Requirement for Both H-2Db and H-2Kd for the Induction of Diabetes by the Promiscuous CD8+ Clonotype AI4

This information is current as Toshiyuki Takaki, Scott M. Lieberman, Thomas M. Holl, of October 1, 2021. Bingye Han, Pere Santamaria, David V. Serreze and Teresa P. DiLorenzo J Immunol 2004; 173:2530-2541; ; doi: 10.4049/jimmunol.173.4.2530 http://www.jimmunol.org/content/173/4/2530 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 © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Requirement for Both H-2Db and H-2Kd for the Induction of Diabetes by the Promiscuous CD8؉ T Cell Clonotype AI41

Toshiyuki Takaki,2* Scott M. Lieberman,2* Thomas M. Holl,‡ Bingye Han,§ Pere Santamaria,§ David V. Serreze,3‡ and Teresa P. DiLorenzo3*†

The NOD mouse is a model for autoimmune type 1 diabetes in humans. CD8؉ T cells are essential for the destruction of the insulin-producing pancreatic ␤ cells characterizing this disease. AI4 is a pathogenic CD8؉ T cell clone, isolated from the islets of a 5-wk-old female NOD mouse, which is capable of mediating overt diabetes in the absence of CD4؉ T cell help. Recent studies using MHC-congenic NOD mice revealed marked promiscuity of the AI4 TCR, as the selection of this clonotype can be influenced by multiple MHC molecules, including some class II variants. The present work was designed, in part, to determine whether similar promiscuity also characterizes the effector function of mature AI4 CTL. Using splenocyte and bone marrow disease transfer models and in vitro islet-killing assays, we report that efficient recognition and destruction of ␤ cells by AI4 requires the Downloaded from ␤ cells to simultaneously express both H-2Db and H-2Kd class I MHC molecules. The ability of the AI4 TCR to interact with both H-2Db and H-2Kd was confirmed using recombinant peptide libraries. This approach also allowed us to define a mimotope peptide recognized by AI4 in an H-2Db-restricted manner. Using ELISPOT and mimotope/H-2Db tetramer analyses, we demonstrate for the first time that AI4 represents a readily detectable T cell population in the islet infiltrates of prediabetic NOD mice. Our identification of a ligand for AI4-like T cells will facilitate further characterization and manipulation of this pathogenic and promiscuous T cell population. The Journal of Immunology, 2004, 173: 2530–2541. http://www.jimmunol.org/

n both humans and NOD mice, type 1 diabetes (T1D)4 is an The CD8ϩ T cells contributing to the earliest stages of islet autoimmune disease that results from T cell-mediated de- inflammation represent several distinct antigenic specificities (7). I struction of insulin (INS)-producing pancreatic ␤ cells, and The AI4 CD8ϩ T cell clone, originally isolated from the islets of involves complex interactions among developmental, genetic, and a 5-wk-old female NOD mouse (3), represents one of these ␤ environmental factors (1). Although there are multiple susceptibil- cell-autoreactive specificities. NOD mice transgenically express- ity loci, the strong association of particular MHC class II mole- ing the AI4 TCR (designated NOD.AI4␣␤ transgenic (Tg)) ϩ cules with disease has led to extensive investigation of CD4 T progress to overt diabetes significantly earlier than nontransgenic cells in T1D (2). However, several studies in NOD mice have NOD mice (8). Strikingly, this accelerated diabetes development is by guest on October 1, 2021 ϩ documented the importance of pathogenic CD8 T cells in the also observed in NOD-scid.AI4␣␤ Tg, NOD.CD4null.AI4␣␤ Tg initial stages of ␤ cell destruction (3–6). (8), and NOD.Rag1null.AI4␣␤ Tg mice (7), all of which lack CD4ϩ T cells. Hence, naive AI4 T cells are able to develop, ma- ␤ Departments of *Microbiology and Immunology, and †Medicine (Division of Endo- ture, and mediate sufficient cell destruction to cause accelerated ϩ crinology), Albert Einstein College of Medicine, Bronx, NY 10461; ‡The Jackson disease in the complete absence of CD4 T cell help. AI4 repre- § Laboratory, Bar Harbor, ME 04609; and Department of Microbiology and Infectious sents the only diabetogenic CD8ϩ T cell clone known to be ca- Diseases, and Julia McFarlane Diabetes Research Centre, Faculty of Medicine, Health ϩ Sciences Centre, University of Calgary, Calgary, Alberta, Canada pable of doing so. In NOD mice, two other pathogenic CD8 T Received for publication April 27, 2004. Accepted for publication June 14, 2004. cell clones, 8.3 and G9C8, have been identified. NOD mice trans- The costs of publication of this article were defrayed in part by the payment of page genically expressing the diabetogenic 8.3 TCR (8.3-NOD) develop ϩ charges. This article must therefore be hereby marked advertisement in accordance accelerated diabetes that is enhanced by CD4 T cell help, as the with 18 U.S.C. Section 1734 solely to indicate this fact. frequency and rate of disease onset are greatly reduced in 8.3-NOD 1 This work was supported by National Institutes of Health Grants DK64315 (to mice lacking CD4ϩ T cells (9). The other known pathogenic T.P.D.), DK52956 (to T.P.D.), DK51090 (to D.V.S.), DK46266 (to D.V.S.), and ϩ DK20541 (Albert Einstein College of Medicine’s Diabetes Research and Training CD8 T cell clone, G9C8, has been shown to cause diabetes in the Center), and by grants from the Juvenile Diabetes Research Foundation (to T.P.D. and absence of CD4ϩ T cell help, but these experiments involved D.V.S.), the Alexandrine and Alexander Sinsheimer Foundation (to T.P.D.), the Ca- nadian Institutes of Health Research (to P.S.), and the Natural Sciences and Engi- transfer of previously activated G9C8 T cells into recipient mice; ϩ neering Research Council of Canada (to P.S.). S.M.L. is supported by National In- thus, their ability to develop and mature in the absence of CD4 T stitutes of Health Medical Scientist Training Grant GM07288. B.H. is supported by cell help is unknown (5). a postdoctoral fellowship from the Alberta Heritage Foundation for Medical Research (AHFMR). P.S. is a scientist of the AHFMR. The flow cytometry facility at Albert 8.3 recognizes an antigenic peptide derived from islet-specific Einstein College of Medicine is supported by National Institutes of Health Cancer glucose-6-phosphatase catalytic subunit-related protein Center Grant CA13330. (IGRP) , while G9C8 recognizes an INS B chain peptide 2 206–214 T.T. and S.M.L. contributed equally to this work. d (INS-B15–23), with both clones being H-2K -restricted (10, 11). 3 Address correspondence and reprint requests to Dr. Teresa P. DiLorenzo, Depart- We recently showed that AI4 recognizes a ␤ cell peptide (still ment of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. E-mail address: [email protected]; or unidentified) that is distinct from these two (7). Thus, AI4 repre- Dr. David V. Serreze, The Jackson Laboratory, 600 Main Street, Bar Harbor, ME sents a third antigenic specificity contributing to early ␤ cell de- 04609. E-mail address: [email protected] struction in NOD mice. 4 Abbreviations used in this paper: T1D, type 1 diabetes; Tg, transgenic; IGRP, islet- ␣␤ specific glucose-6-phosphatase catalytic subunit-related protein; INS, insulin; GAD, Crosses between NOD.AI4 Tg mice and NOD stocks congenic glutamic acid decarboxylase; NRP, NOD-relevant peptide. for a variety of MHC haplotypes were recently used to determine

