IL-10 Is Critical for Regulation of Cytotoxic CD4+NKG7+ T Cells in Lung Allograft Rejection but Is Not Required for Allograft Acceptance.

Lung transplant remains the primary therapeutic option for patients with end-stage lung disease, but long-term survival rates remain suboptimal compared with other solid organ transplants. Acute cellular rejection (ACR) is a significant challenge in lung transplant recipients, with T cell-mediated mechanisms playing a major role. IL-10 is known for its immunoregulatory function, although its specific role in lung allograft rejection remains unclear. Using the mouse orthotopic lung transplant model, we investigated the role of IL-10 in regulating alloeffector T cell responses. Unexpectedly, we found that IL-10 was not required for early costimulation blockade-induced allograft acceptance. However, IL-10 deficiency or blockade resulted in increased CD4+ T cell numbers, proliferation, graft infiltration, and alloeffector responses. In the absence of IL-10, CD4+ T cell responses predominated over CD8 responses during ACR in contrast to wild-type mice. Type 1 immunity (IFN-γ) responses along with elevated CD4+NKG7+ and CD4+CD107a+ responses predominated during ACR, highlighting a critical regulatory role for IL-10 in modulating CD4+ T cell alloimmune responses. We further demonstrated increased colocalization of NKG7 and CD107a in CD4+ T cells from IL-10-deficient allografts, suggesting coordination in cytotoxic activity. Together, our findings highlight a critical role for IL-10 in regulation of cytotoxic CD4+NKG7+ T cells, an effector population that needs further investigation to elucidate their role in lung allograft rejection.

Lung Epithelium Releases Growth Differentiation Factor 15 in Response to Pathogen-mediated Injury.

GDF15 (growth differentiation factor 15) is a stress cytokine with several proposed roles, including support of stress erythropoiesis. Higher circulating GDF15 levels are prognostic of mortality during acute respiratory distress syndrome, but the cellular sources and downstream effects of GDF15 during pathogen-mediated lung injury are unclear. We quantified GDF15 in lower respiratory tract biospecimens and plasma from patients with acute respiratory failure. Publicly available data from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were reanalyzed. We used mouse models of hemorrhagic acute lung injury mediated by Pseudomonas aeruginosa exoproducts in wild-type mice and mice genetically deficient for Gdf15 or its putative receptor, Gfral. In critically ill humans, plasma levels of GDF15 correlated with lower respiratory tract levels and were higher in nonsurvivors. SARS-CoV-2 infection induced GDF15 expression in human lung epithelium, and lower respiratory tract GDF15 levels were higher in coronavirus disease (COVID-19) nonsurvivors. In mice, intratracheal P. aeruginosa type II secretion system exoproducts were sufficient to induce airspace and plasma release of GDF15, which was attenuated with epithelial-specific deletion of Gdf15. Mice with global Gdf15 deficiency had decreased airspace hemorrhage, an attenuated cytokine profile, and an altered lung transcriptional profile during injury induced by P. aeruginosa type II secretion system exoproducts, which was not recapitulated in mice deficient for Gfral. Airspace GDF15 reconstitution did not significantly modulate key lung cytokine levels but increased circulating erythrocyte counts. Lung epithelium releases GDF15 during pathogen injury, which is associated with plasma levels in humans and mice and can increase erythrocyte counts in mice, suggesting a novel lung-blood communication pathway.

Cross-Regulation of F-Box Protein FBXL2 with T-bet and TNF-α during Acute and Chronic Lung Allograft Rejection.

