bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigating the Distinct Tissue Repair and Immunosuppressive Roles of Regulatory T cells in HSV-2 Infection
Authors: Talavera Irene Cruz , Traxinger Brianna R , Arkatkar Tanvi , Lund Jennifer M
Published: August 2023

Herpes Simplex Virus 2 (HSV-2) is the primary cause of genital herpes, affecting 500 million people worldwide of which approximately 64% are women. Despite this disproportionately high burden, the female reproductive tract and its unique mucosal immune environment is surprisingly understudied. Tregs are necessary for CD4+ T cell priming in the draining lymph node (dLN), though their role in shaping tissue-resident memory T cell (Trm) responses during HSV-2 reactivation is unclear. Our preliminary data indicate that activated Tregs accumulate and persist in mouse vaginal tissue (VT) after above baseline out to at least 90 days post-infection (p.i.), thus implicating Tregs in memory recall responses to reinfection. We hypothesize that Tregs restrain robust Trm recall responses to HSV-2 challenge. Additionally, we hypothesize that vaginal Tregs promote tissue healing in the VT in response to inflammatory alarmins through the expression of the epidermal growth factor ligand, amphiregulin (Areg). Female FoxP3DTR mice were infected intravaginally with attenuated HSV-2, then systemically depleted of Tregs via intraperitoneal injection of diphtheria toxin and challenged with WT HSV-2 on day 30 p.i. Systemically Treg-depleted mice show increased activation and proliferation of T cells, and increased frequency of cytotoxic HSV-2-specific CD8+ T cells in the VT by day 3 post-challenge. Treg-depleted mice also have significantly higher pathology scores in H&E-stained VT. We also report that VT Tregs from infected mice express more Areg than dLN Tregs via flow cytometry and single cell RNAseq. Ongoing work includes characterization of Areg expressing VT Tregs and further dissection of the Trm recall responses in Treg-depleted mice.

NIH (T32 AI07140, R01 AI141435)
Plasticity of atypical B cell composition and function in response to malaria
Authors: Reyes Raphael , Batugedara Gayani , Dutta Paramita , Reers Ashley , Greenhouse Bryan , Bol Sebastiaan , Ay Ferhat , Bunnik Evelien
Published: August 2023

Atypical B cells are generated during a normal adaptive immune response, yet the role of these cells in protective immunity is still largely unknown. Here, we used chronic Plasmodium falciparum exposure as a model to gain insight into the function and potential heterogeneity of atypical B cells in the immune response to infection. To this end, we performed single-cell sequencing on circulating B cells from malaria-experienced individuals. Based on distinct gene expression signatures, we identified three novel atypical B cell subsets. Each subset had an effector profile that translated to one of previously proposed roles for atypical B cells: i) quiescent cells ii) antigen-presenting cells or iii) antibody-secreting cell precursors. We identified differentially expressed cell surface markers that allowed us to track each atypical B cell subset by flow cytometry. We observed that the relative abundance of each subset was influenced by the timing of sample collection in relation to infection. Additionally, our analysis revealed an enrichment of CXCR3+ atypical B cells in adults as compared to children, suggesting further heterogeneity in the context of age or chronic antigen stimulation. To explore the contribution of atypical B cells in response to malaria, we used antigen-tetramers to identify parasite-specific cells. All three atypical B cell subsets harbored antigen-specific B cells that decreased in frequency in the absence of exposure, confirming their active and transient role in malaria immunity. Finally, preliminary results suggest that the three subsets differ in their ability to secrete antibodies after stimulation, highlighting the cellular heterogeneity and distinct effector functions of atypical B cells.

National Institutes of Health predoctoral fellowship F31AI169993 (RR) National Institutes of Health grant R35GM128938 (FA) National Institutes of Health grant R01AI153425 (EB)
Strong adaptive immunity in humans with inherited CD4 deficiency
Authors: Guerin Antoine M , Ma Cindy
Published: August 2023

Since the early 1970s when the T cell receptor was first characterized, the role of human CD4 in adaptative immunity has been extensively studied, especially in the context of HIV infection and inborn error of immunity (e.g. HLA class II deficiency). Nonetheless, it was only recently (2019, 2021) that patients with inactivating germline mutations in CD4 has been described. Herein, we present five novel patients from three unrelated kindred with bi-allelic rare variants in CD4. Consistent with previous studies, patients showed a broad spectrum of clinical infectious susceptibility which was rather mild given their lack of detectable CD4+ T cells in peripheral blood mononuclear cells (PBMCs). Despite an almost unremarkable immunophenotype, patient’s PBMCs were characterized by the expansion of an enigmatic CD4−CD8 − (DN) TCRαβ+ T cells population which retained some functional characteristic of typical CD4+ T cells. We observed that the patient’s DN cells transcriptionally (Sc-RNAseq) and functionally (cytokine production/secretion) were comparable to that of healthy donor CD8− T cells. Interestingly, using cutting-edge long-read sc-RNAseq technology we revealed that patient’s DN cells expressed normal TCR repertoires, and paired TCRαβ sequences comparable to healthy donor CD4+ T cells. Furthermore, via biochemical studies we identified a CD4 alternative transcript expressed in patient’s PBMCs (also detected in healthy donor cells) leading to the surface expression of an unreported CD4 shorter isoform which is not detectable with conventional CD4 antibodies. To conclude, our study not only characterizes novel CD4-deficient patients, but also reveals alternative CD4 isoform expression and its compensatory role in immunity.

