Upregulation Of IFN-γ Research Articles

Expression kinetics of cytokines and the humoral antibody response concerning short-term protection induced by radiation-attenuated Trypanosoma evansi in bovine calves.

'Surra', an economically important disease of livestock, is caused by the parasitic blood protozoon Trypanosoma evansi. Both innate and adaptive immunity contribute to the protection against this infection. T-helper cells play a crucial role in the antibody-mediated clearance of T. evansi. We present here the data on the kinetics of expression of important Th1, Th2 and Th17 cytokines, vis-a-vis the dynamics of humoral response in bovine calves following immunization with γ-radiation-attenuated live T. evansi and later challenged with homologous virulent T. evansi. Significant upregulation of the pro-inflammatory Th1 and Th17 cytokines was correlated with the IgG2-mediated protection in the immunized bovine calves post-challenge. The calves were immunized with 5×106 500Gy γ-radiation-attenuated live T. evansi (horse isolate) thrice at 15days intervals through the subcutaneous route and subsequently, challenged with 1×103 virulent T. evansi on day 50. Significantly high serum IgG (1:1600) and IgM (1:800) titres were recorded on week 2 PC, whereas the peak serum IgG2 titre (1:800) was recorded on week 6 PC. Significant upregulation of IFN-γ, TNF, IL-1β, and IL-2 was recorded between days 1 to 3 PC, while the same for IL-17 was recorded on day 14 PC. The immunized calves were free from parasitemia post-challenge and were clinically healthy till the end of the experiment. Significant upregulation of IL-10 and IL-4 transcripts and a corresponding increase of serum IgG1 titre in the placebo group helped patency of the parasite in an anti-inflammatory environment and clinical exacerbation of the disease. The expression of the important Th1 cytokines was crucial for antibody-mediated short-term protection against a lethal challenge of T. evansi in cattle.

Combined immunotherapeutic effect of Leishmania-derived recombinant aldolase and Ambisome against experimental visceral leishmaniasis.

Available therapeutics for visceral leishmaniasis (VL), a deadly parasitic infection, are usually associated with inadequate efficacy and adverse aftereffects. Further, the primary site of Leishmania parasite are host macrophages resulting in compromised immunity; ensuing marked T-cell immunosuppression. Such settings emphasize the exploration of chemo-immunotherapeutic strategies for improvising the infected person's immune status with better resolution of infection. Present work employs the immunization of Leishmania-infected hamsters with Leishmania-derived recombinant aldolase (rLdAld) and enolase (rLdEno) proteins in consort with the sub-optimal dose of Ambisome (2.5mg/kg). After the completion of immunization, hamsters were sacrificed on day 60 and 90 post infection and different organ samples were collected to perform immunological assay for evaluating the therapeutic efficacy and modulation in protective cellular immune responses. Combining these proteins, particularly rLdAld with Ambisome (2.5mg/kg), has significantly reduced the parasitic load (∼80%) with remarkable enhancement in DTH and lymphoproliferative responses compared to the infected control and only Ambisome treated groups. Moreover, cytokine levels at RNA and protein levels were noticed to be inclined towards Th-1 phenotype through up-regulation of IFN-γ and TNF-α with significant down-regulation in IL-10 and TGF-β expression, an indication towards the generation of protective immunity against experimental VL. Our experimental findings demonstrated that the chemo-immunotherapeutic approach could be an effective way of controlling human VL infection at minimal dosages of antileishmanial with reduced side-effects and propensity of drug resistance emergence.

Activated Chicken Gamma Delta T Cells Are Involved in Protective Immunity against Marek's Disease.

Gamma delta (γδ) T cells play a significant role in the prevention of viral infection and tumor surveillance in mammals. Although the involvement of γδ T cells in Marek's disease virus (MDV) infection has been suggested, their detailed contribution to immunity against MDV or the progression of Marek's disease (MD) remains unknown. In the current study, T cell receptor (TCR)γδ-activated peripheral blood mononuclear cells (PBMCs) were infused into recipient chickens and their effects were examined in the context of tumor formation by MDV and immunity against MDV. We demonstrated that the adoptive transfer of TCRγδ-activated PBMCs reduced virus replication in the lungs and tumor incidence in MDV-challenged chickens. Infusion of TCRγδ-activated PBMCs induced IFN-γ-producing γδ T cells at 10 days post-infection (dpi), and degranulation activity in circulating γδ T cell and CD8α+ γδ T cells at 10 and 21 dpi in MDV-challenged chickens. Additionally, the upregulation of IFN-γ and granzyme A gene expression at 10 dpi was significant in the spleen of the TCRγδ-activated PBMCs-infused and MDV-challenged group compared to the control group. Taken together, our results revealed that TCRγδ stimulation promotes the effector function of chicken γδ T cells, and these effector γδ T cells may be involved in protection against MD.

Virus-like particles containing a prefusion-stabilized F protein induce a balanced immune response and confer protection against respiratory syncytial virus infection in mice.

