bluuebunny/crossref_metadata_2025 huggingface dataset:
MAIT cells are imprinted by the microbiota in early life and promote tissue repair
Authors: Constantinides Michael G , Link Verena M. , Tamoutounour Samira , Wong Andrea C. , Perez-Chaparro P. Juliana , Han Seong-Ji , Chen Y. Erin , Li Kelin , Farhat Sepideh , Weckel Antonin , Krishnamurthy Siddharth R. , Vujkovic-Cvijin Ivan , Linehan Jonathan L. , Bouladoux Nicolas , Merrill E. Dean , Roy Sobhan , Cua Daniel J. , Adams Erin J. , Bhandoola Avinash , Scharschmidt Tiffany C. , Aubé Jeffrey , Fischbach Michael A. , Belkaid Yasmine
Published: January 2023

Early-life microbial colonization has been shown to impart long-lasting effects on host fitness. Because mucosal-associated invariant T (MAIT) cells are predominantly located in tissues colonized by the microbiota and characterized by their recognition of microbial-derived riboflavin intermediates, they are thought to be particularly dependent on the microbiota. However, little is known about their development and how they contribute to tissue physiology. MAIT cells accumulated in barrier tissues between 2 and 3 weeks of age, suggesting that MAIT cells develop during a very specific temporal window and in response to defined microbial exposure. The isolation of early-life intestinal commensals and subsequent colonization of neonatal germ-free mice with defined bacteria induced MAIT cell development. Conversely, colonization later in life failed to promote their development within tissues, indicating that microbial exposure must occur during an early-life window. Following their development, MAIT cells represented a dominant type-17 subset in the skin and cutaneous MAIT cells uniquely expressed a transcriptional program associated with tissue repair. Cutaneous MAIT cells responded locally to skin commensals in a manner that required IL-1 and IL-18, as well as MR1-mediated presentation of riboflavin metabolites. Topical application of a riboflavin derivative selectively increased MAIT cells in the skin and was sufficient to promote cutaneous wound healing. Our work demonstrates that MAIT cells are induced during a specific early-life window in response to riboflavin-synthesizing commensals, which permanently imprints the abundance of this subset in tissues, thereby controlling tissue repair and homeostasis.
The Contribution of Pulmonary Dendritic Cells to the Induction and Regulation of T Cell Responses During Respiratory Infection
Authors: Lopez Christopher , Zacharias Zeb Raplh , Waldstein Kody , Legge Kevin L
Published: January 2023

The respiratory tract represents a major portal of entry for pathogens, including Group A Streptococci (GAS). GAS represents a significant burden in cases of primary infection as well as cases of secondary bacterial pneumonia following influenza virus infection. Control of infection and eventual resolution is dependent on the local adaptive immune response. This is facilitated by the uptake and subsequent presentation of antigen by dendritic cells (DC) to T cells. Furthermore, after GAS infection, DC drive T cell development towards a Th17 immune response that is associated with pathogen control. Our hypothesis is that pulmonary DC, both resident and recruited, contribute locally to the maintenance and regulation of an effector CD4 T cell response in the context of a GAS infection. Toward this end, our studies are defining the contribution of local DC: T cell interactions in shaping the phenotype and magnitude of Th17 and Tfh responses within the lungs during GAS infection. An improved understanding of the contribution of these DC to the development and regulation of these CD4 T cells responses during GAS infection should elaborate strategies to the development of better therapies.
A new therapeutic influenza virus vaccine that uses host cell-restricted attenuation by artificial microRNAs
Authors: Guo Hua , Wen Ke , Wang Haiyan , Chen Yanping , Yang Xing , Yang Huixiao , Pena Adilene Realivazquez , Zeng Mingtao
Published: January 2023

New strategies are urgently needed for developing vaccines and/or anti-viral drugs against influenza viruses, because antigenic shift and drift inevitably occurs in circulating strains each year, and new strains resistant to anti-viral drugs have recently emerged. In our study, we designed and incorporated artificial microRNAs (amiRNAs) into the NA segment of rescued influenza viruses to separately target two host genes, CLK1 and SON, which were found to play an essential role in virus replication. Mouse epithelial fibroblast (MEF) or A549 cells infected with engineered influenza PR8 based viruses containing amiR-30CLK1 (PR8-amiR-30CLK1) or amiR-93SON (PR8-amiR-93SON) had reduced expression of host proteins CLK1 and SON, respectively. All engineered influenza viruses functioned as attenuated vaccines, induced significantly higher antibody responses, and provided significantly greater protective efficacy. In addition, they were found to be safe, based on the mouse weight changes and clinical signs observed. In contrast to the engineered viruses targeting SON, mice treated with engineered viruses targeting CLK1 by 6 hours after lethal-dose PR8 infection, recovered from weight loss and survived lethal infection, suggesting that our PR8-amiR-30CLK1-engineered influenza virus could be used as a new post exposure therapeutic influenza vaccine.
ClinicoPathological Correlation (CPC) ImmunoTechnologies for Monitoring Lymphotoxin Alpha
Authors: Harbi Shaghayegh , Wang Rong , Harbi Vahid Dean , Kerbawy John , Park Hannah Lui , Gonzales John
Published: January 2023

Neuroinflammation has been proposed as a target for neuroprotection. Therefore significant opportunities for preventing neurologic decline exist. ClinicoPathological Correlation (CPC) ImmunoTechnologies are critically needed for clinical utility for acute and chronic immunological and inflammatory disorders (including autoimmune neurological disorders such as Parkinson’s disease and Alzheimer’s disease) to determine if an individual has variants associated with a higher risk of disease (Gain of Function [GoF] variants); and elevated levels of analytes (circulating biomarkers).

