Phage therapy in a lung transplant recipient with cystic fibrosis infected with multidrug-resistant Burkholderia multivorans.

BACKGROUND: There is increased interest in bacteriophage (phage) therapy to treat infections caused by antibiotic-resistant bacteria. A lung transplant recipient with cystic fibrosis and Burkholderia multivorans infection was treated with inhaled phage therapy for 7 days before she died. METHODS: Phages were given via nebulization through the mechanical ventilation circuit. Remnant respiratory specimens and serum were collected. We quantified phage and bacterial deoxyribonucleic acid (DNA) using quantitative polymerase chain reaction, and tested phage neutralization in the presence of patient serum. We performed whole genome sequencing and antibiotic and phage susceptibility testing on 15 B. multivorans isolates. Finally, we extracted lipopolysaccharide (LPS) from two isolates and visualized their LPS using gel electrophoresis. RESULTS: Phage therapy was temporally followed by a temporary improvement in leukocytosis and hemodynamics, followed by worsening leukocytosis on day 5, deterioration on day 7, and death on day 8. We detected phage DNA in respiratory samples after 6 days of nebulized phage therapy. Bacterial DNA in respiratory samples decreased over time, and no serum neutralization was detected. Isolates collected between 2001 and 2020 were closely related but differed in their antibiotic and phage susceptibility profiles. Early isolates were not susceptible to the phage used for therapy, while later isolates, including two isolates collected during phage therapy, were susceptible. Susceptibility to the phage used for therapy was correlated with differences in O-antigen profiles of an early versus a late isolate. CONCLUSIONS: This case of clinical failure of nebulized phage therapy highlights the limitations, unknowns, and challenges of phage therapy for resistant infections.

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.

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.

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.

Induction Strategies in Lung Transplantation: Alemtuzumab vs. Basiliximab a Single-Center Experience.

Background: Induction therapy is used in about 80% of lung transplant centers and is increasing globally. Currently, there are no standards or guidelines for the use of induction therapy. At our institution, we have two induction strategies, basiliximab, and alemtuzumab. The goal of this manuscript is to share our experience and practice since this is an area of controversy. Methods: We retrospectively reviewed 807 lung transplants performed at our institution between 2011 and 2020. Indications for the use of the basiliximab protocol were as follows: patients over the age of 70 years, history of cancer, hepatitis C virus or human immunodeficiency virus infection history, and cytomegalovirus or Epstein-Barr virus (donor positive/ recipient negative). In the absence of these clinical factors, the alemtuzumab protocol was used. Results: 453 patients underwent alemtuzumab induction and 354 patients underwent basiliximab. There were significant differences in delayed chest closure (24.7% alemtuzumab vs 31.4% basiliximab, p = 0.037), grade 3 primary graft dysfunction observed within 72 hours (19.9% alemtuzumab vs 29.9% basiliximab, p = 0.002), postoperative hepatic dysfunction (8.8% alemtuzumab vs 14.7% basiliximab, p = 0.009), acute cellular rejection in first year (39.1% alemtuzumab vs 53.4% basiliximab, p < 0.001). The overall survival rate of the patients with alemtuzumab induction was significantly higher than those of the patients with basiliximab induction (5 years survival rate: 64.1% alemtuzumab vs 52.3%, basiliximab, p < 0.001). Multivariate Cox regression analysis confirmed lower 5-year survival for basiliximab induction (HR = 1.41, p = 0.02), recipient cytomegalovirus positive (HR = 1.49, p = 0.01), postoperative hepatic dysfunction (HR = 2.20, p < 0.001), and acute kidney injury requiring renal replacement therapy (HR = 2.27, p < 0.001). Conclusions: In this single center retrospective review, there was a significant difference in survival rates between induction strategies. This outcome may be attributable to differences in recipient characteristics between the groups. However, the Alemtuzumab group experienced less episodes of acute cellular rejection within the first year.

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.

Extracorporeal Membrane Oxygenation to Overcome Graft-Cavity Disparity of Lung Transplantation.

The waitlist mortality rate is higher in patients with severe restrictive pulmonary disease because of the rapid clinical progression of the disease and the challenges of finding lung allografts that fit their small chest cavities in a timely manner. This report describes a case of urgent lung transplantation in which venovenous extracorporeal membrane oxygenation was used after open chest management and volume reduction to allow the lung allograft to accommodate to a smaller chest cavity. The use of venovenous extracorporeal membrane oxygenation in this case facilitated early chest closure in a patient with graft-cavity disparity while lowering the risk of infection without hemodynamic or respiratory compromise.