Exhausted CD8 Research Articles

A spatiotemporal comparative analysis on tumor immune microenvironment characteristics between neoadjuvant chemotherapy and preoperative immunotherapy for ESCC

The average five-year survival rate for esophageal cancer, a common malignant tumor of the digestive system, is barely 20%. The majority of esophageal squamous cell carcinoma (ESCC) patients had already progressed to a locally advanced or even advanced stage at initial diagnosis, making routine surgery ineffective. Chemotherapy and immunotherapy are important neoadjuvant treatments for ESCC, however, it remains unknown how treatment will affect the immunological microenvironment, especially at the spatial level. Here, we presented the TME characters of ESCC from the temporal and spatial dimensions using scRNA-seq and ST, investigated the changes of immune cell clusters in the TME under neoadjuvant chemotherapy and preoperative immunotherapy, and explored the potential mechanisms. It was found that compared with chemotherapy, immunotherapy combined with chemotherapy increased the level of T cell proliferation, partially restored the function of exhausted T cells, induced the expansion of specific exhausted CD8 T cells, increased the production of dendritic cells (DCs), and supported the immune hot microenvironment of the tumor. We also found that CD52 and ID3 have potential as biomarkers of ESCC. Particularly, CD52 may be served as a predictor of the efficacy to screen the advantaged population of different regimens. Through multiple pathways, CAF2 and CAF5’s antigen-presenting role affected the other fibroblast clusters, resulting in malignant transformation. We analyzed the immune microenvironment differences between the two regimens to provide a more thorough description of the ESCC microenvironment profile and serve as a foundation for customized neoadjuvant treatment of ESCC.

Distinct features of immune activation and exhaustion markers in people with perinatally acquired HIV.

The aim of this study was to characterize T-cell activation, exhaustion, maturation and Treg frequencies in individuals who acquire perinatal HIV (PHIV), in individuals who acquired HIV as adult (AHIV), and in healthy controls. This cross-sectional study included people with HIV at least 14 and younger than 40 years, HIV-RNA less than 50 copies/ml on antiretroviral therapy for at least 6 months, and HC. We assessed the expression of PD-1, TIM-3, EOMES, CD38 + DR+, maturation status by CD4 + and CD8 + T cells and the frequency of CD4 + and CD8 + Treg cells. Principal component analysis (PCA) and k-means cluster analysis investigated which combination of immunological parameters better associated with each group. Twenty-six PHIV and 18 AHIV with median ages of 26 (8.0) and 28 (6.8) years were consecutively enrolled. PHIV showed significant higher frequency of naive and lower frequency of terminal effector memory CD4 + and CD8 + T cells than AHIV. AHIV exhibited higher expression of exhaustion and activation markers. The statistical analysis returned two clusters with 94% of specificity and 88% of sensitivity identifying PHIV vs. AHIV. The nine healthy controls had a lower expression of exhaustion markers on both CD4 + and CD8 + T lymphocytes than PHIV and AHIV. These data may exclude major alterations of lymphopoiesis in PHIV, with even lower state of immune-activation and exhaustion compared with AHIV. This suggests that recent lack of virological control, may affect immune activation and exhaustion of CD4 + and CD8 + T cells.

Torque teno viruses exhaust and imprint the human immune system via the HLA-E/NKG2A axis.

The ubiquitous Torque teno virus (TTV) establishes a chronically persistent infection in the human host. TTV has not been associated with any apparent disease, but, as part of the human virome, it may confer a regulatory imprint on the human immune system with as yet unclear consequences. However, so far, only few studies have characterized the TTV-specific immune responses or the overall immunological imprints by TTV. Here, we reveal that TTV infection leads to a highly exhausted TTV-specific CD8+ T-cell response, hallmarked by decreased IFN-γ production and the expression of the inhibitory NKG2A-receptor. On a functional level, we identified a panel of highly polymorphic TTV-encoded peptides that lead to an expansion of regulatory NKG2A+ natural killer, NKG2A+CD4+, and NKG2A+CD8+ T cells via the stabilization of the non-classical HLA-E molecule. Our results thus demonstrate that TTV leads to a distinct imprint on the human immune system that may further regulate overall human immune responses in infectious, autoimmune, and malignant diseases.