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 The Journal of Immunology 2531 whether the development of pathogenic CD8ϩ T cells could be af- Isolation of peptides from immunoaffinity-purified MHC class I fected by MHC variants within haplotypes known to dominantly in- H-2Kd and H-2Db molecules were immunoaffinity purified from 4 ϫ 109 hibit T1D (12). This work revealed that, when expressed on bone IFN-␥-treated NIT-1 pancreatic ␤ cells using the mAbs SF1-1.1 (anti-H- marrow-derived APC, MHC molecules within the H2nb1 and H2b 2Kd) and 28-14-8 (anti-H-2Db) (hybridomas from the American Type Cul- haplotypes, including one or both of the H2nb1-encoded class II mol- ture Collection) and their associated peptides extracted as previously de- ecules, could influence AI4 T cell selection through the induction of scribed (24). Peptide extracts were fractionated by reverse-phase HPLC as described (7). thymic deletion or anergy. Thus, the AI4 TCR is capable of tolero- genic interactions with a number of different MHC molecules during Peptide libraries and synthetic peptides T cell development. We now demonstrate that mature AI4 CTL also Positional scanning combinatorial peptide libraries (25), mimotope candi- exhibit promiscuous recognition of multiple peptide/MHC com- date peptides (including YFIENYLEL, designated Mim), and alanine-sub- plexes, and report the surprising finding that ␤ cell coexpression of stituted Mim peptides (including the F2A variant, designated MimA2) both H-2Db and H-2Kd is required for their efficient recognition and were purchased from Mimotopes (Clayton, Victoria, Australia). NOD–rel- evant peptide (NRP)–V7 (KYNKANVFL), INS-I9 (G9I variant of murine destruction by AI4 T cells. Further, we have identified a synthetic INS-B15–23; LYLVCGERI), INS-BC (murine INS 2 B25-C34; FYTPMSR- ligand for AI4, and we have used this advance to demonstrate that, as REV), murine glutamic acid decarboxylase (GAD)65206 (GAD 65206–214; has been previously shown for 8.3- and G9C8-like T cells, AI4-like T TYEIAPVFV), GAD65546 (murine GAD65546–554; SYQPLGDKV), and b cells constitute a readily detectable population within the islets of TRL9 (a negative control H-2D -binding peptide; TSPRNSTVL) peptides were synthesized by standard solid-phase methods using fluorenylmethoxy- prediabetic NOD mice. This brings to three the number of pathogenic ϩ carbonyl chemistry in an automated peptide synthesizer (model 433A; Applied CD8 T cell populations in NOD mice that can now be manipulated Biosystems, Foster City, CA), and their identities were confirmed by mass and monitored. spectrometry. Downloaded from Pancreatic islet isolation and culture Materials and Methods Islet isolation by collagenase P perfusion of the common bile duct was Mice modified from a previously described protocol (26). Briefly, the bile duct NOD/Lt mice (H2g7 MHC haplotype, i.e., Kd,Ag7,Enull,Db) are main- was cannulated and the pancreas perfused with collagenase P (Boehringer tained by brother-sister mating. Previously described T1D-resistant strains Mannheim, Mannheim, Germany). The inflated pancreas was removed, of -deficient NOD-scid.Emv30null mice (13) and class I MHC- and incubated at 37°C to digest exocrine tissue. Following dispersion of deficient NOD.␤2mnull mice (4) are all maintained at the N11 backcross digested tissue and three washes with HBSS, islets were resuspended in http://www.jimmunol.org/ generation. Previously described stocks (14, 15) of T1D-resistant NOD HBSS containing DNase I (Worthington Biochemical, Lakewood, NJ), and mice congenic for the H2nb1 (Kb,Anb1,Ek,Db)orH2b (Kb,Ab,Enull,Db) handpicked using a silanized micropipet under a dissecting microscope. haplotypes are designated NOD.H2nb1 or NOD.H2b and are maintained at Isolated islets were washed with FBS-containing HBSS, resuspended in the N21 or N15 backcross generation, respectively. T1D-resistant NOD RPMI 1640 medium supplemented with 10% FBS (HyClone, Logan, UT) mice congenic for a previously described (16) class II-deficient H2b hap- and 50 U/ml recombinant human IL-2 (PeproTech), and cultured in 24-well b null null b ϳ lotype (K ,A ,E ,D) are maintained at the N11 backcross generation tissue culture plates ( 50 islets/well) at 37°C, 5% CO2 for 7 days. The b 0 incubation of NOD islets in IL-2-supplemented medium allows for the (designated NOD.H2 -Ab mice). A newly developed N10 backcross gen- ϩ eration stock of NOD mice congenic for the ALR-derived H2gx haplotype expansion of ␤ cell-autoreactive CD8 T cells (27). d g7 null dx (K ,A ,E ,D ) (17) is partially T1D-resistant (female incidence of Splenocyte adoptive transfer

