Bronchiolitis After Lung Transplantation Lymphoid Tissue In

The Role of Intrapulmonary De Novo Lymphoid Tissue in Obliterative Bronchiolitis after Lung Transplantation

This information is current as Masaaki Sato, Shin Hirayama, David M. Hwang, Humberto of September 25, 2021. Lara-Guerra, Dirk Wagnetz, Thomas K. Waddell, Mingyao Liu and Shaf Keshavjee J Immunol 2009; 182:7307-7316; ; doi: 10.4049/jimmunol.0803606

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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

The Role of Intrapulmonary De Novo Lymphoid Tissue in Obliterative Bronchiolitis after Lung Transplantation1

Masaaki Sato,* Shin Hirayama,* David M. Hwang,*† Humberto Lara-Guerra,* Dirk Wagnetz,* Thomas K. Waddell,* Mingyao Liu,* and Shaf Keshavjee2*

Chronic rejection after lung transplantation is manifested as obliterative bronchiolitis (OB). The development of de novo lymphoid tissue (lymphoid neogenesis) may contribute to local immune responses in small airways. Compared with normal lungs, the lung tissue of 13 lung transplant recipients who developed OB demonstrated a significantly larger number of small, airway-associated, peripheral node addressin-positive (PNAd؉) high endothelial venules (HEVs) unique to lymphoid tissue (p < 0.001). HEVs were most abundant in lesions of lymphocytic bronchiolitis and “active” OB infiltrated by lymphocytes compared with those of “inactive” OB. T cells in lymphocytic bronchiolitis and active OB were predominantly of the CD45RO؉CCR7؊ effector memory phenotype. Similar lymphoid tissue was also observed in the rat lung after intrapulmonary transplantation of allograft trachea Downloaded from (Brown Norway (BN) to Lewis), but not after isograft transplantation. Subsequent orthotopic transplantation of the recipient ؋ Lewis lung containing a BN trachea into an F1 (Lewis BN) rat demonstrated stable homing of Lewis-derived T cells in the lung and their Ag-specific effector function against the secondary intrapulmonary BN trachea. In conclusion, we found de novo lymphoid tissue in the lung composed of effector memory T cells and HEVs but lacking delineated T cell and B cell zones. This de novo lymphoid tissue may play a critical role in chronic local immune responses after lung transplantation. The Journal of Immunology, 2009, 182: 7307–7316. http://www.jimmunol.org/

e novo lymphoid tissue formation or lymphoid neogen- However, formation of TLO after lung transplantation has not esis has recently been recognized as a potentially im- been reported and is controversial. Chronic rejection after lung D portant mechanism that supports chronic inflammation transplantation is manifested by obliterative bronchiolitis (OB), a in peripheral tissue (1). The best characterized de novo lymphoid chronic inflammatory and fibroproliferative condition in small air- tissue is the tertiary lymphoid organ (TLO),3 a structure resem- ways, and its clinical correlate, bronchiolitis obliterans syndrome bling a lymph node that includes high endothelial venules (HEVs), (BOS) (10). In the lung, TLO is considered to be represented by an well-defined T cell and B cell zones, and a germinal center ac- inducible form of bronchus-associated lymphoid tissue (BALT), by guest on September 25, 2021 companying follicular dendritic cells (2). TLO has been reported in namely iBALT (1, 11). Formation of iBALT has been reported in various chronic inflammatory conditions such as autoimmune (3, various chronic inflammatory lung diseases such as emphysema 4) and chronic infectious diseases (5, 6). (12), pulmonary fibrosis (13), and chronic hypersensitive pneumo- Chronic rejection after organ transplantation is a condition in nitis (14). In contrast, Hasegawa et al. demonstrated a negative which alloantigen persists and chronic inflammation occurs. In- result regarding the relationship between OB/BOS and BALT, deed, development of TLO has been reported in various tissues and which should theoretically include iBALT (15). Although the organs after transplantation, including human cardiac and renal study using transbronchial biopsies was limited in its sensitivity to allografts (7) and animal models of cardiac (8), skin (9), and vas- detect OB lesions and (i)BALT, it is possible that de novo lym- cular (7) allografts. phoid tissue after lung transplantation does not form the conventional structure of TLO with HEVs, T and B cell zones, and germinal center formation. *Latner Thoracic Surgery Research Laboratories, Toronto General Research Institute Interestingly, an evolving concept proposed by van Panhuys † and Department of Pathology, University Health Network, University of Toronto, et al. indicates the existence of “effector lymphoid tissue” (ELT) Toronto, Ontario, Canada that exerts effector function by collecting effector and effector Received for publication October 27, 2008. Accepted for publication March 31, 2009. memory T cells but does not necessarily take the conventional 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 anatomical form of TLO or iBALT (16). Increasing evidence sug- with 18 U.S.C. Section 1734 solely to indicate this fact. gests that the lung might be an important reservoir of effector and 1 This work was supported by the Canadian Cystic Fibrosis Foundation. M.S. is a effector memory T cells (17, 18). In a s.c. tracheal transplant model recipient of Wyeth/Canadian Institutes of Health Research Rx&D Industrial Partner of OB, preferential localization of effector memory CD4ϩ and Program Fellowship (2005–2007) and Canadian Cystic Fibrosis Foundation Postdoc- ϩ toral Fellowship (2007–2009). CD8 T cells to the lung parenchyma and airways has been dem- 2 Address correspondence and reprint requests to Dr. Shaf Keshavjee, Toronto Lung onstrated (19). Transplant Program, Toronto General Hospital, 200 Elizabeth Street 9N947, Toronto, Thus, we hypothesized that lymphoid neogenesis plays an im- Ontario, Canada M5G 2C4. E-mail address: [email protected] portant role in chronic rejection after lung transplantation. To ad- 3 Abbreviations used in this paper: TLO, tertiary lymphoid organ; BALT, bronchus- dress the hypothesis, we conducted careful histological and immu- associated lymphoid tissue; BN, Brown Norway; BOS, bronchiolitis obliterans; ELT, effector lymphoid tissue; HEV, high endothelial venule; iBALT, inducible BALT; nohistochemical analyses of the lungs that were explanted at the MAdCAM, mucosal addressin cell adhesion molecule; OB, obliterative bronchiolitis; time of retransplantation due to OB/BOS after the initial lung PNAd, peripheral node addressin. transplantation. Furthermore, we have devised an animal model Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 that combines a rat intrapulmonary tracheal transplant model of www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803606 7308 LYMPHOID NEOGENESIS AFTER LUNG TRANSPLANTATION

