bluuebunny/crossref_metadata_2025 huggingface dataset:
Naïve B cell invasion drives longevity of Influenza-primed germinal centers
Authors: de Carvalho Renan , Barbulescu Alexandru , Ersching Jonatan , Hobbs Alvaro , Mesin Luka , Jacobsen Johanne , Victora Gabriel
Published: November 2024

Germinal centers (GCs) form in secondary lymphoid organs in response to infection and immunization and are the source of affinity-matured B cells. The duration of GC reactions spans a wide range, and long-lasting GCs (LLGCs) are potentially a source of highly mutated B cells. We show that, rather than consisting of continuously evolving B cell clones, LLGCs elicited by influenza virus or SARS-CoV-2 infection in mice are sustained by progressive replacement of founder clones by naïve-derived invader B cells that do not detectably bind viral antigens. Rare founder clones that resist replacement for long periods are enriched in clones with heavily mutated immunoglobulins, including some with very high affinity for antigen, that can be recalled by boosting. Our findings reveal underappreciated aspects of the biology of LLGCs generated by respiratory virus infection and identify clonal replacement as a potential constraint on the development of highly mutated antibodies within these structures (de Carvalho et al, Cell, 2023).
Elevation of KIR+CD8+ T cells during human pregnancy
Authors: Li Jing , Narasimhan Purnima , Li Lin , Mallajosyula Vamsee , Prunicki Mary , Kirosingh Adam , Braun Amy , Nankya Felistas , Musinguzi Kenneth , Kakuru Abel , Kamya Moses , Rosenthal Philip , Dorsey Grant , Jagannathan Prasanna , Nadeau Kari , Gaw Stephanie , Winn Virginia
Published: November 2024

Recently, we identified KIR+CD8+ T cells as a previously underappreciated regulatory subset important in humans and mice to suppress self-reactivity, especially in the context of infections. To better understand what other roles these cells might play, we asked whether they are active during pregnancy. We first observed an increased frequency of KIR+CD8+ T cells in the peripheral blood of pregnant women at the second trimester compared to age-matched nonpregnant females. Women with a male fetus had a significantly higher level of KIR+CD8+ T cells than those with a female fetus. In vitro, KIR+CD8+ T cells suppressed HY-specific CD8+ T cells only from mothers with a male fetus. Therefore, the higher induction of KIR+CD8+ T cells in mothers carrying a male fetus may suppress the additional allogenic immune responses triggered by the Y chromosome from the male fetus. Longitudinal analysis over pregnancy showed that KIR+CD8+ T cells undergo expansion and differentiate into functional cytotoxic cells during pregnancy, and that their levels are maintained postnatally. In addition, increased levels of KIR+CD8+ T cells correlated with pregnancy disorders. Taken together, our findings suggest an important role of KIR+CD8+ T cells in the maintenance of maternal tolerance by suppressing fetal-specific alloreactive T cells. They may also be useful as predictive biomarkers or drug targets for human pregnancy disorders.
Deletion of RANKL in regulatory T cells ameliorates bone erosion in arthritis
Authors: Laza Briviesca Raquel , Del Mundo Michael , Zboinski Elissa , Charles Julia , Nigrovic Peter
Published: November 2024

Rheumatoid arthritis (RA) is an autoimmune disease that leads to bone erosion mediated through osteoclasts.Osteoclast differentiation is promoted through engagement of RANK by its ligand (RANKL) expressed by multiple lineages. Whereas regulatory T cells (Tregs) typically suppress osteoclastogenesis, Tregs developing in a pro-inflammatory environment can express RANKL and promote osteoclast function (Levescot et al.2021).To test the importance of osteoclastogenic Tregs in vivo, we developed a mouse selectively lacking RANKL expression in Tregs (FoxP3YFP/creRANKLflox/flox, hereafter RANKLΔTreg). Arthritis was induced by administration of K/BxN serum containing arthritogenic IgG antibodies. Joint inflammation and synovial neutrophil infiltration in RANKLΔTreg mice were similar to WT animals. Compared with Tregs from lymph nodes, Tregs from inflamed joints exhibited increased expression of activation markers including CD25,Foxp3,CD44, and CTLA4, without a difference between RANKLΔTreg and WT animals; RANKLΔTreg mice exhibited a trend for higher Treg PD-1 expression. In WT mice, the percentage of Tregs expressing RANKL was elevated in synovial fluid compared to lymph nodes. Correspondingly, microCT showed a significant reduction in bone erosion in RANKLΔTreg compared to WT mice (p=0.003). These findings are consistent with a pathogenic role of RANKL-expressing Treg for RA bone erosion in arthritis; whether RANKL impacts other Treg functions remains to be established.
The ability of immunomodulatory imide drugs (IMiDs) to inhibit the polarization of T helper cells towards Th2 phenotype depends on cereblon
Authors: Vikhe Pratik , Ferrao Silas , Kong Nikki , Jones Lyn
Published: November 2024

IMiDs are small molecules that can modulate the immune system and cellular process by targeting proteins for degradation. IMiDs hijack the CRL4(CRBN) E3 ligase to ubiquitinate and degrade their neosubstrate, IKZF1 and IKZF3, among others. Thalidomide, Lenalidomide, Pomalidomide, as well as the second generation Iberdomide and Avadomide are current IMiDs used in the clinic. In this study, using 13-plex cytokine profiling, we assessed the ability of these five IMiDs to modulate T helper (Th) cell polarization. Most IMiDs polarize the Th cells towards the Th1 phenotype and inhibited Th2, among these Iberdomide, followed by Avadomide, were the most potent. To confirm that these effects are on-mechanism, we used a VHL-based CRBN-PROTAC, 14a, to degrade cereblon (CRBN). While it had not substantial effect on Th cell polarization, pre-treatment of Th cells with 14a rescued the degradation of neosubstrates and cytokine response stimulated by Iberdomide, with the exception of IL-9 levels, indicating a CRBN-independent effect. Pertaining to the significance of these IMiD drugs that are currently used for cancer and proposed for treating autoimmune diseases, this study shows that these small molecules can program T helper cells independent of influence from other immune cells.
Improved identification of allele-specific MHC class II immunopeptidome
Authors: Rumble Julie , Nyayapathy Seeta , Jones Richard , Good Paul , Ho Bill
Published: November 2024

