CD8α+ DC are not the sole subset cross‐presenting cell‐associated tumor antigens from a solid tumor

One of the clear paradoxes in tumor immunology is the fact that cross‐presentation of cell‐associated tumor antigens to CD8⁺ T cells is efficient, yet CTL generation is weak, and tumors continue to grow. We examined, for the first time whether this may be due to alterations in the phenotype or function of cross‐presenting DC using a solid tumor model expressing a membrane bound neo‐antigen (hemagglutinin, HA). Tumor antigen was constitutively cross‐presented in the tumor‐draining LN throughout tumor progression by CD11c⁺ DC. Further analysis revealed that both CD8α⁺ and CD8α^? DC subsets, but not plasmacytoid DC, were effective at cross‐presenting HA tumor antigen. The proportions of DC subsets in the tumor‐draining LN were equivalent to those seen in the LN of naïve mice; however, a significant increase in the expression of the potential inhibitory B7 molecule, B7‐DC, was noted and appeared to be restricted to the CD8α^– DC subset. Therefore LN resident CD8α⁺ DC are not the sole DC subset capable of cross‐presenting cell‐associated tumor antigens. Migratory tumor DC subsets with altered co‐stimulatory receptor expression may contribute to induction and regulation of tumor‐specific responses.     

HIV gag protein is efficiently cross‐presented when targeted with an antibody towards the DEC‐205 receptor in Flt3 ligand‐mobilized murine DC

DC present exogenous proteins to MHC class I‐restricted CD8⁺ T cells. This function does not require endogenous antigen synthesis within DC, providing the potential to elicit CD8⁺ T‐cell responses to immune complexes, inactivated microbes, dying cells, and proteins such as OVA. In mice, the CD8⁺ or DEC‐205⁺ DC are specialized for cross‐presentation, and this subset can be increased 10‐fold in numbers following Fms‐like tyrosine kinase 3 ligand (Flt3L) treatment in vivo. Therefore, we studied cross‐presentation by abundant Flt3L DC using HIV gag protein. When enriched by positive selection with anti‐CD11c beads, cells from Flt3L mice are not only more abundant but are also more highly enriched in CD11c^high DC, particularly the DEC‐205⁺ subset. DC cross‐present HIV gag to primed CD8⁺ T cells, but when the antigen is delivered within an antibody to DEC‐205 receptor, cross‐presentation becomes 100‐fold more efficient than non‐targeted antigen. This finding requires gag to be engineered into anti‐DEC antibody, not just mixed with antibody. Flt3L DC are a valuable tool to study cross‐presentation, since their use overcomes the obstacle posed by the low number of cross‐presenting DC in the steady state. These findings support future experiments to use Flt3L to enhance presentation of DC‐targeted vaccines.     

The synthetic TLR2 agonist BPPcysMPEG leads to efficient cross‐priming against co‐administered and linked antigens

The property of DC to generate effective CTL responses is influenced by TLR signaling. TLR ligands contain molecular signatures associated with pathogens, have an impact on the antigen processing and presentation by DC, and are being exploited as potential adjuvants. We hypothesized that the TLR2/6 heterodimer agonist S‐[2,3‐bispalmitoyiloxy‐(2R)‐propyl]‐R‐cysteinyl‐amido‐monomethoxyl polyethylene glycol (BPP), a synthetic derivative of the Mycoplasma macrophage activating lipopeptide‐2, is a potent adjuvant for cross‐priming against cellular antigens. Systemic administration of BPP‐induced maturation of CD8α⁺ DC and CD8α^? DC in the spleen and resulted in enhanced cross‐presentation of intravenously co‐administered antigen in mice. In addition, administration of BPP and cell‐associated OVA generated an effective CTL response against OVA in vivo in a CD4⁺ T helper cell‐dependent manner, but independent of IFN‐α. Delivering antigenic peptides directly linked to BPP led to superior CTL immunity as compared to giving antigens and adjuvants admixed. In contrast to other TLR ligands, such as CpG, systemic activation of DC with BPP did not result in shut‐down of antigen presentation by splenic DC subsets, although cross‐priming against subsequently encountered antigens was reduced. Together, our data provide evidence that BPP is a potent stimulus to generate CTL via cross‐priming.     

