Specific Th1 Research Articles

In vivo edited eosinophils reconcile antigen specific Th2 response and mitigate airway allergy

BackgroundImprovement is needed in the remedies used to control Th2 polarization. Bioengineering approaches have modified immune cells that have immunosuppressive functions. This study aims to generate modified eosinophils (Meos) in vivo and use Meos to balance Th2 polarization and reduce airway allergy.MethodsA cell editor was constructed. The editor contained a peptide carrier, an anti-siglec F antibody, MHC II, ovalbumin, and LgDNA (DNA extracted from a probiotic, Lactobacillus rhamnosus GG). Which was designated as Cedit. Meos are eosinophils modified using Cedits. An airway Th2 polarization mouse model was established used to test the effect of Meos on suppressing airway allergy.ResultsThe Cedits remained physically and chemically stable in solution (pH7.2) for at least 96 h. Cedits specifically bound to eosinophils, which are designated as Meos. Meos produced programmed death ligand-1 (PD-L1); the latter induced antigen specific CD4+ T cell apoptosis. Administration of Cedits through nasal instillations generated Meos in vivo, which significantly reduced the frequency of antigen specific CD4+ T cells in the airways, and mitigated airway Th2 polarization.ConclusionsWe constructed Cedit, which could edit eosinophils into Meos in vivo. Meos could induce antigen specific CD4+ T cell apoptosis, and reconcile airway Th2 polarization.

Design of a novel EmTSP-3 and EmTIP based multi-epitope vaccine against Echinococcus multilocularis infection.

Current treatments and prevention strategies for echinococcosis are inadequate. Recent advancements in molecular vaccine development show promise against Echinococcus granulosus; however, Echinococcus multilocularis remains a challenge. A Multi-epitope Vaccine could potentially induce specific B and T lymphocyte responses, thereby offering protection against Echinococcus multilocularis infection. This study aimed to develop a MEV against alveolar echinococcosis. Key epitopes from the Echinococcus multilocularis proteins EmTSP3 and EmTIP were identified using immunoinformatics analyses. These analyses were conducted to assess the MEV feasibility, structural characteristics, molecular docking, molecular dynamics simulations, and immune simulations. The immunogenicity and antigenicity of the vaccine were evaluated through in vitro and in vivo experiments, employing ELISA, Western blotting, FCM, challenge infection experiments, and ELISPOT. The effective antigenicity and immunogenicity of MEV were demonstrated through immunoinformatics, as well as in vitro and in vivo experiments. In vitro experiments revealed that MEV increased the secretion of IFN-γ and IL-4 in PBMC and successfully bound to specific antibodies in patient serum. Furthermore, mice immunized with MEV developed a robust immune response, characterized by elevated levels of CD4+ and CD8+ T-cells, increased secretion of IFN-γ and IL-4 by specific Th1 and Th2 cells, and heightened serum antibody levels. Importantly, MEV reduced the weight of cysts by conferring resistance against echinococcosis. These findings suggest that MEV is a promising candidate for the prevention of Echinococcus multilocularis infection. A total of 7 CTL, 7 HTL, 5 linear B-cell, and 2 conformational B-cell epitopes were identified. The vaccine has demonstrated effective antigenicity and immunogenicity against AE through molecular docking, immune simulation, molecular dynamics studies, and both in vitro and in vivo experiments. It provides effective protection against Echinococcus multilocularis infection, thereby laying a foundation for further development.

201.2: Patients remaining donor specific antibody (DSA) negative post transplantation have more regulated and fewer antigen specific Th-1 cell responses to mismatched donor human leucocyte antigens (HLA) versus those who develop DSA.

201.2: Patients remaining donor specific antibody (DSA) negative post transplantation have more regulated and fewer antigen specific Th-1 cell responses to mismatched donor human leucocyte antigens (HLA) versus those who develop DSA.

Evaluation of a novel intramuscular prime/intranasal boost vaccination strategy against influenza in the pig model.

