Hematopoietic stem cell (HSC) self-renewal and lineage commitment are tightly controlled by post-translational mechanisms, but the contribution of deubiquitination to these processes remains unclear. Here, we define ubiquitin-specific protease 7 (USP7) as a critical regulator of HSC maintenance and hematopoietic homeostasis. Conditional Usp7 deletion in murine HSCs triggered rapid stem cell depletion, multilineage cytopenias, and systemic hematopoietic failure. Usp7-deficient HSCs displayed defective quiescence, reduced competitive repopulation capacity, and aberrant lineage differentiation. Mechanistically, USP7 directly binds and deubiquitinates the transcription factor PU.1, shielding it from proteasomal degradation. Loss of USP7 destabilized PU.1, leading to suppressed expression of PU.1 target genes critical for HSC quiescence and lineage specification. In competitive transplants, USP7-null HSCs exhibited severely impaired self-renewal, marked by diminished engraftment and differentiation. Ectopic PU.1 expression partially restored HSC function, confirming the USP7-PU.1 axis as essential for HSC integrity. Our study identifies USP7 as a post-translational checkpoint in hematopoiesis and reveals a novel deubiquitination-dependent mechanism controlling stem cell fate. These findings highlight the USP7-PU.1 interaction as a potential therapeutic target for hematopoietic disorders.

Aging studies using animal and cellular models have uncovered key proteins and pathways central to organismal aging. However, these models differ genetically and physiologically from human aging, posing challenges in translating discoveries to human contexts. In this study, we present a human normal cell aging model based on the development of cytotrophoblasts (CTBs) to syncytiotrophoblasts (STBs) in the placenta. The in vitro-derived STBs from human trophoblast stem cells (hTSCs) recapitulate the maturation and major cellular aging features of in vivo CTB-STB, including multinucleation, hormone secretion, cell cycle arrest, genome instability, epigenetic changes, activation of endogenous transposable elements, and senescence-associated secretory phenotypes (SASPs). Notably, the progressive senescence in the trophoblast system closely matches the predicted aging trajectory of other human tissue stem cells. Known anti-aging molecules, such as mTOR inhibitors and senolytics, attenuate senescence signals in STBs. The established CGA-EGFP reporter hTSC line enables scalable and quantitative screening and identified candidates with it can be further extended to other context-specific aging processes like that of skin fibroblasts. The hTSC-STB system represents a novel physiologically accelerated cellular aging model, bridges the gap between fundamental aging research and interventions, and prioritizes anti-aging candidates for clinical development.

Immunoscore (IS), based on CD3/CD8, has been proposed to characterize the immune landscape of the tumor immune microenvironment and has demonstrated an association with the prognosis of laryngeal squamous cell carcinoma (LSCC). However, traditional IS does not include immunosuppressive cells. The purpose of this study is to evaluate the prognostic performance of cytotoxic-T-lymphocytes to immunosuppressive cells ratio (CIL) in laryngeal squamous cell carcinoma (LSCC) patients. Two cohorts were included in this study: The training cohort (N = 75) consisted of tumor tissue microarrays from LSCC patients in our department, and the validation cohort (N = 116) utilized bulk RNA-seq data from the TCGA database. Patients with high IS or CIL showed significantly prolonged overall survival and disease-free survival in both cohorts. Upon analyzing the relative contribution of each parameter, it was found that CIL exhibited the highest significance among the factors examined. It emerged as the strongest predictor of overall survival, emphasizing its crucial influence in determining the outcomes. The prognostic ability of IS-TCGA was similar to the original IS. Additionally, high CILM2-TCGA was associated with prolonged survival of patients with LSCC in the TCGA dataset. CIL, which is easier to construct than IS, proves to be reliable in predicting survival outcomes for patients with LSCC.

Inflammation represents an adaptive physiological response of body immune system to infection or tissue damage, which may be regulated by food supplementation. Eicosapentaenoic acid (EPA) has multi-functions and its anti-inflammatory effect has gained great attention. This study aimed to address the exact molecular mechanism underlying its inflammatory control. The results showed that EPA decreased lipopolysaccharide-induced inflammatory response in RAW264.7 cells by modulating the production of cellular cytokines. In addition, EPA downregulated miR-125b-5p, which showed pro-inflammatory effect and its forced expression attenuated EPA's anti-inflammatory activity. Moreover, the cAMP-responsive element-binding protein (CREB) is targeted by miR-125b-5p. CREB overexpression reduced inflammation probably via modulating the PGC-1α/NF-κB pathway, which resembled the effect of EPA pre-treatment. Therefore, EPA exhibited anti-inflammatory activity by targeting the miR-125b-5p/CREB axis, which modulated the production of inflammatory mediators probably via transcription control. This study provides insights into microRNA-mediated action mechanism and facilitates the relief of inflammation-associated diseases by food ingredients.

