The precise nature of the local immune responses in lung tuberculosis (TB) granulomas requires a comprehensive understanding of their environmental complexities. At its most basic level, a granuloma ...is a compact, organized immune aggregate of macrophages surrounded by myeloid, B and T cells. We established two complementary multiplex immunolabeling panels to simultaneously evaluate the myeloid and lymphocytic contexture of 14 human lung TB granulomas in formalin-fixed paraffin-embedded tissue samples. We observed diverse CD3+ and CD8+ T-cell and CD20+ B lymphocyte compositions of the granuloma immune environment and a relatively homogeneous distribution of all myeloid cells. We also found significant associations between CD8+ T-cell densities and the myeloid marker CD11b and phagocytic cell marker CD68. In addition, significantly more CD68+ macrophages and CD8+ T cells were found in Mycobacterium tuberculosis-infected granulomas, as detected by Ziehl–Neelsen staining. FOXP3 expression was predominately found in a small subset of CD4+ T cells in different granulomas. As the success or failure of each granuloma is determined by the immune response within that granuloma at a local and not a systemic level, we attempted to identify the presence of reactive T cells based on expression of the T-cell activation marker CD137 (4-1BB) and programmed cell death-1 (PD-1). Only a small fraction of the CD4+ and CD8+ T cells expressed PD-1. CD137 expression was found only in a very small fraction of the CD4+ T cells in two granulomas. Our results also showed that multinucleated giant cells showed strong PD-L1 but not CTLA-4 membrane staining. This study offers new insights into the heterogeneity of immune cell infiltration in lung TB granulomas, suggesting that each TB granuloma represents a unique immune environment that might be independently influenced by the local adaptive immune response, bacterial state, and overall host disease status.
Background
Programmed death–ligand 1 (PD‐L1) expression, as assessed by immunohistochemistry (IHC), is used to select patients with non–small cell lung cancer (NSCLC) for anti‐programmed cell death ...protein 1 (PD‐1)/PD‐L1 therapy. The current study evaluated the feasibility and efficacy of PD‐L1 immunostaining and quantitation on direct Papanicolaou‐stained cytological smears compared with formalin‐fixed paraffin‐embedded samples (cytological cell blocks and surgical resection specimens) in NSCLC cases using 2 commercially available assays: the PD‐L1 IHC 22C3 pharmDx assay (Agilent Technologies/Dako, Carpinteria, CA, USA) and the Ventana SP263 Assay (Ventana Medical Systems Inc, Tucson, Arizona).
Methods
PD‐L1 immunostaining using either both or one of the assays was tested in 117 sets of paired samples obtained from 62 NSCLC cases. The tumor proportion score was reported in every case following the recommendations of the International Association for the Study of Lung Cancer (IASLC).
Results
In 57 sets of samples, both PD‐L1 assays were used. Due to the availability of samples, only 1 assay was performed in 3 sets of samples and in 2 cases, only cytology smears were used and tested for both assays. A total of 113 sets of paired samples finally were evaluated; 4 cases could not be studied due to intense nonspecific background staining. A significant concordance between the 2 assays on cytological smears was found. Concordance between paired cytological smears and formalin‐fixed paraffin‐embedded samples was observed in 97.3% of the cases.
Conclusions
The quantification of PD‐L1 expression on direct Papanicolaou‐stained cytology smears is feasible and reliable for both PD‐L1 assays.
The quantification of programmed death–ligand 1 (PD‐L1) expression on a direct Papanicolaou‐stained cytology smear is feasible. In the current study, PD‐L1 testing in cytology appears to be mostly concordant with corresponding histology samples.
Objective
Understanding the immune environment of non‐small cell lung cancer (NSCLC) is important for designing effective anticancer immunotherapies. We describe the use of multiplex ...immunofluorescence (mIF) assays to enable characterisation of the tumour‐infiltrating immune cells and their interactions, both across and within immune subtypes.
