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This information is current as Naohiko Fukami, Sabarinathan Ramachandran, Deepti Saini, of September 26, 2021. Michael Walter, William Chapman, G. Alexander Patterson and Thalachallour Mohanakumar J Immunol 2009; 182:309-318; ; doi: 10.4049/jimmunol.182.1.309

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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 © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Antibodies to MHC Class I Induce Autoimmunity: Role in the Pathogenesis of Chronic Rejection1

Naohiko Fukami,* Sabarinathan Ramachandran,* Deepti Saini,* Michael Walter,‡ William Chapman,* G. Alexander Patterson,§ and Thalachallour Mohanakumar2*†

Alloimmunity to mismatched donor HLA-Ags and autoimmunity to self-Ags have been hypothesized to play an important role in immunopathogenesis of chronic rejection of transplanted organs. However, it is not known what role, if any, alloimmune response plays in inducing autoimmunity. To test whether Ab-developed posttransplantation to mismatched donor MHC induces autoim- munity and chronic rejection, we developed a murine model wherein anti-MHC class I Abs or control (C1.18.4/anti-keratin) were administered intrabronchially into native lungs. Animals receiving anti-MHC class I, but not control Abs, developed marked cellular infiltration around vessels and bronchiole of lung by day 15, followed by epithelial hyperplasia, fibrosis, and occlusion of the distal airways similar to chronic rejection following human . Lungs of mice receiving anti-MHC class I Downloaded from showed increased expression of chemokines, their receptors, and growth factors, and induced IL-17 as well as de novo Abs to self-Ags, K-␣1 tubulin, and collagen V. IL-17 neutralization by anti-IL-17 resulted in reduction of autoantibody and lesions induced by anti-MHC class I Abs. Thus, our results indicate that Abs to donor MHC can induce autoimmunity, mediated by IL-17, which plays a pivotal role in chronic rejection postlung transplantation. Therefore, approaches to prevent autoim- munity should be considered for the treatment of chronic rejection postlung transplantation. The Journal of Immunology, 2009, 182: 309–318. http://www.jimmunol.org/

ransplantation is the current treatment of choice for many period with development of chronic rejection (4, 5). A longitudinal end-stage renal, hepatic, cardiac, and pulmonary diseases. study conducted over a 10-year period for HLA Abs demonstrated T Improvement in surgical techniques, postoperative man- a strong association between the production of HLA Abs after agement, and have all resulted in marked im- renal transplantation and failure (6). Similar association of provement in allograft survival. However, long-term functions of posttransplant Abs with rejection of heart (7)and LTx (8) has also the transplanted organs are often limited due to development of been reported. Based upon these reports, it is generally believed that chronic rejection. This is particularly true following lung trans- HLA Ab monitoring during the posttransplant period is an effective 3 by guest on September 26, 2021 plantation (LTx), in which chronic rejection, referred to as bron- way to predict impending chronic rejection of solid organs. chiolitis obliterans syndrome (BOS), occurs in 50% of the trans- Induction of autoimmunity following has plants within 3 years posttransplant. A retrospective analysis of also been demonstrated after and LTx (9, 10). 2190 transplants performed at Washington University/Barnes Hos- It has been postulated that autoreactive T cells to self-Ags occur pital demonstrated that nearly all of the transplanted organs suc- after an alloresponse, and once activated they can induce pathol- cumb to chronic rejection during the course of 10 years posttrans- ogy of chronic rejection (11). Cellular autoreactivity against heat plant (1). It is generally accepted that chronic rejection is a shock protein 60 (HSP-60) has been demonstrated in peripheral manifestation of alloimmune responses against the transplanted blood and graft-infiltrating following renal transplan- (2, 3). More recently, strong correlation has been noted for tation (12). These cells produced both inflammatory and regulatory the development of anti-MHC class I Abs during the posttransplant in response to HSP-60, and IL-10 predominance was noted in the late period posttransplant, suggesting a regulatory role *Department of Surgery, †Department of Pathology and Immunology, ‡Department of for HSP-60 autoreactivity. Cardiac myosin-reactive T and B cells § Internal Medicine, and Department of Cardiothoracic Surgery, Washington Univer- have been demonstrated following murine cardiac allograft rejec- sity School of Medicine, St. Louis, MO 63110 tion (9). Furthermore, anticardiac myosin immune response has Received for publication August 8, 2008. Accepted for publication October 27, 2008. been shown to contribute to the graft rejection in that pretransplant The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance modulation of this response in recipient mice can affect cardiac with 18 U.S.C. Section 1734 solely to indicate this fact. allograft survival (13). It has been demonstrated that anticardiac 1 This work was supported by National Institutes of Health Grant HL66452 (to T.M.) myosin reactivity persists in the late posttransplant period in the from National Heart, Lung, and Blood Institute, and the contents are solely the re- absence of an alloimmune response, suggesting that the autoim- sponsibility of the authors and do not necessarily represent the official views of the National Heart, Lung, and Blood Institute. mune response to myosin may be associated with the pathogenesis 2 Address correspondence and reprint requests to Dr. Thalachallour Mohanakumar, of chronic rejection (14). Furthermore, pretransplant sensitization Washington University School of Medicine, Department of Surgery, Box 1809-3328 with cardiac myosin resulted in accelerated rejection of the graft, CSRB, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail address: [email protected] and more significantly under the same condition, even a syngenic graft was rejected within 40 days (9). Using a rat model of LTx, 3 Abbreviations used in this paper: LTx, lung transplantation; BMP, bone morpho- genetic protein; BOS, obliterans syndrome; ColV, collagen V; EC, en- evidence has been obtained for an immune response to a self-Ag, dothelial cell; FGF, fibroblast growth factor; GC, germinal center; HSP-60, heat shock collagen V (ColV), in the pathogenesis of lung allograft rejection protein 60; O/N, overnight; RT, room temperature. (15). Two lines of evidence supporting the view that autoreactive Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 T cells specific to ColV are sufficient to cause rejection of the www.jimmunol.org 310 Abs TO MHC CLASS I INDUCE AUTOIMMUNITY

