Effective Priming Research Articles

Synthetic cationic helical polypeptides for the stimulation of antitumour innate immune pathways in antigen-presenting cells.

Intracellular DNA sensors regulate innate immunity and can provide a bridge to adaptive immunogenicity. However, the activation of the sensors in antigen-presenting cells (APCs) by natural agonists such as double-stranded DNAs or cyclic nucleotides is impeded by poor intracellular delivery, serum stability, enzymatic degradation and rapid systemic clearance. Here we show that the hydrophobicity, electrostatic charge and secondary conformation of helical polypeptides can be optimized to stimulate innate immune pathways via endoplasmic reticulum stress in APCs. One of the three polypeptides that we engineered activated two major intracellular DNA-sensing pathways (cGAS-STING (for cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes) and Toll-like receptor 9) preferentially in APCs by promoting the release of mitochondrial DNA, which led to the efficient priming of effector T cells. In syngeneic mouse models of locally advanced and metastatic breast cancers, the polypeptides led to potent DNA-sensor-mediated antitumour responses when intravenously given as monotherapy or with immune checkpoint inhibitors. The activation of multiple innate immune pathways via engineered cationic polypeptides may offer therapeutic advantages in the generation of antitumour immune responses.

The medulla controls effector primed γδT-cell development in the adult mouse thymus.

γδT cells are produced in the thymus throughout life and provide immunity at epithelial-rich sites. Unlike conventional αβT cells, γδT-cell development involves intrathymic acquisition of effector function, with priming for either IL17 or IFN-γ production occurring during embryonic or adult life, respectively. How the thymus controls effector-primed γδT-cell generation in adulthood is poorly understood. Here, we distinguished de novo γδT cells from those undergoing thymus recirculation and/or retention using Rag2GFP mice alongside markers of maturation/effector priming including CD24, CD25, CD73, and IFN-γ, the latter by crossing with IFN-γYFP GREAT mice. We categorize newly developing γδT-cells into an ordered sequence where CD25+ CD73- IFN-γYFP- precursors are followed sequentially by CD25- CD73+ IFN-γYFP- intermediates and CD25- CD73+ IFN-γYFP+ effectors. To determine intrathymic requirements controlling this sequence, we examined γδT-cell development in Relb-/- thymus grafts that lack medullary microenvironments. Interestingly, medulla deficiency did not alter CD25+ γδT-cell precursor generation, but significantly impaired development of effector primed stages. This impact on γδT-cell priming was mirrored in plt/plt mice lacking the medullary chemoattractants CCL19 and CCL21, and also Ccl21a-/- but not Ccl19-/- mice. Collectively, we identify the medulla as an important site for effector priming during adult γδT-cell development and demonstrate a specific role for the medullary epithelial product CCL21 in this process.

Versican Proteolytic Fragments (Matrikines) Regulate the Intratumoral Dendritic Cell Milieu In Vivo: Implications for in Situ Tumor Vaccination

