Transduction with a fiber-modified adenoviral vector is superior to non-viral nucleofection for expressing tumor-associated Ag mucin-1 in human DC

BACKGROUND: DC-presenting tumor Ag are currently being developed to be used as a vaccine in human cancer immunotherapy. To increase the chances for successful therapy it is important to deliver full-length tumor Ag instead of loading single peptides. Methodologically, several recombinant DNA delivery techniques have been used. METHODS: In this study we compared nucleofection, an optimized form of electroporation, and adenoviral transduction regarding their efficiency to transduce human monocyte-derived (Mo-) DC in vitro. Expression of the tumor-associated Ag mucin-1 (MUC1) after adenoviral transduction (rAd5Fib35-MUC1) was determined using two MAb. RESULTS: We showed that the viability of cells and percentage of green fluorescent protein (GFP)-positive cells after transduction with a fiber-modified adenoviral vector (rAd5F35-GFP) was much higher than after nucleofection. Furthermore, phenotype and function of DC were not impaired by infection with adenovirus particles. Cells matured normally; up-regulation of CD40, CD80, CD83, CD86 and HLA-DR was not affected by adenoviral transduction. The capacity to stimulate naive T-cell proliferation was preserved and no change in IL-10 production was observed. Production of IL-12 increased up to 500-fold upon adenoviral transduction, considered to contribute positively to an anti-tumor immune response. Non-transduced mature DC expressed low levels of endogenous MUC1. After transduction with the rAd5F35-MUC1 adenoviral vector, a 100-fold increase in MUC1 expression by DC was observed. DISCUSSION: The use of the fiber-modified adenoviral vector presented here may therefore be favorable compared with non-viral gene delivery systems for DC that will be used in cancer immunotherapy.     

Intradermal delivery of adenoviral type-35 vectors leads to high efficiency transduction of mature, CD8+ T cell-stimulating skin-emigrated dendritic cells

Recombinant adenovirus (Ad) type 35 (rAd35) shows great promise as vaccine carrier with the advantage of low pre-existing immunity in human populations, in contrast to the more commonly used rAd5 vector. The rAd35 vector uses CD46 as a high-affinity receptor, which, unlike the rAd5 receptor, is expressed on human dendritic cells (DC), the most powerful APCs identified to date. In this study, we show that in contrast to rAd5, rAd35 infects migrated and mature CD83+ cutaneous DC with high efficiency (up to 80%), when delivered intradermally in an established human skin explant model. The high transduction efficiency is in line with high expression levels of CD46 detected on migratory cutaneous DC, which proved to be further increased upon intradermal administration of GM-CSF and IL-4. As compared with Ad5, these Ad35 infection characteristics translate into higher absolute numbers of skin-emigrated DC per explant that both express the transgene and are phenotypically mature. Finally, we demonstrate that upon intracutaneous delivery of a rAd35 vaccine encoding the circumsporozoite (CS) protein of Plasmodium falciparum, emigrated DC functionally express and process CS-derived epitopes and are capable of activating specific CD8+ effector T cells, as evidenced by activation of an HLA-A2-restricted CS-specific CD8+ T cell clone. Collectively, these data demonstrate the utility of rAd35 vectors for efficient in vivo human DC transduction.     

Differential regulation of antigen-specific CD8+ T cell responses by IL-12p40 in a dose-dependent manner

