TIM-3 expression characterizes regulatory T cells in tumor tissues and is associated with lung cancer progression

Background

T cell immunoglobulin-3 (TIM-3) has been established as a negative regulatory molecule and plays a critical role in immune tolerance. TIM-3 is upregulated in exhausted CD8⁺ T cells in both chronic infection and tumor. However, the nature of TIM-3⁺CD4⁺ T cells in the tumor microenvironment is unclear. This study is to characterize TIM-3 expressing lymphocytes within human lung cancer tissues and establish clinical significance of TIM-3 expression in lung cancer progression.

Methodology

A total of 51 human lung cancer tissue specimens were obtained from pathologically confirmed and newly diagnosed non-small cell lung cancer (NSCLC) patients. Leukocytes from tumor tissues, distal normal lung tissues, and peripheral blood mononuclear cells (PBMC) were analyzed for TIM-3 surface expression by flow cytometry. TIM-3 expression on tumor-infiltrating lymphocytes (TILs) was correlated with clinicopathological parameters.

Conclusions

TIM-3 is highly upregulated on both CD4⁺ and CD8⁺ TILs from human lung cancer tissues but negligibly expressed on T cells from patients'' peripheral blood. Frequencies of IFN-γ⁺ cells were reduced in TIM-3⁺CD8⁺ TILs compared to TIM-3⁻CD8⁺ TILs. However, the level of TIM-3 expression on CD8⁺ TILs failed to associate with any clinical pathological parameter. Interestingly, we found that approximately 70% of TIM-3⁺CD4⁺ TILs expressed FOXP3 and about 60% of FOXP3⁺ TILs were TIM-3⁺. Importantly, TIM-3 expression on CD4⁺ T cells correlated with poor clinicopathological parameters of NSCLC such as nodal metastasis and advanced cancer stages. Our study reveals a new role of TIM-3 as an important immune regulator in the tumor microenvironment via its predominant expression in regulatory T cells.     

B7-H4 expression associates with cancer progression and predicts patient’s survival in human esophageal squamous cell carcinoma

A retrospective cohort study including 112 patients suffering from esophageal squamous cell carcinoma (ESCC) was performed to investigate the expression of B7-H4 in ESCC and determine its association with patient’s clinicopathological parameters and survival. Expression levels of B7-H4 on tumor cells and densities of tumor infiltrating lymphocytes (TILs) in the surgical specimens of ESCC tissues were characterized using immunohistochemical assays. Uni- and multivariate analyses were performed to evaluate the prognostic value of B7-H4 expression levels and densities of TILs in tumor sections. Positive B7-H4 immunostaining was observed in 107 of 112 (95.5%) of ESCC tissue sections. We further divided all patients into two major subgroups, a lower B7-H4 expression group with 46 patients and a higher B7-H4 expression group with 66 patients. We found that expression levels of B7-H4 on tumor cells were significantly correlated with patient’s gender (P = 0.0288), distant metastasis (P = 0.0500), and TNM stage (P = 0.0258). Moreover, tumor cell B7-H4 expression was inversely correlated with densities of CD3⁺ T cells in tumor nest (P = 0.0424) and CD8⁺ T cells in tumor stroma (P = 0.0229). The overall survival rate of the patients with higher B7-H4 expression was significantly worse than that of the patients with lower B7-H4 expression (P = 0.0105, Hazard Ratio: 1.854, 95%CI:1.152–2.902). Markers of cell-mediated immune responses such as CD3, CD8, and T-bet were associated with better patient survival. The present study demonstrated that B7-H4 expression in human ESCC is associated with cancer progression, reduced tumor immunosurveillance and worse patient outcomes. B7-H4 can serve as a novel prognostic predictor for human ESCC and a potential target for the immune therapy against this malignancy.     

