Lymphoid Neogenesis Research Articles

JAK Inhibitors and Modulation of B Cell Immune Responses in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic, systemic immune-mediated inflammatory disease that can lead to joint destruction, functional disability and substantial comorbidity due to the involvement of multiple organs and systems. B cells have several important roles in RA pathogenesis, namely through autoantibody production, antigen presentation, T cell activation, cytokine release and ectopic lymphoid neogenesis. The success of B cell depletion therapy with rituximab, a monoclonal antibody directed against CD20 expressed by B cells, has further supported B cell intervention in RA development. Despite the efficacy of synthetic and biologic disease modifying anti-rheumatic drugs (DMARDs) in the treatment of RA, few patients reach sustained remission and refractory disease is a concern that needs critical evaluation and close monitoring. Janus kinase (JAK) inhibitors or JAKi are a new class of oral medications recently approved for the treatment of RA. JAK inhibitors suppress the activity of one or more of the JAK family of tyrosine kinases, thus interfering with the JAK-Signal Transducer and Activator of Transcription (STAT) signaling pathway. To date, there are five JAK inhibitors (tofacitinib, baricitinib, upadacitinib, peficitinib and filgotinib) approved in the USA, Europe and/ or Japan for RA treatment. Evidence from the literature indicates that JAK inhibitors interfere with B cell functions. In this review, the main results obtained in clinical trials, pharmacokinetic, in vitro and in vivo studies concerning the effects of JAK inhibitors on B cell immune responses in RA are summarized.

Effect of CTLA4-Ig on Obliterative Bronchiolitis in a Mouse Intrapulmonary Tracheal Transplantation Model.

Objectives: One of the serious problems after lung transplantation is chronic lung allograft dysfunction (CLAD). Most CLAD patients pathologically characterized by obliterative bronchiolitis (OB). Cytotoxic T-lymphocyte-associated antigen 4 (CTLA4)-Ig is a combination protein of the Fc fragment of human IgG1 linked to the extracellular domain of CTLA4. The aim of the study was to examine the effect of CTLA4-Ig therapy on OB using a mouse intrapulmonary tracheal transplantation (IPTT) model.Methods: IPTT was performed between BALB/c (donor) and C57BL/6 (recipient) mice. Abatacept, which is a commercially available form of CTLA4-Ig, was intraperitoneally injected in recipient mice immediately after surgery, on days 7, 14, and 21. The mice in the control group received human IgG.Results: We performed semi-quantitative analysis of graft luminal obliteration at post-transplant day 28. We calculated the obliteration ratio of the lumen of the transplanted trachea in each case. The obliteration ratio was significantly lower in the CTLA4-Ig group than that in the control group (91.2 ± 2.1% vs. 47.8 ± 7.9%, p = 0.0008). Immunofluorescent staining revealed significantly decreased lymphoid neogenesis in the lung.Conclusions: CTLA4-Ig therapy attenuated tracheal obliteration with fibrous tissue in the mouse IPTT model. The attenuation of fibrous obliteration was correlated with the inhibition of lymphoid neogenesis.

Positron emission tomography and single photon emission computed tomography imaging of tertiary lymphoid structures during the development of lupus nephritis.

Lymphoid neogenesis occurs in tissues targeted by chronic inflammatory processes, such as infection and autoimmunity. In systemic lupus erythematosus (SLE), such structures develop within the kidneys of lupus-prone mice ((NZBXNZW)F1) and are observed in kidney biopsies taken from SLE patients with lupus nephritis (LN). The purpose of this prospective longitudinal animal study was to detect early kidney changes and tertiary lymphoid structures (TLS) using in vivo imaging. Positron emission tomography (PET) by tail vein injection of 18-F-fluoro-2-deoxy-D-glucose (18F-FDG)(PET/FDG) combined with computed tomography (CT) for anatomical localization and single photon emission computed tomography (SPECT) by intraperitoneal injection of 99mTC labeled Albumin Nanocoll (99mTC-Nanocoll) were performed on different disease stages of NZB/W mice (n = 40) and on aged matched control mice (BALB/c) (n = 20). By using one-way ANOVA analyses, we compared two different compartmental models for the quantitative measure of 18F-FDG uptake within the kidneys. Using a new five-compartment model, we observed that glomerular filtration of 18FFDG in lupus-prone mice decreased significantly by disease progression measured by anti-dsDNA Ab production and before onset of proteinuria. We could not visualize TLS within the kidneys, but we were able to visualize pancreatic TLS using 99mTC Nanocoll SPECT. Based on our findings, we conclude that the five-compartment model can be used to measure changes of FDG uptake within the kidney. However, new optimal PET/SPECT tracer administration sites together with more specific tracers in combination with magnetic resonance imaging (MRI) may make it possible to detect formation of TLS and LN before clinical manifestations.