IL-12p40 is a natural antagonist which inhibits IL-12- and IL-23-mediated biological activity by blocking the binding of IL-12/23 to their receptors. Recently, IL-12p40 was also shown to have immune-enhancing activity through the activation of macrophages or dendritic cells. In this study, we investigated the effects of IL-12p40 as a genetic adjuvant on immune modulation using recombinant adenoviruses expressing IL-12p40 (rAd/IL-12p40) and OVA (rAd/OVA). Coimmunization of rAd/IL-12p40 at a low dose (1 x 10(4) PFU) with rAd/OVA resulted in OVA-specific immune enhancement, while a high dose of rAd/IL-12p40 (1 x 10(8) PFU) caused significant suppression of CD8(+) T cell responses. In addition, the enhancement and suppression of OVA-specific CD8(+) T cell responses correlated with antitumor activity against E.G7-OVA tumor challenge, which subsequently affected the survival rate. Moreover, the differential CD8(+) T cell response by IL-12p40 was still observed in IL-12Rbeta2 knockout (IL-12Rbeta2KO), but not in IL-12Rbeta1 knockout (IL-12Rbeta1KO) mice, indicating that IL-12p40 is a cytokine which can modulate Ag-specific T cell responses depending on IL-12Rbeta1. Our findings provide a novel insight on the physiological role of IL-12p40, which can be informative in the design of vaccine strategies and therapeutic regimens.     

CD154 plays a central role in regulating dendritic cell activation during infections that induce Th1 or Th2 responses

We compared splenic DC activation during infection with either the Th2 response-inducing parasite Schistosoma mansoni or with the Th1 response-inducing parasite Toxoplasma gondii. CD8alpha(+) DC from schistosome-infected mice exhibited a 2- to 3-fold increase in the expression of MHC class II, CD80, and CD40 (but not CD86) compared with DC from uninfected control animals, while CD8alpha(-) DC exhibited a 2- to 3-fold increase in the expression of MHC class II and CD80 and no alteration, compared with DC from uninfected mice, in the expression of CD86 or CD40. Intracellular staining revealed that DC did not produce IL-12 during infection with S. mansoni. In contrast, infection with T. gondii resulted in a more pronounced increase in the expression of activation-associated molecules (MHC class II, CD80, CD86, and CD40) on both CD8alpha(-) and CD8alpha(+) splenic DC and promoted elevated IL-12 production by DC. Analysis of MHC class I and of additional costimulatory molecules (ICOSL, ICAM-1, OX40L, 4-1BBL, and B7-DC) revealed a generally similar pattern, with greater indication of activation in T. gondii-infected mice compared with S. mansoni-infected animals. Strikingly, the activation of DC observed during infection with either parasite was not apparent in DC from infected CD154(-/-) mice, indicating that CD40/CD154 interactions are essential for maintaining DC activation during infection regardless of whether the outcome is a Th1 or a Th2 response. However, the ability of this activation pathway to induce IL-12 production by DC is restrained in S. mansoni-infected, but not T. gondii-infected, mice by Ag-responsive CD11c(-) cells.     

Immature monocyte-derived dendritic cells are productively infected with herpes simplex virus type 1

Herpes simplex viruses (HSV) have developed several immunoevasive strategies. Here we demonstrate a novel mechanism by which HSV type 1 may interfere with the immune response through infection of immature dendritic cells (DC) and selective downmodulation of costimulatory molecules. In our study we show productive infection of immature monocyte-derived DC, which closely resemble sessile Langerhans cells, by sequential expression of immediate-early, early, and late viral proteins and of glycoprotein D mRNA, as well as production of infectious virus of moderate titers. Infection was cytopathic, with the progressive loss of 20 to 45% of cells from 24 to 48 h after infection, with no more than 80% of DC found to be infected. These results are in contrast to those of previous findings of nonpermissive or abortive infection of monocytes and mature monocyte-derived DC. Infection of immature DC also led to selective and asynchronous downregulation of CD1a, CD40, CD54 (ICAM-1) (12 h postinfection), CD80 (24 h postinfection), and CD86 (48 h postinfection) but not of CD11c or major histocompatibility complex class I and II molecules when compared to DC exposed to UV-inactivated virus. Thus, we propose that productive infection of epidermal Langerhans cells in vivo may lead to delayed activation of T cells, allowing more time for replication of HSV type 1 in epidermal cells.     

IL-1 beta enhances CD40 ligand-mediated cytokine secretion by human dendritic cells (DC): a mechanism for T cell-independent DC activation.

