Background: Mitochondrial DNA (MT-DNA) are intrinsically inflammatory nucleic acids released by damaged solid organs. Whether circulating cell-free MT-DNA quantitation could be used to predict the risk of poor COVID-19 outcomes remains undetermined. Methods: We measured circulating MT-DNA levels in prospectively collected, cell-free plasma samples from 97 subjects with COVID-19 at hospital presentation. Our primary outcome was mortality. Intensive care unit (ICU) admission, intubation, vasopressor, and renal replacement therapy requirements were secondary outcomes. Multivariate regression analysis determined whether MT-DNA levels were independent of other reported COVID-19 risk factors. Receiver operating characteristic and area under the curve assessments were used to compare MT-DNA levels with established and emerging inflammatory markers of COVID-19. Results: Circulating MT-DNA levels were highly elevated in patients who eventually died or required ICU admission, intubation, vasopressor use, or renal replacement therapy. Multivariate regression revealed that high circulating MT-DNA was an independent risk factor for these outcomes after adjusting for age, sex, and comorbidities. We also found that circulating MT-DNA levels had a similar or superior area under the curve when compared against clinically established measures of inflammation and emerging markers currently of interest as investigational targets for COVID-19 therapy. Conclusion: These results show that high circulating MT-DNA levels are a potential early indicator for poor COVID-19 outcomes. Funding: Washington University Institute of Clinical Translational Sciences COVID-19 Research Program and Washington University Institute of Clinical Translational Sciences (ICTS) NIH grant UL1TR002345.

Bronchiolitis obliterans syndrome (BOS) is a major impediment to lung transplant survival and is generally resistant to medical therapy. Extracorporeal photophoresis (ECP) is an immunomodulatory therapy that shows promise in stabilizing BOS patients, but its mechanisms of action are unclear. In a mouse lung transplant model, we show that ECP blunts alloimmune responses and inhibits BOS through lowering airway TGF-β bioavailability without altering its expression. Surprisingly, ECP-treated leukocytes were primarily engulfed by alveolar macrophages (AMs), which were reprogrammed to become less responsive to TGF-β and reduce TGF-β bioavailability through secretion of the TGF-β antagonist decorin. In untreated recipients, high airway TGF-β activity stimulated AMs to express CCL2, leading to CCR2⁺ monocyte-driven BOS development. Moreover, we found TGF-β receptor 2–dependent differentiation of CCR2⁺ monocytes was required for the generation of monocyte-derived AMs, which in turn promoted BOS by expanding tissue-resident memory CD8⁺ T cells that inflicted airway injury through Blimp-1–mediated granzyme B expression. Thus, through studying the effects of ECP, we have identified an AM functional plasticity that controls a TGF-β–dependent network that couples CCR2⁺ monocyte recruitment and differentiation to alloimmunity and BOS.

Obliterative bronchiolitis (OB) is a poorly understood airway disease characterized by the generation of fibrotic bronchiolar occlusions. In the lung transplant setting, OB is a pathological manifestation of bronchiolitis obliterans syndrome (BOS), which is a major impediment to long-term recipient survival. Club cells play a key role in bronchiolar epithelial repair, but whether they promote lung transplant tolerance through preventing OB remains unclear. We determined if OB occurs in mouse orthotopic lung transplants following conditional transgene-targeted club cell depletion. In syngeneic lung transplants club cell depletion leads to transient epithelial injury followed by rapid club cell–mediated repair. In contrast, allogeneic lung transplants develop severe OB lesions that are largely devoid of club cells despite immunosuppression treatment. Lung allograft club cell ablation also triggers the recognition of alloantigens, and pulmonary restricted self-antigens reported associated with BOS development. However, CD8 ⁺ T cell depletion restores club cell reparative responses and prevents OB. In addition, ex vivo analysis reveals a specific role for alloantigen-primed CD8 ⁺ T cells in inhibiting club cell proliferation and maintenance. Taken together, our results demonstrate a vital role for club cells in maintaining lung transplant tolerance and propose a model to identify the underlying mechanisms of OB.

Ischemia/reperfusion injury–mediated (IRI-mediated) primary graft dysfunction (PGD) adversely affects both short- and long-term outcomes after lung transplantation, a procedure that remains the only treatment option for patients suffering from end-stage respiratory failure. While B cells are known to regulate adaptive immune responses, their role in lung IRI is not well understood. Here, we demonstrated by intravital imaging that B cells are rapidly recruited to injured lungs, where they extravasate into the parenchyma. Using hilar clamping and transplant models, we observed that lung-infiltrating B cells produce the monocyte chemokine CCL7 in a TLR4-TRIF–dependent fashion, a critical step contributing to classical monocyte (CM) recruitment and subsequent neutrophil extravasation, resulting in worse lung function. We found that synergistic BCR-TLR4 activation on B cells is required for the recruitment of CMs to the injured lung. Finally, we corroborated our findings in reperfused human lungs, in which we observed a correlation between B cell infiltration and CM recruitment after transplantation. This study describes a role for B cells as critical orchestrators of lung IRI. As B cells can be depleted with currently available agents, our study provides a rationale for clinical trials investigating B cell–targeting therapies.

Mechanisms that mediate allograft tolerance differ between organs. We have previously shown that Foxp3 ⁺ T cell–enriched bronchus-associated lymphoid tissue (BALT) is induced in tolerant murine lung allografts and that these Foxp3 ⁺ cells suppress alloimmune responses locally and systemically. Here, we demonstrated that Foxp3 ⁺ cells that reside in tolerant lung allografts differed phenotypically and transcriptionally from those in the periphery and were clonally expanded. Using a mouse lung retransplant model, we showed that recipient Foxp3 ⁺ cells were continuously recruited to the BALT within tolerant allografts. We identified distinguishing features of graft-resident and newly recruited Foxp3 ⁺ cells and showed that graft-infiltrating Foxp3 ⁺ cells acquired transcriptional profiles resembling those of graft-resident Foxp3 ⁺ cells over time. Allografts underwent combined antibody-mediated rejection and acute cellular rejection when recruitment of recipient Foxp3 ⁺ cells was prevented. Finally, we showed that local administration of IL-33 could expand and activate allograft-resident Foxp3 ⁺ cells, providing a platform for the design of tolerogenic therapies for lung transplant recipients. Our findings establish graft-resident Foxp3 ⁺ cells as critical orchestrators of lung transplant tolerance and highlight the need to develop lung-specific immunosuppression.

A greater understanding of chronic lung allograft dysfunction (CLAD) pathobiology, the primary cause of death after lung transplantation (LTx), is needed to improve outcomes. The complement system links innate to adaptive immune responses and is activated early after lung transplantation to form C3 convertase, a critical enzyme that cleaves the central complement component C3. We hypothesized that LTx recipients with a genetic predisposition to enhanced complement activation have worse CLAD-free survival mediated through increased adaptive alloimmunity. We interrogated a known functional C3 polymorphism (C3 R102G) that increases complement activation through impaired C3 convertase inactivation in 2 independent LTx recipient cohorts. C3 R102G, identified in at least 1 of 3 LTx recipients, was associated with worse CLAD-free survival, particularly in the subset of recipients who developed donor-specific antibodies (DSAs). In a mouse orthotopic LTx model, impaired recipient complement regulation led to B cell–dependent CLAD pathology despite moderate differences in graft-infiltrating effector T cells. Dysfunctional complement regulation promoted intragraft accumulation of memory B cells and Ab-secreting cells, leading to increased local and circulating DSA levels in mice. In summary, genetic predisposition to complement activation is associated with an increased humoral response and worse CLAD-free survival.