Higher Levels Of IFN-γ Research Articles

Immune Biomarkers at Birth Predict Lower Respiratory Tract Infection Risk in a Large Birth Cohort.

Lower respiratory tract infections (LRTIs) remain the leading cause of infant morbidity and mortality worldwide and affect long-term respiratory health. Identifying immunological determinants of LRTI susceptibility may help stratify disease risk and identify therapies. This study aimed to identify neonatal immunological factors predicting LRTI risk in infancy. Cord blood plasma from 191 neonates from the Boston Birth Cohort was analyzed for 28 soluble immune factors. LRTI was defined as bronchiolitis, bronchitis, or pneumonia during the first year of life. Welch's t-test demonstrated significantly higher log10 transformed concentrations of IL-17 and IFNγ in the LRTI group compared to neonates without LRTI in the first year of life (p < 0.05). Risk associations were determined using multivariate survival models. There were 29 infants with LRTIs. High cord blood levels of IFNγ (aHR = 2.35, 95% CI 1.07-5.17), TNF-β (aHR = 2.86, 95% CI 1.27-6.47), MIP-1α (aHR = 2.82, 95% CI 1.22-6.51), and MIP-1β (aHR = 2.34, 95% CI 1.05-5.20) were associated with a higher risk of LRTIs. RANTES was associated with a lower risk (aHR = 0.43, 95% CI 0.19-0.97). Soluble immune factors linked to antiviral immunity (IFNγ) and cytokines mediating inflammatory responses (TNF-β), and cell homing (MIP-1α/b), at birth were associated with an increased risk of LRTIs during infancy.

α2,6 sialylation distinguishes a novel active state in CD4+ and CD8+ cells during acute Toxoplasma gondii infection.

Toxoplasmosis is a worldwide parasitosis that is usually asymptomatic; cell-mediated immunity, particularly T cells, is a crucial mediator of the immune response against this parasite. Membrane protein expression has been studied for a long time in T lymphocytes, providing vital information to determine functional checkpoints. However, less is known about the role of post-translational modifications in T cell function. Glycosylation plays essential roles during maturation and function; particularly, sialic acid modulation is determinant for accurate T cell regulation of processes like adhesion, cell-cell communication, and apoptosis induction. Despite its importance, the role of T cell sialylation during infection remains unclear. Herein, we aimed to evaluate whether different membrane sialylation motifs are modified in T cells during acute Toxoplasma gondii infection using different lectins. To this end, BALB/c Foxp3EGFP mice were infected with T. gondii, and on days 3, 7, and 10 post-infection, splenocytes were obtained to analyze conventional (Foxp3-) CD4+ and CD8+ populations by flow cytometry. Among the different lectins used for analysis, only Sambucus nigra lectin, which detects sialic acid α2,6 linkages, revealed two distinctive populations (SNBright and SN-/Dim) after infection. Further characterization of CD4+ and CD8+ SN-/Dim lymphocytes showed that these are highly activated cells, with a TEf/EM or TCM phenotype that produce high IFN-γ levels, a previously undescribed cell state. This work demonstrates that glycan membrane analysis in T cells reveals previously overlooked functional states by evaluating only protein expression.

Microbiota-induced plastic T cells enhance immune control of antigen-sharing tumors.

Therapies that harness the immune system to target and eliminate tumor cells have revolutionized cancer care. Immune checkpoint blockade (ICB), which boosts the anti-tumor immune response by inhibiting negative regulators of T cell activation1-3, is remarkably successful in a subset of cancer patients, yet a significant proportion do not respond to treatment, emphasizing the need to understand factors influencing the therapeutic efficacy of ICB4-9. The gut microbiota, consisting of trillions of microorganisms residing in the gastrointestinal tract, has emerged as a critical determinant of immune function and response to cancer immunotherapy, with multiple studies demonstrating association of microbiota composition with clinical response10-16. However, a mechanistic understanding of how gut commensal bacteria influence the efficacy of ICB remains elusive. Here we utilized a gut commensal microorganism, segmented filamentous bacteria (SFB), which induces an antigen-specific Th17 cell effector program17, to investigate how colonization with it affects the efficacy of ICB in restraining distal growth of tumors sharing antigen with SFB. We find that anti-PD-1 treatment effectively inhibits the growth of implanted SFB antigen-expressing melanoma only if mice are colonized with SFB. Through T cell receptor clonal lineage tracing, fate mapping, and peptide-MHC tetramer staining, we identify tumor-associated SFB-specific Th1-like cells derived from the homeostatic Th17 cells induced by SFB colonization in the small intestine lamina propria. These gut-educated ex-Th17 cells produce high levels of the pro-inflammatory cytokines IFN-γ and TNF-α, and promote expansion and effector functions of CD8+ tumor-infiltrating cytotoxic lymphocytes, thereby controlling tumor growth. A better understanding of how distinct intestinal commensal microbes can promote T cell plasticity-dependent responses against antigen-sharing tumors may allow for the design of novel cancer immunotherapeutic strategies.

