Research ArticleTransplantation

Zbtb7a induction in alveolar macrophages is implicated in anti-HLA–mediated lung allograft rejection

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Science Translational Medicine  12 Jul 2017:
Vol. 9, Issue 398, eaal1243
DOI: 10.1126/scitranslmed.aal1243

Curbing culprits of chronic rejection

De novo donor-specific antibodies generated after organ transplantation can lead to chronic rejection, and Nayak et al. sought to understand the mechanisms leading to production of these antibodies in lung transplantation. Using mouse models and data from human patients, they identified expression of the transcription factor Zbtb7a in alveolar macrophages as a crucial mediator. Patients eventually diagnosed with chronic rejection had higher expression of this transcription factor early on. Preventing macrophages from expressing Zbtb7a ameliorated models of obliterative airway disease and prevented chronic rejection of lung transplants in mice. Interrupting macrophage presentation of donor antigens may be a solution to prevent generation of these destructive antibodies and the ensuing chronic rejection.

Abstract

Chronic rejection significantly limits long-term success of solid organ transplantation. De novo donor-specific antibodies (DSAs) to mismatched donor human leukocyte antigen after human lung transplantation predispose lung grafts to chronic rejection. We sought to delineate mediators and mechanisms of DSA pathogenesis and to define early inflammatory events that trigger chronic rejection in lung transplant recipients and obliterative airway disease, a correlate of human chronic rejection, in mouse. Induction of transcription factor zinc finger and BTB domain containing protein 7a (Zbtb7a) was an early response critical in the DSA-induced chronic rejection. A cohort of human lung transplant recipients who developed DSA and chronic rejection demonstrated greater Zbtb7a expression long before clinical diagnosis of chronic rejection compared to nonrejecting lung transplant recipients with stable pulmonary function. Expression of DSA-induced Zbtb7a was restricted to alveolar macrophages (AMs), and selective disruption of Zbtb7a in AMs resulted in less bronchiolar occlusion, low immune responses to lung-restricted self-antigens, and high protection from chronic rejection in mice. Additionally, in an allogeneic cell transfer protocol, antigen presentation by AMs was Zbtb7a-dependent where AMs deficient in Zbtb7a failed to induce antibody and T cell responses. Collectively, we demonstrate that AMs play an essential role in antibody-induced pathogenesis of chronic rejection by regulating early inflammation and lung-restricted humoral and cellular autoimmunity.

INTRODUCTION

Elicitation of immune responses to mismatched donor human leukocyte antigen (HLA) and breakdown of tolerance to self-antigens pose significant challenges to the initial graft acceptance and its continued function after solid organ transplantation (1, 2). Lung transplantation is a viable lifesaving intervention in many end-stage respiratory diseases such as chronic obstructive pulmonary disease, cystic fibrosis, idiopathic pulmonary fibrosis, α-1 antitrypsin deficiency, pulmonary arterial hypertension, interstitial lung disease, and bronchiectasis. However, its success is limited: More than 50% of recipients develop chronic rejection, clinically diagnosed as bronchiolitis obliterans syndrome (BOS) within 5 years of transplantation (3, 4). The chronic inflammation in BOS is a condition of fibroproliferation with involvement of autoimmune components that often leads to pulmonary lymphoid neogenesis (5). Further, BOS is a state of irreversible loss of respiratory function that does not normally respond to immunosuppressive regimens. Although the etiology of BOS is unclear and the mechanism of lung allograft rejection is poorly defined, de novo donor-specific antibody (DSA) to mismatched donor HLA even when nonpersistent is a significant risk factor for chronic rejection (610). The immunodominant role of DSA in chronic lung allograft rejection is well supported. De novo DSA is associated with recurrent and high-grade cellular rejection and lymphocytic bronchiolitis (8, 11, 12), and an early elicitation of DSA is a significant risk factor for chronic rejection (13). DSA development often precedes de novo antibodies (Abs) and T cell responses to lung-restricted self-antigens [collagen V (Col V) and K-α 1 tubulin (Kα1T)] (9). Moreover, DSA depletion via Ab-directed therapies lowers BOS hazard ratio and improves BOS-free survival in lung transplant recipients (6, 1416).

In addition to the elicitation of DSA and consequent immune responses to lung-restricted self-antigens, pretransplant Col V and Kα1T Abs in prospective lung transplant recipients significantly correlate with increases in primary graft dysfunction, de novo DSA, and BOS (17, 18). In a preclinical mouse model of obliterative airway disease (OAD), exogenous anti–major histocompatibility complex (MHC) delivered to native lungs recapitulated many histopathological correlates of BOS and elicited Col V– and Kα1T-specific immune responses (19, 20). The proximal mechanisms and effectors of immune activation for anti-MHC are unknown and must be defined to facilitate an early detection and possible intervention in Ab-induced lung inflammation. We hypothesize that Ab ligation of MHC elicits a set of “early triggers,” culminating in the activation of immune responses to lung-restricted self-antigens and leading to epithelial metaplasia, airway fibrosis, and airway occlusion—cardinal signs of chronic rejection in lung allografts.

The alveolar macrophage (AM) is a luminal sentinel for pathogens and pollutants in lungs (21, 22). AMs represent the vast majority (>80%) of lung-resident macrophages, occurring at a density of up to 12 cells per alveolus (23, 24), and they are essential in the functional preservation of respiratory epithelium (22, 25), containment of airway infection and inflammation (2629), and elicitation of intranasal vaccine-induced immunity (30, 31). Furthermore, AMs participate in lung allograft rejection by inducing local cytokine and Ab production in a rodent lung transplant model (32). Our analysis of the extractable cells in bronchoalveolar lavage (BAL) (33), supported by evaluation of lung tissue biopsies (34) from lung transplant recipients, revealed long-term sustenance (up to 3.5 years) of donor-derived AMs in the transplanted lungs. Besides serving as a reservoir for donor antigen(s), the donor-derived AMs were capable of inducing DSA and responded to DSA with production of proinflammatory cytokines (33). Adoptive transfer of single antigen–mismatched AM bearing a macrophage-specific MHC class I transgene induced de novo DSA, Col V– and Kα1T-directed T cell and B cell autoimmunity, and OAD. This functional duality in which AMs can initiate and influence pathogenesis of DSA indicated an important role for AMs in Ab-mediated lung graft rejection. Here, we describe induction of a transcription factor, zinc finger and BTB domain containing protein 7a (Zbtb7a), in AM as an indicator and a critical regulator of the anti-MHC–induced inflammation and OAD.

Zbtb7a, initially discovered as a proto-oncogene (35), regulates development and function of lymphocytes and tissue-resident macrophages (36), and its global deficiency triggers embryonic lethality by impairing erythropoiesis (37). Zbtb7a is essential for B cell neogenesis (38), mature B cell function (39), CD4 differentiation (40), and preservation of peripheral T helper 1 (TH1) and TH17 phenotypes (41, 42). It also regulates bone resorption by osteoclasts (43, 44). Functional pleiotropy of Zbtb7a further involves T cell repertoire purging by regulating intrathymic antigen expression (45), oncosuppression in prostate cancer (46), and repression of fetal hemoglobin synthesis in adults (47). Zbtb7a participates in oncogenesis, hematopoiesis, and immunity, but its involvement in pulmonary inflammation and autoimmunity is unknown. Here, we identified Zbtb7a as an early molecular signature and established its dominant role in the initiation and amplification of Ab-induced inflammatory circuits in the cellular and humoral autoimmunity associated with chronic lung graft rejection.