AAI Careers in Immunology Fellowship 2022 (RG222573)
MR1-restricted T cell clonotypes are associated with resistance to Mycobacterium tuberculosis infection
Authors: Cross Deborah L , Layton Erik D , Yu Krystle K Q , Smith Malisa T , Aguilar Melissa S , Li Shamin , Mayanja-Kizza Harriet , Stein Catherine M , Boom Willem Henry , Hawn Thomas R , Bradley Philip , Newell Evan , Seshadri Chetan
Published: August 2023

T-cells are a critical facet of the adaptive immune system and have been shown in both human and animal studies to be essential for control of Mycobacterium tuberculosis (Mtb) infection. T-cells recognize foreign antigens presented by infected cells via a unique T-cell receptor (TCR). Understanding the specific TCRs that mediate recognition and clearance of Mtb-infected cells is essential to inform vaccine design. We recently described a cohort of Ugandan household contacts of tuberculosis cases that appear to ‘resist’ Mtb infection (RSTRs) and showed that these individuals harbor IFN-γ independent T-cell responses to Mtb peptide antigens. The role of unconventional T-cells that recognize non-peptide antigens, such as gd T-cells, CD1-restricted and MR1-restricted T-cells (MR1T), in “resistance” is unknown. In this study, we aimed to characterize the frequencies, functional programs, and repertoire of unconventional T-cells in RSTRs in comparison with latently infected (LTBI) controls. We used multi-parameter flow cytometry, single cell RNA and TCR sequencing and immunosequencing to address these gaps. We observed a 1.65-fold increase in frequency of circulating MR1T cells among RSTRs in comparison with LTBI (p=0.03). Single-cell RNA-sequencing of 18,251 sorted MR1T cells revealed 5,150 clonotypes expressing a common transcriptional program, the majority of which were private. Immunosequencing of the TCR-α/d repertoire revealed several TCRα clonotypes that were expanded in RSTRs (p <0.01), including at least two MR1T clonotypes. Overall, our data reveal unexpected donor-specific diversity in the TCR repertoire of human MR1T cells as well as associations between MR1 clonotypes and ‘resistance’ to Mtb infection.

This work was supported by grants from the NIH (R01-AI124348 to Boom, Stein, and Hawn; R01-AI125189 and R01-AI146072 to Seshadri) and the Bill & Melinda Gates Foundation (OPP1151836 and OPP1109001 to Hawn and GH-VAP-IS-ID5 to Seshadri).
Single Cell Immune Atlas of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Identifies Substantial Transcriptional Dysregulation
Authors: Iu David S , Grimson Andrew
Published: August 2023

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a severe, debilitating disease with striking similarities to long-COVID/PASC, but its etiology remains unclear. Patients typically experience overwhleming fatigue, inflammatory symptoms and cognitive impairment, exacerbated by physical or mental exertion, also known as Post-Exertional Malaise (PEM). Mounting evidence points to immune system dysfunction being a key contributor to ME/CFS pathology, but our current understanding of gene regulation in ME/CFS leukocytes is hampered by a lack of quantitative and temporal resolution.

To comprehensively characterize the gene regulatory state and precisely define the cell types underlying transcriptional dysregulation, we have generated single cell transcriptomes and chromatin profiles of peripheral blood mononuclear cells (PBMCs) using single cell RNA-seq and single cell ATAC-seq in matched patients and controls, at baseline and after exercise challenge. At baseline, ME/CFS patients displayed dysregulation of classical monocytes pointing to increased migration to tissues as well as inappropriate chemokine signaling, as well as dysregulation of certain subsets of effector CD8+ T cells, leading to a more inflammatory transcriptional program. Taken together, these data identify substantial immunological defects and serves as an important resource for understanding ME/CFS pathology.
The histone methyltransferase Whsc1 regulates TGFβ-driven macrophage to myofibroblast transition
Authors: Mangum Kevin D , Joshi Amrita D , Melvin William J , Mazumder Purba , Erturk Asuman , Wolf Sonya , Barrett Emily C , Moon Jadie Yoonjoo , Obi Andrea , Kunkel Steven L , Moore Bethany B , Gallagher Katherine
Published: August 2023

In normal wound healing, macrophages regulate early and late inflammation, allowing for resolution of tissue injury and subsequent healing. During normal tissue repair, macrophages undergo transition to myofibroblasts, which promote wound healing. While this critical transition is impaired in diabetic wounds, the underlying mechanisms responsible are unknown. Our group and others have identified that epigenetic regulation of macrophages controls their phenotype in wounds. Since TGFβ drives macrophage to myofibroblast transition, we performed an epigenetic superarray to identify key chromatin modifying enzymes in macrophages (CD11b+CD3− CD19-NK1.1-Ly6G-) from normal diet (ND) and diabetic-induced obese (DIO) murine wounds treated ex vivo with TGFβ. The histone methyltransferase Whsc1, which mediates di- and tri-methylation at H3K36, was significantly increased in DIO macrophages with TGFβ treatment compared to ND macrophages (p<0.05). siRNA knockdown of Whsc1 in BMDMs decreased TGFβ-dependent expression of fibrotic (Acta2, Col1a1, Col3a1) and inflammatory genes (IL-1b, IL-6, and IL-12) (p<0.05). In ChIP experiments, Whsc1 was enriched at Col1a1 and Col3a1 promoters in ND but not DIO BMDMs, and TGFβ inhibited Whsc1 binding as well as H3K36me2 and H3K36me3 at fibrotic gene promoters. Additionally, H3K36me2 and H3K36me3 were decreased in Whsc1-knockdown BMDMs at Col1a1 and Col3a1, validating Whsc1 as an H3K36 methyltransferase that regulates myofibroblast gene expression. These results suggest that TGFβ regulates macrophage to myofibroblast conversion via Whsc1, which we identify as a critical epigenetic switch in tissue repair and potentially an important therapeutic target in diabetic wounds.