Association between endometrial protein expression of IFN-γ and delayed conception after parturition in dairy cows.

The cytokine context present in the reproductive tract of cows is closely involved in normal uterine functions, including the estrous cycle and the establishment and maintenance of pregnancy. However, the roles of some cytokines in the uterus, and their relation with reproductive performance remain to be elucidated. Thus, this study aimed to examine the protein expression of several cytokines such as TNFα, IL-6, IL-8, IFNγ, IL-4, and TGF-β3 in endometrial biopsies previous to conception, to evaluate the possible association with delayed conception in dairy cows. Protein expression levels were evaluated by immunohistochemistry. Results showed that the protein expression levels of TNFα, IL-6, IL-4 and TGF-β3 were not associated with the parturition-conception interval, whereas the high protein expression levels of IFNγ were associated with the parturition-conception interval. Finally, the low protein expression of IL-8 showed a statistical tendency to be associated with delayed conception. This is the first report about the protein expression of IFN-γ in the endometrium of dairy cows and also, this cytokine could enhance the favorable conditions to achieve an early pregnancy.

Characteristics of SARS-CoV-2 and Opisthorchis viverrini coinfections: insights into immune responses and clinical outcomes.

The effects of co-infections with SARS-CoV-2 and parasitic diseases have been little investigated in terms of immune response, disease dynamics, and clinical outcomes. This study aimed to explore the impact of co-infection with Opisthorchis viverrini and SARS-CoV-2 on the immune response concerning clinical symptoms and the severity of pulmonary abnormalities. A cross-sectional study was conducted, including healthy participants as controls, participants with opisthorchiasis, SARS-CoV-2 infection, and a co-infection group with both diseases. Characteristics of SARS-CoV-2 infection were assessed based on clinical parameters and severity of pulmonary abnormalities, whereas opisthorchiasis burden was evaluated by eggs-per-gram (EPG) counts. Immune responses were assessed by measuring levels of interferon-γ (IFN-γ), SARS-CoV-2 anti-spike receptor binding domain (RBD) IgG, and neutralizing antibody against SARS-CoV-2. In the co-infected group, clinical parameters and hospitalization rates were lower than in the SARS-CoV-2 group. Pulmonary abnormalities, such as bronchial fibrosis, were commonly observed in the SARS-CoV-2 group, leading to hospitalization in some cases. Participants with opisthorchiasis had higher IFN-γ levels than healthy individuals. IFN-γ levels were significantly lower in the co-infection group compared with the SARS-CoV-2 group (P = 0.002). There was a significant (P = 0.044) positive correlation between RBD-specific IgG and percent neutralization levels in the SARS-CoV-2 group. Levels of both were somewhat lower (not statistically significant) in the co-infection group. A negative correlation was observed between opisthorchiasis burden (EPG counts) and IFN-γ and RBD-specific IgG levels in the co-infected group. Following vaccination, the increase in IgG levels against the RBD protein was significantly lower in the co-infected group than in the SARS-CoV-2 group. These results suggest that O. viverrini infection suppresses immune responses and may lead to a reduction in severity in cases of SARS-CoV-2 co-infection.

Therapeutic potential of oral alginate nanoparticles against experimental toxoplasmosis

Side effects and low efficacy of current anti-toxoplasmosis therapeutics against encysted bradyzoites necessitate research into alternative safe therapeutic options. The safety, immunostimulatory, and antimicrobial properties of alginate nanoparticle formulation (Alg-NP) highlight its potential as an oral therapy against acute toxoplasmosis. In the current study, Alg-NP was formulated and characterized and then assessed for its anti-Toxoplasma effects using parasitological, ultrastructural, immunological, and histopathological studies. Treatment with Alg-NP significantly prolonged mice survival and reduced the parasite burden in both peritoneal fluid and tissue impression smears. In addition, it altered parasite viability and caused severe tachyzoite deformities as evidenced by ultrastructural studies. Alg-NP induced high levels of serum IFN-γ in infected mice with significant amelioration in histopathological changes in both hepatic and splenic tissue sections. In conclusion, Alg-NP could be considered a promising therapeutic agent against acute murine toxoplasmosis, and owing to its safety, it could potentially be enlisted for human use.