Tertiary Lymphoid Organs: A Primer for Otolaryngologists.

Lymphoid neogenesis or the development of organised, de novo lymphoid structures has been described increasingly in chronically inflamed tissues. The presence of tertiary lymphoid organs (TLOs) has already been demonstrated to result in significant consequences for disease pathology, severity, prognosis and patient outcomes. Whilst the wider medical community has embraced TLOs as important markers of disease and potential therapeutic targets, the otolaryngology field has only begun turning to these entities in an academic capacity. This review aims to outline the role of tertiary lymphoid organs in disease and summarise key early findings in the ENT field. We also an overview of TLOs, their developmental process and clinicopathological implications. Literature review. A literature search for all relevant peer-reviewed publications pertaining to TLOs and ENT diseases. Search was conducted using PubMed, Embase and CINAHL databases. A total of 24 studies were identified relevant to the topic. The majority of TLO research in ENT fell into the areas of oral squamous cell carcinoma (SCC) and chronic rhinosinusitis (CRS). Early research into both oral SCC and CRS suggests that TLOs have significant roles within ear, nose and throat (ENT) diseases. At this point in time, however, TLOs remain somewhat a mystery amongst otolaryngologists. As information in this field increases, we may develop a better understanding of how lymphoid neogenesis can influence disease outcomes amongst our patients and, ultimately, how they can be utilised in an immunotherapeutic manner. Laryngoscope, 131:1697-1703, 2021.

Inhibition of LTβR signalling activates WNT-induced regeneration in lung.

SummaryLymphotoxin β-receptor (LTβR)-signalling orchestrates lymphoid neogenesis and subsequent tertiary lymphoid structures (TLS)1,2, associated with severe chronic inflammatory diseases spanning multiple organ systems3–6. How LTβR-signalling drives chronic tissue damage particularly in the lung, which mechanism(s) regulate this process, and whether LTβR-blockade might be of therapeutic value has remained unclear. Here we demonstrate increased expression of LTβR-ligands on adaptive and innate immune-cells, enhanced non-canonical NF-κB signalling and enriched LTβR-target gene expression in epithelial cells of lungs from patients with smoking-associated chronic obstructive pulmonary disease (COPD) and mice exposed to chronic cigarette smoke. Therapeutic inhibition of LTβR-signalling in young and aged mice disrupted smoking-related inducible bronchus-associated lymphoid tissue (iBALT), induced lung tissue regeneration, and reverted airway-fibrosis and systemic muscle wasting. Mechanistically, LTβR-signalling blockade dampened epithelial non-canonical NF-κB activation, reduced TGFβ-signalling in airways, induced regeneration by preventing epithelial cell-death and by activating Wnt/β-catenin-signalling in alveolar epithelial progenitor cells. These findings highlight that LTβR-signalling inhibition represents a viable therapeutic option combining anti-TLS, anti-apoptotic with tissue regenerative strategies.

Effective control of Staphylococcus aureus lung infection despite tertiary lymphoid structure disorganisation.

Tertiary lymphoid structures (TLS) are triggered by persistent bronchopulmonary infection with Staphylococcus aureus, but their roles remain elusive. The present study sought to examine the effects of B- and/or T-cell depletion on S. aureus infection and TLS development (lymphoid neogenesis) in mice. C57Bl/6 mice were pre-treated with 1) an anti-CD20 monoclonal antibody (mAb) (B-cell depletion) or 2) an anti-CD4 and/or an anti-CD8 mAb (T-cell depletion) or 3) a combination of anti-CD20, anti-CD4 and anti-CD8 mAbs (combined B- and T-cell depletion) or 4) isotype control mAbs. After lymphocyte depletion, mice were infected by intratracheal instillation of agarose beads containing S. aureus (106 CFU per mouse). 14 days later, bacterial load and lung inflammatory cell infiltration were assessed by cultures and immunohistochemistry, respectively. 14 days after S. aureus-bead instillation, lung bacterial load was comparable between control and lymphocyte-depleted mice. While TLS were observed in the lungs of infected mice pre-treated with control mAbs, these structures were disorganised or abolished in the lungs of lymphocyte-depleted mice. The absence of CD20+ B-lymphocytes had no effect on CD3+ T-lymphocyte infiltration, whereas CD4+/CD8+ T-cell depletion markedly reduced CD20+ B-cell infiltration. Depletion of CD4+ or CD8+ T-cells separately had limited effect on B-cell infiltration, but led to the absence of germinal centres. TLS disorganisation is not associated with loss of infection control in mice persistently infected with S. aureus.