Live-attenuated influenza vaccines (LAIV) offer advantages over the commonly used inactivated split influenza vaccines. However, finding the optimal balance between sufficient attenuation and immunogenicity has remained a challenge. We recently developed an alternative LAIV based on the 2009 pandemic H1N1 virus with a truncated NS1 protein and lacking PA-X protein expression (NS1(1-126)-ΔPAX). This virus showed a blunted replication and elicited a strong innate immune response. In the present study, we evaluated the efficacy of this vaccine candidate in the porcine animal model as a pertinent in vivo system. Immunization of pigs via the nasal route with the novel NS1(1-126)-ΔPAX LAIV did not cause disease and elicited a strong mucosal immune response that completely blocked replication of the homologous challenge virus in the respiratory tract. However, we observed prolonged shedding of our vaccine candidate from the upper respiratory tract. To improve LAIV safety, we developed a novel prime/boost vaccination strategy combining primary intramuscular immunization with a haemagglutinin-encoding propagation-defective vesicular stomatitis virus (VSV) replicon, followed by a secondary immunization with the NS1(1-126)-ΔPAX LAIV via the nasal route. This two-step immunization procedure significantly reduced LAIV shedding, increased the production of specific serum IgG, neutralizing antibodies, and Th1 memory cells, and resulted in sterilizing immunity against homologous virus challenge. In conclusion, our novel intramuscular prime/intranasal boost regimen interferes with virus shedding and transmission, a feature that will help combat influenza epidemics and pandemics.

Evaluation of the suitability of β-lactoglobulin and whey protein in a BALB/c mouse model of cow's milk allergy

Cow's milk allergy (CMA) is common in infants and young children, which can adversely affect their growth and quality of life. Currently, research on CMA is mainly carried out in mouse models due to ethical constraints in population studies, but the determination of the optimal modelling protocol remains controversial. Here, we aim to evaluate the applicability of β-lactoglobulin (BLG) and whey protein from multiple perspectives in the construction of CMA models in BALB/c mice. To this end, the mice were sensitized by oral gavage of allergens, and then challenged by BLG or whey protein orally administered as well as whey intradermally in the ear, respectively. The allergic reactions were determined by the evaluation of anaphylactic symptoms, levels of specific immunoglobulins and Th2 cytokines, and intestinal tissue histopathology. Overall, mice in the BLG group showed more severe allergic symptoms and decreased body temperature than the other groups. The stronger allergic reactions to BLG in mice were also proved in higher levels of mouse mast cell protease-1 (mMCP-1), allergen specific antibodies, and Th2 cytokines as well as more serious intestinal damage than whey protein. These results suggest that BALB/c mice vary in the susceptibility to different cow's milk allergens, and the single component represented by BLG instead of whey protein, may be a more appropriate and effective allergen for the construction of BALB/c mouse model of CMA.

Th1/Th2 associated transcription factors and cytokines in pregnancies with systemic lupus erythematosus.

Th1/Th2 shift occurs during pregnancy. Systemic lupus erythematosus (SLE) flares may induce the dysregulation of Th1 and Th2 cells. We aimed to investigate the dynamic changes of Th1/Th2 associated transcription factors and cytokines in patients with SLE during pregnancy. Twenty-five pregnant patients with SLE and twenty-two healthy age-matched women served as controls from September 2021 to March 2022 were enrolled in the study. Real-time quantitative reverse transcription polymerase chain reaction analysis of peripheral blood mononuclear cells were performed to measure the expression of Th1 specific transcription factors T-bet, cytokine IFN-γ, and Th2 specific transcription factors GATA3, cytokine IL-4. The main statistical analysis methods were t test, Mann-Whitney U-test, Pearson correlation and Spearman rank correlation analysis. The mRNA level of IFN-γ and the relative expression of T-bet/GATA3 and IFN-γ/IL-4 in SLE patients were significantly higher than those in healthy individuals, whereas the GATA3 expression is lower in pregnant patients with SLE (p<0.001, p<0.05, p<0.05 and p<0.01 during the whole pregnancy, respectively; p<0.05, p<0.01, p<0.05 and p<0.05 specifically for the 3rd trimester, respectively). There were significant correlations between T-bet and IFN-γ (r=0.492, p<0.05), and between T-bet/GATA3 and IFN-γ/IL-4 (r=0.482, p<0.05). Our work indicates that in SLE patients Th1/Th2 shift is blocked with up-regulation of Th1 cell function and insufficient Th2 polarisation during pregnancy, which may be involved in adverse pregnancy outcomes.

