Candidate tumor-specific CD8+ T cell subsets identified in the malignant pleural effusion of advanced lung cancer patients by single-cell analysis.

Isolation of tumor-specific T cells and their antigen receptors (TCRs) from malignant pleural effusions (MPE) may facilitate the development of TCR-transduced adoptive cellular immunotherapy products for advanced lung cancer patients. However, the characteristics and markers of tumor-specific T-cells in MPE are largely undefined. To this end, to establish the phenotypes and antigen specificities of CD8+ T cells, we performed single-cell RNA and TCR sequencing of samples from three advanced lung cancer patients. Dimensionality reduction on a total of 4,983 CD8+ T cells revealed 10 clusters including naïve, memory, and exhausted phenotypes. We focused particularly on exhausted T cell clusters and tested their TCR reactivity against neoantigens predicted from autologous cancer cell lines. Four different TCRs specific for the same neoantigen and one orphan TCR specific for the autologous cell line were identified from one of the patients. Differential gene expression analysis in tumor-specific T cells relative to the other T cells identified CXCL13, as a candidate gene expressed by tumor-specific T cells. In addition to expressing CXCL13, tumor-specific T cells were present in a higher proportion of T cells co-expressing PDCD1(PD-1)/TNFRSF9(4-1BB). Furthermore, flow cytometric analyses in advanced lung cancer patients with MPE documented that those with high PD-1/4-1BB expression have a better prognosis in the subset of 57 adenocarcinoma patients (p = .039). These data suggest that PD-1/4-1BB co-expression might identify tumor-specific CD8+ T cells in MPE, which are associated with patients' prognosis. (233 words).

The correlation between TRIM28 expression and immune checkpoints in CRPC.

This study delves into the unexplored realm of castration-resistant prostate cancer (CRPC) by investigating the role of TRIM28 and its intricate molecular mechanisms using high-throughput single-cell transcriptome sequencing and advanced bioinformatics analysis. Our comprehensive examination unveiled dynamic TRIM28 expression changes, particularly in immune cells such as macrophages and CD8+ T cells within CRPC. Correlation analyses with TCGA data highlighted the connection between TRIM28 and immune checkpoint expression and emphasized its pivotal influence on the quantity and functionality of immune cells. Using TRIM28 knockout mouse models, we identified differentially expressed genes and enriched pathways, unraveling the potential regulatory involvement of TRIM28 in the cGAS-STING pathway. In vitro, experiments further illuminated that TRIM28 knockout in prostate cancer cells induced a notable anti-tumor immune effect by inhibiting M2 macrophage polarization and enhancing CD8+ T cell activity. This impactful discovery was validated in an in situ transplant tumor model, where TRIM28 knockout exhibited a deceleration in tumor growth, reduced proportions of M2 macrophages, and enhanced infiltration of CD8+ T cells. In summary, this study elucidates the hitherto unknown anti-tumor immune role of TRIM28 in CRPC and unravels its potential regulatory mechanism via the cGAS-STING signaling pathway. These findings provide novel insights into the immune landscape of CRPC, offering promising directions for developing innovative therapeutic strategies.

Tumor-intrinsic IFNα and CXCL10 are critical for immunotherapeutic efficacy by recruiting and activating T lymphocytes in tumor microenvironment.

