Combination of rapamycin and IL-2 do not affect antigen presentation ability of rat B cell and could promote Tregs proliferation and inhibitory activity

Rapamycin (RPM), a powerful agent used clinically in transplant recipients, induces CD4⁺CD25⁺ regulatory T cells (Tregs) which play an important role in induction of immune tolerance. However, long-term use of RPM has negative side effects. In this report, we found that combination with the low dose RPM and high dose IL-2 did not affect antigen presentation of rat B cells to Tregs, and could efficiently promote Tregs proliferation and enhance their inhibitory activities in vitro. In addition, the combination of low dose RPM and high dose IL-2 enhanced mRNA expression of Foxp3, TGF-β1 and Pim-2 in Tregs but not in CD4⁺CD25⁻ T effector cells (Teffs). The Tregs inhibitory activity is positively associated with mRNA expressions of TGF-β1 and Pim-2 while unrelated to the Foxp3 mRNA expression. Our present study offers one approach to expand functional Tregs in vitro, which maybe used for clinical immune tolerance induction.     

Increase in TGF-β Secreting CD4+CD25+ FOXP3+ T Regulatory Cells in Anergic Lepromatous Leprosy Patients

Background

Lepromatous leprosy caused by Mycobacterium leprae is associated with antigen specific T cell unresponsiveness/anergy whose underlying mechanisms are not fully defined. We investigated the role of CD25⁺FOXP3⁺ regulatory T cells in both skin lesions and M.leprae stimulated PBMC cultures of 28 each of freshly diagnosed patients with borderline tuberculoid (BT) and lepromatous leprosy (LL) as well as 7 healthy household contacts of leprosy patients and 4 normal skin samples.

Methodology/Principle Findings

Quantitative reverse transcribed PCR (qPCR), immuno-histochemistry/flowcytometry and ELISA were used respectively for gene expression, phenotype characterization and cytokine levels in PBMC culture supernatants. Both skin lesions as well as in vitro antigen stimulated PBMC showed increased percentage/mean fluorescence intensity of cells and higher gene expression for FOXP3⁺, TGF-β in lepromatous (p<0.01) as compared to tuberculoid leprosy patients. CD4⁺CD25⁺FOXP3⁺ T cells (Tregs) were increased in unstimulated basal cultures (p<0.0003) and showed further increase in in vitro antigen but not mitogen (phytohemaglutinin) stimulated PBMC (iTreg) in lepromatous as compared to tuberculoid leprosy patients (p<0.002). iTregs of lepromatous patients showed intracellular TGF-β which was further confirmed by increase in TGF-β in culture supernatants (p<0.003). Furthermore, TGF-β in iTreg cells was associated with phosphorylation of STAT5A. TGF-β was seen in CD25⁺ cells of the CD4⁺ but not that of CD8⁺ T cell lineage in leprosy patients. iTregs did not show intracellular IFN-γ or IL-17 in lepromatous leprosy patients.

Conclusions/Significance

Our results indicate that FOXP3⁺ iTregs with TGF-β may down regulate T cell responses leading to the antigen specific anergy associated with lepromatous leprosy.     

Characterization of an in vitro proliferation response to solubilized Trichinella spiralis antigens: Role of Ia antigens and Ly-1+ T cells

An antigen extract from Trichinella spiralis muscle larvae was prepared and used to immunize strains of mice which were either relatively resistant (B10.S) or susceptible (B10.BR) to oral infection with T. spiralis larvae. Proliferation of cells from the draining lymph nodes was then measured in vitro after stimulation with the T. spiralis extract as well as appropriate control antigens. Primed cells from resistant B10.S mice responded better to challenge than did cells from the susceptible B10.BR strain. Cell-depletion experiments involving B10.S cells indicated that the in vitro cell proliferation response is dependent upon Ly-1⁺ T cells. The data were also consistent with a requirement for Ly-1⁺, -2⁺, and -3⁺ amplifier cells. Administration of anti-I^s serum to the cultures specifically inhibited (nearly 75%) the cell proliferation response. The potential applications of this system as a tool in immunogenetic analyses of immunity to T. spiralis are discussed.     