Decreased expression of interleukin-36α correlates with poor prognosis in hepatocellular carcinoma

Interleukin-36α (IL-36α) has been found to have a prominent role in the pathogenesis of inflammatory disorders; however, little is known about the role of IL-36α in cancer. In this study, we investigated the expression, prognostic value, and the underlying antitumor mechanism of IL-36α in hepatocellular carcinoma (HCC). From immunohistochemistry analysis, IL-36α expression was lower in poorly differentiated HCC cells. In clinicopathological analysis, low IL-36α expression significantly correlated with tumor size, histological differentiation, tumor stage, and vascular invasion, and low intratumoral IL-36α expression had significantly worse overall survival rates and shorter disease-free survival rates. Moreover, intratumoral IL-36α expression was an independent risk factor for overall survival. Consecutive sections were used to detect CD3⁺, CD8⁺, and CD4⁺ tumor-infiltrating lymphocytes (TILs), and we found that high-IL-36α-expressing tumor tissues exhibited a significantly higher proportion of intratumoral CD3⁺ and CD8⁺ TILs, but not CD4⁺ TILs. Our in vitro model confirmed that supernatant from IL-36α-overexpressing human HCC cells had an increased capacity to recruit CD3⁺ and CD8⁺ T cells. Consistently, mouse HCC cells engineered to overexpress IL-36α demonstrated markedly delayed growth in vivo, as well as higher levels of intratumoral CD3⁺ and CD8⁺ TILs, compared with control mice. In vitro chemotaxis analysis also showed that mouse HCC cells overexpressing IL-36α could recruit more number of CD3⁺ and CD8⁺ T cells. These results show that IL-36α expression may play a pivotal role in determining the prognosis of patients with HCC, which we attribute to the activation of adaptive T cell immunity, especially CD8⁺ T cell immune response.     

CD8+ tumor-infiltrating lymphocytes predict favorable prognosis in malignant pleural mesothelioma after resection

Defects in human leukocyte antigen (HLA) class I expression may allow tumor cells to escape immune recognition. T cell infiltration is associated with a good prognosis in many cancers. However, the role of HLA class I expression and tumor-infiltrating lymphocytes (TILs) in malignant pleural mesothelioma (MPM) has not been fully analyzed. In the present study, we investigated the immune profiles and conducted outcome analyses of MPM patients. HLA class I expression and TILs (CD4⁺, CD8⁺, and NK cells) were detected by immunohistochemistry in a series of 44 MPM cases. To detect HLA class I expression, specimens were stained with the anti-pan HLA class I monoclonal antibody EMR8-5. The expression of HLA class I was positive in all patients. There was no case that showed negative HLA class I expression. The density of CD4⁺ and CD8⁺ TILs were strongly correlated (R = 0.76, p < 0.001). A high density of CD8⁺ TILs was a significantly better prognostic factor for the survival of patients with extrapleural pneumonectomy (p < 0.05). Multivariate analysis revealed that a high density of CD8⁺ TILs is an independent prognostic factor for patients who underwent extrapleural pneumonectomy. The presence of intratumoral CD8⁺ T cells was correlated with an improved clinical outcome, raising the possibility that CD8⁺ T cells might play a pivotal role in the antitumor immune response against MPMs. Thus, the stimulation of CD8⁺ lymphocytes might be an efficacious immunotherapy for MPM patients.     

IRF4 Regulates the Ratio of T-Bet to Eomesodermin in CD8+ T Cells Responding to Persistent LCMV Infection