transplants have been presented, as follows: 1) instillation of syn- genic BAL cells pulsed with ColV was sufficient to cause rejection of lung allograft, and 2) adoptive transfer of anti-ColV cell lines into naive rats caused peribronchiolar and perivascular inflamma- tion in lung isografts (16). Taken together, the above experiments using animal models of transplantation strongly support the con- clusion that the induction of an autoimmune response after trans- plantation may lead to chronic rejection of allografts. IL-17 has been to shown to be a potent proinflammatory cyto- kine that acts on epithelial cells, endothelial cells (ECs), fibrocytes, and stromal cells, leading to a proinflammatory signal that results in enhanced proinflammatory and chemokine secretion (17). IL-17 has also been shown to play a crucial role in the in- duction of humoral autoimmune responses (18). Analysis of the responses to ColV in LTx recipients has been shown to be dependent on CD4 T cells and monocytes (19), and this response was found to be dependent on IL-17, TNF-␣, and IL-1␤ (19). Furthermore, this report demonstrated the interaction between

IL-17 and monocytes is necessary for the induction of the ColV- Downloaded from specific responses that correlates with the development of BOS. Studies in the literature have also demonstrated that a deficiency in the IL-17 or blocking of IL-17 results in a decrease in the produc- tion of autoantibodies (20, 21). IL-17 has been also shown to play a crucial role in the formation of germinal centers (GCs) and reg-

ulate the movement of the B cells in the GCs as mediated by http://www.jimmunol.org/ chemokines CXCL12 and CXCL13. Blocking of the IL-17 has been shown to result in a reduction in the levels of GCs and de- creases the titer of autoantibodies (22). Taken together, several bodies of evidence strongly support the hypothesis that IL-17 may be an important contributor in the production of autoantibodies. Although there is strong evidence for both allo- and autoim- FIGURE 1. Administration of anti-MHC class I Abs developed signif- mune responses in the pathogenesis of chronic rejection, the rela- icant cellular infiltration around vessel and bronchiole as well as hyper- tionship between and autoimmunity and its role in plasia of the bronchial (H&E stain). Anti-H2Kd or control the pathogenesis of chronic rejection still remains to be elucidated. (C1.18.4) Ab or anti-keratin Ab was administered endobronchially in by guest on September 26, 2021 In our study, we administered anti-MHC class I Abs intrabronchi- BALB/c mice on days 1, 2, 3, and 6, and weekly thereafter. The lungs were harvested on day 15 or 30 and analyzed by H&E and trichrome staining. A ally into the native murine lung so that anti-MHC class I Abs will representative picture from data obtained from five mice is presented in the specifically ligate MHC molecules in the lung parenchyma. Using figure. Original magnification, ϫ100; insets, ϫ400. A, B, C, G, H, and I, this novel model, we demonstrate for the first time that ligation of Represent sections stained with H&E; D, E, F, J, K, and L, represent anti-MHC class I Abs to the lung parenchyma results in an auto- trichrome staining. A and D, Anti-H2Kd-treated mice on day 15; G and J, immune response leading to endotheliitis, cellular infiltration, ep- anti-H2Kd-treated mice on day 30; B and E, C1.18.4 Ab-treated mice on ithelial hyperplasia, and fibrosis, as well as luminal occlusion of day 15; H and K, C1.18.4 Ab-treated mice on day 30; C and F, anti-keratin the small airways. Histopathology of the lesion closely resembles Ab-treated mice on day 15; I and L, anti-keratin Ab-treated mice on day 30. chronic rejection pathology noted following human LTx, thus pro- Lungs from the anti-H2Kd Ab-treated mice showed significant cellular in- viding support for both alloimmunity and autoimmunity in the im- filtration around bronchia and vessel (A), and hyperplasia of the bronchial munopathogenesis of chronic rejection following LTx. Further- epithelium (G) are marked by the arrows. A significant increase in the fibroproliferation, collagen deposition, and luminal occlusion was observed more, our data demonstrates that binding of MHC molecules with in the lungs of the anti-H2Kd Ab-treated mice (J). Mice treated with iso- specific Abs can result in exposure of self-Ags of the epithelial type control Ab or anti-keratin Ab showed no significant morphological cells to , leading to both cellular and humoral au- changes compared with the anti-H2Kd Ab-treated mice (B, C, E, F, H, I, K, toimmunity and immunopathogenesis of chronic rejection that is and L). predominantly mediated by the IL-17-producing cells.

Materials and Methods a murine mAb to H2Kb (C57BL/6, 6 wk, male, IgG2a) as well as murine mAb against the HLA class I molecules (HLA-A2 transgenic C57BL/6 Anti-MHC class I Abs induced obliterative airway disease mice, 6 wk, male) were administered endobronchially to test the ability of development anti-MHC Abs to induce lesions in other strains of mice. In brief, each mice was anesthetized, and the tongue was pulled out. A mosquito clamp We developed a murine model in which obliterative airway disease, cor- was inserted into the mouth, and a 20-gauge catheter was placed into the relate of BOS, takes place in the distal airways following ligation of MHC trachea. Ab (200 ␮g) was administered into the lung on days 1, 2, 3, and molecules in the lung by a mAb to MHC class I Ags. Because we wanted 6, and then weekly thereafter. C1.18.4, same isotype, was given on the days to have good titer of anti-MHC class I Ab in the lung and not absorbed by mentioned above as controls. other tissues, we opted to give anti-MHC class I Abs directly into the lung. All experiments were performed in compliance with the guidelines of the Immunohistochemical analysis Institutional Laboratory Animal Care and Use Committee of Washington University School of Medicine (protocol 20070121). Murine mAb to H2Kd Frozen lung samples were embedded in Freez Tissue matrix (OCT), and (BALB/c, 6 wk, male, IgG2a), which has no detectable endotoxin as mea- sections were cut at 5 ␮m thickness. The sections were fixed in cold al- sured by Limulus amebocyte lysate assay, was given at a dose of 200 cohol for 2 min (Ϫ20°C) and air dried. The sections were treated with 3% ␮ g/administration into mice intubated with a 20-gauge catheter. Similarly, H2O2 in EtOH for 10 min to block endogenous peroxidase activity. The The Journal of Immunology 311