In humans, there are three main MHC class II alleles that present peptides: HLA-DR, HLA-DQ and HLA-DP. While peptides presented by HLA-DR have been well characterized due to the availability of a high-quality antibody to this allele, peptides presented by HLA-DQ and HLA-DP are less well characterized. In this study, we have developed a workflow to improve the experimental identification of allele-specific MHC class II-associated peptides by biotinylating the alpha chain of HLA-DR in live cells. This was accomplished using the HLA-DR-null HL-60 cell line, cotransfecting with BirA and Avi-tagged MHC expression constructs. Supply of exogenous biotin ensured the specific biotinylation of MHC, which was then enriched from lysates using either streptavidin or HLA-DR-specific antibody. Peptides were eluted from these complexes and identified by mass spec. This procedure successfully captured all HLA-DR expressed by the cells and peptides identified by the traditional immunoaffinity approach were compared with the biotinylation approach. Detailed characterization of peptides which bind to MHC class II haplotypes has the potential to aid in the development of therapies for autoimmune disorders in two ways. Better understanding of the antigens driving disease could lead to better therapies, and further, ensuring that biologic drugs used to treat these conditions do not themselves elicit immune responses through class II presentation will improve existing treatments.
Neutrophil hypercitrullination activates monocyte-derived dendritic cells via a TLR4-dependent mechanism
Authors: Ananth Kusuma , Curran Ashley , Antiochos Brendan , Darrah Erika
Published: November 2024

Immunogenic responses against citrullinated antigens—proteins post-translationally modified by peptidylarginine deiminase (PAD) enzymes—are hallmark features of rheumatoid arthritis (RA). An important source of citrullinated autoantigens is leukotoxic hypercitrullination (LTH), a form of neutrophil cell death wherein cell membrane perforation results in PAD activation and subsequent widespread citrullination of antigens. In RA, citrullinated proteins can activate autoreactive adaptive immune cells, but their effect on antigen presenting cells is not well characterized. Here, we utilized differentiated HL-60s (dHL-60s), PAD-expressing neutrophil-like cells, killed by LTH as a cellular source of citrullinated antigens and evaluated their effect on monocyte-derived dendritic cells (moDCs). MoDCs co-cultured with LTH-killed dHL-60s upregulated activation markers, CD80 and CD83, by 2.5-fold more than moDCs alone. This activation was not observed in the absence of hypercitrullination, e.g. with live, apoptotic, or necrotic dHL-60s or with undifferentiated HL-60s. Inhibiting TLR4 and its endogenous ligand S100A9 prevented upregulation of CD83 on moDCs co-cultured with LTH-killed dHL-60s, suggesting that this effect is TLR4-dependent. Thus, LTH-killed neutrophils may create a pro-inflammatory environment by releasing S100A9, allowing moDCs presenting peptides from citrullinated proteins to upregulate co-stimulatory molecules and activate CD4+ T cells more potently.
IgA+ B cells are recruited to the brain after intracerebral hemorrhage
Authors: Ohashi Sarah , Herman Allison , Sansing Lauren , O'Connor Kevin
Published: November 2024

The role of B cells is comparatively understudied in stroke broadly, but particularly in hemorrhagic stroke. There is ample evidence of a B cell response in human stroke patients, and increasing evidence that IgA B cells in particular can serve immune regulatory functions in the context of neuroinflammatory diseases broadly. Utilizing a collagenase injection-based murine model of intracerebral hemorrhage (ICH), we report that B cells are robustly recruited to the ipsilateral hemisphere as early as 2 weeks post-ICH (3248 +/- 726 cells; n=5) and increase by 8 weeks post-ICH (22816 +/- 9715, n=6). Interestingly, at 8 weeks post-ICH the majority of these cells are IgA B cells (85.1% +/- 4.5%; n=6), suggesting a possible gut origin for these cells. Indeed, single cell VDJ sequencing revealed that of 76 clones isolated from 8-week post-ICH brains, 59.6% had matching clonotypes in the small intestine, as compared to only 16.8% overlap with splenic B cells. Finally, spatial sequencing using DBiT-Seq indicates that there is ongoing tissue disruption 8 weeks post-ICH compared to mice that have undergone sham surgery. Our findings suggest that intracerebral hemorrhage induces an IgA-specific B cell response within the affected brain hemisphere, underscoring the need to better understand the role IgA B cells play in the context of neuroinflammation.
Pathogenicity of human CD91 SNPs
Authors: Sedlacek Abigail , Binder Robert
Published: November 2024