Methods
Six cytological samples of NSCLC taken by transoesophageal ultrasound‐guided fine needle aspiration were tested with an mIF assay designed to detect the expression of key immune cell markers such as CD3, CD8, CD20, CD11b, and CD68. Pan‐cytokeratin was used to detect the NSCLC cells. Fluorescence images were acquired on a Vectra‐Polaris Automated Quantitative Pathology Imaging System (Akoya Biosciences).
Results
MIF assay was able to reliably detect and quantify the myeloid cell markers CD11b, CD68, CD3+ and CD8+ T cells, and CD20+ B lymphocytes on cytological samples of NSCLC. Whole‐tissue analysis and its correlation with the corresponding H&E stains allowed a better understanding of the tissue morphology and the relationship between tumour and stroma compartments. Additionally, a uniform, specific, and correct staining pattern was seen for every immune marker.
Conclusion
The implementation of mIF assay on cytological samples taken with minimally invasive methods seems feasible and can be used to explore the immune environment of NSCLC.
Multiplex immunofluorescence (mIF) assays allow better understanding of the interactions between the tumour and its direct immune environment. The implementation of mIF assay on cytological samples taken with minimally invasive methods seems feasible and can be used to explore the immune environment of NSCLC.
Our aim was to assess oncologic, safety, and quality of life-related outcomes of focal therapy with irreversible electroporation in men with localized prostate cancer.
This was a single-center, phase ...II study.
prostate cancer International Society of Urological Pathology grade 1-2, prostate specific antigen ≤15 ng/ml, ≤cT2b. Patients were selected based on multiparametric magnetic resonance imaging and transperineal systematic and targeted magnetic resonance imaging-ultrasound fusion-guided biopsy. Ablation of index lesions with safety margin was performed. Primary end point was cancer control, defined as the absence of any biopsy-proven tumor. A control transperineal biopsy was planned at 12 months and when suspected based on prostate specific antigen and/or multiparametric magnetic resonance imaging information. Quality of life was assessed using Expanded Prostate Cancer Index Composite Urinary Continence domain, International Index of Erectile Function, and International Prostate Symptom Score.
From November 2014 to July 2021, 41 consecutive patients were included with a median follow-up of 36 months. Thirty patients (73%) had International Society of Urological Pathology grade 1 tumors, 10 (24%) grade 2, and 1 (2.4%) grade 3. Recurrence was observed in 16 of 41 (39%) of the whole cohort, and 16 of 33 (48.4%) who underwent biopsy. In-field recurrence was detected in 5 (15%) and out-of-field in 11 (33.3%). Ten of 41 (24.6%) including 3 of 5 (60%) with in-field recurrences had significant tumors (Gleason pattern 4-5; more than 1 core or any >5 mm involved). Median recurrence-free survival was 32 months (95% CI 6.7-57.2). Twenty-six patients (63.4%) were free from salvage treatment. All patients preserved urinary continence. Potency was maintained in 91.8%.
Irreversible electroporation can achieve satisfactory 3-year in-field tumor control with excellent quality of life results in selected patients.
Locoregional failure (LRF) in patients with breast cancer post-surgery and post-irradiation is linked to a dismal prognosis. In a refined new model, we identified ectonucleotide ...pyrophosphatase/phosphodiesterase 1/CD203a (ENPP1) to be closely associated with LRF. ENPP1hi circulating tumor cells (CTC) contribute to relapse by a self-seeding mechanism. This process requires the infiltration of polymorphonuclear myeloid-derived suppressor cells and neutrophil extracellular trap (NET) formation. Genetic and pharmacologic ENPP1 inhibition or NET blockade extends relapse-free survival. Furthermore, in combination with fractionated irradiation, ENPP1 abrogation obliterates LRF. Mechanistically, ENPP1-generated adenosinergic metabolites enhance haptoglobin (HP) expression. This inflammatory mediator elicits myeloid invasiveness and promotes NET formation. Accordingly, a significant increase in ENPP1 and NET formation is detected in relapsed human breast cancer tumors. Moreover, high ENPP1 or HP levels are associated with poor prognosis. These findings unveil the ENPP1/HP axis as an unanticipated mechanism exploited by tumor cells linking inflammation to immune remodeling favoring local relapse.