RESULTS

A distinct lung transcriptional landscape induced by Ab ligation of MHC class I

We sought to evaluate the effect of anti–MHC class I ligation in lungs on gene expression level(s) after intrabronchial Ab administration. Global changes in the gene expression of C57BL/6 lungs treated with anti–H-2Kb were compared with those of the immunoglobulin G (IgG) isotype control on 1, 2, 3, and 6 days after Ab administration. A differential expression was observed, with 14 genes significantly up-regulated [>1.5-fold, log2; P < 0.05 and false discovery rate (FDR) q < 0.1] after MHC class I ligation compared to the isotype control (Fig. 1A). Most significant was the up-regulation of transcription factor Zbtb7a (1.68862-fold, P = 1.08 × 10−5) (Fig. 1B and table S1). Induction of Zbtb7a was detected in lungs on day 2 after anti–H-2Kb administration, and its expression increased further at subsequent time points (Fig. 1C). Concurrently, 97 genes were significantly down-modulated in the anti–H-2Kb–administered lungs (P < 0.05). On the basis of established functions of Zbtb7a, we reasoned that Zbtb7a exerts a major effect on the early events that lead to development of immune responses to lung-restricted self-antigens and OAD.

Fig. 1. Profile of lung transcriptome after MHC class I ligation by specific Ab.

(A) Heat map presentation of differentially expressed genes after intrabronchial administration of H-2Kb or isotype control Abs into C57BL/6 mice. Three mice per group per day were analyzed and plotted. Probe intensity was normalized by z score, and red and blue indicate higher and lower expression levels, respectively. Data have been deposited into the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database (GEO ID: GSE71426). (B) Scatterplot highlighting 23 selected genes differentially expressed between H-2Kb and isotype control groups. Mean signal intensities were transformed into log2, and quantile normalization was applied. Differential expression was determined by two-way analysis of variance (ANOVA) test, and expression level exceeding 1.5-fold (P < 0.05 and FDR q < 0.1) was deemed significant. The central solid line represents line of equality, and genes expressed >1.5-fold demarcated by dashed lines are annotated. Red, up-regulated; blue, down-regulated. (C) Quantitative polymerase chain reaction (qPCR) validation of Zbtb7a expression was conducted in a parallel experiment. Three mice per time point were analyzed for Zbtb7a expression and normalized with that of Actb. Zbtb7a expression in untreated C57BL/6 lungs was set as baseline expression. Data are presented as means ± SEM, multiple t test was applied with multiple comparisons by Holm-Sidak method, and *P < 0.05 is considered significant.

In addition to transcript analyses, we evaluated influence of anti–MHC class I ligation on lung-infiltrating leukocytes (fig. S1). Anti–H-2Kb resulted in a massive neutrophil influx as early as 4 hours after Ab administration and led to a persistent neutrophilia. Accumulation of lung-trophic B cells also occurred, albeit at a slower pace (increase in B cell numbers was significant on day 6; P < 0.05). However, T helper cell (CD4+), cytotoxic T cell (CD8+), and AM compartments remained unchanged.

Reduced accumulation of lung-trophic leukocytes and formation of inducible bronchus-associated lymphoid tissue in Zbtb7a-deficient lungs

Intrabronchial instillation of lentivirus selectively delivers genes to AMs and induces a durable gene expression (48, 49). Here, si-Zbtb7a lentivirus reduced lung Zbtb7a expression by about 15-fold, compared with expression in the si-scrambled control (Fig. 2, A and B). Expression of Zbtb7a remained consistently low in the si-Zbtb7a–transduced lungs for about 5 months (that is, until the conclusion of analysis), indicating persistent repression of the target gene expression. Furthermore, induction of Zbtb7a at the protein level occurred in anti–H-2Kb administered si-scramble–transduced lungs, but not in si-Zbtb7a–transduced lungs (Fig. 2C).

Fig. 2. Disruption of Zbtb7a expression in lungs prevents anti-MHC–induced lung-restricted autoimmunity and OAD.

(A) Schematic representation of siRNA lentivirus transduction and subsequent Ab challenge. Mice were intrabronchially inoculated with lentivirus 3 days before intrabronchial Ab regimen. (B) Repression in the Zbtb7a expression was quantified by qPCR. n = 3. (C) Representative detection of Zbtb7a expression in lung lysates by Western blot analysis. (D) Representative Masson’s trichrome staining on day 45 after Ab administration. Fibrotic areas with deposition of collagenous ECM are stained blue. Scale bars, 100 μm. (E) Histopathologic score for a data point represents average of five fields from each mouse. Median lines are drawn. (F) Titers for serum anti-Kα1T and Col V were evaluated by enzyme-linked immunosorbent assay (ELISA) from eight mice, and mean ± SEM lines are drawn. (G) Development of T cell autoreactivity was analyzed by enzyme-linked immunospot (ELISPOT) on day 45. The data are presented as spot-forming cell (SFC) per million cells. n = 8 per group. Results are presented as means ± SEM, multiple t tests were applied with correction for Holm-Sidak method. P values are indicated. *P < 0.05 is considered significant.

Although B cells and neutrophils were the predominant cell types in Zbtb7a-sufficient (si-scramble–transduced) lungs, the Zbtb7a deficiency (si-Zbtb7a–transduced lungs) resulted in an 83% reduction in total neutrophils (fig. S2). The Zbtb7a-deficient lungs also demonstrated a 59% decrease in total B cells compared to that in Zbtb7a-sufficient lungs. However, deficiency of Zbtb7a did not alter the T cell (CD4+ and CD8+) and AM compartments in response to anti-MHC ligation, which remained unchanged between the treatment groups. These results suggested a proinflammatory role for Zbtb7a in Ab-induced lung inflammation.

Ectopic formation of inducible bronchus-associated lymphoid tissue (iBALT) is known to function as a local depot for immunologic priming and is induced in native as well as transplanted lungs as a response to Abs, infection, inflammation, and graft rejection (5053). Our results demonstrate a marked reduction in the frequency of iBALT formation in anti–H-2Kb–administered mice (>175% reduction for percent iBALT-positive and >600% reduction for iBALT per level) after Zbtb7a knockdown compared to si-scramble (fig. S3).

Reduction of OAD and lung-restricted autoimmunity in response to anti–MHC class I after Zbtb7a deficiency

Induction of OAD was according to our previously described protocol (19), with repeated intrabronchial administration of anti–H-2Kb (Fig. 2A). Mice received siRNA lentivirus 3 days before the beginning of Ab regimen. At 45 days after Ab administration, lung Zbtb7a expression and histopathology were assessed, and immune responses to lung-restricted self-antigens were measured. Anti–H-2Kb administration in si-scramble–treated mice led to bronchiolar hypertrophy, mononuclear leukocytic infiltrations, and intraluminal as well as peribronchiolar fibrosis with near-complete obliteration of the small airways (Fig. 2, D and E). The Zbtb7a-deficient mice, on the other hand, showed a marked reduction in the OAD pathology, with preservation of near-normal bronchiolar architecture and minimal collagen deposition (Fig. 2, D and E).

Development of de novo Abs to Col V and Kα1T is a measure of autoimmunity after human lung transplantation (9, 17) and in animal models of chronic rejection (19, 20). Zbtb7a deficiency resulted in a marked decline in anti-MHC–induced OAD concomitant with a steady rise in the serum anti–Col V and anti-Kα1T titers that occurred in Zbtb7a-sufficient mice (si-scramble), reaching >650 μg/ml at day 45 (Fig. 2F). Remarkably, Zbtb7a-deficient mice failed to generate Abs to Col V and Kα1T. Further, elicitation of Col V– and Kα1T-specific interferon-γ (IFN-γ)– and interleukin-17 (IL-17)–producing T cells was significantly (P < 0.001) lower in si-Zbtb7a–transduced lungs than that in si-scramble, indicating a Zbtb7a-dependent impairment of the cellular immune activation (Fig. 2G). Although there was no change in the number of IL-4–producing cells between treatment groups, marginal increases in Col V– and Kα1T-specific IL-10–producing cells were observed in Zbtb7a knockdown mice (58 and 65% increases, respectively; Fig. 2G).

