PI3Kδ is essential for tumor clearance mediated by cytotoxic T lymphocytes

Background

PI3Kδ is a lipid kinase of the phosphoinositide 3-kinase class 1A family and involved in early signaling events of leukocytes regulating proliferation, differentiation and survival. Currently, several inhibitors of PI3Kδ are under investigation for the treatment of hematopoietic malignancies. In contrast to the beneficial effect of inhibiting PI3Kδ in tumor cells, several studies reported the requirement of PI3Kδ for the function of immune cells, such as natural killer and T helper cells. Cytotoxic T lymphocytes (CTLs) are essential for tumor surveillance. The scope of this study is to clarify the potential impact of PI3Kδ inhibition on the function of CTLs with emphasis on tumor surveillance.

Principal Findings

PI3Kδ-deficient mice develop significantly bigger tumors when challenged with MC38 colon adenocarcinoma cells. This defect is accounted for by the fact that PI3Kδ controls the secretory perforin-granzyme pathway as well as the death-receptor pathway of CTL-mediated cytotoxicity, leading to severely diminished cytotoxicity against target cells in vitro and in vivo in the absence of PI3Kδ expression. PI3Kδ-deficient CTLs express low mRNA levels of important components of the cytotoxic machinery, e.g. prf1, grzmA, grzmB, fasl and trail. Accordingly, PI3Kδ-deficient tumor-infiltrating CTLs display a phenotype reminiscent of naïve T cells (CD69^lowCD62L^high). In addition, electrophysiological capacitance measurements confirmed a fundamental degranulation defect of PI3Kδ−/− CTLs.

Conclusion

Our results demonstrate that CTL-mediated tumor surveillance is severely impaired in the absence of PI3Kδ and predict that impaired immunosurveillance may limit the effectiveness of PI3Kδ inhibitors in long-term treatment.     

Fibro-vascular coupling in the control of cochlear blood flow

Background

Transduction of sound in the cochlea is metabolically demanding. The lateral wall and hair cells are critically vulnerable to hypoxia, especially at high sound levels, and tight control over cochlear blood flow (CBF) is a physiological necessity. Yet despite the importance of CBF for hearing, consensus on what mechanisms are involved has not been obtained.

Methodology/Principal Findings

We report on a local control mechanism for regulating inner ear blood flow involving fibrocyte signaling. Fibrocytes in the super-strial region are spatially distributed near pre-capillaries of the spiral ligament of the albino guinea pig cochlear lateral wall, as demonstrably shown in transmission electron microscope and confocal images. Immunohistochemical techniques reveal the inter-connected fibrocytes to be positive for Na+/K+ ATPase β1 and S100. The connected fibrocytes display more Ca²⁺ signaling than other cells in the cochlear lateral wall as indicated by fluorescence of a Ca²⁺ sensor, fluo-4. Elevation of Ca²⁺ in fibrocytes, induced by photolytic uncaging of the divalent ion chelator o-nitrophenyl EGTA, results in propagation of a Ca²⁺ signal to neighboring vascular cells and vasodilation in capillaries. Of more physiological significance, fibrocyte to vascular cell coupled signaling was found to mediate the sound stimulated increase in cochlear blood flow (CBF). Cyclooxygenase-1 (COX-1) was required for capillary dilation.

Conclusions/Significance

The findings provide the first evidence that signaling between fibrocytes and vascular cells modulates CBF and is a key mechanism for meeting the cellular metabolic demand of increased sound activity.     

Circulating vascular progenitor cells and central arterial stiffness in polycystic ovary syndrome

Objective

Subjects with Polycystic ovarian syndrome (PCOS) are at increased risk of Type 2 diabetes mellitus (T2DM). The mechanism of this enhanced risk is unclear. Circulating vascular progenitor cells (VPC) are immature bone marrow derived cells capable of differentiating into mature endothelial cells. VPC number/function and central arterial stiffness predict cardio-metabolic disease in at-risk populations.

Design

We studied VPC and arterial stiffness measures in non-obese PCOS subjects as compared to age and body mass index (BMI) matched healthy controls in a cross–sectional study.

Methods

Fourteen subjects with PCOS and 12 controls of similar age, BMI (all <30 kg/m²) and metabolic profile were studied. VPC number and in vitro function were studied by flow cytometry and tube formation assays respectively. Augmentation index (AIx), a measure of central arterial stiffness, and central (aortic) blood pressures (BP) were measured by applanation tonometry.