F32 DK131799-01, 2022 American College of Surgeons Resident Research Scholarship, 2022 VESS/Medtronic Resident Research Award
New insight into DC deficiencies in Chronic Lymphocytic Leukemia (CLL)
Authors: Welch Baustin M
Published: August 2023

Dendritic cells (DCs) function as principal sensors of PAMPs and DAMPs, connecting the innate and adaptive arms of the immune system. DC subset deficiencies have been observed in untreated CLL patients compared to age-matched healthy controls. Studies investigating the specific DC subsets that differ in function and localization; conventional DCs (cDCs), plasmacytoid DCs (pDCs), and Axl+ Siglec-6+ (AS) DCs are lacking in CLL. In mice, DC development and maintenance is highly dependent on the Flt3/Flt3L signaling axis. Activated T cells are believed to be the major source of Flt3L to DCs. Previous studies have reported elevated Flt3L in the serum of untreated CLL patients. Interestingly, increased serum levels of Flt3L have been associated with DC deficiencies, as DCs are believed to be the principle immune cell requiring this cytokine. This study will utilize extensive flow cytometry panels to investigate the prevalence and functional status of DCs in untreated CLL patient blood, including cDC1, cDC2, pDCs, and AS DCs. In addition, we will interrogate the ability of CLL-derived exhausted T cells to provide Flt3L upon activation.
A High Parameter Flow Cytometric Assay to Identify Human Myeloid Derived Suppressive Cells
Authors: Wang Xiaoyang , Anderson Keon , Chen Jingyi , Bonilla Diana , Deng Jun , Edinger Mark
Published: August 2023

The concept of myeloid derived suppressive cells (MDSCs) has emerged in recent years as a group of myeloid cells that pocess potent immunosuppressive activity. These cells expand under pathologic conditions and suppress the immune functions of T cells, dendritic cells, macrophages, and natural killer cells. They have been shown to be involved in the pathological states of cancer, autoimmune diseases, and infections, such as COVID-19.

MDSCs are subset into PMN (polymorphonuclear)- and M (monocytic)-MDSCs. Phenotypic markers of MDSCs have been elucidated in recent years. Here Cytek® developed a 14-color, 16-marker, single-tube flow cytometric assay to analyzed MDSCs. The panel includes markers to exclude lymphoid lineage cells, and markers to identify PMN-MDSC and M-MDSC populations, such as CD15, CD14, CD16, CD66b, CD11b and HLA-DR. In addition, PMN- and M-MDSC specific expression markers such as Lox-1, CXCR-1, DR5 are also included. Fresh whole blood samples were stained with erythrocyte lyse wash method and the samples were analyzed on a Cytek® Aurora or 3-laser Northern LightTM instrument. The panel performance was evaluated on blood samples collected in EDTA, Heparin, ACD, and Cytochex blood tubes as well as peripheral blood mononuclear cell (PBMC) samples. Assay precision was assessed by testing on 12 donor samples in triplicates on three consecutive days. Targeted cell populations are well identified in peripheral blood and PBMC samples with good resolution and assay precision with %CV less than 25% for all cell populations of interests.

This 14-color 16-marker preoptimized flow cytometric assay can be used in clinical research and drug discovery in cancer, autoimmune diseases, infections and other inflammatory diseases.
Pituitary immune cells regulate gonadotrope hormone secretion
Authors: Del Mundo Zena Marie , Nguyen Thu , Ujagar Naveena , Nicholas Dequina
Published: August 2023

Polycystic ovary syndrome (PCOS) is one of the most common causes of female infertility in the US and is associated with a combination of reproductive neuroendocrine physiological changes. Previous studies have shown that PCOS patients have elevated serum C reactive peptide and lipopolysaccharide (LPS), which indicates chronic low-grade inflammation. However, it is unknown how inflammation or immune cells impact reproductive outcomes. In this project, I study how inflammation affects gonadotrope regulation of sex hormone secretion. I show that in vitro depletion of CD45+ cells from dispersed primary mouse pituitary increases the luteinizing hormone (LH) to follicle-stimulating hormone (FSH) ratio. In contrast, immune activation through chronic LPS exposure in mice results in decreased serum LH to FSH ratio. Using single-cell RNA sequencing data from CD45+ enriched cells from mouse pituitaries, I identified specific resident immune cells. These cell populations include macrophages with higher RNA expression of heat shock proteins in male mice compared to female mice suggesting inflammatory protection in males. Ongoing experiments include chronic LPS exposure in mice, where pituitary cells are immunophenotyped, sex hormone serum levels measured, and ovulation monitored. Ultimately this work will allow us to understand the contribution of inflammation to PCOS, with the hope of designing immunotherapy for PCOS.

NICHD R00 HD098330-03
An immunosuppressive tumor population drives immunotherapy resistance of heterogeneous tumors
Authors: Tanaka Miho , Lum Lotus , Ledezma-Soto Cecilia , Hu Kenneth , Superville Daphne , Adams Zoe , Kersten Kelly , Krummel Matthew F , Reeves Melissa Q
Published: August 2023

