bluuebunny/crossref_metadata_2025 huggingface dataset:
Uptake of extracellular complement C3 by cardiomyocytes intervenes the intrinsic apoptosis.
Authors: Fang Zhou
Published: January 2023

A recycling pathway of complement C3 was discovered as a component of the intracellular complement system. Human immune cells (B and T lymphocytes) and non-immune cells (endothelial cells) can rapidly internalize C3 from the extracellular milieu, which plays an important role in T cell survival and epithelial cell death. We investigated if the uptake of extracellular C3 can take place in the parenchymal cells which shielded by endothelial/epithelia barriers and further studied the pathophysiological role of such internalized C3. Using an in vitro system of human cardiomyocytes, we found that AC-16 cardiomyocytes, which did not produce matured C3, were readily uptake the exogenous C3 once being exposed to it. Such internalized C3 helped these cells to mitigate apoptosis under oxidative stress condition. Cardiomyocytes may be exposed to circulating C3 after the endothelial barrier being compromised, which happened upon the reperfusion of ischemic myocardium. We further investigated in the effect of internalized C3 in an animal model of myocardial ischemia/reperfusion (IR). Our results showed that C3−/− mice had significant increase of apoptosis compared with WT mice. Furthermore, C3-bound immunocomplex from WT mice undergone IR contained Cyt c, a key apoptotic factor in the intrinsic apoptosis pathway. Biochemically, C3 purified from blood can inhibit the Cyt c mediated cell death in a cell free apoptosis system. Thus, the C3-uptaking mechanism by the parenchymal cells may serve as an innate immune surveillant for intracellular homeostasis by recognition of potential intracellular damage-associated molecular pattern molecules (DAMPs).
Intracellularly Delivered Nuclear Localization Sequence Attenuates The Severity Of COVID-19 By Inhibits Interaction Of Inflammatory Transcription Factors And Importins
Authors: Kim Dongho , Kim Gyunam , Shin Dasom , Yu Sanghyeon , Baek Misuk , Lee Jaewook , Yu Hyemin , Pi Joohyun , Kang Mingu , Kim Jieun , Kim Sujeong , Chung Eunna , Jo Daewoong
Published: January 2023

The outbreak of infectious severe and acute respiratory syndrome (SARS)-CoV-2 which induces coronavirus disease 19 (COVID-19) causes patients to develop acute respiratory distress syndrome (ARDS) and death by triggering uncontrolled immune responses. Dysregulated immune responses impair healthy organs by over-production of pro-inflammatory cytokines, which are called “cytokine storm”. In this study, an improved cell-permeable nuclear import inhibitor (iCP-NI) has been developed by fusing sequence-optimized advanced macromolecule transduction domains (aMTDs) and nuclear localization sequence (NLS) originated from human NF-κB to treat the cytokine storm mediated organ failure and death in COVID-19. Intracellularly delivered NLS inhibits the transport of various inflammation-associated transcription factors (IATFs) such as NF-κB, NFAT, AP-1 and STAT1/3 which results in suppressed expression of pro-inflammatory cytokines. In brief, iCP-NI decreased the lethality of RNA virus infection mimetic pneumonitis animals by protecting the lung structure from inflammation-mediated destruction via reduction of the inflammatory cytokines in the lung. Continuously, iCP-NI significantly attenuates the severity of the SARS-CoV-2 infected syrian hamster model. The inhibition of viral replication is proved with quantitative PCR by reducing the level of RdRp gene (log7.04 vs log5.79). Likewise, the levels of inflammatory cytokines such as TNF-α and IL-17A in the lungs are significantly decreased and thereby, the lung structures are protected from inflammation-mediated damages. As a result, the iCP-NI could be developed as a novel medicine to cure various inflammatory diseases including COVID-19.
Rad52 mediates class-switch DNA recombination to IgD
Authors: Xu Yijiang , Zhou Hang , Post Ginell , Zan Hong , Casali Paolo
Published: January 2023

While the biology of IgD begins to be better understood, the mechanism of expression of this phylogenetically old and highly conserved Ig remains unknown. In B cells, IgD is mainly expressed together with IgM through alternative splicing of primary VHDJH-Cμ-s-m-Cδ-s-m RNAs, but also independently through IgD class switch DNA recombination (CSR) via double-strand DNA breaks (DSBs) and synapse of Sμ with σδ. How such DSBs, however, are resolved is still unknown. Our previous demonstration of a novel role of Rad52 in a Ku70/Ku86-independent “short-range” microhomology-mediated synapsis of intra-Sμ region DSBs led us to hypothesize that this homologous recombination DNA annealing factor is also involved in short-range microhomology-mediated alternative endjoining (A-EJ) recombination of Sμ with σδ. We found that induction of IgD CSR downregulates Zfp318 and promotes Rad52 phosphorylation and recruitment to Sμ and σδ, leading to alternative end-joining (A-EJ)-mediated Sμ-σδ recombination of resected DSB ends with extensive microhomologies, VHDJH-Cδs transcription and sustained IgD secretion. Rad52 ablation in mouse Rad52−/− B cells aborted IgD CSR in vitro and in vivo and dampened the specific IgD antibody response to OVA. Rad52 knockdown in human B cells also abrogated IgD CSR. Finally, Rad52 phosphorylation was associated with high levels of IgD CSR and anti-nuclear IgD autoantibodies in lupus-prone mice and patients with lupus. Our findings thus show that Rad52 mediates IgD CSR through microhomology-mediated A-EJ in concert with Zfp318 downregulation. This is a previously unrecognized and critical role of Rad52 in mammalian DNA repair that provides a mechanistic underpinning to CSR A-EJ.