Regulated proteolysis of the tolerogenic matrix proteoglycan versican (VCAN) through the actions of ADAMTS-proteases, is associated with enhanced CD8+ infiltration in both hematopoietic and solid tumors. However, it is unclear whether the enhanced CD8+ infiltration results from proteolysis-mediated depletion of precursor VCAN at the tumor site or from generation of bioactive proteolytic fragments ("matrikines") (e.g., the 441-aa N-terminal fragment of V1-VCAN isoform, versikine). We have previously shown that versikine promotes Batf3-dendritic cell (DC) generation from FLT3L-mobilized bone marrow (BM) progenitors in vitro. However, the effects of versikine in DC homeostasis in the tumor microenvironment in vivo are unknown. To investigate the effects of versikine in DC homeostasis in vivo, we utilized the first Ras-driven myeloma (MM) model (VQ model- Rajagopalan et al., Blood 132:1006, 2018) as well as transplantable solid tumor models in both C57BL/6J (LLC lung carcinoma) and Balb/c (4T1 mammary carcinoma) backgrounds. Tumor cells were stably engineered to secrete HA-tagged versikine vs. empty-vector (EV) controls. EV-VQ or versikine-VQ myeloma cells were implanted intracardiacally into C57BL/6J syngeneic recipients and mice were monitored until they developed myeloma-related end-organ damage (hindlimb paralysis). Both groups of mice were paralyzed at similar rates. Intratumoral conventional DCs (CD138-CD45+, CD11chi,MHC IIhiLy6C-, CD64-) clustered into two populations: cDC1 (Batf3-DC: CD24hi,CD11blo), a subset with crucial activity in cross-priming anti-tumor CD8+ T cells, and cDC2 (CD24lo, CD11bhi). Versikine enhanced intratumoral Batf3-DC frequency/infiltration, while cDC2 levels were diminished in versikine-VQ BM (Figure 1A)(Batf3-DC: 48% in EV-VQ vs. 72% in versikine-VQ, p-value= 0.0246; cDC2: 52% in EV-VQ vs. 28% in versikine-VQ, p=0.0312). Monocytic-derived DC (Mo-DC: CD11chi, MHC IIhi, Ly6C+, CD64+) frequency remained unchanged. Versikine's effects were replicated in 2 solid tumor models. Versikine-expressing tumors were characterized by significantly enhanced Batf3-DC infiltration (Fig. 1A, p-value= 0.0079 for 4T1 model and <0.0001 for LLC model), whereas cDC2 numbers were diminished (p-value: 0.0079 and <0.0001 respectively). Adoptive transfer of CD45.2+ pre-DC (SIRPaint, FLIT3+, CD11c+, MHC II-, Celltrace+) in LLC-EV and LLC-versikine tumors in CD45.1+ recipients did not show any differences in 3-day differentiation potential of DC precursors, implicating other mechanisms to explain the steady-state imbalance in DC subset frequencies. To examine whether versikine's effects on the intratumoral DC milieu in vivo could be therapeutically harnessed, we compared responses to STING agonist therapy between versikine-expressing and EV tumors. LLC-EV-OVA and LLC-versikine-OVA (ovalbumin, a model antigen) -expressing tumors received therapeutic intratumoral injections of DMXAA, a murine STING agonist. Analysis of splenocytes 5 days later showed a significant increase in the frequency of OVA antigen-specific, CD8+ (MHCI:SIINFEKL tetramer+) splenocytes in LLC-versikine-bearing animals (Figure 1B). Interestingly, there was a marked increase in total central memory T splenocytes (TCM) (CD62LhiCD44hi) harvested from LLC-versikine tumor-bearing mice. We conclude that versikine influences the DC milieu in the tumor bed with promotion of intratumoral cross-presenting Batf3-DC and depletion of the cDC2 subset. Our findings highlight an unappreciated facet of immune regulation of the tumor microenvironment through matrix proteolytic fragments ("matrikines"). Whereas detection of native VCAN proteolysis on myeloma biopsies (see abstract by Dhakal et al.in this meeting) portends adverse outcomes likely due to the tolerogenic effects of accumulated precursor VCAN at the tumor site, therapeutic use of the isolated, purified fragments may promote tumor innate sensing and effector priming. VCAN-matrikines, through their effects on intratumoral Batf3-DC and antigen-specific CD8+ T cell infiltration, may potentiate in situ vaccination strategies across diverse hematopoietic and solid tumor types. Figure 1 Disclosures No relevant conflicts of interest to declare.

Quantitative determination of phagocytosis by bone marrow-derived dendritic cells via imaging flow cytometry.

Immunogenic cell death (ICD), induced by certain anticancer chemotherapeutics, leads to the emission of danger associated molecular patterns (DAMP) by cancer cells, which facilitates the attraction, activation and maturation of dendritic cells (DC) as well as the subsequent priming of effector T cells. In this context calreticulin (CALR) exposed at an early stage of ICD at the surface of the cancer cells serves as phagocytic signal and triggers the formation of immunological synapses between malignant cells and DC. Subsequent phagocytosis facilitates the transfer of tumor associated antigen and thus depicts a fundamental step in the generation of anticancer immunity. Here we provide an imaging flowcytometric protocol for the quantification of ICD-associated DC phagocytosis of cancer cells. As compared to the traditional flowcytometry-based analysis, the presented method offers additional means of differentiation between the transient formation of immunological synapses and the final DC-mediated phagocytosis of cancer cells.

T cell and dendritic cell interactions in lymphoid organs: More than just being in the right place at the right time.

