bluuebunny/crossref_metadata_2025 huggingface dataset:
Rebalancing TGFβ1/BMP Signaling Epigenetically Reprograms Fully Exhausted Human CD8 T Cells into a Functional State
Authors: Ghoneim Hazem E , Saadey Abbey A , Yousif Amir , Osborne Nicole , Chen Yu-Lin , Laster Brooke , Zayed Ahmed , Bauman Parker
Published: January 2023

Epigenetic scarring of exhausted T cells (Tex) remains a major obstacle to achieving durable responses by T cell immunotherapies. While we established that de novo DNA methylation programs are causally linked to T cell’s full exhaustion and poor response to immune checkpoint blockade (ICB), major gaps remain in our current understanding of T cell exhaustion—(1) What are the upstream signals that regulate acquisition of exhaustion-specific epigenetic programs? (2) Can we remodel the epigenetic state of Tex to an ICB-responsive state? To address these questions, we developed a novel in vitro model of human T cell dysfunction. First, we demonstrated that chronic TCR stimulation of CD8 T cells is insufficient to develop T cell exhaustion. Instead, our integrative analyses of epigenetic and transcriptional changes in CD8 T cell subsets during chronic virus infections and cancer revealed TGFβ1 as the most significant upstream regulator linked to full exhaustion in mice and humans. Indeed, we found that post-effector TGFβ1 signaling accelerates full exhaustion in chronically stimulated CD8 T cells through stable epigenetic changes linked to impaired effector function and memory potential. Therapeutic rebalancing of TGFβ1/BMP signals by blocking TGFβ1 while boosting BMP signals not only restored effector function, but also unlocked memory programs in human Tex cells. This new therapeutic approach induced a superior anti-tumor activity of human T cells and synergized the ICB response in a murine model of chronic LCMV infection. Our findings highlight the role of TGFβ1/BMP signals in T cell exhaustion and propose a novel therapeutic strategy to epigenetically reprogram fully exhausted T cells, ultimately enhancing T cell immunotherapies.
Sushi domain containing 2 suppresses CD8+ T cell antitumor immunity by targeting IL-2 receptor signaling
Authors: Wen Haitao , Zhao Bao
Published: January 2023

One major mechanism of immunosuppressive tumor microenvironment (TME) is dysfunctional CD8+ T cells which exhibit defective production of antitumor effector molecules. However, the intrinsic molecular mechanism(s) underlying CD8+ T cell dysfunction in cancer remains incompletely understood. Here, we show that the sushi domain containing 2 (SUSD2) is a negative regulator of CD8+ T cell antitumor function. Genetic deletion of Susd2 (Susd2−/−) results in an enhanced production of antitumor molecules in effector CD8+ T cells, which consequently blunts tumor growth in multiple syngeneic mouse tumor models. Through a quantitative mass spectrometry assay, we find that SUSD2 interacts with interleukin-2 receptor a (IL-2Ra) via sushi domain-dependent protein interaction and their interaction suppresses IL-2Ra binding with IL-2, a well-established cytokine essential for CD8+ T cell effector function. Adoptive transfer of Susd2−/− CD19-targeting chimeric antigen receptor (CAR) T cells induces a robust antitumor effect, highlighting SUSD2 as a potential immunotherapy target for cancer treatment.

Supported by grants from NIH (R01 AI62779-01)
Anti-CD123 Chimeric Antigen Receptor Natural Killer cell therapy to treat Acute Myeloid Leukemia.
Authors: Kizerwetter Monika , Yang Huilin , Rahnama Ruyan , Bonifant Challice , Spangler Jamie
Published: January 2023

Acute myeloid leukemia (AML) is a highly prevalent blood and bone marrow cancer characterized by the uncontrolled growth of abnormal myeloblasts. Current treatments for AML often result in systemic toxicities for patients, many of whom will still experience relapse. There is thus an unmet need to develop safer and more effective therapies targeting AML. Chimeric antigen receptor (CAR) T cells, which are engineered to express a cancer-targeting extracellular antibody single-chain variable fragment (scFv) linked to an intracellular T cell-activating domain, have shown promise in treating B cell leukemias, but their success has been limited in myeloid leukemias. This is due in part to the lack of AML-specific target antigens. Moreover, impaired T cell function, a hallmark of AML, is also problematic when relying on autologous T cell activation upon reinfusion. We overcame the limitations of CAR T therapy in AML by developing CAR-expressing natural killer (NK) cells targeting CD123. While present on both healthy and AML cells, CD123 is expressed at >10-fold higher levels on malignant cells. Furthermore, use of NK cells allows use of fully functional allogeneic cells from healthy donors, since NK cells do not induce graft-versus-host-disease. To optimize the tumor cell selectivity of CAR NK cells, we employed the yeast display directed evolution platform to isolate anti-CD123 scFvs with varying affinities and are identifying clones that maximize the interaction with CD123high AML cells over healthy CD123low cells, as determined by binding, cytotoxicity, and cytokine secretion studies. Collectively, our efforts introduce a new paradigm for engineered cell therapeutics with significant promise for AML treatment.
D2C7 CAR: A novel CAR T cell that simultaneously targets wildtype EGFR and its mutant isoform EGFRvIII for treatment of glioma
Authors: Wilkinson Daniel S , Ryan Katherine , Wilson Joseph , Chandramohan Vidyalakshmi , Landi Daniel , Bigner Darell , Fecci Peter E
Published: January 2023