Results

Subjects with PCOS had a reduced number, mean±SEM, of circulating CD34⁺133⁺ VPCs (317.5±51.0 vs. 558.3±101.2, p = 0.03) and impaired in vitro tube formation (completed tube area 1.0±0.06 vs. 1.2±0.05×10⁶ µm² p = 0.02). PCOS subjects had significantly higher AIx (18.4±1.9% vs. 4.9±2.0%) and this difference remained significant even after adjustments for age, BMI and smoking (p = 0.003) in multivariate analyses. Central systolic and pulse pressure were higher in PCOS subjects but these differences were not statistically significant after adjustment for age. Brachial systolic and pulse pressures were similar. VPC number/function and arterial stiffness or BP measures were not correlated.

Conclusions

Non-obese PCOS is characterized by a reduced VPC number, impaired VPC function and increased central arterial stiffness. These changes in novel vascular risk markers may explain the enhanced risk of T2DM and CVD in PCOS.     

In Vitro Priming Recapitulates In Vivo HIV-1 Specific T Cell Responses,Revealing Rapid Loss of Virus Reactive CD4+ T Cells in Acute HIV-1 Infection

Background

The requirements for priming of HIV-specific T cell responses initially seen in infected individuals remain to be defined. Activation of T cell responses in lymph nodes requires cell-cell contact between T cells and DCs, which can give concurrent activation of T cells and HIV transmission.

Methodology

The study aim was to establish whether DCs pulsed with HIV-1 could prime HIV-specific T cell responses and to characterize these responses. Both infectious and aldrithiol-2 inactivated noninfectious HIV-1 were compared to establish efficiencies in priming and the type of responses elicited.

Findings

Our findings show that both infectious and inactivated HIV-1 pulsed DCs can prime HIV-specific responses from naïve T cells. Responses included several CD4⁺ and CD8⁺ T cell epitopes shown to be recognized in vivo by acutely and chronically infected individuals and some CD4⁺ T cell epitopes not identified previously. Follow up studies of acute and recent HIV infected samples revealed that these latter epitopes are among the earliest recognized in vivo, but the responses are lost rapidly, presumably through activation-induced general CD4⁺ T cell depletion which renders the newly activated HIV-specific CD4⁺ T cells prime targets for elimination.

Conclusion

Our studies highlight the ability of DCs to efficiently prime naïve T cells and induce a broad repertoire of HIV-specific responses and also provide valuable insights to the pathogenesis of HIV-1 infection in vivo.     

Cytokine profiles, CTL response and T cell frequencies in the peripheral blood of acute patients and individuals recovered from hepatitis E infection

Background

Hepatitis E is a major public health problem in the developing countries. Pathogenesis of hepatitis E virus (HEV) infection is poorly understood.

Methods

This case-control study included 124 Hepatitis E patients (46 acute and 78 recovered), 9 with prior exposure to HEV and 71 anti-HEV negative healthy controls. HEV induced CTL response by Elispot, cytokines/chemokines quantitation by Milliplex assay and peripheral CD4+ & CD8+ T cell frequencies by flow cytometry were assessed.

Results

Among the patient categories, HEV specific IFN-γ responses as recorded by Elispot were comparable. Comparisons of cytokines/chemokines revealed significantly high levels of IL-1α and sIL-2Rα during acute phase. Circulating peripheral CD4/CD8+ T-cell subsets in acute and recovered individuals were comparable compared to controls, while among patient categories CD8+T cell subset was significantly higher in recovered individuals.

Conclusions

Our findings suggest that IL-1α and sIL-2Rα play a role in the pathogenesis of acute Hepatitis E infection. Lack of robust HEV ORF2-specific CTL response in the peripheral blood of HEV infected patients during the acute and recovered phases of the disease may be associated with involvement of innate immune cells/localization of the immune events at the site of infection.     

Effects of natalizumab treatment on Foxp3+ T regulatory cells

Background

Natalizumab, a monoclonal humanized antibody targeting the alpha-4 chain of very late activation antigen 4 (VLA-4) exerts impressive therapeutic effects in patients with relapsing-remitting multiple sclerosis. Our objective was to study impacts of Natalizumab therapy on Foxp3+ T regulatory cells (Tregs) in multiple sclerosis (MS) patients.

