Analysis of FoxP3+ T-Regulatory Cells and CD8+T-Cells in Ovarian Carcinoma: Location and Tumor Infiltration Patterns Are Key Prognostic Markers

Purpose

Tumor infiltrating CD4+CD25+FoxP3+ regulatory immune cells (Treg) have been associated with impaired anti- tumor immune response and unfavorable prognosis for patients affected by ovarian carcinoma, whereas CD8+ T-cells have been found to positively influence survival rates in a large panel of solid tumors. Recently, density, location and tumor infiltration patterns of the respective immune cell subtypes have been identified as key prognostic factors for different types of tumors.

Patients and Methods

We stained 210 human ovarian carcinoma samples immunhistochemically for FoxP3 and CD8 to identify the impact different immune cell patterns have on generally accepted prognostic variables as well as on overall survival.

Results

We found that FoxP3+ cells located within lymphoid aggregates surrounding the tumor were strongly associated with reduced survival time (P = 0.007). Central accumulation of CD8+ effector cells within the tumor bed shows a positive effect on survival (P = 0,001).

Conclusion

The distribution pattern of immune cells within the tumor environment strongly influences prognosis and overall survival time of patients with ovarian carcinoma.     

FOXP3 Expression Is Upregulated in CD4+T Cells in Progressive HIV-1 Infection and Is a Marker of Disease Severity

Background

Understanding the role of different classes of T cells during HIV infection is critical to determining which responses correlate with protective immunity. To date, it is unclear whether alterations in regulatory T cell (Treg) function are contributory to progression of HIV infection.

Methodology

FOXP3 expression was measured by both qRT-PCR and by flow cytometry in HIV-infected individuals and uninfected controls together with expression of CD25, GITR and CTLA-4. Cultured peripheral blood mononuclear cells were stimulated with anti-CD3 and cell proliferation was assessed by CFSE dilution.

Principal Findings

HIV infected individuals had significantly higher frequencies of CD4⁺FOXP3⁺ T cells (median of 8.11%; range 1.33%–26.27%) than healthy controls (median 3.72%; range 1.3–7.5%; P = 0.002), despite having lower absolute counts of CD4⁺FOXP3⁺ T cells. There was a significant positive correlation between the frequency of CD4⁺FOXP3⁺ T cells and viral load (rho = 0.593 P = 0.003) and a significant negative correlation with CD4 count (rho = −0.423 P = 0.044). 48% of our patients had CD4 counts below 200 cells/µl and these patients showed a marked elevation of FOXP3 percentage (median 10% range 4.07%–26.27%). Assessing the mechanism of increased FOXP3 frequency, we found that the high FOXP3 levels noted in HIV infected individuals dropped rapidly in unstimulated culture conditions but could be restimulated by T cell receptor stimulation. This suggests that the high FOXP3 expression in HIV infected patients is likely due to FOXP3 upregulation by individual CD4⁺ T cells following antigenic or other stimulation.

Conclusions/Significance

FOXP3 expression in the CD4⁺ T cell population is a marker of severity of HIV infection and a potential prognostic marker of disease progression.     

Functional Improvement of Regulatory T Cells from Rheumatoid Arthritis Subjects Induced by Capsular Polysaccharide Glucuronoxylomannogalactan

Objective

Regulatory T cells (Treg) play a critical role in the prevention of autoimmunity, and the suppressive activity of these cells is impaired in rheumatoid arthritis (RA). The aim of the present study was to investigate function and properties of Treg of RA patients in response to purified polysaccharide glucuronoxylomannogalactan (GXMGal).

Methods

Flow cytometry and western blot analysis were used to investigate the frequency, function and properties of Treg cells.