35% at 25 wk of age, compared with 90% for standard NOD females at The by guest on October 1, 2021 Jackson Laboratory, Bar Harbor, ME). These NOD.H2gx mice were ob- Aliquots of 1 ϫ 107 splenic leukocytes isolated as described (28) from tained from E. Leiter (The Jackson Laboratory) and will be described in prediabetic female NOD.Rag1null.AI4␣␤ Tg donors were injected i.v. into detail elsewhere (E. Leiter, manuscript in preparation). NOD-Kb Tg mice irradiated (700–750 rad from a 137Cs source) mice or nonirradiated scid have been previously described (12). Expression of the H-2Kb transgene mice as indicated in the figures. Recipient mice were monitored for dia- does not alter the incidence of T1D development in these mice. NOD mice betes development for up to 20 wk after transfer. At diabetes onset or the transgenically expressing the TCR of the ␤ cell-autoreactive CD8ϩ T cell end of the observation period, recipient splenocytes were analyzed by mul- clone AI4 (V␣8/V␤2), and a substock congenic for a functionally inacti- ticolor flow cytometry for extent of AI4 T cell reconstitution. vated Rag1 gene (designated NOD.AI4␣␤ Tg or NOD.Rag1null.AI4␣␤ Tg mice), have also both been previously described (7, 8). 8.3-NOD mice Bone marrow transfer ␤ ϩ transgenically express the TCR of the cell-autoreactive CD8 T cell Mice were lethally irradiated (1200–1400 rad from a 137Cs source) at 4–6 ␣ ␤ g7 clone 8.3 (V 17/V 8S1) (9). C57BL/6 mice congenic for the H2 hap- wk of age and reconstituted as previously described (29) with 5 ϫ 106 bone lotype are maintained at the N8 backcross generation and designated null ␣␤ g7 d d d d marrow cells from prediabetic female NOD.Rag1 .AI4 Tg donors. B6.H2 (18). Lymphocyte-deficient CB17-scid (K ,A,E,D) (19) and Recipient mice were monitored for diabetes development for up to 20 wk B6-scid (Kb,Ab,Enull,Db) stocks (20) have been previously described. d d d d d after reconstitution. At diabetes onset or at the end of the observation C57BL/10 mice congenic for the H2 haplotype (K ,A,E,D) derived period, recipient splenocytes were analyzed by multicolor flow cytometry from the DBA/2 strain were obtained from The Jackson Laboratory, and for extent of AI4 T cell reconstitution. are designated B10.D2. All mice are maintained under specific pathogen- free conditions and used in accordance with institutional guidelines for Cytotoxicity assays using intact islets as targets animal welfare. All scid mice receive a sulfamethoxazole-trimethoprim mixture in their drinking water on alternate weeks to prevent infection by NOD pancreatic islets (10/well) were allowed to adhere in 96-well plates Pneumocystis carinii (19). during a 7–10 day incubation at 37°C in low-glucose DMEM (3). Adherent islets were then labeled with 5 ␮Ci/well of 51Crfor3hat37°C. Islets were Cell lines washed and overlaid with 100 ␮l of medium containing various numbers of T cells from NOD.Rag1null.AI4␣␤ Tg mice that had been preactivated for RMA-S/Kd (generously provided by M. Bevan, University of Washington, 72 h with the mimotope peptide YFIENYLEL at a concentration of 10 nM. Seattle, WA) was derived from the TAP-deficient cell line RMA-S (21) and For establishing E:T ratios, each islet was assumed to contain ϳ800 cells. engineered to express H-2Kd in addition to the endogenous H-2Kb and A minimum of three wells were set up for each E:T ratio. Controls con- H-2Db. The TAP-deficient cell line T2 (22) was obtained from the Amer- sisted of at least six wells of labeled NOD islets cultured in the absence of ican Type Culture Collection (Manassas, VA). T2-Kd (generously provided T cells. Following an overnight incubation at 37°C, the radioactivity in two by J. Yewdell, National Institute of and Infectious Diseases, Be- fractions from each well was measured. The first fraction was the culture thesda, MD) was derived from T2 and engineered to express H-2Kd in supernatant, and the second was obtained by solubilizing the remaining addition to the endogenous HLA-A2.1. The NIT-1 ␤ cell line, established islets in 100 ␮l of 2% SDS. The percentage of 51Cr release for each well from an adenoma that arose in an NOD mouse transgenically expressing was calculated by the formula [(supernatant cpm)/(supernatant cpm ϩ SDS SV40 T Ag under the control of a rat INS promoter, was maintained in a lysate cpm)] ϫ 100. In turn, percentage of specific lysis was calculated by complete medium previously described (23). For IFN-␥ treatment, NIT-1 subtracting the percentage of 51Cr release from islets cultured in medium cells were cultured overnight in this complete medium supplemented with alone (i.e., spontaneous release) from the release by each well of islets 10 U/ml IFN-␥ (murine recombinant; PeproTech, Rocky Hill, NJ). cultured with a given number of T cells. 2532 PROMISCUITY OF DIABETOGENIC AI4 T CELL CLONOTYPE

Cytotoxicity assays for screening of HPLC fractions and peptide libraries AI4 CTL were generated by culturing splenocytes from NOD.AI4␣␤ Tg mice with IFN-␥-treated NIT-1 cells and 12.5 U/ml IL-2 as described (7). NY8.3 CTL were generated by culturing splenocytes from 8.3-NOD mice with mitomycin C-treated NOD splenocytes pulsed with 10 nM NRP-A7 peptide as described (7). CTL were used in 16-h 51Cr-release cytotoxicity assays to test for recognition of peptide-pulsed target cells at an E:T ratio of 40:1 as described (7). TAP-deficient cell lines RMA-S, RMA-S/Kd, T2, and T2-Kd were used as targets. Synthetic peptides or peptide library mixes were used at concentrations indicated in the figures, and 1.6 ϫ 109 NIT-1 cell equivalents of peptide were used for HPLC fraction screening assays. H-2Db stabilization assay RMA-S cells, cultured overnight at 28°C, were pulsed with peptides in DMEM with 10% FBS for1hat28°C, incubated at 37°C for 3 h, washed, stained with anti-H-2Db mAb 28-14-8, counterstained with FITC-conju- gated polyclonal goat anti-mouse Ab (BD Pharmingen, San Diego, CA), and analyzed by flow cytometry. Data were calculated by subtracting mean fluorescence intensity of H-2Db on nonpeptide-pulsed cells from that on peptide-pulsed cells.

Tetramer staining and flow cytometry Downloaded from PE-conjugated MimA2/H-2Db tetramers were obtained through the Na- tional Institute of Allergy and Infectious Diseases Tetramer Facility and titrated to determine optimal concentration. PE-conjugated NRP-V7/H-2Kd and INS-L9/H-2Kd tetramers were prepared as previously described (30).

INS-L9 (LYLVCGERL) is the G9L variant of murine INS-B15–23. FITC- conjugated anti-CD8␣ Ab was purchased from BD Pharmingen. Cells were

FIGURE 1. AI4 recognizes a NIT-1 ␤ cell peptide in the context of http://www.jimmunol.org/ prepared to the appropriate density and incubated with tetramer and anti- b CD8␣ Ab in 96-well V-bottom plates at 4°C for 45 min. Samples were H-2D . AI4 cytotoxic activity toward RMA-S target cells pulsed with b d analyzed by flow cytometry using a FACSCalibur instrument and HPLC fractions of ␤ cell peptides eluted from H-2D (A) or RMA-S/K CellQuest software (BD Immunocytometry Systems, San Jose, CA). All target cells pulsed with HPLC fractions of ␤ cell peptides eluted from samples were gated on live cells as determined by propidium iodide H-2Kd (B). Class I MHC molecules were separately immunoaffinity-puri- labeling. fied from IFN-␥-treated NIT-1 ␤ cells. MHC class I-bound peptides were ϫ 9 ELISPOT assay eluted, fractionated by reverse-phase HPLC, and 1.6 10 NIT-1 cell equivalents of peptide were tested for reconstitution activity by ELISPOT plates (MAHA S45 10; Millipore, Billerica, MA) were pre- 51Cr-release cytotoxicity assay at an E:T ratio of 40. Fraction toxicity was coated with anti-murine IFN-␥ Ab (R4-6A2; BD Pharmingen) and blocked determined by incubating HPLC peptide-fraction-pulsed target cells in the with 1% BSA (Fraction V; Sigma-Aldrich, St. Louis, MO) in PBS. APC absence of AI4 T cells. AI4-mediated lysis of nonpeptide-pulsed target by guest on October 1, 2021 ϫ 4 (mitomycin C-treated NOD splenocytes) were added at 2 10 cells/well cells was 4.2% (RMA-S) and 8.5% (RMA-S/Kd). and pulsed for 1 h with 1 ␮M of each peptide (MimA2, NRP-V7, INS-I9,

