Cutting edge: priming of CTL by transcutaneous peptide immunization with imiquimod

CTL are important in combating cancer and viruses. Therefore, triggering the complete potential of CTL effector functions by new vaccination strategies will not only improve prophylaxis of tumor or virus-related diseases, but also open opportunities for effective therapeutic immunizations. Using transcutaneous immunization, we show that epicutaneous (e.c.)(4) application of an ointment containing a CTL epitope and the TLR7 ligand imiquimod is highly effective in activating T cells in mice using TCR-transgenic CTL or in wild-type mice. Transcutaneous immunization-activated CTL mount a full-blown immune response against the target epitope characterized by proliferation, cytolytic activity, and the production of IFN-gamma that is completely restricted to the epitope used for vaccination. Our results obtained by simple e.c. application of an ointment, without further skin irritating procedures, provide the basis for the development of new, easy to use vaccines against cancer or virus-associated diseases.     

Cutting edge: cross-presentation as a mechanism for efficient recruitment of tumor-specific CTL to the brain

The number and localization of effector cells to the tumor site are crucial elements for immune rejection of solid tumors. However, for cerebral malignancies, antitumor responses need to be finely tuned to avoid neuropathologic consequences. In this study, we determine factors that regulate CTL localization and tumoricidal function after intracerebral implantation of tumors expressing model Ag. H-2(bxd) mice implanted with a CW3(+) murine glioma lacking H-2K(d) molecules necessary to present the CW3(170-179) epitope demonstrate cross-priming of H-2K(d)-restricted CTL, and moreover, Ag-dependent accumulation of functional H-2K(d)/CW3(170-179)-specific CTL within the tumor bed. This implicates a role for cross-presentation not only in priming, but also in retention of fully differentiated CTL in the tumor stroma at the effector stage of the response. Modulating cross-presentation of Ag may be the key in regulating specific immune responses in the brain: either by augmenting protective responses or by down-modulating destructive autoimmune reactions.     

Cutting edge: biasing immune responses by directing antigen to macrophage Fc gamma receptors

An immune response can deviate toward either a Th1- or Th2-like response. In this work we examine the contribution that activated macrophages and IgG Abs make toward this deviation. The use of activated macrophages as APCs resulted in a strong polarized T cell response that was predominated by IFN-gamma. However, when Ag was targeted to FcgammaRs on these macrophages, the T cell response was reversed and biased toward a Th2-like response. This Th2-like phenotype was stable and was retained when the T cells were subsequently restimulated under nonbiasing conditions. The T cell biasing and its reversal via FcgammaR was also observed in vivo. Mice vaccinated with IgG-opsonized OVA made high levels of IgG Ab of the IgG1 isotype. These studies demonstrate that the ligation of FcgammaR on activated macrophages can reverse the Th1 biasing that occurs as a result of innate immune responses to microbial products.     

Cutting edge: a crucial role for B7-CD28 in transmitting T help from APC to CTL

Although APC activation via CD40-CD40L signaling plays a critical role in enabling CD4(+) T cells to provide the "help" necessary for cross-priming of naive CTL, it is unclear how this makes the APC competent for priming. We have investigated the roles of B7-1/B7-2 and their receptors [corrected] CD28/CTLA-4 in cross-priming of CD4-dependent CTL in vivo. We find that both CD28 and B7-1/B7-2 are required for CD40-activated APC to cross-prime CTL, and that priming by CD40-activated APC was prevented by blockade of CD28. Conversely, augmenting CD28 signals with an agonistic Ab bypassed the requirement for CD4(+) T help or CD40 activation. Interestingly, blockade of the negative regulatory B7 receptor CTLA-4 failed to prime CTL in the absence of T help. These results support a model in which activation-induced up-regulation of B7 molecules on APC leads to increased CD28 signaling and a commitment to cross-priming of CD4-dependent CTL.     