Methodology

A combined approach of in vitro and ex vivo experiments using T cells isolated from the peripheral blood of healthy donors and Natalizumab treated MS patients was chosen. We determined binding of Natalizumab and its effects on the frequency, transmigratory behaviour and suppressive function of Tregs.

Principal Findings

Binding of Natalizumab and expression of CD49d (alpha-4 chain of VLA-4) differed between non-regulatory and regulatory cells. Albeit Foxp3+ Tregs had lower levels of CD49d, Natalizumab blocked the transmigration of Foxp3+ Tregs similar to non-regulatory T cells. The frequency of peripheral blood Tregs was unaffected by Natalizumab treatment. Natalizumab does not alter the suppressive capacity of CD4+CD25^highCD127^lowFoxp3+ Tregs under in vitro conditions. Furthermore, the impaired function of Tregs in MS patients is not restored by Natalizumab treatment.

Conclusions

We provide a first detailed analysis of Natalizumab effects on the regulatory T cell population. Our prospective study shows that Foxp3+ Tregs express lower levels of VLA-4 and bind less Natalizumab. We further the understanding of the mechanisms of action of Natalizumab by demonstrating that unlike other immunomodulatory drugs the beneficial therapeutic effects of the monoclonal antibody are largely independent of alterations in Treg frequency or function.     

The prognostic value of tumor-infiltrating neutrophils in gastric adenocarcinoma after resection

Background

Several pieces of evidence indicate that tumor-infiltrating neutrophils (TINs) are correlated to tumor progression. In the current study, we explore the relationship between TINs and clinicopathological features of gastric adenocarcinoma patients. Furthermore, we investigated the prognostic value of TINs.

Patients and Methods

The study was comprised of two groups, training group (115 patients) and test group (97 patients). Biomarkers (intratumoral CD15+ neutrophils) were assessed by immunohistochemistry. The relationship between clinicopathological features and patient outcome were evaluated using Cox regression and Kaplan-Meier analysis.

Results

Immunohistochemical detection showed that the tumor-infiltrating neutrophils (TINs) in the training group ranged from 0.00–115.70 cells/high-power microscopic field (HPF) and the median number was 21.60 cells/HPF. Based on the median number, the patients were divided into high and low TINs groups. Chi-square test analysis revealed that the density of CD15+ TINs was positively associated with lymph node metastasis (p = 0.024), distance metastasis (p = 0.004) and UICC (International Union Against Cancer) staging (p = 0.028). Kaplan-Meier analysis showed that patients with a lower density of TINs had a better prognosis than patients with a higher density of TINs (p = 0.002). Multivariate Cox''s analysis showed that the density of CD15+ TINs was an independent prognostic factor for overall survival of gastric adenocarcinoma patients. Using another 97 patients as a test group and basing on the median number of TINs (21.60 cells/HPF) coming from the training group, Kaplan-Meier analysis also showed that patients with a lower density of TINs had a better prognosis than patients with a higher density of TINs (p = 0.032). The results verify that the number of CD15+ TINs can predict the survival of gastric adenocarcinoma surgical patients.

Conclusions

The presence of CD15+ TINs is an independent and unfavorable factor in the prognosis of gastric adenocarcinoma patients. Targeting CD15+ TINs may be a potential intervenient therapy in the future.     

CD8+ T Cells Cause Disability and Axon Loss in a Mouse Model of Multiple Sclerosis

Background

The objective of this study was to test the hypothesis that CD8⁺ T cells directly mediate motor disability and axon injury in the demyelinated central nervous system. We have previously observed that genetic deletion of the CD8⁺ T cell effector molecule perforin leads to preservation of motor function and preservation of spinal axons in chronically demyelinated mice.

Methodology/Principal Findings

To determine if CD8⁺ T cells are necessary and sufficient to directly injure demyelinated axons, we adoptively transferred purified perforin-competent CD8⁺ spinal cord-infiltrating T cells into profoundly demyelinated but functionally preserved perforin-deficient host mice. Transfer of CD8⁺ spinal cord-infiltrating T cells rapidly and irreversibly impaired motor function, disrupted spinal cord motor conduction, and reduced the number of medium- and large-caliber spinal axons. Likewise, immunodepletion of CD8⁺ T cells from chronically demyelinated wildtype mice preserved motor function and limited axon loss without altering other disease parameters.