The initiation of T lymphocyte responses within secondary lymphoid organs involves interactions with different subsets of dendritic cells (DCs). Recent studies have revealed the complexity of microanatomical organization within lymphoid organs. Exactly how T cells and DCs locate each other and the type of cellular interactions required for optimal priming of effector and memory T cell responses are beginning to be unraveled. Here we review advances in our understanding of how T cell priming is choreographed during infections, highlight the importance of cell positioning in this process and discuss how a spectrum of cellular interactions shapes T cell activation and differentiation.

Combination of CD40 Agonism and CSF-1R Blockade Reconditions Tumor-Associated Macrophages and Drives Potent Antitumor Immunity.

Efficacious antitumor immune responses must overcome multiple suppressive mechanisms in the tumor microenvironment to control cancer progression. In this study, we demonstrate that dual targeting of suppressive myeloid populations by inhibiting CSF-1/CSF-1R signaling and activation of antigen-presenting cells with agonist anti-CD40 treatment confers superior antitumor efficacy and increased survival compared with monotherapy treatment in preclinical tumor models. Concurrent CSF-1R blockade and CD40 agonism lead to profound changes in the composition of immune infiltrates, causing an overall decrease in immunosuppressive cells and a shift toward a more inflammatory milieu. Anti-CD40/anti-CSF-1R-treated tumors contain decreased tumor-associated macrophages and Foxp3+ regulatory T cells. This combination approach increases maturation and differentiation of proinflammatory macrophages and dendritic cells and also drives potent priming of effector T cells in draining lymph nodes. As a result, tumor-infiltrating effector T cells exhibit improved responses to tumor antigen rechallenge. These studies show that combining therapeutic approaches may simultaneously remove inhibitory immune populations and sustain endogenous antitumor immune responses to successfully impair cancer progression. Cancer Immunol Res; 5(12); 1109-21. ©2017 AACR.

Timing of in utero malaria exposure influences fetal CD4 T cell regulatory versus effector differentiation.

BackgroundIn malaria-endemic areas, the first exposure to malaria antigens often occurs in utero when the fetal immune system is poised towards the development of tolerance. Children exposed to placental malaria have an increased risk of clinical malaria in the first few years of life compared to unexposed children. Recent work has suggested the potential of pregnancy-associated malaria to induce immune tolerance in children living in malaria-endemic areas. A study was completed to evaluate the effect of malaria exposure during pregnancy on fetal immune tolerance and effector responses.MethodsUsing cord blood samples from a cohort of mother-infant pairs followed from early in pregnancy until delivery, flow cytometry analysis was completed to assess the relationship between pregnancy-associated malaria and fetal cord blood CD4 and dendritic cell phenotypes.ResultsCord blood FoxP3+ Treg counts were higher in infants born to mothers with Plasmodium parasitaemia early in pregnancy (12–20 weeks of gestation; p = 0.048), but there was no association between Treg counts and the presence of parasites in the placenta at the time of delivery (by loop-mediated isothermal amplification (LAMP); p = 0.810). In contrast, higher frequencies of activated CD4 T cells (CD25+FoxP3−CD127+) were observed in the cord blood of neonates with active placental Plasmodium infection at the time of delivery (p = 0.035). This population exhibited evidence of effector memory differentiation, suggesting priming of effector T cells in utero. Lastly, myeloid dendritic cells were higher in the cord blood of infants with histopathologic evidence of placental malaria (p < 0.0001).ConclusionTogether, these data indicate that in utero exposure to malaria drives expansion of both regulatory and effector T cells in the fetus, and that the timing of this exposure has a pivotal role in determining the polarization of the fetal immune response.

MRNA Structural constraints on EBNA1 synthesis impact on in vivo antigen presentation and early priming of CD8+ T cells.