Brain tumors are the leading cause of cancer death in children and a significant cause of morbidity and mortality in adults. Conventional treatments are suboptimal, thus signifying the need for novel therapeutic strategies, such as immunotherapy. Chimeric antigen receptor (CAR) T cells represent a revolutionary class of immunotherapy, achieving considerable success in eliminating hematological cancers but generally failing to control solid tumors in part due to the lack of a suitably-expressed target antigen. Epidermal growth factor receptor (EGFR) is the most ubiquitous and homogeneous antigen on glial brain tumors, and EGFR-directed therapies have been hotly pursued. Moreover, the mutant EGFR variant, EGFRvIII, is present on a subset of pediatric and adult high grade gliomas, representing a targetable, tumor-specific antigen. Unfortunately, CAR T cells targeting EGFRvIII fail to treat tumors possessing as few as 5–10% EGFRvIII-negative cells due to antigen escape. Thus, a CAR T cell that can target both EGFR and EGFRvIII is expected to be superior to a CAR that targets EGFRvIII alone. In this study, we developed a novel third generation CAR T cell consisting of the D2C7scfv targeting moiety that binds a shared epitope between EGFR and EGFRvIII. This D2C7 CAR was able to specifically and potently kill tumor cells expressing wildtype EGFR or EGFRvIII. Importantly, D2C7 CAR significantly prolonged survival of mice bearing EGFR or EGFRvIII-expressing gliomas of both adult (U87) and pediatric (DAOY) origin. Toxicity experiments involving EGFR-expressing human skin grafts provided evidence that D2C7 CAR is safe and effective when administered intracranially to mice bearing intracranial tumors.
Overcoming barriers to solid tumor immunotherapy using natural killer cell therapies designed to mimic intraepithelial group 1 innate lymphoid cells
Authors: Horowitz Nina Beryl , Hickey John , Nolan Garry P , Sunwoo John B
Published: January 2023

Our work aims to design cell-based immunotherapies for maximum solid tumor efficacy by leveraging insights from tissue-resident cells and single-cell RNA sequencing of innate immune cell subsets present within patient tumors. We discovered that by co-culturing peripheral blood natural killer cells (pbNKs) with irradiated epithelial tumor cells, we induced high expression of surface integrins, increased cytotoxicity and IFNg production, decreased sensitivity to TGFb, and significantly enhanced solid tumoroid infiltration. The cells were profiled using CyTOF and we determined that they closely resembled intraepithelial group 1 innate lymphoid cells, so we refer to them as ieILC1-like cells. We quantified the cytotoxicity of ieILC1-like cells against a variety of target cell lines and determined that they are broadly cytotoxic and capable of antibody-dependent cellular cytotoxicity, which was assessed using cetuximab. Tumor-infiltrating capacity was modeled using 3D tumoroids grown from single-cell suspensions of epithelial tumor cell lines in basement membrane extracts; ieILC1-like NK cells or pbNK cells were fluorescently labeled, added to tumoroids, and imaged using confocal microscopy. We stimulated the cells in the presence or absence of overnight TGFb exposure to determine the extent of TGFb-mediated immunosuppression. ieILC1-like cells were more resistant to TGFb and produced significantly more IFNg after stimulation than their pbNK counterparts. Preliminary in vivo work indicates that ieILC1-like cells perform comparably to K562-expanded NK cells. Thus, ieILC1-like NK cells represent a novel class of cell therapy that are more capable of infiltrating solid tumors and resisting their immunosuppressive cues.

Supported by NIH R35DE030054.
Evaluating potential toxicities of cancer immunotherapies using autoimmune animal models.
Authors: Okpasuo Onyekachi Juliet , Cattin-Roy Alexis N , Karasseva Natalia , Mahadevappa Chandrashekara Puthanapura , Radhakrishnan Vijay , Zaghouani Habib , Pages Diana Gil
Published: January 2023

During the past few decades, cancer immunotherapy has recorded breakthroughs involving therapeutic agents that mediate immune response against several tumor types. However, an important consideration with immunotherapy treatment in the clinic has been the emergence of immune related adverse events (irAEs), that could lead to frequent hospitalization, premature therapy cessation and fatalities, thus limiting the efficacy of these therapies. Additionally, despite the clinical benefits of combining therapies, it has been observed that severity of toxicity increases with combination of therapy. Although, these irAEs are commonly seen in patients receiving immunotherapy, such events have been rarely observed in preclinical mouse models due to their nature: inbred, resistant, and short study duration. Here, we report the use of EAE autoimmune mouse model to evaluate potential toxicities of a novel cancer immunotherapy: anti-CD3ɛ mono-Fab, that is effective as a single therapy and could synergize with checkpoint inhibitors for superior anti-metastatic tumor response. EAE model groups treated with combination therapies showed more disease severity recapitulating the higher grade of irAEs seen in patients receiving combination therapies. The data suggest that autoimmune mouse models could serve as an effective tool to study the severity of toxicity associated with immunotherapeutic agents, and to determine optimized combinatorial regimens for best therapeutic performance.

Supported by NCI U01CA244314
Overcoming AML T cell therapy barriers with engineered proteins
Authors: Ruan Aitong , Chiu Edison , Oda Shannon K
Published: January 2023

Leukemia is the most common pediatric cancer and the second leading cause of cancer deaths in children. Adoptive cell therapy (ACT) has been validated in pediatric B cell acute lymphoblastic leukemia, where engineered cancer-targeting T cells can effectively eradicate tumor and tumor recurrence, generally more safely than drug and radiation therapy. However, ACT is not approved for patients with pediatric AML, other leukemias or solid tumors. Of note, pediatric AML patients have a worse 5-year survival rate than ALL.