IL‐10 promotes homeostatic proliferation of human CD8+ memory T cells and,when produced by CD1c+ DCs,shapes naive CD8+ T‐cell priming

IL‐10 is an anti‐inflammatory cytokine that inhibits maturation and cytokine production of dendritic cells (DCs). Although mature DCs have the unique capacity to prime CD8⁺ CTL, IL‐10 can promote CTL responses. To understand these paradoxic findings, we analyzed the role of IL‐10 produced by human APC subsets in T‐cell responses. IL‐10 production was restricted to CD1c⁺ DCs and CD14⁺ monocytes. Interestingly, it was differentially regulated, since R848 induced IL‐10 in DCs, but inhibited IL‐10 in monocytes. Autocrine IL‐10 had only a weak inhibitory effect on DC maturation, cytokine production, and CTL priming with high‐affinity peptides. Nevertheless, it completely blocked cross‐priming and priming with low‐affinity peptides of a self/tumor‐antigen. IL‐10 also inhibited CD1c⁺ DC‐induced CD4⁺ T‐cell priming and enhanced Foxp3 induction, but was insufficient to induce T‐cell IL‐10 production. CD1c⁺ DC‐derived IL‐10 had also no effect on DC‐induced secondary expansions of memory CTL. However, IL‐15‐driven, TCR‐independent proliferation of memory CTL was enhanced by IL‐10. We conclude that DC‐derived IL‐10 selects high‐affinity CTL upon priming. Moreover, IL‐10 preserves established CTL memory by enhancing IL‐15‐dependent homeostatic proliferation. These combined effects on CTL priming and memory maintenance provide a plausible mechanism how IL‐10 promotes CTL responses in humans.     

In vivo targeting of human DC‐SIGN drastically enhances CD8+ T‐cell‐mediated protective immunity

Vaccination is one of the oldest yet still most effective methods to prevent infectious diseases. However, eradication of intracellular pathogens and treatment of certain diseases like cancer requiring efficient cytotoxic immune responses remain a medical challenge. In mice, a successful approach to induce strong cytotoxic CD8⁺ T‐cell (CTL) reactions is to target antigens to DCs using specific antibodies against surface receptors in combination with adjuvants. A major drawback for translating this strategy into one for the clinic is the lack of analogous targets in human DCs. DC‐SIGN (DC‐specific‐ICAM3‐grabbing‐nonintegrin/CD209) is a C‐type lectin receptor with potent endocytic capacity and a highly restricted expression on human immature DCs. Therefore, DC‐SIGN represents an ideal candidate for DC targeting. Using transgenic mice that express human DC‐SIGN under the control of the murine CD11c promoter (hSIGN mice), we explored the efficacy of anti‐DC‐SIGN antibodies to target antigens to DCs and induce protective immune responses in vivo. We show that anti‐DC‐SIGN antibodies conjugated to OVA induced strong and persistent antigen‐specific CD4⁺ and CD8⁺ T‐cell responses, which efficiently protected from infection with OVA‐expressing Listeria monocytogenes. Thus, we propose DC targeting via DC‐SIGN as a promising strategy for novel vaccination protocols against intracellular pathogens.     

Efficient and versatile manipulation of the peripheral CD4+ T‐cell compartment by antigen targeting to DNGR‐1/CLEC9A

DC NK lectin group receptor‐1 (DNGR‐1, also known as CLEC9A) is a C‐type lectin receptor expressed by mouse CD8α⁺ DC and by their putative equivalents in human. DNGR‐1 senses necrosis and regulates CD8⁺ T‐cell cross‐priming to dead‐cell‐associated antigens. In addition, DNGR‐1 is a target for selective in vivo delivery of antigens to DC and the induction of CD8⁺ T‐cell and Ab responses. In this study, we evaluated whether DNGR‐1 targeting can be additionally used to manipulate antigen‐specific CD4⁺ T lymphocytes. Injection of small amounts of antigen‐coupled anti‐DNGR‐1 mAb into mice promoted MHC class II antigen presentation selectively by CD8α⁺ DC. In the steady state, this was sufficient to induce proliferation of antigen‐specific naïve CD4⁺ T cells and to drive their differentiation into Foxp3⁺ regulatory lymphocytes. Co‐administration of adjuvants prevented this induction of tolerance and promoted immunity. Notably, distinct adjuvants allowed qualitative modulation of CD4⁺ T‐cell behavior: poly I:C induced a strong IL‐12‐independent Th1 response, whereas curdlan led to the priming of Th17 cells. Thus, antigen targeting to DNGR‐1 is a versatile approach for inducing functionally distinct CD4⁺ T‐cell responses. Given the restricted pattern of expression of DNGR‐1 across species, this strategy could prove useful for developing immunotherapy protocols in humans.     

