CD80 (B7-1) and CD86 (B7-2) are functionally equivalent in the initiation and maintenance of CD4+ T-cell proliferation after activation with suboptimal doses of PHA

Effective activation of T cells requires engagement of two separate T-cell receptors. The antigen-specific T-cell receptor (TCR) binds foreign peptide antigen-MHC complexes, and the CD28 receptor binds to the B7 (CD80/CD86) costimulatory molecules expressed on the surface of antigen-presenting cells (APC). The simultaneous triggering of these T-cell surface receptors with their specific ligands results in an activation of this cell. In contrast, CTLA-4 (CD152) is a distinct T-cell receptor that, upon binding to B7 molecules, sends an inhibitory signal to T cell activation. Many in vitro and in vivo studies demonstrated that both CD80 and CD86 ligands have an identical role in the activation of T cells. Recently, functions of B7 costimulatory molecules in vivo have been investigated in B7-1 and/or B7-2 knockout mice, and the authors concluded that CD86 could be more important for initiating T-cell responses, while CD80 could be more significant for maintaining these immune responses. In this study, we directly compared the role of CD80 and CD86 in initiating and maintaining proliferation of resting CD4(+) T cells in an in vitro mode system that allowed to provide the first signal-to-effector cells through the use of suboptimal doses of PHA and the second costimulatory signal through cells expressing CD80 or CD86, but not any other costimulatory molecules. Using this experimental system we demonstrate that the CD80 and CD86 molecules can substitute for each other in the initial activation of resting CD4(+) T cells and in the maintenance of their proliferative response.     

Time courses of B7 family molecules expressed on activated T-cells and their biological significance

B7 family molecules are mainly expressed on the outer membrane of antigen-presenting cells. Here, our results demonstrate that CD80, CD86, and PD-L1 molecules are also expressed on T-cells that have been activated by simultaneous exposure to anti-CD3 and anti-CD28 mAbs, but PD-L2 and GL50 molecules were not detectable during the first six days of culture that follow such stimulation. We have analysed the time course of B7 family molecule expression on activated T-cells. CD28 and its ligands, CD80/CD86, have a high degree of co-localization and exhibit compartmental distribution on the membrane of activated T-cells, which is visualized by confocal microscopy. Interestingly, the co-localization of PD-1 and its ligand also exhibit similar phenomenon. Additionally, we provide evidence indicating that the CD80, CD86, and PD-L1 molecules are functional, since T-cells expressing B7 family molecules are able to stimulate the proliferation of highly purified allogeneic or autologous T-cells. Anti-CD80, anti-CD86, and soluble CD28-Ig protein could significantly attenuate the proliferation of T-cells, whereas anti-PD-L1 mAb may lead to the expansion of activated T-cells. We can conclude that activated T-cells expressing B7 family molecules could act as "APC" to trigger purified T-cells, and B7 family molecules play important roles during the activation of T-cells. These results indicate a need for further work, exploring the regulatory roles these molecules may play in immune responses.     

Exposure to apoptotic activated CD4+ T cells induces maturation and APOBEC3G-mediated inhibition of HIV-1 infection in dendritic cells

Dendritic cells (DCs) are activated by signaling via pathogen-specific receptors or exposure to inflammatory mediators. Here we show that co-culturing DCs with apoptotic HIV-infected activated CD4(+) T cells (ApoInf) or apoptotic uninfected activated CD4(+) T cells (ApoAct) induced expression of co-stimulatory molecules and cytokine release. In addition, we measured a reduced HIV infection rate in DCs after co-culture with ApoAct. A prerequisite for reduced HIV infection in DCs was activation of CD4(+) T cells before apoptosis induction. DCs exposed to ApoAct or ApoInf secreted MIP-1α, MIP-1β, MCP-1, and TNF-α; this effect was retained in the presence of exogenous HIV. The ApoAct-mediated induction of co-stimulatory CD86 molecules and reduction of HIV infection in DCs were partially abrogated after blocking TNF-α using monoclonal antibodies. APOBEC3G expression in DCs was increased in co-cultures of DCs and ApoAct but not by apoptotic resting CD4(+) T cells (ApoRest). Silencing of APOBEC3G in DC abrogated the HIV inhibitory effect mediated by ApoAct. Sequence analyses of an env region revealed significant induction of G-to-A hypermutations in the context of GG or GA dinucleotides in DNA isolated from DCs exposed to HIV and ApoAct. Thus, ApoAct-mediated DC maturation resulted in induction of APOBEC3G that was important for inhibition of HIV-infection in DCs. These findings underscore the complexity of differential DC responses evoked upon interaction with resting as compared with activated dying cells during HIV infection.     