Tumor immunotherapies targeting PD-(L)1 exhibit anti-tumor efficacy in only 10-30% of patients with various cancers. Literature has demonstrated that a "hot tumor" which contains high T lymphocytes in the tumor microenvironment exhibits a better response to immunotherapies than a "cold tumor." This study aimed to investigate whether tumor-intrinsic IFNα and CXCL10 determine the recruitment and activation of CD8+ T cells to become "hot tumor." In this study, we found that CXCL10 overexpressed in a variety of tumors including lung, colon, and liver tumors with a correlation with PD-L1. High PD-L1 and CXCL10 are associated with better survival rates in tumor patients receiving immunotherapies. IFNs-downstream transcriptional factor IRF-1 and STAT1 were correlated with PD-L1 and CXCL10 expression. We demonstrated that IRF-1 and STAT1 were both bound with the promoters of PD-L1 and CXCL10, sharing the same signaling pathway and determining IFNs-mediated PD-L1 and CXCL10 expression. In addition, IFNα significantly increased activation marker IFNγ in PBMCs, promoting M1 type monocyte differentiation, CD4+ T, and CD8+ T cell activation. Particularly, we found that CD8+ T lymphocytes abundantly expressed CXCR3, a receptor of CXCL10, by flow cytometry, indicating that tumor-intrinsic CXCL10 potentially recruited CD8+ T in tumor microenvironment. To demonstrate the hypothesis, immunotherapy-sensitive CT26 and immunotherapy-resistant LL/2 were used and we found that CT26 cells exhibited higher IFNα, IFNγ, CXCL10, and PD-L1 levels compared to LL/2, leading to higher IFNγ expression in mouse splenocytes. Moreover, we found that CD8+ T cells were recruited by CXCL10 in vitro, whereas SCH546738, an inhibitor of CXCR3, inhibited T cell migration and splenocytes-mediated anti-tumor effect. We then confirmed that CT26-derived tumor was sensitive to αPD-L1 immunotherapy and LL/2-tumor was resistant, whereas αPD-L1 significantly increased T lymphocyte activation marker CD107a in CT26-derived BALB/c mice. In conclusion, this study revealed that CXCL10 expression is correlated with PD-L1 in tumors, sharing the same signaling pathway and associating with better immunotherapeutic efficacy. Further evidence in the syngeneic tumor models demonstrated that immunotherapy-sensitive CT26 intrinsically exhibited higher IFNα and CXCL10 compared to immunotherapy-resistant LL/2 to recruit and activate CD8+ T cells in the tumor microenvironment, exhibiting "hot tumor" characteristic of sensitizing αPD-L1 immunotherapies.

Tissue-resident memory CD103+CD8+ T cells in colorectal cancer: its implication as a prognostic and predictive liver metastasis biomarker.

BACKGROUND: Tissue-resident memory CD103+CD8+ T cells (CD103+CD8+ TRMs) are important components of anti-tumor immunity. However, the significance of CD103+CD8+ TRMs in colorectal cancer (CRC) and their advantages remain unclear. METHODS: Clinical data and specimens were used to evaluate the significance of CD103+CD8+ TRMs in CRC. A mouse subcutaneous tumorigenesis model and colony-formation assay were conducted to evaluate the anti-tumor effects of CD103+CD8+ TRMs. Finally, the infiltration density and function of CD103+CD8+ TRMs in the tumors were evaluated using flow cytometry. RESULTS: In this study, we showed that highly infiltrated CD103+CD8+ TRMs were associated with earlier clinical stage and negative VEGF expression in CRC patients and predicted a favorable prognosis for CRC/CRC liver metastases patients. Interestingly, we also found that CD103+CD8+ TRMs may have predictive potential for whether CRC develops liver metastasis in CRC. In addition, we found a positive correlation between the ratio of the number of α-SMA+ vessels to the sum of the number of α-SMA+ and CD31+ vessels in CRC, and the infiltration level of CD103+CD8+ TRMs. In addition, anti-angiogenic therapy promoted infiltration of CD103+CD8+ TRMs and enhanced their ability to secrete interferon (IFN)-γ, thus further improving the anti-tumor effect. Moreover, in vivo experiments showed that compared with peripheral blood CD8+ T cells, CD103+CD8+ TRMs infused back into the body could also further promote CD8+ T cells to infiltrate the tumor, and they had a stronger ability to secrete IFN-γ, which resulted in better anti-tumor effects. CONCLUSION: We demonstrated that CD103+CD8+ TRMs have the potential for clinical applications and provide new ideas for combined anti-tumor therapeutic strategies, such as anti-tumor angiogenesis therapy and CAR-T combined immunotherapy.

DOK1 and DOK2 regulate CD8 T cell signaling and memory formation without affecting tumor cell killing.