CD40 ligand (CD40L) is a membrane-bound molecule expressed by activated T cells. CD40L potently induces dendritic cell (DC) maturation and IL-12p70 secretion and plays a critical role during T cell priming in the lymph nodes. IFN-gamma and IL-4 are required for CD40L-mediated cytokine secretion, suggesting that T cells are required for optimal CD40L activity. Because CD40L is rapidly up-regulated by non-T cells during inflammation, CD40 stimulation may also be important at the primary infection site. However, a role for T cells at the earliest stages of infection is unclear. The present study demonstrates that the innate immune cell-derived cytokine, IL-1beta, can increase CD40L-induced cytokine secretion by monocyte-derived DC, CD34(+)-derived DC, and peripheral blood DC independently of T cell-derived cytokines. Furthermore, IL-1beta is constitutively produced by monocyte-derived DC and monocytes, and is increased in response to intact Escherichia coli or CD40L, whereas neither CD34(+)-derived DC nor peripheral blood DC produce IL-1beta. Finally, DC activated with CD40L and IL-1beta induce higher levels of IFN-gamma secretion by T cells compared with DC activated with CD40L alone. Therefore, IL-1beta is the first non-T cell-derived cytokine identified that enhances CD40L-mediated activation of DC. The synergy between CD40L and IL-1beta highlights a potent, T cell-independent mechanism for DC activation during the earliest stages of inflammatory responses.     

Adoptive transfer of CD8alpha+ dendritic cells (DC) isolated from mice infected with Chlamydia muridarum are more potent in inducing protective immunity than CD8alpha- DC

Chlamydial infections are serious public health concerns worldwide. In this study, we examined the role of dendritic cell (DC) subsets in inducing protective immunity against chlamydial infection using an adoptive transfer approach. We found that CD11c+CD8alpha+ (double-positive, DP) DC, compared with CD11c+CD8alpha- (single-positive, SP) DC isolated from infected mice, are more potent inducers of protective immunity. Specifically, mice pretreated with DPDC from infected mice, upon infection with Chlamydia trachomatis mouse pneumonitis (MoPn), experienced significantly less severe body weight loss and in vivo chlamydial growth. Analysis of MoPn-driven cytokine production by immune cells revealed that mice that were treated with DPDC produced significantly higher levels of Th1 (TNF-alpha, IFN-gamma, and IL-12) but lower levels of Th2 (IL-4, IL-5, and IL-13)-related cytokines than the recipients of SPDC following infection challenge. Moreover, DPDC-treated mice displayed significantly higher levels of MoPn-specific IgG2a production and delayed-type hypersensitivity responses compared with SPDC-treated mice. Furthermore, DPDC isolated from infected mice produced higher amounts of IL-12 and IL-10 in vitro in comparison with SPDC. These data indicate that CD8alpha+ DC have a significantly higher capacity in inducing protective immunity compared with CD8alpha- DC, demonstrating the crucial role of DC1-like cells in eliciting protection against C. trachomatis infection.     

Human dendritic cells discriminate between viable and killed Toxoplasma gondii tachyzoites: dendritic cell activation after infection with viable parasites results in CD28 and CD40 ligand signaling that controls IL-12-dependent and -independent T cell production of IFN-gamma

We studied how the interaction between human dendritic cells (DC) and Toxoplasma gondii influences the generation of cell-mediated immunity against the parasite. We demonstrate that viable, but not killed, tachyzoites of T. gondii altered the phenotype of immature DC. DC infected with viable parasites up-regulated the expression of CD40, CD80, CD86, and HLA-DR and down-regulated expression of CD115. These changes are indicative of DC activation induced by T. gondii. Viable and killed tachyzoites had contrasting effects on cytokine production. DC infected with viable T. gondii rather than DC that phagocytosed killed parasites induced secretion of high amounts of IFN-gamma by T cells from T. gondii-seronegative donors. IFN-gamma production in response to DC infected with viable parasites required CD28 and CD40 ligand (CD40L) signaling. In addition, this IFN-gamma response was dependent in part on IL-12 secretion. Production of IL-12 p70 occurred after interaction between T cells and DC infected with viable T. gondii, but not after incubation of T cells with DC plus killed tachyzoites. IL-12 synthesis was inhibited by blockade of CD40L signaling. IL-12-independent IFN-gamma production required CD80/CD86-CD28 interaction and, to a lesser extent, CD40-CD40L signaling. Taken together, T. gondii-induced activation of human DC is associated with T cell production of IFN-gamma through CD40-CD40L-dependent release of IL-12 and through CD80/CD86-CD28 and CD40-CD40L signaling that mediate IFN-gamma secretion even in the absence of bioactive IL-12.     

