϫ 100. Spontaneous release from target cells was always Ͻ20%. pancreatic lymph nodes in adult but not in neonatal (10-day-old) Peptide synthesis The peptides TUM (KYQAVTTTL), NRP-A7 (KYNKANAFL), and OVA (SIINFEKL) were prepared using N-(9-fluorenyl)methoxycarbonyl chem- istry and purified by reversed phase HPLC to Ͼ80% purity at the Nucleic Acid and Peptide Synthesis Facility of the University of British Columbia. STZ treatment Animals were injected i.p. with STZ (Sigma-Aldrich), freshly prepared in citrate buffer (0.01 mmol/L; pH 4.5), and used at the indicated doses. Identification and quantification of apoptotic ␤ cells FIGURE 1. The 8.3 CD8 T cells are naive and respond to mimotope in vitro. The 8.3 CD8 T cells were purified and analyzed immediately ex vivo The TUNEL method for labeling DNA strand breaks was used to quantify (solid line, unstimulated) or cultured for 24 h in the presence of irradiated ␤ the number of apoptotic cells in sections of pancreas from mice aged 2 syngeneic splenocytes and an irrelevant peptide (dotted line, TUM, wk, as has been described (26). Pancreata were removed immediately after killing and were fixed in Bock’s fixative for 48 h at room temperature. The KYQAVTTTL) or a known agonist peptide (gray dotted line, NRP-A7, KYN ␤ tissue was washed in PBS and then in 70% alcohol and was embedded in KANAFL). Live cells were gated for CD8 and TCR V 8.1/8.2 positivity. paraffin using standard histological techniques. Microwave irradiation was Expression of activation markers CD69, CD25, and CD44 was then analyzed used for Ag retrieval. The ApopTag Kit (Intergen, Purchase, NY) was then by fluorescence cytometry. Data are representative of two independent used to identify the apoptotic cells. Visualization of positive cells was experiments. 1468 PRIMING OF DIABETOGENIC CD8 T LYMPHOCYTES mice (14). Similarly, transgenic expression of OVA in pancreatic islets leads to OVA-specific CTLs in adult but not in 10-day-old mice. To determine whether diabetogenic CD8 T cells recognizing an endogenous pancreatic ␤ cell Ag are primed in the pancreatic lymph nodes, we labeled purified naive 8.3 CD8 T cells with the fluorescent dye CFSE. We then adoptively transferred 10 ϫ 106 of these cells into 8-wk-old NOD mice by tail vein injection. Within 72 h after transfer, cells that had proliferated at least once (as indicated by serial CFSE dye dilution) were detected in the pancreatic lymph nodes but not in the mesenteric lymph nodes or spleens of these mice (Fig. 2). Thus, naive 8.3 CD8 T cells pro- liferate and are first activated (primed) in the pancreatic draining lymph nodes of prediabetic NOD mice.

Time course of 8.3 CD8 T cell priming in NOD mice NOD mice develop an inflammatory infiltrate of the islets (insulitis) consisting of T and B lymphocytes, macrophages, and dendritic cells (DCs). Onset of diabetes in female NOD mice occurs FIGURE 3. Time course of 8.3 CD8 T cell priming in NOD mice. Pu- Downloaded from from 12 wk of age onwards (2). The spontaneous onset of insulitis rified CD8 T cells from 8.3-TCR NOD mice were stained with CFSE and seems to be a fixed checkpoint at approximately days 18Ð21 (31). transferred into NOD mice at various ages. After 4 days, the pancreatic and It has been shown that diabetogenic CD4 T cells are primed in the peripheral lymph nodes were harvested for analysis. CFSE-positive cells pancreatic lymph nodes before islet infiltration (14). To establish were gated, and the percentage of CFSE-low cells was defined as the per- the time course of priming of naive diabetogenic CD8 T cells in centage of cells having undergone at least one division. A, Data in the left NOD mice, we adoptively transferred CFSE-labeled naive 8.3 panel are expressed as mean Ϯ SD of four to 10 mice per time point. The

