Clinical significance of regulatory T cells and CD8+ effector populations in patients with human endometrial carcinoma

BACKGROUND:

A study was carried out to determine the functional attributes of CD4⁺ CD25⁺ regulatory T cells in cancer progression by suppressing antitumor immunity.

METHODS:

Triple‐color flow cytometry was used to study the phenotype expression of CD4⁺ CD25⁺ regulatory T cells and CD8⁺ T cells in the peripheral blood lymphocytes (PBLs) and tumor‐infiltrating lymphocytes (TILs) of 57 cases of stage I to IV endometrial carcinoma. The expression of T cell subsets was correlated with clinical prognostic parameters.

RESULTS:

The prevalence of CD4⁺ CD25⁺ T cells was significantly higher in the TILs than PBLs. The expression of CD4⁺ CD25⁺ regulatory T cells in cancer milieu correlated with the tumor grade, stage, and myometrium invasion. The expression of FOXP3 and GITR in CD4⁺ CD25⁺ regulatory T cells was lower in PBLs than TILs. Most tumor‐infiltrating CD8⁺ T cells were CD28^? CD45RA^? CD45RO⁺ CCR7^?, suggesting good terminal differentiation. Most of them had an activated role with CD69⁺ CD103⁺ CD152⁺. Functionally, both granzyme B and perforin were scarcely expressed in peripheral regulatory T cells but were highly expressed in peripheral regulatory T cells in the tumor microenvironment. In contrast, CD8⁺ cytotoxic T cells derived from PBLs expressed both granzyme B and perforin, and at significantly higher levels than in TILs. Further functional assays demonstrated that Th1 cytokines and cytotoxic molecules can be synchronously up‐regulated in CD8⁺ cytotoxic T cells.

CONCLUSIONS:

Regulatory T cells in the tumor microenvironment may abrogate CD8⁺ T cell cytotoxicity in a granzyme B‐ and perforin‐dependent conduit. Decreases in both Th1 cytokines and cytotoxic enzymes are relevant for regulatory T cell‐mediated restraint of tumor clearance in vivo. Of clinical significance, the expression of regulatory T cells in TILs may mediate T cell immune repression within cancer milieu and thus greatly correlate with cancer progression. Cancer 2010. © 2010 American Cancer Society.     

Tumour-infiltrating CD4+ and CD8+ lymphocytes as predictors of clinical outcome in glioma

Background:

T lymphocyte infiltration has been detected in glioma, although its significance remains unclear. The purpose of the present study was to explore the prognostic value of CD4⁺ and CD8⁺ tumour-infiltrating lymphocytes (TILs) in glioma, and the prognostic value of infiltrating Forkhead box protein 3 (FoxP3⁺) regulatory T cells were also investigated.

Methods:

CD4⁺, FoxP3⁺ and CD8⁺ TILs were assessed by immunohistochemical staining of tissue microarray cores from 284 gliomas. Kaplan–Meier analysis and Cox proportional hazards models were used to examine the survival function of these TILs in 90 glioblastoma patients.

Results:

The number of CD8⁺ TILs was inversely correlated with tumour grade (P=0.025), whereas the number of CD4⁺ TILs was positively correlated with tumour grade (P=0.002). FoxP3⁺ TILs were only observed in glioblastomas, but not in low-grade astrocytomas or oligodendroglial tumours. Among patients with glioblastoma, none of CD4⁺ TILs, FoxP3⁺ TILs and CD8⁺ TILs alone was significantly associated with patient prognosis. However, the presence of high CD4⁺ and low CD8⁺ TIL levels was an independent predictor of poor progress-free survival (multivariate hazard ratio (HR) 1.618, 95% confidence interval (CI) 1.245–2.101, P<0.001) and poor overall survival (multivariate HR 1.508, 95% CI 1.162–1.956, P=0.002). Moreover, pseudoprogression was more often found in patients with high CD4⁺ TILs and high CD8⁺ TILs.

Conclusions:

The combination of CD4⁺ and CD8⁺ TILs is a predictor of clinical outcome in glioblastoma patients, and a high level of CD4⁺ TILs combined with low CD8⁺ TILs was associated with unfavourable prognosis.     

