Circulating Angiogenic Cells can be Derived from Cryopreserved Peripheral Blood Mononuclear Cells

Background

Cell transplantation for regenerative medicine has become an appealing therapeutic method; however, stem and progenitor cells are not always freshly available. Cryopreservation offers a way to freeze cells as they are generated, for storage and transport until required for therapy. This study was performed to assess the feasibility of cryopreserving peripheral blood mononuclear cells (PBMCs) for the subsequent in vitro generation of their derived therapeutic population, circulating angiogenic cells (CACs).

Methods

PBMCs were isolated from healthy human donors. Freshly isolated cells were either analyzed immediately or cryopreserved in media containing 6% plasma serum and 5% dimethyl sulfoxide. PBMCs were thawed after being frozen for 1 (early thaw) or 28 (late thaw) days and analyzed, or cultured for 4 days to generate CACs. Analysis of the cells consisted of flow cytometry for viability and phenotype, as well as functional assays for their adhesion and migration potential, cytokine secretion, and in vivo angiogenic potential.

Results

The viability of PBMCs and CACs as well as their adhesion and migration properties did not differ greatly after cryopreservation. Phenotypic changes did occur in PBMCs and to a lesser extent in CACs after freezing; however the potent CD34⁺VEGFR2⁺CD133⁺ population remained unaffected. The derived CACs, while exhibiting changes in inflammatory cytokine secretion, showed no changes in the secretion of important regenerative and chemotactic cytokines, nor in their ability to restore perfusion in ischemic muscle.

Conclusion

Overall, it appears that changes do occur in cryopreserved PBMCs and their generated CACs; however, the CD34⁺VEGFR2⁺CD133⁺ progenitor population, the secretion of pro-vasculogenic factors, and the in vivo angiogenic potential of CACs remain unaffected by cryopreservation.     

Sprint Interval and Sprint Continuous Training Increases Circulating CD34+ Cells and Cardio-Respiratory Fitness in Young Healthy Women

Introduction

The improvement of vascular health in the exercising limb can be attained by sprint interval training (SIT). However, the effects on systemic vascular function and on circulating angiogenic cells (CACs) which may contribute to endothelial repair have not been investigated. Additionally, a comparison between SIT and sprint continuous training (SCT) which is less time committing has not been made.

Methods

12 women (22±2 yrs) completed 12 sessions of either SIT (n = 6) or work-matched SCT (n = 6) on 3 days/week. Pre and post-training assessments included brachial artery endothelial function and peripheral blood analysis for CAC number (CD34⁺/CD34⁺CD45^dim). CAC function was measured by migration and adhesion assays. Cardio-respiratory fitness, carotid arterial stiffness and carotid-radial and brachial-foot pulse wave velocity (PWV) were also evaluated.

Results

CD34⁺ CACs increased following training in both groups but CD34⁺CD45^dim did not (Pre CD34⁺: 40±21/10⁵ leukocytes, Post CD34⁺: 56±24/10⁵ leukocytes, main time effect p<0.05). Brachial artery flow-mediated dilation (FMD) increased following SIT but SCT had no effect (Pre SIT: 5.0±3.4%, Post SIT: 5.9±3.0%, Pre SCT: 7.2±2.7%, Post SCT: 6.5±2.9%; group x time interaction p = 0.08). increased in both training groups (Pre: 34.6±4.6 ml•kg•ml⁻¹, Post: 36.9±5.4 ml•kg•ml⁻¹, main time effect p<0.05). CAC function, carotid arterial stiffness and PWV did not change after training (p>0.05).

Discussion

SCT involving little time commitment is comparable to SIT in increasing CD34⁺ cell number and . An increased mobilisation of CD34⁺ CACs suggests that sprint training may be an effective method to enhance vascular repair.     