Intratumoral heterogeneity (ITH) – cellular and molecular diversity within a tumor – is linked to failure of immunotherapy in multiple cancer types. A high degree of ITH is associated with poor infiltration of T cells into the tumor and resistance to immune checkpoint blockade (ICB) therapy. To determine how distinct tumor populations within heterogeneous tumors shape the immune microenvironment and how this impacts therapy response, we modeled heterogeneous tumors composed of an RFP-tagged immunosuppressive tumor population and a YFP-tagged pro-inflammatory tumor population. The resulting tumors contained a patchwork of distinct regions with YFP+ cells, RFP+ cells, or a mixture of YFP+ and RFP+ cells. Analysis of the immune infiltrates in each region revealed a higher frequency of total CD4 T cells, Th1 cells and IFNg+ CD8 T cells in YFP regions compared to RFP regions, whereas macrophages exhibited the opposite pattern. PD-1 blockade and CD40 agonist combinatorial antibody therapy induced an increase in Th1 abundance in RFP regions, but the treatment did not clear the tumors. Together, these results reveal that distinct regional immune infiltration pattens within the tumor are driven by the local tumor cells present in each region, and that the treatment-induced global improvement of Th1 infiltration alone is not sufficient to induce tumor clearance. Moreover, we identified Cx3cl1 as a driver of a dominant immunosuppressive tumor microenvironment, marked by an increase in immunosuppressive CD206+ macrophages and decrease in anti-tumoral neutrophils and monocytes. These results suggest that an immunosuppressive tumor population drives immunotherapy resistance of heterogeneous tumors and Cx3cl1 as a new therapeutic target.

Supported by grants from Parker Institute for Cancer Immunotherapy (Project Grant)
Enzymatic depletion of adenosine to overcome resistance to immune checkpoint inhibitors in non-small cell lung cancer
Authors: Sarkar Omar Sadi , Donninger Howard , Chew Lewis C , Eaton John , Yaddanapudi Kavitha
Published: August 2023

Despite the remarkable outcome of immune checkpoint inhibitors (ICIs) in non-small cell lung cancer (NSCLC), a large number of patients remain unresponsive to this therapy. A key factor of the unresponsiveness to ICIs is intrinsic therapeutic resistance where monocytic myeloid-derived suppressor cells (M-MDSCs) contribute significantly via suppressing CD8+ T cell mediated anti-tumor response. Our results showed that in tumor microenvironment (TME), tumor cells derived PGE2; a prostaglandin that directly induces CD73 (an ecto-nucleotidase that converts AMP to immunosuppressive adenosine) expression in M-MDSCs; consequently, CD73 expressing M-MDSCs suppress T cells via adenosine thus confer resistance to ICIs. In addition, we have shown that the depletion of adenosine via PEGylated Adenosine Deaminase (PEG-ADA) can reinvigorate CD8+ T cells, ultimately enhance the anti-tumor immunity.
Targeting the intrinsic signaling of PD-L1 in human monocytes by a novel PD-L1 antibody to promote anti-tumor immunity
Authors: Hsu Michelle A , Liu Xin , Barham Whitney , Hirdler Jacob , Mao Zhiming , Lucien Fabrice , Dong Haidong
Published: August 2023

Although immune checkpoint blockade that targets the PD-L1 has revolutionized the treatment of human cancers, only a small proportion of patients positively respond to this form of treatment. A limitation of these therapies is that they only target and inhibit PD-L1’s extracellular function, which is to bind its receptor on PD-1 on T cells and inhibit T cell cytotoxicity. Recent discoveries have shown that PD-L1 expressed on tumor cells can signal intrinsically and independently of PD-1 ligation to promote tumor cell survival. Besides tumor cells, host myeloid cells have been shown to have high expression of PD-L1, therefore contributing to the immunosuppressive nature of the tumor. However, PD-L1 intrinsic signaling in human myeloid cells has not been studied. Therefore, the purpose of our work is to determine the intrinsic functions of PD-L1 in myeloid cells and how this impacts anti-tumor immunity. To study this, we used a novel PD-L1 antibody (H1A) that does not block PD-L1’s binding with PD-1, but directs PD-L1 for degradation, thereby decreasing total PD-L1 expression. Additionally, we developed a chimeric mouse model in which the murine PD-1/PD-L1 has been replaced with the human version. This allows us to evaluate PD-L1’s intrinsic function in vivo. In our preliminary studies, the degradation of PD-L1 in human monocytes led to increased chemokine release as well as increased conversion of effector T cells. Additionally, H1A treatment of tumor-bearing mice led to lower tumor burden in a CD8 T cell dependent manner. Based on these results, PD-L1 intrinsic signaling has the potential to regulate the immune-stimulatory response of monocytes, thereby limiting effector T cell responses against cancer.

Supported by grants from NIH (R0192389) and Mayo Clinic graduate school of biomedical sciences
Assessment of the effectiveness of team-based learning activities on learning outcomes in the undergraduate immunology classroom
Authors: Bruns Heather A , Baty Joshua
Published: August 2023

Immunology is inherently interdisciplinary. Understanding how the immune system functions requires knowledge from several scientific disciplines including molecular biology, cellular biology, genetics, and biochemistry. Furthermore, immunology is conceptually complex, requiring the identification of a plethora of immune components and mastery of a large volume of new vocabulary. These attributes can pose challenges to student learning in the undergraduate immunology classroom. Team-based learning (TBL) is a pedagogical method used to increase student engagement in learning, improve student collaboration, and develop communication skills. In a variety of educational settings, TBL activities have been shown to foster a deeper understanding of complex topics, increase student confidence in course content, and improve learning outcomes. In this study, we examined differences in the impact of traditional lecture versus TBL activities on student learning outcomes for four different topics presented in an undergraduate adaptive immunity course. We matched content, across two student cohorts, delivered via team-based learning methodology (T cell development and antibody-mediated function) and traditional lecture (B cell development and T cell effector functions). Student learning was assessed using content questions across a range of Bloom’s taxonomy levels, which demonstrated that the TBL activities increased learning. Additionally, students indicated that the TBL activities assisted with preparation for exams and provided a necessary opportunity to address misconceptions. Taken together, TBL intervention increased student performance on basic content, and students found this learning tool to be valuable.