CD8 T cells into male and female NOD mice aged 1Ð8 wk. The bar on the abscissa has been added for illustrative purpose and indicates the http://www.jimmunol.org/ ␤ percentage of CFSE-labeled cells that had undergone at least one period of maximal neonatal cell apoptosis in NOD mice (33). The right panel compares the time course of priming in female (solid line, as shown cell division was then quantified (Fig. 3). The priming of diabe- Ϯ Ϯ in the right panel) and male (dotted line) mice (mean SD of three to four togenic CTLs in the pancreatic lymph nodes was negligible (3% mice per time point). B, Representative CFSE expression profiles from the ϭ 0.9% of adoptively transferred cells at 1 wk, n 8) until 4 wk of pancreatic lymph nodes of mice aged 2, 4, and 8 wk are shown. Percent- age (at which time it was 11% Ϯ 6%, n ϭ 7). Priming then pro- ages correspond to the fraction of CFSE-low cells, as determined by the gressively increased until 8 wk of age (when it reaches a maximum gating shown. FL1, Fluorescence. of 42% Ϯ 11%, n ϭ 6). Autoantigen presentation to diabetogenic CD8 T cells between 4 and 8 wk of age was not influenced by by guest on September 26, 2021 gender, as shown by the fact that priming of adoptively transferred cilitate the priming of cells to self-Ag. Cell death may also act as cells into male and female NOD recipients was similar. Thus, as is a stimulus to create mature immunogenic DCs (24, 34). To deter- the case for diabetogenic CD4 T cells, there is a developmental mine whether cell death has adjuvant properties in addition to pro- regulation of the presentation of endogenous pancreatic ␤ cell Ags viding a means of accessing cell-associated Ag, C57BL/6 mice to diabetogenic CD8 T cells with minimal presentation of Ag be- were immunized with the nominal soluble Ag OVA in the pres- fore 3Ð4 wk of age. Interestingly, this period of activation follows ence or absence of either apoptotic or necrotic cells. We chose to a period of remodeling and physiologic apoptosis of the pancreatic use OVA as the nominal Ag because its immunodominant CTL b islets that peaks at 2 wk of age (32, 33). Thus, in situ ␤ cell death epitope, SIINFEKL, in the context of the H-2K class I MHC precedes the priming of diabetogenic CD8 T cells. Whether this molecule, is well defined (35). CTL responses were monitored by local wave of cell death plays a role in the initiation of autoim- the enumeration of OVA (SIINFEKL)-specific CTLs using fluo- b munity (33) or whether Ag presentation is developmentally rescence cytometry and K -OVA multimeric complexes. CTL re- blocked to minimize autoimmunity is not clear (16). sponses were confirmed using a standard chromium release assay (Fig. 4). Whereas C57BL/6 mice immunized with soluble protein did Cell death enhances CTL priming to nominal Ag not mount significant CTL responses, mice immunized with soluble Cell death by either apoptosis or necrosis can make cellular con- OVA co-injected with either irradiated (apoptotic) or freeze thawed tents available for uptake by professional APCs and may thus fa- (necrotic) EL4 thymoma cells mounted good CTL responses. These responses were not due to tumor-specific factors because similar results were obtained with the co-injection of autologous irradiated or freeze thawed fibroblasts (data not shown). These results suggest that cellular material released by either apoptotic or necrotic cells can po- tentiate the priming of CTLs to soluble co-administered Ag.

