Mouse Splenocytes Research Articles

Preclinical development of the lipid nanoparticle–based mRNA vaccine with multiple natural T cell epitopes for pancreatic cancer.

752 Background: Immunotherapy has recently emerged as a promising avenue in cancer treatment; however, its effectiveness on pancreatic ductal adenocarcinoma (PDAC) is hindered by the lack of effector T cells in the tumor microenvironment. One of the strategies to overcome the resistance of PDAC to immunotherapy involves identifying immunogenic tumor-associated and specific antigens for the development of personalized cancer vaccines. The mRNA platform has become an attractive platform for antigen presentation. The delivery of mRNA cancer vaccines via lipid nanoparticles (LNPs) enables intracellular delivery and controlled release of the payload, offering improved vaccine efficacy. However, mRNA vaccines presenting human T cell epitopes often lack a preclinical model for antitumor immune efficacy testing. Methods: A syngeneic mouse model of PDACs was established by implanting KPC tumor cells into the livers by hemispleen surgery. mRNA vaccine expressing five 25-mer peptide regions were synthesized. Two mouse CD8+ T cell epitopes were predicted. Their MHC binding was tested by the T2 binding assay. CD8+ T cells specific for these two epitopes were examined by ELISpot assays and were quantified by ImmunoSpot ELISpot Reader to identify changes in IFN-g secretion. Results: Using mass spectrometry analysis, we successfully identified natural HLA class I and class II-binding epitopes in the neoplastic tissues of human PDACs. For the design of mRNA vaccine, we chose five 25-mer peptide regions that each contains both class I and class II epitopes punctuated by AAY and GPGPG linkers and followed by an MITD sequence, which all aim to improve the efficiency of antigen presentation to dendritic cells. Using NetMHC, we predicted H2Kb or H2Db-binding epitopes LYPFPLAL and SMVQMTFL, within these five human peptide regions. The T2 binding assay showed a lack of evident binding of both peptides to the T2-Kb or T2-Db cells. However, the ELISpot assay showed that LYPFPLAL, but not SMVQMTFL, can induce the IFN-g expression from CD8+ T cells derived from splenocytes of the KPC tumor mice vaccinated by a peptide vaccine containing both peptides. Therefore, this result confirmed that LYPFPLAL is a mouse CD8+ T cell epitope and can be used to monitor antigen-specific CD8+ T cell response in response to the mRNA vaccine treatment in the syngeneic mouse model of PDACs. Conclusions: Antitumor efficacy of our above-designed mRNA vaccine can be tested in the syngeneic mouse hemispleen model of KPC tumors. Mouse LYPFPLAL epitope-specific CD8+ T cell response can serve as a surrogate marker for the antitumor efficacy of this mRNA vaccine in mouse models for PDAC.

Enhanced Immune Functions of In Vitro Human Natural Killer Cells and Splenocytes in Immunosuppressed Mice Supplemented with Mature Silkworm Products

Objectives: The immune-enhancing properties of steamed mature silkworm, known as HongJam (HJ), were investigated using human interleukin-2-independent Natural Killer 92 (NK92-MI) cells and a cyclophosphamide intraperitoneal injection-induced immunosuppressed mice model (CPA-IP). White Jade variety mature silkworm HJ (WJ-HJ) was used to prepare WJ-HJ supercritical fluid extracts (WJ-SCE) and WJ-HJ-supplemented feeds. Results: Treatment with WJ-SCE significantly enhanced proliferation, migration, and cytotoxicity of NK92-MI cells against various cancer cells while improving mitochondrial function and ATP production (p < 0.05). In CPA-IP mice, consumption of WJ-HJ-supplemented feeds restored immune function by improving body weight, immune organ indices, immunoglobulin levels, and blood cytokines. Splenocyte proliferation and cytotoxicity were significantly elevated in both saline intraperitoneal injection (Sal-IP) and CPA-IP groups with WJ-HJ supplementation, independent of mitogen activation (p < 0.05). Conclusions: These results suggest that WJ-HJ enhances immune modulation and immune surveillance functions of NK cells by improving mitochondrial and cytotoxic functions. WJ-HJ holds promise as a functional food for immune enhancement, pending clinical validation.