Supported by NIH grants AI 079705, AI 105813, AAI 167416 and LRA grant 641363 to PC.
The mutation PTPN6Ala455Thr causing pulmonary emphysema affects B lymphocyte populations and causes spontaneous pulmonary tertiary lymphoid tissue formation
Authors: Pineault Marie , Bouffard Gabrielle , Milad Nadia , Tremblay Félix , Lechasseur Ariane , Routhier Joanie , Aubin Sophie , Beaulieu Marie-Josée , Bossé Yohan , Maltais François , Morissette Mathieu C
Published: January 2023

Background

A new mutation responsible for causing severe and early pulmonary emphysema was recently discovered in a French-Canadian family. The mutation is located in the PTPN6 gene (PTPN6Ala455Thr) leading to a reduction in the activity of SHP-1, a phosphatase that regulates numerous pathways including B cell differentiation and activation. Here, we aimed to characterize B cell abnormalities in both humans and mice carrying this mutation.

Methods

Circulating B cell populations in humans carrying the PTPN6Ala455Thr mutation were analyzed by flow cytometry and immunoglobulin levels were measured in serum. In mice carrying the same mutation (Ptpn6Ala457Thr) aged up to 12 months, B cell populations were analyzed by flow cytometry and lung tissue histology performed. Mice were immunized with the T cell-independent antigen NP-Ficoll and plasma was collected to assess NP-specific antibodies.

Results

Human carriers of the PTPN6Ala455Thr mutation had reduced IgG1 and IgG4, and increased IgG3 levels as well as high circulating immature/mature B cell ratio compared to controls. Aging Ptpn6Ala457Thr mice developed spontaneous pulmonary tertiary lymphoid tissues and exhibited higher B-1 cells and lower B-2 cells proportions in the lung and spleen. Ptpn6Ala457Thr mice had lower levels of NP-specific IgG1 antibodies compared to the wild-type group, but normal IgG3 in response to NP-Ficoll immunization.

Conclusion

SHP-1 appears to be important to B cell development and functions, as the PTPN6Ala455Thr mutation seems to cause a form of subclinical immunodeficiency that affects B cell populations and immunoglobulin levels. The link between B cell abnormalities and development of pulmonary emphysema requires further investigation.

Supported by grants from Canadian Institutes of Health and Research and Fonds de Recherche du Québec- Santé
Effect of post-exercise cupping therapy on the inflammatory markers in taekwondo athletes
Authors: Fang Shih-Hua , Yeh Yu-An
Published: January 2023

Cupping therapy has been claimed to drain toxins, loosen adhesions, and bring blood flow to skin and muscles. Moreover, cupping therapy is believed to modulate the immune system. We investigated the acute effects of post-exercise cupping on the fatigue, mucosal and blood immune responses of atheletes. Fourteen elite taekwondo athletes were recruited, and randomly allocated to two groups including placebo group (PLA) who lied down and rested for 15 min and the treatment group (CUP) who received cupping treatment for 10 min and rested for 5 min. A rated perceived exertion (RPE) was used to evaluate how hard the athletes felt at the time before training (Pre), after training (Post) and 30 min after cupping treatment (Post30). Furthermore, the concentrations of tumor necrosis factor alpha (TNF-a), white blood cells (WBC) and neutrophils, neutrophil–lymphocyte ratio (NLR), and platelet–lymphocyte ratio (PLR) in blood were measured. In addition, the salivary immunoglobulin A (SIgA) and a-amylase activity were also analyzed. The CUP group showed significantly lower RPE than PLA group at Post30. The concentrations of WBC, neutrophil, NLR and PLR were markedly increased after training in both groups, but PLR was significantly decreased at Post30 in CUP group. In addition, the concentration of TNF-a was also decreased significantly at Post30. Furthermore, the activity of a-amylase of CUP group was significantly higher than PLA group at Post30. After high intensity of training, cupping treatment decreased the concentrations of PLR and TNF-a, but increased the salivary a-amylase activity. This study suggested that cupping therapy improved the mucosal immune function and decreased inflammation.

Supported by MOST 106-2410-H-028-002 & MOST109-2410-H-028-009-MY3
Treatment with an orally delivered non-replicating, non-colonizing strain of Veillonella parvula resolves systemic inflammation in murine models of disease
Authors: Ramani Kritika , Cormack Taylor , Cartwright Adam , Alami Aula , Argueta Shannon , Raghunathan Divya , Abdou Mary , Romano Fabian B , Kravitz Valeria , Rommel Tyler , Kiran Esra Uckun , Pradeep Pallavi , Ponichtera Holly , Sizova Maria , Ganguly Tanmoy , Itano Andrea
Published: January 2023

Introduction

The mucosal immune system comprises of a complex network of intestinal epithelial cells and immune cells. We report that an orally delivered microbial strain of Veillonella parvula (EDP1867) that is non-replicating and non-colonizing acts through pattern recognition receptor mediated modulation of small intestinal immune responses to induce potent systemic inflammation resolution.

Methods

EDP1867 is a strain of Veillonella parvula isolated from mucosal tissue of a human donor and rendered non replicating by gamma irradiation. In vitro experiments were performed in primary human immune cells. In vivo efficacy was done using mouse models of delayed-type hypersensitivity (DTH) and psoriasis. Fluorescently labeled EDP1867 was used in biodistribution experiments.

Results

EDP1867 induced IL-10 in primary human immune cells. In a DTH model, orally delivered EDP1867 reduced inflammation and diminished production of pro-inflammatory cytokines ex vivo. Fluorescently labeled EDP1867 transited through the gut without systemic exposure in biodistribution studies to demonstrate gut restriction. EDP1867 activity is dependent on TLR2 and migration of immune cells through the gut. In an imiquimod-induced psoriasis model, EDP1867 reduced skin inflammation and psoriasis score and Th17 cytokines in the skin.