Up-regulation of ZBTB7A in human lung transplant recipients with de novo DSA and BOS

To evaluate the clinical relevance of ZBTB7A as a measure of response(s) to de novo DSA, we analyzed BAL specimens from 139 lung transplant recipients (table S2). ZBTB7A expression at 3 months after transplantation was the designated baseline level. Cohorts of lung transplant recipients were based on clinical characteristics (that is, posttransplant time frame, DSA, autoAb, and graft rejection status). We compared 21 lung transplant recipients who developed DSA and were diagnosed with BOS against 21 lung transplant recipients at matched posttransplant time points with no detectable DSA with stable pulmonary function. We found a significantly (P < 0.001) higher ZBTB7A expression in patients with BOS at the time of diagnosis compared to that of stable patients (2.44-fold versus 1.02-fold, respectively; Fig. 3A). Furthermore, kinetics of ZBTB7A expression were evaluated at 6 and 12 months after transplantation in 12 lung transplant recipients who later developed BOS (onset between 36 and 59 months after transplantation) and in 11 BOS-free lung transplant recipients with stable pulmonary function up to 65 months after transplantation (Fig. 3, B and C). In this cohort, nine lung transplant recipients who developed BOS had documented de novo DSA to mismatched donor HLA, whereas BOS-free lung transplant recipients did not develop DSA. An elevated ZBTB7A (2.9-fold) was evident at 6 months; this increased to 5.6-fold at 12 months in BOS patients, whereas the corresponding increase varied between 1.1- and 1.2-fold in stable patients.

Fig. 3. Early induction of ZBTB7A in lung transplant recipients who developed de novo DSA and BOS.

BAL cell ZBTB7A expression was evaluated by qPCR after normalization with ACTB, CD68, and PPARG. ΔCt values at 3 months after transplantation were set as baseline for respective lung transplant recipients (table S2), and relative expression for subsequent time points was calculated as 2−ΔΔCt. (A) ZBTB7A induction in a cohort of 21 BOS+ lung transplant recipients and time-matched 21 lung transplant recipients who remained BOS-free, (B) kinetic study at 6 ± 1 and 12 ± 1 posttransplant months on 12 lung transplant recipients who later developed BOS, and (C) 11 lung transplant recipients who remained stable were evaluated. (D) Ten lung transplant recipients who developed de novo DSA (MFI > 2000) and received Ab-directed therapies were stratified as pre-DSA (MFI < 2000), DSA-peaked (highest MFI observed during follow-up of individual patient), DSA-reduced (2000 < MFI < peak), and DSA-resolved (MFI < 2000). (E) Serial samples from eight stable lung transplant recipients (DSA and not treated by Ab-directed therapy) were evaluated. (F) Role of DSA and autoAb (anti-Kα1T and anti–Col V) in ZBTB7A induction evaluated in lung transplant recipients DSA, autoAb (n = 11); DSA, autoAb+ (either anti-Kα1T or anti–Col V; n = 5); DSA+, autoAb (n = 10); DSA+, one autoAb (either anti-Kα1T or anti–Col V; n = 11), and DSA+, two autoAbs (anti-Kα1T and anti–Col V; n = 19). Two-tailed Mann-Whitney U test was applied, and P values are indicated. *P < 0.05 is considered significant.

Elicitation of de novo DSA is an independent and significant risk factor for development of BOS after human lung transplantation (17). The kinetics of ZBTB7A induction followed the trend of DSA in which high ZBTB7A expression coincided with the peak DSA level, after which it progressively declined with reduction in DSA (Fig. 3D). This cohort of lung transplant recipients developed DSA at 7 ± 3 months after transplantation and was treated with Ab-directed therapies [that is, rituximab and intravenous immunoglobulin (Ig)]. However, in lung transplant recipients who did not develop DSA and were stable without evidence of rejection, there was no induction of ZBTB7A up to 2 years after transplantation (Fig. 3E). Because DSA and Abs to lung-restricted self-antigens are predisposing factors for BOS, we differentially assessed the association of DSA and Abs to Col V and Kα1T with ZBTB7A expression. Lung transplant recipients who developed DSA expressed significantly (P < 0.05) higher ZBTB7A than lung transplant recipients who did not develop DSA (Fig. 3F). In the DSA group (at the time of analysis), those who developed Abs to lung-restricted self-antigens elicited greater ZBTB7A expression compared to lung transplant recipients who did not develop these autoimmune responses. Moreover, DSA+ lung transplant recipients who developed Abs to self-antigens registered a far greater ZBTB7A induction than those who developed DSA alone (that is, without Abs to Col V or Kα1T), suggesting a synergetic effect. It is noteworthy that peripheral blood leukocytes collected from lung transplant recipients at the time of BAL collection revealed no significant changes in ZBTB7A expression, irrespective of BOS or DSA outcomes (table S3). This suggests that ZBTB7A induction may be a local phenomenon. Collectively, these results demonstrate a positive association between dynamics of posttransplant DSA and BAL cell ZBTB7A expression.

Localization of anti-MHC–induced Zbtb7a expression in AMs

To ascertain the cellular origin of Zbtb7a expression in the lungs, we fractionated BAL leukocytes into Siglec-F+ and CD11c+ (AM) and Siglec-F and CD11c (non-AM) and isolated respiratory epithelial cells on day 7 after anti-MHC administration. Lentiviral delivery of siRNAs did not induce Zbtb7a expression per se (Fig. 4A). Transcript for Zbtb7a was detectable only in the AM fraction from si-scramble lungs after anti-MHC administration. Furthermore, no expression of Zbtb7a was detected in non-AM BAL cells or in respiratory epithelial cells. These results indicate that the predominant Zbtb7a signal after MHC ligation was localized to AMs. Furthermore, AMs with induced Zbtb7a also expressed chemokine (C-X-C motif) ligand 13 [(Cxcl13)] and Cxcl15 involved, respectively, in B cell and neutrophil chemotaxis (54, 55), and matrix metallopeptidase 9, a type IV collagenase involved in the remodeling of extracellular matrix (ECM) (56).

Fig. 4. Induced Zbtb7a in lungs is localized to AMs.

(A) Representative reverse transcription PCR analysis on total RNA isolated from AMs (lanes 1, 4, 7, and 10), non-AM BAL cells (lanes 2, 5, 8, and 11), and alveolar respiratory epithelial cells (REC; lanes 3, 6, 9, and 12) on day 7 from siRNA lentivirus–transduced and Ab-treated mice (n = 6). bp, base pair. (B) Freshly isolated human AMs (from stable lung transplant recipients; DSA, autoAb, and with no documented rejection) and mouse AMs (from C57BL/6) were cultured at 1 × 106 cells per well (33). Surface MHC class I was ligated by anti–H-2Kb and anti-HLA, respectively, in mouse and human AM cultures, and MHC class II on mouse AMs was ligated by anti–I-A/I-E. Isotype-matched controls for MHC Abs and ligation of a non-MHC molecule (keratin) were also evaluated. Zbtb7a expression was normalized to that of Actb and presented as relative expression compared to no Ab–treated AMs. n = 5. Data are presented as means ± SEM, two-tailed unpaired t tests are applied, *P < 0.05 is considered significant, and P values are indicated. (C) BAL cells from five BOS+ lung transplant recipients (P-1 to P-5) and four BOS-free lung transplant recipients (P-6 to P-9) with stable lung function were fractionated into AM and non-AM cells. The lung biopsy specimens were obtained on the same day of BAL fluid collection. Fold induction in ZBTB7A expression compared to the peripheral blood leukocytes for individual lung transplant recipient is plotted as heat map.