PULMONARY ARTERIAL HYPERTENSION AS A MANIFESTATION OF SYSTEMIC LUPUS ERYTHEMATOUS

SESSION TITLE: Medical Student/Resident Pulmonary Manifestations of Systemic Disease Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: October 18-21, 2020 INTRODUCTION: Systemic Lupus Erythematosus (SLE) has long been associated with pulmonary manifestations such as pleural effusion, pulmonary hemorrhage, and pleuritis. Rarely, pulmonary arterial hypertension (PAH) develops as a consequence of SLE, usually presenting on average 3.2 years after diagnosis. We present a case of PAH as the initial manifestation of a patient who would later be diagnosed with SLE. CASE PRESENTATION: A 59 year old female with no medical history presented to the pulmonary clinic with dyspnea for two years. She stated that her dyspnea is present at rest but worsened with exertion. She had no other symptoms aside from generalized fatigue and denied cough, sputum production, chest pain, skin lesions or sores. Her symptoms had progressively worsened and previous treatment with prednisone 40mg po daily for a presumptive diagnosis of sarcoidosis had not led to any benefit. Previous biopsies for pulmonary nodules of undetermined significance did not render a diagnosis due to lack of available tissue. Normal spirometry and lung volumes were noted on pulmonary function testing. 6 minute walk test showed an SpO2 of 94% at the start, and 89% at completion and heart rate increased from 113 to 117 beats per minute. High resolution Chest CT was performed that showed no change in pulmonary nodules from previous. There was evidence of pulmonary artery enlargement. A right and left heart catheterization was performed revealing no coronary artery disease, but mean pulmonary artery pressure was elevated at 52mmHg, pulmonary capillary wedge pressure was 4mmHg and pulmonary vascular resistance was 15mmHg. She had adequate reactivity to adenosine. Rheumatologic evaluation later revealed positive ANA with a titer of 1:640, RF IgM, anti-cardiolipin IgM and anti-phospholipid IgM. Complements were decreased. The patient underwent a surgical wedge resection, and pathology revealed bronchial-associated lymphoid tissue. She was diagnosed with SLE as well as group 1 PAH, and initiated on hydroxychloroquine, ambrisentan and nifedipine with subjective and objective improvement in her clinical course. DISCUSSION: Pre-capillary PAH is the form closely linked with SLE-associated histopathological changes affecting small lung vessels. PAH is diagnosed by right heart catheterization revealing a mean pulmonary artery pressure >25mmHg, and can be defined as pre-capillary if the pulmonary capillary wedge pressure is <15mmHg. Bronchus-associated lymphoid tissues have been found in animal studies to be auto-immunologically active, and contribute to pulmonary vascular remodeling and pulmonary hypertension. CONCLUSIONS: This case highlights the close link between autoimmune conditions, small-vessel pulmonary vascular changes related to lymphoid aggregation and pulmonary hypertension. Targeted treatment for both group 1 PAH and SLE should be considered early in such patients, and initiated without delay. Reference #1: Colvin, Kelly L, et al. “Bronchus-Associated Lymphoid Tissue in Pulmonary Hypertension Produces Pathologic Autoantibodies.” American Journal of Respiratory and Critical Care Medicine, vol. 188, 2013, pp. 1126–1136. Reference #2: Perros, Frederic, et al. “Pulmonary Lymphoid Neogenesis in Idiopathic Pulmonary Arterial Hypertension.” American Journal of Respiratory and Critical Care Medicine, vol. 185, 2012, pp. 311-321. Reference #3: Badlam, Jessica B, et al. “Steps forward in the treatment of pulmonary arterial hypertension: latest developments and clinical opportunities.” Therapeutic Advances in Chronic Disease, 8(2-3), pp. 47-64. DISCLOSURES: No relevant relationships by Alexandra Gastesi, source=Web Response No relevant relationships by Julia Ladna, source=Web Response No relevant relationships by Glenn Singer, source=Admin input No relevant relationships by Kenneth Wojnowski, source=Web Response

Vascular Remodeling and Immune Cell Infiltration in Splenic Artery Aneurysms.