Lipopolysaccharide modulation of dendritic cells is insufficient to mature dendritic cells to generate CTLs from naive polyclonal CD8+ T cells in vitro, whereas CD40 ligation is essential.

Many cytotoxic CD8+ T cell responses are dependent on the interactions between CD40 ligand on the helper CD4+ T cell and CD40 on the APC. Although CD40 triggering of dendritic cells (DC) has been shown to mature the DC by increasing the level of expression of costimulatory molecules and inducing IL-12 secretion, the precise mechanisms by which CD40-CD40 ligand interactions allow DC to drive CTL responses remain unknown. We have used an in vitro model in which naive polyclonal CD8+ T cells can be activated by bone marrow-derived DC to investigate factor(s) that are responsible for this CD40-dependent generation of CTLs. DC modulated with agonistic anti-CD40 mAb (aCD40) are able to generate Ag-specific CTL responses while DC modulated with the microbial stimulus LPS alone do not. We compared the Ag-presenting capacity, levels of costimulatory molecules, and release of cytokines and chemokines of DC modulated with aCD40 to that of DC modulated by LPS. None of the factors assayed account for the unique capacity of anti-CD40-matured DC to drive CTL but this model provides a simplified system for further investigation. Although we attempted to use an LPS-free system for these studies, we are unable to rule out the possibility that very low levels of endotoxin (<20 pg/ml) may synergize with CD40 ligation in the generation of CTLs.     

IFN-beta differentially regulates CD40-induced cytokine secretion by human dendritic cells

We have previously shown that IFN-beta, a key cytokine associated with the early phase of the innate host defense, can prevent the generation of human Th1 cells. Specifically, we demonstrated that IFN-beta prevents the in vitro monocyte-derived mature dendritic cell (DC)-dependent differentiation of naive Th cells into IFN-gamma-secreting Th cells, as a result of its ability to inhibit DC IL-12 secretion. The goal of the present study was to identify how IFN-beta negatively regulates IL-12 secretion by DC. We report that in our Th cell differentiation model, DC IL-12 secretion is dependent on the CD40L/CD40 accessory pathway, and, utilizing a Th cell-free system, we find that IFN-beta inhibits anti-CD40 mAb-induced DC secretion of the p40 chain of the IL-12 heterodimer. In addition, we show that IFN-beta-mediated inhibition of CD40 signaling does not interfere with all signaling pathways emanating from CD40, since anti-CD40 mAb-induced DC IL-6 secretion is augmented by IFN-beta. Thus, our results demonstrate that signaling from CD40 is differentially regulated by IFN-beta. A second critical element of innate immunity involves the response against components of bacterial membranes such as LPS. DC respond to LPS by secreting IL-6 and IL-12. In contrast to CD40-dependent IL-6 and IL-12 secretion, we find that LPS-induced DC secretion of p40 IL-12 and IL-6 is not affected by IFN-beta. Our findings show that IFN-beta influences the generation of acquired immune responses through its regulation of CD40-dependent DC functions.     