AML cells can severely diminish ACT efficacy by expressing ligands that activate T cell inhibitory receptors and by downregulating costimulatory ligand expression. To address these issues, we developed fusion proteins that combine the ectodomain of an inhibitory T cell receptor with an intracellular costimulatory signaling domain. We have shown that this effectively “replaces a brake with an accelerator” in CD8 T cells, and have now developed FPs with different costimulatory domains to confer different attributes to T cells. Despite improved outcomes with single FP T cell therapy, we hypothesized that combining differently costimulated T cells would provide a synergistically diverse T cell response and improve outcomes. We tested 1:1 pairwise combinations of differently costimulated T cells that are also transduced with a tumor-specific TCR in serial killing over-time (SKO) assays and in vivo persistence studies. In survival studies, dual T cell therapy resulted in enhanced survival. We propose that a synergistically diverse T cell therapy provides enhanced antitumor efficacy over a monogenic cell product.
Adjuvant AS01 induces monocyte activation that favors T cell expansion and diversification in primary human cells
Authors: Smith Carson L , Canaday David H
Published: January 2023

Vaccines are less effective in older adults, and efforts to improve vaccine efficacy generally show limited clinical improvement. However, the vaccine Shingrix, developed by GlaxoSmithKline, is FDA approved and recommended for adults over the age of 50 for the prevention of shingles. Shingrix contains the novel adjuvant AS01 and shows remarkably high (>90%) effectiveness at both preventing shingles and reducing the development of postherpetic neuralgia, even in adults over 80. We seek to examine AS01-induced activation of myeloid cells and determine their impacts on T cell memory differentiation and function in an effort to understand how this vaccine’s high clinical efficacy is produced.

We use peripheral blood samples to examine AS01-induced innate immune cell activation, including interactions with T cells once activated. We find that AS01 induces costimulatory marker upregulation and cytokine production in myeloid dendritic cells and monocytes in a synergistic manner, with the most upregulation seen in monocytes. Naïve and memory CD4 T cells incubated with AS01-treated monocytes have increased frequencies of TH17 (CCR6+ CXCR3−) cells and decreased frequencies of TH1 cells compared to controls. Naïve CD4 T cells proliferate more when incubated with AS01-treated monocytes than with controls. AS01-treated monocytes additionally induce 2x less IFN-γ and IL-5 in response to mitogen (SEB) from CD4 T cells, suggesting a modulation of response.

We propose that AS01 influences monocytes to diversify the existing memory T cell repertoire and skews naïve T cells towards more cytotoxic phenotypes such as TH17, contributing to greater clinical efficacy in preventing shingles in vivo.

Supported by VA Merit grant (CX002060)
Improving the metabolic efficiency of human T cells through AMPK agonist treatment
Authors: Braverman Erica , McQuaid Margaret , Byersdorfer Craig A
Published: January 2023

BACKGROUND

Adoptive immunotherapies are limited by the increased differentiation and metabolic exhaustion of the T cell product. The cellular energy sensor AMP-activated protein kinase (AMPK) controls many metabolic pathways. We hypothesized that increasing AMPK signaling during T cell expansion would create more metabolically efficient T cells.

RESULTS

Human T cells were stimulated in vitro, expanded with or without AMPK agonist A769662, and restimulated under varying glucose concentrations. Agonist treatment increased T cell expansion by manual cell counts (1.5× increase in cell number, p<0.01), and produced a higher percentage of CD62L+CD27+ cells by flow cytometry (10% increase, p<0.01). Twenty-four hours after re-stimulation, agonist-treated cells exhibited decreased basal oxygen consumption and extracellular acidification rates (45% and 20% decrease, p<0.001) but increased spare respiratory capacity (SRC) (97% increase, p<0.001). At 72 hours, control cells decreased proliferation in a glucose dose-dependent manner, as measured by BrDU uptake, whereas treated cells maintained a higher level of proliferation despite limited glucose availability.

DISCUSSION

Increasing AMPK signaling during T cell expansion improves doubling time while maintaining higher CD27 positivity, suggesting a greater yield of less differentiated cells. Upon re-stimulation, treated cells initiate less metabolic activity but maintain greater proliferation and SRC under glucose-limiting conditions. These results suggest that greater metabolic efficiency in agonist-treated cells allows for continued function despite nutrient restriction, a trait which could confer significant advantages to adoptive immunotherapies.

Supported by grants from NIH (5K12 HD052892) and the St. Baldrick’s Foundation (St. Baldrick’s Fellowship Grant)
Phenotypic and transcriptomic profiles of tumor-conditioned macrophages in dogs, and the translational value of canine cancer immunology research.
Authors: Kerboeuf Mikael , Haaland Anita Haug , Moe Lars , Argyle David , Boysen Preben
Published: January 2023