Cutting edge: Dendritic cells prime a high avidity CTL response independent of the level of presented antigen

CTL that possess a high functional avidity are known to be optimal for the clearance of pathogens in vivo. We have shown that the amount of peptide encountered by a CD8+ CTL determines its functional avidity. Notably, in these studies nonprofessional APC were used. However, it is mature dendritic cells (DC) that are predominantly responsible for the activation of naive T cells in vivo. Whether DC also direct dose dependent-differences in avidity is unknown. In this work we examined the ability of mature DC presenting a high vs low level of peptide to generate CTL of distinct avidities. In contrast to what was observed with nonprofessional APC, CTL generated by stimulation with mature DC were of high avidity regardless of the amount of peptide presented. This DC property may promote generation of highly effective CTL that retain plasticity, which would allow the tuning of avidity in the periphery to promote optimal pathogen recognition and clearance.     

Cutting edge: IL-18-transgenic mice: in vivo evidence of a broad role for IL-18 in modulating immune function

IL-18 has been shown to be a strong cofactor for Th1 T cell development. However, we previously demonstrated that when IL-18 was combined with IL-2, there was a synergistic induction of a Th2 cytokine, IL-13, in both T and NK cells. More recently, we and other groups have reported that IL-18 can potentially induce IgE, IgG1, and Th2 cytokine production in murine experimental models. Here, we report on the generation of IL-18-transgenic (Tg) mice in which mature mouse IL-18 cDNA was expressed. CD8+CD44high T cells and macrophages were increased, but B cells were decreased in these mice while serum IgE, IgG1, IL-4, and IFN-gamma levels were significantly increased. Splenic T cells in IL-18 Tg mice produced higher levels of IFN-gamma, IL-4, IL-5, and IL-13 than control wild-type mice. Thus, aberrant expression of IL-18 in vivo results in the increased production of both Th1 and Th2 cytokines.     

Cutting edge: differentiation of antitumor CTL in vivo requires host expression of Stat1.

Several lines of evidence suggest that an IFN-gamma-producing, Th1/Tc1 phenotype may be optimal for tumor rejection. Recent work has indicated that IFN signaling on tumor cells is important for protection against carcinogenesis. However, the potential involvement of IFN signaling among host immune cells has not been carefully examined. To this end, Stat1-deficient mice were employed as tumor recipients. In contrast to wild-type mice, Stat1-/- mice failed to reject immunogenic tumors and did not support regression of poorly immunogenic tumors when treated with an IL-12-based vaccine. T cells from immunized Stat1-/- mice produced 50% of the levels of IFN-gamma and lacked cytolytic activity compared with wild-type mice, and NK lytic activity also was not observed. Lack of cytolytic function correlated with a failure to up-regulate serine esterase activity. Thus, IFN-mediated signaling on host cells is required for the development of antitumor lytic effector cells.     

Cutting edge: role of Toll-like receptor 1 in mediating immune response to microbial lipoproteins

The Toll-like receptor (TLR) family acts as pattern recognition receptors for pathogen-specific molecular patterns (PAMPs). TLR2 is essential for the signaling of a variety of PAMPs, including bacterial lipoprotein/lipopeptides, peptidoglycan, and GPI anchors. TLR6 associates with TLR2 and recognizes diacylated mycoplasmal lipopeptide along with TLR2. We report here that TLR1 associates with TLR2 and recognizes the native mycobacterial 19-kDa lipoprotein along with TLR2. Macrophages from TLR1-deficient (TLR1(-/-)) mice showed impaired proinflammatory cytokine production in response to the 19-kDa lipoprotein and a synthetic triacylated lipopeptide. In contrast, TLR1(-/-) cells responded normally to diacylated lipopeptide. TLR1 interacts with TLR2 and coexpression of TLR1 and TLR2 enhanced the NF-kappaB activation in response to a synthetic lipopeptide. Furthermore, lipoprotein analogs whose acylation was modified were preferentially recognized by TLR1. Taken together, TLR1 interacts with TLR2 to recognize the lipid configuration of the native mycobacterial lipoprotein as well as several triacylated lipopeptides.     

