bluuebunny/crossref_metadata_2025_split huggingface dataset:
Application of individual HLA-binding prediction thresholds increases the detection of target cell surface-presented HPV16 E6- and E7-derived epitopes as a basis for therapeutic HPV vaccine design
Authors: Bonsack Maria , Mohan Nitya , Öhlenschläger Kathrin , Lotsch Catharina , Heinze Julia M. , Schmitt Janika , Hoppe Stephanie , Förster Jonas , Blatnik Renata , Riemer Angelika B. 
Published: January 2023

Abstract
For the development of a therapeutic vaccine against human papillomavirus (HPV), viral epitopes bona fide presented on the surface of HPV-transformed cells need to be identified. The constitutively expressed HPV oncoproteins E6 and E7 represent ideal targets for immunotherapeutic vaccination approaches. However, viral epitopes are low abundant and human leukocyte antigen (HLA)-restricted, posing a challenge for developing peptide-based vaccines.
For the identification of novel epitopes, in silico HLA-binding prediction was used to preselect HPV16 E6- and E7-derived peptides for 7 major HLA-types representing 5 HLA supertypes. Based on performance evaluation of predictors, new individual binding affinity thresholds were applied. HLA-binding of synthesized predicted binders was validated in vitro in competitive cellular binding assays. Interferon-γ ELISpot assays performed with blood samples from HLA-characterized healthy donors identified memory T cell responses against HLA-binding HPV peptides. T cells of responding donors were further characterized for epitope-specific mediation of specific target cell lysis by CD8+ CTLs using a flow cytometry based cytotoxicity assay. Complementary, an immunopeptidomics approach was used to determine the surface presentation of epitopes on HPV16-transformed cancer cells. Targeted liquid chromatography-mass spectrometry was capable of detecting epitopes predicted within the new individual affinity thresholds but missed by common thresholds.
Completing the identification of bona fide target cell surface-presented E6- and E7-derived T cell epitopes will provide a map of targets for the development of a therapeutic vaccine to treat HPV16-induced malignancies.
Preclinical studies of the off-target reactivity of AFP158-specific TCR engineered T cells
Authors: He Yukai , Cai Lun , Caraballo-Galva Leidy D , Peng Yibing 
Published: January 2023

Abstract
Autologous T cells engineered with T receptor genes (TCR-T) are being studied to treat cancers. We have recently identified a panel of mouse TCRs specific for the HLA-A0201/alpha fetoprotein epitope (AFP158) complex and have shown that the TCR-engineered human T cells can eradicate hepatocellular carcinoma xenografts in NSG mice. In this current study, we evaluate their off-target toxicity. We found that 3 AFP158-specific TCR-Ts could be cross-activated by ENPP1436 peptide and that the TCR3-Ts could also be activated by another off-target peptide, RCL1215. However, compared to AFP158, it requires 250 times more ENPP1436 and 10,000 times more RCL1215 peptides to achieve the same level of activation. The EC50 of ENPP1436 peptide for activating TCR-Ts is approximately 17–33 times higher than AFP158. Importantly, the ENPP1+ tumor cells did not activate TCR1-Ts and TCR2-Ts, and only weakly activated TCR3-Ts. The IFNγ produced by TCR3-Ts after ENPP1+ cell stimulation was 22 times lower than that after HepG2 cells. And, all TCR-Ts did not kill ENPP1+ tumor cells. Furthermore, ectopic over-expression of ENPP1 protein in HLA-A0201+ tumor cells did not activate TCR-Ts. In silico analysis showed that the ENPP1436 peptide affinity for HLA-A0201 was ranked 40 times lower than AFP158 and the chance of ENPP1436 peptide being processed and presented by HLA-A0201 was 100 times less likely than AFP158. In contrast, the two off-targets (Titin and MAGE-A3) that did cause severe toxicity in previous trials have the same or higher MHC-binding affinity and the same or higher chance of being processed and presented. In conclusion, our data showed that these TCR-Ts, especially TCR1-Ts and TCR2-Ts, will unlikely cause significant off-target toxicity.
Improved trafficking and tumor control with regional, pressure-enabled drug delivery of anti-CEA CAR-T cells for colorectal liver metastases in a murine model
Authors: Chai Louis F. , Hardaway John C. , Heatherton Kara R. , O’Connell Kyle P. , LaPorte Jason P. , Guha Prajna , Saied Abdul , Knight Robert , Jaroch David , Cox Bryan F. , Katz Steven C. 
Published: January 2023

Abstract

Introduction
Efficacy of cellular immunotherapy for liver metastases (LM) has been limited by poor trafficking and elevated intra-tumoral pressures. In a phase 1b study, we found that regional, pressure-enabled drug delivery (PEDD, TriSalus Life Sciences, Inc.) improved LM CAR-T delivery. We hypothesized that PEDD of CAR-T in a murine model can improve LM control.