Rupture of splenic artery aneurysms (SAAs) is associated with a high mortality rate. The aim of this study was to identify the features of SAAs. Tissue sections from SAAs were compared to nonaneurysmal splenic arteries using various stains. The presence of intraluminal thrombus (ILT), vascular smooth muscle cells (VSMCs), cluster of differentiation (CD)-68+ phagocytes, myeloperoxidase+ neutrophils, CD3+, and CD20+ adaptive immune cells were studied using immunofluorescence microscopy. Analysis of SAAs revealed the presence of atherosclerotic lesions, calcifications, and ILT. Splenic artery aneurysms were characterized by a profound vascular remodeling with a dramatic loss of VSMCs, elastin degradation, adventitial fibrosis associated with enhanced apoptosis, and increased matrix metalloproteinase 9 expression. We observed an infiltration of immune cells comprising macrophages, neutrophils, T, and B cells. The T and B cells were found in the adventitial layer of SAAs, but their organization into tertiary lymphoid organs was halted. We failed to detect germinal centers even in the most organized T/B cell follicles and these lymphoid clusters lacked lymphoid stromal cells. This detailed histopathological characterization of the vascular remodeling during SAA showed that lymphoid neogenesis was incomplete, suggesting that critical mediators of their development must be missing.

Human Lung Conventional Dendritic Cells Orchestrate Lymphoid Neogenesis during Chronic Obstructive Pulmonary Disease.

Rationale: Emerging evidence supports a crucial role for tertiary lymphoid organs (TLOs) in chronic obstructive pulmonary disease (COPD) progression. However, mechanisms of immune cell activation leading to TLOs in COPD remain to be defined.Objectives: To examine the role of lung dendritic cells (DCs) in T follicular helper (Tfh)-cell induction, a T-cell subset critically implicated in lymphoid organ formation, in COPD.Methods: Myeloid cell heterogeneity and phenotype were studied in an unbiased manner via single-cell RNA sequencing on HLA-DR+ cells sorted from human lungs. We measured the in vitro capability of control and COPD lung DC subsets, sorted using a fluorescence-activated cell sorter, to polarize IL-21+CXCL13+ (IL-21-positive and C-X-C chemokine ligand type 13-positive) Tfh-like cells. In situ imaging analysis was performed on Global Initiative for Chronic Obstructive Lung Disease stage IV COPD lungs with TLOs.Measurements and Main Results: Single-cell RNA-sequencing analysis revealed a high degree of heterogeneity among human lung myeloid cells. Among these, conventional dendritic type 2 cells (cDC2s) showed increased induction of IL-21+CXCL13+ Tfh-like cells. Importantly, the capacity to induce IL-21+ Tfh-like cells was higher in cDC2s from patients with COPD than in those from control patients. Increased Tfh-cell induction by COPD cDC2s correlated with increased presence of Tfh-like cells in COPD lungs as compared with those in control lungs, and cDC2s colocalized with Tfh-like cells in TLOs of COPD lungs. Mechanistically, cDC2s exhibited a unique migratory signature and (transcriptional) expression of several pathways and genes related to DC-induced Tfh-cell priming. Importantly, blocking the costimulatory OX40L (OX40 ligand)-OX40 axis reduced Tfh-cell induction by control lung cDC2s.Conclusions: In COPD lungs, we found lung EBI2+ (Epstein-Barr virus-induced gene 2-positive) OX-40L-expressing cDC2s that induced IL-21+ Tfh-like cells, suggesting an involvement of these cells in TLO formation.

Bronchiolitis obliterans syndrome and restrictive allograft syndrome after lung transplantation: why are there two distinct forms of chronic lung allograft dysfunction?

