S. Juvet

Harnessing Regulatory T Cells for Clinical Use in Transplantation: The End of the Beginning

Owing to the adverse effects of immunosuppression and an inability to prevent chronic rejection, there is a pressing need for alternative strategies to control alloimmunity. In three decades, regulatory T cells (Tregs) have evolved from a hypothetical mediator of adoptively transferred tolerance to a well‐defined population that can be expanded ex vivo and returned safely to patients in clinical trials. Herein, we review the historical developments that have permitted these advances and the current status of clinical trials examining Tregs as a cellular therapy in transplantation. We conclude by discussing the critical unanswered questions that face this field in the coming years.

Regulation of antigen-expressing dendritic cells by double negative regulatory T cells

TCRαβ+CD3+CD4−CD8−NK1.1− double negative (DN) Tregs comprise 1–3% of peripheral T lymphocytes in mice and humans. It has been demonstrated that DN Tregs can suppress allo‐, xeno‐ and auto‐immune responses in an Ag‐specific fashion. However, the mechanisms by which DN Tregs regulate immune responses remain elusive. Whether DN Tregs can regulate DCs has not been investigated previously. In this study, we demonstrate that DN Tregs express a high level of CTLA4 and are able to down‐regulate costimulatory molecules CD80 and CD86 expressed on Ag‐expressing mature DCs (mDCs). DN Tregs from CTLA4 KO mice were not able to downregulate CD80 and CD86 expression, indicating that CTLA4 is critical for DN Treg‐mediated downregulation of costimulatory molecule expression on Ag‐expressing mature DCs. Furthermore, DN Tregs could kill both immature and mature allogeneic DCs, as well as Ag‐loaded syngeneic DCs, in an Ag‐specific manner in vitro and in vivo, mainly through the Fas‐FasL pathway. These data demonstrate, for the first time, that DN Tregs are potent regulators of DCs and may have the potential to be developed as a novel immune suppression treatment.

Rare Lung Diseases II: Pulmonary Alveolar Proteinosis

The present article is the second in a series on rare lung diseases. It focuses on pulmonary alveolar proteinosis (PAP), a disorder in which lipoproteinaceous material accumulates in the alveolar space. PAP was first described in 1958, and for many years the nature of the material accumulating in the lungs was unknown. Major insights into PAP have been made in the past decade, and these have led to the notion that PAP is an autoimmume disorder in which autoantibodies interfere with signalling through the granulocyte-macrophage colony-stimulating factor receptor, leading to macrophage and neutrophil dysfunction. This has spurred new therapeutic approaches to this disorder. The discussion of PAP will begin with a case report, then will highlight the classification of PAP and review recent insights into the pathogenesis of PAP. The approach to therapy and the prognosis of PAP will also be discussed.

Desquamative Interstitial Pneumonitis and Systemic Lupus Erythematosus

Desquamative interstitial pneumonia (DIP) is a rare form of interstitial lung disease (ILD) commonly found among healthy smokers. ILD is a rare manifestation of systemic lupus erythematosus (SLE), and typically associated with a histopathological pattern of nonspecific interstitial pneumonia (NSIP). The present article describes an unusual case of DIP in a nonsmoking patient with SLE presenting as NSIP. DIP can occur in the context of SLE in patients with a negative smoking history, and clinicians should consider lung biopsy to correctly classify ILD with unusual presentation on computed tomography scan.

Successful Lung Transplantation from Hepatitis C Positive Donor to Seronegative Recipient

Lung transplantation using RNA+ hepatitis C (HCV+) donors to seronegative recipients is not currently performed due to the very high risk of transmission. Previous reports have shown poor survival when this practice was applied. The emergence of new direct‐acting antiviral drugs (DAA) suggests a high chance of sustained virologic response in immunocompetent patients. We report here successful transplantation of lungs from HCV+ donor to HCV− recipient. The recipient was an HCV− patient with chronic lung allograft dysfunction. Viral transmission occurred early posttransplant but excellent clinical outcomes were observed including elimination of HCV after 12 weeks of treatment using DAAs.

Halofuginone treatment reduces interleukin-17A and ameliorates features of chronic lung allograft dysfunction in a mouse orthotopic lung transplant model.