P1114 Engineering and Development of a Novel Bispecific Antibody Targeting IL-23 and TL1A

Abstract Background Both IL23 and TL1A are clinically validated targets with genetical linkage to various human autoimmune diseases. Here we engineered and produced HXN-1003, a bispecific antibody (bsAb) simultaneously targeting both IL23 and TL1A, with the aim to further enhance clinical efficacy, and to overcome refractory and resistance associated with single-target based therapies. Methods The binding and blocking efficacy of HXN-1003 for each target were assessed using various in vitro assays, including IL23 reporter and mouse splenocyte activation assays for IL23, and TF-1 apoptosis and DR3-NFκB-Luc reporter assays for TL1A. The synergistic effect of dual targeting was measured by IL22 secretion in mouse splenocytes co-stimulated with IL23 and TL1A. In vivo efficacy was evaluated in hIL23/hTL1A transgenic mice with DSS-induced colitis and IMQ-induced psoriasis models, as well as in SD rats with subcutaneous injections of hTL1A and hIL23 to induce dermatitis. Results HXN-1003 features a tetravalent structure, with the anti-IL23 arm specifically binds to the p19 subunit of IL23, demonstrating superior affinity and blocking activity compared to benchmark Risankizumab. For TL1A, HXN-1003 binds to both the TL1A trimer and monomer with sub-nanomolar affinity, exhibiting similar blocking activity to its parent antibody HXN-1001 and RVT-3101. In the mouse splenocyte activation assay induced by IL23 and TL1A, HXN-1003 showed greater activity than each individual antibody and was equally active to their combination. In animal models, HXN-1003 significantly alleviated symptoms of DSS-induced colitis, and demonstrated superior therapeutic effects in the IMQ-induced psoriasis model compared to the IL23 monoclonal antibody. Additionally, in the dermatitis model induced by hTL1A and hIL23, the bsAb outperformed each individual antibody and their combination. Conclusion HXN-1003 simultaneously blocks IL23 and TL1A, with each arm exhibiting activity comparable to the parent monoclonal antibodies. It has demonstrated synergistic/additive therapeutic effects in both in vitro and in vivo experiments, highlighting its potential to enhance clinical efficacy and benefit autoimmune patients in multiple clinical settings.

P0640 Development and characterization of LQ080, a novel extended half-life bispecific TL1A/IL-23 p19 single domain antibody for the treatment of IBD

Abstract Background Despite the effectiveness of current anti-TL1A treatments, unmet needs remain for effective therapeutics for IBD. Therapies simultaneously neutralizing TL1A and IL-23 might be better choices owing to their synergistic anti-inflammatory effects in the pathogenesis of IBD. Methods TL1A and IL-23 single domain antibodies (sdAbs) were selected from immunized sdAb Phage display libraries. A human whole blood-based assay measuring IFNγ secretion was used to assess functional blockade of TL1A. Mouse splenocytes based assay measuring mouse IL-17 release was used to evaluate functional blockade of IL-23. Dextran Sulfate Sodium (DSS) induced mouse bowel inflammatory model was used to judge the in vivo effect of anti-TL1A antibodies. Synergistic anti-inflammatory effect was evaluated in mouse dermatitis model. Half-life extension was measured via pharmacokinetic analysis in non-human primate (NHP) given a single bolus of LQ080 by subcutaneous administration. Results The TL1A sdAb, which binds to both soluble and membrane-bound TL1A in a pH dependent pattern, potently blocked TL1A/DR3 while leaving the TL1A/DcR3 binding undisturbed and illustrated better IFNγ release in human whole blood induced by TL1A than RVT-3101 and PRA023. In DSS model, the TL1A sdAb also demonstrated better anti-inflammation effect than RVT-3101. The TL1A/IL-23 p19 biparatropic sdAb, LQ080, showed better IFNγ secretion inhibition than RVT-3101 and PRA023, and better inhibition of mouse IL-17 induction than Risankizumab and Guselkumab. Synergistic effects were found using LQ080 in both IFNγ release inhibition in human PBMCs induced by IL-23 and TL1A and in the mouse dermatitis model. The half-life of LQ080 significantly extended of 2-3 folds in non-human primate (NHP) compared to that of RVT-3101. Besides, LQ080 did not form large immune complex in vitro when mixed with TL1A and IL-23. Moreover, LQ080 did not stimulate the release of pro-inflammatory cytokines in the cytokines release assay. Conclusion The TL1A sdAb demonstrated better bioactivity than RVT-3101 and PRA023. LQ080, a TL1A/IL-23 p19 biparatropic sdAb, illustrated synergistic anti-inflammation effect both in vitro and iv vivo for its simultaneous neutralization of TL1A/DR3 and IL-23/IL-23R pathways, which would greatly improve the clinical results of IBD patients. The pH dependent TL1A binding makes LQ080 very different from other anti-TL1A mAbs. LQ080 demonstrated extended half-life in NHP making infrequent dosing possible for IBD treatment. No large immune complex formation when mixed with TL1A and IL-23 would make LQ080 safer when administrated by SC route. Overall, LQ080 is a promising TL1A/IL-23 p19 sdAb for IBD treatment.