CD91, expressed by antigen presenting cells, is a necessary receptor for cancer immunosurveillance. It binds multiple ligands including immunogenic heat shock proteins (HSP) and functions as both an endocytic receptor for antigen capture and a signaling receptor to upregulate cytokines and costimulatory molecules. Due to its size, there are more than 3500 identified single nucleotide polymorphisms (SNPs) for human CD91, however the functional consequences for these SNPs have not been addressed. Thus, we sought to investigate if certain SNPs result in altered function of CD91 and if these SNPs are linked with cancer higher incidence. Using a cohort of sarcoma patients, we identified 11 SNPs that resulted in missense mutations of CD91. Correlations between these SNPs and the immunophenotype of the respective tumors has identified several SNPs which correlate with poor immune infiltration and increased tumor antigenicity. Alternatively, a small number of SNPs correlate with above average immune infiltration and reduced tumor antigenicity, potentially enhancing CD91 function. In addition to our patient data, predictive folding algorithms were used to assess the potential effects of individual SNPs on protein folding. In order to directly assess the function of these SNPs, we have generated an in vitro model system using THP1 cells. Cells carrying single CD91 SNPs will be assessed for protein expression levels, HSP binding and internalization as well as costimulatory function.
Probiotic-derived exopolysaccharide interferes with Th2 priming and inhibits allergic inflammation
Authors: Hollinger Maile , Velez Tania , Grayson Emily , Sureshkumar Rishi , Zamora-Pineda Jesus , Knight Katherine , Sperling Anne
Published: November 2024

Allergic asthma is an increasing burden in industrialized countries, where treatments are limited to treating symptoms rather than root causes. In recent years, the use of inhaled probiotic cocktails has shown promise in preventing allergic asthma. However, the mechanisms by which such probiotics prevent sensitization remain to be elucidated. In this study, we focused on exopolysaccharide (EPS) from B. subtilis, a ubiquitous soil bacterium. We found that EPS treatment prior to sensitization with house dust mite (HDM) limited lung mucus hypersecretion and eosinophilia. Furthermore, EPS treatment inhibited accumulation of TH2, but not TH1 or TH17 subsets. To determine whether EPS was affecting TH2 cell priming by dendritic cells (DCs), we compared the composition and phenotype of DCs in the lung and lung-draining lymph node (LDLN) after EPS and HDM treatment. Surprisingly, pre-treatment with EPS induced DC activation, as measured by an increase in CD86, and a decrease in the ability to uptake HDM and migrate to the LDLN. Additionally, EPS-pretreated BMDCs were unable to induce antigen-specific T cell proliferation in vitro. Together, our data suggest that EPS inhibits allergic sensitization by inducing DC activation prior to HDM exposure, thereby leading to decreased TH2 priming and allergic inflammation. Thus, we provide a potential mechanism by which inhalation of a single prebiotic molecule can prevent allergic sensitization.
DOCK8 and STAT3 cooperate to restrain IgE-inducing T follicular helper cells.
Authors: Williams Adam , Siniscalco Emily , Grassmann Jessica , Craft Joseph , Eisenbarth Stephanie , Uthaman Gowthaman
Published: November 2024

DOCK8 deficient patients have elevated food specific IgE and severe food allergy. In Dock8-/- mice, we showed that T cell intrinsic DOCK8 restrains the development of IL-13 expressing T follicular helper (Tfh13) cells that drive anaphylactic IgE production. Oral exposure to food results in tolerance, yet T cell intrinsic loss of DOCK8 (T-Dock8-/-) is sufficient to drive Tfh13 differentiation and peanut specific IgE. How DOCK8 prevents Tfh13 differentiation is unknown. In human T cells, DOCK8 promotes STAT3 phosphorylation, an inhibitor of GATA3. We show that mouse DOCK8 promotes STAT3 activity in T cells and STAT3-/- T cells differentiate into Tfh13-like cells in vitro. Yet, T cell specific loss of STAT3 (T-Stat3-/-) did not drive peanut specific IgE or Tfh13 differentiation in vivo after oral peanut. STAT3 patients have high IgE, but not severe food allergies and we found elevated levels of Tfh13s in donors with DOCK8 but not STAT3 mutations, suggesting additional mechanisms to inhibit Tfh13 differentiation. DOCK8 promotes Tregs and we show that the analogous regulatory population for Tfh cells, T follicular regulatory (Tfr) cells were reduced in T-Dock8-/- but not T-Stat3-/- mice. To overcome tolerance, we used oral peanut with cholera toxin; levels of Tfh13s and peanut specific IgE where higher in T-Stat3-/- mice than controls. We propose that DOCK8 and STAT3 cooperate in CD4+ T cells to prevent Tfh13 differentiation.
Neurokinin A reduces the influx of inflammatory cells into the skin in a mast-cell dependent manner
Authors: Barreto Joharis , Bharadwaj Shreya , Hay Ahnika , Sumpter Tina
Published: November 2024

Cutaneous mast cells promote immune responses but are also involved in reducing inflammation and in promoting immune resolution. Mechanisms initiating the anti-inflammatory function of mast cells are poorly defined. Mast cells reside in close proximity to neurons and their activation is modulated by neuropeptides, including the tachykinin family members. Neurokinin A (NKA) is an understudied tachykinin family member that limits cutaneous inflammation in a murine contact hypersensitivity (CHS) model but increases inflammation in colitis. In this study, the regulatory function of NKA in cutaneous inflammation is mechanistically evaluated using a robust model of DNFB-induced CHS. Administration of NKA prior to sensitization inhibited the expression of inflammatory cytokines and chemokines, including Il1b, Il6 and Ccl2. Correspondingly, NKA reduced recruitment of CCR2+ neutrophils and inflammatory monocytes to the skin. In mast cell-deficient Mcpt5Cre+ x Rosa26DTA and in mice in which mast cells do not produce IL-10 (Mcpt5Cre+ x Il10fl/fl) the capacity for NKA to reduce cytokines and chemokines was diminished. Lastly, in skin explant NKA diminished IL-1b and CCL2, genes upregulated after DNFB-treatment. Collectively, these data suggest that NKA reduces inflammation in the skin in a mast cell/IL-10 dependent manner and demonstrate a novel regulatory pathway for NKA in the skin that acts on mast cells to unleash their potential to reduce cutaneous inflammation.
Mice harboring a human Mediterranean G6PD gene variant causing G6PD deficiency have decreased B1b cells and atheroprotective IgM, and increased atherosclerosis.
Authors: Andrews Patrick , Srikakulapu Prasad , Zhou Yilun , Ransegnola Brett , Blackburn Cassidy , Dennis Emily , Loeb Skyler , Ozsvar Kozma Maria , Hay Ariel , Root Katherine , Marshall Melissa , Binder Christoph , Zimring James , McNamara Coleen
Published: November 2024