Tumor-associated macrophages (TAMs) are heterogeneous and abundantly present in the tumor stroma. They derive from tissue-resident macrophages and recruited monocytes and play a central role in several aspects of cancer. In murine models, TAMs promote invasion, intravasation, circulatory survival and extravasation of tumor cells. TAMs also promote tumor progression and angiogenesis, remodel the tumor microenvironment, and modulate the adaptive immune system. Evidence suggests TAMs are vital for establishing the premetastatic niche. Historically, family-owned dogs have proven to be reliable cancer models. In contrast to laboratory animals, dogs develop cancer naturally and commonly, co-inhabit our environment, are genetically outbred and immunologically experienced. We hypothesized that canine tumor conditioned monocyte-derived macrophages (TCMs) share many characteristics with M2 macrophages and have a tumor-promoting phenotype as in humans. We collected blood from healthy dogs, isolated peripheral blood monocytes and differentiated them in vitro using tumor conditioned media from three canine cancer lines and M-CSF. Three TCM populations were generated and compared with M1 and M2 macrophages. We assessed the macrophages using multicolor flow cytometry and RNA-sequencing. All TCM showed an increased expression of two M2 markers (CD209a and FcɛRI), while 2/3 TCM populations had increased expression of two additional M2 markers (CD206 and CD11d). Transcriptomic analysis is ongoing. Results so far support the hypothesis that canine TCMs share many similarities with human TCMs, further warranting the use of dogs as cancer immunology models.

Supported by grant from Agria Animal Insurances (N-2020-0061) and The Research fund for cancer in dogs (Oslo, Norway)
Discovery of an organized nasopharynx-associated lymphoid tissue in the nasal cavity of rainbow trout and its role in secondary adaptive immune responses to nasal vaccines
Authors: Salinas Irene , Garcia Benjamin , Dong Fen , Casadei Elisa
Published: January 2023

The olfactory organ of all vertebrates is constantly exposed to pathogens. The nasal mucosa is therefore equipped with a local immune system that protects the host from infection. In teleost fish, we previously described the presence of a diffuse nasopharynx-associated lymphoid tissue (NALT) found in the olfactory lamellae. This diffuse NALT (d-NALT) consists of myeloid and lymphoid cells but lacks an organized structure. Rainbow trout d-NALT mounts quick innate immune responses as well as adaptive immune responses to nasal vaccines. Here we report the discovery of a novel lymphoid structure located in the inner epithelial lining of the nasal cavity, opposite to the tips of the olfactory lamellae, in rainbow trout. This lymphoid structure represents the first description of an organized NALT (O-NALT) in teleost fish. Rainbow trout O-NALT is rich in B and T lymphocytes at the steady state with a proportion of approximately 8:3:1 IgM+ B cells, CD8+ T cells and IgT+ cells, respectively. Using laser capture microdissection we show that compared to d-NALT, O-NALT is highly enriched in expression of CD4, CD8 and IgM but not IgT. Strikingly, O-NALT expresses 8-fold higher levels of activation-induced cytidine deaminase aidc mRNA compared to d-NALT. Following primary and secondary intranasal vaccination with a live attenuated viral vaccine against infectious hematopoietic necrosis (IHN), trout O-NALT was expanded with increased numbers of B and T cell lymphocytes. Our results indicate that teleost fish posses organized lymphoid structures in the nasal cavity that participate in the maturation of the adaptive immune response and that need to be considered as important players in the response to mucosal vaccines.

This work was supported by USDA NIFA award # 2020-67015-31457
Altered transcriptome responses in the lungs of preweaned calves supplemented with a yeast fermentate during a viral-bacterial coinfection
Authors: McDonald Paiton O , Maina Teresia Wangari , Samuel Beulah Esther Rani , McGill Jodi L
Published: January 2023

Bovine respiratory disease is a complex syndrome which contributes to severe and often fatal pneumonia in beef and dairy cattle. Saccharomyces cerevisiae fermentation products (SCFP) are feed ingredients that have been previously shown to alter the immune response and outcome of a respiratory viral infection in calves. The objective of this study was to determine the effect of SCFP supplementation on the lung transcriptome in calves responding to a co-infection with bovine respiratory syncytial virus (BRSV) and Pasteurella multocida (PM). Twenty-eight, 1–2 day old calves were assigned to two groups: 1) control diet; or 2) SCFP treated diet. Calves were infected with BRSV on day 21, followed 6 days later by intratracheal inoculation with PM. Calves were euthanized on day 10 post-viral infection. There were no differences in viral or bacterial burden between treatment groups, but SCFP treated calves tended to have less severe lung pathology. Transcriptome analysis of lung tissue and bronchoalveolar lavage samples revealed over-represented genes related to membrane integrity, pathogen binding, and complement activation which presumably contributes to the host’s resiliency and response to the coinfection. Results from this study suggest that supplementing preweaned calves with SCFP may modulate the innate and adaptive immune response in mucosal sites. These findings will contribute to a better understanding of the underlying host response to bovine respiratory disease.

Supported by funding from Diamond V Mills, Inc., Cedar Rapids, IA
Mapping the spatial biology of COVID-19 immunopathology in placenta tissues
Authors: Ma Ning , Braubach Oliver , Rebutini Patricia Zadorosnei , Machado-Souza Cleber , Stonoga Emanuele Therezinha Schueda , de Noronha Lucia , Souza-Fonseca-Guimaraes Fernando , Kulasinghe Arutha
Published: January 2023