Targeting intracellular inhibiting proteins has been revealed to be a promising strategy to improve CD8+ T cell anti-tumor efficacy. Here, we are focusing on intracellular inhibiting proteins specific to TCR signaling: DOK1 and DOK2 expressed in T cells. We hypothesized that depletion of intracellular inhibition checkpoint DOK1 and DOK2 could improve CD8+ T-cell based cancer therapies. To evaluate the role of DOK1 and DOK2 depletion in physiology and effector function of CD8+ T lymphocytes and in cancer progression, we established a transgenic T cell receptor mouse model specific to melanoma antigen hgp100 (pmel-1 TCR Tg) in WT and Dok1/Dok2 DKO (double KO) mice. We showed that both DOK1 and DOK2 depletion in CD8+ T cells after an in vitro pre-stimulation induced a higher percentage of effector memory T cells as well as an up regulation of TCR signaling cascade- induced by CD3 mAbs, including the increased levels of pAKT and pERK, two major phosphoproteins involved in T cell functions. Interestingly, this improved TCR signaling was not observed in naïve CD8+ T cells. Despite this enhanced TCR signaling essentially shown upon stimulation via CD3 mAbs, pre-stimulated Dok1/Dok2 DKO CD8+ T cells did not show any increase in their activation or cytotoxic capacities against melanoma cell line expressing hgp100 in vitro. Altogether we demonstrate here a novel aspect of the negative regulation by DOK1 and DOK2 proteins in CD8+ T cells. Indeed, our results allow us to conclude that DOK1 and DOK2 have an inhibitory role following long term T cell stimulations.

Unique immune characteristics and differential anti-PD-1-mediated reinvigoration potential of CD8+ TILs based on BRCA1/2 mutation status in epithelial ovarian cancers.

BACKGROUND: We aimed to investigate the distinct immunological characteristics of the tumor immune microenvironment in epithelial ovarian cancer (EOC) according to BRCA1/2 mutations status and differential PD-1 expression levels. METHODS: Tumor-infiltrating lymphocytes (TILs) were collected from patients with newly diagnosed advanced-stage EOC (YUHS cohort, n=117). This YUHS cohort was compared with The Cancer Genome Atlas (TCGA) data for ovarian serous cystadenocarcinoma (n=482), in terms of survival outcomes and immune-related gene profiles according to BRCA1/2 status. We used multicolor flow cytometry to characterize the immune phenotypes and heterogeneity of TILs with or without BRCA1/2 mutations. In vitro functional assays were conducted to evaluate the reinvigorating ability of CD8+ TILs on anti-PD-1 treatment. RESULTS: We found that EOC patients with BRCA1/2 mutations (BRCA1/2mt) exhibited better survival outcomes and significantly higher tumor mutation burden (TMB), compared with BRCA1/2 non-mutated (BRCA1/2wt) patients. Furthermore, CD8+ TILs within BRCA1/2mt tumors displayed characteristics indicating more severe T-cell exhaustion than their BRCA1/2wt counterparts. Notably, the capacity for anti-PD-1-mediated reinvigoration of CD8+ TILs was significantly greater in BRCA1/2wt tumors compared with BRCA1/2mt tumors. Additionally, within the BRCA1/2wt group, the frequency of PD-1highCD8+ TILs was positively correlated with the reinvigoration capacity of CD8+ TILs after anti-PD-1 treatment. CONCLUSION: Our results highlight unique immune features of CD8+ TILs in EOC and a differential response to anti-PD-1 treatment, contingent on BRCA1/2 mutation status. These findings suggest that immune checkpoint blockade may be a promising frontline therapeutic option for selected BRCA1/2wt EOC patients.

TCF1-positive and TCF1-negative TRM CD8 T cell subsets and cDC1s orchestrate melanoma protection and immunotherapy response.