Conclusions

EDP1867 drives broad based resolution of inflammation and can reestablish normal homeostasis. These data demonstrate that EDP1867 can act as a new oral anti-inflammatory drug that is gut restricted and dependent on local immune recognition within the small intestine, to induce systemic anti-inflammatory effects. EDP1867 is currently in clinical development for treatment of inflammatory diseases.
The role of immune checkpoint PD-1 in the submandibular gland
Authors: Borys Samantha , Brossay Laurent
Published: January 2023

Immune checkpoints play an important role in restraining the immune response, maintaining self-tolerance and preventing excessive collateral damage. Immune checkpoint blockade has revolutionized cancer immunotherapy but can lead to immune related adverse events (IrAEs). Patients receiving programmed cell death protein 1 (PD-1) blockade can develop IrAEs including vitiligo, colitis, endocrinopathies, and Sicca Syndrome, an autoimmune disease affecting the salivary and lacrimal glands. After immune cell activation, PD-1 is upregulated, and PD-1 signaling results in reduced proliferation, cytokine production, and cytotoxicity. PD-1 is expressed on infiltrating lymphocytes of the submandibular gland (SMG) including natural killer (NK) and T cells, and recognizes the ligand, PD-L1. Hyporesponsive immune cells in the SMG are thought to retain the integrity of this delicate tissue, while inadvertently contributing to viral latency. We therefore hypothesized that signaling through coinhibitory receptors, including PD-1, may impact these hyporesponsive effector cells. Our preliminary data indicate that genetic loss of PD-1 results in increased CD8+ T cell number and frequency in the SMG, with greater proliferation status. Given the interplay between self-tolerance and chronic viral control, we also examined the role of PD-1 during latent murine cytomegalovirus (MCMV) infection in the SMG. Interestingly, an increased infiltration of CD8+ T cells in the SMG was also observed during MCMV latency period. These studies of PD-1 on SMG immune cells should provide insights for prevention and treatments of IrAEs and chronic viral infections.

This work was supported by the following NIH research Grants: R01 AI46709 (L.B.), R01 AI122217 (L.B.) and a Brown University Presidential Award (S.B.).
Androgens protect ILC2s from interferon-mediated functional suppression during influenza virus infection
Authors: Kovats Susan , Karlik Anna , Turner Sean , Miller Reegan , Ainsua-Enrich Erola , Hatipoglu Ibrahim , Bagavant Harini , Alberola-Ila Jose , Pelikan Richard , Kadel Sapana
Published: January 2023

Biological sex differences in morbidity upon influenza A virus (IAV) infection are linked to stronger interferon-centered immune responses in females, yet the regulatory role of sex hormone receptors in immune cell subsets is incompletely understood. Lung-resident group 2 innate lymphoid cells (ILC2s) express notably high levels of androgen receptors (AR). In IAV infection, ILC2s produce type 2 cytokines and facilitate tissue repair, but they also may be functionally suppressed by type 1 inflammation. Here we report sex differences in the magnitude of lung ILC2 functional suppression at the peak of sublethal IAV infection. Relative to males, ILC2s in female lungs showed attenuated proliferation, decreased propensity for IL-5 and amphiregulin production and reduced expression of GATA3 and IL-33R. Single cell RNA sequencing revealed sex-divergent transcriptomes, with female cells preferentially present in clusters of stressed, functionally suppressed ILC2s and male cells dominating in clusters of canonically activated ILC2s. Equivalent inflammatory cytokine levels and viral load suggested sex differences in ILC2-intrinsic factors. Indeed, naïve female ILC2s showed elevated IFNGR expression and higher phospho-STAT1 levels following IFNG stimulation, and lymphocyte-restricted STAT1 deficiency reversed IAV-induced suppression of female ILC2s. Lymphocyte-restricted AR deficiency or loss of androgens via orchiectomy led to increased IFNGR expression and suppression of male ILC2s. Collectively, these data show that ILC2-intrinsic AR activity regulates IFNGR-STAT1 signaling pathways to preserve canonical ILC2 function in males during IAV infection.

Supported by grants from NIH (HL 119501) and the Presbyterian Health Foundation
Functional tuning of commensal-specific lymphocytes by nociceptive sensory neurons
Authors: Kulalert Warakorn , Wells Alexandria , Enamorado Michel , Link Verena , Kabat Juraj , Kamenyeva Olena , Belkaid Yasmine
Published: January 2023

The somatosensory nervous system surveils external stimuli at barrier tissues, regulating innate immune cells under infection and inflammation. The roles of nociceptive nerves in homeostatic adaptive immunity to the microbiota, however, remain elusive. Here, we identified a novel mechanism for direct neuro-immune interaction between commensal-specific T lymphocytes and skin-innervating nociceptive neurons through secretion of calcitonin gene-related peptide (CGRP). We observed upregulation of the CGRP co-receptor, receptor activity modifying protein 1 (RAMP1), in T lymphocytes induced by topical colonization of the human skin commensal Staphylococcus epidermidis, but not in T cell populations elicited by cutaneous infection or inflammation. Utilizing intravital imaging, we observed that commensal-specific T cells interact intimately and dynamically with skin nociceptive nerves in a CGRP-dependent manner. Importantly, CGRP secreted from nociceptors acts directly on commensal-specific T cells via RAMP1 to regulate their accumulation in the tissues, cytokine production and motility in vivo. Activation or inhibition of nociceptive neurons functionally tune commensal-specific T cells and optimize their responses to ensuing heterologous infections or damaging challenges in the skin. The ability of the somatosensory neurons to participate in the highly specific and potentially long-lasting adaptive immune response to the microbiota underscores the functional versatility and plasticity of commensal-specific T lymphocytes that can be swiftly tuned to diverse arrays of sensory modalities ranging from thermosensation to noxious pain under steady state and pathology.