To validate the in vivo findings and delineate sufficiency of MHC ligation in Zbtb7a induction, we analyzed purified human and mouse AMs in an in vitro culture system. Stimulation by anti-HLA framework Ab, but not isotype-matched control, elicited ZBTB7A expression in AMs from human lung transplant recipients (Fig. 4B). However, such an induction was not evident in non-AM BAL cells that mostly consisted of lymphocytes and granulocytes. In parallel, mouse AMs were evaluated for their responses to MHC class I and class II ligations. Both of the MHC stimuli resulted in higher Zbtb7a expression in murine AMs compared to respective isotype controls (Fig. 4B).

Further analysis of the fractionated AM and non-AM BAL cells and lung tissue collected by needle biopsies revealed higher induction of ZBTB7A in AMs. Purified AMs from BOS patients recorded a >5-fold higher ZBTB7A expression over the non-AM BAL cells and biopsies from the same patient collected on the same day (Fig. 4C). Moreover, none of the specimens from BOS-free lung transplant recipients with stable pulmonary function had enhanced ZBTB7A expression.

Induction of Zbtb7a and OAD after Ab ligation of AM MHC class I

We used a C57BL/6-based transgenic (Tg) mouse model, huCD68-Kd Tg C57BL/6, which expresses full-length H-2Kd transgene in C57BL/6 (H-2Kb) macrophages (57), to delineate the specific role(s) of AM in anti-MHC pathogenesis. The BAL cells from naïve lungs were predominantly (>80%) AM that concurrently expressed H-2Kd and H-2Kb on the cell surface (Fig. 5A). Additionally, expression of CD11blow and F4/80int in Tg AMs was comparable with their wild-type counterparts (C57BL/6 AMs) and was consistent with the AM lineage markers (22, 25). After a lentivirus-delivered siRNA transduction (as described in Fig. 2A), a 30-day intrabronchial anti–H-2Kd administration was conducted to elicit OAD. In si-scramble huCD68-Kd Tg C57BL/6 lungs, anti–H-2Kd induced intraluminal and peribronchiolar fibrosis and de novo Abs and TH17 responses to Col V and Kα1T (Fig. 5, B to E).

Fig. 5. Induction of lung-restricted autoimmunity and OAD by anti-MHC ligation of AM is Zbtb7a-dependent.

(A) Expression of lineage markers and H-2Kd transgene in huCD68-Kd Tg C57BL/6 AMs. BAL cells were pregated for CD45+, CD19, and CD3 leukocytes. (B) Representative trichrome-stained images showing fibrotic scar and occluded bronchiole in anti–H-2Kd–induced OAD on day 30 and (C) histopathology assessment of the OAD lesion. n = 6. Scale bar, 100 μm. (D) Development of de novo Ab titers and (E) stimulation of TH17 cells specific for Kα1T and Col V were evaluated on day 30. n = 6 to 7. (F) Representative surface expression of MHC (class I and II) and costimulatory molecules in Zbtb7a-sufficient or Zbtb7a-deficient AMs after anti–H-2Kd administration was analyzed on day 30. n = 3. Data were analyzed by multiple t tests, and statistical significance (*P < 0.05) was determined using the Holm-Sidak method.

The Zbtb7a-deficient lungs (si-Zbtb7a–transduced), in contrast, exhibited intact bronchiolar architecture with nonobstructed lumen and reduced Col V– and Kα1T-specific immune responses in response to anti–H-2Kd (Fig. 5, D and E). To assess the role(s) of AM in anti-MHC pathogenesis as an antigen-presenting cell, we analyzed expression of MHC class II and costimulatory molecules (Fig. 5F). The Zbtb7a-sufficient huCD68-Kd Tg C57BL/6 AMs exhibited an activated phenotype with higher cell surface expression of I-Ab and CD40, indicating greater antigen-presenting ability compared to that of Zbtb7a-deficient AMs. The effect of Zbtb7a deficiency on the phagocytic properties of AM was further evaluated; however, no noticeable differences were observed in AMs from untreated, si-scramble, and si-Zbtb7a groups because they were capable of dextran endocytosis and latex bead phagocytosis at equivalent rates regardless of their Zbtb7a expression (fig. S4).

To further understand the pathogenesis of MHC ligation and to investigate the protective effects of Zbtb7a knockdown in Ab-induced OAD, we analyzed a detailed panel of molecules involved in anti-inflammation, granulocyte growth, leukocyte traffic, vesicular mobilization and cytoskeletal stability, and remodeling of ECM (fig. S5). Consistent with other observations of anti-MHC ligation, Zbtb7a was up-regulated in huCD68-Kd Tg C57BL/6 lungs after anti–H-2Kd administration. Furthermore, Zbtb7a deficiency resulted in a higher expression of anti-inflammatory molecules, such as leukemia inhibitory factor, and chemoattractants, chemokine C-C motif ligand 27a, Cxcl11, Cxcl13, and Cxcl15. In addition, Zbtb7a-sufficient lungs induced a higher expression of Col V (Col5a1), the core component of collagen fibrillogenesis (58), whereas Zbtb7a-deficient lungs produced greater amounts of ECM remodeling and cytoskeletal stabilizing factors.

Requirement of Zbtb7a signaling in elicitation of AM-directed alloimmunity

We evaluated the influence of Zbtb7a deficiency on the development of spontaneous allogeneic response and OAD after an adoptive AM transfer. In our earlier study, transfer of single antigen–mismatched AMs resulted in alloimmunity (directed to the mismatched antigen), autoimmunity (directed to lung-restricted self-antigens), and OAD (33). The purpose of the present study on intrabronchial transfer of AMs (si-scramble– or si-Zbtb7a–transduced) into Zbtb7a-sufficient or Zbtb7a-deficient lungs was to assess the significance of donor and recipient Zbtb7a signaling in the allogeneic AM-induced immune responses. Transfer of huCD68-Kd Tg AMs (unmodified or si-scramble–transduced) into Zbtb7a-sufficient lungs elicited higher anti–H-2Kd titers (Fig. 6A). In contrast, transfer of Zbtb7a-deficient AMs into Zbtb7a-sufficient lungs and vice versa resulted in lower titers [nearly 50% reduction in median fluorescence intensity (MFI)]. However, complete resolution of the anti–H-2Kd titer (~95% MFI decline) was achieved when both the donor AM and recipient lungs were Zbtb7a-deficient. Additionally, induction of alloimmune (H-2Kd–reactive) TH17 cells exhibited a similar trend where Zbtb7a knockdown in both donor and recipient registered a maximum reduction compared to other AM transfer combinations (Fig. 6B). The protective effects of Zbtb7a deficiency in donor and recipient were additive, whereas knockdown in both AM donor and recipient induced near-complete abrogation of AM-directed alloimmune responses.

Fig. 6. Zbtb7a expression is essential in elicitation of AM-directed humoral and cellular alloimmunity.