Radiotherapy (RT) is a commonly used clinical management for hypopharyngeal squamous cell carcinoma (HPSCC), which represents the most unfavorable prognosis among all subtypes of head and neck squamous cell carcinoma. However, radiation may cause lymphopenia, a significantly adverse event with detrimental prognostic implications for patients. While CD8(+) T cells are vital in tumor immunity, the specific effects of RT on CD8(+) T cells as well as the underlying mechanisms have not been clearly elucidated. Here we found that subpopulations of peripheral T lymphocytes exhibited differential profiles in patients with HPSCC compared to healthy individuals both pre- and post-irradiation. Importantly, CD8(+) T cells from HPSCC patients showed greater reduction of cytokine production, more severe proliferation defect, and increased apoptosis compared to those from healthy individuals after in vitro irradiation. Mechanistically, the ATM-Chk2 pathway mediated the enhanced apoptosis of CD8(+) T lymphocytes from HPSCC patients upon irradiation. Therefore, our study demonstrated that CD8(+) T cells in patients with HPSCC exhibit a higher susceptibility to radiation-induced damage compared to those in healthy individuals. The ATM-Chk2 pathway represents a potential immunotherapeutic target for safeguarding CD8(+) T cells in HPSCC patients against radiation-induced apoptosis.

Tumor-Infiltrating Lymphocytes (TILs) immunotherapy is a highly promising treatment for Non-small Cell Lung Cancer (NSCLC), which is responsible for 18% of all cancer-related deaths. The heterogeneity of TILs remains poorly understood. Here, we utilized combined single-cell RNA (scRNA)/T cell receptor sequencing (scTCR-seq) data from lung adenocarcinoma (LUAD) patients. Naïve CD4+ and effector memory CD8+ T cells were increased in tumor tissue compared with circulating blood samples. Activated signaling pathways were detected, and GZMA was identified as a potential novel diagnostic biomarker. During the transitional phase, macrophages (FTL) and dendritic (AIF1) cells transported the most CD3 TCR clones to T cells, while cytotoxicity CD8+ T (NKG7) cells transported to terminal exhausted CD8+ T cells. In both transition and expansion phases, T helper cells (CXCL13) are transported to regulatory T cells (Tregs). Additionally, we investigated the expression profiles of key cytokines, checkpoint receptors, and their ligands. Cytotoxicity CD8+ T cells (CCL5 and IFNG), T helper cells (FTL, TNFRSF4, and TIGIT), and regulatory T cells (CTLA4, TIGIT and FTL) exhibited functional roles in both primary and metastatic tumor stages. Taken together, our study provides a single-cell resolution of the TIL immune landscape and suggests potential treatment strategies to overcome drug resistance.

Donor selection determines the occurrence of acute graft-versus-host-disease (aGVHD) following allogeneic hematopoietic stem cell transplantation (allo-HSCT). To optimize the current clinical donor selection criteria and identify putative donor lymphocyte subsets associated with better recipient outcomes, we analyzed the peripheral CD4+ and CD8+ subsets in 80 granulocyte colony-stimulating factor (G-CSF) mobilized donors and examined the aGVHD incidence of the corresponding 80 haploidentical and identical allo-HSCT recipients. The G-CSF-induced expansion of subsets varied among donors. We discovered a novel PD-1+CD8+CD45RA+CCR7+ T lymphocyte subset in suitable donors that was significantly correlated with lower incidence of aGVHD and post-transplant anti-infection. The anti-aGVHD activity of this subset was confirmed in a validation cohort (n = 30). Single-cell RNA sequencing revealed that this T cell subset exhibited transcriptomic features of stem cell-like memory T cell (TSCM) with both Treg and Teff activities which indicated its dual functions in aGVHD inhibition and graft-versus-leukemia (GVL) effect. Intriguingly, upon G-CSF mobilization, the donor PD-1+CD8+ TSCM-like regulatory cells increased the PD-1 expression in a BCL6-dependent manner. Next, we showed that the mouse counterpart of this subset (PD-1+CD8+CD44−CD62L+) ameliorated aGVHD, and confirmed the existence of this subset in clinical recipients. In summary, we, for the first time, identified a novel donor peripheral T cell subset suppressing aGVHD while promoting the immune reconstitution of recipients. It may serve as an indicator for optimal haploidentical and identical donor selection. Importantly, the dual Treg and Teff function of these T cells makes it a promising treatment for not only aGVHD but also auto-immune diseases.