Results

GXMGal was able to: i) induce strong increase of FOXP3 on CD4⁺ T cells without affecting the number of CD4⁺CD25⁺FOXP3⁺ Treg cells with parallel increase in the percentage of non-conventional CD4⁺CD25⁻FOXP3⁺ Treg cells; ii) increase intracellular levels of TGF-β1 in CD4⁺CD25⁻FOXP3⁺ Treg cells and of IL-10 in both CD4⁺CD25⁺FOXP3⁺ and CD4⁺CD25⁻FOXP3⁺ Treg cells; iii) enhance the suppressive activity of CD4⁺CD25⁺FOXP3⁺ and CD4⁺CD25⁻FOXP3⁺ Treg cells in terms of inhibition of effector T cell activity and increased secretion of IL-10; iv) decrease Th1 response as demonstrated by inhibition of T-bet activation and down-regulation of IFN-γ and IL-12p70 production; v) decrease Th17 differentiation by down-regulating pSTAT3 activation and IL-17A, IL-23, IL-21, IL-22 and IL-6 production.

Conclusion

These data show that GXMGal improves Treg functions and increases the number and function of CD4⁺CD25⁻FOXP3⁺ Treg cells of RA patients. It is suggested that GXMGal may be potentially useful for restoring impaired Treg functions in autoimmune disorders and for developing Treg cell-based strategies for the treatment of these diseases.     

T regulatory cells in cord blood--FOXP3 demethylation as reliable quantitative marker

Background

Regulatory T-cells (Tregs), characterized as CD4+CD25^hi T-cells expressing FOXP3, play a crucial role in controlling healthy immune development during early immune maturation. Recently, FOXP3 demethylation was suggested to be a novel marker for natural Tregs in adults. In cord blood, the role and function of Tregs and its demethylation is poorly understood. We assessed FOXP3 demethylation in cord blood in relation to previously used Treg markers such as CD4⁺CD25^hi, FOXP3 mRNA, protein expression, and suppressive Treg function.

Methodology

Cord blood mononuclear cells (CBMC) were isolated from 70 healthy neonates, stimulated for 3 days with the microbial stimulus lipid A (LpA), and allergen Dermatophagoides pteronyssinus (Derp1). Tregs (CD4+CD25^hi, intracellular, mRNA FOXP3 expression, isolated cells), DNA methylation of the FOXP3-locus and suppressive Treg function were assessed.

Principal Findings

Demethylation of FOXP3 in whole blood was specific for isolated CD4+CD25^hi Tregs. Demethylation of FOXP3 was positively correlated with unstimulated and LpA-stimulated FOXP3 mRNA-expression (p≤0.05), and CD4⁺CD25^hi T-cells (p≤0.03). Importantly, increased FOXP3 demethylation correlated with more efficient suppressive capacity of Tregs (r = 0.72, p = 0.005). Furthermore, FOXP3 demethylation was positively correlated with Th2 cytokines (IL-5, IL-13) following LpA-stimulation (p = 0.006/0.04), with Th2 and IL-17 following Derp1+LpA-stimulations (p≤0.009), but not Th1 cytokines (IFN-γ).

Conclusions

FOXP3 demethylation reliable quantifies Tregs in cord blood. FOXP3 demethylation corresponds well with the suppressive potential of Tregs. The resulting strict correlation with functionally suppressive Tregs and the relative ease of measurement render it into a valuable novel marker for large field studies assessing Tregs as qualitative marker indicative of functional activity.     

Increased Frequency of CD4 and CD8 Regulatory T Cells in Individuals under 15 Years with Multibacillary Leprosy

Background

Leprosy is a chronic disease, caused by Mycobacterium leprae, which poses a serious public health problem worldwide. Its high incidence in people under 15 years old in Ceará state, Brazil, reflects the difficulty of its control. The spectrum of clinical manifestations is associated with the immune response developed, with the Th1 and Th2 responses being related to the paucibacillary and multibacillary forms, respectively. Regulatory T cells (Treg), which can suppress Th1 and Th2 response, have received special attention in the literature and have been associated with development of chronic infections. However, their role in leprosy in individuals under 15 years old has not yet been elucidated. We evaluated the frequency of CD4⁺/CD8⁺CD25^highFOXP3⁺ and CD4⁺/CD8⁺CD25^highFOXP3^high cells in leprosy patients and household contacts, in both cases under 15 years old.