Bhlhe40 Promotes CD4+ T Helper 1 Cell and Suppresses T Follicular Helper Cell Differentiation during Viral Infection.

In response to acute infection, naive CD4+ T cells primarily differentiate into T helper 1 (Th1) or T follicular helper (Tfh) cells that play critical roles in orchestrating cellular or humoral arms of immunity, respectively. However, despite the well established role of T-bet and BCL-6 in driving Th1 and Tfh cell lineage commitment, respectively, whether additional transcriptional circuits also underlie the fate bifurcation of Th1 and Tfh cell subsets is not fully understood. In this article, we study how the transcriptional regulator Bhlhe40 dictates the Th1/Tfh differentiation axis in mice. CD4+ T cell-specific deletion of Bhlhe40 abrogates Th1 but augments Tfh differentiation. We also assessed an increase in germinal center B cells and Ab production, suggesting that deletion of Bhlhe40 in CD4+ T cells not only alters Tfh differentiation but also their capacity to provide help to B cells. To identify molecular mechanisms by which Bhlhe40 regulates Th1 versus Tfh lineage choice, we first performed epigenetic profiling in the virus specific Th1 and Tfh cells following LCMV infection, which revealed distinct promoter and enhancer activities between the two helper cell lineages. Furthermore, we identified that Bhlhe40 directly binds to cis-regulatory elements of Th1-related genes such as Tbx21 and Cxcr6 to activate their expression while simultaneously binding to regions of Tfh-related genes such as Bcl6 and Cxcr5 to repress their expression. Collectively, our data suggest that Bhlhe40 functions as a transcription activator to promote Th1 cell differentiation and a transcription repressor to suppress Tfh cell differentiation.

Vaccine-elicited IL-1R signaling results in Th17 TRM-mediated immunity

Lung tissue resident memory (TRM) cells are thought to play crucial roles in lung host defense. We have recently shown that immunization with the adjuvant LTA1 (derived from the A1 domain of E. coli heat labile toxin) admixed with OmpX from K. pneumoniae can elicit antigen specific lung Th17 TRM cells that provide serotype independent immunity to members of the Enterobacteriaceae family. However, the upstream requirements to generate these cells are unclear. Single-cell RNA-seq showed that vaccine-elicited Th17 TRM cells expressed high levels of IL-1R1, suggesting that IL-1 family members may be critical to generate these cells. Using a combination of genetic and antibody neutralization approaches, we show that Th17 TRM cells can be generated independent of caspase-1 but are compromised when IL-1α is neutralized. Moreover IL-1α could serve as a molecular adjuvant to generate lung Th17 TRM cells independent of LTA1. Taken together, these data suggest that IL-1α plays a major role in vaccine-mediated lung Th17 TRM generation.

Sublingual allergen immunotherapy prevents house dust mite inhalant type 2 immunity through dendritic cell-mediated induction of Foxp3+ regulatory T cells

Sublingual allergen immunotherapy (SLIT) is an emerging treatment option for allergic asthma, and a potential disease modifying strategy for asthma prevention. The key cellular events leading to such long term tolerance remains to be fully elucidated. We administered prophylactic SLIT in a mouse model of house dust mite (HDM) driven allergic asthma. HDM extract was sublingually administered over 3 weeks followed by intratracheal sensitization and intranasal challenges with HDM. Prophylactic SLIT prevented allergic airway inflammation and hyperreactivity with a low lab-to-lab variation. The HDM specific Th2 (CD4 T helper) response was shifted by SLIT towards a regulatory and Th17 response in lung and mediastinal lymph node (MLN). By using Der p 1 specific CD4+ T cells (1-DER), we found that SLIT blocked 1-DER T cell recruitment to the MLN and dampened IL-4 secretion following intratracheal HDM sensitization. Sublingually administered Der p 1 protein activated 1-DER T cells in the cervical lymph node (CLN) via CCR7+ migratory dendritic cells (DC). DCs migrating from the oral submucosa to the CLN after SLIT induced Foxp3+ regulatory T cells. When mice were sensitized with HDM, prior prophylactic SLIT increased Der p 1 specific Tregs and lowered Th2 recruitment in the lung. By using Foxp3-DTR mice, Tregs were found to contribute to the immunoregulatory prophylactic effect of SLIT on type 2 immunity. These findings in a mouse model suggest that DC-mediated functional Treg induction in oral mucosa draining LNs is one of the driving mechanisms behind the disease modifying effect of prophylactic SLIT.