Allogeneic reconstitution of AM was achieved by intrabronchial transfer of 4 × 105 huCD68-Kd Tg C57BL/6 AM into C57BL/6 recipients. (A) Development of H-2Kd–specific Abs in BAL fluid was measured by flow cytometry at 15 weeks. Representative anti–H-2Kd titers (in table S5) are presented as overlaid histograms (n = 5 to 7), and individual MFI values are indicated. (B) ELISPOT assay measured alloreactive (H-2Kd–specific) IL-17–producing cells per million lung leukocytes (mean ± SEM) at 15 weeks. One-way ANOVA was applied to test statistical significance (*).

Reduced production of donor-derived exosomes by Zbtb7a-deficient AMs

Nanovesicular/exosome-mediated trafficking of donor-derived antigen is recognized in the allorecognition of transplanted antigens (59). We evaluated the influence of Zbtb7a on the production of H-2Kd+ exosomes after allogeneic transfer of huCD68-Kd Tg AMs into C57BL/6 recipients (from Fig. 6). Using anti-CD63–coated beads, total exosomes in cell-free BAL fluid were identified, and composition of the Siglec-F+ (AM-derived) exosomes was further analyzed for the alloantigen-containing exosomes (that is, H-2Kd+). Although the total exosomes found in BAL fluid did not differ significantly (P < 0.05) between the AM transfer groups (Fig. 7A), production of allogeneic exosomes by Zbtb7a-deficient donor AM was markedly lower, constituting ~3% of the exosome pool compared with >19% in the Zbtb7a-sufficent donor AMs (Fig. 7B). Although Zbtb7a deficiency in the recipient AM resulted in a marginal decline in total BAL exosomes, it did not affect the alloexosome production. Zbtb7a deficiency in both donor and recipient AMs resulted in a decline in AM-derived total exosomes and alloexosomes (~9 and <1%, respectively). This reduction of H-2Kd+ exosome preponderance associated with donor Zbtb7a deficiency may be indicative of a lower availability of transferable donor antigen(s) in alloimmune sensitization.

Fig. 7. Zbtb7a deficiency in donor AMs reduces donor-derived exosome production.

(A) Quantitation of total exosomes isolated from cell-free BAL fluid after adoptive transfer of allogeneic AMs from Fig. 6. Data from three mice per AM transfer group were individually analyzed and are presented as means ± SEM. One-way ANOVA was applied with Brown-Forsythe test that compares exosomes from the AM transfer groups with those from C57BL/6 as the baseline expression. Significance (P values) of differences is indicated. (B) Representative flow cytometric analysis on BAL exosomes from AM transfer groups (n = 3 to 4) was performed by anti-CD63–coated Exo-Flow magnetic beads. The AM-derived exosomes (Siglec-F+) were analyzed for frequency of donor or recipient origin as defined by H-2Kd and H-2Kb staining (summary in table S6). Constitution of the BAL exosomes (mean) derived from AMs, non-AM cells, and donor AMs (alloexosomes) is also shown. SSC-A, side scatter area; FSC-A, forward scatter area; APC, allophycocyanin.

Impaired antigen presentation by AMs due to Zbtb7a deficiency

To obtain insight into possible impairment in the antigen-processing ability of Zbtb7a-deficient AMs that did not induce alloimmunity (Fig. 6), we cotransferred antigen-pulsed AM and T cell receptor Tg T cells into naïve recipients. Proliferation of OT-II T cell receptor Tg T cells [I-Ab–restricted chicken ovalbumin (OVA) 323-339–specific] was measured by carboxyfluorescein succinimidyl ester (CFSE) dilution in the lungs as well as in the spleen after intrabronchial transfer of AMs primed with latex bead (L)–coated OVA (L-OVA) or bovine serum albumin (L-BSA) or OVA 323-339 peptide. The OT-II T cells proliferated in lungs that received L-OVA or OVA 323-339 loaded to Zbtb7a-sufficient AMs. Furthermore, cotransfer of Zbtb7a-deficient AMs pulsed with whole-protein antigen or the T cell epitope peptide failed to induce proliferation of OT-II cells. Impaired T cell proliferation (Fig. 8, A and B) along with reduced AM-directed alloimmunity (Fig. 6) after Zbtb7a knockdown is indicative of a Zbtb7a-dependent antigen-presenting cell function for AM and suggests an important role for AM in the in vivo priming of alloantigen and lung-restricted self-antigen–reactive T cells.

Fig. 8. Zbtb7a deficiency impairs antigen presentation by AMs.

Adoptively transferred Zbtb7a-sufficient or Zbtb7a-deficient huCD68-Kd Tg AMs in Fig. 6 were antigen-pulsed (with L-OVA or L-BSA delivering 5 μg of protein or 1 μg of OVA 323-339 peptide) in vivo at 15 weeks after transfer, harvested at 6 hours after pulsing, and transferred (1 × 105) with 5 × 106 CFSE-labeled naïve OT-II T cells into C57BL/6 recipients. (A) Representative plot of OT-II T cell proliferation in lungs and spleen measured by CFSE dilution at 72 hours after AM and T cell transfer. Cells were pregated for CFSE+ and CD3+. n = 5. SSC-H, side scatter height. (B) Summary of the CFSE dilution. Mean ± SEM is plotted for T cell proliferation in the lungs. n = 5.

Induction of Zbtb7a in lung allograft by systemic alloantibody

Because influence of systemic Abs could not be evaluated in the OAD model that necessitated local administration of Abs to lungs, we assessed intraperitoneally administered anti-MHC on Zbtb7a expression in the accepted lung allograft after mouse orthotopic lung transplantation. Exogenous administration of anti–H-2Kd induced Zbtb7a in the transplanted lung (fig. S6), whereas isotype control did not stimulate such a response (table S4). Further, no induction of Zbtb7a was observed in the native lung, peripheral blood leukocytes, and splenocytes from the corresponding transplant recipient mouse (fig. S6 and table S4).

Induction of Zbtb7a after anti-MHC ligation in the absence of lymphocytes

We assessed the requirement of T cells and B cells in Zbtb7a induction after anti–MHC class I ligation in the lungs. The anti–H-2Kb–treated Rag1−/− mice mounted a significantly higher Zbtb7a expression than that of isotype-treated mice (fig. S7). Further, the anti-MHC ligation did not induce bronchiolar obliteration and peribronchiolar or intrabronchial fibrosis. Therefore, although lymphoid cells are dispensable in anti-MHC–induced Zbtb7a expression in the lungs, their participation is essential in the development of OAD and lung-restricted autoimmunity (Fig. 2).

DISCUSSION

The incidence of BOS remains a primary obstacle to successful lung transplantation (4). Immunologic factors, particularly the number of HLA mismatches between a lung graft donor and the recipient (60), and an early appearance of DSA (13) are risk factors for the development of BOS. A clinical association between de novo DSA and lung graft rejection has been demonstrated; however, a lack of understanding of the early mediators and key regulators in DSA-induced lung inflammation has hindered the understanding of—and possible intervention for—Ab-induced chronic lung graft rejection. In our preclinical murine model, ligation of surface MHC by specific Abs elicited OAD, a clinical correlate of BOS in humans (19, 20). Because the OAD model necessitated exogenous Ab administration to the lung milieu without interference of immunosuppression, it afforded us unperturbed access to kinetic studies at the molecular and cellular levels that is currently not feasible in any experimental models of chronic rejection, including the orthotopic murine lung transplantation (61, 62). Gene microarray analysis, followed by functional validations, identified Zbtb7a as an early indicator and a critical modulator in anti-MHC pathogenesis. Strikingly, the lung transplant recipients who developed de novo DSA and BOS exhibited greater induction of ZBTB7A compared to those who remained BOS-free. This finding is particularly meaningful considering the lack of a viable molecular marker that predicts BOS. Induction of ZBTB7A as early as 6 months after transplantation in lung transplant recipients who developed BOS and its localization to AMs offers prognostic significance because the half-life of lung allografts remains as low as 5 years, and the progressive decline in pulmonary function at diagnosis of BOS is an irreversible and untreatable phenomenon.