Antigen receptor triggered upregulation of CD86 and CD80 in human B cells: augmenting role of the CD21/CD19 co-stimulatory complex and IL-4

The impact of BCR:CD21 co-engagement on B cell expression of molecules critical for T cell activation was investigated with receptor-specific mAbs conjugated to high MW dextran as stimulatory ligands. In the absence of IL-4, BCR:CD21 co-ligation augmented BCR-triggered CD86 only under conditions of very low BCR ligand dose or affinity, and CD80 was minimally induced by BCR and/or CD21 crosslinking. In the presence of IL-4, BCR:CD21 co-ligation augmented CD86 and CD80 expression under conditions of greater BCR engagement. However, with very high level BCR engagement, no bonus effect of BCR:CD21 crosslinking was observed. Co-ligation-promoted CD86 and CD80 expression was associated with heightened B cell activation of resting allogeneic T cells. The data suggest that co-clustering of BCR and the CD21/CD19 co-stimulatory complex following B cell engagement with C3d-bound microbial or self-antigens will enhance B cell recruitment of T cell help only when IL-4 is present and/or BCR engagement is very limiting.     

Interactions between osteosarcoma cell lines and dendritic cells immune function: An in vitro study

Dendritic cells (DCs) might be partly responsible for the defective immune response in tumor bearing hosts, but no study in osteosarcoma patients is still available. Therefore, we investigated in vitro whether human osteosarcoma cell lines have an inhibitor effect on different types of DCs: CD14+DCs, DC1 and DC2. DCs derived from healthy donors were cultured with osteosarcoma cell lines and appropriate cytokine cocktails and analysed for the expression of co-stimulatory molecules (CD40, CD80, CD83, CD86, HLA-DR). Each interaction resulted in a lower phenotypic expression of the DCs maturation markers, especially on DC2. Moreover, the addition of various cytokines and compounds (rhIL-12, CD40L, Indometacin) induced the DC1 and DC2 subsets towards the Th1 pattern as shown by ELISA. Osteosarcoma highly interferes with an in vitro DCs immune function as antigen presenting cells. The understanding of tumor biology underlines the need for a specific osteosarcoma immunotherapy able to reverse this immune-surveillance inhibition.     

Release of endogenous danger signals from HIFU-treated tumor cells and their stimulatory effects on APCs

The effects of high-intensity focused ultrasound (HIFU) on the release of endogenous danger signals from tumor cells and subsequent activation of antigen-presenting cells (APCs) were evaluated in vitro. MC-38 mouse tumor cells were treated using a 1.1 MHz HIFU transducer under two different protocols (P-=6.7 MPa, 30% duty cycle, 5 s vs. P-=10.7 MPa, 3% duty cycle, 30 s) to produce either thermal necrosis or mechanical lysis of the tumor cells. Here, we report that HIFU treatment can cause the release of endogenous danger signals (ATP and hsp60) and exposure of dendritic cells (DCs) and macrophages to the supernatants of HIFU-treated tumor cells leads to an increased expression of co-stimulatory molecules (CD80 and CD86) with enhanced secretion of IL-12 by the DCs and elevated secretion of TNF-alpha by the macrophages. The potency in APC activation produced by mechanical lysis is much stronger than thermal necrosis of the tumor cells. These findings suggest that optimization of treatment strategy may help to enhance HIFU-elicited anti-tumor immunity.     

