SARS-CoV-2 DNA Vaccine INO-4800 Induces Durable Immune Responses Capable of Being Boosted in a Phase 1 Open-Label Trial.

BACKGROUND: Additional severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines that are safe and effective as primary vaccines and boosters remain urgently needed to combat the coronavirus disease 2019 (COVID-19) pandemic. We describe safety and durability of immune responses following 2 primary doses and a homologous booster dose of an investigational DNA vaccine (INO-4800) targeting full-length spike antigen. METHODS: Three dosage strengths of INO-4800 (0.5 mg, 1.0 mg, and 2.0 mg) were evaluated in 120 age-stratified healthy adults. Intradermal injection of INO-4800 followed by electroporation at 0 and 4 weeks preceded an optional booster 6-10.5 months after the second dose. RESULTS: INO-4800 appeared well tolerated with no treatment-related serious adverse events. Most adverse events were mild and did not increase in frequency with age and subsequent dosing. A durable antibody response was observed 6 months following the second dose; a homologous booster dose significantly increased immune responses. Cytokine-producing T cells and activated CD8+ T cells with lytic potential were significantly increased in the 2.0-mg dose group. CONCLUSIONS: INO-4800 was well tolerated in a 2-dose primary series and homologous booster in all adults, including elderly participants. These results support further development of INO-4800 for use as primary vaccine and booster. CLINICAL TRIALS REGISTRATION: NCT04336410.

Establishment of a Pig Influenza Challenge Model for Evaluation of Monoclonal Antibody Delivery Platforms.

mAbs are a possible adjunct to vaccination and drugs in treatment of influenza virus infection. However, questions remain whether small animal models accurately predict efficacy in humans. We have established the pig, a large natural host animal for influenza, with many physiological similarities to humans, as a robust model for testing mAbs. We show that a strongly neutralizing mAb (2-12C) against the hemagglutinin head administered prophylactically at 15 mg/kg reduced viral load and lung pathology after pandemic H1N1 influenza challenge. A lower dose of 1 mg/kg of 2-12C or a DNA plasmid-encoded version of 2-12C reduced pathology and viral load in the lungs but not viral shedding in nasal swabs. We propose that the pig influenza model will be useful for testing candidate mAbs and emerging delivery platforms prior to human trials.

SARS-CoV-2 Assays To Detect Functional Antibody Responses That Block ACE2 Recognition in Vaccinated Animals and Infected Patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of COVID-19, resulting in cases of mild to severe respiratory distress and significant mortality. The global outbreak of this novel coronavirus has now infected >20 million people worldwide, with >5 million cases in the United States (11 August 2020). The development of diagnostic and research tools to determine infection and vaccine efficacy is critically needed. We have developed multiple serologic assays using newly designed SARS-CoV-2 reagents for detecting the presence of receptor-binding antibodies in sera. The first assay is surface plasmon resonance (SPR) based and can quantitate both antibody binding to the SARS-CoV-2 spike protein and blocking to the Angiotensin-converting enzyme 2 (ACE2) receptor in a single experiment. The second assay is enzyme-linked immunosorbent assay (ELISA) based and can measure competition and blocking of the ACE2 receptor to the SARS-CoV-2 spike protein with antispike antibodies. The assay is highly versatile, and we demonstrate the broad utility of the assay by measuring antibody functionality of sera from small animals and nonhuman primates immunized with an experimental SARS-CoV-2 vaccine. In addition, we employ the assay to measure receptor blocking of sera from SARS-CoV-2-infected patients. The assay is shown to correlate with pseudovirus neutralization titers. This type of rapid, surrogate neutralization diagnostic can be employed widely to help study SARS-CoV-2 infection and assess the efficacy of vaccines.

In Vivo Delivery of a DNA-Encoded Monoclonal Antibody Protects Non-human Primates against Zika Virus.

Zika virus (ZIKV) infection is endemic to several world regions, and many others are at high risk for seasonal outbreaks. Synthetic DNA-encoded monoclonal antibody (DMAb) is an approach that enables in vivo delivery of highly potent mAbs to control infections. We engineered DMAb-ZK190, encoding the mAb ZK190 neutralizing antibody, which targets the ZIKV E protein DIII domain. In vivo-delivered DMAb-ZK190 achieved expression levels persisting >10 weeks in mice and >3 weeks in non-human primate (NHPs), which is protective against ZIKV infectious challenge. This study is the first demonstration of infectious disease control in NHPs following in vivo delivery of a nucleic acid-encoded antibody, supporting the importance of this new platform.