CD8⁺ T cell exhaustion commonly occurs in chronic infections and cancers. During T cell exhaustion there is a progressive and hierarchical loss of effector cytokine production, up-regulation of inhibitory co-stimulatory molecules, and eventual deletion of antigen specific cells by apoptosis. A key factor that regulates T cell exhaustion is persistent TCR stimulation. Loss of this interaction results in restoration of CD8⁺ T cell effector functions in previously exhausted CD8⁺ T cells. TCR stimulation is also important for the differentiation of Eomes^hi anti-viral CD8⁺ effector T cells from T-bet^hi precursors, both of which are required for optimal viral control. However, the molecular mechanisms regulating the differentiation of these two cell subsets and the relative ratios required for viral clearance have not been described. We show that TCR signal strength regulates the relative expression of T-bet and Eomes in antigen-specific CD8⁺ T cells by modulating levels of IRF4. Reduced IRF4 expression results in skewing of this ratio in the favor of Eomes, leading to lower proportions and numbers of T-bet⁺ Eomes^- precursors and poor control of LCMV-clone 13 infection. Manipulation of this ratio in the favor of T-bet restores the differentiation of T-bet⁺ Eomes^- precursors and the protective balance of T-bet to Eomes required for efficient viral control. These data highlight a critical role for IRF4 in regulating protective anti-viral CD8⁺ T cell responses by ensuring a balanced ratio of T-bet to Eomes, leading to the ultimate control of this chronic viral infection.     

Critical role of spatial interaction between CD8+ and Foxp3+ cells in human gastric cancer: the distance matters

Purpose

In various cancer types, an abundance of FoxP3⁺ regulatory T cells (Treg) has been associated with an unfavorable outcome. Yet, the role of Treg on cancer immunity has been shown to be complex. In single cell marker technique, other tumor-infiltrating lymphocytes (TILs) such as cytotoxic CD8⁺ T cells (CTL) also influenced prognosis. This study for the first time investigates the concurrent spatial distribution pattern of CD8⁺ and FoxP3⁺ TILs and their prognostic impact in human gastric cancer.

Materials and methods

Tumor tissue microarrays of 50 patients with surgically treated adenocarcinoma of the cardia were studied. An immunohistochemical double staining of CD8⁺ and FoxP3⁺ TILs was performed. Cell counts and cell-to-cell distances in tumor epithelium and stroma were evaluated with image-processing software. Metastasis-free survival, no-evidence-of-disease survival, and overall survival were investigated (mean follow-up time 6.9 years).

Results

High intraepithelial infiltration of CD8⁺ and FoxP3⁺ TIL was associated with the improved 10-year metastasis-free survival (83 vs. 54 %, p = 0.04 and 85 vs. 59 %, p = 0.09, respectively). Considering cell-to-cell distance and comparing patients with functional (30–110 μm) versus nonfunctional distances of CD8⁺ and FoxP3⁺ TILs, 10-year survival rates differed between 89 and 55 % (p = 0.009), respectively.

Conclusion

Prognostic influence of tumor-infiltrating immune cells in gastric cancer critically depends on their cell-to-cell distance. FoxP3⁺ TILs must be located within a distance between 30 and 110 μm of CD8⁺ T cells to positively impact on prognosis.     

Generation of colon cancer–derived tumor-infiltrating T cells (TILs) for adoptive cell therapy

Adoptive cell therapy (ACT) using specific immune cells and stem cells has emerged as a promising treatment option that could complement traditional cancer therapies in the future. In particular, tumor-infiltrating lymphocytes (TILs) have been shown to be effective against solid tumors in various clinical trials. Despite the enormous disease burden and large number of premature deaths caused by colorectal cancer (CRC), studies on TILs isolated from tumor tissue of patients with CRC are still rare. To date, studies on ACT often lack controlled and comparable expansion processes as well as selected ACT-relevant T-cell populations. We describe a procedure for generating patient-specific TILs, which are prerequisites for clinical trials of ACT in CRC. The manufacturing and characteristics of these TILs differ in important modalities from TILs commonly used for this therapeutic approach. Tumor tissue samples were obtained from 12 patients undergoing surgery for primary CRC, predominantly with low microsatellite instability (pMMR-MSI-L). Tumors in the resected specimens were examined pathologically, and an approved volume of tumor tissue was transferred to a disposable perfusion bioreactor. Tissue samples were subjected to an automatically controlled and highly reproducible cultivation process in a GMP-conform, closed perfusion bioreactor system using starting medium containing interleukin-2 and interleukin-12. Outgrowth of TIL from tissue samples was initiated by short-term supplementation with a specific activation cocktail. During subsequent expansion, TILs were grown in interleukin-2–enriched medium. Expansion of TILs in a low-scaled, two-phase process in the Zellwerk ZRP bioreactor under hyperoxic conditions resulted in a number of approximately 2 × 10⁹ cells. The expanded TILs consisted mainly (73%) of the ACT-relevant CD3⁺/CD8⁺ effector memory phenotype (CD45RO⁺/CCR7⁻). TILs harvested under these conditions exhibited high functional potential, which was confirmed upon nonspecific stimulation (interferon-γ, tumor necrosis factor-α cytokine assay)     