Chronic lung allograft dysfunction is the major barrier to long-term survival in lung transplant recipients. Evidence supports type 1 alloimmunity as the predominant response in acute/chronic lung rejection, but the immunoregulatory mechanisms remain incompletely understood. We studied the combinatorial F-box E3 ligase system: F-box protein 3 (FBXO3; proinflammatory) and F-box and leucine-rich repeat protein 2 (FBXL2; anti-inflammatory and regulates TNFR-associated factor [TRAF] protein). Using the mouse orthotopic lung transplant model, we evaluated allografts from BALB/c → C57BL/6 (acute rejection; day 10) and found significant induction of FBXO3 and diminished FBXL2 protein along with elevated T-bet, IFN-γ, and TRAF proteins 1-5 compared with isografts. In the acute model, treatment with costimulation blockade (MR1/CTLA4-Ig) resulted in attenuated FBXO3, preserved FBXL2, and substantially reduced T-bet, IFN-γ, and TRAFs 1-5, consistent with a key role for type 1 alloimmunity. Immunohistochemistry revealed significant changes in the FBXO3/FBXL2 balance in airway epithelia and infiltrating mononuclear cells during rejection compared with isografts or costimulation blockade-treated allografts. In the chronic lung rejection model, DBA/2J/C57BL/6F1 > DBA/2J (day 28), we observed persistently elevated FBXO3/FBXL2 balance and T-bet/IFN-γ protein and similar findings from lung transplant recipient lungs with chronic lung allograft dysfunction versus controls. We hypothesized that FBXL2 regulated T-bet and found FBXL2 was sufficient to polyubiquitinate T-bet and coimmunoprecipitated with T-bet on pulldown experiments and vice versa in Jurkat cells. Transfection with FBXL2 diminished T-bet protein in a dose-dependent manner in mouse lung epithelial cells. In testing type 1 cytokines, TNF-α was found to negatively regulate FBXL2 protein and mRNA levels. Together, our findings show the combinatorial E3 ligase FBXO3/FBXL2 system plays a role in the regulation of T-bet through FBXL2, with negative cross-regulation of TNF-α on FBXL2 during lung allograft rejection.

Impact of enzymatic digestion on single cell suspension yield from peripheral human lung tissue.

An increasing number of translational investigations of lung biology rely on analyzing single cell suspensions obtained from human lungs. To obtain these single cell suspensions, human lungs from biopsies or research-consented organ donors must be subjected to mechanical and enzymatic digestion prior to analysis with either flow cytometry or single cell RNA sequencing. A variety of enzymes have been used to perform tissue digestion, each with potential limitations. To better understand the limitations of each enzymatic digestion protocol and to establish a framework for comparing studies across protocols, we performed five commonly published protocols in parallel from identical samples obtained from 6 human lungs. Following mechanical (gentleMACS™) and enzymatic digestion, we quantified cell count and viability using a Nexcelom Cellometer and determined cell phenotype using multiparameter spectral flow cytometry (Cytek™ Aurora). We found that all protocols were superior in cellular yield and viability when compared to mechanical digestion alone. Protocols high in dispase cleaved immune markers CD4, CD8, CD69, and CD103 and contributed to an increased monocyte to macrophage yield. Similarly, dispase led to a differential epithelial cell yield, with increased TSPN8+ and ITGA6+ epithelial cells and reduced CD66e+ cells. When compared to collagenase D, collagenase P protocols yielded increased AT1 and AT2 cells and decreased endothelial cells. These results provide a framework for selecting an enzymatic digestion protocol best suited to the scientific question and allow for comparison of studies using different protocols.

CD4+ T-Cell Dysfunction in Severe COVID-19 Disease Is Tumor Necrosis Factor-α/Tumor Necrosis Factor Receptor 1-Dependent.

Rationale: Lymphopenia is common in severe coronavirus disease (COVID-19), yet the immune mechanisms are poorly understood. As inflammatory cytokines are increased in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we hypothesized a role in contributing to reduced T-cell numbers. Objectives: We sought to characterize the functional SARS-CoV-2 T-cell responses in patients with severe versus recovered, mild COVID-19 to determine whether differences were detectable. Methods: Using flow cytometry and single-cell RNA sequence analyses, we assessed SARS-CoV-2-specific responses in our cohort. Measurements and Main Results: In 148 patients with severe COVID-19, we found lymphopenia was associated with worse survival. CD4+ lymphopenia predominated, with lower CD4+/CD8+ ratios in severe COVID-19 compared with patients with mild disease (P < 0.0001). In severe disease, immunodominant CD4+ T-cell responses to Spike-1 (S1) produced increased in vitro TNF-α (tumor necrosis factor-α) but demonstrated impaired S1-specific proliferation and increased susceptibility to activation-induced cell death after antigen exposure. CD4+TNF-α+ T-cell responses inversely correlated with absolute CD4+ counts from patients with severe COVID-19 (n = 76; R = -0.797; P < 0.0001). In vitro TNF-α blockade, including infliximab or anti-TNF receptor 1 antibodies, strikingly rescued S1-specific CD4+ T-cell proliferation and abrogated S1-specific activation-induced cell death in peripheral blood mononuclear cells from patients with severe COVID-19 (P < 0.001). Single-cell RNA sequencing demonstrated marked downregulation of type-1 cytokines and NFκB signaling in S1-stimulated CD4+ cells with infliximab treatment. We also evaluated BAL and lung explant CD4+ T cells recovered from patients with severe COVID-19 and observed that lung T cells produced higher TNF-α compared with peripheral blood mononuclear cells. Conclusions: Together, our findings show CD4+ dysfunction in severe COVID-19 is TNF-α/TNF receptor 1-dependent through immune mechanisms that may contribute to lymphopenia. TNF-α blockade may be beneficial in severe COVID-19.