Abstract Rationale: HMGB family plays an important role in homeostasis of immunity and regulation of inflammation. However, the role of HMGB2 in atherosclerosis remains uncharacterized. Objective The current study aimed to ascertain the relation of HMGB2 levels in macrophages of atherosclerotic plaques and in circulatory monocytes/macrophages with coronary artery disease (CAD), and to investigate the influence of HMGB2 on atherogenesis in mice and inflammatory reaction in vitro. Methods/Results We evaluated HMGB2 levels in macrophages of atherosclerotic plaques in coronary endarterectomy tissue from patients with angiographically documented severe coronary artery disease (CAD)(n=23), and in coronary artery specimens from autopsy cases with no or mild atherosclerosis (n=11) by immunohistochemistry. HMGB2 levels were significantly decreased in macrophages of progressive atherosclerotic plaques, as compared with those from mild plaques. Moreover, we also analyzed HMGB2 levels in peripheral blood monocytes from patients with CAD (n=44) or age- and sex-matched non-CAD controls (n=44). The results showed that decreased intracellular HMGB2 protein levels in monocytes were associated with CAD. In experiments, HMGB2 deficiency significantly promoted atherosclerosis in ApoE-/- mice, whereas such increment of atherosclerosis was markedly attenuated after transplantation of bone marrow derived macrophages from ApoE-/-mice. Mechanistically, intracellular HMGB2 repressed IL-1 transcription and induced autophagic degradation of NLRP3, thus suppressing NLRP3 inflammasome priming and activation. Extracellular HMGB2 levels increased in progressive atherosclerotic plaques with macrophage apoptosis. Recombinant HMGB2 protein promoted inflammation in vitro via RAGE and intraperitoneal administration of this protein exacerbated atherosclerosis in mice. Conclusion This study indicates that decreased HMGB2 level in macrophage is associated with inflammation and atherosclerosis in patients with CAD. Intracellular HMGB2 inhibits NLRP3 inflammasome and atherosclerosis in mice, whereas released HMGB2 promotes inflammation and atherosclerosis in mice.

Hypopharyngeal squamous cell carcinoma (HPSCC) has the worst prognosis among head and neck squamous cell carcinomas. The lack of available tumor cell lines poses a significant obstacle to the development of efficient treatments for HPSCC. In this study, we successfully established a novel cell line, named CZH1, from the postcricoid region of a Chinese male patient with a T3N0M0 HPSCC. Short tandem repeat analysis confirmed the uniqueness of CZH1. The cell line was characterized by its phenotypes, biomarkers, and genetics. Importantly, CZH1 cells retained the typical features of epithelial malignancy, similar to the primary tumor tissue. Furthermore, CZH1 demonstrated a greater capacity for invasion and increased susceptibility to irradiation in comparison to FaDu, which is the most commonly used HPSCC cell line. Whole-exome sequencing analysis revealed that CZH1 cells had typical genomic features of HNSCC, including mutations of TP53 and amplifications of multiple transcripts. Therefore, our newly developed CZH1 cell line could serve as an efficient tool for the in vitro investigation of the etiology, pathogenesis, and preclinical treatment of HPSCC.

Tumor-infiltrating neutrophils play a crucial role in the progression of head and neck squamous cell carcinoma (HNSCC). Here, we aimed to statistically quantify the plasticity of HNSCC-infiltrating N2/N1 neutrophils and examine its impacts on survival and immune infiltration landscape. A retrospective study of 80 patients who underwent curative surgical resection for HNSCC between 2014 and 2017 was conducted in this study. HNSCC-infiltrating neutrophil phenotypes were classified using immunofluorescence staining, and the N2/N1 neutrophil plasticity was evaluated via the ratio of N2/N1 neutrophils. We then assessed the correlations between N2/N1 neutrophil plasticity, clinicopathological characteristics, and immune infiltration landscape using rigorous statistical methods. Infiltration variations of N1 and N2 neutrophils were observed between the tumor nest (TN) and tumor stroma (TS), with TN exhibiting higher N2 neutrophil infiltration and lower N1 neutrophil infiltration. High ratios of N2/N1 neutrophils were correlated with advanced TNM stage, large tumor size and invasion of adjacent tissue. High infiltration of N2 neutrophils was associated with decreased overall and relapse-free survival, which were opposite for N1 neutrophils. The independent prognostic role of N2/N1 neutrophil plasticity, particularly within the TN region, was confirmed by multivariate analyses. Moreover, the ratio of N2/N1 neutrophils within the TN region showed correlations with high CD8+ T cells infiltration and low FOXP3+ Tregs infiltration. We identify HNSCC-infiltrating N2/N1 neutrophil plasticity as a crucial prognostic indictor which potentially reflects the tumor microenvironment (TME) and immune escape landscape within HNSCC tissues. Further investigations and validations may provide novel therapeutic strategies for personalized immunomodulation in HNSCC patients.