Targeting Common Inflammatory Mediators in Experimental Severe Asthma and Acute Lung Injury.

Neutrophils, known to be mobilized and activated in high amounts through Il-17 stimulation, are a key factor for clinical manifestation and imbalance of redox systems favoring a dominant oxidative state in both severe asthma and acute lung injury (f). The aim of this study was to evaluate in mice, the effect of Secukinumab (SECU) in a model of ovalbumin-induced asthma exacerbated with LPS administration to induce ALI, compared to dexamethasone (DEXA), already known for its benefit in both asthma and ALI. Results on cytokine levels for specific Th1, Th2 and Th17 revealed an interplay of immune responses. For Th1 effector cytokines in BALF, DEXA treatment increased TNF-α levels, but TNF-α was not modified by SECU; DEXA and SECU significantly decreased IFN-γ and IL-6 levels. For typical Th2 cytokines, DEXA significantly increased Il-4, Il-5 and Il-13 levels, while SECU significantly inhibited Il-5 levels. Both SECU and DEXA significantly decreased Il-17 levels. Cytokine level changes in lung tissue homogenate were partly similar to BALF cytokines. Conclusion: in addition to DEXA, SECU possesses the ability to modulate inflammatory cytokine release and to decrease Th17 responses in ALI overlapped on exacerbated asthma in mice.

Th17 cell promotes apoptosis of IL-23R+ neurons in experimental autoimmune encephalomyelitis

Myelin antigen-reactive Th1 and Th17 cells are critical drivers of central nervous system (CNS) autoimmune inflammation. Transcription factors T-bet and RORγt play a crucial role in the differentiation and function of Th1 and Th17 cells, and impart them a pathogenic role in CNS autoimmune inflammation. Mice deficient in these two factors do not develop experimental autoimmune encephalomyelitis (EAE). While T-bet and RORγt are known to regulate the expression of several cell adhesion and migratory molecules in T cells, their role in supporting Th1 and Th17 trafficking to the CNS is not completely understood. More importantly, once Th1 and Th17 cells reach the CNS, how the function of these transcription factors modulates the local inflammatory response during EAE is unclear. In the present study, we showed that myelin oligodendrocyte glycoprotein 35–55 peptide (MOG35–55)-specific Th1 cells deficient in RORγt could cross the blood-brain barrier (BBB) but failed to induce demyelination, apoptosis of neurons, and EAE. Pathogenic Th17 cell-derived cytokines GM-CSF, TNF-α, IL-17A, and IL-21 significantly increased the surface expression of IL-23R on neuronal cells. Furthermore, we showed that, in EAE, neurons in the brain and spinal cord express IL-23R. IL-23-IL-23R signaling in neuronal cells caused phosphorylation of STAT3 (Ser727 and Tyr705) and induced cleaved caspase 3 and cleaved poly (ADP-ribose) polymerase-1 (PARP-1) molecules in an IL-23R-dependent manner and caused apoptosis. Thus, we provided a mechanism showing that T-bet is required to recruit pathogenic Th17 cells to the CNS and RORγt-mediated inflammatory response to drive the apoptosis of IL-23R+ neurons in the CNS and cause EAE. Understanding detailed molecular mechanisms will help to design better strategies to control neuroinflammation and autoimmunity. One Sentence SummaryIL-23-IL-23R signaling promotes apoptosis of CNS neurons.

Mucosal and systemic immune responses after a single intranasal dose of nanoparticle and spore-based subunit vaccines in mice with pre-existing lung mycobacterial immunity.

