bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural variants of Bacteroides fragilis-derived alpha-galactosylceramides induce unique natural killer T cell responses.
Authors: Jung Da-Jung , Lee Jesang , Son Sumin , Park Seung Bum , Oh Sungwhan
Published: November 2024

Natural killer T (NKT) cell is a unique subset of thymus-derived T cells that recognizes glycolipids presented by CD1d molecule expressed on antigen-presenting cells. The most studied agonistic antigen of NKT cells is alpha-galactosylceramide (αGC). It has been shown that structural features of lipid antigens determine distinct NKT cell responses. In order to drive context-dependent NKT cell responses, understanding the structural characteristics of lipid antigens is important. In the previous study, we have shown that human gut symbiont Bacteroides fragilis (B.fragilis) uniquely produces αGCs (BfaGC), which can normalize the overproliferation of neonatal colonic NKT cells in germ-free mice, and chemically-synthesized structural isomers of BfaGCs induce structure-specific NKT cell responses. In this study, to further investigate the essential structural moiety of BfaGC for the unique immunomodulatory activity, we synthesized BfaGC analogues by modifying their chain lengths and functional groups. We have examined how the analogues differently stimulate NKT cells by analyzing in vitro cytokine production, NKT cell proliferation and effect on specific NKT cell subpopulations. We found the critical features and functional groups on the BfaGC structure leading to immunomodulatory responses. It is expected that our findings may advance further development of lipid antigens to fine-tune context-specific NKT cell responses in immunological diseases.
IL-7 improves the T cell IFNγ response in sepsis patients
Authors: Kim Caleb , Swanson Whitney , Xu Julie , Kucaba Tamara , Kancans Kristine , Badovinac Vladimir , Gould Robert , Griffith Thomas
Published: November 2024

Sepsis is the life-threatening organ dysfunction stemming from the severe inflammatory response to systemic microbial infection. Immune consequences for post-septic survivors include acute lymphopenia and prolonged susceptibility to infection. Stratifying Patient Immune Endotypes in Sepsis (‘SPIES’) is a prospective observational study testing the utility of ELISpot as a bioassay to identify the immunosuppressed endotype, and test potential immune stimulants for clinical use. Our data show a whole blood IFNγ ELISpot assay can identify: 1) septic patients at increased risk of late mortality, and 2) immunosuppressed sepsis patients. We wanted to determine which cells in the blood were producing IFNγ. Flow cytometry revealed memory CD4 and CD8 T cells were making the majority of IFNγ after ex vivo stimulation with anti-CD3/CD28 mAb. We next defined the breadth of cytokines produced by whole blood samples from sepsis patients after stimulation. Last, we tested the potential for IL-7 to improve the functional activity of T cells from sepsis patients. Using anti-CD3/CD28 mAb-stimulated PBMC from healthy and septic donors, we found adding IL-7 to the stimulation milieu improves both the number of cytokine-producing cells, and magnitude/variety of cytokines produced from T cells. In sum, these data reveal important functional information about the degree of immune suppression experienced by sepsis patients, along with a potential means of restoring T cell function in these patients.
Artificial Targets: a versatile cell-free platform to characterize CAR T cell function in vitro
Authors: Evaristo Cesar , Wang Xueting , Tokarew Nicholas , Borgelt Nadine , Siemer Ramona , Casonato Melo Cristiane , Langer Christian , Kasampalidis Ioannis , Ogusuku Isabella , Cathomen Toni , Gessner Isabel , Dose Christian , Fauerbach Jonathan , Richter Anne
Published: November 2024

Chimeric antigen receptor (CAR) T cells have proven efficacy against a number of cancers, but further research is needed to extend their therapeutic potential. Functional characterization of CAR T cells plays is central across fundamental research and therapeutic development, with increasing needs for standardization. Typically, effector functions are assessed following co-culture with target-expressing cell lines. This poses several limitations, including high variability, allogeneic-induced background, and work and time intensive workflows. To overcome these limitations, we developed a synthetic bead-based platform (“Artificial Targets”, or "AT") to characterize CAR T cell function in vitro. AT could specifically induce CAR T cell activation, and multiple effector functions of CAR T cells, mimicking the response triggered by target cell lines. These include cytotoxic activity, expression and secretion of cytokines, and cell proliferation. Finally, AT demonstrated flexibility to engage multiple co-stimulatory molecules to enhance CAR T cell function, representing a powerful tool for modulating CAR T cell responses. Collectively, our results show that ATs can specifically activate CAR T cells for essential effector functions that could significantly advance standardization of functional assessment of CAR T cells, from early development to clinical applications.
Affinity and formulation of a phenyl glycolipid for stimulation of invariant natural killer T cells to inhibit tumor growth
Authors: Hung Jung-Tung , Chiou Shih-Pin , Yu Alice
Published: November 2024

α-galactosylceramide (α-GalCer) can activate invariant natural killer T (iNKT) cells to modulate immune system. We have synthesized and tested many α-GalCer analogs and found the most potent iNKT-stimulating agent C34, which has two phenyl rings on the acyl chain. The complex of C34 and CD1d showed the highest binding affinity to the T cell receptor of iNKT cells. Moreover, C34 can induce strong Th1-biased cytokines and exhibit potent anticancer efficacy in tumor animal models. However, poor water solubility of C34 limits its clinical application. In this study, we incorporated C34 into PEGylated lipid nanocarriers (PLN), which improves the water solubility of C34. The PLN-C34 shortened the activation time of iNKT cells and maintained C34's ability to activate iNKT cells to secrete cytokines in vitro. Furthermore, PLN-C34 showed similar anticancer efficacy as C34 dissolved in dimethyl sulfoxide to prolong the survival of mice bearing with TC1 or B16F10 tumor. In general, the PLN-C34 preserved the ability to activate iNKT cells and inhibited tumor growth, suggesting that PLN-C34 is a promising candidate for cancer therapy in clinical settings.
Loss of Trex1 in intestinal epithelial cells prevents enteric viral infections
Authors: Zhang Li , Song Jaewon , Gheshm Newsha , Orvedahl Anthony , Lee Sanghyun
Published: November 2024