Immunogenicity of HIV-1 Env mRNA and Env-Gag VLP mRNA Vaccines in Mice.

The development of a protective vaccine is critical for conclusively ending the human immunodeficiency virus (HIV) epidemic. We constructed nucleotide-modified mRNA vaccines expressing HIV-1 Env and Gag proteins. Env-gag virus-like particles (VLPs) were generated through co-transfection with env and gag mRNA vaccines. BALB/c mice were immunized with env mRNA, env-gag VLP mRNA, env plasmid DNA vaccine, or lipid nanoparticle (LNP) controls. HIV Env-specific binding and neutralizing antibodies in mouse sera were assessed via enzyme-linked immunosorbent assay (ELISA) and pseudovirus-based neutralization assays, respectively. Env-specific cellular immune responses in mouse splenocytes were evaluated using an Enzyme-linked immunosorbent assay (ELISpot) and in vivo cytotoxic T cell-killing assays. The Env-specific humoral and cellular immune responses elicited by HIV-1 env mRNA and env-gag VLP mRNA vaccine were stronger than those induced by the DNA vaccine. Specific immune responses induced by the env mRNA vaccine were significantly stronger in the high-dose group than in the low-dose group. Immunization with co-formulated env and gag mRNAs elicited superior cellular immune responses compared to env mRNA alone. These findings suggest that the env-gag VLP mRNA platform holds significant promise for HIV-1 vaccine development.

Cystatin A promotes the antitumor activity of T helper type 1 cells and dendritic cells in murine models of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a disease with poor prognosis due to diagnostic and therapeutic limitations. We previously identified cystatin A (CSTA) as a PDAC biomarker and have conducted the present study to investigate the antitumor effects of CSTA. PDAC murine models were established with genetically modified PAN02 tumor cell lines to evaluate the antitumor immune response. PDAC mouse survival was significantly longer with CSTA, and its antitumor effect was mediated mainly by CD4+ cells and partly by CD8+ cells. We also observed an increased infiltration of CD4+ and CD8+ cells in tumors of mice overexpressing CSTA. Phenotypically, we confirmed higher T helper type 1 (Th1) cell activity and increased frequency and activity of M1 macrophages and dendritic cells (DCs) in CSTA-overexpressing mice. Gene expression analysis highlighted pathways related to interferon gamma (IFN-γ) induction and Th1 lymphocyte activation that were induced by CSTA. Macrophages and DCs shifted toward proinflammatory antitumor phenotypes. Furthermore, activated splenocytes of PDAC model mice expressing CSTA had increased proapoptotic activity. CSTA also promoted the selective migration of CD4+ and CD11c+ immune cells in an in vitro migration assay. In conclusion, CSTA exerts antitumor effects by enhancing Th1-mediated antitumor effects through promotion of DC and M1 macrophage activity, thereby increasing immune cell chemotaxis. CSTA could be a novel therapeutic candidate for PDAC.

Immunostimulatory effects of Heyndrickxia coagulans SANK70258.

Here, we examined the immunomodulating effects of Heyndrickxia coagulans SANK70258 (SANK70258). Mouse splenocytes treated with γ-ray-irradiated SANK70258 produced higher levels of IFN-γ than those with 7 types of lactic acid bacteria. IFN-γ was mainly produced by NK cells, involving IL-12/IL-23, dendritic cells (DCs), and NFκB signaling. SANK70258 induced the release of IL-6, IL-10, and IL-12p40 from mouse DCs and the expression of cytokine genes in the human monocyte. Cytokine release from SANK70258-treated DCs was partially reduced by the knockdown of Tlr2 or Nod2, and was abolished by Myd88 knockout. DC-stimulating components of SANK70258 were enriched in ether- and butanol-insoluble peptidoglycan-related fractions. SANK70258 component induced high levels of IgA production in Peyer's patch cells, and its oral intake significantly increased intestinal IgA and IgA-expressing B cells in Peyer's patches in mice. We conclude that SANK70258 component exhibits high activity as an immunostimulant that induces the production of IFN-γ and IgA.

Immunopeptidomic MHC-I profiling and immunogenicity testing identifies Tcj2 as a new Chagas disease mRNA vaccine candidate.

