A novel in vivo inducible dendritic cell ablation model in mice

Dendritic cells (DCs) are involved in T cell activation via their uptake and presentation of antigens. In vivo function of DCs was analyzed using transgenic mouse models that express diphtheria toxin receptor (DTR) or the diphtheria toxin-A subunit (DTA) under the control of the CD11c/Itgax promoter. However, CD11c⁺ cells are heterogeneous populations that contain several DC subsets. Thus, the in vivo function of each subset of DCs remains to be elucidated. Here, we describe a new inducible DC ablation model, in which DTR expression is induced under the CD11c/Itgax promoter after Cre-mediated excision of a stop cassette (CD11c-iDTR). Crossing of CD11c-iDTR mice with CAG-Cre transgenic mice, expressing Cre recombinase under control of the cytomegalovirus immediate early enhancer-chicken beta-actin hybrid promoter, led to the generation of mice, in which DTR was selectively expressed in CD11c⁺ cells (iDTRΔ mice). We successfully deleted CD11c⁺ cells in bone marrow-derived DCs in vitro and splenic CD11c⁺ cells in vivo after DT treatment in iDTRΔ mice. This mouse strain will be a useful tool for generating mice lacking a specific subset of DCs using a transgenic mouse strain, in which the Cre gene is expressed by a DC subset-specific promoter.     

Characterization of distinct mesenchymal-like cell populations from human skeletal muscle in situ and in vitro

Human skeletal muscle is an essential source of various cellular progenitors with potential therapeutic perspectives. We first used extracellular markers to identify in situ the main cell types located in a satellite position or in the endomysium of the skeletal muscle. Immunohistology revealed labeling of cells by markers of mesenchymal (CD13, CD29, CD44, CD47, CD49, CD62, CD73, CD90, CD105, CD146, and CD15 in this study), myogenic (CD56), angiogenic (CD31, CD34, CD106, CD146), hematopoietic (CD10, CD15, CD34) lineages. We then analysed cell phenotypes and fates in short- and long-term cultures of dissociated muscle biopsies in a proliferation medium favouring the expansion of myogenic cells. While CD56⁺ cells grew rapidly, a population of CD15⁺ cells emerged, partly from CD56⁺ cells, and became individualized. Both populations expressed mesenchymal markers similar to that harboured by human bone marrow-derived mesenchymal stem cells. In differentiation media, both CD56⁺ and CD15⁺ cells shared osteogenic and chondrogenic abilities, while CD56⁺ cells presented a myogenic capacity and CD15⁺ cells presented an adipogenic capacity. An important proportion of cells expressed the CD34 antigen in situ and immediately after muscle dissociation. However, CD34 antigen did not persist in culture and this initial population gave rise to adipogenic cells. These results underline the diversity of human muscle cells, and the shared or restricted commitment abilities of the main lineages under defined conditions.     

Lifelong dynamics of human CD4CD25 regulatory T cells: Insights from in vivo data and mathematical modeling

Despite their limited proliferation capacity, regulatory T cells (T_regs) constitute a population maintained over the entire lifetime of a human organism. The means by which T_regs sustain a stable pool in vivo are controversial. Using a mathematical model, we address this issue by evaluating several biological scenarios of the origins and the proliferation capacity of two subsets of T_regs: precursor CD4⁺CD25⁺CD45RO⁻ and mature CD4⁺CD25⁺CD45RO⁺ cells. The lifelong dynamics of T_regs are described by a set of ordinary differential equations, driven by a stochastic process representing the major immune reactions involving these cells. The model dynamics are validated using data from human donors of different ages. Analysis of the data led to the identification of two properties of the dynamics: (1) the equilibrium in the CD4⁺CD25⁺FoxP3⁺T_regs population is maintained over both precursor and mature T_regs pools together, and (2) the ratio between precursor and mature T_regs is inverted in the early years of adulthood. Then, using the model, we identified three biologically relevant scenarios that have the above properties: (1) the unique source of mature T_regs is the antigen-driven differentiation of precursors that acquire the mature profile in the periphery and the proliferation of T_regs is essential for the development and the maintenance of the pool; there exist other sources of mature T_regs, such as (2) a homeostatic density-dependent regulation or (3) thymus- or effector-derived T_regs, and in both cases, antigen-induced proliferation is not necessary for the development of a stable pool of T_regs. This is the first time that a mathematical model built to describe the in vivo dynamics of regulatory T cells is validated using human data. The application of this model provides an invaluable tool in estimating the amount of regulatory T cells as a function of time in the blood of patients that received a solid organ transplant or are suffering from an autoimmune disease.     