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that emerged in late 2019 has spread globally, causing a pandemic of respiratory illness. The virus primarily affects the lungs, where it induces respiratory distress syndrome ranging from mild to acute. However, a growing body of evidence shows COVID-19 pathology in other organs that also carry the ACE-2 receptor, including the placenta. Most newborns delivered from COVID-19 positive mothers test negative following delivery, suggesting that there are protective mechanisms within the placenta. In this study, we compared a unique collection of placental tissue samples from COVID-19 positive and gestational age-matched non-COVID-19 samples. Using AKOYA’s ultrahigh-plex Phenocycler assay, we mapped dozens of protein biomarkers in situ with single-cell and subcellular spatial resolution to produce unbiased and comprehensive spatial biomarker maps of potential placental pathobiology associated with COVID-19 infections. Our antibody panel was aimed at in-depth identification of immune cell lineages, activation states, immune checkpoints, and tissue structures; infected cells were identified via the deployment of two antibodies directed at COVID-19 spike and capsid proteins, as well as an antibody directed against the ACE-2 receptor. All imaging data were obtained at single-cell resolution across whole tissue samples, and cellular phenotypes were subsequently identified via unsupervised clustering methods. These data are the first of their kind and will provide additional insight into the pathobiology of COVID-19.
TLR2 senses spike protein of SARS-CoV-2 to trigger inflammation
Authors: Zaki Hasan , Khan Shahanshah
Published: January 2023

The pathogenesis of COVID-19 involves cell death caused by SARS-CoV-2 infection and hyperinflammatory response. Therefore, understanding the mechanism of inflammatory response during SARS-CoV-2 is the key to develop strategies to reduce COVID-19 complications. We investigated what viral ligand is responsible for activation of innate immune cells by stimulating human and mouse macrophages with recombinant spike (S), membrane (M), envelope (E), and neucleocapsid (N) proteins. Interestingly, we observed that S protein, its subunits and S-trimer, but not M, N, and E proteins, induced inflammatory cytokines and chemokines. Similar to immune cells, human and mouse lung epithelial cells also responded to S protein to produce inflammatory molecules. Since SARS-CoV-2 dose not infect immune cells, we interrogated whether virally infected epithelial cells stimulate macrophages. We observed that S protein-expressed lung epithelial cells induced inflammatory responses in macrophages when cocultured. Notably, S protein induced inflammation independent of ACE2 receptor. Digging dipper, we wanted to explore what pattern recognition receptor senses S protein to trigger inflammation. Our investigation revealed that TLR2, but not TLR4, is the immune sensor for S protein which in association with either TLR1 or TLR6 activates the NF-kB pathway via MyD88. Consistently, S-induced inflammatory response was completely absent in Tlr2−/− mice. Blocking TLR2 chemically or though gene manipulation also abrogated S-induced inflammation in vitro. Together these data demonstrate the mechanism of cytokine storm during SARS-CoV-2 infection, and suggest that TLR2 could be a potential therapeutic target for COVID-19.

Supported by R01 DK125352
Children with Multi-System Inflammatory Syndrome (MIS-C) develop functionally competent T cell memory against SARS-CoV-2 following recovery
Authors: Rybkina Ksenia , Bradley Marissa C , Bell Joseph N , Meng Wenzhao , DiLorenzo Michael P , Anderson Brett R , Pethe Kalpana , Luning-Prak Eline , Farber Donna L , Connors Thomas J
Published: January 2023

While pediatric SARS-CoV-2 infection is largely spared from severe COVID-19, a small fraction of children develop a life-threatening condition known as MIS-C, which is associated with an aberrant peripheral blood immunophenotype; namely within adaptive immune cell populations. Little is known about how MIS-C impacts memory generation that would be imperative for future SARS-CoV-2 responses. Here, we used high dimensional flow cytometry and TCR sequencing to assess the peripheral blood T cell profile during acute MIS-C (n=17), following recovery (MIS-C-R) up to 9 months post-MIS-C (n=9), and relative to age-matched convalescent, mild pediatric COVID-19 (non-MIS-C; n=5) and uninfected controls (n=3). We also applied antigen-specific analyses to determine the magnitude and functional capacity of SARS-CoV-2-specific T cells in MIS-C-R (n=10) and non-MIS-C children (n=16). Flow cytometry showed unique T cell composition in MIS-C which resolved in recovery: lacking persistence of exhausted CD8+ T cells observed during MIS-C, and reestablishment of the naïve T cell pool. TCR sequencing showed decreased clonal diversity and distinct TCR Vβ usage in acute MIS-C compared to MIS-C-R. Results from antigen-specific analyses show that while T cell memory is similar phenotypically, MIS-C-R children have greater frequencies of SARS-CoV-2-specific CD4+ T cells than non-MIS-C up to 9 months since diagnosis. Meanwhile, SARS-CoV-2-specific CD8+ T cells in MIS-C children were more cytokine-producing, while more cytotoxic in non-MIS-C children. Together, these results reveal a restoration in global T cell phenotype in MIS-C-R children, as well as the robust production of functionally competent SARS-CoV-2 specific T cell memory.

This work is supported by grants from NIH (K23 AI141686, 3U01 AI100119-10S1)
Investigating the role of inflammasome activation by dengue virus non-structural protein 1 during dengue infection.
Authors: Wong Marcus P , Juan Evan Y W , Blanc Sophie F , Biering Scott B , Wang Phoebe , Beatty Robert , Harris Eva
Published: January 2023

Dengue virus (DENV), consisting of serotypes 1–4, is the most medically important flavivirus, causing an estimated 50–100 million dengue cases globally every year. Most symptomatic DENV infections present as an acute febrile illness; however, severe complications include vascular leakage leading to hemorrhage and hypovolemic shock. DENV non-structural protein 1 (NS1) is secreted from infected cells and has been implicated as a major driver of dengue pathogenesis, activating immune cells and acting directly on endothelial barriers, causing them to become pathologically hyperpermeable. While recent work has delved into the mechanisms behind the endothelial cell-intrinsic pathway of DENV NS1-induced vascular leak, much less is known about how DENV NS1 interacts with immune cells and what role this activation plays in DENV infection. We have discovered that DENV NS1 can trigger activation of the inflammasome, a family of cytosolic innate immune sensors that react to danger-associated molecular patterns. DENV NS1 induces the release of the pro-inflammatory cytokine IL-1β in human and murine monocytes in a caspase-1 dependent manner that is independent of NLRP3. Additionally, we found that caspase-1-deficient, but not NLRP3-deficient, mice are more susceptible to infection in a murine model of DENV infection. These results indicate that the inflammasome acts as a sensor of the DENV viral toxin NS1 and plays a protective role during DENV infections

