VX-659-Tezacaftor-Ivacaftor in Patients with Cystic Fibrosis and One or Two Phe508del Alleles.

BACKGROUND: The next-generation cystic fibrosis transmembrane conductance regulator (CFTR) corrector VX-659, in triple combination with tezacaftor and ivacaftor (VX-659-tezacaftor-ivacaftor), was developed to restore the function of Phe508del CFTR protein in patients with cystic fibrosis. METHODS: We evaluated the effects of VX-659-tezacaftor-ivacaftor on the processing, trafficking, and function of Phe508del CFTR protein using human bronchial epithelial cells. A range of oral VX-659-tezacaftor-ivacaftor doses in triple combination were then evaluated in randomized, controlled, double-blind, multicenter trials involving patients with cystic fibrosis who were heterozygous for the Phe508del CFTR mutation and a minimal-function CFTR mutation (Phe508del-MF genotypes) or homozygous for the Phe508del CFTR mutation (Phe508del-Phe508del genotype). The primary end points were safety and the absolute change from baseline in the percentage of predicted forced expiratory volume in 1 second (FEV1). RESULTS: VX-659-tezacaftor-ivacaftor significantly improved the processing and trafficking of Phe508del CFTR protein as well as chloride transport in vitro. In patients, VX-659-tezacaftor-ivacaftor had an acceptable safety and side-effect profile. Most adverse events were mild or moderate. VX-659-tezacaftor-ivacaftor resulted in significant mean increases in the percentage of predicted FEV1 through day 29 (P<0.001) of up to 13.3 points in patients with Phe508del-MF genotypes; in patients with the Phe508del-Phe508del genotype already receiving tezacaftor-ivacaftor, adding VX-659 resulted in a further 9.7-point increase in the percentage of predicted FEV1. The sweat chloride concentrations and scores on the respiratory domain of the Cystic Fibrosis Questionnaire-Revised improved in both patient populations. CONCLUSIONS: Robust in vitro activity of VX-659-tezacaftor-ivacaftor targeting Phe508del CFTR protein translated into improvements for patients with Phe508del-MF or Phe508del-Phe508del genotypes. VX-659 triple-combination regimens have the potential to treat the underlying cause of disease in approximately 90% of patients with cystic fibrosis. (Funded by Vertex Pharmaceuticals; VX16-659-101 and VX16-659-001 ClinicalTrials.gov numbers, NCT03224351 and NCT03029455 .).

Models of Lung Transplant Research: a consensus statement from the National Heart, Lung, and Blood Institute workshop.

Lung transplantation, a cure for a number of end-stage lung diseases, continues to have the worst long-term outcomes when compared with other solid organ transplants. Preclinical modeling of the most common and serious lung transplantation complications are essential to better understand and mitigate the pathophysiological processes that lead to these complications. Various animal and in vitro models of lung transplant complications now exist and each of these models has unique strengths. However, significant issues, such as the required technical expertise as well as the robustness and clinical usefulness of these models, remain to be overcome or clarified. The National Heart, Lung, and Blood Institute (NHLBI) convened a workshop in March 2016 to review the state of preclinical science addressing the three most important complications of lung transplantation: primary graft dysfunction (PGD), acute rejection (AR), and chronic lung allograft dysfunction (CLAD). In addition, the participants of the workshop were tasked to make consensus recommendations on the best use of these complimentary models to close our knowledge gaps in PGD, AR, and CLAD. Their reviews and recommendations are summarized in this report. Furthermore, the participants outlined opportunities to collaborate and directions to accelerate research using these preclinical models.

IL-17A Blockade Attenuates Obliterative Bronchiolitis and IFN-γ Cellular Immune Response in Lung Allografts.