CpG-C immunostimulatory oligodeoxyribonucleotide activation of plasmacytoid dendritic cells in rhesus macaques to augment the activation of IFN-gamma-secreting simian immunodeficiency virus-specific T cells

There are two principle subsets of dendritic cells (DCs); CD11c(+)CD123(-) myeloid DCs (MDCs) and CD11c(-)CD123(+) plasmacytoid DCs (PDCs). DC activation via TNF-TNFRs (e.g., CD40L) and TLRs (e.g., immunostimulatory oligodeoxyribonucleotides (ISS-ODNs)) is crucial for maximal stimulation of innate and adaptive immunity. Macaque DC biology is being studied to improve HIV vaccines using the SIV macaque model. Using lineage (Lin) markers to exclude non-DCs, Lin(-)HLA-DR(+)CD11c(+)CD123(-) MDCs and Lin(-)HLA-DR(+)CD11c(-)CD123(+) PDCs were identified in the blood of uninfected macaques and healthy macaques infected with SIV or simian-human immunodeficiency virus. Overnight culture of DC-enriched Lin-depleted cells increased CD80 and CD86 expression. IL-12 production and CD80/CD86 expression by MDC/PDC mixtures was further enhanced by CD40L and ISS-ODN treatment. A CpG-B ISS-ODN increased CD80/CD86 expression by PDCs, but resulted in little IFN-alpha secretion unless IL-3 was added. In contrast, a CpG-C ISS-ODN and aldrithiol-2-inactivated (AT-2) SIV induced considerable PDC activation and IFN-alpha release without needing exogenous IL-3. The CpG-C ISS-ODN also stimulated IL-12 release (unlike AT-2 SIV) and augmented DC immunostimulatory activity, increasing SIV-specific T cell IFN-gamma production induced by AT-2 SIV-presenting MDC/PDC-enriched mixtures. These data highlight the functional capacities of MDCs and PDCs in naive as well as healthy, infected macaques, revealing a promising CpG-C ISS-ODN-driven DC activation strategy that boosts immune function to augment preventative and therapeutic vaccine efficacy.     

Myeloid and plasmacytoid dendritic cells are susceptible to recombinant adenovirus vectors and stimulate polyfunctional memory T cell responses

Although replication-incompetent recombinant adenovirus (rAd) type 5 is a potent vaccine vector for stimulating T and B cell responses, high seroprevalence of adenovirus type 5 (Ad5) within human populations may limit its clinical utility. Therefore, alternative adenovirus serotypes have been studied as vaccine vectors. In this study, we characterized the ability of rAd5 and rAd35 to infect and induce maturation of human CD11c(+) myeloid dendritic cells (MDCs) and CD123(+) plasmacytoid dendritic cells (PDCs), and their ability to stimulate Ag-specific T cells. Both MDCs and PDCs were found to express the primary receptor for Ad35 (CD46) but not Ad5 (coxsackie-adenovirus receptor; CAR). Both dendritic cell (DC) subsets were also more susceptible to rAd35 than to rAd5. MDCs were more susceptible to both rAd35 and rAd5 than were PDCs. Whereas rAd35 used CD46 for entry into DCs, entry of rAd5 may be through a CAR-independent pathway. Exposure to rAd35 but not rAd5 induced high levels of IFN-alpha in PDCs and phenotypic differentiation in both DC subsets. MDCs and PDCs exposed to either rAd5 or rAd35 encoding for CMV pp65 were able to present pp65 and activate CMV-specific memory CD8(+) and CD4(+) T cells in a dose-dependent manner, but MDCs stimulated the highest frequencies of pp65-specific T cells. Responding T cells expressed multiple functions including degranulation (CD107a surface mobilization) and production of IFN-gamma, IL-2, TNF-alpha, and MIP-1beta. Thus, the ability of rAd35 to naturally target important DC subsets, induce their maturation, and appropriately present Ag to T cells may herald greater in vivo immunogenicity than has been observed with rAd5.     