The host intestinal microbiota comprises bacteria, viruses, fungi, multicellular parasites, and archaea, which directly impact host’s health. Norovirus and rotavirus are agents of acute infectious gastroenteritis with symptoms of vomiting and watery diarrhea. These viruses are restrained by interferons, especially IFN-α and IFN-λ. The Tree-prime Repair Exonuclease 1 (Trex1) is a nonprocessive 3’→5’ exonuclease that degrades cytosolic nucleic acids. Mutations in Trex1 are associated with a broad spectrum of inflammatory and autoimmune diseases. Failure to degrade cytosolic DNA activates the cGAS-STING DNA sensing pathway and ultimately drives chronic type I and III interferon signaling pathway activation. We found persistent murine norovirus (MNoV) and rotavirus infections were prevented in the Trex1-/- mice. However, Trex1-/-cGAS-/- and Trex1-/-Ifnlr1-/- lost the protection against enteric viral infection seen in Trex1-/- mice. Moreover, tissues from the Trex1-/- mice exhibited a robust interferon stimulated genes (ISG) induction in response to MNoV infection compared to their WT controls. The ISG levels were dampened in both Trex1-/-cGAS-/- and Trex1-/-Ifnlr1-/- mice. Furthermore, Trex1 deficiency in gut epithelial cells is sufficient to suppress MNoV infection. In conclusion, Trex1, as a gatekeeper enzyme of the cGAS-STING pathways, has been demonstrated to be a therapeutic target to restrict enterovirus infection.
102
Authors: Zeleniak Ann , Martinez-Martin Nadia , Laddha Saurabh Vishnu , Kurtagic Elma , Greving Carrie , Hammaker Deepa , Dudas Paul , Perrigoue Jacqueline
Published: November 2024

Inflammation-associated fibroblasts (IAFs) are a cell population that emerges in tissue inflammation. In inflammatory bowel diseases (IBD), IAFs correlate with disease severity and are enriched in colon tissue of patients refractory to anti-TNF therapy. IAFs can amplify tissue inflammation and mediate pro-fibrotic matrix remodeling and therefore represent a novel cellular target for treating inflammatory diseases like IBD. To explore the cellular and molecular pathways that mediate transition to an IAF-like phenotype, we developed a co-culture of human PBMCs and primary colonic fibroblasts to study stromal-immune crosstalk in promoting IAFs. Culturing primed PBMCs with fibroblasts in direct contact drives fibroblast transition to an IAF phenotype through upregulation of inflammatory mediators, evidenced by modulation of gene expression and secreted protein profiles. Single-cell RNAseq revealed IAF development is influenced by PBMC priming conditions. Exposure to LPS-primed PBMCs promoted a matrix-remodeling IAF phenotype while PHA-primed PBMCs promoted an immunomodulatory IAF. IAF activation can be dampened by inhibitors, including TNF and JAK inhibitors. This co-culture system can therefore be used to define mechanisms mediating IAF transition and identify IAF states that can be tailored to accentuate pathways for focused interrogation. We aim to leverage this system to identify novel targets and test prospective therapies to modulate IAF transition and downstream activity.
Single chain MHC-I-β2 microglobulin-peptide trimer constructs identify CD8 T cells recognizing low-affinity MHC-I-binding neoepitopes
Authors: Maji Stabonia , Chawla Arshmeet , Singhaviranon Summit , Dempsey Joesph , Chou Steven , Srivastava Pramod
Published: November 2024

Cancer-specific neoepitopes (mutated peptides in tumor cells presented by MHC-I) are under intense scrutiny to understand the anti-tumor CD8 T cell response. Only a small minority of such potential neoepitopes actually elicit tumor control in vivo. A majority of the neoepitopes, that do mediate tumor control in vivo, have very low affinity for MHC-I, ranging from IC50 values between 500 nM and 50,000 nM (Duan et al., 2014; Brennick and George et al., 2021; Ebrahimi-Nik et al., 2021). This contrasts with the strong MHCI-binding (IC50 <100 nM) epitopes of viruses and model antigens (Peters et al., 2020). As expected, we have found it impossible to create MHC-I-tetramers for low-affinity tumor-controlling neoepitopes. Here, we have genetically created single chain trimers that contain MHC-I alleles, β2 microglobulin as well as the low-affinity peptide, for each of the nine low-affinity neoepitopes identified previously in our laboratory (Brennick and George et al., 2021). Such trimers have been purified and characterized and have been used to identify, for the first time, CD8 T cells that recognize low-affinity cancer neoepitopes presented by tumor MHC-I molecules. The characteristics (abundance, amplification, specificity etc) of these low-affinity MHC-I binding neoepitopes shall be presented.
β-adrenergic signaling regulates erythropoiesis and through it; cancer immunity
Authors: Chawla Arshmeet , Hagymasi Adam , Geyer Rory , Mandoiu Ion , Peterson Kenneth , Scanlon Vanessa , Srivastava Pramod , Nevin James
Published: November 2024

The intricate interplay between the nervous and immune systems significantly influences the fate of immune cells in homeostasis as well as under pathological conditions such as cancer. Notably, adrenergic signals from the sympathetic nervous system have been identified as regulators of hematopoiesis. Disruption of sympathetic signaling has been linked to mal-differentiation of myeloid and lymphoid lineages leading to the emergence of Tregs, myeloid-derived suppressor cells, and the exhaustion of CD8+ T cells. We show here that disrupted sympathetic signaling also leads to mal-differentiation of erythroid lineage via the emergence of erythroid precursor cells (EPCs), which, apart from their role in maintenance of the red blood cell pool, contribute significantly to suppression of the anti-tumor CD8+ T cell response.