BACKGROUND: Melanoma, the most lethal form of skin cancer, has undergone a transformative treatment shift with the advent of checkpoint blockade immunotherapy (CBI). Understanding the intricate network of immune cells infiltrating the tumor and orchestrating the control of melanoma cells and the response to CBI is currently of utmost importance. There is evidence underscoring the significance of tissue-resident memory (TRM) CD8 T cells and classic dendritic cell type 1 (cDC1) in cancer protection. Transcriptomic studies also support the existence of a TCF7+ (encoding TCF1) T cell as the most important for immunotherapy response, although uncertainty exists about whether there is a TCF1+TRM T cell due to evidence indicating TCF1 downregulation for tissue residency activation. METHODS: We used multiplexed immunofluorescence and spectral flow cytometry to evaluate TRM CD8 T cells and cDC1 in two melanoma patient cohorts: one immunotherapy-naive and the other receiving immunotherapy. The first cohort was divided between patients free of disease or with metastasis 2 years postdiagnosis while the second between CBI responders and non-responders. RESULTS: Our study identifies two CD8+TRM subsets, TCF1+ and TCF1-, correlating with melanoma protection. TCF1+TRM cells show heightened expression of IFN-γ and Ki67 while TCF1- TRM cells exhibit increased expression of cytotoxic molecules. In metastatic patients, TRM subsets undergo a shift in marker expression, with the TCF1- subset displaying increased expression of exhaustion markers. We observed a close spatial correlation between cDC1s and TRMs, with TCF1+TRM/cDC1 pairs enriched in the stroma and TCF1- TRM/cDC1 pairs in tumor areas. Notably, these TCF1- TRMs express cytotoxic molecules and are associated with apoptotic melanoma cells. Both TCF1+ and TCF1- TRM subsets, alongside cDC1, prove relevant to CBI response. CONCLUSIONS: Our study supports the importance of TRM CD8 T cells and cDC1 in melanoma protection while also highlighting the existence of functionally distinctive TCF1+ and TCF1- TRM subsets, both crucial for melanoma control and CBI response.

Construction and validation of prognostic signature for transcription factors regulating T cell exhaustion in hepatocellular carcinoma.

In the tumor microenvironment (TME), CD8+ T cells showed stage exhaustion due to the continuous stimulation of tumor antigens. To evaluate the status of CD8+ T cells and reverse the exhaustion is the key to evaluate the prognosis and therapeutic effect of tumor patients. The aim of this study was to establish a prognostic signature that could effectively predict prognosis and response to immunotherapy in patients with hepatocellular carcinoma (HCC). We used univariate Cox analysis to obtain transcription factors associated with CD8+ T cell exhaustion from The Cancer Genome Atlas dataset. Then, the prognostic signature for transcription factors basic leucine zipper ATF-like transcription factor, Eomesodermin, and T-box protein 21 regulating T cell exhaustion was constructed using LASSO Cox regression. The relative expression levels of the mRNA of the 3 transcription factors were detected by reverse transcription-quantitative polymerase chain reaction in 23 pairs of HCC and paracancer tissues, and verified internally in The Cancer Genome Atlas dataset and externally in the International Cancer Genome Consortium dataset. Cox regression analysis showed that risk score was an independent prognostic variable. The overall survival of the high-risk group was significantly lower than that of the low-risk group. The low-risk group had higher immune scores, matrix scores, and ESTIMATE scores, and significantly increased expression levels of most immune checkpoint genes in the low-risk group. Therefore, patients with lower risk scores benefit more from immunotherapy. The combination of the 3 transcription factors can evaluate the exhaustion state of CD8+ T cells in the TME, laying a foundation for evaluating the TME and immunotherapy efficacy in patients with HCC.

ATP6V0A1-dependent cholesterol absorption in colorectal cancer cells triggers immunosuppressive signaling to inactivate memory CD8+ T cells.

Obesity shapes anti-tumor immunity through lipid metabolism; however, the mechanisms underlying how colorectal cancer (CRC) cells utilize lipids to suppress anti-tumor immunity remain unclear. Here, we show that tumor cell-intrinsic ATP6V0A1 drives exogenous cholesterol-induced immunosuppression in CRC. ATP6V0A1 facilitates cholesterol absorption in CRC cells through RAB guanine nucleotide exchange factor 1 (RABGEF1)-dependent endosome maturation, leading to cholesterol accumulation within the endoplasmic reticulum and elevated production of 24-hydroxycholesterol (24-OHC). ATP6V0A1-induced 24-OHC upregulates TGF-ß1 by activating the liver X receptor (LXR) signaling. Subsequently, the release of TGF-ß1 into the tumor microenvironment by CRC cells activates the SMAD3 pathway in memory CD8+ T cells, ultimately suppressing their anti-tumor activities. Moreover, we identify daclatasvir, a clinically used anti-hepatitis C virus (HCV) drug, as an ATP6V0A1 inhibitor that can effectively enhance the memory CD8+ T cell activity and suppress tumor growth in CRC. These findings shed light on the potential for ATP6V0A1-targeted immunotherapy in CRC.

Establishment of an ovarian cancer exhausted CD8+T cells-related genes model by integrated analysis of scRNA-seq and bulk RNA-seq.