INS-BC, GAD65206, and GAD65546). Cultured islet T cells were added at 2 ϫ 104 cells/well and plates were incubated at 37°C for 40 h. IFN-␥ ␥ secretion was detected with a second biotinylated anti-murine IFN- Ab peak previously noted in HPLC fraction 22 of the acid-stripped (XMG1.2; BD Pharmingen). Spots were developed using streptavidin-alkaline phosphatase (Zymed Laboratories, South San Francisco, CA) and 5-bromo-4- combined-peptide pool (7) was clearly reproduced when HPLC chloro-3-indolyl-phosphate/NBT chloride substrate (Sigma-Aldrich). fractions of peptides purified from H-2Db (Fig. 1A), but not H-2Kd (Fig. 1B), were assayed. A second active peak, possibly represent- Results ing a modified version of the antigenic peptide detected in frac- ␤ b AI4 T cells target a cell peptide in the context of H-2D tions 22 and 23, was also detected when the H-2Db-eluted peptide AI4 was originally reported to be restricted to the MHC class I fractions were examined (Fig. 1A). Thus, AI4 recognizes a peptide molecule H-2Kd, based on its cytotoxic activity against pancreatic from NIT-1 ␤ cells in an H-2Db-restricted fashion. d b islet cells from NOD-scid (expressing H-2K and H-2D class I d b MHC molecules) but not NOD.H2nb1 mice (expressing H-2Kb and Both H-2K and H-2D molecules are required for AI4 T cells H-2Db) (3). We have previously shown that AI4 CTL recognize an to cause diabetes NOD-derived ␤ cell peptide distinct from those targeted by the To test whether AI4 CTL are also restricted to H-2Db in terms of other diabetogenic CD8ϩ T cell clones 8.3 and G9C8 (7). This was their in vivo effector function, we used a splenocyte transfer model done through mild acid stripping of the NOD-derived ␤ cell line of disease. Splenocytes were isolated from prediabetic female NIT-1, resulting in a mixture of ␤ cell peptides eluted from both NOD.Rag1null.AI4␣␤ Tg donors and transferred to a variety of cell surface H-2Kd and H-2Db combined. In this earlier work, we mouse strains as indicated (Fig. 2). NOD.Rag1null.AI4␣␤ Tg mice then fractionated the peptides by HPLC and performed 51Cr-re- were used as the AI4 T cell source, because T cells from these lease cytotoxicity assays using target cells expressing both H-2Db mice can only express the Tg AI4 TCR, due to their inability to and H-2Kd. However, in subsequent experiments, an epitope rec- rearrange endogenous TCR genes. As expected, at 20 wk post- ognized by AI4 was surprisingly reconstituted using a target cell transfer, the majority of standard NOD recipients had developed line lacking H-2Kd expression, but expressing H-2Db (T.P.D., un- disease (Fig. 2A). Surprisingly, however, congenic strains express- published observations). ing H-2Db in the absence of H-2Kd (NOD.H2b and NOD.H2nb1) In an attempt to resolve the apparent contradiction between the remained disease-free, as did NOD.H2gx recipients, which express islet cytotoxicity and epitope reconstitution data, we next assayed H-2Kd in the absence of H-2Db. Disease could only be transferred peptides eluted separately from H-2Kd and H-2Db molecules im- to recipients homozygously expressing both H-2Kd and H-2Db munoaffinity purified from NIT-1 ␤ cells (Fig. 1). The positive (NOD, NOD-Kb Tg, and B6.H2g7). Failure to transfer disease did The Journal of Immunology 2533 Downloaded from http://www.jimmunol.org/

FIGURE 2. Both H-2Kd and H-2Db are required for recipient mice to develop diabetes after transfer of splenocytes from NOD.Rag1null.AI4␣␤ Tg mice. Aliquots of 1 ϫ 107 splenic leukocytes isolated from prediabetic female NOD.Rag1null.AI4␣␤ Tg donors were injected i.v. into irradiated (700–750 rad) mice (A) or nonirradiated scid mice (B) as indicated. Recipient mice were monitored for diabetes development for up to 20 wk after reconstitution. Recipient splenocytes were assessed for proportions of AI4 TCR-positive (V␣8) CD8 T cells by costaining with V␣8- and CD8-specific Abs at diabetes onset or at by guest on October 1, 2021 the end of the observation period. not correlate with the overall genetic T1D resistance of the recip- Interestingly, despite efficient AI4 T cell reconstitution, diabetes g7 ient strains. For example, 100% of the B6.H2 recipients devel- did not develop in any of the F1 recipients. This finding is consis- oped diabetes upon AI4 T cell transfer, even though unmanipu- tent with the hypothesis that the AI4 ligands are of low abundance, lated B6.H2g7 mice are T1D-resistant. Also, although the and that heterozygous expression of H-2Kd or H-2Db reduces them gx NOD.H2 strain is only partially genetically resistant to sponta- to a level that is insufficient to lead to ␤ cell elimination by AI4. neous diabetes, none of these recipients of AI4 T cells developed This is consistent with the earlier suggestion that low-avidity in- disease. Failure to transfer disease did not correlate with the pres- teractions between T cells and ␤ cells may not be pathogenic (27). ence of non-NOD class II MHC molecules, as the only class II Although NOD.H2nb1, NOD.H2b, and NOD.H2gx mice were gx g7 molecule expressed by NOD.H2 recipients is H-2A , yet they poorly reconstituted with AI4 T cells (Fig. 2A), perhaps due to did not develop disease upon AI4 transfer. The presence of the b allogeneic rejection of the transferred cells, the splenocyte transfer non-NOD class I MHC molecule H-2K also cannot explain the data (taken in sum) suggested that both H-2Kd and H-2Db are failure to transfer disease to NOD.H2b and NOD.H2nb1 recipients, required for AI4 T cells to cause diabetes. However, to eliminate as disease was efficiently transferred to the NOD-Kb Tg strain. the possible complications of allogeneic rejection of the trans- Our finding that disease could only be transferred to recipients ferred AI4 cells or regulatory effects from recipient , expressing both H-2Kd and H-2Db is most consistent with the idea we next did splenocyte transfers to scid recipients (Fig. 2B). Here that efficient destruction of islets by AI4 requires recognition of d b again, no diabetes development was observed if either H-2K or two peptide/MHC complexes, one containing H-2D and the other b H-2Kd. Individually, each of the complexes is apparently of too H-2D were expressed alone. Unfortunately, however, B6-scid re- low an abundance on the surface of ␤ cells to trigger sufficient AI4 cipients were poorly reconstituted, perhaps due to NK cell-medi- d T cell activation for T1D development, but together they provide ated rejection of the transferred cells or a requirement for H-2K a sufficient antigenic stimulus for disease induction. To further for homeostatic expansion (31) by AI4. explore this idea, we next transferred AI4 splenocytes to (NOD ϫ In an attempt to minimize the problems of poor reconstitution b ϫ gx ϫ due to rejection or lack of expansion, we next used a bone marrow NOD.H2 )F1, (NOD NOD.H2 )F1, or (NOD B10.D2)F1 re- cipients, all of which express both H-2Kd and H-2Db. However, all transfer model of disease. Bone marrow from prediabetic female d b null ␣␤ of the F1 mice are heterozygous for either H-2K or H-2D , thus NOD.Rag1 .AI4 Tg donors was reconstituted into lethally ir- presumably leading to a decrease in the abundance of the corre- radiated mice as indicated (Table I). In all recipients, AI4 T cells sponding antigenic peptide/MHC complexes recognized by AI4. represented at least 30% of CD8ϩ splenocytes at the time of T1D 2534 PROMISCUITY OF DIABETOGENIC AI4 T CELL CLONOTYPE

Table I. Both H-2Kd and H-2Db are required for recipient mice to develop diabetes after transfer of bone marrow from NOD.Rag1null.AI4␣␤ Tg mice

Percentage of AI4 TCRϩ Cells Percentage Diabetes among CD8ϩ Splenocytesb Recipient MHC Class I Alleles MHC Class II Alleles Incidencea (%) (% Ϯ SEM)