Increased tumor-infiltrating CD8+Foxp3+ T lymphocytes are associated with tumor progression in human gastric cancer

Background

CD8⁺Foxp3⁺ T lymphocytes have been detected in tumors. However, the distribution, phenotypic features, and regulation of these cells in gastric cancer remain unknown.

Methods

The levels of CD8⁺Foxp3⁺ T lymphocytes in the peripheral blood, tumor-draining lymph nodes, non-tumor tissues, and tumor tissues of patients with gastric cancer were detected by flow cytometry. Foxp3 induction in CD8⁺Foxp3^? T cells was investigated in vitro. The suppressive function of CD8⁺Foxp3⁺ T lymphocytes was analyzed by their effect on CD4⁺ T-cell proliferation and IFN-γ production. The percentages of CD8⁺Foxp3⁺ T lymphocytes were evaluated for the association with tumor stage.

Results

The frequency of CD8⁺Foxp3⁺ T lymphocytes in tumor tissues was significantly higher than that in non-tumor tissues, and similar results were also observed in tumor-draining lymph nodes compared with peripheral blood. Most intratumoral CD8⁺Foxp3⁺ T lymphocytes were activated effector cells (CD45RA^?CD27^?). TGF-β1 levels were positively correlated with the frequency of CD8⁺Foxp3⁺ T lymphocytes in tumor tissues, and in vitro TGF-β1 could induce the generation of CD8⁺Foxp3⁺ T lymphocytes in a dose-dependent manner. Furthermore, intratumoral CD8⁺Foxp3⁺ T lymphocytes suppressed the proliferation and IFN-γ production of CD4⁺ T cells. Finally, intratumoral CD8⁺Foxp3⁺ T lymphocytes were significantly increased with tumor progression in terms of tumor-node-metastasis (TNM) stage.

Conclusions

Our data have shown that increased intratumoral CD8⁺Foxp3⁺ T lymphocytes are associated with tumor stage and potentially influence CD4⁺ T-cell functions, which may provide insights for developing novel immunotherapy protocols against gastric cancer.     

Immune and Inflammatory Cell Composition of Human Lung Cancer Stroma

Recent studies indicate that the abnormal microenvironment of tumors may play a critical role in carcinogenesis, including lung cancer. We comprehensively assessed the number of stromal cells, especially immune/inflammatory cells, in lung cancer and evaluated their infiltration in cancers of different stages, types and metastatic characteristics potential. Immunohistochemical analysis of lung cancer tissue arrays containing normal and lung cancer sections was performed. This analysis was combined with cyto-/histomorphological assessment and quantification of cells to classify/subclassify tumors accurately and to perform a high throughput analysis of stromal cell composition in different types of lung cancer. In human lung cancer sections we observed a significant elevation/infiltration of total-T lymphocytes (CD3⁺), cytotoxic-T cells (CD8⁺), T-helper cells (CD4⁺), B cells (CD20⁺), macrophages (CD68⁺), mast cells (CD117⁺), mononuclear cells (CD11c⁺), plasma cells, activated-T cells (MUM1⁺), B cells, myeloid cells (PD1⁺) and neutrophilic granulocytes (myeloperoxidase⁺) compared with healthy donor specimens. We observed all of these immune cell markers in different types of lung cancers including squamous cell carcinoma, adenocarcinoma, adenosquamous cell carcinoma, small cell carcinoma, papillary adenocarcinoma, metastatic adenocarcinoma, and bronchioloalveolar carcinoma. The numbers of all tumor-associated immune cells (except MUM1⁺ cells) in stage III cancer specimens was significantly greater than those in stage I samples. We observed substantial stage-dependent immune cell infiltration in human lung tumors suggesting that the tumor microenvironment plays a critical role during lung carcinogenesis. Strategies for therapeutic interference with lung cancer microenvironment should consider the complexity of its immune cell composition.     