CTC exploit the ENPP1/HP axis to promote local recurrence post-surgery and post-irradiation by subduing myeloid suppressor cells in breast tumors. Blocking this axis impairs tumor engraftment, impedes immunosuppression, and obliterates NET formation, unveiling new opportunities for therapeutic intervention to eradicate local relapse and ameliorate patient survival. This article is highlighted in the In This Issue feature, p. 1171.
Locoregional failure (LRF) in breast cancer patients post-surgery and post-irradiation (IR) is linked to a dismal prognosis. In a refined new model, we identified Enpp1 (Ectonucleotide ...pyrophosphatase /phosphodiesterase 1/CD203a) to be closely associated with LRF. Enpp1high circulating tumor cells (CTC) contribute to relapse by a self-seeding mechanism. This process requires the infiltration of PMN-MDSC and neutrophil extracellular traps (NET) formation. Genetic and pharmacological Enpp1 inhibition or NET blockade extend relapse-free survival. Furthermore, in combination with fractionated irradiation (FD), Enpp1 abrogation obliterates LRF. Mechanistically, Enpp1-generated adenosinergic metabolites enhance Haptoglobin (Hp) expression. This inflammatory mediator elicits myeloid invasiveness and promotes NET formation. Accordingly, a significant increase in ENPP1 and NET formation is detected in relapsed human breast cancer tumors. Moreover, high ENPP1 or HP levels are associated with poor prognosis. These findings unveil the ENPP1/HP axis as an unanticipated mechanism exploited by tumor cells linking inflammation to immune remodeling favoring local relapse.
Multiple Myeloma (MM) is a malignant plasma cell (PC) disorder characterized by a heterogeneous distribution of PCs in the bone marrow (BM) where the interactions between the PCs and the BM ...microenvironment guide the pathogenesis of the disease. The development of single cell technologies has contributed to understand the transcriptional heterogeneity of the disease both from the tumor and the microenvironment point of view, yet the spatial organization of cellular states and niche-specific regulatory programs remain to be investigated. To address the role of spatially resolved interactions in MM, we performed spatial transcriptomic analysis in BM from the MI cγ1mice strain (Larrayoz et al Nat Med. 2023) a recently described mouse model that recapitulates the characteristics of a MYC driven human MM disease using the Visium Spatial Gene Expression analysis (10x Genomics). Using formalin-fixed paraffin-embedded (FFPE) BM tissues from the femur of control mice, we first characterized the healthy BM. Considering that each spot contains an averaged transcriptomic profile of 3-10 cells, we generated a single-cell reference set that included the most prevalent cell types in the BM to identify the cell type contribution per spot. The relative proportions of the estimations are consistent with percentage estimations observed by previous works (Hongzhe et al, eLife. 2023). Next, using the “cell proportion estimations per spot” we identified four different clusters of spots. A deeper characterization of those clusters revealed a positive correlation between erythroblasts and B cells, and a negative correlation between both cell types and neutrophils. Additionally, we spatially defined clusters 1 and 2, those with larger proportion of erythroblasts, in the metaphysis trabecular regions of the bone marrow. We followed the same analysis framework to characterize the spatial configuration in the MI cγ1 mice BM with a developed MM. To this end, we incorporated the MM-derived single-cell PCs in the single-cell reference set. Importantly, the pathological PCs were identified and organized in large groups in the BM space. Cell proportion estimations in these samples revealed a distribution in 7 clusters, 4 of them concentrated in the periphery of the PC hot spots and 3 mirroring the PC gradient observed in previous analysis (Figure 1A). We next performed a targeted differential analysis to confirm previous observed markers identities of different MM processes as Cd44 (de Jong et al, Nat Immunol. 