Cell-mediated Research Articles

MCRS1 sensitizes T cell-dependent immunotherapy by augmenting MHC-I expression in solid tumors.

Dampened antigen presentation underscores the resistance of pancreatic cancer to T cell-mediated anti-tumor immunity, rendering immunotherapy largely ineffective. By high-throughput CRISPR activation perturbation, we discovered that the transcriptional regulator MCRS1 significantly augmented the sensitivity of mouse pancreatic cancer cells to T cell immunity in vitro and in vivo. Mechanistically, MCRS1 interacted with the transcription factor and genome organizer YY1 to coordinately increase the chromatin accessibility and expression of MHC-I genes. Elevated MCRS1 subverted MHC-I suppression and activated anti-tumor T cells, which sensitized mouse pancreatic cancer to α-PD-1 therapy. Remarkably, high MCRS1 expression was associated with increased T cell infiltration and extended survival of patients with pancreatic cancer and was predictive of favorable responses to α-PD-1 therapy in patients with lung cancer. Together, our study uncovers that MCRS1 sensitizes cancer cells to T cell immunity by transcriptionally subverting MHC-I suppression, which enhances the effectiveness of α-PD-1 therapy in mice and humans, paving the way to further improve immunotherapy against solid tumors.

The HDAC6 inhibitor AVS100 (SS208) induces a pro-inflammatory tumor microenvironment and potentiates immunotherapy.

Histone deacetylase 6 (HDAC6) inhibition is associated with an increased pro-inflammatory tumor microenvironment and antitumoral immune responses. Here, we show that the HDAC6 inhibitor AVS100 (SS208) had an antitumoral effect in SM1 melanoma and CT26 colon cancer models and increased the efficacy of anti-programmed cell death protein 1 treatment, leading to complete remission in melanoma and increased response in colon cancer. AVS100 treatment increased pro-inflammatory tumor-infiltrating macrophages and CD8 effector T cells with an inflammatory and T cell effector gene signature. Acquired T cell immunity and long-term protection were evidenced as increased immunodominant T cell clones after AVS100 treatment. Last, AVS100 showed no mutagenicity, toxicity, or adverse effects in preclinical good laboratory practice studies, part of the package that has led to US Food and Drug Administration clearance of an investigational new drug application for initiating clinical trials. This would be a first-in-human combination therapy of pembrolizumab with HDAC6 inhibition for locally advanced or metastatic solid tumors.

Ro 90-7501 suppresses colon cancer progression by inducing immunogenic cell death and promoting RIG-1-mediated autophagy.

Colon cancer is one of the leading causes of cancer-related mortality worldwide. Current treatments, including surgery, chemotherapy, and radiation therapy, often have limited efficacy due to tumor heterogeneity and resistance mechanisms. Therefore, there is a critical need for novel therapeutic strategies that can enhance the immune response against colon cancer cells while promoting their efficient clearance. In this study, we investigated the anti-tumor effects of Ro 90-7501, a specific small molecule, in colon cancer cell lines (HCT8 and SW480) and in vivo models. MTS, EdU, Scratch Wound and Transwell assays were performed to detect the cell proliferation and metastasis. Additionally, flow cytometry and immunofluorescence staining were used to analyze changes in apoptosis and necrosis. Furthermore, we examined its ability to induce immunogenic cell death (ICD) and promote retinoic acid-inducible gene I (RIG-I)-mediated autophagy. The expression levels of key molecules involved in ICD and autophagy were assessed using Western blot analysis, immunofluorescence staining, and enzyme-linked immunosorbent assay (ELISA). Our findings demonstrated that Ro 90-7501 significantly suppressed colon cancer cell proliferation and metastasis, inducing apoptosis and necrosis. Mechanistically, Ro 90-7501 triggered ICD by upregulating the exposure of calreticulin (CRT) on the cell surface and increasing the release of high mobility group box 1 (HMGB1) and extracellular ATP levels. Concurrently, it promoted RIG-I-mediated autophagy via the MAVS-TRAF6 signaling axis, evidenced by increased expression of autophagy-related genes, such as microtubule-associated protein 1 light chain 3 (LC3) and Beclin 1 proteins, coupled with a reduction in P62 protein. Additionally, Ro 90-7501 treatment activated the RIG-I-MAVS-TRAF6 signaling axis in colon cancer cells. Furthermore, Ro 90-7501 enhanced the secretion of pro-inflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNFα), thereby activating the immune response. In conclusion, Ro 90-7501 exhibits potent anti-tumor activity against colon cancer by inducing ICD and promoting RIG-I-mediated autophagy. These results suggest that Ro 90-7501 may serve as a promising therapeutic candidate for colon cancer treatment by enhancing both intrinsic cellular processes and adaptive immune responses. Further studies are warranted to elucidate the detailed mechanisms underlying these effects and to evaluate its therapeutic potential in clinical settings.