Recent studies have shown that virally encoded mRNA sequences of genome maintenance proteins from herpesviruses contain clusters of unusual structural elements, G-quadruplexes, which modulate viral protein synthesis. Destabilization of these G-quadruplexes can override the inhibitory effect on self-synthesis of these proteins. Here we show that the purine-rich repetitive mRNA sequence of Epstein-Barr virus encoded nuclear antigen 1 (EBNA1) comprising G-quadruplex structures, limits both the presentation of MHC class I-restricted CD8+ T cell epitopes by CD11c+ dendritic cells in draining lymph nodes and early priming of antigen-specific CD8+ T-cells. Destabilization of the G-quadruplex structures through codon-modification significantly enhanced in vivo antigen presentation and activation of virus-specific T cells. Ex vivo imaging of draining lymph nodes by confocal microscopy revealed enhanced antigen-specific T-cell trafficking and APC-CD8+ T-cell interactions in mice primed with viral vectors encoding a codon-modified EBNA1 protein. More importantly, these antigen-specific T cells displayed enhanced expression of the T-box transcription factor and superior polyfunctionality consistent with the qualitative impact of translation efficiency. These results provide an important insight into how viruses exploit mRNA structure to down regulate synthesis of their viral maintenance proteins and delay priming of antigen-specific T cells, thereby establishing a successful latent infection in vivo. Furthermore, targeting EBNA1 mRNA rather than protein by small molecules or antisense oligonucleotides will enhance EBNA1 synthesis and the early priming of effector T cells, to establish a more rapid immune response and prevent persistent infection.

NK cell activation by the heat shock protein gp96 (VAC12P.1018)

Abstract Heat Shock Proteins (HSPs) are key mediators in the development of immune responses. Following immunization with HSPs chaperoning tumor antigens, anti-tumor immune responses are elicited. The current model of HSP mediated anti-tumor immunity suggests HSPs bind to and are internalized by antigen presenting cells (APCs), which results in the priming of effector T cells that subsequently inhibit tumor growth. In addition to APCs and T cells, NK cells have also been demonstrated to be essential for HSP mediated anti-tumor immunity. However what role NK cells play in eliciting immunity is poorly understood. In these studies, we aim to characterize the activation of NK cells following stimulation with the prototypical HSP, gp96. Our results show that, following incubation with gp96 in vitro, we observe an increase in the frequency of NK cells containing the intracellular effector proteins granzyme B and IFNγ. Although intracellular levels of IFNγ are increased, we do not detect IFNγ secretion and the intracellular levels are less than what is observed following cytokine mediated activation. Due to these responses, we are currently investigating NK cells for the presence of the HSP receptor CD91. Collectively, these data indicate that directly, HSP stimulation results in partial NK cell activation, and other mechanisms, such as indirect activation via HSP activated APCs, may be necessary to observe optimal NK cell effector functions.

Dynamic Simulation and Static Matching for Action Prediction: Evidence From Body Part Priming

Accurately predicting other people's actions may involve two processes: internal real-time simulation (dynamic updating) and matching recently perceived action images (static matching). Using a priming of body parts, this study aimed to differentiate the two processes. Specifically, participants played a motion-controlled video game with either their arms or legs. They then observed arm movements of a point-light actor, which were briefly occluded from view, followed by a static test pose. Participants judged whether this test pose depicted a coherent continuation of the previously seen action (i.e., "action prediction task"). Evidence of dynamic updating was obtained after compatible effector priming (i.e., arms), whereas incompatible effector priming (i.e., legs) indicated static matching. Together, the results support action prediction as engaging two distinct processes, dynamic simulation and static matching, and indicate that their relative contributions depend on contextual factors like compatibility of body parts involved in performed and observed action.

Pointing hand stimuli induce spatial compatibility effects and effector priming.

The present study investigated the automatic influence of perceiving a picture that indicates other’s action on one’s own task performance in terms of spatial compatibility and effector priming. Participants pressed left and right buttons with their left and right hands respectively, depending on the color of a central dot target. Preceding the target, a left or right hand stimulus (pointing either to the left or right with the index or little finger) was presented. In Experiment 1, with brief presentation of the pointing hand, a spatial compatibility effect was observed: responses were faster when the direction of the pointed finger and the response position were spatially congruent than when incongruent. The spatial compatibility effect was larger for the pointing index finger stimulus compared to the pointing little finger stimulus. Experiment 2 employed longer duration of the pointing hand stimuli. In addition to the spatial compatibility effect for the pointing index finger, the effector priming effect was observed: responses were faster when the anatomical left/right identity of the pointing and response hands matched than when the pointing and response hands differed in left/right identity. The results indicate that with sufficient processing time, both spatial/symbolic and anatomical features of a static body part implying another’s action simultaneously influence different aspects of the perceiver’s own action. Hierarchical coding, according to which an anatomical code is used only when a spatial code is unavailable, may not be applicable if stimuli as well as responses contain anatomical features.