Epidemiological studies suggest a role of glucose 6-phosphate dehydrogenase deficiency (G6PDd) in exacerbated atherosclerosis. To date, no studies have established a causal relationship between the two. We herein report the first direct evidence of G6PDd causing atherosclerosis and deficits in B cells. Mice harboring the human Mediterranean G6PDd variant (hG6PDMed-) or the nondeficient human G6PD allele (hG6PDND) were rendered hyperlipemic via PCSK9 AAV and 12 weeks of high fat diet (HFD). Sudan-IV en face staining revealed increased atherosclerosis in the G6PDd mice when normalized to non-HDL cholesterol (hG6PDND n=9, hG6PDMed- n=8, P=0.0339). Next, flow cytometry was used to identify alterations in immune cells involved in atherosclerosis and revealed reduced atheroprotective B1b cells (CD19+B220-IgM+CD5-) in the peritoneal cavity and spleen (hG6PDND n=9, hG6PDMed- n=8, P=0.0111 & P=0.0207). B1b cells secrete atheroprotective IgM to oxidation-specific epitopes (IgMOSE), and thus ELISA was used to measure IgMOSE levels. Despite normal total IgM levels compared to non-G6PDd mice, G6PDd mice had reduced IgMOSE to CuOx-LDL and PC-BSA when normalized to total IgM at baseline (hG6PDND n=8, hG6PDMed- n=7, P=0.0289 & P=0.0022) and at 2 weeks of HFD (hG6PDND n=9, hG6PDMed- n=6, P=0.0016 & P=0.0028). Together, these data demonstrate that G6PDd worsens atherosclerosis and suggest that this effect may be partly mediated by associated deficits in B1b cells and IgMOSE.
Genetic screening to identify novel targets enhancing regulatory T-cell (Treg) phenotype.
Authors: Walsh Ariane , Kramer Marina , Wahmes Jan , Komadina Dana , Luu Sabine , Cornils Hauke , Dangl Markus , Cernecka Hana
Published: November 2024

Tregs possess immunosuppressive properties that can restore and maintain immune homeostasis via various suppressive mechanisms. A marker of Tregs is the transcription factor Forkhead box P3 (FoxP3), which regulates Treg development and function. Autoimmune patients have shown dysfunction or reduction in FoxP3+ Tregs, highlighting the critical role of these cells in preventing pathogenic immune responses. There is great interest in developing medical interventions against autoimmune diseases by enhancing Treg function and/or numbers. Currently, a major limitation to generate efficient Treg therapies is the lack of selective targets as starting points for drug discovery.

To identify novel Treg targets, Evotec developed a CRISPR/Cas9-based arrayed screening platform using human CD4+ T-cells. About 8,000 genes were screened with a FACS read-out and the effect of gene knockout (KO) on the frequency of FoxP3+ Tregs was assessed. We identified ~450 targets where the KO led to an increase of FoxP3+ cells within the CD4+ T-cell compartment, indicating their potential for drug development starting points. In addition to FoxP3, the effect of KO on CD4+ conventional as well as Treg viability, proliferation and markers such as, CTLA-4, PD-1 or CD25 was monitored. The hits are being confirmed and the impact of the target KO on Treg function and phenotype assessed in a panel of in vitro assays. The goal is to identify novel Treg targeting product candidates to treat autoimmune diseases.
Regulation of interferon alpha production by MAGUK-family protein CASK in H5N1-infected Macrophage
Authors: Huang Jing-Ying , Sung Pei-Shan , Hsieh Shie-Liang
Published: November 2024

To investigate the involvement of CASK, a MAGUK-family scaffold protein, in H5N1-infected macrophages, we generated mice with myeloid-specific CASK knockout (CASKfloxLysMCre). Using bone marrow-derived GM macrophages (BMDM-GM), we explored the impact of CASK KO on H5N1-induced cytokine storm.

We found that CASK KO in H5N1-infected BMDM-GM selectively reduces interferon alpha production by half, attributed to interference with Ifna2/4 mRNA nuclear export, as confirmed by ELISA and RNA FISH. We also found that H5N1-triggered CASK nucleus translocation mirrors responses to RIG-I and MDA-5 ligand stimulation. LC-MS-MS analysis identified HCK as a promoter of CASK phosphorylation at the S395 site, likely through CDK5. Co-overexpression of EGFP-CASK with S395A mutation and constitutively active HCK resulted in significantly reduced nuclear EGFP signal. CASK Interactome analysis (by IP-LC-MS/MS) and NCBI-interactome meta-analysis indicated that nuclear CASK is recruited to the Ifna promoter by IRF7-binding CCT4. This facilitates co-transcriptional loading of the CASK complex onto Ifna transcripts and supports its CRM-1-dependent nuclear export during H5N1 infection. In vivo studies demonstrated that myeloid-specific CASK KO exacerbates H5N1-induced lung pathology. Our results strongly suggest that CASK plays a crucial role as a host factor, with its nucleus translocation in response to H5N1 infection promoting the nuclear export of anti-viral Ifna mRNA.
The immunological aftermath of SARS-CoV-2: How prior infection impacts subsequent pulmonary injury
Authors: Roberts Lydia , Wehrly Tara , Schwarz Benjamin , Bohrnsen Eric , Shaia Carl , Bosio Catharine
Published: November 2024

