bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and function of a C3hiIL-33+ fibroblast population in the lungs
Authors: Atakkatan Aishwarya , Gour Naina , Magesh Aishwarya , Yong Hwan Mee , Lajoie Stephane
Published: August 2023

Complement component C3 is known to be predominantly expressed by epithelial and immune cells, however, we have found that fibroblasts are a primary source of C3 in murine and human lungs. Specifically, C3 is majorly expressed in adventitial fibroblasts. Moreover, we found that C3hi fibroblasts are characterized by the expression of IL-33. Further, C3 and IL-33 are not only markers of a novel subset of pulmonary adventitial fibroblasts but form a functional axis where C3 drives the expression of IL-33. As compared to other adventitial fibroblasts, this C3hiIL-33+ subset is enriched for matrix genes, especially type I collagen (COL1A1). These cells require a functional C3− IL-33 axis to maintain their matrix identity. Impairing C3 not only reduces their ability to make collagen but guides them towards a lipofibroblast program characterized by lower collagen content and higher lipid accumulation. As we and others have published that C3 is a key driver of allergic airway inflammation, we wanted to understand how exposure to allergen affected C3hiIL-33+ fibroblasts. Using a murine model of airway allergy, we observed a significant increase in the C3hiIL33+ fibroblast population in lung samples from house dust mite (HDM)-treated mice compared to the PBS control group. Recently, IL-33+ fibroblasts were found to support ILC2 recruitment during helminth infection. As we have found C3 is critical to maintain IL-33 in these cells, we aim to understand if C3hiIL-33+ fibroblasts participate in allergy by mediating cellular crosstalk with ILC2s and T cells during type 2 immune responses.

Supported by grants from NIH R01AI27644 and the Johns Hopkins Catalyst Award to Dr. Stephane Lajoie.
Manipulation of Leucocytic NADPH Oxidase Maturation May Drive Macrophage Differentiation and Affect Atherogenesis
Authors: Cheng Wan-Ting , Shieh Chi-Chang
Published: August 2023

Atherosclerosis is a process of arterial intima thickening caused by lipid deposition as a consequence of insufficient efferocytosis of apoptotic cells by M2 macrophages and excessive inflammatory cytokine secretion from M1 macrophages. NADPH oxidase 2 (NOX2) is recognized as a major source of ROS for enhancing macrophage efferocytosis and functional polarization. We previously found that human macrophages contain significant amount of endoplasmic reticulum (ER)-retained gp91phox, a major component of NOX2, which may hinder the formation of functional NOX2. To address the role of macrophage NOX2 in atherosclerosis, we established a mouse model bearing Apoe−/− and the CYBB point mutation (C1024T), which leads to ER-retained gp91phox in immune cells. We also developed a method for targeted delivery of a SERCA inhibitor with poly (lacticcoglycolic acid) (PLGA) nanoparticles (NPs), which may promote the glycosylation and maturation of ER-retained gp91phox. We first found that oxLDL-administrated M1 macrophages expressed increased M2 marker CD163 and increased efferocytotic ability after being treated with NPs. We also found that there was an increased glycosylation of gp91phox in those macrophages from human and mice with CYBB point mutation (C1024T) after the treatment of NPs. Macrophage ATP-binding cassette transporters, which could decrease cholesterol overloading in macrophages, were upregulated in macrophages after macrophages were treated with NPs. Our results demonstrated that NPs not only can recover the maturation of ER-retained gp91phox in macrophages but also may facilitate the differentiation of M1 to M2-like macrophages with enhanced efferocytotic activity and cholesterol efflux.
Microanatomical lipid niche controls intestinal memory B cell migration and effector function
Authors: Raso Fiona Ann , Howley Kelsey , Reboldi Andrea
Published: August 2023

Enteric infections are a leading global burden, yet few oral vaccines elicit long-lasting immunity and prevent disease, likely due to our lack of understanding of intestinal immunological memory. Long-lived memory B cells can be rapidly reactivated to differentiate into plasma cells, thus further increasing antibody titers and mediating host protection. Such long-term protection requires memory B cells to migrate and persist in sub-anatomical niches within secondary lymphoid organs. While survival niches have been described for memory B cells in lymph nodes and spleen, no niche have been identified in mucosal lymphoid organs, such as Peyer’s patches (PPs).

Here, we show that memory B cells require positioning in the PP subepithelial dome (SED) for maintenance and effector function. In PPs, memory B cells express the migratory receptors EBI2, which binds the oxysterol 7alpha, 25-hydroxycholesterol, and CCR6, which recognizes the chemokine CCL20. Both ligands are present in PPs and drive memory B cell migration in vitro. Conditional removal of EBI2 from memory B cells, either in a constitutive or acute fashion, alters their zonation in PPs, reduces their persistency, and impairs their antigen-specific response. While most memory B cells in PPs are IgA+, IgA B cell receptor expression is dispensable for memory B cell positioning in the SED.

Using conditional mice for the enzymes generating the oxysterol gradient and cell specific Cre lines, we are currently mapping the cellular and metabolic requirements for EBI2 dependent memory B cell maintenance and effector function in PPs.

Understanding the mechanisms for memory B cell persistence is fundamental to our understanding of the intestinal B cell memory response against pathogens.