Table I. Demographics of lung transplant recipients who were diagnosed with OB/BOSa

Age at Time from 1st Primary Examined Lung No. of Lymphocytic No. of Active OB No. of Inactive OB Sex 2nd Tx Tx (mo) Diagnosis Area (cm2) Bronchiolitis (area in cm2) (area in cm2) (area in cm2)

Male 20 10.4 CF 4.30 3 (2.6) 2 (0.5) 2 (0.5) Female 36 10.7 CF 5.91 3 (2.0) 6 (1.0) 0 (0) Female 20 21.5 CF 1.15 0 (0) 1 (0.9) 0 (0) Male 36 34.2 BE 1.93 2 (0.4) 3 (1.6) 7 (3.6) Female 26 35.0 IPF 8.62 7 (4.0) 7 (0.8) 12 (1.4) Male 57 40.6 COPD 12.82 5 (5.8) 4 (0.3) 10 (0.8) Female 60 52.2 IPF 12.08 0 (0) 0 (0) 23 (1.9) Male 23 69.3 OB 3.85 6 (3.3) 2 (0.5) 0 (0) Female 40 82.2 IPF 3.76 4 (2.1) 2 (0.5) 4 (1.1) Female 53 88.2 OB 17.67 0 (0) 1 (0.1) 0 (0) Female 48 92.2 BE 3.90 6 (2.1) 5 (1.3) 0 (0) Female 35 132.1 OB 6.37 7 (2.2) 9 (1.4) 11 (1.7) Female 39b 18.6b BE 3.32 0 (0) 1 (1.2) 7 (2.1) Average Ϯ SEM 38.0 Ϯ 3.8 52.9 Ϯ 10.4 6.59 Ϯ 1.4 3.31 Ϯ 0.8 (1.89 Ϯ 0.5) 3.31 Ϯ 0.8 (0.77 Ϯ 0.1) 5.85 Ϯ 1.9 (1.00 Ϯ 0.3)

a Tx, Transplantation; BE, bronchiectasis; CF, cystic ﬁbrosis; COPD, chronic obstructive pulmonary disorder; IPF, idiopathic pulmonary ﬁbrosis; OB, obliterative bronchiolitis (idiopathic or secondary to hematopoietic stem cell transplantation). b This patient underwent open lung biopsy. The age at 2nd Tx indicates the age at the time of open lung biopsy. The other 12 patients underwent retransplantation for diagnosed OB/BOS after the initial lung transplantation. Downloaded from

OB (20) and orthotopic lung transplantation to examine the effec- Histology and immunofluorescence labeling tor function of intrapulmonary de novo lymphoid tissue after Formalin-fixed, paraffin-embedded tissues cut into 5-␮m-thick sections transplantation. were used for standard H&E stain, elastic trichrome stain, and all human immunofluorescence labeling studies. CD3 (rabbit anti-human), CD20 (mouse anti-human), Ki-67 (mouse anti-human) (DakoCytomation), Ki-67