Following pulmonary insults there are persistent alterations in the lung that influence responsiveness to subsequent injury. Over 600 million individuals have survived SARS-CoV-2 (SCV2) infection. Thus, the impact of SCV2 on the ability of the newly remodeled lung to handle various pulmonary challenges is of significant interest and therapeutic importance. Using the K18-hACE2 mouse model, we found that SCV2 resulted in an expanded pool of recruited alveolar macrophages along with persistent lymphocytic perivascular cuffs up to 90 days post-infection in the absence of detectable viral antigen. SCV2 resolved lungs had only modest changes following an OVA-specific allergic response compared to mock infected controls. In contrast, despite no differences in bacterial burdens, SCV2 experienced lungs had significantly less inflammation following infection with Bordetella pertussis compared to mock controls. However, this was not consistent for all bacterial driven inflammatory responses. Specifically, we observed an exacerbation of LPS derived inflammation in mice that had recovered from SCV2. The balance of inflammatory responses in these models was associated with specific changes in lipid mediators (LMs). Together, our data reveal the impact of SCV2 infection on the ability of the lung to optimally respond to a variety of inflammation provoking stimuli and underscore the potential role of LMs in the trained innate immune response.
Aryl hydrocarbon receptor ligand supplementation impacts colitis-associated depressive-like behavior
Authors: Bogdon Raymond , Roark Kasie , Saxena Archana , Mitchell Chandani , Hailey Michele , Staley Shanieka , Nagarkatti Prakash , Nagarkatti Mitzi , Busbee Philip
Published: November 2024

Colitis is an inflammatory bowel disease (IBD) with increased incidences of depression in patients. The mechanisms that define this comorbidity remain poorly understood. Our lab previously showed supplementation with indole-3-carbinol (I3C), an aryl hydrocarbon receptor (AhR) ligand, can reduce disease severity in models of colitis. We aimed to determine the behavioral impact I3C supplementation has on colitis-induced depression via alterations in the gut metabolome. Colitis was induced in using the dextran sodium sulfate (DSS) method and treatment groups were given a regimen of 40 mg/kg I3C as previously reported. Untargeted metabolomic studies revealed quinolinic acid (QA), a metabolite produced by the kynurenine (KYN) pathway and linked to depression, was found to be significantly reduced in I3C-treated mice when compared to colitis controls. To determine the effects of I3C and QA modulation on depressive-like behavior, colitis mice were treated with either I3C or an inhibitor of a major enzyme involved in QA production. Depressive-like behavior was measured using the Tail Suspension Test (TST) method, along with evaluation of neural biomarkers of depression (dopamine, BDNF, GFAP) and stabilization of the blood brain barrier (BBB). Results showed I3C reduced depressive-like behavior and altered select biomarkers associated with depression. These studies provide evidence that AhR can be a potential therapeutic target for both colitis and colitis-associated depression.
Determination of IgA antibodies colostrum against Mycobacterium antigens.
Authors: Romero-Ramírez Héctor , Baltierra-Uribe Shantal Lizbeth , Pedraza-Romero Martha Raquel , Barreto-Lesmes Natalia , Sandoval-Montes Claudia , Santos-Argumedo Leopoldo
Published: November 2024

Colostrum, the first milk secretion, is characterized by a high concentration of immunologically active molecules with neutralizing and antibacterial capabilities. Mycobacterium tuberculosis is a microorganism primarily infecting the lungs but can also affect non-pulmonary tissues. Despite a BCG vaccination coverage exceeding 97% in Mexico, pulmonary and meningeal tuberculosis cases have shown a yearly increase. This study aimed to determine the presence of IgA antibodies against soluble extracts from H37Rv M. tuberculosis and BCG antigens in colostrum samples from Mexican mothers. Quantification of IgA subclasses and colostrum reactivity to Mycobacterium were performed via ELISA using 106 colostrum samples from healthy Mexican mothers. Results indicated that 87.7% of the analyzed colostrum samples contained total IgA antibodies, with 77% exhibiting IgA2 antibodies against H37Rv, of which 22.6% and 17.9% showed high titers, respectively. Among the H37Rv antigen-positive samples, 80% recognized the BCG antigen. In conclusion, most of the analyzed colostrum samples from women contained IgA antibodies against M. tuberculosis antigens and demonstrated BCG reactivity. Most of these antibodies belonged to the IgA2 subclass, suggesting a potential protective effect against tuberculous meningitis in nursing infants.
Chimeric Antigen Receptor T-Cell Recruitment and Killing can be Evaluated on an Organ-Chip model system
Authors: MEHTA ANITA , Jacob Irene , Downs Isaac , Kanellias Marianne , Cespedes Adriana , Heng Anthony , Ewart Lorna
Published: November 2024

Objective. Chimeric antigen (CAR) T-cell therapies for solid tumors are challenging to develop due to the lack of human relevant models that adequately capture the mechanisms of CAR T-cell recruitment-a critical yet often overlooked rate-limiting step. The current study aimed to develop a novel system for investigating both the recruitment and killing capacity of CAR T cells in Organ-Chips.

Methods. Chip-S1™ Stretchable Chips (Emulate) were seeded with a human HER2+ non-small cell lung cancer cell line (NSCLC) in the top channel and lung microvascular endothelial cells in the vascular (bottom) channel to create a tumor-vascular interface. Both channels were primed with an NSCLC-specific mix of chemokines and cytokines. HER2-specific CAR T cells were perfused through the vascular channel. Total CAR T-cell recruitment into the top channel and CAR T-mediated killing were measured over 48 hours. The IL-2 cytokine was also co-administered with CAR T cells as immunotherapy.

Results. HER2 CAR T cells demonstrated significant antigen-specific killing when recruited to the top channel as measured by caspase activity and cytokine release compared to the control. Furthermore, IL-2 co-therapy increased CAR T-cell recruitment and antigen-specific killing compared to the control group.