Final maturation of dendritic cells is associated with impaired responsiveness to IFN-gamma and to bacterial IL-12 inducers: decreased ability of mature dendritic cells to produce IL-12 during the interaction with Th cells

Activation of immature CD83- dendritic cells (DC) in peripheral tissues induces their maturation and migration to lymph nodes. Activated DC become potent stimulators of Th cells and efficient inducers of Th1- and Th2-type cytokine production. This study analyzes the ability of human monocyte-derived CD1a+ DC at different stages of IL-1 beta and TNF-alpha-induced maturation to produce the major Th1-driving factor IL-12. DC at the early stages of maturation (2 and 4 h) produced elevated amounts of IL-12 p70 during interaction with CD40 ligand-bearing Th cells or, after stimulation with the T cell-replacing factors, soluble CD40 ligand and IFN-gamma. The ability to produce IL-12 was strongly down-regulated at later time points, 12 h after the induction of DC maturation, and in fully mature CD83+ cells, at 48 h. In contrast, the ability of mature DC to produce IL-6 was preserved or even enhanced, indicating their intact responsiveness to CD40 triggering. A reduced IL-12-producing capacity of mature DC resulted mainly from their impaired responsiveness to IFN-gamma, a cofactor in CD40-induced IL-12 p70 production. This correlated with reduced expression of IFN-gamma R (CD119) by mature DC. In addition, while immature DC produced IL-12 and IL-6 after stimulation with LPS or Staphylococcus aureus Cowan I strain, mature DC became unresponsive to these bacterial stimuli. Together with the previously described ability of IL-10 and PGE2 to stably down-regulate the ability to produce IL-12 in maturing, but not in fully mature, DC, the current data indicate a general resistance of mature DC to IL-12-modulating factors.     

IL-10- and IL-12-independent down-regulation of allergic sensitization by stimulation of CD40 signaling

Interaction between CD154 (CD40 ligand) on activated T lymphocytes and its receptor CD40 has been shown to be critically involved in the generation of cell-mediated as well as humoral immunity. CD40 triggering activates dendritic cells (DC), enhances their cytokine production, up-regulates the expression of costimulatory molecules, and induces their maturation. It is unknown how stimulation of CD40 during sensitization to an airborne allergen may affect the outcome of allergic airway inflammation. We took advantage of a mouse model of allergic asthma and a stimulatory mAb to CD40 (FGK45) to study the effects of CD40-mediated DC activation on sensitization to OVA and subsequent development of OVA-induced airway inflammation. Agonistic anti-CD40 mAb (FGK45) injected during sensitization with OVA abrogated the development of allergic airway inflammation upon repeated airway challenges with OVA. Inhibition of bronchial eosinophilia corresponded with reduced Th2 cytokine production and was independent of IL-12, as evidenced by a similar down-regulatory effect of anti-CD40 mAb in IL-12 p40-deficient mice. In addition, FGK45 equally down-regulated allergic airway inflammation in IL-10-deficient mice, indicating an IL-10-independent mechanism of action of FGK45. In conclusion, our results show that CD40 signaling during sensitization shifts the immune response away from Th2 cytokine production and suppresses allergic airway inflammation in an IL-12- and IL-10-independent way, presumably resulting from enhanced DC activation during sensitization.     

Cytoplasmic entry of Listeria monocytogenes enhances dendritic cell maturation and T cell differentiation and function

Protective immunity to the intracellular bacterial pathogen, Listeria monocytogenes, is mediated by a vigorous T cell response. In particular, CD8(+) cytolytic T cells provide essential effector function in the clearance of bacterial infection. The cytoplasmic entry of Listeria facilitated by listeriolysin O is an essential feature not only of the bacteria's virulence, but of the ability of the bacteria to elicit protective immunity in the host. To determine how cytoplasmic entry of Listeria regulates the development of protective immunity, we examined the effects of this process on the maturation of murine dendritic cells (DC) and on their ability to prime naive CD8(+) T cell responses. Costimulatory molecules (CD40, CD80, and CD86) were induced by listerial infection only when the bacteria invaded the cytoplasm. In addition, the production of IL-12, IL-10, IL-6, and TNF-alpha was most efficiently triggered by cytosolic Listeria. Naive T cells primed by peptide-loaded DC infected with either wild-type or nonhemolytic mutant Listeria proliferated equivalently, but a much larger proportion of those primed by wild-type Listeria monocytogenes produced IFN-gamma. Costimulatory molecules induced by cytosolic entry regulated T cell proliferation and, as a result, the number of functional T cells generated. DC-produced cytokines (specifically IL-12 and IL-10) were the major factors determining the proportion of T cells producing IFN-gamma. These data highlight the requirement for listerial cytoplasmic invasion for the optimal priming of T cell cytokine production and attest to the importance of this event to the development of protective CTL responses to this pathogen.     