2021), a signature of dormant cells (Khoo et al, Blood. 2019), the 38 MM-associated surface-protein-encoding genes (Yao et al, Cancer Res. 2023), Mmp9 and different profiles of T cell exhaustion markers within the 3 clusters with higher PC concentration. These analyses allowed us to verify two different markers, Cd44 and Mmp9 localized in the periphery of the hot spots of PC which may explain an invasive mechanism of the cells localized in the outer areas of the MM combining an inflammatory environment, manifested by a Cd44 increment, with a degradative mechanism driven by Mmp9. Simultaneously, an untargeted differential analysis aimed to identify markers characterizing the clusters with high PC prevalence, revealed novel marker genes never described before in MM and not associated with PC proportion, as the Transmembrane Immune Signaling Adaptor ( Tyrobp) in the cluster 5 (peripheral cluster), which is predictive of a poor prognosis and high tumor immune infiltration in other described tumors (Lu et al BMC Cancer 2021) (Fig 1B). Finally, as a proof of principle of this technology potential in human samples, spatial transcriptomic was applied to FFPE samples from BM biopsies of 7 MM patients with different degrees of PC infiltration, providing a source for validation of the results obtained in the mouse model. In conclusion, our findings demonstrate that the application of spatial transcriptomics represents a useful tool for understanding the spatial architecture and niche specific interactions in human diseases, offering a systemic approach to dissect the role of the spatial interactions in the pathogenesis of MM.
Introduction Follicular lymphomas (FL) and diffuse large B cell lymphomas (DLBCL) share a common germinal center (GC) B cell precursor. Nevertheless, these lymphomas appear to originate from ...different developmental stages, through distinct pathogenetic mechanisms and their clinical behavior varies significantly, even among FL or DLBCL patients. To understand the molecular and transcriptional heterogeneity underlying this clinical diversity we conducted a multi-omic study in GC B cell lymphomas. Methods Our study comprised 3 DLBCL patients (1 non-GCB (germinal center B), 1 non-GCB CD5+, and 1 GCB) and 2 FL (1 with no transformation (ntFL) and 1 patient with transformation (tFL)) at diagnosis. We performed single-cell multiome-sequencing (scDNA-seq + scProtein) ( Tapestri platform -Mission Bio-) and single cell RNA-sequencing (scRNA-seq) ( Chromium system -10xGenomics-) from cell suspensions and spatial transcriptomics ( Visium technology -10xGenomics-) from paraffin blocks. Results Genotyping data were available for 19,700 cells. A total of 311 variants across the 5 samples passed all quality controls. We selected probably pathogenic and pathogenic variants for downstream analysis. Germline variants were discarded. In 3 of 5 patients, we detected 1 somatic and nonsynonymous variant defining the first clone (variants in KMT2D and NOTCH2 mutated in 6-38% of the cells) and another somatic and nonsynonymous variant, defining a subclone (variants in KMT2D, ATM, EZH2 mutated in 1-15% of the cells). In 1 of 5 patients (ntFL), we detected up to 4 mutations, acquired linearly ( B2M, GNA13, EZH2, RHOA mutated clone represents 8% of the cells). Remarkably, all patients showed the same pathogenic variant in TET2 (1-2% of the cells), as an independent clone. The highest molecular similarity was observed between the GCB DLBCL patient and the tFL patient. Both had the same variant in KMT2D as the first hit (later GCB-DLBCL acquired ATM, while tFL acquired EZH2 as a second hit). ATM and EZH2 mutations have been described in “bulk” studies conducted on DLBCL and FL patients, respectively. Regarding protein expression, there was a good correlation between GCB and non-GCB phenotypes studied by immunohistochemistry and scProtein (the expression of CD10 by proteomics was lower in non-GCB patients). TET2 clones described by scDNA-seq were enriched in CD5+ and CD19- cells, suggesting