BACKGROUND Increasing evidence suggests that interleukin (IL)-17A plays an important role in chronic lung allograft dysfunction (CLAD), characterized by airway and lung parenchymal fibrosis, after lung transplantation. Halofuginone is a plant derivative that has been shown to inhibit Th17 differentiation. The purpose of this study was to examine the effect of halofuginone on CLAD development using a minor alloantigen‒mismatched mouse orthotopic lung transplant model. METHODS C57BL/6 recipient mice received an orthotopic left lung transplant from C57BL/10 donors, mismatched for minor antigens. Lung transplant recipients received daily intraperitoneal injections of 2.5 μg halofuginone or vehicle alone. Lung grafts were assessed on Days 7, 14, and 28 post-transplant. RESULTS Compared with control mice, on Day 28 post-transplant, lung grafts of mice treated with halofuginone showed a significant reduction in the percentage of obliterated airways (6.8 ± 4.7% vs 52.5 ± 13.8%, p < 0.01), as well as significantly reduced parenchymal fibrosis (5.5 ± 2.3% vs 35.9 ± 10.9%, p < 0.05). Immunofluorescent staining for IL-17A demonstrated a decreased number and frequency of IL-17A‒positive cells in halofuginone-treated lung grafts on Day 28, as compared with controls. Halofuginone treatment also decreased IL-17A and IL-22 transcripts at Day 14, transforming growth factor-β1 and matrix metalloproteinase-2 transcripts at Days 14 and 28. CONCLUSION The beneficial effect of halofuginone on development of airway and lung parenchymal fibrosis in the mouse lung transplant model highlights the important role of IL-17A in CLAD and merits further pre-clinical and clinical studies.

Rare lung diseases I – Lymphangioleiomyomatosis

The present article is the first in a series that will review selected rare lung diseases. The objective of this series is to promote a greater understanding and awareness of these unusual conditions among respirologists. Each article will begin with a case that serves as a focal point for a discussion of the pathophysiology and management of the particular condition. The first article is on lymphangioleiomyomatosis (LAM); subsequent articles will focus on pulmonary alveolar proteinosis, alpha-1-antitrypsin deficiency and primary ciliary dyskinesia. LAM is a rare, progressive and (without intervention) often fatal interstitial lung disease that predominantly affects women of childbearing age. LAM is characterized by progressive interstitial infiltration of the lung by smooth muscle cells, resulting in diffuse cystic changes of the lung parenchyma. The molecular basis of this disorder has been delineated over the past five years and LAM is now known to be a consequence of mutations in the tuberous sclerosis genes. This knowledge, combined with advances in our understanding of the signalling pathways regulated by these genes, has given rise to potential molecular therapies that hold great promise for treating this devastating disease.

Rejection of tracheal allograft by intrapulmonary lymphoid neogenesis in the absence of secondary lymphoid organs.

Background Obliterative bronchiolitis after lung transplantation is associated with intrapulmonary lymphoid neogenesis. The purpose of this study was to examine the role of lymphoid neogenesis, especially its relationship with secondary lymphoid organs (SLOs) in allograft airway rejection. Methods A murine intrapulmonary tracheal transplant model and a conventional subcutaneous tracheal transplant model were tested using wild-type control mice and splenectomized lymphotoxin &agr; knockout (LT−/−) mice deficient in SLOs as recipients. Results In both subcutaneous and intrapulmonary tracheal transplant models using wild-type animals, tracheal isografts remained open without rejection, whereas allografts showed progressive luminal obliteration after transplantation. Lymphoid neogenesis containing alloreactive T cells was observed in the lungs, which received an intrapulmonary tracheal allograft. Despite a lack of SLOs, intrapulmonary allografts in splenectomized LT−/− mice were rejected and obliterated by day 28, but the rejection of subcutaneous allografts was significantly delayed. Extensive lymphoid neogenesis was observed in the lungs of both intrapulmonary and subcutaneous allograft LT−/− recipients. Increased proliferation of CD4+ T cells and B220+ B cells was observed in the lungs but not in the thymus or bone marrow. Conclusions Intrapulmonary lymphoid neogenesis is capable of mounting alloimmune responses without SLOs. Tracheal allograft rejection occurs as efficiently as in wild-type animals when it is placed in the lungs. Tracheal allograft rejection in the subcutaneous tissue occurs in a delayed manner without SLO in association with intrapulmonary lymphoid neogenesis.