A Meta-Analysis of Association Between Interleukin Polymorphisms (rs4073, rs1800925, rs1179251, rs1179246, rs2227485, rs17855750, and rs153109) and Colorectal Cancer Risk.

Interleukins (ILs) play a significant role in triggering the inflammatory response in blood vessels and immune cells. A systematic review and meta-analysis were conducted to investigate the relationship between IL-8 (rs4073), IL-13 (rs1800925), IL-22 (rs1179251, rs1179246, and rs2227485), and IL-27 (rs17855750 and rs153109) polymorphisms and the risk of developing colorectal cancer (CRC). Four databases were searched up until October 13, 2023, without any restrictions, to find relevant studies. The association was evaluated using crude odds ratios (ORs) and 95% confidence intervals in five genetic models. A total of twenty-three articles were entered into the meta-analysis. The pooled ORs (p-values) for the IL-8 (rs4073) polymorphism were 0.98 (0.63), 0.93 (0.44), 0.89 (0.13), 0.94 (0.38), and 0.99 (0.90) for studies following HWE without heterogeneity, and for all studies with high heterogeneity were 1.03 (0.69), 1.30 (0.07), 1.04 (0.71), 1.12 (0.20), and 1.23 (0.06). For the IL-13 (rs1800925) polymorphism, the pooled ORs were 1.44 (0.06), 2.58 (0.0004), 1.72 (0.16), 1.82 (0.09), and 2.37 (0.001) in AHHDR models, respectively. The pooled ORs of IL-22 (rs1179251) polymorphism for AHHDR models were 0.97 (0.92), 0.92 (0.90), 0.98 (p = 0.95), 1.08 (0.87), and 0.96 (0.82), respectively. The pooled ORs of IL-22 (rs1179246) polymorphism for AHHDR models were 0.98 (0.67), 0.97 (0.80), 0.92 (0.36), 0.93 (0.42), and 1.02 (0.84), respectively. The pooled ORs of IL-22 (rs2227485) polymorphism for AHHDR models were 1.47 (0.02), 2.03 (0.02), 1.28 (0.29), 1.52 (0.06), and 1.70 (0.04), respectively. The pooled ORs of IL-27 (rs17855750) polymorphism for AHHDR models were 0.53 (0.46), 0.19 (0.28), 1.10 (0.60), 0.55 (0.58), and 0.27 (p = 0.05), respectively. The pooled ORs of IL-27 (rs153109) polymorphism for AHHDR models were 1.28 (0.007), 1.45 (0.002), 1.40 (0.0002), 1.41 (< 0.0001), and 1.20 (0.09), respectively. The results reported that just the TT genotype of IL-13 (rs1800925), the T allele and TT genotype of IL-22 (rs2227485), and the G allele and GG, AG and GG + AG genotypes of IL-27 (rs153109) polymorphisms had an elevated risk in CRC patients.

The Photocleavable Protein PhoCl-Based Dynamic Hydrogels.