Tumor‐infiltrating dendritic cells exhibit defective cross‐presentation of tumor antigens,but is reversed by chemotherapy

Cross‐presentation defines the unique capacity of an APC to present exogenous Ag via MHC class I molecules to CD8⁺ T cells. DCs are specialized cross‐presenting cells and as such have a critical role in antitumor immunity. DCs are routinely found within the tumor microenvironment, but their capacity for endogenous or therapeutically enhanced cross‐presentation is not well characterized. In this study, we examined the tumor and lymph node DC cross‐presentation of a nominal marker tumor Ag, HA, expressed by the murine mesothelioma tumor AB1‐HA. We found that tumors were infiltrated by predominantly CD11b⁺ DCs with a semimature phenotype that could not cross‐present tumor Ag, and therefore, were unable to induce tumor‐specific T‐cell activation or proliferation. Although tumor‐infiltrating DCs were able to take up, process, and cross‐present exogenous cell‐bound and soluble Ags, this was significantly impaired relative to lymph node DCs. Importantly, however, systemic chemotherapy using gemcitabine reversed the defect in Ag cross‐presentation of tumor DCs. These data demonstrate that DC cross‐presentation within the tumor microenvironment is defective, but can be reversed by chemotherapy. These results have important implications for anticancer therapy, particularly regarding the use of immunotherapy in conjunction with cytotoxic chemotherapy.     

Antigen‐specific airway IL‐33 production depends on FcγR‐mediated incorporation of the antigen by alveolar macrophages in sensitized mice

Although interleukin (IL)‐33 is a candidate for the aggravation of asthma, the mechanisms underlying antigen‐specific IL‐33 production in the lung are unclear. Therefore, we analysed the mechanisms in mice. Intra‐tracheal administration of ovalbumin (OVA) evoked increases in IL‐33 and IL‐33 mRNA in the lungs of both non‐sensitized and OVA‐sensitized mice, and the increases in the sensitized mice were significantly higher than in the non‐sensitized mice. However, intra‐tracheal administration of bovine serum albumin did not increase the IL‐33 level in the OVA‐sensitized mice. Depletion of neither mast cells/basophils nor CD4⁺ cells abolished the OVA‐induced IL‐33 production in sensitized mice, suggesting that the antigen recognition leading to the IL‐33 production was not related with either antigen‐specific IgE‐bearing mast cells/basophils or memory CD4⁺ Th2 cells. When a fluorogenic substrate‐labelled OVA (DQ‐OVA) was intra‐tracheally administered, the lung cells of sensitized mice incorporated more DQ‐OVA than those of non‐sensitized mice. The lung cells incorporating DQ‐OVA included B‐cells and alveolar macrophages. The allergic IL‐33 production was significantly reduced by treatment with anti‐FcγRII/III mAb. Depletion of alveolar macrophages by clodronate liposomes significantly suppressed the allergic IL‐33 production, whereas depletion of B‐cells by anti‐CD20 mAb did not. These results suggest that the administered OVA in the lung bound antigen‐specific IgG Ab, and then alveolar macrophages incorporated the immune complex through FcγRII/III on the cell surface, resulting in IL‐33 production in sensitized mice. The mechanisms underlying the antigen‐specific IL‐33 production may aid in development of new pharmacotherapies.     

Long‐lasting cross‐presentation of tumor antigen in human DC

DC cross‐present exogenous antigens on MHC class I molecules, a process required for the onset of anti‐tumor immune responses. In order to study the cross‐presentation of tumor antigens by human DC, we compared the pathways of cross‐presentation of long peptides requiring internalization and intracellular processing with the direct presentation of short peptides, which does not require intracellular processing. We found that, after brief incubations with DC, short peptides were presented to CD8⁺ T cells with higher efficiencies than long peptides. After longer times of chase in the absence of peptide, however, the efficiency of presentation of the two types of peptides was reversed. After 2–3 days, DC pulsed with long peptides still activated T cells efficiently, while DC pulsed with short peptides failed to do so. Long‐lasting presentation of the long peptides was, at least in part, due to a stored persistent pool of antigen, which was still available for loading on MHC class I molecules after several days of chase. These results show that the use of long synthetic peptides allows the efficient, long‐lasting, presentation of tumor antigens, suggesting that long peptides represent an interesting approach for active anti‐tumor vaccination.     