VCAM-1 and VLA-4 modulate dendritic cell IL-12p40 production in experimental visceral leishmaniasis

Vascular cell adhesion molecule-1 (VCAM-1) interacts with its major ligand very late antigen-4 (VLA-4) to mediate cell adhesion and transendothelial migration of leukocytes. We report an important role for VCAM-1/VLA-4 interactions in the generation of immune responses during experimental visceral leishmaniasis caused by Leishmania donovani. Our studies demonstrate that these molecules play no direct role in the recruitment of leukocytes to the infected liver, but instead contribute to IL-12p40-production by splenic CD8(+) dendritic cells (DC). Blockade of VCAM-1/VLA-4 interactions using whole antibody or anti-VCAM-1 Fab' fragments reduced IL-12p40 mRNA accumulation by splenic DC 5 hours after L. donovani infection. This was associated with reduced anti-parasitic CD4(+) T cell activation in the spleen and lowered hepatic IFNgamma, TNF and nitric oxide production by 14 days post infection. Importantly, these effects were associated with enhanced parasite growth in the liver in studies with either anti-VCAM-1 or anti-VLA-4 antibodies. These data indicate a role for VCAM-1 and VLA-4 in DC activation during infectious disease.     

The Glycosylated Rv1860 Protein of Mycobacterium tuberculosis Inhibits Dendritic Cell Mediated TH1 and TH17 Polarization of T Cells and Abrogates Protective Immunity Conferred by BCG

We previously reported interferon gamma secretion by human CD4⁺ and CD8⁺ T cells in response to recombinant E. coli-expressed Rv1860 protein of Mycobacterium tuberculosis (MTB) as well as protection of guinea pigs against a challenge with virulent MTB following prime-boost immunization with DNA vaccine and poxvirus expressing Rv1860. In contrast, a Statens Serum Institute Mycobacterium bovis BCG (BCG-SSI) recombinant expressing MTB Rv1860 (BCG-TB1860) showed loss of protective ability compared to the parent BCG strain expressing the control GFP protein (BCG-GFP). Since Rv1860 is a secreted mannosylated protein of MTB and BCG, we investigated the effect of BCG-TB1860 on innate immunity. Relative to BCG-GFP, BCG-TB1860 effected a significant near total reduction both in secretion of cytokines IL-2, IL-12p40, IL-12p70, TNF-α, IL-6 and IL-10, and up regulation of co-stimulatory molecules MHC-II, CD40, CD54, CD80 and CD86 by infected bone marrow derived dendritic cells (BMDC), while leaving secreted levels of TGF-β unchanged. These effects were mimicked by BCG-TB1860His which carried a 6-Histidine tag at the C-terminus of Rv1860, killed sonicated preparations of BCG-TB1860 and purified H37Rv-derived Rv1860 glycoprotein added to BCG-GFP, but not by E. coli-expressed recombinant Rv1860. Most importantly, BMDC exposed to BCG-TB1860 failed to polarize allogeneic as well as syngeneic T cells to secrete IFN-γ and IL-17 relative to BCG-GFP. Splenocytes from mice infected with BCG-SSI showed significantly less proliferation and secretion of IL-2, IFN-γ and IL-17, but secreted higher levels of IL-10 in response to in vitro restimulation with BCG-TB1860 compared to BCG-GFP. Spleens from mice infected with BCG-TB1860 also harboured significantly fewer DC expressing MHC-II, IL-12, IL-2 and TNF-α compared to mice infected with BCG-GFP. Glycoproteins of MTB, through their deleterious effects on DC may thus contribute to suppress the generation of a TH1- and TH17-dominated adaptive immune response that is vital for protection against tuberculosis.     

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.