Tuberculosis (TB) is a major global health threat that claims more than one million lives annually. With a quarter of the global population harbouring latent TB, post-exposure vaccination aimed at high-risk populations that could develop active TB disease would be of great public health benefit. Mucosal vaccination is an attractive approach for a predominantly lung disease like TB because it elicits both local and systemic immunity. However, the immunological consequence of mucosal immunisation in the presence of existing lung immunity remains largely unexplored. Using a mycobacterial pre-exposure mouse model, we assessed whether pre-existing mucosal and systemic immune responses can be boosted and/or qualitatively altered by intranasal administration of spore- and nanoparticle-based subunit vaccines. Analysis of lung T cell responses revealed an increasing trend in the frequency of important CD4 and CD8 T cell subsets, and T effector memory cells with a Th1 cytokine (IFNγ and TNFα) signature among immunised mice. Additionally, significantly greater antigen specific Th1, Th17 and IL-10 responses, and antigen-induced T cell proliferation were seen from the spleens of immunised mice. Measurement of antigen-specific IgG and IgA from blood and bronchoalveolar lavage fluid also revealed enhanced systemic and local humoral immune responses among immunised animals. Lastly, peripheral blood mononuclear cells (PBMCs) obtained from the TB-endemic country of Mozambique show that individuals with LTBI showed significantly greater CD4 T cell reactivity to the vaccine candidate as compared to healthy controls. These results support further testing of Spore-FP1 and Nano-FP1 as post-exposure TB vaccines.

Mettl3 induced miR-338-3p expression in dendritic cells promotes antigen-specific Th17 cell response via regulation of Dusp16.

Pathogenic Th17 cells are critical drivers of multiple autoimmune diseases, including uveitis and its animal model, experimental autoimmune uveitis (EAU). However, how innate immune signals modulate pathogenic Th17 responses remains largely unknown. Here, we showed that miR-338-3p endowed dendritic cells (DCs) with an increased ability to activate interphotoreceptor retinoid-binding protein (IRBP)1-20 -specific Th17 cells by promoting the production of IL-6, IL-1β, and IL-23. Invivo administration of LV-miR-338-infected DCs promoted pathogenic Th17 responses and exacerbated EAU development. Mechanistically, dual-specificity phosphatase 16 (Dusp16) was a molecular target of miR-338-3p. miR-338-3p repressed Dusp16 and therefore strengthened the mitogen-activated protein kinase (MAPK) p38 signaling, resulting in increased production of Th17-polarizing cytokines and subsequent pathogenic Th17 responses. In addition, methyltransferase like 3 (Mettl3), a key m6A methyltransferase, mediated the upregulation of miR-338-3p in activated DCs. Together, our findings identify a vital role for Mettl3/miR-338-3p/Dusp16/p38 signaling in DCs-driven pathogenic Th17 responses and suggest a potential therapeutic avenue for uveitis and other Th17 cell-related autoimmune disorders.

Sensing of protease activity as a triggering mechanism of Th2 cell immunity and allergic disease.

CD4 T-helper cell type 2 (Th2) cells mediate host defense against extracellular parasites, like helminths. However, Th2 cells also play a pivotal role in the onset and progression of allergic inflammatory diseases such as atopic dermatitis, allergic rhinitis, asthma, and food allergy. This happens when allergens, which are otherwise harmless foreign proteins, are mistakenly identified as "pathogenic." Consequently, the encounter with these allergens triggers the activation of specific Th2 cell responses, leading to the development of allergic reactions. Understanding the molecular basis of allergen sensing is vital for comprehending how Th2 cell responses are erroneously initiated in individuals with allergies. The presence of protease activity in allergens, such as house dust mites (HDM), pollen, fungi, or cockroaches, has been found to play a significant role in triggering robust Th2 cell responses. In this review, we aim to examine the significance of protease activity sensing in foreign proteins for the initiation of Th2 cell responses, highlighting how evolving a host protease sensor may contribute to detect invading helminth parasites, but conversely can also trigger unwanted reactions to protease allergens. In this context, we will explore the recognition receptors activated by proteolytic enzymes present in major allergens and their contribution to Th2-mediated allergic responses. Furthermore, we will discuss the coordinated efforts of sensory neurons and epithelial cells in detecting protease allergens, the subsequent activation of intermediary cells, including mast cells and type 2 innate lymphoid cells (ILC2s), and the ultimate integration of all signals by conventional dendritic cells (cDCs), leading to the induction of Th2 cell responses. On the other hand, the review highlights the role of monocytes in the context of protease allergen exposure and their interaction with cDCs to mitigate undesirable Th2 cell reactions. This review aims to provide insights into the innate functions and cell communications triggered by protease allergens, which can contribute to the initiation of detrimental Th2 cell responses, but also promote mechanisms to effectively suppress their development.