Supported by Damon Runyon Cancer Research Foundation
Analysis of the mechanisms that maintain intestinal regulatory T cells
Authors: Morales Elisa Cruz , Hart Andrew P , Laufer Terri M
Published: January 2023

Regulatory T cells (Tregs) play an important role maintaining intestinal homeostasis. The Treg pool from the small intestine lamina propria (siLP) include two subpopulations: Helios+ Tregs, comprised of mainly thymic derived Tregs, and the microbiota-induced RORgt+ Tregs. We used K14 transgenic mice lacking peripheral TCR-MHCII to examine the phenotype and maintenance of Helios+ Tregs in siLP. We find that the siLP of K14 mice fills with Helios+ Tregs that adopt an effector phenotype independent of TCR signaling.

To dissect the mechanisms that maintain siLP Helios+ and RORgt+ siLP Treg subpopulations, we used blocking antibodies to inhibit costimulatory signals. We find that the composition of the Treg pool is dynamic; both subpopulations expand to compensate for the loss of the other to maintain intestinal Treg numbers. Moreover, we found that the balance between RORgt+ and Helios+ Tregs in siLP is differentially modulated by ICOS and CD28 signals. Thus, while Helios+ Tregs rely only on CD28 but neither ICOS nor MHCII signaling for their expansion; MHCII, CD28, and ICOS signaling play a role in RORgt+ Treg maintenance. Together, our data highlight the intricate signaling network that regulates the intestinal Treg homeostasis.

Supported by grants from: AAI Careers in Immunology Fellowship VA NIH
Parenteral vaccination strategies target protective immunity to the small and large intestine mucosa
Authors: Chen Ziqi , Tierney Flora , Raposo Bruno , Gonzalez Rodrigo , Sadeghi Ilin , Schudel Alex , Hu Quanyin , Garcia Johnny , Langer Robert , Jaklenec Ana , von Andrian Ulrich H
Published: January 2023

Prophylactic vaccines are most commonly given by a parenteral route. However, this strategy often confers poor protection against GI pathogens because antigens are primarily presented in peripheral lymph nodes, which do not provide the prerequisite ‘tissue imprinting signals’ to target memory T and B cells to the gut. Here, we describe a platform that enables parenteral vaccines to target protection to the GI tract. This was achieved by subcutaneous (s.c.) delivery of compounds known to imprint gut tropism onto lymphocytes. Am80, a retinoic acid receptor agonist, targeted immunity to the small intestine through up-regulation of gut-homing receptors α4β7 and CCR9, and promotion of IgA class-switching. S.c. injection of OVA + CpG combined with repeated injections of Am80 generated significantly higher and durable fecal anti-OVA IgA titers, compared to OVA + CpG without Am80. Vaccination with Am80 also increased antigen-specific CD4 and CD8 T cell trafficking to the small intestine and generated tissue resident memory T (Trm) cells. To overcome the need for multiple doses of Am80, which can cause tissue toxicity at the injection site and hinder clinical translation, we developed a polymeric nanocarrier that showed equivalent imprinting efficacy upon a single injection, lowering the cumulative dose to one-third. Furthermore, we also developed a modified vaccination strategy that could augment effector and memory T cell responses not only in the small intestine, but also in the cecum, colon and rectum, conferring protection to the entire intestinal tract. We conclude that supplementation with mucosal imprinting signals is a promising approach to enhance parenteral vaccines by generating mucosal protection against GI infections.

Supported by the Bill & Melinda Gates Foundation
TGF-β signaling is important for female reproductive tract resident memory CD8 T cell development
Authors: Hasan Mohammad H , Mithila Farha J , Pacia Jessica , Zhang Nu , Beura Lalit K
Published: January 2023

Tissue-resident memory T cells (TRM) patrol non-lymphoid barrier tissues and provide rapid protection against reinvading pathogens. While TRM formation in several barrier mucosal tissues including intestine and respiratory tract has been investigated, factors that control TRM establishment in the female reproductive tract (FRT) remain unclear. Here we established a model of local FRT infection, characterized CD8 TRM development and probed the role of cytokine TGF-β in TRM formation. We adopted a mouse model of intravaginal lymphocytic choriomeningitis virus (LCMV) infection that generated robust local CD8 T cell response in both the lower FRT (cervix, vagina) and upper FRT (ovaries and uterine horn). Upon viral clearance, CD8 T cells in the tissue gradually adopted mature TRM phenotype. Parabiosis confirmed most antiviral T cells in the FRT were truly resident. There were marked differences in the phenotypes of TRM in upper and lower FRT with CD103+ epithelial TRM predominating the lower FRT mucosa. Longitudinal analysis revealed TRM in both compartments undergoes attrition. The cytokine TGF-β has been implicated in TRM development in number of barrier and visceral organs. Using TGF-β RII deficient transgenic CD8 T cells, we observed that lack of TGF-β receptor signaling impairs acquisition of CD103 and CD69 expression by FRT TRM. However, TGF-β RII deficient CD8 T cells showed elevated expression of effector molecules granzyme-B and interferon-gamma upon in vivo stimulation. Taken together, our data suggest a key role of TGF-β in the formation and function of virus specific TRM in the FRT. Further investigation will provide mechanistic insight on the nature of TGF-β signaling pathway impacting FRT TRM formation.

