Peikert, Tobias

Abstract Introduction Several studies have shown a positive association between Obstructive Sleep Apnea (OSA) and malignancies such as lung cancer. However, the effects of intermittent hypoxemia in cancer-free survival following curative therapy are not known. We propose that OSA-related episodic hypoxemia and hypoxic burden are independent risk factors for accelerated lung cancer reoccurrence (< 2 years). Methods We performed a retrospective record review of patients 18 years and older from January 2016 to September 2023 with history of non-small cell lung cancer who received an overnight oximetry study (OXY) within three years prior to undergoing curative malignancy treatment. Subjects with history of central sleep apnea, baseline oxygen saturation (SpO2) below 90%, receiving supplemental oxygen or treatment for OSA were excluded. Results 403 patients met inclusion criteria (52% female vs 48% males; median age 74 y). The most common histologic subtypes were adenocarcinoma (68%) followed by squamous cell carcinoma (22%). Most patients fell into Stage IA1 (35%) followed by IA2 (24.1%) and IB (19%) as per TNM 8th edition classification. Over this period, 68 patients (22%) had lung cancer recurrence (median 19 months). A 4% oxygen desaturation index (ODI) of >15 and time spent in desaturation events were found to be a risk factor for cancer reappearance in less than 2 years (p< 0.001). Measures of hypoxic burden such as time spent below 89% SpO2, average SpO2 value below 89%, and single nadir oxygen levels, showed a similar association (p< 0.001). This was irrespective of histologic subtype, stage at diagnosis, and all studied demographic variables. Basal SpO2 demonstrated no correlation. A multivariate proportional hazard survival model showed that ODI>15 was no longer significant, however average SpO2 below 89% and single minimum oxygen level remained strongly correlated with accelerated recurrence. Conclusion This study showed a strong, positive association between accelerated lung cancer recurrence and OSA-related episodic hypoxemia and hypoxic burden on our univariate analysis. The multivariate model demonstrated correlation with hypoxic load measures only, suggesting the need for a larger sample to elucidate the involvement of this highly treatable risk factor. To our knowledge, this is the first study revealing this potential link. Support (if any)

The optimal detection strategies for effective convalescent immunity after SARS-CoV-2 infection and vaccination remain unclear. The objective of this study was to characterize convalescent immunity targeting the SARS-CoV-2 spike protein using a multiparametric approach. At the beginning of the pandemic, we recruited 30 unvaccinated convalescent donors who had previously been infected with COVID-19 and 7 unexposed asymptomatic controls. Peripheral blood mononuclear cells (PBMCs) were obtained from leukapheresis cones. The humoral immune response was assessed by measuring serum anti-SARS-CoV-2 spike S1 subunit IgG via semiquantitative ELISA, and T-cell immunity against S1 and S2 subunits were studied via IFN-γ enzyme-linked immunosorbent spot (ELISpot) and flow cytometric (FC) activation-induced marker (AIM) assays and the assessment of cytotoxic CD8+ T-cell function (in the subset of HLA-A2-positive patients). No single immunoassay was sufficient in identifying anti-spike convalescent immunity among all patients. There was no consistent correlation between adaptive humoral and cellular anti-spike responses. Our data indicate that the magnitude of anti-spike convalescent humoral and cellular immunity is highly heterogeneous and highlights the need for using multiple assays to comprehensively measure SARS-CoV-2 convalescent immunity. These observations might have implications for COVID-19 surveillance, and the determination of optimal vaccination strategies for emerging variants. Further studies are needed to determine the optimal assessment of adaptive humoral and cellular immunity following SARS-CoV-2 infection, especially in the context of emerging variants and unclear vaccination schedules.

Abstract Optimal detection strategies for effective convalescent immunity after SARS-CoV-2 infection and vaccination remain unclear. The objective of this study was to characterize convalescent immunity targeting the SARS-CoV-2 spike protein using a multiparametric approach. At the beginning of the pandemic, between April 23, 2020, to May 11, 2020, we recruited 30 COVID-19 unvaccinated convalescent donors and 7 unexposed asymptomatic donors. Peripheral blood mononuclear cells (PBMCs) were obtained from leukapheresis cones. The humoral immune response was assessed by measuring serum anti-SARS-CoV-2 spike S1 subunit IgG semiquantitative ELISA and T cell immunity against S1 and S2 subunits were studied by IFN-γ Enzyme-Linked Immune absorbent Spot (ELISpot), flow cytometric (FC) activation-induced marker (AIM) assays and the assessment of cytotoxic CD8+ T-cell function (in the subset of HLA-A2 positive patients). No single immunoassay was sufficient in identifying anti-spike convalescent immunity among all patients. There was no consistent correlation between adaptive humoral and cellular anti-spike responses. Our data indicate that the magnitude of anti-spike convalescent humoral and cellular immunity is highly heterogeneous and highlights the need for using multiple assays to comprehensively measure SARS-CoV-2 convalescent immunity. These observations might have implications for COVID-19 surveillance, and optimal vaccination strategies for emerging variants. Further studies are needed to determine the optimal assessment of adaptive humoral and cellular immunity following SARS-CoV-2 infection, especially in the context of emerging variants and unclear vaccination schedules.