The ␤ cell toxin STZ enhances the priming of 8.3 CD8 T cells in 2-wk-old NOD mice Next we wanted to know whether increased cell death at the tissue FIGURE 2. Proliferating 8.3 CD8 T cells are detected in 8-wk-old NOD level could promote the priming of autoreactive CD8 T cells. STZ, pancreatic lymph nodes after adoptive transfer. A total of 10 ϫ 106 CD8 T cells from 8.3-TCR NOD mice were labeled with CFSE and adoptively trans- a D-glucopyranose derivative of N-methyl-N-nitrosourea, is cyto- ␤ ferred i.v. into NOD mice at 8 wk of age. Lymphoid organs were collected for toxic to pancreatic cells. When delivered at high doses (200 analysis 3 days later. Proliferation as detected by serial dilution of cytoplasmic mg/kg), STZ causes complete ␤ cell destruction and insulin de- dye is detected in the pancreatic lymph nodes only. Values are representative pletion. At low concentrations and when administered in multiple of three independent experiments. FL1, Fluorescence. low doses, STZ can initiate a delayed diabetic state in susceptible The Journal of Immunology 1469 Downloaded from FIGURE 4. Cell death enhances priming to soluble Antigen. A, C57BL/6 mice were injected with OVA protein in the presence or absence of irradiated (Irr) or freeze thawed (F-T) EL4 cells. Animals were boosted 1 wk later and sacriﬁced for analysis on d 14. Spleen cells were stained for Kb-OVA tetramer and CD8 expression. The numbers correspond to the percentages of CD8 T cells that stain with tetramer. B, Scatter diagram

showing the results of three mice per group. Bars reflect the mean per- http://www.jimmunol.org/ centages of OVA-specific CTLs detected in the spleens of treated animals. C, Representative data from a chromium release assay showing induction of CTL activity in animals treated with freeze-thawed EL4 (hatched line), irradiated EL4 (dotted line), or OVA alone (solid line). The results are representative of three separate experiments. FIGURE 5. STZ enhances the priming of 8.3 CD8 T cells in NOD mice. A, Two-week-old NOD mice were treated with STZ (40 mg/kg body weight) or citrate buffer daily for 3 days before adoptive transfer of 8.3 strains that is largely dependent on immune mechanisms (36Ð38). CD8 T cells. Pancreatic lymph node cells were analyzed 4 days later for High doses of STZ in vitro can cause necrosis of ␤ cells (39). Low proliferation and CD69 expression. Live cells were analyzed for CD8 and doses of STZ, however, initiate an apoptotic pathway of ␤ cell CFSE expression. Cells expressing both CD8 and CFSE were then gated by guest on September 26, 2021 death. Multiple low doses of STZ have been shown to increase in for CD69 expression analysis. In this representative experiment, STZ in- situ apoptotic cell death in the pancreas (26). Because cell death duced proliferation and up-regulation of CD69. B, Two- and 4-wk-old o ϭ may increase cellular Ag availability and have adjuvant properties, NOD mice were treated as in A with STZ (40 mg/kg body weight, , n 11) or citrate buffer (f, n ϭ 5) daily for 3 days, and ability to prime 8.3 we treated 2-wk-old mice with STZ and assessed ␤ cell-specificT CD8 T cells was then assessed. Values represent the means Ϯ SEM. STZ cell priming in the pancreatic lymph nodes. Mice were treated with significantly enhanced priming in 2-wk-old mice (p ϭ 0.007) and 4-wk-old either STZ (40 mg/kg) daily for 3 days or with citrate buffer alone mice (n ϭ 5 each; p ϭ 0.016). FL1, Fluorescence. before adoptive transfer of naive 8.3 CD8 T cells. Adoptively transferred cells were analyzed 4 days later (Fig. 5). Priming was detected by CFSE dilution and up-regulation of the early activation oped diabetes (blood glucose Ն 14 mmol/L over two consecutive marker, CD69. Although there was minimal priming in 2- to 3-wk- readings) at age 6 wk. Nine of 10 developed diabetes by age 7 wk old animals treated with citrate buffer alone (n ϭ 5), both prolif- and 10 of 10 did so at 8 wk, compared with zero of 10 citrate eration and CD69 up-regulation of 8.3 CD8 T cells were observed buffer-treated mice (Fig. 6.). A delay of 1Ð2 wk between STZ in the pancreatic lymph nodes of mice treated with STZ (n ϭ 11). administration and diabetes onset suggests an immune contribution Comparison of the means of CFSE-labeled cells having undergone to diabetes development and is consistent with the delayed onset at least one division revealed that STZ significantly enhanced observed in standard susceptible strains. Our