sections were blocked with biotin/avidin system components for 15 min by blocking reagent (Avidin/Biotin Blocking Kit; Vector Laboratories). Pri- mary and secondary Abs were diluted with Ab dilution solution (BD Bio- sciences). The sections were incubated overnight (O/N) at 4°C with puri- fied rat anti-mouse CD4, CD8, CD11b, and CD19 mAbs (5.0 ␮g/ml; BD

Pharmingen). Then, the sections were incubated for 30 min at room tem- 3.4% ( Ϯ 2.8) 4.1% ( Ϯ 3.9) perature (RT) with diluted biotin-conjugated goat anti-rat IgG (1:50; BD Pharmingen). The sections were incubated with streptavidin-HRP and in- cubated for 30 min at RT (BD Pharmingen). The presence of positive cells was detected with the diaminobenzidine substrate kit (BD Pharmingen), counterstained with hematoxylin, and examined using a light microscope. Positive cells were counted by random sampling. Lungs were fixed in 10% formaldehyde. Sections were cut at 5 ␮m thickness and stained with Masson’s trichrome and H&E. Lesions that displayed cellular infiltration, epithelial abnormalities, and fibro prolifera- tion were analyzed by random sampling. Percentage of fibrosis was cal- culated using Optimas software version 6.5.172 (Media Cybernetics). Per- centage of fibrosis was quantified by dividing the total area enclosed by basement membrane. Percentage of cellular infiltration and epithelial ab- normalities was calculated by dividing the total bronchiole and vessel, respectively. Epithelial Hyperplasia Luminal Occlusion Area

Gene expression array Downloaded from To determine chemokines and their receptors, lungs were harvested at 4 and 15 days after treatment, respectively. Expression of 96 genes, including chemokines, their receptors, and growth factors, was analyzed by RT2 Pro- filer PCR Array for mouse chemokines and receptors and RT2 Profiler PCR

Array for mouse growth factor (SuperArray). Lung tissues were homoge- 51.4% (14.7)15.1% ( Ϯ 5.8)93.1% ( Ϯ 5.7) 66.1%21.2% ( Ϯ 15.9) ( Ϯ 11.7) 2.4% ( Ϯ 1.1) 40.5% ( Ϯ 20.2) 1.8% ( Ϯ 1.5) 0.0% 14.6% ( Ϯ 4.3) 0.0% 35.4% 0.0% ( Ϯ 7.4) 15.2% ( Ϯ 6.1) nized with TRIzol reagent (Life Technologies), and total RNA was iso- lated. cDNA was synthesized by extension of primers using the Reaction- http://www.jimmunol.org/ Ready First Strand cDNA Synthesis Kit (SuperArray). ( Ϯ 0.3) ( Ϯ 0.4) ( Ϯ 2.1) ( Ϯ 0.4) a a a a 2.0 Isolation of lung-infiltrating lymphocytes 2.0 1.0 13.0 Lung tissues were mechanically cut and stirred in a suspension of RPMI 1640 medium (Invitrogen) supplemented with HEPES buffer (25 mM), sodium pyruvate (1 mM), nonessential amino acid (100 ␮M), penicillin ( Ϯ 4.6) ␮ ( Ϯ 16.3) ( Ϯ 4.4) ( Ϯ 16.7) a a a (100 U/ml), and streptomycin (100 g/ml), along with 0.1% collagenase a type XI (Sigma-Aldrich) and 0.002% DNase (Sigma-Aldrich) for enzy- 8.0 18.0 50.0

matic digestion O/N at RT. The suspension was filtered through a cell 145.5 strainer and washed with PBS twice. Lymphocytes were separated using by guest on September 26, 2021 Ficoll-Paque density centrifugation. Buffy coat enriched in lymphocytes was collected and washed with PBS twice. Lymphocytes isolated from ( Ϯ 4.6) lung tissues were suspended in medium. ( Ϯ 2.8) ( Ϯ 4.9) ( Ϯ 9.2) a a a a ELISPOT assay ELISPOT assays were performed, as described previously (23). Briefly, MultiScreen 96-well filtration plates (Millipore) were coated O/N at 4°C with 5.0 ␮g/ml capture mouse cytokine-specific mAb (BD Biosciences) in 0.05 M carbonate-bicarbonate buffer (pH 9.6). The plates were blocked with 5% BSA for 2 h and washed three times with PBS. Subsequently, 3 ϫ 105 cells were cultured in triplicate in the presence of ColV (20 ␮g/ml) or 71.1% ( Ϯ 15.3)15.4% ( Ϯ 5.9) 48.0 81.8% ( Ϯ 12.2) 17.5 12.5% ( Ϯ 7.1) 52.0 8.0 human K-␣1 tubulin (1 ␮g/ml) and irradiated feeder autologous spleno- cytes (1:1 ratio). After 48–72 h, the plates were washed three times with ␮