Supported by NIH grants (P20GM121298 and P20GM109035) and Searle Scholars Foundation
Interactions between the Environment and Genetics determines immune variation in rewilded mice
Authors: Oyesola Oyebola Oluwakemi , Downie Alexander E , Barre Ramya Smithaveni , Chen Ying-Han , Kiwanuka Kasalina N , Zaldana Kimberly , Howard Nina , Lee Soo Ching , Devlin Joseph , Mondragon Octavio Paloma , Herrmann Christin , Zhao Mingming , Koralov Sergie B , Cadwell Kenneth , Graham Andrea L , Loke P’ng
Published: January 2023

Immune responses to pathogens and vaccination can be varied with some individuals inducing optimal responses while others do not. The host genetic profile, environment and previous microbial experience could influence an individual’s response, but the relative contribution, and interactions of these different factors remains largely unknown. Here, using various multi-omics, ecological and single cell approaches, we show that release of genetic inbred strains of mice, 129-SL, PWK and C57/B6 mice, to a rewilded environment and exposure of these rewilded and laboratory specific pathogen free control mice to a helminth parasite, Trichuris muris allowed us to assess the contribution and interaction of host genotype and environment to the immune cell landscape in the blood and secondary lymphoid organs. Critically, we find that the environment has the greatest effect on circulating blood immune cells while the genetic profile has the greatest effect on the mesenteric lymph node. We also observed significant interactions between the host genetic profile, environment, and infection status in their contribution to immune cell composition, with most of the effect driven by the cells of the adaptive immune system. These findings provide a model for contribution and interactions between genetics, environment, and helminth infection in the inter-individual variation of immune responses.
A novel digital device-based adjuvant-like effect for influenza vaccine: A feasibility and pilot study
Authors: Hahm Jarang , Choi Jun-Pyo , Ayoub Ghazal , Kim Sae-Hoon , Kim Eu Suk , Noh Ji Yun , Song Joon Young , Chang Yoon-Seok
Published: January 2023

Viral infections such as influenza and COVID-19 have a huge impact on humans. Previous studies showed that some exercise could enhance the anti-viral antibody titers after vaccination. We developed a novel digital device designed to enhance host immune system against viral infection by acting like adjuvants. The digital device, SAT-008, is a mobile application based on an algorithm to regulate physical activity. To assess the feasibility of SAT-008, a randomized, open-label, and controlled study was conducted for 13 weeks (Oct 20 to Jan 21). A total of 42 healthy adults (mostly healthcare workers) aged 24 to 46 years were recruited and finally 32 subjects served for analysis. A single-dose of quadrivalent inactivated influenza vaccine was administered for each subject. The control group maintained daily life without using SAT-008, while the experimental group (Exp) used SAT-008 during the study. As a result, compared to the controls, the Exp showed a significant increase in hemagglutination-inhibition (HI) titers of antigen subtype B Yamagata lineage after 4 weeks of vaccination and antigen subtype B Victoria lineage after 12 weeks of vaccination (P < 0.05), whereas the controls did not. As for type A influenza, there was no significant difference in HI titers between groups. In addition, the Exp was divided into high performers (HP) and low performers (LP) based on the adherence rate. The HP showed significantly higher HI titers of B Yamagata lineage in 12 weeks after vaccination than controls and LP (P < 0.05) and of B Victoria lineage than controls (P < 0.01). Our findings indicate a novel approach using digital device may play an important role to enhance host immune system to act as a vaccine adjuvant against viral diseases such as influenza.

Supported by grants from S-Alpha Therapeutics, Inc.
A feeder cell-free activation and expansion strategy to generate memory-like NK cells sufficient for off-the-shelf multi-dose adoptive cell therapy.
Authors: Wong Hing C , Dee Michael J , Shrestha Niraj , Chaturvedi Pallavi , Leclerc Giles M , Zhu Xiaoyun , Liu Bai , Kong Lin , Echeverri Christian A , You Lijing , Egan Jack O , Jiao Jin-An , Rhode Peter R , Becker-Hapak Michelle K , Berrien-Elliott Melissa M , McClain Ethan , Foster Mark , Pence Patrick , Neal Carly C , Kersting-Schadek Samantha , Fehniger Todd A
Published: January 2023

Adoptive cell therapy (ACT) using NK cells is a promising armament in the fight against cancer. Cytokine induced memory like (CIML) NK cells have been shown in clinical studies to have potent antitumor activity with superior in vivo persistence. Currently, the expansion of NK cells for clinical development is mainly based on feeder cells, which imposes significant regulatory hurdles and increases the costs for manufacturing. We have developed fusion proteins, HCW9201 and HCW9206 comprising of IL-15/IL-18/IL-12 and IL15/IL-7/IL-21, respectively, capable of priming memory-like differentiation and expanding CIML NK cell products without using feeder cells. This “Kick and Expand” strategy allows greater than 100x expansion of CIML NK cells from donor PBMCs in as little as 14 days without the use of exogenous feeder cells. Continued expansion can yield sufficient CIML NK cells for cryopreservation and multiple ACT infusions. The NK cells generated have bona fide memory-like properties: enhanced antitumor activity across multiple cancer cell lines, higher metabolic capacity, stable epigenetic demethylation of the IFN-γ promoter and increased persistence in NSG mice, when compared to conventional NK cells. In conclusion, this “Kick and Expand” process supports generation of abundant CIML NK cells for multiple ACT infusions and provides simpler, more regulatory friendly, off-the-shelf platform for generating NK cell products, including those with chimeric antigen receptor (CAR) constructs.
Cell membrane-anchored and tumor-targeted IL-12 (attIL12)-T cell therapy for eliminating large and heterogeneous solid tumors
Authors: Hu Jiemiao , Yang Qing , Zhang Wendong , Du Hongwei , Dao Long , Xia Xuqing , Fowlkes Natalie , Mahadeo Kris , Gorlick Richard , Dotti Gianpietro , Li Shulin
Published: January 2023

Background

Adoptive T-cell transfer has become an attractive therapeutic approach for hematologic malignancies but shows poor activity against large and heterogeneous solid tumors. Interleukin-12 (IL-12) potent antitumor efficacy against solid tumors, but its clinical application has been stalled because of toxicity. Here, we aimed to develop a safe approach to IL-12 T-cell therapy for eliminating large solid tumors.