Tumor-conditioned Gr-1CD11b myeloid cells induce angiogenesis through the synergistic action of CCL2 and CXCL16 in vitro

Gr-1⁺CD11b⁺ cells can suppress innate and adaptive immunity, and the functional immunosuppressive characteristics of these cells can be modulated by the tumor microenvironment. Since Gr-1⁺CD11⁺ cells are also involved in tumor-associated angiogenesis, we hypothesized that the angiogenic nature of Gr-1⁺CD11b⁺ cells could be regulated by the tumor milieu. To address this hypothesis, we imitated a tumor microenvironment by exposing Gr-1⁺CD11b⁺ cells isolated from spleen of 4T1 mammary carcinoma-bearing mice to tumor-conditioned medium. Supernatants from tumor-conditioned Gr-1⁺CD11b⁺ cells significantly induced capillary-like tube formation and migration of human umbilical vein endothelial cells (HUVECs) compared to naive Gr-1⁺CD11b⁺ cells. Incubation of Gr-1⁺CD11b⁺ cells with tumor-conditioned medium induced production of pro-angiogenic chemokines CCL2 and CXCL16. Pretreatment with an anti-CCL2 antibody, but not an anti-CXCL16 antibody, suppressed the angiogenic effects of tumor-conditioned Gr-1⁺CD11b⁺ cells on HUVECs. Simultaneous neutralization of CCL2 and CXCL16 significantly inhibited tube formation and migration of HUVECs compared to the sole neutralization against CCL2. Supernatants from tumor-conditioned Gr-1⁺CD11b⁺ cells induced phosphorylation of ERK1/2 in HUVECs, and inhibition of the ERK pathway blocked angiogenic effects. ERK pathway activity was partially abrogated by neutralization of CCL2 and more suppressed by simultaneous neutralization of CCL2 and CXCL16. These results collectively indicate that CCL2 and CXCL16 chemokines produced by tumor-conditioned Gr-1⁺CD11b⁺ myeloid cells synergistically induce angiogenesis in vitro by stimulating the ERK1/2 signaling pathway. Thus, regulation of Gr-1⁺CD11b⁺ cells in the tumor microenvironment may contribute to angiogenesis through the secretion of pro-angiogenic chemokines.     

Functional Dichotomy between NKG2D and CD28-Mediated Co-Stimulation in Human CD8+ T Cells

Both CD28 and NKG2D can function as co-stimulatory receptors in human CD8⁺ T cells. However, their independent functional contributions in distinct CD8⁺ T cell subsets are not well understood. In this study, CD8⁺ T cells in human peripheral blood- and lung-derived lymphocytes were analyzed for CD28 and NKG2D expression and function. We found a higher level of CD28 expression in PBMC-derived naïve (CD45RA⁺CD27⁺) and memory (CD45RA⁻CD27⁺) CD8⁺ T cells (CD28^Hi), while its expression was significantly lower in effector (CD45RA⁺CD27⁻) CD8⁺ T cells (CD28^Lo). Irrespective of the differences in the CD28 levels, NKG2D expression was comparable in all three CD8⁺ T cell subsets. CD28 and NKG2D expressions followed similar patterns in human lung-resident GILGFVFTL/HLA-A2-pentamer positive CD8⁺ T cells. Co-stimulation of CD28^Lo effector T cells via NKG2D significantly increased IFN-γ and TNF-α levels. On the contrary, irrespective of its comparable levels, NKG2D-mediated co-stimulation failed to augment IFN-γ and TNF-α production in CD28^Hi naïve/memory T cells. Additionally, CD28-mediated co-stimulation was obligatory for IL-2 generation and thereby its production was limited only to the CD28^Hi naïve/memory subsets. MICA, a ligand for NKG2D was abundantly expressed in the tracheal epithelial cells, validating the use of NKG2D as the major co-stimulatory receptor by tissue-resident CD8⁺ effector T cells. Based on these findings, we conclude that NKG2D may provide an expanded level of co-stimulation to tissue-residing effector CD8⁺ T cells. Thus, incorporation of co-stimulation via NKG2D in addition to CD28 is essential to activate tumor or tissue-infiltrating effector CD8⁺ T cells. However, boosting a recall immune response via memory CD8⁺ T cells or vaccination to stimulate naïve CD8⁺ T cells would require CD28-mediated co-stimulation.     

Expression of regulatory receptors on γδ T Cells and their cytokine production in Behcet's disease

Introduction

Behcet''s disease (BD) is a multi-systemic disorder with muco-cutaneous, ocular, arthritic, vascular or central nervous system involvement. The role of γδ T cells is implicated in BD. The activation status of γδ T cells and their cytokine secretion against phosphoantigens are evaluated in BD.