Previously supported by training grants from NIH (5 T32 AI 132152-2, 5 T32 AI 007349-29)
Does MHC Heterozygosity Promote IgA Plasmablast Diversity in the Gut?
Authors: Peacock Tori , Kubinak Jason
Published: August 2023

The diversity of protein antigens the adaptive immune system can respond to is governed by polymorphic major histocompatibility complex (MHC) molecules. Because MHC alleles are co-dominantly expressed, MHC heterozygosity should allow for a wider diversity of antigens to activate T cells. B cells present antigen via MHC molecules to T cells, and through the ensuing T cell interaction, differentiate into plasma cells. Because of this, MHC heterozygosity should result in the generation of a plasma cell pool able to generate antibodies that target a wider array of antigens. To investigate this claim, we compared B cell selection dynamics between homozygote and heterozygote MHC congenic mice within the germinal centers (GCs) of Peyer’s patches; the major secondary lymphoid tissues of the gut where plasma cells develop in response to the microbiota. I have observed differences in the distribution of GC B cells between the dark zone (DZ) and light zone (LZ) of GCs. Specifically, GC B cells in MHC heterozygotes are found in greater abundance in the LZ and lower abundance in the DZ compared the GC B cells from MHC homozygote mice, which suggests that B cells are more capable of interacting with T cells in MHC heterozygotes. MHC heterozygotes also have significantly higher abundance of GC-TFH cells in their Peyer’s patches. I have observed that MHC heterozygote mice show enhanced binding of IgA to SI-resident bacteria. Future experiments seek to sort-purify IgA-bound SI-resident bacteria from MHC heterozygotes and homozygotes for 16S analysis to determine if MHC heterozygotes bind a more diverse array of bacterial species. We will also perform IgH-sequencing to define the effect of MHC heterozygosity on repertoire diversity of plasma cell populations.

Supported by grants from the NIH (R21AI142409, R01AII55887)
Bruton’s tyrosine kinase impacts IgA repertoire and microbiome composition in non-obese diabetic mice
Authors: Olin Ilana Rae , Cohen Lucy , Brown Alana C , Verma Sonam , Kendall Peggy L
Published: August 2023

The microbiome has emerged as a contributor to type 1 diabetes (T1D), but how interactions between the microbiome and the mucosal immune system lead to or prevent T1D is not clear. Previously, we found that deletion of the B cell signaling protein Bruton’s Tyrosine Kinase (BTK) selectively eliminates autoreactive B cells and autoantibodies, preventing T1D development. However, we discovered that this disease protection is microbiome-dependent, as it disappears in cleaner facilities. We hypothesize that disruption of BTK-mediated B cell receptor (BCR) signaling promotes the growth of particular microbes that are protective against T1D by altering IgA production. Thus, the objective of this study is to determine how BTK-deficiency affects the IgA repertoire, shapes the gut microbiome, and synergistically prevents T1D. First, we performed IgA-seq to assess how BTK-deficiency alters IgA-coating of the microbiota. Then, we characterized the microbiomes of non-obese diabetic (NOD) mouse colonies living in distinct environments using 16S sequencing and conducted disease studies to track T1D development. We found that BTK-deficiency altered the ability of IgA to coat microbiota in the small intestine and colon. We also found that BTK-deficiency significantly impacts the composition of the microbiome in NOD mice living in distinct facilities. BTK-deficient mice have increased commensal diversity and enrichment of specific microbes. Based on these findings, we conclude that disruption of BTK-mediated BCR signaling alters IgA production and microbiome composition, with downstream effects on T1D pathogenesis.

Supported by grants from NIH (R01-DK-084246I) and Veteran’s Affairs (101-BX-002882)
Segmented filamentous bacteria (SFB) drives enhanced T cell-dependent IgA and IgG2b responses in Peyer’s patches
Authors: Kang Byunghyun , Kim Eun-Do , Kim Bong-Hyun , Tomachi Tomohiro , He Jianping , Kelsall Brian L
Published: August 2023

Peyer’s patches (PPs) are the major site for T-cell dependent IgA B-cell differentiation. Segmented filamentous bacteria (SFB) are commensal bacteria that preferentially colonize the ileum, mainly over PPs. SFB colonization of germ-free mice induces polyclonal IgA responses by unclear mechanisms. Using scmRNAseq and flow cytometry we found SFB colonization of SPF mice induced an increase in the size and overall cell number in PPs that includes enhanced numbers of naïve, follicular, and germinal center B-cells as well as Th 17 and Tfh cells. SFB colonization increased the numbers of IgA+ and IgG2b+, but not IgG1+ B-cells in PPs. Enhanced class-switching to IgA and IgG2b was dependent on T-cells, CCR6, CD40-CD40L and DC-expression of MHCII, as well as to some degree on IL-6. An increased number of activated CD11c+ SIRPa+ ST2− CD11b+ DCs with CCR6+ Th17 of SFB+ compared to SFB− mice suggests increased initiation of IgA class switching in the PP subepithelial dome (SED). SFB colonization increased the conversion of Th17 into Tfh cells, however unlike previous reports, SFB-colonized IL-17R−/− and CD4CRE-RORgtfl/fl mice indicated that neither IL-17, nor the enhanced Tfh conversion from Th17 cells is required for increased IgA and IgG2b responses. Finally, B cell repertoire analysis from IgA+, IgG1+ and IgG2b+ PP B cells showed SFB colonization affected the B-cell repertoire qualitatively as well as quantitatively. Together, these data indicate that SFB colonization enhances the recruitment of naïve B cells, as well as drives IgA and IgG2b antibody responses through enhanced T cell-dependent class switching in PPs, and contribute to our understanding of how commensal microbes affect humoral immune responses to infection and vaccination.