Methodology/Principal Findings

PBMC from 12 patients and 17 contacts were cultured for 72 hours with anti-CD3 and anti-CD28 (activators) or with activators associated with total sonicated fraction of M. leprae. After culture, the frequency of CD4⁺/CD8⁺ Treg was identified by flow cytometry. Cells stimulated by activators and antigen from multibacillary patients showed Treg frequencies almost two times that of the contacts: CD4⁺FOXP3⁺ (21.93±8.43 vs. 13.79±8.19%, p = 0.0500), CD4⁺FOXP3^high (10.33±5.69 vs. 5.57±4.03%, p = 0.0362), CD8⁺FOXP3⁺ (13.88±9.19 vs. 6.18±5.56%, p = 0.0230) and CD8⁺FOXP3^high (5.36±4.17 vs. 2.23±2.68%, p = 0.0461). Furthermore, the mean fluorescence intensity of FOXP3 in Treg was higher in multibacillary patients than in the contacts. Interestingly, there was a positive correlation of the bacillary index and number of lesions with the frequency of all Treg evaluated in patients.

Conclusions/Significance

We have demonstrated for the first time that multibacillary leprosy patients under 15 years old have greater CD4⁺ and CD8⁺ Treg frequencies and these correlate with clinical and laboratorial aspects of disease. These findings suggest the involvement of these cells in the perpetuation of M. leprae infection.     

Tumor-Derived Microvesicles Induce,Expand and Up-Regulate Biological Activities of Human Regulatory T Cells (Treg)

Background

Tumor-derived microvesicles (TMV) or exosomes are present in body fluids of patients with cancer and might be involved in tumor progression. The frequency and suppressor functions of peripheral blood CD4⁺CD25^highFOXP3⁺ Treg are higher in patients with cancer than normal controls. The hypothesis is tested that TMV contribute to induction/expansion/and activation of human Treg.

Methodology/Principal Findings

TMV isolated from supernatants of tumor cells but not normal cells induced the generation and enhanced expansion of human Treg. TMV also mediated conversion of CD4⁺CD25^neg T cells into CD4⁺CD25^highFOXP3⁺ Treg. Upon co-incubation with TMV, Treg showed an increased FasL, IL-10, TGF-β1, CTLA-4, granzyme B and perforin expression (p<0.05) and mediated stronger suppression of responder cell (RC) proliferation (p<0.01). Purified Treg were resistant to TMV-mediated apoptosis relative to other T cells. TMV also increased phospho-SMAD2/3 and phospho-STAT3 expression in Treg. Neutralizing Abs specific for TGF-β1 and/or IL-10 significantly inhibited TMV ability to expand Treg.

Conclusions/Significance

This study suggests that TMV have immunoregulatory properties. They induce Treg, promote Treg expansion, up-regulate Treg suppressor function and enhance Treg resistance to apoptosis. Interactions of TMV with Treg represent a newly-defined mechanism that might be involved in regulating peripheral tolerance by tumors and in supporting immune evasion of human cancers.     

Increased T Regulatory Cells Are Associated with Adverse Clinical Features and Predict Progression in Multiple Myeloma

Background

Regulatory T (Treg) cells play an important role in the maintenance of immune system homeostasis. Multiple myeloma (MM) is a plasma cell disorder frequently associated with impaired immune cell numbers and functions.

Methods

We analyzed Treg cells in peripheral blood (n = 207) and bone marrow (n = 202) of pre-malignant and malignant MM patients using flow cytometry. Treg cells and their subsets from MM patients and healthy volunteers were functionally evaluated for their suppressive property. A cohort of 25 patients was analyzed for lymphocytes, CD4 T cells and Treg cells before and after treatment with cyclophosphamide, thalidomide plus dexamethasone (CTD).