Trypanosoma cruzi is a protozoan parasite that causes Chagas disease. Globally 6 to 7 million people are infected by this parasite of which 20-30% will progress to develop Chronic Chagasic Cardiomyopathy (CCC). Despite its high disease burden, no clinically approved vaccine exists for the prevention or treatment of CCC. Developing vaccines that can stimulate T. cruzi-specific CD8+ cytotoxic T cells and eliminate infected cells requires targeting parasitic antigens presented on major histocompatibility complex-I (MHC-I) molecules. We utilized mass spectrometry-based immunopeptidomics to investigate which parasitic peptides are displayed on MHC-I of T. cruzi infected cells. Through duplicate experiments, we identified an array of unique peptides that could be traced back to 17 distinct T. cruzi proteins. Notably, six peptides were derived from Tcj2, a trypanosome chaperone protein and member of the DnaJ (heat shock protein 40) family, showcasing its potential as a viable candidate vaccine antigen with cytotoxic T cell inducing capacity. Upon testing Tcj2 as an mRNA vaccine candidate in mice, we observed a strong memory cytotoxic CD8+ T cell response along with a Th1-skewed humoral antibody response. In vitro co-cultures of T. cruzi infected cells with splenocytes of Tcj2-immunized mice restricted the replication of T. cruzi, demonstrating the protective potential of Tcj2 as a vaccine target. Moreover, antisera from Tcj2-vaccinated mice displayed no cross-reactivity with DnaJ in lysates from mouse and human indicating a decreased likelihood of triggering autoimmune reactions. Our findings highlight how immunopeptidomics can identify new vaccine targets for Chagas disease, with Tcj2 emerging as a promising new mRNA vaccine candidate.

Differential Immune Responses of Th1 Stimulatory Chimeric Antigens of Leishmania donovani in BALB/c Mice.

Visceral leishmaniasis (VL) is the third most severe infectious parasitic disease and is caused by the protozoan parasite Leishmania. To control the spread of the disease in endemic areas where the asymptomatic patients act as reservoirs as well as in nonendemic areas, an effective vaccine is indispensable. In this direction, we have developed three chimeric proteins by the combination of three already known Th1 stimulatory leishmanial antigens, i.e., enolase, aldolase, and triose phosphate isomerase (TPI). The newly developed chimeric proteins, i.e., enolase-aldolase, TPI-enolase, and aldolase-TPI along with BCG as an adjuvant were assessed and compared, examining humoral and cellular adaptive immune responses elicited in BALB/c mice. The three chimeric antigens exhibited differential immune responses shown by differences in Th1 and Th2 cytokine production in ex vivo stimulated splenocytes of immunized mice. It was observed that all three chimeric proteins are more immunogenic than their component proteins. However, while comparing the immune response of the three chimeric proteins, aldolase-TPI exhibited a better immunogenic (Th1-type) response, as evidenced by the highest IFN-γ production, a high IgG2a antibody isotype switching, a high % population of CD8+ and CD4+ T-cells, and a significantly high expression of iNOS2. Thus, the results suggest the potential of these chimeric antigens as strong immunogens that can be harnessed in vaccine development against VL.

Determination of the Content of FAD Cofactor and NAD(P)H-Oxidase Complexes in Mouse Splenocytes and Lewis Carcinoma Cells Under Conditions of Apoptosis by Confocal Microscopy Method

Determination of the Content of FAD Cofactor and NAD(P)H-Oxidase Complexes in Mouse Splenocytes and Lewis Carcinoma Cells Under Conditions of Apoptosis by Confocal Microscopy Method

Protection against N. gonorrhoeae induced by OMV-based Meningococcal Vaccines are associated with cross-species directed humoral and cellular immune responses.

Limited protective immunologic responses to natural N. gonorrhoeae infection and a lack of knowledge about mechanisms of protection have hampered development of an effective vaccine. Recent studies in humans and mice have found meningococcal outer membrane vesicle-containing vaccines (OMV) induce cross species immune responses against gonococci and are associated with protection. The exact mechanisms or how humoral and cellular immunity are related to protection, remain unclear. To study this, we immunized mice with two meningococcal OMV-containing vaccines known to accelerate clearance of N. gonorrhoeae , 4CMenB and OMV from an engineered N. meningitidis strain lacking major surface antigens PorA, PorB, and Rmp (MC58 ΔABR). We assessed serologic and cellular immune signatures associated with these immunizations and assessed bacterial clearance in the mice using a vaginal/cervical gonococcal infection model. Mice immunized with 4CMenB or MC58 ΔABR demonstrated shortened courses of recovery of vaginal N. gonorrhoeae compared to control mice immunized with alum alone. Vaccination with 4CMenB or MC58ΔABR OMV elicited serum and vaginal cross-reactive anti-Ng-OMV antibody responses that were augmented after vaginal challenge with N. gonorrhoeae . Further, splenocytes in 4CMenB and MC58 ΔABR immunized mice exhibited elevated cytokine production after restimulation with heterologous N. gonorrhoeae OMV when compared to splenocytes from Alum immunized mice. We further tested for correlations between bacterial burden and the measured anti-gonococcal immune responses within each vaccination group and found different immunologic parameters associated with reduced bacterial burden for each vaccine. Our findings suggest the cross-protection against gonococcal infection induced by different meningococcal OMV vaccines is likely multifactorial and mediated by different humoral and cellular immune responses induced by these two vaccines.