Contribution of TCR signaling strength to CD8+ T cell peripheral tolerance mechanisms.

Peripheral tolerance mechanisms are in place to prevent T cells from mediating aberrant immune responses directed against self and environmental Ags. Mechanisms involved in the induction of peripheral tolerance include T cell-intrinsic pathways, such as anergy or deletion, or exogenous tolerance mediated by regulatory T cells. We have previously shown that the density of peptide-MHC class I recognized by the TCR determines whether CD8(+) T cells undergo anergy or deletion. Specifically, using a TCR-transgenic CD8(+) T cell model, we demonstrated that persistent peripheral exposure to low- or high-dose peptides in the absence of inflammatory signals resulted in clonal deletion or anergy of the T cell, respectively. In this study, by altering the affinity of the peptide-MHC tolerogen for TCR, we have confirmed that this mechanism is dependent on the level of TCR signaling that the CD8(+) T cell receives. Using altered peptide ligands (APLs) displaying high TCR affinities, we show that increasing the TCR signaling favors anergy induction. Conversely, using APLs displaying a decreased TCR affinity tilted our system in the direction of deletional tolerance. We demonstrate how differential peripheral CD8(+) T cell tolerance mechanisms are controlled by both the potency and density of MHC class I-peptide tolerogen.

Ammonium Acetate Enhances the Attractiveness of a Variety of Protein-Based Baits to Female Ceratitis capitata (Diptera: Tephritidae).

Ammonia and its derivatives are used by female fruit flies (Diptera: Tephritidae) as volatile cues to locate protein-rich food needed to produce their eggs. This need for external protein sources has led to the development of behaviorally based control strategies such as food-based lures and insecticidal baits targeting pestiferous fruit fly species. In field cage studies conducted in Hawaii, we examined the behavioral response of laboratory-reared male and female Mediterranean fruit fly, Ceratitis capitata (Wiedemann), to seven commercially available protein baits and to beer waste, a relatively inexpensive and readily available substance. Each material was tested alone or in combination with either ammonium acetate or ammonium carbonate. For the majority of baits evaluated, the presence of ammonium acetate, but not ammonium carbonate, elicited a significantly greater level of response of female C. capitata compared with the protein baits alone. The addition of ammonium acetate to selected baits increased bait attractiveness to a level comparable with that elicited by the most widely used spinosad-based protein bait, GF-120. Our findings indicate that the addition of ammonium acetate to commercially available proteinaceous baits and to beer waste can greatly improve their attractiveness to C. capitata, potentially increasing the bait's effectiveness for fruit fly monitoring and suppression.

Attraction and mortality of Bactrocera dorsalis (Diptera: Tephritidae) to STATIC Spinosad ME weathered under operational conditions in California and Florida: a reduced-risk male annihilation treatment.

Studies were conducted in 2013-2014 to quantify attraction, feeding, and mortality of male oriental fruit flies, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), to STATIC Spinosad ME a reduced-risk male annihilation treatment (MAT) formulation consisting of an amorphous polymer matrix in combination with methyl eugenol (ME) and spinosad compared with the standard treatment of Min-U-Gel mixed with ME and naled (Dibrom). Our approach used a behavioral methodology for evaluation of slow-acting reduced-risk insecticides. ME treatments were weathered for 1, 7, 14, 21, and 28 d under operational conditions in California and Florida and shipped to Hawaii for bioassays. In field tests using bucket traps to attract and capture wild males, and in toxicity studies conducted in 1-m(3) cages using released males of controlled ages, STATIC Spinosad ME performed equally as well to the standard formulation of Min-U-Gel ME with naled for material aged up to 28 d in both California and Florida. In laboratory feeding tests in which individual males were exposed for 5 min to the different ME treatments, mortality induced by STATIC Spinosad ME recorded at 24 h did not differ from mortality caused by Min-U-Gel ME with naled at 1, 7, 14, and 21 d in California and was equal to or higher for all weathered time periods in Florida during two trials. Spinosad has low contact toxicity, and when mixed with an attractant and slow release matrix, offers a reduced-risk alternative for eradication of B. dorsalis and related ME attracted species, without many of the potential negative effects to humans and nontargets associated with broad-spectrum contact insecticides such as naled.

Real-Time Assessment of Rodent Engagement Using ArUco Markers: A Scalable and Accessible Approach for Scoring Behavior in a Nose-Poking Go/No-Go Task.