Understanding Delayed T-Cell Priming, Lung Recruitment, and Airway Luminal T-Cell Responses in Host Defense against Pulmonary Tuberculosis

Mycobacterium tuberculosis (M.tb), the causative bacterium of pulmonary tuberculosis (TB), is a serious global health concern. Central to M.tb effective immune avoidance is its ability to modulate the early innate inflammatory response and prevent the establishment of adaptive T-cell immunity for nearly three weeks. When compared with other intracellular bacterial lung pathogens, such as Legionella pneumophila, or even closely related mycobacterial species such as M. smegmatis, this delay is astonishing. Customarily, the alveolar macrophage (AM) acts as a sentinel, detecting and alerting surrounding cells to the presence of an invader. However, in the case of M.tb, this may be impaired, thus delaying the recruitment of antigen-presenting cells (APCs) to the lung. Upon uptake by APC populations, M.tb is able to subvert and delay the processing of antigen, MHC class II loading, and the priming of effector T cell populations. This delay ultimately results in the deferred recruitment of effector T cells to not only the lung interstitium but also the airway lumen. Therefore, it is of upmost importance to dissect the mechanisms that contribute to the delayed onset of immune responses following M.tb infection. Such knowledge will help design the most effective vaccination strategies against pulmonary TB.

Targeting 4-1BB (CD137) to enhance CD8 T cell responses with poxviruses and viral antigens

Attenuated vaccinia virus (VACV) vectors are considered prime vaccine candidates for use in immunotherapy of infectious disease. In spite of this, recent data show that the level of attenuation may hamper the efficient generation of protective CD8 T cells. This suggests that additional adjuvant-like activities may need to be combined with attenuated VACV for optimal vaccination. Stimulatory reagents to the TNFR family molecule 4-1BB (CD137) may represent such an adjuvant for vaccination. Previous murine studies have found that 4-1BB can participate in optimal priming of effector and memory CD8 T cells in response to several virus infections, and concordantly direct stimulation of 4-1BB with agonist reagents effectively boosts the CD8 T cell response against those viruses. In contrast, we recently reported that 4-1BB plays no role in the response to a virulent strain of VACV, questioning whether agonists of 4-1BB will be useful adjuvants for vaccination with VACV vectors. Here we show that agonist anti-4-1BB strongly enhanced the primary viral-specific effector CD8 T cell response during infection with live virulent VACV and attenuated VACV, and during immunization with VACV peptides given in IFA. However, accumulation of memory CD8 T cells was enhanced only following infection with virulent VACV or with peptide vaccination, but not with attenuated VACV, correlating in part with more transient expression of 4-1BB on CD8 T cells with attenuated virus. Our data therefore suggest that 4-1BB may be a promising candidate for targeting as an adjuvant for short-term enhancement of CD8 T cell responses with VACV vaccine strategies, but additional receptors may need to be engaged with 4-1BB to allow long-term CD8 T cell immunity with attenuated VACV vectors.

Batf3-dependent CD11b(low/-) peripheral dendritic cells are GM-CSF-independent and are not required for Th cell priming after subcutaneous immunization.

Dendritic cells (DCs) subsets differ in precursor cell of origin, functional properties, requirements for growth factors, and dependence on transcription factors. Lymphoid-tissue resident CD8α+ conventional DCs (cDCs) and CD11blow/−CD103+ non-lymphoid DCs are developmentally related, each being dependent on FMS-like tyrosine kinase 3 ligand (Flt3L), and requiring the transcription factors Batf3, Irf8, and Id2 for development. It was recently suggested that granulocyte/macrophage colony stimulating factor (GM-CSF) was required for the development of dermal CD11blow/−Langerin+CD103+ DCs, and that this dermal DC subset was required for priming autoreactive T cells in experimental autoimmune encephalitis (EAE). Here, we compared development of peripheral tissue DCs and susceptibility to EAE in GM-CSF receptor deficient (Csf2rb −/−) and Batf3 −/− mice. We find that Batf3-dependent dermal CD11blow/−Langerin+ DCs do develop in Csf2rb −/− mice, but that they express reduced, but not absent, levels of CD103. Further, Batf3 −/− mice lacking all peripheral CD11blow/− DCs show robust Th cell priming after subcutaneous immunization and are susceptible to EAE. Our results suggest that defective T effector priming and resistance to EAE exhibited by Csf2rb −/− mice does not result from the absence of dermal CD11blow/−Langerin+CD103+ DCs.