Transport and immunomodulatory effect of Hericium erinaceus peptide LPGKVIAS

Abstract Hericium erinaceus–derived peptides have been found to exhibit various bioactivities, including immunoregulatory properties. This study investigated the transport, absorption, and potential immunomodulatory activities of a new peptide, Leu-Pro-Gly-Lys-Val-Ile-Ala-Ser (LPGKVIAS), derived from H. erinaceus. Transport and absorption of LPGKVIAS were analyzed by near-infrared fluorescence in vivo imaging in mice injected with a fluorescence probe–labeled LPGKVIAS. RNA sequencing was used to explore the immunological effects of the peptide on mouse splenocytes. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that LPGKVAS upregulated differentially expressed genes involved in immune regulation. Notably, activation of the lysosome–phagosome pathway in splenocytes of mice treated with LPGKVIAS was identified as a crucial immune defense mechanism against pathogen infection. Furthermore, reverse transcription–polymerase chain reaction studies validated the gene expression data, supporting the potential application of the peptide LPGKVIAS as an immunomodulator.

Design of the conserved epitope peptide of SARS-CoV-2 spike protein as the broad-spectrum COVID-19 vaccine

Our previous study has found that monoclonal antibodies targeting a conserved epitope peptide spanning from residues 1144 to 1156 of SARS-CoV-2 spike (S) protein, namely S(1144–1156), can broadly neutralize all of the prevalent SARS-CoV-2 strains, including the wild type, Alpha, Epsilon, Delta, and Gamma variants. In the study, S(1144–1156) was conjugated with bovine serum albumin (BSA) and formulated with Montanide ISA 51 adjuvant for inoculation in BALB/c mice to study its potential as a vaccine candidate. Results showed that the titers of S protein-specific IgGs and the neutralizing antibodies in mouse sera against various SARS-CoV-2 variants, including the Omicron sublineages, were largely induced along with three doses of immunization. The significant release of IFN-γ and IL-2 was also observed by ELISpot assays through stimulating vaccinated mouse splenocytes with the S(1144–1156) peptide. Furthermore, the vaccination of the S(1143–1157)- and S(1142–1158)-EGFP fusion proteins can elicit more SARS-CoV-2 neutralizing antibodies in mouse sera than the S(1144–1156)-EGFP fusion protein. Interestingly, the antisera collected from mice inoculated with the S(1144–1156) peptide vaccine exhibited better efficacy for neutralizing Omicron BA.2.86 and JN.1 subvariants than Omicron BA.1, BA.2, and XBB subvariants. Since the amino acid sequences of the S(1144–1156) are highly conserved among various SARS-CoV-2 variants, the immunogen containing the S(1144–1156) core epitope can be designed as a broadly effective COVID-19 vaccine.Key points• Inoculation of mice with the S(1144–1156) peptide vaccine can induce bnAbs against various SARS-CoV-2 variants.• The S(1144–1156) peptide stimulated significant release of IFN-γ and IL-2 in vaccinated mouse splenocytes.• The S(1143–1157) and S(1142–1158) peptide vaccines can elicit more SARS-CoV-2 nAbs in mice.

Evaluation of Immunomodulatory Activity of a Polyherbal Formulation (Phytocee™) Using ex vivo and in vitro Models

Background The immune system plays a crucial role in protecting the host by regulating biological activities in multiple organs and circulatory fluids. The phytoextracts are evident to modulate a series of biological activities and play a protective role in managing healthy conditions through interaction with the immune system and exhibit antioxidant, antitumor, anti-inflammatory, antimicrobial, and immunomodulatory activities. Objectives Understanding the function and regulation of immunomodulation may provide potential strategies for the development of natural extracts for the management of adverse conditions. Materials and Methods In the present study, we have investigated the immunomodulatory activity of proprietary polyherbal formulation (Phytocee™) containing Emblica officinalis, Ocimum sanctum, and Withania somnifera using in vitro macrophage phagocytosis and ex vivo natural killer (NK) cell activity. The effect of water and methanolic extracts of Phytocee on NK cell activity was performed using mice splenocytes as effector cells and YAC-1 fibroblast as target cells. The effect of Phytocee on phagocytosis in murine macrophage (J774A.1) cells against fluorescein-labeled bioparticles was also investigated. Results The significant increase in NK cell activity of 35% at 200 µg/mL of water extract and dose-dependent increase with methanol extract confirmed the NK cell-mediated immunomodulation of Phytocee. A phagocytic accumulation of bioparticles within macrophages at 100 and 200 µg/mL of extract group suggests a macrophage activating role and immuno-effectiveness of Phytocee herbal formulation. Conclusion Based on the current data and the historical use of ingredients in the polyherbal formulation support the immunoprotective role of Phytocee.