We hypothesize the neurovascular compartment plays a critical role in neurodegeneration. Using a multipronged approach, we identified specific immune cell subsets and inflammatory factors in the blood, which can potentially play a critical role in the early stages of neurodegeneration by disrupting homeostatic regulation, destabilizing the neurovascular compartment; and elevated levels of analytes (proteomic multiplexed quantification) in the cerebrospinal fluid (CSF) and plasma, which can potentially serve as robust, indicative biomarkers consistent with model of neuroinflammation contributing to neurovascular dysfunction, potentially leading to neurodegeneration. More specifically, we identified analytes (lymphotoxin/TNF-beta/LTA) in the plasma (low levels in CSF); TNF-alpha levels were low in plasma and CSF. Next-generation sequencing enabled the identification of risk variants, pathogen/virus profiles and LTA GoF variant strongly associated with increased protein production. We included distinct LTA inflammatory/autoimmune disease controls (rheumatoid arthritis, multiple sclerosis, uveitis or vasculitis).
Comprehensive profiling of TCR and BCR repertoire of infiltrated lymphocytes in the mouse injured skeletal muscle
Authors: Iwabuchi Kumiko A. , Sasakawa Noriko , Hotta Akitsu
Published: January 2023

Lymphocytes infiltrating to the injured skeletal muscle have been shown to play an important role in muscle regeneration. Site-specific infiltrating/expanding lymphocytes and its molecular features are potential resources for bioengineering such as a drug delivery system. Although some previous studies showed the accumulation of lymphocytes to the injured skeletal muscle, the actual frequency of distinct lymphocyte clones in situ is still unclear. Hence, a comprehensive picture of the lymphocyte repertoire has yet to be described in detail. To this end, we conducted a comprehensive profiling of TCR and BCR repertoire of infiltrating lymphocytes in the mouse injured skeletal muscle model by next-generation sequencing. In the TCR repertoire profiling, we found that the expression level of TCR transcripts contains unique CDR3 α and distinct population of CDR3 β were increased (7.6–17% and 6.7–15%) in infiltrating T cells in both of cardiotoxin- or cryo-injured skeletal muscle, whereas no clonal expansion of specific TCR α/β transcripts observed in control splenic T cells (<1% respectively). While in the BCR repertoire profiling, various transcripts of immunoglobulin kappa chains showed expansion in infiltrated fraction, despite the proportion of immunoglobulin heavy and lambda chain were remained similar to control splenic B cells. Together these data have allowed us to identify TCR/BCR candidates targeting injured skeletal muscles. Furthermore, we aim to utilize the T cells/B cells expressing high-affinity TCR/BCR for the drug delivery system, especially for large protein complexes.
Antibody-suppressor CD8+ T cells ameliorate antibody-mediated rejection following kidney transplant in mice
Authors: Han Jing Light , Zimmerer Jason M , Zeng Qiang , Ringwald Bryce A , Cassol Clarissa , Warren Robert T , Abdel-Rasoul Mahmoud , Breuer Christopher K , Bumgardner Ginny L
Published: January 2023

Purpose

CCR5 KO kidney transplant (KT) mice produce high titer alloantibody (alloAb) associated with severe antibody-mediated rejection (AMR) that reproduces AMR histology observed in human KT recipients. We previously reported the novel alloAb suppressor activity of alloprimed CXCR5+IFNγ+CD8+ T (Tab-supp) cells following hepatocellular transplant in mice. Here, we investigated the biologic impact of alloprimed CD8+ Tab-supp cells and their capacity to suppress alloAb following adoptive cell transfer (ACT) into CCR5 KO KT mice.

Methods

CCR5 KO (H-2b) mice were transplanted with allogeneic A/J (H-2a) kidneys, and on postoperative day (POD) 5 underwent ACT of alloprimed CD8+ Tab-supp cells (retrieved from C57BL/6 mice alloprimed with A/J alloantigen). A second cohort received concomitant bilateral native nephrectomy, and allograft survival was monitored with serial serum creatinine (>100 μmol/L defines KT graft loss). Untreated CCR5 KO KT mice served as controls.

Results

CCR5 KO KT mice developed high alloAb titer compared to wild-type (WT) recipients (5,800±700 vs 1,200±100; p=0.0003). ACT significantly inhibited alloAb production by 5-fold (1,200±200; p<0.0001) and POD14 AMR pathology (peritubular capillary margination and C4d deposition, arteritis) in CCR5 KO KT mice (composite histologic score 3.6±1.5 vs 8.4±0.2 in untreated controls; p=0.006). Following ACT, alloAb remained suppressed beyond POD 30, correlating with enhanced allograft survival (MST 52 vs 14 days; p=0.006).