Abstract Background Biomarkers that predict the patients most likely to develop progressive NTM-LD are urgently needed. We hypothesize flow cytometric (FC) detection of T cell markers after ex vivo antigen challenge in PBMC can differentiate patients with progressive vs. nonprogressive NTM-LD. We also measured the serum anti-glycopeptidolipid core IgA antibody by commercial ELISA kit and compared with FC immunoprofiling results. Methods PBMCs were isolated from patients with NTM-LD and stimulated with purified protein derivative (PPD), mycobacteria-specific peptide pools (MTB300, RD1-peptides). We have performed ELISA and FC T-cell phenotypic assays to measure diagnostic accuracy of these assays by ROC and non-parametric statistics analysis. Results A total of 29 NTM-LD patients were studied and classified into 15 non-progressive and 14 progressive NTM-LD based on radiological and clinical evaluation. Patients with progressive NTM-LD showed a statistically significantly higher frequency of PPD-specific CD3+/CD8+ IFN-γ+HLA-DR+ subsets and high diagnostic accuracy compared with non-progressors: Sensitivity = 80% specificity = 71.4% with the ROC of 0.72. There were no other statistically significant differences in other antigen-stimulated T-cell subsets but a trend was observed with HLA-DR+TNF-α+, CD25+CD134+ and CD25+PDL1+ T-cell subsets in progressors. The anti-GPL IgA levels did not show a significant difference and might not be useful in distinguishing progressive from non-progressive NTM-LD. Conclusion Our study findings suggest that PPD-specific CD8+ IFN-γ+HLA-DR+ can distinguish progressive and nonprogressive NTM-LD patients, which can identify those patients at risk of disease progression.

Abstract Background: Binding of the human IgM antibody B7-DC XAb to B7-DC on dendritic cells (DC) has profound immune modulatory effects. It enhances antigen presentation by DC, repolarizes T-helper (Th) 2 to Th1 cell responses, rapidly generates cytotoxic effectors and reprograms regulatory T-cells (Treg) into non-suppressive, cytokine producing autoimmune effectors. We hypothesized that co-culture of B7-DC XAb treated DC (DCXAb) with Treg results in the rapid generation of cytotoxic effectors. Methods: Bone marrow derived DC from wild type C57BL6 mice (WT-B6) were treated for 24 hours with either B7-DC XAb or a control human IgM antibody in the presence of melanoma cell lysate (B16). Subsequently, DCXAb were co-cultured with Treg (CD4+CD25+FOXP3+), Non-Treg (CD4+, CD25-), cytotoxic CD8+ T-cells isolated from WT-B6 mice and Treg from CD8 knockout mice (CD8 -/-). After re-isolation these T-cells were used in cytotoxic T-lymphocyte (CTL) assays targeting B16 and Thymic lymphoma (EL4) tumor cells. Results: Treg isolated from either WT-B6 mice or CD8 -/- mice co-cultured with B16 lysate treated DCXAb but not control antibody treated DC transformed into cytotoxic effectors targeting B16 but not EL4 tumor cells. Their level of cytotoxicity was comparable to that of CD8+ cells co-cultured with DCXAb. In contrast Non-Treg co-cultured with DCXAb did not kill in similar CTL assays. Conclusion: Co-culture of Treg with antigen treated DCXAb rapidly generates antigen specific cytotoxic effectors.

Abstract B-cell activation is increasingly linked to numerous fibrotic lung diseases, and it is well known that aggregates of lymphocytes form in the lung of many of these patients. Activation of B-cells by pattern recognition receptors (PRRs) drives the release of inflammatory cytokines, chemokines, and metalloproteases important in the pathophysiology of pulmonary fibrosis. However, the specific mechanisms of B-cell activation in patients with idiopathic pulmonary fibrosis (IPF) are poorly understood. Herein, we have demonstrated that B-cell activation by microbial antigens contributes to the inflammatory and profibrotic milieu seen in patients with IPF. B-cell stimulation by CpG and β-glucan via PRRs resulted in activation of mTOR-dependent and independent pathways. Moreover, we showed that the B-cell–secreted inflammatory milieu is specific to the inducing antigen and causes differential fibroblast migration and activation. B-cell responses to infectious agents and subsequent B-cell–mediated fibroblast activation are modifiable by antifibrotics, but each seems to exert a specific and different effect. These results suggest that, upon PRR activation by microbial antigens, B-cells can contribute to the inflammatory and fibrotic changes seen in patients with IPF, and antifibrotics are able to at least partially reverse these responses.

7568 Background: The molecular events in the initiation and progression of invasive lung adenocarcinoma remain poorly characterized. These tumors are thought to develop through an adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA), invasive adenocarcinoma (AD) sequence. The goal of our study was to assess lineage relationships between in situ and invasive components within adenocarcinomas with prominent lepidic growth patterns. Methods: Frozen tissue from fourteen lung adenocarcinomas with mixtures of AD together with an adjacent in situ component (AIS), varying in ratio between 40 to 80%, were selected from our Lung Specimen Registry. Laser capture microdissection (LCM) of each component was performed separately for each tumor. Genomic DNA was isolated using a direct in situ whole genome amplification (WGA) methodology, and Next Generation Sequencing performed using an Illumina Mate Pair (MP) library protocol. MP sequence reads were mapped to the human genome and primers spanning the fusion junctions were used in validation PCRs. Results: Genomic break points identical and unique to a specific patient were identified between the AIS and AD components in 13 (of 14) cases. The total number of events per case ranged from 4-215, and the number of identical events shared between the AIS and AD components ranged from 30 to 90%. Recurrent genomic breakpoints between cases were also observed, with the 2 most common involving 8q24.3 in 5 cases and FAM19A2 on chromosome 12 in 4 cases. PCR validation of selected genomic alterations confirmed the genomic break points identified from sequencing in both the AIS and AD in all cases. Interestingly, we also observed a limited number of genomic alterations present in a zonal distribution of surrounding normal lung around the tumor mass. Conclusions: Our study demonstrates unique chromosomal alterations present in both the AIS and AD components of individual lung tumors with features of lepidic growth. These data provide evidence for lineage relationship and clonal relatedness between the two components, and provide genomic evidence for a model of stepwise progression from AIS to invasive AD in this subset of lung adenocarcinomas.