Respiratory syncytial virus (RSV) is a serious respiratory pathogen in infants and young children worldwide. Currently, no licensed RSV vaccines are available. In this study, we explored stable prefusion conformation virus-like particles (Pre-F VLPs) as RSV vaccine candidates. RSV fusion (F) protein mutants were constructed to form stabilized Pre-F or postfusion (Post-F) configurations. VLPs containing Pre-F or Post-F protein were generated using a recombinant baculovirus (rBV)-insect cell expression system. The assembly and immunological properties of Pre-F or Post-F VLPs were investigated. Pre-F and Post-F VLPs contained antigenic sites Ø and I of pre- and postfusion conformations, respectively. Compared with Post-F VLPs, immunization with Pre-F VLPs elicited upregulation of IFN-γ, IL-2 and IL-10 and downregulation of IL-4 and IL-5 cytokine production in mice. A high percentage of CD25+ Foxp3+ cells or a low percentage of IL-17A-producing cells among CD4+ T cells was observed in the lungs of mice vaccinated with Pre-F VLPs. Importantly, immunization with Pre-F VLPs induced a high level of RSV neutralizing antibody and a balanced immune response, which protected mice against RSV infection without evidence of immunopathology. Our results suggested that Pre-F VLPs generated from rBV-insect cells represent promising RSV vaccine candidates.

A Novel Human OX40L-CAR-Treg Specifically Targets Activated Antigen-Presenting Cells and Effectively Controls T Cell Alloreactivity and Xeno-GvHD

Background: Graft-versus-host disease (GvHD) remains the leading cause of non-relapse mortality after allogeneic hematopoietic stem cell transplantation (HCT). Adoptive transfer of regulatory T cells (Tregs) has demonstrated promising results in controlling GvHD in both animal models and phase I/II clinical trials. However, strategies to increase the stability of ex-vivo expanded Tregs, and to optimize their potency, are needed. Recent efforts to accomplish these goals have focused on engineering Chimeric Antigen Receptor (CAR) Tregs, with the first attempts using CARs targeted against disparate HLA antigens. However, Tregs targeting HLA have several limitations, including the downregulation of these antigens that accompanies some diseases, as well as the lack of utility of this approach in the HLA-matched setting. In this study, we tested a novel GvHD prophylaxis approach consisting of infusing donor Tregs engineered to express an anti-OX40L CAR that, upon engagement with activated OX40L+ host antigen-presenting cells (APCs), increases their suppressor potency to dampen APC-mediated activation of alloreactive T cells. Methods and Results: We designed a CAR construct containing an scFv targeting OX40L, a CD28 costimulatory domain, along with CD3ζ, as well as the fluorescent reporter NeonGreen, all under the control of a synthetic FOXP3 promoter. This design enabled the selective and sustained expression of the anti-OX40L CAR on CD25+CD127- FoxP3+ Treg cells. We also created Tregs transduced with a non-targeting NeonGreen-only control construct (Neon-Tregs). Engineered OX40L CAR-Tregs maintained expression of the canonical Treg markers FOXP3, CD25, and CTLA-4, and demonstrated robust activation in the presence of OX40L-expressing cells. In co-culture assays of OX40L CAR-Tregs with OX40L-expressing K562 target cells, we demonstrate upregulation of Treg suppressive proteins CTLA-4, LAP, CD71, GARP and LAG3 without inducing expression of pro-inflammatory cytokines (no upregulation of IFNγ, TNFα, IL17a) (Figure 1A). Activation of Tregs via CAR recognition of OX40L+ cells resulted in 2.5-fold enhancement in their potency to suppress anti-CD3/CD28-driven T cell proliferation in vitro compared to Neon-Tregs at a 1:4 Treg-to-Teff ratio (p<0.05). Furthermore, OX40L-CAR-Tregs demonstrated a greater capacity to inhibit monocyte-derived dendritic cell activation, highlighting the superior suppressive activity of these OX40L-CAR-Tregs compared to non-targeting Neon-Tregs. Importantly, in a human xenograft model of GvHD, induced by transplantation of human PBMC into sublethally irradiated NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice, OX40L CAR-Treg demonstrated superior GvHD disease control compared to both PBMC alone and Neon-Tregs. Clinical scores were assessed by a combinatorial system including five parameters: weight loss, posture, mobility, skin, and fur conditions, and curves were compared using a 2-way ANOVA mixed-effects analysis, which demonstrated improvement in clinical xeno-GvHD with the OX40L CAR-Treg > Neon-Treg > PBMC alone over the entire length of analysis (p <0.001). An example of the substantial improvement in the clinical score on Day +14 is as follows: combined GvHD clinical score = 6.5 for PBMC vs 2.5 for Neon-Treg vs 0.15 for OX40L CAR-Treg, p <0.0001 for all comparisons. This control of clinical GvHD resulted in prolonged recipient survival when compared to both PBMC alone and to control Neon-Tregs (MST = 34 days for OX40L-CAR-Treg, 20 days for Neon-Treg, 14 days for PBMC alone, p<0.001, Figure 1B). Conclusions: Collectively, these results demonstrate a novel and efficacious approach using OX40L-CAR Tregs to enhance Treg suppressive function and to control GvHD. Because OX40L is upregulated on APCs in inflammatory environments, and its expression is limited to APCs and activated endothelial cells, this approach provides a unique strategy for the control of allo-immunity after HCT. Moreover, these OX40L-CAR Treg may be broadly applicable beyond HCT, to control allo-immunity after solid organ transplant and to suppress T cell activation in autoimmune diseases. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