Monitoring the efficacy of dendritic cell vaccination by early detection of 99mTc‐HMPAO‐labelled CD4+ T cells

DC vaccines have been used to induce tumour‐specific cytotoxic T cells 1 . However, this approach to cancer immunotherapy has had limited success. To be successful, injected DCs need to migrate to the LNs where they can stimulate effector T cells 1 . We and others have previously demonstrated by MRI that tumour antigen‐pulsed‐DCs labelled ex vivo with superparamagnetic iron oxide nanoparticles migrated to the draining LNs and are capable of activating antigen‐specific T cells 2 , 3 . The results from our study demonstrated that ex vivo superparamagnetic iron oxide nanoparticles‐labelled and OVA‐pulsed DCs prime cytotoxic CD8⁺ T‐cell responses to protect against a B16‐OVA tumour challenge. In the clinic, a possible noninvasive surrogate marker for efficacy of DC vaccination is to image the specific migration and accumulation of T cells following DC vaccination.     

Fcγ receptor‐mediated antigen uptake by lung DC contributes to allergic airway hyper‐responsiveness and inflammation

During asthma, lung DC capture and process antigens to initiate and maintain allergic Th2 cell responses to inhaled allergens. The aim of the study was to investigate whether allergen‐specific IgG, generated during sensitization, can potentiate the acute airway inflammation through Fcγ receptor (FcγR)‐mediated antigen uptake and enhance antigen presentation resulting in augmented T‐cell proliferation. We examined the impact of antigen presentation and T‐cell stimulation on allergic airway hyperresponsiveness and inflammation using transgenic and gene‐deficient mice. Both airway inflammation and eosinophilia in bronchoalveolar lavage fluid were markedly reduced in sensitized and challenged FcγR‐deficient mice. Lung DC of WT, but not FcγR‐deficient mice, induced increased antigen‐specific CD4⁺ T‐cell proliferation when pulsed with anti‐OVA IgG immune complexes. Intranasal application of anti‐OVA IgG immune complexes resulted in enhanced airway inflammation, eosinophilia and Th2 cytokine release, mediated through enhanced antigen‐specific T‐cell proliferation in vivo. Finally, antigen‐specific IgG in the serum of sensitized mice led to a significant increase of antigen‐specific CD4⁺ T‐cell proliferation induced by WT, but not FcγR‐deficient, lung DC. We conclude that FcγR‐mediated enhanced antigen presentation and T‐cell stimulation by lung DC has a significant impact on inflammatory responses following allergen challenge in asthma.     

Dendritic cells activated by an anti‐inflammatory agent induce CD4+ T helper type 2 responses without impairing CD8+ memory and effector cytotoxic T‐lymphocyte responses

Prevalence of pro‐inflammatory diseases is rising in developed country populations. The increase in these diseases has fuelled the search for new, immune suppressive, anti‐inflammatory therapies, which do not impact, or minimally impact, CD4⁺ and/or CD8⁺ T‐cell‐mediated immunity. The goal of this study was to determine if antigen‐presenting cells (APCs) activated by the anti‐inflammatory oligosaccharide, lacto‐N‐fucopentaose III (LNFPIII), would have an impaired ability to drive CD4⁺ T helper (Th) or CD8⁺ memory and effector T‐cell responses. To investigate this we activated splenic dendritic cells (SDCs) with LNFPIII and examined their ability to drive antigen‐specific CD4⁺ Th, and CD8⁺ memory and cytotoxic T‐cell (CTL) responses compared with lipopolysaccharide (LPS) ‐stimulated SDCs. The LNFPIII‐activated SDCs had altered co‐stimulatory molecule expression compared with LPS‐stimulated SDCs, while the levels of SDC chemokines following activation by either compound were similar. LNFPIII‐activated SDCs produced significantly lower levels of interleukin‐12 but surprisingly higher levels of interleukin‐6 than LPS‐activated SDCs. Similar to previous studies using bone‐marrow‐derived DCs, LNFPIII‐activated SDCs induced strong Th2 responses in vivo and ex vivo. LNFPIII activation of APCs was independent of the Toll‐interleukin‐1 receptor adaptor myeloid differentiating factor 88. Importantly, LNFPIII‐matured DCs induced CD8⁺ memory and effector CTL responses similar to those driven by LPS‐matured DCs, including the frequency of interferon‐γ‐producing CD8⁺ T cells and induction of CTL effectors. Treatment of APCs by the anti‐inflammatory glycan LNFPIII did not impair their ability to drive CD8⁺ effector and memory cell‐mediated immunity.     