Bronchiolitis obliterans syndrome (BOS) had been considered to be the representative form of chronic rejection or chronic lung allograft dysfunction (CLAD) after lung transplantation. In BOS, small airways are affected by chronic inflammation and obliterative fibrosis, whereas peripheral lung tissue remains relatively intact. However, recognition of another form of CLAD involving multiple tissue compartments in the lung, termed restrictive allograft syndrome (RAS), raised a fundamental question: why there are two phenotypes of CLAD? Increasing clinical and experimental data suggest that RAS may be a prototype of chronic rejection after lung transplantation involving both cellular and antibody-mediated alloimmune responses. Some cases of RAS are also induced by fulminant general inflammation in lung allografts. However, BOS involves alloimmune responses and the airway-centered disease process can be explained by multiple mechanisms such as external alloimmune-independent stimuli (such as infection, aspiration and air pollution), exposure of airway-specific autoantigens and airway ischemia. Localization of immune responses in different anatomical compartments in different phenotypes of CLAD might be associated with lymphoid neogenesis or the de novo formation of lymphoid tissue in lung allografts. Better understanding of distinct mechanisms of BOS and RAS will facilitate the development of effective preventive and therapeutic strategies of CLAD.

Chronic Inflammation: A Common Promoter in Tertiary Lymphoid Organ Neogenesis.

Tertiary lymphoid organs (TLOs) frequently develop locally in adults in response to non-resolving inflammation. Chronic inflammation leads to the differentiation of stromal fibroblast cells toward lymphoid tissue organizer-like cells, which interact with lymphotoxin immune cells. The interaction initiates lymphoid neogenesis by recruiting immune cells to the site of inflammation and ultimately leads to the formation of TLOs. Mature TLOs harbor a segregated T-cell zone, B-cell follicles with an activated germinal center, follicular dendritic cells, and high endothelial venules, which architecturally resemble those in secondary lymphoid organs. Since CXCL13 and LTα1β2 play key roles in TLO neogenesis, they might constitute potential biomarkers of TLO activity. The well-developed TLOs actively regulate local immune responses and influence disease progression, and they are thereby regarded as the powerhouses of local immunity. In this review, we recapitulated the determinants for TLOs development, with great emphasis on the fundamental role of chronic inflammation and tissue-resident stromal cells for TLO neogenesis, hence offering guidance for therapeutic interventions in TLO-associated diseases.

Docosahexaenoic Acid Consumption Impedes Early Interferon- and Chemokine-Related Gene Expression While Suppressing Silica-Triggered Flaring of Murine Lupus.

Exposure of lupus-prone female NZBWF1 mice to respirable crystalline silica (cSiO2), a known human autoimmune trigger, initiates loss of tolerance, rapid progression of autoimmunity, and early onset of glomerulonephritis. We have previously demonstrated that dietary supplementation with the ω-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) suppresses autoimmune pathogenesis and nephritis in this unique model of lupus flaring. In this report, we utilized tissues from prior studies to test the hypothesis that DHA consumption interferes with upregulation of critical genes associated with cSiO2-triggered murine lupus. A NanoString nCounter platform targeting 770 immune-related genes was used to assess the effects cSiO2 on mRNA signatures over time in female NZBWF1 mice consuming control (CON) diets compared to mice fed diets containing DHA at an amount calorically equivalent to human consumption of 2 g per day (DHA low) or 5 g per day (DHA high). Experimental groups of mice were sacrificed: (1) 1 d after a single intranasal instillation of 1 mg cSiO2 or vehicle, (2) 1 d after four weekly single instillations of vehicle or 1 mg cSiO2, and (3) 1, 5, 9, and 13 weeks after four weekly single instillations of vehicle or 1 mg cSiO2. Genes associated with inflammation as well as innate and adaptive immunity were markedly upregulated in lungs of CON-fed mice 1 d after four weekly cSiO2 doses but were significantly suppressed in mice fed DHA high diets. Importantly, mRNA signatures in lungs of cSiO2-treated CON-fed mice over 13 weeks reflected progressive amplification of interferon (IFN)- and chemokine-related gene pathways. While these responses in the DHA low group were suppressed primarily at week 5, significant downregulation was observed at weeks 1, 5, 9, and 13 in mice fed the DHA high diet. At week 13, cSiO2 treatment of CON-fed mice affected 214 genes in kidney tissue associated with inflammation, innate/adaptive immunity, IFN, chemokines, and antigen processing, mostly by upregulation; however, feeding DHA dose-dependently suppressed these responses. Taken together, dietary DHA intake in lupus-prone mice impeded cSiO2-triggered mRNA signatures known to be involved in ectopic lymphoid tissue neogenesis, systemic autoimmunity, and glomerulonephritis.