Specifically, we show here that EPCs are highly regulated by β-adrenergic signaling in naïve as well as tumor-bearing mice. We observe that EPCs actively promote tumor growth in vivo and influence the fate of tumor infiltrating CD8+ T cells by suppressing their proliferation and effector functions. We have reported several aspects of the mechanisms involved in the β-adrenergic regulation of erythropoiesis including the specific receptors involved, the signaling pathway and the kinetics of the effect. This discovery unveils a novel dimension of neuroimmune communication and offers insights that could catalyze the development of new therapeutic approaches to cancer.
Secondary hemophagocytic lymphohistiocytosis with associated central pontine myelinolysis
Authors: Philip Rachel , Alam Mariam
Published: November 2024

A 34-year-old previously healthy male presented to the emergency department with an altered mental state and unexplained intermittent fevers persisting for three weeks. Initially, his symptoms mimicked an ear infection, for which he received a prescription for Augmentin. As his condition deteriorated, additional symptoms emerged, including bilateral ear pain, fever, and diffuse headaches. Initial evaluations revealed acute hepatic encephalopathy, mastoiditis, and pancytopenia. Further tests uncovered hepatosplenomegaly, lymphadenopathy, and reactivated EBV. The patient met six of the eight HLH criteria, including fever above 38.3°C, cytopenia, elevated triglyceride and ferritin levels, and a bone marrow biopsy confirming hemophagocytosis. The treatment plan followed the HLH-2004 protocol, involving an eight-week induction therapy using etoposide, dexamethasone, and cyclosporine. Monitoring of disease-specific markers such as ferritin and IL-2R showed improvement. One month later, the patient experienced a throbbing headache, nausea, photophobia, and vision changes. MRI showed abnormal signals consistent with central pontine myelinolysis. Lumbar puncture findings included pleocytosis, elevated total protein, and a substantial EBV viral load, indicative of progressive CNS-HLH with persistent EBV reactivation. The patient is currently undergoing salvage therapy for EBV reactivation, with a poor prognosis for CNS involvement and allogeneic transplant preparations.
Dendritic cell CTLA-4+ extracellular vesicles as regulators of T cell polarization
Authors: Bisht Nalini
Published: November 2024

Dendritic cells (DC) are professional antigen-presenting cells that prime de novo T cell responses. DC release extracellular vesicles (EV) to communicate environmental context to other cells of the immune system. Our published data demonstrate that CTLA-4 is expressed on the outer surface of DC-secreted EV. DC-released CTLA-4+ EV are internalized by bystander DC and result in downregulation of costimulatory B7 expression on these bystander DC. TH2 polarization of DC results in enhanced secretion of CTLA-4+ EV as compared to TH1 polarized DC which abrogate secretion of CTLA-4+ EV almost entirely. Furthermore, knockdown of CTLA-4 in DC resulted in upregulated CD8+ T-cell proliferation and enhanced antitumor immunity. Here we report additional characterization of how DC-derived EV modulate adaptive immunity. Co-culture of CTLA-4neg EV with naïve T cells induced significant T cell blast formation and enhanced effector functionality of T cells in the form of upregulated granzyme B and IFN-g in comparison to naïve T-cells cultured with CTLA-4+ EV, indicative of a TH1 response. Additionally, CTLA-4neg EV downregulated Treg proliferation compared to CTLA-4+ EV. These data support the hypothesis that DC CTLA-4+ EV are important effector vehicles through which DC propagate TH polarization signals. Future investigations will determine the therapeutic application of the EV-stimulated T cells, and the corresponding transcriptional changes induced.
Characterization of immune population of model murine of muscular dystrophy.
Authors: Romero Hector , García-Pérez Blanca Estela , Vélez-Caballero Sara H , Cano-Martínez Luis Javier , Martínez-Damas Mirna G , Castillo-Cruz Juan , Coral-Vázquez Ramón M , Baltierra-Uribe Shantal Lizbeth
Published: November 2024

Muscular dystrophy comprises a group of genetic diseases leading to progressive weakness and muscle mass loss due to protein deficiency, such as delta-sarcoglycan protein involved in stabilization of the sarcolemma and in signal transduction. Dystrophic muscles exhibit a predominant inflammatory environment characterized by cell infiltrate capable of producing immunostimulatory signals. While it has been reported that myeloid cell infiltrate may contribute to the pathogenesis of chronic myopathies, the specific role of delta-sarcoglycan protein deficiency in the overall immune response profile has not been previously assessed. In this study, we used flow cytometry to evaluate B and T cell populations, macrophages, monocytes, and neutrophils in the spleens of delta-sarcoglycan null and wildtype mice. We observed significant differences only in the F4/80 macrophage population. These findings suggest that delta-sarcoglycan deficiency may play a role in the differentiation of immune cells, particularly macrophages.
An early burst of IL-2 production by CD8 T cells biases their developmental fate decisions
Authors: Ingram Jennifer , Kahan Shannon , Weaver Casey , Harrington Laurie , Zajac Allan
Published: November 2024