OX40 ligand regulates splenic CD8 dendritic cell-induced Th2 responses in vivo

In mice, splenic conventional dendritic cells (cDCs) can be separated, based on their expression of CD8α into CD8⁻ and CD8⁺ cDCs. Although previous experiments demonstrated that injection of antigen (Ag)-pulsed CD8⁻ cDCs into mice induced CD4 T cell differentiation toward Th2 cells, the mechanism involved is unclear. In the current study, we investigated whether OX40 ligand (OX40L) on CD8⁻ cDCs contributes to the induction of Th2 responses by Ag-pulsed CD8⁻ cDCs in vivo, because OX40–OX40L interactions may play a preferential role in Th2 cell development. When unseparated Ag-pulsed OX40L-deficient cDCs were injected into syngeneic BALB/c mice, Th2 cytokine (IL-4, IL-5, and IL-10) production in lymph node cells was significantly reduced. Splenic cDCs were separated to CD8⁻ and CD8⁺ cDCs. OX40L expression was not observed on freshly isolated CD8⁻ cDCs, but was induced by anti-CD40 mAb stimulation for 24 h. Administration of neutralizing anti-OX40L mAb significantly inhibited IL-4, IL-5, and IL-10 production induced by Ag-pulsed CD8⁻ cDC injection. Moreover, administration of anti-OX40L mAb with Ag-pulsed CD8⁻ cDCs during a secondary response also significantly inhibited Th2 cytokine production. Thus, OX40L on CD8⁻ cDCs physiologically contributes to the development of Th2 cells and secondary Th2 responses induced by Ag-pulsed CD8⁻ cDCs in vivo.     

Local immune response against larvae of Rhipicephalus (Boophilus) microplus in Bos taurus indicus and Bos taurus taurus cattle

Bos taurus indicus cattle are less susceptible to infestation with Rhipicephalus (Boophilus) microplus than Bos taurus taurus cattle but the immunological basis of this difference is not understood. We compared the dynamics of leukocyte infiltrations (T cell subsets, B cells, major histocompatibility complex (MHC) class II-expressing cells, granulocytes) in the skin near the mouthparts of larvae of R. microplus in B. t. indicus and B. t. taurus cattle. Previously naïve cattle were infested with 50,000 larvae (B. t. indicus) or 10,000 larvae (B. t. taurus) weekly for 6 weeks. One week after the last infestation all of the animals were infested with 20,000 larvae of R. microplus. Skin punch biopsies were taken from all animals on the day before the primary infestation and from sites of larval attachment on the day after the first, second, fourth and final infestations. Infiltrations with CD3⁺, CD4⁺, CD8⁺ and γδ T cells followed the same pattern in both breeds, showing relatively little change during the first four weekly infestations, followed by substantial increases at 7 weeks post-primary infestation. There was a tendency for more of all cell types except granulocytes to be observed in the skin of B. t. indicus cattle but the differences between the two breeds were consistently significant only for γδ T cells. Granulocyte infiltrations increased more rapidly from the day after infestation and were higher in B. t. taurus cattle than in B. t. indicus. Granulocytes and MHC class II-expressing cells infiltrated the areas closest to the mouthparts of larvae. A large volume of granulocyte antigens was seen in the gut of attached, feeding larvae.     