Conclusion

ACT of CD8+ Tab-supp cells effectively inhibits alloAb production and AMR not only after allogeneic hepatocellular transplant, but also after vascularized solid organ transplant such as in CCR5 KO KT recipients.
Posttransplant cyclophosphamide as a platform for immunotherapy after allogeneic stem cell transplantation for multiple myeloma
Authors: Minnie Simone A , Kuns Rachel D , Ensbey Kathleen S , Samson Luke D , Chesi Marta , Gartlan Kate H , Smyth Mark J , Vuckovic Slavica , Hill Geoffrey R
Published: January 2023

Allogeneic stem cell transplantation (alloSCT) is a highly effective, curative therapy for leukemia yet does not provide a survival benefit above autologous SCT in patients with multiple myeloma (MM). To explore this, we developed preclinical models of SCT using C57Bl/6 recipient mice and either C57Bl/6 (ASCT) or C3H.SW (alloSCT) donor grafts. Importantly, these models recapitulated the clinical setting whereby alloSCT provided superior outcomes, compared to ASCT, in recipients bearing MLL-AF9-driven acute myeloid leukemia (AML) but not in those bearing Vk*MYC-MM. Interestingly, we found that MM-specific, T cell-mediated immunity was generated after ASCT, which failed due to MM-induced T cell exhaustion. MM relapse after ASCT could be prevented by TIGIT or PD-1 targeted immune checkpoint inhibition or via depletion of suppressive CSF1-R+ myeloid cells. Conversely, after alloSCT, T cell exhaustion was driven principally by alloantigen and CD8 T cells expressed high levels of PD-1, TIGIT and TIM-3. Furthermore, Vk*MYC myeloma exploited this alloantigen-driven T cell exhaustion via expression of high levels of PD-L1 and CD155 (the cognate ligands for PD-1 and TIGIT), which were expressed minimally on MLL-AF9 AML. To exploit alloSCT in MM, we used post-transplant cyclophosphamide (PT-Cy) to delete high affinity alloreactive T cells that generate the exhausted donor T cell pool. Subsequent administration of CD137 agonists enhanced T cell activation and cytolytic activity within bone marrow, without exacerbating GVHD. Thus, PT-Cy provides a platform for optimizing immunotherapy after alloSCT.
Exopolysaccharide from a commensal bacterium ameliorates aGVHD by decreasing alloreactive donor T cell activation
Authors: Kalinina Olga , Talley Sarah , Zamora-Pineda Jesus Bernabe , Paik Won , Campbell Edward , Knight Katherine L
Published: January 2023

Acute graft versus host disease (aGVHD) is a severe and often lethal complication of allogeneic bone marrow transplantation (BMT). We found that mice injected with exopolysaccharide (EPS) secreted by a commensal bacterium, Bacillus subtilis, significantly improved survival, reduced weight loss and decreased production of systemic pro-inflammatory cytokines in GVHD mice. We utilized a novel biosensor mouse model to assess the activation of donor T cells in live mice during aGVHD and found that EPS inhibited the activation of alloreactive donor T cells. EPS also inhibited alloreactive T cell activation in mixed lymphocyte reactions (MLR) in vitro and this inhibition required DCs. Treating DCs with EPS led to upregulation of the inhibitory molecule PD-L1 and secretion of the anti-inflammatory cytokine IL-10, characteristic of inhibitory DCs. We determined that EPS induces inhibitory DCs through TLR4, signaling through both MyD88 and TRIF signaling pathways. The inhibition of alloreactive T cells by these inhibitory DCs is mediated by indoleamine 2,3-dioxygenase (IDO). We hypothesized that inhibition of alloreactive donor T cell activation in GVHD mice by EPS is also mediated by DCs and that EPS induces inhibitory DCs in vivo as well as in vitro. The data demonstrate the potential of EPS or EPS-treated DCs as novel reagents for preventing GVHD.
Circulating tumor cell clusters exhibit enhanced immune evasion from natural killer cells
Authors: Lo Hin Ching , Xu Zhan , Kim Ik Sun , Muscarella Aaron , Liu Jun , Hein Sarah , Wang Hai , Krupnick Alexander , Neilson Joel , Paust Silke , Rosen Jeffrey , Zhang Xiang
Published: January 2023

During cancer metastasis, the ability to evade immunosurveillance is key to the survival of circulating tumor cells (CTCs). Recent studies have found that in addition to single CTCs, multicellular clusters of CTCs can be detected in patient blood. Strikingly, CTC clusters show higher metastatic potential than single CTCs, but the molecular mechanism is unclear. We hypothesize that CTC clusters adopt immune evasive strategies, which enhance their metastatic ability. In mouse models of breast cancer, CTC clusters show enhanced metastatic growth over single cells in immunocompetent mice, but this difference is abrogated in immunodeficient mice lacking NK cells or mice treated with NK cell-depleting antibodies. Adoptive transfer of NK cells into NK cell-deficient hosts selectively suppresses metastatic growth by single CTCs, suggesting that single CTCs are more sensitive to NK cells than clusters. Analysis of cell line and patient RNA-seq data reveal that CTC clusters have elevated expression of cell-cell adhesion and epithelial genes as compared to single cells, which is in turn associated with decreased expression of NK cell-activating NKG2D ligands. Furthermore, modulation of tumor cell epithelial status alters NK ligand expression and sensitivity to NK cell killing, but this effect is attenuated by the addition of an NKG2D-blocking antibody. Taken together, we propose a model in which the epithelial characteristics of CTC clusters promotes their resistance to NK cell attack and thus their overall metastatic success. These findings reveal that NK cells play an important role in determining the fate of CTCs of different epithelial/mesenchymal states, and illuminate our understanding of immune evasion mechanisms by CTCs.
A novel, non-feeder-cell approach to generate large numbers of Cytokine-Induced Memory-Like NK cells for adoptive cells therapies
Authors: Shrestha Niraj , Dee Michael , Chaturvedi Pallavi , Leclerc Gilles , Zhu Xiaoyun , George Varghese , Prendes Caitlin , You Lijing , Spanoudis Catherine , Rhode Peter , Wong Hing C
Published: January 2023