Ovarian cancer (OC) was the fifth leading cause of cancer death and the deadliest gynecological cancer in women. This was largely attributed to its late diagnosis, high therapeutic resistance, and a dearth of effective treatments. Clinical and preclinical studies have revealed that tumor-infiltrating CD8+T cells often lost their effector function, the dysfunctional state of CD8+T cells was known as exhaustion. Our objective was to identify genes associated with exhausted CD8+T cells (CD8TEXGs) and their prognostic significance in OC. We downloaded the RNA-seq and clinical data from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. CD8TEXGs were initially identified from single-cell RNA-seq (scRNA-seq) datasets, then univariate Cox regression, the least absolute shrinkage and selection operator (LASSO), and multivariate Cox regression were utilized to calculate risk score and to develop the CD8TEXGs risk signature. Kaplan-Meier analysis, univariate Cox regression, multivariate Cox regression, time-dependent receiver operating characteristics (ROC), nomogram, and calibration were conducted to verify and evaluate the risk signature. Gene set enrichment analyses (GSEA) in the risk groups were used to figure out the closely correlated pathways with the risk group. The role of risk score has been further explored in the homologous recombination repair deficiency (HRD), BRAC1/2 gene mutations and tumor mutation burden (TMB). A risk signature with 4 CD8TEXGs in OC was finally built in the TCGA database and further validated in large GEO cohorts. The signature also demonstrated broad applicability across various types of cancer in the pan-cancer analysis. The high-risk score was significantly associated with a worse prognosis and the risk score was proven to be an independent prognostic biomarker. The 1-, 3-, and 5-years ROC values, nomogram, calibration, and comparison with the previously published models confirmed the excellent prediction power of this model. The low-risk group patients tended to exhibit a higher HRD score, BRCA1/2 gene mutation ratio and TMB. The low-risk group patients were more sensitive to Poly-ADP-ribose polymerase inhibitors (PARPi). Our findings of the prognostic value of CD8TEXGs in prognosis and drug response provided valuable insights into the molecular mechanisms and clinical management of OC.

Interconnected lineage trajectories link conventional and natural killer (NK)-like exhausted CD8+ T cells beneficial in type 1 diabetes.

Distinct Natural Killer (NK)-like CD57+ and PD-1+ CD8+ exhausted-like T cell populations (Tex) have both been linked to beneficial immunotherapy response in autoimmune type 1 diabetes (T1D) patients. The origins and relationships between these cell types are poorly understood. Here we show that while PD-1+ and CD57+ Tex populations are epigenetically similar, CD57+ Tex cells display unique increased chromatin accessibility of inhibitory Killer Cell Immunoglobulin-like Receptor (iKIR) and other NK cell genes. PD-1+ and CD57+ Tex also show reciprocal expression of Inhibitory Receptors (IRs) and iKIRs accompanied by chromatin accessibility of Tcf1 and Tbet transcription factor target sites, respectively. CD57+ Tex show unappreciated gene expression heterogeneity and share clonal relationships with PD-1+ Tex, with these cells differentiating along four interconnected lineage trajectories: Tex-PD-1+, Tex-CD57+, Tex-Branching, and Tex-Fluid. Our findings demonstrate new relationships between Tex-like populations in human autoimmune disease and suggest that modulating common precursor populations may enhance response to autoimmune disease treatment.

Clonal associations between lymphocyte subsets and functional states in rheumatoid arthritis synovium.

Rheumatoid arthritis (RA) is an autoimmune disease involving antigen-specific T and B cells. Here, we perform single-cell RNA and repertoire sequencing on paired synovial tissue and blood samples from 12 seropositive RA patients. We identify clonally expanded CD4 + T cells, including CCL5+ cells and T peripheral helper (Tph) cells, which show a prominent transcriptomic signature of recent activation and effector function. CD8 + T cells show higher oligoclonality than CD4 + T cells, with the largest synovial clones enriched in GZMK+ cells. CD8 + T cells with possibly virus-reactive TCRs are distributed across transcriptomic clusters. In the B cell compartment, NR4A1+ activated B cells, and plasma cells are enriched in the synovium and demonstrate substantial clonal expansion. We identify synovial plasma cells that share BCRs with synovial ABC, memory, and activated B cells. Receptor-ligand analysis predicted IFNG and TNFRSF members as mediators of synovial Tph-B cell interactions. Together, these results reveal clonal relationships between functionally distinct lymphocyte populations that infiltrate the synovium of patients with RA.