Bordetella Adenylate Cyclase Toxin Differentially Modulates Toll-Like Receptor-Stimulated Activation,Migration and T Cell Stimulatory Capacity of Dendritic Cells

Adenylate cyclase toxin (CyaA) is a key virulence factor of the whooping cough agent Bordetella pertussis. The toxin targets CD11b-expressing phagocytes and delivers into their cytosol an adenylyl cyclase (AC) enzyme that subverts cellular signaling by increasing cAMP levels. In the present study, we analyzed the modulatory effects of CyaA on adhesive, migratory and antigen presenting properties of Toll-like receptor (TLR)-activated murine and human dendritic cells (DCs). cAMP signaling of CyaA enhanced TLR-induced dissolution of cell adhesive contacts and migration of DCs towards the lymph node-homing chemokines CCL19 and CCL21 in vitro. Moreover, we examined in detail the capacity of toxin-treated DCs to induce CD4⁺ and CD8⁺ T cell responses. Exposure to CyaA decreased the capacity of LPS-stimulated DCs to present soluble protein antigen to CD4⁺ T cells independently of modulation of co-stimulatory molecules and cytokine production, and enhanced their capacity to promote CD4⁺CD25⁺Foxp3⁺ T regulatory cells in vitro. In addition, CyaA decreased the capacity of LPS-stimulated DCs to induce CD8⁺ T cell proliferation and limited the induction of IFN-γ producing CD8⁺ T cells while enhancing IL-10 and IL-17-production. These results indicate that through activation of cAMP signaling, the CyaA may be mobilizing DCs impaired in T cell stimulatory capacity and arrival of such DCs into draining lymph nodes may than contribute to delay and subversion of host immune responses during B. pertussis infection.     

in vitro generation of IFN-γ-producing Listeria-specific T cells is dependent on IFN-γ production by non-NK cells

In vitro 5-day cultures of naive spleen cells with viable Listeria monocytogenes (VLM), but not heat-killed L. monocytogenes, induced CD4⁺ T cells that produced IFN-γ upon secondary antigen stimulation. The VLM-induced Listeria-specific T cells produced IFN-γ but lacked expression of IL-2 and IL-4. To study the role of IFN-γ in the induction of the IFN-γ-producing T cells, we added anti-IFN-γ mAb to the primary culture and analyzed IFN-γ production upon secondary antigen stimulation. Addition of anti-IFN-γ mAb to the culture suppressed generation of IFN-γ-producing CD4⁺ T cells, suggesting that IFN-γ is important in the induction of IFN-γ-producing CD4⁺ T cells. Furthermore, our results showed that depletion of NK cells from spleen cells by anti-asialo GM1 antibody plus complement before culture enhanced induction of IFN-γ-producing CD4⁺ T cells. Although NK cells are known to produce IFN-γ, the results indicate that NK cell-derived IFN-γ may not be important in induction of the Listeria-specific IFN-γ-producing CD4⁺ T cells in the culture system. In addition, we demonstrated that IFN-γ expression was high in CD4⁺ T cells from cultures of spleen cells with VLM at the primary culture level. These results suggest that IFN-γ derived from T cells may enhance production of IFN-γ by CD4⁺ T cells, while NK cells rather suppress the induction of IFN-γ-producing CD4⁺ T cells.     

Prostaglandin-mediated regulation of the mixed lymphocyte culture and generation of cytotoxic cells

We examined the role of prostaglandins or prostaglandin-producing cells in the regulation of proliferation and generation of specific cytotoxicity in one-way mixed lymphocyte cultures of mouse spleen cells. Cultures treated with indomethacin or other prostaglandin synthesis inhibitors resulted in enhanced proliferation and cytotoxicity. The level of prostaglandins produced in vitro, as measured by RIA, was 10^?8M and was found to be completely blocked by indomethacin. Adding back 10^?8M PG restored baseline (control) proliferative responses. Kinetics of the enhanced MLC response were unchanged from controls as were the specificities of the cytotoxic cells. Cells from indomethacin-treated cultures were more efficient at killing targets than those from control cultures. Prostaglandins appear to have a preferential effect on the induction of cytotoxic cells.     