Results

We found elevated frequencies of Treg cells in newly diagnosed (P<0.01) and relapsed MM patients (P<0.0001) compared to healthy volunteers. Also, Treg subsets including naïve (P = 0.015) and activated (P = 0.036) Treg cells were significantly increased in MM patients compared to healthy volunteers. Functional studies showed that Treg cells and their subsets from both MM and healthy volunteers were similar in their inhibitory function. Significantly increased frequencies of Treg cells were found in MM patients with adverse clinical features such as hypercalcemia (>10 mg/dL), decreased normal plasma cell (≤5%) count and IgA myeloma subtype. We also showed that MM patients with ≥5% of Treg cells had inferior time to progression (TTP) (13 months vs. median not reached; P = 0.013). Furthermore, we demonstrated the prognostic value of Treg cells in prediction of TTP by Cox regression analysis (P = 0.045). CTD treatment significantly reduced frequencies of CD4 T cells (P = 0.001) and Treg cells (P = 0.018) but not Treg cells/CD4 T cells ratio compared to pre-treatment.

Conclusions

Our study showed immune deregulation in MM patients which is evidenced by elevated level of functionally active Treg cells and patients with increased Treg cells have higher risk of progression.     

Low Frequency Magnetic Fields Enhance Antitumor Immune Response against Mouse H22 Hepatocellular Carcinoma

Objective

Many studies have shown that magnetic fields (MF) inhibit tumor growth and influence the function of immune system. However, the effect of MF on mechanism of immunological function in tumor-bearing mice is still unclear.

Methods

In this study, tumor-bearing mice were prepared by subcutaneously inoculating Balb/c mice with hepatocarcinoma cell line H22. The mice were then exposed to a low frequency MF (0.4 T, 7.5 Hz) for 30 days. Survival rate, tumor growth and the innate and adaptive immune parameters were measured.

Results

MF treatment could prolong survival time (n = 28, p<0.05) and inhibit tumor growth (n = 9, p<0.01) in tumor-bearing mice. Moreover, this MF suppressed tumor-induced production of cytokines including interleukin-6 (IL-6), granulocyte colony- stimulating factor (G-CSF) and keratinocyte-derived chemokine (KC) (n = 9–10, p<0.05 or 0.01). Furthermore, MF exposure was associated with activation of macrophages and dendritic cells, enhanced profiles of CD4⁺ T and CD8⁺ T lymphocytes, the balance of Th17/Treg and reduced inhibitory function of Treg cells (n = 9–10, p<0.05 or 0.01) in the mice model.

Conclusion

The inhibitory effect of MF on tumor growth was related to the improvement of immune function in the tumor-bearing mice.     

Identification of Biological Properties of Intralymphatic Tumor Related to the Development of Lymph Node Metastasis in Lung Adenocarcinoma

Background

Intralymphatic tumors in the extratumoral area are considered to represent the preceding phase of lymph node metastasis. The aim of this study was to clarify the biological properties of intralymphatic tumors susceptible to the development of lymph node metastasis, with special reference to the expression of cancer initiating/stem cell (CIC/CSC) related markers in cancer cells and the number of infiltrating stromal cells.

Material and Methods

Primary lung adenocarcinomas with lymphatic permeation in the extratumoral area were retrospectively examined (n = 107). We examined the expression levels of CIC/CSC related markers including ALDH1, OCT4, NANOG, SOX2 and Caveolin-1 in the intralymphatic cancer cells to evaluate their relationship to lymph node metastasis. Moreover, the number of infiltrating stromal cells expressing CD34, α-smooth muscle actin, and CD204 were also evaluated.

Results

Among the intralymphatic tissues, low ALDH1 expression in cancer cells, high SOX2 expression in cancer cells, and a high number of CD204(+) macrophages were independent predictive factors for lymph node metastasis (P = 0.004, P = 0.008, and P = 0.028, respectively). Among these factors, only low ALDH1 expression in cancer cells was significantly correlated with the farther spreading of lymph node metastasis (mediastinal lymph node, pathological N2) (P = 0.046) and the metastatic lymph node ratio (metastatic/resected) (P = 0.028). On the other hand, in the primary tumors, ALDH1 expression in the cancer cells was not associated with lymph node metastasis. Intralymphatic cancer cells expressing low ALDH1 levels exhibited lower E-cadherin expression levels than cancer cells with high levels of ALDH1 expression (P = 0.015).