Obesity induces PD-1 on macrophages to suppress anti-tumour immunity.

Obesity is a leading risk factor for progression and metastasis of many cancers1,2, yet can in some cases enhance survival3-5 and responses to immune checkpoint blockade therapies, including anti-PD-1, which targets PD-1 (encoded by PDCD1), an inhibitory receptor expressed on immune cells6-8. Although obesity promotes chronic inflammation, the role of the immune system in the obesity-cancer connection and immunotherapy remains unclear. It has been shown that in addition to T cells, macrophages can express PD-19-12. Here we found that obesity selectively induced PD-1 expression on tumour-associated macrophages (TAMs). Type I inflammatory cytokines and molecules linked to obesity, including interferon-γ, tumour necrosis factor, leptin, insulin and palmitate, induced macrophage PD-1 expression in an mTORC1- and glycolysis-dependent manner. PD-1 then provided negative feedback to TAMs that suppressed glycolysis, phagocytosis and T cell stimulatory potential. Conversely, PD-1 blockade increased the level of macrophage glycolysis, which was essential for PD-1 inhibition to augment TAM expression of CD86 and major histocompatibility complex I and II molecules and ability to activate T cells. Myeloid-specific PD-1 deficiency slowed tumour growth, enhanced TAM glycolysis and antigen-presentation capability, and led to increased CD8+ T cell activity with a reduced level of markers of exhaustion. These findings show that obesity-associated metabolic signalling and inflammatory cues cause TAMs to induce PD-1 expression, which then drives a TAM-specific feedback mechanism that impairs tumour immune surveillance. This may contribute to increased cancer risk yet improved response to PD-1 immunotherapy in obesity.

Peripheral Blood CD8+ T-Lymphocyte Immune Response in Benign and Subpopulations of Breast Cancer Patients.

Peripheral blood CD8+ T lymphocytes play a crucial role in cell-mediated immunity and tumor-related immune responses in breast cancer. In this study, label-free quantification analysis and gene set enrichment analysis (GSEA) of CD8+ T lymphocytes in the peripheral blood of benign patients and patients with different breast cancer (BC) subtypes, i.e., luminal A, luminal B, and triple-negative breast cancer (TNBC), were performed using nano-UHPLC and Orbitrap mass spectrometry. Differential protein expression in CD8+ T lymphocytes revealed significant downregulation (log2 FC ≥ 0.38 or ≤-0.38, adj. p < 0.05), particularly in proteins involved in cytotoxicity, cytolysis, and proteolysis, such as granzymes (GZMs) and perforin 1 (PRF1). This downregulation was observed in the benign group (GZMH, GZMM, and PRF1) and luminal B (GZMA, GZMH) subtypes, whereas granzyme K (GZMK) was upregulated in TNBC in comparison to healthy controls. The RNA degradation pathway was significantly downregulated (p < 0.05, normalized enrichment score (NES) from -1.47 to -1.80) across all BC subtypes, suggesting a potential mechanism for regulating gene expression during T cell activation. Also, the Sm-like proteins (LSM2, LSM3, and LSM5) were significantly downregulated in the RNA degradation pathway. Proteomic analysis of CD8+ T lymphocytes in peripheral blood across different breast cancer subtypes provides a comprehensive view of the molecular mechanisms of the systemic immune response that can significantly contribute to advancements in the diagnosis, treatment, and prognosis of this disease.