This work was supported by the Division of Intramural Research, NIAID, NIH
Small intestine and colon tissue-resident memory CD8+ T cells exhibit molecular heterogeneity and differential dependence on Eomesodermin
Authors: Lin Yun Hsuan Elena , Duong Han G , Limary Abigail E , Kim Eleanor S , Hsu Paul , Patel Shefali A , Wong William H , Indralingam Cynthia S , Liu Yi Chia , Yao Priscilla , Chiang Natalie R , Vandenburgh Sara A , Ferry Amir , Takehara Kennidy K , Tsai Matthew S , Yeo Gene W , Goldrath Ananda W , Chang John T
Published: August 2023

Tissue-resident memory CD8+T (TRM) cells are a subset of memory T cells positioned at barrier sites, critical for host defense against infection. Recent studies have begun to reveal substantial heterogeneity in CD8+ TRM populations across different tissues. The gut consists of four anatomically distinct compartments, the epithelia and lamina propria in the small intestine (siIEL, siLPL) and colon (cIEL, cLPL). While CD8+ TRM in the small intestine epithelia are well-described, findings are often generalized to the whole gut, thus obscuring potential differences in CD8+ TRM in the much-less studied small intestine lamina propria and colon compartments. We thereby characterized CD8+ TRM in the four intestinal layers using single cell RNA-sequencing and flow cytometry. We show that CD8+ TRM across the intestinal compartments exhibit differences in the expression of surface molecules, cytokines, granzymes, and transcription factors. For example, small intestine intraepithelial CD8+ TRM produce the highest levels of granzymes, whereas colon intraepithelial CD8+ TRM have the greatest capacity for cytokine production. We also uncover an unexpected, tissue-specific role for the T-box transcription factor Eomesodermin (Eomes), known to suppress TRM formation in the skin, liver, and kidney. Our data demonstrate that Eomes is dispensable for CD8+ TRM formation in both the small intestine and colon. By contrast, following CD8+ TRM formation, Eomes plays a critical role in promoting the maintenance of established CD8+ TRM in the small intestine, but not in the colon. Together, our study provides new insights into intestinal CD8+ TRM heterogeneity and differential transcriptional regulation of intestinal TRM formation vs. maintenance.

Presenting author Y.H.L. is supported by the Canadian Institutes of Health Research (CIHR) Doctoral Foreign Study Award. This study is supported by grants from NIH (P01 AI132122).
The genomic landscape of infant CD4 T cells and monocytes after BCG vaccination
Authors: Kandiyil Vidya Vijayan Karuvan , Kamthunzi Portia , Tegha Gerald , Hosseinipour Mina , De Paris Kristina
Published: August 2023

The live-attenuated Bacille Calmette-Guérin (BCG) vaccine has been introduced a century ago to prevent tuberculosis and is still administered to >80% of newborns worldwide. Independent of its effect on TB, BCG introduction markedly reduced childhood mortality. Although BCG vaccine is routinely administered to infants, there is a gap in understanding the influence of BCG on the neonatal immune system. In adults, BCG induces trained or heterologous immunity. Here, to better understand the molecular changes in the infant immune system after BCG immunization, we performed RNA sequencing and ATAC sequencing in circulating CD4 T cells and monocytes of 28 infants prior to and 6–12 weeks after administering BCG.

Consistent with data from BCG-vaccinated adults, the expression profile of 168 differentially expressed genes (DEG) supports the activation of monocytes after BCG vaccination, and ATAC sequencing data identified epigenetic modification that impact accessibility of genes in Nod1 and Nod 2 signaling pathways. In CD4 T cells, we identified a total of 322 DEG, including 95 upregulated and 226 downregulated genes. The functional enrichment and transcription factor analysis showed the enrichment of ErbB signaling pathways which regulate cell proliferation and differentiation mediated through MAPK signaling pathways. These results combined with peaks identified by ATAC sequencing were indicative of the expansion of Th1 CD4 T cells in BCG vaccinated infants. This study provides an understanding of molecular changes in monocytes and CD4 T cells after BCG vaccination that may explain the improved capacity of the infant immune system to respond to intracellular pathogens.
Adjuvant effects of peptidoglycan on CD4+ T cell activation by MHC-II+ fibroblast-like synoviocytes
Authors: Rouse Joe , Danner Rebecca , Wahhab Amanda , Jutras Brandon , Lochhead Robert B
Published: August 2023

Lyme arthritis (LA) is the predominant late-stage manifestation of Lyme disease, caused by tick-borne spirochete Borrelia burgdorferi. LA can resolve with antibiotic therapy or persist after treatment in a subset of patients, known as post-antibiotic LA. Post-antibiotic LA is characterized by proliferative synovitis, dysregulated Th1/ IFNɣ responses and MHC-II+ fibroblast-like synoviocytes (FLS), a common cell type in inflamed joints. High levels of B. burgdorferi peptidoglycan (PG) have been found in LA patient synovia, yet the effects of PG on host immune responses are not well characterized. We hypothesize that PG acts as an adjuvant to enhance antigen presentation and CD4+ T cell activation in the joint. To test this, primary murine macrophages and FLS, both abundant in LA synovia, were treated in vitro with IFNɣ, PG, or both. Stimulation of macrophages led to upregulation of MHC-II in an IFNɣ-dependent manner, as expected. FLS stimulated with IFNɣ led to upregulation of MHC-II and production of proinflammatory cytokines, which was enhanced by PG as well as the muramyl-dipeptide subunit of PG, a known NOD2 ligand. To examine T cell responses, IFNɣ-primed MHC-II+ FLS were co-cultured with OT-II mouse T cells (all with identical antigen-specific T cell receptors) and supplemented with the cognate OVA323–339 peptide. MHC-II+ FLS activated naïve CD4+ T cell proliferation, which was enhanced by PG stimulation. Co-culture supernatants showed upregulated IFNɣ and IL-12 production, indicating a Th1 response. Negative controls using an alternative OVA257–264 peptide or NOD2-deficient FLS failed to induce T cell responses. These data show that PG acts as an immune adjuvant enhancing peptide-specific CD4+ T cell activation by MHC-II+ FLS.
Heterogeneous Specificities of Nickel Presentation to Nickel Specific CD4 T Cells
Authors: Dai Shaodong , Zhang Yan , Chen Lan , Wang Yang , Li Wei
Published: August 2023