IL-10 alters DC function via modulation of cell surface molecules resulting in impaired T-cell responses

IL-10 is a potent inhibitor of T-cell activation and has tolerizing effects on these cells. These effects are primarily mediated via modulation of antigen presenting cell function. Here, it is demonstrated that IL-10 completely inhibits LPS-induced DC maturation, resulting in altered DC-T-cell interactions and reduced T-cell responses. IL-10 inhibited LPS-induced upregulation of costimulatory molecules, MHC Class II, and the secretion of IL-12, TNF-alpha, IL-6, and IL-1beta by DCs, although it upregulated the SLAM (CD150) expression at both the mRNA and protein levels. IL-10 pre-treated DC did not respond to subsequent LPS activation and its stimulatory ability for allogeneic and antigen-specific T-cells was severely impaired. Importantly, T-cells derived from co-cultures with Ag-pulsed, IL-10-treated DC were impaired in their responses to subsequent Ag-specific restimulation. Transwell and DC-derived plasma membrane experiments indicated that the capacity of IL-10-treated DC to induce T-cell unresponsiveness results from alterations in the cell surface molecules rather than modulation of cytokine secretion.     

Opisthorchis viverrini antigens up-regulates the expression of CD80 and MHC class II in JAWSII mouse dendritic cells and promotes IL-10 and TGF-β secretions

Dendritic cells (DCs) are antigen-presenting cells (APC) involved in the initiation of immune responses. Maturation of DCs is characterized by the high expression of major histocompatibility complex (MHC) class II and co-stimulatory clusters of differentiation (CD) 40, CD80, and CD86 molecules. Matured DCs are required for T cell differentiation and proliferation. However, the response of DCs to Opisthorchis viverrini antigens has not yet been understood. Therefore, this study sought to determine the expression of surface molecules of JAWSII mouse DCs stimulated by crude somatic (CS) and excretory-secretory (ES) antigens of O. viverrini. ES antigen significantly induced only mRNA expression of CD80 and MHC class II in JAWSII mouse DCs, while CS antigen promoted up-regulation of both mRNA and protein levels of CD80 and MHC class II, indicating relative maturation of JAWII mouse DCs. Moreover, the secreted cytokines from the co-cultures of O. viverrini antigens stimulated JAWSII DC with naïve CD4⁺ T cells was determined. Significantly increased levels of immunosuppressive cytokines interleukin (IL)-10 and transforming growth factor beta (TGF-β) were found. The up-regulation of these cytokines may indicate the response of regulatory T cells (Treg) to CS antigen-stimulated JAWSII DC. These findings may lead to a better understanding of the role that DCs play in O. viverrini infection.     

Oxidized phospholipids negatively regulate dendritic cell maturation induced by TLRs and CD40

Maturation of dendritic cells (DCs) induced by pathogen-derived signals via TLRs is a crucial step in the initiation of an adaptive immune response and therefore has to be well controlled. In this study, we demonstrate that oxidized phospholipids (ox-PLs), which are generated during infections, apoptosis, and tissue damage, interfere with DC activation, preventing their maturation. ox-PLs blocked TLR-3- and TLR-4-mediated induction of the costimulatory molecules CD40, CD80, CD83, and CD86, the cytokines IL-12 and TNF, as well as lymphocyte stimulatory capacity. CD40 and TLR-2-mediated cytokine production was also inhibited, whereas up-regulation of costimulatory molecules via these receptors was not affected by ox-PLs. Thus, formation of ox-PLs during the course of an inflammatory response may represent a negative-feedback loop preventing excessive and sustained immune reactions through regulating DC maturation.     