Expansion of regulatory T cells via IL‐2/anti‐IL‐2 mAb complexes suppresses experimental myasthenia

Human autoimmune diseases are often characterized by a relative deficiency in CD4⁺CD25⁺ regulatory T cells (Treg). We therefore hypothesized that expansion of Treg can ameliorate autoimmune pathology. We tested this hypothesis in an experimental model for autoimmune myasthenia gravis (MG), a B‐cell‐mediated disease characterized by auto‐Ab directed against the acetylcholine receptor within neuromuscular junctions. We showed that injection of immune complexes composed of the cytokine IL‐2 and anti‐IL‐2 mAb (JES6‐1A12) induced an effective and sustained expansion of Treg, via peripheral proliferation of CD4⁺CD25⁺Foxp3⁺ cells and peripheral conversion of CD4⁺CD25^?Foxp3^? cells. The expanded Treg potently suppressed autoreactive T‐ and B‐cell responses to acetylcholine receptor and attenuated the muscular weakness that is characteristic of MG. Thus, IL‐2/anti‐IL‐2 mAb complexes can expand functional Treg in vivo, providing a potential clinical application of this modality for treatment of MG and other autoimmune disorders.     

Activation of invariant natural killer T cells in regional lymph nodes as new antigen‐specific immunotherapy via induction of interleukin‐21 and interferon‐γ

Invariant natural killer T (iNKT) cells play important immunoregulatory functions in allergen‐induced airway hyperresponsiveness and inflammation. To clarify the role of iNKT cells in allergic rhinitis (AR), we generated bone marrow‐derived dendritic cells (BMDCs), which were pulsed by ovalbumin (OVA) and α‐galactosylceramide (OVA/α‐GalCer‐BMDCs) and administered into the oral submucosa of OVA‐sensitized mice before nasal challenge. Nasal symptoms, level of OVA‐specific immunoglobulin (IgE), and T helper type 2 (Th2) cytokine production in cervical lymph nodes (CLNs) were significantly ameliorated in wild‐type (WT) mice treated with OVA/α‐GalCer‐BMDCs, but not in WT mice treated with OVA‐BMDCs. These anti‐allergic effects were not observed in Jα18^–/– recipients that lack iNKT cells, even after similar treatment with OVA/α‐GalCer‐BMDCs in an adoptive transfer study with CD4⁺ T cells and B cells from OVA‐sensitized WT mice. In WT recipients of OVA/α‐GalCer‐BMDCs, the number of interleukin (IL)‐21‐producing iNKT cells increased significantly and the Th1/Th2 balance shifted towards the Th1 dominant state. Treatment with anti‐IL‐21 and anti‐interferon (IFN)‐γ antibodies abrogated these anti‐allergic effects in mice treated with α‐GalCer/OVA‐BMDCs. These results suggest that activation of iNKT cells in regional lymph nodes induces anti‐allergic effects through production of IL‐21 or IFN‐γ, and that these effects are enhanced by simultaneous stimulation with antigen. Thus, iNKT cells might be a useful target in development of new treatment strategies for AR.     

Effective therapeutic anticancer vaccines based on precision guiding of cytolytic T lymphocytes

Summary: In natural immune responses CD4⁺ T helper (Th) cells, reactive with peptide antigens presented by major histocompatibility complex (MHC) class II molecules on dendritic cells (DC), can drive the maturation of DC that is required for induction of CD8⁺ cytolytic T‐lymphocyte (CTL) immunity. Proper induction, expansion and maintenance of CTL responses are achieved through delicate interactions between CD4⁺ T cells, DC and CD8⁺ T cells involving several ligand–receptor pairs. Th cells to a large extent operate through up‐regulation of CD40L, which then interacts with CD40 on DC to cause DC maturation. Subsequent CTL induction by activated DC requires CD80/CD86 on the DC to interact with the CD28 costimulatory receptor on CD8⁺ T cells. For maintenance and full expansion of CTL, interaction of the DC‐expressed 4–1BB ligand with its receptor 4–1BB on CTL is also important. Alternative molecular triggers of DC activation that can support induction of powerful CTL responses include agonistic anti‐CD40 antibody or ligands of Toll‐like receptors (TLR) such as LPS (TLR4 ligand) or oligodeoxynucleotides containing CpG‐motifs (TLR9 ligand). The combination of CpG adjuvant with a 35 amino acid long synthetic peptide comprising both tumor‐specific CTL and Th epitopes proved to be a highly effective vaccine formulation capable of inducing therapeutic immunity against human papillomavirus‐induced mouse tumors. The recently acquired insights into antigen presentation and costimulatory signals have made possible the development of a new generation of therapeutic anticancer vaccines.     

