bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of CISH-knockout NK cells derived from human peripheral blood and evaluated the antitumor effects in allogeneic glioblastoma
Authors: Nakazawa Tsutomu , Morimoto Takayuki , Maeoka Ryosuke , Matsuda Ryosuke , Nishimura Fumihiko , Tsujimura Takahiro , Nakagawa Ichiro
Published: November 2024

Background: Glioblastoma (GBM) is the most common malignant brain tumor. Allogeneic natural killer cells (NKCs) have gained considerable attention as promising immunotherapeutic tools against cancer, where gene-edited NKCs would result in effective anti-cancer treatment. This study focused on the immune checkpoint molecule cytokine-inducible SH2-containing protein (CISH) as a critical negative regulator in NKCs.

Methods: We generated human primary CISH-deleted NKCs (NKC dCISH) by combining our specific NKC expansion method and CRISPR/Cas9. The genome-edited NKCs underwent microarray analysis. The anti-GBM activity of the genome-edited NKCs was evaluated in vitro and further detected in vivo antitumor effects using xenograft brain tumor mice.

Results: We successfully induced NKC dCISH derived from human peripheral blood. Gene set enrichment analysis (GSEA) using microarrays revealed that the enriched genes were involved in effector functions in NKC dCISH. CISH deletion enhanced NKC-mediated apoptosis induction against allogeneic GBM cells and spheroids. Intracranial administration of the allogeneic CISH-deleted NKCs prolonged the overall survival of xenograft brain tumor mice.

Conclusion: CISH deletion enhanced NKCs' effector functions and NKC-mediated antitumor effects in allogeneic GBM. The genome-editing NKCs could be a promising immunotherapeutic alternative for patients with GBM.
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Authors: Velu Vijayakumar , Govindaraj Sakthivel , Babu Hemalatha , Ali Syed , Rahman Sheikh , Ribeiro Susan , Tomalka Jeff , Sharma Ashish , Sekaly Rafick , Amara Rama , Villinger Francois , Velu Vijayakumar
Published: November 2024

Immunotherapeutic cytokines such as IL-15 enhance follicular CD8 and IL-15 + IL-12 enhances follicular NK cells during chronic SIV infection. Here we tested the therapeutic effects of IL-15 and IL-12 administered alone or in combination during chronic SIV in rhesus macaques (RMs).

Twenty-two RMs infected with SIVmac251 were initiated on antiretroviral therapy (ART) at 8 weeks post-SIV for 9 months. Three groups of RMs received cytokine therapy - IL-15/IL-15Ra (n=6), IL-12 (n=5), IL-15/IL-15Ra+IL-12 (n=6) - in 2 phases (5 doses once a week). The 1st phase was administered at 6 weeks post-SIV (2 weeks before ART) and the 2nd phase was during ART at 12 weeks pre-ATI. ART alone (n=5) group served as the control.

IL-15/IL-15Ra and IL-15/IL-15R+IL-12 therapies induced significant expansion of Ki-67+ (proliferative), CXCR5 (follicular homing) SIV-specific CD8 T and CD16+ NK cells in blood and LN. Importantly, IL-15/IL-15Ra therapy resulted in the expansion of CD107a+CD8 T cells pre-ATI (P=0.03). Further, the blood transcriptomic profile displayed induction of cytolytic molecules (granzyme-B, perforin), Jak/Stat signaling pathway. Post-ATI, the IL-15/IL-15Ra treated animals showed 3-log lower viremia compared to other groups (p=0.004) with 83% of RMs <500 copies/ml at 20 weeks. SIV-specific CXCR5+, CD28+ CD8 T , CD16+ NK in blood, LN were associated with enhanced viral control. These studies define IL-15/IL-15Ra as a potentially effective immune therapy for HIV cure strategy.
SARS-CoV-2 adaptive responses in Confirmed Positive (CP) and non-infected household contacts (HC)
Authors: Lee Yeji , Goodwin Benjamin , Tarke Alison , Frazier April , Grifoni Alba , Dan Jennifer , Sette Alessandro , da Silva Antunes Ricardo
Published: November 2024

Objective: Many studies have explored the protective mechanism from infection of SARS-CoV-2. However, studies of couples living together in which one individual experienced symptomatic Confirmed Positive (CP) while the other consistently tested negative for infection (Household contact; HC) are scarce. Herein, we investigated humoral and T-cell responses in pre- and post-infection samples from CP and HC.