Conclusions/Significance

In multiple sclerosis patients, CD8⁺ T cells outnumber CD4⁺ T cells in active lesions and the number of CD8⁺ T cells correlates with the extent of ongoing axon injury and functional disability. Our findings suggest that CD8⁺ T cells may directly injure demyelinated axons and are therefore a viable therapeutic target to protect axons and motor function in patients with multiple sclerosis.     

Human cellular immune response to the saliva of Phlebotomus papatasi is mediated by IL-10-producing CD8+ T cells and Th1-polarized CD4+ lymphocytes

Background

The saliva of sand flies strongly enhances the infectivity of Leishmania in mice. Additionally, pre-exposure to saliva can protect mice from disease progression probably through the induction of a cellular immune response.

Methodology/Principal Findings

We analysed the cellular immune response against the saliva of Phlebotomus papatasi in humans and defined the phenotypic characteristics and cytokine production pattern of specific lymphocytes by flow cytometry. Additionally, proliferation and IFN-γ production of activated cells were analysed in magnetically separated CD4+ and CD8+ T cells. A proliferative response of peripheral blood mononuclear cells against the saliva of Phlebotomus papatasi was demonstrated in nearly 30% of naturally exposed individuals. Salivary extracts did not induce any secretion of IFN-γ but triggered the production of IL-10 primarily by CD8+ lymphocytes. In magnetically separated lymphocytes, the saliva induced the proliferation of both CD4+ and CD8+ T cells which was further enhanced after IL-10 blockage. Interestingly, when activated CD4+ lymphocytes were separated from CD8+ cells, they produced high amounts of IFN-γ.

Conclusion

Herein, we demonstrated that the overall effect of Phlebotomus papatasi saliva was dominated by the activation of IL-10-producing CD8+ cells suggesting a possible detrimental effect of pre-exposure to saliva on human leishmaniasis outcome. However, the activation of Th1 lymphocytes by the saliva provides the rationale to better define the nature of the salivary antigens that could be used for vaccine development.     

Relationships between Levels of Serum IgE,Cell-Bound IgE,and IgE-Receptors on Peripheral Blood Cells in a Pediatric Population

Background

Elevated serum immunoglobulin (Ig) E is a diagnostic marker of immediate-type allergic reactions. We hypothesize that serum IgE does not necessarily reflect total body IgE because in vivo IgE can be bound to cell surface receptors such as FcεRI and FcεRII (CD23). The aim of this study was to analyze the relationships between levels of serum IgE, cell-bound IgE, and IgE-receptors on peripheral blood cells in a pediatric population.

Methodology

Whole blood samples from 48 children (26 boys, 22 girls, mean age 10,3±5,4 years) were analyzed by flow cytometry for FcεRI, CD23, and cell-bound IgE on dendritic cells (CD11c+MHC class II+), monocytes (CD14+), basophils (CD123+MHC class II-) and neutrophils (myeloperoxidase+). Total serum IgE was measured by ELISA and converted into z-units to account for age-dependent normal ranges. Correlations were calculated using Spearman rank correlation test.

Principal Findings

Dendritic cells, monocytes, basophils, and neutrophils expressed the high affinity IgE-receptor FcεRI. Dendritic cells and monocytes also expressed the low affinity receptor CD23. The majority of IgE-receptor positive cells carried IgE on their surface. Expression of both IgE receptors was tightly correlated with cell-bound IgE. In general, cell-bound IgE on FcεRI+ cells correlated well with serum IgE. However, some patients carried high amounts of cell-bound IgE despite low total serum IgE levels.

Conclusion/Significance

In pediatric patients, levels of age-adjusted serum IgE, cell-bound IgE, and FcεRI correlate. Even in the absence of elevated levels of serum IgE, cell-bound IgE can be detected on peripheral blood cells in a subgroup of patients.     

Local induction of immunosuppressive CD8+ T cells in the gut-associated lymphoid tissues

Background

In contrast to intestinal CD4⁺ regulatory T cells (T_regs), the generation and function of immunomodulatory intestinal CD8⁺ T cells is less well defined. To dissect the immunologic mechanisms of CD8⁺ T cell function in the mucosa, reactivity against hemagglutinin (HA) expressed in intestinal epithelial cells of mice bearing a MHC class-I-restricted T-cell-receptor specific for HA was studied.