Conclusions

Intralymphatic cancer cells expressing low levels of ALDH1 and infiltrating macrophages expressing CD204 have a critical impact on lymph node metastasis. Our study also highlighted the significance of evaluating the biological properties of intralymphatic tumors for tumor metastasis.     

Increased Sensitivity of CD4+ T-Effector Cells to CD4+CD25+ Treg Suppression Compensates for Reduced Treg Number in Asymptomatic HIV-1 Infection

Background

In HIV infection, uncontrolled immune activation and disease progression is attributed to declining CD4+CD25+FoxP3+ regulatory T-cell (Treg) numbers. However, qualitative aspects of Treg function in HIV infection, specifically the balance between Treg cell suppressive potency versus suppressibility of effector cells, remain poorly understood. This report addresses this issue.

Methodology/Principal Findings

A classic suppression assay to measure CD4+CD45RO+CD25hi Treg cells to suppress the proliferation of CD4+CD45RO+CD25− effectors cells (E) following CD3/CD28 polyclonal stimulation was employed to compare the suppressive ability of healthy volunteers (N = 27) and chronic, asymptomatic, treatment naïve, HIV-infected subjects (N = 14). HIV-infected subjects displayed significantly elevated Treg-mediated suppression compared to healthy volunteers (p = 0.0047). Cross-over studies comparing Treg cell potency from HIV-infected versus control subjects to suppress the proliferation of a given population of allogeneic effector cells demonstrated increased sensitivity of CD4+CD25− effector cells from HIV-infected subjects to be suppressed, associated with reduced production of the Treg counter-regulatory cytokine, IL-17, rather than an increase in the suppressive potential of their CD4+CD25+ Treg cells. However, compared to controls, HIV+ subjects had significantly fewer absolute numbers of circulating CD4+CD25+FoxP3+ Treg cells. In vitro studies highlighted that one mechanism for this loss could be the preferential infection of Treg cells by HIV.

Conclusions/Significance

Together, novel data is provided to support the contention that elevated Treg-mediated suppression may be a natural host response to HIV infection     

A Decreased Frequency of Regulatory T Cells in Patients with Common Variable Immunodeficiency

Introduction

Common variable immunodeficiency disorder (CVID) is a heterogeneous syndrome, characterized by deficient antibody production and recurrent bacterial infections in addition abnormalities in T cells. CD4⁺CD25^high regulatory T cells (Treg) are essential modulators of immune responses, including down-modulation of immune response to pathogens, allergens, cancer cells and self-antigens.

Objective

In this study we set out to investigate the frequency of Treg cells in CVID patients and correlate with their immune activation status.

Materials and Methods

Sixteen patients (6 males and 10 females) with CVID who had been treated with regular intravenous immunoglobulin and 14 controls were enrolled. Quantitative analyses of peripheral blood mononuclear cells (PBMC) were performed by multiparametric flow cytometry using the following cell markers: CD38, HLA-DR, CCR5 (immune activation); CD4, CD25, FOXP3, CD127, and OX40 (Treg cells); Ki-67 and IFN-γ (intracellular cytokine).

Results

A significantly lower proportion of CD4⁺CD25^highFOXP3 T cells was observed in CVID patients compared with healthy controls (P<0.05). In addition to a higher proportion of CD8⁺ T cells from CVID patients expressing the activation markers, CD38⁺ and HLA-DR⁺ (P<0.05), we observed no significant correlation between Tregs and immune activation.

Conclusion

Our results demonstrate that a reduction in Treg cells could have impaired immune function in CVID patients.     