Cutting edge: rapid in vivo CTL activity by polyoma virus-specific effector and memory CD8+ T cells

For viruses that establish persistent infection, continuous immunosurveillance by effector-competent antiviral CD8(+) T cells is likely essential for limiting viral replication. Although it is well documented that virus-specific memory CD8(+) T cells synthesize cytokines after short term in vitro stimulation, there is limited evidence that these T cells exhibit cytotoxicity, the dominant antiviral effector function. Here, we show that antiviral CD8(+) T cells in mice acutely infected by polyoma virus, a persistent mouse pathogen, specifically eliminate viral peptide-pulsed donor spleen cells within minutes after adoptive transfer and do so via a perforin-dependent mechanism. Antiviral memory CD8(+) T cells were similarly capable of rapidly mobilizing potent Ag-specific cytotoxic activity in vivo. These findings strongly support the concept that a cytotoxic effector-memory CD8(+) T cell population operates in vivo to control this persistent viral infection.     

Cutting edge: conventional CD8 alpha+ dendritic cells are generally involved in priming CTL immunity to viruses

Dendritic cells (DCs) play a central role in initiating immune responses. Despite this, there is little understanding how different DC subsets contribute to immunity to different pathogens. CD8alpha(+) DC have been shown to prime immunity to HSV. Whether this very limited capacity of a single DC subset priming CTL immunity is restricted to HSV infection or is a more general property of anti-viral immunity was examined. Here, we show that the CD8alpha(+) DCs are the principal DC subset that initiates CTL immunity to s.c. infection by influenza virus, HSV, and vaccinia virus. This same subset also dominated immunity after i.v. infection with all three viruses, suggesting a similar involvement in other routes of infection. These data highlight the general role played by CD8alpha(+) DCs in CTL priming to viral infection and raises the possibility that this DC subset is specialized for viral immunity.     

Effect of cytokine-encoding plasmid delivery on immune response to Japanese encephalitis virus DNA vaccine in mice

We have previously shown that immunization of mice with plasmid pMEa synthesizing Japanese encephalitis virus (JEV) envelope protein induced anti-JEV humoral and cellular immune responses. We now show that intra-muscular co-administration of mice with pMEa and pGM-CSF, encoding murine granulocyte-macrophage colony-stimulating factor or pIL-2, encoding murine interleukin-2 given 4 days after pMEa, augmented anti-JEV antibody titers. This did not enhance the level of protection in immunized mice against JEV. However, intra-dermal co-administration of pMEa and pGM-CSF in mice using the gene gun, enhanced anti-JEV antibody titers resulting in an increased level of protection in mice against lethal JEV challenge.     

Cutting edge: critical role of intracellular osteopontin in antifungal innate immune responses

We found that absence of osteopontin (OPN) in immunocompromised Rag2(-/-) mice, which lack T and B cells, made the mice extremely susceptible to an opportunistic fungus Pneumocystis, although immunocompetent OPN-deficient mice could clear Pneumocystis as well as wild-type mice. OPN has been studied as an extracellular protein, and the role of an intracellular isoform of OPN (iOPN) is still largely unknown. In this study, we elucidated the mechanism by which iOPN was involved in antifungal innate immunity. First, iOPN was essential for cluster formation of fungal receptors that detect Pneumocystis, including dectin-1, TLR2, and mannose receptor. Second, iOPN played a role as an adaptor molecule in TLR2 and dectin-1 signaling pathways and mediated ERK activation and cytokine production by zymosan, which simultaneously activates TLR2 and dectin-1 pathways. Third, iOPN enhanced phagocytosis and clearance of Pneumocystis. Our study suggests the critical involvement of iOPN in antifungal innate immunity.     

Cutting edge: enhanced pulmonary clearance of Pseudomonas aeruginosa by Muc1 knockout mice

MUC1 (MUC1 in human and Muc1 in nonhumans) is a membrane-tethered mucin that interacts with Pseudomonas aeruginosa (PA) through flagellin. In this study, we compared PA pulmonary clearance and proinflammatory responses by Muc1(-/-) mice with Muc1(+/+) littermates following intranasal instillation of PA or flagellin. Compared with Muc1(+/+) mice, Muc1(-/-) mice showed increased PA clearance, greater airway recruitment of neutrophils, higher levels of TNF-alpha and KC in bronchoalveolar lavage fluid, higher levels of TNF-alpha in media of flagellin-stimulated alveolar macrophages, and higher levels of KC in media of tracheal epithelial cells. Knockdown of MUC1 enhanced flagellin-induced IL-8 production by primary human bronchial epithelial cells. Expression of MUC1 in HEK293T cells attenuated TLR5-dependent IL-8 release in response to flagellin, which was completely ablated when its cytoplasmic tail was deleted. We conclude that MUC1/Muc1 suppresses pulmonary innate immunity and speculate its anti-inflammatory activity may play an important modulatory role during microbial infection.     