Higher M30 and high mobility group box 1 protein levels in ex vivo lung perfusate are associated with primary graft dysfunction after human lung transplantation

BACKGROUND Ex vivo lung perfusion (EVLP) enables assessment of marginal donor lungs for transplantation, with similar clinical outcomes to conventional lung transplantation. We investigated whether cell death-related proteins in the EVLP perfusate could predict primary graft dysfunction (PGD) after transplantation. METHODS M30 (indicating epithelial apoptosis), M65 (indicating total epithelial cell death), and high mobility group box 1 (HMGB-1, related to cell death and inflammation) protein levels in EVLP perfusate were measured by enzyme-linked immunosorbent assay and correlated with clinical outcomes. RESULTS From 100 sequential EVLP patients, 79 lungs were transplanted. Patients who were bridged with extracorporeal life support (ECLS, n = 6) or who received lobar/single lung (n = 25) were excluded. PGD grade 3 (partial pressure of arterial oxygen/fraction of inspired oxygen <200 or need for ECLS) developed in 11 at any time within 72 hours after transplantation (PGD Group). PGD grade 3 did not develop in 34 patients (Control Group). M30 was significantly higher in the PGD Group than in the Control Group at 1 hour (PGD: 73.3 ± 24.9, control: 53.9 ± 15.9 U/liter; p < 0.01) and at 4 hours (PGD: 137.0 ± 146.6, Control: 72.4 ± 40.0 U/liter; p = 0.046) of EVLP. The increase of HMGB-1 from 1 to 4 hours of EVLP was significantly greater in the PGD Group (PGD: 37.0 ± 25.4, Control: 7.2 ± 16.8 ng/ml; p < 0.001). Higher levels of or a greater increase in M30 and a greater increase in HMGB-1 were associated with higher mortality in Cox regression. CONCLUSIONS Levels of M30 and HMGB-1 in the EVLP perfusate correlate with PGD after lung transplantation and might therefore be useful biomarkers to improve donor lung assessment during EVLP.

Syk Regulates Neutrophilic Airway Hyper-Responsiveness in a Chronic Mouse Model of Allergic Airways Inflammation

Background Asthma is a chronic inflammatory disease characterized by airways hyper-responsiveness (AHR), reversible airway obstruction, and airway inflammation and remodeling. We previously showed that Syk modulates methacholine-induced airways contractility in naïve mice and in mice with allergic airways inflammation. We hypothesize that Syk plays a role in the pathogenesis of AHR; this was evaluated in a chronic 8-week mouse model of house dust mite (HDM)-induced allergic airways inflammation. Methods We used the Sykflox/flox//rosa26CreERT2 conditional Syk knock-out mice to assess the role of Syk prior to HDM exposure, and treated HDM-sensitized mice with the Syk inhibitor, GSK143, to evaluate its role in established allergic airways inflammation. Respiratory mechanics and methacholine (MCh)-responsiveness were assessed using the flexiVent® system. Lungs underwent bronchoalveolar lavage to isolate inflammatory cells or were frozen for determination of gene expression in tissues. Results MCh-induced AHR was observed following HDM sensitization in the Syk-intact (Sykflox/flox) and vehicle-treated BALB/c mice. MCh responsiveness was reduced to control levels in HDM-sensitized Sykdel/del mice and in BALB/c and Sykflox/flox mice treated with GSK143. Both Sykdel/del and GSK143-treated mice mounted appropriate immune responses to HDM, with HDM-specific IgE levels that were comparable to Sykflox/flox and vehicle-treated BALB/c mice. HDM-induced increases in bronchoalveolar lavage cell counts were attenuated in both Sykdel/del and GSK143-treated mice, due primarily to decreased neutrophil recruitment. Gene expression analysis of lung tissues revealed that HDM-induced expression of IL-17 and CXCL-1 was significantly attenuated in both Sykdel/del and GSK143-treated mice. Conclusion Syk inhibitors may play a role in the management of neutrophilic asthma.