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Background: Bronchoalveolar lavage (BAL) is essential in determining the efficacy of novel therapies in alpha-1 antitrypsin deficiency (AATD). These require initial proof-of-concept demonstration that treatment administration increases alpha-1 antitrypsin (AAT) levels and/or anti-neutrophil elastase inhibitory capacity (ANEC) in the lung. Early-phase studies often encounter high interindividual variability of BAL results, primarily stemming from the inherent dilution characteristics of returned BAL fluid. A BAL protocol that minimises this variability is needed for reliable comparison of biochemical endpoints in the lung., Methods: The study population included 21 severe AATD (ZZ), 22 moderate AATD (MZ) and 23 non-AATD (MM) individuals, further categorised as healthy, unobstructed current smokers or patients with chronic obstructive pulmonary disease (COPD). An additional six ZZ individuals were receiving intravenous alpha-1 augmentation therapy. We compared common BAL correction methods-albumin, total protein and epithelial lining fluid (ELF) volume measured by urea-when reporting early-phase biochemical endpoints, AAT and ANEC., Results: BAL performed with a paediatric bronchoscope (PB) improved alveolar sampling compared with a traditional adult bronchoscope. Both uncorrected and ELF-corrected BAL demonstrated high interindividual variability regardless of lung health status. BAL total protein correction minimised interindividual variability, producing significant differences in AAT and ANEC between all genotypes, the strongest relationship with plasma AAT levels (r 2 =0.83), greatest inter-lobar concordance in AAT levels (r 2 =0.76) and strong correlation between BAL AAT and ANEC (r 2 =0.88)., Conclusions: By capitalising on the marked consistency in AAT levels between AAT genotypes, and the close relationship between plasma and lung AAT levels, we demonstrate reliable alveolar sampling that aligns closely with plasma., Competing Interests: Competing interests: NMcE: In the past 36 months, unrestricted research grant: Grifols, CSL Behring research grant in alpha-1 antitrypsin deficiency; advisory board: Intellia, Vertex, Inhibrx, Takeda, Dicerna, Centessa in the area of alpha-1 antitrypsin deficiency; speaker honorarium: CS Behring ERS; patent for development of resistant form of AAT in CHO cells., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Immunosuppressive treatment in patients with rheumatic diseases can maintain disease remission but also increase risk of infection. Their response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination is frequently blunted. In this study we evaluated the effect of immunosuppression exposure on humoral and T cell immune responses to SARS-CoV-2 infection and vaccination in two distinct cohorts of patients; one during acute SARS-CoV-2 infection and 3 months later during convalescence, and another prior to SARS-CoV-2 vaccination, with follow up sampling 6 weeks after vaccination. Results were compared between rituximab-exposed (in previous 6 months), immunosuppression-exposed (in previous 3 months), and non-immunosuppressed groups. The immune cell phenotype was defined by flow cytometry and ELISA. Antigen specific T cell responses were estimated using a whole blood stimulation interferon-γ release assay. A focused post-vaccine assessment of rituximab-treated patients using high dimensional spectral cytometry was conducted. Acute SARS-CoV-2 infection was characterised by T cell lymphopenia, and a reduction in NK cells and naïve CD4 and CD8 cells, without any significant differences between immunosuppressed and non-immunosuppressed patient groups. Conversely, activated CD4 and CD8 cell counts increased in non-immunosuppressed patients with acute SARS-CoV-2 infection but this response was blunted in the presence of immunosuppression. In rituximab-treated patients, antigen-specific T cell responses were preserved in SARS-CoV-2 vaccination, but patients were unable to mount an appropriate humoral response.