Methods

We generated a cell membrane-anchored IL-12 (aIL12), a tumor-targeted IL-12 (ttIL12), and a cell membrane-anchored and tumor-targeted IL-12 (attIL12) armed T cells, CAR-T cells, and TCR-T cells with each. We compared the antitumor efficacy of these armed T cells in treating osteosarcoma PDX tumors and mouse melanoma tumors, and addressed the safety concerns of IL-12 expressing T cell therapy after intravenous infusions of the armed T cells.

Results

attIL12-T cell infusion showed remarkable antitumor efficacy in human and mouse large solid tumor models. Mechanistically, attIL12-T cells target tumor cells that express cell-surface vimentin (CSV) and accumulate in tumor sites. Such CSV-attIL12 interaction stimulates effector T cell to produce high levels of inflammatory cytokines (IFNγ, Flt3L, GM-CSF and etc.) only in tumors, which in turn triggers dendritic cell maturation for activating secondary T-cell responses and tumor antigen spreading. Both attIL12- and aIL12-T-cell transfer eliminated peripheral cytokine release and the associated toxic effects due to tumor-specific T cell distribution and compartment-dependent cytokine secretion.

Conclusions

This novel approach sheds light on the safe application of IL-12–based T-cell therapy for large and heterogeneous solid tumors.

Supported by R01 CA200574
Close proximity of immune and tumor cells underlies response to BRAF/MEK-targeted therapies in metastatic melanoma patients
Authors: Yan Chi , Chen Sheau-Chiann , Ayers Gregory , Nebhan Caroline , Roland Joseph , Weiss Vivian , Johnson Douglas , Richmond Ann
Published: January 2023

Acquired resistance to BRAF/MEK-targeted therapy occurs in the majority of melanoma patients that harbor BRAF-mutated tumors, leading to relapse or progression and the underlying mechanism is unclear in many cases. The tumoral spatial heterogeneity is a potential key to the understanding of the therapy-response relationships, but they also bring challenges of proper parameterization and model validation. In this study, we developed a workflow to detect immune and tumor cells using multiplex immunohistochemistry and spatial imaging analysis from whole-tumor imaging data. Simultaneous characterization of CD11c+, CD8+, CD40+, CD80+ and SOX10+ cells using multispectral imaging was performed on paired tumor sections from eleven melanoma patients prior to BRAF/MEK-targeted therapy and when disease progressed on therapy. We observed a significant increase of tumor cellularity in the progressed tumors, and the close association of SOX10+ melanoma cells with CD8+ cytotoxic T cells (Tc) negatively correlated with patient’s progression-free survival, which may indicate a potential mechanism for acquired resistance to BRAF/MEK-targeted therapy. Using preclinical mouse model of YUMM3.3 BRAF-mutated tumors, we showed the detrimental role of PD-L1:PD-1 axis between melanoma-cell:Tc-cell in melanoma. In TCGA-melanoma dataset (n=445), SOX10hi is associated with a significant reduction of IFNG and GZMB, as well as poor overall survival (OS) in melanoma. Tumor cellularity (SOX10 and MLANA expression) exhibited additive prognostic value in the immune score signature to predict OS in patients with early-stage melanoma and prognoses OS independent of immune score in patients with late-stage melanoma.

This work was supported by a VA Merit Award (I01BX002301) and SRCS award to AR and by R01-CA116021 (AR).
Neutrophil dynamics in the tumor microenvironment determines therapy efficacy and is regulated by microbiota
Authors: Araya Romina E , Lam Khiem C , Huang April , Chen Quanyi , Di Modica Martina , Lopes Amelie , Yang Howard , Liu Huaitian , Lee Maxwell P , Goldszmid Romina S
Published: January 2023

Neutrophils play an important role in cancer progression and both pro- and antitumorigenic functions have been described. However, their role in response to therapy and whether environmental signals modulate their function in the tumor microenvironment (TME) remain unclear. Here, we show that neutrophil content in the TME defines the response to chemo- and immunotherapy. Neutrophil depletion or recruitment blockade impaired the response, while overexpression of neutrophil-attracting chemokines rendered non-responder tumors susceptible to therapy. Importantly, we demonstrate that tumor neutrophils are a heterogeneous and dynamic population that differ from those found in blood or bone marrow, and we identified a neutrophil subset that correlates with therapy response. Additionally, we found that mice lacking microbiota have impaired neutrophil recruitment and function associated with reduced therapy efficacy. Mechanistically, administration of a microbiota-derived NOD2 ligand to mice lacking microbiota was sufficient to restore neutrophil dynamics and response to therapy. We confirmed tumor neutrophil heterogeneity in cancer patients and showed that while total neutrophil content had no predictive value for patient’s overall survival, two specific neutrophil subsets associated with better outcome for pancreatic cancer patients. Remarkably, the subset enriched in non-responder germ-free tumors in our preclinical studies associated with a similar poor outcome in colorectal cancer patients. Collectively, our findings highlight the importance of characterizing neutrophil heterogeneity in the TME and the contribution of microbiota in shaping these cells to predict a successful therapy outcome.