The differentiation of CD8 T cell responses is guided by a combination of antigenic, costimulatory, and cytokine signals. Here we show that naïve CD8 T cells undergo a heterogeneous burst of cytokine synthesis within hours following activation. This divergent early response develops with every specificity of CD8 T cells we have checked, manifests in vivo and in vitro, and occurs even prior to the first cell division. Using cytokine reporter systems, we have demonstrated that these early functional differences are consequential for establishing fate biases and directing the gain of effector and memory T cell traits. Furthermore, we have discovered that the initial cell intrinsic synthesis of IL-2 attenuates IL-2-dependent STAT5 signaling. The bifurcation between the abilities of IL-2-producing and non-producing subsets to elaborate STAT5 signaling are consistent with a model in which recently activated CD8 T cells that manufacture IL-2 become less permissive to cytokine-driven differentiation signals, thereby preserving their “stem-like” properties and allowing them to serve as precursors for memory T cells as well as future effectors. By contrast, the non-IL-2-producing contingent remains receptive to extrinsic IL-2 signals allowing a more terminally-differentiated series of effector CD8 T cells, that are legitimized by cytokine signals, to rapidly form in order to facilitate clearance of the antigenic insult.
Metagenomic investigation of intestinal bacterial and parasitic infections in urban canines in Seoul, Korea
Authors: Kim Ju Yeong
Published: November 2024

Approximately 25% of South Koreans own pets, predominantly dogs. Despite this, there's limited research on the fecal organisms of these dogs and the identification of zoonotic pathogens. This study employs metabarcoding analysis to explore these bacterial and parasitic infections in dog feces in Seoul. Fecal samples were collected from 16 pet and 25 stray dogs in the Mapo district of Seoul. Next-generation sequencing was utilized, employing 16S and 18S rRNA amplicon sequencing to identify prokaryotic and eukaryotic pathogens. Significant differences in bacterial and parasitic composition were found between stray and pet dogs, and putative prokaryotic and eukaryotic pathogens were identified, including Helicobacter, Campylobacter, Giardia, Pentatrichomonas, and Cystoisospora. Interestingly, Campylobacter, Giardia, and Pentatrichomonas were found to be significantly more prevalent in stray dogs than in pet dogs. Additionally, subsequent PCR analysis confirmed that some of these pathogens were strains capable of infecting humans. This study offers valuable insights into the zoonotic risks associated with dog populations residing in diverse environments. By identifying and characterizing potential pathogens in dog feces, this study highlights the need for further research into the immunological mechanisms that enable specific parasites to infect both humans and animals simultaneously.
Immune-modifying cold plasma for treatment of periprosthetic orthopedic infection
Authors: Smith Carly , Watkins Amanda , Melvage Autumn , Lucas Abigail , Schaer Thomas , Freeman Theresa
Published: November 2024

Non-antibiotic treatment options are needed to combat biofilm formation in clinical settings, and cold plasma, an energized gas containing a cocktail of reactive oxygen and nitrogen species, may be such a strategy. In vitro, cold plasma is antimicrobial through direct bacterial killing and modulation of virulence factors. Cold plasma treatment may also be immunostimulatory by enhancement of innate immune effector responses.

We evaluated the efficacy of cold plasma for treating periprosthetic infection in an in vivo rat model. Infection mitigation following cold plasma treatment was assessed by measurement of colony forming units (CFU) of infected rats. Immune modulation was also determined using qPCR, Westerns, IHC, RNA sequencing, and histology.

While we observed no reduction in CFUs, RNAseq revealed cold plasma treatment significantly enriched genes associated with neutrophil activation, chemotaxis, and inflammasome activation. IL-1β was increased at the site of infection, coordinating with increased myeloperoxidase protein and positive cells. Draining lymph nodes in plasma-treated animals were markedly smaller than untreated animals, which had significant lymphadenomegaly.

Cold plasma treatment leads to enhanced innate immune cell recruitment and activation at the site of infection. Additional studies will determine if cold plasma treatment in combination with standard clinical antibiotic therapy will improve bacterial clearance following revision surgery.
A Pro-Drug Strategy Enhances Butyrate Bioavailability and Therapeutic Efficacy In Atherosclerosis
Authors: Beckman Taryn , Volpatti Lisa , Cao Shijie , Slezak Anna , Reda Joe , Budina Erica , De Matos Salvador , DeLeon Orlando , Borjas Gustavo , Chang Eugene , Hubbell Jeffrey
Published: November 2024

Butyrate is a gut microbiota derived short chain fatty acid with pleiotropic positive effects on inflammation and metabolism. In atherosclerosis, sodium butyrate has been shown to significantly reduce atherosclerotic lesions, rectify routine metabolic parameters such as LDL-C in the serum, and reduce systemic inflammation. However, its foul odor, low absorption, and fast metabolism limit its therapeutic effectiveness. We engineered a derivate of butyrate attached to the amino acid serine (SerBut) to mask the taste and odor and co-opt amino acid transporters in the gut to increase its bioavailability. In Vitro, SerBut maintains the anti-inflammatory, NFkB suppressing, capacity of sodium butyrate while allowing higher dosing without inducing cytotoxicity. SerBut can be stably, easily, and odorlessly administered in drinking water at higher doses than previously possible. Over 6 weeks of HFD in concurrence with SerBut water, SerBut reduces LDL-C in the serum, plaque accumulation in the aortic root, and monocytes in the aorta of an apolipoprotein-E-/- murine model of atherosclerosis. Bioinformatic analysis of fecal 16S rRNA sequencing reveals Seryl modification of butyrate to be associated with less plaque in the aortic root of mice, while general butyrate treatment was not. SerBut reduces enzymes associated with liver damage and has a systemic tolerogenic effect. SerBut is a promising therapeutic that lowers lipids and lipid driven inflammation without inducing liver toxicity.
Development of conditionally active IL-10 INDUKINE TM molecules for the treatment of inflammatory bowel disease
Authors: Sullivan Jenna , Aderhold Pamela , Brodkin Heather , Nagy-Domonkos Celesztina , Economides Kyriakos , Hicklin Daniel , Lewis Yuka , Magness Leigh , Siedel-Dugan Cynthia , Steuert Zoe , Stieglitz Jessica , Winston William , Salmerson Andres
Published: November 2024