Genetic immunization based on the ubiquitin-fusion degradation pathway against Trypanosoma cruzi

Cytotoxic CD8⁺ T cells are particularly important to the development of protective immunity against the intracellular protozoan parasite, Trypanosoma cruzi, the etiological agent of Chagas disease. We have developed a new effective strategy of genetic immunization by activating CD8⁺ T cells through the ubiquitin-fusion degradation (UFD) pathway. We constructed expression plasmids encoding the amastigote surface protein-2 (ASP-2) of T. cruzi. To induce the UFD pathway, a chimeric gene encoding ubiquitin fused to ASP-2 (pUB-ASP-2) was constructed. Mice immunized with pUB-ASP-2 presented lower parasitemia and longer survival period, compared with mice immunized with pASP-2 alone. Depletion of CD8⁺ T cells abolished protection against T. cruzi in mice immunized with pUB-ASP-2 while depletion of CD4⁺ T cells did not influence the effective immunity. Mice deficient in LMP2 or LMP7, subunits of immunoproteasomes, were not able to develop protective immunity induced. These results suggest that ubiquitin-fused antigens expressed in antigen-presenting cells were effectively degraded via the UFD pathway, and subsequently activated CD8⁺ T cells. Consequently, immunization with pUB-ASP-2 was able to induce potent protective immunity against infection of T. cruzi.     

Impaired pro-inflammatory cytokine production and increased Th2-biasing ability of dendritic cells exposed to Taenia excreted/secreted antigens: A critical role for carbohydrates but not for STAT6 signaling

In cysticercosis, a parasitic disease caused by cestodes, the details of early interactions between parasite antigens and innate cells from the host are not well understood. In this study, the role of cestode-conditioned dendritic cells (DCs) in priming Th1 versus Th2 responses to bystander antigen was examined by using CD11c⁺ DCs as antigen-presenting cells and naive CD4⁺ DO11.10 lymphocytes specific to ovalbumin (OVA) as responding cells. No conventional maturation was induced in DCs exposed to Taenia crassiceps excreted/secreted antigens (TcES). The ability of TcES to affect Toll-like receptor (TLR)-mediated maturation and the pro-inflammatory response was analyzed by co-pulsing DCs with TcES and TLR ligands. DCs exposed to TcES blocked TLR4, TLR9 and Toxoplasma soluble antigen-induced phenotypic maturation. TcES-exposed DCs also blocked secretion of pro-inflammatory cytokines and alloreactive T cell proliferation, while preserving IL-10 production. DCs pulsed with TcES + OVA suppressed IFN-γ, whereas they induced greater IL-4 production by CD4⁺ DO11.10 cells. TcES with chemically-altered glycans failed to modulate TLR-mediated activation of DCs and their Th1-inhibitng ability, which was STAT6-independent. Our results reflect the capacity of TcES glyco-antigens to modulate Th1-type and inflammatory responses mediated through DC activation.     

Sclareol modulates the Treg intra-tumoral infiltrated cell and inhibits tumor growth in vivo

A regulatory or suppressor T cell is functionally defined as a T cell that inhibits an immune response by influencing the activity of another cell type. On the other hand, Th1 cells express IFN-γ and mediate cellular immunity.Sclareol exhibits growth inhibition and cytotoxic activity against a variety of human cancer cell lines. In the first set of experiments, Sclareol was isolated from the plant Salvia sclarea and our study assessed the immuno-therapeutic effectiveness of Sclareol by direct intra-tumoral injection. Secondly, several immunological parameters such as splenocytes proliferation, intra-tumor CD4⁺CD25⁺Foxp3⁺ Treg cells, IFN-γ and IL-4 secretion and tumor size were assessed to evaluate the anti-tumoral immune response. By all means, the findings confirmed that the activity of Sclareol could reduce the tumor growth in vivo against breast cancer.     

Quantitative and qualitative features of heterologous virus-vector-induced antigen-specific CD8 T cells against Trypanosoma cruzi infection

We studied some aspects of the quantitative and qualitative features of heterologous recombinant (re) virus-vector-induced, antigen-specific CD8⁺ T cells against Trypanosoma cruzi. We used three different, highly attenuated re-viruses, i.e., influenza virus, adenovirus and vaccinia virus, which all expressed a single, T. cruzi antigen-derived CD8⁺ T-cell epitope. The use of two out of three vectors or the triple virus-vector vaccination regimen not only confirmed that the re-vaccinia virus, which was placed last in order for sequential immunisation, was an effective booster for the CD8⁺ T-cell immunity in terms of the number of antigen-specific CD8⁺ T cells, but also demonstrated that (i) the majority of cells exhibit the effector memory (T_EM) phenotype, (ii) robustly secrete IFN-γ, (iii) express higher intensity of the CD122 molecule and (iv) present protective activity against T. cruzi infection. In contrast, placing the re-influenza virus last in sequential immunisation had a detrimental effect on the quantitative and qualitative features of CD8⁺ T cells. The triple virus-vector vaccination was more effective at inducing a stronger CD8⁺ T-cell immunity than using two re-viruses. The different quantitative and qualitative features of CD8⁺ T cells induced by different immunisation regimens support the notion that the refinement of the best choice of multiple virus-vector combinations is indispensable for the induction of a maximum number of CD8⁺ T cells of high quality.     