finding that multiple priming ( p ϭ 0.007). Similar results were found using 4-wk-old low-dose STZ can accelerate the onset of diabetes in NOD mice is mice (n ϭ 5 for both test and control groups; p ϭ 0.016). These in agreement with previous observations (45). To confirm that the results suggest that STZ enhances the priming of diabetogenic administered STZ was inducing apoptotic cell death of pancreatic CD8 T cells to endogenous pancreatic ␤ cell Ag and allows this ␤ cells, 2-wk-old mice were treated with three repeated injections priming to occur at a time when there is normally minimal Ag of STZ using varying doses. Pancreatic tissue was then removed, presentation and priming. processed for histology, and double stained for insulin to label ␤ cells and by the TUNEL technique to identify apoptotic cells. The ␤ ␤ CTL priming to a cell autoantigen correlates with cell death number of apoptotic pancreatic ␤ cells correlated positively with STZ-induced pancreatic ␤ cell toxicity is dependent on initial glu- the dose of STZ (Fig. 7). Identically treated littermates received cose transporter-2-mediated STZ transport into the cell (40, 41) naive 8.3 CD8 T cells after the three injections of STZ to assess and subsequent poly(A)DP-ribose polymerase (PARP)-dependent levels of priming in the pancreatic lymph nodes. Again, priming depletion of NADϩ within the ␤ cells (42Ð44). Because suscep- increased with increasing doses of STZ, and the percentage of tibility to low-dose STZ is strain specific, we first determined proliferating cells correlated with the incidence of apoptotic pan- whether 4-wk-old NOD mice given five daily doses of STZ de- creatic ␤ cells in situ. We conclude that low doses of STZ induce velop accelerated diabetes. Seven of 10 STZ-treated mice devel- apoptotic death of pancreatic ␤ cells. Furthermore, the magnitude 1470 PRIMING OF DIABETOGENIC CD8 T LYMPHOCYTES

were next treated with a single injection of STZ at a dose of 80 mg/kg. Adoptive transfer of CFSE-labeled 8.3 CD8 T cells was performed the next day, with analysis 4 days later. Although this treatment was toxic to the mice, in the two of ﬁve mice that sur- vived this treatment, 14 and 34% of adoptively transferred cells detectable in the pancreatic lymph nodes had proliferated. We conclude that chemically induced pancreatic ␤ cell death can facilitate priming of diabetogenic CD8 T cells at a time point when it does not occur naturally and before the peak of physiologic apoptosis.

Discussion This is the first study in which adoptive transfer of naive self-Ag- specific CD8 T cells has allowed determination of the location and timing of priming to self in the NOD mouse model of type 1 diabetes. We first observed significant priming of adoptively trans- FIGURE 6. STZ accelerates diabetes in NOD female mice. Four-week- ferred naive CD8 T cells in the pancreatic lymph nodes of NOD old NOD female mice were treated with STZ 40 mg/kg daily for 5 days mice at 4 wk of age. Previous studies have shown that this priming (solid line) or with buffer alone (hatched line) (10 mice/group). Blood is the result of cross-presentation of islet Ags by professional glucose was monitored twice weekly, and mice were termed diabetic after APCs and is not due to direct presentation by ␤ cells (19, 46). Downloaded from two consecutive blood glucose measurements of Ͼ14 mmol/L. Consistent with previous reports, we find that there is a developmental regulation of priming to Ags present in the pancreata of NOD mice (14, 15). Developmental regulation of priming exists in of in situ cell death correlates with CD8 T cell priming to a natural both NOD mice and in nonautoimmune mouse strains and is spe- pancreatic ␤ cell self-Ag in the pancreatic lymph nodes. To deter- cific for the pancreas. Although priming of OVA-specific CD8 T mine whether increased ␤ cell death at an early time point could cells to transgenically expressed Ag in the pancreas does not occur http://www.jimmunol.org/ result in the priming of diabetogenic T cells, 9-day-old NOD mice in the pancreatic lymph nodes in 10-day-old mice, it does occur in the lymph nodes draining the kidneys, which also express the Ag (14). This suggests organ-specific mechanisms for the developmental regulation of Ag presentation. The priming of naive diabetogenic CD8 T cells that we noted in NOD mice precedes the commonly observed onset of insulitis in genetically unmanipulated mice (47). This is also subsequent to a natural