Dendritic cell homeostasis is maintained by nonhematopoietic and T‐cell‐produced Flt3‐ligand in steady state and during immune responses

Lymphoid‐tissue dendritic cells (DCs) are short‐lived and need to be continuously replenished from bone marrow‐derived DC progenitor cells. Fms‐related tyrosine kinase 3 is expressed during cellular development from hematopoietic progenitors to lymphoid‐tissue DCs. Fms‐related tyrosine kinase 3 ligand (Flt3L) is an essential, nonredundant cytokine for DC progenitor to lymphoid tissue DC differentiation and maintenance. However, which cells contribute to Flt3L production and how Flt3L cytokine levels are regulated in steady state and during immune reactions remains to be determined. Here we demonstrate that besides nonhematopoietic cells, WT T cells produce Flt3L and contribute to the generation of both classical DCs (cDCs) and plasmacytoid DCs in Flt3L^?/? mice. Upon stimulation in vitro, CD4⁺ T cells produce more Flt3L than CD8⁺ T cells. Moreover, in vivo stimulation of naïve OT‐II CD4⁺ T cells with OVA leads to increase of pre‐cDCs and cDCs in draining lymph nodes of Flt3L^?/? mice in a partially Flt3L‐dependent manner. Thus, Flt3L‐mediated lymphoid tissue DC homeostasis is regulated by steady‐state T cells as well as by proliferative T cells, fostering local development of lymphoid organ resident DCs.     

Interleukin‐33 and alveolar macrophages contribute to the mechanisms underlying the exacerbation of IgE‐mediated airway inflammation and remodelling in mice

Allergen‐specific IgE has long been regarded as a major molecular component of allergic asthma. Additionally, there is increasing evidence of the important roles of interleukin‐33 (IL‐33) in the disease. Here, we show that IL‐33 and alveolar macrophages play essential roles in the exacerbation of IgE‐mediated airway inflammation and remodelling. BALB/c mice passively sensitized with ovalbumin (OVA)‐specific IgE monoclonal antibody (mAb) were challenged with OVA seven times intratracheally. The seventh challenge exacerbated airway inflammation and remodelling compared with the fourth challenge; furthermore, markedly increased expression of IL‐33 in the lungs was observed at the fourth and seventh challenges. When anti‐IL‐33 or anti‐ST2 antibody was administered during the fourth to seventh challenge, airway inflammation and remodelling were significantly inhibited at the seventh challenge. Because increases of IL‐33⁺ and ST2⁺ alveolar macrophages and ST2⁺ CD4⁺ T cells in the lungs were observed at the fourth challenge, the roles of macrophages and CD4⁺ cells were investigated. Depletion of macrophages by 2‐chloroadenosine during the fourth to seventh challenge suppressed airway inflammation and remodelling, and IL‐33 production in the lung at the seventh challenge; additionally, anti‐CD4 mAb inhibited airway inflammation, but not airway remodelling and IL‐33 production. Meanwhile, treatment with 2‐chloroadenosine or anti‐CD4 mAb decreased IL‐33‐induced airway inflammation in normal mice; airway remodelling was repressed only by 2‐chloroadenosine. These results illustrate that macrophage‐derived IL‐33 contributes to the exacerbation of IgE‐mediated airway inflammation by mechanisms associated with macrophages and CD4⁺ cells, and airway remodelling through the activation of macrophages.     