Dynamic protein hydrogels have attracted increasing attention owing to their tunable physiochemical and mechanical properties, customized functionality, and biocompatibility. Among the different types of dynamic hydrogels, photoresponsive hydrogels are of particular interest. Here, we report the engineering of a photoresponsive protein hydrogel by using the photocleavable protein PhoCl. We employed the well-developed SpyTag and SpyCatcher chemistry to engineer PhoCl-containing covalently cross-linked hydrogels. In the hydrogel network, PhoCl, which can be cleaved into two fragments upon violet irradiation, is employed as a dynamic structural motif to regulate the cross-linking density of the hydrogel network. The resultant PhoCl-containing hydrogels showed photoresponsive viscoelastic properties. Upon violet irradiation, the PhoCl hydrogels soften, leading to an irreversible reduction in the storage moduli. However, no gel-sol transition was observed. Leveraging this light-induced stiffness change, we employed this hydrogel as a cell culture substrate to investigate the mechanobiological response of NIH-3T3 fibroblast cells. Our results showed that 3T3 cells can change their morphologies in response to the stiffness change of the PhoCl hydrogel substrate dynamically, rendering PhoCl-based hydrogels a useful substrate for other mechanobiological studies.

Insulin-like growth factor-binding protein-3 is induced by tamoxifen and fulvestrant and modulates fulvestrant response in breast cancer cells

IntroductionInsulin-like growth factor binding protein-3 (IGFBP-3) exerts varying effects on estrogen receptor alpha (ERα)-positive and triple-negative breast cancer (TNBC) cells. In ERα-positive cells, IGFBP-3 is antiproliferative and proapoptotic. In contrast, IGFBP-3 stimulates proliferation in triple-negative breast cancer (TNBC) cells via EGFR activation.MethodsTo identify potential mechanisms that underlie the opposing effects of IGFBP-3 on these two breast cancer subtypes, IGFBP-3 expression was determined in cell line models of both ERα-positive breast cancer and TNBC, and cells were treated with antiestrogens tamoxifen and fulvestrant. Results and discussionMCF-7 and T-47D cells expressed low levels of IGFBP-3 when compared to MDA-MB-231 and MDA-MB-468 cells. MCF-7 cells with acquired resistance to the selective estrogen receptor degrader fulvestrant expressed high IGFBP-3 and MCF-7 cells with constitutive IGFBP-3 expression were fulvestrant resistant. IGFBP-3 expression was increased in all cell lines upon treatment with fulvestrant or the selective estrogen receptor modulator tamoxifen and both fulvestrant and tamoxifen increased TNBC cell proliferation. Further, IGFBP-3 expression was increased by treatment with the GPER1 agonist G-1 and attenuated upon treatment with P17, a YAP/TAZ inhibitor. These data suggest that IGFBP-3 modulates breast cancer cells and is a mediator of breast cancer cell response to fulvestrant and tamoxifen.

Resveratrol improves follicular development in PCOS rats by inhibiting the inflammatory response and pyroptosis of granulosa cells.

Chronic inflammation is a key characteristic of polycystic ovary syndrome (PCOS) and is associated with follicular dysplasia in PCOS. PCOS patients treated with 1000mg resveratrol (RES) daily for 3months showed significant improvement in menstrual cycle regularity compared to the placebo group. This investigation explores potential impact of RES on a rat model of PCOS. Sprague-Dawley (SD) rats were subjected to a 30-day letrozole/high-fat diet interventions for PCOS model establishment, followed by RES intervention (20mg/kg/d) for an additional 30days. RES intervention mitigated obesity, estrous cycle irregularities, and ovulation disorders while decreasing serum testosterone and lipopolysaccharide (LPS) levels in PCOS rats. Concurrently, inflammatory markers (TNF-α, NLPR3, IL-6,) and pyroptosis-related markers (GSDMD, cleaved-Caspase-1, IL-1β, IL-18) were downregulated. Additionally, KGN cells (a human granulosa-like cell line) were treated with LPS and RES for in vitro assays. It was observed that RES (15μM) significantly reduced ROS production and downregulated inflammatory cytokine expression in LPS-intervened KGN cells. Additionally, RES downregulated the expression levels of pyroptosis-related factors (GSDMD and cleaved-Caspase-1) and attenuated IL-18 and IL-1β secretion in LPS-induced KGN cells. Furthermore, RES intervention improved the pyroptosis-associated morphology of KGN cells after LPS treatment. In conclusion, RES may restore follicular development in PCOS rats by inhibiting inflammation and NLRP3/GSDMD/Caspase-1-mediated pyroptosis of ovarian granulosa cells, providing new insights into potential therapeutic approaches for PCOS.