Tumor Microenvironment Macrophage Inhibitory Factor Directs the Accumulation of Interleukin-17-producing Tumor-infiltrating Lymphocytes and Predicts Favorable Survival in Nasopharyngeal Carcinoma Patients

The accumulation of an intratumoral CD4⁺ interleukin-17-producing subset (Th17) of tumor-infiltrating lymphocytes (TILs) is a general characteristic in many cancers. The relationship between the percentage of Th17 cells and clinical prognosis differs among cancers. The mechanism responsible for the increasing percentage of such cells in NPC is still unknown, as is their biological function. Here, our data showed an increase of Th17 cells in tumor tissues relative to their numbers in normal nasopharynx tissues or in the matched peripheral blood of NPC patients. Th17 cells in tumor tissue produced more IFNγ than did those in the peripheral blood of matched NPC patients and healthy controls. We observed high levels of CD154, G-CSF, CXCL1, IL-6, IL-8, and macrophage inhibitory factor (MIF) out of 36 cytokines examined in tumor tissue cultures. MIF promoted the generation and recruitment of Th17 cells mediated by NPC tumor cells in vitro; this promoting effect was mainly dependent on the mammalian target of rapamycin pathway and was mediated by the MIF-CXCR4 axis. Finally, the expression level of MIF in tumor cells and in TILs was positively correlated in NPC tumor tissues, and the frequency of MIF-positive TILs was positively correlated with NPC patient clinical outcomes. Taken together, our findings illustrate that tumor-derived MIF can affect patient prognosis, which might be related to the increase of Th17 cells in the NPC tumor microenvironment.     

Recruitment of CCR6+ Foxp3+ regulatory gastric infiltrating lymphocytes in Helicobacter pylori gastritis

Helicobacter pylori (H. pylori) infection is associated with an inflammatory response in the gastric mucosa, leading to chronic gastritis, peptic ulcers, and gastric cancer. Increased T‐cell infiltration is found at sites of H. pylori infection. The CCR6⁺ subset of CD4⁺ regulatory T cells (Tregs), a newly characterized subset of Tregs, has been reported to contribute to local immune inhibition. However, whether CCR6⁺ Tregs are present in H. pylori gastritis, and what their relationship is to disease prognosis, remains to be elucidated. In this study, gastric infiltrating lymphocytes were isolated from endoscopic biopsy specimens of H. pylori gastritis patients and analyzed. We found that in gastric infiltrating lymphocytes, CCR6⁺ CD4⁺ CD25^high Tregs, which express high levels of CD45RO, are positively associated with more severe inflammation in gastric mucosa during H. pylori infection. Furthermore, the frequency of CCR6⁺ Tregs in gastric infiltrating lymphocytes, but not CCR6^? Tregs, is significantly increased in inflamed gastric tissues, which is inversely correlated with significantly lower expression of IFN‐γ⁺ CD8⁺ T cells. We also found that the frequency of CCR6⁺ Tregs is positively correlated with the frequency of CD4⁺ IFN‐γ⁺ T cells. In addition, the frequency of CCR6⁺ Tregs, but not that of CCR6^? Tregs, is significantly correlated with increased inflammation in H. pylori gastritis. This study demonstrates that immunosuppression in H. pylori gastritis might be related to the activity of CCR6⁺ Tregs, which could influence disease prognosis.     