Methodology and Principal Findings

HA-specific CD8⁺ T cells were isolated from gut-associated tissues and phenotypically and functionally characterized for the expression of Foxp3⁺ and their suppressive capacity. We demonstrate that intestinal HA expression led to peripheral induction of HA-specific CD8⁺Foxp3⁺ T cells. Antigen-experienced CD8⁺ T cells in this transgenic mouse model suppressed the proliferation of CD8⁺ and CD4⁺ T cells in vitro. Gene expression analysis of suppressive HA-specific CD8⁺ T cells revealed a specific up-regulation of CD103, Nrp1, Tnfrsf9 and Pdcd1, molecules also expressed on CD4⁺ T_reg subsets. Finally, gut-associated dendritic cells were able to induce HA-specific CD8⁺Foxp3⁺ T cells.

Conclusion and Significance

We demonstrate that gut specific antigen presentation is sufficient to induce CD8⁺ T_regs in vivo which may maintain intestinal homeostasis by down-modulating effector functions of T cells.     

"Negative vaccination" by specific CD4 T cell tolerisation enhances virus-specific protective antibody responses

Background

Cooperation of CD4⁺ T helper cells with specific B cells is crucial for protective vaccination against pathogens by inducing long-lived neutralizing antibody responses. During infection with persistence-prone viruses, prolonged virus replication correlates with low neutralizing antibody responses. We recently described that a viral mutant of lymphocytic choriomeningitis virus (LCMV), which lacks a T helper epitope, counterintuitively induced an enhanced protective antibody response. Likewise, partial depletion of the CD4⁺ T cell compartment by using anti-CD4 antibodies enhanced protective antibodies.

Principal Findings

Here we have developed a protocol to selectively reduce the CD4⁺ T cell response against viral CD4⁺ T cell epitopes. We demonstrate that in vivo treatment with LCMV-derived MHC-II peptides induced non-responsiveness of specific CD4⁺ T cells without affecting CD4⁺ T cell reactivity towards other antigens. This was associated with accelerated virus-specific neutralizing IgG-antibody responses. In contrast to a complete absence of CD4⁺ T cell help, tolerisation did not impair CD8⁺ T cell responses.

Conclusions

This result reveals a novel “negative vaccination” strategy where specific CD4⁺ T cell unresponsiveness may be used to enhance the delayed protective antibody responses in chronic virus infections.     

Subcellular Min Oscillations as a Single-Cell Reporter of the Action of Polycations,Protamine, and Gentamicin on Escherichia coli

Background

In Escherichia coli, MinD-GFP fusion proteins show rapid pole to pole oscillations. The objective was to investigate the effects of extracellular cations on the subcellular oscillation of cytoplasmic MinD within Escherichia coli.

Methodology/Principal Findings

We exposed bacteria to the extracellular cations Ca⁺⁺, Mg⁺⁺, the cationic antimicrobial peptide (CAP) protamine, and the cationic aminoglycoside gentamicin. We found rapid and substantial increases in the average MinD oscillation periods in the presence of any of these polyvalent cations. For Ca⁺⁺ and Mg⁺⁺ the increases in period were transient, even with a constant extracellular concentration, while increases in period for protamine or gentamicin were apparently irreversible. We also found striking interdependence in the action of the small cations with protamine or gentamicin, distorted oscillations under the action of intermediate levels of gentamicin and Ca⁺⁺, and reversible freezing of the Min oscillation at high cationic concentrations.

Conclusions/Significance

Intracellular Min oscillations provide a fast single-cell reporter of bacterial response to extracellular polycations, which can be explained by the penetration of polycations into cells.     

Granulysin-expressing CD4+ T cells as candidate immune marker for tuberculosis during childhood and adolescence

Background

Granulysin produced by cytolytic T cells directly contributes to immune defense against tuberculosis (TB). We investigated granulysin as a candidate immune marker for childhood and adolescent TB.

Methods

Peripheral blood mononuclear cells (PBMC) from children and adolescents (1–17 years) with active TB, latent TB infection (LTBI), nontuberculous mycobacteria (NTM) infection and from uninfected controls were isolated and restimulated in a 7-day restimulation assay. Intracellular staining was then performed to analyze antigen-specific induction of activation markers and cytotoxic proteins, notably, granulysin in CD4⁺ CD45RO⁺ memory T cells.