This work was supported by NIAID/NIH grant R01 AI24493 (E.H.)
Multiplex imaging to reveal the dynamics of MAVS-mediated control of Ebola virus replication in tissues.
Authors: Bettis Natalia , Speranza Emily , Sturdevant Gail , Robertson Shelly , Germain Ronald , Best Sonja
Published: January 2023

The RIG-I like receptor pathway that signals through the mitochondrial antiviral signaling protein (MAVS) is important in sensing RNA viruses, driving production of interferons, and inhibiting viral spread. How this signaling coordinates immune responses in tissues is incompletely understood. To understand the mechanisms of MAVS-mediated control, we examined the spatial relationship between EBOV antigen and cells in livers. C57BL/6J (B6) or MAVS−/− mice were inoculated intraperitoneally with 100 FFU of either wild-type (WT-EBOV) or mouse-adapted EBOV (MA-EBOV) with samples collected at 3 and 5 days post infection (dpi). This resulted in a spectrum of disease outcomes: complete survival (B6 WT-EBOV), weight loss and survival (B6 MA-EBOV and MAVS−/− WTEBOV) and 100% lethality (MAVS−/− MA-EBOV). Macrophage (MF), dendritic cell (DC) and T-Cell (TC) responses were examined by Opal-Plex, a multiplex staining method developed in house, combined with quantitative image analysis through object-based segmentation and spatial statistics. In B6 mice at 3 dpi, there was an increase in cellular recruitment of monocytes and CD8+ T-cells. An association between EBOV nucleoprotein (NP) and F4/80+ cells was observed in the survival phenotype and was especially prominent at 3 dpi. Further development of LysMCre MAVSfl/fl mice will determine if MAVS signaling in the macrophages is required for viral control to the macrophage compartment or if the phenotypes observed could have resulted from a bystander effect. This work will help to define dynamics of host control of viral dissemination and further define the relationship between tissue architecture and disease outcome.

All studies were done with approval from the ICUC of the NIH/NIAID.

Supported by The Intramural Program of the NIH/NIAID
Elucidating neuronal innate immune mechanisms in the central nervous system.
Authors: Negatu Seble G , Jurado Kellie A
Published: January 2023

Neurotropic viruses are the most common cause of infectious encephalitis. Published studies have increased our understanding of how resident glial cells and infiltrating leukocytes contribute to neuroimmune responses during pathogen invasion. However, neuron-specific immune responses remain poorly described. Neurons are readily infected by neurotropic viruses, but studies show neuronal stimulation by viral components poorly induces pathways that are critical for viral control, such as the type I interferon (IFN) response. This leads our central question of how do neurons orchestrate effective antiviral immunity during CNS infection? To probe type I IFN responses in the CNS, I have developed an intracranial neonatal infection model using La Crosse Virus (LACV), an emerging RNA virus. Preliminary data show that infections of C57Bl/6 wild-type, Type I IFN receptor (IFNAR) receptor knock-out (KO), and non-canonical type I IFN pathway (STING) KO neonates with both wild-type LACV and LACV-ΔNSs (lacks the known type I IFN antagonist, NSs) viruses causes severe disease and reduced survival of neonates. IFNAR KO and STING KO mice succumb to disease earlier than C57Bl/6 mice. Additionally, in all genetic backgrounds, viral titers have an inverse relationship with survival. Future work will assess cell-specific infectivity of LACV and type I IFN responses in the CNS. I have also developed an in vitro infection pipeline where primary neurons isolated and cultured from mice are infected and further characterized using fluorescence in situ hybridization and RNA-sequencing. I plan to infect neurons of differential maturation states with LACV-ΔNSs and evaluate changes in gene expression compared to uninfected, bystander cells.

Supported by Penn Presidential Fellowship.
A granular view of X-linked chronic granulomatous disease exploiting single-cell transcriptomics
Authors: Muzumdar Sukalp , Ballouz Sara , Lam Fung , Degrange Maureen , Kreuzburg Samantha , Chong Hey , Zerbe Christa , Jongco Artemio , Gillis Jesse
Published: January 2023

X-linked chronic granulomatous disease (X-CGD) is a rare monogenetic immunodeficiency primarily affecting phagocytes. Precipitated by mutations in the CYBB gene, patients exhibit a compromised oxidative burst, leading to recurrent infections which can be life-threatening. Curiously, autoimmune manifestations are also common in patients and carriers. Here, exploiting the cell type-specific nature of this disorder, we characterize X-CGD on a transcriptional level using single-cell sequencing. Peripheral blood from 14 X-CGD probands and 10 carriers signed onto IRB approved protocol NCT00404560, as well as from 15 controls was sampled, and PBMCs and isolated monocytes were subjected to single-cell sequencing. Probands exhibited a strong differential expression signal relative to controls. This was composed of not only genes previously described to be up-regulated in X-CGD such as IFI27, and indeed an autoimmunity-associated broader type I interferon response, but also previously undescribed genes involved in monocyte function (ARG1), antimicrobial proteins (CAMP, SLPI), and inflammasome components (AIM2). Surprisingly, expression variability was not greater in carriers relative to probands or controls, indicating a lack of cell autonomous effects from the deletion of CYBB. Interestingly, aggregate expression of differentially expressed genes in the probands was able to classify carriers from sex-matched controls with high accuracy (AUROC = 0.92), indicating the presence of an X-CGD-specific gene signature. This gene signature was also strongly co-expressed across 17 chordate species, pointing towards the disruption of ancestral pathways important in antimicrobial immunity in X-CGD probands and carriers.

