Lymphocyte-Directed Immunomodulation Remits Thymoma-Associated Autoimmune Pneumonitis

Background Thymoma presents with several autoimmune manifestations and is associated with secondary autoimmune regulator (AIRE) deficiency. Pneumonitis has recently been described as an autoimmune manifestation associated with thymoma presenting with similar clinical, radiographic, histological, and autoantibody features as seen in patients with inherited AIRE deficiency who suffer from Autoimmune PolyEndocrinopathy-Candidiasis-Ectodermal Dystrophy (APECED) syndrome. Objectives To treat two patients with biopsy-proven thymoma-associated pneumonitis with lymphocyte-directed immunomodulation. Methods Two patients with thymoma were enrolled on IRB-approved protocols at the NIH Clinical Center. We performed history and physical examination; laboratory, radiographic, histologic and pulmonary function evaluations; and measurement of the lung-directed autoantibodies KCNRG and BPIFB1 prior to and at 1- and 6-months following initiation of lymphocyte-directed immunomodulation with azathioprine with or without rituximab. Results Combination T- and B-lymphocyte-directed immunomodulation resulted in improvement of clinical, functional, and radiographic parameters at 6-month follow-up evaluations in both patients with sustained remission up to 12–36 months following treatment initiation. Conclusion Lymphocyte-directed immunomodulation remitted autoimmune pneumonitis in two patients with thymoma.


Introduction
The thymus is essential for the development of T-lymphocytes [1].Patients with thymoma have an abnormal thymic microenvironment and decreased AIRE expression associated with impaired negative selection of T-cells and a broad spectrum of autoimmune manifestations [2,3].Among those, myasthenia gravis is well-recognized to affect 30-44% of thymoma patients, but other autoimmune diseases have also been reported including Addison's disease, erythroblastopenia, systemic lupus erythematosus, inflammatory myopathies, alopecia, vitiligo, thyroiditis, type 1 diabetes, hepatitis, pure red cell aplasia, pneumonitis, and Good's syndrome [2,[4][5][6][7][8].We previously reported that patients with thymoma-associated autoimmune pneumonitis exhibit similar clinical, radiographic, and histological features with autoimmune pneumonitis observed in the setting of Autoimmune PolyEndocrinopathy-Candidiasis-Ectodermal Dystrophy (APECED), also known as Autoimmune Polyendocrine Syndrome type 1 (APS-1), which is caused by loss-of-function AIRE variants [5,9].In addition, some thymoma patients carry autoantibodies against the lung-targeted bactericidal/permeabilityincreasing fold-containing B1 (BPIFB1), the potassium channel regulator (KCNRG), or both, which are also seen in APECED-associated pneumonitis [4,5].We previously demonstrated that lymphocyte-directed immunomodulatory treatment can remit APECED-associated autoimmune pneumonitis with improvement in clinical symptoms and radiographic and pulmonary function abnormalities [5].The shared features between APECED-associated and thymoma-associated autoimmune pneumonitis suggest common immunopathogenic mechanisms.Thus, we postulated that the lymphocyte-targeted immunomodulatory regimen that remits APECED-associated autoimmune pneumonitis may also remit thymoma-associated autoimmune pneumonitis.Here, we describe the clinical presentation and response to lymphocyte-directed immunomodulatory treatment of two patients with thymoma-associated autoimmune pneumonitis.

Methods
Patients provided written informed consent in accordance with the Declaration of Helsinki to participate in IRBapproved protocols NCT00001355 and NCT00018044 at the NIH Clinical Center.Pulmonary function testing (PFT) was performed in accordance with American Thoracic Society/European Respiratory Society standards [10].Lung and liver tissue biopsies were routinely processed and sectioned for hematoxylin and eosin (H&E) and immunohistochemistry staining as previously described [5,11].A luciferase immunoprecipitation systems immunoassay was used to detect autoantibody immunoreactivity against BPIFB1 and KCNRG.A particle-based multiplex assay was used to detect immunoreactivity against anti-IFNα, anti-IFNβ, anti-IFNγ, anti-IFNω, anti-IL-12p70, anti-IL-17 A, anti-IL-17 F, anti-IL-22, anti-IL-23, and anti-GM-CSF.Seropositivity was determined based on the mean plus three standard deviations of control patients as previously described [5].