Methods

NKG2A, NKG2C, NKG2D, CD16 and CCR7 molecules on γδ T cells were analyzed in 70 BD, 27 tuberculosis (TB) patients and 26 healthy controls (HC). Peripheral γδ T cells were expanded with a phosphoantigen (BrHPP) and IL-2, restimulated with BrHPP and a TLR3 ligand, and cytokine production was measured.

Results

γδ T cells were not increased in both BD and TB patients, but the proportions of TCRVδ2⁺ T cells were lower (58.9 and 50.7 vs. 71.7%, P = 0.04 and P = 0.005) compared to HC. Higher proportion of TCRVδ2⁺ T cells were CD16⁺ (26.2 and 33.9 vs. 16.6%, P = 0.02 and P = 0.001) and CCR7^- (32.2 and 27.9 vs. 17.7%, P < 0.0001 and P = 0.014) in BD and TB patients compared to HC. NKG2C⁺ γδ⁺ T cells were relatively increased (0.5 and 0.6 vs. 0.3%, P = 0.008 and 0.018), whereas NKG2D positivity was decreased in patients with BD and TB (77.7 and 75.8 vs. 87.5%, P = 0.001 and 0.004). Expansion capacity of γδ T cells in BD and TB as well as production of IL-13, IFN-γ, granulocyte monocyte colony stimulating factor (GM-CSF), TNF-α, CCL4 and CCL5 in BD was lower compared to HC, when restimulated by TLR3 ligand and BrHPP.

Conclusion

The changes on γδ T cells of BD as well as TB patients implicate that γδ T cells have already been exposed to regulatory effects, which changed their activity. Lower cytokine response of γδ T cells implicates down modulation of these cells in BD.     

Differential Capacity of Human Skin Dendritic Cells to Polarize CD4+T Cells into IL-17, IL-21 and IL-22 Producing Cells

Accumulating evidence suggests a contribution of T cell-derived IL-17, IL-21 and IL-22 cytokines in skin immune homeostasis as well as inflammatory disorders. Here, we analyzed whether the cytokine-producing T lymphocytes could be induced by the different subsets of human skin dendritic cells (DCs), i.e., epidermal Langerhans cells (LCs), dermal CD1c⁺CD14⁻ and CD14⁺ DCs (DDCs). DCs were purified following a 2-day migration from separated epidermal and dermal sheets and co-cultured with allogeneic T cells before cytokine secretion was explored. Results showed that no skin DCs could induce substantial IL-17 production by naïve CD4⁺ or CD8⁺T lymphocytes whereas all of them could induce IL-17 production by memory T cells. In contrast, LCs and CD1c⁺CD14⁻DDCs were able to differentiate naïve CD4⁺T lymphocytes into IL-22 and IL-21-secreting cells, LCs being the most efficient in this process. Intracellular cytokine staining showed that the majority of IL-21 or IL-22 secreting CD4⁺T lymphocytes did not co-synthesized IFN-γ, IL-4 or IL-17. IL-21 and IL-22 production were dependent on the B7/CD28 co-stimulatory pathway and ICOS-L expression on skin LCs significantly reduced IL-21 level. Finally, we found that TGF-β strongly down-regulates both IL-21 and IL-22 secretion by allogeneic CD4⁺ T cells. These results add new knowledge on the functional specialization of human skin DCs and might suggest new targets in the treatment of inflammatory skin disorders.     

Vaccine-Elicited CD8+ T Cells Protect against Respiratory Syncytial Virus Strain A2-Line19F-Induced Pathogenesis in BALB/c Mice