IMMUNOMODULATORY MECHANISMS OF GRASS ALLERGEN IMMUNOTHERAPY ON DIFFERENTIAL T CELLS TRANSCRIPTOME POLARIZATION IN EXPLORATORY TRIAL

<h3>Introduction</h3> A short pre-seasonal course of grass subcutaneous immunotherapy (SCIT) using MicroCrystalline Tyrosine (MCT) and Monophosphoryl Lipid A showed a significant improvement in combined symptom and medication scores (CSMS) in an exploratory field study (PQGrass309). Moreover, molecular mechanisms associated clinical benefit remain to be fully determined. <h3>Methods</h3> Peripheral blood mononuclear cells were obtained from participants, who underwent subcutaneous grass immunotherapy or placebo with MCT at baseline, end of treatment and end of grass pollen season (GPS). A panel of 58 genes was profiled. Ingenuity Pathway Analysis was deployed to elucidate molecular signature and functional relationships for observed gene expression. <h3>Results</h3> CSMS was significantly decreased (-33%) following grass SCIT compared to placebo with MCT. Transcriptome profiling showed transient upregulation of Th2/Th2A and Th17 signature cytokines at the start of GPS and steady downregulation at the end of GPS. Immune modulation towards Th1/Treg at pre-GPS and post-GPS with upregulation of IFNγ, IL-12A, EBI3 (IL-35), IL-27, IL-10, TGFβ was indicative of immune tolerance induction. T helper cell differentiation was one of the most significantly enriched pathways with Th17 mapped as a top enriched canonical pathway, which was transiently upregulated at the start of GPS and downregulated at the end of GPS. Upstream regulator analysis predicted IL-4, IL17A/IL17F, IL-13 and IFNγ as top upstream regulators at pre-GPS and IFNγ, IL-12A, IL-27 at post-GPS. <h3>Conclusion</h3> A distinct immunomodulatory profile of grass SCIT induced responses revealed clusters of differentially expressed genes, which supports restoration of the Th1/Th2 balance and provides the underpinning mechanisms associated with the robust clinical efficacy outcome.

Infectious pancreatic necrosis virus (IPNV) recombinant viral protein 1 (VP1) and VP2-Flagellin fusion protein elicit distinct expression profiles of cytokines involved in type 1, type 2, and regulatory T cell response in rainbow trout (Oncorhynchus mykiss)

In this study, we examined the cytokine immune response against two proteins of infectious pancreatic necrosis virus (IPNV) in rainbow trout (Oncorhynchus mykiss), the virion-associated RNA polymerase VP1 and VP2-Flagellin (VP2-Flg) fusion protein. Since VP1 is not a structural protein, we hypothesize it can induce cellular immunity, an essential mechanism of the antiviral response. At the same time, the fusion construction VP2-Flg could be highly immunogenic due to the presence of the flagellin used as an adjuvant. Fish were immunized with the corresponding antigen in Montanide™, and the gene expression of a set of marker genes of Th1, Th2, and the immune regulatory response was quantified in the head kidney of immunized and control fish. Results indicate that VP1 induced upregulation of ifn-γ, il-12p40c, il-4/13a, il-4/13b2, il-10a, and tgf-β1 in immunized fish. Expression of il-2a did not change in treated fish at the times tested. The antigen-dependent response was analysed by in vitro restimulation of head kidney leukocytes. In this assay, the group of cytokines upregulated after VP1-restimulation was consistent with those upregulated in the head kidney in vivo. Interestingly, VP1 induced il-2a expression after in vitro restimulation. The analysis of sorted lymphocytes showed that the increase of cytokines occurred in CD4-1+ T cells suggesting that Th differentiation happens in response to VP1. This is also consistent with the expression of t-bet and gata3, the master regulators for Th1/Th2 differentiation in the kidneys of immunized animals. A different cytokine expression profile was found after VP2-Flg administration, i.e., upregulation occurs for ifn-γ, il-4/13a, il-10a, and tgf-β1, while down-regulation was observed in il-4/13b2 and il-2a. The cytokine response was due to flagellin; only the il-2a effect was dependent upon VP2 in the fusion protein. To the best of our knowledge this study reports for the first-time characteristics of the adaptive immune response induced in response to IPNV VP1 and the fusion protein VP2-Flg in fish. VP1 induces cytokines able to trigger the humoral and cell-mediated immune response in rainbow trout. The analysis of the fish response against VP2-Flg revealed the immunogenic properties of Aeromonas salmonicida flagellin, which can be further tested for adjuvanticity. The novel immunogenic effects of VP1 in rainbow trout open new opportunities for further IPNV vaccine development using this viral protein.