In the field of behavioral neuroscience, the classification and scoring of animal behavior play pivotal roles in the quantification and interpretation of complex behaviors displayed by animals. Traditional methods have relied on video examination by investigators, which is labor-intensive and susceptible to bias. To address these challenges, research efforts have focused on computational methods and image-processing algorithms for automated behavioral classification. Two primary approaches have emerged: marker- and markerless-based tracking systems. In this study, we showcase the utility of "Augmented Reality University of Cordoba" (ArUco) markers as a marker-based tracking approach for assessing rat engagement during a nose-poking go/no-go behavioral task. In addition, we introduce a two-state engagement model based on ArUco marker tracking data that can be analyzed with a rectangular kernel convolution to identify critical transition points between states of engagement and distraction. In this study, we hypothesized that ArUco markers could be utilized to accurately estimate animal engagement in a nose-poking go/no-go behavioral task, enabling the computation of optimal task durations for behavioral testing. Here, we present the performance of our ArUco tracking program, demonstrating a classification accuracy of 98% that was validated against the manual curation of video data. Furthermore, our convolution analysis revealed that, on average, our animals became disengaged with the behavioral task at ∼75 min, providing a quantitative basis for limiting experimental session durations. Overall, our approach offers a scalable, efficient, and accessible solution for automated scoring of rodent engagement during behavioral data collection.

Gastroenterology trainee experience, confidence and satisfaction in nutrition training: a cross-sectional survey in the UK.

Introduction: Nutrition is an essential part of gastroenterology specialist training. There is limited evidence of trainee experience in this area. The shorter training programme introduced in 2022 may lead to reduced exposure to this subspecialty. We aimed to explore and describe current nutrition training experiences, confidence and satisfaction to inform future improvements. Methods: Gastroenterology trainees were invited to participate in an online survey from 20 May 2022 to 18 July 2022. The questionnaire consisted of 27 questions with a range of free-text and Likert scale responses. Results: 86 responses were received. 39.5% had undertaken an advanced training programme or core placement in nutrition. 52.9% of these felt 'fairly confident' or 'very confident' in managing intestinal failure vs 5.8% of those who had not completed a nutrition placement. Obesity and eating disorders management received the lowest ratings. Nutrition training was described as 'fairly important' or 'very important' by 98.8% and 47.0% included nutrition as part of their preferred future practice. 53.1% of ST6/7 trainees were 'fairly confident' or 'very confident' their training offered adequate experience in nutrition. Participants reported barriers including a lack of education and training opportunities, and limited early rotations offering nutrition training. Conclusion: Gastroenterology trainees believe nutrition training to be important. Nutrition placements increase trainee confidence, knowledge and experiences overall, but there is variability in this. Improved structuring of placements, increased educational opportunities and exposure to this subspecialty at an earlier stage are required to ensure competency in nutrition is reliably achieved during gastroenterology training.

In vivo delivery of engineered synthetic DNA-encoded SARS-CoV-2 monoclonal antibodies for pre-exposure prophylaxis in non-human primates.

COVID-19 remains a major public health concern. Monoclonal antibodies have received emergency use authorization (EUA) for pre-exposure prophylaxis against COVID-19 among high-risk groups for treatment of mild to moderate COVID-19. In addition to recombinant biologics, engineered synthetic DNA-encoded antibodies (DMAb) are an important strategy for direct in vivo delivery of protective mAb. A DMAb cocktail was synthetically engineered to encode the immunoglobulin heavy and light chains of two different two different Fc-engineered anti-SARS-CoV-2 antibodies. The DMAbs were designed to enhance in vivo expression and delivered intramuscularly to cynomolgus and rhesus macaques with a modified in vivo delivery regimen. Serum levels were detected in macaques, along with specific binding to SARS-CoV-2 spike receptor binding domain protein and neutralization of multiple SARS-CoV-2 variants of concern in pseudovirus and authentic live virus assays. Prophylactic administration was protective in rhesus macaques against signs of SARS-CoV-2 (USA-WA1/2020) associated disease in the lungs. Overall, the data support further study of DNA-encoded antibodies as an additional delivery mode for prevention of COVID-19 severe disease. These data have implications for human translation of gene-encoded mAbs for emerging infectious diseases and low dose mAb delivery against COVID-19.

Delineation of DNA and mRNA COVID-19 vaccine-induced immune responses in preclinical animal models.