CD8⁺ T cells may contribute to vaccines for respiratory syncytial virus (RSV). Compared to CD8⁺ T cells responding to RSV infection, vaccine-elicited anti-RSV CD8⁺ T cells are less well defined. We used a peptide vaccine to test the hypothesis that vaccine-elicited RSV-specific CD8⁺ T cells are protective against RSV pathogenesis. BALB/c mice were treated with a mixture (previously termed TriVax) of an M2_82-90 peptide representing an immunodominant CD8 epitope, the Toll-like receptor (TLR) agonist poly(I·C), and a costimulatory anti-CD40 antibody. TriVax vaccination induced potent effector anti-RSV CD8⁺ cytotoxic T lymphocytes (CTL). Mice were challenged with RSV strain A2-line19F, a model of RSV pathogenesis leading to airway mucin expression. Mice were protected against RSV infection and against RSV-induced airway mucin expression and cellular lung inflammation when challenged 6 days after vaccination. Compared to A2-line19F infection alone, TriVax vaccination followed by challenge resulted in effector CD8⁺ T cells with greater cytokine expression and the more rapid appearance of RSV-specific CD8⁺ T cells in the lung. When challenged 42 days after TriVax vaccination, memory CD8⁺ T cells were elicited with RSV-specific tetramer responses equivalent to TriVax-induced effector CD8⁺ T cells. These memory CD8⁺ T cells had lower cytokine expression than effector CD8⁺ T cells, and protection against A2-line19F was partial during the memory phase. We found that vaccine-elicited effector anti-RSV CD8⁺ T cells protected mice against RSV infection and pathogenesis, and waning protection correlated with reduced CD8⁺ T cell cytokine expression.     

Differential Contribution of Acute and Chronic Inflammation to the Development of Murine Mammary 4T1 Tumors

Based on the notion that inflammation favors tumorigenesis, our experiments comparatively assessed the influence of acute and chronic inflammation on the development of a murine mammary tumor (4T1). In addition, we characterized angiogenic and inflammatory markers in the tumor tissue and systemically. Subcutaneous implantation of polyether-polyurethane sponge discs in Balb/c mice was used to host 4T1 tumor cells (1x10⁶), which were inoculated intraimplant 24h or 10 days post implantation. Flow cytometric analysis of enzyme-digested implants revealed that, after 24 hours, the population of leukocytes was primarily characterized by neutrophils (42.53% +/- 8.45) and monocytes (37.53% +/- 7.48), with some lymphocytes (16.27% +/- 4.0) and a few dendritic cells (1.82% +/- 0.36). At 10 days, macrophages were predominant (37.10% +/- 4.54), followed by lymphocytes (28.1% +/- 4.77), and monocytes (22.33% +/- 3.05), with some dendritic cells (13.60% +/- 0.55) and neutrophils (11.07% +/- 2.27). A mammary tumor grown in a chronic inflammatory environment was 2-fold when compared with one grown in acute inflammation and 5-fold when compared with tumor alone. The levels of pro-angiogenic cytokine (VEGF-Vascular Endothelial Growth Factor) were higher in implant-bearing tumor when 4T1 cells were grown in 10-day old implants as compared to the VEGF levels of the two other groups. Overall, the levels of the inflammatory markers evaluated (NAG -N-acetylglucosaminidase, TNF-α –Tumor Necrosis Factor- α) were higher in both groups of implant-bearing tumors and in serum from those animals when compared with the tumor alone levels. This inflammation-related difference in tumor growth may provide new insights into the contribution of different inflammatory cell populations to cancer progression.     

Feasibility of repairing skin defects by VEGF165 gene-modified iPS-HFSCs seeded on a 3D printed scaffold containing astragalus polysaccharide

The preparation of biodegradable scaffolds loaded with cells and cytokine is a feature of tissue-engineered skin. IPSCs-based tissue-engineered skin treatment for wound repair is worth exploring. Healthy human skin fibroblasts were collected and reprogrammed into iPSCs. After gene modification and induction, CK19⁺/Integrinβ1⁺/CD200⁺ VEGF₁₆₅ gene-modified iPS-HFSCs^GFP were obtained and identified by a combination of immunofluorescence and RT-qPCR. Astragalus polysaccharide-containing 3D printed degradable scaffolds were prepared and co-cultured with VEGF₁₆₅ gene-modified iPS-HFSCs^GFP, and the biocompatibility and spatial structure of the tissue-engineered skin was analysed by cell counting kit-8 (CCK8) assay and scanning electron microscopy. Finally, the tissue-engineered skin was transplanted onto the dorsal trauma of nude mice, and the effect of tissue-engineered skin on the regenerative repair of total skin defects was evaluated by a combination of histology, immunohistochemistry, immunofluorescence, RT-qPCR, and in vivo three-dimensional reconstruction under two-photon microscopy. CK19⁺/Integrinβ1⁺/CD200⁺ VEGF₁₆₅ gene-modified iPS-HFSCs^GFP, close to the morphology and phenotype of human-derived hair follicle stem cells, were obtained. The surface of the prepared 3D printed degradable scaffold containing 200 μg/mL astragalus polysaccharide was enriched with honeycomb-like meshwork, which was more conducive to the proliferation of the resulting cells. After tissue-engineered skin transplantation, combined assays showed that it promoted early vascularization, collagen and hair follicle regeneration and accelerated wound repair. VEGF₁₆₅ gene-modified iPS-HFSCs^GFP compounded with 3D printed degradable scaffolds containing 200 μg/mL astragalus polysaccharide can directly and indirectly participate in vascular, collagen, and hair follicle regeneration in the skin, achieving more complete structural and functional skin regenerative repair.     