IL-7Rα on CD4+ T cells is required for their survival and the pathogenesis of experimental autoimmune encephalomyelitis

BackgroundThe IL-7 receptor alpha (IL-7Rα) binds both IL-7 and thymic stromal lymphopoietin (TSLP). IL-7Rα is essential for the development and survival of naive CD4+ T cells and their differentiation to effector/memory CD4+ T cells. Mice lacking IL-7Rα have severe lymphopenia and are resistant to experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. However, it has been reported that IL-7Rα on peripheral CD4+ T cells is disposable for their maintenance and EAE pathogenesis, which does not align with the body of knowledge on the role of IL-7Rα in the biology of CD4+ T cells. Given that a definitive study on this important topic is lacking, we revisited it using a novel approach, an inducible knockout of the IL-7Rα gene in CD4+ T cells.MethodsWe generated Il7rafl/fl/CD4CreERT2 double transgenic mouse line (henceforth CD4ΔIl7ra), susceptible to tamoxifen-induced knockout of the IL-7Rα gene in CD4+ T cells. CD4ΔIl7ra mice were immunized with MOG35 − 55 for EAE induction and monitored for disease development. The expression of IL-7Rα, CD4+ T cell numbers, and MOG35 − 55-specific CD4+ T cell response was evaluated in the central nervous system (CNS) and lymphoid tissues by flow cytometry. Additionally, splenocytes of CD4ΔIl7ra mice were stimulated with MOG35 − 55 to assess their proliferative response and cytokine production by T helper cells.ResultsLoss of IL-7Rα from the surface of CD4+ T cells in CD4ΔIl7ra mice was virtually complete several days after tamoxifen treatment. The loss of IL-7Rα in CD4+ T cells led to a gradual and substantial decrease in their numbers in both non-immunized and immunized CD4ΔIl7ra mice, followed by slow repopulation up to the initial numbers. CD4ΔIl7ra mice did not develop EAE. We found a decrease in the total numbers of TNF-, IFN-γ-, IL-17 A-, and GM-CSF-producing CD4+ T cells and regulatory T cells in the spleens and CNS of immunized CD4ΔIl7ra mice. Tracking MOG35 − 55-specific CD4+ T cells revealed a significant reduction in their numbers in CD4ΔIl7ra mice and decreased proliferation and cytokine production in response to MOG35 − 55.ConclusionOur study demonstrates that IL-7Rα on peripheral CD4+ T cells is essential for their maintenance, immune response, and EAE pathogenesis.

7967 Ruxolitinib Prevents Immune-Checkpoint Inhibitor-Related Diabetes Mellitus Via Inhibition Of Pathogenic IL-21 And IFNγ-Producing CD4+ T Cells

Abstract Disclosure: N.L. Huang: None. J. Ortega: None. S.J. Wang: None. L.N. Scott: None. K. Kimbrell: None. E. McCarthy: None. J. Olay: None. J.N. Nguyen: None. K. Kim: None. J. Lee: None. M.G. Lechner: None. The use of immune checkpoint inhibitor (ICI) therapy has revolutionized the treatment landscape for many advanced cancers by leveraging immune activation through the blockade of checkpoint proteins, including programmed death protein-1 (PD-1). Unfortunately, autoimmune attack of healthy tissue can occur in up to 60% of patients. ICI-induced Type 1 diabetes mellitus (ICI-T1DM) is a rare but life-threatening adverse event during ICI therapy that leads to rapid destruction of pancreatic islets, hyperglycemia, and lifetime dependence on insulin. While cytotoxic CD8+ T cells are recognized as important players in ICI-T1DM, the role for CD4+ helper T cells in disease pathogenesis is less well understood. Using a mouse model of ICI-autoimmunity, in which male and female NOD mice treated with anti-PD-1 (10mg/kg/day, i.p., twice weekly) develop multi-organ autoimmune infiltrates including insulitis and DM, we identified interleukin-21 (IL-21) and interferon gamma (IFNγ) CD4+ T cells as key contributors to ICI-T1DM. Antibody depletion of CD4+ T cells in NOD mice significantly delayed the onset of ICI-T1DM. Immunophenotyping of pancreatic islet infiltrates in ICI-treated mice revealed increased IL-21+ IFNγ+ CD4+ T follicular helper (Tfh; ICOS+ PD1+ CXCR5+) and T peripheral helper (Tph; ICOS+PD1+ CXCR5- CXCR6+) cells. In vitro, IL-21 increased effector CD8+ T cell function and IFNγ promoted production of T cell-attracting chemokines CXCL9, CXCL10, and CXCL16 by macrophages. Given the potential role for IL-21 and IFNγ to amplify ICI-DM progression, we sought to target these signaling pathways as potential interventions. Genetic inhibition of IL-21 or IFNγ signaling in NOD mice reduced insulitis and DM in anti-PD-1 treated mice. Considering that IL-21 and IFNγ act through JAK/STAT signaling in immune cells, we proposed to block this pathway to prevent ICI-DM using ruxolitinib, a clinically available JAK1/2 inhibitor. Indeed, ruxolitinib blocked STAT3 and STAT1 phosphorylation in mouse splenocytes in response to IL-21 or IFNγ, respectively, and completely protected ICI-treated NOD mice from DM. Finally, in a syngeneic tumor model, use of ruxolitinib after initiation of anti-PD-1 therapy did not abrogate ICI anti-tumor effects. Taken together, these data suggest that IL-21 and IFNγ producing CD4+ T cells are important drivers of ICI-T1DM, and that the use of JAK inhibitors may be a promising clinical intervention to reduce ICI-T1DM toxicities in patients. Presentation: 6/2/2024