increase in the incidence of apoptotic pancreatic ␤ cells in the pancreas that peaks at 2 wk of age (32). Acceleration of diabetes and insulitis in NOD by guest on September 26, 2021 mice over-expressing TNF-␣ on ␤ cells is preceded by increased apoptotic death of these cells (47). It is possible that this endogenous ␤ cell death subsequently allows the priming of naive diabetogenic CD8 T cells. We chose to explore this possibility in subsequent experiments. The observation that priming does not differ between male (which have an incidence of diabetes of ϳ30%) and female (80% of which will develop diabetes) NOD mice suggests that the timing of presentation of self-Ag does not identify mice that will later develop diabetes. This finding is consistent with the observation that the onset of insulitis does not differ between male and female NOD mice. Therefore, the difference between male and female NOD mice (with respect to their equal priming ability and differing susceptibilities to diabetes development) must reside either within the avidity/affinity of self-reactive T cells generated or in the ex- FIGURE 7. The 8.3 CD8 T cell priming correlates with ␤ cell apoptosis. Two-week-old NOD mice were treated with varying doses of STZ for 3 istence of differential suppressor or homeostatic mechanisms to days. Pancreata were then removed, and apoptotic ␤ cells were quantitated control these self-reactive T cell populations. It recently has been using a combination of TUNEL staining to identify apoptotic cells and shown that the progression of benign to destructive insulitis is immunohistochemistry to identify ␤ cells. Representative photomicro- characterized by an increase in the avidity/affinity of diabetogenic graphs of pancreatic sections of mice treated with either buffer (A)orSTZ CD8 T cells for a mimotope target (48). Furthermore, CD8 T cells at a dose of 80 mg/kg/day (B) are shown. Apoptotic cells, as determined by that exhibit high avidity for a model pancreatic Ag can be isolated morphology and TUNEL staining of insulin-positive cells, are indicated by from NOD mice bearing the model Ag on the pancreas. In contrast, arrows (original magnification, ϫ1000). C, Dose response in ␤ cell apo- BALB/c mice, bearing the same model Ag on the pancreas, retain Ϯ ptosis induction. Data points represent means SEM for three mice. D, only low-avidity CD8 T cells for the immunodominant epitope Littermates (n ϭ 2Ð3) received CFSE-labeled 8.3 CD8 T cells to assess (49). Although priming to self-Ag must occur to initiate autoim- priming. Increasing doses of STZ resulted in increasing numbers of apoptotic ␤ cells in situ. Percentage of ␤ cell apoptosis obtained with 0, 40, and munity, these observations underscore the importance of events 80 mg/kg body weight STZ is plotted against the percent of 8.3 CD8 T cells subsequent to the priming of autoreactive T cells in the develop- primed at these dosages of STZ as detected by serial CFSE dilution. Values ment of a full autoimmune phenotype. represent the means Ϯ SEM. The percentage of apoptotic ␤ cells correlates Apoptotic cell death can enable Ag uptake by professional positively with the priming of diabetogenic CD8 T cells (r ϭ 0.99). APCs (21, 22). Cell death can thus enhance the availability and The Journal of Immunology 1471 quantity of self-Ag to be sampled and detected by the immune after low-dose STZ administration, we cannot rule out a concom- system. For instance, apoptotic cells derived from the gut can be itant increase in necrotic cell death. transported to the T cell areas of the mesenteric lymph nodes by a Tissue damage induced by activated cytotoxic T cells can pro- subpopulation of DCs (50). Such sampling of apoptotic cells has mote priming to tissue Ags (57). Autoantibodies to epitopes gen- been associated with tolerance induction. An apoptotic cell load erated by granzyme B released during cytotoxic cell assault are a can also promote the maturation of DCs (23). Indeed, injection of unifying theme in many autoimmune diseases (58). Although these large numbers of apoptotic cells has been associated with the in- observations argue for the importance of cellular cytotoxic mech- duction of autoimmune responses (51). Therefore, we sought to anisms in the development of autoimmunity, these observations do determine whether cell death can also enhance immune responses not explain how the initial wave of CTLs may become activated. to co-administered Ag in an