Partial protective responses induced by a recombinant cysteine proteinase from Leishmania (Leishmania) amazonensis in a murine model of cutaneous leishmaniasis

A 500 bp fragment encoding an isoform of cysteine proteinase from Leishmania (Leishmania) amazonensis was subcloned and expressed in the pHis vector, resulting in a recombinant protein of 24 kDa, rLacys24. In Western blots of L. (L.) amazonensis extracts, antibodies directed to rLacys24 recognized a cysteine proteinase isoform of 30 kDa. Analysis by fluorescence-activated cell sorter showed a significantly higher expression of CD8⁺ lymphocytes in animals immunized with rLacys24 plus CFA, whereas a low expression of CD4⁺ lymphocytes was observed in these animals. The cytotoxicity of lymphocytes isolated from mice immunized with rLacys24 plus CFA on L. (L.) amazonensis-infected macrophages was significantly higher than that observed in the presence of lymphocytes from control animals. Immunization of BALB/c mice with rLacys24 plus CFA resulted in a low but significant decrease of foot lesions after challenge with L. (L.) amazonensis compared to those exhibited by control mice.     

A novel in vivo regulatory role of P-glycoprotein in alloimmunity

P-glycoprotein (P-gp) is required for adaptive immunity through defined functions in T cell activation and antigen presenting cell (APC) maturation. The potential role of P-gp as an in vivo regulator of alloimmunity is currently unknown. Here we show that P-gp blockade prolongs graft survival in a murine heterotopic cardiac allotransplantation model through in vivo inhibition of the T helper 1 (Th1) cytokine IFN-γ and the Th2 product IL-4, and via downregulation of the APC-expressed positive costimulatory molecule CD80. In vitro, the P-gp antagonist PSC833, a non-calcineurin-inhibitory cyclosporine A analogue, specifically inhibited cellular efflux of the P-gp substrate rhodamine-123 in wild-type CD3⁺ T cells and MHC class II⁺ APCs but not their P-gp knockout counterparts that lacked rhodamine-123 efflux capacity. Additionally, P-gp blockade significantly inhibited murine alloimmune T cell activation in a dose-dependent fashion. In vivo, P-gp blockade significantly prolonged graft survival in Balb/c recipients of C57BL/6 cardiac allografts from 8.5 ± 0.5 to 11.7 ± 0.5 days (P < 0.01), similar in magnitude to the effects of monotherapy with cyclosporine A. Moreover, P-gp blockade, compared to controls, attenuated intragraft expression of CD3 and CD80, but not CD86, and inhibited IFN-γ and IL-4 production (P < 0.05). In the setting of systemic CD86 inhibition, P-gp blockade suppressed IFN-γ and IL-4 production significantly further (to 98% and 89% inhibition, respectively) compared to either P-gp or anti-CD86 blockade alone, and markedly prolonged allograft survival compared to anti-CD86 blockade alone (40.5 ± 4.6 versus 22.5 ± 2.6 days, respectively, P < 0.01). Our findings define a novel in vivo regulatory role of P-gp in alloimmunity and identify P-gp as a potential therapeutic target in allotransplantation.     