Inflammatory bowel disease (IBD) encompasses two chronic inflammatory diseases of the gastrointestinal tract, Ulcerative Colitis (UC) and Crohn’s disease. Although great strides have been made in the treatment of IBD, subsets of patients do not respond or lose responsiveness to therapy, underlining a need for new treatments. Interleukin 10 (IL-10) is a immunoregulatory cytokine, known to suppress and inhibit inflammatory responses of a variety of immune cells including monocytes, macrophages, and T cells. Polymorphisms associated with reduced expression of IL-10 and IL-10R have been linked with IBD. Clinical trials using recombinant IL-10 in IBD have been unsuccessful, highlighting a need for further engineering to utilize IL-10 as a therapeutic agent. We have developed IL-10 INDUKINE molecules that are designed as prodrugs containing human IL-10, an inactivation domain and a half-life extension domain tethered together by protease-sensitive linkers. IL-10 INDUKINE molecules are peripherally inactive but due to dysregulation of the protease milieu in the inflamed colon, the linkers are cleaved, and IL-10 is released locally. We have identified proprietary linkers which are cleaved by UC and Crohn’s human colon samples. In a mouse model of colitis, IL-10 INDUKINE molecules prevent weight loss, intestinal damage and inhibit inflammatory cytokine production within the colon. Together these data support the potential of an IL-10 INDUKINE molecule for the treatment of IBD.
Targeting IRF3 to increase the efficacy of oncolytic herpesvirus in mouse cancer cells
Authors: Benencia Fabian , Matocha Grace , Courreges Maria , McCall Kelly
Published: November 2024

Oncolytic viruses (OV) specifically infect and kill tumor cells because these cells harbor a defective IFN response or express proteins that block apoptosis pathways. Oncolytic herpesvirus (HSV) infection activates pathogen recognition receptors as part of the innate immune response. HSV genome contains un-methylated CpG motifs which activate toll-like receptor TLR9, and during infection it generates dsRNA intermediates which can activate TLR3, RIG-I, MDA5, and STING. Signaling pathways downstream of dsRNA recognition activate pro-apoptotic IRF3 leading to the production of type-I IFN, induction of co-stimulatory molecules and caspase cascade activation. We hypothesize that inhibiting IRF3 will diminish the antiviral response, increasing the efficacy of the OV. Here, we inhibited IRF3 expression in murine ovarian cancer cells with phenylmethimazole (C10), which inhibits dsRNA-triggered IRF3 signaling, preventing induction of type-I IFN gene expression, and type-I IFN production and signaling. Cancer cells treated with C10 were more susceptible to oncolytic HSV, as measured by increased cell death. The innate antiviral response in those cells was diminished as shown by lower levels of CXCL10 produced by infected cells. The levels of immunogenic cell death markers, e.g., HGMB1, were not decreased in C10-treated infected cells, indicating that the immunogenicity of the killed tumor cells is preserved. Thus, targeting IRF3 can be a strategy to increase the efficacy of OV.
Effect of extracellular matrix components and tumors factors from breast cancer on the biological properties of myeloid cells in the mouse model
Authors: Benencia Fabian , Matocha Grace , Cunningham Aiden , Courreges Maria
Published: November 2024

The tumor microenvironment (TME) is composed of cells and extracellular matrix (ECM) components that oftentimes help cancer cells to survive and growth. Immune cells, whose role is to protect against infections or malignancies, can be coopted by the TME or reprogrammed by tumor factors rendering them incapable of fighting the tumor inducing a protumor phenotype. Key immune cells affected in this process are antigen presenting cells (APCs) such as dendritic cells (DC) and macrophages (Macs). The effect of soluble factors produced by tumors (i.e., VEGF and TGF beta) on these cells has been broadly studied, but other factors such as extracellular matrix components can also alter their biology. Herewith, we were able to determine that APCs infiltrating the TME of the 4T1 model of breast cancer express angiogenic factors and chemokine receptors. In addition, we observed that tumor factors were able to induce migration of DCs and Macs. Furthermore, we detected markers of proliferation in tumor-infiltrating APCs, indicating that these cells might also be able to proliferate within the TME. We observed that tumor supernatants can activate these APCs invitro, and that this activation is partially mediated via TLR4 signaling. Finally, we observed that tumor cells and mouse tumors produce both fibronectin and fibronectin-extracellular domain (a known DAMP and activator of TLR4). Thus, production of ECM components by tumor cells can help shape the phenotype of tumor-associated APCs.
Gamma-Delta (γδ) CAR-T Cells targeting CD33 and lacking the CD3ζ signaling domain enhance killing of AML cells without toxicity to normal hematopoietic cells
Authors: Lamb Lawrence , Ding Lei , Li Yanjie , Li Jixue , ter Haak Mariska
Published: November 2024