Results

CD4⁺ CD45RO⁺ T cells co-expressing granulysin with specificity for Mycobacterium tuberculosis (Mtb) were present in high frequency in TB-experienced children and adolescents. Proliferating memory T cells (CFSE_lowCD4⁺CD45RO⁺) were identified as main source of granulysin and these cells expressed both central and effector memory phenotype. PBMC from study participants after TB drug therapy revealed that granulysin-expressing CD4⁺ T cells are long-lived, and express several activation and cytotoxicity markers with a proportion of cells being interferon-gamma-positive. In addition, granulysin-expressing T cell lines showed cytolytic activity against Mtb-infected target cells.

Conclusions

Our data suggest granulysin expression by CD4⁺ memory T cells as candidate immune marker for TB infection, notably, in childhood and adolescence.     

Trans-epithelial immune cell transfer during suckling modulates delayed-type hypersensitivity in recipients as a function of gender

Introduction

Breast feeding has long term effects on the developing immune system which outlive passive immunization of the neonate. We have investigated the transfer of milk immune cells and examined the result of transfer once the recipients were adult.

Methods

Non-transgenic mouse pups were foster-nursed by green fluorescent protein (GFP) transgenic dams for 3 weeks and the fate of GFP+ cells was followed by FACS analysis, immunohistochemistry and RT-PCR for GFP and appropriate immune cell markers. Pups suckled by non-transgenic dams served as controls.

Results

Despite a preponderance of B cells and macrophages in the stomach contents of the pups, most cells undergoing trans-epithelial migration derived from the 3–4% of milk cells positive for T lymphocyte markers. These cells homed to the spleen and thymus, with maximal accumulation at 3–4 weeks. By sensitizing dams with an antigen which elicits a T cell-mediated delayed-type-hypersensitivity (DTH) response, we determined that nursing by a sensitized dam (compared to a non-sensitized dam) amplified a subsequent DTH response in females and yet suppressed one in males.

Discussion

These results suggest that clinical evaluation weighing the pros and cons of nursing male versus female children by mothers with genetically-linked hypersensitivity diseases, such as celiac disease and eczema, or those in regions of the world with endemic DTH-eliciting diseases, such as tuberculosis, may be warranted.     

Resting regulatory CD4 T cells: a site of HIV persistence in patients on long-term effective antiretroviral therapy

Background

In HIV-infected patients on long-term HAART, virus persistence in resting long-lived CD4 T cells is a major barrier to curing the infection. Cell quiescence, by favouring HIV latency, reduces the risk of recognition and cell destruction by cytotoxic lymphocytes. Several cell-activation-based approaches have been proposed to disrupt cell quiescence and then virus latency, but these approaches have not eradicated the virus. CD4⁺CD25⁺ regulatory T cells (Tregs) are a CD4⁺ T-cell subset with particular activation properties. We investigated the role of these cells in virus persistence in patients on long-term HAART.

Methodology/Principal Findings

We found evidence of infection of resting Tregs (HLADR⁻CD69⁻CD25^hiFoxP3⁺CD4⁺ T cells) purified from patients on prolonged HAART. HIV DNA harbouring cells appear more abundant in the Treg subset than in non-Tregs. The half-life of the Treg reservoir was estimated at 20 months. Since Tregs from patients on prolonged HAART showed hyporesponsiveness to cell activation and inhibition of HIV-specific cytotoxic T lymphocyte-related functions upon activation, therapeutics targeting cell quiescence to induce virus expression may not be appropriate for purging the Treg reservoir.

Conclusions

Our results identify Tregs as a particular compartment within the latent reservoir that may require a specific approach for its purging.     

Fetal microchimeric cells in blood of women with an autoimmune thyroid disease

Context

Hashimoto''s thyroiditis (HT) and Graves'' disease (GD), two autoimmune thyroid diseases (AITD), occur more frequently in women than in men and show an increased incidence in the years following parturition. Persisting fetal cells could play a role in the development of these diseases.

Objective

Aim of this study was to detect and characterize fetal cells in blood of postpartum women with and without an AITD.

Participants

Eleven patients with an AITD and ten healthy volunteers, all given birth to a son maximum 5 years before analysis, and three women who never had been pregnant, were included. None of them had any other disease of the thyroid which could interfere with the results obtained.

Methods

Fluorescence in situ hybridization (FISH) and repeated FISH were used to count the number of male fetal cells. Furthermore, the fetal cells were further characterized.