T cell responses in fresh and cryopreserved peripheral blood mononuclear cells: kinetics of cell viability, cellular subsets, proliferation, and cytokine production

Polyclonal stimuli like phorbol myristate acetate (PMA) plus calcium ionophore (Ca-I), concanavalin A (ConA) or anti-CD3 plus anti-CD28 (αCD3/αCD28) are widely used T cell stimuli. All three stimuli act at different sites and in different ways to activate the T cell receptor pathway and are widely used in different concentrations, stimulation durations and read-out systems. This study was designed to establish the most suitable polyclonal stimulus in human peripheral blood mononuclear cells (PBMC) experiments by assessing the kinetics of cell viability, present immunophenotypes, proliferation, and cytokine production of the PBMC. In addition, changes in these read-out parameters due to cryopreservation have been investigated by comparing fresh and cryopreserved PBMC cultures at days 1, 3, 5, and 7. This study showed a reduction in the cytokine levels after cryopreservation of PMA/Ca-I stimulated PBMC, whereas no significant differences due to the cryopreservation were observed in ConA or αCD3/αCD28 stimulated PBMC. Cryopreservation did not alter the maximal proliferation capacity of ConA or αCD3/αCD28 stimulated PBMC, whereas it did delay the proliferation. Although cryopreservation had no effect on the CD3⁺CD4⁺ or CD3⁺CD8⁺ T cell subsets, PMA/Ca-I significantly reduced the amount of both T cell subsets over time.In conclusion, PMA/Ca-I is suitable as a positive control in experiments where high cytokine production is expected and only fresh PBMC are used. Proliferation and effects on the T cell subsets in long-term PBMC cultures should use ConA or αCD3/αCD28 as positive control.     

The Polyunsaturated Fatty Acids Arachidonic Acid and Docosahexaenoic Acid Induce Mouse Dendritic Cells Maturation but Reduce T-Cell Responses In Vitro

Long-chain polyunsaturated fatty acids (PUFAs) might regulate T-cell activation and lineage commitment. Here, we measured the effects of omega-3 (n-3), n-6 and n-9 fatty acids on the interaction between dendritic cells (DCs) and naïve T cells. Spleen DCs from BALB/c mice were cultured in vitro with ovalbumin (OVA) with 50 μM fatty acids; α-linolenic acid, arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid or oleic acid and thereafter OVA-specific DO11.10 T cells were added to the cultures. Fatty acids were taken up by the DCs, as shown by gas chromatography analysis. After culture with arachidonic acid or DHA CD11c⁺ CD11b⁺ and CD11c⁺ CD11b^neg DCs expressed more CD40, CD80, CD83, CD86 and PDL-1, while IA^d remained unchanged. However, fewer T cells co-cultured with these DCs proliferated (CellTrace Violet^low) and expressed CD69 or CD25, while more were necrotic (7AAD⁺). We noted an increased proportion of T cells with a regulatory T cell (Treg) phenotype, i.e., when gating on CD4⁺ FoxP3⁺ CTLA-4⁺, CD4⁺ FoxP3⁺ Helios⁺ or CD4⁺ FoxP3⁺ PD-1⁺, in co-cultures with arachidonic acid- or DHA-primed DCs relative to control cultures. The proportion of putative Tregs was inversely correlated to T-cell proliferation, indicating a suppressive function of these cells. With arachidonic acid DCs produced higher levels of prostaglandin E₂ while T cells produced lower amounts of IL-10 and IFNγ. In conclusion arachidonic acid and DHA induced up-regulation of activation markers on DCs. However arachidonic acid- and DHA-primed DCs reduced T-cell proliferation and increased the proportion of T cells expressing FoxP3, indicating that these fatty acids can promote induction of regulatory T cells.     