Role of immune-inflamed phenotype in the prognosis of hypopharyngeal carcinoma following primary surgery.

The immune profile in primary resected hypopharyngeal squamous cell carcinoma (HPSCC) and its prognostic value remain to be defined. We enrolled 100 patients with HPSCC underwent primary surgical resection at our department. HPSCC samples were examined using immunohistochemistry for the expressions of CD8, Foxp3, CD163, CD66B, programmed death ligand-1 (PD-L1), and interferon (IFN)-γ. The immune pattern of the tumor microenvironment (TME) was discriminated into inflamed and non-inflamed tumors based on the presence or absence of parenchymal CD8+ T cells. We found that 74% of HPSCC cases in our cohort were characterized by an immune-inflamed TME. Immune-inflamed patterns demonstrated an inferior survival with a significantly increased density of CD163+ tumor-associated macrophages and Foxp3+ regulatory T cells. Additionally, the inflamed tumor showed increased expression of PD-L1, without IFN-γ upregulation. The immune-inflamed pattern is the predominant preexisting immune phenotype in HPSCC and demonstrates immunosuppressive immune cell recruitment.

Tracheal cellular immune response in chickens inoculated with Mycoplasma synoviae vaccine, MS-H or its parent strain 86079/7NS

Mycoplasma synoviae causes respiratory tract disease in chickens characterised by mild to moderate lymphoplasmacytic infiltration of the tracheal mucosa. MS-H (Vaxsafe1 MS, Bioproperties Pty Ltd.) is an effective live attenuated vaccine for M. synoviae, but the immunological basis for its mechanism of protection has not been investigated, and the phenotypes of lymphocytes and associated cytokines involved in the local adaptive immune response have not been described previously. In this study, specific-pathogen-free chickens were inoculated intra-ocularly at 3 weeks of age with either M. synoviae vaccine strain MS-H or vaccine parent strain 86079/7NS (7NS), or remained uninoculated. At 2-, 7- and 21 days post-inoculation (dpi), tracheal mucosal pathology, infiltrating lymphocytes subsets and transcription levels of mRNA encoding 8 cytokines were assessed using light microscopy, indirect immunofluorescent staining and RT-qPCR, respectively. After inoculation, tracheal mucosal thickness, tracheal mucosal lesions, and numbers of infiltrating CD4+CD25- cells, B-cells, and macrophages were greater in MS-H- and 7NS-inoculated chickens compared with non-inoculated. Inoculation with 7NS induced up-regulation of IFN-γ, while vaccination with MS-H induced up-regulation of IL-17A, when compared with non-inoculated birds. Both inoculated groups had a moderate infiltrate of CD4+CD25+ T cells in the tracheal mucosa. These findings reveal that the tracheal local cellular response after MS-H inoculation is dominated by a Th-17 response, while that of 7NS-inoculated chickens is dominated by a Th-1 type response.

A time-course comparative clinical and immune response evaluation study between the human pathogenic Orientia tsutsugamushi strains: Karp and Gilliam in a rhesus macaque (Macaca mulatta) model.

BackgroundScrub typhus is a vector-borne febrile illness caused by Orientia tsutsugamushi transmitted by the bite of Trombiculid mites. O. tsutsugamushi has a high genetic diversity and is increasingly recognized to have a wider global distribution than previously assumed.Methodology/principle findingsWe evaluated the clinical outcomes and host immune responses of the two most relevant human pathogenic strains of O. tsutsugamushi; Karp (n = 4) and Gilliam (n = 4) in a time-course study over 80 days post infection (dpi) in a standardized scrub typhus non-human primate rhesus macaque model. We observed distinct features in clinical progression and immune response between the two strains; Gilliam-infected macaques developed more pronounced systemic infection characterized by an earlier onset of bacteremia, lymph node enlargement, eschar lesions and higher inflammatory markers during the acute phase of infection, when compared to the Karp strain. C-reactive protein (CRP) plasma levels, interferon gamma (IFN-γ, interleukin-1 receptor antagonist (IL-1ra), IL-15 serum concentrations, CRP/IL10- and IFN-γ/IL-10 ratios correlated positively with bacterial load in blood, implying activation of the innate immune response and preferential development of a T helper-type 1 immune response. The O. tsutsugamushi-specific immune memory responses in cells isolated from skin and lymph nodes at 80 dpi were more markedly elevated in the Gilliam-infected macaques than in the Karp-infected group. The comparative cytokine response dynamics of both strains revealed significant up-regulation of IFN-γ, tumor necrosis factor (TNF), IL-15, IL-6, IL-18, regulatory IL-1ra, IL-10, IL-8 and granulocyte-colony-stimulating factor (G-CSF). These data suggest that the clinical outcomes and host immune responses to scrub typhus could be associated with counter balancing effects of pro- and anti-inflammatory cytokine-mediated responses.Currently, no data on characterized time-course comparisons of O. tsutsugamushi strains regarding measures of disease severity and immune response is available. Our study provides evidence for the strain-specificity of host responses in scrub typhus, which supports our understanding of processes at the initial inoculation site (eschar), systemic disease progression, protective and/or pathogenic host immune mechanisms and cellular immune memory function.Conclusions/significanceThis study characterised an improved intradermal rhesus macaque challenge model for scrub typhus, whereby the Gilliam strain infection associated with higher disease severity in the rhesus macaque model than the previous Karp strain infection. Difficulties associated with inoculum quantitation for obligate-intracellular bacteria were overcome by using functional inoculum titrations in outbred mice. The Gilliam-based rhesus macaque model provides improved endpoint measurements and contributes towards the identification of correlates of protection for future vaccine development.