Transcriptional regulation of proteolytic enzymes in dendritic cells by effector versus regulatory T cells: a new mechanism of maintaining peripheral tolerance (52.15)

Abstract MUC1.Tg mice express the human tumor associated antigen MUC1 in a physiological pattern that is analogous to its expression on normal human epithelial cells and tumors. Vaccine-induced responses to MUC1 peptide in these animals are weak compared to WT mice. Gene expression profiling post vaccination revealed differential regulation in spleen dendritic cells (DC) of several proteolytic enzymes that were previously thought to function exclusively in pancreatic catabolism (i.e. Trypsin-1, Trypsin-4, Carboxypeptidase B1). These enzymes were upregulated in DC when WT mice were vaccinated with the MUC1 peptide (more foreign) and highly downregulated after the same vaccination in MUC1.Tg mice (more self). Using an in vitro co-culture system, we show that this regulation is dependent on CD4 T cell interactions with DC, such that activated effector T cells (Teff), but not TLR agonists, upregulate these enzymes, whereas activated regulatory T cells (Treg) actively suppress their expression. Cell-cell contact appears to be necessary. The Treg mediated suppression is dominant and it occurs even when DC are cultured simultaneously with Treg and Teff. We propose a new mechanism of maintaining peripheral tolerance whereby self-antigen specific Treg recognize the presence of antigen on a few DC and temporarily shut down antigen processing by all DC to avoid efficient priming of effector T cells.

Social attitudes differentially modulate imitation in adolescents and adults

Previous studies have demonstrated a bidirectional relationship between social attitudes and imitation in adults: pro-social attitudes promote imitation, and imitation further increases positive social attitudes. Social attitudes and the social brain are developing throughout the adolescent years. Thus, the aim of this study was to test whether pro-social attitudes promote imitation in an Adolescent Group to the same extent as in an Adult Group. Participants were primed with pro-social or non-social words in a Scrambled Sentence Priming task. They then completed an Imitation task wherein participants were required to perform a lift action with either the index or middle finger, whilst observing either a compatible action (e.g. index finger response and observed index finger lift) or an incompatible action (e.g. index finger response and observed middle finger lift). In an Effector Priming control condition, observed fingers remained stationary but a semi-transparent green mask was added to either the compatible or incompatible finger. The magnitude of the Imitation Effect and Effector Priming Effect was calculated by subtracting reaction times on compatible trials from those on incompatible trials. In the Adult Group, social priming specifically modulated the Imitation Effect: pro-social priming produced a larger Imitation Effect but did not modulate the Effector Priming Effect. In adolescents, however, no effect of social priming was seen on either the Imitation or Effector Priming measures. We consider possible explanations for these results including the immature development of social brain regions and reduced experience of the relationship between social attitudes and imitation in adolescence.

Gap junctions between regulatory T cells and dendritic cells prevent sensitization of CD8+ T cells

Regulatory T (Treg) cells suppress the sensitization phase of experimental contact hypersensitivity (CHS) reactions when injected before hapten application. Our aim was to analyze the mechanisms by which Treg cells suppress the sensitization phase of CHS reactions. Treg cells were labeled with different fluorescent dyes and injected into naive mice directly before sensitization with the hapten 2,4,6-trinitro-1-chlorobenzene. Two days after sensitization, the lymphoid organs were analyzed for the presence of Treg cells and engagement of gap junctions with other cells. Dendritic cells (DCs) and effector CD8(+)T cells were isolated from the draining lymph nodes (LNs) of the differently treated groups, analyzed by using FACS for activation markers, and assessed for the T-cell stimulatory capacity of the DCs and the priming of effector T cells. Only the LN-homing Treg cells suppressed the sensitization phase in CHS reactions by means of establishing gap junctions with DCs in the dLNs. This gap junctional intercellular communication led to downregulation of T-cell costimulatory molecules on the surface of the DCs, abrogating the priming, activation, and proliferation of hapten-specific CD8(+)T cells. Consequently, the ear-swelling response induced by challenge with the respective hapten was prevented. Treg cells not only modulate ongoing CD4(+)T cell-mediated immune reactions at tissue sites but also abrogate the de novo induction of CD8(+)T cell-driven immune reactions by interfering with T-cell stimulatory activity of DCs through gap junctional intercellular communication.