Rate of recipient-derived alveolar macrophage development and major histocompatibility complex cross-decoration after lung transplantation in humans.

Alveolar macrophages (AM) play critical roles in lung tissue homeostasis, host defense, and modulating lung injury. The rate of AM turnover (donor AM replacement by circulating monocytes) after transplantation has been incompletely characterized. Furthermore, the anatomic pattern of recipient-derived lung macrophages repopulation has not been reported, nor has their ability to accumulate and present donor major histocompatibility complex (a process we refer to as MHC cross-decoration). We longitudinally characterized the myeloid content of bronchoalveolar lavage (BAL) and biopsy specimens of lung transplant recipients and found a biphasic rate in AM turnover in the allograft, with a rapid turnover perioperatively, accelerated by both the type of induction immunosuppression and the presence of primary graft dysfunction. We found that recipient myeloid cells with cell surface AM phenotype repopulated the lung in a disorganized pattern, comprised mainly of large clusters of cells. Finally, we show that recipient AM take up and present donor peptide-MHC complexes yet are not able to independently induce an in vitro alloreactive response by circulating recipient T cells.

Human Lung-Resident Macrophages Colocalize with and Provide Costimulation to PD1hi Tissue-Resident Memory T Cells.

Rationale: Tissue-resident memory T cells (TRM) play a critical role in the defense against inhaled pathogens. The isolation and study of human lung tissue-resident memory T cells and lung-resident macrophages (MLR) are limited by experimental constraints. Objectives: To characterize the spatial and functional relationship between MLR and human lung tissue-resident memory T cells using ex vivo lung perfusion (EVLP). Methods: TRM and MLR were isolated using EVLP and intraperfusate-labeled CD45 antibody. Cells isolated after 6 hours of EVLP were analyzed using spectral flow cytometry. Spatial relationships between CD3+ and CD68+ cells were explored with multiplexed immunohistochemistry. Functional relationships were determined by using coculture and T-cell-receptor complex signal transduction. Measurements and Main Results: Lungs from 8 research-consenting organ donors underwent EVLP for 6 hours. We show that human lung TRM and MLR colocalize within the human lung, preferentially around the airways. Furthermore, we found that human lung CD8+ TRM are composed of two functionally distinct populations on the basis of PD1 (programed cell death receptor 1) and ZNF683 (HOBIT) protein expression. We show that MLR provide costimulatory signaling to PD1hi CD4+ and CD8+ lung TRM,, augmenting the effector cytokine production and degranulation of TRM. Conclusions: EVLP provides an innovative technique to study resident immune populations in humans. Human MLR colocalize with and provide costimulation signaling to TRM, augmenting their effector function.

Type-1 immunity and endogenous immune regulators predominate in the airway transcriptome during chronic lung allograft dysfunction.

Chronic lung allograft dysfunction (CLAD) remains the major complication limiting long-term survival among lung transplant recipients (LTRs). Limited understanding of CLAD immunopathogenesis and a paucity of biomarkers remain substantial barriers for earlier detection and therapeutic interventions for CLAD. We hypothesized the airway transcriptome would reflect key immunologic changes in disease. We compared airway brush-derived transcriptomic signatures in CLAD (n = 24) versus non-CLAD (n = 21) LTRs. A targeted assessment of the proteome using concomitant bronchoalveolar lavage (BAL) fluid for 24 cytokines/chemokines and alloimmune T cell responses was performed to validate the airway transcriptome. We observed an airway transcriptomic signature of differential genes expressed (DGEs) in CLAD marked by Type-1 immunity and striking upregulation of two endogenous immune regulators: indoleamine 2, 3 dioxygenase 1 (IDO-1) and tumor necrosis factor receptor superfamily 6B (TNFRSF6B). Advanced CLAD staging was associated with a more intense airway transcriptome signature. In a validation cohort using the identified signature, we found an area under the curve (AUC) of 0.77 for CLAD LTRs. Targeted proteomic analyses revealed a predominant Type-1 profile with detection of IFN-γ, TNF-α, and IL-1ß as dominant CLAD cytokines, correlating with the airway transcriptome. The airway transcriptome provides novel insights into CLAD immunopathogenesis and biomarkers that may impact diagnosis of CLAD.