Deciphering the role of KLRB1: a novel prognostic indicator in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) represents a significant global health challenge with high incidence and mortality rates. T cells and natural killer (NK) cells are pivotal in this context, yet HCC can evade immune surveillance. CD161 (KLRB1), a C-type lectin receptor, modulates immune responses and is expressed on NK cells and a subset of T cells. Its relevance in HCC remains poorly understood, with conflicting findings regarding its impact on patient prognosis. METHODS: Utilizing TCGA data and single-cell analysis, we investigated the biological functions of KLRB1 in HCC. Peripheral blood samples from 126 HCC patients were collected to assess KLRB1 expression on NK and T cells. The diagnostic performance of KLRB1 on NK and CD8 + T cells was evaluated using receiver operating characteristic curve (ROC) analysis, while its prognostic significance was assessed using Kaplan-Meier analysis and COX regression models. RESULTS: Analysis of TCGA data revealed a significant correlation between KLRB1 expression and immune activation, particularly T cell activation. Single-cell data further demonstrated elevated KLRB1 expression in tissue-resident NK and T cells within HCC, which co-expressed markers of immune activation. Clinical data showed downregulated KLRB1 expression on NK and T cells in HCC patients compared to health individuals, with lower expression levels correlating with poorer prognosis. CONCLUSION: KLRB1 emerges as a promising biomarker in HCC, with its downregulation on peripheral blood NK and T cells suggesting potential prognostic value. Further elucidation of KLRB1's role in HCC may pave the way for the development of targeted immunotherapies and the improvement of patient outcomes.

PLGA micro/nanoparticle vaccination elicits non-tumor antigen specific resident memory CD8+ T cell protection from hepatocellular carcinoma.

Together, tumor and virus-specific tissue-resident CD8+ memory T cells (TRMs) of hepatocellular carcinoma (HCC) patients with Hepatitis B virus (HBV) infection can provide rapid frontline immune surveillance. The quantity and activity of CD8+ TRMs were correlated with the relapse-free survival of patients with improved health. However, HBV-specific CD8+ TRMs have a more exhausted phenotype and respond more actively under anti-PDL1 or PD1 treatment of HBV+HCC patients. Vaccination strategies that induce a strong and sustained CD8+ TRMs response are quite promising. Herein, a biodegradable poly(D,L-lactide-co-glycolide) microsphere and nanosphere particle (PLGA N.M.P) delivery system co-assembled by anti-PD1 antibodies (aPD1) and loaded with ovalbumin (OVA-aPD1 N.M.P) was fabricated and characterized for size (200 nm and 1 µm diameter), charge (-15 mV), and loading efficiencies of OVA (238 µg mg-1 particles) and aPD1 (40 µg mg-1 particles). OVA-aPD1 N.M.P could stimulate the maturation of BMDCs and enhance the antigen uptake and presentation by 2-fold compared to free OVA. The nanoparticles also induced the activation of macrophages (RAW 264.7) to produce a high level of cytokines, including TNF-α, IL-6 and IL-10. In vivo stimulation of mice using OVA-aPD1 N.M.P robustly enhanced IFN-γ-producing-CD8+ T cell infiltration in tumor tissues and the secretion of IgG and IgG2a/IgG1 antibodies. OVA-aPD1 N.M.P delivered OVA to increase the activation and proliferation of OVA-specific CD8+ TRMs, and its combination with anti-PD1 antibodies promoted complete tumor rejection by the reversal of tumor-infiltrating CD8+ T cell exhaustion. Thus, PLGA N.M.P could induce a strong CD8+ TRMs response, further highlighting its therapeutic potential in enhancing an antitumor immune response.

NCF4 attenuates colorectal cancer progression by modulating inflammasome activation and immune surveillance.

The spatiotemporal regulation of inflammasome activation remains unclear. To examine the mechanism underlying the assembly and regulation of the inflammasome response, here we perform an immunoprecipitation-mass spectrometry analysis of apoptosis-associated speck-like protein containing a CARD (ASC) and identify NCF4/1/2 as ASC-binding proteins. Reduced NCF4 expression is associated with colorectal cancer development and decreased five-year survival rate in patients with colorectal cancer. NCF4 cooperates with NCF1 and NCF2 to promote NLRP3 and AIM2 inflammasome activation. Mechanistically, NCF4 phosphorylation and puncta distribution switches from the NADPH complex to the perinuclear region, mediating ASC oligomerization, speck formation and inflammasome activation. NCF4 functions as a sensor of ROS levels, to establish a balance between ROS production and inflammasome activation. NCF4 deficiency causes severe colorectal cancer in mice, increases transit-amplifying and precancerous cells, reduces the frequency and activation of CD8+ T and NK cells, and impairs the inflammasome-IL-18-IFN-γ axis during the early phase of colorectal tumorigenesis. Our study implicates NCF4 in determining the spatial positioning of inflammasome assembly and contributing to inflammasome-mediated anti-tumor responses.