Results
The first patient is a 42-year-old male with a history of thymoma, nontuberculous mycobacterial (NTM) pulmonary infection, hypothyroidism, type 2 diabetes, and iatrogenic adrenal insufficiency who developed chronic cough productive of green sputum, dyspnea, pleuritic chest pain, and night sweats (Table 1).He was diagnosed at age 40 with a modified Masaoka stage 11 A, World Health Organization (WHO) type-B2 thymoma and bronchiolitis obliterans organizing pneumonia (BOOP; now known as cryptogenic organizing pneumonia) by lung wedge resection.He was treated with high-dose prednisone for BOOP.He was referred to the NIH for surgical resection where pre-surgical chest computed tomography (CT) identified bilateral lower lung infiltrates concerning for infection as well as bronchiectasis and treein-bud abnormalities throughout the lung fields.Mycobacterium abscessus grew from multiple sputum cultures.Thus, it became apparent that 'BOOP' was a misdiagnosis, and that the pathology was consistent with pulmonary NTM disease.Anti-mycobacterial therapy was initiated with azithromycin, linezolid, amikacin, and imipenem.One month later, he underwent right hemi-clamshell thoracotomy with resection of thymoma and pericardial reconstruction using Gore-Tex.Amikacin was discontinued secondary to mild ototoxicity, and clofazimine was added.Antibiotics were discontinued after 12 months of repeatedly negative mycobacterial sputum cultures and improved cough and radiographic abnormalities.He returned to NIH 3 months later with worsening respiratory symptoms and radiographic abnormalities concerning for recurrence of NTM infection.Sputum cultures grew Mycobacterium avium complex (MAC), M. abscessus with a different susceptibility profile from his initial isolate, and methicillin-susceptible Staphylococcus aureus.He improved symptomatically and sputum cultures became negative following 8-weeks of treatment with vancomycin, imipenem, azithromycin, and rifampin; however, his chest CT abnormalities of right middle and bibasilar focal consolidations, bronchial wall thickening, and nodular opacities (Fig. 1A) were largely unchanged despite antibiotics.

Lung tissue histology Endobronchial biopsy
Bronchial mucosa with chronic inflammation.Predominately lymphocytic infiltrate in the lamina propria both singly and in aggregates made up of CD3/CD4 T-cells and CD20 B-cells and mixed with a few CD8 T-cells and CD4 histiocytes.

Deeper lung biopsy
Bronchial tissue with peribronchial lymphocytic inflammation, scattered plasma cells and prominent infiltration of lymphocytes into bronchial epithelium.Respiratory mucosa with dense chronic inflammation and thick epithelial basement membrane.

Microbiology findings
Negative FITE and AFB stains.BAL negative for infectious organisms.symptom-free, had no radiographic abnormalities, and had stably improved PFT and 6MWT (Table 3).