Regulatory T-Cell Suppression of CD8+ T-Cell-Mediated Graft-Versus-Host Reaction Requires Their Presence During Priming

Despite the promising therapeutic potential of regulatory T cells (Treg) in animal studies of graft-versus-host disease (GVHD), little is known about their effect on human GVHD. Whether Treg are capable of ameliorating GVHD tissue damage has never been demonstrated in humans. It is also unknown whether Treg modulation of GVH histopathologic damage relies on their presence during effector T-cell priming, or whether allogeneic Treg are safe to use clinically. To address these questions, we used an in vitro human skin explant GVHD model, which mimics the physiopathology of GVHD. First, "donor"-derived CD8 T cells were stimulated with human leukocyte antigen-unmatched "recipient" dendritic cells (priming phase), then primed "donor" CD8 T cells were co-cultured with "recipient" skin to induce GVH tissue damage (effector phase). "Donor"-derived Treg were added at the priming or effector phase of the GVH response. Histopathologic changes in the skin were evaluated using a clinically validated GVHD scoring system. "Donor"-derived Treg significantly reduced the severity of GVH histopathologic damage when present during T-cell priming. In contrast, Treg failed to prevent GVH tissue damage when added to the skin co-culture (effector phase), concurrently with primed T cells. Importantly, "donor" Treg alone did not induce GVH tissue damage. Delayed Treg addition led to reduced and impaired Treg suppression of CD8 T-cell activation and their cytolytic function. "Donor"-derived Treg effectively suppress CD8 T-cell-mediated GVH tissue damage but are critically required during priming of effector T cells. "Donor"-derived Treg seem to be safe and do not induce GVH histopathologic damage.

Absence of IFN-β Impairs Antigen Presentation Capacity of Splenic Dendritic Cells via Down-Regulation of Heat Shock Protein 70

Type I IFNs play a key role in linking the innate and adaptive arms of the immune system. Although produced rapidly in response to pathogens, IFNs are also produced at low levels in the absence of infection. In the present study, we demonstrate that constitutively produced IFNs are necessary in vivo to maintain dendritic cells in an "Ag presentation-competent" state. Conventional dendritic cells (cDCs) isolated from spleens of IFN-beta or IFNAR-deficient mice exhibit a highly impaired ability to present Ag and activate naive T cells. Microarray analysis of mRNA isolated from IFN-beta(-/-) and IFNAR(-/-) cDCs revealed diminished expression of two genes that encoded members of the heat shock protein 70 (Hsp70) family. Consistent with this observation, pharmacological inhibition of Hsp70 in cDCs from wild-type mice impaired their T cell stimulatory capacity. Similarly, the Ag presentation ability of splenic cDCs isolated from Hsp70.1/3(-/-) mice was also severely impaired in comparison to wild-type cDCs. Thus, constitutive IFN-beta expression regulates Hsp70 levels to help maintain dendritic cells in a competent state for efficient priming of effector T cells in vivo.

House dust mite allergen induces asthma via Toll-like receptor 4 triggering of airway structural cells

Barrier epithelial cells and airway dendritic cells (DCs) make up the first line of defense against inhaled substances such as house dust mite (HDM) allergen and endotoxin (lipopolysaccharide, LPS). We hypothesized that these cells need to communicate with each other to cause allergic disease. We show in irradiated chimeric mice that Toll-like receptor 4 (TLR4) expression on radioresistant lung structural cells, but not on DCs, is necessary and sufficient for DC activation in the lung and for priming of effector T helper responses to HDM. TLR4 triggering on structural cells caused production of the innate proallergic cytokines thymic stromal lymphopoietin, granulocyte-macrophage colony-stimulating factor, interleukin-25 and interleukin-33. The absence of TLR4 on structural cells, but not on hematopoietic cells, abolished HDM-driven allergic airway inflammation. Finally, inhalation of a TLR4 antagonist to target exposed epithelial cells suppressed the salient features of asthma, including bronchial hyperreactivity. Our data identify an innate immune function of airway epithelial cells that drives allergic inflammation via activation of mucosal DCs.