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The diagnostic utility of surveillance transbronchial biopsies (sTBBx) collected beyond the first post LTx year is unclear. Our aim was to compare the diagnostic utility of sTBBx performed in the second post LTx year to sTBBx performed in the first post LTx year. All sTBBx collected from LTx recipients between 2007 and 2019 were grouped by collection time (2 weeks, 6 weeks, 3 months, 6 months, 9 months, 12 months, 18 months, or 24 months post LTx). sTBBx were defined as collected in the absence of acute allograft dysfunction (> 10% decline in FEV1 from prior) or other clinical indications. sTBBx were considered to have diagnostic utility if acute cellular rejection (ACR) was identified (A score ≥ A1 and/or B score ≥ B1); otherwise they had no diagnostic utility (A score AX or A0 and B score B0 or BX). Mixed effects logistic regression models were used to determine the association between sTBBx collection time and diagnostic utility, as well as the impact of prior ACR and the chronic lung allograft dysfunction (CLAD) status on the diagnostic utility of the sTBBx collected in the second post LTx year. The diagnostic utility of sTBBx performed at 24 months post LTx is not significantly different from sTBBx performed 6-12 months post LTx, but is significantly less than sTBBx performed at ≤ 3 months post LTx. Among sTBBx performed in the second post LTx year, prior ACR may be associated with diagnostic utility (OR 1.5, 95% CI 0.96-2.3, p=0.08), but CLAD status is not (OR 0.79, 95% CI 0.33-1.9, p=0.6). There is a role for sTBBx beyond the first post LTx year, as the diagnostic utility is comparable to sTBBx performed 6-12 months post LTx. [ABSTRACT FROM AUTHOR]

The Toronto Lung Transplant Program uses a desensitization protocol that includes intraoperative therapeutic plasma exchange (iTPE) for virtual crossmatch (VXM) positive lung transplants (LTx), which improves access to LTx for sensitized candidates while successfully mitigating immunologic risk. Concern for peri-operative bleeding has reduced broader adoption of this protocol. We conducted a retrospective cohort study to quantify the impact of iTPE on peri-operative bleeding. All first-time LTx recipients from 2014-2019 who received iTPE were compared to those who did not. Logistic regression was used to determine the association between iTPE and high peri-operative transfusion requirements (>5 packed red blood cell (pRBC) units within 24 hours of transplant start), adjusted for disease type, LTx type, and extracorporeal membrane oxygenation (ECMO)/cardiopulmonary bypass (CPB) use. The margins command (Stata/SE15.1) was used to determine the impact of iTPE on the predicted probability of high peri-operative transfusion requirements. The incidence of hemothorax (need for re-operation within 7 days of LTx) was compared between the 2 groups using Chi square test. 142 patients (16%) received iTPE and 755 patients (84%) did not. iTPE was associated with an increased odds of high peri-operative transfusion requirements (OR 1.6, 95% CI: 0.9-2.8, p=0.08), but was not associated with an increased incidence of hemothorax (5% in both groups, p=0.99). The predicted probabilities of high peri-operative transfusion requirements for a double LTx for restrictive lung disease based on iTPE and ECMO/CPB use are shown in Table 1. This study demonstrates that the use of iTPE in LTx may increase peri-operative bleeding, but not to a clinically meaningful degree. The addition of iTPE allows sensitized candidates to undergo LTx safely without changing the most probable peri-operative transfusion requirements (even for LTx performed on ECMO or CPB), or increasing the risk of hemothorax. [ABSTRACT FROM AUTHOR]

CC10 is an abundant immune modulator produced by club cells that is reportedly reduced in lung transplant recipients diagnosed with CLAD. Previous work shows that rhCC10 treatment promotes anti-inflammatory effects in models of acute and chronic injury but whether it can mitigate CLAD development remains unknown. Using a recently developed mouse orthotopic lung transplant model of CLAD, we asked if rhCC10 ameliorates airway epithelial injury and alloimmunity. C57BL/6 recipients of FVB major-mismatched left lung transplants that encode a diptheria toxin transgene under the control of the CC10 promoter and the reverse tetracycline transactivator were induced to partially deplete allograft club cells between post-operative days (PODs) 7-9. Recipients were treated daily by intratracheal instillation (IT) or intravenous (IV) routes with rhCC10 (0.6, 1.2, 2.4, and 4.8 mg/kg body weight) or vehicle on PODs 8-15. On POD 16, recipients were euthanized and blindly assessed for allograft injury and lymphocyte activation. IT but not IV rhCC10 significantly improved lung airway histopathology scores (ISHLT B & C, Ashcroft), reduced hydroxyproline content, and increased club cell regeneration. The IT optimal effective dose was 1.2 mg/kg (Figure 1). IT rhCC10 also sharply reduced donor specific antibodies and intragraft IL-17A+CD4+ and IFN-γ+CD8+ T cell abundance. An intermediate dose of IT rhCC10 was effective in mitigating airway injury and preventing adaptive immune responses known to drive CLAD development. Work is ongoing to determine the precise mechanisms by which CC10 promotes lung transplant tolerance. [ABSTRACT FROM AUTHOR]