Nucleic acid vaccines are designed based on genetic sequences (DNA or mRNA) of a target antigen to be expressed in vivo to drive a host immune response. In response to the COVID-19 pandemic, mRNA and DNA vaccines based on the SARS-CoV-2 Spike antigen were developed. Surprisingly, head-to-head characterizations of the immune responses elicited by each vaccine type has not been performed to date. Here, we have employed a range of preclinical animal models including the hamster, guinea pig, rabbit, and mouse to compare and delineate the immune response raised by DNA, administered intradermally (ID) with electroporation (EP) and mRNA vaccines (BNT162b2 or mRNA-1273), administered intramuscularly (IM), expressing the SARS-CoV-2 WT spike antigen. The results revealed clear differences in the quality and magnitude of the immune response between the two vaccine platforms. The DNA vaccine immune response was characterized by strong T cell responses, while the mRNA vaccine elicited robust humoral responses. The results may assist in guiding the disease target each vaccine type may be best matched against and suggest mechanisms to further enhance the breadth of each platform's immune response.

Engineered antibody cytokine chimera synergizes with DNA-launched nanoparticle vaccines to potentiate melanoma suppression in vivo.

Cancer immunotherapy has demonstrated great promise with several checkpoint inhibitors being approved as the first-line therapy for some types of cancer, and new engineered cytokines such as Neo2/15 now being evaluated in many studies. In this work, we designed antibody-cytokine chimera (ACC) scaffolding cytokine mimetics on a full-length tumor-specific antibody. We characterized the pharmacokinetic (PK) and pharmacodynamic (PD) properties of first-generation ACC TA99-Neo2/15, which synergized with DLnano-vaccines to suppress in vivo melanoma proliferation and induced significant systemic cytokine activation. A novel second-generation ACC TA99-HL2-KOA1, with retained IL-2Rß/γ binding and attenuated but preserved IL-2Rα binding, induced lower systemic cytokine activation with non-inferior protection in murine tumor studies. Transcriptomic analyses demonstrated an upregulation of Type I interferon responsive genes, particularly ISG15, in dendritic cells, macrophages and monocytes following TA99-HL2-KOA1 treatment. Characterization of additional ACCs in combination with cancer vaccines will likely be an important area of research for treating melanoma and other types of cancer.

Use of Pyriproxyfen to Induce Oogenesis in Diapausing Megacopta cribraria (Heteroptera: Plataspidae), and Evaluation of Pyriproxyfen-Induced Eggs for Rearing the Parasitoid Paratelenomus saccharalis (Hymenoptera: Scelionidae).

The mass rearing of hymenopteran egg parasitoids requires an abundant supply of host eggs. The onset of reproductive diapause and subsequent decline in egg production poses a challenge for parasitoid rearing when using host colonies augmented by field-collected insects. We investigated the application of pyriproxyfen, a juvenile hormone analog, to induce oviposition in diapausing adult kudzu bugs, Megacopta cribraria (Fabricius) (Heteroptera: Plataspidae), and the use of eggs produced by pyriproxyfen-treated kudzu bugs to rear the egg parasitoid, Paratelenomus saccharalis (Dodd) (Hymenoptera: Scelionidae). The effects of pyriproxyfen and photoperiod treatments on host mortality, egg production, and rates of parasitoid eclosion from the eggs were used to calculate the parasitoid yield for the different treatment regimes. A combination of pyriproxyfen and a long-day photoperiod increased the parasitoid yield by 87% compared to acetone and a long-day photoperiod. The general applicability of JH-analog mediated egg production for parasitoid rearing is discussed.

Impact of direct-access IBD physician delivered endoscopy on clinical outcomes: a pre-implementation and post-implementation study.