Single cell RNA sequencing identifies IGFBP5 and QKI as ciliated epithelial cell genes associated with severe COPD.

BACKGROUND: Whole lung tissue transcriptomic profiling studies in chronic obstructive pulmonary disease (COPD) have led to the identification of several genes associated with the severity of airflow limitation and/or the presence of emphysema, however, the cell types driving these gene expression signatures remain unidentified. METHODS: To determine cell specific transcriptomic changes in severe COPD, we conducted single-cell RNA sequencing (scRNA seq) on n = 29,961 cells from the peripheral lung parenchymal tissue of nonsmoking subjects without underlying lung disease (n = 3) and patients with severe COPD (n = 3). The cell type composition and cell specific gene expression signature was assessed. Gene set enrichment analysis (GSEA) was used to identify the specific cell types contributing to the previously reported transcriptomic signatures. RESULTS: T-distributed stochastic neighbor embedding and clustering of scRNA seq data revealed a total of 17 distinct populations. Among them, the populations with more differentially expressed genes in cases vs. controls (log fold change >|0.4| and FDR = 0.05) were: monocytes (n = 1499); macrophages (n = 868) and ciliated epithelial cells (n = 590), respectively. Using GSEA, we found that only ciliated and cytotoxic T cells manifested a trend towards enrichment of the previously reported 127 regional emphysema gene signatures (normalized enrichment score [NES] = 1.28 and = 1.33, FDR = 0.085 and = 0.092 respectively). Among the significantly altered genes present in ciliated epithelial cells of the COPD lungs, QKI and IGFBP5 protein levels were also found to be altered in the COPD lungs. CONCLUSIONS: scRNA seq is useful for identifying transcriptional changes and possibly individual protein levels that may contribute to the development of emphysema in a cell-type specific manner.

Body composition and mortality after adult lung transplantation in the United States.

RATIONALE: Obesity and underweight are contraindications to lung transplantation based on their associations with mortality in studies performed before implementation of the lung allocation score (LAS)-based organ allocation system in the United States Objectives: To determine the associations of body mass index (BMI) and plasma leptin levels with survival after lung transplantation. METHODS: We used multivariable-adjusted regression models to examine associations between BMI and 1-year mortality in 9,073 adults who underwent lung transplantation in the United States between May 2005 and June 2011, and plasma leptin and mortality in 599 Lung Transplant Outcomes Group study participants. We measured body fat and skeletal muscle mass using whole-body dual X-ray absorptiometry in 142 adult lung transplant candidates. MEASUREMENTS AND MAIN RESULTS: Adjusted mortality rates were similar among normal weight (BMI 18.5-24.9 kg/m(2)), overweight (BMI 25.0-29.9), and class I obese (BMI 30-34.9) transplant recipients. Underweight (BMI < 18.5) was associated with a 35% increased rate of death (95% confidence interval, 10-66%). Class II-III obesity (BMI ≥ 35 kg/m(2)) was associated with a nearly twofold increase in mortality (hazard ratio, 1.9; 95% confidence interval, 1.3-2.8). Higher leptin levels were associated with increased mortality after transplant surgery performed without cardiopulmonary bypass (P for interaction = 0.03). A BMI greater than or equal to 30 kg/m(2) was 26% sensitive and 97% specific for total body fat-defined obesity. CONCLUSIONS: A BMI of 30.0-34.9 kg/m(2) is not associated with 1-year mortality after lung transplantation in the LAS era, perhaps because of its low sensitivity for obesity. The association between leptin and mortality suggests the need to validate alternative methods to measure obesity in candidates for lung transplantation. A BMI greater than or equal to 30 kg/m(2) may no longer contraindicate lung transplantation.