We have constructed two heterodimeric multi-cytokine fusions, HCW9201 and HCW9206, to support a “Kick and Expand” approach to activate and induce proliferation of purified NK cells for adoptive cell therapy (ACT). HCW9201 is a heterodimeric fusion protein complex comprising human IL-15 (complexed with an IL-15Rα-sushi domain), IL-18 and IL-12. HCW9206 is heterodimeric fusion protein comprising human IL-15 (complexed with an IL-15Rα-sushi domain), IL-7, and IL-21. When purified human NK cells from peripheral blood were activated with HCW9201 for three hours and then incubated with HCW9206 in combination with a capture antibody, the HCW9201-activated NK cells expanded approximately 100–300 folds within 14 days. The expanded NK cells exhibit a cytokine-induced memory-like (CIML) phenotype with a high metabolic rate and respiratory capacity, remarkable anti-tumor activity, and persistence when they were adoptively transferred into NSG mice. They also retained their heightened responsiveness when re-stimulated with tumor targets. Cytokine dependent epigenetic demethylation imprints of the Ifng promoter region were also observed for at least 10 days after the NK cells were adoptively transferred to NSG mice. In conclusion, a simple, scalable, non-feeder-cell-based “Kick and Expand” process was developed to support the generation of large numbers of CIML NK cells for multiple rounds of ACT using peripheral blood NK cell. We also provide data to show that these CIML NK cells are an excellent source for generation of CAR-NK cells.
Satb1 deficiency licenses TFH-differentiation and Tertiary Lymphoid Structure formation in cancer
Authors: Gonzalez Ricardo A Chaurio , Payne Kyle K , Galindo Carmen Maria Anadon , Costich Tara Lee , Harro Carly , Birwas Subir , Rigolizzo Kristen , Mine Jessica A , Mandal Gunjan , Powell Chase , Martin Alexandra L , Wang Zhitao , Kroeger Jodi , Robinson John , Melendez Johana , Rodriguez Paulo C , Conejo-Garcia Jose R
Published: January 2023

Tertiary Lymphoid Structures (TLS) are commonly identified in human tumors with improved outcome, but how they are orchestrated remains elusive. Here we show that silencing of the master genomic organizer Satb1 results in enhanced antigen-specific T Follicular Helper (TFH) differentiation. Increased TFH thereby promoted antigen-specific intra-tumoral CD19+B220+ B cell responses and spontaneous TLS assembly upon ovarian tumor challenge. Mechanistically, Satb1 deficiency drives increased TFH formation through de-repression of ICOS and PD-1. Accordingly, TGF-β1-driven downregulation of Satb1 licenses activated human CD4+ T-cells for enhanced antigen-specific T Follicular Helper (TFH) differentiation. Furthermore, Satb1 deficiency abrogates the generation of PD-1highCXCR5+Foxp3+ T Follicular Regulatory (TFR) cells during the TFH differentiation process. Importantly, functional TFH cell accumulation, in the absence of Satb1 specifically in CD4+ T cells, resulted in corresponding isotype-switched B cell responses and spontaneous formation of TLS, while B cell depletion accelerated malignant progression. Our results indicate that the formation of TLS in cancer depends on enhanced B cell responses driven by TFH cells generated through Satb1 down-regulation.
Analysis of stability CRISPR/Cas9 engineered B cells using flow cytometry
Authors: Inwood Sarah L , Tian Linhua , Maragh Samantha , Wang Lili
Published: January 2023

Objective

CRISPR/Cas9, a widely used genome editing system, has huge potential and is being used to improve protein, cell and gene therapies. Though an exciting tool, there is not a lot of long-term data about the genomic and phenotypic stability and off-target effects that may arise during the editing process. Since the cells are used in the patient with cell and gene therapies, cell characterization is essential for safety.

In support of production of safe and effective CRISPR/Cas9 engineered therapies, the objective of this work is to study both on- and off-target effects of CRISPR/Cas9 using flow cytometry on a B-lymphoblast cell line, GM24385, whose genome sequence has been well characterized.

Methods

The cultured B cells were characterized with a B cell panel using flow cytometry. The CD19 marker was knocked out using CRISPR/Cas9 RNPs with a nucleofection method using four gRNAs. The resulting pools were then evaluated with sequencing and a flow cytometry B cell panel and sorted using a FACSAria II flow sorter.

Results

The CD19− B cells were created from nucleofection of cultured GM24385 cells with CRISPR/Cas9 RNPs. The characteristics obtained from a B cell subtyping panel showed different efficiencies of CD19 knockout and different B cell subtypes. The growth rate of CD19− B cells was unpredictable over time post transfection. Additionally, different gRNAs against CD19 resulted in different editing efficiencies evaluated at genomic and proteomic levels.

Conclusions

Using CRISPR/Cas9 with different gRNAs to knock out CD19 in B lymphoblasts, GM24385, influences the B cell subtype profile. The work with well sequenced GM24385 enables the identification of off-target effects of the editing method, the next step of the study.
characterization of newly isolated porcine epidemic diarrhea virus (PEDV) and its resistance to interferon and neutralizing antibody
Authors: Shin Hyun-Jin
Published: January 2023