Autoantigen-specific CD4+ T cells acquire an exhausted phenotype and persist in human antigen-specific autoimmune diseases

Pro-inflammatory autoantigen-specific CD4+ T helper (auto-Th) cells are central orchestrators of autoimmune diseases (AIDs). We aimed to characterize these cells in human AIDs with defined autoantigens by combining human leukocyte antigen (HLA)-tetramer-based and activation-based multidimensional exvivo analyses. In aquaporin4-antibody-positive neuromyelitis optica spectrum disorder (AQP4-NMOSD) patients, auto-Th cells expressed CD154, but proliferative capacity and pro-inflammatory cytokines were strongly reduced. Instead, exhaustion-associated co-inhibitory receptors were expressed together with FOXP3, the canonical regulatory Tcell (Treg) transcription factor. Auto-Th cells responded invitro to checkpoint inhibition and provided potent B cell help. Cells with the same exhaustion-like (ThEx) phenotype were identified in soluble liver antigen (SLA)-antibody-autoimmune hepatitis and BP180-antibody-positive bullous pemphigoid, AIDs of the liver and skin, respectively. While originally described in cancer and chronic infection, our data point to Tcell exhaustion as a common mechanism of adaptation to chronic (self-)stimulation across AID types and link exhausted CD4+ Tcells to humoral autoimmune responses, with implications for therapeutic targeting.

Blockade of PD-1 and TIM-3 Ameliorates CD8+ T Cell Exhaustion in a Mouse Model of Chronic Myeloid Leukemia.

The immune system plays a pivotal role in controlling chronic myeloid leukemia (CML). CD8+ T cell exhaustion results in reduced effectiveness of T cell-mediated immunity, thereby contributing to disease progression. This study intends to figure out whether the combined blockade of inhibitory molecules TIM-3/PD-1 can affect CD8+ T cell exhaustion in CML. A CML mouse model was established via transplantation of bone marrow cells transduced with BCR-ABL-expressing retrovirus vectors. PD-1 and TIM-3 signaling were blocked using corresponding molecular antibodies. Flow cytometry analysis was conducted to detect cell surface molecules and intracellular cytokines. ELISA was employed for measuring cytokine concentrations in the culture medium. The results showed that TIM-3 and PD-1 were coexpressed on exhausted CD8+ T cells from CML mice. Combined blockade of PD-1/TIM3 synergistically delayed CML progression in mice. Moreover, ex vivo experiments showed that their co-blockade promoted the proliferation and cytokine secretion of CD8+ T cells isolated from CML mice. In conclusion, blocking TIM-3 and PD-1 improves exhausted CD8+ T cell function in CML.

Targeting IL-33 reprograms the tumor microenvironment and potentiates antitumor response to anti-PD-L1 immunotherapy

BackgroundThe main challenge against patients with cancer to derive benefits from immune checkpoint inhibitors targeting PD-1/PD-L1 appears to be the immunosuppressive tumor microenvironment (TME), in which IL-33/ST2 signal fulfills critical...

Intrahepatic Exhausted Antiviral Immunity in an Immunocompetent Mouse Model of Chronic Hepatitis B

Targeting exhausted immune systems would be a promising therapeutic strategy to achieve a functional cure for HBV infection in patients with chronic hepatitis B (CHB). However, animal models recapitulating the immunokinetics of CHB are very limited. We aimed to develop an immunocompetent mouse model of CHB for intrahepatic immune profiling. CHB mice were created by intrahepatic delivery of the Sleeping Beauty transposon vector tandemly expressing the hepatitis B virus (HBV) genome and fumarylacetoacetate hydrolase (FAH) cDNA into C57BL/6J congenic FAH knockout mice via hydrodynamic tail vein injection. We profiled the viral and intrahepatic immune kinetics in CHB mice with or without treatment with recombinant IFNα or the hepatotropic Toll-like receptor 7 agonist SA-5 using single-cell RNA-seq. CHB mice exhibited sustained HBV viremia and persistent hepatitis. They showed intrahepatic expansion of exhausted CD8+ T (Tex) cells, the frequency of which was positively associated with viral load. Recruited macrophages increased in number but impaired inflammatory responses in the liver. The cytotoxicity of mature natural killer (NK) cells also increased in CHB mice. IFNα and SA-5 treatment both resulted in viral suppression with mild hepatic flares in CHB mice. Although both treatments activated NK cells, SA-5 had the capacity to revitalize the impaired function of Tex cells and liver-recruited macrophages. Our novel CHB mouse model recapitulated the intrahepatic exhausted antiviral immunity in patients with CHB, which might be able to be reinvigorated by a hepatotropic TLR7 agonist.