Sensitization to nickel (Ni2+) is the most common T cell mediated type IV hypersensitivity, although the mechanisms by which Ni2+ alters antigen presentation are not understood. To study how the TCR then recognizes Ni2+ bound to the MHC molecule, we performed paired single cell RNA sequencing of PBMCs from two highly Ni2+ sensitized subjects to orthopedic implants and characterized three Ni2+ specific CD4+ T cell clones. One Ni2+ specific CD4+ T cell is highly restricted to HLA-DR7. But the other highly cross-reactive CD4+ T cell clones was restricted to multiple HLA alleles, including HLA-DR, HLA-DP, and HLA-DQ expressing antigen presenting cells. We identified a HLA-DQ8 restricted Ni2+ dependent mimotope reactive to the highly cross-reactive T cell clone. This mimotope appeared to present Ni2+ ions with the Glu at P2 position, involving βH81. The mutational studies of HLA-DP2 molecule showed that the same T cell recognized the Ni2+ ion bound to a completely different location in the P4-P7 pocket. Ni2+ ion was also can be presented efficiently by HLA-DR53 to the same T cells. mutational and structural study of HLA-DR53 molecules suggested that HLA-DR53 presented Ni2+ in the P4-P7 pocket. This is the first evidence showing that a metal ion can be presented by HLA using the different sites. The metal binding sites are HLA and peptide dependent. The heterogeneous specificities of Ni2+ specific TCRs are determined by different alleles of HLAs together with particular self-peptides.
NEI supplementation improves alum-based immunity in CFTR KO mice
Authors: Yoo Sungjun , Kim Eunsoo , Mazik Logan , Steiner Haley E , Cormet-Boyaka Estelle , Boyaka Prosper N
Published: August 2023

Cystic fibrosis (CF) is a genetic disorder that causes severe damage to the lungs and digestive system. CF affects the cells that produce mucus, sweat, and digestive enzymes and is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). Expression and function of CFTR have been extensively studied on epithelial cells and several recent publications have shown that CFTR also regulates the functions of myeloid cells. We addressed whether CFTR is expressed by B cells and the role of CFTR in B cell functions. We found that CFTR is expressed on murine and human B cells. Compared to control wild-type (WT) mice, CFTR KO exhibited reduced frequency of B cells in lymphoid tissues and lower levels of serum immunoglobulin isotypes. Upon systemic immunization with an alum-based vaccine, CFTR KO mice developed lower antibody responses than control mice. Interestingly, vaccine supplementation with an inhibitor of the serine protease elastase (NEI) enhanced the titers of vaccine-specific serum antibody responses in CFTR KO mice and CFTR heterozygote (CFTR Het) mice. We have previously reported that NEI supplementation could promote mucosal immunity in mice immunized with the alum-based injected vaccine. In this regard, CFTR KO and CFTR Het mice immunized with NEI-supplemented alum-based injected developed higher IgA and IgG responses in saliva than control WT mice. These results provide new insights on the regulatory role of CFTR for B cell differentiation and function. They also suggest that vaccine supplementation with NEI could improve protection of individuals leaving with CF against respiratory pathogens.

NIH R01 DK101323
Investigating the immunomodulatory effects of histotripsy ablation for osteosarcoma
Authors: Patwardhan Manali Nitin , Hay Alayna , Vickers Elliana , Hixson Haleigh , Ruger Lauren , Allen Irving C , Vlaisavljevich Eli , Tuohy Joanne
Published: August 2023

Osteosarcoma (OS) is the most common primary bone cancer in humans and dogs. Canine OS shares many biological similarities with human OS and is a valuable comparative oncology research model. In both species, metastasis remains a major hurdle against improved survival, thus warranting a novel therapeutic approach such as histotripsy, a non-thermal, non-invasive, focused ultrasound ablation modality. Histotripsy can mechanically disintegrate gross tumor and potentially induces an anti-tumor immune response to inhibit metastatic development. The objective of this in vitro study was to investigate the immunomodulatory effects of histotripsy ablation of canine OS. We hypothesized that histotripsy ablation of OS promotes a pro-inflammatory immune signature in primary monocytes and macrophages. We isolated CD14+ primary monocytes using positive selection immunomagnetic cell separation from peripheral blood mononuclear cells of healthy dogs, and differentiated primary macrophages by culturing primary monocytes with canine GM-CSF for 72 hours. We co-cultured primary monocytes and macrophages with histotripsy-ablated canine D17 OS cells for 24 hours, then characterized their cell surface markers using flow cytometry and performed multiplex supernatant cytokine analysis. We observed upregulation of CD80 and CD62L on primary monocytes and a downregulation of CD206 on macrophages exposed to histotripsy-ablated D17 cells compared to controls, suggesting immune activation. We observed decreased levels of the pro-tumor cytokine, TGF-β, from primary monocytes exposed to histotripsy-ablated D17s compared to controls. Our results indicate that histotripsy ablation of OS can induce a pro-inflammatory immune signature.