The development of efficient treatments for laryngeal squamous cell carcinoma (LSCC) is hindered by the lack of applicable tumor cell lines and animal models of the disease, especially those related to cancer stem-like cells (CSCs). CSCs play critical roles in tumor propagation and pathogenesis whereas no CSCs lines have been developed to date. In this study, we establish an LSCC cell line (FD-LS-6) from primary LSCC tumor tissue (not experienced single-cell cloning) and adapted a culturing condition for the expansion of potential stem cells (EPSCs) to isolate CSCs from FD-LS-6. We successfully derived novel CSCs and named them as LSCC sphere-forming cells (LSCSCs) which were subsequently characterized for their CSC properties. We showed that LSCSCs shared many properties of CSCs, including CSC marker, robust self-renewal capacity, tumorigenesis ability, potential to generate other cell types such as adipocytes and osteoblasts, and resistance to chemotherapy. Compared to parental cells, LSCSCs were significantly more potent in forming tumors in vivo in mice and more resistant to chemotherapy. LSCSCs have higher expressions of epithelial–mesenchymal transition proteins and chemotherapy resistance factors, and exhibit an activated COX2/PEG2 signaling pathway. Altogether, our work establishes the first CSCs of LSCC (FD-LS-6) and provides a tool to study tumorigenesis and metastasis of LSCC and help the development of anticancer therapies.

Oncolytic viruses (OVs) possess the unique ability to selectively replicate within tumor cells, leading to their destruction, while also reversing the immunosuppression within the tumor microenvironment and triggering an antitumor immune response. As a result, OVs have emerged as one of the most promising approaches in cancer therapy. However, the effective delivery of intravenously administered OVs faces significant challenges imposed by various immune cells within the peripheral blood, hindering their access to tumor sites. Notably, neutrophils, the predominant white blood cell population comprising approximately 50%-70% of circulating white cells in humans, show phagocytic properties. Our investigation revealed that the majority of oncolytic vaccinia viruses (VV) are engulfed and degraded by neutrophils in the bloodstream. The depletion of neutrophils using the anti-LY6G Ab (1-A8) resulted in an increased accumulation of circulating oncolytic VV in the peripheral blood and enhanced deposition at the tumor site, consequently amplifying the antitumor effect. Neutrophils heavily rely on PI3K signaling to sustain their phagocytic process. Additionally, our study determined that the inhibition of the PI3Kinase delta isoform by idelalisib (CAL-101) suppressed the uptake of oncolytic VV by neutrophils. This inhibition led to a greater presence of oncolytic VV in both the peripheral blood and at the tumor site, resulting in improved efficacy against the tumor. In conclusion, our study showed that inhibiting neutrophil functions can significantly enhance the antitumor efficacy of intravenous oncolytic VV.

The infiltration and prognostic significance of tumor-infiltrating plasmacytoid dendritic cells (TI-pDC) have been elucidated in various human solid cancers. However, the infiltrating patterns and functional importance of TI-pDC in laryngeal squamous cell carcinoma (LSCC) remain unknown. In this study, flow cytometric analyses were conducted to characterize the infiltration of dendritic cells and T lymphocytes, along with their respective subgroups in tumor tissues (TT), para-carcinoma tissues (PT), and peripheral blood (PB) from LSCC patients. Immunohistochemical staining for CD4 and CD8, as well as immunofluorescence staining for CD123, were performed on serial tissue sections to investigate the co-localization of TI-pDC and tumor-infiltrating T lymphocytes (TIL) within the tumor microenvironment (TME). Our results demonstrated significantly lower percentages of all three DC subsets in PB compared to TT and PT. Notably, the pDC percentage was markedly higher in TT than in PT. Moreover, TI-pDC percentage was significantly elevated in N+ stage patients compared to those with N0 stage. The results of survival analysis consistently demonstrated that high levels of TI-pDC infiltration were indicative of a poor prognosis. Further investigation revealed a significant negative correlation between TI-pDC and CD8+ TILs; notably, pDCs expressed an inhibitory surface molecule PD-L2 rather than PD-L1 within PT. Collectively, our findings suggest that increased TI-pDC is associated with adverse outcomes in LSCC patients while exhibiting an inhibitory phenotype that may play a crucial role in suppressing CD8+ TILs within LSCC tumors. These results highlight the potential therapeutic strategy targeting PD-L2+ pDCs for immunotherapies against LSCC.

