FOXP3 Staining Research Articles

Activin levels correlate with lymphocytic infiltration in epithelial ovarian cancer.

The TGF-β superfamily member activin, a dimer of the gene products of INHBA and/or INHBB, has been implicated in immune cell maturation and recruitment, but its immune impact within epithelial ovarian cancer (EOC) is not well characterized. We sought to explore differences in activin (INHBA/ Inhibin-βA and INHBB/ Inhibin-βB) between malignant and ovarian tissues at the RNA and protein level and assess the relationship between activin and immune cells in EOC. Publicly available RNA sequencing data were accessed from GEO (#GSE143897) with normalization and quantification performed via DESeq2. Immune gene expression profile was further explored within the TCGA-OV cohort derived from The Cancer Genome Atlas (TCGA). Immunohistochemical analysis was performed to evaluate activin A and T-cell markers CD8 and FoxP3 at the protein level. ELISA to activin-A was used to assess levels in the ascites of advanced EOC patients. Kaplan-Meier curves were generated to visualize survival outcomes. Gene expression levels of components of the activin signaling pathway were elevated within EOC when compared to a benign cohort, with differences in activin type I/II receptor gene profiles identified. Additionally, INHBA gene expression was linked to lymphocytic immune markers in EOC samples. Immunohistochemistry analysis revealed a positive correlation of CD8 and FOXP3 staining with activin A at the protein level in both primary and metastatic epithelial ovarian cancer samples. Furthermore, Activin-A (inhibin-βA) is significantly elevated in EOC patient ascites. INHBA expression is elevated within EOC, correlating with worse survival, with activin protein levels correlating with specific immune infiltration. Our findings suggest that activin-A may play a role in suppressing anti-tumor immunity in EOC, highlighting its potential as a therapeutic target.

Prognostic Value of Insulin Growth Factor-Like Receptor 1 (IGFLR1) in Stage II and III Colorectal Cancer and Its Association with Immune Cell Infiltration.

IGFLR1 is a novel biomarker, and some evidences suggested that is involved in the immune microenvironment of CRC. Here, we explored the expression of IGFLR1 and its association with the prognosis as well as immune cell infiltration in CRC, with the aim to provide a basis for further studies on IGFLR1. Immunohistochemical staining for IGFLR1, TIM-3, FOXP3, CD4, CD8, and PD-1 was performed in eligible tissues to analyze the expression of IGFLR1 and its association with prognosis and immune cell infiltration. Then, we screened colon cancer samples from TCGA and grouped patients according to IGFLR1-related genes. We also evaluated the co-expression and immune-related pathways of IGFLR1 to identify the potential mechanism of it in CRC. When P < 0.05, the results were considered statistically significant. IGFLR1 and IGFLR1-related genes were associated with the prognosis and immune cell infiltration (P < 0.05). In stage II and III CRC tissue and normal tissue, we found (1) IGFLR1 was expressed in both the cell membrane and cytoplasm and which was differentially expressed between cancer tissue and normal tissue. IGFLR1 expression was associated with the expression of FOXP3, CD8, and gender but was not associated with microsatellite instability. (2) IGFLR1 was an independent prognostic factor and patients with high IGFLR1 had a better prognosis. (3) A model including IGFLR1, FOXP3, PD-1, and CD4 showed good prognostic stratification ability. (4) There was a significant interaction between IGFLR1 and GATA3, and IGFLR1 had a significant co-expression with related factors in the INFR pathway. IGFLR1 has emerged as a new molecule related to disease prognosis and immune cell infiltration in CRC patients and showed a good ability to predict the prognosis of patients.

Combined oral low-dose cyclophosphamide endocrine therapy may improve clinical response among patients with metastatic breast cancer via Tregs in TLSs

Despite limited research on refractory and/or endocrine therapy failure in elderly metastatic breast cancer (MBC) patients, a prior study showed that low-dose oral cyclophosphamide (CY) can improve the overall survival rate of MBC patients, possibly through the immunoregulation of regulatory T cells (Tregs). We preliminarily investigated the combination of endocrine therapy (ET) with oral low-dose CY as salvage therapy in elderly patients via peripheral blood regulatory T-cell analyses. In addition, we evaluated the associations of tumor tertiary lymphoid structures (TLSs) with therapeutic outcomes. HR+/HER2− advanced breast cancer patients who received low-dose CY combined with ET or ET only from April 2015 to August 2021 were enrolled in this retrospective study. The primary outcome was the clinical control rate (CCR), and the secondary outcome was progression-free survival (PFS). Circulating T lymphocyte subpopulations represented by Tregs were monitored during treatment by flow cytometry methods. TLSs wereconfirmed by hematoxylin–eosin staining of pretreatment specimens, and CD3, CD4, and Foxp3 were detected using Opal multicolor immunofluorescence. A total of 85 patients who received CY + ET and 50 patients who received ET only were enrolled, the percentage of patients who received CCR was 73% (62/85) vs. 70% (45/50), and the objective response rate (ORR) was 28% (24/85) vs. 24% (12/50). No deaths occurred during the study period. The mean PFS time was 13 vs. 11 months (P = 0.03). In the CY + ET group, decreases in CD4+/CD25+/Foxp3+ T cells (P < 0.001) were favorable for both clinical control and prolonged PFS (P < 0.001). Compared with patients without TLSs, those with TLSs were more likely to have better clinical control and PFS (mean time = 6 months), and a greater number of Treg cells during TLS pretreatment correlated with longer PFS (P = 0.043). Oral low-dose CY combined with standard ET exerts immunological effects by decreasing Treg levels to achieve improved clinical responses. Moreover, patients with TLSs might benefit more from such therapy than those without TLSs, and a high Treg cell count in TLSs before treatment predicts better therapeutic efficacy.