NOD (n ϭ 13) Kd,Db Ag7,Enull 53.8 49.0 Ϯ 6.6 NOD-Kb Tg (n ϭ 8) Kd,Db,TgKb Ag7,Enull 87.5 30.5 Ϯ 5.0 NOD.H2b (n ϭ 7) Kb,Db Ab,Enull 0 75.2 Ϯ 3.5 NOD.H2nb1 (n ϭ 5) Kb,Db Anb1,Ek 0 66.4 Ϯ 0.71 NOD.H2gx (n ϭ 9) Kd,Ddx Ag7,Enull 0 40.0 Ϯ 6.9 NOD.H2b-Ab0 (n ϭ 4) Kb,Db Anull,Enull 0 77.4 Ϯ 5.1 ϫ b 0 ϭ d/b b g7 null Ϯ (NOD NOD.H2 -Ab )F1 (n 4) K ,D A ,E 0 70.2 2.2 a All recipient mice were followed for diabetes development for up to 20 wk after bone marrow transfer. b Splenocytes were assessed for proportions of AI4 TCR-positive (V␣8) CD8 T cells by costaining with V␣8- and CD8-specific Abs at diabetes onset or at the end of the observation period (20 wk after bone marrow transfer). development or at 20 wk posttransfer. As before, diabetes devel- unknown antigenic specificities, mimotope peptides have proven opment was only observed in recipients that homozygously ex- their utility. For example, mimotope peptides were used to dem- pressed both H-2Kd and H-2Db. Note that heterozygous expression onstrate the importance of the diabetogenic 8.3-like T cell popu- Downloaded from of H-2Kd led to failure to transfer disease in (NOD ϫ NOD.H2b- lation even before IGRP was identified as its Ag (27, 30). Mimo- 0 Ab )F1 mice, again supporting the idea that abundances of the AI4 tope peptides can also be used to study thymic selection of specific ligands are low, and heterozygous expression results in an insuf- T cells and may even help to identify natural peptide ligands. ficient level of ␤ cell destruction by AI4 for T1D to develop. Im- We screened a peptide library designed to bind H-2Db, namely, b 0 ϫ b 0 portantly, both NOD.H2 -Ab and (NOD NOD.H2 -Ab )F1 peptides of 9 aa with anchors N at position 5 and L at position 9 recipients did not develop disease. This suggests that the presence (32). The library is composed of seven peptide sets (one for each of non-NOD MHC class II molecules in NOD.H2b and nonanchor position), each containing 19 different peptide mixes. In http://www.jimmunol.org/ NOD.H2nb1 mice is not responsible for the lack of disease devel- each mix, 1 aa is fixed at a nonanchor position; thus, the 19 mixes opment in these recipients. in each set cover all natural amino acids (cysteine excluded) at the We also used in vitro islet-killing assays to examine more di- specified position. The six remaining nonfixed positions in each rectly the MHC requirements for AI4 T cell recognition of ␤ cells mix are composed of equimolar amounts of the 19 aa under con- (Fig. 3). These experiments confirmed the necessity for both sideration. Using this positional scanning format, the potential H-2Kd and H-2Db expression on the ␤ cell surface for efficient contribution of each of the 19 aa to T cell recognition can be killing by AI4 T cells. Interestingly, if we added a mimotope pep- evaluated at each position of the peptide individually, and, ideally, b tide recognized by AI4 in the context of H-2D (described below), a dominant amino acid can be identified for each nonanchor po- by guest on October 1, 2021 AI4 T cells could efficiently kill islet ␤ cells in the presence of only sition (33). With three of the nine positions of the peptide fixed as H-2Db, indicating that these islets are not intrinsically resistant to single amino acids, and an equimolar mixture of the 19 amino CTL lysis. acids at each of the other six positions, each peptide mix is com- 7 posed of 196, or 4.7 ϫ 10 , different peptides. A positive response to AI4 T cells exhibit promiscuous peptide recognition behavior any one mix is likely due to T cell recognition of multiple peptides Because our ongoing attempts to identify AI4’s target Ag(s) by within that mix, and suggests that the fixed nonanchor amino acid sequence analysis of peptides eluted from purified MHC or by in that mix is important for T cell recognition. Screening of our cDNA expression cloning have not yet been successful, we used H-2Db-binding peptide library in a 51Cr-release cytotoxicity assay positional scanning synthetic combinatorial peptide libraries to with AI4 CTL and a total peptide concentration of 60 ␮g/ml per identify mimotope peptides recognized by AI4 CTL. For T cells of mix resulted in the activity profile shown in Fig. 4A. AI4 showed