Extending CAR-T therapy to myeloid malignancies has proven challenging due to co-expression of targetable antigens on hematopoietic progenitors. We leveraged the enhanced targeting conferred by CAR-T technology with the broad stress-associated antigen recognition and killing capabilities of γδ T cells by employing a non-signaling CAR (nsCAR) that excludes the CD3ζ domain, thereby facilitating targeted tumor recognition while sparing healthy tissues. Jurkat cells expressing a CD3ζ+ CD33+ CAR showed significant upregulation of CD69 in co-culture with KG1a AML cells but nsCAR-expressing Jurkat cells did not upregulate CD69. A time-dependent reduction (43%) in the CD3ζ+CD33CAR+ Jurkat population was also noted over a 7-day coculture while the nsCAR+ Jurkat population remained stable, suggesting improved durability of the nsCAR modified Jurkat cells. nsCAR-expressing expanded and activated γδ T cells from healthy donors exhibited enhanced killing against AML lines HL-60 (1.3x) and K-562 (1.6x) compared to unmodified γδ T cells (UTD) in a 24-hour cytotoxicity assay. Incorporation of sIL-15 also increased killing (1.8x for HL-60, 2.6x for KG-1a, 2.0x for MOLM-13 2.0x for K-562). Minimal cytotoxicity (<10%) was observed for nsCARs or UTD γδ T cells against CD34+ HPCs. Our findings suggest that combining nsCAR constructs with γδ T cells may widen the therapeutic window to enable of CAR-T therapy for cancers with target antigen expression on healthy tissues.
A viral-specific CD4+ T cell response protects female mice from Coxsackievirus B3 infection
Authors: Pattnaik Aryamav , Dhalech Adeeba , Condotta Stephanie , Corn Caleb , Richer Martin , Snell Laura , Robinson Christopher
Published: November 2024

Biological sex significantly influences the ability of humans to respond to various pathogens. Males are frequently more susceptible to viral, bacterial, and fungal infections, often attributed to weaker immune responses. In contrast, a heightened immune response in females enables better pathogen elimination but leaves females more predisposed to autoimmune diseases. Unfortunately, the underlying basis for sex-specific immune responses remains poorly understood. Here, we show that Coxsackievirus B3 (CVB3) elicits a distinctive CD4+ T cell response in female but not male mice. We found that CVB3 elicits the expansion of CD62Llo CD4+ T cells in the mesenteric lymph node and spleen of female but not male mice five days post-inoculation, signifying early activation. Using a recombinant CVB3 expressing a model CD4+ T cell epitope, we found that this response is due to viral antigen. Further, we found that depleting CD4+ T cells before CVB3 infection increases mortality in females, underscoring their protective role against CVB3. Currently, we are characterizing the differentiation of CD4+ T cells to examine the role of specific subtypes in the sex bias. Overall, our data demonstrate that CVB3 induces a sex -specific early CD4+ T cell response and emphasizes the impact of sex differences on immune-mediated disease outcomes.
Identification of T cell subpopulations exhibiting a high-degree of TCR-independent, IL-15-induced activation in pateints with acute hepatitis A
Authors: Yu Hosun , Lee Hoyoung , Kim So-Young , Moon Bae-Hyeon , Shin Eui-Cheol
Published: November 2024

During acute hepatitis A, pre-existing memory CD8+ T cells specific to hepatitis A virus-unrelated antigens are activated by IL-15 in a T-cell receptor (TCR)-independent manner, which is termed bystander activation, and these T cells contribute to liver injury (Immunity 2018, 48:161). However, it has been remained to be elucidated if other types of T cells are also activated by IL-15 without TCR engagement during acute hepatitis A. Here, we performed CITE-seq analysis in combination with single-cell RNA sequencing (scRNA-seq) using peripheral blood mononuclear cells (PBMCs) from patients with acute hepatitis A at acute and convalescent phases and healthy controls. In the scRNA-seq analysis, we used a gene set that was previously known to be upregulated by IL-15 in the absence of TCR stimulation to identify T-cell clusters that undergo TCR-independent, IL-15-induced activation. Among CD8+ T and γδ T cells, subclusters expressing CD45RO, CD57, and KLRG1 exhibited the highest module score of TCR-independent, IL-15-induced activation. Among MAIT cells, subclusters expressing CD8 and CD226 showed the highest module score of TCR-independent, IL-15-induced activation. Notably, these subclusters with the highest module score were exclusively found at the acute stage compared to the convalescent phase. In summary, T cell subpopulations exhibiting a high-degree of TCR-independent, IL-15-induced activation express distinct cell surface protein markers in patients with acute hepatitis A.
Differential activation of human lung epithelial cell subsets by virus-specific CD8+ T cell derived IFNg
Authors: Mattingly Cameron , Jimenez Ariana , Williams Mark , Kost Kirsten , Michalets Sarah , Thomas Jeronay , Kohlmeier Jacob
Published: November 2024

Lung CD8+ tissue resident memory T cells (TRM) act as sentinels that rapidly respond to respiratory viruses. While mouse models have shown that TRM mediate protection at barrier sites, our understanding in human lungs remains limited. We examined the functionality of virus-specific CD8+ TRM from non-transplantable, healthy human lungs by first quantifying their frequency and phenotype against prevalent viruses. While CD8+ T cells targeting respiratory viruses displayed a heightened tissue residency profile, RNAseq revealed that Flu and CMV specific lung CD8+ T cells are functionally and transcriptionally similar. Next, we investigated how virus-specific CD8s impact cells in the local lung environment. Following antigen stimulation, we noted activation of both innate and epithelial cells, dependent on IFNg. Examination of epithelial subsets showed that basal cells were more activated than ciliated cells. Upon stimulating lung CD8+ TRM with flu peptide +/- anti-IFNg, we conducted scRNAseq on epithelial cells and observed that stimulated CD8+ TRM robustly activated anti-viral genes in basal cells and to a lesser degree in secretory cells. Using Air-Liquid Interface (ALI) cultures, we modeled the effects of IFNg mediated epithelial activation on viral replication. Our findings show how crosstalk between CD8+ TRM and respiratory epithelium activates ISG pathways in epithelial cells and by doing so significantly decreases the frequency of infection and overall viral load.
CD2 costimulation as a method to improve mouse T cell activation by soluble antibody complexes
Authors: Rogers Trevor , Eaves Allen , Louis Sharon , Antignano Frann
Published: November 2024