Time-dependent changes in genome-wide gene expression and post-transcriptional regulation across the post death process in silkworm.

Despite death marking the end of life, several gene expression and miRNA-mediated post-transcriptional regulation events may persist or be initiated. The silkworm (Bombyx mori) is a valuable model for exploring life processes, including death. In this study, we combined transcriptomics and miRNAomics analyses of young, old, and postmortem silkworms across the entire process after death to unravel the dynamics of gene expression and miRNA-mediated post-transcriptional regulation. In total, 171 genes exhibited sustained differential expression in postmortem silkworms compared to the pre-death state, which are primarily involved in nerve signaling, transport, and immune response. Postmortem time-specific genes were associated with cell cycle regulation, thermogenesis, immunity, and zinc ion homeostasis. We found that the down-regulated expression of 36 genes related to transcription, epigenetic modification, and homeostasis resulted in a significant shift in global gene expression patterns at 2h post-death. We also identified five mRNA-miRNA pairs (i.e., bmo-miR-2795-mhca, 2784-achi, 2762-oa1, 277-5p-creb, and 1000-tcb1) associated with stress hormone regulation, transcription activity, and signal transduction. The roles of these pairs were validated through in vivo experiments using miRNA mimics in silkworms. The findings provide valuable insights into the intricate mechanisms underlying the transcriptional and miRNA-mediated post-transcriptional regulation events in animals after death.

Cell response to extracellular matrix viscous energy dissipation outweighs high-rigidity sensing.

The mechanics of the extracellular matrix (ECM) determine cell activity and fate through mechanoresponsive proteins including Yes-associated protein 1 (YAP). Rigidity and viscous relaxation have emerged as the main mechanical properties of the ECM steering cell behavior. However, how cells integrate coexisting ECM rigidity and viscosity cues remains poorly understood, particularly in the high-stiffness regime. Here, we have exploited engineered stiff viscoelastic protein hydrogels to show that, contrary to current models of cell-ECM interaction, substrate viscous energy dissipation attenuates mechanosensing even when cells are exposed to higher effective rigidity. This unexpected behavior is however readily captured by a pull-and-hold model of molecular clutch-based cell mechanosensing, which also recapitulates opposite cellular response at low rigidities. Consistent with predictions of the pull-and-hold model, we find that myosin inhibition can boost mechanosensing on cells cultured on dissipative matrices. Together, our work provides general mechanistic understanding on how cells respond to the viscoelastic properties of the ECM.

B cell adapter for PI 3-kinase (BCAP) coordinates antigen internalization and trafficking through the B cell receptor.

B cell adapter for PI 3-kinase (BCAP) is an adaptor molecule associated with signaling through multiple immune receptors, including the B cell receptor (BCR). However, B cell-intrinsic role of BCAP in antibody responses is unclear. We investigated the role of BCAP in B cell response to viral particles and found a previously unidentified mechanism by which BCAP regulates antigen-specific responses. B cell-specific deletion of BCAP in mice leads to decreases in antigen-specific responses through defects in BCR-antigen endocytosis. BCAP is necessary to orchestrate actin reorganization around the antigen for efficient endocytosis through BCR and intracellular processing of antigens. Therefore, loss of BCAP from B cells leads to defects in antigen endocytosis, hampering the propagation of antigen-derived signals and decreasing the ability of B cells to present antigens to T cells. Thus, our study clarifies how BCAP regulates B cell responses to complex antigens and elucidates that antigen positioning inside B cells determines different B cell activation outcomes.