PBS and three times with 0.05% PBS/Tween 20. Then, 2.0 g/ml biotin- ( Ϯ 0.4) ( Ϯ 0.2) ( Ϯ 1.2) ( Ϯ 0.2)

ylated mouse cytokine-specific mAb (BD Biosciences) in PBS/BSA/ a a a a

Tween 20 was added to the wells. After an O/N incubation at 4°C, the 2.0 2.0 6.7 2.0 plates were washed three times, and HRP-labeled streptavidin (BD Bio- sciences) diluted 1/100 in PBS/BSA/Tween 20 was added to the wells. After 2 h, the assay was developed with 3-amino-9-ethylcarbazole sub-

strate reagent (BD Biosciences) for 5–10 min. The plates were washed with ( Ϯ 5.6) ( Ϯ 13.9) ( Ϯ 20.9) ( Ϯ 7.6) tap water to stop the reaction and air dried. Spots were analyzed in an a a a a

ImmunoSpot Series I analyzer (Cellular Technology), and the results were 33.0 17.7 74.0 22.7

expressed as spots per million cells Ϯ SE. Any spots obtained by culturing Cellular Infiltration around Bronchiole Cellular Infiltration around Vessel T cell lines with APCs alone without ColV or human K-␣1 tubulin were subtracted from the number of spots in the test cultures. ( Ϯ 17.9) ( Ϯ 4.2) ( Ϯ 17.9) ( Ϯ 2.1) a a a a ELISA CD11b CD4 CD8 Total CD11b CD4 CD8 Total ELISA plate (Nunc) was coated with pure ColV (BD Biosciences; 1 ␮g/ 34.3 54.3 ml) in PBS O/N at 4°C. Serum samples were collected from mice treated with anti-H2Kd Ab or isotype Ab and nontreated mice. Serum samples d d were tested (1:250 and 1:500) for binding to ColV. Detection was done C1.18.4 18.3 C1.18.4 12.3 with anti-mouse IgG, IgM-HRP, and development using tetramethylben- Morphometric analysis of lesions developed in lungs by anti-MHC class I Ab zidine substrate and read at 450 nm. A sample was considered as positive Per high power field. 30 H2K Day15 Ab H2K if the values were over an average cutoff values obtained from normal sera a

from 10 different mice. Concentration of Abs was calculated based on a Table I. 312 Abs TO MHC CLASS I INDUCE AUTOIMMUNITY Downloaded from http://www.jimmunol.org/

FIGURE 2. Administration of anti-MHC class I Abs induces significant increase in the CD4ϩ and CD11bϩ cells in the lungs. Anti-H2Kd (A, D, G, J, and M) or control (C1.18.4) Ab (B, E, H, K, and N) or anti-keratin Ab (C, F, I, L, and O) was administered endobronchially in BALB/c mice on days 1, 2, 3, and 6, and weekly thereafter. The lungs were harvested on day 15 or 30 and analyzed for infiltration of CD4ϩ T cells, CD11b, and C4d deposition by immunohistochemistry using mouse anti-CD4, anti-mouse CD11b Abs, and anti-mouse C4d Abs. CD4ϩ cells infiltrated around by guest on September 26, 2021 the bronchiole (arrowhead) and vessel at both 15 and 30 days (A and G), along with significant infiltration of CD11bϩ cells around the bronchiole (arrowhead) and vessel at 15 and 30 days (D and J) in anti-H2Kd-administered mice, whereas no cellular infiltration around the bronchiole and vessel was observed in isotype control (B, E, H, and K) or anti-keratin Ab-administered mice (C, F, I, and L). A significant increase in the C4d deposition was observed in the lungs of anti-H2Kd-administered mice (M) when compared with control Ab-administered mice (N and O). A significant increase in the levels of CD4ϩ T cells and CD11bϩ cells was observed in the lungs of mice treated with anti-H2Kd Ab compared with mice treated with isotype control Ab. standard curve using the binding of known concentration of anti-ColV Abs (7), and lung (8) allografts. To determine the consequences of (Santa Cruz Biotechnology). anti-MHC class I Ab binding to the organ, we administered intra- ELISA to detect anti-K-␣1 tubulin Abs was done in a similar manner ␣ ␮ bronchially into the lungs of BALB/c mice mAb with specificity to using purified recombinant human K- 1 tubulin (1 g/ml). Concentration d of anti-K-␣1 tubulin Abs was expressed as ␮g/ml based on a standard H2K (anti-MHC class I Ab). C1.18.4 control Ab of the same curve using the binding of known concentration of anti-K-␣1 tubulin Abs isotype was administered in control animals. (Santa Cruz Biotechnology). Fifteen and 30 days after administration of Abs, the lung tissue Neutralization of IL-17 was harvested and histopathological analysis was performed. Lungs from animals administered with anti-MHC class I Ab after To study the role of IL-17 in induction of autoimmunity following ligation 15 days demonstrated significant inflammatory cells around the of anti-MHC class I Abs, we administered on days 1, 3, and 5, and weekly thereafter 50 ␮g of anti-IL-17 mAb (eBioscience) i.v. into BALB/c mice vessels and bronchiole (Fig. 1A). In addition, there was epithelial treated with anti-MHC class I Ab on days 1, 2, 3, and 6, and weekly hyperplasia (Fig. 1D) and an increase in fibrosis. Morphometric thereafter (24). Serum from these mice was collected on day 30 following analysis of these lesions demonstrated 51.4% cellular infiltration ␣ anti-MHC class I Ab treatment to quantitate the levels of Abs against K- 1 around the vessels, 71.1% cellular infiltration around the bronchi- tubulin and ColV, as described earlier. Histopathological analysis of the lungs was also performed to determine the levels of cellular infiltration and ole, 71.1% epithelial hyperplasia, and 15.2% fibrosis (Table I). In epithelial hyperplasia by H&E staining. Fibrosis in the lungs was analyzed contrast, C1.18.4- or anti-keratin Ab-administered lungs did not by trichrome staining. show any significant lesions (Fig. 1, B, C, E, and F). All of the above lesions were markedly increased on day 30. Results Cellular infiltration around the vessels and bronchiole was in- Ligation of MHC class I molecules of the lung parenchyma by creased to 93.1 and 81.8%, respectively (Fig. 1G and Table I). specific anti-MHC class I Abs results in autoimmune cellular More significant is our finding of an increase in fibrosis (35.4%) infiltration, endotheliitis, epithelial hyperplasia, and fibrosis (Fig. 1J) along with 14.6% lumen occlusion of the small airways Development of anti-donor MHC Abs has been strongly associated (Fig. 1J and Table I). Lungs from animals treated with control with early development of chronic rejection in (6), heart C1.18.4 Ab or the anti-keratin Abs did not demonstrate any of the The Journal of Immunology 313 Downloaded from http://www.jimmunol.org/