Chronic lung allograft dysfunction (CLAD) is the leading cause of lung transplant failure, with 50% of patients developing it by five years post-transplant. CLAD is characterized by progressive fibrosis, and it has no effective therapies. Nintedanib is a multiple tyrosine kinase inhibitor used to treat idiopathic pulmonary fibrosis. Here we assess nintedanib's efficacy, with tacrolimus, as a potential preventative CLAD therapy in the heterotopic tracheal transplant mouse model of CLAD-like airway fibrosis. We performed Balb/C (H-2d)-to-C57BL/6 (H-2b) transplants. To arrive at effective doses of nintedanib and tacrolimus alone, we treated nine groups of n=10 recipients with one of three doses of nintedanib (0, 25, 50mg/kg), or six doses of tacrolimus (0, 0.1, 0.25, 0.5, 1, 2mg/kg). Mice were given either drug daily for 28 days. Following sacrifice, tracheas underwent Masson-Trichrome staining and were graded for luminal fibrotic occlusion and epithelial necrosis. Kruskal-Wallis one-way analyses of variance were performed on both grades within drug treatments. We found that compared to the control (Fig. 1C), fibrotic occlusion was significantly reduced in nintedanib treatment groups, but that epithelial necrosis was unaffected (Fig. 1 A, B, D). In contrast, we found that high doses of tacrolimus eliminated both pathologies (Fig. 1 A, B, E). Lower doses of tacrolimus had a moderate effect on luminal occlusion, and no effect on epithelial necrosis. Our results demonstrate nintedanib's potential as an anti-fibrotic drug in lung transplantation. Nintedanib caused a significant decrease in fibrotic occlusion, but epithelial necrosis was reduced only by tacrolimus. Based on this, we will examine the combined effects of the drugs to arrive at a treatment strategy with both immunosuppressive and antifibrotic components. [ABSTRACT FROM AUTHOR]

Summary of Objectives Bronchoalveolar lavage (BAL) is a key tool for clinical monitoring and research in lung transplantation (LTx). However, BAL collection and processing are not standardized across LTx centers, resulting in significant variability of practices. This ISHLT-supported consensus statement on BAL standardization aims to clarify definitions and propose common approaches to improve: a) clinical and research practice standards, b) comparison and validation of research data, and c) collaboration between centers. Methods After an open invitation to ISHLT members, an international workgroup of 68 LTx specialists from 10 countries (75% North America, 16% Europe, 9% Australia/Asia) was created and divided into 9 subgroups covering the following topics: 1) bronchoscopy procedure and BAL collection, 2) sample handling in the bronchoscopy department, 3) sample processing for clinical microbiology, 4) clinical cytology, 5) research and 6) microbiome, 7) sample inventory and tracking, 8) donor bronchoscopy and 9) pediatric considerations. The workgroup designed and administered an online survey to capture practices of BAL collection and processing in LTx, according to the 9 subgroup topics. Results from 112 of 159 invited LTx centers (response rate 70%) indicate significant inter-center discrepancies in the definitions and methodology of BAL collection and processing. A systematic literature analysis performed by the workgroup identified 525 relevant articles; a specifically designed questionnaire was used to a) identify relevant articles for each subgroup and b) assess the quality of published BAL methodology. Endpoints Based on the large-scale international survey and a systematic literature review, the ISHLT BAL Standardization Workgroup is currently preparing consensus statements relevant to the 9 subgroup topics above. The statements will be subjected to three rounds of voting by the workgroup according to the Delphi method (consensus goal >80% of responders within two integers of the median). This abstract will be updated in March 2019 with key excerpts from finalized statements, which will be presented at the ISHLT 2019 annual meeting and submitted for publication. This consensus document will be instrumental for the harmonization of clinical and research practices and will help improve the performance of the international LTx community in this regard. [ABSTRACT FROM AUTHOR]

Purpose Regulatory T cells (Tregs) are a potential therapeutic option in lung transplantation (LTx). We have shown that expanded recipient Tregs can be seeded pre-transplant in the donor lung graft using normothermic ex vivo lung perfusion (EVLP). Here, we examined whether this therapy can alleviate lung injury early post-transplant. Methods Study design is shown in Fig A. CD4+CD25hi Tregs were isolated from Wistar Kyoto rats (WKy, RT1l) and expanded for 7d-14d. Fischer 344 (F344 RT1lv1) heart-lung blocks were flushed and placed on EVLP. Lungs were perfused with SteenTM solution (37°C, 4h). Expanded Tregs (5-120 × 106) were infused after 1h. Control lungs received no Tregs. The right lung was used for assessment at end-EVLP, and left LTx into WKy recipients was performed after 60min cold preservation. Necropsy was performed on day 3 post-LTx. Results Wet-dry ratio was lower in Treg-treated grafts compared to controls (Fig B). Transcripts for FoxP3, IL-10, CTLA-4 and GITR were elevated in Treg-treated grafts (Fig B). Tregs isolated from the right donor lung at end-EVLP suppressed CD4+CD25− T cell proliferation in vitro (Fig C). On day 3 post-LTx, ICAM1 and CD44 (adhesion molecules) expression on both CD4+ and CD8+ T cells were lower in Treg-treated grafts compared to untreated controls (Fig D). Histologically, both Treg-treated and control grafts had minor lung injury and cellular rejection on day 3 post-LTx: acute lung injury scores (4.10 ± 1.66 and 3.88 ± 1.36), ISHLT A grades (0.60 ± 0.516 and 0.625 ± 0.744), B grades (0.400 ± 0.516 and 0.313 ± 0.458). Conclusion Expanded recipient Tregs directly delivered to the lung allograft prior to transplantation exert immune regulatory effects in vivo and in vitro. Further work is needed to optimize this pre-transplant therapeutic approach. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) can lead to end stage lung disease for which lung transplantation can be a life-saving therapy. CF has systemic manifestations that can lead to other end organ damage. In addition, immune suppression following single organ transplantation has adverse effects including end stage renal disease. Multi-organ transplant in CF has had a significant impact on long-term survival of CF patients. In this study, we aim to assess the impact of multi-organ transplant on the survival of CF patients. This is a retrospective review of all CF patients who have been multi-organ recipients after their initial lung transplant (March 1988 to July 2020). Lung re- transplant only were excluded. From 435 lung transplant recipients we identified 19 patients with CF who had received multi-organ transplant either simultaneously with the lung transplant or consecutively over time. 7 recipients received simultaneously 15 organs that included: 1 heart and lung, 5 lung and liver and 1 lung, liver, pancreas. The median survival for this group was: 8.9 years. 12 recipients received consecutively 30 organs that included: 6 lung followed by kidney transplant; 1 lung followed by liver transplant; 1 lung followed by combined kidney+liver; 1 lung followed by combined kidney + pancreas; 1 lung followed by liver and then kidney transplant; 1 lung transplant followed by a second lung transplant and later on kidney transplant and finally 1 lung followed by another lung transplant and later on combined kidney+ pancreas.The median survival of these multiorgan recipients is 14 years. The current median survival for CF patients in our program excluding re-transplants is 8.2 years. Lung transplant for CF offers survival benefit and improved quality of life, such that a subgroup of these patients qualify for a second and even a third organ transplant to further prolong life. [ABSTRACT FROM AUTHOR]