CD19+CD73+ B cells infiltration indicates poor prognosis and unfavorable responses to immunotherapy in gastric cancer

ObjectivesCluster of Differentiation 73 (CD73) is expressed on immune cells and plays a significant role in tumor inhibition by suppressing antitumor immunity. The objectives of this study were to explore the expression and functional mechanisms of CD73 on B cells in patients with gastric cancer (GC). MethodsThe prognostic significance of CD19+CD73+ B cells was evaluated in 390 GC patients through dual immunohistochemistry staining. Flow cytometry was employed to analyze the phenotype of the CD19 subpopulation using fresh tumor and non-tumor tissue samples from 8 GC patients. A bioinformatics analysis of CD19+CD73+ B cells was also performed within the scRNA-seq cohort, and the CD19+ B cell subtype was assessed using multiple immunofluorescence staining. ResultsThe infiltration of CD19+CD73+ B cells was observed to be elevated in gastric cancer (GC) tissue compared to normal tissues. A strong correlation was observed between high CD19+CD73+ B cell infiltration, poor overall survival, and diminished responsiveness to neoadjuvant immunotherapy in GC. These cells emerged as a novel subset of regulatory B cells (Bregs) linked to adenosine metabolism and the exhaustion of CD8+ T cells. The CD19+CD73+ B cells also correlated with the production of immunosuppressive cytokines IL-10 and TGFB1. Further analysis indicated an association between CD19+CD73+ B cells and advanced-stage GC. ConclusionsThe presence of CD19+CD73+ B cells in GC may serve as a prognostic indicator for clinical outcomes and a predictive marker for poor responsiveness to neoadjuvant immunotherapy. The correlation between the presence of CD19+CD73+ B cells and CD8+ T cell exhaustion, along with immunosuppression, highlights the tumor-promoting function of these cells.

Combination anti-PD-1 and anti-CTLA-4 therapy generates waves of clonal responses that include progenitor-exhausted CD8+ T cells

Combination checkpoint blockade with anti-PD-1 and anti-CTLA-4 antibodies has shown promising efficacy in melanoma. However, the underlying mechanism in humans remains unclear. Here, we perform paired single-cell RNA and Tcell receptor (TCR) sequencing across time in 36 patients with stage IV melanoma treated with anti-PD-1, anti-CTLA-4, or combination therapy. We develop the algorithm Cyclone to track temporal clonal dynamics and underlying cell states. Checkpoint blockade induces waves of clonal Tcell responses that peak at distinct time points. Combination therapy results in greater magnitude of clonal responses at 6 and 9weeks compared to single-agent therapies, including melanoma-specific CD8+ Tcells and exhausted CD8+ Tcell (TEX) clones. Focused analyses of TEX identify that anti-CTLA-4 induces robust expansion and proliferation of progenitor TEX, which synergizes with anti-PD-1 to reinvigorate TEX during combination therapy. These next generation immune profiling approaches can guide the selection of drugs, schedule, and dosing for novel combination strategies.