Human CD8+ T cells display a differential ability to undergo cytokine-driven bystander activation

A subset of CD44^hiCD8⁺ T cells in some, but not all mice, can be induced to rapidly secrete IFNγ during infection with Listeria monocytogenes. This response is dependent on the presence of both IL-12 and IL-18 and does not require engagement of the T cell receptor. In this study, we demonstrate that human CD8⁺ T cells also vary widely in their ability to secrete IFNγ within 15 h of either Listeria infection or cytokine stimulation. The magnitude of the rapid IFNγ response correlated more closely with the intrinsic responsiveness of the T cells to cytokine stimulation rather than the amount of IL-12 produced. CD8⁺ T cells from 2 out of 16 blood donors (12.5%) failed to generate a significant IFNγ response. These results demonstrate that bystander activation of CD8⁺ T cells varies among individuals and validate further study of the differential responses observed using BALB/c vs. C57BL/6 mice.     

The beta2 integrin CD11c distinguishes a subset of cytotoxic pulmonary T cells with potent antiviral effects in vitro and in vivo

Background

The integrin CD11c is known as a marker for dendritic cells and has recently been described on T cells following lymphotropic choriomeningitis virus infection, a systemic infection affecting a multitude of organs. Here, we characterise CD11c bearing T cells in a murine model of localised pulmonary infection with respiratory syncytial virus (RSV).

Methods

Mice were infected intranasally with RSV and expression of β2 integrins and T lymphocyte activation markers were monitored by flow cytometry. On day 8 post RSV infection CD11c⁺ CD8⁺ and CD11c^- CD8⁺ T cells were assessed for cytokine production, cytotoxic activity and migration. Expression of CD11c mRNA in CD8⁺ T cells was assessed by quantitative PCR.

Results

Following RSV infection CD11c⁺ CD8⁺ T cells were detectable in the lung from day 4 onwards and accounted for 45.9 ± 4.8% of CD8⁺ T cells on day 8 post infection, while only few such cells were present in mediastinal lymph nodes, spleen and blood. While CD11c was virtually absent from CD8⁺ T cells in the absence of RSV infection, its mRNA was expressed in CD8⁺ T cells of both naïve and RSV infected mice. CD11c⁺, but not CD11c^-, CD8⁺ T cells showed signs of recent activation, including up-regulation of CD11a and expression of CD11b and CD69 and were recruited preferentially to the lung. In addition, CD11c⁺ CD8⁺ T cells were the major subset responsible for IFNγ production, induction of target cell apoptosis in vitro and reduction of viral titres in vivo.

Conclusion

CD11c is a useful marker for detection and isolation of pulmonary antiviral cytotoxic T cells following RSV infection. It identifies a subset of activated, virus-specific, cytotoxic T cells that exhibit potent antiviral effects in vivo.     

Helminth Infections Coincident with Active Pulmonary Tuberculosis Inhibit Mono- and Multifunctional CD4+ and CD8+ T Cell Responses in a Process Dependent on IL-10