Functional maturation of lamina propria dendritic cells by activation of NKT cells mediates the abrogation of oral tolerance

We previously showed that although systemic administration of α‐galactosylceramide (αGalCer) or agonistic anti‐CD40 induced functional maturation of dendritic cells (DC) in mesenteric lymph nodes, only the former treatment succeeded in breaking the induction of oral tolerance. In this study, we looked for the essential factor responsible for the disruption of oral tolerance. We found that lamina propria (LP)‐DC was responsible for the oral OVA presentation and that Peyer's patch was not essential for the induction of oral tolerance. Therefore, we investigated the role of LP‐DC. Treatment with αGalCer but not with anti‐CD40 induced the full maturation of LP‐DC at an early time point. This functional activation of LP‐DC was mediated by strong activation of NKT cells that reside abundantly in the small intestinal lamina propria (SI‐LP) and interferon‐γ partially contributed to the LP‐DC activation. LP‐DC isolated from αGalCer‐treated OVA‐fed mice induced the differentiation of naïve CD4⁺ T cells into Th1 and Th2 and was associated with the reduced Foxp3⁺ population. In contrast, LP‐DC isolated from anti‐CD40‐treated OVA‐fed mice failed to generate Th cell differentiation but induced more Foxp3⁺ CD4⁺ T cells. Our results demonstrate that triggered by NKT cells in SI‐LP, functional maturation of Ag‐capturing DC from SI‐LP is necessary for the abrogation of oral tolerance induction.     

DNA vaccination with T‐cell epitopes encoded within Ab molecules induces high‐avidity anti‐tumor CD8+ T cells

Stimulation of high‐avidity CTL responses is essential for effective anti‐tumor and anti‐viral vaccines. In this study we have demonstrated that a DNA vaccine incorporating CTL epitopes within an Ab molecule results in high‐avidity T‐cell responses to both foreign and self epitopes. The avidity and frequency was superior to peptide, peptide‐pulsed DC vaccines or a DNA vaccine incorporating the epitope within the native Ag. The DNA Ab vaccine was superior to an identical protein vaccine that can only cross‐present, indicating a role for direct presentation by the DNA vaccine. However, the avidity of CTL responses was significantly reduced in Fc receptor γ knockout mice or if the Fc region was removed suggesting that cross presentation of Ag via Fc receptor was also important in the induction of high‐avidity CTL. These results suggest that generation of high‐avidity CTL responses by the DNA vaccine is related to its ability to both directly present and cross‐present the epitope. High‐avidity responses were capable of efficient anti‐tumor activity in vitro and in vivo. This study demonstrates a vaccine strategy to generate high‐avidity CTL responses that can be used in anti‐tumor and anti‐viral vaccine settings.     

Differential requirements of CD4+ T‐cell signals for effector cytotoxic T‐lymphocyte (CTL) priming and functional memory CTL development at higher CD8+ T‐cell precursor frequency

Increased CD8⁺ T‐cell precursor frequency (PF) precludes the requirement of CD4⁺ helper T (Th) cells for primary CD8⁺ cytotoxic T‐lymphocyte (CTL) responses. However, the key questions of whether unhelped CTLs generated at higher PF are functional effectors, and whether unhelped CTLs can differentiate into functional memory cells at higher PF are unclear. In this study, ovalbumin (OVA) ‐pulsed dendritic cells (DC_OVA) derived from C57BL/6, CD40 knockout (CD40^?/?) or CD40 ligand knockout (CD40L^?/?) mice were used to immunize C57BL/6, Ia^b?/?, CD40^?/? or CD40L^?/? mice, whose PF was previously increased with transfer of 1 × 10⁶ CD8⁺ T cells derived from OVA‐specific T‐cell receptor (TCR) transgenic OTI, OTI(CD40^?/?) or OTI(CD40L^?/?) mice. All the immunized mice were then assessed for effector and memory CTL responses. Following DC immunization, relatively comparable CTL priming occurred without CD4⁺ T‐cell help and Th‐provided CD40/CD40L signalling. In addition, the unhelped CTLs were functional effectors capable of inducing therapeutic immunity against established OVA‐expressing tumours. In contrast, the functional memory development of CTLs was severely impaired in the absence of CD4⁺ T‐cell help and CD40/CD40L signalling. Finally, unhelped memory CTLs failed to protect mice against lethal tumour challenge. Taken together, these results demonstrate that CD4⁺ T‐cell help at higher PF, is not required for effector CTL priming, but is required for functional memory CTL development against cancer. Our data may impact the development of novel preventive and therapeutic approaches in cancer patients with compromised CD4⁺ T‐cell functions.