FIGURE 3. Administration of anti-MHC class I Ab increases the frequency of IFN-␥-, IL-4-, and IL-17-secreting T cells against K-␣1 tubulin and ColV in the lung. A, Frequency of the IFN-␥-secreting T cells against K-␣1 tubulin and ColV. B, Frequency of the IL-4-secreting T cells against K-␣1 tubulin and ColV; C, frequency of the IL-17-secreting T cells against K-␣1 tubulin and ColV. Anti-H2Kd or control Ab was administered endobronchially in BALB/c mice on days 1, 2, 3, and 6, and weekly thereafter. The lungs were harvested on days 15 and 30. T cells infiltrating the lungs were harvested by collagenase digestion, and the frequency of T cells secreting IFN-␥, IL-4, and IL-17 on stimulation with K-␣1 tubulin and collagen was analyzed by ELISPOT. The values are represented as means Ϯ SD using data obtained from five ELISPOT assays. u, Represent the isotype control; patterned bars, represent the T cells isolated from lungs on day 15; and f, represent the T cells isolated on day 30. A significant increase in the frequency of IFN-␥-, IL-4-, by guest on September 26, 2021 ;p Ͻ 0.05 ,ء) and IL-17-secreting T cells against K-␣1 tubulin and ColV was observed in mice administered anti-H2Kd Ab when compared with controls control vs anti-MHC class I Ab).

above changes (Fig. 1, H, I, K, and L). Similarly, administration of anti-MHC class I Ab administration and stimulated with K-␣1 tu- anti-H2Kb Abs into the C57BL/6 mice or the W6/32 Abs in the bulin or ColV in the presence of autologous splenocytes. As shown HLA-A2 transgenic C57BL/6 mice resulted in epithelial hyperpla- in Fig. 3, A and B, anti-MHC class I Ab-administered animals had sia, induction of fibrosis, and occlusion of the small airways with a significant increase in K-␣1 tubulin- and ColV-reactive T cells in a similar kinetics as observed in the anti-H2Kd-administered comparison with C1.18.4-administered animals. These autoreac- BALB/c mice (data not shown). Moreover, histopathological anal- tive T cells include K-␣1 tubulin-reactive cells capable of secret- ysis of the other organs, i.e., kidney, heart, liver, and pancreas, ing IFN-␥ (80.0 vs 43.3) and IL-4 (33.9 vs 0), and Col V-reactive showed no significant morphological changes following adminis- cells secreting IFN-␥ (71.1 vs 17.2) and IL-4 (44.2 vs 0). The tration of the anti-MHC class I Abs (data not shown). response of T cells isolated from the lungs on day 30 both against Immunohistochemical staining of the cellular infiltration dem- K-␣1 tubulin and ColV, respectively, was significantly elevated, as onstrated a predominance of CD4ϩ cells (Fig. 2, A and G, and follows: IFN-␥ (171.1 vs 65.0), IL-4 (38.3 vs 6.3), IFN-␥ (101.4 vs Table I) along with CD11bϩ (Fig. 2, D and J, and Table I) mac- 28.6), and IL-4 (45.1 vs 5.0). This observation indicates that ad- rophages around the bronchiole on days 15 and 30. In addition, a ministration of anti-MHC class I Abs increases the activation of small percentage of CD8ϩ T cells was seen around the vessel, but autoimmune responses against the self-Ags, ColV and K-␣1 not around the bronchiole when compared with controls treated tubulin. with C1.18.4 (Fig. 2, B, E, H, and K) or anti-keratin- Ab-administered mice (Fig. 2, C, F, I, and L). Ab binding and Increased ColV-specific IL-17 production by the lung-infiltrating complement deposition (C4d) on the lung parenchyma were noted T lymphocytes (Fig. 2M) in anti-H2Kd-treated mice, whereas no C4d deposition T cells, isolated from lung tissue at 15 and 30 days, after anti-MHC was observed in the controls (Fig. 2, N and O). class I Ab administration and stimulation with K-␣1 tubulin or ColV in the presence of autologous splenocytes, were analyzed by ␥ Lung-infiltrating lymphocytes secrete increased levels of IFN- ELISPOT to enumerate the frequency of IL-17-secreting T cells. ␣ and IL-4 upon stimulation with self-Ags K- 1 tubulin or ColV As shown in Fig. 3C, anti-MHC class I Ab-administered animals K-␣1 tubulin- and ColV-specific T lymphocytes that infiltrate the had a significant increase in K-␣1 tubulin/ColV-reactive T cells in lung following administration of anti-MHC class I Abs were enu- comparison with C1.18.4-administered animals, indicating that au- merated. T cells were isolated from lung tissue at 15 days after toreactive T cells include cells capable of secreting IL-17 at 15 314 Abs TO MHC CLASS I INDUCE AUTOIMMUNITY Downloaded from http://www.jimmunol.org/