Abstract 2898: HPN424, a Tri-specific T-cell Activating Construct, induces T cell-mediated cytotoxicity against human metastatic castration-resistant prostate cancers (mCRPC)

Abstract Background: Advanced prostate cancers (PC) are largely insensitive to existing immunotherapies. T-cell-engaging antibodies, linking tumor cells to T cells, are being developed to treat malignancies. HPN424 is a Tri-specific T-cell Activating Construct (TriTAC), binding PSMA, CD3ε, and albumin to improve drug half-life. We evaluated the activity of HPN424 to induce T-cell-mediated cytotoxicity against organoids generated from patient-derived xenograft (PDX-Os) established from human mCRPC biopsies. Design: PDX-Os from three models were evaluated; their membranous PSMA immunohistochemical expression was quantified using H-scores (HS). PDX-Os were co-cultured with: i) healthy donor allogeneic T cells, ii) healthy donor allogeneic T cells combined with HPN424 (1pM, 10pM or 100pM), or iii) healthy donor allogeneic T cells combined with PL711, a non-targeting TriTAC control (1pM, 10pM, 100 pM). Cytotoxicity was determined by LDH release and visualisation of T cell/PDO co-localisation and PDO disruption using confocal microscopy. Early and mid-late T cell activation was determined by FACS for CD69 and CD25 expression, respectively. Cytolytic (CTL) and CD4 T cell cytokine release (IFNγ, TNF∝, IL-1β/2/4/6/8/10) was evaluated using the AlphaLISA kit (MSD). FACS studies were performed after 48- and 72-hours, while microscopy, LDH and cytokine assays were performed after 24, 48, and 72-hours, of co-culture respectively. Results: In the highest PSMA-expressing PDX-Os (CP50c, HS: 225; CP142c, HS: 165), co-culture of resting healthy donor T cells with CRPC PDX-Os led to dose-dependent increases in LDH release at 48-hours (p&amp;lt;0.0001) and 72-hours (p&amp;lt;0.0001), in the presence of HPN424 but not PL711. Correspondingly, in these models, confocal microscopy showed T cells co-localising with and disrupting the PDX-Os by 24-hours across all 3 HPN424 concentrations tested. HPN424 induced T-cell expression of CD25 and CD69 within 48-hours of co-culture across all 3 concentrations in both models. When compared with PL711, HPN424 induced dose-dependent upregulation of IFNγ, IL-1β, IL-2, IL-4, IL-10, and TNF∝ in the PSMA-high PDX-Os, CP50c and CP142c (all p&amp;lt;0.0001), indicating T cell activation and induction of CTL and Th1/Th2/Treg cytokines by HPN424. Interestingly, in co-cultures of T cells and CP89c PDX-Os, which have lower PSMA expression (HS of 25), HPN424 did not significantly increase LDH release when compared with PL711; in this model CD25/CD69 induction and the upregulation of IFNγ, IL-1β/2, and TNF∝ was only observed at the highest concentration of HPN424 (100pM). Conclusions: HPN424, a PSMA-targeting TriTAC antibody construct, induced T cell activation and elicited T-cell mediated cytotoxicity against PSMA expressing mCRPC PDX-Os. Our data support further evaluation of this agent in advanced PC with membranous PSMA expression. Citation Format: Veronica Gil, Christina Guo, George Seed, Antje Neeb, Lorenzo Buroni, Bora Gurel, Ines Figueiredo, Golzar Hemmati, Wade Aaron, Johann S. de Bono. HPN424, a Tri-specific T-cell Activating Construct, induces T cell-mediated cytotoxicity against human metastatic castration-resistant prostate cancers (mCRPC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2898.

Comprehensive Testing of Chemotherapy and Immune Checkpoint Blockade in Preclinical Cancer Models Identifies Additive Combinations