Immunomodulatory properties of splenocytes treated with chlorpromazine in experimental aggression

Aggressive behavior is considered to be as one of the central symptoms of many neuropsychiatric disorders. It is a serious medical and biological problem associated both with high frequency and severity of manifestations and lack of selective correction means. The purpose of this study was to evaluate the functional phenotype of immune cells treated with chlorpromazine in aggressive animals in vitro, as well as the effect of transplantation of these cells on syngeneic aggressive recipient’s immune cells functional activity. Aggressive behavior was formed in active mice as a result of repeated experience of victories in agonistic interactions with animals with a submissive partner. Further, aggressive animals were isolated into individual cells and used as donors and recipients of immune cells. Immune cells were obtained under sterile conditions from suspension of spleen cells precultured with chlorpromazine. The level of spontaneous and mitogen-induced cell proliferation was assessed using a standard method of radioactive label incorporation. The quantitative content of cytokines in samples of treated with chlorpromazine cell cultures supernatant was determined by enzyme immunoassay using appropriate test systems. Further aggressive syngeneic recipients were intravenously injected with splenocytes precultured with chlorpromazine. The control group of animals was injected with splenocytes precultured under similar conditions, without the chlorpromazine addition. The recipients were assessed for the spleen cells proliferative activity and the intensity of humoral and cellular immune response using standard methods, by the number of antibody-forming spleen cells and severity of a delayed-type hypersensitivity reaction in response to T-dependent antigen introduction. It was found that treatment with chlorpromazine in vitro suppressed mitogen-stimulated proliferation of splenocytes in aggressive mice, without changing of spontaneous proliferation. It was also accompanied by some cytokines production decrease: IL-6, IL-2 and IFNã. When studying the humoral and cellular immune response intensity in aggressive recipients after transplantation of donor’s syngeneic splenocytes treated with chlorpromazine, a decrease in the intensity of humoral immune response was recorded. Transplantation of donor’s splenocytes treated with chlorpromazine was also accompanied by a decrease in spontaneous and mitogen-stimulated proliferation of splenocytes from aggressive recipients. Thus, the obtained data indicate the inhibitory effect of chlorpromazine on immune cell’s functional activity in aggressive mice, as well as the positive immunomodulatory effect of chlorpromazine-treated immune cells transplantation at aggressive behavior in experimental animals.

The immune response formed by the introduction of a DNA vector containing a cDNA fragment of the human thyroid-stimulating hormone receptor gene into transgenic mice