Accumulation of CCR4+ CTLA-4hi FOXP3+CD25hi Regulatory T Cells in Colon Adenocarcinomas Correlate to Reduced Activation of Conventional T Cells

Background

Colorectal cancer usually gives rise to a specific anti-tumor immune response, but for unknown reasons the resulting immunity is not able to clear the tumor. Recruitment of activated effector lymphocytes to the tumor is important for efficient anti-tumor responses, while the presence of regulatory T cells (Treg) down-modulate tumor-specific immunity. We therefore aimed to determine homing mechanisms and activation stage of Treg and effector T cell infiltrating colon tumors compared to cells from the unaffected mucosa in patients suffering from colon adenocarcinoma.

Methodology/Principal Findings

Lymphocytes were isolated from unaffected and tumor mucosa from patients with colon adenocarcinoma, and flow cytometry, immunohistochemistry, and quantitative PCR was used to investigate the homing mechanisms and activation stage of infiltrating Treg and conventional lymphocytes. We detected significantly higher frequencies of CD25^highFOXP3⁺CD127^low putative Treg in tumors than unaffected mucosa, which had a complete demethylation in the FOXP3 promotor. Tumor-associated Treg had a high expression of CTLA-4, and some appeared to be antigen experienced effector/memory cells based on their expression of αEβ7 (CD103). There were also significantly fewer activated T cells and more CTLA-4⁺ conventional T cells susceptible to immune regulation in the tumor-associated mucosa. In contrast, CD8⁺granzyme B⁺ putative cytotoxic cells were efficiently recruited to the tumors. The frequencies of cells expressing α4β7 and the Th1 associated chemokine receptor CXCR3 were significantly decreased among CD4⁺ T cells in the tumor, while frequencies of CD4⁺CCR4⁺ lymphocytes were significantly increased.

Conclusions/Significance

This study shows that CCR4⁺CTLA4^hi Treg accumulate in colon tumors, while the frequencies of activated conventional Th1 type T cells are decreased. The altered lymphocyte composition in colon tumors will probably diminish the ability of the immune system to effectively attack tumor cells, and reducing the Treg activity is an important challenge for future immunotherapy protocols.     

Depletion of Regulatory T Lymphocytes Reverses the Imbalance between Pro- and Anti-Tumor Immunities via Enhancing Antigen-Specific T Cell Immune Responses

Background

The regulatory T cells (Tregs) can actively suppress the immune responses. However, literature about detailed changes of host effective and suppressive immunities before and after depletion of Tregs in ovarian carcinomas, is rare.

Materials and Methods

Ovarian cancer patients and the ascitogenic animal model were employed. Immunologic profiles with flow cytometric analyses, immunohistochemistric staining, RT-PCR, ELISA, and ELISPOT assays were performed. In vivo depletion of Treg cells with the mAb PC61was also performed in the animal model.

Results

The cytokines, including IL-4 (p = 0.017) and TNF-α (p = 0.046), significantly decreased while others such as TGF-β (p = 0.013), IL-6 (p = 0.016), and IL-10 (p = 0.018) were elevated in ascites of ovarian cancer patients, when the disease progressed to advanced stages. The ratio of CD8⁺ T cell/Treg cell in ascites was also lower in advanced diseases than in early diseases (advanced 7.37±0.64 vs. early 14.25±3.11, p = 0.037). The kinetic low-dose CD25 Ab depletion group had significantly lower intra-peritoneal tumor weight (0.20±0.03 g) than the sequential high-dose (0.69±0.06 g) and sequential low-dose (0.67±0.07 g) CD25 Ab deletion groups (p = 0.001) after 49 days of tumor challenge in the animal. The kinetic low-dose CD25 Ab depletion group generated the highest number of IFN-γ-secreting, mesothelin-specific T lymphocytes compared to the other groups (p<0.001).

Conclusions

The imbalance between effective and suppressive immunities becomes more severe as a tumor progresses. The depletion of Treg cells can correct the imbalance of immunologic profiles and generate potent anti-tumor effects. Targeting Treg cells can be a new strategy for the immunotherapy of ovarian carcinoma.     