Methods: 52 CP and 44 HC individuals were recruited. Humoral responses were measured from plasma. Antigen-specific T cells against spike, non-spike, and common cold coronaviruses (CCC) were measured using an AIM+ICS from PBMCs.

Results: Humoral and T responses were similar between CP and HC before infection. In longitudinal analyses, except for the spike T responses, those responses were significantly increased after infection in CP but not in HC. One-third of the HC pairs were also associated with increased non-spike reactivity besides displaying increased spike reactivity in post-infection. 41.7% of HC pairs showed high pre-existing or CCC-specific T cells in pre-infection. No obvious pattern was found in the remaining HC pairs.

Conclusions: Measurement of antibody and T cell responses before infection does not predict protective mechanisms in household discordant pairs. Despite testing negative for COVID-19, some HC might have experienced asymptomatic/abortive infections and the other HC might have been protected due to the pre-existing or cross-reactive T cells.
The gastrointestinal tract-restricted helminth H. polygyrus bakeri protects against SARS-CoV-2-induced lung disease in mice
Authors: Karl Courtney , Desai Pritesh , Liu Meizi , Mackin Samantha , Chong Zhenlu , Yu Jinsheng , Thackray Larissa , Diamond Michael
Published: November 2024

Helminth-virus coinfection models can provide valuable insights into the regulation and modulation of host antiviral immunity. Epidemiological evidence suggests that pre-existing helminth infection is associated with reduced risk of severe SARS-CoV-2 infection. Several prior studies have reported that Heligmosomoides polygyrus bakeri (Hpb), an enteral murine helminth, protects against infection by respiratory viruses. Here, we developed an Hpb-SARS-CoV-2 coinfection model and show that coinfection with Hpb protects against mouse-adapted SARS-CoV-2 (MA10) induced weight loss and mortality. Compared to mice infected with MA10 alone, coinfected mice have a small reduction in peak lung viral load, increased eosinophil infiltrates in the lung and bronchoalveolar lavage fluid, and better lung function and blood oxygen levels. Using a series of knockout mice, we found that Hpb protects against SARS-CoV-2 independently of several canonical type 2 immunity pathways including STAT6, tuft cells, and IL-33 signaling as well as adaptive immunity. Coinfected mice that are depleted of eosinophils have a partial loss of protection. Together, these findings suggest that enhanced eosinophil production induced by Hpb infection protects mice from MA10-induced inflammation in a STAT6-independent manner.
PPAR delta promotes tumor-infiltrating Treg plasticity
Authors: Bunk Paul , Maher Timothy , Beyaz Semir
Published: November 2024

Regulatory T-cells (Tregs) accumulate within the tumor microenvironment and are exploited to suppress anti-tumor immune responses. Lipid metabolism has been implicated as a regulator of Treg function. Unexpextedly, we found the master regulator of fatty acid catabolism PPARd as a Treg-intrinsic switch that restricts tumor growth by inducing Treg plasticity. Activation of PPARd in Tregs specifically leads to inflammatory reprograming of the tumor immune microenvironment without adverse effects in the periphery. We identified PPARd-induced transcriptional, epigenetic and metabolic alterations in Tregs that drive this plasticity response. Finally, using an inducible model of PPARd gain-of-function in Tregs, we demonstrate therapeutic potential of targeting this transcription factor to enhance immunotherapy against cancer. Collectively, these results indicate that inducing Treg plasticity in tumors can be harnessed to promote anti-tumor immunity.
Time-resolved analysis of CAR-T Cell induced apoptosis of low frequency target cells using an Image-enhanced flow cytometer was enabled by fluorescent protein compensation with BrightComp eBeads.
Authors: Narayanan Jayanth , Taylor Erin
Published: November 2024