Early Stage HER2-Positive Breast Cancers Not Achieving a pCR From Neoadjuvant Trastuzumab- or Pertuzumab-Based Regimens Have an Immunosuppressive Phenotype

Background

Stromal tumor-infiltrating lymphocytes (TILs) might predict pathologic complete response (pCR) in patients with HER2-positive (HER2⁺) breast cancer treated with trastuzumab (H). Docetaxel (T), carboplatin (C), H, and pertuzumab (P) have immune-modulating effects. Pre- and post-treatment immune biomarkers in cancers treated with neoadjuvant TCH with or without P are lacking. In this study we quantified baseline and changes in TILs, cluster of differentiation (CD) 4⁺, CD8⁺, FoxP3⁺, and PD-L1⁺ cells using immunohistochemistry (IHC) and quantified productive T-cell receptor β (TCRβ) rearrangements and TCRβ clonality using next-generation sequencing (NGS) in 30 HER2⁺ breast cancer tissues treated with neoadjuvant H with or without P regimens.

Prognostic significance of FOXP3+ tumor-infiltrating lymphocytes in breast cancer depends on estrogen receptor and human epidermal growth factor receptor-2 expression status and concurrent cytotoxic T-cell infiltration

Introduction

The infiltration of FOXP3⁺ regulatory T cells into invasive tumors has been reported to be associated with survival in a variety of cancers. The prognostic significance of FOXP3⁺ tumor-infiltrating lymphocytes (TILs) in breast cancer, however, remains controversial.

Methods

FOXP3⁺ TILs were assessed by immunohistochemistry on tissue microarrays constructed from a well-defined cohort of 3,992 breast cancer patients linked to detailed demographic, biomarker, treatment and outcome data. Survival analyses were performed using the Kaplan-Meier function and Cox proportional hazards regression models to evaluate the association of FOXP3⁺ TILs with breast cancer-specific survival, stratified by intrinsic subtype and cytotoxic T-cell infiltration status (as defined by CD8 immunohistochemistry).

Results

The presence of high numbers of FOXP3⁺ TILs was significantly associated with young age, high grade, estrogen receptor (ER) negativity, concurrent CD8⁺ cytotoxic T-cell infiltration, and human epidermal growth factor receptor-2 positive (HER2⁺)/ER^? and core basal subtypes. On multivariate survival analysis, a high level of FOXP3⁺ TILs was significantly associated with poor survival in ER⁺ breast cancers that lacked CD8⁺ T-cell infiltrates (hazard ratio (HR)?=?1.30, 95% confidence interval (CI)?=?1.02 to 1.66). However, in ER^? breast cancers, FOXP3⁺ TILs were strongly associated with improved survival in the HER2⁺/ER^? subgroup, particularly in those with co-existent CD8⁺ T-cell infiltrates (HR?=?0.48, 95% CI?=?0.23 to 0.98), for which the presence of high levels of FOXP3⁺ TILs was independent of standard clinical prognostic factors.

Conclusions

FOXP3⁺ regulatory TILs are a poor prognostic indicator in ER⁺ breast cancer, but a favorable prognostic factor in the HER2⁺/ER^? subtype. The prognostic value of FOXP3⁺ TILs in breast cancer differs depending on ER and HER2 expression status and CD8⁺ T-cell infiltration.     

Influence of neoadjuvant radio-chemotherapy on tumor-infiltrating lymphocytes in squamous esophageal cancer

Introduction

Data on influence of radio-chemotherapy (RCT) on tumor-infiltrating lymphocytes (TILs) is scarce and no study addressed this issue in esophageal squamous cell cancer (SCC) so far.

Methods

Tumor specimens of 49 patients with SCC were re-evaluated with immunohistochemical staining with anti-CD3, anti-CD4, anti-CD8, anti-CD25 and anti-FOXP3 antibodies. Lymphocytes were counted in one high power field (0.189 mm²) at the periphery and in the centre of tumors.