1241 Sustained Localized Delivery of Anti-PD-1 to Tumor Draining Lymph Nodes Increases Survival and Reverses Immunosuppression in Murine Glioblastoma Models

INTRODUCTION: Despite the advent of immunotherapy as a promising therapeutic, glioblastoma (GBM) remains resistant to using checkpoint blockade against programmed cell death protein 1 (PD-1) on T cells. The highly immunosuppressive tumor milieu of GBM prevents rescue of inactivated T cells due to decreased numbers of infiltrating T cells at the tumor site as well as increased recruitment of myeloid cells. Moreover, the current strategy of using antibodies against PD-1 (anti-PD-1) requires multiple intravenous infusions every two or three weeks with debilitating systemic effects. METHODS: Mice orthotopically implanted with GL261 glioma cells were injected with PCL:PEG:PCL hydrogel polymers loaded with anti-PD-1 in one of the following locations: cervical lymph nodes, inguinal lymph nodes, and the tumor site. Mice treated systemically with anti-PD-1 were used as comparative controls. Kaplan-Meier curves were generated for all arms, with subsequent ex vivo flow cytometric staining for CD4, CD8, IFN-y, TNF-a, Foxp3, and PD-1. RESULTS: Mice implanted with PCL:PEG:PCL hydrogels carrying anti-PD-1 at the site of their lymph nodes showed significantly improved survival outcomes compared to mice systemically treated with three doses of anti-PD-1 (p &lt; 0.001). Flow cytometric analysis of lymph nodes and brain tissue in mice injected with this gel demonstrated increased levels of IFN-y, indicating greater reversal of immunosuppression compared to standard treatment. CONCLUSIONS: Our data demonstrates proof of principle of the advantages of using localized therapy that targets lymph nodes for GBM. We propose a paradigm shift for developing new sustained local treatments with immunotherapy that are able to eliminate the need for multiple systemic infusions and their off-target effects.

Abstract 7667: Neutrophil to lymphocyte ratio (NLR) as an indicator of tumor immune status in non-small cell lung cancer

Abstract Background: Neutrophil to lymphocyte ratio (NLR) has been reported as a prognostic biomarker in non-small cell lung cancer (NSCLC), while its biological rationale has yet to be clarified. In cancer patients, inflammation and immune status plays crucial roles in tumor growth and the prognosis. While several studies have explored the relationship between NLR and inflammation, there are few reports on the relationship between NLR and immune status in the tumor microenvironment of cancer patient so far. The purpose of this study is to explore the potential utility of NLR as a surrogate biomarker for assessing the immune response to tumors. Methods: The medical records of NSCLC patients who underwent surgery at our institution in 2012 were retrospectively reviewed. The patients’ clinical information including age, sex, performance status, smoking history, histology, clinical stage (7th), and NLR, was obtained. NLR was calculated based on blood tests conducted within a month before surgery. Tumor immune status was evaluated by Immunohistochemical staining for CD3, CD8, and FOXP3 in their surgical specimens. The relationship between NLR, clinicopathology, and tumor immune status was examined. The survival curves were estimated using the Kaplan-Meier method and compared using the log-rank test. Blood cell counts and ratios between different groups were compared using nonpaired t test or Mann-Whitney U test, with a significance threshold set at p = 0.05. Results: A total of 120 patients were included. The 5-year overall survival rate (OS) in all 120 patients was 63.4%. The 5-year OS of the patients with lower NLR was significantly better than those with high NLR (low &amp;lt; 2.2 vs high ≥ 2.2; 70.1% vs 56.8%, p = 0.042). The 5-year OS of the patients with high density of CD3+ TILs was significantly better than that in patients with low density of CD3+ TILs (high ≥ 242 vs low &amp;lt; 242; 70% vs 56.8%, p = 0.019). The 5-year OS of the patients with high density of CD8+ TILs was significantly better than that in patients with low density of CD8+ TILs (high ≥ 112 vs low &amp;lt; 112; 72.4% vs 54.1%, p = 0.011). Conversely, the 5-year OS of the patients with low density of FOXP3+ TILs was significantly better than that in patients with high density of FOXP3+ TILs (high ≥ 7.5 vs low &amp;lt; 7.5; 46.2.% vs 79.6%, p = 0.0006). The number of CD3+ TILs had negative correlation with NLR (p = 0.005), while the number of CD8+ TILs and FOXP3+ TILs didn’t exhibit an association with NLR. Conclusion: Our results suggest that NLR might potentially reflect immune status in the tumor microenvironment, explaining its impact on the prognosis of NSCLC patients. While further research is needed, these findings may offer valuable insights for guiding treatment options. Citation Format: Ryo Yoshichika, Kazuma Iwata, Ken Suzawa, Yin Min Thu, Daisuke Mizuno, Tomoaki Higashihara, Naohiro Hayashi, Atsushi Matsuoka, Mao Yoshikawa, Fumiaki Mukohara, Kazuhiko Shien, Hiromasa Yamamoto, Shinichi Toyooka. Neutrophil to lymphocyte ratio (NLR) as an indicator of tumor immune status in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7667.