Introduction: Patients with suspected inflammatory bowel disease (IBD) referred from primary care often face diagnostic and treatment delays. This study aimed to compare a novel direct-access IBD endoscopy pathway with the traditional care model. Method: Single centre real-world study analysing primary care referrals with suspected IBD. Group A: patients triaged to direct-access IBD endoscopy. Group B: patients undergoing traditional outpatient appointments before the availability of direct-access IBD endoscopy. Demographics, fecal calprotectin (FCP), C-reactive protein (CRP), disease activity score, endoscopy findings, treatment and follow-up were collected and statistically analysed. Ranked semantic analysis of IBD symptoms contained within referral letters was performed. Results: Referral letters did not differ significantly in Groups A and B. Demographic data, FCP and CRP values were similar. Referral to treatment time (RTT) at the time of IBD endoscopy was reduced from 177 days (Group B) to 24 days (Group A) (p<0.0001). Diagnostic yield of IBD was 35.6% (Group B) versus 62.0% (Group A) (p=0.0003). 89.2% of patients underwent colonoscopy in Group B versus 46.4% in Group A. DNA rates were similar in both groups. The direct to IBD endoscopy pathway saved 100% of initial IBD consultant clinics with a 2.5-fold increase in IBD nurse-led follow-up. Conclusion: Our novel pathway resulted in an 86% reduction in RTT with associated increased diagnostic yield while saving 100% of initial IBD consultant outpatient appointments. Replication in other trusts may improve patient experience and accelerate time to diagnosis/treatment while optimising the use of healthcare resources.

Clinically relevant dosing and pharmacokinetics of DNA-encoded antibody therapeutics in a sheep model.

DNA-encoded delivery and in vivo expression of antibody therapeutics presents an innovative alternative to conventional protein production and administration, including for cancer treatment. To support clinical translation, we evaluated this approach in 18 40-45 kg sheep, using a clinical-matched intramuscular electroporation (IM EP) and hyaluronidase-plasmid DNA (pDNA) coformulation setup. Two cohorts of eight sheep received either 1 or 4 mg pDNA encoding an ovine anti-cancer embryonic antigen (CEA) monoclonal antibody (mAb; OVAC). Results showed a dose-response with average maximum serum concentrations of respectively 0.3 and 0.7 µg/ml OVAC, 4-6 weeks after IM EP. OVAC was detected in all 16 sheep throughout the 6-week follow-up, and no anti-OVAC antibodies were observed. Another, more exploratory, cohort of two sheep received a 12 mg pOVAC dose. Both animals displayed a similar dose-dependent mAb increase and expression profile in the first two weeks. However, in one animal, an anti-OVAC antibody response led to loss of mAb detection four weeks after IM EP. In the other animal, no anti-drug antibodies were observed. Serum OVAC concentrations peaked at 4.9 µg/ml 6 weeks after IM EP, after which levels gradually decreased but remained detectable around 0.2 to 0.3 µg/ml throughout a 13-month follow-up. In conclusion, using a delivery protocol that is currently employed in clinical Phase 1 studies of DNA-based antibodies, we achieved robust and prolonged in vivo production of anti-cancer DNA-encoded antibody therapeutics in sheep. The learnings from this large-animal model regarding the impact of pDNA dose and host immune response on the expressed mAb pharmacokinetics can contribute to advancing clinical translation.

Mucosal chemokine adjuvant enhances synDNA vaccine-mediated responses to SARS-CoV-2 and provides heterologous protection in vivo.

The global coronavirus disease 2019 (COVID-19) pandemic has claimed more than 5 million lives. Emerging variants of concern (VOCs) continually challenge viral control. Directing vaccine-induced humoral and cell-mediated responses to mucosal surfaces may enhance vaccine efficacy. Here we investigate the immunogenicity and protective efficacy of optimized synthetic DNA plasmids encoding wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (pS) co-formulated with the plasmid-encoded mucosal chemokine cutaneous T cell-attracting chemokine (pCTACK; CCL27). pCTACK-co-immunized animals exhibit increased spike-specific antibodies at the mucosal surface and increased frequencies of interferon gamma (IFNγ)+ CD8+ T cells in the respiratory mucosa. pCTACK co-immunization confers 100% protection from heterologous Delta VOC challenge. This study shows that mucosal chemokine adjuvants can direct vaccine-induced responses to specific immunological sites and have significant effects on heterologous challenge. Further study of this unique chemokine-adjuvanted vaccine approach in the context of SARS-CoV-2 vaccines is likely important.

Prime-boost vaccination regimens with INO-4800 and INO-4802 augment and broaden immune responses against SARS-CoV-2 in nonhuman primates.