GMS stain negative for fungi and
Of interest, over the course of the next 6 months, he developed gradually worsening dyspnea, particularly upon climbing stairs.Chest CT (Fig. 1B) was notable for new scattered tree-in-bud opacities in the bilateral bases and sputum culture was positive for methicillin-susceptible Staphylococcus aureus for which he was prescribed a 14-day course of levofloxacin.Despite treatment, his symptoms did not improve, he continued to have radiographic abnormalities, and he demonstrated a severe mixed obstructive-restrictive spirometry pattern with moderate diffusion defect on PFTs in combination with a 6% decline in SpO 2 on 6-minute walk test (Table 4).He underwent bronchoscopy with endobronchial biopsy for which histological examination revealed chronic inflammation composed of mixed CD4 + and CD8 + T lymphocytes and aggregates of B lymphocytes consistent with recurrence of autoimmune pneumonitis.We performed the same panel of microbiological studies on the BAL that were all negative.Given the treatment response we had observed in Patient 2 to combination T and B cell-targeted therapy and the favorable response with the combination of azathioprine and rituximab in APECED-associated autoimmune pneumonitis, we administered two intravenous infusions of rituximab (1 gram each) within 2 weeks and followed the patient clinically and radiographically at 1-month and 12-months post-treatment.By 1-month, he reported resolved cough and sputum production and improved dyspnea on exertion.Chest CT demonstrated clearing of treein-bud abnormalities in the bilateral bases.At 12-months, of predicted, FEV 1 /FVC 0.58, and a moderate diffusion defect [diffusing capacity of the lung adjusted for hemoglobin (DLCO adj ), 60%] (Table 3).He had a 6-minute walk (6MWT) distance of 488 m, with 3% decrease in oxygen saturation.
Before starting treatment for autoimmune pneumonitis, we tested for thiopurine methyltransferase (TPMT) activity to avoid the risk for azathioprine (AZA)-induced toxicity, which was normal [12].We initiated AZA at 100 mg (1 mg/ kg) daily and continued prednisone 20 mg (0.25 mg/kg) daily.Three weeks later, he developed herpes zoster infection presenting with a painful dermatomal rash affecting the right shoulder and extending to the neck, chest, and back and associated right phrenic nerve neuropathy resulting in right hemi-diaphragm elevation documented by chest X-ray and positive sniff test.He was treated with intravenous acyclovir while continuing azathioprine and prednisone was tapered to 17.5 mg daily (Table 3).At 1-month followup, he had improvement in cough, dyspnea, and chest CT findings of bibasilar infiltrates and tree-in-bud nodularity (Fig. 1A).By 6-months, his respiratory symptoms had resolved with further improvement noted in chest CT abnormalities (Fig. 1A).DLCO adj improved to 69% and 6MWT distance increased to 531 m.At 1-year post-treatment initiation, attempts to wean prednisone resulted in recurrence of respiratory complaints.Consequently, over the next 1.5 years, AZA was titrated up to 100 mg twice daily allowing for tapering of prednisone to 9 mg daily, which he remained on for treatment of iatrogenic adrenal insufficiency.At 3-years post-treatment initiation, he remained respiratory negative IgG and IgG4 staining.He underwent thymectomy and initiated pancreatic enzyme replacement, which resolved his abdominal symptoms.Following thymectomy, he developed persistent dry cough and recurrent sinusitis symptoms, which were unresponsive to several courses of antibiotics to treat Pseudomonas and MAC including rifampin, ethambutol, azithromycin, inhaled tobramycin, doxycycline, and levofloxacin for 6 months.Evaluation for vocal cord dysfunction and cystic fibrosis were negative.Autoantibody evaluations showed high titers of KCNRG, but not BPIFB1, autoantibodies (Fig. 2A) as well as high titers for anti-IFN-α, anti-IFN-ω, and IL-12p70 (Table 2).Screening for autoantibodies against IFN-β, IFN-γ, IL-17 A, cough had resolved, and mild dyspnea occurred only when climbing > 3 flights of stairs.He demonstrated resolution of tree-in-bud abnormalities on CT and pulmonary function improvement on PFTs with FVC 86% of predicted, FEV 1 63% of predicted, and DLCO adj of 72%.
The second patient is a 33-year-old male with no significant past medical history until age 20 when he was diagnosed and treated for guttate psoriasis, which resolved with UV therapy, and alopecia areata (Table 1).At age 30, he developed severe abdominal pain and weight loss.Abdominal CT revealed an atrophic pancreas and a lung mass.A transbronchial biopsy showed a WHO B1 thymoma and a pancreatic biopsy revealed atrophic pancreatitis with and ethambutol and inhaled amikacin was initiated 4 weeks prior to starting immunosuppression.During the first month of antibiotic induction period, routine surveillance labs noted persistently elevated transaminase levels for which he underwent liver biopsy that revealed chronic hepatitis with autoimmune hepatitis-like features of moderate activity with periportal and perivenular fibrosis such as that seen in APECED-associated autoimmune hepatitis (Fig. 2G) [11].He received two doses of rituximab (1 gram each) within 2 weeks and, given normal TPMT activity, was started on AZA at 75 mg (1 mg/kg) daily.AZA was increased to 100 mg (1.3 mg/kg) once daily after one month of therapy (Table 5).At 3-months following AZA and rituximab IL-17 F, IL-22, IL-23, and GM-CSF was negative (Table 2).Chest CT revealed diffuse peribronchial thickening, mucus plugging, and tree-in-bud nodularity throughout the lungs (Fig. 1C).PFTs demonstrated a mild restrictive pattern (Table 5).He underwent bronchoscopy with BAL, which revealed airway neutrophilia (73% of nucleated cells).BAL cultures grew Mycobacterium intracellulare/chimaera. Endobronchial biopsies showed basement membrane thickening and profound lymphocyte infiltration in intraepithelial and submucosal areas.His lung biopsy showed chronic peri-bronchiolitis composed predominately of CD3 + T lymphocytes (CD4 + > CD8 + T lymphocytes) (Fig. 2B-F).Anti-mycobacterial therapy was started with azithromycin  5), and his alopecia had resolved (Table 1).KCNRG antibodies remained highly positive despite rituximab administration (Fig. 2H) suggesting they may not be directly pathogenic [5].He completed 18 months of treatment for MAC and remained on AZA 100 mg daily and has not necessitated re-dosing with rituximab.