BAL GM has higher sensitivity than traditional culture methods. However, smaller studies have documented higher false positivity in the early post-transplant period. This study aimed to evaluate the rate of IA in patients who had a negative fungal culture and positive BAL GM in the first 30 days post lung transplant. We retrospectively identified LTRs from January 2010 to January 2017, who had a negative Aspergillus culture and positive BAL GM with an index cutoff value of ≥1.0 in the first 30 days post-transplant. Development of IA within 30 days after the initial positivity was evaluated. ISHLT definitions were used to define IA and colonization. We reviewed 791 LTRs; 119 patients had negative fungal culture and a positive BAL GM in the first 30 days post-transplant. 59% (70/119) were male, mean age 53 years old, 15% (18/119) had cystic fibrosis. 3% (4/119) of the patients had IA at the time of initial positive BAL GM. Of the other 115 patients, 112 were noted to be positive only once, 3 were positive two or more times. 63% (73/115) received preemptive antifungal therapy. None of them developed IA. 37% (42/115) did not receive preemptive antifungal therapy, and only 7% (3/42) developed IA. In the absence of antifungal therapy, 7% (3/42) of LTRs subsequently developed IA following positive BAL GM >1.0 in the first 30 days post-transplant. Further characterization of patients with positive BAL GM during the first month may be helpful in devising the pre-emptive antifungal therapy in the early period after lung transplantation. [ABSTRACT FROM AUTHOR]

CD4+CD25highFoxp3+ regulatory T cells (Treg) can establish immunologic tolerance in animal models. We have developed a method to deliver recipient-derived Tregs to donor lungs during ex-vivo lung perfusion (EVLP). Our goal is to maximize the effectiveness of this therapy by developing a Treg-permissive post transplant immunosuppression protocol. We hypothesized that combination therapy with interleukin-2 (IL-2)/anti-IL-2 antibody complexes (IL-2c) and tacrolimus (Tac) would facilitate the expansion of endogenous Tregs and attenuate acute rejection, thus creating an environment permissive to Treg therapy. We performed rat left lung transplantation (LTx) experiments using Fischer 344 (F344, RT-1lv1) donors and Wistar Kyoto (WKy, RT-1l) recipients with or without antecedent 3h EVLP. Animals received: no treatment (NT); IL-2c (a single low [30ug] or high [60ug] dose) alone; Tac (0.1mg/kg/day, 7 doses) alone; or both IL-2c and Tac, intraperitoneally starting on the day of transplantation. Allografts that underwent EVLP had lower acute rejection grades in IL-2c/Tac-treated rats than in NT and Tac-treated rats (A Grade p = 0.046; B Grade p = 0.064; Fig1). In contrast, there were no differences among these groups in allografts that had not undergone EVLP. In the grafts, the frequency of CD4+CD25highFoxp3+ cells increased in both low and high dose IL-2c/Tac groups compared with Tac group, regardless of EVLP (p = 0.012, p = 0.107; Fig 2). Although the frequency of CD4+CD25+Foxp3- effector T cells also increased with high dose IL-2c in the non-EVLP group, this was not seen with EVLP (p = 0.804) (Fig 2). Despite the presence of Tac, IL-2c increased Tregs in the graft after transplantation and attenuated acute rejection in EVLP-treated lungs. This work lays the groundwork for pre-implantation allograft-directed regulatory T cell therapy. [ABSTRACT FROM AUTHOR]