Methods
LM bearing CD45.2 mice were treated with CD45.1 anti-CEA CAR-T via systemic delivery (SD, tail vein) or regional delivery (RD, portal vein) at low pressure (LPRD) vs. high pressure (HPRD) to model PEDD. Flow rates were calibrated to achieve LPRD or HPRD. Tumor bioluminescence (TB), flow cytometry (FC), immunofluorescence (IF), & quantitative polymerase chain reaction (qPCR) were assessed on various post-treatment days (PTD).

Results
FC of liver non-parenchymal cells on PTD7 showed increased CAR-T density in HPRD & LPRD (9.4% & 11.0%, p = 0.03 & 0.002) vs. SD (3.7%). qPCR of the CAR transgene supported this observation, but was not significant (HPRD 135.8, p = 0.76, LPRD 108.6, p = 0.40 vs. SD 69.8). TB was lower in RD vs. SD up to PTD7 (log2 fold-change HPRD −1.3 & LPRD −0.9 vs. SD 4.3, p = 0.005 & 0.006). Tumor proliferation by Ki67 IF was significantly reduced with RD vs. control (HPRD & LPRD, p &lt; 0.001 & p = 0.05) with HPRD superior to LPRD (p = 0.03). Serum liver enzymes were not significantly elevated using HPRD vs. LPRD at PTD7 (AST/ALT 154.7/36.3 vs. 139.1/29.1, p = 0.84/0.67), but did rise at PTD17, correlating with tumor progression (AST/ALT 800.7/229.3 vs. 387.5/139.5, p = 0.10/0.28).

Conclusions
RD increased LM CAR-T delivery and HPRD showed evidence of improved CAR-T trafficking and tumor control relative to LPRD. Further clinical testing of PEDD is planned for LM cellular therapy.
Engineering CAR T cells with a GM-CSF:IL-18 cytokine switch receptor results in potent antitumor activity in solid tumors
Authors: Lange Shannon Marie , Sand Laurens , Gottschalk Stephen 
Published: January 2023

Abstract
Immunotherapy in the form of chimeric antigen receptor (CAR) T cells has emerged as a promising treatment option for various cancers following its success for the treatment of B cell hematological malignancies. However, the solid tumor microenvironment (TME) has presented many challenges to CAR T cell efficacy and persistence, in part due to lack of T cell-supportive cytokines at the tumor site. We hypothesized that we could improve the efficacy of CAR T cell infusion by engineering a self-sustaining cytokine receptor that would fuel CAR T cells in the TME. One cytokine that is abundantly produced by CAR T cells upon stimulation and is abundant in the TME is the myeloid cytokine GM-CSF. We exploited this biology to design a chimeric cytokine switch receptor that would bind GM-CSF, but signal intracellularly through the IL-18 receptor (GM18). This switch receptor encoded by a retroviral vector was transduced into second generation CAR T cells targeting different solid tumor antigens in order to test the effect of GM18 in multiple model systems. In culture, GM18-expressing CAR T cells initially killed tumor cells to the same degree as unmodified CAR T cells, but displayed greater expansion when repeatedly challenged with tumor cells over time. In vivo, GM18+ CAR T cells exhibited enhanced antitumor activity compared to unmodified CAR T cells in NSG experimental models of solid tumors. These findings paralleled greater expansion and persistence by GM18+ CAR T cells, leading to improved survival. Overall, this work demonstrates the GM18 cytokine switch receptor as an advantageous modification to CAR T cells in solid tumors, suggesting this receptor could be a novel mechanism to modify existing cellular therapies to enhance efficacy in solid tumors.
Type I interferons mediate pathogenic reactive oxygen species during neonatal influenza virus infection
Authors: Kumova Ogan K , Carey Alison J 
Published: January 2023