Immunology research using mouse T cells requires cell activation that is often achieved using anti-CD3/CD28 antibodies immobilized (imm) on beads or cultureware. This immobilization can be tedious, leading us to investigate mouse T cell activation using soluble (sol) antibody complexes. Purified mouse T cells were activated by immCD3/CD28 or solCD3/CD28 complexes for 2 days, resulting in an average of 85% and 68% CD25+ T cells by flow cytometry, respectively (n = 12). However, adding CD2 to the solCD3/CD28 complex increased the %CD25+ T cells to 99% after 2 days (n = 12), suggesting that CD2 enhances activation. Purified T cells activated by solCD3/CD28/CD2 complexes showed improved fold expansion (28-fold) after 7 - 8 days compared to activation by immCD3/CD28 (15-fold) or solCD3/CD28 (18-fold) complexes (n = 17). As well, purified CD4+ T cells activated by solCD3/CD28 complexes exhibited increased fold expansion from 1- to 20-fold after 8 days by adding CD2 to the complexes (n = 3). Lastly, we investigated how activation methods impact a 6-day polarization of naive CD4+ T cells to T helper subsets. Cells activated by solCD3/CD28 complexes resulted in 53% Th1, 29% Th2, or 67% Treg cells, whereas solCD3/CD28/CD2 complexes resulted in 77% Th1, 24% Th2, or 79% Treg cells (n = 6). In summary, these soluble ImmunoCult™ Mouse T Cell Activators effectively activate, expand, and polarize mouse T cell subsets, which is enhanced by CD2 costimulation.
Application of Deep Learning Models on Single-Cell RNA Sequencing Analysis Uncovers Novel Markers of Double Negative T Cells
Authors: Xu Tian , Xu Qin , Lu Ran , Oakland David , Li Song , Luo Xin
Published: November 2024

Double negative T (DNT) cells, a unique subset of CD3+TCRαβ+ T lymphocytes lacking CD4, CD8, or NK1.1 expression, constitute 3-5% of the total T cell population in C57BL/6 mice. Traditional machine learning models such as principal component analysis in single-cell RNA sequencing (scRNA-seq) analysis have been utilized to characterize this subset, but only deep learning models such as Single Cell Variational Inference (SCVI) can capture nonlinear gene expression in the sequencing data. In this study, using the deep learning approach, we have uncovered novel markers of splenic DNT cells in C57BL/6 mice. We classified DNT cells into two subgroups, naïve DNT (nDNT) cells and activated DNT (aDNT) cells, which could be differentiated by the expression of Ly6C and MHC-II, respectively. In addition, our data identified CD153 and CD153 as unique markers for aDNT cells. SCVI analysis suggested, and flow cytometry analysis confirmed, that Ly49G2 and CCR2 are respective markers for two distinct subsets within nDNT and aDNT populations. In addition to cell surface markers, we further discovered that nDNT cells expressed higher Rgs2 that is required for T cell activation and that aDNT cells expressed higher Rbpj involved in Notch signaling. Together, our comprehensive analysis has uncovered and validated novel markers for different subpopulations of DNT cells that can be used in the phenotypic and/or functional characterization of these relatively rare cells in health and disease.
B cell-intrinsic IL-27 and IFNγ receptor signaling drive PD-L1 expression, IgG2a autoantibody production, VEGF-dependent tumor angiogenesis and cancer progression
Authors: Hinton Karli , Cervantes Christian , Viswanadhapalli Suryavathi , Vankamamidi Sai Eashan , Yan Hui , Luo Weiwei , Vadlamudi Ratna , Xu Zhenming
Published: November 2024

B effector cells produce Abs and autoantibodies, while B regulatory cells use cytokines and transmembrane proteins to fine tune immune functions. Our lab recently revealed that tumor-infiltrating B cells produce IL-27 to promote immune evasion, partly by upregulating tumoral PD-L1, including that expressed by B cells per se. To investigate the autocrine activity of IL-27 in tumor progression, we generated μMT:Il27ra–/– bone marrow chimera mice lacking the IL-27 receptor specifically in B cells. These mice displayed substantially reduced tumor growth from orthotopically engrafted ER+ E0771 breast cancer cells, as associated with only moderately decreased PD-L1 levels but, unexpectedly, severely compromised blood vessel integrity. The residual PD-L1 levels and tumor growth were virtually abolished by additional B cell-specific deficiency in IFNγ receptor signaling, as occurring in double KO μMT:Il27ra–/–Ifngr1–/– mice, in which the microvessel permeability was also worsened. Mechanistically, this aligned with our findings that IL-27-producing B cells expressed high levels of VEGFA, whose transcription in activated B cells could be further enhanced by IL-27 and IFNγ. Double KO mice also lacked anti-dsDNA IgG2a autoantibodies, as elicited by tumor engraftment. Thus, B cell-intrinsic IL-27 and IFNγ receptor signaling synergize to promote B-cell PD-L1 expression, IgG2a autoantibody production, and vascular integrity, all of which may contribute to cancer development.
Therapeutic potential of VBY-825 in MSU crystal-induced NLRP3 inflammasome activation
Authors: Orellano Laura , Rock Kennet , Lai Jiann-Jyh
Published: November 2024