Donor aspiration of gastric contents represents a significant risk for lung injury and severe primary graft dysfunction after lung transplantation. Concern about possible aspiration is a common reason for clinicians to decline donor lungs for transplantation. The presence of bile acid in donor bronchial wash (BW) or bronchoalveolar lavage (BAL) fluid may indicate that a donor has aspirated. Previous reports have also shown that aspiration of bile acids cause increased inflammation in the lung. Ex vivo lung perfusion (EVLP) has become an important platform to assess questionable or "marginal" donors. In this study, we examined the relationship between possible donor aspiration and physiologic and biologic assessment of inflammation on EVLP. Large airway BW samples were collected from 270 donors (n=111 non-EVLP, n=159 EVLP) prior to transplantation. Bile acid levels were measured in BW samples using a commercially available colorimetric assay and clinically approved platform. Protein and biochemical measurements were made in perfusate samples collected hourly after the start of EVLP. The majority of donor lungs with clinically suspected aspiration were successfully transplanted after EVLP assessment (58% [23/40]). Donor bile acid levels were significantly higher in donor lungs that were ultimately declined for transplantation at the end of EVLP (0.28 vs. 0.12 μM, p<0.05, n=143). During EVLP there was a positive correlation between bile acid in the donor BW and inflammation (IL-8 levels (r=0.19, p<0.05, n=159)). Similarly, there was an increase in biochemical markers of cell death ([Ca2+]) in the EVLP perfusate of donors with high bile acid levels in BW (r=0.27, p<0.001, n=142). Following EVLP and transplantation, we observed no significant effects of donor bile acid levels on recipient ICU length-of-stay (r=0.09, p=0.35, n=117). Suspected donor aspiration can be assessed using the EVLP platform. Bile acid levels measured in the donor lung are associated with increased inflammation and lung injury observed during EVLP, but not recipient outcomes post-EVLP. Therefore, EVLP may be an ideal platform to identify marginal donor lungs suspected of aspiration but are ultimately suitable for clinical transplantation. [ABSTRACT FROM AUTHOR]

Purpose Airway inflammation after lung transplantation (LTX), resulting from infection, pollution, or aspiration, is an important risk factor for chronic lung allograft dysfunction. Classical dendritic cells (cDCs) link innate immunity and adaptive immunity, and exhibit regional and functional specialization in the lung. CD103+ cDCs reside in the airway epithelium. Their role in lung transplantation (LTX) has not been extensively investigated. We hypothesized that CD103+ cDCs augment adaptive immune responses under conditions of lipopolysaccharide (LPS)-induced airway inflammation inamouse minor alloantigen-mismatched orthotopic LTX model (C57BL/10 [B10] →C57BL/6 [B6] and vice versa). Methods BATF3-/- mice (B6 background), lacking CD103+ cDCs, were used. Three comparisons were made: 1) B10 → 6 vs. B10 → ATF3-/-, 2) B10 →B6 vs. B10 → ATF3-/- with 6 doses of intra-tracheal (i.t.) LPS (5μg in 50μlPBS)on serial postoperative days (POD), 3)B6 → 10 and BATF3-/- → 10 with 8 doses of i.t. LPS on serial PODs. The grafts were assessed on POD 28. Results Acute rejection (ISHLT criteria) did not differ significantly between B10 → 6 and B10 → ATF3-/- without LPS, or between B6 → 10 and BATF3-/- → 10 with LPS. In contrast, with LPS exposure, acute rejection scores were lower in B10àBATF3-/- compared to B10 àB6 (Figure A). Surprisingly, lung cell analysis by flow cytometry showed that monocytes and neutrophils were significantly higher in BATF3-/- recipients than wild-type recipients under repeated airway LPS exposure (Figure B). Conclusion Our data show that recipient, but not donor, CD103+ cDCs contribute to the augmentation of acute rejection after LTx in the setting of ongoing airway inflammation. As CD103+ cDCs can promote immune regulation, they may be an important therapeutic target after LTX. [ABSTRACT FROM AUTHOR]

Purpose Chronic lung allograft dysfunction (CLAD) is the primary obstacle to long-term survival after lung transplantation (LTX). Ischemia-reperfusion injury (IRI) increases the risk of CLAD in humans but the underlying mechanisms are not well understood. In a mouse minor alloantigen mismatched LTX model (C57BL/10 [B10] →C57BL/6 [B6]), we observed that IRI augments formation of B cell-rich tertiary lymphoid organs (TLOs) and chronic rejection in allografts but not isografts. We therefore hypothesized that B cells may link IRI with chronic rejection. Methods B10→B6 LTX with extended graft preservation (6 hours at 4°C followed by 45 minutes at 37°Cand 15 minutes at room temperature during anastomosis), which augments acute rejection, fibrosis, and TLO formation, was employed. Anti-CD20 antibody (aCD20: n=9) or isotype control (Iso: n=10) were given i.p. on days -2, +2, +7, +14, and +21. Peripheral blood was longitudinally assessed by flow cytometry after LTX. Grafts were assessed with histology, flow cytometry and immunofluorescence 28 days after LTX. Results aCD20 treatment effectively depleted CD19+ or B220+ B cells in peripheral blood (Figure A) and lung grafts (Figure B). Acute rejection (ISHLT A grade), peribronchial fibrosis, and airways with obliterative bronchiolitis (%OB) were lower in aCD20 mice (1.8, 1.4, and 13.1% respectively) than in Iso mice (3.1, 2.6, and 46.7% respectively) (Figure C). TLOs containing CD3+, CD19+, and peripheral node addressin (PNAd)+cells were also reduced (Figure D). Conclusion In this model of IRI-augmented chronic rejection, B cell depletion attenuated acute rejection, airway fibrosis, and lymphoid neogenesis. Our observations suggest that B cell activation and recruitment may be directly involved in the augmentation of CLAD in LTX recipients experiencing IRI. [ABSTRACT FROM AUTHOR]