Conclusion. These findings suggest that human-centric Organ-Chips can evaluate CAR T-cell therapies for solid tumors by integrating the critical rate-limiting steps of CAR T recruitment and killing.
Transcriptional profiling of primary human-skin derived mast cell activation by various MRGPRX2 agonists and inhibition with EVO756
Authors: Bagchi Sreya , Patel Jeegar , Harden Jamie
Published: November 2024

Mas-related G-protein coupled receptor X2 (MRGPRX2) is a GPCR expressed predominantly by mast cells and has been implicated to play an important role in diseases such as chronic spontaneous urticaria, atopic dermatitis, and asthma. Cationic ligands, either endogenous or exogenous, can activate the receptor and induce IgE-independent mast cell activation, resulting in allergic responses and non-histaminergic itch. We have previously shown that EVO756, a novel small molecule antagonist of MRGPRX2, demonstrates potent, concentration dependent inhibition of MRGPRX2 mediated activation of transfected CHO-cells, ROSA and LAD2 mast cell lines, and primary human skin-derived mast cells. Furthermore, EVO756 does not affect mast cell viability. Here, we describe, for the first time, the transcriptional landscape of primary human skin-derived mast cells cultured in the presence of several MRGPRX2 agonists and analyze how the mast cell phenotype is altered in response to MRGPRX2 inhibition by EVO756.
Exploring Pre-Pandemic SARS-CoV-2-Specific B Cell Responses in the West African Population.
Authors: Sharma Pragati , Hurtado Jonathan , Messmer Terrence , Briney Bryan
Published: November 2024

The emergence of the novel coronavirus, SARS-CoV-2, during the COVID-19 pandemic prompted investigations into pre-existing immunity in human populations. Despite being a new coronavirus, individuals had previous exposure to common seasonal human coronaviruses. In this study, we examined SARS-CoV-2-specific memory B cells in six pre-pandemic Lassa virus-infected patients from West Africa.

By incorporating a SARS-CoV-2 Spike Antigen (WA-1 strain) into a broader antigen panel that included Lassa virus GPC and a control, we identified and sorted antigen-positive B cells. Further analysis revealed a distinct population of SARS-CoV-2-specific memory B cells. Native paired monoclonal antibody (mAb) sequences were recovered from 598 SARS-CoV-2 Spike-positive B cells, demonstrating a surprisingly high frequency of pre-existing SARS-CoV-2-specific B cells.

Remarkably, Spike-specific mAbs were recovered from all six donors, each contributing to at least one expanded clonal lineage. Notably, most IgM antibodies belonged to the same lineages. These findings suggest a notable prevalence of pre-existing immunity to SARS-CoV-2 in pre-pandemic patients, potentially influencing susceptibility to the virus and contributing to adverse COVID-19 outcomes. This study provides insights for risk management and supports the development of universal coronavirus vaccines by highlighting the complex interplay between pre-existing immunity and susceptibility to SARS-CoV-2.
Whole slide imaging modes for Imaging Mass Cytometry reveal cellular diversity of the tumor immune microenvironment in mouse glioblastoma
Authors: Motta Vinicius , Raza Qanber , Zabinyakov Nick , Pfister Thomas , Parsotam Nikesh , Howell David , Lim Liang , Loh Christina
Published: November 2024

Mouse models are used for translational research and permit detection of therapeutic-induced immune-related modification in the tumor immune microenvironment (TIME). Imaging Mass Cytometry™ (IMC™) is a spatial biology technique capable of quantitative evaluation of multiparametric tumor and immune cell composition in brain tissue.

Here we demonstrate the application of a 40-marker panel composed of tumor and immune markers on mouse normal and glioblastoma (GBM) tissue using two new whole slide imaging methods for IMC. Tissue mode (TM) rapidly scans the entire tissue followed by pixel-based analysis (PBA) to uncover spatial distribution of relevant markers. Preview mode (PM) samples the entire tissue at predefined spacing to rapidly capture a low-resolution image of all expressed markers, improving informed identification of areas for high-resolution cell mode (CM) imaging on the same slide.

TM imaging with PBA provided quantitative spatial expression patterns of tumor and immune markers across the GBM tissue. Necrotic cores, areas with high immune infiltration, extracellular matrix deposits, and activated tumor cells were quantified. PM in combination with CM imaging and single-cell analysis revealed extensive tissue vascularization, replicating tumor cells, and a variety of infiltrating immune cells.

Overall, we highlight the capability of IMC to provide quantitative insights into the spatial biology of the TIME.

For Research Use Only. Not for use in diagnostic procedures
RNA sequencing reveals differential gene expression by hypoxia in γδ T cells.
Authors: Park WonHyung , Lee Heung Kyu
Published: November 2024

Hypoxia not only enhances tumor survival but also inhibits the anti-tumor response. Particularly, hypoxia is known to promote exhaustion of T cells. So, overcome hypoxia becomes imperative, especially in T cell therapy. One prominent T cell therapy is CAR T cell therapy. However, it has limitation because of autologous applications. Recent attention has shifted towards γδ T cell therapy, especially when autologous options are limited by senescence or expansion suppression in tumor patients. In the context of γδ T cell therapy, investigating the effects of hypoxia on γδ T cells is pivotal for optimizing their functionality. Therefore, this study aims to investigate how γδ T cells change under hypoxic conditions. The ultimate goal is to uncover specific targets that can mitigate the negative consequences of hypoxia, thereby improving the overall efficacy of γδ T cell-based therapies.

γδ T cells is stimulated with γδ TCR antibodies for ex vivo expansion. Subsequently, total RNA is purified from γδ T cells following a 24-hour culture under hypoxic condition (1.0% O2). Bulk RNA sequencing is then conducted on the collected samples.