Characterizing Foxp3+ and Foxp3- T cells in the homeostatic state and after allo-activation: resting CD4+Foxp3+ Tregs have molecular characteristics of activated T cells.

Due to the intracellular expression of Foxp3 it is impossible to purify viable Foxp3+ cells on the basis of Foxp3 staining. Consequently CD4+Foxp3+ regulatory T cells (Tregs) in mice have mostly been characterized using CD4+CD25+ T cells or GFP-Foxp3 reporter T cells. However, these two populations cannot faithfully represent Tregs as the expression of CD25 and Foxp3 does not completely overlap and GFP+Foxp3+ reporter T cells have been reported to be functionally altered. The aim of this study was to characterize normal Tregs without separating Foxp3+ and Foxp3- cells for the expression of the main functional molecules and proliferation behaviors by flow cytometry and to examine their gene expression characteristics through differential gene expression. Our data showed that the expressions of Foxp3, CD25, CTLA-4 (both intracellular and cell surface) and PD-1 was mostly confined to CD4+ T cells and the expression of Foxp3 did not completely overlap with the expression of CD25, CTLA-4 or PD-1. Despite higher levels of expression of the T cell inhibitory molecules CTLA-4 and PD-1, Tregs maintained higher levels of Ki-67 expression in the homeostatic state and had greater proliferation in vivo after allo-activation than Tconv. Differential gene expression analysis revealed that resting Tregs exhibited immune activation markers characteristic of activated Tconv. This is consistent with the flow data that the T cell activation markers CD25, CTLA-4, PD-1, and Ki-67 were much more strongly expressed by Tregs than Tconv in the homeostatic state.

Multiplexed high-throughput immune cell imaging in patients with high-risk triple negative early breast cancer: Analysis from the International Breast Cancer Study Group (IBCSG) Trial 22-00

BackgroundTriple-negative breast cancer (TNBC) is the most aggressive breast cancer (BC) subtype, with dismal prognosis and limited option in advanced settings, yet stromal tumor infiltrating lymphocytes (sTILs) in this subtype has a predictive role. Patients and methods: The International Breast Cancer Study Group (IBCSG) Trial 22–00 is a randomized phase III clinical trial testing the efficacy of low-dose metronomic oral Cyclophosphamide-Methotrexate (CM) maintenance following standard adjuvant chemotherapy treatment for early-stage hormone receptor-negative breast cancer patients. A case-cohort sampling was used. We characterized immune cells infiltrates in patients with TNBC by 6 plex immunofluorescence (IF) staining for CD4, FOXP3, CD3, cytokeratine and CD8 ResultsWe confirmed that high immune CD3+ T cells as well as stromal and intra-epithelial Tregs (CD4+Foxp3+ T cells) infiltrates were associated with a better Distant Recurrence-Free Interval (DRFI), especially in LN+ patient, regardless of the treatment. More importantly, we showed that the spatial distribution of immune cells at baseline is crucial, as CM maintenance was detrimental for T cells excluded LN+ TNBC patients. Conclusionsimmune spatial classification on immune cells infiltrates seems crucial and could help patients’ selection in clinical trial and greatly improve responses to specific therapies.

Clinical Usefulness of Immune Profiling for Differential Diagnosis between Crohn's Disease, Intestinal Tuberculosis, and Behcet's Disease.