Molecular evidence of anti-leukemia activity of gypenosides on human myeloid leukemia HL-60 cells in vitro and in vivo using a HL-60 cells murine xenograft model

We have shown that gypenosides (Gyp) induced cell cycle arrest and apoptosis in many human cancer cell lines. However, there are no reports showing that show Gyp acts on human leukemia HL-60 cells in vitro and in a murine xenograft model in vivo. In the present study effects of Gyp on cell morphological changes and viability, cell cycle arrest and induction of apoptosis in vitro and effects on Gyp in an in vivo murine xenograft model. Results indicated that Gyp induced morphological changes, decreased cell viability, induced G0/G1 arrest, DNA fragmentation and apoptosis (sub-G1 phase) in HL-60 cells. Gyp increased reactive oxygen species production and Ca²⁺ levels but reduced mitochondrial membrane potential in a dose- and time-dependent manner. Gyp also changed one of the primary indicators of endoplasmic reticulum (ER) stress due to the promotion of ATF6-α and ATF4-α associated with Ca²⁺ release. Gyp reduced the ratio of Bcl-2 to Bax due to an increase in the pro-apoptotic protein Bax and inhibited levels of the anti-apoptotic protein Bcl-2. Oral consumption of Gyp reduced tumor size of HL-60 cell xenograft mode mice in vivo. These results provide new information on understanding mechanisms by which Gyp induces cell cycle arrest and apoptosis in vitro and in vivo.     

Differential in vivo response of soft-shell clam hemocytes against two strains of Vibrio splendidus: Changes in cell structure, numbers and adherence

Host-pathogen interaction models in aquatic species are useful tools for understanding the pathogenicity of diseases in cultured and wild populations. In this study we report the differential in vivo response of soft-shell clam (Mya arenaria) hemocytes against two strains of Vibrio splendidus. Responses were measured 24 h after injecting into the posterior adductor muscle either an endemic wild-type strain (7SHRW) or a strain associated with oyster mortalities (LGP32-GFP). Changes in hemocyte structure (percentage of rounded cells) were assessed microscopically. Changes in adherence and hemocyte numbers were analyzed by flow-cytometric cell counting. Increased percentages of rounded cells were found in response to both strains. However, values from the group infected with LGP32-GFP were significantly higher (p < 0.01) than with 7SHRW. The cell adherence was markedly diminished (p < 0.001) by LGP32-GFP whereas 7SHRW did not change it significantly. Increased numbers of hemocytes (p < 0.001) were induced by LGP32-GFP, while no significant changes were found after infection with 7SHRW. These results show the regulatory capacity of soft-shell clams hemocytes to perform specific responses against different strains of V. splendidus.     

The roles of IL-12 and IL-23 in CD8 T cell-mediated immunity against Listeria monocytogenes: Insights from a DC vaccination model

Listeria monocytogenes infection induces a strong inflammatory response characterized by the production of IL-12 and IFN-γ and protective immunity against this pathogen is dependent on CD8⁺ T cells (CTL). Recent studies have suggested that these inflammatory cytokines affect the rate of memory CD8⁺ T cell generation as well as the number of short-lived effector cells generated. The role of the closely related cytokine, IL-23, in this response has not been examined. We hypothesized that IL-12 and IL-23 produced by dendritic cells collectively enhance the generation and function of memory cells. To test this hypothesis, we employed a DC vaccination approach. Mice lacking IL-12 and IL-23 were vaccinated with wild-type (WT), IL-12^−/−, or IL-12/23^−/− DC and protection to Lm was monitored. Mice vaccinated with WT and IL-12^−/− DC were resistant to lethal challenge with Lm. Surprisingly, mice vaccinated with IL-12/23^−/− DC exhibited significantly reduced protection when challenged. Protection correlated with the relative size of the memory pools generated. In summary, these data indicate that IL-23 can partially compensate for the lack of IL-12 in the generation protective immunity against Lm.     

Plasmodium: Mammalian codon optimization of malaria plasmid DNA vaccines enhances antibody responses but not T cell responses nor protective immunity

We have evaluated the effect of mammalian codon optimization on the immunogenicity and protective efficacy of plasmid DNA vaccines encoding pre-erythrocytic stage Plasmodium falciparum and Plasmodium yoelii antigens in mice. Codon optimization significantly enhanced in vitro expression and in vivo antibody responses for P. falciparum circumsporozoite protein (PfCSP) and P. yoelii hepatocyte erythrocyte protein 17 kDa (PyHEP17) but not for P. yoelii circumsporozoite protein (PyCSP). Unexpectedly, more robust CD4⁺ and CD8⁺ T cell responses as measured by IFN-γ ELIspot, lymphoproliferation, and cytotoxic T lymphocyte assays were noted with native as compared with codon optimization constructs. Codon optimization also failed to enhance CD8⁺ T cell dependent protection against P. yoelii sporozoite challenge as measured by liver-stage parasite burden. These data demonstrate that the effect of mammalian codon optimization is antigen-dependent and may not be beneficial for vaccines designed to induce T cell dependent protective immunity in this malaria model.     