Porcine epidemic diarrhea virus (PEDV) infects pigs and causes enteric disease characterized by watery diarrhea and dehydration. Despite the extensive vaccination, PEDV strains with variations in the spike gene have been reported in several countries, including Korea, China, and the United States. Variant PEDV belonging to genogroup G2 has higher pathogenicity and morbidity than conventional PEDV belonging to genogroup G1. To understand the pathogenesis of the variant PEDV, we examined the susceptibility of the variant PEDV to interferon and PEDV specific neutralizing antibodies, which are known to important for the control of PEDV infection. We found that variant PEDV (WG strain) showed higher resistance to interferon than conventional PEDV (KPEDV-9 strain). Similarly, KPEDV-9 strain grew well in interferon-deficient Vero cells but did not grow in interferon-releasing porcine alveolar macrophages, whereas WG strain showed similar infectivity in both cells. To elucidate the possible mechanism of resistance to interferon and neutralizing antibody in variant PEDV, sequencing analysis was performed. We found huge variations in spike and ORF3 proteins. To test the impacts of these mutations on the acquisition of interferon and neutralizing antibody resistance, we are currently generating pseudotyped PEDV and recombinant PEDVs with mutations. Our results indicate that the recently introduced variant PEDV exhibits higher resistance to the interferon than the existing PEDV and avoids the neutralizing antibody against the existing PEDV. These results also provide understanding of the occurrence of variant PEDV and its pathogenesis.
Inflammatory reactions and melanogenesis in salmon during virus infection
Authors: Bjoergen Haavard , Malik Muhammad Salman , Rimstad Espen , Koppang Erling Olaf
Published: January 2023

Melanised skeletal muscle changes (black spots) are a serious problem for the salmon industry world-wide, causing extensive production losses. Piscine orthoreovirus (PRV) is associated with the condition. The initial cause for the focal changes remains obscure. Early changes start as muscle bleedings and extensive muscle necrosis, and so far, these changes cannot be explained, However, from this acute phase, the changes may progress into melanised focal changes appearing as chronic granulomatous reactions. Granulomatous melanised changes have been consistently observed in PRV infected fish, and in serious changes, the tissue persistence of virus dominate the changes despite the inflammatory changes. Here, we address the pathogenesis of PRV infection and phases of melanised focal changes development. Fluorescent in situ hybridisation (FISH) assay confirmed the colocalisation of iNOS2 transcripts (M1 activated macrophages) with PRV in red spot whereas both M1 and M2 (Arg2 transcripts) macrophages localized at PRV infected clusters in black spots. Melano-macrophages were found positive with Arg2 transcripts demonstrating their role in tissue repair mechanism. In PRV negative fish low expression of M1 and M2 specific transcripts suggest PRV act as an accelerator for the melanisation in these focal changes. Transcripts specific for melanin production were observed in non-melanised cells in early stages of the condition and in melano-macrophages in late stages. The nature of the melano-macrophages is disputed and the rationale for their melanin production is not understood. Our investigations reveal new characteristics both of the pathogenesis of focal melanised changes as well as of the melano-macrophages involved.
Hypothetical evolutionary models of the variable lymphocyte receptor genes in jawless vertebrates
Authors: Das Sabyasachi , Hirano Masayuki , Rast Jonathan , Cooper Max D
Published: January 2023

Both humoral and cellular arms of an adaptive immune system are common to jawed and jawless vertebrates. However, whereas antigen recognition in the jawed vertebrates is mediated by immunoglobulin domain-based B cell receptors and T cell receptors, antigen recognition in jawless vertebrates is mediated by leucine-rich repeat (LRR) sequences in variable lymphocyte receptors (VLRs). Three types of VLR genes (VLRA, VLRB and VLRC) have been characterized in the extant jawless vertebrates, lampreys and hagfishes. VLRA- and VLRC-producing cells are T cell-like, while VLRB-producing cells are B cell-like. The VLRs are assembled by copying neighboring LRR sequences into the incomplete germline genes. In searching for the evolutionary origin of the VLRs we identified hypothetical VLR-like progenitors in amphioxus and tunicates (VLRPs). Molecular cladistics markers and phylogenetic analysis suggest the VLRPs are structurally more similar than other ancestral candidates to assembled VLRs. Based on our further analysis we propose alternative models for the origin of VLRs: (A) Among the VLRs, VLRB is most similar to the ancestral state and two subsequent duplication events gave rise to VLRC and VLRA-like genes; each of the three ancestral VLR genes in a common jawless vertebrate ancestor was then modified by cut and paste transposition of the internal DNA region encoding multiple LRR modules. (B) A cut and paste transposition event in an ancestral VLRB-like gene was followed by sequential duplication events which gave rise to VLRC and VLRA. In both models, subsequent duplications and fragmentations have generated a large array of genomic donor cassettes. Thus, after the emergence of the three VLR loci, each has undergone independent evolution.
Genetic variation in the RSV fusion protein controls RSV strain-dependent inflammasome activation and the development of a pathogenic Th17 response
Authors: Waldstein Kody , Weiss Kayla A , Hartwig Stacey M , Varga Steven M
Published: January 2023

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in young children worldwide. RSV-induced disease ranges from mild to severe, with severe disease correlated with elevated levels of pro-inflammatory cytokines, including the Th17-associated cytokine, IL-17. To better understand the viral factors that influence the induction of a Th17 response, we compared the inflammatory responses elicited by different RSV strains. In contrast to the commonly used RSV A2 strain, RSV 2-20 infection resulted in increased protein levels of IL-6 and IL-1β, which were required to induce a pathogenic Th17 response. RSV 2–20 infection increased mucus production and airway hyperreactivity indicating strain-specific disease induction. RSV 2–20 and A2 induced similar protein levels of caspase-1, however, 2–20 infection resulted in increased protein levels of pro-IL-1β suggesting differential activation of inflammasome signal 1. Increased production of IL-1β following RSV 2–20 infection was dependent on TLR4 expression and signaling, a canonical inflammasome signal 1 activator. A recombinant A2 virus expressing the 2–20 fusion (F) protein induced IL-1β to similar levels as wild-type 2–20 indicating the 2–20 F protein is sufficient to increase pro-inflammatory cytokine production. Reversion of three-point mutations in a region likely to interact with host cells in the RSV A2/2–20 F recombinant virus to the A2 sequence significantly reduced IL-6 and IL-1β protein production. Our results demonstrate that variation in the RSV F protein plays a crucial role in the induction of pro-inflammatory cytokines and modulating disease severity in the animal model and may contribute to differential RSV pathogenesis.
Generating protective immunity to severe influenza disease in Indigenous Australians
Authors: Hensen Luca , Illing Patricia , Rowntree Louise , Habel Jennifer , Mifsud Nicole , Koutsakos Marios , Loh Liyen , van de Sandt Carolien , Nguyen Andrea , Gras Stephanie , Rockman Steve , Miller Adrian , Nguyen Oanh , Clemens Bridie , Tong Steven , Purcell Anthony , Kedzierska Katherine
Published: January 2023