Through bulk RNA sequencing, it is established that hypoxia inhibits the proliferation of γδ T cells. This finding holds particular significance in the context of therapy maintenance. Furthermore, the analysis reveals upregulation in cytokine genes, notably Il10 and Tgfb3, suggesting a potential regulatory role in anti-tumor immune responses.
IL-33 mediated regulation of Tregs is tissue specific and dispensable in the omental visceral adipose tissue.
Authors: Desai Sweta , Meza-Perez Selene , Liu Mingyong , Randall Troy
Published: November 2024

The omentum, a visceral adipose tissue in the peritoneal cavity, is a frequent site of metastasis for gastric, ovarian and various other cancers. Currently available therapies are ineffective in providing long-term tumor control, with a higher plausibility for relapse. The rapid growth of tumors in the omentum is supported by the expansion and accumulation of immunosuppressive Tregs. Additionally, we detect persistent and continuously expanding tumor specific CD8 T cells despite Treg accumulation in the omentum. Omental Tregs are a specialized population of visceral adipose tissue associated Tregs (VAT-Tregs). Some of them express the interleukin-33 receptor ST2, suggesting IL-33 produced in the omentum promotes Treg mediated immunosuppression. However, our findings show that absence of IL-33 mediated signaling in VAT-Tregs does not alter their accumulation or expansion in the tumor bearing omentum. Moreover, we demonstrate that absence of IL-33 signaling in the omental VAT-Tregs does not reduce tumor growth, enhance the persistence of tumor specific CD8 T cells, or CD8 T cell mediated tumor killing. Our results show that IL-33 is dispensable for Treg maintenance or accumulation in the omental visceral adipose tissue. We are investigating the pathways involved in regulation of Tregs in the omentum.
Gelatinases are important for CD4+ T-cell metabolic reprogramming
Authors: Protz Ella , Onwuha-Ekpete Lillian , Fields Gregg
Published: November 2024

Matrix metalloproteinases (MMP) are a family of proteolytic enzymes essential for various processes such as development and healing. Alternatively, the dysregulation of the gelatinases, MMP-2 and MMP-9, is implicated in immune cell migration and inflammation associated with multiple sclerosis (MS). MS is an autoimmune disease in which CD4+ T-cells play a key role both in humans and its animal model, experimental autoimmune encephalomyelitis (EAE). CD4+ T-cells are known to rely on both oxidative phosphorylation and aerobic glycolysis during various stages of activation. We have recently demonstrated that gelatinase inhibition can impact CD4+ T-cell activation and mitigate EAE progression. The present study utilized the Seahorse assay to characterize the cellular energetics of CD4+ T-cells in response to gelatinase inhibition. The resultant data suggests that gelatinases are important for both oxidative phosphorylation and the effective induction of glycolysis in CD4+ T-cells.
Signaling flow cytometry: One-step staining for phenotyping and functional characterization of immune cells
Authors: Alonzo Emily , Manning Christopher , MacDonald Robert , Cunningham Lori
Published: November 2024

Understanding how signaling pathways in immune cells are modulated by various perturbations can reveal potential therapeutic targets and uncover mechanisms of therapeutic response. Immune cells are typically observed in the context of a heterogeneous mixture of cells, and techniques that enable single cell analysis, such as flow cytometry, are essential. However, observing changes in pathway activation by flow cytometry can be challenging as antibodies used for immune phenotyping are generally validated in live cell assays, while antibodies targeting signaling readouts usually require fixation and permeabilization. To simplify multiplexing with signaling and phenotyping readouts, we tested many commonly used immune phenotyping antibodies in multiple fixation and permeabilization protocols. We compiled the data into a table to enable design of flow cytometry panels that include both phenotyping and signaling antibodies and can be used as a single staining antibody mix on cells following fixation and permeabilization. To illustrate this functionality, we designed and generated data for two different panels on fixed, permeabilized cells. Using one panel, we identify B cells among PBMCs and observe phospho-Syk and phospho-CD79a following BCR activation. With the other panel, we compare phospho-STING across different myeloid subsets from PBMCs treated with the STING ligand cGAMP and observe upregulation of STING activation in classical monocytes, with very little change among DCs.
Nociceptor sensory neurons promote pneumonic sepsis during carbapenem-resistant Klebsiella pneumoniae lung infection.
Authors: Joshi Prabhu , Adhikari Sandeep , Judd Abigail , Baral Pankaj
Published: November 2024

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a leading cause of Gram-negative pneumonia and pneumonic sepsis for which limited therapeutic options are available. The lungs are densely innervated by nociceptive sensory neurons (nociceptors), which mediate breathing, cough, and bronchoconstriction. However, the role of these neurons in host defense against CRKP-induced pneumonic sepsis is unknown. We found that nociceptors play a host deleterious role to promote pneumonic sepsis. Nociceptor-ablated mice showed an enhanced survival and bacterial clearance after CRKP lung infection. Furthermore, nociceptors ablation resulted in the increased influx of neutrophils and Ly6Chi monocytes and cytokine induction. Depletion of Ly6Chi monocytes, but not of neutrophils, impaired lung bacterial clearance and bacterial dissemination in nociceptor-ablated mice, indicating that Ly6Chi monocytes are a critical cellular population downstream of nociceptors to regulate pneumonic sepsis. Moreover, nociceptive neuropeptide calcitonin gene-related peptide (CGRP) suppressed induction of reactive oxygen species and intracellular bacterial killing. Collectively, our data suggest that the nociceptor neuronal signaling could be a therapeutic target for treating multidrug-resistant CRKP lung infections and pneumonic sepsis.