Role of Serum IFN-γ and IL-10 as Predictive Biomarkers of Vitiligo Disease Activity: A Case-Control Study

Abstract Background and Objectives: IFNγ is a pleiotropic cytokine and through the regulation of immunologically relevant genes, they coordinate a wide range of cellular programs.[1] As a potent pro-inflammatory cytokine, it plays a pivotal role to induce depigmentation in vitiligo. In this study, we aim to assess the role of IFNγ in the activity, duration and extent of the disease and the antagonistic action of IL-10 is also assessed in vitiligo. Materials and Methods: A case-control study was conducted with 100 study participants with 50 cases clinically diagnosed as Vitiligo and 50 controls. All patients underwent complete evaluation with detailed demographic parameters, history and physical examination. The severity of the disease was assessed clinically by Vitiligo Area Scoring Index (VASI) and Vitiligo Disease Activity Score (VIDA). And blood investigations done were IFN-γ and IL-10. Results: We observed significantly higher levels of serum IFNγ levels in the patient group when compared with those of the normal controls (p=0.002) and showed a positive correlation with the activity and severity of the disease with a significant VASI (p=0.05) and VIDA score (p=&lt;0.001). The mean serum IL-10 (p&lt;0.001) in patients with vitiligo was significantly lower than that in the control group. Conclusion: This study showed significantly high serum levels of IFN-γ and correspondingly low levels of IL-10. New strategies, such as the combination of IFNγ blockade with inhibition of other signalling pathways, may further improve IFNγ targeted immunotherapy of the disease.

Immuno-informatics study identifies conserved T cell epitopes in non-structural proteins of Bluetongue virus serotypes: formulation of a computationally optimized next-generation broad-spectrum multi-epitope vaccine.

Bluetongue (BT) poses a significant threat to the livestock industry, affecting various animal species and resulting in substantial economic losses. The existence of numerous BT virus (BTV) serotypes has hindered control efforts, highlighting the need for broad-spectrum vaccines. In this study, we evaluated the conserved amino acid sequences within key non-structural (NS) proteins of BTV and identified numerous highly conserved murine- and bovine-specific MHC class I-restricted (MHC-I) CD8+ and MHC-II-restricted CD4+ epitopes. We then screened these conserved epitopes for antigenicity, allergenicity, toxicity, and solubility. Using these epitopes, we developed in silico-based broad-spectrum multiepitope vaccines with Toll-like receptor (TLR-4) agonists. The predicted proinflammatory cytokine response was assessed in silico using the C-IMMSIM server. Structural modeling and refinement were achieved using Robetta and GalaxyWEB servers. Finally, we assessed the stability of the docking complexes through extensive 100-nanosecond molecular dynamics simulations before considering the vaccines for codon optimization and in silico cloning. We found many epitopes that meet these criteria within NS1 and NS2 proteins and developed in silico broad-spectrum vaccines. The immune simulation studies revealed that these vaccines induce high levels of IFN-γ and IL-2 in the vaccinated groups. Protein-protein docking analysis demonstrated promising epitopes with strong binding affinities to TLR-4. The docked complexes were stable, with minimal Root Mean Square Deviation and Root Mean Square Fluctuation values. Finally, the in silico-cloned plasmids have high % of GC content with > 0.8 codon adaptation index, suggesting they are suitable for expressing the protein vaccines in prokaryotic system. These next-generation vaccine designs are promising and warrant further investigation in wet lab experiments to assess their immunogenicity, safety, and efficacy for practical application in livestock. Our findings offer a robust framework for developing a comprehensive, broad-spectrum vaccine, potentially revolutionizing BT control and prevention strategies in the livestock industry.