Heparanase-induced endothelial glycocalyx degradation exacerbates lung ischemia/reperfusion injury in male mice.

The endothelial glycocalyx (eGC) is a carbohydrate-rich layer on the vascular endothelium, and its damage can lead to endothelial and organ dysfunction. Heparanase (HPSE) degrades the eGC in response to cellular stress, but its role in organ dysfunction remains unclear. This study investigates HPSE's role in lung ischemia-reperfusion (I/R) injury. A left lung hilar occlusion model was used in B6 wildtype (WT) and HPSE genetic knockout (-/-) mice to induce I/R injury in vivo. The left lungs were ischemic for 1 h followed by reperfusion for 4 h prior to investigations of lung function and eGC status. Data were compared between uninjured lungs and I/R-injured lungs in WT and HPSE-/- mice. WT lungs showed significant functional impairment after I/R injury, whereas HPSE-/- lungs did not. Inhibition or knockout of HPSE prevented eGC damage, inflammation, and cellular migration after I/R injury by reducing matrix metalloproteinase activities. HPSE-/- mice exhibited compensatory regulation of related gene expressions. HPSE facilitates eGC degradation leading to inflammation and impaired lung function after I/R injury. HPSE may be a therapeutic target to attenuate graft damage in lung transplantation.

Impact of variable titer COVID-19 convalescent plasma and recipient SARS-CoV2-specific humoral immunity on survival in hospitalized patients.

COVID-19 convalescent plasma (CCP) was one of the first therapies to receive emergency use authorization for management of COVID-19. We assessed the effectiveness of CCP in a propensity-matched analysis, and whether the presence of antibodies in the recipient at the time of treatment or the titer of antibodies in the administered CCP influenced clinical effectiveness. In an inpatient population within a single large health system, a total of 290 CCP patients were matched to 290 controls. While CCP increased titers of anti-SARS-CoV-2 RBD IgG titers post-CCP (p = <0.0001), no differences in 30-day survival were observed between CCP patients and controls in univariate and multivariate analyses. Survival at 30 days was numerically lower in recipients who were seronegative prior to CCP administration, compared to those with low titer and high titer anti-SARS-CoV-2 RBD IgG, respectively, but did not reach statistical significance (56% vs 82% vs 75%, p = 0.16). Patients who received 2 units of high-titer CCP had numerically better survival versus those who received fewer high-titer units, but this was not statistically significant (p = 0.08). CCP did not improve 30-day survival compared to propensity matched controls. Together these data support that CCP therapy provides limited benefit to hospitalized patients with SARS-CoV-2 infection.

Single center study investigating the clinical association of donor-derived cell-free DNA with acute outcomes in lung transplantation.

Background: Circulating donor-derived cell-free DNA (dd-cfDNA) levels have been proposed as a potential tool for the diagnosis of graft injury. In this study, we prospectively investigated dd-cfDNA plasma levels and their association with severe primary graft dysfunction (PGD) and graft rejection after lung transplant. Methods: A total of 40 subjects undergoing de-novo lung transplants at our institution were recruited in this study. Blood samples were collected at various time points before and after lung transplant for 1 year. Dd-cfDNA in samples was determined using AlloSure assay (CareDx Inc.). The correlation of the value of %dd-cfDNA was investigated with the incidence of PGD, acute cellular rejection (ACR), and donor-specific antibody. Results: We observed a rapid increase of %dd-cfDNA in the blood of recipients after lung transplantation compared to baseline. The levels of dd-cfDNA decreased during the first two weeks. The peak was observed within 72 h after transplantation. The peak values of %dd-cfDNA varied among subjects and did not correlate with severe PGD incidence. We observed an association between levels of %dd-cfDNA from blood collected at the time of transbronchial biopsy and the histological diagnosis of ACR at 3 weeks. Conclusion: Our data show that circulating dd-cfDNA levels are associated with ACR early after transplantation but not with severe PGD. Plasma levels of dd-cfDNA may be a less invasive tool to estimate graft rejection after lung transplantation however larger studies are still necessary to better identify thresholds.

Rabbit Antithymocyte Globulin for Treatment of Corticosteroid Refractory Acute Cellular Rejection After Lung Transplantation.