Abstract
Infants less than six months of age are particularly susceptible to respiratory viral infection-associated morbidity and mortality. Type I Interferons, IFN I, a potent anti-viral cytokine which controls viral replication, can also result in the production of pathogenic reactive oxygen species, ROS. Infants are particularly susceptible to ROS because of immature antioxidant production, such as superoxide dismutase 3, and demonstrate increased pathology under oxidative stress conditions. Using our previously established neonatal mouse influenza virus model, we sought to determine if IFN I was protective or deleterious in neonatal influenza virus infection. Three-day old neonatal IFN I receptor, IFNAR-deficient and C57Bl/6 mice were infected with PR8, H1N1 influenza virus and tracked for survival. IFNAR deficient neonatal mice had improved survival, 80% survival versus 20% survival in C57Bl/6 pups. To determine the mechanism of IFN I toxicity, pulmonary type II epithelial cells were isolated 48 hours post-infection and IFNb was applied to the epithelial cells ex vivo. A flow cytometric CellROX assay was performed to determine production of ROS. When exposed to IFNb, neonatal lung epithelial cells produce more ROS than adult lung cells. To determine if an antioxidant could mitigate oxidative stress during in vivo neonatal influenza virus infection, 3 day old murine neonates were treated with an antioxidant, N-acetyl cysteine, NAC, after influenza virus infection. Survival improved from 10% in sham-treated pups to 60% in NAC-treated pups. We speculate that IFN I-induced ROS, coupled with the neonatal inability to neutralize ROS, increases mortality in this vulnerable population.
Early IFN-I limits virus-specific immunity
Authors: Palacio Nicole M , Dangi Tanushree , Chung Young Rock , Wang Yidan , Loredo Juan , Zhang Zhongyao , Penaloza-MacMaster Pablo 
Published: January 2023

Abstract
Type I interferons (IFN-I) play a major role in the innate immune response. These cytokines are induced within hours of infection, conferring rapid protection before the onset of adaptive immunity. IFN-I are also critical for the priming of adaptive immune responses, but it is not clear if early antiviral restriction by IFN-I can also limit the amount of antigen available to prime the adaptive immune system. Here we demonstrate that reinforcement of IFN-I signaling early after viral immunization limits the priming of adaptive immunity, whereas blocking IFN-I signaling improves adaptive immunity and enhances subsequent host protection following viral re-infection. Paradoxically, short-term blockade of IFN-I improved the efficacy of clinically approved vaccines, such as the yellow fever virus vaccine (YFV-17D) and the smallpox virus vaccine, as well as experimental HIV vaccines in mice. Short-term blockade of IFN-I also improved immune responses following acute viral infections, such as those with Zika virus, lymphocytic choriomeningitis virus (LCMV), and vesicular stomatitis virus (VSV). This unexpected adjuvant effect of blocking early IFN-I responses was associated with a hyperacute increase in antigen availability, which could not be recapitulated by increasing the dose of the virus inoculum. The improvement of virus-specific immunity following acute IFN-I blockade was mechanistically due to an increase in antigen presentation and costimulation. These findings warrant a re-evaluation of the immunoregulatory roles of IFN-I during immune memory differentiation, which may provide a framework for rational vaccine design.
GILT in thymic epithelial cells facilitates central CD4 T cell tolerance to a tissue-restricted, melanoma-associated self antigen
Authors: Hastings Karen , Rausch Matthew P. , Meador Lydia R. , Metzger Todd C. , Li Handong , Qiu Shenfeng , Anderson Mark S 
Published: January 2023

Abstract
Central tolerance prevents autoimmunity, but also limits T cell responses to potentially immunodominant tumor epitopes with limited expression in healthy tissues. In peripheral antigen presenting cells (APCs), gamma-interferon-inducible lysosomal thiol reductase (GILT) is critical for MHC class II-restricted presentation of disulfide bond-containing proteins, including the self and melanoma antigen tyrosinase-related protein 1 (TRP1). The role of GILT in thymic antigen processing and generation of central tolerance has not been investigated. We found that GILT enhanced the negative selection of TRP1-specific thymocytes. GILT expression was enriched in thymic APCs capable of mediating deletion, namely medullary thymic epithelial cells (mTECs) and dendritic cells, while TRP1 expression was restricted solely to mTECs. GILT facilitated MHC class II-restricted presentation of endogenous TRP1 by pooled thymic APCs. Using bone marrow chimeras, GILT expression in thymic epithelial cells (TECs), but not hematopoietic cells, was sufficient for complete deletion of TRP1-specific thymocytes. An increased frequency of TRP1-specific regulatory T (Treg) cells was present in chimeras with increased deletion of TRP1-specific thymocytes. Only chimeras that lacked GILT in both TECs and hematopoietic cells had a high conventional T:Treg cell ratio and were protected from melanoma challenge. Thus, GILT expression in thymic APCs, and mTECs in particular, preferentially facilitates MHC class II-restricted presentation, negative selection and increased Treg cells, resulting in a diminished anti-tumor response to a tissue-restricted, melanoma-associated self antigen.
TLR9 dependent regulation of AXL expression impacts migration of autoreactive B cells
Authors: Nundel Kerstin (Kiefer) , Moses Stephanie , Richmond Jillian M , Rothlin Carla V. , Marshak-Rothstein Ann 
Published: January 2023