The enhanced transmissibility and immune evasion associated with emerging SARS-CoV-2 variants demands the development of next-generation vaccines capable of inducing superior protection amid a shifting pandemic landscape. Since a portion of the global population harbors some level of immunity from vaccines based on the original Wuhan-Hu-1 SARS-CoV-2 sequence or natural infection, an important question going forward is whether this immunity can be boosted by next-generation vaccines that target emerging variants while simultaneously maintaining long-term protection against existing strains. Here, we evaluated the immunogenicity of INO-4800, our synthetic DNA vaccine candidate for COVID-19 currently in clinical evaluation, and INO-4802, a next-generation DNA vaccine designed to broadly target emerging SARS-CoV-2 variants, as booster vaccines in nonhuman primates. Rhesus macaques primed over one year prior with the first-generation INO-4800 vaccine were boosted with either INO-4800 or INO-4802 in homologous or heterologous prime-boost regimens. Both boosting schedules led to an expansion of T cells and antibody responses which were characterized by improved neutralizing and ACE2 blocking activity across wild-type SARS-CoV-2 as well as multiple variants of concern. These data illustrate the durability of immunity following vaccination with INO-4800 and additionally support the use of either INO-4800 or INO-4802 in prime-boost regimens.

DNA-delivered antibody cocktail exhibits improved pharmacokinetics and confers prophylactic protection against SARS-CoV-2.

Monoclonal antibody therapy has played an important role against SARS-CoV-2. Strategies to deliver functional, antibody-based therapeutics with improved in vivo durability are needed to supplement current efforts and reach underserved populations. Here, we compare recombinant mAbs COV2-2196 and COV2-2130, which compromise clinical cocktail Tixagevimab/Cilgavimab, with optimized nucleic acid-launched forms. Functional profiling of in vivo-expressed, DNA-encoded monoclonal antibodies (DMAbs) demonstrated similar specificity, broad antiviral potency and equivalent protective efficacy in multiple animal challenge models of SARS-CoV-2 prophylaxis compared to protein delivery. In PK studies, DNA-delivery drove significant serum antibody titers that were better maintained compared to protein administration. Furthermore, cryo-EM studies performed on serum-derived DMAbs provide the first high-resolution visualization of in vivo-launched antibodies, revealing new interactions that may promote cooperative binding to trimeric antigen and broad activity against VoC including Omicron lineages. These data support the further study of DMAb technology in the development and delivery of valuable biologics.

Nucleic acid delivery of immune-focused SARS-CoV-2 nanoparticles drives rapid and potent immunogenicity capable of single-dose protection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines may target epitopes that reduce durability or increase the potential for escape from vaccine-induced immunity. Using synthetic vaccinology, we have developed rationally immune-focused SARS-CoV-2 Spike-based vaccines. Glycans can be employed to alter antibody responses to infection and vaccines. Utilizing computational modeling and in vitro screening, we have incorporated glycans into the receptor-binding domain (RBD) and assessed antigenic profiles. We demonstrate that glycan-coated RBD immunogens elicit stronger neutralizing antibodies and have engineered seven multivalent configurations. Advanced DNA delivery of engineered nanoparticle vaccines rapidly elicits potent neutralizing antibodies in guinea pigs, hamsters, and multiple mouse models, including human ACE2 and human antibody repertoire transgenics. RBD nanoparticles induce high levels of cross-neutralizing antibodies against variants of concern with durable titers beyond 6 months. Single, low-dose immunization protects against a lethal SARS-CoV-2 challenge. Single-dose coronavirus vaccines via DNA-launched nanoparticles provide a platform for rapid clinical translation of potent and durable coronavirus vaccines.

CD8alpha+ dendritic cells prime TCR-peptide-reactive regulatory CD4+FOXP3- T cells.

CD4(+) T cells with immune regulatory function can be either FOXP3(+) or FOXP3(-). We have previously shown that priming of naturally occurring TCR-peptide-reactive CD4(+)FOXP3(-) Treg specifically controls Vbeta8.2(+)CD4(+) T cells mediating EAE. However, the mechanism by which these Treg are primed to recognize their cognate antigenic determinant, which is derived from the TCRVbeta8.2-chain, is not known. In this study we show that APC derived from splenocytes of naïve mice are able to stimulate cloned CD4(+) Treg in the absence of exogenous antigen, and their stimulation capacity is augmented during EAE. Among the APC populations, DC were the most efficient in stimulating the Treg. Stimulation of CD4(+) Treg was dependent upon processing and presentation of TCR peptides from ingested Vbeta8.2TCR(+)CD4(+) T cells. Additionally, DC pulsed with TCR peptide or apoptotic Vbeta8.2(+) T cells were able to prime Treg in vivo and mediate protection from disease in a CD8-dependent fashion. These data highlight a novel mechanism for the priming of CD4(+) Treg by CD8alpha(+) DC and suggest a pathway that can be exploited to prime antigen-specific regulation of T-cell-mediated inflammatory disease.