DQA mismatches, defined by the presence of a donor DQA antigen not present in the recipient, and risk epitope mismatches, defined by the presence of a donor DQA1*05/DQB1*03:01 or DQA1*05/DQB1*02 antigen not present in the recipient, are associated with de novo donor specific antibodies (dnDSA). Our aim was to stratify lung transplant recipients (LTR) by their DQA and risk epitope mismatches to determine the impact on dnDSA and chronic lung allograft dysfunction (CLAD). First-time, adult LTR from 2014-2019 with complete human leukocyte antigen (HLA) typing and post-transplant HLA antibody testing were included. LTR were divided into 4 risk categories: green if no DQA or risk epitope mismatches present; yellow if only a DQA mismatch present; orange if only one of the risk epitope mismatches present; or red if 2 risk epitope mismatches or one risk epitope mismatch and DQA mismatch present. dnDSA-free and CLAD-free survival were compared among the risk categories using log rank test. Of the 859 LTR included in the study, 158 (18%) were risk category green, 410 (48%) risk category yellow, 29 (3%) risk category orange, and 262 (31%) risk category red. dnDSA-free survival was significantly shorter among LTR in risk category red (Figure 1, log-rank p-value<0.001). CLAD-free survival did not differ among the 4 risk categories (Figure 2, log-rank p-value=0.57). Stratifying LTR by DQA and risk epitope mismatches predicts dnDSA but not CLAD development, which may indicate a "two-hit hypothesis" for CLAD driven by dnDSA. Further studies are required to determine if avoiding risk category red lung transplants is a feasible strategy to reduce dnDSA. [ABSTRACT FROM AUTHOR]

Despite years of study using conventional cellular molecular approaches applied to both humans and animal models, the biology of lung transplantation remains incompletely understood. We aimed to develop a new strategy to study molecular mechanisms using human lung transplant samples and modern sequencing technology. The interaction between donor and recipient cells is unique to transplantation. As such, we developed a sequencing and bioinformatic strategy to help deconvolute recipient from donor specific gene expression in a frozen lung tissue biopsy. We performed whole exome sequencing on frozen biopsies of pure donor and recipient lungs, followed by RNA-seq on a frozen lung biopsy taken at 2h after reperfusion. For each patient, donor/recipient specific single nucleotide variations (SNVs) were identified and used to quantify donor/recipient expression in the RNA-seq data. The resultant genes were compared to known marker genes from the lung and immune cell types. Recipient-specific transcripts identified from the RNA-seq data were enriched for immune cell specific transcripts and gene ontology analysis depicted neutrophil activation and degranulation (Fig 1). Donor-specific transcripts were enriched in lung parenchymal cell types. Allele fractions of the recipient specific transcripts had a median between 0.04-0.06, while donor specific transcripts had the expected median of 0.5. This indicates that the majority of gene expression is derived from the donor lung and that there is only a small fraction of recipient derived transcripts as would be expected in the early reperfusion period. We show that it is possible to deconvolute donor and recipient transcripts at the earliest stages of reperfusion by taking advantage of SNVs. The resultant pathway analysis corresponds with our current knowledge of reperfusion biology. Following validation, we aim to apply this to less well-defined phenomena such as CLAD and to generalize the technique to solid-organ transplantation. [ABSTRACT FROM AUTHOR]

CMV infection following lung transplant (LTx) is associated with increased morbidity and mortality. Inflammation, infection and longer ischemic times are important risk factors for CMV infection. Ex vivo lung perfusion (EVLP) has helped to successfully increase utilization of high-risk donors over the last decade. However, the impact of EVLP on post-transplant CMV infection is unknown. Single-center, retrospective analysis of all LTx recipients from 2010 to 2020. The primary endpoint was comparison of CMV viremia in recipients that received EVLP vs. non-EVLP donor lungs. CMV viremia was defined as CMV PCR > 1000 IU/mL within two years post-transplant. Secondary endpoints were time from LTx to CMV viremia, peak CMV PCR and survival. Outcomes were also compared between the different donor (D)-recipient (R) CMV serostatus matching groups. Included were 862 recipients of non-EVLP lungs and 389 recipients of EVLP lungs. There was no significant difference in the distribution of the CMV serostatus matching groups. 35.8% of patients in the non-EVLP group developed CMV viremia vs. 31.9% in the EVLP group (p=0.18). Median time to CMV viremia was 234 days [IQR, 179-318] in non-EVLP and 249 days [IQR, 186-313] in EVLP group (p=0.5). The mean±SD peak viremia was 4.1±0.8 log 10 IU/mL in the non-EVLP group and 4.2±0.8 log 10 IU/mL in the EVLP group (p=0.4). There was also no difference in survival of the viremic patients between the groups (log-rank p=0.8). All outcomes were similar when comparing non-EVLP and EVLP patients within each serostatus matching group (Fig.1). Lung transplant activities have significantly increased with the use of high-risk donor lungs evaluated on EVLP. The practice of utilizing more injured donor organs via EVLP has not affected CMV viremia rates and severity in lung transplant recipients. EVLP could further provide the opportunity to pre-treat donor lungs prior to implantation, perhaps decreasing incidence of post-transplant CMV infection. [ABSTRACT FROM AUTHOR]