Results

21 patients received preoperative RCT, 28 proceeded directly to surgery. There was no significant difference in survival between the two groups (median survival 23.2 months vs. 22.1 months, log rank test p = n.s.). Cox regression analysis showed that no variable had a significant effect on survival. The infiltrating pattern of TILs revealed higher numbers peripherally independent of the administration of RCT. There was a significant decrease in all cell numbers except CD4⁺ cells in the centre of the tumors after RCT (CD3⁺p = 0.005; CD8⁺p = 0.02; CD25⁺p = 0.01; FoxP3⁺p = 0.01). There were fewer TILs in the periphery after RCT; however, this difference only reached significance in FoxP3⁺ cells (p = 0.01).

Conclusion

Neoadjuvant RCT reduced the number of TILs in esophageal SCC. This was primarily seen in the centre of tumors and suggests that the effect of RCT on immunological response is located in the centre of tumors.     

Intraepithelial T cells and tumor proliferation

BACKGROUND:

The aim of the study was to determine whether tumor‐infiltrating lymphocytes and/or tumor mitotic activity could identify subgroups of patients with advanced serous epithelial ovarian cancer who would maximally benefit from aggressive surgical cytoreduction.

METHODS:

Snap‐frozen specimens from 134 consecutive patients with stage III or IV serous or poorly differentiated ovarian adenocarcinoma undergoing primary debulking surgery from a single US institution were characterized based on CD3⁺, CD8⁺, FoxP3⁺ tumor‐infiltrating lymphocytes, and Ki67 expression. Kaplan‐Meier survival curves were estimated and compared using a log‐rank statistic. A multivariate Cox model was used to estimate adjusted hazard ratios. Interactions were modeled using recursive partitioning based on maximal prognostic differentiation.

RESULTS:

Brisk intraepithelial CD8⁺ cells (P = .035) and low Ki67 expression (P = .042) portended prolonged survival. The T‐cell infiltration was more likely to occur in tumors with high proliferation index. Patients whose tumors exhibited low Ki67 expression and high intraepithelial CD8⁺ frequency had a 5‐year survival rate of 73.3%. Patients with aggressive tumor behavior, that is, whose tumors exhibited low frequency of intraepithelial CD8⁺ T cells or high Ki67 expression were more likely to draw benefit from aggressive surgical cytoreduction. Survival was similar for patients with brisk CD8⁺ T cells who had optimal or suboptimal debulking. Likewise, survival was similar for patients with low Ki67 expression who had optimal or suboptimal debulking.

CONCLUSIONS:

For the first time, these novel interactions of T cells, tumor proliferation index, and surgical treatment reveal that biological prognosticators may be useful for surgical decision making in ovarian cancer. Cancer 2009. © 2009 American Cancer Society.     

Comprehensive immune characterization and T‐cell receptor repertoire heterogeneity of retroperitoneal liposarcoma

Retroperitoneal liposarcoma (RLPS) is one of the most common subtypes of retroperitoneal soft tissue sarcomas and lacks effective treatment. This study aimed to provide a thorough profile of immune characteristics of RLPS. This study included 56 RLPS patients. Multisite tumor tissues were collected from 16 patients. Immunohistochemistry was carried out to identify CD4⁺, CD8⁺, FoxP3⁺, CD20⁺, or programmed cell death‐1 (PD‐1)⁺ tumor infiltrating lymphocytes (TILs) and  Programmed cell death ligand‐1 (PD‐L1) expression in tumor tissues. Ultradeep sequencing of T‐cell receptor (TCR) β‐chain gene was carried out in 42 tumor samples as well as peripheral blood samples collected from 6 patients. In RLPS, TILs were distributed in 3 patterns and T cells were more prevalent than B cells. Generally, the proportion of TILs decreased and PD‐L1 expression increased with tumor progression. Patients with higher PD‐1/PD‐L1 expression tended to have poorer prognosis, whereas patients with tertiary lymphoid structure tended to have a favorable disease‐free survival. Although T‐cell clones in tumors were quite different from those in peripheral blood, TCR sequencing showed low TCR repertoire reads as well as polyclonal status within tumors, which indicated limited T cell response in the tumors. Both TILs distribution and TCR repertoires suggested spatial immune heterogeneity in RLPS. Our research described the immune landscape of RLPS, and suggested RLPS might be a kind of tumor with low T cell infiltration as well as great immune heterogeneity. Therefore, strategies that can facilitate lymphocytic infiltration and immune reactivity need to be developed in the future to improve the efficacy of immunotherapy.     