Effect of plasma viremia on apoptosis and immunophenotype of dendritic cells subsets in acute SIVmac239 infection of Chinese rhesus macaques

Non-human primates such as Chinese rhesus macaques (Ch Rhs) provide good animal models for research on human infectious diseases. Similar to humans, there are two principal subsets of dendritic cells (DCs) in the peripheral blood of Ch Rhs: myeloid DCs (mDCs) and plasmacytoid DCs (pDCs). In this study, two-color fluorescence-activated cell sorting (FACS) analyses were used to identify the main DC subsets, namely CD1c(+) mDCs and pDCs from Ch Rhs. Then, the apoptosis and immunophenotype changes of DCs subsets were first described during the acute phase of SIVmac239 infection. Both the DCs subsets showed decreased CD4 expression and enhanced CCR5 expression; in particular, those of pDCs significantly changed at most time points. Interestingly, the plasma viral loads were negatively correlated with CD4 expression, but were positively correlated with CCR5 expression of pDCs. During this period, both CD1c(+) mDCs and pDCs were activated by enhancing expressions of co-stimulatory molecules, accompanied with increase in CCR7. Either CD80 or CD86 expressed on CD1c(+) mDCs and pDCs was positively correlated with the plasma viral loads. Our analysis demonstrates that the pDCs were more prone to apoptosis after infection during the acute phase of SIVmac239 infection, which may be due to their high expressions of CD4 and CCR5. Both DCs subsets activated through elevating the expression of co-stimulatory molecules, which was beneficial in controlling the replication of SIV. However, a mere broad immune activation initiated by activated DCs may lead to tragic AIDS progression.     

Increased extracellular pressure provides a novel adjuvant stimulus for enhancement of conventional dendritic cell maturation strategies

Dendritic cell (DC)-based vaccine strategies have gained increasing popularity in recent years. Methods for ex vivo generation of immunocompetent mature DCs still require optimization. DCs have been shown to phenotypically mature under elevated pressure. We compared the effects of pressure on DC maturation with LPS- and cytokine-stimulation. Human monocyte-derived immature or LPS- and cytokine-matured DCs were exposed to ambient or 40 mmHg increased pressure for 12 h, then assessed for expression of CD80, CD86, CD40, MHC-I/II, and inflammatory cytokine production. DCs were also evaluated for capacity to stimulate T-cell proliferation by co-culture with allogeneic lymphocytes. Pressure significantly increased cytokine production and expression of all surface molecules on immature DC other than MHC-I and CD40. Pressure/LPS-treated DCs displayed further upregulation of MHC-I, CD40, and IL-12p70. Cytokine-matured DCs appeared less responsive to pressure. T-cell proliferation correlated with MHC expression. Results suggest mechanical stimulation of DCs may provide a useful adjuvant to TLR-agonist maturation strategies.     

Gap junction-mediated intercellular communication between dendritic cells (DCs) is required for effective activation of DCs

Gap junctions, formed by members of the connexin (Cx) family, are intercellular channels allowing direct exchange of signaling molecules. Gap junction-mediated intercellular communication (GJIC) is a widespread mechanism for homeostasis in organs. GJIC in the immune system is not yet fully understood. Although dendritic cells (DC) reportedly form cell-to-cell contact between DCs in nonlymphoid and lymphoid organs, GJIC between DCs remains unknown. In this study we examined whether DCs form GJIC. XS52 and bone marrow-derived DCs (BMDCs) were tested for GJIC by counting intercellular transfer of Lucifer Yellow microinjected into a cell. Either DC became effectively dye-coupled when activated with LPS plus IFN-gamma or TNF-alpha plus IFN-gamma. LPS- plus IFN-gamma-induced dye-coupling was mediated by DC-derived TNF-alpha. In addition, CpG plus IFN-gamma induced dye-coupling in BMDCs, which was also mediated by DC-derived TNF-alpha. LPS- plus IFN-gamma-induced activation of DCs (assessed by CD40 expression) was observed when there was cell-to-cell contact and was significantly blocked by heptanol, a gap junction blocker. These results indicate that cell-to-cell contact and GJIC are required for effective DC activation. In addition, heptanol significantly inhibited the LPS- plus IFN-gamma-induced up-regulation of the other costimulatory (i.e., CD80 and CD86) and MHC class II molecules expressed by BMDCs, and it significantly reduced their allostimulatory capacity. Among Cx members, Cx43 was up-regulated in dye-coupled BMDCs, and Cx mimetic peptide, a blocker of Cx-mediated GJIC, significantly inhibited the dye-coupling and activation, suggesting the involvement of Cx43. Thus, our study provides the first evidence for GJIC between DCs, which is required for effective DC activation.     