Profiling calcium signals of in vitro polarized human effector CD4+ T cells

Differentiation of naïve CD4⁺ T cells into effector subtypes with distinct cytokine profiles and physiological roles is a tightly regulated process, the imbalance of which can lead to an inadequate immune response or autoimmune disease. The crucial role of Ca²⁺ signals, mainly mediated by the store operated Ca²⁺ entry (SOCE) in shaping the immune response is well described. However, it is unclear if human effector CD4⁺ T cell subsets show differential Ca²⁺ signatures in response to different stimulation methods. Herein, we provide optimized in vitro culture conditions for polarization of human CD4⁺ effector T cells and characterize their SOCE following both pharmacological store depletion and direct T-cell receptor (TCR) activation. Moreover, we measured whole cell Ca²⁺ release activated Ca²⁺ currents (I_CRAC) and investigated whether the observed differences correlate to the expression of CRAC genes. Our results show that Ca²⁺ profiles of helper CD4⁺ Th1, Th2 and Th17 are distinct and in part shaped by the intensity of stimulation. Regulatory T cells (Treg) are unique being the subtype with the most prominent SOCE response. Analysis of in vivo differentiated Treg unraveled the role of differential expression of ORAI2 in fine-tuning signals in Treg vs. conventional CD4⁺ T cells.     

Peripheral CD4CD8 cells are the activated T cells expressed granzyme B (GrB), Foxp3, interleukin 17 (IL-17), at higher levels in Th1/Th2 cytokines

Peripheral CD4⁺CD8⁺ T cells have been identified as a T cell subset existing in animals and humans. However, the characterization of CD4⁺CD8⁺ T cells, their relationship with T memory (T_M), T effector (T_E), Th1/Th2, Treg and Th-17, remain unclear. This study was to characterize the CD4⁺CD8⁺ T cells. The results from human subjects showed that activated T cells were CD4⁺CD8⁺ T cells, comprised CD4^hiCD8^lo, CD4^hiCD8^hi and CD4^loCD8^hi subsets. They expressed CD62L^hi/lo, granzyme B (GrB), CD25, Foxp3, interleukin 17 (IL-17) and the cytokines of both Th1 and Th2, and had cytolytic function. These findings suggested that CD4⁺CD8⁺ T cells had over-lap function while they kept diversity, and that T cells could be divided into two major populations: activated and inactivated. Hence, the hypotheses of Th1/Th2, Treg and Th-17 might reflect the positive/negative feedback regulation of immune system. When compared to GrB⁺CD62L^lo T effector (T_E) cells, GrB⁺CD62L^hi T central memory effector (T_CME) cells had a quicker response to virus without CD62L loss.     

In vitro activation and differentiation of naïve CD4 and CD8 T cells into HCV Core- and NS3-specific armed effector cells: A new role for CD4 T cells

Viral clearance in hepatitis C virus (HCV) infection has been correlated with strong, multi-specific and sustained T cell responses. The number of functionally active effector T cells determines the outcome of infection. Only a small number of antigen-specific naïve T cells are originally present. Upon infection, they undergo activation, clonal expansion and differentiation to become effector cells. In this study, we determined the ability of dendritic cells (DCs) to prime T cells in vitro to become effector cells upon stimulation with various TLR ligands or IFNα. T cell priming and activation was determined by proliferation and production of effector molecules, IFN-γ and Granzyme B (GrB). HCV Core-specific T cells showed significant increase in proliferation, and the number of HCV Core-specific CD4⁺ and CD8⁺ T cells producing IFN-γ and GrB was higher than control or NS3-specific T cells. These in vitro-primed CD4⁺ and CD8⁺ T cells exhibit the phenotype of just-activated and/or armed effector lymphocytes confirming the transition of naïve T cells to effector cells. This is the first study demonstrating the activation of GrB⁺CD4⁺ T cells against antigen(s) derived from HCV. Our study suggests a novel role of CD4⁺ T cells in immunity against HCV.