The clinical impact of tumour‐infiltrating lymphocytes in colorectal cancer differs by anatomical subsite: A cohort study

Accumulating evidence demonstrates an association between dense infiltration of lymphocytes and prognosis in colorectal cancer (CRC), but whether this prognostic impact differs by tumour location remains unknown. This study investigated the prognostic impact of cytotoxic and regulatory T cells in CRC, with particular reference to the anatomical subsite of the primary tumour. The density of CD3⁺, CD8⁺ and FoxP3⁺ tumour‐infiltrating T cells was calculated in tissue microarrays with tumours from 557 incident CRC cases from a prospective population‐based cohort. Kaplan–Meier and Cox regression analyses were applied to determine the impact of high and low lymphocyte density on 5‐year overall survival, in subgroup analysis of right colon, left colon and rectum. High CD8⁺ cell density was a favourable prognostic factor for patients with right‐sided colon tumours (hazard ratio [HR]=0.53, 95% confidence interval [CI] 0.29–0.95), independent of age, sex, TNM stage, differentiation grade and vascular invasion, with a significant prognostic interaction between CD8⁺ cells and right‐sidedness (p = 0.031). High FoxP3⁺ cell density was an independent favourable prognostic factor only in patients with rectal tumours (HR = 0.54, 95% CI 0.30‐0.99), and CD3⁺ cell density was an independent favourable prognostic factor for tumours in the right colon and rectum, but there was no significant prognostic interaction between CD3⁺ or FoxP3⁺ cells and sidedness. These results demonstrate that the prognostic impact of tumour‐infiltrating lymphocytes in CRC differs by primary tumour site, further indicating that tumour location may be an important factor to take into consideration in therapeutic decisions, including eligibility for immunotherapy.     

Anti‐inflammatory M2 type macrophages characterize metastasized and tyrosine kinase inhibitor‐treated gastrointestinal stromal tumors

We have made a detailed inventory of the immune infiltrate of gastrointestinal stromal tumors (GISTs), which originate from mesenchymal cells in the intestinal tract. These sarcomas are heavily infiltrated with macrophages and T cells, while immune cells of other lineages were much less abundant. Dissecting the functional subtypes of T cells with multicolor fluorescent microscopy revealed substantial populations of cytotoxic T cells, helper T cells and FoxP3⁺ regulatory T cells. The balance of cytotoxic T cells and FoxP3⁺ T cells was toward immune suppression. Analysis of the macrophage population also showed a dominance of anti‐inflammatory cells, as the M2 type scavenger receptor CD163 was abundantly present. Other subsets of macrophages (CD14⁺CD163⁻) were occasionally detected. M2 type CD163⁺ macrophages were associated with the number of infiltrating FoxP3⁺ regulatory T cells and twice as many macrophages were found in metastatic GIST compared to primary lesions. Most metastatic GISTs had been treated with the tyrosine kinase inhibitors imatinib and sunitinib, but the high macrophage infiltrate was not related to this treatment. However, imatinib and sunitinib did induce secretion of anti‐inflammatory IL‐10 in macrophage cultures, indicating that treatment with these inhibitors might contribute to an immune suppressive microenvironment in GIST. Overall, our data reveal a picture of GIST as an active site of tumor‐immune interaction in which suppressive mechanisms overrule potential antitumor responses. Tyrosine kinase inhibitors might promote this negative balance.     