Materials and Methods http://www.jimmunol.org/ (rabbit anti-human, Lab Vision), CD45RO (Serotec), peripheral node ad- Tissue samples of BOS lungs and normal control lungs dressin (PNAd; BD Biosciences), and CCR7 (Novus Biologicals) were Human tissue samples of OB/BOS lungs were obtained from patients dur- used with the appropriate isotype-specific Ig as negative control. For rat ing their follow-up at Toronto General Hospital (Toronto, Canada). Twelve specimens, the following primary Abs reported to react with rat tissue were lung specimens were obtained from patients with established diagnoses of used: rabbit anti-human CD3 (DakoCytomation) and mouse anti-human BOS at the time of retransplantation and one lung specimen was obtained CD79a (Abcam) for formalin-fixed paraffin-embedded or frozen sections; at the time of open lung biopsy, which confirmed the diagnosis of OB. and mouse anti-rat mucosal addressin cell adhesion molecule (MAd- Fifteen control lung tissue samples were obtained from patients with early CAM)-1 (BD Biosciences), rabbit anti-human von Willebrand factor (Mil- u stage lung cancer without any evidence of other previous or current pul- lipore), and anti-rat RT1A (OX27; Serotec) for frozen sections. Immuno- monary disorders (age, 57.85 Ϯ 4.0 (mean Ϯ SEM); seven males and eight fluorescence labeling was conducted as described previously (22). female). The human study was approved by the Research Ethics Board of by guest on September 25, 2021 University Health Network, University of Toronto (Toronto, Canada). Evaluation and quantification of airway lesions and lymphocyte aggregates Animal models In the human study, serial sections were cut from two to five blocks ob- Male Brown Norway (BN; RT1Au) and Lewis rats (RT1Al) were pur- tained from different areas of a single lung specimen and stained for OB/ chased from Charles River Laboratories. F1 rats were bred from male BN BOS, H&E, elastic trichrome, and CD3/CD20. H&E and trichrome stain- and female Lewis rats at the Toronto General Research Institute (Toronto, ing was used to identify bronchioles, whereas for individual bronchioles Canada). Intrapulmonary tracheal transplantation was conducted as previ- H&E and CD3/CD20 staining was used to evaluate the extent of inflam- ously described (20). Animals were sacrificed at postoperative days 7, 28, mation and lymphocyte infiltration. Trichrome staining was used to eval- 60, and 120 for the following analyses (n ϭ 5 for each time point and uate lumenal obliteration. Bronchioles with lymphocyte infiltration and group). without lumenal obliteration were classified as lymphocytic bronchiolitis; Intrapulmonary tracheal transplantation followed by orthotopic lung bronchioles with obliteration were classified as OB, among which those transplantation (Fig. 5A). Rat intrapulmonary tracheal transplantation was with lymphocyte infiltration in or around the bronchiole were classified as conducted as described above at day 0. After 7 or 28 days, the left lung of “active OB” and those without such infiltration were classified as “inactive the intrapulmonary tracheal transplant recipient was used as a donor lung OB.” Each bronchiole was also evaluated for HEVs using PNAd staining. for orthotopic left lung transplantation using surgical techniques described In the animal study, the size of lymphocyte aggregates was morpho- previously (21). The recipient animals of orthotopic lung transplantation metrically classified using ImageJ, version 1.30 (W. Rasband, National ϩ were sacrificed 28 days after lung transplantation (i.e., day 35 or 56 after Institute of Mental Health, Bethesda, MD). The number of CD3 T cells the initial intrapulmonary tracheal transplantation; n ϭ 4 for each group). infiltrating into the tracheal graft was counted in 10 randomly selected high power fields confined to the obliterated area. Intrapulmonary tracheal transplantation followed by orthotopic lung ϩ transplantation in combination with concurrent second intrapulmonary To standardize the number of HEV bronchioles (human study) and the and s.c. tracheal transplantation (Fig. 8A). Rat intrapulmonary allograft size of lymphocyte aggregates (animal study), the whole lung area was tracheal transplantation (BN to Lewis) was conducted as described above. scanned using SNAPSCAN e50 (Agfa) and the size was calculated using After 28 days, the left lung of the initial intrapulmonary tracheal transplant ImageJ software. recipient was used as a donor lung for orthotopic left lung transplantation. After reperfusion of the left lung graft, a secondary tracheal graft from a Flow cytometric analysis BN rat was implanted in the left lung adjacent to the first intrapulmonary The following anti-rat Abs and appropriate IgG isotype controls were pur- tracheal graft using the same technique as regular intrapulmonary tracheal chased from BD Pharmingen: FITC-labeled CD3 (G4.18), PE-Cy5-labeld transplantation. Another tracheal graft from a BN rat was implanted in the CD4 (OX35), Alexa Fluor 647-labeled CD8 (OX8), PE-labeled CD45RC dorsal s.c. tissue through the site of thoracotomy. The recipient animals of (OX22), and PE-labeled RT1Au (OX27). Cell surface staining was con- orthotopic lung transplantation were sacrificed 28 days after the concurrent ducted as described previously (22). transplantation (i.e., 56 days after the initial intrapulmonary tracheal transplantation; n ϭ 4). Statistics All animals received care in compliance with the Guide to the Care and Use of Experimental Animals formulated by the Canadian Council on An- Data are expressed as means Ϯ SEM. When comparing two groups, data imal Care. The experimental protocol was approved by the Animal Care were analyzed with t tests. When comparing three groups, one-way Committee of the Toronto General Research Institute. ANOVA was followed by post hoc Tukey tests. All statistical analyses The Journal of Immunology 7309 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 1. Effector memory T cells in airways after lung transplantation. A, H&E staining of a normal airway and lesions of lymphocytic bronchiolitis, active OB, and inactive OB (top panels) and the corresponding immunofluorescence labeling for T cell (CD3) and B cell (CD20) staining (bottom panels). B, Immunofluorescence labeling for CD3 and CD45RO, a marker of memory T cells. Pictures were taken from the same active OB lesions as in A. C, Immunofluorescence labeling for CD45RO and CCR7, a marker for naive and central memory T cells. Scale bar, 50 ␮m. NC, Negative control.