Cutting edge: perforin down-regulates CD4 and CD8 T cell-mediated immune responses to a transplanted organ

Perforin mediates target cell apoptosis by CTLs and NK cells. Although perforin expression correlates strongly with acute allograft rejection, perforin-deficient mice reject allografts with the same kinetics as wild-type recipients. In this study, we tested the hypothesis that while perforin is dispensable for acute rejection, it is essential for down-regulating the alloimmune response by inducing the apoptosis of host immune cells. Using a skin transplantation model, we found that perforin-deficient mice are resistant to the induction of allograft acceptance by agents that block T cell costimulation. Failure to induce allograft acceptance in these mice was observed irrespective of whether the alloimmune response was CD4 or CD8 T cell-mediated and could be attributed to defective apoptosis of activated CD4 and CD8 T cells. In contrast, perforin did not influence T cell proliferation. Therefore, perforin is an essential immunoregulatory molecule that may be required for the induction of transplantation tolerance.     

Cutting edge: activation of murine TLR8 by a combination of imidazoquinoline immune response modifiers and polyT oligodeoxynucleotides

Synthetic immune response modifiers (IRM) such as imidazoquinolines can selectively activate human TLR7 or TLR8. Although these endosomal TLRs are close relatives, TLR7-deficient mice are unresponsive to TLR8 agonist IRMs. Similarly, natural ssRNA cannot activate murine TLR8, leading to the belief that murine TLR8 is nonfunctional. In this study, we transfected HEK293 cells with murine TLR8 and NF-kappaB reporter constructs and stimulated them with combinations of IRM and oligodeoxynucleotides (ODNs). When stimulated with TLR7 or TLR8 agonists alone, no NF-kappaB response was observed. However, a combination of polyT ODN plus the TLR8 agonist activated NF-kappaB, whereas polyT ODN plus the TLR7 agonist did not activate. Primary mouse cells responded to the IRM/polyT ODN by secreting TNF. Cells from TLR7(-/-) and TLR9(-/-) mice responded to the IRM/polyT ODN combination, whereas MyD88(-/-) cells did not respond. In conclusion, this study demonstrates for the first time that mouse TLR8 is functional.     

The induction of protective immune response in mice vaccinated by recombinant avian adenovirus CELO expressing glycoprotein G of the rabies virus

The recombinant avian adenovirus CELO-gpRb expressing glycoprotein G of rabies virus (strain TS-80, ARRIW&M, Pokrov, Russia) was used for mice vaccination against rabies. Double intramuscular immunization by recombinant CELO-gpRb adenovirus in a dose 10(9) pfu per mouse caused the induction of virus neutralizing antibodies (VNA) synthesis in 78% of mice, while twice repeated intradermal injections of the recombinant adenovirus failed to induce the VNA production. The protection level in groups of vaccinated mice after intracerebral injection of CVS rabies virus in a dose of 100 MLD50 was equal to 45% at single intramuscular immunization and to 91% after twice repeated intramuscular immunization. The recombinant adenoviral vaccine against rabies, based on CELO viral genome, has a good perspective for domestic and wild animal vaccination, not only due to rather high protection level, but also because the production of adenoviral CELO vaccine in chicken embryos is of high technology and inexpensive.     

Enhancement of gene delivery to cancer cells by a retargeted adenovirus

The inefficiency of in vivo gene transfer using currently available vectors reflects a major hurdle in cancer gene therapy. Both viral and non-viral approaches that improve gene transfer efficiency have been described, but suffer from a number of limitations. Herein, a fiber-modified adenovirus, carrying the small peptide ligand on the capsid, was tested for the delivery of a transgene to cancer cells. The fiber-modified adenovirus was able to mediate the entry and expression of a beta-galactosidase into cancer cells with increased efficiency compared to the unmodified adenovirus. Particularly, the gene transfer efficiency was improved up to 5 times in OVCAR3 cells, an ovarian cancer cell line. Such transduction systems hold promise for delivering genes to transferrin receptor overexpressing cancer cells, and could be used for future cancer gene therapy.     