Results

In patients with HT, 7 to 11 fetal cells per 1.000.000 maternal cells were detected, compared to 14 to 29 fetal cells in patients with GD (p = 0,0061). In patients with HT, mainly fetal CD8⁺ T cells were found, while in patients with GD, fetal B and CD4⁺ T cells were detected. In healthy volunteers with son, 0 to 5 fetal cells were observed, which was significantly less than the number observed in patients (p<0,05). In women who never had been pregnant, no male cells were detected.

Conclusion

This study shows a clear association between fetal microchimeric cells and autoimmune thyroid diseases.     

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.     

The presence of IL-17A and T helper 17 cells in experimental mouse brain tumors and human glioma

Background

Recently, CD4⁺IL-17A⁺ T helper 17 (Th17) cells were identified and reported in several diseased states, including autoimmunity, infection and various peripheral nervous system tumors. However, the presence of Th17 in glia-derived tumors of the central nervous system has not been studied.

Methodology/Principal Findings

In this report, we demonstrate that mRNA expression for the Th17 cell cytokine IL-17A, as well as Th17 cells, are present in human glioma. The mRNA expression for IL-17A in glioma was recapitulated in an immunocompetent mouse model of malignant glioma. Furthermore, the presence of Th17 cells was confirmed in both human and mouse glioma. Interestingly, some Th17 cells present in mouse glioma co-expressed the Th1 and Th2 lineage markers, IFN-γ and IL-4, respectively, but predominantly co-expressed the Treg lineage marker FoxP3.

Conclusions

These data confirm the presence of Th17 cells in glia-derived CNS tumors and provide the rationale for further investigation into the role of Th17 cells in malignant glioma.     

Increased bone marrow interleukin-7 (IL-7)/IL-7R levels but reduced IL-7 responsiveness in HIV-positive patients lacking CD4+ gain on antiviral therapy

Background

The bone marrow (BM) cytokine milieu might substantially affect T-lymphocyte homeostasis in HIV-positive individuals. Interleukin-7 (IL-7) is a bone marrow-derived cytokine regulating T-cell homeostasis through a CD4+-driven feedback loop. CD4+ T-lymphopenia is associated with increased free IL-7 levels and reduced IL-7R expression/function, which are only partially reverted by highly active antiretroviral therapy (HAART). We investigated the BM production, peripheral expression and signaling (pStat5+ and Bcl-2+ CD4+/CD8+ T cells) of IL-7/IL-7Rα in 30 HAART-treated HIV-positive patients who did not experience CD4+ recovery (CD4+ ≤200/µl) and who had different levels of HIV viremia; these patients included 18 immunological nonresponders (INRs; HIV-RNA≤50), 12 complete failures (CFs; HIV-RNA>1000), and 23 HIV-seronegative subjects.

Methods

We studied plasma IL-7 levels, IL-7Rα+CD4+/CD8+ T-cell proportions, IL-7Rα mRNA expression in PBMCs, spontaneous IL-7 production by BM mononuclear cells (BMMCs), and IL-7 mRNA/IL-7Rα mRNA in BMMC-derived stromal cells (SCs). We also studied T-cell responsiveness to IL-7 by measuring the proportions of pStat5+ and Bcl-2+ CD4+/CD8+ T cells.

Results

Compared to HIV-seronegative controls, CFs and INRs presented elevated plasma IL-7 levels and lower IL-7Rα CD4+/CD8+ cell-surface expression and peripheral blood production, confirming the most relevant IL-7/IL-7R disruption. Interestingly, BM investigation revealed a trend of higher spontaneous IL-7 production in INRs (p = .09 vs. CFs) with a nonsignificant trend toward higher IL-7-Rα mRNA levels in BMMC-derived stromal cells. However, upon IL-7 stimulation, the proportion of pStat5+CD4+ T cells did not increase in INRs despite higher constitutive levels (p = .06); INRs also displayed lower Bcl-2+CD8+ T-cell proportions than controls (p = .04).

Conclusions

Despite severe CD4+ T-lymphopenia and a disrupted IL-7/IL-7R profile in the periphery, INRs display elevated BM IL-7/IL-7Rα expression but impaired T-cell responsiveness to IL-7, suggesting the activity of a central compensatory pathway targeted to replenish the CD4+ compartment, which is nevertheless inappropriate to compensate the dysfunctional signaling through IL-7 receptor.