Obliterative bronchiolitis (OB), characterized by fibrous obliteration of the small airways, is a major impediment to long-term survival in lung allograft recipients. We found previously that IL-17A is produced primarily by CD4+ T cells and γδ T cells after lung transplant in a mouse model of orthotopic lung transplant. The absence of either subset of T cells was compensated for by expansion of the other subset, which suggested that systemic blockade of IL-17A was necessary. To determine the specific role of IL-17A in the development of OB, we treated lung allograft recipients with an IL-17A antagonistic antibody. After IL-17A blockade, the incidence of OB was significantly reduced in lung allografts. IL-17A blockade also significantly attenuated the severity of acute rejection and overall lung fibrosis. The decreased OB incidence was associated with reduced lymphocyte recruitment, particularly CD8+ T cells and other IFN-γ-producing lymphocytes, to the lung allograft. Interestingly, IL-17A blockade led to an increase in the frequency of IL-17A-producing T-helper cell type 17 cells and γδ T cells in lung allografts, suggesting that IL-17A is a negative regulator of these T cells. Our data suggest that blocking IL-17A after lung transplant reduces the overall IFN-γ-mediated lymphocyte response and decreases the development of OB.

Th17 cells are not required for maintenance of IL-17A-producing γδ T cells in vivo.

γδ T cells producing interleukin-17A (γδT17) are thought to develop spontaneously in the thymus and to be maintained in the periphery. Previous studies suggested a role for T-helper 17 (Th17) cells in the maintenance of γδT17 via the expression of transforming growth factor-ß1 (TGFß1). However, we have previously found that Th17 cells were not required for expansion of γδT17 cells after lung transplant in a mouse model. Using mice deficient in signal transducer and activator of transcription 3 (STAT3) in CD4+ T cells, which are unable to develop Th17 cells, we investigated the requirement for Th17 cells and TGFß1 to maintain γδT17 cells in the lung and lymphoid tissues. At steady state, we found no defect in γδT17 cells in the thymus or periphery of these mice. Further, STAT3-deficient CD4+ T cells produced significantly higher levels of TGFß1 than wild-type CD4+ T cells under Th17 differentiation conditions in vitro. To determine whether STAT3-deficient CD4+ T cells could expand γδT17 cells in vivo, we used TCRß-/- mice, which are known to have a defect in γδT17 cells that can be rescued by Th17 cells. However, adoptive transfer of wild-type Th17 cells or bulk CD4+ T cells did not expand γδT17 cells in TCRß-/- mice. In contrast, interferon-γ+ γδ T cells preferentially expanded, particularly in the lungs. Interestingly, we found in vivo and in vitro that TGFß1 may negatively regulate the pool of γδT17 cells. Our data suggest that Th17 cells and TGFß1 are not required for the maintenance of γδT17 cells.

Propofol Increases Host Susceptibility to Microbial Infection by Reducing Subpopulations of Mature Immune Effector Cells at Sites of Infection.

Anesthetics are known to modulate host immune responses, but separating the variables of surgery from anesthesia when analyzing hospital acquired infections is often difficult. Here, the bacterial pathogen Listeria monocytogenes (Lm) was used to assess the impact of the common anesthetic propofol on host susceptibility to infection. Brief sedation of mice with physiologically relevant concentrations of propofol increased bacterial burdens in target organs by more than 10,000-fold relative to infected control animals. The adverse effects of propofol sedation on immune clearance of Lm persisted after recovery from sedation, as animals given the drug remained susceptible to infection for days following anesthesia. In contrast to propofol, sedation with alternative anesthetics such as ketamine/xylazine or pentobarbital did not increase susceptibility to systemic Lm infection. Propofol altered systemic cytokine and chemokine expression during infection, and prevented effective bacterial clearance by inhibiting the recruitment and/or activity of immune effector cells at sites of infection. Propofol exposure induced a marked reduction in marginal zone macrophages in the spleens of Lm infected mice, resulting in bacterial dissemination into deep tissue. Propofol also significantly increased mouse kidney abscess formation following infection with the common nosocomial pathogen Staphylococcus aureus. Taken together, these data indicate that even brief exposure to propofol severely compromises host resistance to microbial infection for days after recovery from sedation.

CD4 T Cells but Not Th17 Cells Are Required for Mouse Lung Transplant Obliterative Bronchiolitis.