Tissue invasive helminth infections and tuberculosis (TB) are co-endemic in many parts of the world and can trigger immune responses that might antagonize each other. We have previously shown that helminth infections modulate the Th1 and Th17 responses to mycobacterial-antigens in latent TB. To determine whether helminth infections modulate antigen-specific and non-specific immune responses in active pulmonary TB, we examined CD4⁺ and CD8⁺ T cell responses as well as the systemic (plasma) cytokine levels in individuals with pulmonary TB with or without two distinct helminth infections—Wuchereria bancrofti and Strongyloides stercoralis infection. By analyzing the frequencies of Th1 and Th17 CD4⁺ and CD8⁺ T cells and their component subsets (including multifunctional cells), we report a significant diminution in the mycobacterial–specific frequencies of mono- and multi–functional CD4⁺ Th1 and (to a lesser extent) Th17 cells when concomitant filarial or Strongyloides infection occurs. The impairment in CD4⁺ and CD8⁺ T cell cytokine responses was antigen-specific as polyclonal activated T cell frequencies were equivalent irrespective of helminth infection status. This diminution in T cell responses was also reflected in diminished circulating levels of Th1 (IFN-γ, TNF-α and IL-2)- and Th17 (IL-17A and IL-17F)-associated cytokines. Finally, we demonstrate that for the filarial co-infections at least, this diminished frequency of multifunctional CD4⁺ T cell responses was partially dependent on IL-10 as IL-10 blockade significantly increased the frequencies of CD4⁺ Th1 cells. Thus, co-existent helminth infection is associated with an IL-10 mediated (for filarial infection) profound inhibition of antigen-specific CD4⁺ T cell responses as well as protective systemic cytokine responses in active pulmonary TB.     

PD-1 and Tim-3 Pathways Regulate CD8+ T Cells Function in Atherosclerosis

T cell-mediated immunity plays a significant role in the development of atherosclerosis (AS). There is increasing evidence that CD8⁺ T cells are also involved in AS but their exact roles remain unclear. The inhibitory receptors programmed cell death-1 (PD-1) and T cell immunoglobulin and mucin domain 3 (Tim-3) are well known inhibitory molecules that play a crucial role in regulating CD8⁺ T cell activation or tolerance. Here, we demonstrate that the co-expression of PD-1 and Tim-3 on CD8⁺ T cells is up-regulated in AS patients. PD-1⁺ Tim-3⁺ CD8⁺ T cells are enriched for within the central T (T_CM) cell subset, with high proliferative activity and CD127 expression. Co-expression of PD-1 and Tim-3 on CD8⁺ T cells is associated with increased anti-atherogenic cytokine production as well as decreased pro-atherogenic cytokine production. Blockade of PD-1 and Tim-3 results in a decrease of anti-atherogenic cytokine production by PD-1⁺ Tim-3⁺ CD8⁺ T cells and in an augmentation of TNF-α and IFN-γ production. These findings highlight the important role of the PD-1 and Tim-3 pathways in regulating CD8⁺ T cells function in human AS.     

Increased Carotid Intima-Media Thickness and Reduced Distensibility in Human Class III Obesity: Independent and Differential Influences of Adiposity and Blood Pressure on the Vasculature

Carotid intima-media-thickness (cIMT) and carotid distensibility (distensibility), structural and functional properties of carotid arteries respectively, are early markers, as well as strong predictors of cardiovascular disease (CVD). The characteristic of these two parameters in individuals with BMI>40.0 kg/m² (Class III obesity), however, are largely unknown. The present study was designed to document cIMT and distensibility in this population and to relate these to other factors with established association with CVD in obesity. The study included 96 subjects (65 with BMI>40.0 kg/m² and 31, age- and gender-matched, with BMI of 18.5 to 30.0 kg/m²). cIMT and distensibility were measured by non-invasive high resolution ultrasonography, circulatory CD133⁺/KDR⁺ angiogenic cells and endothelial microparticles (EMP) by flow cytometry, and plasma levels of adipokines, growth factors and cytokines by Luminex immunoassay kits. The study results demonstrated increased cIMT (0.62±0.11 mm vs. 0.54±0.08 mm, P = 0.0002) and reduced distensibility (22.52±10.79 10⁻³kpa⁻¹ vs. 29.91±12.37 10⁻³kpa⁻¹, P<0.05) in individuals with BMI>40.0 kg/m². Both cIMT and distensibility were significantly associated with traditional CVD risk factors, adiposity/adipokines and inflammatory markers but had no association with circulating angiogenic cells. We also demonstrated, for the first time, elevated plasma EMP levels in individuals with BMI>40.0 kg/m². In conclusion, cIMT is increased and distensibility reduced in Class III obesity with the changes predominantly related to conventional CVD risk factors present in this condition, demonstrating that both cIMT and distensibility remain as CVD markers in Class III obesity.     