they belong to the T-lymphoid lineage. The other clones and subclones were enriched in CD19+ and CD5- cells ( Figure 1). Data from 54,024 single cells were obtained for scRNA-seq analysis, with an average of 10,805 cells (7,335-18,205) for each sample. Cell type annotation was performed based on the expression of canonical markers. We identified 13 different clusters, the main ones being: GC B cells, post-GC B cells, naïve B cells, myeloid cells, CD8+ cytotoxic T cells, CD4+ naïve T cells, CD4+ reg T cells, CD4+ helper T cells. All samples contained subpopulations of both GC and post-GC B cell clusters. However, GCB DLBCL and FL patients were significantly enriched in GC B cells (63.2 vs 22.2%; p<0.01), whereas non-GCB patients were significantly enriched in post-GC B cells (35.5 vs 2.9%; p<0.01). Part of the normal B-cell transcriptional function was preserved in all samples ( Figure 2). The analysis of the infiltrating T cells revealed that the CD4+/CD8+ T cells ratio progressively increased from ntFL (1.14) to tFL (2.25) and DLBCL (2.58). Furthermore, we estimated the exhaustion status of T cells by the expression of these five markers ( TIGIT, LAG3, CTLA4, HAVCR2, PDCD1). Exhaustion T cell markers were highly expressed in CD8+ cytotoxic T cells and cycling T cells and lower expressed in CD4+ reg T cells. Both DLBCL and tFL have a stronger pattern of exhaustion than ntFL. Finally, by the spatial transcriptomics analysis, we have identified distinct cell type proportions according to their matched single-cell RNA data. Conclusions Our results confirm the intra and intertumor heterogeneity in GC B cell lymphomas. Notably, the greatest molecular and transcriptional similarities are observed between the GCB DLBCL and tFL patients at diagnosis. Moreover, our findings support the critical role of the tumor microenvironment for the persistence and development of the tumor clone. The finding of a TET2 mutated clone in the infiltrating T cells of all the patients is particularly noteworthy, as it may suggest the possible presence of clonal lymphopoiesis.
Excessive inflammation is pathogenic in the pneumonitis associated with severe COVID-19. Neutrophils are among the most abundantly present leukocytes in the inflammatory infiltrates and may form ...neutrophil extracellular traps (NETs) under the local influence of cytokines. NETs constitute a defense mechanism against bacteria, but have also been shown to mediate tissue damage in a number of diseases.
Could NETs and their tissue-damaging properties inherent to neutrophil-associated functions play a role in the respiratory failure seen in patients with severe COVID-19, and how does this relate to the SARS-CoV-2 viral loads, IL-8 (CXCL8) chemokine expression, and cytotoxic T-lymphocyte infiltrates?
Sixteen lung biopsy samples obtained immediately after death were analyzed methodically as exploratory and validation cohorts. NETs were analyzed quantitatively by multiplexed immunofluorescence and were correlated with local levels of IL-8 messenger RNA (mRNA) and the density of CD8+ T-cell infiltration. SARS-CoV-2 presence in tissue was quantified by reverse-transcriptase polymerase chain reaction and immunohistochemistry analysis.
NETs were found in the lung interstitium and surrounding the bronchiolar epithelium with interindividual and spatial heterogeneity. NET density did not correlate with SARS-CoV-2 tissue viral load. NETs were associated with local IL-8 mRNA levels. NETs were also detected in pulmonary thrombi and in only one of eight liver tissues. NET focal presence correlated negatively with CD8+ T-cell infiltration in the lungs.
Abundant neutrophils undergoing NETosis are found in the lungs of patients with fatal COVID-19, but no correlation was found with viral loads. The strong association between NETs and IL-8 points to this chemokine as a potentially causative factor. The function of cytotoxic T-lymphocytes in the immune responses against SARS-CoV-2 may be interfered with by the presence of NETs.
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