Optimization of CAR-T therapies requires an understanding the dynamic interactions between the CAR-T and target cells. Flow cytometry analysis of CAR-T cell interaction with dimly expressed fluorescent protein in target cells presents a major challenge for compensation especially in the presence of high numbers of non-target cells. Also, short, transient interaction between CAR-T and target cells makes it challenging to observe and characterize CAR-T cell candidate interactions. Here we demonstrate the use of the Attune CytPix imaging cytometer and BrighComp eBead in CFP and mCherry to analyze CAR-T target cell interactions and compensate for dim fluorescent proteins expression. We also leverage the the Caspase 3/7 to report on apoptosis over the entire course CAR-T mediated cell killing. Attune Cytpix image-enhanced flow cytometer enabled the simultaneous characterization of various cellular parameters, including cell morphology, fluorescent protein expression and apoptosis progression. Although apoptosis increased overtime, the frequency of true CAR-T and Target cell interaction did not change with time, however, these interactions were greatly altered by altering the CAR-T cell:Target cell ratios. This comprehensive methodology employed in our study and findings will contribute to the optimization and development of Car-T cell therapy approaches for improved treatment outcomes.
Langerhans cell histiocytosis (LCH) preceding ulcerative proctitis: Immunological insights
Authors: Jafri Ali , Gerardot Shori , Shaya Justin
Published: November 2024

Langerhans cell histiocytosis (LCH) is a disorder of abnormal myeloid dendritic cell proliferation; this process is thought to be linked to mitogen-activated protein kinase (MAPK) pathway mutations. This case report will examine the unusual sequence where LCH preceded the diagnosis of ulcerative proctitis. Interestingly, interleukin-23 (IL-23) is a shared mediator in both processes.

A 25 year-old white male presented with worsening right flank pain and eczematous rash. Skin biopsy revealed numerous Langerhans cells with surrounding inflammatory cells; Immunohistochemical staining was positive for CD1a, s100, and cyclin D1. PET-CT scans revealed pulmonary and bony involvement. Multi-system LCH was diagnosed and complete metabolic response was achieved with cytarabine therapy. Social history included marijuana usage.

Curiously, the patient developed frequent non-bloody diarrhea, and crampy abdominal pain a year later. Colonoscopy and biopsy revealed chronic active proctitis with ulcerative proctitis being the primary differential diagnosis. Mesalamine was started and remission was achieved.

The unusual history of both LCH and ulcerative proctitis raises the possibility of a shared immunological pathway; specifically, if IL-23 is a common mediator. This warrants further research. Additionally, cigarette smoking is a known risk factor for LCH while potentially decreasing risk of ulcerative colitis. As marijuana is increasingly legalized, its profile is also worth exploring.
The production of CXCL13 in CD4+ T cells is influenced by adjacent Tregs in the tertiary lymphoid structures of chronic pemphigus blisters
Authors: Lee A Yeong , Han Dawoon , Kim Taehee , Min SeoYeon , Kim DongJun , Jung Ju Hyun , Kim Changhyeon , Park Kyemyung , Kim Do-Young , Kim Soo-Chan , Kim Jong Hoon
Published: November 2024

Pemphigus is a rare and life-threatening autoimmune blistering disease mediated by autoreactive B cells targeting desmogleins (DSGs) in keratinocytes. Anti-DSG autoantibodies disrupt cell-cell adhesion, leading to suprabasal acantholytic blisters. Our investigation reveals the presence of tertiary lymphoid structures (TLSs) harboring DSG-specific B cells and plasma cells in chronic skin blisters of pemphigus. TLSs, resembling B-cell follicles of secondary lymphoid organs, are detected in various inflammatory diseases. CXCL13 is an important chemokine for recruiting CXCR5-expressing naïve B and T follicular helper cells. Through immunofluorescence studies, we found that CXCL13+ cells were mainly produced by CD4+ T cells. Single-cell RNA sequencing revealed clonally expanded CXCL13+CD4+ T cells containing DSG3-specific memory T cells with activated cytotoxic features. Paradoxically, these cells exhibit an attenuation of T-cell receptor (TCR) signaling when expressing high levels of CXCL13. Spatial proteomics reveals that Tregs are spatially adjacent to CXCL13+CD4+ T cells within the skin TLSs. In vitro depletion of Tregs leads to decreased CXCL13 production by CD4+ T cells. These data suggest that adjacent Tregs play a role in regulating CXCL13+CD4+ T cells. In conclusion, skin TLSs are associated with the maintenance of chronic blisters, and the microenvironmental network involving CXCL13+CD4+ T cells and Tregs plays a crucial role in CXCL13 production.
Dissecting signaling parameters in autoreactive B cells
Authors: Pozovskiy Rita , Atisha - Fregoso Yemil
Published: November 2024