Antibodies that target immune checkpoints such as cytotoxic T lymphocyte antigen 4 (CTLA‐4) and the programmed cell death protein 1/ligand 1 (PD-1/PD-L1) are now a treatment option for multiple cancer types. However, as a monotherapy, objective responses only occur in a minority of patients. Chemotherapy is widely used in combination with immune checkpoint blockade (ICB). Although a variety of isolated immunostimulatory effects have been reported for several classes of chemotherapeutics, it is unclear which chemotherapeutics provide the most benefit when combined with ICB. We investigated 10 chemotherapies from the main canonical classes dosed at the clinically relevant maximum tolerated dose in combination with anti‐CTLA-4/anti-PD-L1 ICB. We screened these chemo-immunotherapy combinations in two murine mesothelioma models from two different genetic backgrounds, and identified chemotherapies that produced additive, neutral or antagonistic effects when combined with ICB. Using flow cytometry and bulk RNAseq, we characterized the tumor immune milieu in additive chemo-immunotherapy combinations. 5-fluorouracil (5-FU) or cisplatin were additive when combined with ICB while vinorelbine and etoposide provided no additional benefit when combined with ICB. The combination of 5-FU with ICB augmented an inflammatory tumor microenvironment with markedly increased CD8+ T cell activation and upregulation of IFNγ, TNFα and IL-1β signaling. The effective anti‐tumor immune response of 5-FU chemo-immunotherapy was dependent on CD8+ T cells but was unaffected when TNFα or IL-1β cytokine signaling pathways were blocked. Our study identified additive and non-additive chemotherapy/ICB combinations and suggests a possible role for increased inflammation in the tumor microenvironment as a basis for effective combination therapy.

Listeria monocytogenes Inoculation Impedes the Development of Brain Pathology in Experimental Cerebral Malaria by Inhibition of Parasitemia.

Cerebral malaria (CM) is a serious central nervous system dysfunction caused by Plasmodium falciparum infection. In this study, we investigated the effect of Listeria monocytogenes (Lm) inoculation on experimental cerebral malaria (ECM) using Plasmodium berghei ANKA (PbA)-infected C57BL/6 mice. Live Lm inoculation inhibited the parasitemia and alleviated ECM symptoms. The protective effect against ECM symptoms was connected with improved brain pathology manifested as a less-damaged blood-brain barrier, decreased parasite sequestration, and milder local inflammation. Meanwhile, Lm inoculation decreased expression of cell adhesion molecules (ICAM-1 and VCAM-1) and accumulation of pathogenic CD8+ T cells in the brain. In keeping with the suppression of parasitemia, there was an upregulation of IFN-γ, IL-12, MCP-1, and NO expression in the spleen by Lm inoculation upon PbA infection. Early treatment with exogenous IFN-γ exhibited a similar effect to Lm inoculation on PbA infection. Taken together, Lm inoculation impedes the development of brain pathology in ECM, and early systemic IFN-γ production may play a critical role in these protective effects.

Use of two freshwater macrophytes, water hyacinth ( Eichhornia crassipes ) and coontail ( Ceratophyllum demersum ), as carbohydrate sources in biofloc system for Nile tilapia ( Oreochromis niloticus )

Two freshwater macrophytes, water (Eichhornia crassipes) and coontail (Ceratophyllum demersum), were evaluated as carbon sources in biofloc system for Nile tilapia (Oreochromis niloticus), in comparison with molasses and a RAS with 10% water exchange daily (control). All-male tilapia (9.35 g) were stocked in 70-L fibreglass tanks at 10 fish tank−1 and fed on a commercial diet (30% cp) for 60 days. The average water temperature, pH and dissolved oxygen (DO) did not significantly differ (p > 0.05) among all treatments. Total ammonia–nitrogen (TAN) was significantly lower in the control group (0.11 mg L−1) than in biofloc treatments (0.15–0.17 mg L−1). Nitrite (NO2–N) concentrations were significantly lower, while nitrates (NO3–N) higher, in biofloc-based treatments (20.23–25.77 mg L−1) than in the control group (12.85 mg L−1). Biofloc volume (BFV) (28.17–33.50 ml L−1) and total suspended solids (TSS) (691–824 mg L−1) were not significantly different among molasses and macrophytes treatments. The control group displayed lower growth rates and feed efficiency than macrophytes-based groups. Blood parameters, immunological responses, digestive enzymes activities, liver function enzymes and upregulation of IFN-γ, TNF-α and IL-8 genes were significantly better in molasses- and macrophytes-treated tanks than the control group. These results suggest that water hyacinth and coontail can be used as costless, eco-friendly carbon sources in biofloc systems.

Prompt Antiviral Action of Pulmonary CD8+ TRM Cells Is Mediated by Rapid IFN-γ Induction and Its Downstream ISGs in the Lung.

Growing lines of evidence supported the importance of CD8+ lung tissue resident memory T (TRM) cells in protection against respiratory viruses, exemplified by influenza A virus. However, the underlying in vivo mechanism remains largely undetermined. Here, we used mouse infection models to dissect in vivo cross-protective activity of lung CD8+ TRM cells. By simultaneously interrogating transcriptional dynamics in lung CD8+ TRM cells and surrounding tissues during the early course of infection, we demonstrated that lung CD8+ TRM cells react to antigen re-exposure within hours, manifested by IFN-γ upregulation, and a tissue-wide interferon-stimulated gene (ISG) program is subsequently elicited. Using antibody-mediated IFN-γ neutralization and IFN-γ receptor knockout mice, we could show that the induction of several important antiviral ISGs required IFN-γ signaling, so did the suppression of key inflammatory cytokines. Interestingly, there were also examples of ISGs unaffected in the absence of IFN-γ activity. Collectively, focusing on in situ characterization of lung CD8+ TRM cells during very early stage of infection, a critical period of host antiviral defense that has been poorly investigated, our studies highlight that these cells, once triggered by antigen re-exposure, are programmed to produce IFN-γ expeditiously to promote a lung-wide antiviral response for effective virus control.