Inhibition of DNMTs increases neoantigen-reactive T-cell toxicity against microsatellite-stable colorectal cancer in combination with radiotherapy

The therapeutic efficacy of immunotherapy is limited in the majority of colorectal cancer patients due to the low mutational and neoantigen burdens in this immunogenically “cold” microsatellite stability-colorectal cancer (MSS-CRC) cohort. Here, we showed that DNA methyltransferase (DNMT) inhibition upregulated neoantigen-bearing gene expression in MSS-CRC, resulting in increased neoantigen presentation by MHC class I in tumor cells and leading to increased neoantigen-specific T-cell activation in combination with radiotherapy. The cytotoxicity of neoantigen-reactive T cells (NRTs) to DNMTi-treated cancer cells was highly cytotoxic, and these cells secreted high IFNγ levels targeting MSS-CRC cells after ex vivo expansion of NRTs with DNMTi-treated tumor antigens. Moreover, the therapeutic efficacy of NRTs further increased when NRTs were combined with radiotherapy in vivo. Administration of DNMTi-augmented NRTs and radiotherapy achieved an ∼50 % complete response and extended survival time in an immunocompetent MSS-CRC animal model. Moreover, remarkably, splenocytes from these mice exhibited neoantigen-specific T-cell responses, indicating that radiotherapy in combination with DNMTi-augmented NRTs prolonged and increased neoantigen-specific T-cell toxicity in MSS-CRC patients. In addition, these DNMTi-augmented NRTs markedly increase the therapeutic efficacy of cancer vaccines and immune checkpoint inhibitors (ICIs). These data suggest that a combination of radiotherapy and epi-immunotherapeutic agents improves the function of ex vivo-expanded neoantigen-reactive T cells and increases the tumor-specific cytotoxic effector population to enhance therapeutic efficacy in MSS-CRC.

Engineered dendritic cells-derived exosomes harboring HIV-1 Nefmut-Tat fusion protein and heat shock protein 70: A promising HIV-1 safe vaccine candidate

Antigen presenting cells (APCs)-derived exosomes are nano-vesicles that can induce antigen-specific T cell responses, and possess therapeutic effects in clinical settings. Moreover, dendritic cells (DCs)-based vaccines have been developed to combat human immunodeficiency virus-1 (HIV-1) infection in preclinical and clinical trials. We investigated the immunostimulatory effects (B- and T-cells activities) of DCs- and exosomes-based vaccine constructs harboring HIV-1 Nefmut-Tat fusion protein as an antigen candidate and heat shock protein 70 (Hsp70) as an adjuvant in mice. The modified DCs and engineered exosomes harboring Nefmut-Tat protein or Hsp70 were prepared using lentiviral vectors compared to electroporation, characterized and evaluated by in vitro and in vivo immunological tests. Our data indicated that the engineered exosomes induced high levels of total IgG, IgG2a, IFN-γ, TNF-α and Granzyme B. Moreover, co-injection of exosomes harboring Hsp70 could significantly increase the secretion of antibodies, cytokines and Granzyme B. The highest levels of IFN-γ and TNF-α were observed in exosomes harboring Nefmut-Tat combined with exosomes harboring Hsp70 (Exo-Nefmut-Tat + Exo-Hsp70) regimen after single-cycle replicable (SCR) HIV-1 exposure. Generally, Exo-Nefmut-Tat + Exo-Hsp70 regimen can be considered as a promising safe vaccine candidate due to high T-cells (Th1 and CTL) activity and its maintenance against SCR HIV-1 exposure.

Immunotherapy Combining Mimotopes Selected by Phage Display Plus Amphotericin B Is Effective for Treatment Against Visceral Leishmaniasis.

The treatment for visceral leishmaniasis (VL) causes toxicity in patients, entails high cost and/or leads to the emergence of resistant strains. No human vaccine exists, and diagnosis presents problems related to the sensitivity or specificity of the tests. Here, we tested two phage clones, B1 and D11, which were shown to be protective against Leishmania infantum infection in a murine model as immunotherapeutics to treat mice infected with this parasite species. The phages were used alone or with amphotericin B (AmpB), while other mice received saline, AmpB, a wild-type phage (WTP) or WTP/AmpB. Results showed that the B1/AmpB and D11/AmpB combinations induced polarised Th1-type cellular and humoral responses, which were primed by high levels of parasite-specific IFN-γ, IL-12, TNF-α, nitrite and IgG2a antibodies, which reflected in significant reductions in the parasite load in distinct organs of the animals when analyses were performed 1 and 30 days after the treatments. Reduced organic toxicity was also found in these animals, as compared with the controls. In conclusion, preliminary data suggest the potential of the B1/AmpB and D11/AmpB combinations as immunotherapeutics against L. infantum infection.