Interruption of gene function by siRNA resulted in a persistent decline of Zbtb7a expression in lungs. With less accumulation of lung-trophic B cells and neutrophils in si-Zbtb7a–transduced lungs, anti-MHC failed to induce pulmonary inflammation, autoimmunity, and OAD. The appearance of “early” neutrophil and “late” B cell accumulations is considered key cellular responses—albeit nonspecific—to MHC ligation, because B cells have an obligatory role in airway obliteration (51) and neutrophils regulate development of secondary lymphoid clusters consistent with iBALT (50). Additionally, a concurrent decrease in the Col V– and Kα1T-specific IL-17– and IFN-γ–producing cells further confirmed establishment of an “anti-inflammatory state” associated with the loss of Zbtb7a. Further, we observed Zbtb7a induction in lymphopenic mice after MHC ligation. Because anti–H-2Kb resulted in Zbtb7a up-regulation, nonlymphoid cells in lungs, without participation of lymphocytes, were sufficient to induce Zbtb7a in response to MHC Ab. AM was the predominant cell type responsible for lung Zbtb7a expression. Further, stimulation of Zbtb7a by MHC ligations in primary murine and human AM cultures implies Zbtb7a induction as an early response to Abs. Development of OAD and lung-restricted, self-antigen–directed autoimmunity after MHC class I ligation on huCD68-Kd Tg C57BL/6 AM was a proof of concept that demonstrated a key role for AM in the anti-MHC pathogenesis. Furthermore, AM’s downstream effector functions were Zbtb7a-dependent because the targeted gene disruption ameliorated bronchiolar occlusion and reduced exacerbation of lung-restricted autoimmunity.

The current study focused on characterization of molecular mechanisms critical in the initiation and amplification of AM-induced DSA responses. Naïve AMs from noninflamed lungs are poor antigen presenters due to a lack of costimulation (63), whereas AMs function as efficient antigen-presenting cells after pulmonary infection, vaccination, or lung transplantation (27, 30, 31, 64). Uninterrupted Zbtb7a signaling induced the antigen presentation ability of AMs in response to MHC ligation by stimulating greater expression of MHC class II (I-Ab) and costimulatory (CD40) molecules. The Zbtb7a-deficient AMs, in contrast, did not exhibit such stimulations—indicating their diminished capacity for antigen presentation. Additionally, intrabronchial transfer of Zbtb7a-deficient allogeneic AMs into Zbtb7a-sufficient lungs or vice versa resulted in decreased alloAbs and alloimmune T cells. To elucidate this reduced immunogenicity of Zbtb7a-deficient AMs, we evaluated intercellular transfer of transplant antigen(s) via exosomes. Exosome-mediated passage of donor antigens into recipient’s antigen-presenting cells has been recognized in semidirect and indirect alloimmune sensitization (59), and exosomes produced by antigen-presenting cells are known to be inflammatory (65, 66). We have shown an association of donor-derived exosomes with lung graft rejection. The exosomes from human lung transplant recipients experiencing lung graft rejection contained donor HLA molecules, whereas exosomes from stable lung transplant recipients did not (67). Demonstration of an ~85% decrease in alloexosome production by Zbtb7a-deficient donor AMs indicates a reduced availability of transferable alloantigen, and along with diminished antigen presentation, it correlates with reduced alloimmunity due to Zbtb7a deficiency in donor AMs. However, a complete abrogation of alloimmune priming was achieved when both the donor and recipient AMs were Zbtb7a-deficient. Additionally, the Zbtb7a-deficient AMs were unable to stimulate antigen-specific T cell proliferation. Together, protection from Ab-induced lung inflammation achieved by selective loss of Zbtb7a in the AM was multifactorial, where Zbtb7a deficiency stimulated anti-inflammatory signals, dampened proinflammatory and profibrotic signals, produced fewer alloexosomes, and rendered AMs as less-efficient antigen-presenting cells.

Limitations to the current study include lack of a multicenter randomized controlled trial, due to which confounding variables including demographics of donor and lung transplant recipients and pre- and posttransplant care at transplant centers were not balanced. Further, we did not stratify lung transplant recipients based on acute rejection; therefore, significance of ZBTB7A in suspected Ab-mediated acute rejection was not evaluated.

The present study documents the complexity of MHC Ab-induced pathogenesis in which an early and unique transcriptional landscape regulated airway inflammation, autoimmunity, and remodeling of airways, ultimately leading to chronic small airway obstruction. An early induction of Zbtb7a in lung-resident AMs was an indicator and an important mediator in the anti-MHC disease development. Overall, Ab ligation of MHC class I on AM cell surface stimulated an early entry of inflammatory cells into lung milieu and development of a “conditioned state” for subsequent priming and expansion of lung-restricted autoimmunity. Given the long-term persistence of allogeneic donor AMs in transplanted lungs (33, 34), the efficacy of local ZBTB7A disruption needs to be evaluated in BOS prevention in lung transplant recipients. Because B cell priming and production of de novo DSA precede detectable serum DSA, and because BAL analysis is a standard postoperative care across transplant centers, evaluation of AM ZBTB7A may serve as an early indicator for de novo DSA and BOS and may measure the efficacy of DSA-directed therapies.

MATERIALS AND METHODS

Study design

Our focus in this study was to delineate early trigger(s) in DSA pathogenesis. By using an Ab-induced murine OAD model, a correlate of human BOS, we analyzed the lung transcriptome by gene microarray and found Zbtb7a as one of the induced genes in response to anti-MHC ligation. We determined Zbtb7a’s functional significance by targeted gene knockdown, where Zbtb7a deficiency in lungs rendered protection from Ab assaults and blunted the lung-restricted autoimmunity. Further, we evaluated lung transplant recipients for ZBTB7A expression and found that transplant recipients who developed BOS had an early induction of BAL ZBTB7A before their clinical diagnosis. We ascertained AMs to be the cellular origin for DSA-induced Zbtb7a expression. We further assessed the effect of Zbtb7a deficiency on AM functionality with regard to endocytosis, antigen presentation, and exosome production. Experimental group sizes were selected on the basis of our experience with intrabronchial Ab administration and AM transfer protocols. All male mice were used and were age-matched between treatment groups. Cohorts of lung transplant recipients were retrospectively analyzed, and all consented clinical specimens collected/available during the study period meeting the study stratification based on sampling time frame, immunosuppression, DSA, autoAb, and BOS status were used. Experimental groups were not blinded, and no randomization was necessary. No data were excluded from this study. Primary data are shown in table S7.

Mice and Ab-induced OAD

Wild-type C57BL/6 and Balb/c mice (6 to 8 weeks old, male) were procured from Charles River Laboratories, Rag1−/− mice were obtained from The Jackson Laboratory, and Rag2/OT-II mice were from Taconic Biosciences. C57BL/6 mice that express H-2Kd transgene on macrophage (huCD68-Kd Tg C57BL/6 mice) were described earlier (57). Mice were maintained at Washington University School of Medicine according to the institutional guidelines and Animal Studies Committee–approved protocols. For induction of OAD, anti–H-2Kb (AF6-88.5.5.3), anti–H-2Kd (SF1.1.10), or isotype control (C1.18.4) Abs tested for being endotoxin-free [protein (<2 EU/mg) determined by LAL gel clotting test] were administered intrabronchially as described earlier (19). Briefly, 200 μg of Ab (40 μl) was instilled through a 22-gauge catheter on days 1, 2, 3, and 6 and weekly thereafter. Histopathologic evaluation of lung tissue sections was assessed by hematoxylin and eosin and Masson’s trichrome staining. The images were captured by Aperio VERSA scanner (Leica Biosystems) and analyzed by Aperio ImageScope (Leica Biosystems).