HIV specific Th1 responses are altered in Ugandans with HIV and Schistosoma mansoni coinfection

BackgroundFishing communities surrounding Lake Victoria in Uganda have HIV prevalence of 28% and incidence rates of 5 per 100 person years. More than 50% of the local fishermen are infected with Schistosoma mansoni (S. mansoni). We investigated the role of S. mansoni coinfection as a possible modifier of immune responses against HIV. Using polychromatic flow cytometry and Gran-ToxiLux assays, HIV specific responses, T cell phenotypes, antibody-dependent cell-mediated cytotoxic (ADCC) potency and titres were compared between participants with HIV-S. mansoni coinfection and participants with HIV infection alone.ResultsS. mansoni coinfection was associated with a modified pattern of anti-HIV responses, including lower frequency of bifunctional (IFNγ + IL-2 − TNF-α+) CD4 T cells, higher overall CD4 T cell activation and lower HIV ADCC antibody titres, compared to participants with HIV alone.ConclusionsThese results support the hypothesis that S. mansoni infection affects T cell and antibody responses to HIV in coinfected individuals.

T cell deletional tolerance restricts AQP4 but not MOG CNS autoimmunity

Aquaporin-4 (AQP4)-specific Th17 cells are thought to have a central role in neuromyelitis optica (NMO) pathogenesis. When modeling NMO, only AQP4-reactive Th17 cells from AQP4-deficient (AQP4-/-), but not wild-type (WT) mice, caused CNS autoimmunity in recipient WT mice, indicating that a tightly regulated mechanism normally ensures tolerance to AQP4. Here, we found that pathogenic AQP4 T cell epitopes bind MHC II with exceptionally high affinity. Examination of T cell receptor (TCR) α/β usage revealed that AQP4-specific T cells from AQP4-/- mice employed a distinct TCR repertoire and exhibited clonal expansion. Selective thymic AQP4 deficiency did not fully restore AQP4-reactive T cells, demonstrating that thymic negative selection alone did not account for AQP4-specific tolerance in WT mice. Indeed, AQP4-specific Th17 cells caused paralysis in recipient WT or B cell-deficient mice, which was followed by complete recovery that was associated with apoptosis of donor T cells. However, donor AQP4-reactive T cells survived and caused persistent paralysis in recipient mice deficient in both T and B cells or mice lacking T cells only. Thus, AQP4 CNS autoimmunity was limited by T cell-dependent deletion of AQP4-reactive T cells. In contrast, myelin oligodendrocyte glycoprotein (MOG)-specific T cells survived and caused sustained disease in WT mice. These findings underscore the importance of peripheral T cell deletional tolerance to AQP4, which may be relevant to understanding the balance of AQP4-reactive T cells in health and in NMO. T cell tolerance to AQP4, expressed in multiple tissues, is distinct from tolerance to MOG, an autoantigen restricted in its expression.

A phase II study of concurrent WOKVAC vaccination with neoadjuvant chemotherapy and HER2-targeted monoclonal antibody therapy.