This work was partially supported by a Swiss National Science Foundation fellowship to S.M.
Phenotypic Heterogeneity in Patients and Mice with Immunodeficiency Caused by SASH3 Mutations
Authors: Majumdar Shamik , McDermott David , Jaiswal Hemant , Cho Kyoungin , Khillan Jaspal , Murphy Philip
Published: January 2023

SASH3 encodes a putative adaptor protein designated SAM and SH3 domain-containing 3 (SASH3), also known as SH3-containing Lymphocyte Protein 1 (SLY1), that is expressed in the cytoplasm and nucleus of lymphocytes. Recently we reported mutations in SASH3 in 4 unrelated male patients as the cause of X-linked recessive immunodeficiency presenting with B, NK and CD4+ T cell lymphopenia; recurrent sinopulmonary, cutaneous and mucosal infections; and refractory autoimmune cytopenias. Patient P1 was hemizygous for a cysteine substitution at the highly conserved Arg347 located in the C-terminus resulting in a protein-positive phenotype, while the other three patients were hemizygous for nonsense protein-negative variants. P1’s immunophenotype was confounded by concomitant large granular lymphocyte leukemia (LGL) and was clinically associated with severe neutropenia, low IgM, significant wart burden on all four extremities, recurrent aphthous stomatitis, and a history of pneumonia. To study the effects p.Arg347Cys, we developed a knock in (KI) mouse model in the C57BL/6 strain designated SASH3KI by CRISPR-Cas9 gene editing. Similar to the patient, SASH3KI mice displayed significant T cell lymphopenia in blood, whereas B cell numbers were normal. CD8+ T cells in SASH3KI mice were reduced in frequency and absolute number in bone marrow, spleen and both mesenteric and inguinal lymph nodes, but were normal in thymus. After infection with LCMV, T cells in SASH3KI mice proliferated normally and produced normal levels of effector molecules. These findings contrast with those reported previously in Sly1−/− and Sly1d/d mutant mice, which both exhibit thymic and splenic hypocellularity and B and T cell lymphopenia.
Human G Protein-Coupled Receptor 15 mutations associated with inflammatory bowel disease.
Authors: Cheng Yan , Cui Jing , Ravell Juan C , Lenardo Michael J
Published: January 2023

G Protein-Coupled Receptor 15 (GPR15) is a chemokine receptor, primarily expressed in T cells based on human single-cell RNA sequencing data. Its natural ligand, GPR15L, is found in the gastrointestinal basal epithelial region and presumably guide GPR15+ T cells to that location. Previous studies showed that Gpr15−/− mice were more susceptible to C. rodentium-induced colitis but the function of GPR15 and whether its deficiency plays a role in inflammatory bowel disease (IBD) in humans are unclear. We recruited three pediatric patients who developed severe early onset IBD and found novel GPR15 mutations using whole-exome sequencing. A reduction in T cells was observed in patient colon biopsies. Surface staining of patient T cells after TCR stimulation further revealed low expression of the mutant GPR15 alleles compared to the wildtype. Characterizing the GPR15 mutants in transduced stable cell lines, we found that the mutations led to lower GPR15L-dependent signaling, causing decreased cell chemotaxis towards the ligand. Accordingly, Gpr15−/− mouse colon also exhibited reduced T cell phenotype observed in patients. Our results indicate that GPR15 is critical for T cell and migration to the human colon and its absence may lead to immunodeficiency in the region, predisposing individuals to IBD induced by specific pathogenic microbiota. This research was supported NIAID by the Intramural Research Program of the NIH.

This research was supported NIAID by the Intramural Research Program of the NIH.
From genetic variants to diseases: the role of LACC1 in T cells
Authors: Li Yingcong , Chandra Vivek , Vijayanand Pandurangan , Kronenberg Mitchell
Published: January 2023

Expression quantitative trait loci (eQTLs) studies provide associations of genetic variants with the expression of particular genes. But how these eQTLs directly affect gene expression and in what cell types of the immune system they act remain largely unknown. One hypothesis is that functional eQTLs in the cis-regulatory elements may affect cell type-specific transcription factor binding, thus reducing gene expression and even contributing to the cause of autoimmune and inflammatory diseases. Previously, we identified T cell-specific eQTLs of a gene known as laccase domain containing 1 (LACC1), which are GWAS hits for Crohn’s disease. Four of them are located in the LACC1 promoter region in the same haploblock. Direct association of disease-risk variants with lower LACC1 expression was confirmed by comparing pre-mRNA quantity of the different alleles in LACC1 heterozygous human CD4 T cells. We further validated the functional role of the eQTLs using CRISPR Cas9-mediated base-pair editing. LACC1 function has been analyzed primarily in macrophages and shown to affect their function, but the GWAS hits do not affect expression in macrophages. Moreover, in our in vitro studies, human CD4 T cells with LACC1 knockdown showed altered metabolic programming and proliferation, which may contribute to inflammatory diseases. Overall, our study provides an excellent model for analyzing and explaining how genetic variants contribute to diseases by influencing gene expression in specific cell types and changing cell functions.
Targeting the NLRP3 inflammasome in Rare hereditary blood disorder Fanconi Anemia
Authors: Beesetti Swarna , Sumpter Rhea , Green Douglas R
Published: January 2023