Lung transplant survival is limited by obliterative bronchiolitis (OB), but the mechanisms of OB development are unknown. Previous studies in a mouse model of orthotopic lung transplantation suggested a requirement for IL-17. We have used this orthotopic mouse model to investigate the source of IL-17A and the requirement for T cells producing IL-17A. The major sources of IL-17A were CD4(+) T cells and γδ T cells. Depletion of CD4(+) T cells led to a significantly decreased frequency and number of IL-17A(+) lymphocytes and was sufficient to prevent acute rejection and OB. However, mice with STAT3-deficient T cells, which are unable to differentiate into Th17 cells, rejected lung allografts and developed OB similar to control mice. The frequency of IL-17A(+) cells was not decreased in mice with STAT3-deficient T cells due mainly to the presence of IL-17A(+) γδ T cells. Deficiency of γδ T cells also did not affect the development of airway fibrosis. Our data suggest that CD4(+) T cells are required for OB development and expansion of IL-17A responses in the lung, while Th17 and γδ T cells are not absolutely required and may compensate for each other.

ICOS-expressing lymphocytes promote resolution of CD8-mediated lung injury in a mouse model of lung rejection.

Acute rejection, a common complication of lung transplantation, may promote obliterative bronchiolitis leading to graft failure in lung transplant recipients. During acute rejection episodes, CD8(+) T cells can contribute to lung epithelial injury but the mechanisms promoting and controlling CD8-mediated injury in the lung are not well understood. To study the mechanisms regulating CD8(+) T cell-mediated lung rejection, we used a transgenic model in which adoptively transferred ovalbumin (OVA)-specific cytotoxic T lymphocytes (CTL) induce lung injury in mice expressing an ovalbumin transgene in the small airway epithelium of the lungs (CC10-OVA mice). The lung pathology is similar to findings in humans with acute lung transplant. In the presence of an intact immune response the inflammation resolves by day 30. Using CC10-OVA.RAG(-/-) mice, we found that CD4(+) T cells and ICOS(+/+) T cells were required for protection against lethal lung injury, while neutrophil depletion was not protective. In addition, CD4(+)Foxp3 (+) ICOS(+) T cells were enriched in the lungs of animals surviving lung injury and ICOS(+/+) Tregs promoted survival in animals that received ICOS(-/-) T cells. Direct comparison of ICOS(-/-) Tregs to ICOS(+/+) Tregs found defects in vitro but no differences in the ability of ICOS(-/-) Tregs to protect from lethal lung injury. These data suggest that ICOS affects Treg development but is not necessarily required for Treg effector function.

Autoreactive T and B cells induce the development of bronchus-associated lymphoid tissue in the lung.

Rheumatoid arthritis-related interstitial lung disease (RA-ILD) is associated with significant morbidity and mortality. Studies in humans have found that the incidence of bronchus-associated lymphoid tissue (BALT) correlates with the severity of lung injury. However, the mechanisms underlying the development of BALT during systemic autoimmunity remain unknown. We have determined whether systemic autoimmunity in a murine model of autoimmune arthritis can promote the development of BALT by generating a novel murine model derived from K/BxN mice. Transgenic mice with the KRN T-cell receptor specific for the autoantigen, glucose-6-phosphate isomerase (GPI), were crossed with GPI-specific immunoglobulin heavy and light chain knock-in mice, producing mice with a majority of T and B cells specific for the same autoantigen. We found that 67% of these mice demonstrated lymphocytic infiltration in the lungs, localized to either the perivascular or peribronchial regions. Fifty percent of the mice with lymphocytic infiltration manifested lymphoid-like lesions resembling BALT, with distinct T and B cell follicles. The lungs from mice with lymphoid infiltrates had increased numbers of cytokine-producing T cells, including IL-17A(+) T cells and increased major histocompatibility complex Class II expression on B cells. Interestingly, challenge with bleomycin failed to elicit a significant fibrotic response, compared with wild-type control mice. Our data suggest that systemic autoreactivity promotes ectopic lymphoid tissue development in the lung through the cooperation of autoreactive T and B cells. However, these BALT-like lesions may not be sufficient to promote fibrotic lung disease at steady state or after inflammatory challenge.

Protective effector memory CD4 T cells depend on ICOS for survival.