TPS636 Background: HER2+ breast cancer (BCa) is an aggressive breast cancer subtype. The treatment of Stage I-III HER2+ BCa with neoadjuvant chemotherapy and HER2 monoclonal antibodies (trastuzumab and pertuzumab) induces a pathologic complete response in 43-67% of patients. Residual HER2+ disease (i.e. residual cancer burden) after neoadjuvant therapy is associated with a significantly higher risk of recurrence. Evidence supports that HER2 immunity is progressively lost in HER2 oncogenesis and that restoration of HER2 specific CD4+ Th1 immunity is associated with a lower risk of recurrence. Trastuzumab can partially restore HER2 specific immunity, but only in a minority of patients. We have demonstrated that HER2 specific vaccination can augment HER2 specific immunity. While taxane chemotherapy can deplete immunosuppressive cells (MDSCs) 13 – 17 days after treatment creating an optimal time for vaccination. The WOKVAC vaccine is a plasmid vaccine encoding Th1 selective epitopes from HER2 and two other high-risk cancer antigens IGFBP-2 and IGF-1R. The Phase I trial of this vaccine reported that it is safe and all doses induced antigen specific Type I immune responses. We hypothesize that addition of WOKVAC to neoadjuvant chemo/HER2 targeted therapy will induce a significant increase in the number of patients with Th1 CD4+ and CD8+ T cells after neoadjuvant vaccine/chemo/HER2 therapy. Methods: Trial Design: Phase II single arm trial of WOKVAC vaccination with concurrent neoadjuvant taxane based chemotherapy, trastuzumab, and pertuzumab in patients with Stage I-III HER2+ (HR+/-) breast cancer. Enrolled patients will receive WOKVAC vaccine 150mcg on day 13 of cycles 1-3. WOKVAC vaccination may be given with either TCHP or THP. A maximum of 16 patients will be enrolled in this study. Objectives: The primary objective is to evaluate whether concurrent WOKVAC vaccine with chemotherapy and HER2 antibody therapy can significantly increase T-bet+ CD4+ and CD8+ TIL. Secondary objectives are to determine: (1) whether concurrent WOKVAC with neoadjuvant chemo/HER2 therapy is safe, (2) if the magnitude of vaccine induced antigen specific Th1 immunity after neoadjuvant therapy is associated with pathologic response. Statistical Methods: (1) In order to detect a 30% increase in the number of patients with Tbet+ CD4+ and CD8+ TIL in the enrolled patients with 80% power and a one-side alpha of 0.05, our enrollment goal for this study will be 16 patients. (2) Safety will be assessed per CTCAE v5.0, Benchmarks for safety needed to advance this trial concept to a Phase III study will be a grade 3 toxicity rate of ≤13% and a grade 4 toxicity rate of ≤ 7%. (3) The induction of Th1 immunity will be compared against the presence or absence of a complete pathologic response to determine if there is a significant correlation using Pearson r correlation analysis. Targeted Accrual: The study will accrue up to 16 patients. Clinical trial information: NCT04329065 .

The role of protease activity and monocyte subsets in initiating a Th2 response following allergen exposure

Abstract Allergy diseases are primarily caused by aberrant T helper 2 (Th2)-driven immune responses to allergens. During the primary sensitization phase, allergen specific Th2 cells are activated and become central players in orchestrating the subsequent development of allergic inflammation following secondary exposure to the same allergen. Still, the mechanisms by which the innate immune system detects allergens and initiates Th2 responses remain unclear. Protease activity is a feature common to many allergens, including house dust mite (HDM) and papain, a protein-digesting enzyme found in latex and papaya. Using models of Th2 airway inflammation induced by the allergens HDM and papain, we show that protease activity on allergens is required for the stimulation and clonal expansion of antigen specific T cells and their polarization towards a Th2 cell profile. Mechanistically, we found that protease activity was, in particular, required for enhanced migration of allergen-carrying dendritic cells (DCs) from the lung to draining lymph nodes and the subsequent activation of T cells throughout both phases of allergic disease. Our results suggest this occurs through CCR7 and CD86 expression. Further, we observed a decrease in monocytes and monocyte derived DCs when no protease activity was present within the allergens. Our results determine the early events involved in innate immune recognition of allergens and highlight a potential role for monocytic cells in initiating Th2 responses to allergens containing protease activity. Supported by National Institutes of Health grant 2R01AI116584 to B. León.