Supported by NIH Intramural Program
Intertumoral CD4+ T-cells instruct monocyte differentiation in pancreatic ductal adenocarcinoma
Authors: Patterson Mike , Burrack Adam L , Schmiechen Zoe C , Xu Yingzheng , Firulyova Maria , Miller Ebony , Schrank Patricia , Zaitsev Konstantin , Williams Jesse , Stromnes Ingunn
Published: January 2023

Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy that is currently the third leading cause of cancer related deaths. PDA creates a suppressive fibroinflammatory tumor microenvironment (TME) composed of stromal and immune cells that inhibit anti-tumor immune responses. Tumor associated macrophages (TAMs) account for a large percentage of the TME and have remarkably heterogeneous functions, with some subsets promoting anti-tumor responses while others suppress tumor specific T-cells. While many studies have highlighted macrophage heterogeneity in PDA, it remains unclear mechanisms regulating monocyte differentiation into pro vs anti-tumor populations. To understand monocyte differentiation in PDA, we performed trajectory analysis on scRNA-seq data from mouse and human tumors and identified predicted monocyte differentiation pathways toward either MHCII-hi anti-tumor TAMs or Trem2-hi pro-tumor TAMs. Using a newly designed monocyte tracking mouse (CCR2CreER x R26tdTomato) implanted with orthotopic tumors that express the click beetle luciferase (CB) neoantigen, we temporally tracked monocyte differentiation within tumors into TAM populations. Furthermore, using antibody depletion, we found that CD4 depletion led to increased monocyte differentiation into pro-tumor macrophages and a dramatic downregulation of PDL1 on monocyte derived TAMs. Finally, using Ifngr1−/− mice implanted with CB+ tumors, we identify that polarization toward anti-tumor macrophages was driven by IFNy, but TAM PDL1 expression was IFNy independent. Together these data are the first to address monocyte differentiation within PDA and identify previously unexpected role for CD4 T cells governing TAM differentiation program.
The role of the Thrombin/PAR axis in modulating CD8+ T cell anti-tumor immunity
Authors: Cantrell Rachel , Rosenfeldt Leah , Sharma Bal Krishan , Gourley Benjamin , Revenko Alexey , Monia Brett , Palumbo Joseph
Published: January 2023

CD8+ tumor infiltrating lymphocytes (TIL) are critical for tumor clearance following immune checkpoint inhibitor (ICI) therapy. Thrombin, the central hemostatic protease, is activated in the tumor microenvironment and CD8+ T cells express the thrombin-cleavable protease activated receptors (PAR-1, -2, -3, -4). Recent studies showed that thrombin can enhance CD8+ T cell effector functions, but the role of thrombin in ICI is unknown. We hypothesized that thrombin enhances CD8+ TIL anti-tumor effector functions (αTEF) during ICI therapy. Consistent with this hypothesis, we found that reducing circulating prothrombin to ~10% of normal completely abrogated the efficacy of PD-1 blockade therapy to reduce tumor growth, reduced CD8+ TIL infiltration into tumor tissue, and limited αTEF.

Mechanistic in vitro studies showed that thrombin stimulation of isolated murine or human CD8+ T cells resulted in a dose-dependent increase in survival, proliferation and cytokine production (activation profile). Surprisingly, when we stimulated CD8+ T cells lacking PAR-1 or PAR-2, we observed an increase in their activation profile relative to WT CD8+T cells in the absence of thrombin, which was further enhanced with thrombin. Moreover, stimulation of PAR-2 deficient CD8+ T cells with a specific PAR-1 activating peptide resulted in an increased activation profile compared to WT CD8+ T cells stimulated with the same peptide. Together these data suggest that 1) thrombin promotes adaptive tumor immunity in the context of ICI therapy, 2) thrombin promotes αTEF through PAR-1 dependent and independent mechanisms and 3) PAR-2 suppresses CD8+ T cell functions independently of thrombin and limits the capacity of PAR-1 activation to enhance effector functions.

Supported by grants from NIH T32 (5T32AI118697-04)
Utilizing distinct CAR and TCR signaling to generate enhanced cellular immunotherapy
Authors: Gascoigne Nicholas R J , Wu Ling , Brzostek Joanna , Wei Qianru , Vale Previtha Dawn Sakthi , Koh Clara K T , Chua Yen Long , Yap Jiawei , Tan Triscilla Y Y , Lai Junyun , MacAry Paul A
Published: January 2023

CAR-T cells utilize TCR signaling cascades and the recognition functions of antibodies. CAR-T technology has achieved significant success in treatment of certain, primarily liquid, cancers. Nonetheless, many challenges hinder the development of this therapy, for example the efficacy for solid tumors. These challenges show our inadequate understanding of this technology, particularly regarding CAR signaling, and how it may differ from TCR signaling. To dissect CAR signaling, CAR and TCR targeting the same antigen were compared directly. This comparison revealed that CAR was sufficient to bind monomeric antigens due to its high affinity but required oligomeric antigens for its activation. CAR sustained the transduced signal significantly longer than did TCR. CD8 coreceptor was recruited to the CAR synapse but played a negligible role in signaling, unlike for TCR signaling. Surprisingly, we identified a non-canonical CAR signaling triggered in the absence of SRC family kinase (SFK) LCK, which is essential for TCR signaling. We show that LCK-deficient CAR-T cells are strongly signaled through CAR and have a better in vivo efficacy because of reduced exhaustion phenotype and enhanced induction of memory. This non-canonical signaling of CAR-T cells provides new insight into the initiation of TCR and CAR signaling as well as important clinical implications for improvement of CAR function.