Anti-tumor activity of <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> outer membrane protein A on dendritic cell-based immunotherapy against murine melanoma

Acinetobacter baumannii outer membrane protein A (AbOmpA) is a major surface protein that is an important pathogen-associated molecular pattern. Based on our previous findings that AbOmpA induced the phenotypic maturation of dendritic cells (DCs) and drove the Th1 immune response in vitro, we investigated the therapeutic efficacy of AbOmpA-pulsed DC vaccines in a murine melanoma model. The surface expression of co-stimulatory molecules (CD80 and CD86) and major histocompatibility complex class I and II molecules was higher in DCs pulsed with AbOmpA alone or with a combination of B16F10 cell lysates than that of DCs pulsed with B16F10 cell lysates. AbOmpA stimulated the maturation of murine splenic DCs in vivo. In a therapeutic model of murine melanoma, AbOmpA-pulsed DCs significantly retarded tumor growth and improved the survival of tumor-bearing mice. AbOmpA-pulsed DCs significantly enhanced CD8+, interleukin-2+ T cells and CD4+, interferon-gamma+ T cells in tumor-bearing mice. These results provide evidence that AbOmpA may be therapeutically useful in adjuvant DC immunotherapy against poorly immunogenic melanoma without tumor-specific antigens.     

Endocytosis of micro- and nanosized particles in vitro by human dendritic cells

The ability of human dendritic cells (DC) to uptake synthetic micro- and nanosized particles was assessed by flow cytometry and fluorescent microscopy. DCs were differentiated in vitro from blood monocytes in the presence of recombinant cytokines. Further maturation of DC in culture after the addition of maturation factors resulted in the increased expression of HLA-DR and co-stimulatory molecules CD80, CD83, CD86, in comparison with immature DC. Active internalization of Fluoresbrite-YG fluorescent microbeads (0.2 μm) was noted for immature but not mature DCs. The decrease of endocytic activity after DC maturation correlated with the reduced expression of CD209, the surface membrane receptor participating in phagocytosis. Unlike microparticles, the uptake of nanoscale Quantum dots-655 did not depend on the stage of DC maturation and probably was mediated by a different endocytosis mechanism.     

MyD88 and IFN-alphabeta differentially control maturation of bystander but not Salmonella-associated dendritic cells or CD11cintCD11b+ cells during infection

The interface between dendritic cells (DCs) and T cells is critical to elicit effective immunity against pathogens. The maturation state of DCs determines the quality of the interaction and governs the type of response. DCs can be matured directly through activating Toll-like receptors (TLRs) or indirectly by cytokines. We explore the role of the TLR adaptor MyD88 on DC maturation during Salmonella infection. Using Salmonella expressing GFP, we also examine the phenotype and function of bacteria-associated DCs matured in the absence of bacteria-mediated TLR signalling. MyD88 was required for upregulation of CD80 on DCs during infection, whereas CD86 and CD40 were upregulated independently of MyD88, although requiring a higher bacterial burden in the MLN. MyD88-independent upregulation was mediated by IFN-αβ produced during infection. In infected MyD88^−/−IFN-αβR^−/− mice, which lack most bacteria-driven TLR signalling, indirect DC maturation was abolished. In contrast, DCs containing Salmonella upregulated co-stimulatory molecules independently of MyD88 and IFN-αβ, revealing a pathway of phenotypic maturation active in infected DCs. However, despite high co-stimulatory molecule expression, Salmonella -containing DCs from MyD88^−/− or MyD88^−/−IFN-αβR^−/− mice had a compromised capacity to activate T cells. Thus, bacterial stimulation of TLRs influences DC function at multiple levels that modulates their capacity to direct antibacterial immunity.     