Abstract
Migration of autoreactive B cells to target tissues and the formation of tertiary lymphoid structures (TLS) is an important step in end-organ damage in autoimmune disease. TLSs are thought to be the main site of autoantibody production by autoreactive B cells, leading to immune complex formation and deposition in the affected organ. The formation of TLS was reported to be dependent on B cells as well as chemokines like CXCL13. Hence it is of importance to understand the migratory behavior of autoreactive B cells to these sites and how it is regulated. The receptor tyrosine kinase AXL was shown to play a role in the migration of cancer cells and expression of AXL in cancer cells is usually associated with worse prognoses. We found that AXL expression on B cells is highly induced by crosslinking of the B cell receptor (BCR) and not by TLR ligation as in dendritic cells and macrophages. Intriguingly, co-engagement of TLR9 together with the BCR inhibits AXL upregulation. This is unique to TLR9 and does not occur following co-engagement with other TLRs. Furthermore, we have found that AXL deficient B cells have a diminished potential to migrate in response to the B cell chemokine CXCL13. Also, in the pristane induced model of SLE, mice with AXL deficient B cells have lower numbers of TLS in the peritoneum compared to mice with AXL sufficient B cells, further supporting a role of AXL in the migration of B cells. Our data suggests that TLR9 plays an important role in suppressing AXL expression and the concurrent inhibition of B cell migration to TLS. Our studies have the potential to identify a novel and pharmacologically tractable mechanism for restricting the migration of autoreactive B cells and ensuing tissue damage.
Autoantibodies targeting apolipoprotein A-I are associated with cardiovascular disease outcomes
Authors: Venditto Vincent J , Pitts Michelle , Henson David 
Published: January 2023

Abstract
Cardiovascular disease (CVD) is characterized by an inflammatory immune response associated with disease progression. Apolipoprotein A-I (ApoA-I), the major protein of high-density lipoprotein (HDL), has been identified as the target of antibodies in patients, but their role in disease progression is poorly understood. Recently, we identified immune complexes composed of ApoA-I and IgG (ApoA-I/IgG ICs) in human plasma, which are associated with delayed disease progression. Studies in a large prospective cohort of cardiovascular disease are currently underway. The mechanistic impact of these observations and the dichotomy between free and bound antibodies urge continued exploration of the anti-ApoA-I antibody response in mouse models of inflammation. To complement our efforts with plasma from human subjects, we evaluated anti-ApoA-I antibody induction in mice in response to immune stimuli including western diet, immunization with ApoA-I epitopes, induction of hypercholesterolemia, or combinations of all three. Anti-ApoA-I antibody profiles and subclass composition were characterized by ELISA and compared between groups. Most notably, mice exhibit elevated levels of free antibody responses toward ApoA-I with age, irrespective of gender or diet composition. These data indicate that autoantibodies targeting ApoA-I are naturally occurring in both mice and humans and prompt continued investigation into their role in cardiovascular disease progression.
Autoantibodies targeting components of neutrophil extracellular traps are elevated in cystic fibrosis
Authors: Yadav Ruchi , Fejes Zsolt , Li Quan-Zhen , Nagy Bela , Rada Balazs 
Published: January 2023

Abstract
Cystic fibrosis (CF) lung disease is characterized by the presence of neutrophils and released neutrophil extracellular traps (NETs). NETs are composed of a genomic DNA scaffold decorated with histones and neutrophil granule proteins. The long-term presence of NETs could lead to an autoimmune response in CF. This is supported by our published results showing elevated levels of anti-PAD4 autoantibodies in a U.S. CF cohort. To confirm and expand this observation, here we hypothesized that anti-PAD4 autoantibodies are also elevated in another CF cohort from Hungary, and additional, NET-targeting autoantibodies can be identified in CF.
Fifty-seven CF patients (class I–III CFTR mutations, stable lung function, 26±−5.4 years of age (mean±SD), 54% female) and forty healthy subjects (26.2±3.6 years of age, 57% female) were recruited into this study in Hungary. Anti-PAD4 and anti-dsDNA autoantibodies were measured by ELISA while additional autoantibodies were determined by autoantigen microarray. Increased levels of anti-PAD4 autoantibodies were confirmed in this CF cohort. Serum levels of anti-dsDNA IgA autoantibodies were significantly higher in CF patients compared to control subjects. The autoantigen microarray analysis revealed significantly elevated levels of several, novel, NET-targeting autoantibodies in CF: anti-chromatin IgA, anti-core Histone IgA and IgE, anti-Histone H1 IgA, anti-Histone H2A IgA and IgM. No significant correlations of these autoantibodies with lung function (FEV1%pred), age or inflammatory markers (human epididymis protein 4, C-reactive protein) were found. Overall, these results indicate that chronic presence of NETs leads to a systemic autoimmune response in CF.
Conditional deletion of the actin-regulatory protein hematopoietic protein-1 alters B cell maturation, migration and antibody production
Authors: Avalos Alan Vinicio , Park Heon , Suwankitwat Nutthakarn , Iwata Terri , Iritani Brian M 
Published: January 2023