The 2009 influenza pandemic caused generally mild infections due, at least partially, to broadly cross-reactive pre-existing CD8+ T cell immunity. In contrast, severe disease was observed in Indigenous populations worldwide, as shown by disproportionate hospitalisation rates. Human leukocyte antigen-I (HLA-I) molecules present viral peptides to CD8+ T cells, eliciting anti-viral responses that contribute to accelerated viral clearance. We identified several HLA-I alleles which are highly prevalent in Indigenous Australians (HLA-A*11:01, A*24:02, A*34:01, B*13:01 and B*15:21) but largely understudied. Using specific HLA-allomorph expressing C1R cell lines, we identified influenza A and B immunopeptidomes by mass spectrometry. A total of 653 influenza peptides derived from various viral proteins were presented during infection by different HLA-Is. Using virus-specific PBMC expansions, we screened these peptides for their potential to reactivate influenza-specific memory CD8+ T cells. We identified on average 4.6 (2–8) immunogenic epitopes per HLA-I for influenza A and B. We defined epitope-specific CD8+ T cells ex vivo across different human tissues and determined their memory subsets, phenotype and activation in healthy donors and influenza-infected patients. Overall, our approach is effective in detection of immunogenic epitopes and understanding CD8+ T cell pools in Indigenous populations, thus providing potential vaccine targets to protect Indigenous populations globally, from severe influenza disease. This is the first study to identify hallmarks of immunogenic responses and reveal key targets of an effective CD8+ T cell vaccine in Indigenous people.
Chronic ethanol consumption causes lesions in CD8 T cell memory and is associated with increased susceptibility to influenza A virus infections
Authors: Zacharias Zeb Raplh , Legge Kevin L
Published: January 2023

Chronic alcohol consumption is associated with increased risk of severe disease during both bacterial and viral infections. We have previously shown that this increased disease severity during a primary influenza A virus (IAV) infection is due to deficits in the developing IAV-specific CD8 T cell response. Recent studies have highlighted that human alcoholics have increased susceptibility to IAV infections, even though they likely possess pre-existing immune memory to IAV. However, the effects of chronic alcohol consumption on pre-existing immune memory responses have not been explored. Here, our results show that mice previously exposed to IAV infection that then consumed alcohol (IAV-immune➞EtOH) exhibited increased morbidity and mortality to secondary heterologous IAV challenge compared to IAV-immune mice that had consumed water (IAV-immune➞H2O). This increased susceptibility in IAV-immune➞EtOH mice was associated with reduced IAV-specific killing of target cells. Furthermore, we observed a universal numerical reduction of all memory CD8 T cell subsets within the lungs of IAV-immune➞EtOH mice compared to IAV-immune➞H2O mice. Finally, our results show that IAV-specific CD8 T cell recall responses upon secondary IAV challenge are altered in IAV-immune➞EtOH mice, particularly within the lung tissue, and were associated with reduced expression of CXCL10 and CXCL11, suggesting defects in cell trafficking. Overall, these data demonstrate that the negative impacts of chronic alcohol consumption occur across the full CD8 T cell compartment, including both the naïve and memory response, contributing to lesions in the development of de novo CD8 T cell and pre-existing memory CD8 T cell responses.
Lung-resident memory CD8+ T cells prevent transmission of respiratory viruses
Authors: Uddback Ida Elin Maria , Laccette Kristiana H , Li Zheng-Rong Tiger , Kohlmeier Jacob E
Published: January 2023

Lung tissue-resident memory CD8+ T cells (TRM) reduce viral replication and limit pathology following respiratory virus infections. In particular, TRM are crucial for protection against infections with heterosubtypic influenza viruses, where pre-existing antibody does not provide sterilizing immunity. In addition to providing individual host immunity, vaccination programs are designed to limit pathogen spread at population level. Despite the interest in lung TRM for intra-host protective immunity, their effect on the transmission respiratory viruses has been understudied due to the lack of animal models where influenza infection can be tracked longitudinally, and where the immunological parameters are easily identified and manipulated. To circumvent these issues, we used a luciferase-expressing Sendai virus, a natural mouse parainfluenza virus that readily transmits via the aerosol and contact routes, to evaluate the efficacy of lung TRM against transmission of respiratory viruses. Using a recombinant influenza virus expressing the immunodominant SenNP324–332Kb epitope to generate SenNP+ memory CD8+ T cells and IVIS imaging to non-invasively measure Sendai virus infection over time, we found that mice with pre-existing SenNP+ lung TRM don’t transmit Sendai virus to naïve mice when co-housed. In contrast, immunized mice that had pre-existing circulating effector memory CD8+ T cells (TEM), but no lung TRM, failed to prevent transmission of Sendai virus to naïve mice when co-housed. These results show the ability of lung TRM to contribute to herd immunity by preventing respiratory virus transmission and underscore the potential of a TRM-inducing vaccine for protection against respiratory viruses.
A BAFF-ling new role for NK cells in promoting marginal zone antibacterial resistance during chronic virus infection
Authors: Ali Ayad , Cranert Stacey A , Moran Michael T , Rajaram Sanjeeth , Waggoner Stephen N
Published: January 2023