It is important to make a differential diagnosis between inflammatory diseases of the bowel with similar clinical and endoscopic features. The profiling of immune cells could be helpful for accurately diagnosing inflammatory bowel diseases. We compared immune marker expression between Crohn's disease (CD), intestinal Behcet's disease (BD), and intestinal tuberculosis (TB) and evaluated the usefulness of immune profiling in differentiating between these diseases. Biopsy specimens were acquired around ulcerations on the terminal ileum or cecum from five patients with each disease. Panel 1 included multiplex immunohistochemistry staining for CD8, CD4, Foxp3, CD20, programmed death-1, and granzyme B. CD56, CD68, CD163, CD11c, and HLA-DR were analyzed in panel 2. The differences in cytotoxic T cells (CD8+CD4-Fopx3-CD20-), helper T cells (CD8-CD4+Fopx3-CD20-), and regulatory T cells (CD8-CD4+Fopx3+CD20-) were also not significant. However, M1 macrophage (CD68+CD163-HLA-DR-) cell densities were significantly higher in intestinal BD than in other diseases. The expression level of dendritic cells (CD56-CD68-CD163-CD11c+HLA-DR+) was highest in intestinal TB and lowest in intestinal BD. The expression of immune cells, including M1 macrophages and dendritic cells, was different between CD, intestinal BD, and intestinal TB. Immune profiling can be helpful for establishing differential diagnoses of inflammatory bowel diseases.

Autologous olfactory mucosa mesenchymal stem cells treatment improves the neural network in chronic refractory epilepsy

Background and aimsRefractory epilepsy is also known as drug-resistant epilepsy with limited clinical treatment. Benefitting from its safety and easy availability, olfactory mucosa mesenchymal stem cells (OM-MSCs) are considered a preferable MSC source for clinical application. This study aims to investigate whether OM-MSCs are a promising alternative source for treating refractory epilepsy clinically and uncover the mechanism by OM-MSCs administration on an epileptic mouse model.MethodsOM-MSCs were isolated from turbinal and characterized by flow cytometry. Autologous human OM-MSCs treatment on a patient was carried out using intrathecal administration. Epileptic mouse model was established by 1 mg/kg scopolamine and 300 mg/kg pilocarpine treatment (intraperitoneal). Stereotaxic microinjection was employed to deliver the mouse OM-MSCs. Mouse electroencephalograph recording was used to investigate the seizures. Brain structure was evaluated by magnetic resonance imaging (MRI). Immunohistochemical and immunofluorescent staining of GFAP, IBA1, MAP2, TUBB3, OLIG2, CD4, CD25, and FOXP3 was carried out to investigate the neural cells and Treg cells. QRT-PCR and ELISA were performed to determine the cytokines (Il1b, Il6, Tnf, Il10) on mRNA and protein level. Y-maze, the object location test, and novel object recognition test were performed to measure the cognitive function. Footprint test, rotarod test, balance beam test, and grip strength test were conducted to evaluate the locomotive function. Von Frey testing was carried out to assess the mechanical allodynia.ResultsMany beneficial effects of the OM-MSC treatment on disease status, including seizure type, frequency, severity, duration, and cognitive function, and no apparent adverse effects were observed at the 8-year follow-up case. Brain MRI indicated that autologous OM-MSC treatment alleviated brain atrophy in epilepsy patients. A study in an epileptic mouse model revealed that OM-MSC treatment recruited Treg cells to the brain, inhibited inflammation, rebuilt the neural network, and improved the cognitive, locomotive, and perceptive functions of epileptic mice.ConclusionsAutologous OM-MSC treatment is efficacious for improving chronic refractory epilepsy, suggesting a future therapeutic candidate for epilepsy.Trial registration: The study was registered with Chinese Clinical Trial Registry (ChiCTR2200055357).

Nominating molecular/immunologic drivers of central nervous system (CNS) metastases in squamous cell carcinoma of the head/neck (SCCHN).

e18002 Background: In the ~1% of patients with SCCHN who experience CNS metastases, median overall survival (mOS) is &lt;1 year. We investigated whether molecular/immunologic features distinguish SCCHN cases that metastasize to the CNS. Methods: We performed a case-control study on a convenience sample of individuals with SCCHN treated at two academic institutions. Next generation targeted sequencing and/or immunoprofiling of primary and metastatic tumor sites were performed. Results: We identified 39 patients with SCCHN (21 with CNS metastases (cases), 18 without (controls)). Median age at diagnosis 60, 79% male, 46% current/former smokers, 51% oropharyngeal primary, 51% HPV+/31% HPV-/18% not tested (NT) across all cases, and 69% stage III or IV disease at diagnosis. HPV+ frequency trended higher among cases (52%+, 14%-, 33% NT) vs. controls (50%+, 50%-, 0% NT) (p=0.08). mOS from diagnosis was 34.4 months (95% CI 14.5-41.0) in cases and 44.4 months (95% CI 19.0-82.3) in controls. Genomic data was available for 18 (86%) cases and 12 (67%) controls. Most common mutations in cases were KMT2D (39%) and NOTCH1 (39%). There were no genes for which alteration frequency differed significantly between groups. Tumors of 18 (86%) cases and 18 (100%) controls underwent immunoprofiling. Mean PD-1 density at the tumor-stroma interface (TSI), CPS, and TPS were higher in controls (Table). In cases, TSI PD-1 density tended to be higher at primary sites than at sites of CNS metastases. No significant difference was seen in CD8 or FOXP3 staining. Conclusions: SCCHN with CNS metastases shows markers of lower immunogenicity at both the primary site and site of CNS metastasis vs. other SCCHN tumors, regardless of HPV status. This suggests that localized tumors with low immune infiltration and poor responsiveness to immunotherapy may be at higher risk of CNS metastasis and could imply reduced benefit from immunotherapy for patients with CNS metastases. [Table: see text]