Lung transplantation in the presence of donor specific antibodies (DSA) is safe with peri-operative desensitization, although persistence of DSA is common. Herein, we present a unique case of a highly sensitized lung transplant recipient with selective loss of DSA post-transplantation. A 65-year-old female with a calculated PRA (cPRA) value of of 17% (class I) and 100% (class II), underwent bilateral lung transplantation for chronic obstructive pulmonary disease. Virtual crossmatch (VCM) was positive, involving DPB1*04:01, DR17, and DR52. The subsequent flow cytometry B-cell crossmatch was strongly positive (median channel shift 296). She received peri-operative desensitization protocol for VCM positive patients, which includes intra-operative and post-operative plasmapheresis, intravenous immune globulin, and anti-thymocyte globulin. Maintenance immunosuppression included cyclosporine (target trough 250-350 umol/L), mycophenolic acid 900mg BID and prednisone 20 mg OD. An increase in DSA reactivity was observed one month after transplant (DPB1*04:01 (MFI=17639), DR17 (MFI=17812), and DR52 (MFI=19414). Repeat HLA testing at 3, 4, and 6 months post-transplant showed sequential reduction of DSAs to below the laboratory cut-off for positivity point despite persistence of non-DSA alloantibodies. The patient continues to maintain good lung function with an FEV1 of 2L (86% of predicted) at 6 months post-transplant. Surveillance transbronchial biopsies at 3 and 6 months were free of cellular rejection. Despite initial rebound of pre-formed DSA following peri-operative desensitization, sustained selective clearance of the DSA subsequently occurred in this case, which likely benefited graft function. Similar cases have been reported after combined kidney-liver transplant. Robust laboratory research and prospective studies are required to understand the mechanism of selective DSA elimination in organ transplantation. [ABSTRACT FROM AUTHOR]

Despite years of study using conventional approaches applied to human and animal models, the biology of lung transplantation remains incompletely understood. We aimed to develop a new strategy to study the molecular mechanisms using human lung transplant samples and modern sequencing. The interaction between donor and recipient cells is a unique feature to transplantation. As such, we developed a sequencing and bioinformatic strategy to deconvolute recipient from donor specific gene expression in a frozen lung tissue biopsy. We performed whole exome sequencing (40x coverage) on frozen biopsies of pure donor and recipient lungs, followed by RNA-seq (200M 2x125 bp) on a frozen lung biopsy taken at 2h after reperfusion. For each patient, donor/recipient specific single nucleotide variations (SNVs) were identified and used to quantify donor/recipient expression in the RNA-seq data. The genes were compared to known marker genes from the lung and immune cell types. Recipient-specific transcripts identified from the RNA-seq data were enriched for immune cell specific transcripts and gene ontology analysis depicted neutrophil activation and degranulation (Fig 1). Donor-specific transcripts were enriched in lung parenchymal cell types. Allele fractions of the recipient specific transcripts had a median ∼0.05, while donor specific transcripts had the expected median of 0.5. This indicates that the majority of gene expression is derived from the donor lung and there is only a small fraction of recipient derived transcripts as would be expected in the early reperfusion period. We have demonstrated that it is possible to deconvolute donor and recipient transcripts at the earliest stages of reperfusion by taking advantage of SNVs. The resultant pathway analysis corresponds with our current knowledge of reperfusion biology. Following validation, we aim to apply this to less well-defined phenomena such as CLAD and to generalize the technique to solid-organ transplantation [ABSTRACT FROM AUTHOR]

There is a paucity of studies assessing the incidental finding of histoplasmosis in the explanted tissue from lung transplant recipients (LTRs). Thus, there are currently no recommendations for the management of Histoplasmosis in the explanted organ of LTRs. This study aims to describe our experience in the management of histopathologic evidence of Histoplasmosis, encountered unexpectedly in the explanted tissue of LTRs at the time of transplantation. We retrospectively identified LTRs at the Toronto program from 2010-2017, with histopathologic evidence of Histoplasma in the lung explanted tissue. Of 810 LTRs, 6 recipients had Histoplasmosis in the explanted organ (incidence 0.75%, clinical characteristics shown in Table 1. None of the 6 patients had a previously known diagnosis of Histoplasmosis. All of them lived in Ontario, Canada, and only one had traveled abroad in the past five years (to Florida, USA). All 6 patients had lung parenchyma and lymph node involvement in the histopathology findings, with necrotizing granulomas and fungal spores compatible with Histoplasmosis. All patients received antifungal pre-emptive therapy after transplant: 4 (67%) Itraconazole and 2 (33%) voriconazole for a mean duration of 4.8 months (IQR, 2.82-8.1 months). No patients in this group developed posttransplant Histoplasmosis (median follow up of 3.0 years [IQR 2.0-4.1 years]). The unexpected finding of histoplasmosis in the explanted tissue of LTRs was infrequent (0.75%) in this non-endemic area. None of the patients, all of whom received antifungal therapy, developed active Histoplasmosis post-transplant. [ABSTRACT FROM AUTHOR]