In systemic lupus erythematosus (SLE), IgG antibodies (Abs) directed against nuclear antigens (ANA) are pathogenic. Healthy individuals (HC) harbor a similar frequency of ANA+ mature B cells without producing pathogenic IgG Abs. Differences in behavior between ANA+/- B cells in SLE patients and HC have not been explored. The aim of this study was to measure the response to BCR activation in the ANA+/- B cell subpopulations in SLE patients and HC. B cells from 7 SLE patients and 5 HC were isolated and activated with F(ab)'2 fragments of anti-IgG Abs. Calcium flux was measured using indo-1 AM. The response was quantified by area under the curve (AUC), using the bound/unbound ratio of the marker for 5 minutes after activation. Values were compared using Wilcoxon rank sum test. In SLE patients, ANA+ IgG memory cells have a stronger calcium influx than ANA- (AUC for ANA + vs -: 89.46±18.51 vs 76.17±12.65; p=0.05). This difference is not present in HC (AUC for ANA + vs -: 88.02±16.51 vs 89.52±14.49; p=ns). In both HC and SLE patients, ANA+ naïve B cells are less responsive to BCR activation, suggesting anergy. IgM+ B cells overall, irrespective of ANA status, seem to be less responsive in patients with SLE. These results show that ANA+ IgG memory B cells are hyperreactive in SLE patients. The data also suggests a mechanism for the breach in tolerance that is observed in SLE. Further studies are warranted to explore the mechanism and effects on autoantibody generation in SLE.
CCR2, CCR4 and CCR9 expression on T cells is required for homing to colon cancer as demonstrated by Zr-89 T cell labeling
Authors: Sato Noriko , Olkowski Colleen , Minor Hannah , Lin Qiaoya , Choyke Peter
Published: November 2024

Adoptive T cell therapy relies on trafficking of infused T cells to the tumor. Chemokine gradients and chemokine receptors expressed on T cells mediate this recruitment of T cells. However, due to the high complexity of the tumor microenvironment, regulation of T cell homing to tumors remains poorly understood. We examined how various chemokine receptors expressed on T cells impact their ability to home to cancer using zirconium-89 (89Zr)-oxine cell labeling. Ex vivo activated mouse T cells, wild type (WT) or deficient (KO) for a chemokine receptor, were labeled with 89Zr-oxine and intravenously infused into WT mice bearing subcutaneous syngeneic MC38 colon cancer. Bulk T cells, not tumor antigen specific T cells, were used to determine homing capability of the cells independent of their antigen interaction. Serial PET imaging demonstrated initial lung accumulation of the T cells followed by trafficking to the liver, spleen, and to a much lesser extent other tissues, including the tumor. Ex vivo biodistribution analyses on Day 2 showed CCR2KO, CCR4KO and CCR9KO T cells homed significantly less to the tumor compared to WT T cells, indicating that CCR2, CCR4 and CCR9 expression on T cells is important for homing to colon cancer. Augmenting expression of these chemokine receptors on T cells, or the relevant ligands in the tumor, could enhance T cell homing to colon cancer thereby improving efficacy of T cell therapy.
Correlates of susceptibility in household influenza studies
Authors: St. James Deryn , Souquette Aisha , Mettelman Robert , Van de Velde Lee-Ann , Wilson Taylor , Vegesana Kasi , Allen E Kaitlynn , Petrie Joshua , Gordon Aubree , Martin Emily , Monto Arnold , Thomas Paul
Published: November 2024