FIGURE 4. Administration of anti-MHC class I Abs increases the ex- pression of chemokines, cytokines, and growth factor in the lungs. A, Ex- FIGURE 5. Administration of anti-MHC class I Abs leads to develop- by guest on September 26, 2021 pression levels of chemokines and their receptors. B, Expression levels of ment of autoantibodies against self-Ags K-␣1 tubulin and ColV. Anti- the cytokines. C, Expression levels of the growth factor. Anti-H2Kd or H2Kd or control Ab was administered endobronchially in BALB/c mice control Ab was administered endobronchially in BALB/c mice on days 1, (n ϭ 5 each) on days 1, 2, 3, and 6, and weekly thereafter. Sera were 2, 3, and 6, and weekly thereafter. The lungs were harvested on days 4 collected from the mice on days 15, 30, and 60. ELISA was performed for (chemokines) and 15 (cytokines and growth factor) and analyzed using a Abs to self-Ags in the serum of mice treated with anti-MHC Abs or control quantitative real-time PCR array. RNA was extracted from the lung tissue, Abs using purified K-␣1 tubulin or ColV as Ag. Administration of anti- and 1 ␮g of the RNA was reverse transcribed and used as template for the H2Kd Ab results in the increased concentrations of anti-K-␣1 tubulin (A)- quantitative real-time PCR array (chemokines and PCR array, and ColV (B)-specific Abs on days 15, 30, and 60 compared with control common cytokines PCR array, and growth factor PCR array; Superarray). mice treated with C1.18.4 Abs. Cycle threshold values obtained from the quantitative real-time PCR were analyzed by using the PCR array analysis software from Superarray. The samples were normalized using the expression levels of the housekeeping genes GAPDH and hypoxanthine phosphoribosyltransferase 1. The data into the lung, we analyzed for up-regulation of the genes for che- represent the average values obtained from three different experiments. A mokines and their receptors that may be involved in promoting significant (p Ͼ 0.05) increase in the expression of chemokines (CCL9, cellular infiltration into the lung and causing epithelial hyperplasia. 9.2; CCR2, 10.6; CXCL1, 6.35; and CXCL12, 8.0), cytokines (IL-10, 4.0; As shown in Fig. 4A, lungs harvested on day 4 following treatment IL-12b, 5.66; IL-17f, 12.17; Inhba, 13.0; Tnfsf11, 6.5; Tnfsf15, 8.0; Tnfsf with anti-MHC class I Ab showed a several fold increase in rela- 4, 21.11; CD40 lg, 5.28), and growth factors (Bmp6, 4.30; Bmp8a, 10.88; tion to C1.18.4 in the expression of results in an increased expres- Bmp8b, 5.28; Fgf6, 5.07; Fgf2, 37.46; and IL-4, 290.82) was observed in sion of CCL9 (9.2-fold, p Ͻ 0.01), CCR2 (10.6-fold, p Ͻ 0.01), mice administered anti-H2Kd compared with controls. CXCL1 (6.35-fold, p Ͻ 0.01), and CXCL12 (8.0-fold, p Ͻ 0.01). Other genes analyzed in this panel, which include genes for CC, CXC motif ligands, and their receptors, did not demonstrate any days (101.1 vs 13.6 for K-␣1 tubulin, and 39.2 vs 0 for ColV in significant change in comparison with C1.18.4-treated animals. anti-MHC class I Ab compared with isotype control, respectively) and at 30 days (173.0 vs 10.3 for K-␣1 tubulin, and 123.7 vs 8.2 Specific binding of MHC class I molecules of the lung for ColV). parenchyma with Abs results in up-regulation of several cytokines Specific binding of MHC class I molecules with Abs results in To analyze the genes that are activated following anti-MHC class up-regulation of chemokines and its receptors on the lung I Ab treatment, the expression of 96 cytokine genes in lympho- parenchyma cytes purified from lung tissues treated with anti-MHC class I Ab Because marked cellular infiltration and epithelial hyperplasia and C1.18.4 at 15 days after treatment were determined by RT2- were seen following the administration of anti-MHC class I Abs PCR array. As shown in Fig. 4B, lymphocytes purified from lung The Journal of Immunology 315

post-anti-MHC class I Ab administration, mice reacted with tubu- lin. In addition, sera obtained at 15, 30, and 60 days post-anti- MHC class I Ab administration also reacted to another self-Ag, ColV (Fig. 5B). These results concur with earlier findings from both human and animal LTx studies indicating that autoantibodies to self-Ags may play a role in the pathogenesis of chronic rejec- tion. Furthermore, our results indicate that anti-MHC class I Abs can indeed expose otherwise cryptic Ags, and immune responses to these Ags can be elicited following the development of anti- donor MHC Abs.

Neutralization of IL-17 results in decreased autoantibody production and fibrosis in the lung IL-17 is a pleiotropic cytokine implicated in autoimmune disorders and in development of autoantibodies. To determine the role of IL-17 in the mechanism by which anti-MHC class I Abs induce autoimmunity, we administered neutralizing anti-IL-17 Abs (eBioscience) or control Abs (50 ␮g) i.v. on day 1, 3, and 5, and