Lentivirus transduction

Zbtb7a-targeted knockdown was achieved by a lentiviral delivery of siRNA targeting four sequences (fig. S2A) on mouse Zbtb7a. Lentiviruses with si-Zbtb7a (iV036281) and scrambled siRNA (LVP015-G) were obtained from Applied Biological Materials. Briefly, the siRNAs were cloned into piLenti-siRNA-GFP vector, and replication-deficient pantropic lentiviral particles were produced with vesicular stomatitis virus glycoprotein (VSV-G) as envelope protein. The in vivo transduction was performed according to Wilson et al. (49). Briefly, mice were anesthetized with ketamine/xylazine cocktail (100 mg/kg body), and siRNA lentivirus (107 infectious units per mouse) was delivered intrabronchially. Three days after lentivirus transduction, mice were infused with Abs for OAD development.

Isolation of lung leukocytes

Mice were anesthetized with ketamine/xylazine cocktail, and lungs were perfused with low-endotoxin phosphate-buffered saline (PBS; pH 7.4; Thermo Fisher Scientific) (3 × 10 ml) via the right ventricle to deplete circulating blood cells. Single-cell suspension was prepared from the PBS-perfused lungs by digestion with Liberase TL (300 μg/ml; Roche Life Science) and deoxyribonuclease I (DNase I) (5 U/ml; Sigma-Aldrich) at 37°C for 25 min. The lung digests were filtered through 100-μm membrane and washed thrice in PBS supplemented with fetal bovine serum (FBS) (2%) and EDTA (2 mM), after which leukocytes were isolated by a Ficoll-Paque (Sigma-Aldrich) gradient centrifugation and resuspended at 1 × 107/ml.

ELISA, ELISPOT, and Western blot

Mouse Abs to lung-restricted self-antigens were detected by our ELISA protocol (19). Briefly, ELISA plates were coated with Col V or Kα1T proteins (100 ng per well), and detection of the specific Abs in serum was done by probing with horseradish peroxidase–conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories). Analyses of donor-specific HLA Abs and Col V or Kα1T autoAbs in lung transplant recipients were described earlier (68). Estimation of self-antigen–specific TH1, TH2, TH17, and regulatory T cells in the lungs was conducted by quantifying cytokine (IFN-γ, IL-4, IL-17A, and IL-10)–secreting ELISPOT assays (BD Biosciences and eBioscience) following our earlier optimized protocol with modification (19). One million lung leukocytes were added per well and were supplemented with 5 × 104 C57BL/6 splenocytes [irradiated at 30 gray (Gy)] as antigen-presenting cells. The cells were primed with Col V or Kα1T proteins at 1 μg/ml, and cytokine-producing spots were enumerated by an Immunospot analyzer (Cellular Technology Limited). Enumeration of alloreactive (H-2Kd–specific) and autoreactive (H-2Kb–specific) T cells was conducted by stimulating splenocytes from recipient mice with irradiated (30 Gy) T cells (EasySep mouse T cell isolation kit; STEMCELL Technologies) from Balb/c and C57BL/6, respectively, following our established protocol (33). For Western blot analysis, lung tissue was lysed in radioimmunoprecipitation assay lysis buffer (Santa Cruz Biotechnology) with protease inhibitors. The lysates (2 μg of protein equivalent) were electrophoresed on 12% SDS–polyacrylamide gel, then blotted onto polyvinylidene fluoride membrane, and probed with anti-Zbtb7a (466407) or anti–β-actin (C4). The electrochemiluminescence images were captured with Odyssey Fc Imaging System (LI-COR Biosciences).

Lung transplant recipients and sampling strategy

Human studies were approved by the Institutional Review Board at Washington University, and lung transplant recipients gave consent for enrollment in the study. We retrospectively identified a total of 139 patients who underwent primary uni/bilateral lung transplantation at Barnes-Jewish Hospital/Washington University School of Medicine (BJC/WU) between January 2009 and December 2013. Routine care for lung transplant recipients at BJC/WU includes follow-up visits at 1, 2, 3, 6, and 12 months and then yearly posttransplant visits, unless more are needed for clinical reasons.

Blood, needle biopsy, and BAL fluid were collected in clinic during follow-up visits as a standard procedure, and cellular profiles of BAL fluid at the time of collection were obtained from the patients’ charts. Serum and peripheral blood leukocytes were fractionated from whole blood. BAL cells were isolated by centrifugation at 1500g for 10 min at 4°C and stored in RNAlater (Thermo Fisher Scientific) at −80°C. We evaluated 21 lung transplant recipients who developed BOS and compared them with a time-matched cohort of 21 lung transplant recipients with stable pulmonary function with no evidence of rejection. DSA was determined using LABScreen single-antigen assay (Thermo Fisher Scientific), and Abs to lung-restricted self-antigens (Col V and Kα1T) were determined using ELISA developed in our laboratory (68).

We retrospectively analyzed lung transplant recipients who developed BOS, and those remained BOS-free (stable) at 3, 6, and 12 months after transplantation. Lung transplant recipients who developed de novo DSA (MFI > 2000) received Ab-directed therapy (that is, intravenous Ig and rituximab), and the matched post–lung transplant BAL samples were stratified as pre-DSA, DSA-peaked, DSA-reduced, and DSA-resolved on the basis of their serum MFI for DSA. The lung transplant recipients who did not develop DSA (DSA-negative) had no episodes of rejection and did not develop de novo Abs to Col V and Kα1T and remained stable without any decrease in pulmonary function. We further evaluated elicitation of DSA in combination with de novo Abs to lung-restricted self-antigens.

Isolation of mouse BAL fluid and respiratory epithelial cells

Mouse BAL fluid was collected via a 22-gauge catheter installed into the trachea followed by five subsequent washes (1 ml per wash) with ice-chilled Ca+2- and Mg+2-free PBS with EDTA (1 mM). The BAL cells were pelleted by centrifugation at 250g at 4°C for 10 min. Isolation of mouse respiratory epithelial cells was performed according to the methods described by Lam et al. (69). Briefly, after collection of BAL cells, the lungs were digested overnight by 0.15% pronase (Sigma-Aldrich) at 4°C, and the epithelial cells were harvested after DNase I treatment (50 U/ml) for 5 min on ice.

Immunofluorescence microscopy

Immunofluorescence microscopy was performed on serial lung sections cut from paraffin-embedded tissue blocks at 200-μm intervals at five to seven levels according to Foo et al. (50) and stained with Alexa Fluor 594–conjugated anti-B220 (RA3-6B2) and Alexa Fluor 488–conjugated anti-CD3 (17A2) to identify B cells and T cells, respectively. Zeiss Observer Z1 with AxioVision 4.8.2 (Carl Zeiss Meditec Inc.) was used to acquire and analyze the immunofluorescence images. A lymphoid cluster with >30 detectable B cells and T cells was recorded as an iBALT, and the sum of the lymphoid clusters (from five to seven levels) was averaged out to calculate iBALT per level.