Noncanonical immunomodulatory activity of complement regulator C4BP(β-) limits the development of lupus nephritis

Lupus nephritis is a chronic autoimmune-inflammatory condition that can lead to end-stage kidney disease. Presently available immunosuppressive treatments for lupus nephritis are suboptimal and can induce significant side effects. Recently, we characterized a novel immunomodulatory activity of the minor isoform of the classical pathway complement inhibitor, C4BP(β-). We show here that C4BP(β-) treatment prevented the development of proteinuria and albuminuria, decreased significantly the formation of anti-dsDNA antibodies and, locally, mitigated renal glomerular IgG and C3 deposition and generation of apoptotic cells. There was a consequent histological improvement and increased survival in lupus-prone mice. The therapeutic efficacy of C4BP(β-) was analogous to that of the broad-acting immunosuppressant cyclophosphamide. Remarkably, a comparative transcriptional profiling analysis revealed that the kidney gene expression signature resulting from C4BP(β-) treatment turned out to be 10 times smaller than that induced by cyclophosphamide treatment. C4BP(β-) immunomodulation induced significant downregulation of transcripts relevant to lupus nephritis indicating immunopathogenic cell infiltration, including activated T cells (Lat), B cells (Cd19, Ms4a1, Tnfrsf13c), inflammatory phagocytes (Irf7) and neutrophils (Prtn3, S100a8, S100a9). Furthermore, cytokine profiling and immunohistochemistry confirmed that C4BP(β-), through systemic and local CXCL13 downregulation, was able to prevent ectopic lymphoid structures neogenesis in aged mice with lupus nephritis. Thus, due to its anti-inflammatory and immunomodulatory activities and high specificity, C4BP(β-) could be considered for further clinical development in patients with systemic lupus erythematosus.

Progression or suppression: Two sides of the innate lymphoid cells in cancer.

Innate lymphoid cells (ILCs) as key players in innate immunity have been shown to be significantly associated with inflammation, lymphoid neogenesis, tissue remodeling, mucosal immunity and lately have been considered a remarkable nominee for either tumor-promoting or tumor-inhibiting functions. This dual role of ILCs, which is driven by intrinsic and extrinsic factors like plasticity of ILCs and the tumor microenvironment, respectively, has aroused interest in ILCs subsets in past decade. So far, numerous studies in the cancer field have revealed ILCs to be key players in the initiation, progression and inhibition of tumors, therefore providing valuable insights into therapeutic approaches to utilize the immune system against cancer. Herein, the most recent achievements regarding ILCs subsets including new classifications, their transcription factors, markers, cytokine release and mechanisms that led to either progression or inhibition of many tumors have been evaluated. Additionally, the available data regarding ILCs in most prevalent cancers and new therapeutic approaches are summarized.

CXCL13 and Its Receptor CXCR5 in Cancer: Inflammation, Immune Response, and Beyond.

It is well-established that the chemokine C-X-C motif ligand 13 (CXCL13) and its receptor, the G-protein coupled receptor (GPCR) CXCR5, play fundamental roles in inflammatory, infectious and immune responses. Originally identified as a B-cell chemoattractant, CXCL13 exerts important functions in lymphoid neogenesis, and has been widely implicated in the pathogenesis of a number of autoimmune diseases and inflammatory conditions, as well as in lymphoproliferative disorders. Current evidence also indicates that the CXCL13:CXCR5 axis orchestrates cell-cell interactions that regulate lymphocyte infiltration within the tumor microenvironment, thereby determining responsiveness to cytotoxic and immune-targeted therapies. In this review, we provide a comprehensive perspective of the involvement of CXCL13 and its receptor in cancer progression. Studies in recent years postulated novel roles for this chemokine in controlling the cancer cell phenotype, and suggest important functions in the growth and metastatic dissemination of solid tumors. Carcinogens have been found to induce CXCL13 production, and production of this chemokine within the tumor milieu has been shown to impact the proliferation, migration, and invasive properties of cancer cells. Thus, the complex networks of cellular interactions involving tumoral CXCL13 and CXCR5 integrate to promote cancer cell autonomous and non-autonomous responses, highlighting the relevance of autocrine and paracrine interactions in dictating the cancer phenotype. Dissecting the molecular and signaling events regulated by CXCL13 and how this chemokine dynamically controls the interaction between the cancer cell and the tumor microenvironment is key to identify novel effectors and therapeutic targets for cancer treatment.