CD8+tumor‐infiltrating lymphocytes at primary sites as a possible prognostic factor of cutaneous angiosarcoma

Tumor‐infiltrating lymphocytes (TILs) have been reported as a prognostic factor in various cancers and are a promising target for immunotherapy. To investigate whether TILs have any impact on the prognosis of angiosarcoma patients, 55 non‐treated patients (40 patients at stage 1 with cutaneous localized tumors, 4 patients at stage 2 with lymph node metastases and 11 patients at stage 3 with distant metastases) with angiosarcoma were evaluated retrospectively by immunohistochemistry stained CD4, CD8, FOXP3 and Ki67. The Kaplan–Meier method was used to estimate overall survival with patients at stage 1. Survival differences were analyzed by the log‐rank test. Patients with higher numbers of CD8⁺ TILs in their primary tumors survived significantly longer compared with patients with lower values. Moreover, the number of CD8 in TILs was positively correlated with a distant metastasis‐free period. The total number of primary TILs (CD4 plus CD8) and CD8⁺ primary TILs of stage 3 patients with distant metastases was positively correlated with their overall survival. To evaluate whether CD8⁺ effector T cells are activated or differentiated, flow cytometric analysis of peripheral blood mononuclear cells (PBMC) was performed. The percentages of CD8⁺ T cells producing IFN‐γ in PBMC were significantly higher in patients with angiosarcoma (n = 10) compared not only with that of healthy controls (n = 20) but also patients with advanced melanoma (n = 11). These results suggest that anti‐tumor immunity is clinically relevant in angiosarcoma.     

Chronologically changing patterns in the survival of korean patients with breast cancer and related clinical factors: a nationwide registry-based study

Purpose

The purpose of the study was to evaluate protein expression of PD-L1 and CD20 as prognostic biomarkers of patient outcome in inflammatory breast cancer (IBC) samples.

Methods

PD-L1 and CD20 protein expression was measured by immunohistochemistry in 221 pretreatment IBC biopsies. PD-L1 was assessed in tumor cells (PD-L1⁺ tumor cells) and tumor stromal infiltrating lymphocytes (PD-L1⁺ TILs); CD20 was scored in tumor-infiltrating B cells. Kaplan–Meier curves and Cox proportional hazard models were used for survival analysis.

Results

PD-L1⁺ tumor cells, PD-L1⁺ TILs, and CD20⁺ TILs were found in 8%, 66%, and 62% of IBC, respectively. PD-L1⁺ tumor cells strongly correlated with high TILs, pathological complete response (pCR), CD20⁺ TILs, but marginally with breast cancer-specific survival (BCSS, P?=?0.057). PD-L1⁺ TILs strongly correlated with high TILs, CD20⁺ TILs, and longer disease-free survival (DFS) in all IBC and in triple-negative (TN) IBC (P?<?0.035). IBC and TN IBC patients with tumors containing both CD20⁺ TILs and PD-L1⁺ TILs (CD20⁺TILs/PD-L1⁺TILs) showed longer DFS and improved BCSS (P?<?0.002) than patients lacking both, or those with either CD20⁺ TILs or PD-L1⁺ TILs alone. In multivariate analyses, CD20⁺TILs/PD-L1⁺TILs status was an independent prognostic factor for DFS in IBC (hazard ratio (HR): 0.53, 95% CI 0.37–0.77) and TN IBC (HR: 0.39 95% CI 0.17–0.88), and for BCSS in IBC (HR: 0.60 95% CI 0.43–0.85) and TN IBC (HR: 0.38 95% CI 0.17–0.83).

Conclusion

CD20⁺TILs/PD-L1⁺TILs status represents an independent favorable prognostic factor in IBC and TN IBC, suggesting a critical role for B cells in antitumor immune responses. Anti-PD-1/PD-L1 and B cell-activating immunotherapies should be explored in these settings.

     

Stromal infiltration of CD8 T cells is associated with improved clinical outcome in HPV-positive oropharyngeal squamous carcinoma

Background:

Patients with human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) have a better prognosis than those with HPV-negative tumours. There is interest in de-escalating their treatment but strategies are needed for risk stratification to identify subsets with a poor prognosis. This study investigated tumour-infiltrating lymphocytes (TILs) in relation to HPV tumour status and patient survival.

Methods:

Biopsies from 218 patients diagnosed with OPSCC between 2002 and 2011, who underwent chemo/radiotherapy were analysed for HPV by PCR, in-situ hybridisation and p16 immunohistochemistry (IHC). One hundred and thirty-nine samples with concordant HPV detection were analysed for CD3, CD4, CD8 and FoxP3 expression in tumour and stromal regions using multiplexIHC and multispectral image analysis. Labelling of smooth muscle actin (SMA) identified activated stroma.