FIGURE 3. AI4 T cells only show in vitro cytotoxic responses to islets expressing both H-2Kd and H-2Db. Splenocytes from NOD.Rag1null.AI4␣␤ Tg mice were cultured for 72 h in the presence of the AI4 mimotope peptide YFIENYLEL (10 nM) and 50 U/ml IL-2. Effector AI4 T cells were seeded at the indicated E:T ratios into wells containing 51Cr-pulsed islets from the indicated mice. Percentage of specific lysis was determined as described in Materials and Methods. Two single data points for the E:T ratio of 50 indicate cytotoxic responses to the NOD.H2nb1 (●) and NOD.H2b (Œ) islets pulsed with the mimotope peptide. The Journal of Immunology 2535 Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 4. AI4 exhibits promiscuous peptide recognition behavior. AI4 (A) and 8.3 (D) cytotoxic responses toward RMA-S target cells pulsed with peptide mixes from the H-2Db-binding peptide library. 8.3 (B) and AI4 (C) cytotoxic responses toward T2-Kd target cells pulsed with peptide mixes from the H-2Kd-binding peptide library. Lysis of RMA-S by AI4 and 8.3 in the absence of peptide was 5.3 and 2.8%, respectively. Lysis of T2-Kd by AI4 and 8.3 in the absence of peptide was 5.3 and 7%, respectively. For all panels, 19 different peptide mixes were tested for each amino acid position, and 1 aa was fixed at each nonanchor position in each mix (A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, in order from left to right). Each black bar represents the cytotoxic response toward the corresponding peptide mix. Peptide recognition was determined by 51Cr-release assay at an E:T ratio of 40. Letters in A and B denote the AI4- and 8.3-preferred amino acids at the indicated positions, based on those mixes eliciting the highest CTL responses. Each peptide mix was tested at a final total peptide concentration of 60 ␮g/ml. No lysis was observed when peptide-pulsed target cells were cultured in the absence of T cells. high reactivity to most amino acids fixed at positions 1, 2, 3, 7, and each fixed position consisted of an equimolar mix of two similar 8. When peptide position 6 was fixed, AI4 tolerated only large amino acids rather than an individual amino acid as in our library. hydrophobic amino acids with aromatic side chains (F, W, or Y). To test whether the concentration was appropriate with our library The AI4 response to amino acids fixed at position 4 was also design, we screened a peptide library designed to bind H-2Kd (an- somewhat restricted. chor residues fixed as Y at position 2 and L at position 9 (32)) with The initial peptide concentration of 60 ␮g/ml was chosen based H-2Kd-restricted 8.3 CTL. This library was similar to our H-2Db- on previous peptide library screens, such as was used to identify binding peptide library except for the differences in fixed anchor res- the mimotope peptide recognized by 8.3 CTL (34). However, the idues. Upon screening this H-2Kd-binding peptide library with 8.3 library used in that study was designed slightly differently, in that CTL at a concentration of 60 ␮g/ml, we observed a dramatically 2536 PROMISCUITY OF DIABETOGENIC AI4 T CELL CLONOTYPE different activity profile (Fig. 4B) than that seen when we screened the peptide library screen (Fig. 4A). However, while not as robust as H-2Db library with AI4 CTL. Whereas AI4 responded to almost all the H-2Db-mediated responses, AI4 also responded to H-2Kd- mixes tested (Fig. 4A), the 8.3 response resulted in clear dominant bound peptides in a promiscuous fashion. For the sake of compar- amino acid contributions at six of seven positions tested, and the one ison, we also tested the H-2Db-binding peptide library with 8.3 position that did not show obvious dominance among the amino acids CTL, but we could not detect any reproducible significant re- fixed at that position (position 1) resulted in a profile with no activity, sponses in this screening (Fig. 4D). Taken together, these results rather than with activity toward all mixes (Fig. 4B). In fact, only one indicate that AI4 is a promiscuous clonotype. Although degener- or two mixes elicited clearly positive 8.3 lysis responses for positions acy in Ag recognition by TCRs is now well accepted (35, 36), the 3, 4, 5, 6, 7, and 8. Importantly, our results agree with the sequences extreme promiscuity demonstrated in this study by AI4 is clearly of the superagonist 8.3 mimotope NRP-V7 (KYNKANVFL) (30) and not characteristic of all autoreactive T cells, as diabetogenic 8.3 the original 8.3 mimotope NRP (KYNKANWFL) (34) at all positions CTL exhibited a far more restricted response. other than position 1. Identification of mimotope peptides recognized by AI4 CTL in Considering our splenocyte and bone marrow transfer data (Fig. b 2, and Table I), along with our in vitro islet-killing results (Fig. 3), the context of H-2D all of which suggested that AI4 recognizes ␤ cell peptides in the Because our data suggested that AI4 recognizes a NIT-1 ␤ cell context of both H-2Db and H-2Kd, we next screened the H-2Kd- peptide in the context of H-2Db (Fig. 1A), we continued to screen binding peptide library with AI4 CTL to independently test the H-2Db-binding peptide library to identify a mimotope peptide whether AI4 can interact with peptide/H-2Kd complexes. The re- recognized by AI4. To do this, we reduced the peptide concentra- sults showed that an AI4 response could indeed be elicited by tion by 250-fold dilution (0.24 ␮g/ml) compared with the initial H-2Kd-bound peptides (Fig. 4C), although, in this case, the re- screen (60 ␮g/ml). Even at this greatly reduced concentration (Fig. Downloaded from sponses were not as robust as those observed in the H-2Db-binding 5A), the AI4 response was still more broad than that seen when 8.3 http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 5. Identification of a mimotope peptide recognized by AI4 in the context of H-2Db. A, AI4 cytotoxic response toward RMA-S target cells pulsed with peptide mixes from the H-2Db-binding peptide library at a final total peptide concentration of 0.24 ␮g/ml. Lysis of target cells by AI4 in the absence of peptide was 7%. Nineteen different peptide mixes were tested for each amino acid position, and 1 aa was fixed at each nonanchor position in each mix (A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, in order from left to right). Each black bar represents the cytotoxic response toward the corresponding peptide mix. Peptide recognition was determined by 51Cr-release assay at an E:T ratio of 40. Letters denote amino acids chosen to be included in candidate mimotope peptides. B, AI4 cytotoxic response toward RMA-S target cells pulsed with varying concentrations of candidate mimotope peptides or an H-2Db-binding negative control peptide (TRL9). The cytotoxic activity of the T cells was determined by 51Cr-release assay at an E:T ratio of 40. The Journal of Immunology 2537

CTL were used to screen the H-2Kd library at 60 ␮g/ml (Fig. 4B). 39). Strikingly, the A2 substitution resulted in a peptide that elic- Despite the lack of single dominant amino acids for each position, ited a near maximal lysis response at all concentrations tested (as however, this screen did result in a smaller set of AI4-preferred low as 0.1 nM). This response was confirmed to be dose-dependent amino acids at each position, compared with the initial screen. upon screening lower concentrations, with half-maximal activity at Again, AI4 demonstrated a clear preference for F, W, or Y at a concentration of 10 pM (Fig. 6B). Position 2 is not expected to position 6 (Fig. 5A). In addition, E was dominant at position 4 (Fig. be a key TCR contact residue, so we hypothesized that alanine at 5A). To choose amino acids for each of the other nonanchor po- position 2 allows for a more stable peptide-MHC interaction. To sitions to include in our candidate mimotope peptides, we consid- evaluate this possibility, we tested the ability of these alanine- ered reproducibility among the top responders in multiple screens. substituted mimotope peptides, along with the original Mim pep- However, we avoided combinations of residues that would result tide, to bind to H-2Db in an MHC stabilization assay. The A2- in highly hydrophobic peptides, as their lack of solubility in culture substituted mimotope peptide clearly demonstrated a much greater medium would make them difficult to assay. Using these criteria, ability to stabilize H-2Db (Fig. 6C). It is possible that steric inter- three mimotope peptide candidates, differing only at position 6, were actions involving the bulky F side chain at position 2 in the orig- chosen: YFIENFLEL, YFIENWLEL, and YFIENYLEL. These pep- inal and all other alanine-substituted mimotope peptides hindered tides were synthesized and tested for recognition by AI4 CTL (Fig. their ability to bind MHC as tightly as the A2-substituted mimo- 5B). All peptides demonstrated dose-dependent recognition by AI4, tope peptide. Interestingly, F appeared favored over A when the compared with an irrelevant H-2Db-binding peptide, TRL9 (TSPRN- position 2 peptide mixes of the H-2Db-binding peptide library STVL) (37). However, YFIENYLEL demonstrated the best response were tested (Figs. 4A and 5A). This suggests that the number of from AI4 CTL, as it elicited greater specific lysis at concentrations of individual peptides recognized by AI4 within the mix where po- 10 and 1 nM compared with the other candidate peptides. Thus, sition 2 is fixed as A may be limited, whereas the number of pep- Downloaded from YFIENYLEL (Mim) is the most active AI4 mimotope peptide of the tides recognized by AI4 within the mix where position 2 is fixed as three examined. Recognition of Mim was restricted to H-2Db,asAI4 F may be higher. did not lyse Mim-pulsed T2-Kd cells (data not shown). Peptide/MHC tetramers composed of YAIENYLEL/H-2Db To better understand the contributions of each amino acid po- complexes were shown to specifically stain splenocytes from sition within this AI4 mimotope peptide, we tested a set of alanine- NOD.AI4␣␤Tg mice, with minimal staining of nontransgenic substituted mimotope peptides (Fig. 6A). As expected, A substi- NOD splenocytes (Fig. 6D). Thus, YAIENYLEL (MimA2) is a http://www.jimmunol.org/ tuted for Y at position 6 (designated A6 in Fig. 6) and A superagonist mimotope peptide recognized by the diabetogenic substituted for E at position 4 (A4 in Fig. 6) resulted in signifi- AI4 T cell clone, and MimA2/H-2Db tetramers may be used to cantly lower responses compared with the original Mim peptide. detect AI4-like T cells. Similarly, the responses toward A7- and A8-substituted peptides were decreased compared with the original Mim peptide response. Characterization of the AI4-like T cell population in NOD islet Based on crystallographic data, amino acids at positions 4, 6, 7, infiltrates and 8 of peptides bound to H-2Db are expected to point away from Until now, AI4 was only known to be present within the islet the MHC molecule, and thus, are expected to contact the TCR (38, infiltrates of the one nondiabetic 5-wk-old NOD mouse from by guest on October 1, 2021