Fanconi anemia (FA) patients are at higher risk of developing bone marrow failure (BMF) or cancer upon exposure to potential threats like viral infections, bacterial infections, carcinogens, and mutagens. Deleterious mutations in FA genes cause multiple cellular defects which include impaired ability to repair DNA interstrand crosslink lesions in the nucleus and impaired removal of damaged mitochondria via mitophagy in the cytoplasm. Fancc-deficient mice exhibit enhanced inflammatory response and are hypersensitive to lipopolysaccharide-induced septic shock as a result of hematopoietic suppression. Considering the role of inflammation in pathogenesis of Fanconi anemia and bone marrow failure diseases in general, we asked whether inflammasome inhibition could be a promising therapeutic for the treatment of FA. Our studies demonstrate that MCC950, a small molecule inhibitor of the NLRP3 inflammasome, can protect Fancc null mice from LPS-induced hemopoietic suppression and septic shock as evidenced by increased survival rate of Fancc−/− mice. MCC950 partially rescues the thrombocytopenia in peripheral blood and partially restores the loss of megakaryocyte-erythroid progenitor (MEP) and granulocyte-macrophage progenitor (GMP) pools upon LPS challenge. Further, Treatment with MCC950 significantly improved the bone marrow colony-forming capacity of Fancc−/− bone marrow cells in vitro. Our studies demonstrate that MCC950, a small molecule inhibitor of the NLRP3 inflammasome, can protect Fancc null mice from LPS-induced hemopoietic suppression and septic shock and warrants further evaluation as a potential therapeutic strategy in FA.
Augmentation of lymphoid tissues in the human immune system mouse to advance host directed therapies for HIV
Authors: Holloway Alex , Saito Tais B , Naqvi Kubra F , Huante Matthew , Fan Xiuzhen , Lisinicchia Joshua , Gelman Benjamin B , Endsley Janice , Endsley Mark
Published: January 2023

The study of HIV infection and pathogenicity in physical reservoirs requires a biologically relevant model which resembles the human immune system and physiology. The human immune system (HIS) mouse is well established as a model of HIV, but defects in immune tissue reconstitution remain a challenge for examining pathology in tissues. Herein we show that exogenous injection of the human cytokine FLT-3L into the HSC cord blood HIS mouse model significantly expanded the total area of axial lymph nodes and the circulating percentage of human T cells, thus enabling us to visualize and quantify HIV infectivity in the secondary lymphoid tissues of the spleen and axial node. Further, we detected cell death and T cell depletion in tissues, consistent with HIV pathogenesis. Treatment with the caspase-1 inhibitor VX-765 significantly decreased viral load as measured by gag qPCR, and apoptosis as measured by TUNEL assay, in the spleen. A significant restoration of CD4+ T cells in the spleen due to VX-765 treatment was observed via flow cytometry. In situ hybridization further demonstrated a significant decrease in viral RNA in both the spleen and axial lymph nodes. Transcriptomic analysis further revealed an upregulation in host HIV restriction factors such as APOBR, SAMHD1 and APOBEC3A as a result of VX-765 administration. These findings demonstrate FLT-3L as a mechanism whereby the human immune environment in HIS mice can be enhanced to support investigations of HIV pathogenesis and immune outcomes in tissue compartments. Preliminary results indicate that targeting inflammasome pathways with VX-765 in HIS mice treated with FLT-3L, and infected with HIV, preserved T cell populations and decreased viral load.

Supported by R01 A1HL129881, NIH/NHLBI
The effect of miRNAs targeting TGFβ-Signaling in Multiple Sclerosis
Authors: Rau Christina N , Severin Mary E , Lovett-Racke Amy E
Published: January 2023

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) that is mediated by a dysregulated immune system. It can lead to severe neurological issues and is one of the major causes for disability in young adults. The cause of the disease is unknown, the immune and neurodegenerative mechanisms underlying the pathophysiology of the disease are poorly understood. To this date there is no cure and current therapies are not able to completely slow down disease progression.

In a large miRNA profiling study on naïve and effector/memory CD4 T cell in untreated MS patients, our lab found a number of miRNAs differently expressed. One of the main pathways affected by this dysregulation of miRNAs is the TGFβ signaling pathway. This impairment limits the differentiation of regulatory T cells. To study the effect of TGFβ-targeting miRNAs in a murine model of MS, neonatal mice were injected with miRNAs that were found to be upregulated in MS patients at day 0 and 21 of life and experimental autoimmune encephalomyelitis (EAE) was induced using conditions under which the controls would only get a mild form of the disease.

Mice, injected with these miRNAs had an earlier onset of EAE and disease severity was significantly increased compared to control mice. Not only did an injection of TGFβ-targeting miRNAs caused mice to have reduced numbers of Tregs in the spleen and thymus compared to control mice, but these Tregs also showed a dramatic loss of TCR diversity in most Vβ genes as TCR Vβ deep sequencing of sorted Tregs revealed. Our findings contribute to the understanding of Treg dysfunction in MS patients and determining candidate markers for disease susceptibility and treatment efficacy.

Supported by grants from NIH (R01 AI152435-01)