The number of patients with autoimmune thyroid diseases (Graves’disease, Hashimoto’sthyroiditis) is increasing globally. The most important part in the diagnosis of Graves’ disease (GD) is the detection of autoantibodies to the thyrotropin receptor (TSHR) in Graves’ patients’ sera. For the differential diagnosis of antibodies to thyroid antigens, it is promising to use tests based on monoclonal antibodies to TSHR, which can be obtained not only as a result of immunization with native or recombinant TSHR protein, but also through DNA immunization with genetically engineered constructs containing fragments of the TSHR gene. Based on mRNA we isolated from the thyroid tissue in GD, a number of fragments of the thyrotropin receptor gene were cloned, suitable for DNA immunization of animals. The purpose of this work is to evaluate the immunogenic properties of one of the constructed vectors, pVAX1-TSHR (1160), in a mouse model. The successful inclusion of the extracellular domain gene fragment of the human TSHR (1160), which was transfected into CHO cells as a part of the pVAX1 vector was confirmed by immunoblotting and ELISA. The immune response formed to the injection of the pVAX1 vector into BALB/c mice, containing a fragment of the human TSHR gene, was detected in different versions of ELISA. Immunization of animals with the DNA vector pVAX1-TSHR according to an experimentally selected scheme was effective for the formation of mouse splenocytes, secreting antibodies to TSHR, which were used for successful hybridization. This was confirmed by the results of determining antibody production to TSHR in murine blood sera. The level of antibody production remained high (titer more than 1:10.000) at the 8th week of the experiment. As a result of selection of individual clones according to the criteria of proliferative activity and stability of antibody production, the most stable cultures secreting mAbs against TSHR were selected.

Nonclinical Profile of PF-06952229 (MDV6058), a Novel TGFβRI/Activin Like Kinase 5 Inhibitor Supports Clinical Evaluation in Cancer.

The development of transforming growth factor βreceptor inhibitors (TGFβRi) as new medicines has been affected by cardiac valvulopathy and arteriopathy toxicity findings in nonclinical toxicology studies. PF-06952229 (MDV6058) selected using rational drug design is a potent and selective TGFβRI inhibitor with a relatively clean off-target selectivity profile and good pharmacokinetic properties across species. PF-06952229 inhibited clinically translatable phospho-SMAD2 biomarker (≥60%) in human and cynomolgus monkey peripheral blood mononuclear cells, as well as in mouse and rat splenocytes. Using an optimized, intermittent dosing schedule (7-day on/7-day off/cycle; 5 cycles), PF-06952229 demonstrated efficacy in a 63-day syngeneic MC38 colon carcinoma mouse model. In the pivotal repeat-dose toxicity studies (rat and cynomolgus monkey), PF-06952229 on an intermittent dosing schedule (5-day on/5-day off cycle; 5 cycles, 28 doses) showed no cardiac-related adverse findings. However, new toxicity findings related to PF-06952229 included reversible hepatocellular (hepatocyte necrosis with corresponding clinically monitorable transaminase increases) and lung (hemorrhage with mixed cell inflammation) findings at ≥ targeted projected clinical efficacious exposures. Furthermore, partially reversible cartilage hypertrophy (trachea and femur in rat; femur in monkey) and partially to fully reversible, clinically monitorable decreases in serum phosphorus and urinary phosphate at ≥ projected clinically efficacious exposures were observed. Given the integral role of TGFβ in endochondral bone formation, cartilage findings in toxicity studies have been observed with other TGFβRi classes of compounds. The favorable cumulative profile of PF-06952229 in biochemical, pharmacodynamic, pharmacokinetic, and nonclinical studies allowed for its evaluation in cancer patients using the intermittent dosing schedule (7-day on/7-day off) and careful protocol-defined monitoring. SIGNIFICANCE STATEMENT: Only a few TGFβRi have progressed for clinical evaluation due to adverse cardiac findings in pivotal nonclinical toxicity studies. The potential translations of such findings in patients are of major concern. Using a carefully optimized intermittent dosing schedule, PF-06952229 has demonstrated impressive pharmacological efficacy in the syngeneic MC38 colon carcinoma mouse model. Additionally, a nonclinical toxicology package without cardiovascular liabilities and generally monitorable toxicity profile has been completed. The compound presents an acceptable International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use S9-compliant profile for the intended-to-treat cancer patients.

Development of Hypoallergenic Derivatives of Cra a 1 with B Cell Epitope Deletion and T Cell Epitope Retention.

Tropomyosin was reported as an important allergen in Crassostrea angulata and designated as Cra a 1. The localization of the T cell epitopes and the reduction of the immunoreactivity of Cra a 1 are still lacking. In this study, four T cell epitopes were identified by using wild-type Cra a 1 (wtCra a 1)-immunized mouse splenocytes cultured with synthetic peptides. The immunoreactivity was maintained after chemical denaturation treatment, indicating that the linear epitope is an immunodominant epitope of wtCra a 1. Furthermore, the hypoallergenic derivative (mCra a 1) was developed by the deletion of linear B cell epitopes and retention of T cell epitopes. mCra a 1 could stimulate CD4+T cell proliferation and upregulate interleukin-10 secretion. Overall, basophil activation by mCra a 1 was low, but its ability to induce T cell proliferation was retained, suggesting that mCra a 1 may serve as a viable candidate for treating oyster allergy.