Immunological correlates of response and immune-mediated toxicity in checkpoint inhibitor (ICI)-treated metastatic urothelial carcinoma (mUC) patients (pts).

526 Background: Myeloid derived suppressors cells (MDSC) are immune cells that create an immunosuppressive microenvironment. Increased expression of MDSC subsets is associated with worse overall survival in ICI-treated mUC pts, but their role in immune-related adverse events (irAE) is unknown. Immune profiles associated with irAE are also unknown. We investigated associations of MDSC and –omics profiles with response and irAE in ICI-treated mUC pts. Methods: Baseline (B) and on-treatment (Tx) blood samples were collected from ICI-treated mUC pts. MDSC were measured in fresh unfractionated whole blood (WB) and in peripheral blood mononuclear cells (PBMC). MDSC were identified by flow cytometry in WB, defined as LinloCD33+/HLADR-, and subclassified as polymorphonuclear (PMN)-MDSC (CD15+/CD14-), monocytic (M)-MDSC (CD15-/CD14+), and uncommitted (UC)-MDSC (CD15-/CD14-). MDSC populations were presented as % of live nucleated blood cells and as absolute numbers from WB. irAE severity was graded by CTCAE v5. In a subcohort of 17 pts, proteomics and transcriptomics were analyzed via Olink and Bulk RNAseq, respectively. Wilcoxon rank sum test compared MDSC and –omics among response and irAE groups. Kruskal-Wallis test compared –omics results between irAE responders (irAE-R), irAE non-responders (irAE-NR), and no irAE/non-responders (noAE-NR). Results: 41 ICI-treated mUC pts (25 anti-PD-L1, 16 anti-PD-1) had at least 1 MDSC sample: 28 pts at B, 30 pts at Tx, and 17 pts at both B and Tx. Primary UC sites were bladder (78%) and upper tract (22%); 73% male; median age 72 (range, 28-82); 85% had KPS &gt; 80%; 51% had visceral metastasis. ICI was first and second-line therapy in 37% and 63% of pts, respectively. 13 pts were responders (R); 26 pts were non-responders (NR); 2 pts were not evaluable. 22 pts developed irAE. Median time to irAE was 84 days (range, 21-145); 10 pts required steroids; 3 required ICI discontinuation. UC-MDSC was predominant in WB and PMN-MDSC in PBMC in both B and Tx. Between B and Tx, WB UC-MDSC and PB UC-MDSC increased in R (n = 13; p = 0.04), but decreased in NR (n = 26; p = 0.02). In the subcohort of 17 pts, 11 had irAE (7 irAE-R; 4 irAE-NR), 6 had noAE-NR. Proteomic analysis showed increased expression of CXCL12 in noAE-NR pts (p = 0.006) and increased expression of IL-8 (p = 0.016), IL-18 (p = 0.012), and IL-18R1 (p = 0.016) in all irAE pts. At the transcriptome level, upregulation of IFN-γ was associated with response, whereas upregulation of both IFN-γ and IFN-α differentiated irAE-R from irAE-NR. Conclusions: In ICI-treated mUC pts, WB &amp; PB UC-MDSC increased in R and decreased in NR between B and Tx. Increased expression of pro-inflammatory chemokines was observed in irAE pts, independent of response. A distinct inflammatory pathway was observed in irAE-R. Prospective investigation of blood-based biomarkers of response and irAE development is warranted.

TIRAP drives myelosuppression through an Ifnγ-Hmgb1 axis that disrupts the endothelial niche in mice.

Inflammation is associated with bone marrow failure syndromes, but how specific molecules impact the bone marrow microenvironment is not well elucidated. We report a novel role for the miR-145 target, Toll/interleukin-1 receptor domain containing adaptor protein (TIRAP), in driving bone marrow failure. We show that TIRAP is overexpressed in various types of myelodysplastic syndromes (MDS) and suppresses all three major hematopoietic lineages. TIRAP expression promotes up-regulation of Ifnγ, leading to myelosuppression through Ifnγ-Ifnγr-mediated release of the alarmin, Hmgb1, which disrupts the bone marrow endothelial niche. Deletion of Ifnγ blocks Hmgb1 release and is sufficient to reverse the endothelial defect and restore myelopoiesis. Contrary to current dogma, TIRAP-activated Ifnγ-driven bone marrow suppression is independent of T cell function or pyroptosis. In the absence of Ifnγ, TIRAP drives myeloproliferation, implicating Ifnγ in suppressing the transformation of MDS to acute leukemia. These findings reveal novel, noncanonical roles of TIRAP, Hmgb1, and Ifnγ in the bone marrow microenvironment and provide insight into the pathophysiology of preleukemic syndromes.