Every year there is an estimated one billion cases of seasonal influenza, with varying disease severity. While some have asymptomatic infections, others are hospitalized which can result in fatal disease. It is still unclear why only some will develop symptomatic or severe illness. Household studies allow us to investigate infection status after a known exposure to influenza. The Nicaragua Household study, and the Household Influenza Vaccine Effectiveness (HIVE) study have been formed to further understand of protection and severity. To this end, we characterized distinct cellular profiles in our subjects with diverse infection outcomes. Acute infection sampling in the Nicaragua study identified high frequencies of basophils, gamma delta T cells, and interferon gamma producing natural killer (NK) cells in subjects with mild disease, whereas granzyme B producing NK cell populations were correlated with severe disease. In the HIVE study, pre-infection samples with higher cytotoxic and cytokine producing NK cells as well as high neutrophil cell populations were correlated with protection from influenza infection and severe disease. Further analysis is in progress to train and test models to predict susceptibility to influenza and disease severity. These analyses expand the diversity of subjects analyzed for immune features in influenza disease to identify more universal correlates of susceptibility.
Heme drives proliferation of naïve and memory B cells by enhancing G1/S phase cell cycle progression
Authors: Padilla-Quirarte Herbey , Scharer Christopher , Kania Anna , Janto Nic , Hicks Sakeenah , Maurer Jeffrey , Boss Jeremy
Published: November 2024

Iron is elemental for multiple cellular biochemical processes like DNA synthesis and energy generation and recently the role of it has been reported to be important for B cells to produce antibodies. B cell differentiation to plasma cells is tightly regulated with cell division, where several epigenetic and metabolic changes occur, as cell divides. RNAseq performed on iron/heme treated differentiating naïve B cells (nB) after activation with LPS showed that heme regulated cell cycle, proliferation, and DNA synthesis genes when compared to cells treated with vehicle. Genes like Cdkn1a, Cdkn2a and Cdkn2b, which codify for cyclin-dependent kinase inhibitors that control the retinoblastoma protein activity were DEG with heme treatment, as was as Rb1 gene itself. To understand better how heme regulates cell division, ex vivo cultures of naïve and memory B cells (MBC) were stained with the cell tracing dye CTY and followed activation after T dependent or T independent activating signals. Both nB and MBC showed increased proliferative capacity against two antigens when treated with heme that correlated with higher levels of replicating cells as BrDu stain indicated. Furthermore, the Rb protein levels of total form were decreased on nB treated with heme compared to vehicle control. In summary, this work provides more insight to the mechanism of heme promoting plasma cell differentiation by enhancing the proliferation by enhancing G1/S phase cell cycle progression.
Corneal GR signaling is critical for preventing neovascularization and immune cell infiltration of the cornea
Authors: Diaz-Jimenez David , O'Neill Erin , Aguayo-Abarca Felipe , Kadmiel Mahita , Cidlowski John
Published: November 2024

The cornea protects the interior of the eye from external agents such as bacteria, viruses and debris. Synthetic glucocorticoids are widely prescribed in the treatment of ocular infections and disorders. The actions of glucocorticoids are mediated by the glucocorticoid receptor (GR); however, the molecular and physiological functions of GR signaling in the cornea are poorly understood. In this study, we show that treatment of mice with glucocorticoid eye drops led to regulation of the corneal transcriptome associated with multiple biological functions including the immune response. To understand the direct role of GR signaling in the cornea, we developed mice with conditional knockout of GR in the corneal epithelium. We discovered that an intact corneal GR signaling pathway is required for ocular development. Mice lacking corneal GR exhibited microphthalmia, loss of pupils, and mislocalization of key structural proteins within the corneal epithelial layers. Global transcriptomic approaches revealed that loss of GR signaling in the cornea also resulted in the dysregulation of a large cohort of genes strongly associated with an enhanced inflammatory response. Finally, corneal GR signaling was required for preventing neovascularization of blood and lymphatic vessels and thereby immune cell infiltration of the cornea. These results reveal that corneal GR signaling plays a critical role in ocular development and in maintaining the homeostasis of the eye.
Understanding immune responses in children who remain aparasitemic despite exposure to Plasmodium falciparum malaria
Authors: Holla Prasida , Bhardwaj Jyoti , Gaskin Erik , Doumbo Safiatou , Ongoiba Aissata , Felgner Philip , Hopp Christine , Xuei Xiaoling , Kayentao Kassoum , Traore Boubacar , Crompton Peter , Tran Tuan
Published: November 2024