T cell factor 1 (TCF-1) defines T cell differentiation in colorectal cancer

The presence of precursor to exhausted (Tpex) CD8+ Tcells is important to maintain robust immunity following treatment with immune checkpoint inhibition (ICI). Impressive responses to ICI are emerging in patients with stage II-III mismatch repair (MMR)-deficient (dMMR) colorectal cancer (CRC). We found 64% of dMMR and 15% of mismatch repair-proficient (pMMR) stage III CRCs had a high frequency of tumor infiltrating lymphocytes (TIL-hi). Furthermore, expression of TCF-1 (Tcf7) by CD8+ Tcells predicted improved patient prognosis and Tpex cells (CD3+CD8+TCF-1+PD-1+) were abundant within lymphoid aggregatesof stage III CRCs. In contrast, CD3+CD8+TCF-1-PD-1+ cells were more abundant at the invasive front and tumor core, while γδ Tcells were equally abundant in all tumor areas. Interestingly, no differencesin the frequency of Tpex cells were observed between TIL-hi dMMR and TIL-hi pMMR CRCs. Therefore, Tpex cell function and ICI response rates in TIL-hi CRC warrants further investigation.

Somatic mutations in tumor-infiltrating lymphocytes impact on antitumor immunity

Immune checkpoint inhibitors (ICIs) exert clinical efficacy against various types of cancers by reinvigorating exhausted CD8+ T cells that can expand and directly attack cancer cells (cancer-specific T cells) among tumor-infiltrating lymphocytes (TILs). Although some reports have identified somatic mutations in TILs, their effect on antitumor immunity remains unclear. In this study, we successfully established 18 cancer-specific T cell clones, which have an exhaustion phenotype, from the TILs of four patients with melanoma. We conducted whole-genome sequencing for these T cell clones and identified various somatic mutations in them with high clonality. Among the somatic mutations, an SH2D2A loss-of-function frameshift mutation and TNFAIP3 deletion could activate T cell effector functions in vitro. Furthermore, we generated CD8+ T cell-specific Tnfaip3 knockout mice and showed that Tnfaip3 function loss in CD8+ T cell increased antitumor immunity, leading to remarkable response to PD-1 blockade in vivo. In addition, we analyzed bulk CD3+ T cells from TILs in additional 12 patients and identified an SH2D2A mutation in one patient through amplicon sequencing. These findings suggest that somatic mutations in TILs can affect antitumor immunity and suggest unique biomarkers and therapeutic targets.

Identification of the novel exhausted T cell CD8 + markers in breast cancer

Cancer is one of the most concerning public health issues and breast cancer is one of the most common cancers in the world. The immune cells within the tumor microenvironment regulate cancer development. In this study, single immune cell data sets were used to identify marker gene sets for exhausted CD8 + T cells (CD8Tex) in breast cancer. Machine learning methods were used to cluster subtypes and establish the prognostic models with breast cancer bulk data using the gene sets to evaluate the impacts of CD8Tex. We analyzed breast cancer overexpressing and survival-associated marker genes and identified CD8Tex hub genes in the protein–protein-interaction network. The relevance of the hub genes for CD8 + T-cells in breast cancer was evaluated. The clinical associations of the hub genes were analyzed using bulk sequencing data and spatial sequencing data. The pan-cancer expression, survival, and immune association of the hub genes were analyzed. We identified biomarker gene sets for CD8Tex in breast cancer. CD8Tex-based subtyping systems and prognostic models performed well in the separation of patients with different immune relevance and survival. CRTAM, CLEC2D, and KLRB1 were identified as CD8Tex hub genes and were demonstrated to have potential clinical relevance and immune therapy impact. This study provides a unique view of the critical CD8Tex hub genes for cancer immune therapy.

Treatment with oncolytic vaccinia virus infects tumor-infiltrating regulatory and exhausted T cells

BackgroundOncolytic viruses (OVs) are an attractive way to increase immune infiltration into an otherwise cold tumor. While OVs are engineered to selectively infect tumor cells, there is evidence that they...