Unraveling the immune dynamics of lung tissue infected with Francisella tularensis in a spatial context

Abstract Francisella tularensis (Ftt) is a highly virulent bacterial pathogen whose vaccine offers only short-lived immunity. While CD4+ T cells are required for protection, the mechanism behind waning immunity remains unclear. One potential reason is that the local environment induced by the bacteria inhibits T cell function. We therefore carried out a multi-omics study of the infection microenvironment following vaccination and secondary challenge in mice. Flow cytometry and single cell sequencing analysis revealed that during Ftt secondary challenge, the CD4+ T cells in vaccinated animals show a delayed proliferative response and produced high levels of IFN-γ which correlated with an up-regulation of indoleamine 2,3-dioxygenase (IDO1) by endothelial cells. Importantly, increased IDO1 expression corresponded with decreased levels of tryptophan in the lung as measured by LC/MS. Given the requirement for tryptophan in optimal T cell proliferation, we investigated if IDO1 expression was spatially associated with sites of bacterial replication. We performed multiplex immunohistochemistry targeting bacterial antigen, immune cells, and nutrient depleting enzymes and quantified the proximity of CD4+ T cells to IDO1-expressing cells which arose just outside the points of bacterial replication. Overall, this research uncovered a potential systems level of regulation that is inhibiting T cell function. All studies were approved by the IACUC and was supported by the IRP of the NIH/NIAID

Construction of novel multi-epitope-based diagnostic biomarker HP16118P and its application in the differential diagnosis of Mycobacterium tuberculosis latent infection

Tuberculosis (TB) is an infectious disease that significantly threatens human health. However, the differential diagnosis of latent tuberculosis infection (LTBI) and active tuberculosis (ATB) remains a challenge for clinicians in early detection and preventive intervention. In this study, we developed a novel biomarker named HP16118P, utilizing 16 helper T lymphocyte (HTL) epitopes, 11 cytotoxic T lymphocyte (CTL) epitopes, and 8 B cell epitopes identified from 15 antigens associated with LTBI-RD using the IEDB database. We analyzed the physicochemical properties, spatial structure, and immunological characteristics of HP16118P using various tools, which indicated that it is a hydrophilic and relatively stable alkaline protein. Furthermore, HP16118P exhibited good antigenicity and immunogenicity, while being non-toxic and non-allergenic, with the potential to induce immune responses. We observed that HP16118P can stimulate the production of high levels of IFN-γ+ T lymphocytes in individuals with ATB, LTBI, and health controls. IL-5 induced by HP16118P demonstrated potential in distinguishing LTBI individuals and ATB patients (p=0.0372, AUC=0.8214, 95% CI [0.5843 to 1.000]) with a sensitivity of 100% and specificity of 71.43%. Furthermore, we incorporated the GM-CSF, IL-23, IL-5, and MCP-3 induced by HP16118P into 15 machine learning algorithms to construct a model. It was found that the Quadratic discriminant analysis model exhibited the best diagnostic performance for discriminating between LTBI and ATB, with a sensitivity of 1.00, specificity of 0.86, and accuracy of 0.93. In summary, HP16118P has demonstrated strong antigenicity and immunogenicity, with the induction of GM-CSF, IL-23, IL-5, and MCP-3, suggesting their potential for the differential diagnosis of LTBI and ATB.

477 Evaluating the therapeutic efficacy of combination IL-12 and trabectedin for the treatment of triple negative breast cancer

OBJECTIVES/GOALS: IL-12 has potent immune effects but the presence of myeloid-derived suppressor cells (MDSC) can inhibit IL-12-induced NK cell cytotoxicity. Thus, we hypothesized that combining IL-12 with trabectedin, an immunosuppressive myeloid cell depleting agent, would improve its therapeutic efficacy in triple negative breast cancer (TNBC). METHODS/STUDY POPULATION: Combination IL-12 and trabectedin was tested in the 4T1 mouse model of TNBC. 4T1 cells were injected into the mammary fat pad of female BALB/cj mice. When tumors reached 50 mm3, mice were randomly divided into 4 groups and treated with PBS, IL-12 (0.5 μg/mouse 3x/wk), 0.15 mg/kg trabectedin weekly or the combination. Tumor volumes were measured by calipers. Mass cytometry was performed on spleens and tumors using a 35-antibody panel. Plasma IFN-γ levels were measured by ELISA. The role of NK cells was evaluated via depletion with anti-asialo-GM1. The Luminex Discovery Assay was used to measure plasma cytokines and immunohistochemistry was performed for CD4 and CD8a. Linear/nonlinear mixed effects modeling was used for in vivo data analysis and applicable t- or ANOVA tests were used for in vitrodata analysis. RESULTS/ANTICIPATED RESULTS: Combination IL-12 and trabectedin led to a significant reduction in tumor burden compared to single-agent IL-12, trabectedin and control treatments (all p&lt;0.001), as well as higher levels of IFN-γ (all p&lt;0.04). One combination treated mouse had complete tumor regression. Splenic MDSC were significantly decreased in combination treated mice. NK depletion abrogated the effects of combination therapy. Compared to mice receiving a control antibody, NK depletion of combination treated mice led to lower levels of CCL5 (p&lt;0.01) and CXCL10 (p&lt;0.001) and significantly higher tumor burden (p=0.001). CD8+T cell levels were significantly higher in combination treated mice compared to those receiving IL-12 (p&lt;0.01), and these levels were decreased when mice were depleted of NK cells (p=0.01). DISCUSSION/SIGNIFICANCE: TNBC represents 15% of all breast cancer diagnoses and is associated with a worse prognosis compared to other subtypes. Black women are twice as likely to be diagnosed with TNBC and more likely to die from disease than White women. Thus, there is an increasing need to develop additional therapeutic options for this disease.