The Significant Role of PA28αß in CD8+ T Cell-Mediated Graft Rejection Contrasts with Its Negligible Impact on the Generation of MHC-I Ligands.

The proteasome generates the majority of peptides presented on MHC class I molecules. The cleavage pattern of the proteasome has been shown to be changed via the proteasome activator (PA)28 alpha beta (PA28αß). In particular, several immunogenic peptides have been reported to be PA28αß-dependent. In contrast, we did not observe a major impact of PA28αß on the generation of different major histocompatibility complex (MHC) classI ligands. PA28αß-knockout mice infected with the lymphocytic choriomeningitis virus (LCMV) or vaccinia virus showed a normal cluster of differentiation (CD) 8 response and viral clearance. However, we observed that the adoptive transfer of wild-type cells into PA28αß-knockout mice led to graft rejection, but not vice versa. Depletion experiments showed that the observed rejection was mediated by CD8+ cytotoxic T cells. These data indicate that PA28αß might be involved in the development of the CD8+ T cell repertoire in the thymus. Taken together, our data suggest that PA28αß is a crucial factor determining T cell selection and, therefore, impacts graft acceptance.

Exploring the role of CD8+ T cells in clear renal cell carcinoma metastasis.

Clear cell renal cell carcinoma (ccRCC) accounts for approximately 75-80% of all patients with renal cell carcinoma. Despite its prevalence, little is known regarding the key components involved in ccRCC metastasis. In this study, scRNA-seq analysis was employed to classify CD8+ T cells into four sub-clusters based on their genetic profiles and immunofluorescence experiments were used to validate two key clusters. Through gene set enrichment analysis, these newly identified sub-clusters were found to exhibit distinct biological characteristics. Notably, TYMP, TOP2A, CHI3L2, CDKN3, CENPM, and RZH2 were highly expressed in these sub-clusters, indicating a correlation with poor prognosis. Among these sub-clusters, CD8+ T cells (MT-ND4) were identified as potentially playing a critical role in mediating ccRCC metastasis. These results contribute to our understanding of CD8+ T cell heterogeneity in ccRCC and shed light on the mechanisms underlying the loss of immune response against cancer.

The Density of CD8+ Tumor-infiltrating Lymphocytes Correlated With Akt Activation and Ki-67 Index in Canine Soft Tissue Sarcoma.

BACKGROUND/AIM: The activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway has been implicated in canine soft tissue sarcoma (STS) and may serve as a prognostic marker. This study investigated the correlation between PI3K/Akt activation in tumor cells and tumor-infiltrating lymphocytes (TILs). MATERIALS AND METHODS: A total of 59 STS samples were labeled via immunohistochemistry to calculate the density of TILs, including CD3+ T cells, CD8+ T cells, CD20+ B cells, and FOXP3+ regulatory T cells. RESULTS: Forty-eight samples (81.3%) had intra-tumoral TILs with a high density of CD3+ T cells (mean: 283.3 cells/mm2) and CD8+ T cells (mean: 134.8 cells/mm2). Conversely, CD20+ B cells (mean: 73.6 cells/mm2) and FOXP3+ regulatory T cells (mean: 9.2 cells/mm2) were scarce. The abundance of CD3+/CD8+, CD3+/CD20+, and CD8+/CD20+ TILs were highly correlated in multivariate analyses (r=0.895, 0.946, and 0.856, respectively). Nonetheless, TIL density was unrelated to clinicopathological parameters (sex, age, tumor location, breed) and tumor grade. The abundance of CD8+ T cells was positively correlated with the activation of PI3K/Akt, indicating that samples with high levels of phospho-Akt and phospho-S6 tend to have a higher CD8+ T cell density (p=0.0032 and 0.0218, respectively). Furthermore, TIL density was correlated with the Ki-67 index, a tumor proliferation and growth marker. Samples with a high Ki-67 index had a significantly higher abundance of CD3+ T cells, CD8+ T cells, and CD20+ B cells (p=0.0392, 0.0254, 0.0380, respectively). CONCLUSION: PI3K/Akt pathway activation may influence the infiltration of CD8+ T cells within the tumor microenvironment in canine STS. Prospective studies involving a higher number of cases are warranted to confirm these findings.