Passive transfer of experimental allergic encephalomyelitis in the Lewis rat with activated spleen cells: differential activation with mitogens

It has previously been shown that spleen cell transfer of clinical EAE requires donor cells to be cultured in vitro prior to transfer. Donor cells must be stimulated when cultured, and either Con A or the encephalitogen, guinea pig myelin basic protein (BP), satisfies this stimulation requirement. Following recovery from passive disease, recipients of these in vitro cultured cells will subsequently develop clinical symptoms of EAE sooner than controls when challenged with BP in complete Freund's adjuvant (BP-CFA). In the present study, three T-cell mitogens were evaluated as donor cell stimulants in the required in vitro culture period. Pokeweed mitogen (PWM) as well as Con A stimulated the donor cell population to the degree that clinical EAE could be transferred with 5 × 10⁶ cultured viable cells. Con A at culture levels below 0.25 μg/ml did not yield transfer active cells even though proliferation levels were similar to those found at concentrations of Con A that did yield transfer active cells. Phytohemagglutinin (PHA)-stimulated cultures did not transfer clinical disease even though the degree of lectin induced proliferation ([³H]thymidine uptake as well as recovered cells from culture) was equivalent to the PWM- or Con A-stimulated, transfer positive, cultures. Mixing experiments suggested that the inability of PHA or low doses of Con A to induce transfer active cells was not due to the induction of suppressor cells. Although cells cultured with PHA do not transfer clinical EAE, recipients of these cells as well as recipients of either PWM- or Con A-stimulated donor cells develop an early appearance of disease upon subsequent challenge with BP-CFA. This included cells incubated with a concentration of Con A (0.1 μg/ml) which did not induce cells capable of transferring clinical EAE. These results suggest that PHA and perhaps the low dose of Con A may stimulate the proliferation of the EAE effector cell precursor population without causing the additional differentiation of this precursor population into the effector cell population which is capable of transferring clinical disease. Alternatively, PHA may expand only the helper cell population while effective doses of Con A and PWM would expand both helper and effector cell populations.     

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.     

Regulatory CD8 T cells induced by exposure to all-trans retinoic acid and TGF-β suppress autoimmune diabetes

Antigen-specific regulatory CD4⁺ T cells have been described but there are few reports on regulatory CD8⁺ T cells. We generated islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-specific regulatory CD8⁺ T cells from 8.3-NOD transgenic mice. CD8⁺ T cells from 8.3-NOD splenocytes were cultured with IGRP, splenic dendritic cells (SpDCs), TGF-β, and all-trans retinoic acid (ATRA) for 5 days. CD8⁺ T cells cultured with either IGRP alone or IGRP and SpDCs in the absence of TGF-β and ATRA had low Foxp3⁺ expression (1.7 ± 0.9% and 3.2 ± 4.5%, respectively). In contrast, CD8⁺ T cells induced by exposure to IGRP, SpDCs, TGF-β, and ATRA showed the highest expression of Foxp3⁺ in IGRP-reactive CD8⁺ T cells (36.1 ± 10.6%), which was approximately 40-fold increase compared with that before induction culture. CD25 expression on CD8⁺ T cells cultured with IGRP, SpDCs, TGF-β, and ATRA was only 7.42%, whereas CD103 expression was greater than 90%. These CD8⁺ T cells suppressed the proliferation of diabetogenic CD8⁺ T cells from 8.3-NOD splenocytes in vitro and completely prevented diabetes onset in NOD-scid mice in cotransfer experiments with diabetogenic splenocytes from NOD mice in vivo. Here we show that exposure to ATRA and TGF-β induces CD8⁺Foxp3⁺ T cells ex vivo, which suppress diabetogenic T cells in vitro and in vivo.     