were performed using JMP 5.0 (SAS Institute). Values of p Յ 0.05 were whereas inactive OB lesions and normal lungs contained only a considered to be significant. small number of T cells and B cells (Fig. 1A). In lymphocytic bronchiolitis and active OB lesions, the memory T cell marker Results CD45RO was largely colocalized to CD3ϩ T cells, demonstrat- Effector memory T cells in small airways of BOS lungs ing that T cells in active inflammatory lesions in BOS lungs are We examined 13 human lungs from patients that developed mostly memory T cells (Fig. 1B). Furthermore, CCR7, a che- BOS after lung transplantation and compared them with 15 nor- mokine receptor expressed by naive and central memory T cells mal lung controls. Patient demographics are shown in Table I. but not by effector and effector memory T cells (23), was not ϩ Histologically, all of the posttransplant lungs contained multi- localized to CD45RO cells, demonstrating that memory T cells in ple OB lesions and 11 of them also contained airway inflam- BOS lungs are of an effector memory phenotype (Fig. 1C). mation without obliteration (lymphocytic bronchiolitis). Among 164 OB lesions examined, 65 (39.6%) were classified as “active” lesions while the others were classified as “inactive” Development of high endothelial venules in small airways after based on H&E, elastic trichrome, and CD3/CD20 staining as lung transplantation described in Materials and Methods. The aggregates of effector memory T cells in BOS lungs do not In immunofluorescence labeling for T cells and B cells, we completely meet the anatomical criteria of secondary or tertiary observed a number of T cells and a relatively small number of lymphoid tissue because they do not include segregated T cell and B cells in lymphocytic bronchiolitis and active OB lesions, B cell zones (Fig. 1A) or B cell follicles positive for CD21ϩ 7310 LYMPHOID NEOGENESIS AFTER LUNG TRANSPLANTATION

FIGURE 2. Development of HEVs

in small airways after lung transplan- Downloaded from tation. A, H&E staining, trichrome staining, and double immunofluorescence labeling for CD3 and PNAd, a marker of high endothelial venules. Representative pictures of a normal bronchiole (top) and lesions of lymphocytic bronchiolitis (second from http://www.jimmunol.org/ top), active OB with lymphocyte infiltration in the lumen (third from top) active OB with lumenal fibrosis and peribronchiolar lymphocyte aggregates (fourth from top), and two inactive OB lesions (fifth and sixth from top, only one of which shows positive PNAd staining). B, Higher magnification of a PNAdϩ HEV in an active by guest on September 25, 2021 OB lesion. Scale bar, 100 ␮m. NC, Normal control.

follicular dendritic cells (data not shown). Conversely, immuno- PNAd, a marker specific for HEVs, demonstrated a large number fluorescence labeling for Ki-67 (a cell proliferation marker) dem- of HEVs composed of characteristic cuboidal endothelial cells in onstrated a relatively small number of proliferating B cells as well the airways of BOS lungs, whereas bronchioles in normal lungs as T cells in the lymphoid tissue (data not shown). Although his- did not show HEVs (Fig. 2). Because HEVs positive for PNAd are tologically obvious germinal centers were not identified in the considered to be unique to secondary and tertiary lymphoid organs, study, T cell-dependent B cell maturation is still theoretically a a similar finding in a nonlymphoid organ was surprising to us. possible component of the intrapulmonary lymphoid tissue. Through quantitative evaluation we found that HEVs existed in HEVs are specialized endothelial lined vessels that exist almost all of the lesions of lymphocytic bronchiolitis and active uniquely in lymphoid tissue and play critical roles in lymphocyte OB lesions in the bronchiolar wall (Fig. 2A), whereas a portion of trafficking (24). Interestingly, immunofluorescence labeling for inactive OB lesions were also accompanied by a small number of The Journal of Immunology 7311

cipient lungs at day 28 demonstrated that Ͼ 90% of T cells in the lung tissue are of the CD45RCϪ memory phenotype after allograft tracheal transplantation at day 28, whereas mediastinal lymph nodes and spleen tissue showed smaller numbers of memory T cells (Fig. 4E). Although demonstration of HEVs in rat lungs was limited by the unavailability of an anti-rat PNAd Ab, we used MadCAM-1, another marker of HEVs, in combination with von Willebrand factor, a marker of vascular endothelial cells. Indeed, we observed MadCAM-1ϩvon Willebrand factorϩ HEVs in well- developed lymphocyte aggregates (Fig. 4F). These observations indicate that lymphocyte aggregates that develop after intrapulmonary allograft tracheal transplantation are likely to be de novo lymphoid tissue, similar to those observed in transplanted human FIGURE 3. HEVs are associated with lymphocytic bronchiolitis and active OB. A, Quantification of the number of bronchioles positive for PNAd lungs. demonstrates that most of the lesions of lymphocytic bronchiolitis (LB) Early lymphocyte aggregates are insufficient for allograft and active OB accompany HEVs. B, Quantification of the number of bron- airway rejection chioles positive for PNAd demonstrates a significantly larger number of bronchioles accompanying HEVs in the lung affected by BOS compared We subsequently addressed the question regarding whether or not with that of normal lung control (p Ͻ 0.01; n ϭ 15 and 13 in control and the lymphocyte aggregates become a stable structure in association Downloaded from BOS, respectively; the mean values are indicated by filled bars). with the stable homing of memory T cells. Following initial intrapulmonary allograft tracheal transplantation (BN to Lewis), we conducted orthotopic transplantation of the Lewis lung containing ϫ HEVs (Fig. 3A). In some active OB lesions, HEVs were also ob- a BN tracheal graft into an F1 (BN Lewis) rat at day 7 or day 28 served in the lumen of obliterated airways, suggesting that the (Fig. 5A). Because F1 rats should accept both Lewis-derived and induction of HEVs can occur through angioneogenesis. BN-derived grafts, the only components that could reject BN-de- http://www.jimmunol.org/ The percentages of lymphocytic bronchiolitis lesions, active rived grafts are Lewis-derived lymphocytes that may persist in the