Abstract
Hematopoietic protein-1 is a hematopoietic cell specific member of the actin-regulatory WAVE complex 2, which acts downstream of multiple immune receptors to stimulate F-actin nucleation. Loss-of-function mutations in the gene encoding Hem-1 (NCKAP1L) have recently been found to result in Primary Immunodeficiency Disease (PID) in humans, characterized by recurrent bacterial and viral respiratory infections, asthma, skin infections, otitis media, and bacteremia. However, the cellular and molecular mechanisms of how loss of Hem-1 results in PID are not known. In this study, we generated constitutive and B cell specific Nckap1l knockout mice using the Cre-LoxP system to dissect the importance of Hem-1 in B cell development and functions. Mice with constitutive deletions of the NCKAP1L gene are severely immunodeficient, characterized by reduced total B and T cells, dysregulated cytokine production and impaired neutrophil migration. To determine why B cells are reduced in the mouse, we bred Nckap1lfloxed mice to Mb1Cre which expresses Cre throughout B cell development. Utilizing flow cytometry, we found that B cell specific deletion of Hem-1 in Nckap1lfl/fl Mb1Cre mice resulted in a reduction in mature B cells in the bone marrow, reduced follicular and marginal zone B cells in the spleen, and reduced innate-like B1a cells. Peripheral B cells from Hem1fl/fl Mb1Cre mice had decreased abilities to migrate to lymphoid tissues in vivo, and altered antibody production following immunization with T-dependent and T-independent immunogens. These results suggest that Hem-1 is essential for normal B cell development and Ab production, and provide novel information regarding why mutations in NCKAP1L result in severe immunodeficiency in humans.
Neonatal exposure to commensal bacteria-derived antigens directs polysaccharide-specific B-1 B cell repertoire development
Authors: New James Stewart , Dizon Brian L.P. , Fucile Christopher , Rosenberg Alexander F. , Kearney John F , King Rodney G 
Published: January 2023

Abstract
Mouse B-1 B lymphocytes play essential roles in defense against infectious microorganisms and maintenance of self-tolerance by producing natural antibodies. However, whether exogenous factors influence B-1 B cell repertoire formation remains poorly understood. Herein, we demonstrate that early immunization with N-acetyl-D-glucosamine (GlcNAc)-bearing Streptococcus pyogenes elicits GlcNAc-reactive B-1 B cells in C57BL/6 mice expressing immunoglobulin heavy chains distinct from those of naïve mice and mice immunized with S. pyogenes in adulthood. Moreover, germ-free mice exhibit greatly reduced GlcNAc-reactive serum antibodies and deficiencies in oligoclonal GlcNAc-reactive B-1 cell clonotypes dominant in conventionally raised mice. These phenotypes are also observed in Myd88-deficient mice and reversed following conventionalization of adult germ-free mice. We show that colonization by the microbiota promotes development of GlcNAc-reactive B cells and IgA-secreting plasma cells in the small intestine that are clonally related to PerC and splenic B-1 B cells. These results indicate that environmental and microbiota-derived antigens influence B-1 clonotype selection into the mature repertoire.
Altered 3D chromatin structure permits inversional recombination at the IgH locus
Authors: Du Hansen , Qiu Xiang , Zhao Mingming , Singh Amit , Sen Ranjan 
Published: January 2023

Abstract
Immunoglobulin heavy chain (IgH) genes are assembled by two sequential DNA rearrangement events that are initiated by recombinase activating gene products (RAG) 1 and 2. Diversity gene segments (DH) rearrange first, followed by variable (VH) gene rearrangements. Here we provide evidence that each rearrangement step is guided by different rules of engagement between rearranging gene segments. DH gene segments, that recombine by deletion of intervening DNA, must be located within a RAG1/2 scanning domain for efficient recombination. In the absence of intergenic control region 1, a regulatory sequence that delineates the RAG scanning domain on WT IgH alleles, VH and DH gene segments can recombine with each other by both deletion and inversion of intervening DNA. We propose that VH gene segments find their targets by diffusion-controlled mechanisms. These distinct mechanisms may underlie differential allelic choice associated with each step of IgH gene assembly.