CD4+CD62L+ Central Memory T Cells Can Be Converted to Foxp3+ T Cells

The peripheral Foxp3⁺ Treg pool consists of naturally arising Treg (nTreg) and adaptive Treg cells (iTreg). It is well known that naive CD4⁺ T cells can be readily converted to Foxp3⁺ iTreg in vitro, and memory CD4⁺ T cells are resistant to conversion. In this study, we investigated the induction of Foxp3⁺ T cells from various CD4⁺ T-cell subsets in human peripheral blood. Though naive CD4⁺ T cells were readily converted to Foxp3⁺ T cells with TGF-β and IL-2 treatment in vitro, such Foxp3⁺ T cells did not express the memory marker CD45RO as do Foxp3⁺ T cells induced in the peripheral blood of Hepatitis B Virus (HBV) patients. Interestingly, a subset of human memory CD4⁺ T cells, defined as CD62L⁺ central memory T cells, could be induced by TGF-β to differentiate into Foxp3⁺ T cells. It is well known that Foxp3⁺ T cells derived from human CD4⁺CD25^- T cells in vitro are lack suppressive functions. Our data about the suppressive functions of CD4⁺CD62L⁺ central memory T cell-derived Foxp3⁺ T cells support this conception, and an epigenetic analysis of these cells showed a similar methylation pattern in the FOXP3 Treg-specific demethylated region as the naive CD4⁺ T cell-derived Foxp3⁺ T cells. But further research showed that mouse CD4⁺ central memory T cells also could be induced to differentiate into Foxp3⁺ T cells, such Foxp3⁺ T cells could suppress the proliferation of effector T cells. Thus, our study identified CD4⁺CD62L⁺ central memory T cells as a novel potential source of iTreg.     

Tumor-Targeted Human T Cells Expressing CD28-Based Chimeric Antigen Receptors Circumvent CTLA-4 Inhibition

Adoptive T cell therapy represents a promising treatment for cancer. Human T cells engineered to express a chimeric antigen receptor (CAR) recognize and kill tumor cells in a MHC-unrestricted manner and persist in vivo when the CAR includes a CD28 costimulatory domain. However, the intensity of the CAR-mediated CD28 activation signal and its regulation by the CTLA-4 checkpoint are unknown. We investigated whether T cells expressing an anti-CD19, CD3 zeta and CD28-based CAR (19-28z) displayed the same proliferation and anti-tumor abilities than T cells expressing a CD3 zeta-based CAR (19z1) costimulated through the CD80/CD28, ligand/receptor pathway. Repeated in vitro antigen-specific stimulations indicated that 19-28z⁺ T cells secreted higher levels of Th1 cytokines and showed enhanced proliferation compared to those of 19z1⁺ or 19z1-CD80⁺ T cells. In an aggressive pre-B cell leukemia model, mice treated with 19-28z⁺ T cells had 10-fold reduced tumor progression compared to those treated with 19z1⁺ or 19z1-CD80⁺ T cells. shRNA-mediated CTLA-4 down-regulation in 19z1-CD80⁺ T cells significantly increased their in vivo expansion and anti-tumor properties, but had no effect in 19-28z⁺ T cells. Our results establish that CTLA-4 down-regulation may benefit human adoptive T cell therapy and demonstrate that CAR design can elude negative checkpoints to better sustain T cell function.     

Age-Dependent Changes in the Sphingolipid Composition of Mouse CD4+ T Cell Membranes and Immune Synapses Implicate Glucosylceramides in Age-Related T Cell Dysfunction

To determine whether changes in sphingolipid composition are associated with age-related immune dysfunction, we analyzed the core sphingolipidome (i.e., all of the metabolites through the first headgroup additions) of young and aged CD4⁺ T cells. Since sphingolipids influence the biophysical properties of membranes, we evaluated the compositions of immune synapse (IS) and non-IS fractions prepared by magnetic immuno-isolation. Broadly, increased amounts of sphingomyelins, dihydrosphingomyelins and ceramides were found in aged CD4⁺ T cells. After normalizing for total sphingolipid content, a statistically significant decrease in the molar fraction of glucosylceramides was evident in both the non-IS and IS fractions of aged T cells. This change was balanced by less dramatic increases in the molar fractions of sphingomyelins and dihydrosphingomyelins in aged CD4⁺ T cells. In vitro, the direct or enzymatic enhancement of ceramide levels decreased CD4⁺ T cell proliferation without regard for the age of the responding T cells. In contrast, the in vitro inhibition of glucosylceramidase preferentially increased the proliferation of aged CD4⁺ T cells. These results suggest that reductions in glucosylceramide abundance contribute to age-related impairments in CD4⁺ T cell function.     