VR09 Cell Line: An EBV-Positive Lymphoblastoid Cell Line with In Vivo Characteristics of Diffuse Large B Cell Lymphoma of Activated B-Cell Type

Background

small B-cell neoplasms can show plasmacytic differentiation and may potentially progress to aggressive lymphoma (DLBCL). Epstein-Barr virus (EBV) infection may cause the transformation of malignant cells in vitro.

Design and Method

we established VR09 cell line with plasmacytic differentiation, obtained from a case of atypical, non-CLL B-cell chronic lymphoproliferative disease with plasmacytic features. We used flow cytometry, immunohistochemistry, polymerase chain reaction, cytogenetic analysis and florescence in situ hybridization in the attempt at thoroughly characterizing the cell line. We showed VR09 tumorigenic potential in vivo, leading to the development of activated DLBCL with plasmacytic features.

Results

VR09 cells displayed plasmacytic appearance and grew as spherical tumors when inoculated subcutaneously into immunodeficient Rag2^−/− γ-chain^−/− mice. VR09 cell line and tumors displayed the phenotype of activated stage of B cell maturation, with secretory differentiation (CD19+ CD20+ CD79a+ CD79b+/− CD138+ cyclin D1- Ki67 80% IgM+ IgD+ MUM1+ MNDA+ CD10- CD22+ CD23+ CD43+ K+, λ- Bcl2+ Bcl6-) and they presented episomal EBV genome, chromosome 12 trisomy, lack of c-MYC rearrangement and Myd88 gene mutation, presence of somatic hypermutation in the VH region, and wild-type p53.

Conclusion

This new EBV-positive cell line may be useful to further characterize in vivo activated DLBCL with plasmacytic features.     

Markers of T Cell Infiltration and Function Associate with Favorable Outcome in Vascularized High-Grade Serous Ovarian Carcinoma

Background

When T cells infiltrate the tumor environment they encounter a myriad of metabolic stressors including hypoxia. Overcoming the limitations imposed by an inadequate tumor vasculature that contributes to these stressors may be a crucial step to immune cells mounting an effective anti-tumor response. We sought to determine whether the functional capacity of tumor infiltrating lymphocytes (TIL) could be influenced by the tumor vasculature and correlated this with survival in patients with ovarian cancer.

Methodology and Principal Findings

In 196 high-grade serous ovarian tumors, we confirmed that the tumor vascularity as measured by the marker CD31 was associated with improved patient disease-specific survival. We also found that tumors positive for markers of TIL (CD8, CD4 and forkhead box P3 (FoxP3)) and T cell function (granzyme B and T-cell restricted intracellular antigen-1 (TIA-1)) correlated significantly with elevated vascularity. In vitro, hypoxic CD8 T cells showed reduced cytolytic activity, secreted less effector cytokines and upregulated autophagy. Survival analysis revealed that patients had a significant improvement in disease-specific survival when FoxP3 expressing cells were present in CD31-high tumors compared to patients with FoxP3 expressing cells in CD31-low tumors [HR: 2.314 (95% CI 1.049–5.106); p = 0.0377]. Patients with high vascular endothelial growth factor (VEGF) expressing tumors containing granzyme B positive cells had improved survival compared to patients with granzyme B positive cells in VEGF-low tumors [HR: 2.522 (95% CI 1.097–5.799); p = 0.0294].

Significance

Overall, this data provides a rationale for developing strategies aimed at improving the adaptability and function of TIL to hypoxic tumor conditions.     

Detection of Circulating Tumor Cell Subpopulations in Patients with Head and Neck Squamous Cell Carcinoma (HNSCC)

Background

Since image based diagnostic tools fail to detect early metastasis in head and neck squamous cell carcinoma (HNSCC) it is crucial to develop minimal invasive diagnostic methods. A promising approach is to identify and characterize circulating tumor cells (CTC) in the peripheral blood of HNSCC patients. In this pilot study, we assessed which non-hematopoietic cell types are identifiable and whether their numbers differ in pre- and postoperative blood samples.