Supported by grants from NIH/NIGMS P20GM113117, American Lung Association, and JCRC Heart Research Award.
Investigating the role of Natural Killer cells in the control of latent Epstein-Barr virus infection
Authors: Pena Josselyn , Zhang Wenming , Esquivel Carlos , Martinez Olivia , Krams Sheri
Published: November 2024

Epstein–Barr virus (EBV) is a ubiquitous gamma-herpesvirus that persists as a chronic, asymptomatic infection in over 90% of the adult population with the virus transitioning to latency in a subset of memory B cells. Failure to control latent EBV can result in a variety of malignancies, including lymphoproliferative diseases. Studies in experimental models and humans suggest that NK cells are critical in the host defense against EBV. Previously, we demonstrated NK cells expressing the inhibitory receptor NKG2A were specifically able to recognize and kill autologous EBV-lymphoblastoid cell lines (EBV-LCL). We also showed that HLA-E-presented peptides derived from latent EBV proteins can impair NKG2A recognition and signaling, potentially leading to NK cell activation. To better understand the phenotype of NK cells that respond to latent EBV, we generated a panel of EBV-LCL and performed co-culture experiments with primary NK cells and autologous EBV-LCL. We developed a novel mass cytometry panel and found that in addition to NKG2A, EBV-LCL-responsive NK cells express increased NKp30 and CD32 and decreased CD16 which may contribute to NK cell activation and killing of EBV-LCL. Further, we generated HLA-E knockout EBV-LCL using CRISPR and determined that autologous NK cell cytotoxicity increased, suggesting that disrupting the NKG2A:HLA-E immune checkpoint axis enhances killing of EBV-infected B cells and would be an attractive therapeutic option for EBV-associated malignancies.
NFIL3/E4BP4 regulates the developmental stage-specific acquisition of basophil function
Authors: Park Jiyeon , Cho Yuri , Yang Dongchan , Yang Hanseul , Lee Daeyoup , Kubo Masato , Kang Suk-Jo
Published: November 2024

Basophils are essential effector cells in many allergic disorders, but the terminal developmental processes through which basophils acquire their unique functional properties are not fully understood. Here, we report the identification of a novel population of late-stage mitotic basophil precursor cells expressing high levels of the IgE receptor FcεRI alpha chain, termed ‘transitional basophils’ (tBasos). These tBasos produce type 2 cytokines more robustly than mature basophils, despite being less responsive to IgE stimulation. Transcriptome analysis revealed dynamic gene expression shifts during basophil development that enables precursor stages to proliferate and late-stage basophils to perform effector functions. Furthermore, we found that the transcription factor NFIL3/E4BP4 is essential for basophils to acquire IgE receptor-mediated signaling, cytokine secretion, and degranulation, critical basophil functions, as well as for the regulation of basophil-mediated allergic skin reactions. Collectively, our results unveil a new population of mitotic basophil precursors and elucidate the role of NFIL3/E4BP4 in establishing basophils’ IgE-mediated functions.
Inhibition of neutrophil elastase by the bacterial serine protease inhibitor ecotin in cystic fibrosis airway samples
Authors: Son Yeongseo , Fantone Kayla , Nothaft Harold , Stecenko Arlene , Szymanski Christine , Rada Balazs
Published: November 2024

Cystic fibrosis (CF), a prevalent fatal genetic condition in North America, disrupts respiratory function by impeding chloride ion transport across mucosal surfaces. This leads to the thickening of mucus, resulting in constant lung inflammation and elevated levels of neutrophils. Neutrophil elastase (NE), a potent serine protease crucial for host defense, is released by these neutrophils. However, in excess, NE can cause detrimental host tissue damage. Abundant in CF airways, NE is a key predictor of lung disease progression. While past studies have explored targeting NE in CF, no optimal inhibitors have reached clinics. Our study tested ecotin, a bacterial periplasmic serine protease inhibitor, for its potential to impede NE activity in CF airway samples and curb NE release from human neutrophils. Results show that ecotin significantly reduces NE activity in sputum samples obtained from several CF patients. Additionally, we found that ecotin inhibits NE release from neutrophils exposed to CF lung stimuli like Staphylococcus aureus bacteria. These results establish ecotin as a promising NE inhibitor in CF with clinical potential.
Adjuvant conditioning reprograms the innate and adaptive immune system through the NLRP3/IL-1 pathway
Authors: Boccia da Costa Thais , Pan Weikang , Fattori Victor , Rogers Michal , Zanoni Ivan , Cuenca Alex
Published: November 2024

While adjuvants are typically employed to boost immune responses, we have shown that multiple administrations of the adjuvant alum, adjuvant conditioning (AC), creates an immunosuppressive milieu that delays allograft rejection by expanding myeloid-derived suppressor cells (MDSCs). Since alum activates NLRP3, we performed AC in C57BL/6 and NLRP3-/- mice, with or without Anakinra before analyzing MDSC populations and immunizing mice with OVA. AC induces the expansion of highly suppressive MDSCs in the spleen of C57BL/6 mice, which is significantly decreased in the absence of NLRP3 (fold increase 2.1 vs 0.72, p<0.0001), or when IL-1 signaling is blocked (fold increase 4.46 vs 0.48, p<0.0001). AC before OVA immunization significantly decreased the proliferation of OVA-specific CD4 T cells and OVA-specific IgG titers compared to saline controls (p=0.0057, and p<0.0001 respectively). In the absence of NLRP3, we observed the increased expansion of Th1, Th2, and Th17, and IFN-g and IL-4 production in OVA-specific CD4 T cells from AC-NLRP3-/- mice, and also higher OVA-IgG and IgE titers compared to the saline group. In a pancreatic alloislet transplantation model, AC-treated NLRP3-/- mice showed no difference in allograft survival compared to saline-treated controls, highlighting the requirement for NLRP3 signaling in AC. In summary, these data suggest that AC induces an immunosuppressive milieu through the NLRP3/IL-1 axis that shapes innate and adaptive immunity.