Purpose Cytomegalovirus (CMV) infection and development of CMV drug resistance can cause significant morbidity and mortality in lung transplantation (LTX) patients. We investigated the incidence of CMV drug resistance in adult LTX patients and characterized this patient group and its outcomes. Methods We analyzed a single center retrospective cohort of LTX patients who had undergone CMV UL97 and UL54 genotyping for clinically suspected drug resistance. Of the 852 patients who received LTX between January 2012 and May 2018, 27 patients were genotyped, and 11 were confirmed to have drug resistance. Case-matched controls (3 control patients for each resistant patient) were identified by matching for CMV serostatus, development of CMV disease or significant CMV viremia (over 3000 IU/ml), and transplantation date. Results The cumulative incidence of drug resistant CMV was 1.29% (11/852). Altogether 27 patients were tested for drug resistance and 40.7% (11/27) of strains were resistant, 29.6% (8/27) sensitive, and 29.6% (8/27) inconclusive. The median follow-up time was 630 days (range 100-1950 days). Most patients (7/10) who developed resistant CMV infection were seronegative recipients who received a lung from a CMV seropositive donor. Comparison of characteristics and clinical outcomes between resistant CMV patients and control patients is provided in Table 1. No differences in immunosuppression, acute rejection, or pre-transplant sensitization were seen between groups. The peak CMV viral load and duration of CMV viremia were significantly higher in the resistant group. The resistant group had a significantly increased overall mortality after onset of viremia compared to the controls. Conclusion We conclude that drug resistant CMV infection is rare but patients who develop it have decreased overall survival. Risk factors for developing resistant CMV infection were peak CMV viral load and duration of CMV viremia. [ABSTRACT FROM AUTHOR]

Purpose We have applied cutting-edge mass cytometry (MC) technology to serial bronchoalveolar lavage (BAL) cells obtained from lung transplant recipients (LTRs) and have used it to compare the cellular composition of the BAL compartment in patients who remain stable or go on to develop lung allograft dysfunction (LAD), defined here as a drop, from any cause, in the forced expiratory volume in 1 second of 20% from the best post-transplant baseline value. Methods We have collected and cryopreserved BAL cells from 50 consecutive LTRs at their 3-, 6- and 9-month post-transplant surveillance bronchoscopies. Cryopreserved samples were then recovered and labeled with a panel of 37 heavy metal-tagged antibodies against surface markers and intracellular cytokines before MC. We applied t-distributed stochastic neighbor embedding (tSNE; Fig1 A) which assists with better visualization of multi-dimensinal analysis to identify both common and rare cell populations in the BAL. We further applied an algorithm called cluster identification, characterization and regression (CITRUS; Fig1 B) to allow unsupervised identification of differentially represented BAL cellular subsets between stable and LAD patients. Results Longitudinal analysis of the first 25 patients demonstrated different cell subsets and cytokine-producing cells, with substantial intra- and inter-patient variation. Our data identified an elevation in the frequency of CD4+ CD57+ PD-1+ cells separates LAD (18.41±4.89%) from stable patients (4.43±2.11%; Fig1 C). Emergence of CD57+ PD-1+ events among CD4+ T cells preceded LAD by up to several weeks (Fig1 D), suggesting that these cells may contribute to LAD development rather than simply correlating with it. Conclusion Our data suggest that emergence of CD4+ CD57+ PD-1+ T cells may predict and/or contribute to allograft dysfunction. Molecular and functional studies on this cell population are underway to obtain a better understanding of their role in lung transplant immunobiology. [ABSTRACT FROM AUTHOR]

CLAD is the main limitation to long-term survival after lung transplantation. Reduced epithelial club cells and lung CCSP levels have been associated with CLAD. Recombinant human (rh)CCSP has been shown to have anti-inflammatory and anti-fibrotic properties in animals and humans. However, CCSP roles in CLAD pathogenesis and its capacity to prevent or reverse CLAD features remain unknown. Thus, we hypothesized that rhCCSP would prevent CLAD via decreasing inflammation and fibrosis. A minor alloantigen-mismatched orthotopic lung transplant mouse model of C57BL/10 donors to C57BL/6 recipients was used. 8 doses of lipopolysaccharide (LPS) were administered intratracheally (IT) from postoperative day (POD) 3 to POD21. rhCCSP (doses of 0.6, 1.2, 2.4, or 5 mg/kg) was administered intravenously (IV) or IT on the day following each LPS, with an additional 5 doses on POD 22-27. Lung samples were collected on POD28. CLAD-like features of fibrosis and inflammation were graded using H&E and Masson-Trichrome-stained histology samples. RT-qPCR and immunofluorescence were performed. IV administration of rhCCSP at 2.4mg/kg resulted in greatest changes, with a trend towards decreased ISHLT A-grade rejection, B-grade rejection, bronchiolitis obliterans scores, percent parenchymal fibrosis, and epithelial hyperplasia, a marker epithelial injury, without statistically-significant differences (Fig 1A-F). IV rhCCSP treatment was also associated with trends towards reduced Collagen1α1, TIMP1 and IL-17A transcripts (Fig 1G-I), but no change in CD3+ T cells (Fig 1J). IT rhCCSP did not have significant effects on measured endpoints. Our pilot dose-finding experiment suggests that IV rhCCSP treatment at 2.4mg/kg may attenuate inflammation, fibrosis, and epithelial injury in an LPS-induced mouse model of CLAD. Further optimally-powered validation experiments are underway, in order to elucidate whether rhCCSP treatment might be an effective therapy for CLAD. [ABSTRACT FROM AUTHOR]