Results:

Human papillomavirus-positive compared with HPV-negative OPSCC had higher infiltration in both tumour and stromal areas of CD4 and CD8 T cells but not FoxP3 T regulatory cells. Only CD3+CD8+ stromal and not tumour area infiltration was associated with increased survival (P=0.02). There was significantly higher SMA expression in HPV-positive compared with -negative tumours, which did not correlate with survival.

Conclusions:

Studies of TILs for risk stratification in OPSCC should assess stromal infiltration.     

The prognostic influence of tumour-infiltrating lymphocytes in cancer: a systematic review with meta-analysis

Background:

Tumour-infiltrating lymphocytes (TILs) are often found in tumours, presumably reflecting an immune response against the tumour. We carried out a systematic review and meta-analysis, aiming to establish pooled estimates for survival outcomes based on the presence of TILs in cancer.

Methods:

A Pubmed and Embase literature search was designed. Studies were included, in which the prognostic significance of intratumoural CD3+, CD4+, CD8+, and FoxP3+ lymphocytes, as well as ratios between these subsets, were determined in solid tumours.

Results:

In pooled analysis, CD3+ TILs had a positive effect on survival with a hazard ratio (HR) of 0.58 (95% confidence interval (CI) 0.43–0.78) for death, as did CD8+ TILs with a HR of 0.71 (95% CI 0.62–0.82). FoxP3+ regulatory TILs were not linked to overall survival, with a HR of 1.19 (95% CI 0.84–1.67). The CD8/FoxP3 ratio produced a more impressive HR (risk of death: HR 0.48, 95% CI 0.34–0.68), but was used in relatively few studies. Sample size and follow-up time seemed to influence study outcomes.

Conclusion:

Any future studies should be carefully designed, to prevent overestimating the effect of TILs on prognosis. In this context, ratios between TIL subsets may be more informative.     

Stromal PD-L1–Positive Regulatory T cells and PD-1–Positive CD8-Positive T cells Define the Response of Different Subsets of Non–Small Cell Lung Cancer to PD-1/PD-L1 Blockade Immunotherapy

Introduction

Inhibition of programmed cell death-1 (PD-1) and its ligand programmed death ligand 1 (PD-L1) by using an immune checkpoint inhibitor has emerged as a promising immunotherapy for NSCLC. The correlation of PD-L1 expression in tumor cells with treatment outcomes has been reported in many pivotal trials; however, the relationship remains unclear. Here, we demonstrate that those patients with both high density of PD-1–positive CD8 and PD-L1–positive CD4-positive CD25-positive (PD-1^hi PD-L1^hi) regulatory T cells (Tregs) have a better response to PD1/PD-L1 blockade.

Presence of circulating Her2-reactive CD8?+?T-cells is associated with lower frequencies of myeloid-derived suppressor cells and regulatory T cells,and better survival in older breast cancer patients

Introduction

Breast cancer is one of the most common cancers among women. Its incidence is increasing in many countries and a higher number of older women are now being diagnosed with the disease. Immune parameters are implicated in disease progression, and the frequencies of both myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs), associated with tumour burden, have been suggested to be indicators of poor prognosis in cases of metastatic breast cancer.

Methods

Here, we have assessed the frequency of peripheral Tregs and MDSCs in relation to in vitro T cell responses to Her2 antigen in 40 untreated breast cancer patients 65 to 87 years of age at diagnosis.

Results

The five-year survival rate of patients who mounted a CD8+ T cell response to Her2 peptides and had a lower frequency of Lin⁻CD14⁺HLA-DR⁻MDSCs was 100% compared to only 38% in patients without Her2-reactive CD8+ T cells and with higher frequencies of MDSCs (P = 0.03). Patients who lacked a CD8 response to Her2 tended to have higher frequencies of MDSCs. Similarly, patients who lacked a CD8 response to Her2 and had higher frequencies of CD4⁺Foxp3⁺CD127^lowCD25⁺ Tregs had only 50% survival compared to the 100% survival of patients who did mount a CD8 response and had lower frequencies of Tregs (P = 0.03). A similar trend was observed for activated (CD4⁺CD45RA⁻Foxp3^hi) but not resting Tregs (CD4⁺CD45RA⁺FoxP3⁺). This survival advantage was observed in both metastatic and non-metastatic patients.