Abstract 4017: CAR T cell-mediated cytotoxicity and cytokine release in response to varying levels of antigen expression on target cells

Abstract The development of immunotherapies relies on the use of in vitro potency assays, which are crucial for understanding the complex interactions between immune cells (effectors) and cancer cells (targets). Chimeric antigen receptor (CAR) T-cell therapy is an immunotherapeutic approach that exhibits promise; however, varying levels of antigen expression on the surface of tumor cells can influence the efficacy of CAR T cell-mediated killing. To fully understand the impact of target antigen density on CAR T cell activity, we utilized a matrix approach to assess both target cell death, using an impedance-based in vitro potency assay to quantify CAR T cell-mediated cytotoxicity, as well as cytokine release with homogeneous LumitTM immunoassays. SKOV3 (high HER2 expression), A549 (low HER2 expression), or MDA-MB-231 (no HER2 expression) target cells were seeded into a 96-well microplate with embedded electrodes in the substrate that detect the attachment and proliferation of target cells. HER2 CAR T cells were added after 24 hours at effector-to-target (E:T) ratios of 1:5 or 1:1 and target cell cytolysis was recorded continuously by the Maestro Z for 72 hours. Cytolysis of the target cells was calculated by comparing the treated wells to no treatment control wells and full lysis wells (1% TritonX). At 72 hours post CAR T cell addition, complete killing was observed in SKOV3 cells at the 1:1 ratio, while A549 cells exhibited only 80% cytolysis. MDA-MB-231 cells showed 20% cytolysis, likely due to nonspecific killing by non-engineered T cells, as approximately only 78% of the CAR T cell population was CAR positive. Because the cytolysis assay was label-free, cytokine analysis was able to be multiplexed with the same plates. To assess release of pro-inflammatory cytokines, TNF-α and IFN-γ, we collected supernatant post-antigen exposure at 24, 48, and 72 h. At the 1:1 E:T ratio, CAR T cells co-cultured with target cells expressing low (A549) or high (SKOV3) levels of HER2 had the highest TNF-α production at 24 hours, with CAR T cells co-cultured with SKOV3 cells releasing 30.6% more TNF-α when compared to A549 cells. The highest levels of IFN-γ were detected in both A549 and SKOV3 groups at the 1:1 E:T ratio, with the highest levels observed from CAR T cells co-cultured with SKOV3 cells at 72 hours, releasing approximately 1459.6 +/- 357.3 pg/mL of IFN-γ, compared to 1093.8 +/- 387.5 pg/mL IFN-γ released by CAR T cells co-cultured with A549 cells. As expected, CAR T cells co-cultured with MDA-MB-231 cells did not release any detectable TNF-α or IFN-γ at either E:T ratios during the duration of the experiment. Future work will continue to explore multiplexed potency assays for the evaluation of CAR T cell therapies. Citation Format: Sierra Barnes, Denise Sullivan, Dan Lazar, Kim Haupt, Danielle Califano, Stacie Chvatal, Daniel Millard. CAR T cell-mediated cytotoxicity and cytokine release in response to varying levels of antigen expression on target cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4017.