A Case of Paraneoplastic Autoimmune Retinopathy in a Young Man with Testicular Cancer

Purpose: To report a case of paraneoplastic autoimmune retinopathy in a patient with a history of testicular cancer.Case summary: A 29-year-old man presented with photopsia and floaters. Initial fundus examination revealed no abnormal findings. However, he returned 1 month later with complaints of visual field defects. Fundus examination revealed diffuse white spots in the macula and midperipheral retina and fundus autofluorescence demonstrated hyper-autofluorescence. Optical coherence tomography showed disruption of the ellipsoid zone sparing the fovea. Visual field examination revealed peripheral visual field defects and an electroretinogram showed reduced rod and cone cell responses. Considering his history of testicular cancer, serum paraneoplastic autoantibody panel testing was performed which revealed borderline levels of anti-recoverin antibody leading to a diagnosis of paraneoplastic autoimmune retinopathy. The patient was treated with oral steroids and mycophenolate mofetil for 1 year. However, there was no improvement in the subjective symptoms or ophthalmologic findings.Conclusions: This case of paraneoplastic autoimmune retinopathy in a young man with a history of testicular cancer highlights an early clinical presentation of the disease. It is crucial to recognize that the initial clinical presentation of autoimmune retinopathy can be nonspecific.

Antibody isotyping and cytokine profiling in natural cases of Burkholderia mallei infection (glanders) in equines.

Immunological aspects of B. mallei infection were rarely studied in natural cases of equines. The present study was conducted to determine IgG, IgM, IgA and IgG (T) titre against recombinant Hcp1, TssA and TssB proteins and PilA-Hcp1-TssN-BipD and BpaB-BpaC- BMAA0553 chimeras and cytokine responses in glanders affected equine serum. The study was conducted on serum samples collected from 151 glanders positive equines which include horses (n=134), mules (n=16), and donkeys (n=01). The IgM, IgG, IgA and IgG (T) titre were determined against recombinant antigens by indirect ELISA and interleukin(IL)-1β, IL-17, IL-6, tumor necrosis factor alpha(TNF-α), monocyte chemoattractant protein 1 (MCP-1) and interferon gamma (IFN-γ) responses were measured by commercial ELISA kits. The study showed that glanders affected equines elicited strong antibody response against Hcp1, moderate responses against TssA and TssB, and weak responses against two chimeras. Among the cytokines, IL-1β, MCP-1, IL-17 and IL-6 concentration were significantly higher in glanders affected equine serum. We found that IgG antibody titre was higher than IgM, IgG (T) and IgA isotypes and Hcp1 was most predominant antibody inducers in comparison to TssA, TssB, PilA-Hcp1-TssN-BipD and BpaB-BpaC-T2SS proteins. The elevated level of IL-1β, MCP-1, IL-17, IL-6, IFN-γ and TNF-α observed in this study support the important role of this cytokines for augmenting cellular defence by recruitment of macrophages, neutrophil and dendritic cells against B. mallei infection. Further studies should be conducted to determine memory cell responses in natural cases of equine glanders using recombinant B. mallei proteins for identifying well-characterized immuno-protective vaccine candidates.

Divergent clinical and immunologic outcomes based on STK11 co-mutation status in resectable KRAS-mutant lung cancers following neoadjuvant immune checkpoint blockade.

Co-mutations of the KRAS and STK11 genes in advanced non-small cell lung cancer (NSCLC) are associated with immune checkpoint blockade (ICB) resistance. While neoadjuvant chemoimmunotherapy is now a standard of care treatment for resectable NSCLC, the clinical and immunologic impact of KRAS andSTK11 co-mutations in this setting are unknown. We evaluated and compared recurrence-free survival of resectable KRAS-mutated NSCLC tumors, with or without co-occuring STK11 mutations, treated with neoadjuvant ICB. Single cell transcriptomics was performed on tumor-infiltrating T cells from 7 KRASmut/STK11wttumors and 6 KRASmut/STK11mut tumors. Relative to KRASmut/STK11wttumors, KRASmut/STK11mut exhibited significantly higher recurrence risk. Single-cell transcriptomics showed enhanced oxidative phosphorylation with evidence of decreased PGE-2 signaling and increased IL-2 signaling in CD8+ tumor-infiltrating lymphocytes (TIL) from KRASmut/STK11mut tumors, a finding that was mirrored in KRASwt tumors that relapsed. TIL from KRASmut/STK11mut tumors expressed high levels of molecules associated with tumor residence, including CD39 and ZNF683 (HOBIT). These divergent T cell transcriptional fates suggest T cell maintenance and residence may be detrimental to anti-tumor immunity in the context of neoadjuvant ICB for resectable NSCLC, regardless of KRAS mutation status. Our work provides a basis for future investigations into the mechanisms underpinning PGE-2 and IL-2 signaling as they relate to T cell immunity to cancer and to divergent clinical outcomes in KRASmut/STK11mut NSCLC treated with neoadjuvant ICB.