OSCC in Never-Smokers and Never-Drinkers Is Associated with Increased Expression of Tumor-Infiltrating Lymphocytes and Better Survival

The aim of this study was to investigate the clinical, histopathologic, and immunologic differences of oral squamous cell carcinoma of never-smokers/never-drinkers and smokers/drinkers. Immunohistochemical staining for CD4, CD8, FoxP3, CD1a, and p16 was performed in 131 oral squamous cell carcinomas from smokers/drinkers and never-smokers/never-drinkers. Associations of smoking/drinking status with clinicopathologic data, immunohistochemical antibody expression, and survival were examined. Oral squamous cell carcinoma in never-smokers/never-drinkers was associated with the female gender (p &lt; 0.001). Never-smokers/never-drinkers were older at diagnosis than smokers/drinkers (p &lt; 0.001). Never-smokers/never-drinkers had more tumors in the maxilla, mandible, and tongue (p &lt; 0.001). Pre-existing oral potentially malignant disorders appeared to be more common in never-smokers/never-drinkers (p &lt; 0.001). Perineural invasion was more common in smokers/drinkers (p = 0.039). Never-smoking/never-drinking was associated with better overall survival (p = 0.004) and disease-specific survival (p = 0.029). High CD4+ T cell infiltration was associated with never-smoking/never-drinking (p = 0.008). Never-smokers/never-drinkers also showed increased CD8+ T cell infiltration (p = 0.001) and increased FoxP3+ Treg infiltration (p = 0.023). Furthermore, the total group of tumor-infiltrating lymphocytes was associated with never smoking/never drinking (p = 0.005). To conclude oral squamous cell carcinoma of the never-smokers/never-drinkers appears to be a distinct type of tumor, as it appears to have unique clinical and pathologic features and a more immunogenic microenvironment.

Mocravimod, a S1P receptor modulator, increases T cell counts in bone marrow biopsies from patients undergoing allogeneic hematopoietic stem cell transplantation

Graft-versus-leukemia (GvL) effects are critical to prevent relapses after allogeneic hematopoietic cell transplantation (allo-HCT). However, the success of allo-HCT is limited by graft-versus-host disease (GvHD). Both, CD4+ and CD8+ T cells contribute to GvHD and GvL. The sphingosine-1-phosphate receptor (S1PR) signaling plays a crucial role in lymphocyte trafficking. Mocravimod is an S1PR modulator and its administration leads to blocking lymphocyte egress from lymphoid organs. We hypothesized that this applies to the bone marrow (BM) too, and analyzed BM biopsies from the clinical study with mocravimod (phase I trial in allo-HCT patients; NCT01830010) by immunohistochemical staining for CD3, CD4, CD8, TIA1, FoxP3, PD1, T-Bet, GATA3, and ROR-γt to identify and quantify T cell subsets in situ. Allo-HCT patients without receiving mocravimod were used as controls. BM from 9 patients in the mocravimod group and 10 patients in the control group were examined. CD3+ T cells were found to accumulate in the BM of mocravimod-treated patients compared to controls, both on day 30 and 90 post-transplant. The effect was stronger for CD4+ T cells, than CD8+ T cells, which is in line with data from murine studies showing that CD4+ T cells are more sensitive to mocravimod treatment than CD8+ T cells. Clinically-relevant acute GvHD events (grade II-IV) were slightly lower, but comparable to controls when mocravimod was administered. Taken together, data are supportive of mocravimod’s mode of action and bring additional evidence of fewer relapses for allo-HCT patients treated with S1PR modulators.

Leukocytic Infiltration of Intraductal Carcinoma of the Prostate: An Exploratory Study.