Abstract 5251: RNA primed SMAR-T cells against multiple driver mutations, all HLA's designed for first line therapy

Abstract Success of immune checkpoint inhibitors (e.g., anti-PD1 antibodies) have revolutionized cancer immunotherapy by demonstrating that a patient’s own T-cells recognize and treat cancer. The efficacy of PD-1 blockade is driven by recruitment of new T-cells from blood rather than via activation of pre-existing tumor infiltrating lymphocytes (Yost K.E., et al. Nat. Med. 2019). However, anti-PD1 therapy is most effective in ~5% of malignancies i.e., cancers with high mutational burden. Hence, the challenge of addressing most lower mutational burden cancers (the ~95%) needs an alternate treatment strategy. We address this challenge by using RNA to prime and expand peripheral T-cells to cancer-specific mutations ex-vivo. Using our proprietary, patented, and robust manufacturing method, we can generate T-cell populations reactive to as low as 8 and as high as 40 cancer-specific mutant proteins. In-vitro, these T-cells have cancer mutation-specific cytotoxicity and do not kill the normal cells. Further, the T-cells express homing receptors enabling them to infiltrate tumors and express high levels of TNFα and IFNγ, which are associated with effective tumor cytotoxicity and pro-inflammatory modification of tumor microenvironment (TME). Additional characterization shows these cells to be predominantly CD4+ and CD8+ T-cells bearing central and effector memory phenotypic with negligible regulatory or exhausted T-cells. We believe our T-cells can be used for cellular therapy in conjunction with, or as an alternative to, immune checkpoint inhibitors to treat lower mutational burden cancers present in most patients. Citation Format: Alfred E. Slanetz, Sriram Srivatsa, Farzona Muzaffar, Walter Barry, David Ryan, Salim Metri, Michael Slanetz. RNA primed SMAR-T cells against multiple driver mutations, all HLA's designed for first line therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5251.

Abstract 7260: An HSV-1-based vector for local delivery of IL-12 and IL-2 reshapes the immune landscape leading to tumor clearance and systemic immune surveillance

Abstract Clinical use of recombinant interleukin (IL)-12 and -2 therapy has been hindered due to unfavorable kinetics and toxicity associated with systemic exposure. Durable targeted delivery to the tumor microenvironment (TME) is a promising therapeutic strategy to overcome these clinical limitations, prompting the development of KB707, a replication-defective herpes simplex virus type 1 (HSV-1)-based vector encoding human IL-12 and -2, for the treatment of solid tumors. Prior studies in mice demonstrated that local vector-encoded cytokine administration enhanced murine IL-12/IL-2 expression while limiting systemic exposure compared to conventional protein therapy. In a syngeneic, B16-F10 melanoma mouse model, intratumoral murine KB707-equivalent treatment led to improved survival in a unilateral tumor model and an abscopal effect in a bilateral tumor model. Additionally, the majority of previously treated animals survived rechallenge with B16-F10 in the absence of further therapy, indicative of systemic anti-tumor immune memory. Together, these results suggested that vector-mediated delivery of IL-12 and -2 could elicit both local and systemic anti-tumor immune responses. To elucidate the underlying changes to the immune landscape, B16-F10 tumor-bearing animals were treated intratumorally with vector or vehicle control on day 0 and were sacrificed on day 7; a second cohort of tumor-bearing animals received two vector doses (days 0 and 7) and were sacrificed on days 8, 10, 12, or 14. Immune cell profiling of samples taken from the tumor, tumor draining lymph node (dLN), spleen, and whole blood were analyzed by flow cytometry, and serum cytokine analysis was conducted by multiplex ELISA. Flow cytometric analysis of tumors revealed that vector treatment resulted in increased frequencies of proliferating and activated effector CD8+ and CD4+ T cells, peaking 24 hours post-second dose. These results correlated with high levels of the proinflammatory cytokines IFNγ and TNFα in the serum of vector recipient animals. The blood, spleens, and dLNs also showed increased frequencies of activated effector CD8+ and CD4+ T cells, peaking 24-72 hours post-second dose. Interestingly, although IL-2 alone has been shown to promote immune suppression and tumor outgrowth by mediating regulatory T cell (Treg) expansion and infiltration in certain contexts, the frequency of Tregs in treated vs. control tumors was substantially reduced after first and repeat dosing. Together, these results support a mechanism by which vector-driven local expression of IL-12 and IL-2 can expand and activate effector T cells both in the TME and systemically while simultaneously repressing Tregs, consistent with the previously described inhibition of primary tumor outgrowth, abscopal effects on secondary tumors, and prevention of recurrence observed with this therapeutic approach. Citation Format: Dana M. Previte, Trevor J. Parry, Suma M. Krishnan. An HSV-1-based vector for local delivery of IL-12 and IL-2 reshapes the immune landscape leading to tumor clearance and systemic immune surveillance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7260.