Culture of AM

Freshly isolated human and mouse AMs were cultured, respectively, in RPMI 1640 (Thermo Fisher Scientific) or Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific) supplemented with 10% FBS and 20% L929 culture supernatant (as source of macrophage colony-stimulating factor; final concentration, 100 U/ml) and seeded in a 12-well plate at 1 × 106 cells per well as described (33). For ligation of surface MHC, anti–H-2Kb (AF6-88.5.5.3, IgG2a) or anti–I-A/I-E (M5/114, IgG2b) was added to mouse AM, and anti-HLA (W6/32, IgG2a) was added to human AM cultures. The Abs were used at 100 μg/ml, and the responses to MHC ligation were compared with that of isotype-matched Igs (C.1.18.4, IgG2a or LTF-2, IgG2b). Ligation of keratin by anti-keratin (AE3, IgG1) was also evaluated. Thirty minutes before Ab stimulation, cells were incubated with Fc block (clones 2.4G2 and 93 for mouse AMs and Human BD Fc Block for human AMs) at 2 μg/ml, and the Fc block was maintained at 2 μg/ml during the experiment. The cells were cultured at 37°C at 6% CO2 for 4 hours and harvested for mRNA analysis.

Assessment of particle endocytosis by AMs was conducted for 20 min at 37°C with pH-sensitive pHrodo Red dextran (50 μg/ml, 10,000 MW; Thermo Fisher Scientific). AMs were seeded at 1 × 104 cells/cm2 (Lab-Tek II Chamber Slide, Thermo Fisher Scientific), stained with NucBlue Live ReadyProbe to visualize nuclei, and analyzed by EVOS FL Cell Imaging System (Thermo Fisher Scientific). Phagocytosis assay on AM was conducted by incubation with varying-size polystyrene microspheres (0.1, 0.5, 1.0, and 5.0 μm; Bangs Laboratories) at 100 microspheres per AM for 30 min at 37°C. Noninternalized beads were removed by three washes with cold PBS. Subsequently, cells were fixed with 4% paraformaldehyde, and phagocytosis index was calculated on the basis of number of internalized microspheres per cell.

Isolation and analysis of BAL exosome

Exosome isolation and analysis were carried out as per our described protocol (67). Briefly, exosomes from cell-free BAL fluid were precipitated by ExoQuick-TC (System Biosciences). The total protein quantification and enumeration of exosomes from BAL fluid were performed by FluoroProfile Protein Quantification Kit (Sigma-Aldrich) and a FluoroCet kit (System Biosciences), respectively, according to the manufacturer’s guidelines. Anti-CD63–coated beads (Exo-Flow, System Biosciences) were used to capture exosomes, and bead-bound exosomes were analyzed for exosome-specific staining (Exo-APC, System Biosciences) by LSRFortessa X-20 (BD Biosciences) flow cytometer. Positive staining with anti–Siglec-F was done to ascertain cellular origin, and staining with anti–H-2Kd and anti–H-2Kb was done to distinguish the origin (that is, donor versus recipient) for BAL fluid exosomes.

Adoptive transfer and proliferation of T cells

Aldehyde/Amidine Latex Beads (1.5 μm; Thermo Fisher Scientific) were conjugated with OVA or BSA according to the manufacturer’s protocol and were maintained at a final concentration of 1% solid that contained 0.95 to 1.02 μg of protein/μl. Lungs were antigen-pulsed with intrabronchial administration of 5 μl of the antigen-coated beads or with 1 μg of OVA 323-339 peptide (AnaSpec). At 6 hours after pulsing, BAL cells were collected and AMs were purified as described earlier. The purified AMs (>99% viability) were intrabronchially transferred into C57BL/6 recipients (1 × 105per mouse). Naïve CD4 T cells were isolated from Rag2/OT-II spleen (EasySep mouse CD4+ T cell enrichment kit, STEMCELL Technologies) and were labeled with CFSE (5 μM; Thermo Fisher Scientific). The CFSE-labeled cells were intravenously transferred into C57BL/6 recipients (5 × 106 per mouse) that received intrabronchial AMs immediately after the AM transfers. Proliferation of OT-II T cells was measured by CFSE dilution in lungs and spleen at 72 hours after AM–T cell transfer.

Orthotopic murine lung transplantation

Orthotopic vascularized left lung transplant was performed using cuff techniques with Balb/c donors and C57BL/6 recipients (70). All recipients were treated with anti-CD154 (MR1, 250 μg on day 0 postoperatively; Bio X Cell) and CTLA4-Ig (200 μg on day 2 postoperatively; Bio X Cell) to induce allograft acceptance (61). On day 28 after transplantation anti–H-2Kd (200 μg, SF1.1.10) was administered intraperitoneally, and the native lung, transplanted lung, peripheral blood leukocytes, and splenocytes from the lung transplant recipient mice were harvested for analysis after 6 hours.

Statistical analysis

All results are presented as mean ± SEM, and ANOVA, multiple, paired or unpaired t tests were used to evaluate statistical significance. Study designs, sample sizes with biological replicates, and statistical analyses are indicated in respective experiments. P < 0.05 was considered statistically significant. Data were analyzed and plotted with Prism 7.0 (GraphPad Software Inc.).

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/9/398/eaal1243/DC1

Materials and Methods

Fig. S1. Ab ligation of MHC class I increases lung-infiltrating B cells and neutrophils.

Fig. S2. Zbtb7a knockdown in lungs reduces anti-MHC–induced pulmonary inflammation.

Fig. S3. Zbtb7a knockdown reduces development of anti-MHC–induced iBALT in lungs.

Fig. S4. Zbtb7a deficiency does not diminish endocytosis by AMs.

Fig. S5. Zbtb7a deficiency in AM alters molecular responses to anti-MHC ligation.

Fig. S6. Exogenous alloAb induces Zbtb7a expression in the accepted lung allograft after mouse orthotopic lung transplantation.

Fig. S7. Lymphocytes are dispensable in anti-MHC–induced Zbtb7a expression but are required for OAD development.

Table S1. Differential gene expression in lungs after Ab ligation of MHC class I.

Table S2. Clinical characteristics of the lung transplant recipients.

Table S3. ZBTB7A expression in lung transplant recipients.

Table S4. Summary of Zbtb7a expression in response to exogenous Abs after mouse orthotopic lung transplantation.

Table S5. Summary of the allogeneic AM transfer protocol.

Table S6. Summary of the AM-derived alloexosome production.

Table S7. Primary data.

References (71, 72)

REFERENCES AND NOTES

  1. Acknowledgments: We thank C. Prendergast for assistance with manuscript editing and M. Marchionni for assistance with illustration. Funding: This research was supported by Flinn Foundation (award 2095 to D.K.N. and M.A.S.) and NIH (R01HL056643 and R01HL092514 to T.M. and R01AI102891 to M.T.). Author contributions: D.K.N. and T.M. conceived the idea and designed the research studies. D.K.N. and F.Z. conducted the experiments. J.H. and M.T. developed/provided reagents used in the study. M.X. assisted with the immunofluorescence studies. J.Y. evaluated the microarray data. R.H. assisted with patient selection and clinical sample procurement. D.K.N., F.Z., R.H., and T.M. evaluated the clinical history of lung transplant recipients. A.E.G. assisted with the mouse lung transplant study. D.K.N., F.Z., and T.M. analyzed the data. D.K.N. and T.M. wrote and revised the manuscript. J.H., M.T., A.E.G., R.M.B., M.A.S., and T.M. reviewed the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: The microarray data for this study are available at the NCBI GEO database (GSE71426). The huCD68-Kd Tg mouse is available from Rockefeller University under a material transfer agreement.
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