Intraductal carcinoma of the prostate (IDC-P) is an aggressive histological subtype of prostate cancer (PCa) detected in approximately 20% of radical prostatectomy (RP) specimens. As IDC-P has been associated with PCa-related death and poor responses to standard treatment, the purpose of this study was to explore the immune infiltrate of IDC-P. Hematoxylin- and eosin-stained slides from 96 patients with locally advanced PCa who underwent RP were reviewed to identify IDC-P. Immunohistochemical staining of CD3, CD8, CD45RO, FoxP3, CD68, CD163, CD209 and CD83 was performed. For each slide, the number of positive cells per mm2 in the benign tissues, tumor margins, cancer and IDC-P was calculated. Consequently, IDC-P was found in a total of 33 patients (34%). Overall, the immune infiltrate was similar in the IDC-P-positive and the IDC-P-negative patients. However, FoxP3+ regulatory T cells (p < 0.001), CD68+ and CD163+ macrophages (p < 0.001 for both) and CD209+ and CD83+ dendritic cells (p = 0.002 and p = 0.013, respectively) were less abundant in the IDC-P tissues compared to the adjacent PCa. Moreover, the patients were classified as having immunologically "cold" or "hot" IDC-P, according to the immune-cell densities averaged in the total IDC-P or in the immune hotspots. The CD68/CD163/CD209-immune hotspots predicted metastatic dissemination (p = 0.014) and PCa-related death (p = 0.009) in a Kaplan-Meier survival analysis. Further studies on larger cohorts are necessary to evaluate the clinical utility of assessing the immune infiltrate of IDC-P with regards to patient prognosis and the use of immunotherapy for lethal PCa.

SPP1 exacerbates ARDS via elevating Th17/Treg and M1/M2 ratios through suppression of ubiquitination-dependent HIF-1α degradation.

Acute respiratory distress syndrome (ARDS) is a severe inflammatory pulmonary condition that leads to respiratory failure. The imbalance of Th17/Treg and M1/M2 is implicated in ARDS. A better understanding of the regulation of the balance of Th17/Treg and M1/M2 may provide novel therapeutic targets for ARDS. Plasma and BALF samples were collected from ARDS patients. Inflammatory cytokines were examined by ELISA. Th17, Treg, M1 and M2 were identified via immunofluorescence staining of RORγt, Foxp3, iNOS and Arg-1. H&E and Masson's trichrome staining were applied for evaluating pulmonary damage and fibrosis. A mouse model of ARDS was established through LPS administration. HIF-1α was immunoprecipitated and subjected to ubiquitination analysis via western blotting. The expression of SPP1, VHL and HIF-1α was examined by RT-qPCR and western blotting. ARDS patients showed elevated levels of inflammatory cytokines and ratios of Th17/Treg and M1/M2. SPP1 was upregulated in ARDS mice, and silencing of SPP1 alleviated lung injury and fibrosis. SPP1 inhibited VHL expression to reduce the ubiquitination and degradation of HIF-1α in ARDS. Overexpression of SPP1 facilitated Th17, Treg and M1 polarization but inhibited M2 polarization through upregulation of HIF-1α. SPP1 elevates Th17/Treg and M1/M2 ratio by suppressing VHL expression and ubiquitination-dependent HIF-1α degradation, thus exacerbating ARDS. Our study provides novel mechanistic insights into ARDS pathogenesis and promising therapeutic targets.

Spontaneous Murine Model of Anaplastic Thyroid Cancer.

Anaplastic thyroid cancer (ATC) is a rare but lethal malignancy with a dismal prognosis. There is an urgent need for more in-depth research on the carcinogenesis and development of ATC, as well as therapeutic methods, since standard treatments are essentially depleted in ATC patients. However, low prevalence has hampered thorough clinical studies and the collection of tissue samples, so little progress has been achieved in creating effective treatments. We used genetic engineering to create a conditionally inducible ATC murine model (mATC) in a C57BL/6 background. The ATC murine model was genotyped by TPO-cre/ERT2; BrafCA/wt; Trp53ex2-10/ex2-10 and induced by intraperitoneal injection with tamoxifen. With the murine model, we investigated the tumor dynamics (tumor size ranged from 12.4 mm2 to 32.5 mm2 after 4 months of induction), survival (the median survival period was 130 days), and metastasis (lung metastases occurred in 91.6% of mice) curves and pathological features (characterized by Cd8, Foxp3, F4/80, Cd206, Ki67, and Caspase-3 immunohistochemical staining). The results indicated that spontaneous mATC possesses highly similar tumor dynamics and immunological microenvironment to human ATC tumors. In conclusion, with high similarity in pathophysiological features and unified genotypes, the mATC model resolved the shortage of clinical ATC tissue and sample heterogeneity to some extent. Therefore, it would facilitate the mechanism and translational studies of ATC and provide an approach to investigate the treatment potential of small molecular drugs and immunotherapy agents for ATC.

Myofibroblast stroma differentiation in infiltrative basal cell carcinoma is accompanied by regulatory T-cells.