This work is supported by grants from the American Kennel Club Canine Health Foundation, the Focused Ultrasound Foundation.
TGFβ1 and BMP signals modulate CD8+ T cell functionality and responsiveness to immune checkpoint blockade therapy
Authors: Saadey Abbey , Yousif Amir , Osborne Nicole , Shahinfar Roya , Castillo Chelsea , Laster Brooke , Rajeev Meera , Kumar Shrea , Webb Amy , Ghoneim Hazem
Published: August 2023

T cell dysfunctionality is a major challenge during chronic infections and cancer, limiting durable responses to T-cell based immunotherapies including immune checkpoint blockade (ICB). However, the role of upstream environmental signals in modulating the development of terminal dysfunction in CD8+ T cells remains to be explored. We have recently established that persistent TGFβ1 exposure drives an epigenetically stabilized, terminal dysfunction program in chronically stimulated human and mouse CD8+ T cells. In contrast, boosting BMP signaling induces a striking ability of T cells to maintain effector functionality and survival under chronic stimulation. Here, we show how rebalancing TGFβ1/BMP-signals in dysfunctional CD8+ T cells retains functionality in chronically stimulated human cord blood or tumor-infiltrating CD8+ T cells in vitro, while enhancing chronic virus or tumor control in mice. We also explore the therapeutic potential of BMP-signals in promoting durability of CD8+ T cell responses. Altogether, these findings provide an exciting new approach to enhance T cell responses to immunotherapy.
Sulfides in the gut mediate protection against gastrointestinal infection via alterations to local immunity and the microbiome
Authors: Band Victor I , Stacy Apollo , Chau Joanna , Belkaid Yasmine
Published: August 2023

Sulfide is a gaseous molecule, which has toxic effects at high concentrations yet plays key roles in homeostasis throughout the body. Sulfides are produced endogenously by both host tissues and the bacterial cells of the gut microbiome, which results in the gut containing the highest concentrations of sulfide in the body. We sought to assess the role of the highly abundant sulfide molecule in gut immunity, microbiome homeostasis and resistance to enteric infections. Local sulfides can be depleted by the compound bismuth subsalicylate (BSS), a common anti-diarrheal medication, which acts locally by sequestering sulfides in the gut. Key gut commensals such as Lactobacillus and segmented filamentous bacteria, major mediators of gut immunity and resistance to pathogen colonization, were profoundly depleted following sulfide sequestration. Additionally, we observed significant downstream immune effects, specifically within the local immunity of the small intestine. Depletion of gut sulfides resulted in profound collapse of CD4 T cells, especially among Th1 cells in the lamina propria of the small intestine. Using a mouse model of Salmonella Typhimurium, mice treated with BSS were extremely susceptible to infection, with a 5-log increase in fecal bacterial load at 24 hours post infection. These data reveal a central role for sulfides in gut homeostasis and prevention of enteric infection. Strategies to manage gut sulfide levels, including diet supplementation and microbiome engineering, could be a possible intervention to promote gut health.

NIH Office of Dietary Supplements (Research Scholar Grant 2022)
Jedi Mindset: Discussing Effective Learning Strategies with Students
Authors: Alard Pascale
Published: August 2023

Students’ ability to retrieve acquired knowledge long-term is critical for every discipline, especially Immunology. The research on learning shows undergraduate students use ineffective learning strategies, misjudging passive vs active ways of learning. Moreover, first generation graduates may be at a disadvantage as they have less opportunity to learn from family member experience. Sixty percent of students who attend medical, dental or graduate school continue to use ineffective strategies, e.g., re-reading. Our goal was to develop a bookclub for our incoming graduate students to discuss effective learning strategies, and assess student’s willingness to switch to more effective learning strategies as well as evaluate the benefit of the bookclub.

We held three consecutive weekly sessions before courses started to discuss the book by Dr. Saundra McGuire entitled, “Teach yourself how to learn”. Incoming graduate students read a few chapters for each session and answered questions about their reading. A discussion was facilitated with senior students, who shared their experience about the program and provided advice. Short podcasts from the UCLA Lasting Learning website were viewed to revisit the important points.

Students recognized that re-reading is not effective and were willing to modify their study habits and continued to use effective learning strategies the following semester. In addition, students agreed that the bookclub was beneficial and provided tools to help them succeed in their coursework, and emphasized the positive impact of senior students’ participation. Our study suggested that a bookclub about effective learning strategies is beneficial and can help incoming graduate students reach their full potential.
Polyomavirus ependyma infection induces region-specific and Stat1-dependent expression of chemokines in the CNS
Authors: Spencer Samantha A , Lukacher Aron
Published: August 2023

Immunity of the central nervous system to pathogens is partly established by critical barrier sites. One barrier within the four ventricles, the blood-cerebrospinal fluid (CSF)-brain barrier, is comprised of the choroid plexus epithelial cells separating the vasculature from the CSF, and the CSF from the brain parenchyma by the ependymal cells lining the ventricles. Intracerebroventricular delivery of mouse polyomavirus (MuPyV) in wild-type mice leads to infection of ependymal cells. In Stat1 null mice, viral infection is increased, and ependyma effacement is associated with hydrocephalus. Unknown is whether innate immunity and immune cell recruitment via Stat1 signaling by the ependyma limits MuPyV infection of the parenchyma. Dcdc2α-Cre mice, which show recombination in the ependyma and choroid plexus, were crossed with Stat1fl/fl mice to yield ependyma-specific Stat1 knockout. MuPyV infection was greater in Dcdc2αCre+ x Stat1fl/fl mice than in wild-type (WT) controls at 4 days post infection (dpi), but by 7 dpi the Dcdc2α-Cre+ x Stat1fl/fl mice exhibited higher levels of MuPyV infection in the periventricular region. Prior studies using the MuPyV-infected Stat1 null mice showed increased monocyte and T cell recruitment. Several chemokines (Ccl2, Cxcl10, Cxcl11, and Cxcl16) were elevated at 7 dpi in Dcdc2αCre+ x Stat1fl/fl mice relative to WT mice, while Cxcl13 was reduced at 4 and 7 dpi. Of these, Ccl2, Cxcl10 and Cxcl11 showed greater levels near the ventricles in both wild-type and Dcdc2αCre+ x Stat1fl/fl mice. Our data highlight the ependyma as a critical barrier protecting the brain from MuPyV infection that is dependent on Stat1 signaling.