departmental support only
Immune system perturbations in patients with severe long COVID
Authors: Rodriguez Lucie , Tan Ziyang , Lakshmikanth Tadepally , Wang Jun , Barcenilla Hugo , Gonzalez Laura , Mugabo Constantin , Johnsson Annette , Chen Yang , Mikes Jaromir , Landegren Nils , Runold Michael , Bruchfeld Judith , Brodin Petter
Published: August 2023

SARS-CoV-2 infections have different outcomes ranging from mild to severe acute infection as well as post-infectious multi-system inflammatory syndromes (MIS-C/A). Post-acute sequelae of COVID-19 (PASC) or long COVID is another outcome, which is poorly understood and range from mild to very severe persistent organ dysfunction and damages. To maximize our chances of identifying underlying mechanisms of long COVID we focus on the most severe cases with objective measures of disease such as lung damages, microvascular and autonomic nerve dysfunction. We are testing the following hypotheses that I) long COVID is a super-antigen mediated disease, II) an autoimmune condition driven by pathogenic autoantibodies, and/or III) a disease caused by viral persistence. A cohort of over 100 long COVID patients, 24 recovered long COVID patients as well as 9 COVID convalescent controls have been recruited thus far and blood samples analyzed for plasma proteome (Olink assays), immune cell composition and phenotypes (mass cytometry), T-cell repertoire (scTCR-seq), innate cell activation (circulating nucleosomes), and auto-antibody analyses (Luminex arrays) in order to understand immune perturbations in severe long COVID patients. Unlike children with hyperinflammatory MIS-C, we have not found any evidence of superantigen-mediated T-cell activation in long COVID, but instead substantial clonal expansion of memory T cell clones with effector memory states and shared clonality across patients. Additionally, cytokine perturbations such as elevated levels of IL8 and elevated levels of circulating nucleosomes indicate persistent activation of neutrophils in severe long COVID.
Non-fatal outcomes of COVID-19 disease in children with solid organ transplants associates with down-regulation of senescent pathways
Authors: Subramanian Kumar , Tesfai Ivy , Muralidaran Vinona , Seeley Christina , Corbert Lauren , Yazigi Nada , Khan Khalid , Kaufman Stuart , Vitola Bernadette , Matsumoto Cal , Kroemer Alexander , Fishbein Thomas , Ekong Udeme
Published: August 2023

Objective

To study the immune landscape during acute infection (AI) & COVID-19 recovery in children with solid organ transplants (SOTs).

Method

Blood collected between Nov 2020 – June 2022 from pediatric SOT recipients 1–14-days after a positive PCR for SARS-CoV-2 (AI), & 15–60-days after a positive PCR (convalescence), for Ficoll of PBMC. Cryopreserved PBMC taken from pediatric SOT recipients before 2019 used as non-infected controls (ctrls). PBMCs stimulated with 15-mer peptides from spike protein & co-stimulation with anti-CD49d & anti-CD28. Immunophenotyping by mass cytometry. Data analyzed using the Astrolabe Cytometry Platform, which follows a standardized pipeline for population identification & statistics & visualized using the t-SNE dimensionality reduction method. Disease severity stratified using WHO criteria. Subjects unvaccinated against COVID-19.

Results

38 children included: median age & time from transplant (TX) at infection: 5.5 (2.1–15.1) & 3.3 (0.2–14.7)-years for AI (n=10); 2.4 (0.6–15.6) & 0.5 (0.2–11.2)-years for ctrls (n=20); 6.7 (1.0–12.9) & 4.3 (0.2–9.7)-years for convalescence (n=8). NK & CD4+T cell (EM, Th2) frequency increased during AI {p<0.05}. CD57 expression low in T cells with EM phenotype, NKT, & gdT cells in AI vs. ctrls (p<0.05) & in NK cells in severe disease (p<0.05). CD161 expression high in CD4+ T cell (CM, Th1) in convalescence vs. ctrls {p=0.04}.

Conclusion

contraction in NK cells is not a feature of COVID-19 disease in pediatric SOT & global characteristics of NK, NKT & T cell compartments not compatible with senescence. Upregulation of cytolytic effectors in AI. Above a plausible mechanism for low mortality from COVID-19 disease in this patient population.
Structure of pathogenic anti-PF4 autoantibodies reveals the mechanism of immune response formation in vaccine-induced thrombotic thrombocytopenia (VITT)
Authors: Ivanov Daniil G , Ngyuen Son N , Le Si-Hung , Du Yi , Ivetic Nikola , Nazy Ishac , Kaltashov Igor A
Published: August 2023

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe, but rare, side effect of adenoviral-vectored COVID-19 vaccines. It is attributed to formation of autoantibodies recognizing Platelet Factor 4 (PF4) and capable of triggering platelet activation, which leads to thrombosis. We used mass spectrometry (MS) to determine the structure of a VITT patient-derived anti-PF4 antibodies (VITT IgG) and identified their unique properties underlying pathogenicity.

The VITT IgGs studied in this work were extracted from the plasma of a ChAdOx1 vaccine recipient, who developed VITT. Intact-mass MS analysis of these antibodies revealed one major clone, and its complete structural characterization was carried out using MS-based de novo sequencing. This allowed us to establish its subclass (IgG2), determine the amino acid sequence, and identify an N-glycan in the variable region.