Aging of the murine immune system is reflected by declining ability to generate antibodies that promote elimination of Trypanosoma musculi

Trypanosoma musculi established extracellular infections in aged BC3F1 and C57BL/6 mice that were approximately 10 times greater and twice as long in duration as those in young adult mice. Elimination of T. musculi infections was found to be an antibody-dependent, cell-mediated process involving effector (presumably phagocytic) cells in the liver and, to a lesser extent, the spleen. A major difference between young and aged mice of the C57BL/6 strain was the deficiency in the ability of aged animals to generate antibodies of appropriate specificity and/or isotype in sufficient amount to promote trypanosome elimination. Indeed, at the time when infected young-adult mice began to produce antibodies that facilitated rapid trypanosome clearance (in young adult, but not in aged animals), the serum of aged mice was found to contain substances that inhibited parasite clearance. Overall, however, the development of antibodies of different isotypes, capable of reacting with intact trypanosomes, was about the same in young and aged animals. Hepatic and splenic effector cells of old mice were at least as efficient as those of young adults. The immunoblotting procedure was used to try to detect trypanosome Ag against which aged animals failed to generate antibodies of one or more isotypes. The complexity of the reactions of infected mouse serum antibodies with the spectrum of trypanosome Ag precluded a precise analysis. However, it was apparent that a delay in the appearance of antibodies of IgG2a and IgG2b isotypes, against Ag of relatively high m.w., was typical of aged, in comparison to young, mice. More exacting analyses, involving fractionated trypanosome extracts and mAb of varying isotypes, are underway to identify key trypanosome Ag that elicit antibodies of isotypes that can facilitate hepatic and splenic clearance of the parasites. This line of investigation will provide information that, in addition to its intrinsic interest, may be vital in judging the future impact of endemic parasitic infections on the emerging cohort of elderly persons in developing tropical nations.     

Cutting edge: a role for CD1 in the pathogenesis of lupus in NZB/NZW mice

Since anti-CD1 TCR transgenic T cells can activate syngeneic B cells via CD1 to secrete IgM and IgG and induce lupus in BALB/c mice, we studied the role of CD1 in the pathogenesis of lupus in NZB/NZW mice. Approximately 20% of B cells from the spleens of NZB/NZW mice expressed high levels of CD1 (CD1high B cells). The latter subset spontaneously produced large amounts of IgM anti-dsDNA Abs in vitro that was up to 25-fold higher than that of residual CD1int/low B cells. T cells in the NZB/NZW spleen proliferated vigorously to the CD1-transfected A20 B cell line, but not to the parent line. Treatment of NZB/NZW mice with anti-CD1 mAbs ameliorated the development of lupus. These results suggest that the CD1high B cells and their progeny are a major source of autoantibody production, and activation of B cells via CD1 may play an important role in the pathogenesis of lupus.     

Cutting edge: Immunity against a "silent" salivary antigen of the Lyme vector Ixodes scapularis impairs its ability to feed

Ixodes scapularis ticks transmit the Lyme disease agent in the United States. Although strong antitick immunity mediates tick rejection by certain vertebrates, only a few Ags have been molecularly characterized. We show that guinea pig vaccination against a secreted tick salivary immunomodulator, sialostatin L2, can lead to decreased feeding ability of I. scapularis nymphs. Increased rejection rate, prolonged feeding time, and apparent signs of inflammation were observed for nymphs attached to vaccinated animals, indicating a protective host immune response. Interestingly, sialostatin L2 humoral recognition does not take place upon repeated tick exposure in control animals, but only in the vaccinated animals that neutralize sialostatin L2 action. Therefore, we demonstrate an essential sialostatin L2 role upon nymphal infestation that can be blocked by vertebrate immunity and propose the discovery of similarly "silent" Ags toward the development of a multicomponent vaccine that will protect against tick bites and the pathogens they transmit.