FIGURE 6. Identification and characterization of a superagonist mimotope peptide for AI4. A, AI4 cytotoxic response toward RMA-S target cells pulsed with varying concentrations of alanine-substituted mimotope peptides in which each amino acid of the original mimotope peptide (YFIENYLEL, Mim) was individually substituted with alanine. The cytotoxic activity of T cells was determined by 51Cr-release assay at an E:T ratio of 40. B, AI4 cytotoxic response toward RMA-S target cells pulsed with lower concentrations of the original mimotope peptide, the A2-substituted variant (YAIENYLEL, A2), or a negative control peptide (TRL9). C, H-2Db stabilization assay for the alanine-substituted mimotope peptides. RMA-S cells were pulsed with the indicated peptides, stained with an anti-H-2Db Ab, and analyzed by flow cytometry as described in Materials and Methods. Each bar represents mean fluorescence intensity (MFI). D, YAIENYLEL/H-2Db tetramer staining of AI4 T cells. Splenocytes were isolated from NOD.AI4␣␤ Tg or NOD mice, stained with FITC- conjugated anti-CD8 Ab and PE-conjugated YAIENYLEL/H-2Db tetramers, and analyzed by flow cytometry. 2538 PROMISCUITY OF DIABETOGENIC AI4 T CELL CLONOTYPE

which it was originally isolated (3). Whether the AI4 clonotype We thus set out to determine whether the tetramer-positive CD8ϩ represents a population of T cells detectable in islet infiltrates of cells isolated from NOD islets were able to produce IFN-␥ in re- other NOD mice was unknown. The use of MimA2/H-2Db tetram- sponse to peptide. To do this, we incubated an aliquot of the same ers allowed us to address this issue. To do this, we cultured islets T cells used for the tetramer analysis in IFN-␥ ELISPOT plates from four nondiabetic, 11-wk-old, female, NOD mice for 7 days in along with peptide-pulsed APC. Importantly, the ELISPOT data the presence of 50 U/ml IL-2. The expanded T cells were stained agreed with the tetramer staining (Fig. 7B). Spots were detectable with FITC-conjugated anti-CD8 Ab and PE-conjugated tetramers, in response to NRP-V7 (8.3-like T cells), MimA2 (AI4-like), and and analyzed by flow cytometry. In addition to the MimA2/H-2Db INS-I9 (G9C8-like). The relative numbers of spots detected for tetramer, NRP-V7/H-2Kd and INS-L9/H-2Kd tetramers were in- each of these were consistent with the tetramer staining data. No

cluded to detect the IGRP206–214-reactive 8.3-like (11) and INS- spots were detected in response to a second INS peptide (INS-BC), ϩ ϩ B15–23-reactive G9C8-like CD8 T cell populations (10), respec- proposed to be a potentially important CD8 T cell epitope in tively. Of the CD8ϩ cells, 8% were stained with the MimA2/H- NOD mice (40), or to either of two H-2Kd-binding GAD65 pep- 2Db tetramer, clearly indicating that AI4-like T cells constitute a tides previously shown to be recognized by NOD splenocytes (41). detectable population in the islet infiltrates of NOD mice (Fig. 7A). Thus, the pathogenic AI4-like, 8.3-like, and G9C8-like T cells de- In addition to the AI4-like T cells, IGRP-reactive T cells accounted tected in islet infiltrates with tetramers represent functional IFN- for 41% of the CD8ϩ cells, and INS-reactive T cells accounted for ␥-producing CD8ϩ T cell populations. Ͼ ϩ 3%. Thus, 50% of the CD8 T cells are accounted for with these d three antigenic specificities. Preferred residues in H-2K -binding peptides recognized by AI4 Detecting Ag specific CD8ϩ T cell populations with peptide/ We have defined an H-2Db-binding AI4 mimotope peptide, but MHC tetramers is a widely used method. However, it is important data presented above also suggested the existence of a peptide(s) Downloaded from to know, in addition, if the tetramer-positive cells are functional. recognized by AI4 in the context of H-2Kd (Figs. 2, 3, and 4C, and Table I). Next, we used a more elaborate screening of the H-2Kd- binding peptide library with AI4 CTL to obtain further evidence for the existence of such a peptide. For this H-2Kd screening, we used both T2 and T2-Kd as APC to insure that responses seen to peptide-pulsed T2-Kd cells were indeed dependent on this class I http://www.jimmunol.org/ variant. At a 10-fold dilution (6 ␮g/ml) of the peptide concentra- tion used for the initial screening depicted in Fig. 4C, it remained difficult to choose a dominant amino acid for each position using T2-Kd as APC (Fig. 8A). However, now AI4 did demonstrate a preference for G, H, or W at position 6, and the recognition profile became more clear at positions 5, 7, and 8. In contrast, AI4 did not show any significant response when T2 cells were used as APC (Fig. 8B). Taken together, our results indicate that AI4 is a pro- by guest on October 1, 2021 miscuous clonotype capable of productively interacting with both H-2Kd and H-2Db peptide/MHC complexes.

Discussion AI4 is one of a panel of ␤ cell-autoreactive CD8ϩ T cell clones that we previously isolated from the earliest insulitic lesions of young NOD mice (3). It is able to mediate ␤ cell destruction suf- ficient to cause overt diabetes development even in the complete absence of CD4ϩ T cell help or CD8ϩ T cells of other antigenic specificities (8). In this study, we have shown, using ELISPOT and peptide/MHC tetramer analyses, that AI4 represents a clearly measurable population among islet-infiltrating T cells in FIGURE 7. AI4-like T cells form a detectable population in cultured NOD mice (Fig. 7). islet infiltrates from NOD mice. A, Profile of T cells cultured from pooled We recently reported that the H-2Anb1 and/or H-2Ek class II islets of 11-wk-old NOD mice. Intact islets were isolated from four 11- MHC molecules encoded by the diabetes-protective H2nb1 haplo- wk-old, nondiabetic, female, NOD mice, pooled, cultured in the presence of 50 U/ml IL-2, stained with FITC-conjugated anti-CD8 Ab and PE-con- type can mediate the negative selection of AI4 when expressed on jugated tetramer as indicated, and analyzed by flow cytometry. Samples are bone marrow-derived APC, and that this clonotype is anergized b gated on CD8ϩ cells, and numbers indicate percent of CD8ϩ cells staining when developing in the presence of H-2K class I molecules (12). q with the indicated tetramer. MimA2, YAIENYLEL/H-2Db; NRP-V7, Heterozygous expression of the H2 haplotype during AI4 T cell KYNKANVFL/H-2Kd; INS-L9, LYLVCGERL/H-2Kd. INS-L9 is the G9L development results in reduced CD8 expression and functional im- ␥ variant of the INS-B15–23 (LYLVCGERG) peptide. B, IFN- ELISPOT pairment (12). Thus, the AI4 TCR demonstrates remarkable pro- assay of T cells cultured from pooled islets of 11-wk-old NOD mice. Mi- miscuity during T cell development. In this report, we have pre- 4 tomycin C-treated NOD splenocytes were seeded at 2 ϫ 10 /well into a sented several independent lines of evidence indicating that mature ␮ 96-well ELISPOT plate and pulsed with 1 M peptide as indicated. Cul- AI4 CTL can interact with both H-2Db and H-2Kd, and that ex- tured islet infiltrates were seeded at 2 ϫ 104/well. Separate aliquots of the pression of both of these molecules is required for efficient recog- same islet culture were used in both A and B. After incubation at 37°C for ␤ 40 h, the plate was developed as described in Materials and Methods. nition and destruction of islet cells. Thus, the AI4 clonotype MimA2, YAIENYLEL; NRP-V7, KYNKANVFL; INS-I9, G9I variant of exhibits promiscuous behavior both during the selection process and while exerting its effector function. INS-B15–23; INS-BC, INS 2 B25–C34; GAD65206, GAD65206–214; GAD65546, GAD65546–554. Both INS-I9 and INS-L9 show more stable Our results obtained using three different in vivo disease transfer d binding to H-2K than INS-B15–23 (58). models (Table I, and Fig. 2) all indicate that AI4 must interact with The Journal of Immunology 2539 Downloaded from