In vitro-irradiated cancer vaccine enhances anti-tumor efficacy of radiotherapy and PD-L1 blockade in a syngeneic murine breast cancer model

Background and PurposeLocal radiotherapy (RT) exerts immunostimulatory effects by inducing immunogenic cell death. However, it remains unknown whether in vitro–irradiated tumor cells can elicit anti-tumor responses and enhance the efficacy of local RT and immune checkpoint inhibitors when injected in vivo. Methods and MaterialsWe tested the “in vitro–irradiated cancer vaccine (ICV)”, wherein tumor cells killed by varying doses of irradiation and their supernatants are intravenously injected. We examined the efficacy of combining local RT (24 Gy in three fractions), PD-L1 blockade, and the ICV in a murine breast cancer model. The immune cell profiles were analyzed via flow cytometry and immunohistochemistry. The cytokine levels were measured by multiplex immunoassays. ResultsThe ICV significantly increased the effector memory phenotype and interferon-γ production capacity in splenic CD8+ T cells. The in vitro–irradiated products contained immune response-related molecules. When combined with local RT and PD-L1 blockade, the ICV significantly delayed the growth of irradiated and non-irradiated tumors. The triple combination therapy increased the proportions of CD8+ T cells and effector memory CD8+ T cells while decreasing the proportion of CTLA-4+ exhausted CD8+ T cells within tumor microenvironment. Additionally, plasma level of interferon-γ and proliferation of effector T cells in the spleen and tumor-draining lymph nodes were significantly increased by the triple combination therapy. ConclusionsThe ICV enhanced the therapeutic efficacy of local RT and PD-L1 blockade by augmenting anti-tumor immune responses. Our findings suggest a therapeutic potential of in vitro–irradiation products of tumor cells.

EPDR1 promotes PD-L1 expression and tumor immune evasion by inhibiting TRIM21-dependent ubiquitylation of IkappaB kinase-β.

While immune checkpoint blockade (ICB) has shown promise for clinical cancer therapy, its efficacy has only been observed in a limited subset of patients and the underlying mechanisms regulating innate and acquired resistance to ICB of tumor cells remain poorly understood. Here, we identified ependymin-related protein 1 (EPDR1) as an important tumor-intrinsic regulator of PD-L1 expression and tumor immune evasion. Aberrant expression of EPDR1 in hepatocellular carcinoma is associated with immunosuppression. Mechanistically, EPDR1 binds to E3 ligase TRIM21 and disrupts its interaction with IkappaB kinase-b, suppressing its ubiquitylation and autophagosomal degradation and enhancing NF-κB-mediated transcriptional activation of PD-L1. Further, we validated through a mouse liver cancer model that EPDR1 mediates exhaustion of CD8+ T cells and promotes tumor progression. In addition, we observed a positive correlation between EPDR1 and PD-L1 expression in both human and mouse liver cancer samples. Collectively, our study reveals a previously unappreciated role of EPDR1 in orchestrating tumor immune evasion and cancer progression.

Survival benefit and spatial properties of tertiary lymphoid structures in esophageal squamous cell carcinoma with neoadjuvant therapies

Tertiary lymphoid structures (TLSs) were associated with survival in esophageal squamous cell carcinoma (ESCC) undergoing surgery alone (SA). However, their clinical relevance in neoadjuvant therapies remains less known. Here, we firstly investigated the presence, maturation and spatial distribution of TLSs in 359 ESCC patients receiving neoadjuvant chemotherapy (NCT), neoadjuvant immunotherapy (NCI), neoadjuvant chemoradiotherapy (NCRT) or SA. We found mature TLS (MTLS) was an independent prognostic factor in ESCC. NCI group had the lowest immature TLS cases. NCRT group had the lowest MTLSs. MTLSs mostly located in stromal and normal compartments; these MTLSs were positively correlated with neoadjuvant therapy outcomes. NCI group displayed the highest T cells within 150 μm proximity of TLSs among the four groups. Most T cells were dispersed up to more than 150 μm from TLSs, while B cells remained concentrated within TLSs. Innate lymphoid cells and follicular dendritic cells infiltrated and connected with survival differently in NCRT and NCI groups compared with SA group. The novel PD-L1 combined positive score, NCPS, was positively connected with MTLSs and neoadjuvant therapy efficacy. ScRNA-seq analysis revealed TLS+ tumors had increased plasma cells, B cells, Th17, Tfh and Th1, and elevated exhausted CD8+ T cells that highly expressed checkpoint molecules and granzymes. Conclusively, MTLSs favored treatment outcome in ESCC patients receiving multiple neoadjuvant therapies. The spatial distribution of MTLSs was associated with multiregional immune status modified by the neoadjuvant therapies.