The implications of infiltrative compared to non-infiltrative growth of cutaneous basal cell carcinoma (BCC) on the tumor stroma and immune cell landscape are unknown. This is of clinical importance, because infiltrative BCCs, in contrast to other BCC subtypes, are more likely to relapse after surgery and radiotherapy. This descriptive cross-sectional study analyzed 38 BCCs collected from 2018 to 2021. In the first cohort (n=28), immune cells were characterized by immunohistochemistry and multiplex immunofluorescence staining for CD3, CD8, CD68, Foxp3, and α-SMA protein expression. In the second cohort (n=10) with matched characteristics (age, sex, location, and BCC subtype), inflammatory parameters, including TGF-β1, TGF-β2, ACTA2, IL-10, IL-12A, and Foxp3, were quantified via RT-qPCR after isolating mRNA from BCC tissue samples and perilesional skin. Infiltrative BCCs showed significantly increased levels of α-SMA expression in fibroblasts (p=0.0001) and higher levels of Foxp3+ (p=0.0023) and CD3+ (p=0.0443) T-cells compared to non-infiltrative BCCs. CD3+ (p=0.0171) and regulatory T-cells (p=0.0026) were significantly increased in α-SMA-positive tumor stroma, whereas CD8+ T-cells (p=0.1329) and CD68+ myeloid cells (p=0.2337) were not affected. TGF-β1 and TGF-β2 correlated significantly with ACTA2/α-SMA mRNA expression (p=0.020, p=0.005). Infiltrative growth of BCCs shows a myofibroblastic stroma differentiation and is accompanied by an immunosuppressive tumor microenvironment.

Tumor P70S6K hyperactivation is inversely associated with tumor-infiltrating lymphocytes in triple-negative breast cancer

PurposeTriple-negative breast cancer (TNBC) is characterized by large heterogeneity and relative lack of available targeted therapies. To find therapeutic strategies for distinct patients with TNBC, several approaches have been used for TNBC clustering, including recently immune and phosphoproteomic patterns. Based on 70-kDa ribosomal protein S6 kinase (P70S6K)-TNBC clustering, the current study explores the immune profiling in TNBC tumors.MethodsStromal tumor-infiltrating lymphocytes (sTILs) were evaluated in human TNBC tumor samples. Furthermore, immunohistochemistry staining for CD8, CD4, Foxp3, and CD20 was performed in tissue microarrays (TMA) sections.ResultsHistological analysis showed decreased sTILs, CD20+ cells, and CD8+/CD4+ ratio in high phosphorylated P70S6K (p-P70S6K) tumors. Moreover, p-P70S6K score was directly correlated with CD4+ and Foxp3+ T cells, while it was inversely correlated with CD8+/CD4+ and CD8+/Foxp3+ ratios.ConclusionsTIL infiltration and lymphocyte profiling vary in the context of hyperactivation of P70S6K in TNBC tumors.

Programmed death-ligand 1 expression in the immune compartment of colon carcinoma

AbstractProgrammed cell death ligand 1 (PD-L1) on tumor cells is a significant prognostic biomarker for a number of malignancies, although less is known about the significance of PD-L1 positive immune cells in colon carcinoma. The purpose of this study is to evaluate the role of PD-L1 in a large cohort of colon carcinomas to identify patterns of PD-L1 expression in the tumor microenvironment and its correlation with other key immune subsets to better understand the impact of these immune cells. We assessed 1218 colon carcinomas on representative tissue microarray sections, gathered relevant clinicopathologic information, and performed immunohistochemical staining for mismatch repair proteins, CD8, CD163, LAG3, PD-L1, FoxP3, and BRAF V600E. We then performed automated quantification; manual quantification was used for PD-L1 tumor cells and immune cells. Dual PD-L1/PU.1 immunostain was also performed. The majority of PD-L1 positive cells expressed PU.1 thus representing tumor-associated macrophages. Based on the median number of PD-L1 positive immune cells (7.6/mm2), we classified tumors into two classes: (1) PD-L1 immune cell low and (2) PD-L1 immune cell high. PD-L1 immune cell high colon carcinomas showed favorable prognostic pathologic features including less frequent extramural venous invasion (p = 0.0001) and lower AJCC stage (p = 0.0001); they were also more commonly associated with deficient mismatch repair (dMMR) (p = 0.0001) and BRAF V600E reactivity. PD-LI immune cell high tumors were associated with high CD8, CD163, and FoxP3 positive cells (p = 0.0001, respectively). PD-L1 immune cell high and LAG3 high colon carcinomas were associated with improved disease-specific survival (p = 0.0001 and 0.001, respectively). PD-L1 expression on tumor cells was not associated with disease-specific survival. On multivariate analysis of chemotherapy naïve stage 2 colon carcinomas, only extramural venous invasion (p = 0.002), perineural invasion (p = 0.001) and PD-L1 immune cell expression (p = 0.032) correlated with disease-specific survival. Resected colonic carcinomas with high expression of PD-L1 and LAG3 proteins on immune cells were associated with improved prognosis in colon carcinoma. The mechanism underlying the improved prognosis of colon carcinomas bearing high numbers of immunoregulatory cells needs further investigation.