BACKGROUND: Chronic lung allograft dysfunction (CLAD) remains a major cause of death after the first year posttransplant, with acute cellular rejection (ACR) being a major risk factor for CLAD. We evaluated the use of rabbit antithymocyte globulin (rATG) for corticosteroid refractory ACR in lung transplant recipients. METHODS: We retrospectively identified 112 adult lung transplant recipients who received rATG for refractory ACR after lung transplantation. The primary endpoint was the incidence of ACR on follow-up transbronchial biopsy. Secondary endpoints included freedom from ACR within 1 y post-rATG, CLAD progression at 1 y post-rATG, and all-cause mortality at 1 y post-rATG. RESULTS: A complete resolution of ACR was observed in 60.2% of patients, an improvement but not complete resolution in 22.1%, and no response on follow-up biopsy in 17.8%. Mean A grade 1 y post-rATG was 0.51 in complete responders, 1.01 in partial responders, and 2.19 in nonresponders ( P < 0.001). Complete responders had significantly less new or worsening CLAD at 1 y than partial responders (17% versus 40%; P = 0.02). All-cause mortality rate was 14.9% in complete responders, 40% in partial responders, and 30% in nonresponders ( P < 0.01). CONCLUSIONS: rATG appears to be an effective treatment of refractory ACR in lung transplant recipients. Failure to respond to rATG carries an increased risk of early CLAD and death.

Incidence of Acute Cellular Rejection After Granulocyte Colony-Stimulating Factor in Lung Transplant Recipients.

BackgroundNeutropenia is a common complication in lung transplant recipients (LTRs). Filgrastim may be used to treat neutropenia in LTRs, but its consequences on acute cellular rejection (ACR) remain controversial. Objective: The purpose was to examine the association between filgrastim and incidence of ACR 6 months after filgrastim administration in LTRs. Secondary outcomes included burden of ACR, infections, chronic lung allograft dysfunction (CLAD), and survival. Methods: This was a matched cohort study of patients transplanted between January 2010 and October 2019. LTRs who received filgrastim for neutropenia were compared to a cohort who did not. LTRs were matched on transplant indication, sex, age, and time post-transplant and multivariable logistic regression models were used to evaluate the likelihood of ACR. Results: 212 patients were included in the analysis (106 in each group). 50 patients (47.2%) in the filgrastim group experienced ACR compared to 37 patients (34.9%) in the no filgrastim group (P = .070). In multivariable analysis, filgrastim use was not associated with ACR at 6 months (OR 1.409, 95% CI 0.772-2.571). Time to first ACR was shorter (P = .049) and 6-month ACR score was higher in the filgrastim group (.49 vs .33, P = .047). LTRs in the filgrastim group had higher incidence of bacterial pneumonia and 1-year mortality. Conclusions: Although not associated with increased likelihood of ACR at 6 months, our study found that filgrastim is associated with increased ACR burden and decreased time to ACR. This study can help inform clinicians of ACR risk after filgrastim use in LTRs.

Impact of age and telomere length on circulating T cells and rejection risk after lung transplantation for idiopathic pulmonary fibrosis.

BACKGROUND: Most idiopathic pulmonary fibrosis (IPF) lung transplant recipients (IPF-LTRs) have short telomere (ST) length. Inherited mutations in telomere-related genes are associated with the development of T cell immunodeficiency. Despite this, IPF-LTRs with telomere-related rare variants are not protected from acute cellular rejection (ACR). We set out to determine the impact of both age and telomere length on the circulating T cell compartment and ACR burden of IPF-LTRs. METHODS: We identified 106 IPF-LTRs who had telomere length testing using flowFISH (57 with short telomeres and 49 with long telomeres) as well as a subset from both cohorts who had cryopreserved PBMC at least 1 time point, 6 months posttransplantation. Circulating T cells from before transplantation and at 6 and 12 months posttransplantation were analyzed using multiparameter flow cytometry to study phenotype and functional capacity, and bulk T cell receptor sequencing was performed to study repertoire diversity. Linear regression was used to study the relationship of age and telomere length on early (within 1 year) and late (between 1 and 2 years) ACR. RESULTS: IPF-LTRs with ST were found to have premature "aging" of their circulating T cell compartment, with age-agnostic elevations in posttransplant terminal differentiation of CD8+ T cells, increased granzyme B positivity of both CD8+ and CD4+ T cells, upregulation of the exhaustion marker, CD57, and chemotactic protein CCR5, and enhanced T cell receptor clonal expansion. Additionally, we found a significant decline in early ACR burden with increasing age, but only in the ST cohort. CONCLUSIONS: IPF-LTRs with ST have premature "aging" of their circulating T cell compartment posttransplantation and a clear age-related decline in ACR burden.