Memory CD4 T cells play a vital role in protection against re-infection by pathogens as diverse as helminthes or influenza viruses. Inducible costimulator (ICOS) is highly expressed on memory CD4 T cells and has been shown to augment proliferation and survival of activated CD4 T cells. However, the role of ICOS costimulation on the development and maintenance of memory CD4 T cells remains controversial. Herein, we describe a significant defect in the number of effector memory (EM) phenotype cells in ICOS(-/-) and ICOSL(-/-) mice that becomes progressively more dramatic as the mice age. This decrease was not due to a defect in the homeostatic proliferation of EM phenotype CD4 T cells in ICOS(-/-) or ICOSL(-/-) mice. To determine whether ICOS regulated the development or survival of EM CD4 T cells, we utilized an adoptive transfer model. We found no defect in development of EM CD4 T cells, but long-term survival of ICOS(-/-) EM CD4 T cells was significantly compromised compared to wild-type cells. The defect in survival was specific to EM cells as the central memory (CM) ICOS(-/-) CD4 T cells persisted as well as wild type cells. To determine the physiological consequences of a specific defect in EM CD4 T cells, wild-type and ICOS(-/-) mice were infected with influenza virus. ICOS(-/-) mice developed significantly fewer influenza-specific EM CD4 T cells and were more susceptible to re-infection than wild-type mice. Collectively, our findings demonstrate a role for ICOS costimulation in the maintenance of EM but not CM CD4 T cells.

Inducible costimulator controls migration of T cells to the lungs via down-regulation of CCR7 and CD62L.

We and others reported that inducible costimulator-deficient (ICOS(-/-)) mice manifest a defect in Th2-mediated airway inflammation, which was attributed to reduced Th2 differentiation in the absence of ICOS signaling. Interestingly, the number of CD4 T cells present in the airways and lungs after sensitization and challenge is significantly reduced in ICOS(-/-) mice. We now show that this reduction is not attributable simply to a reduced proliferation of ICOS(-/-) cells, because significantly more ICOS(-/-) than wild-type activated CD4 T cells are present in the lymph nodes, suggesting that more ICOS(-/-) CD4 T cells than wild-type CD4 T cells migrated into the lymph nodes. Further investigation revealed that activated ICOS(-/-) CD4 T cells express higher concentrations of the lymph node homing receptors, CCR7 and CD62L, than do wild-type CD4 T cells, leading to a preferential return of ICOS(-/-) cells to the nondraining lymph nodes rather than the lungs. Blocking reentry into the lymph nodes after the initiation of Th2-mediated airway inflammation equalized the levels of CD4 and granulocyte infiltration in the lungs of wild-type and ICOS(-/-) mice. Our results demonstrate that in wild-type CD4 T cells, co-stimulation with ICOS promotes the down-regulation of CCR7 and CD62L after activation, leading to a reduced return of activated CD4 T cells to the lymph nodes and a more efficient entry into the lungs.

Role of Th17 cells and IL-17 in lung transplant rejection.

In the past decade, advances in immunology have led to the recognition that T cell differentiation is not simply Th1 or Th2 but involves differentiation to other subsets, such as T regulatory cells, T follicular helper cells, and Th17 cells. Th17 cells, characterized by production of IL-17, IL-22, and IL-21, have been implicated in the pathogenesis of autoimmune diseases, like rheumatoid arthritis and multiple sclerosis, but also play an important role in host defense and mucosal immunity. IL-17, with its pleiotropic effects on stromal cells, as well as hematopoietic cells, has long been recognized as a possible mediator of rejection after lung transplantation. Recent data have implicated IL-17 and Th17 cells in the development of autoimmunity and chronic rejection after lung transplantation in both animal models and humans. In this review, we will discuss the current data on Th17 and the prospects for the future for lung transplantation.

Endogenous matrix metalloproteinases 2 and 9 regulate activation of CD4+ and CD8+ T cells.

We reported that inhibiting matrix metalloproteinases (MMP), known to remodel the extracellular matrix, also down-regulated antigen-specific T-cell responses. However, the direct role of MMP2 and MMP9 in regulating intracellular function in T cells is unknown. Markers of cellular activation and cytokine profiles were examined in anti-CD3-stimulated wild-type C57BL/6 mouse-derived CD4(+) or CD8(+) T cells, or MMP2- or MMP9-deficient (-/-) mice. MMP-sufficient T cells were also treated with SB-3CT, a highly selective inhibitor of MMP2 and MMP9. The effect of MMP-specific inhibition on T cell-dependent, antigen-specific murine lung injury was examined in vivo. SB-3CT induced dose-dependent reductions in anti-CD3-stimulated T-cell proliferation. Although MMP2(-/-) cells were reduced 20%, anti-CD3-induced proliferation was down-regulated 80-85% in MMP9(-/-) or in SB-3CT-treated wild-type CD4(+) and CD8(+) T cells. Intracellular calcium flux was augmented in response to MMP inhibition or deficiency in the same cells, and IL-2 production was reduced in CD4(+) and CD8(+) MMP9(-/-) T cells. SB-3CT-mediated MMP2 and MMP9 inhibition abrogated antigen-specific CD8(+) T cell-mediated lung injury in vivo. MMPs, particularly MMP9, may function intracellularly to regulate T-cell activation. T cell-targeted MMP inhibition may provide a novel approach of immune regulation in the treatment of T cell-mediated diseases.