Discussion
Like in APECED, patients with thymoma have an abnormal thymic microenvironment leading to decreased AIRE expression, impaired negative selection of T-cells, and an enrichment of anti-cytokine antibodies against type-1 interferons, particularly anti-IFN-ω, that collectively result in a broad spectrum of autoimmune manifestations [13].
Although we had previously reported patients with thymoma-associated and APECED-associated autoimmune pneumonitis display common clinical, radiographic, histological, and bronchial-directed autoantibodies (BPIFB1 and KCNRG), there has been no published literature on how to treat thymoma-associated pneumonitis.In both cases, our patients initially presented with mycobacterial disease in the setting of chronic, uncontrolled airway inflammation that persisted despite anti-mycobacterial therapy, which we have also observed in some patients with uncontrolled APECED-associated autoimmune pneumonitis [5,14,15].Importantly, the histological presentation of autoimmune pneumonitis differs from that of patients without thymoma infected with NTM.While both patient groups may have chronic inflammation of the small airways, non-thymoma patients with NTM infection display necrotizing or nonnecrotizing granulomas surrounded by giant cells, a finding initiation, the patient had no pulmonary complaints, had no radiographic abnormalities on chest CT imaging, had normalized transaminase levels, and demonstrated stability in PFTs and 6MWT.At 6-months, he remained asymptomatic thymoma-associated autoimmune pneumonitis and hepatitis.Importantly, they expand upon the previously reported therapeutic role of AZA and rituximab in APECED-associated autoimmune pneumonitis and CVID-associated granulomatous and lymphocytic interstitial lung disease.Additional longitudinal follow-up of our patients will be needed to establish the durability of the observed clinical and radiographic responses.Evaluation of AZA and rituximab-based therapy in a larger number of patients will be required to establish the safety and efficacy of this immunomodulatory therapy in the management of autoimmune lung disease in thymoma patients.
Foci of organizing pneumonia are seen at the edges of the peribronchial inflammation.There is a diffuse peribronchial infiltration of CD3 positive T-cells with CD4 positive T-cells outnumbering CD8 positive T-cells.Both B and T-cells infiltrate the bronchial epithelium.Small aggregates of CD20 positive B-cells are present around the edges of bronchi.

Fig. 1
Fig. 1 Radiographic, autoantibody, and histological features of thymoma-associated pneumonitis.Representative chest CT images for A) Patient 1 following azathioprine monotherapy at baseline and at 1-month and 6-months post-treatment initiation, B) Patient 1 following azathioprine with rituximab at baseline (before rituximab) and at 1.5-months and 12-months post-treatment initiation, and C) Patient 2 at baseline and at 3-months and 6-months post-treatment initiation.Baseline CT scans depict tree-in-bud nodularity (orange circles), bron-

Fig. 2 A
Fig. 2 A) Autoantibody immunoreactivity against BPIFB1 and KCNRG presented as light units (LU).Dotted lines represent the cutoff values for determining autoantibody seropositivity.B) Hematoxylin and eosin (H&E) staining of endobronchial biopsy from patient 2 depicting chronic inflammatory infiltrate within intraepithelial and submucosal areas and thickened epithelial basement membrane.C) CD3 immunostaining showing that the infiltrate is composed primarily of CD3 + T lymphocytes.D) CD4 and CD8 double immunostaining showing that CD4 + T lymphocytes (brown) outnumber CD8 + T lymphocytes (magenta).E) CD20 immunostaining showing aggregates of

Table 1
Summary of patients' clinical characteristics

Table 2
Summary of patient 1's radiographic and pulmonary function results at baseline and in response to azathioprine immunosuppression

Table 3
Summary of patient 1's radiographic and pulmonary function results at baseline and in response to combination azathioprine and

Table 4
Anti-cytokine autoantibody profiles in the patients with thy-

Table 5
Summary of radiographic and pulmonary function results of patient 2 at baseline and in response to immunosuppression