Measurement of CD8+ and CD4+ T Cell Frequencies Specific for EBV LMP1 and LMP2a Using mRNA-Transfected DCs

An EBV-specific cellular immune response is associated with the control of EBV-associated malignancies and lymphoproliferative diseases, some of which have been successfully treated by adoptive T cell therapy. Therefore, many methods have been used to measure EBV-specific cellular immune responses. Previous studies have mainly used autologous EBV-transformed B-lymphoblastoid cell lines (B-LCLs), recombinant viral vectors transfected or peptide pulsed dendritic cells (DCs) as stimulators of CD8⁺ and CD4⁺ T lymphocytes. In the present study, we used an interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) assay by using isolated CD8⁺ and CD4⁺ T cells stimulated with mRNA-transfected DCs. The frequency of latent membrane protein 1 (LMP1)-specific IFN-γ producing CD4⁺ T cells was significantly higher than that of LMP2a. The frequency of IFN-γ producing CD4⁺ T cells was significantly correlated with that of CD8⁺ T cells in LMP1-specific immune responses (r = 0.7187, Pc < 0.0001). To determine whether there were changes in LMP1- or LMP2a-specific immune responses, subsequent peripheral blood mononuclear cells (PBMCs) samples were analyzed. Significant changes were observed in 5 of the 10 donors examined, and CD4⁺ T cell responses showed more significant changes than CD8⁺ T cell responses. CD8⁺ and CD4⁺ T cells from EBV-seropositive donors secreted only the Th1 cytokines IFN-γ, TNF-α, and IL-2, while Th2 (IL-4) and Th17 (IL-17a) cytokines were not detected. CD4⁺ T cells secreted significantly higher cytokine levels than did CD8⁺ T cells. Analysis of EBV-specific T cell responses using autologous DCs transfected with mRNA might provide a comprehensive tool for monitoring EBV infection and new insights into the pathogenesis of EBV-associated diseases.     

IL-10 signaling in CD4+ T cells is critical for the pathogenesis of collagen-induced arthritis

Introduction

IL-10 is a very important anti-inflammatory cytokine. However, the role of this cytokine in T cells in the pathogenesis of collagen-induced arthritis is unclear. The purpose of this study was to define the role of IL-10 signaling in T cells in the pathogenesis of collagen-induced arthritis.

Methods

IL-10 receptor dominant-negative transgenic (Tg) and control mice were immunized with bovine type II collagen to induce arthritis. The severity of arthritis was monitored and examined histologically. T-cell activation and cytokine production were analyzed using flow cytometry. T-cell proliferation was examined by [³H]thymidine incorporation. Antigen-specific antibodies in serum were measured by ELISA. Foxp3 expression in CD4⁺ regulatory T cells (Tregs) was determined by intracellular staining or Foxp3-RFP reporter mice. The suppressive function of Foxp3⁺CD4⁺ Tregs was determined in vitro by performing a T-cell proliferation assay. The level of IL-17 mRNA in joints was measured by real-time PCR. A two-tailed nonparametric paired test (Wilcoxon signed-rank test) was used to calculate the arthritis and histological scores. Student's paired or unpaired t-test was used for all other statistical analyses (InStat version 2.03 software; GraphPad Software, San Diego, CA, USA).

Results

Blocking IL-10 signaling in T cells rendered mice, especially female mice, highly susceptible to collagen-induced arthritis. T-cell activation and proliferation were enhanced and produced more IFN-γ. The suppressive function of CD4⁺Foxp3⁺ regulatory T cells was significantly impaired in Tg mice because of the reduced ability of Tregs from Tg mice to maintain their levels of Foxp3. This was further confirmed by transferring Foxp3-RFP cells from Tg or wild-type (Wt) mice into a congenic Wt host. The higher level of IL-17 mRNA was detected in inflammatory joints of Tg mice, probably due to the recruitment of IL-17⁺γδ T cells into the arthritic joints.

Conclusion

IL-10 signaling in T cells is critical for dampening the pathogenesis of collagen-induced arthritis by maintaining the function of Tregs and the recruitment of IL-17⁺γδ T cells.     

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.