In gout patients, MSU crystals formed in the joints cause inflammation, and the activation of the NLRP3 inflammasome is believed to mediate these responses. Cathepsins play an important role in crystal-induced NLRP3 activation that results in subsequent speck formation, caspase-1 activation, IL-1β secretion, and pyroptosis. In this study, we investigated if cathepsins can also influence MSU-induced NLRP3 activation in a gout mouse model. VBY-825 is a reversible pan-cathepsin inhibitor, and we found that VBY-825 significantly suppressed IL-1β secretion and LDH levels from peritoneal naïve macrophages upon MSU stimulation in vito. In a mouse peritonitis model, we found that VBY-825 suppressed MSU-induced inflammation when compared to the control treatment. Interestingly, the inflammation demonstrated by histology and MPO activity in the joints was also reduced by VBY-825 treatment in MSU-induced arthritis. These findings suggest that cathepsins play a critical role in MSU-induced inflammation and could potentially be a target for gout treatment.
Reshaping immune cells and the antigen-specific repertoire by anti-CD3 mAb teplizumab in Type 1 diabetes
Authors: lledo delgado ana , Preston-Hurlburt paula , Currie Sophia , Clark Pamela , Herold Kevan
Published: November 2024

Teplizumab, a FcR non-binding anti-CD3 mAb, is approved to delay progression of type 1 diabetes (T1D) at-risk patients. Previous investigations described the immediate effects of the 14-day treatment, but longer-term effects of the drug remain unknown. We analyzed the phenotypes, transcriptome, and repertoire of peripheral blood and autoreactive CD8+ T cells from individuals at-risk for T1D, before and after teplizumab.

There were changes in CD4+ and CD8+ T cells at 3 months and 18 months after treatment in genes related to immune regulation. The differential gene expression in the T cells increased after drug was cleared. Persistent EOMES expression was a predictor for the time to clinical T1D diagnosis. Our trajectory analysis showed differentiation into cells with gene signatures related to immunoregulation and exhaustion after teplizumab treatment. This analysis aligned with inhibition of expansion of autoreactive CD8+, which was observed with the placebo, but not following teplizumab.

These findings illustrate that a single course of teplizumab can instigate substantial T cell changes at 3 months, with evolution of CD8+ T cell exhaustion up to 18 months. Teplizumab restrains the expansion of autoantigen reactive CD8+ cells, suggesting mechanisms underlying operational tolerance.
Targeting Hippo pathway diminishes disease signaling in scleroderma skin.
Authors: Plazyo Olesya , Wasikowski Rachael , Gharaee-Kermani Mehrnaz , Tsou Eliza Pei-Suen , Tsoi Lam , Billi Allison , Kahlenberg J , Varga John , Khanna Dinesh , Gudjonsson Johann
Published: November 2024

Scleroderma (SSc), a female-biased autoimmune disease that causes inflammation and fibrosis in skin and internal organs, lacks effective treatment options. Our previous work, including single-cell RNA sequencing (scRNA-Seq) of skin from a large cohort of SSc patients, suggested that Hippo pathway regulates a female-specific immune gene network and drives myofibroblast differentiation in SSc skin. Our objective herein was to assess if inhibiting Hippo pathway pharmacologically can suppress disease signaling in SSc. We targeted distinct steps of Hippo pathway using 3 inhibitors (TRULI, Verteporfin, and MGM-CP1) that were applied to SSc skin biopsies ex vivo, followed by in-depth scRNA-Seq analyses to characterize molecular changes in affected cell populations. We found that Verteporfin, which prevents interaction of TEADs with YAP and VGLL3, selectively depleted myofibroblasts and endothelial-mesenchymal transition cells – the stromal cells pathologically activated in SSc. It also reduced expression of inflammatory, pro-fibrotic, and endothelial activation markers. Moreover, cell-cell interactions that sustain disease signaling were markedly hindered by Verteporfin. We saw a striking overlap between the genes downregulated by Verteporfin and those upregulated in SSc vs healthy patients. Thus, targeting Hippo pathway by Verteporfin effectively reverses molecular hallmarks of SSc. Our data further showcase the utility of this novel approach for modeling drug targeting ex vivo.
Unique T cell signatures are associated with reduced Chlamydia trachomatis reinfection in a highly exposed cohort.
Authors: Yount Kacy , Chen Chi-Jane , Kollipara Avinash , Liu Chuwen , Mokashi Neha , Zheng Xiaojing , O'Connell Catherine , Bagwell C , Iannone Marie , Wiesenfeld Harold , Hillier Sharon , Stanley Natalie , Darville Toni
Published: November 2024

Chlamydia trachomatis (CT) is the most common bacterial sexually transmitted infection (STI) in the U.S. despite effective antibiotics. Understanding natural immunity to CT is crucial to inform vaccine design. This study aimed to identify immune cell populations and functional features associated with reduced risk of CT reinfection. PBMCs were collected from 82 CT-exposed women tested for CT and other STIs upon enrollment, and repeatedly over 1 year of follow-up. Immune responses were profiled by mass cytometry. Women with CT at enrollment exhibited higher frequencies of CD4+ effector memory T cells (TEM) than uninfected women. Specifically, Th2, Th17, and Th17 DN CD4 TEM were increased. However, functional features indicated diminished expression of T cell activation and differentiation markers, underscoring weaknesses of natural immunity that could be addressed by vaccine design. Comparing responses in women who remained follow-up negative (FU-) to those who were reinfected (FU+), higher frequencies of Th1, Th17, and Th17 DN CD4 T cells were observed in FU- women. Conversely, Th2 CD4 T cells were increased in FU+ women. Furthermore, markers of T cell differentiation, memory, and cytotoxicity were increased in FU- women. These data indicate that peripheral T cells exhibit distinct features associated with natural immunity to CT. Notably, the highly plastic Th17 lineage appears to contribute to protection. Addressing these immune nuances could promote efficacy of CT vaccines.