Sirtuin inhibitors reduce intracellular growth of M. tuberculosis in human macrophages via modulation of host cell immunity.

Host-directed therapies aiming to strengthen the body's immune system, represent an underexplored opportunity to improve treatment of tuberculosis (TB). We have previously shown in Mycobacterium tuberculosis (Mtb)-infection models and clinical trials that treatment with the histone deacetylase (HDAC) inhibitor, phenylbutyrate (PBA), can restore Mtb-induced impairment of antimicrobial responses and improve clinical outcomes in pulmonary TB. In this study, we evaluated the efficacy of different groups of HDAC inhibitors to reduce Mtb growth in human immune cells. A panel of 21 selected HDAC inhibitors with different specificities that are known to modulate infection or inflammation was tested using high-content live-cell imaging and analysis. Monocyte-derived macrophages or bulk peripheral blood cells (PBMCs) were infected with the green fluorescent protein (GFP)-expressing Mtb strains H37Ra or H37Rv and treated with HDAC inhibitors in the micromolar range in parallel with a combination of the first-line antibiotics, rifampicin, and isoniazid. Host cell viability in HDAC inhibitor treated cell cultures was monitored with Cytotox-red. Seven HDAC inhibitors were identified that reduced Mtb growth in macrophages > 45-75% compared to average 40% for PBA. The most effective compounds were inhibitors of the class III HDAC proteins, the sirtuins. While these compounds may exhibit their effects by improving macrophage function, one of the sirtuin inhibitors, tenovin, was also highly effective in extracellular killing of Mtb bacilli. Antimicrobial synergy testing using checkerboard assays revealed additive effects between selected sirtuin inhibitors and subinhibitory concentrations of rifampicin or isoniazid. A customized macrophage RNA array including 23 genes associated with cytokines, chemokines and inflammation, suggested that Mtb-infected macrophages are differentially modulated by the sirtuin inhibitors as compared to PBA. Altogether, these results demonstrated that sirtuin inhibitors may be further explored as promising host-directed compounds to support immune functions and reduce intracellular growth of Mtb in human cells.

A multi-epitope subunit vaccine providing broad cross-protection against diverse serotypes of Streptococcus suis.

Streptococcus suis infection represents a major challenge in pig farming and public health due to its zoonotic potential and diverse serotypes, while existing vaccines lack effective cross-protection. This study employed reverse vaccinology and immunoinformatics to identify 8 conserved proteins across 11 prevalent serotypes of S. suis. 16 candidate epitopes were selected to design three multi-epitope antigens against S. suis (designated as MEASs), which fused with a dendritic cell-targeting peptide to improve antigen presentation in host. Purified MEASs displayed favorable cross-reactogenicity against 29 serotype-specific antiserums. Robust humoral and cellular immune responses can be induced by MEAS 1 and MEAS 3 in a mouse model, which provided substantial protection against virulent strains from two different serotypes. In particular, their immune serums exhibited positive opsonization effects within bloodstream and macrophage phagocytosis. Taken together, we identified two promising MEASs with excellent cross-protection, offering potential in preventing S. suis infections in a mouse model.

A self-amplifying RNA RSV prefusion-F vaccine elicits potent immunity in pre-exposed and naïve non-human primates.

