bluuebunny/crossref_metadata_2025_split huggingface dataset:
CRISPR Screens Identify Key Regulators of NK Cell Cytotoxicity in Cancer Therapy
Authors: Rethacker Louise , Romero Hugo , Dulong Joshua , Sasseville Marc , Lalonde Marie-Eve , Gadoury Christine , Beland Kathie , Beausejour Christian , Marcotte Richard , Haddad Elie 
Published: November 2024

Abstract
Natural Killer (NK) cells are innate lymphocytes that exhibit cytotoxic activity against cancer cells and are associated with a good prognosis when present in solid tumors. In the past, NK cells have been used in adoptive cell transfer to treat various cancers with modest relative success. More recently, NK cells modified with chimeric antigen receptor (CAR-NK cells) are being explored as a potential off-the-shelf therapy. However, mechanisms preventing the full cytotoxic anti-tumor activity of NK cells remain understudied.
To unveil the key genes that govern the activation and inhibition of the cytotoxic activity of NK cells towards cancer cells, we performed whole-genome CRISPR screens in NK cells. Primary activated NK cells from the NK-cell Activation and Expansion System (NKAES) were transduced with the Yusa CRISPR library and then electroporated with Cas9 protein, inducing the knockout (KO) of 18,010 genes. These NK cells were exposed to the colorectal cancer cell line HT-29 for 4 hours. After co-culture, NK cells were sorted based on the expression of the degranulation marker CD107a and the production of IFNg, both associated with NK cell activation. To identify genes involved in the inhibition of NK cell activity, we will compare sgRNA enriched in CD107a+ and IFNg+ cells to unactivated negative cells.
In turn, these genes can be therapeutically targeted or genetically modified in future iPSC-derived NK and CAR-NK cells to improve the outcome of cancer patients.
Natural Killer T cell subsets exhibit tissue-specific transcript factor expression following bacterial challenge
Authors: Rodriguez Luisa , Torres Cameron , Spencer Charles 
Published: November 2024

Abstract
Natural Killer T cells are a population of lymphocyte immune cells that have regulatory effects that influence the inflammatory response. There are two subsets of NKT cells, type I and II which can be experimentally differentiated based on their antigen recognition of CD1d-tetramers loaded with α-galactosylceramide (αGalCer-tetramer). NKTTK cells become signaled upon infection of pathogens and have been observed to respond to pathogenic challenges. Francisella Tularensis (Ft) is a bacterial zoonotic bacterial pathogen that, when transmitted to human hosts, can cause severe clinical disease. Infection with Ft leads to an over-response of the inflammatory immune reaction, causing dysregulation of cytokines, known as the cytokine storm. Type I NKT cells have been observed to express exhibited transcription factor profiles similar to T helper cells such as T-bet, GATA3, and RORγT, that represent Th1, Th2, or Th17 phenotypes. In this study, we utilize flow cytometry to characterize both type I and type II NKT transcription factor profiling in response to Ft infection. We identify Th17 responses by type II in respiratory tularemia and Th1 responses by type I in ulceroglandular tularemia to be key factors contributing to disease outcome.
Blood immune receptor repertoire and transcription profiling to characterize disease progression in a murine tuberculosis model
Authors: Kobzik Lester , Chenchik Alex , Hu Dongfang , Makhanov Mikhail , Paraiso Kitt , Ghias Khadija , Diehl Paul , Yabaji Shivraj , Kramnik Igor 
Published: November 2024

Abstract
Murine models of tuberculosis (TB) can reveal mechanisms and allow testing of drug/vaccine interventions. Current approaches often rely on the evaluation of lung lesions after euthanasia, with a large number of animals needed for time course and dose-response studies. Measuring blood biomarkers in small volumes obtained serially could provide useful insights and reduce the number of animals needed. We evaluated the utility of small-volume blood samples for profiling T- and B-cell receptor repertoires and transcriptomes in mice (n=16) at different stages of TB progression (early, middle, and advanced—defined by lung histopathology and mycobacterial loads). We isolated RNA from 100 uL of whole blood for TCR/BCR profiling using the Cellecta DriverMap adaptive immune receptor (AIR) assay as well as transcriptome quantitation of ~4700 key mouse genes using a targeted sequencing panel. The results showed substantial changes in clonotype usage and diversity over the course of TB disease, most prominent in the BCR IgH (e.g, top 10 clonotype abundance count in control, early, advanced disease, respectively: 62 + 47, 1792 + 589, 3559 + 286, p &lt; .01, mean + SD, n=3-4). Blood transcriptome profiling showed abundant up-regulation of immune and inflammatory genes (e.g, up-regulated genes vs control: 182 (early), 382 (advanced), q.05 FC1.5). The results support direct testing of non-terminal serial blood samples to characterize disease progression & therapeutic or vaccine interventions.
CXCR6+ polyfunctional CD4 T cells are essential for protective immunity against Chlamydia in the female reproductive tract
Authors: Mercado Miguel Antonio , Li Qiang , Kim Yejin , Li Lin-Xi 
Published: November 2024

Abstract
The sexually transmitted Chlamydia infection has been increasing in the last few decades in the United States and worldwide. A better understanding of CD4 T cell effector function during infection is essential to develop a protective vaccine. Recently, we reported that mice lacking the transcription factor basic helix-loop-helix family member e40 (BHLHE40) had a significant defect in their ability to clear infection from the female reproductive tract (FRT). An increase in IL-10-producing T regulatory type 1 (Tr1) cells and a decrease in polyfunctional CD4 T cells co-producing IFN-γ, IL-17A, and GM-CSF in Bhlhe40-/- mice correlated with greater bacterial burden and reduced protective immunity in these mice. Using scRNA-seq, we found that Cxcr6, which encodes the chemokine receptor CXCR6, was enriched in the polyfunctional CD4 T cell clusters in WT mice but not in the stem-like clusters in Bhlhe40-/- mice. Here, we found that both CXCR6- and CXCR6+ CD4 T cells were detected in the FRT following infection, but CXCR6+ CD4 T cells contained most of the polyfunctional T cells. Notably, anti-CXCR6-treated mice exhibited a marked decrease in polyfunctional CD4 T cells, particularly, those capable of producing IL-17A. This corresponded to increased bacterial burden and delayed clearance following intravaginal infection. Overall, our results demonstrate that CXCR6 marks a polyfunctional CD4 T population required for protective immunity against Chlamydia in the FRT.
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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

Abstract
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.