Study of clinical factors, focus score, lymphocyte type and NF-κB pathway in Sjögren's syndrome.

Sjögren's syndrome (SS) is an autoimmune disease that occurs predominantly in middle-aged and older women. Although focus score (FS) and lesion grade are determined at pathological diagnosis, few reports have examined whether these results reflect clinical symptoms. In this study, we examined and compared the results of comprehensive immunohistochemical staining of lymphocytes and NF-κB pathway in labial gland biopsies, clinical test data, and radionuclide imaging findings. One hundred labial gland biopsy specimens obtained from 20 female patients with primary SS (5 specimens per patient) were studied. Hematoxylin-eosin-stained specimens were reviewed and FS were calculated. Immunohistochemical staining of CD4, CD8, CD20, CD25, Foxp3, NF-κB, TNFAIP3 and IκBα was performed, and the results were compared with anti-SS-A/Ro (SS-A), anti-SS-B/La (SS-B) and antinuclear antibodies (ANA), and salivary gland scintigraphy findings. FS were significantly higher in the SS-A-, SS-B- and ANA-positive groups than in the respective -negative groups (p < 0.05). Of eight SS-A-positive and SS-B-negative cases, mean FS was 1.9 (seven cases: FS ≥ 1.0) and six cases were ANA-positive. In four SS-A-positive and SS-B-positive cases, mean FS was 3.2 (all cases: FS ≥ 1.0) and all cases were ANA-positive. In immunohistochemical staining, CD4-positive T cells tended to be more abundant than CD8-positive T cells. Small numbers of Foxp3-positive cells were found in all cases. NF-κB, TNFAIP3 and IκBα were positive in the acini, ductal epithelium, and lymphocytes in all cases. The above findings indicated a relationship between FS and clinical test data, and the association of NF-κB pathway with the pathophysiology of primary SS.

Multiomics profiling and association with molecular and immune features in association with benefits from immunotherapy for patients with previously treated stage IV or recurrent squamous cell lung cancer from the phase III SWOG LungMAP S1400I trial.

9046 Background: Immune checkpoint blockade (ICB) has become a standard pillar of treatment for lung cancer. However, only ̃20% of unselected patients can achieve durable clinical benefits. We performed immunogenomic profiling of tissue specimens from a randomized Phase III trial S1400I on metastatic lung squamous cell carcinoma (SCC) to evaluate if there were factors associated with better prognoses with ICB from single-agent versus combined targeting PD-1/CTLA-4 and evaluate if any differentiated between the treatments. Methods: We utilized FFPE tumor tissue submitted for Lung-MAP screening provided by the SWOG bank. SCC samples from 82 eligible patients treated with combined nivolumab+ipilimumab (N+I) or single agent nivolumab (N) were subjected to multiplex immunofluorescence (mIF, n = 82) and NanoString (ncounter PanCancer Immune Profiling Panel, n = 32). Cell density phenotypes (cells/mm2) were defined using image analysis of staining for cytokeratin, CD3, CD8, granzyme B, CD45RO, FOXP3, PD1, PD-L1, and CD68. Immunogenomic features were associated with response, PFS, and OS derived from data provided by the LungMap team to the CIDC portal. For statistical analyses, non-parametric tests were utilized to assess associations of cell phenotypes versus continuous or categorical variables, and log-rank test analysis was performed to identify cell phenotypes or genes correlated with survival. Results: In both arms higher densities of total CD3+CD45RO+ T cells ( P= 0.041), CD3+PD-1+ T cells ( P= 0.024) and CD3+CD8+PD-1 T cells in stroma ( P= 0.042) and CD3+CD8+GZMB+ T cells in the tumor compartment ( P= 0.011) were positively associated with PFS. In the N+I arm but not in the N arm, higher densities of CD3+CD8+GZMB+ T cells in the tumor compartment were associated with better PFS ( P= 0.015) and higher densities of stroma CD3+CD8-FOXP3+ T cells with worse OS. Spatial analysis showed that the presence of CD8+GZMB+ T cells close to malignant cells (median, ≤19.27 µm) was associated with better PFS ( P= 0.037) in N+I arm and cluster analysis showed low clustering of cells in TMB-high vs. TMB-low tumors (P &lt; 0.01). Gene expression profiling demonstrated that myeloid infiltration, immune recruitment, and inflammation genes were associated with a positive clinical outcome ( P&lt; 0.05). In both arms, BLNK, CD163, FCGR2A were associated with better OS ( P&lt; 0.01), IRF1 and BLNK were associated with increased PFS ( P&lt; 0.01). In the N+I arm but not in the N arm, we observed significantly higher CD45 immune cell scores, including CD8 T cells and neutrophils, in responders versus non-responders. Conclusions: Our findings suggest a potential advantage in PFS and OS with an increased presence of cytotoxic immune cells and genes associated with the recruitment and proliferation of these cell types before therapy.