Tumor-associated neutrophils (TANs) play a crucial role in tumor progression and exhibit prolonged survival. However, the mechanism underlying their extended lifespan and significance in laryngeal squamous cell carcinoma (LSCC) remains unclear. Herein, it is observed that apoptosis of TANs is significantly delayed owing to induction by tumor-derived G-CSF and GM-CSF through the activation of the PI3K-AKT signaling pathway, upregulation of anti-apoptotic Mcl-1 expression, and downregulation of activated Caspase-3 levels. It is found that prolonged survival of TANs leads to the accumulation of aged CXCR4+ neutrophils that exhibit potent immunosuppressive properties and are associated with poor patient prognosis. Furthermore, extended survival promotes the enhanced immunosuppressive function of CD8+ T cells by TANs, thereby facilitating the in vitro and in vivo progression and growth of human LSCC tumors. Importantly, this effect could be reversed by blocking G-CSF and GM-CSF stimulation of neutrophils. These findings elucidate the pivotal role of pathologically prolonged neutrophil survival in impairing CD8+ T cell immunity and suggest targeting it as a potential therapeutic strategy for tumors.

Introduction Trichosanthin (TK) is a glycoprotein extracted from the Chinese medicinal herb Trichosanthes kirilowi, which has anti-virus and anti-tumor activity. However, the target and detailed mechanism of TK remains elusive. Objectives We aimed to identify novel antitumor targets of TK in lung adenocarcinoma and study its anti-tumor mechanism. Methods We utilized a Lewis lung carcinoma mouse model to evaluate the inhibition of TK on tumor growth. CCK8 assay was utilized to calculate IC50 of trichosanthin on A549 and H1299. In-vitro cellular assays and in-vivo xenograft mice studies were used to investigate MICU3 overexpression and TK treatment on tumor growth. Fluo-4 dye and JC-1 staining was used to measure the mitochondrial calcium levels and membrane potential. H&E and immunohistochemistry staining were applied the asses the effect of TK on tumor and microenvironment. RNA sequencing was applied to analyze transcriptome changes in TK-treated and MICU3-overexpressed tumor cells. The influence of trichosanthin on DNMT3B expression and MICU3 methylation were detected by qPCR and Western blotting. Transcriptional activity of the MICU3 gene was measured by ChIP-PCR and luciferase assays. Results Trichosanthin ihibited the tumor growth in vivo, resulting cancer cell growth inhibition and cell death, with almost no effect on normal cells. IC50 of trichosanthin in A549 and H1299 cells were 62.8 μg/ml and 39.7 μg/ml, respectively. Mitochondrial Calcium Uptake Family complex MICU3 was shown to associated with favorable prognosis and was upregulated upon trichosanthin treatment, along with reduces tumor cell growth and migration, and increased cell death both in vitro and in vivo. Increased mitochondrial calcium level was observed in MICU3 overexpression cells. Pathway analysis of RNA-seq data revealed that cytokine and receptor pathways were enriched in MICU3-overexpressing cells. Trichosanthin decreased DNMT3B expression and altered MICU3 methylation while increased FOSL2 expression and reduced methylation that correlated with increased transcription of the MICU3 gene. Conclusion Trichosanthin elicits antitumor activity in lung adenocarcinoma via repressing DNMT3B and increasing FOSL2, which in turn induces MICU3-mediated mitochondrial calcium influx and tumor cell death.