Conclusions

Our data demonstrate a negative role of both MDSCs and Tregs in the prognosis of breast cancer patients, the mechanism of which might be through dampening favourable CD8+ T cell immune responses to tumour-associated antigens.

Electronic supplementary material

The online version of this article (doi:10.1186/s13058-015-0541-z) contains supplementary material, which is available to authorized users.     

Proliferation of CD4+CD25high+Foxp3+ regulatory T lymphocytes in ex vivo expanded ascitic fluid from primary and recurrent ovarian carcinoma

Objective

Regulatory T lymphocytes evoke the immune tolerance by suppressing and inactivating cytotoxic T lymphocytes. The objective of this study was to compare the proportion of regulatory T lymphocytes, precisely defined as CD4⁺CD25^high+Foxp3⁺ T lymphocytes, in primary and recurrent ovarian carcinoma before and after ex vivo expansion of ascites with interleukin-2 (IL-2).

Methods

Ascitic fluid samples were obtained from 26 patients with ovarian carcinoma. Lymphocytes were isolated from ascites and cell markers were analyzed by flow cytometry using anti-CD3/CD4/CD8/CD16/CD56/CD25 and anti-Foxp3 antibodies. Lymphocytes were incubated for 2 to 3 weeks and expanded ex vivo by IL-2 stimulation and their phenotypes were analyzed by flow cytometry.

Results

Following ex vivo expansion, ascitic fluid lymphocytes increased by a greater extent in the recurrent group than in the primary group. The proportion of ex vivo-expanded lymphocytes changed as follows; CD4⁺ T lymphocytes increased, CD8⁺ T lymphocytes decreased, and the proportion of CD3^-CD16⁺56⁺ NK cells was unchanged. The proportion of CD4⁺CD25^high+Foxp3⁺ regulatory T lymphocytes in CD4⁺ T lymphocytes increased after ex vivo expansion in both groups, but to a greater degree in the recurrent group.

Conclusion

This study showed that regulatory T lymphocytes, neither cytotoxic T lymphocytes nor NK cells, were extensively increased after ex vivo expansion, especially in recurrent ovarian carcinoma. These results may provide information that helps to guide the future development of adoptive immunotherapy against ovarian carcinoma.     

Predictive factors of the tumor immunological microenvironment for long‐term follow‐up in early stage breast cancer

The aim of this research was to investigate the correlation of immunologic factors in the tumor environment of breast cancer, using immunohistological staining to evaluate the expression of programmed death 1/programmed death ligand 1 (PD‐1/PD‐L1), phosphatase and tensin homolog (PTEN), tumor infiltrating lymphocytes (TILs), and macrophages, and to analyze the association between the immunologic factors and clinical outcome for patients with early stage breast cancer (EBC). A total of 97 EBC patients who underwent standard surgery were investigated. Expression of PD‐1/PD‐L1 and PTEN and the density of CD3⁺ TILs, CD8⁺ TILs, and CD163⁺ macrophages were evaluated by immunohistochemical analysis. The association between the immunologic factors and clinical outcome was statistically analyzed. The density of CD3⁺ TILs, CD8⁺ TILs, and CD163⁺ macrophages and non‐expression of PTEN was significantly higher in cases of triple negative breast cancer. CD8⁺ TIL density and CD8⁺/PD‐L1⁺ expression were predictive factors for disease‐free survival and overall survival (OS). Human epidermal growth factor 2 (HER2)‐positive patients with PTEN expression and luminal/HER2‐negative patients without PD‐L1 expression had significantly longer OS compared to patients without PTEN expression (P = 0.049) and with PD‐L1 expression (P = 0.036), respectively. Furthermore, patients with PD‐L1⁺/CD8⁺ expression had worse median progression‐free survival (P = 0.022) and median OS (P = 0.037) compared with patients without PD‐L1⁺/CD8⁺ expression. The CD3⁺ TILs, CD8⁺ TILs, and CD163⁺ macrophages were shown to infiltrate the tumor area of EBC. In particular, triple negative breast cancer had a higher rate of TIL infiltration within the tumor environment. Expression of PTEN and lack of PD‐L1 expression were associated with favorable survival in HER2‐positive and luminal/HER2‐negative EBC patients, respectively. The PD‐L1 expression combined with CD8⁺ density was significantly associated with an aggressive clinical outcome.     