OB lesions, and inactive OB lesions that are accompanied by lung after orthotopic lung transplantation into an F1 rat. HEVs is shown in Fig. 3A. The number of bronchioles with In the first set of experiments wherein orthotopic lung trans- accompanying HEVs per unit lung area was significantly larger plantation was conducted at day 7, peribronchiolar lymphocyte in BOS lungs than in normal lungs (Fig. 3B; p Ͻ 0.01). The aggregates in the Lewis lung disappeared and the BN graft showed small number of HEVs observed in normal lungs is considered recovery of the epithelium with little subepithelial fibrosis at day to represent constitutive BALT. 35 (Fig. 5B). The “negative” control of Lewis-Lewis-F1 transplantation also demonstrated minimum lymphocyte aggregates and Alloantigen-dependent ELT formation after rat intrapulmonary complete recovery of the tracheal graft in contrast to the “positive” by guest on September 25, 2021 tracheal transplantation control of BN-Lewis-Lewis transplantation in which graft rejec- A rat intrapulmonary tracheal transplant model of OB is an animal tion should continue, and indeed lymphocyte aggregates were still model of OB that reflects the influences of the pulmonary milieu observed in the lung with obliterated BN trachea (Fig. 5B). Mor- (20, 22). This model enables examination of the pulmonary im- phometric quantification demonstrated significantly larger lym- mune system’s reaction to allogenic stimuli. In the present study, phocyte aggregates in BN-Lewis-Lewis transplantation at day 35 we examined whether lymphoid tissue also develops in the lung in compared with the other two groups (Fig. 5C). Thus, the intrapul- fully MHC-mismatched intrapulmonary tracheal transplantation monary lymphocyte aggregates at day 7 are still immature and (BN donors to Lewis recipients) and compared them with syngenic incapable of stably harboring memory T cells. intrapulmonary tracheal transplantation (Lewis to Lewis). Conversely, the second set of experiments wherein orthotopic In normal lungs of Lewis rats (i.e., without transplantation), we lung transplantation was conducted at day 28 demonstrated stable observed a small number of lymphocyte aggregates that are con- homing of memory T cells. In BN-Lewis-F1 transplantation, the sistent with the reported constitutive BALT in rats (25). After in- lymphocyte aggregates in the peribronchiolar tissue were obvi- trapulmonary tracheal transplantation, the recipient lungs of both ously enlarged compared with those in the other two groups and isografts and allografts showed lymphocyte aggregates around extended into the perigraft tissue at day 56 (i.e., 28 days after bronchioles at day 7 (Fig. 4A). By day 28, the lymphocyte aggre- orthotopic lung transplantation) (Fig. 6A). The “negative” control gates were diminished in isograft recipients, whereas allograft re- of Lewis-Lewis-F1 transplantation showed the small size of lym- cipient lungs exhibited dense lymphocyte aggregates composed of phocyte aggregates, whereas those of the “positive” control of BN- T cells as well as B cells (Fig. 4B). At day 28, isografts showed Lewis-Lewis transplantation were similar to those of the untreated complete recovery of the epithelial cells with no evidence of oblit- allograft transplantation control. Morphometric quantification con- erative fibrosis, while allografts showed total loss of the epithelium firmed the significantly larger size of lymphocyte aggregates in the Ͻ with obliterative airway fibrosis (Fig. 4B, insets). The lymphocyte group of BN-Lewis-F1 transplantation ( p 0.05; Fig. 6B). aggregates in allograft recipient lungs persisted until the end of Flow cytometric analyses demonstrated that a significant per- observation at day 120 (Fig. 4C). Morphometric quantification centage of Lewis-derived RT1Au(Ϫ) CD4ϩ T cells and CD8ϩ T demonstrated a significantly larger number of lymphocyte aggre- cells exist in the left lung in BN-Lewis-F1 transplantation as com- gates in allograft recipients at day 28 and after (Fig. 4D). These pared with the other groups (Fig. 7A). Immunofluorescence label- results demonstrate that the persistence of lymphocyte aggregates ing for RT1Au in the lymphocyte aggregates in the lung after BN- in the lung is an alloantigen-dependent process. Lew-F1 transplantation demonstrated chimerism of F1-derived In rats, loss of the CD45RC expression is considered to repre- RT1Au(ϩ) cells and Lewis-derived RT1Au(Ϫ) cells (Fig. 7B). sent maturation into memory T cells, equivalent to CD45RO ex- Moreover, the majority of CD4ϩ T cells in the lung of the BN- pression in humans (23). Flow cytometric analysis of allograft re- Lewis-F1 transplantation group demonstrated a higher ratio of the 7312 LYMPHOID NEOGENESIS AFTER LUNG TRANSPLANTATION Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 4. Development of effector lymphoid tissue in airways after intrapulmonary tracheal transplantation. A and B, H&E staining (top panels) and double immunofluorescence labeling for CD3 (T cell) and CD79a (B cell) (bottom panels). Representative pictures of recipient lungs of intrapulmonary isograft and allograft tracheal transplantation at days 7 and 28 are shown. InsetsinB are corresponding tracheal grafts (original magnification, ϫ40). C, H&E staining of recipient lungs of intrapulmonary isograft and allograft tracheal transplantation at day 120. Arrows indicate peribronchiolar lymphocyte aggregates. D, Morphometric quantification of lymphocyte aggregates in the recipient lungs that received an isograft and an allograft. Allograft recipient lungs demonstrated a significantly larger number of lymphocyte aggregates than those of isograft recipient lungs at day 28 and after (p Ͻ 0.05). n ϭ 4–5; mean Ϯ SEM. E, Flow cytometric analysis of CD4 and CD45RC expression on CD3ϩ T cells in the spleen, mediastinal lymph nodes, and the left lung at day 28 after intrapulmonary allograft tracheal transplantation. Cells were first gated by CD3 positivity. F, Double immunofluorescence labeling for von Willebrand factor (vWF) and MadCAM-1 (top panel) and CD3 and CD79a (bottom panel) taken from a similar area of a well-developed lymphocyte aggregates at day 56 after intrapulmonary allograft tracheal transplantation. The arrow indicates a HEV-like vessel observed in the aggregate of T cells. Scale bar, 100 ␮m. NC, Negative control. The Journal of Immunology 7313 Downloaded from