Abstract 1482: Tissue specific splicing program of hMENA: impact on tumor immune microenvironment in node-negative NSCLC

Abstract Deciphering the complexity of the tumor microenvironment (TME)is essential to unveil mechanisms of therapy resistance and develop novel microenvironment-related anti-tumor treatment. Actin cytoskeleton dynamics act as platforms for gene regulation and key signaling transduction pathways involved in the cross-talk among tumor cells and cellular and non-cellular components of TME.The actin regulatory protein hMENA undergoes tissue specific splicing, generating two alternatively expressed isoforms hMENA11a and hMENAΔv6 with a crucial role in EMT. We have previously demonstrated that hMENA11a and hMENAΔv6, respectively inhibit or increase cell invasiveness, TGFβ and β1 integrin signaling and the secretion of several key extracellular matrix (ECM) proteins. Early node-negative NSCLC patients show a prolonged disease-free survival (DFS) when expressing high tumor hMENA11a/low stromal FN1. Tertiary Lymphoid Structures (TLS), sites of transient lymphoid neo-genesis and determinants of antitumor immunity, have been associated with a favorable clinical outcome in NSCLC patientsandfound in responding lesions of ICB-treated melanoma patients. The aim of the present study was to analyzethe pattern ofhMENA isoforms as biomarker of EMT signature in the context of ECM composition and TLS presence and localization. We evaluated by gain and loss of function experiments the role of hMENA isoforms in TLS neogenesis. hMENA isoforms expression, TLS presence and stromal fibronectin were evaluated in 110primary tumors of node negative NSCLC patients by immunohistochemical analysis using pan-hMENA, hMENA11a, CD3, CD20 and fibronectin (FN) antibodies. The Chi-Square or Fisher Exact tests were used to estimate associations among categorical variables. We found, by RNA-SEQ analysisand subsequent validation by QRT-PCR, in NSCLC cell lines depleted for the expression of ‘epithelial’ hMENA11a isoform,that hMENA11asustains the expression of lymphotoxin beta receptor (LTBR), a regulator of TLS formation. The evaluation of TLS presence and spatial distribution in the primary tumors indicatedthat the presence of TLS within the tumor core is significantly correlatedwith hMENA11a expression in tumor cells, whereas the presence of TLS at the margin oftumor nests correlates with the absence of hMENA11a. When we evaluated also the fibronectin we found a trend of association between low stromal fibronectin and intratumoral TLS, however a low level of stromal FN in concomitance with the expression of hMENA11ain tumor cells,strongly associated with intra-tumoralTLS presence. Our findings indicate that the alternative splicing of hMENA is crucial in the reciprocal signaling between tumor cells and their immune microenvironment, by participating in tertiary lymphoid structure neo genesis and spatial distribution. Funded by Airc Citation Format: Francesca Di Modugno, Sheila Spada, Anna Di Carlo, Paola Trono, Isabella Sperduti, Barbara Antoniani, Enzo Gallo, Giulia Campo, Francesco Facciolo, Paolo Visca, Paola Nisticò. Tissue specific splicing program of hMENA: impact on tumor immune microenvironment in node-negative NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1482.

Tertiary lymphoid structures in the era of cancer immunotherapy.

Tertiary lymphoid structures (TLSs) are ectopic lymphoid organs that develop in non-lymphoid tissues at sites of chronic inflammation including tumours. Key common characteristics between secondary lymphoid organogenesis and TLS neogenesis have been identified. TLSs exist under different maturation states in tumours, culminating in germinal centre formation. The mechanisms that underlie the role of TLSs in the adaptive antitumour immune response are being deciphered. The description of the correlation between TLS presence and clinical benefit in patients with cancer, suggesting that TLSs could be a prognostic and predictive factor, has drawn strong interest into investigating the role of TLSs in tumours. A current major challenge is to exploit TLSs to promote lymphocyte infiltration, activation by tumour antigens and differentiation to increase the antitumour immune response. Several approaches are being developed using chemokines, cytokines, antibodies, antigen-presenting cells or synthetic scaffolds to induce TLS formation. Strategies aiming to induce TLS neogenesis in immune-low tumours and in immune-high tumours, in this case, in combination with therapeutic agents dampening the inflammatory environment and/or with immune checkpoint inhibitors, represent promising avenues for cancer treatment.

Implications and mechanisms of pulmonary lymphoid neogenesis in chronic obstructive pulmonary disease

获得性免疫在慢性阻塞性肺疾病（慢阻肺）发病机制中发挥重要作用，肺淋巴组织新生（淋巴滤泡形成）是获得性免疫发生的重要机制。研究慢阻肺中淋巴滤泡形成的细胞和分子机制及其病理生理作用，对于认识慢阻肺慢性炎症和肺组织破坏（肺气肿）的发生机制，具有重要的科学意义。本文就目前关于肺淋巴滤泡形成与慢阻肺的关系研究进展作简要综述。.