Fas ligand expression on T cells is sufficient to prevent prolonged airway inflammation in a murine model of asthma.

Our previous studies revealed that, in a murine model of asthma, mice that received Fas-deficient T cells developed a prolonged phase of airway inflammation, mucus production, and airway hyperreactivity that failed to resolve even 6 weeks after the last challenge. To investigate how Fas-Fas ligand (FasL) interaction occurs between T cells and other cells in vivo, Gld mice with abnormalities of the FasL signaling pathway were used. The reconstituted mice were made by transferring T cells from B6 or Gld mice to Rag(-/-) or FasL-deficient Rag(-/-) mice. We found that Rag(-/-) mice that received B6 T cells resolved the airway inflammation, whereas FasL-deficient Rag(-/-) mice that received Gld T cells developed a prolonged airway inflammation at Day 28, with decreased IFN-gamma production. Both FasL-deficient Rag(-/-) mice that received B6 T cells and Rag(-/-) mice that received Gld T cells also had completely resolved their airway inflammation by Day 28 after challenge. Interestingly, FasL-deficient Rag(-/-) mice that received Gld T cells eventually resolved airway inflammation at Day 42, with a similar level of IFN-gamma production to that of control group. These results demonstrate that FasL expression on either T cells only or non-T cells only was sufficient for the eventual resolution of airway inflammation, and the prolonged airway inflammation in FasL-deficient Rag(-/-) mice that received Gld T cells was correlated with decreased IFN-gamma production by Gld T cells.

Inducible costimulator expression regulates the magnitude of Th2-mediated airway inflammation by regulating the number of Th2 cells.

BACKGROUND: Inducible Costimulator (ICOS) is an important regulator of Th2 lymphocyte function and a potential immunotherapeutic target for allergy and asthma. A SNP in the ICOS 5' promoter in humans is associated with increased atopy and serum IgE in a founder population and increased ICOS surface expression and Th2 cytokine production from peripheral blood mononuclear cells. However, it is unknown if increased ICOS expression contributes to disease progression or is a result of disease pathology. METHODOLOGY/PRINCIPAL FINDINGS: We developed a mouse model in which ICOS surface expression levels are genetically predetermined to test our hypothesis that genetic regulation of ICOS expression controls the severity of Th2 responses in vivo. Using ICOS+/+ and ICOS+/- mice in a Th2 model of airway inflammation, we found that T cells from the ICOS+/- mice had reduced ICOS expression and decreased Th2-mediated inflammation in vivo. Although the activation status of the T cells did not differ, T cells isolated from the lungs and draining lymph nodes of ICOS+/- mice at the peak of inflammation produced less Th2 cytokines upon stimulation ex vivo. Using 4get mice, which express GFP upon IL-4 transcription, we determined that the decreased Th2 cytokines in ICOS+/- is due to reduced percentage of Th2 cells and not a defect in their ability to produce IL-4. CONCLUSION: These data suggest that in both mice and humans, the level of ICOS surface expression regulates the magnitude of the in vivo Th2 response, perhaps by influencing Th2 differentiation.

CD28 and ICOS play complementary non-overlapping roles in the development of Th2 immunity in vivo.