FIGURE 6. Lymphocyte aggregates become mature by day 28 after intrapulmonary allograft tracheal transplantation. A, Result of intrapulmonary tracheal transplantation followed by orthotopic lung transplantation at http://www.jimmunol.org/ day 28. Representative pictures (H&E staining) of a recipient lung (top panels) and the corresponding intrapulmonary tracheal graft (bottom panels) at day 56 are shown. Scale bar, 500 ␮m. B, Morphometric quantiﬁ- cation at day 56 demonstrates a signiﬁcantly larger number of peribron-

chiolar lymphocyte aggregates in the recipient lung of BN-Lewis-F1 transplantation as compared with the other two groups (p Ͻ 0.05). n ϭ 4; mean Ϯ SEM. Lew, Lewis. by guest on September 25, 2021

because an F1 rat should not reject either Lewis or BN-derived tissue; however, the opposite is not true and, if there were significant Lewis-derived lymphocytes they could theoretically be reac-

tive to the F1 host-derived cells through a mechanism similar to graft-vs-host disease, which can lead to OB after hematopoietic FIGURE 5. Lymphocyte aggregates are immature at day 7 after in- stem cell transplantation (26). trapulmonary allograft tracheal transplantation. A, Study design of com- bined intrapulmonary tracheal transplantation (red) and orthotopic lung Memory lymphocytes homing to the lung exert effector function ء transplantation (blue), which was conducted at day 7 or day 28. , Lym- The previous experiment could not demonstrate the effector func- phoid tissue formation is minimal after isograft tracheal transplantation as tion of intrapulmonary lymphocytes against the airway graft be- shown in Fig. 4. B, A result of intrapulmonary tracheal transplantation followed by orthotopic lung transplantation at day 7. Representative pic- cause the allograft trachea was already obliterated when orthotopic tures (H&E staining) of a recipient lung (top panels) and the corresponding lung transplantation was conducted at day 28. Thus, we added a intrapulmonary tracheal graft (bottom panels) at day 35 are shown. Scale second intrapulmonary tracheal transplantation at the time of or- bar, 500 ␮m. C, Morphometric quantification at day 35 demonstrates a thotopic lung transplantation at day 28 (Fig. 8A). At day 56 after significantly larger number of peribronchiolar lymphocyte aggregates in the initial intrapulmonary tracheal transplantation (i.e., 28 days the recipient lung of BN-Lewis-Lewis transplantation as compared with the after orthotopic lung transplantation), the first intrapulmonary BN- other two groups (p Ͻ 0.05); n ϭ 4, mean Ϯ SEM. Lew, Lewis. derived tracheal grafts showed total loss of the epithelium and lumenal obliteration with fibrous tissue (Fig. 8Ba, top graft). The histological characteristics of the fibrous tissue of the first BN CD45RClow memory phenotype as compared with the other grafts were similar to those of untreated control allografts (Fig. groups (Fig. 7C). This ratio was also higher than those of the 3A). In contrast, the second intrapulmonary BN-derived tracheal opposite lung, mediastinal lymph nodes, and spleen (Ͻ60%), in- grafts showed a massive infiltration of inflammatory cells with dicating that memory T cells dominantly exist in the orthotopically total loss of the epithelium (Fig. 8Bb). Immunofluorescence label- transplanted lungs in association with lymphocyte aggregates. ing for CD3 demonstrated that the majority of the infiltrating cells This overexpansion of lymphocyte aggregates, which was even are T cells (Fig. 8Bb, inset). Conversely, the second intrapulmo- greater than that of the BN-Lewis-Lewis “positive” control trans- nary Lewis-derived trachea (i.e., isograft) implanted adjacent to plantation, was somewhat surprising to us. We speculate that the first BN-derived trachea showed minimum inflammatory infil- Lewis-derived RT1Au(Ϫ) lymphocytes homing to the lung reacted tration (Fig. 8Bc), loose fibrosis, and partial preservation of the u ϩ to F1-derived RT1A ( ) lymphocytes. The Lewis lung containing epithelium (Fig. 8Bd, arrow) with minimal T cell infiltration (Fig. the BN trachea was orthotopically transplanted into an F1 host 8Bd, inset), whereas the first BN-derived trachea was obliterated 7314 LYMPHOID NEOGENESIS AFTER LUNG TRANSPLANTATION Downloaded from http://www.jimmunol.org/