weekly thereafter into BALB/c mice administered anti-H2Kd Abs Downloaded from endobronchially. Analysis of the sera from these mice collected on day 30 showed a significant reduction in the development of au- toantibodies against K-␣1 tubulin and ColV by ELISA similar to the levels observed in the control Ab-treated mice (Fig. 6A). More FIGURE 6. Neutralization of IL-17 blocks the induction of autoimmune significantly, histopathological analysis of the lung showed a sig- responses and development of bronchial occlusion. Anti-H2Kd or control nificant reduction in the levels of cellular infiltration, epithelial http://www.jimmunol.org/ Ab was administered endobronchially in BALB/c mice on days 1, 2, 3, and hyperplasia, and fibrosis (Fig. 6B) compared with those animals 6, and weekly thereafter. A total of 50 ␮g of neutralizing anti-IL-17 or treated with anti-MHC class I Ab alone. These results indicate that control Abs was administered i.v. on days 1, 3, and 5, and weekly thereafter (n ϭ 5 each). Serum and lungs of these mice were harvested on day 30 and IL-17 plays a crucial role in the induction of autoimmunity fol- analyzed for the development of autoantibodies and histopathological anal- lowing administration of anti-MHC class I Abs into the lung. ysis. A, Quantitation of the anti-tubulin Abs by ELISA showed a 3-fold reduction in the concentrations of anti-tubulin Abs in IL-17-neutralized Discussion mice compared with controls. B, H&E staining of the lungs showed a Understanding the mechanisms for the immunopathogenesis of significant reduction in T cell infiltration and bronchial occlusion similar to chronic allograft rejection remains a major goal toward improving that observed in control mice treated with isotype control Ab. the long-term success of transplanted organs. There is considerable by guest on September 26, 2021 evidence that alloimmune response against the mismatched MHC Ags plays a pivotal role in the pathogenesis of chronic rejection tissues treated with anti-MHC class I Ab showed a significant in- (26). In the past, it was presumed that mostly T cell immune re- crease in the expression of IL-10, 4.0-fold; IL-12b, 5.66-fold; IL- sponses, including direct of the organ (27) or indirect 17f, 12.17-fold; Inhba, 13.0-fold; Tnfsf11, 6.5-fold; Tnfsf15, 8.0- Ag presentation of the mismatched allopeptides (28) resulting in fold; Tnfsf 4, 21.11-fold; and CD40 lg, 5.28-fold. both cytokine-mediated (23) and alloantibody complement-medi- ated damage of the organ (29), played a significant role in the Specific binding of MHC class I molecules of the lung pathogenesis of chronic rejection. There is emerging evidence for parenchyma with Abs results in up-regulation of growth factors an important role for alloantibodies in the pathogenesis of chronic on the lung parenchyma rejection of kidneys (6), heart (7), and lung (8). Previous studies Lungs harvested on day 30 postadministration of anti-MHC class from our laboratory have also shown not only a significant corre- I Abs demonstrated significant fibrosis and luminal occlusion of lation between the development of Abs during the posttransplant the bronchioles. To determine the growth factors that may be in- period, but also the emergence of detectable anti-HLA Abs months volved in this process, we analyzed expression of 96 well-known before the onset of BOS following LTx (30). It has been demon- growth factor genes in lungs harvested after 15 days of adminis- strated that anti-MHC class I Ab binding to the endothelial lining tering either anti-MHC class I or control Ab C1.18.4. As presented or to the epithelial surface can activate signaling cascades resulting in Fig. 4C, lungs treated with H2Kd demonstrated significant fold in growth factor production, leading to fibrosis and occlusion, a increase in the expression of many growth factors, including cardinal feature of all chronic rejection (31, 32). However, persis- Bmp6, 4.30-fold; Bmp8a, 10.88-fold; Bmp8b, 5.28-fold; Fgf6, tence of detectable anti-MHC alloantibodies is not a general fea- 5.07-fold; Igf2, 37.46-fold; and Ngfb, 290.82-fold. ture in allograft recipients undergoing chronic rejection (33), and there is controversy that the presence of Abs during the posttrans- ␣ Production of autoantibodies against self-Ags ColV and K- 1 plant period is merely a marker for other immunological events tubulin following administration of anti-MHC class I Abs occurring. In this communication, we present evidence that alloan- Development of autoantibodies against ColV and K-␣1 tubulin has tibody binding leads to autoantibody production, which is respon- been demonstrated in LTx recipients with chronic rejection, as sible for the pathogenesis of chronic rejection. We provide evi- well as in animal models of lung allograft rejection (25). To de- dence for the first time that anti-MHC class I Ab binding to the termine whether anti-MHC class I Ab administration into the lung lung parenchyma results in cellular infiltration of the lung, result- can result in autoantibody formation, we analyzed serial serum ing in endotheliitis and epithelial hyperplasia, leading to autoan- samples from the animals that developed lesions similar to chronic tibody production of two epithelial self-Ags, i.e., ColV and K-␣1 rejection following human LTx. As shown in Fig. 5A, 30 days tubulin. 316 Abs TO MHC CLASS I INDUCE AUTOIMMUNITY