Design of a multi-epitope vaccine against goatpox virus using an immunoinformatics approach.

Goatpox, a severe infectious disease caused by goatpox virus (GTPV), leads to enormous economic losses in the livestock industry. Traditional live attenuated vaccines cause serious side effects and exist a risk of dispersal. Therefore, it is urgent to develop efficient and safer vaccines to prevent and control of GTPV. In the present study, we are aimed to design a multi-epitope subunit vaccine against GTPV using an immunoinformatics approach. Various immunodominant cytotoxic T lymphocytes (CTL) epitopes, helper T lymphocytes (HTL) epitopes, and B-cell epitopes from P32, L1R, and 095 proteins of GTPV were screened and liked by the AAY, GPGPG, and KK connectors, respectively. Furthermore, an adjuvant β-defensin was attached to the vaccine's N-terminal using the EAAAK linker to enhance immunogenicity. The constructed vaccine was soluble, non-allergenic and non-toxic and exhibited high levels of antigenicity and immunogenicity. The vaccine's 3D structure was subsequently predicted, refined and validated, resulting in an optimized model with a Z-value of -3.4. Molecular docking results demonstrated that the vaccine had strong binding affinity with TLR2(-27.25 kcal/mol), TLR3(-39.84 kcal/mol), and TLR4(-59.42 kcal/mol). Molecular dynamics simulation results indicated that docked vaccine-TLR complexes were stable. Immune simulation analysis suggested that the vaccine can induce remarkable increase in antibody titers of IgG and IgM, higher levels of IFN-γ and IL-2. The designed GTPV multi-epitope vaccine is structurally stable and can induce robust humoral and cellular immune responses, which may be a promising vaccine candidate against GTPV.

Controversial Issues of Immunopathogenesis of Psoriasis and Atopic Dermatitis

Psoriasis (PsO) and atopic dermatitis (AD have much in common: both diseases are widespread, characterized by a chronic relapsing course, primarily affect the skin and lead to a quality reduction of life of patients, regardless of their age. The pathogenesis of these two dermatoses, which are the most common in the practice of a pediatric dermatologist, is quite different. PsO is a chronic inflammatory skin disease, the pathogenesis of which is associated with the involvement of the Th1 pathway: Th17 cells and the IL-23/IL-17 axis. AD, in turn, is usually associated with high levels of IL-4, IL-5, IL-13, IL-31 and IFN-γ produced by activated T-helper 2 (Th2) cells. The clinical symptoms and immunopathological responses of these two skin conditions tend to differ. However, patients with PsO may sometimes present with a skin rash resembling AD combined with intense itching and laboratory increase in immunoglobulin E (IgE) which may indicate the need to change the paradigm of dominance of only one type of T-inflammation in patients with these diseases.

Transdiagnostic dimensions of symptoms and experiences associated with immune proteins in the continuity of psychosis.

There is limited evidence as to whether the immune protein profile is associated with a particular symptomatology pattern across the psychosis continuum. We estimated two bifactor models of general and specific dimensions of psychotic experiences in unaffected siblings of patients (n = 52) and community controls (n = 200), and of psychotic symptoms in first-episode psychosis (FEP) patients (n = 110). We evaluated associations between these transdiagnostic dimensions and trait (TNF-α, IFN-γ), state (IL-6, IL-1β), and regulatory (TGF-β, IL-10, IL-4) cytokines. We explored whether schizophrenia genetic liability (schizophrenia polygenic risk score; SZ-PRS) modified the associations. High levels of trait marker IFN-γ were associated with the severity of general psychosis dimension in the unaffected siblings and community controls, expanding to the depressive dimension in siblings and to the manic dimension in FEP. High TNF-α levels were associated with more positive psychotic experiences in unaffected siblings and manic symptoms in FEP. Low levels of state markers IL-6 and IL-1β were observed in unaffected siblings presenting more depressive experiences. Still, high levels of IL-6 and IL-1β were associated with the severity of the depressive and negative symptom dimensions at FEP. The severity of transdiagnostic dimension scores across the three groups was associated with lower regulatory cytokines. Exploratory analysis suggested that a high SZ-PRS contributed mostly to associations with psychotic dimensions. IFN-γ mapped onto the multidimensional expression of psychosis, reinforcing the trait concept. State markers IL-6 and IL-1β may fluctuate along the spectrum. Dysfunction in the regulatory arm may disinhibit the inflammatory system. Associations with psychotic dimensions may be more prone to SZ-PRS susceptibility.