This work was supported by grants from the NIH (R35NS127217-01, T32CA60395-25).
Immunological and genetic analysis of vaccine strains and regional variants of infectious bronchitis virus is examined
Authors: Lee Rangyeon , Jung Ji Seung , Kwon Hyuk Moo , Sung Haan Woo , Park Jeongho
Published: August 2023

Infectious bronchitis virus (IBV) is an acute and highly contagious coronavirus characterized by respiratory and systemic pathology in chickens. Frequent genetic mutation in the spike (S) glycoprotein repeatedly induces regional variants. Although IBV vaccines are extensively utilized in chicken flocks, their efficacy is diminished by variant serotypes and poor cross-protection. To provide restored strategy for the IBV protection, we analyzed the genetic distance between local IBV variants and other coronaviruses, including SARS-CoV-2. We also compared the genetic sequence of the S1 glycoprotein in three commercial vaccine strains. Then, we examined immune responses by vaccine and regional strains in primary chicken embryonic kidney cells and splenocytes. Featured pathways for innate immunity and gene expressions for innate immune response was observed. Our study is expected to provide valid information for vaccine development against emerging variants.

Supported by funding from the office of Research Support, Kangwon National University, a 2020 Research Grant from Kangwon National University (no. 520200065), and a National Research Foundation of Korean (NRF) grant funded by the Korea government (MSIT) (no. 2020R1G1A1099715)
Amniotic fluid derived extracellular vesicles as novel anti-inflammatory therapeutics against SARS-CoV-2 infection
Authors: Chanda Debarati , Bellio Michael A , del Rivero Tania , Mitrani Maria Ines , Channappanavar Rudragouda
Published: August 2023

The ongoing Covid-19 pandemic caused by SARS-CoV-2 is associated with acute respiratory distress syndrome (ARDS) and fatal pneumonia. Excessive inflammation caused by SARS-CoV-2 is the key driver of ARDS and lethal disease. Despite strong evidence for the role of virus induced inflammation in severe COVID19, no effective anti-inflammatory drug is available to control robust inflammation. Although corticosteroids are in clinical use to suppress excessive inflammation, global immunosuppressive effects of these drugs have limited their clinical success. Moreover, cytotoxicity caused by several chemically derived drugs is a cause of concern. Therefore, there is an urgent need to identify biologically derived immunomodulators that suppress inflammation without affecting antiviral immunity. In this study, we evaluated human amniotic fluid derived extracellular vesicles (hAF-EVs) as a potential safe biologic for immunomodulation during Covid-19. Our results showed that hAF-EVs significantly reduced inflammatory cytokine production in TLR2/4/7 and CoV structural protein stimulated mouse macrophages, in-vitro. Importantly, we also found that intraperitoneal administration of hAF-EVs in mice infected with a mouse-adapted SARS-CoV-2 reduced morbidity and mortality. A detailed examination of infected lungs showed that the increased protection in hAF-EV-treated mice was associated with reduced myeloid cell accumulation and inflammatory cytokine production. Collectively, we identify a novel biologic that has potential to suppress excessive inflammation and promote survival following SARS-CoV-2 infection, highlighting the translational potential of hAF-EVs against Covid-19 and other viral lung infections.

This study was supported in part by institutional research funds from Oklahoma State University—Stillwater and NIGMS 5P20GM103648-10.
Fc properties of SARS-CoV-2 spike-specific antibodies elicited by vaccines and infection
Authors: Hioe Catarina E , Klingler Jeromine , Bandres Juan , Emami-Gorizi Rozita , Amanat Fatima , Kowdle Shreya , Gleason Charles , Srivastava Komal , Simon Viviana , Perandones Claudia , Lee Benhur
Published: August 2023

Since its emergence in 2019, SARS-CoV-2 has infected millions of people and several vaccines have been approved for clinical use worldwide. Antibodies against the spike protein of SARS-CoV-2 are a critical immune determinant for protection against this virus. While virus neutralization is a key function of spike-specific antibodies, antibodies also mediate Fc-dependent activities that can play a role in protection or pathogenesis. This study compared the Fc properties and functions of anti-spike antibodies elicited by Pfizer/BioNtech or Moderna mRNA vaccines, Sputnik rAd26+rAd5 vaccines, or natural SARS-CoV-2 infection. We first examined immunoglobulin (Ig) isotypes in sera or plasma from the three groups. IgG1 antibodies against the spike protein were the predominant isotype, produced to high levels by the vast majority of the subjects in the three groups. In addition to IgG1, the mRNA vaccines also elicited IgM, IgG2-4, and IgA1-2, whereas the Sputnik vaccines induced only IgG4. Convalescent COVID-19 patients had yet a distinct set of isotypes, displaying mainly IgM and IgA1 alongside IgG1. When we examined antibody functions, spike-specific antibodies from both mRNA and Sputnik vaccine groups showed greater capacities to bind and activate complement than those from the convalescent COVID-19 patients. However, the Sputnik vaccine-induced antibodies had a greater potency of binding the ADCC-activating FcγRIIIa receptor, although the Sputnik vaccines induced lower Ig levels against spike than the mRNA vaccines. These data demonstrate that antibodies with distinct Ig isotypes/subtypes and Fc functions were elicited by different COVID-19 vaccines and by natural SARS-CoV-2 infection.