In an allograft, all of the Abs directed to the mismatched MHC toantibodies can bind to the epithelial cells and other cells and Ags will have the capacity to bind to the allograft. To mimic this, activate them to produce bone morphogenetic protein (BMP), fi- we directly administered anti-MHC class I Ab to the lung via en- broblast growth factor (FGF) family of growth factors that may dobronchial route. Therefore, we believe that most, if not all, of the play an important role in the pathogenesis of BOS. These results Abs will have a chance to bind and saturate the lung MHC mol- are consistent with the notion that autoantibodies play a significant ecules before getting into the circulation. This assumption turned role in the pathogenesis of chronic rejection (10), as reported for a out to be valid because we found lesions in the lung and not in role for cardiac myosin and vimentin in the pathogenesis of cor- other organs. Specificity of the MHC Abs to cause the lesion was onary artery diseases following heart transplantation (13, 41). confirmed because isotype-matched C1.18.4 Abs failed to cause There is also strong evidence for autoreactivity to ColV in the the lesions (Fig. 1, B, E, H, and K). In addition, we also did not see pathogenesis of chronic rejection following human LTx (19), as any lesions when Abs to keratin, which binds to the epithelial cell well as animal models of lung allograft rejection (15). Both direct surface, were administered endobronchially (Fig. 1, C, F, I, and L). and indirect allorecognition pathways have been shown to contrib- Therefore, Ab binding alone was not sufficient to induce the lesion, ute to rejection following transplantation (42). Studies analyzing and specificity to MHC class I was required. Moreover, similar the immune responses to self-Ags following transplantation have lesions were observed in the C57BL/6 mice and the HLA-A2 clearly demonstrated that the indirect allorecognition pathway b transgenic C57BL/6 mice following administration of anti-H2K plays a crucial role (15, 43). Moreover, studies in the chronic lung and W6/32 Ab, respectively, with a similar kinetics demonstrating disease models have shown that there is an increase in the fre- that the observation is independent of strain and Ab. Therefore, the quency of the B cells in the lung following chronic injury, and the data presented in this study along with the earlier findings (32) B cells can provide help to the T cells by acting as APCs (44). Our Downloaded from indicate that anti-donor MHC Abs developed during the posttrans- preliminary analysis of the cells infiltrating the lung indicates that plant period will bind specifically to the allograft, resulting in the they are activated and secrete IFN-␥, IL-4, as well as IL-17 (Fig. lesions seen in chronic rejection. 3). In vitro studies demonstrate that this population consists of Cellular infiltration into the lung 15 days following treatment cells responding to K-␣1 tubulin as well as ColV. It is of interest with anti-MHC class I Ab was impressive. Initial infiltration was that the autoantigen ColV has been shown to participate in human noted around the small vessels, leading to endotheliitis (Fig. 1A). and animal models of lung allograft rejection (16, 43, 45, 46). This http://www.jimmunol.org/ This is consistent with the reports demonstrating that endotheliitis was followed by the development of Abs to both of these autoan- precedes clinical signs of chronic rejection in human LTx recipi- tigens. It is of interest that cells infiltrating the lung produced large ents (34). Analysis of the chemokine expression in the lungs by amounts of IL-17 following stimulation with both K-␣1 tubulin quantitative real-time PCR demonstrated a significant increase in and ColV (Fig. 3). IL-17 has been shown to be a potent proin- the expression of several chemokines (CCL9, 9.2-fold; CCR2, flammatory cytokine that acts on epithelial cells, ECs, fibrocytes, 10.6-fold; CXCL1, 6.3-fold; and CXCL12, 8.0-fold). CCL9 is a and stromal cells, leading to a proinflammatory signal that results member of the CC motif chemokine ligand that is expressed by the in enhanced proinflammatory cytokine and chemokine secretion epithelial cells and plays a critical role in mucosal immunity, tissue

(17). IL-17 has also been shown to play a crucial role in the in- by guest on September 26, 2021 homeostasis (35), and recruitment of dendritic cells (36). CCL9 duction of humoral autoimmune responses (18). Analysis of the T through its receptor CCR1 has been shown to play a role in in- cell responses to ColV in LTx recipients has been shown to be duction of autoimmunity as well as in transplant rejection (37). dependent on CD4 T cells and monocytes (19). This response was CCR2, a monocyte chemoattractant, has also been shown to be ␣ ␤ expressed in the lung epithelial cells and is thought to be involved found to be dependent on IL-17, TNF- , and IL-1 . The risk of in IL-13-mediated pathogenesis as well as in lung re-epithelializa- BOS development was observed to correlate with the severity of tion following injury (38). Studies in experimental animal model the ColV-specific T cell responses (19). Furthermore, in this study, have also shown that CCR2 plays a crucial role in recruitment of it was also shown that the interaction between IL-17 and mono- the leukocytes in the lung and contributes to rejection and devel- cytes is crucial in induction of the ColV-specific responses. IL-17 opment of BOS (39). CXCL12 has been shown to be critically has also been shown to play a crucial role in the formation of GCs involved in the GC formation, especially in response to self-Ags and to regulate the movement of the B cells in the GCs through (22). Based on our findings of up-regulation of several chemokines chemokine CXCL12 and CXCL13 (22). Studies have also dem- and their receptors (Fig. 4A) following administration of anti-MHC onstrated that a deficiency in the IL-17 or blocking of IL-17 results class I, it is reasonable to assume that anti-MHC class I binding in a decrease in the production of autoantibodies (20, 21). There- induces the signals for infiltration of inflammatory cells into the fore, our finding of IL-17-producing T cells following stimulation organ, which begins at the endothelial parenchymal junction. with self-Ags indicates that these T cells provide signals toward Alloimmune response to mismatched HLA Ags has been con- activation of B cells, resulting in Abs to self-Ags, such as K-␣1 sidered to be the primary insult leading to chronic rejection fol- tubulin and ColV. Moreover, as shown in Fig. 6A, neutralization of lowing human LTx (40). Frequency of indirect Ag presentation of IL-17 resulted in a significant reduction in the levels of Abs to the the allo-MHC has been shown to be increased in patients self-Ag K-␣1 tubulin. More significantly, neutralization of IL-17 undergoing chronic rejection (26). In addition, Abs to mismatched also resulted in abrogation of all of the lesions induced by anti- HLA Ags have been demonstrated in patients with BOS (5), and MHC class I Abs, i.e., a decreased cellular infiltration, epithelial more significantly, precede the development of clinical evidence of hyperplasia, and fibrosis in the lungs following administration of BOS (8, 30). Surprisingly, many patients with BOS have no de- anti-MHC class I Abs. These observations are in agreement with tectable Abs to mismatched HLA Ags, giving rise to the possibility the report that in IL-17-deficient mice, lack of IL-17 signaling that immune responses to non-HLA Ags may play a significant significantly reduces the induction of autoimmunity and down role in the pathogenesis of BOS. Our results clearly demonstrate modulates the formation of GCs leading to a reduction in the titer that anti-MHC class I Abs developed during the posttransplant of autoantibodies (22). Hence, we propose that IL-17 is a critical period upon binding to the organ expose otherwise cryptic deter- mediator in the induction of autoimmunity following ligation of minants on self-Ags, leading to immune responses against them. the anti-MHC class I Abs, resulting in the development of chronic Data presented in this study (Fig. 4C) also indicate that these au- rejection following LTx. The Journal of Immunology 317

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