Geographic disparities in lung transplantation in the United States before and after the November 2017 allocation change.

BACKGROUND: The primary lung allocation unit was expanded from the donation service area to a 250-mile radius in 2017. Prior to the change, geographic disparities in donor lung availability impacted waitlist outcomes. We sought to determine if the new allocation system improved these disparities. METHODS: We conducted a retrospective cohort study comparing the 2-year period before and after the change. Donor lung availability was defined as the ratio of donor lungs to waitlist candidates in the primary allocation unit. Transplant centers were divided into quartiles by donor lung availability. Multivariable competing risk models were used to determine the association between lung availability and waitlist outcomes. Multivariable Cox proportional hazards models compared post-transplant survival. RESULTS: Prior to the allocation change, the unadjusted transplant rate at centers in the lowest and highest quartiles was 132 and 607 transplants per 100 waitlist years. Candidates in the lowest quartile of donor lung availability had a 61% adjusted lower transplantation rate compared to candidates in highest quartile (sub-hazard ratio [sHR]: 0.39, 95% confidence interval [CI]: 0.34-0.44). After the allocation change, the disparity decreased resulting in an unadjusted transplant rate of 141 and 309 among centers in the lowest and highest quartiles. Candidates in the lowest quartile had a 38% adjusted lower transplantation rate compared to those in the highest (sHR: 0.62, 95% CI: 0.57-0.68). There was no significant difference in 1-year post-transplant survival. CONCLUSIONS: Although the expansion of the primary allocation unit improved disparities in waitlist outcomes without any change in post-transplant survival, there still remain significant differences due to geography.

Modulation of tissue resident memory T cells by glucocorticoids after acute cellular rejection in lung transplantation.

Acute cellular rejection is common after lung transplantation and is associated with an increased risk of early chronic rejection. We present combined single-cell RNA and TCR sequencing on recipient-derived T cells obtained from the bronchoalveolar lavage of three lung transplant recipients with rejection and compare them with T cells obtained from the same patients after treatment of rejection with high-dose systemic glucocorticoids. At the time of rejection, we found an oligoclonal expansion of cytotoxic CD8+ T cells that all persisted as tissue resident memory T cells after successful treatment. Persisting CD8+ allograft-resident T cells have reduced gene expression for cytotoxic mediators after therapy with glucocorticoids but accumulate around airways. This clonal expansion is discordant with circulating T cell clonal expansion at the time of rejection, suggesting in situ expansion. We thus highlight the accumulation of cytotoxic, recipient-derived tissue resident memory T cells within the lung allograft that persist despite the administration of high-dose systemic glucocorticoids. The long-term clinical consequences of this persistence have yet to be characterized.

Immune and epithelial determinants of age-related risk and alveolar injury in fatal COVID-19.

Respiratory failure in COVID-19 is characterized by widespread disruption of the lung's alveolar gas exchange interface. To elucidate determinants of alveolar lung damage, we performed epithelial and immune cell profiling in lungs from 24 COVID-19 autopsies and 43 uninfected organ donors ages 18-92 years. We found marked loss of type 2 alveolar epithelial (T2AE) cells and increased perialveolar lymphocyte cytotoxicity in all fatal COVID-19 cases, even at early stages before typical patterns of acute lung injury are histologically apparent. In lungs from uninfected organ donors, there was also progressive loss of T2AE cells with increasing age, which may increase susceptibility to COVID-19-mediated lung damage in older individuals. In the fatal COVID-19 cases, macrophage infiltration differed according to the histopathological pattern of lung injury. In cases with acute lung injury, we found accumulation of CD4+ macrophages that expressed distinctly high levels of T cell activation and costimulation genes and strongly correlated with increased extent of alveolar epithelial cell depletion and CD8+ T cell cytotoxicity. Together, our results show that T2AE cell deficiency may underlie age-related COVID-19 risk and initiate alveolar dysfunction shortly after infection, and we define immune cell mediators that may contribute to alveolar injury in distinct pathological stages of fatal COVID-19.