Circulating and tumor-infiltrating Foxp3⁺ regulatory T cell subset in Chinese patients with extranodal NK/T cell lymphoma

Foxp3⁺ regulatory T lymphocytes (Tregs) usually act as an immune suppressor and correlate with poorer survival in malignancies. This study aims to investigate the distribution and characterization of Foxp3⁺ subset in peripheral blood mononuclear cells (PBMCs) and tumor tissues from extranodal NK/T cell lymphoma (ENKTL). Our study showed the percentage of Foxp3⁺ subset from PBMC was significantly higher than that of healthy individuals (P<0.001). The Foxp3⁺ subset from PBMCs expressed CD45RO, CTLA4, GITR, CCR7, and had an IL-10^highIFNγ⁺TGFβ⁺IL-2^lowIL-17^low cytokine secreting phenotype. Interestingly, the existence of EBV antigen-specific CD8⁺Foxp3⁺ Tregs was discovered in ENKTL. Furthermore, the high density of Foxp3⁺ TILs was associated with improved progression-free survival (PFS) in ENKTL patients (P<0.05). Collectively, our study implicates that EBV antigens could induce antigen-specific CD8⁺Foxp3⁺ Tregs in ENKTL, and Foxp3⁺ TILs is an independent factor for PFS in ENKTL.     

Coupling programmed cell death 1-positive tumor-infiltrating T cells with anti-programmed cell death 1 antibody improves the efficacy of adoptive T-cell therapy

Background aimsAdoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TILs) has shown great success in clinical trials. Programmed cell death 1 (PD-1)-expressing TILs show high specificity to autologous tumor cells. However, limited therapeutic efficiency is observed as a result of the tumor immune microenvironment (TIME).MethodsCoupling PD-1⁺ ex vivo-derived TILs with a monoclonal antibody against anti-PD-1 (aPD-1) reinvigorated the anti-tumor response of TILs against solid tumor without altering their high tumor targeting ability.ResultsUsing a melanoma-bearing mouse model, PD-1⁺ TILs blocked with aPD-1 (PD-1⁺ TILs-aPD-1) exhibited a high capability for tumor targeting as well as improved anti-tumor response in TIME. Tumor growth was substantially delayed in the mice treated with PD-1⁺ TILs-aPD-1.ConclusionsThe strategy utilizing TIL therapy coupled with immune checkpoint antibodies may extend to other therapeutic targets of ACT.     

Phenotypic characterization of chronic inflammation in a rare case of endobronchial carcinoma

This report presents a phenotypical characterization of the immune cell infiltrate in a rare case of endobronchial carcinoma. A patient initially treated for an adenocarcinoma of the esophagus developed an endobronchial carcinoma surrounded by gastric metaplasia distal to a suspected gastrobronchial fistula, 11 years after esophagectomy. Our hypothesis is that the sustained exposure of the bronchial mucosa to a mixed acid and pancreatobiliary refluxate led to chronic inflammation and promoted malignant transformation. We performed an immunohistochemical study of the tumor microenvironment evaluating the density of CD3⁺, CD8⁺ T lymphocytes, CD20⁺ B lymphocytes, CD68⁺ macrophages and FoxP3⁺ regulatory T cells. Quantification of immune cell density was completed using a novel software-based analysis method. Our results suggest that, within all the tissues analyzed, FoxP3⁺ regulatory T cells were present at their highest density in the malignant and metaplastic tissues. The endobronchial metaplasia biopsied several years prior to the detection of the endobronchial adenocarcinoma was already densely infiltrated by B cells and macrophages, when compared to the immune cell infiltrate of the endobronchial carcinoma. Altogether, these observations support the current understanding of carcinogenesis promoted by chronic inflammation.