This work was supported in part by the Department of Veterans Affairs [Merit Review Grant I01BX005794] (C.E.H.) and [Research Career Scientist Award 1IK6BX004607] (C.E.H.); the National Institutes of Health [grant AI139290] to C.E.H., [grants R01 AI123449, R21 AI1498033] to B.L, the Department of Medicine of the Icahn School of Medicine at Mount Sinai (C.E.H.); the Department of Microbiology and the Ward-Coleman estate for endowing the Ward-Coleman Chairs at the Icahn School of Medicine at Mount Sinai (B.L.), and institutional funding as well as anonymous philanthropic donations (V.S.).
Biomarkers of Neurological Injury for Emerging Viral Threats and Post Viral Disease
Authors: Bartlett Maggie , Poeck-Goux Heather , Johnson Linwood Johnson , Scully Kevin , Vittor Amy , Hayes Ronald , Carrera Jean-Paul , Smith Darci R
Published: August 2023

Neurotropic viral infection and the ensuant immune response are a significant cause of morbidity and mortality worldwide, which can range in severity from mild to permanent central nervous system (CNS) damage and death. Encephalitic alphaviruses include Venezuelan and eastern equine encephalitis viruses (VEEV and EEEV; Alphavirus; Togaviridae). Injury to the CNS is an important determinant of poor outcome and tools to predict this outcome are lacking. Neurons are the primary target cells of encephalitic alphaviruses where cytopathology plays a major role in CNS dysfunction. Proteins are released following cell death, such as ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) and glial fibrillary acidic protein (GFAP) are neuromarkers of CNS tissue injury which could serve as biomarkers to assess injury severity, monitor disease progression, direct treatment, and as reliable endpoints to help develop novel medical countermeasures. Recent advances in the use of blood-based biomarkers for diagnosis of traumatic brain injury (TBI) which have FDA approved assays have provided a scientific foundation for expanding the biomarker technology to brain damage caused by other CNS pathologies like viral encephalitis. Here we evaluated the ability to detect these biomarkers for encephalitic alphaviruses, encephalitis of unknown origin, and hospitalized patients with severe coronavirus disease (COVID-19). Higher levels of specific biomarkers were detected in patients diagnosed with alphavirus or unknown encephalitis which may be useful in prognosis and treatment guidance of post viral disease. Collectively, our results suggest that these blood-based biomarkers may be a good indicator for brain injury resulting from viral infection.
Tim-3 expression in Treg controls viral persistence and effector T cell response during chronic LCMV infection.
Authors: Nieves-Rosado Hector M , Banerjee Hridesh , Kane Lawrence P
Published: August 2023

Tim-3, a surface protein, is upregulated on Treg during chronic viral infections. During HCV infection in humans there is an increase in frequency of Tim-3+ Treg, which have increased proliferation, survival, and expression of immunosuppressive cytokines like IL-10, compared to Tim-3− Treg. Similarly, it was recently reported that there is an increase in Tim-3+ Treg in people with HIV regardless of viral suppression. However, the role of regulatory T cells (Treg) during chronic viral infection has not been fully defined. We hypothesize that Tim-3 promotes an effector phenotype on Treg during chronic viral infection, which limits the virus-specific T cell response and impairs viral clearance. Using an inducible Treg-specific Tim-3 loss-of-function (Tim-3 Treg KO) model, we found a significant decrease in morbidity during LCMV chronic infection. Tim-3 Treg KO mice mounted a higher virus-specific T cell response and had lower viral burden in liver, and kidney at 30 days post-infection. Interestingly, we found that there was a decrease in the frequency of PD-1+Tim-3+ and PD-1+Tox+ LCMV-specific CD8 T cells. Lack of Tim-3 expression on Treg limited their ability to upregulate IL-10, TGFb, CD39, PD-1, CD25, CTLA-4 and CD44. Our findings demonstrate that modulation of a single surface protein in Treg can lead to a reduction in viral burden and enhance LCMV-specific T cell response. These studies may also help to identify Tim-3 directed therapies for the management of persistent infections and other chronic illnesses.

Supported by grants from NIH (T32GM008208, R01 AI138504)
Role of germinal center response in the antibody responses against SARS-CoV-2 spike protein
Authors: Khalil Jumana A T , Miyauchi Kosuke , Yamamoto Yuichiro , Noguchi Kohji , Takayama Kazuo , Kubo Masato
Published: August 2023

Spike-based vaccination strategies, including mRNA, effectively induce antibody responses against SARS-CoV-2. However, the breadth of antibody responses raised by Wuhan spike covering newly emerging variants remains unclear. Here, we investigate how the loss of function of GC responses and IL-4 signal influence the antibody and memory B cell responses against spike proteins of SARS-CoV-2 variants. In mouse model, immunization of C57BL/6J and C3H mice with trimeric recombinant spike protein of Wuhan-CoV-2 efficiently induced the production of antibodies capable of recognizing the receptor binding domain of delta, and omicron (BA1, and BA5) variants. The third boosting with Wuhan spike protein was also effective in inducing the antibodies to recognize omicron variants, but the boosting with BA5 was only effective for BA5, not Wuhan. We tested the neutralizing activity of these antibodies by the in vitro binding assay using psuedo-virus and human bronchial organoids infection system. These antibodies effectively blocked the infection of Wuhan and delta variants, but the protection was reduced for omicron variants. We further evaluated the contribution of the GC response and IL-4 using Bcl6 deficient mice (Bcl6f/f CD4-cre, Bcl6f/f CD4ER-cre) or B cell-specific deficient mice of the IL-4 receptor(R) (Il4rΔB mice). The results indicated that Bcl6 deficient mice and the mice lacking IL-4R signaling in B cells markedly reduced initial antibody responses and diminished protectivity of the antibodies against the variant virus. In conclusion, our results indicated that the GC response and IL-4 signal provides a quantitative checkpoint optimizing the breadth of anti-SARS-CoV2 protective antibody response.
Inulin enriched diet reinforces cancer immunosurveillance via γδ T cell activation
Authors: Boucher Emilie , Plazy Caroline , Lavie-Richard Mathias , Suau Antonia , Mangin Irène , Cornet Muriel , Aldebert Delphine , Toussaint Bertrand , Hannani Dalil
Published: August 2023