FIGURE 8. AI4 T cells can recognize peptides in the context of H-2Kd. AI4 cytotoxic responses toward T2-Kd (A)orT2(B) target cells pulsed with peptide mixes from the H-2Kd-binding peptide library at a final total peptide concentration of 6 ␮g/ml. Lysis of T2-Kd and T2 cells by AI4 in the absence of peptide was 21.6 and 13.5%, respectively, and has been subtracted. Nineteen different peptide mixes were tested for each amino acid position, and 1 http://www.jimmunol.org/ aa was fixed at each nonanchor position in each mix (A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y, in order from left to right). Each black bar represents the cytotoxic response toward the corresponding peptide mix. Letters denote the AI4-preferred amino acids at the indicated positions based on those mixes eliciting the highest AI4 responses. No lysis was observed when peptide-pulsed target cells were cultured in the absence of T cells. The cytotoxic activity of T cells was determined by 51Cr-release assay at an E:T ratio of 40. both an H-2Db and an H-2Kd complex to efficiently destroy islet ␤ opment and survival. In some cases, positive selection has been cells. An alternative explanation for our disease transfer results is shown to be mediated by the same MHC molecule to which the T that non-NOD MHC molecules in certain of the recipients induced cell is restricted in terms of its recognition of a foreign peptide, but by guest on October 1, 2021 and rendered AI4 T cells unable to cause T1D. the selecting peptide is a different (self) peptide (43). Besides these However, we do not favor this idea, as our in vitro islet cyto- and multiple other examples (35, 36) of the ability of a TCR to toxicity assays (Fig. 3), using AI4 CTL generated from recognize different peptides bound to the same MHC molecule, NOD.Rag1null.AI4␣␤ Tg mice, demonstrate that AI4 can only kill there are also examples of recognition of the same peptide pre- ␤ cells that express both H-2Kd and H-2Db. Further, we previously sented by two different MHC molecules (44–50). In addition, the reported that NOD.Rag1null.AI4␣␤ Tg splenocytes are not stimu- frequency of alloreactive T cells, which can respond to two dif- lated in vitro by splenic APC from NOD.H2b or NOD.H2b-Ab0 ferent MHC molecules (one self and one not), is high (51–53). mice (12), suggesting that peripheral tolerance induction by the However, examples of recognition of two different peptides bound H2b or H2b-Ab0 MHC haplotypes is unlikely. to two different self MHC molecules, as proposed in this study for Our ability to detect a peak of activity when screening peptides AI4, are quite limited (54–56). They include a recent report that eluted from purified H-2Db but not H-2Kd molecules of NIT-1 ␤ negative selection of a particular CD8ϩ T cell clonotype requires cells (Fig. 1) is not necessarily at odds with our conclusion that recognition of both an MHC class I and an MHC class II molecule AI4 must interact with both an H-2Db and an H-2Kd complex to (56). Thus, there is some precedent, albeit limited, for our finding cause disease. The abundance of the antigenic peptide eluted from that AI4 can respond to ␤ cell peptides bound to H-2Db as well as H-2Kd molecules of NIT-1 cells might simply be too low to elicit H-2Kd self MHC molecules. We are not aware of any other ex- an AI4 response in our assay system. Alternatively, the NIT-1 cell ample of a T cell that requires the simultaneous recognition of two line might no longer express the ␤ cell protein from which the different MHC molecules in order for its effector function to be- antigenic H-2Kd-binding peptide is derived, due to altered differ- come apparent. entiation brought about by its transformation by SV40 T Ag (23) Although the importance of CD8ϩ T cells in T1D is becoming or repeated passage in culture. For example, expression of the ␤ more widely recognized (57), our knowledge of their antigenic ϩ cell Ags recognized by two different diabetogenic CD4 T cell specificities remains incomplete. Besides IGRP206–214 and INS- ␤ clones is rapidly lost from cell adenoma cells upon growth in B15–23, which are targeted by 8.3 and G9C8, respectively (10, 11), culture (42). Screening of the synthetic H-2Kd-binding peptide li- no other natural peptide targets for islet-infiltrating CD8ϩ T cells brary clearly indicates that AI4 is able to productively interact with in NOD mice have been identified to date. Three other candidate H-2Kd complexes (Figs. 4C and 8). peptides of potential relevance have previously been described (40,

TCR cross-reactivity or promiscuity (i.e., the ability of a single 41). GAD65206 and GAD65546 have both been shown to bind TCR to interact with more than one ligand) is now a well-docu- H-2Kd and to permit the generation of peptide-specific IFN-␥-pro- mented phenomenon. The current understanding of the processes ducing CTL from NOD splenocytes (41). However, the ability of of T cell-positive selection (43) and homeostasis (31) indicates that these GAD-reactive CD8ϩ T cells to kill ␤ cells was not evaluated. some degree of cross-reactivity is a requirement for T cell devel- Compelling, though indirect, evidence for the importance of a third 2540 PROMISCUITY OF DIABETOGENIC AI4 T CELL CLONOTYPE

H-2Kd-binding peptide, INS-BC, has also previously been pro- derived diabetogenic genetic intervals: an approach for the genetic dissection of vided (40). Using T cells propagated from islet infiltrates and complex traits. Mamm. Genome. 7:331. 19. Shultz, L. D., P. A. Schweitzer, E. J. Hall, J. P. Sundberg, S. Taylor, and IFN-␥ ELISPOT (Fig. 7B), we were unable to detect responses to P. D. Walzer. 1989. Pneumocystis carinii pneumonia in scid/scid mice. Curr. Top. Microbiol. Immunol. 152:243. GAD65206, GAD65546, or INS-BC. However, we only tested T cells from a pool of islets from 11-wk-old NOD mice, so it is still 20. Christianson, S. W., D. L. Greiner, I. B. Schweitzer, B. Gott, G. L. Beamer, P. A. Schweitzer, R. M. Hesselton, and L. D. Shultz. 1996. Role of natural killer possible that these peptides are targets of islet-infiltrating T cells in cells on engraftment of human lymphoid cells and on metastasis of human T- mice of other ages. 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