Overexpression of hypoxia‐inducible factor 1 alpha impacts FoxP3 levels in mycosis fungoides—Cutaneous T‐cell lymphoma: Clinical implications

Mycosis fungoides (MF) is the most common variant of primary cutaneous T‐cell lymphoma, and decreased forkhead box P3 (FoxP3) expression has been reported in MF late stages. Hypoxia‐inducible factor 1 alpha (HIF‐1α) may regulate FoxP3 expression; however, it is unknown whether HIF‐1α is expressed in the CD4⁺ T cells of MF patients and how it could affect the expression of FoxP3. Therefore, we evaluated the expression of HIF‐1α and FoxP3 in CD4⁺ T cells obtained from the skin lesions of MF patients. We found increased cell proliferation and an increase in CD4⁺ T cells with an aberrant phenotype among early stage MF patients. HIF‐1α was overexpressed in these CD4⁺ T cells. In addition, we found a decrease in the percentage of FoxP3⁺ cells both in the skin of MF patients, when compared with control skin samples, and with disease progression. In addition, a negative correlation was established between HIF‐1α and FoxP3 expression. Skin HIF‐1α expression in MF patients correlated with the extent of the affected area and increased with the disease progression. Finally, we showed that ex vivo inhibition of HIF‐1α degradation increases the percentage of FoxP3⁺ T cells in skin lesions. Our results suggest that overexpression of HIF‐1α affects the levels of FoxP3 in MF patients, which could have relevant implications in terms of disease outcome.     

Clinical Implications of Cytotoxic T Lymphocyte Antigen-4 Expression on Tumor Cells and Tumor-Infiltrating Lymphocytes in Extrahepatic Bile Duct Cancer Patients Undergoing Surgery Plus Adjuvant Chemoradiotherapy

Background

There currently is only limited knowledge on the role of tumor-specific immunity in cholangiocarcinoma.

Objective

This study evaluated the clinical implications of cytotoxic T lymphocyte antigen-4 (CTLA-4) expression levels and CD4⁺ and CD8⁺ tumor-infiltrating lymphocytes (TILs) in extrahepatic bile duct (EHBD) cancer.

Patients and Methods

Immunohistochemistry of CTLA-4, CD4, and CD8 was performed for 77 EHBD cancer patients undergoing surgery plus adjuvant chemoradiotherapy. CTLA-4 expression on tumor cells and TILs were assessed by using H-scores and the proportion of CTLA-4⁺ lymphocytes, respectively.

Results

With optimal cutoff values determined by a maximal chi-square method with overall survival (OS) data, patients with CTLA-4 H-score >70 and a proportion of CTLA-4⁺ TILs >0.15 showed higher mean density of CD8⁺ and CD4⁺ TILs, respectively (P?=?0.025 for CD8⁺ and P?=?0.055 for CD4⁺ TILs). The high CTLA-4 H-score level was associated with prolonged OS and disease-free interval (DFI) (P?=?0.025 and 0.004, respectively). With differential levels of CTLA-4 H-score according to hilar and non-hilar locations (high rate 32 vs. 68%, respectively; P?=?0.013), an exploratory subgroup analysis demonstrated that the associations between the CTLA-4 expression and OS and DFI were confined to hilar tumors (P?=?0.003 and <0.001, respectively), but not to non-hilar ones (P?=?0.613 and 0.888, respectively).

Conclusions

This study demonstrates a potential prognostic relevance of CTLA-4 expression in EHBD cancer. We suggest a differential survival impact of the CTLA-4 expression level according to different tumor locations.

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