This information was used to build a 3D model of the VITT IgG, which revealed the presence of a large polyanionic patch within the paratope involving four CDR regions. Molecular dynamics simulations highlight the role of electrostatics as the major driver of the VITT IgG binding to PF4. The large equatorial belt of the positive charge circumscribing the PF4 tetramer serves as a distributed epitope, allowing it to cross-link up to three VITT IgG molecules, giving rise to large immune complexes capable of FcyRIIa-mediated platelet activation. The molecular mechanism of VITT pathogenesis emerging from this work explains how VITT IgGs and PF4 form large immune complexes in the absence of heparin, which is essential to for platelet activation in a similar disease, heparin-induced thrombocytopenia, and sheds light on the etiology of this devastating condition.

Supported by grant from NIH R01 GM112666
Multi-omic profiling in coeliac disease reveals somatic driver mutations in rogue T cell clones
Authors: Singh Manu , Louie Raymond , Milthopre Claire , Adikari Thiruni , Hardy Melinda , Faulks Megan , Field Matt , Ahlenstiel Golo , Tye-Din Jason , Luciani Fabio , Goodnow Chris C
Published: August 2023

It has long been proposed that the pathogenesis of human autoimmune diseases may share common origins with lymphoid cancer. However, a major hurdle in understanding the immune pathogenesis of autoimmune diseases is distinguishing self-reactive “rogue” lymphocytes from normal immune cells. In Coeliac disease, both the environmental trigger (gluten) and major autoantigen (transglutaminase 2) are well characterised, but the underlying mechanisms by which rogue lymphocytes initiate and drive disease are unknown.

Here, we apply multi-omic technologies that enable detailed DNA, RNA and protein measurements at the single-cell level to profile tens of thousands of immune cells isolated from small intestine duodenum biopsies from individuals with Coeliac disease. We utilise the T-cell receptor as a natural barcode across multiple single-cell experiments to identify expanded rogue T cell clones and their gene and cell-surface protein expression profiles, along with any somatic DNA driver mutations. Strikingly, we identify in multiple patients expanded T cell clones with mRNA and cell-surface protein expression profiles of cytotoxic effector cells, that harboured missense somatic mutations in T cell lymphoma driver genes STAT3, STAT5B and DDX3X. These mutations have been described as strong gain-of-function mutations in multiple T cell lymphomas, highlighting a novel mechanism by which T cell clones escape immune tolerance and adopt a rogue phenotype in Coeliac disease.

Overall, our results highlight the power of single-cell multi-omics in identifying and characterising rare pathogenic clones in a common human autoimmune disease and provide direct evidence for a shared pathogenesis of autoimmunity and lymphoid malignancy.
In silico and multi-dimensional functional interrogation of tissue Tregs reveals novel therapeutics for autoimmunity and inflammation
Authors: Valle Jose , Guillen Jovanny , DiMichele Sarah , Pauli Mariela , Tan Corey , Lam Caroline , Dominguez Claudia X , Labastida Rosario , Nganje Charmaine , Peabody Phillip , Yan Hannah , Navas Ashley , Nishimura Koki , Yard Parker , Abraham Ryan , Meury Marcel , Aglietti Robin , Bowers Peter , McKay Austin , Taylor Ian , Eng Nicolas W L , Mitchell Keith , Chen James , Yohn Christopher B , Astarita Jillian , Lyons Jesse , Kleinschek Melanie A , Zarrin Ali A
Published: August 2023

Many inflammatory and autoimmune diseases present with specific organ manifestations. Understanding the immune process at the site of inflammation may offer important insights into novel therapeutic strategies. To this end, we established a biology platform that allows us to develop a deep understanding of human tissue Treg behavior to enable a next generation of therapeutics aimed at activating these regulatory circuits at the location where the disease manifests. We apply single-cell RNA sequencing and computational tools to our proprietary collection of healthy and diseased tissues to generate a high-resolution map of immune-regulatory pathways. Based on these learnings, we established in vitro culturing conditions that induce the expression of these tissue pathways in primary human Treg. Lastly, we have developed a suite of disease relevant phenotypic assays that allows for multi-dimensional functional interrogation of the mapped tissue Treg pathways in our in vitro-conditioned human primary Treg. This combination of complex in silico tools and translational in vitro assays allows us to identify novel regulatory nodes and forms the foundation of our growing pipeline of a new class of tissue Treg-focused therapeutics for immune-mediated diseases.
Galectin-9 mediates gut-associated inflammation and disruption of tight junctions
Authors: Moar Preeti , Kaur Urvinder , Bhagchandani Tannu , Tandon Ravi , Ndhlovu Lishomwa C
Published: August 2023

Background

High levels of Galectin-9 (Gal-9), a β-galactoside-binding mammalian lectin, are associated with systemic inflammation in chronic viral infections like HIV, despite viral suppression by ART. Gut-systemic microbial translocation is a major contributor of persistent inflammation. Whether Gal-9 influences the integrity of the gut during viral infections remains unknown.

Objective

Here we investigated the impact of Gal-9 in the interplay between gut-systemic microbial translocation in the setting of well-treated chronic HIV infection.

Method

We assessed the levels of Gal-9 and sCD14 in the plasma of people with HIV on ART (PWH; n=25) and people without HIV (PWOH; n=15) by ELISA. The integrity of the gut epithelium in the presence and absence of rGal-9 was assessed in the HT-29 cell line by immunofluorescence. TNFa was used as a positive control to disrupt gut integrity. The levels of TNFa were measured by qPCR. Non parametric tests were used in the analysis.

Results

Plasma Gal-9 and sCD14 were elevated in PWH compared to PWOH (all p<0.0001). Further, high Gal-9 levels correlated with high sCD14 in both the PWH (r=0.43, p=0.0317) and PWOH (r=0.79, p=0.0006) groups. Immunofluorescence revealed disruption of the Claudin tight junctions in vitro after 24 hrs of rGal-9 treatment. rGal-9 also induced a 6-fold increase in the expression of TNFα in HT-29 cells.