Newly approved subunit and mRNA vaccines for respiratory syncytial virus (RSV) demonstrate effectiveness in preventing severe disease, with protection exceeding 80% primarily through the generation of antibodies. An alternative vaccine platform called self-amplifying RNA (saRNA) holds promise in eliciting humoral and cellular immune responses. We evaluate the immunogenicity of a lipid nanoparticle (LNP)-formulated saRNA vaccine called SMARRT.RSV.preF, encoding a stabilized form of the RSV fusion protein, in female mice and in non-human primates (NHPs) that are either RSV-naïve or previously infected. Intramuscular vaccination with SMARRT.RSV.preF vaccine induces RSV neutralizing antibodies and cellular responses in naïve mice and NHPs. Importantly, a single dose of the vaccine in RSV pre-exposed NHPs elicits a dose-dependent anamnestic humoral immune response comparable to a subunit RSV preF vaccine. Notably, SMARRT.RSV.preF immunization significantly increases polyfunctional RSV.F specific memory CD4+ and CD8+ T-cells compared to RSV.preF protein vaccine. Twenty-four hours post immunization with SMARRT.RSV.preF, there is a dose-dependent increase in the systemic levels of inflammatory and chemotactic cytokines associated with the type I interferon response in NHPs, which is not observed with the protein vaccine. We identify a cluster of analytes including IL-15, TNFα, CCL4, and CXCL10, whose levels are significantly correlated with each other after SMARRT.RSV.preF immunization. These findings suggest saRNA vaccines have the potential to be developed as a prophylactic RSV vaccine based on innate, cellular, and humoral immune profiles they elicit.

Mycobacterium tuberculosis Antigen Rv1471 Induces Innate Immune Memory and Adaptive Immunity against Infection.

Protein subunit vaccines form a key pipeline for developing novel tuberculosis (TB) vaccines. Mycobacterium tuberculosis (Mtb) contains approximately 4000 individual proteins. However, only approximately 100 have been evaluated as antigens in the clinical and preclinical stages of vaccine development. Trained immunity-targeting vaccines induce innate immune memory against heterologous infections and enhance antigen-specific adaptive immune responses. However, no trained immunity-targeting subunit TB vaccine has been reported yet. This study tested Rv1471, a thioredoxin secreted by Mtb, as a candidate TB vaccine antigen due to its capacity to stimulate mouse bone marrow-derived macrophages (BMDMs). Rv1471 induced functional and phenotypic maturation of BMDMs in vitro, reflected by the increased production of inflammatory cytokines and surface expression of co-stimulatory molecules. Transcription analysis of Rv1471-trained BMDMs indicated that innate immune memory was activated through pathways of Akt-mTOR-HIF-1α and aerobic glycolysis. Rv1471 also enhanced innate immune memory responses and protection against intracellular infections of different mycobacteria. In a murine model of TB, immunization with Rv1471 formulated with liposomal DDA/MPLA adjuvant produced robust antigen-specific multi-functional CD4+ and CD8+ T-cell immune responses and had substantial protective efficacy against Mtb challenge. Analysis of recall immunity showed that the Rv1471 subunit vaccine triggered robust T-cell immunity post Mtb infection. These findings support the development of an innate immune memory-targeting subunit TB vaccine to increase TB vaccine efficacy.

Staphylococcal superantigens evoke temporary and reversible T cell anergy, but fail to block the development of a bacterium specific cellular immune response.

Superantigens (sAgs) are bacterial virulence factors that induce a state of immune hyperactivation by forming a bridge between certain subsets of T cell receptor (TCR) β chains on T lymphocytes, and class II major histocompatibility complex (MHC-II) molecules; this cross-linking leads to indiscriminate T cell activation, cytokine storm and toxic shock. Here we show that sAg exposure drives the preferential expansion of naive and central memory T cell subsets, but not effector or resident memory T cells, which instead, hyper release pro-inflammatory cytokines. A targeted therapeutic approach to minimise cytokine release by effector memory T cells attenuated sAg-induced cytokine release. Irrespective of antigen experience, sAg activation does not render mature T cells permanently dysfunctional, and full restoration of effector function is observed following a transient and reversible anergy. Moreover, we show that in the face of sAg induced immune hyperactivation, an intact bacterium-specific CD4+ T cell response can be mounted.