Investigation of a tamoxifen-RACK7/KDM5C-IFN-I axis for ER+ breast cancer immunomodulation.

9 Background: ER+ breast cancer patients generally have good prognosis. However, significant relapse rates (i.e., hormone drugs, Tamoxifen/TMX) and the poor response to immune checkpoint blockade (ICB) remain critical issues to be addressed. Thus, exploration of anti-ICB resistant mechanisms and alternative targets is needed to improve therapeutic efficacy. Methods: To investigate potential cross-resistant mechanisms between hormone and ICB therapy, ER+ cell lines T47D and MCF7 cells were chronically-treated with TMX for 6 months, and subjected to RNA-Seq and pathway enrichment analysis. Dysregulation of identified factors were validated via western blotting, RT-qPCR, and flow cytometry. Target factors were then inhibited or depleted by shRNA knockdown (KD) or small-compound inhibition. Identified ICB resistance mechanisms were further elaborated by in vitro co-culture assays, syngeneic mouse models, and immune-profiling of tumor microenvironment (TME). Results: Chronic exposure to TMX activates Type I interferon (IFN-I) signaling, induces IFN I-stimulated gene (ISG) expression, and downregulates the H3K4 demethylase and ISG repressor complex known as RACK7/KDM5C. Chronic TMX also upregulates CEACAM1, a well-known ligand for the immune checkpoint receptor TIM3. Results prompt us to evaluate whether loss of RACK7 combined with TMX treatment may modulate a lymphocyte-attractive (via IFN-I), but T-cell exhaustive (via CEACAM1-TIM3) TME. Indeed, in vitro treatment of TMX under RACK7-KD conditions triggers STING upregulation, TBK1 hyperphosphorylation, and a more pronounced activation of ISGs and CEACAM1. In vivo, TMX combined with RACK7-KD enhances tumor growth in the TS/A (ER+) syngeneic mouse model, while immune-profiling reveal that this combination promotes lymphocyte infiltration with a higher population of terminally exhausted CD8+ T-cells in the TME. Conclusions: Our data demonstrate that the TMX-induced activation of cGAS/STING pathway and RACK7 downregulation coordinately shape the IFN-I and immune checkpoint signaling axes in ER+ breast cancer. This TMX–RACK7–IFN-I regulatory network promotes a lymphocyte-attractive TME via IFN-I signaling activation, whereas induction of inhibitory ligands renders T-cells to be terminally-exhausted and unable to kill tumors. These findings highlight the potential use of TMX’s STING-agonistic effect in re-shaping the “cold” breast cancer TME, and the use of combined TMX and anti-TIM3 or anti-CEACAM1 blockade to improve ICB therapy.

Tumor-associated macrophage clusters linked to immunotherapy in a pan-cancer census

Transcriptional heterogeneity of tumor-associated macrophages (TAMs) has been investigated in individual cancers, but the extent to which these states transcend tumor types and represent a general feature of cancer remains unclear. We performed pan-cancer single-cell RNA sequencing analysis across nine cancer types and identified distinct monocyte/TAM composition patterns. Using spatial analysis from clinical study tissues, we assessed TAM functions in shaping the tumor microenvironment (TME) and influencing immunotherapy. Two specific TAM clusters (pro-inflammatory and pro-tumor) and four TME subtypes showed distinct immunological features, genomic profiles, immunotherapy responses, and cancer prognosis. Pro-inflammatory TAMs resided in immune-enriched niches with exhausted CD8+ T cells, while pro-tumor TAMs were restricted to niches associated with a T-cell-excluded phenotype and hypoxia. We developed a machine learning model to predict immune checkpoint blockade response by integrating TAMs and clinical data. Our study comprehensively characterizes the common features of TAMs and highlights their interaction with the TME.

Cholesterol Depletion-Enhanced Ferroptosis and Immunotherapy via Engineered Nanozyme.