Gut microbiota is now recognized as a key player impacting the host’s anti-cancer immunosurveillance and ability to respond to immunotherapy. Its optimal modulation for preventive and therapeutic purposes is very appealing. Since diet is one of the most powerful modulators of the microbiota, nutritional interventions could be leveraged to improve host immunity. The present study aims to analyze the anticancer properties of inulin, a fiber known to promote bacteria described as immunostimulatory. The effects of inulin complementation were tested in mice fed an inulin-enriched diet for 2 weeks before tumor implantation. In the three tumor models used (melanoma, fibrosarcoma, and colorectal cancer), inulin consumption inhibited tumor growth by 80% compared to controls. Tumor-infiltrated immunity analysis showed that inulin triggers an enhanced Th1-polarized CD4+ CD8+ and γδ T cell-mediated antitumor response. In the gut, inulin consumption favored Bifidobacterium relative abundance, and strengthened gut immunity, by increasing the proportion of colonic IFNγ-producing γδ intra-epithelial T lymphocytes as well as the expression of genes involved in inflammation. In addition, we showed that the inulin-mediated anti-tumor effect is gut microbiota- and γδ T cell-dependent, as both antibiotic treatment and in vivo blocking of γδ TcR abrogate inulin efficacy. Overall, our data identified γδ T cells as a critical immune subset, mandatory for inulin-mediated antitumor immunity in vivo. This supports and rationalizes the use of a prebiotic approache as well as the development of immunotherapies targeting γδ T cells in cancer prevention and treatment.

This work is supported by GEFLUC Dauphiné-Savoie, Ligue contre le Cancer Comité Isère, Ligue contre le Cancer Comité Savoie, Université Grenoble Alpes IDEX Initiatives de Recherche Stratégiques. Emilie Boucher is supported by a grant salary from the French Ministry of Higher Education, Research and Innovation.
Ex vivo activation and fold-expansion of human regulatory T cells negatively correlates with preservation of beta cell function in adolescents with type 1 diabetes
Authors: Bender Christine , Hu Alex , Wiedeman Alice , De Castro Grace , Sietsema William K , Gitelman Stephen , Griffin Kurt , Long Alice
Published: August 2023

Type 1 diabetes (T1D) is an autoimmune disease marked by immune destruction of insulin-producing β-cells of the pancreas. In an effort to halt disease progression, autologous polyclonal expanded regulatory T cells (expTregs) were adoptively transferred into adolescents with recent-onset T1D (Sanford/Lisata Therapeutics Trex Phase 2 clinical trial, NCT02691247). Overall, expTreg therapy (1–24×106 cells/kg) did not preserve residual β-cell function, as measured by change in insulin C-peptide AUC-mean over 2 years compared to placebo (p=0.39) regardless of dose (p=0.27). Consistent with a more activated phenotype, expTregs expressed more CD25, CD38, HLA-DR, and CXCR3 than endogenous Tregs by flow cytometry, and bulk-RNAseq revealed 2377 genes (related to metabolism, cell cycle, and immune response) up-regulated in expTregs compared to endogenous Tregs. In subjects from the high-dose group (>10×106 cells/kg), there was a transient increase of total Tregs one week after infusion (p=0.003), which had an activated memory phenotype, consistent with infused expTreg accounting for this difference. However, the increase in the activation of Tregs post-treatment was associated with poorer outcome (p=0.03), and the extent of expansion of the expTregs ex vivo (range 24.3–643-fold) negatively correlated with C-peptide change (p=0.01) in treated subjects regardless of Treg dose. Yet, neither purity nor suppressive function correlated with outcome, suggesting differences in endogenous Treg qualities leading to increased expansion may influence outcome. These results suggest that expTreg quantity alone does not influence outcome; instead, both endogenous and expTreg quality may be important factors modulating T1D progression.
High-throughput Western Blot for in vitro and in vivo assessment of human Ig specific cleaving enzymes
Authors: Le Nam , Pellerin Alex , Sanmarco Liliana , Green Tobias , Borah Minasri , Mande Purvi , Newton Andita , Hoyt Emily , Anderson Jordan , Bengeri Soumya , Higginson-Scott Nathan , Otipoby Kevin , Mascanfroni Ivan
Published: August 2023

Antibodies play a vital role in the body’s immune defense mechanism. However, pathogenic Ig antibodies are thought to contribute to several autoimmune conditions and transplant rejection. Proteases derived from human pathogens can specifically cleave IgG into F(ab’)2 and Fc fragments, thus allowing for rapid clearance of IgG. This unique trait suggests a novel opportunity to use these molecules to treat autoantibody mediated diseases.

Using our IMPACT platform leveraging machine learning, we have engineered Ig proteases that specifically cleave IgG, and treatment with these proteases causes cleavage below the hinge region, which can result in the generation of the following degradation products: 1) Fab’2 and Fc fragments lacking effector function; or 2) single chain cleaved fragments with reduced effector functions. To detect these species, we developed a method using JESS, an automated high throughput western blot instrument. Using this system, we can detect intact, single cleaved and fully cleaved IgG in a time and dose dependent manner in both in vitro and in vivo assays.

In conclusion, we successfully optimized a high throughput assay format that is highly specific and sensitive in evaluating the cleavage activity of different Ig proteases and could be a powerful tool to assess efficacy of IgG cleaving enzymes in the clinic.