Conclusion

Our results show that Gal-9 is associated with markers of gut-systemic microbial translocation irrespective of HIV status and given the direct effects of rGal-9 on tight junction barrier function, targeting Gal-9 either by exploiting its secretory pathways or competitive blockade may serve as a novel therapeutic approach to control gut-associated inflammation.
Improved control of SARS-CoV-2 infection with nucleocapsid-specific antibodies
Authors: Penaloza-MacMaster Pablo A , Dangi Tanushree , Sanchez Sarah , Class Jacob , Richner Michelle , Visvabharathy Lavanya , Chung Young Rock , Bentley Kirsten , Stanton Richard , Koralnik Igor Jerome , Richner Justin , Penaloza-MacMaster Pablo
Published: August 2023

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein is the main antigen in all approved COVID-19 vaccines and is also the only target for monoclonal antibody (mAb) therapies. Immune responses to other viral antigens are generated after SARS-CoV-2 infection, but their contribution to the antiviral response remains unclear. Here, we interrogated whether nucleocapsid-specific antibodies can improve protection against SARS-CoV-2. We first immunized mice with a nucleocapsid-based vaccine and then transferred sera from these mice into naive mice, followed by challenge with SARS-CoV-2. We show that mice that received nucleocapsid-specific sera or a nucleocapsid-specific mAb exhibited enhanced control of SARS-CoV-2. Nucleocapsid-specific antibodies elicited NK-mediated, antibody-dependent cellular cytotoxicity (ADCC) against infected cells. To our knowledge, these findings provide the first demonstration in the coronavirus literature that antibody responses specific to the nucleocapsid protein can improve viral clearance, providing a rationale for the clinical evaluation of nucleocapsid-based mAb therapies to treat COVID-19.

NIH (DP2DA051912)
Prostaglandin D2 receptor expression in the lung and the effect of PPAR-γ, DP1, and DP2 receptor agonist treatment during respiratory viral infection
Authors: Abysalamah Hazzar marwan , Holloway Kathleen , Shornick Laurie P
Published: August 2023

Prostaglandin D2 (PGD2) may play an anti-inflammatory role in the lung during respiratory viral infection, but little is known about the expression of its receptors (PPAR-γ, DP1, and DP2) in the lung or its mechanism of action. Adult C57BL/6 mice were infected with Sendai virus (500 pfu /g body weight), a natural mouse pathogen that infects airway epithelial cells. Immunostaining of uninfected lungs showed PPAR-γ protein in epithelial cells of larger airways, but not in the alveolar epithelial cells. After viral infection, PPAR-γ protein was no longer detectable in the lungs. Immunofluorescence showed DP1 present in parenchymal cells as well as in some CCSP+ airway epithelial cells, but levels did not appear to change during infection. DP2 expression increased after viral infection and was predominantly in CCSP+ epithelial cells 24 hours post-infection. DP2 also co-localized with MHC Class II+ cells in the parenchyma on days 3 and 7 post-infection. Viral infection of primary tracheal epithelial cells showed PPAR-γ mRNA expression was increased 48 and 72 hours post-infection. There was no change in DP1 or DP2 expression in vitro. To test the effect of each receptor in vivo, mice were infected and treated with PPAR-γ, DP1, and DP2 agonists, or vehicle every 12 hours. Mice treated with the PPAR-γ agonist were protected from body weight loss and had reduced IFN-γ expression on day 7, suggesting that PGD2 may be acting via PPAR-γ to reduce inflammation in the lung.

Supported by grants from Sigma Xi Grant in Aid of Research and Saint Louis University College of Arts and Sciences Brennan Award.
Characterizing effects of HB-EGF on H1N1 infection in mice
Authors: Acosta-Vilanova Heizel Karina , Carter Hannah , Moore Bethany
Published: August 2023

Heparin-Binding EGF-like Growth Factor (HB-EGF) is a ligand for the Epidermal Growth Factor Receptor (EGFR), a tyrosine kinase receptor which promotes cell proliferation and migration. In the epithelium of the lung, HB-EGF is an important factor for wound repair, however little is known about the effects of HB-EGF on viral infection in vivo. Literature suggests that in human alveolar epithelial cell lines, EGFR is an important cofactor in Influenza A infection, and that treatment with HB-EGF decreases both EGFR and viral M1 protein in cells. We use a mouse model to examine this in vivo. In our model, we use a HBEGFff/Lyz2Cre mouse to knock out HB-EGF in myeloid and epithelial cells and infect with a mouse adapted H1N1 influenza virus. We then harvest lungs on day 5 and analyze RNA transcripts by qPCR. We expected to see increased H1N1 polymerase transcript in our KO mice. We saw that increase in our male mice in our initial experiment, but not in females. We plan to repeat this experiment and examine changes in the immunological milieu between our HB-EGFff mice and our controls, and between males and females.