FIGURE 7. Stable homing of T cells derived from the initial tracheal transplant recipient and memory T cells in the lung. A, Representative u results of flow cytometric analysis for the expression of RT1A (expressed by guest on September 25, 2021 ϩ ϩ ϩ ϩ by BN and F1)byCD3 CD4 cells (top panels) and CD3 CD8 cells (bottom panels) in the lung. B, Immunofluorescence labeling for RT1Au demonstrating chimerism of peribronchiolar lymphocyte aggregates at day 56. Scale bar, 100 ␮m. C, A representative result of flow cytometric analysis for the expression of CD3ϩCD4ϩCD45RClow memory T cells in the lung. These results were obtained at day 56 from the left lung after intrapulmonary tracheal transplantation (day 0) followed by orthotopic left lung transplantation (day 28). Lew, Lewis; NC, negative control. with fibrous tissue similarly to that of the other group (data not shown). Semiquantification of CD3ϩ T cells demonstrated a significantly larger number of T cells infiltrating into the secondary FIGURE 8. The effector function of lymphocytes in the lung. A, Study BN-derived “allo” graft compared with that of the Lewis-derived design of intrapulmonary tracheal transplantation followed by concurrent “iso” graft (Fig. 8C). orthotopic lung transplantation, secondary intrapulmonary tracheal trans- Moreover, another BN graft implanted in the s.c. tissue at the plantation, and s.c. tracheal transplantation. Ba, Representative picture of time of orthotopic lung transplantation showed complete recovery the first and second intrapulmonary BN-derived tracheal grafts in the of the epithelium with no evidence of T cell infiltration (Fig. 8D). Lewis-derived lung implanted in an F1 host. Bb, Higher magnification of Thus, the effector function of the intrapulmonary de novo lym- the boxed area in a. Inset indicates CD3 staining demonstrating massive phoid tissue has a significant impact on the local immune response infiltration of T cells in the secondary BN graft. Bc, Representative picture against allogenic airways. of secondary intrapulmonary Lewis-derived tracheal graft implanted adjacent to the first BN-derived graft. Bd, Higher magnification of the boxed area in c. Arrow indicates partially preserved epithelium. Inset indicates Discussion CD3 staining demonstrating minimum infiltration of T cells in the second- In the present study, we hypothesized that lymphoid neogenesis ary Lewis graft. C, Semiquantification of the number of CD3ϩ T cells plays an important role in chronic rejection after lung transplan- infiltrating in Lewis-derived secondary “iso” graft (Iso) and BN-derived p Ͻ ,ء) tation. The human study for the first time demonstrated lymphoid secondary “allo” graft (Allo) demonstrated significant difference neogenesis in the lung affected by chronic rejection. The animal 0.05). D, The s.c. BN-derived tracheal graft. “M” indicates mucus pro- study demonstrated that intrapulmonary lymphoid tissue has an duced by the airway epithelium indicated by the arrow. The insets show effector function sufficient to reject allograft airways. These results CD3 staining demonstrating infiltration of T cells in the second intrapul- support our hypothesized role of lymphoid neogenesis in chronic monary tracheal graft. Lew, Lewis. rejection after lung transplantation. The Journal of Immunology 7315

In the human study, we observed a number of lymphoid tissues persistent, poorly resolvable inflammation lies in phenotypical al- composed of T cells, B cells, and HEVs (Fig. 1) in association with terations of resident cells, including endothelial cells (33, 34). lesions of OB and lymphocytic bronchioles after lung transplan- These cells begin to express adhesion molecules (e.g., PNAd) and tation (Fig. 2), suggesting lymphoid neogenesis in chronic rejec- ectopic lymphoid chemokines (e.g., CCL21) in response to inflam- tion after lung transplantation. Although lymphoid neogenesis has matory stimuli (35) such as TNF (36), and the altered tissue mi- been observed in chronically rejected human kidney, liver (7), and croenvironment allows for a large influx and survival of Ag-spe- heart (27, 28), lymphoid neogenesis has not been reported in lung cific lymphocytes in peripheral tissue (35). Interestingly, we found transplantation. It is possible that the presumed existence of “con- that lymphocytic bronchiolitis, a presumed precursor of OB (37, stitutive BALT” has made lymphoid neogenesis in the lung less 38) but still a reversible condition (39), accompanies abundant obvious. However, BALT has been demonstrated not to exist in HEVs and lymphocytes (Fig. 2), indicating that lymphoid neogen- normal human lungs (29). We also observed a significantly larger esis is initiated before the development of OB. Moreover, consid- number of lymphoid tissues containing HEVs in BOS lungs ering alloantigen-independent inflammatory stimuli such as isch- than in normal control lungs (Fig. 3), suggesting the process of emia-reperfusion injury at the time of transplantation (40), lymphoid neogenesis in transplanted lungs. bacterial colonization in airways (41, 42), and bile/acid aspiration Compared with reported lymphoid neogenesis (3–6), lymphoid (43, 44) that contribute to OB/BOS, these Ag-independent factors tissue in transplanted lungs is unique in the lack of delineated T might be enough to activate resident cells such as endothelial cells cell and B cell zones and follicular dendritic cells (Fig. 1). In the in airways to initiate lymphoid neogenesis. An interesting question lung, similar, less organized lymphoid tissue that does not meet the to be answered in the future is how soon such phenotypical conventional criteria of secondary and tertiary lymphoid tissue has changes in the resident cells (e.g., endothelial cells) leading to Downloaded from been reported (13). However, the functional significance of such lymphoid neogenesis start after lung transplantation. anatomical variations among intrapulmonary lymphoid tissue re- The initial set of retransplantation experiments (Figs. 5–7), how- mains unclear. Indeed, the most important factor in lymphoid neo- ever, did not clarify the role of the mature intrapulmonary lym- genesis may be the distribution of effector lymphocytes in periph- phoid tissue in allograft airway rejection, because the first tracheal eral organs, particularly at the sites of pathogen entry such as graft was already rejected and obliterated at day 28 when the or-