Chronic lung allograft dysfunction (CLAD) is the major barrier to long-term survival following lung transplantation and results from repeated acute injuries to the graft epithelium. Epithelial club cells are known to have protective, anti-inflammatory, and regenerative roles and are depleted in CLAD. Mechanisms driving club cell loss in CLAD are unknown. Our single cell RNA sequencing (scRNAseq) data showed that TRAIL transcript was upregulated in CLAD club cells compared to control donor lung club cells. TRAIL induces apoptotic cell death by binding to its receptors (TRAIL-R1 and TRAIL-R2). To validate the scRNAseq data, we here further investigate the expression of TRAIL and its receptors at the protein level. We hypothesized that CLAD develops as a consequence of club cell death induced by TRAIL signaling during episodes of acute lung allograft dysfunction (ALAD). Lung transplant recipients' (LTR) airway brushings were obtained 3-12 months post-LT and processed for flow cytometry. TRAIL, TRAIL-R1, TRAIL-R2 expression was examined in club cells in LTRs with or without ALAD, defined as a >=10% drop in forced expiratory volume in 1 second from prior. After exclusion of non-epithelial cells, EpCAM+ epithelial cells and EpCAM+CCSP+ club cells were identified. Our findings show that the frequencies of both TRAIL+ club cells and TRAIL+ non-club epithelial cells were significantly higher in ALAD LTRs compared to non-ALAD LTRs (Fig. 1A-C). Further, TRAIL-R1 expression on club and non-club epithelial cells tended to be higher in ALAD compared to non-ALAD LTRs (Fig. 1A, D-E). TRAIL-R2 expression did not differ between ALAD and non-ALAD LTRs. Our preliminary data show an upregulation of TRAIL within epithelial cells found in ALAD airway brushings compared to control. The co-localization of TRAIL and TRAIL-R1 in club cell suggests that autocrine or paracrine cell death might be potential mechanisms for club cell loss in CLAD. [ABSTRACT FROM AUTHOR]

Lymphocyte-mediated acute rejection after lung transplant (LTx) is one of the main risk factors for chronic lung allograft dysfunction (CLAD). We hypothesized that a high peripheral absolute lymphocyte count (ALC) after LTx would correlate with a higher prevalence of acute rejection, as well as increased risk of CLAD and death. We performed a retrospective single-center cohort study of adult recipients of a first single or double LTx 2005-2018. The area under the curve (AUC) of ALC between 1 day and 395 days (1 year + 1 month to capture patients around their 1-year follow-up) after LTx was calculated. We evaluated the average of acute rejection A-grades in patients in the highest tertile of ALC AUC compared to the lower 2 tertiles. We used Cox proportional hazards (PH) models to assess the association between ALC AUC with time to CLAD and time to death/retransplant from 395 days post-LTx. 1103 patients were included in this study with a median of 68 ALC measurements (SD 47.3) per patient in the first 395 days post LTx. The average of rejection A-grades in the highest tertile of ALC AUC was significantly higher than those in the two lower tertiles (0.31 vs. 0.2 and 0.25, P = 0.004). Cox PH models restricted follow-up to the first 7 years post-LTx only, in order to satisfy the PH assumption. ALC AUC in the first year post-LTx was not significantly associated with CLAD (HR 1.102, 95% CI 0.943-1.287, P = 0.222). However, in univariable as well as multivariable analyses adjusting for age, sex, cytomegalovirus serostatus mismatch, primary lung disease, and transplant type (single or double LTx), ALC AUC - modeled as both a continuous variable or highest tertile - was associated with a lower risk of death (continuous; HR 0.994, 95% CI 0.989-0.998, P = 0.006; highest tertile: HR 0.762, 95% CI 0.582-0.997, P = 0.048). Higher ALC after LTx correlates with a higher average acute rejection grade but not with CLAD, possibly due to the protective effects of ALC against infection. Higher ALC in the first year post-LTx is associated with a lower risk of death/retransplant in the first 7 years. Further validation is needed to determine whether post-LTx ALC could be an effective biomarker for predicting risks for acute rejection and death. [ABSTRACT FROM AUTHOR]

Lung transplantation (LT) is limited by the lack of suitable donor lungs, and early-post LT survival is hampered by primary graft dysfunction (PGD). Both neurological determination of death (NDD) and donation after circulatory death (DCD) are associated with donor lung injuries, and ex vivo lung perfusion (EVLP) can be used to assess donor lung suitability. The relationship of donor leukocytes within the EVLP perfusate to donor mode of death and PGD is not well studied. Using CITRUS, an algorithm designed to identify stratifying biological signatures, we hypothesized that there would be differences in perfusate leukocyte composition based on donor mode of death and recipient PGD status. Clinical EVLP cases from donor lungs of NDD (n = 20) and DCD (n = 20) origin were retrospectively selected. In a separate cohort, clinical EVLP cases were selected and grouped based on recipient PaO2/FiO2 (P/F) ratio at 72h post-LT into two groups: PGD3 (n = 33) and PGD0/1 (n = 25). Cryopreserved perfusate cells, from 1h and 4h of perfusion, were subjected to flow cytometry and then underwent CITRUS analysis, comparing NDD to DCD and PGD0/1 to PGD3 to identify significantly stratifying cell populations (p < 0.01). When comparing perfusate cells between NDD and DCD, NK cells and macrophages were observed to be higher in NDD whereas memory T cells were enriched in DCD (Fig A). In the comparison of PGD0/1 and PGD3, neutrophils (Nφ) were found to be significantly higher in the PGD3 whereas NK cells were significantly higher in the PGD0/1 group (Fig B). We have shown that certain perfusate cell populations are enriched based on donor mode of death, possibly due to distinct donor lung injury. The association of Nφ with PGD3 suggests a potential biomarker and therapeutic target. This work illustrates the value of cellular analysis of acellular EVLP perfusate, as all cells in the perfusate emanate from the donor lung and reflect its status. [ABSTRACT FROM AUTHOR]