Supported by grants from Singapore Ministry of Health’s National Medical Research Council: OFIRG19nov-0066; and Ministry of Education, NUHSRO/2020/110/T1/SEED-MAR/06, and NUS ILO TAP Grant: TAP2002019-04-25. LW, QW and JL were supported by research scholarships from Yong Loo Lin School of Medicine.
Engineering anti-CD229 CAR T cell selectivity for multiple myeloma
Authors: Baker Jillian , Radhakrishnan Sabarinath Venniyil , Olson Michael , Atanackovic Djordje , Luetkens Tim , Mause Erica Vander
Published: January 2023

T cells expressing high affinity chimeric antigen receptors (CAR) against CD229 are able to eradicate multiple myeloma (MM) cells, which express high CD229 levels. However, they also target healthy T cells expressing low CD229 levels. Reducing the affinity of the parental CD229 CAR may increase the selectivity of CD229 CAR T cells, sparing T cells while maintaining activity against MM cells. No robust approaches to engineer minimally altered low affinity variants from existing high-affinity antibodies have been developed.

We generated a site-saturation mutagenesis library of the parental CD229 antibody targeting both CDR3 domains resulting in 305 variants. Variants were expressed as soluble single-chain variable fragments and screened using high-throughput assays to determine binding and expression. Affinities were determined by biolayer interferometry. A subset of 26 binders was further characterized functionally in CAR format using primary human T cells.

Variant CD229 antibodies carrying single amino acid substitutions showed substantially altered expression and binding of recombinant CD229. Affinities of candidate antibodies varied from 150–10,000nM with mainly off-rate driven reductions in affinity. Analyzing the cytotoxic activity of CD229 CAR variants we found that, in contrast to the parental cells, two variant CAR T cells spared normal T cells while maintaining equal cytotoxic activity against MM cells.

Using a novel unbiased mutagenesis approach, we show that generation of minimally altered low affinity variants from existing antibodies is feasible and results in binders with substantially reduced affinity. Low affinity variants show increased selectivity eliminating a key liability of CD229 CAR T cells.

E.R.V received funding from the American Foundation for Pharmaceutical Education (AFPE, Pre-Doctoral Fellowship) and the ALSAM Foundation (Skaggs Fellowship). D.A. received funding from the Huntsman Cancer Institute (HCI, Translational Scholar Award). T.L. received funding from the National Comprehensive Cancer Network (NCCN, Young Investigator Award).
Identifying novel epigenetic modifiers that sensitize T cell malignancies to CD8+ T cell mediated cytotoxicity by high throughput drug screen
Authors: Bradeen Xander Gordon Ray , Christians Allison J , Haverkos Bradley , Davila Eduardo
Published: January 2023

T Cell Lymphomas are a rare subtype of Lymphoma, occurring in ~1% of all cancers and response rates to current therapies reach only ~23%. The lack of unique targetable T cell antigens as well as their tendency to overcome chemotherapies results in a high incidence of relapse in most patients leading to remission rates of approximately two years. Immunotherapies, especially immune-checkpoint blockade, have slightly improved outcomes in refractory disease, but there remains a need for stronger and more durable responses. The chemotherapeutic potential of epigenetic modifiers is just beginning to be explored. HDAC inhibitors, DNA demethylating agents, and histone methyltransferase inhibitors reshape the transcriptome, potentially altering the expression of immune modulatory pathways. We hypothesized that pretreating T cell malignancies with epigenetic modifiers will alter key genetic signatures that sensitize them to CD8+ T cell-mediated cytotoxicity. We pretreated five T cell lymphoma cell lines, representative of different T cell malignant diseases with compounds from an FDA-approved 700+ epigenetic modifying drug library at four different concentrations and then co-cultured them with a tumor-reactive HLA-A2-matched primary (derived from PBMCs) cytotoxic CD8+ T cell line. Changes in T cell mediated cytotoxicity and alterations in immune modulating surface markers and potential CAR-T cell targets (CD30, CCR4, and CD47) were assessed by flow cytometry. Several promising compounds were identified by their ability to improve T cell mediated cytotoxicity, increase expression of immune stimulatory markers, decrease expression of immune inhibitory markers, and augment expression of existing CAR-T target antigens.

Supported by NIH and the Colorado Clinical and Translational Sciences Institute (CCTSI)
TCR-T cells engineered to overexpress c-Jun have better functionality with improved tumor infiltration and persistence for treatment of Hepatocellular Carcinoma
Authors: Hussein Mohamed Shawky , He Yukai
Published: January 2023

Liver cancer is the sixth most common cancer in the world and the third highest cause of cancer death in the world, indicating the treatment gap and the urgent need for novel effective therapies. The majority (80–90%) of liver cancers are hepatocellular carcinoma (HCC), with a general 5-year survival rate is 20%. Adoptive cell therapy (ACT) targeting malignant tumors have been generally unsuccessful due to lack of T cell activation in vivo with poor expansion and short persistence of the tumor infiltrating T cells. We proposed that engineering T cells to overexpress c-Jun, a transcription factor required for T cell development and activation, would activate T cells, enhance their function, and anti-tumor efficacy. In this study, using T-cell receptor–engineered T-cell (TCR-T) against HCC-associated antigen α-fetoprotein (AFP), we demonstrated that human TCR-T cells engineered to overexpress c-Jun (TCR-JUN) have better expansion potential in culture with enhanced functional capacity against HepG2 tumor cells. Additionally, TCR-JUN cells were less apoptotic and more resistant to exhaustion after HepG2 tumor stimulation. In HCC xenograft tumor model, c-Jun overexpression enhanced TCR+ cells expansion and overall survival of the tumor bearing mice. Interestingly, the tumor infiltrating lymphocytes (TILs) of TCR-JUN cells demonstrated enhanced tumor infiltration, persistence, functionality, and were less exhausted. We conclude that engineering T cells to overexpress c-Jun could be a potential approach to enhance T cell cancer therapy.

This work is supported by NIH Grant R01CA235159 to Dr. Yukai He.