PIM1 Kinase Phosphorylates the Human Transcription Factor FOXP3 at Serine 422 to Negatively Regulate Its Activity under Inflammation

Previous reports have suggested that human CD4⁺ CD25^hiFOXP3⁺ T regulatory cells (Tregs) have functional plasticity and may differentiate into effector T cells under inflammation. The molecular mechanisms underlying these findings remain unclear. Here we identified the residue serine 422 of human FOXP3 as a phosphorylation site that regulates its function, which is not present in murine Foxp3. PIM1 kinase, which is highly expressed in human Tregs, was found to be able to interact with and to phosphorylate human FOXP3 at serine 422. T cell receptor (TCR) signaling inhibits PIM1 induction, whereas IL-6 promotes PIM1 expression in in vitro expanded human Tregs. PIM1 negatively regulates FOXP3 chromatin binding activity by specifically phosphorylating FOXP3 at Ser⁴²². Our data also suggest that phosphorylation of FOXP3 at the Ser⁴¹⁸ site could prevent FOXP3 phosphorylation at Ser⁴²² mediated by PIM1. Knockdown of PIM1 in in vitro expanded human Tregs promoted FOXP3-induced target gene expression, including CD25, CTLA4, and glucocorticoid-induced tumor necrosis factor receptor (GITR), or weakened FOXP3-suppressed IL-2 gene expression and enhanced the immunosuppressive activity of Tregs. Furthermore, PIM1-specific inhibitor boosted FOXP3 DNA binding activity in in vitro expanded primary Tregs and also enhanced their suppressive activity toward the proliferation of T effector cells. Taken together, our findings suggest that PIM1 could be a new potential therapeutic target in the prevention and treatment of human-specific autoimmune diseases because of its ability to modulate the immunosuppressive activity of human Tregs.     

Ex vivo expansion of human CD8+ T cells using autologous CD4+ T cell help

Background

Using in vivo mouse models, the mechanisms of CD4⁺ T cell help have been intensively investigated. However, a mechanistic analysis of human CD4⁺ T cell help is largely lacking. Our goal was to elucidate the mechanisms of human CD4⁺ T cell help of CD8⁺ T cell proliferation using a novel in vitro model.

Methods/Principal Findings

We developed a genetically engineered novel human cell-based artificial APC, aAPC/mOKT3, which expresses a membranous form of the anti-CD3 monoclonal antibody OKT3 as well as other immune accessory molecules. Without requiring the addition of allogeneic feeder cells, aAPC/mOKT3 enabled the expansion of both peripheral and tumor-infiltrating T cells, regardless of HLA-restriction. Stimulation with aAPC/mOKT3 did not expand Foxp3⁺ regulatory T cells, and expanded tumor infiltrating lymphocytes predominantly secreted Th1-type cytokines, interferon-γ and IL-2. In this aAPC-based system, the presence of autologous CD4⁺ T cells was associated with significantly improved CD8⁺ T cell expansion in vitro. The CD4⁺ T cell derived cytokines IL-2 and IL-21 were necessary but not sufficient for this effect. However, CD4⁺ T cell help of CD8⁺ T cell proliferation was partially recapitulated by both adding IL-2/IL-21 and by upregulation of IL-21 receptor on CD8⁺ T cells.

Conclusions

We have developed an in vitro model that advances our understanding of the immunobiology of human CD4⁺ T cell help of CD8⁺ T cells. Our data suggests that human CD4⁺ T cell help can be leveraged to expand CD8⁺ T cells in vitro.     

A Trifunctional Dextran-Based Nanovaccine Targets and Activates Murine Dendritic Cells,and Induces Potent Cellular and Humoral Immune Responses In Vivo

Dendritic cells (DCs) constitute an attractive target for specific delivery of nanovaccines for immunotherapeutic applications. Here we tested nano-sized dextran (DEX) particles to serve as a DC-addressing nanocarrier platform. Non-functionalized DEX particles had no immunomodulatory effect on bone marrow (BM)-derived murine DCs in vitro. However, when adsorbed with ovalbumine (OVA), DEX particles were efficiently engulfed by BM-DCs in a mannose receptor-dependent manner. A DEX-based nanovaccine containing OVA and lipopolysaccharide (LPS) as a DC stimulus induced strong OVA peptide-specific CD4⁺ and CD8⁺ T cell proliferation both in vitro and upon systemic application in mice, as well as a robust OVA-specific humoral immune response (IgG1>IgG2a) in vivo. Accordingly, this nanovaccine also raised both a more pronounced delayed-type hypersensitivity response and a stronger induction of cytotoxic CD8⁺ T cells than obtained upon administration of OVA and LPS in soluble form. Therefore, DEX-based nanoparticles constitute a potent, versatile and easy to prepare nanovaccine platform for immunotherapeutic approaches.     