Previous work has shown ICOS can function independently of CD28, but whether either molecule can compensate for the other in vivo is not known. Since ICOS is a potent inducer of Th2 cytokines and linked to allergy and elevated serum IgE in humans, we hypothesized that augmenting ICOS costimulation in murine allergic airway disease may overcome CD28 deficiency. While ICOS was expressed on T cells from CD28(-/-) mice, Th2-mediated airway inflammation was not induced in CD28(-/-) mice by increased ICOS costimulation. Further, we determined if augmenting CD28 costimulation could compensate for ICOS deficiency. ICOS(-/-) mice had a defect in airway eosinophilia that was not overcome by augmenting CD28 costimulation. CD28 costimulation also did not fully compensate for ICOS for antibody responses, germinal center formation or the development of follicular B helper T cells. CD28 and ICOS play complementary non-overlapping roles in the development of Th2 immunity in vivo.

Bronchiolitis obliterans in lung transplantation: the good, the bad, and the future.

Lung transplantation remains the hope for many incurable pulmonary diseases, such as cystic fibrosis, pulmonary fibrosis, and chronic obstructive pulmonary disease. Remarkable progress has been made in improving outcomes, although the incidence of acute rejection remains more than 50% in the 1st year, and the 5-year graft survival is still less than 50% primarily because of the development of chronic rejection and graft dysfunction. Chronic rejection is characterized by the development of obliterative bronchiolitis in allografts and manifests as bronchiolitis obliterans syndrome in humans with no effective treatment. Previous studies support a role for alloreactive T cells in the development of bronchiolitis obliterans syndrome, but the specific mechanisms are unknown. One major stumbling block to research in the field of lung transplantation has been the lack of physiologic models to study the disease in the laboratory. We will review the current understanding of the immunology of the pathogenesis of obliterative bronchiolitis and will discuss exciting new advances from the laboratory as well as the implications for future research in lung transplantation.

ICOS costimulation expands Th2 immunity by augmenting migration of lymphocytes to draining lymph nodes.

The T cell costimulatory molecule ICOS regulates Th2 effector function in allergic airway disease. Recently, several studies with ICOS(-/-) mice have also demonstrated a role for ICOS in Th2 differentiation. To determine the effects of ICOS on the early immune response, we investigated augmenting ICOS costimulation in a Th2-mediated immune response to Schistosoma mansoni Ags. We found that augmenting ICOS costimulation with B7RP-1-Fc increased the accumulation of T and B cells in the draining lymph nodes postimmunization. Interestingly, the increased numbers were due in part to increased migration of undivided Ag-specific TCR transgenic T cells and surprisingly B cells, as well as non-TCR transgenic T cells. B7RP-1-Fc also increased the levels of the chemokines CCL21 and CXCL13 in the draining lymph node, suggesting ICOS costimulation contributes to migration by direct or indirect effects on dendritic cells, stromal cells and high endothelial venules. Further, the effects of B7RP-1-Fc were not dependent on immunization. Our data support a model in which ICOS costimulation augments the pool of lymphocytes in the draining lymph nodes, leading to an increase in the frequency of potentially reactive T and B cells.

Signaling through Fc gamma RIII is required for optimal T helper type (Th)2 responses and Th2-mediated airway inflammation.

Although inhibitory Fc gamma receptors have been demonstrated to promote mucosal tolerance, the role of activating Fc gamma receptors in modulating T helper type (Th)2-dependent inflammatory responses characteristic of asthma and allergies remains unclear. Here, we demonstrate that signaling via activating Fc gamma receptors in conjunction with Toll-like receptor 4 stimulation modulated cytokine production from bone marrow-derived dendritic cells (DCs) and augmented their ability to promote Th2 responses. Ligation of the low affinity receptor Fc gamma RIII was specifically required for the enhanced Th2 responses, as Fc gamma RIII(-/-) DCs failed to augment Th2-mediated airway inflammation in vivo or induce Th2 differentiation in vitro. Further, Fc gamma RIII(-/-) mice had impaired Th2 cytokine production and exhibited reduced airway inflammation, whereas no defect was found in Fc gamma RI(-/-) mice. The augmentation of Th2 immunity was regulated by interleukin 10 production from the DCs but was distinct and independent of the well-established role of Fc gamma RIII in augmenting antigen presentation. Thus, our studies reveal a novel and specific role for Fc gamma RIII signaling in the regulation of Th cell responses and suggest that in addition to immunoglobulin (Ig)E, antigen-specific IgG also contributes to the pathogenesis of Th2-mediated diseases such as asthma and allergies.