Supported by grant from NIH Postbaccalaureate Research Education Program (PREP) (R25)
Anti-PPIL-5 antibody exacerbates disease of severe influenza in a murine experimental model
Authors: Huang Ching-Tai , Hung Chen-Yiu , Hsiao Sung-Han , Chen Tse-Ching , Chang Chia-Shiang , Huang Yu-Lin , Lin Yung-Chang , Lin Chun-Yen , Dutta Avijit
Published: August 2023

Respiratory viruses such as SARS-CoV2 cause serious emerging infectious diseases. Even seasonal influenza results in significant morbidity and mortality. Host immunity responds to influenza virus infection to eradicate the pathogen. However, dysregulated responses such as the cytokine storm contribute to severe disease. Mechanistic dissection of immune regulation may offer practical solutions for severe influenza. In our mouse model of severe influenza, a LAG-3 regulatory response evolves with the pro-inflammatory IFN-γ response in influenza virus hemagglutinin (HA)-specific CD4+ T cells. Adoptive transfer of LAG-3(+) IFN-γ(-) HA-specific CD4+ T cells suppressed lung inflammation without compromised virus eradication. Genomic study of LAG-3(+) IFN-γ(−) CD4+ T cells revealed up-regulation of the PPIL-5 [Peptidylprolyl isomerase (Cyclophilin)-like 5] gene. PPIL-5(+) HA-specific CD4+ T cells accumulated in the lung with time after infection. PPIL-5 protein suppressed HA-specific CD4+ T cell activation in vitro in a dose dependent manner. PPIL-5 blockade in vitro with siRNA reversed immune suppression and unleashed activation of HA-specific CD4+ T cells. PPIL-5 blockade in vivo with monoclonal anti-PPIL-5 antibodies attenuated LAG-3 and PD-1 and augmented IFN-γ responses of the lung-infiltrating HA-specific CD4+ T cells, with aggravated disease and intensified mortality of severe influenza. The HA-specific CD4+ T cells took less glucose and produced lactate with anti-PPIL-5 blockade. They adopted anaerobic glycolysis in this reverted inflammatory response. Our results suggest that PPIL-5, similar to LAG-3, is also an immune checkpoint and a possible target to be manipulated for alleviation of severe influenza.

The Research Center for Emerging Viral Infections receives support from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE), Taiwan and NSTC 111-2634-F-182-001 from the National Science and Technology Council, Taiwan. This work was supported by Projects MOST 111-2314-B-182-029 (AD) and MOST-105-2321-B-182A-003-MY3 (AD) from the National Science and Technology Council, Taiwan, and by CMRPG3L1671 (CTH), CMRPVVJ0052 (CTH), and CMRPG3J1771 (CYH) from the Medical Research Project Fund, Chang Gung Memorial Hospital, Taiwan.
Hospitalized COVID-19 patients exhibit increased numbers of low density neutrophils expressing the transcription factor ARID3a
Authors: Webb Carol F , Hocker James A , Shankar Malini A , Booth John Leland , Sinko Lauren A , Wu Wenxin , Metcalf Jordan
Published: August 2023

We previously showed that individuals with systemic lupus erythematosus (SLE) and high levels of Type I inflammatory cytokines had increased numbers of low density neutrophils (LDNs) in their peripheral blood compared to healthy donors. Increased numbers of LDNs were associated with increased expression of the transcription factor ARID3a and increased interferon alpha (IFNα) production. Data from other cell types suggest that ARID3a expression occurs upstream of IFNα expression. Therefore, we sought to determine if increases in ARID3a-expressing LDNs might also occur in other severe inflammatory responses, such as infection with SARS-CoV2 where IFNα expression was known to be increased. Subjects admitted to the hospital with a confirmed diagnosis of SARS-CoV2 infection were enrolled in our IRB approved study and peripheral blood mononuclear cells from 24 patients were evaluated via flow cytometry for numbers of LDNs and ARID3a expression at the time of admission and at various time points after hospitalization. Numbers of circulating LDNs were dramatically increased in COVID patient samples at the time of hospitalization, as were percentages of those cells expressing ARID3a protein. IL-6 expression was also high in the LDNs, while IFNα expression was more variable. Over the course of two weeks, IFNα expression appeared to be associated with, or to lag just behind ARID3a expression in most patients, while IL-6 expression levels were not associated with ARID3a or IFNα expression. These data suggest that ARID3a expression is induced in LDNs in association with severe viral infection and may implicate ARID3a as a potential regulator of inflammatory pathways in infection as well as autoimmunity.

Supported in part by grant NIH U19 AI06269
Characterization of CD8+ T cells recognizing a dominant epitope of SARS-CoV-2 spike protein in COVID-19 convalescent, vaccinated, and unexposed individuals
Authors: Zou Ainslee J , Li Jiangyuan , Choy Cecily , Chen Joseph , Hemani Humza , Baptiste Beverly , McKelvey Julia , Melvin Denise , Ruffolo Jeannie , Zukley Linda , Wallace Tonya , Dunn Christopher , Nguyen Cuong , Fan Jinshui , De Supriyo , Chia Chee , Ferrucci Luigi , Egan Josephine , Weng Nan-ping
Published: August 2023

COVID-19, an infectious disease caused by SARS-CoV-2, has given rise to the current global pandemic. Despite the number of infections reaching over 600 million people worldwide, CD8+ T cells against SARS-CoV-2 in COVID-19 convalescent, vaccinated, and unexposed adults have not been fully characterized. Here, we report the frequency, phenotype, and in vitro expansion capacity of circulating CD8+ T cells recognizing 10 epitopes from the SARS-CoV-2 spike (S) protein. Through multi-color flow cytometry using antigen-specific tetramers, we found that the frequencies of circulating CD8+ T cells ranged from undetectable to 1% in HLA-A2+ unexposed, convalescent, and vaccinated individuals. In vitro stimulation showed robust CD8+ T cell expansion (in percentage and magnitude) against the S-YLQ (S-269-277, YLQPRTFLL) epitope compared to other epitopes in convalescent and vaccinated individuals. Lastly, the scTCRseq analysis revealed limited V gene usage for S-YLQ-specific CD8+ T cells. These results identified a dominant S-protein epitope (S-YLQ) of SARS-CoV-2, showing that convalescent and vaccinated individuals have robust specific CD8+ T cell response against this epitope.