Dysregulated lipid metabolism in liver is an important hallmark of non-alcoholic fatty liver disease (NAFLD), which may be modulated by dietary polyphenols or microRNAs (miRNAs). However, the underlying epigenetic regulatory mechanism of polyphenols remain unclear. The current study aimed to address how miRNA mediates hepatic lipid metabolic control of curcumin, a polyphenolic food supplement. The results showed that 24 h treatment with 5-20 mu M curcumin prevented free fatty acid -induced lipid accumulation by around 10-50% in HepG2 cells, which was attenuated by pre-transfection with 40 nM miR-22-3p mimic for 48 h. In consequence, transfection with 40 nM miR-22-3p inhibitor for 48 h significantly reduced lipid accumulation by around 10%. And, 48 h overexpression of miR-22-3p targeting cardiolipin synthase 1 (CRLS1) gene, which encodes a mitochondrial phospholipid synthase, showed a similar regulatory effect. Thus, miR-22-3p and CRLS1 showed opposite effects in modulating lipid metabolism, which probably involved mitochondrial control. In summary, this study demonstrated that curcumin improved hepatic lipid metabolism via targeting the miR-22-3p/CRLS1 pathway. Identification of the epigenetic regulatory mechanism underlying lipid metabolism may thereby facilitate alleviation of metabolic disorders by natural polyphenols.

This study investigates the role of USP47, a deubiquitinating enzyme, in the tumor microenvironment and its impact on antitumor immune responses. Analysis of TCGA database revealed distinct expression patterns of USP47 in various tumor tissues and normal tissues. Prostate adenocarcinoma showed significant downregulation of USP47 compared to normal tissue. Correlation analysis demonstrated a positive association between USP47 expression levels and infiltrating CD8+ T cells, neutrophils, and macrophages, while showing a negative correlation with NKT cells. Furthermore, using Usp47 knockout mice, we observed a slower tumor growth rate and reduced tumor burden. The absence of USP47 led to increased infiltration of immune cells, including neutrophils, macrophages, NK cells, NKT cells, and T cells. Additionally, USP47 deficiency resulted in enhanced activation of cytotoxic T lymphocytes (CTLs) and altered T cell subsets within the tumor microenvironment. These findings suggest that USP47 plays a critical role in modulating the tumor microenvironment and promoting antitumor immune responses, highlighting its potential as a therapeutic target in prostate cancer.

The cGAS-STING pathway has long been recognized as playing a crucial role in immune surveillance and tumor suppression. Here, we show that when the pathway is activated in a cancer-cell-autonomous response manner, it confers drug resistance. Targeted or conventional chemotherapy drugs promoted cytosolic DNA accumulation in cancer cells, activating the cGAS-STING pathway and downstream TBK1-IRF3/NF-κB signaling. This cancer cell-intrinsic response enabled the cells to counteract drug stress, allowing treatment resistance to be acquired and maintained. Blockade of stimulator of interferon genes (STING) signaling delayed and overcame resistance in models in vitro and in vivo. This finding uncovers an alternative face of cGAS-STING signaling other than the well-reported modulation of microenvironmental immune cells. It also implies a caution for the combination of STING agonist with targeted or conventional chemotherapy drug treatment, a strategy prevailing in current clinical trials.

Background Tumor-associated macrophages (TAMs) are major component in the tumor microenvironment (TME) and play regulatory role in tumor progression. We aimed to investigate the infiltration and prognostic value of TAMs in laryngeal squamous cell carcinoma (LSCC) and to reveal the underlying mechanism of TAM subgroups in tumorigenesis. Methods Hematoxylin and eosin (HE) staining were performed to define the tumor nest and stroma of LSCC tissue microarrays. CD206 + /CD163 + and iNOS + TAM infiltrating profiles were obtained and analyzed through double-labeling immunofluorescence and immunohistochemical staining. The recurrence-free (RFS) and overall survival (OS) curves based on the infiltration of TAMs were plotted using the Kaplan-Meier method. Infiltration of macrophages, T lymphocytes and their corresponding subgroups were analyzed in fresh LSCC tissue samples by flow cytometry. Results We found that CD206 + rather than CD163 + M2-like TAMs were the most enriched population in the TME of human LSCC. CD206 + macrophages localized mostly in the tumor stroma (TS) rather than the tumor nest (TN) region. In contrast, relatively low infiltration of iNOS + M1-like TAMs were found in the TS and almost none in the TN region. High level of TS CD206 + TAM infiltration correlated with poor prognosis. Interestingly, we identified a HLA-DR high CD206 + macrophage subgroup that was significantly associated with the tumor-infiltrating CD4 + T lymphocytes and showed different surface costimulatory molecule expression than that of the HLA-DR low/ -CD206 + subgroup. Taken together, our results indicate that HLA-DR high -CD206 + is a highly activated subgroup of CD206 + TAMs that may interact with CD4 + T cells through MHC-II axis and promote tumorigenesis.