The Immunosuppressant Protosappanin A Promotes Dendritic Cell-Mediated Expansion of Alloantigen-Specific Tregs and Prolongs Allograft Survival in Rats

Protosappanin A (PrA), an immunosuppressive ingredient of the medicinal herb Caesalpinia sappan L, prolongs heart allograft survival in rats, possibly by impairing the function of antigen-presenting cells (APCs). We examined the effects of PrA on the maturation and function of dendritic cells (DCs), a potent class of APCs, and the downstream cell–cell and intracellular signaling pathways mediating the immunosuppressive activity of PrA. PrA inhibited LPS-stimulated maturation of Wistar rat DCs in vitro as reflected by reduced expression of costimulatory molecules (CD80 and CD86) and reduced expression of TLR4 and NF-κB, two critical signaling components for antigen recognition. PrA also enhanced the release of IL-10 and decreased the release of IL-12 from DCs, but had no effect on the production of TGF-ß. In mixed cultures, Wistar DCs pretreated with PrA impaired the proliferation of Sprague Dawley (SD) rat T cells while promoting the expansion of SD rat CD4⁺CD25⁺ regulatory T cells (Tregs). Both oral PrA treatment and infusion of PrA-pretreated Wistar DCs prolonged cardiac allograft survival (Wistar donor, SD recipient) and expanded recipient CD4⁺CD25⁺Foxp3⁺ Tregs. Donor spleen cells, but not spleen cells from a third rat strain (DA), supported the expansion of recipient CD4⁺CD25⁺Foxp3⁺ Tregs and suppressed recipient T cell proliferation. We conclude that PrA triggers a tolerogenic state in DCs that allows for the induction of alloantigen-specific Tregs and the suppression of allograft rejection in vivo.     

Characterization of Effector and Memory T Cell Subsets in the Immune Response to Bovine Tuberculosis in Cattle

Cultured IFN-γ ELISPOT assays are primarily a measure of central memory T cell (Tcm) responses with humans; however, this important subset of lymphocytes is poorly characterized in cattle. Vaccine-elicited cultured IFN-γ ELISPOT responses correlate with protection against bovine tuberculosis in cattle. However, whether this assay measures cattle Tcm responses or not is uncertain. The objective of the present study was to characterize the relative contribution of Tcm (CCR7⁺, CD62L^hi, CD45RO⁺), T effector memory (Tem, defined as: CCR7^-, CD62L^low/int, CD45RO⁺), and T effector cells (CCR7^-, CD62L^-/low, CD45RO^-), in the immune response to Mycobacterium bovis. Peripheral blood mononuclear cells (PBMC) from infected cattle were stimulated with a cocktail of M. bovis purified protein derivative, rTb10.4 and rAg85A for 13 days with periodic addition of fresh media and rIL-2. On day 13, cultured PBMC were re-stimulated with medium alone, rESAT-6:CFP10 or PPDb with fresh autologous adherent cells for antigen presentation. Cultured cells (13 days) or fresh PBMCs (ex vivo response) from the same calves were analyzed for IFN-γ production, proliferation, and CD4, CD45RO, CD62L, CD44, and CCR7 expression via flow cytometry after overnight stimulation. In response to mycobacterial antigens, ~75% of CD4⁺ IFN-γ⁺ cells in long-term cultures expressed a Tcm phenotype while less than 10% of the ex vivo response consisted of Tcm cells. Upon re-exposure to antigen, long-term cultured cells were highly proliferative, a distinctive characteristic of Tcm, and the predominant phenotype within the long-term cultures switched from Tcm to Tem. These findings suggest that proliferative responses of Tcm cells to some extent occurs simultaneously with reversion to effector phenotypes (mostly Tem). The present study characterizes Tcm cells of cattle and their participation in the response to M. bovis infection.