Mapping of Dynamic Transcriptome Changes Associated With Silica-Triggered Autoimmune Pathogenesis in the Lupus-Prone NZBWF1 Mouse.

Crystalline silica (cSiO2) is a widely recognized environmental trigger of autoimmune disease. In the lupus-prone female NZBWF1 mouse, airway exposure to cSiO2 triggers pulmonary ectopic lymphoid neogenesis, systemic autoantibody elevation, and glomerulonephritis. Here we tested the hypothesis that upregulation of adaptive immune function genes in the lung precedes cSiO2-triggering of autoimmune disease in this model. The study include three groups of mice, as follows: (1) necropsied 1 d after a single intranasal instillation of 1 mg cSiO2 or vehicle, (2) necropsied 1 d after four weekly single instillations of 1 mg cSiO2 or vehicle, or (3) necropsied 1, 5, 9, or 13 weeks after four weekly single instillations of 1 mg cSiO2 or vehicle. NanoString nCounter analysis revealed modest transcriptional changes associated with innate and adaptive immune response as early as 1 d after a single cSiO2 instillation. These responses were greatly expanded after four weekly cSiO2 instillations. Concurrent with ectopic lymphoid neogenesis, dramatic increases in mRNAs associated with chemokine release, cytokine production, sustained interferon activity, complement activation, and adhesion molecules were observed. As disease progressed, expression of these genes persisted and was further amplified. Consistent with autoimmune pathogenesis, the time between 5 and 9 weeks post-instillation reflected an important transition period where considerable immune gene upregulation in the lung was observed. Upon termination of the chronic study (13 weeks), cSiO2-induced changes in transcriptome signatures were similarly robust in kidney as compared to the lung, but more modest in spleen. Transcriptomic signatures in lung and kidney were indicative of infiltration and/or expansion of neutrophils, macrophages, dendritic cells, B cells, and T cells that corresponded with accelerated autoimmune pathogenesis. Taken together, airway exposure to cSiO2 elicited aberrant mRNA signatures for both innate and adaptive immunity that were consistent with establishment of the lung as the central autoimmune nexus for launching systemic autoimmunity and ultimately, kidney injury.

B Cells Drive Chronic Lung Allograft Rejection after Ischemia-Reperfusion Injury in Mouse Lung Transplantation

Purpose Chronic lung allograft dysfunction (CLAD) is the primary obstacle to long-term survival after lung transplantation (LTX). Ischemia-reperfusion injury (IRI) increases the risk of CLAD in humans but the underlying mechanisms are not well understood. In a mouse minor alloantigen mismatched LTX model (C57BL/10 [B10] →C57BL/6 [B6]), we observed that IRI augments formation of B cell-rich tertiary lymphoid organs (TLOs) and chronic rejection in allografts but not isografts. We therefore hypothesized that B cells may link IRI with chronic rejection. Methods B10→B6 LTX with extended graft preservation (6 hours at 4°C followed by 45 minutes at 37°Cand 15 minutes at room temperature during anastomosis), which augments acute rejection, fibrosis, and TLO formation, was employed. Anti-CD20 antibody (aCD20: n=9) or isotype control (Iso: n=10) were given i.p. on days -2, +2, +7, +14, and +21. Peripheral blood was longitudinally assessed by flow cytometry after LTX. Grafts were assessed with histology, flow cytometry and immunofluorescence 28 days after LTX. Results aCD20 treatment effectively depleted CD19+ or B220+ B cells in peripheral blood (Figure A) and lung grafts (Figure B). Acute rejection (ISHLT A grade), peribronchial fibrosis, and airways with obliterative bronchiolitis (%OB) were lower in aCD20 mice (1.8, 1.4, and 13.1% respectively) than in Iso mice (3.1, 2.6, and 46.7% respectively) (Figure C). TLOs containing CD3+, CD19+, and peripheral node addressin (PNAd)+cells were also reduced (Figure D). Conclusion In this model of IRI-augmented chronic rejection, B cell depletion attenuated acute rejection, airway fibrosis, and lymphoid neogenesis. Our observations suggest that B cell activation and recruitment may be directly involved in the augmentation of CLAD in LTX recipients experiencing IRI.