Monkeypox virus quadrivalent mRNA vaccine induces immune response and protects against vaccinia virus

Monkeypox has been declared a public health emergency by the World Health Organization. There is an urgent need for efficient and safe vaccines against the monkeypox virus (MPXV) in response to the rapidly spreading monkeypox epidemic. In the age of COVID-19, mRNA vaccines have been highly successful and emerged as platforms enabling rapid development and large-scale preparation. Here, we develop two MPXV quadrivalent mRNA vaccines, named mRNA-A-LNP and mRNA-B-LNP, based on two intracellular mature virus specific proteins (A29L and M1R) and two extracellular enveloped virus specific proteins (A35R and B6R). By administering mRNA-A-LNP and mRNA-B-LNP intramuscularly twice, mice induce MPXV specific IgG antibodies and potent vaccinia virus (VACV) specific neutralizing antibodies. Further, it elicits efficient MPXV specific Th-1 biased cellular immunity, as well as durable effector memory T and germinal center B cell responses in mice. In addition, two doses of mRNA-A-LNP and mRNA-B-LNP are protective against the VACV challenge in mice. And, the passive transfer of sera from mRNA-A-LNP and mRNA-B-LNP-immunized mice protects nude mice against the VACV challenge. Overall, our results demonstrate that mRNA-A-LNP and mRNA-B-LNP appear to be safe and effective vaccine candidates against monkeypox epidemics, as well as against outbreaks caused by other orthopoxviruses, including the smallpox virus.

Abstract 4056: Intratumoral delivery of autologous tumor antigen specific CD4 Th1 cells combined with dendritic cells eradicates HER2 mammary carcinoma

Abstract Human epidermal growth factor receptor 2 (HER2) overexpression accounts for 30% of invasive breast cancer (BC) and is critically associated with aggressive disease, recurrence and metastasis. Adoptive cell therapy approaches using cytotoxic CD8 T cells and natural killer cells have been shown to trigger anti-tumor immunity in BC. However, the immunosuppressive tumor microenvironment (TME) can inhibit response to these therapies and diminish the presence and function of tumor-infiltrating lymphocytes. Recently, we have shown a critical role for anti-tumor CD4 Th1 cells in dendritic cells (DC) intratumoral (i.t.) delivery in combination with anti-HER2 therapy. This combination therapy enhanced systemic and local anti-tumor immunity and eradicated tumors in HER2 positive BC with a requirement for CD4 Th1 cells. Here we investigated the efficacy of i.t. delivery of both autologous anti-tumor CD4 Th1 cells and tumor antigen pulsed type 1 polarized dendritic cells (HER2-DC1) in a HER2 mammary carcinoma model. CD4 T cells were isolated from BALB/c mice that had completely regressed (pCR) from orthotopic TUBO tumors following HER2-DC1 i.t. or combination therapy with anti-HER2 antibody. CD4 Th1 cells were then expanded by co-culturing with HER2-DC1 in the presence of interleukin (IL)-2 and IL-7 cytokines. BALB/c mice bearing orthotopic TUBO tumors were treated weekly for six weeks with anti-tumor CD4 Th1 cells i.t., non-specific CD4 Th1 cells i.t., HER2-DC1 i.t. or combination therapy. The i.t. delivery of anti-tumor CD4 Th1 cells combined with HER2-DC1 induced a strong anti-tumor response with survival benefit and complete tumor eradication in 50% of treated mice. Importantly, the i.t. delivery of anti-tumor CD4 Th1 cells were critical for providing priming signals to HER2-DC1 within the TME via CD40/CD40L engagement (licensing). This was supported by a strong anti-tumor response and complete tumor regression in 60% of orthotopic TUBO tumor bearing mice treated with CD40/CD40L licensed HER2-DC1 i.t. therapy compared to un-licensed HER2-DC1 i.t. therapy. Additionally, enhanced survival and functionality of human and mouse HER2-DC1 was observed following activation of CD40/CD40L signaling using anti-CD40 agonistic antibody in vitro. Collectively, these results suggest a new promising therapeutic strategy using DC1-anti-tumor CD4 Th1 adoptive cell therapy intratumoral delivery to induce DC1 priming and create robust anti-tumor immunity within the TME in BC. Citation Format: Ganesan Ramamoorthi, Amy Aldrich, Colin Snyder, Brian Czerniecki. Intratumoral delivery of autologous tumor antigen specific CD4 Th1 cells combined with dendritic cells eradicates HER2 mammary carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4056.