Natural killer (NK) cells are critical for both direct control of virus replication and regulation of potentially pathogenic antiviral responses. Yet, whether NK cells also contribute to immune control of bacterial superinfections during viral infection, a significant clinical cause of morbidity and mortality, remains unknown. We find that NK cells are vital to prevent a near complete loss of B cells and macrophages associated with the marginal zone (MZ) during chronic virus infection in mice. Establishment of chronic infection in the absence of NK cells results in hyper-susceptibility to Listeria monocytogenes infection that cannot be recapitulated by NK-cell depletion prior to bacterial challenge of chronically infected mice. Therefore, we posit that preservation of MZ leukocytes by NK cells, and not direct NK cell activity, curtails post-viral susceptibility toward bacterial superinfection. Mechanistically, we show that NK cells begin expressing the crucial MZ survival factor, B-cell activating factor (BAFF), during chronic virus infection, gradually becoming a prominent source of BAFF in the spleen. In vivo blockade of BAFF in NK-sufficient mice recapitulated the infection-associated loss of MZ cells seen in NK-cell deficient mice. These results reveal a new mechanism whereby NK cells promote bacterial resistance during chronic infection by maintaining BAFF-dependent immune structures. Disruption of this activity of NK cells during certain virus infections in humans may provoke post-viral pneumonia or invasive pneumococcal disease.
DNA Methylation Tightly Regulates TIGIT Expression in CD8+ T cells During Chronic HIV Infection
Authors: Corley Michael J , Chew Glen , Pang Alina PS , Ndhlovu Lishomwa
Published: January 2023

The T cell Ig and ITIM domain (TIGIT) receptor is a next generation negative immune checkpoint that is unregulated on exhausted CD8+ T cells in cancer and in chronic HIV-1 infection. TIGIT is currently being pursued in Phase 1 human clinical studies for immune restoration using targeted monoclonal antibodies. However, the regulation of TIGIT expression on T cells still remain unclear. We characterized and compared genome-wide epigenetic and transcriptional profiles in sorted TIGIT+ and TIGIT− CD4 and CD8 T cells in peripheral blood obtained from three virally-suppressed chronic HIV participants on antiretroviral therapy. Unsupervised hierarchical clustering analysis of differentially methylated loci indicated TIGIT+ T cells stratified from TIGIT− T cells based on cellular DNA methylation profiles. Compared to cell sorted purified TIGIT− T cells, 86.5% of CpG loci exhibiting hypomethylated states in TIGIT+ T cells. Specifically, the levels of DNA methylation at the nine CpG loci on the methylation array related to the TIGIT gene showed significant demethylation in TIGIT+ compared to TIGIT− T cells. We found 6/7 sites located at the TIGIT promotor were significantly decreased in methylation in TIGIT + compared to TIGIT− cells (cg19959917: TIGIT+ = 0.605, TIGIT− = 0.715, P = 0.002). Moreover, the CpG located intergenic (cg19959917: TIGIT+ = 0.605, TIGIT− = 0.715, P = 0.002 and at the 3′ UTR (cg19959917: TIGIT+ = 0.605, TIGIT− = 0.715, P = 0.002) were significantly demethylated in TIGIT+ compared to TIGIT− T cells. DNA methylation changes correlated significantly with mRNA expression and surface protein expression. These reference methylomes expose a distinct DNA methylation state of TIGIT+ T cells and reference for epigenetic targeting.
Anaphylaxis
Authors: Stoland O. O. , Sherwood N. P. , Woodbury R. A.
Published: January 2023

Summary and Conclusions

The chronaxie of the vagus nerves in dogs sensitized to horse serum is lower than for normal animals indicating an increased irritability associated with the development of the hypersensitive state. The decrease in chronaxie may reach such a state that nerve block or apparent decreased irritability occurs. The vagus nerve irritability is not appreciably affected by anaphylactic shock. Anaphylactic shock as shown by fall of blood pressure and prolonged coagulation time does not occur in all dogs supposedly sensitized to horse serum. Vagus nerve stimulation before injection of shocking dose of antigen may or may not modify the anaphylactic shock. The increased irritability and permeability of tissue in sensitized animals is not the primary factor in anaphylactic shock.
Age and the Susceptibility of Rabbits to Scarlet Fever Toxin
Authors: Trask James D.
Published: January 2023

A relation between age and susceptibility to scarlet fever toxin was described by Parish and Okell (1). They found that young rabbits survived longer than mature ones following the inoculation of living scarlatinal streptococci and also that doses of scarlatinal toxin which were fatal to most rabbits of more than 2000 grams were not fatal to most rabbits weighing less than 1000 grams. Although, in the experiments with toxin, the ages of the animals were unknown, nevertheless, considering the weights mentioned there can be no doubt but that these investigators observed a lack of susceptibility to scarlatinal toxin in young as compared to older rabbits. This phenomenon was noted by us independently while studying another phase of the scarlet fever problem. It seems worth while to publish our results for their corroborative value. Accordingly our experiments which have to do with the relation of age and susceptibility follow.
Studies in Anaphylaxis in the Rabbit
Authors: Grove Ella F.
Published: January 2023