mucosal surfaces of the lung and gut (30). ELT has been proposed thotopic lung transplantation was conducted. Thus, we further im- http://www.jimmunol.org/ to describe a functionally unique subset of lymphoid tissue that planted secondary tracheal graft at the time of orthotopic lung collects effector and effector memory T cells regardless of ana- transplantation into an F1 rat (Fig. 8A). In this experiment, we tomically defined structures (16). Our finding of predominant observed massive infiltration of T cells into allografts but not CD45ROϩCCR7Ϫ effector memory T cells supports the formation isografts (Fig. 8), suggesting an allospecific effector function of the of ELT after lung transplantation (Fig. 1). Furthermore, even in a lymphoid tissue in the lung or in ELT in general (16). Moreover, s.c. tracheal transplant model of OB, the trafficking of effector T because the s.c. allograft remained intact, the effector function cells to the lung has been reported (31). This evidence suggests the of intrapulmonary lymphoid tissue was more important in local important role of the lung as a “reservoir” of effector and effector than in systemic immune response as indicated in another report on memory T cells that may contribute to allograft airway rejection iBALT (11). Interestingly, we found that the secondary isografts by guest on September 25, 2021 after lung transplantation (19). implanted in the lung containing lymphoid tissue also show patho- Our human study was, however, limited in the functional as- logical changes such as epithelial loss and loose fibrosis formation, sessment of lymphoid neogenesis. To overcome the challenge, we albeit free of T cell infiltration (Fig. 8). It is possible that the used an animal model of OB in which the allograft airway is ex- inflammatory microenvironment created by intrapulmonary lym- posed to the pulmonary milieu (22). In the model, we observed a phoid tissue affected the isograft in an alloantigen-independent larger number of lymphoid tissues in the lung after allograft trans- manner and delayed normal epithelial regeneration. plantation compared with that of isografts (Fig. 4), demonstrating To further clarify the role of memory T cells, Ab-mediated T that allograft airway rejection is accompanied by significant in- cell depletion would be the ideal experiment. However, the effi- trapulmonary lymphoid neogenesis. In contrast, the lymphoid tis- cacy of Ab-mediated T cell depletion was limited in rats in our sue did not result in obliteration of the airways around which lym- own experience as well as in the literature (45). A mouse intrapul- phoid tissue is formed, even after 120 days (Fig. 4). This is likely monary tracheal transplant model that we have recently developed explained by the nature of the intrapulmonary tracheal transplan- (46) may help to overcome the challenge. tation in which lymphoid tissue is formed in the native (recipient) We acknowledge that the animal model we used has multiple lung instead of the donor lung as seen in human lung transplan- limitations, including the initial intrapulmonary tracheal transplantation. Because the epithelium is considered to be the primary tar- tation (not transplantation of the entire lung) and the lack of im- get of allograft airway rejection (32), the airway that is not allo- munosuppression. Differences among species are another impor- genic may not be significantly affected. tant limitation of an animal experiment, particularly in the To further assess the role of lymphoid neogenesis in allograft organization of intrapulmonary lymphoid tissues such as BALT airway rejection, we orthotopically transplanted the lung contain- (29, 47). Despite these limitations, we emphasize that the animal ing de novo lymphoid tissue into an F1 rat, which dissected the experiments in the present study complemented the limitations in immune response mediated by the de novo lymphoid tissue from the human study in many ways and provided important insights that mediated by conventional secondary lymphoid tissue such as regarding the role of intrapulmonary de novo lymphoid tissue in regional lymph node tissue (Fig. 5A). Interestingly, using this OB after lung transplantation. newly devised animal model of OB, we found that lymphocyte In conclusion, we found de novo lymphoid tissue in the lung aggregates at day 7 after intrapulmonary allograft transplantation affected by chronic rejection after human lung transplantation. The are still reversible (Fig. 5) while the lymphocyte aggregates be- de novo lymphoid tissue likely plays a role in chronic local im- come more stable after day 28 (Fig. 6 and 7), suggesting that there mune responses after lung transplantation. Further studies are ob- is a maturation process in lymphoid neogenesis. Recent studies in viously necessary to improve our understanding of this novel form chronic inflammatory disorders such as rheumatoid arthritis sug- of lymphoid tissue and its contribution to the development of OB gest that the critical switch from acute, resolvable inflammation to after lung transplantation. 7316 LYMPHOID NEOGENESIS AFTER LUNG TRANSPLANTATION

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