Methods

20 ml citrated peripheral blood was taken from 10 HNSCC patients before and after curative resection. CTC were enriched using density gradient centrifugation. CTC presence was verified by multi-immunofluorescence staining against cytokeratin (CK; epithelial), N-cadherin (mesenchymal); CD133 (stem-cell), CD45 (hematopoietic) and DAPI (nucleus). Individual cell type profiles were analyzed.

Results

We were able to detect cells with epithelial properties like CK+/N-cadherin−/CD45− and CK+/CD133−/CD45− as well as cells with mesenchymal features such as N-cadherin+/CK−/CD45− and cells with both characteristics like N-cadherin+/CK+/CD45−. We also observed cells showing stem cell-like features like CD133+/CK−/CD45− and cells with both epithelial and stem cell-like features such as CD133+/CK+/CD45−. The number of CK positive cells (p = 0.002), N-cadherin positive cells (p = 0.002) and CD133 positive cells (p = 0.01) decreased significantly after resection. Kaplan-Meier test showed that the survival was significantly shorter when N-cadherin+ cells were present after resection (p = 0.04; 474 vs. 235 days; [HR] = 3.1).

Conclusions

This is - to the best of our knowledge- the first pilot study identifying different CTC populations in peripheral blood of HNSCC patients and showing that these individual cell type profiles may have distinct clinical implications.     

Regulatory T Lymphocytes Are Associated with Less Aggressive Histologic Features in Microsatellite-Unstable Colorectal Cancers

Background

Colorectal cancers (CRCs) with microsatellite instability (MSI) are associated with a good prognosis and a high density of tumor-infiltrating lymphocytes (TILs). We have undertaken to determine the link between TIL densities and MSI CRC histologic features.

Patients and Methods

Using tissue microarrays, T-cell sub-population infiltration, including T cells (CD3), cytotoxic T cells (CD8) and regulatory T cells (FoxP3) were studied in 86 MSI CRCs. We separately analyzed TILs of the stromal and epithelial compartments in the tumor center, the tumoral invasion margin and associated normal tissue.

Results

For FoxP3+ TIL density in the tumor center stromal compartment, we found a strong negative correlation with T4 stage (p = 0.01), node invasion (p<0.001) and VELIPI (vascular emboli, lymphatic invasion and perinervous invasion) criteria (p = 0.002).

Conclusion

The strong correlation between regulatory T cell density and the absence of VELIPI criteria suggests that this sub-group of T cells is preferentially associated with less invasive tumors.     

The Ratio of Treg/Th17 Cells Correlates with the Disease Activity of Primary Immune Thrombocytopenia

Background

Primary immune thrombocytopenia (ITP) is an autoimmune heterogeneous disorder that is characterized by decreased platelet count. Regulatory T (Treg) cells and T helper type 17 (Th17) cells are two subtypes of CD4⁺ T helper (Th) cells. They play opposite roles in immune tolerance and autoimmune diseases, while they share a common differentiation pathway. The imbalance of Treg/Th17 has been demonstrated in several autoimmune diseases. In this study, we aimed to investigate the ratio of the number of Treg cells to the number of Th17 cells in ITP patients and evaluate the clinical implications of the alterations in this ratio.

Methods

Thirty adult patients with newly diagnosed ITP enrolled in this study. Twelve patients had been clinically followed up for 12 months. The percentages of CD4⁺CD25^hiFoxp3⁺ Treg cells and CD3⁺CD4⁺IL-17-producing Th17 cells in these patients and healthy controls (n = 17) were longitudinally analyzed by flow cytometry.

Results

The percentage of Treg cells in ITP patients was significantly lower than that of healthy controls, and the percentage of Th17 cells increased significantly at disease onset. The ratio of Treg/Th17 correlated with the disease activity.

Conclusion

The ratio of Treg/Th17 might be relevant to the clinical diversity of ITP patients, and this Treg/Th17 ratio might have prognostic role in ITP patients.