Of 53 rabbits treated with the Grove series of injections with egg white (described in the text), 70 per cent were made maximally sensitive to intravenous test, 5.6 per cent exhibited slight symptoms of anaphylaxis and 24.4 per cent failed to show any symptoms. Similar results were obtained with horse serum. Variations in the number or the selected routes of the injections lessened the effectiveness of the treatment in establishing anaphylactic sensitiveness in the animals. The established sensitiveness persists for at least two or three weeks. An additional subcutaneous injection given one week after the period of highest sensitiveness usually causes no diminution in the concentration of the blood-borne antibodies; nevertheless the sensitiveness of the animals is always greatly diminished or entirely extinguished. Active sensitization of rabbits does not depend entirely upon the degree of antibody productivity because animals showing a high power of antibody production may be wholly insensitive and vice versa.
Studies in Phagocytosis
Authors: Wittenberg Joseph , Lederer Max , Mollov Mollie
Published: January 2023

The series of experiments here reported show: That phagocytosis of gonococci by leucocytes depends on the influence of gonotoxin on the organisms rather than on the presence of the organisms themselves.That the union between the gonotoxin and the leucocytes is labile and can easily be disassociated by washing with saline.That avidity of the phagocytes for gonococci can be satisfied, at least partially, by supplying gonotoxin to the leucocytes before exposing the gonococci to their action.
The Urticarial Skin Reaction in Normal and Sensitized Guinea Pigs
Authors: Pilcher, Lewis S.
Published: January 2023

Typical urticarial skin reactions exactly resembling in character and time sequence those described in man by Lewis and Grant were produced in guinea pigs by light skin puncture through a drop of 1:100 histamine solution. The vascular changes preceding and accompanying the urticarial reaction could be accurately observed by looking through the ear of the guinea pig while the reaction was in progress. “Immediate” anaphylactic skin reactions to antigenic horse serum were produced in sensitized guinea pigs by light skin puncture through a drop of undiluted horse serum. These reactions were typically urticarial resembling the histamine wheals in character but having a more gradual and chronic sequence of development. No general anaphylactic reactions of any kind accompanied any of the skin reactions. The anaphylactic sensitivity of all the test animals was checked at the end of the experiment by intracardiac injection of 2 cc. of normal horse serum. There appeared to be a fairly definite relation between the presence of general anaphylactic sensitivity and the production or non-production of the typical anaphylactic skin reaction. There was no relation between general anaphylactic sensitivity and the degree of production of the histamine skin reaction.
Local Organ Hypersensitiveness
Authors: Seegal Beatrice Carrier , Seegal David
Published: January 2023

Repeated intravenous injections of egg white in rabbits whose right eye is inflamed by the introduction of glycerin into the anterior chamber, results in a specific local sensitization of the eye to egg white in 12 of 21 animals tested. Control rabbits injected intravenously with egg white alone and other animals whose right eye is injured with glycerin alone do not develop the eye reaction when the test dose of egg white is injected intravenously. Repeated injections of egg white intravenously in normal animals may produce a general reactivity such that later injections of egg white produce bilateral inflammation of the eyes.
Studies on Tissue Reactions in Immunity
Authors: Kahn Reuben L.
Published: January 2023

In the previous article (1) quantitative data were presented dealing with the degree of skin sensitivity in rabbits following sensitizing injections of bacterial vaccines. This sensitivity was subsequently found to be influenced by the method of administering the sensitizing injections. When administered intravenously, antibody formation seemed to exceed the response toward hypersensitiveness, whereas if administered intradermally, the hypersensitive response appeared to be the outstanding one. Another aspect was observed in connection with the sensitization of rabbits to bacterial suspensions, namely, that frequently repeated injections did not increase the sensitivity of rabbits to the same extent as infrequent injections. This article will briefly consider 2 experiments. In one, a group of rabbits was injected with typhoid vaccine, some intracutaneously and others intravenously, and the skin sensitivity and serum agglutinins were determined at definite intervals.
Studies in Microbic Dissociation
Authors: Mackenzie G. M. , Fitzgerald Helen
Published: January 2023

Studies of microbic dissociation have usually taken as the point of departure either some deviation from the familiar colony type of the culture under investigation or instability of emulsions in normal salt solution. Variants have usually been, and still are, spoken of chiefly as “rough” or “smooth” even though abundant evidence has accumulated that in colonial morphology the variant culture may be indistinguishable from the original culture, yet differing profoundly in serological, morphological, or biochemical properties. Conversely, rough colony variants may manifest complete serological identity (Krumwiede, Cooper and Provost (1) and Savage and White (2)) with the original smooth colony culture. Perhaps the “R” and “S” terminology is the most convenient one now available but its inadequacies are constantly encountered in investigations of the phenomena of microbic dissociation. In this paper and in those which follow we have preferred to designate the original culture, from which both “smooth” and “rough” variants have been derived, as the O-culture.
Meningococcus Toxin and Antitoxin
Authors: Ferry Newell S. , Schornack Philip J.
Published: January 2023

Live cultures, whole killed cultures, washed killed cultures, washings of live cultures, washings of killed cultures and toxin (meningococcus bouillon filtrate) have more or less of a similar effect when injected intracisternally in 250-gram guinea pigs.

Experimental meningococcus antitoxin has a neutralizing effect against meningococcus toxin, the washings of killed cultures and to some degree against washed cultures, when mixed with these substances before injection, but shows no evidence of neutralizing live cultures or killed cultures when the mixtures are injected intracisternally.

Antimeningococcic serum has evidenced some neutralizing effect against meningococcus toxin when the mixture of toxin and serum was injected intracisternally.

Exposure for two hours at incubator temperature has no appreciable deleterious effect on the toxin or mixtures of toxin and antitoxin.