Ferroptosis, an iron- and reactive oxygen species (ROS)-dependent cell death, holds significant promise for tumor therapy due to its ability to induce lipid peroxidation (LPO) and trigger antitumor immune responses. However, elevated cholesterol levels in cancer cells impede ferroptosis and compromise immune function. Here, a novel nanozyme, Fe-MOF/CP, composed of iron metal-organic framework (Fe-MOF) nanoparticles loaded with cholesterol oxidase and PEGylation for integrated ferroptosis and immunotherapy is introduced. Fe-MOF/CP depletes cholesterol and generates hydrogen peroxide, enhancing ROS levels and inducing LPO, thereby promoting ferroptosis. This process disrupts lipid raft integrity and downregulates glutathione peroxidase 4 and ferroptosis suppressor protein 1, further facilitating ferroptosis. Concurrently, Fe-MOF/CP augments immunogenic cell death, reduces programmed death-ligand 1 expression, and revitalizes exhausted CD8+ T cells. In vivo studies demonstrate significant therapeutic efficacy in abscopal, metastasis, and recurrent tumor models, highlighting the robust antitumor immune responses elicited by Fe-MOF/CP. This study underscores the potential of Fe-MOF/CP as a multifunctional therapeutic agent that combines ferroptosis and immunotherapy, offering a promising strategy for effective and durable cancer treatment.

Unlocking the potential of HHLA2: identifying functional immune infiltrating cells in the tumor microenvironment and predicting clinical outcomes in laryngeal squamous cell carcinoma

BackgroundHHLA2 (human endogenous retrovirus-H long terminal repeat-associating protein 2) represents a recently identified member of the B7 immune checkpoint family, characterized by limited expression in normal tissues but notable overexpression in various cancer types. Nevertheless, the precise function and interaction with immune cells remain poorly understood, particularly in laryngeal squamous cell carcinoma (LSCC). This investigation endeavored to elucidate the biological significance of HHLA2 within the tumor microenvironment of human LSCC tissues and delineate the clinical relevance and functional roles of HHLA2 in LSCC pathogenesis.MethodsThrough multiplexed immunohistochemistry analyses conducted on tissue microarrays sourced from LSCC patients (n = 72), the analysis was executed to assess the expression levels of HHLA2, density and spatial patterns of CD68+HLA-DR+CD163− (M1 macrophages), CTLA-4+CD4+FoxP3+ (CTLA-4+Treg cells), CTLA-4+CD4+FoxP3− (CTLA-4+Tcon cells), exhausted CD8+T cells, and terminally exhausted CD8+T cells in LSCC tissues. Survival analysis was conducted to evaluate the prognostic significance of HHLA2 and these immune checkpoints or immune cell populations, employing COX regression analysis to identify independent prognostic factors.ResultsKaplan–Meier (K–M) survival curves revealed a significant association between HHLA2 expression and overall survival (OS) in LSCC. Elevated levels of HHLA2 were linked to reduced patient survival, indicating its potential as a prognostic marker (HR: 3.230, 95%CI 0.9205–11.34, P = 0.0067). Notably, increased infiltration of CD68+ cells (total macrophages), STING+CD68+HLA-DR+CD163− (STING+M1 macrophages), CTLA-4+CD4+FoxP3+, CTLA-4+CD4+FoxP3−, PD-1+LAG-3+CD8+T cells, and PD-1+LAG-3+TIM-3+CD8+T cells strongly linked to poorer survival outcomes (P < 0.05). A discernible trend was observed between the levels of these immune cell populations, STING+CD68+ (STING+ total macrophages), CD68+HLA-DR+CD163−, STING+CD68+CD163+HLA-DR− (STING+M2 macrophages), PD-1+LAG-3−CD8+T cells, PD-1+TIM-3+CD8+T cells, and PD-1+LAG-3+TIM-3−CD8+T cells and prognosis. Importantly, multivariate COX analysis identified HHLA2 as an independent predictive factor for OS in LSCC patients (HR = 3.86, 95%CI 1.08–13.80, P = 0.038). This underscored the potential of HHLA2 as a critical marker for predicting patient outcomes in LSCC.ConclusionsHHLA2 emerged as a detrimental prognostic biomarker for assessing OS in LSCC patients. Relative to other immune checkpoints, HHLA2 exhibited heightened predictive efficacy for the prognosis of LSCC patients.