CD28 costimulatory signals in T lymphocyte activation: Emerging functions beyond a qualitative and quantitative support to TCR signalling

CD28 is one of the most important co-stimulatory receptors necessary for full T lymphocyte activation. By binding its cognate ligands, B7.1/CD80 or B7.2/CD86, expressed on the surface of professional antigen presenting cells (APC), CD28 initiates several signalling cascades, which qualitatively and quantitatively support T cell receptor (TCR) signalling. More recent data evidenced that human CD28 can also act as a TCR-independent signalling unit, by delivering specific signals, which regulate the expression of pro-inflammatory cytokine/chemokines. Despite the enormous progresses made in identifying the mechanisms and molecules involved in CD28 signalling properties, much remains to be elucidated, especially in the light of the functional differences observed between human and mouse CD28. In this review we provide an overview of the current mechanisms and molecules through which CD28 support TCR signalling and highlight recent findings on the specific signalling motifs that regulate the unique pro-inflammatory activity of human CD28.     

Differential Susceptibility and Response of Primary Human Myeloid BDCA1+ Dendritic Cells to Infection with Different Enteroviruses

Coxsackie B viruses (CVBs) and echoviruses (EVs) form the Human Enterovirus-B (HEV-B) species within the family Picornaviridae. HEV-B infections are widespread and generally cause mild disease; however, severe infections occur and HEV-B are associated with various chronic diseases such as cardiomyopathy and type 1 diabetes. Dendritic cells (DCs) are the professional antigen-presenting cells of our immune system and initiate and control immune responses to invading pathogens, yet also maintain tolerance to self-antigens. We previously reported that EVs, but not CVBs, can productively infect in vitro generated monocyte-derived DCs. The interactions between HEV-B and human myeloid DCs (mDCs) freshly isolated from blood, however, remain unknown. Here, we studied the susceptibility and responses of BDCA1⁺ mDC to HEV-B species and found that these mDC are susceptible to EV, but not CVB infection. Productive EV7 infection resulted in massive, rapid cell death without DC activation. Contrary, EV1 infection, which resulted in lower virus input at the same MOI, resulted in DC activation as observed by production of type I interferon-stimulated genes (ISGs), upregulation of co-stimulatory and co-inhibitory molecules (CD80, CD86, PDL1) and production of IL-6 and TNF-α, with a relative moderate decrease in cell viability. EV1-induced ISG expression depended on virus replication. CVB infection did not affect DC viability and resulted in poor induction of ISGs and CD80 induction in part of the donors. These data show for the first time the interaction between HEV-B species and BDCA1⁺ mDCs isolated freshly from blood. Our data indicate that different HEV-B species can influence DC homeostasis in various ways, possibly contributing to HEV-B associated pathology.     

CD40 stimulation induces differentiation of acute lymphoblastic leukemia cells into dendritic cells

Despite the very high percentage of long-term remissions in acute lymphoblastic leukemia (ALL) in children, some of them suffer from recurrence of the disease. New treatment modalities, e.g. effective geno- and immunotherapy are needed. The use of neoplasmatic cells to present tumor antigens is one of the approaches in cancer vaccines. ALL cells lack the expression of costimulatory molecules and are poor antigen presenting cells (APCs) for T-cell activation. CD40/40L interaction stimulates B-cells to proliferate, differentiate, upregulate costimulatory molecules and increase antigen presentation. The aim of the study was to test the hypothesis that ALL cells can be turned into professional APCs by CD40L activation. Children with B-cell precursor ALL were enrolled into the study. Mononuclear cells from bone marrow or peripheral blood were stimulated with CD40L and interleukin 4. Results: 1) after culture we noted upregulation of all assessed costimulatory, adhesion and activatory molecules i.e. CD1a, CD11c, CD40, CD54, CD80, CD83, CD86, CD123, HLA class I and II; 2) CD40L activated ALL cells induced proliferation of allogeneic T-cells (measured by [(3)H]thymidine incorporation). These results confirm the possibility of enhancing the immunogenicity of ALL cells with the CD40L system and indicate that this approach can be used in immunotherapeutic trials.