Neoantigen DNA vaccines are safe, feasible, and induce neoantigen-specific immune responses in triple-negative breast cancer patients

Abstract Background Neoantigen vaccines can induce or enhance highly specific antitumor immune responses with minimal risk of autoimmunity. We have developed a neoantigen DNA vaccine platform capable of efficiently presenting both HLA class I and II epitopes and performed a phase 1 clinical trial in triple-negative breast cancer patients with persistent disease on surgical pathology following neoadjuvant chemotherapy, a patient population at high risk of disease recurrence. Methods Expressed somatic mutations were identified by tumor/normal exome sequencing and tumor RNA sequencing. The pVACtools software suite of neoantigen prediction algorithms was used to identify and prioritize cancer neoantigens and facilitate vaccine design for manufacture in an academic GMP facility. Neoantigen DNA vaccines were administered via electroporation in the adjuvant setting (i.e., following surgical removal of the primary tumor and completion of standard of care therapy). Vaccines were monitored for safety and immune responses via ELISpot, intracellular cytokine production via flow cytometry, and TCR sequencing. Results Eighteen subjects received three doses of a neoantigen DNA vaccine encoding on average 11 neoantigens per patient (range 4–20). The vaccinations were well tolerated with relatively few adverse events. Neoantigen-specific T cell responses were induced in 14/18 patients as measured by ELISpot and flow cytometry. At a median follow-up of 36 months, recurrence-free survival was 87.5% (95% CI: 72.7–100%) in the cohort of vaccinated patients. Conclusion Our study demonstrates neoantigen DNA vaccines are safe, feasible, and capable of inducing neoantigen-specific immune responses. Clinical trial registration number NCT02348320.

Abstract PR005: Modulating the immunosuppressive pancreas tumor microenvironment through intratumoral delivery of cytokine-encoding mRNAs

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive cancer, with a mere 13% average 5-year survival rate. The hallmark desmoplastic stromal response in PDAC leads to poor vascularization, drug delivery challenges, and impaired cytotoxic immune cell infiltration, contributing to de novo therapy resistance. Our previous research has shown therapeutic induction of cellular senescence with RAS pathway targeting therapies can induce cytokine and chemokine production through the senescence-associated secretory phenotype (SASP) that can effectively activate anti-tumor T cell immunity and responses to anti-PD-1 immune checkpoint blockade. To build on these findings as well as overcome limitations associated with senescence-inducing therapy toxicity and induction of pro-tumorigenic SASP cytokines, here we leveraged mRNA technology to deliver specific SASP cytokines and chemokines we have found to stimulate immune responses directly into the suppressive PDAC TME as an immune oncology platform. We created an in vitro transcription pipeline for mRNA production and multiplexing, and demonstrate we can intratumorally deliver multiple mRNAs simultaneously into the TME of transplanted and autochthonous PDAC mouse models, stimulating the local production of cytokines normally absent in PDAC. We further characterized a novel combination of five cytokines and chemokines that can effectively recruit and activate both innate and adaptive immune responses against PDAC. Through repeated bi-weekly dosing, this mRNA combination also promotes tumor destruction and improves survival outcomes. Considering the recent success of off-the-shelf neoantigen mRNA vaccines in early human PDAC patient trials, we have now developed mRNAs that encode PDAC-specific antigens. By integrating our cytokine mRNA platform with antigen mRNAs, we observe a significant increase in intratumoral antigen-presenting dendritic cell populations and a substantially enhanced T-cell mediated anti-tumor immune response in orthotopic transplant models of PDAC. Overall, this study not only unveils pivotal insights into the cytokines absent in PDAC that are crucial for promoting anti-tumor immunity, but also pioneers an innovative immunotherapy approach. This platform has the potential to be integrated with existing immunotherapy modalities, addressing the longstanding challenge of therapy resistance in PDAC. Citation Format: Chaitanya Naimesh Parikh, Kelly De Marco, Boyang Ma, Katherine Murphy, Loretah Chibaya, Lin Zhou, Youwei Qiao, Griffin Kane, Li Li, Wen Xue, Marcus Ruscetti. Modulating the immunosuppressive pancreas tumor microenvironment through intratumoral delivery of cytokine-encoding mRNAs [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr PR005.