adjuvant-like fashion. Our results It has been proposed that increased cell death of keratinocytes after show that co-injection of dead cells with soluble Ag does indeed UV exposure may be one trigger for systemic autoimmunity in result in the enhanced generation of effector cytotoxic CD8 T cells individuals predisposed to photosensitive lupus erythematosus (59, specific for the co-injected Ag. The mechanism of this adjuvancy is 60). Recently, the in vivo prevention of apoptosis using the irre- still unclear, but the release of endogenous heat shock proteins from versible caspase inhibitor carbobenzoxyvalylalanylaspartyl-(␤-O- necrotic cells (52) and the release of cytokines and other “danger” methyl)fluoromethylketone has been shown to decrease the sever- signals from cells undergoing apoptosis may be contributory. ity of glomerular disease in a mouse model of systemic lupus Whereas priming of diabetogenic CD8 T cells is negligible in erythematosus (61). Our present study is the first to link increased 2-wk-old animals, it is clearly detected in 4-wk-old animals. En- parenchymal cell death (in the absence of CTL assault or viral Downloaded from hanced priming at 4 wk of age compared with 2 wk of age may be damage) to the increased priming of autoreactive T cells in the attributable to multiple factors. Activation of APCs by interaction draining lymph nodes of affected tissues and the subsequent ac- with CD4 T cells and subsequent DC licensing after CD40-CD40 celeration of spontaneous autoimmmune diabetes. Our findings ␤ ligand interactions is one such possibility (53Ð55). However, nei- suggest that strategies aimed at decreasing pancreatic cell death ther co-injection of naive diabetogenic CD4 T cells nor co-injec- may delay the onset of autoimmunity. Clinical trials of one such tion of an activating CD40 Ab resulted in enhanced or earlier CD8 strategy, using nicotinamide, are already underway (62). Nicotin-

␤ http://www.jimmunol.org/ T cell priming in 2-wk-old mice (data not shown). This suggests amide can prevent cyclophosphamide-induced pancreatic cell apoptosis (63) and can delay or prevent spontaneous diabetes in that other mechanisms, such as Ag availability or the presence of NOD mice (63, 64). Our observations suggest that one mechanism currently undefined signals that promote DC maturation, are op- contributing to the protective actions of nicotinamide may be to erant. Autoreactive CD8 T cell priming was found to occur sub- diminish T cell priming by decreasing in situ ␤ cell death. This sequent to the natural increase in ␤ cell death in the pancreas (32). hypothesis is currently being tested in our laboratory. The early Whether this sequencing is causally related is unclear. Because cell timing of the ␤ cell-specific T cell priming that we have observed death can both increase sampling of self-Ag and have adjuvant would suggest that such strategies, to be optimally effective, may properties, we next determined whether increasing cell death by need to be initiated in very young predisposed individuals several administration of a pancreatic ␤ cell toxin could enhance priming. by guest on September 26, 2021 years before the clinical onset of diabetes. STZ has been shown to increase the incidence of pancreatic ␤ cell apoptosis both in situ (26) and in a ␤ cell line in vitro (39). The resistance of PARP-deficient mice to the ␤ cell cytotoxic effects of Acknowledgments STZ (42Ð44) suggests that a primary mode of action of this toxin We thank D. Finegood for helpful comments, M. Deuma for excellent animal care, Y. Hu for excellent technical assistance, and Dr. Ruth Mulner for statis- is via PARP-mediated NADϩ depletion, which subsequently in- tical advice. duces ␤ cell death. Our results demonstrate that multiple low doses of STZ facilitate priming of diabetogenic CD8 T cells in 2-wk-old References mice, at an age when negligible priming otherwise occurs. 1. Tisch, R., and H. McDevitt. 1996. Insulin-dependent diabetes mellitus. Cell Because of the chemical reactivity of STZ, it has been suggested 85:291. that one possible mechanism by which STZ may accelerate dia- 2. Delovitch, T. L., and B. Singh. 1997. The nonobese diabetic mouse as a model of autoimmune diabetes: immune dysregulation gets the NOD. Immunity 7:727. betes is through the generation of neoantigens. As the 8.3 CD8 T 3. Bendelac, A., C. Carnaud, C. Boitard, and J. F. Bach. 1987. 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