Lidocaine relieves murine allergic rhinitis by regulating the NF-κB and p38 MAPK pathways.

Allergic rhinitis (AR) is one of the most common chronic inflammatory diseases and its main feature is nasal mucositis. It has been recently revealed that lidocaine demonstrates optimal effects in the treatment of various diseases. However, a limited number of studies have examined the association between lidocaine and AR. In the present study, the AR mouse model was established to explore the effects of lidocaine in AR and to further analyze its molecular mechanism. Subsequently, different concentrations of lidocaine were provided to the animals by intranasal administration and a series of indices were assessed. The data indicated that the frequencies of mouse sneezing and nose rubbing were suppressed following an increase in lidocaine concentration. Subsequently, the number of inflammatory cells was measured. Wright's-Giemsa staining results indicated that lidocaine significantly decreased the numbers of leukocytes, eosinophils, neutrophils and lymphocytes in the nasal lavage fluid (NLF) of AR mice. In addition, the expression levels of ovalbumin (OVA)-specific immunoglobulin E (IgE), leukotriene C4 (LTC4) and certain inflammatory factors were assessed by ELISA. Lidocaine reduced OVA-specific IgE and LTC4 expression in NLF and plasma derived from AR mice. It also decreased the expression levels of IL-4, IL-5, IL-13, IL-17 and TNF-α. Lidocaine caused upregulation of IFN-γ and IL-2 expression levels. Subsequently, western blot analysis indicated that lidocaine suppressed phosphorylated (p)-p38 and p-p65 expression levels in AR mice. Collectively, the results indicated that the NF-κB and p38 MAPK signaling pathways were involved in the lidocaine-mediated relief of AR in mice. In order to further verify the association between the NF-κB and p38 MAPK signaling pathways and AR in mice, the effects of the NF-κB inhibitor IMD-0354 and the p38 MAPK inhibitor SB 203580 were assessed on AR mice. The results indicated that these two compounds exhibited similar inhibitory effects on AR mice as those noted with the use of lidocaine. These findings suggested that lidocaine represented a novel therapeutic agent for AR.

Rare variants in Toll-like receptor 7 results in functional impairment and downregulation of cytokine-mediated signaling in COVID-19 patients

Toll-like receptors (TLR) are crucial components in the initiation of innate immune responses to a variety of pathogens, triggering the production of pro-inflammatory cytokines and type I and II interferons, which are responsible for innate antiviral responses. Among the different TLRs, TLR7 recognizes several single-stranded RNA viruses including SARS-CoV-2. We and others identified rare loss-of-function variants in X-chromosomal TLR7 in young men with severe COVID-19 and with no prior history of major chronic diseases, that were associated with impaired TLR7 signaling as well as type I and II IFN responses. Here, we performed RNA sequencing to investigate transcriptome variations following imiquimod stimulation of peripheral blood mononuclear cells isolated from patients carrying previously identified hypomorphic, hypofunctional, and loss-of-function TLR7 variants. Our investigation revealed a profound impairment of the TLR7 pathway in patients carrying loss-of-function variants. Of note, a failure in IFNγ upregulation following stimulation was also observed in cells harboring the hypofunctional and hypomorphic variants. We also identified new TLR7 variants in severely affected male patients for which a functional characterization of the TLR7 pathway was performed demonstrating a decrease in mRNA levels in the IFNα, IFNγ, RSAD2, ACOD1, IFIT2, and CXCL10 genes.

Effect of Auraptene on angiogenesis in Xenograft model of breast cancer.

Angiogenesis is the most important challenge in breast cancer treatment. Recently, scientists become interesting in rare natural products and intensive researches was performed to identify their pharmacological profile. Auraptene shows helpful effects such as cancer chemo-preventive, anti-inflammatory, anti-oxidant, immuno-modulatory. In this regard, we investigated the anti-angiogenesis effect of Auraptene in in-vitro and in-vivo model of breast cancer. In this study, 4T, MDA-MB-231 and HUVEC cell lines were used. The proliferation study was done by MTT assay. For tube formation assay, 250 matrigel, 1×104 HUVEC treated with Auraptene, 20ng/mL EGF, 20ng/mL bFGF and 20ng/mL VEGF were used. Gene expression of important gene related to angiogenesis in animal model of breast cancer was investigated by Real-time PCR. Protein expression of VCAM-1 and TNFR-1 gene related to angiogenesis in animal model of breast cancer was investigated by western-blot. Auraptene treatment led to reduction in cell viability of MDA-MB-231 in a concentration-dependent manner. Also, we observed change in the number of tubes or branches formed by cells incubated with 40 and 80μM Auraptene. Auraptene effect the gene expression of important gene related to angiogenesis (VEGF, VEGFR2, COX2, IFNɣ). Moreover, the western blot data exhibited that Auraptene effect the protein expression of VCAM-1 and TNFR-1. Overall, this study shows that Auraptene significantly suppressed angiogenesis via down-regulation of VEGF, VEGFR2, VCAM-1, TNFR-1, COX-2 and up-regulation of IFNγ.