Reliable immune correlates of protection against Plasmodium falciparum (Pf) malaria remain elusive, partly because sustained protection from parasitemia through natural exposure is rarely observed. In a prospective cohort study in an area of intense malaria transmission in Mali, we identified a subset of young children who remained aparasitemic during 7 months of active and passive surveillance, despite boosting of Pf-specific IgG. To identify immune responses associated with apparent elite control of infection, we carried out CITE-seq of PBMCs from 7 pairs of age- and sex- matched aparasitemic and parasitemic children. We included Pf antigens MSP1- and AMA1- specific B cell tetramers in the CITE-seq cocktail and sequenced B cell receptors (BCRs) from these cells. To identify epigenetic changes, we performed scATAC-seq on additional samples. Immune cell types from aparasitemic children exhibited markedly different transcriptional states and epigenetic landscapes. Notably, CD14+ monocytes in aparasitemic children showed enrichment of inflammatory signatures including TNFα signaling via NF-κB along with differently accessible chromatin in the target genes of these pathways. We also identified 44 antigen-specific B cells, primarily from aparasitemic children, and cloned their BCRs into expression plasmids to produce Pf-specific monoclonal antibodies in vitro to test their ability to control parasite growth and enhance the opsonophagocytosis capacity of monocytes.
Rapid non-invasive tracking of splenomegaly with hands-free 3D ultrasound for on-demand assessment of immune disorders and cancer
Authors: Houston Zachary , Braza Julie , Tseng Jen-Chieh , Stanley Alexis , McMannus Craig , Czernuszewicz Tomasz , Gessner Ryan , Peterson Jeffrey
Published: November 2024

Splenomegaly is an important measure of cancer progression and immune disorders. Magnetic resonance imaging (MRI) and computed tomography (CT) are the standard in the field but are not optimal for preclinical studies due to their high cost, low throughput and reliance on contrast agents for soft tissue. Here, we report the use of a novel 3D automated ultrasound scanner (Vega, Revvity Inc.) for the rapid assessment of spleen volume in two murine models (cancer and lupus).

Cancer cohort: Tumor-bearing (4T1 breast cancer, right flank) mice were imaged alongside control mice at five timepoints over 25 days. Spleen volume was found to increase 18-fold (30±3 mm3 to 545±105 mm3) after 25 days of tumor growth (938±77 mm3).

Lupus cohort: MRL/lpr mice were allowed to age until they developed lymphoproliferation (12-14 weeks) and then imaged biweekly until 20 weeks of age. A 300-fold increase in calculated spleen volume by 18 weeks of age in MRL/ lpr mice was observed. Kidney volume doubled and a statistical increase (p<0.05) in vascular density was observed (using acoustic angiography), indicating systemic disease progression.

Average acquisition times for both cohorts were 30 seconds per mouse, showing excellent applicability of spleen volume as a measurement of disease progression compared to more laborious techniques (e.g. MRI). Future work in this area will explore the application of machine learning and AI-assisted auto segmentation tools for higher throughput data analysis.
Solutions for quantifying cytopathic effects after viral infection in vitro
Authors: Preyer Rosemarie , Kevin Strecker , Gladyschuk Vitaly , Klimovich Alexei , Ettischer-Schmid Nicole
Published: November 2024

Individual virological assays require a maximum of experience. For different experiments, we introduce an outstanding technology for evaluating them in a reliable way.

In viral infections in vitro, the cytopathic effect (CPE) occurs. In the beginning, only few cells show a CPE, which makes it different to estimate the success. Quanti-and qualification of CPE in stages 1-4 of cells can be helpful to assess the right timepoint for different approaches.

With the AID MultiSpot, an inverted microscope and a fluorescence EliSpot reader combined, the CPE can be monitored and quantified without cellular staining and w/o interrupting any experiment with liquid like medium.

Further assays like plaque assays are performed in platesfrom 6-384 wells, stained either with colorings like crystal violet or, mostly in 96-well format, with fluorescent dyes. The reader type for these (and many more) applications is the vSpot spectrum.

We treated cells with DMSO (Fig.1) or virus (Fig. 2), measured the CPE or counted the plaques. As the MultiSpot is automated, one well can be scanned that the whole well view is possible in magnifications from 4- to 20-fold. The vSpot is able to generate whole-well pictures from all plate formats due to its unique composition.

These readers enable to quantify easily the CPE to get an overview of the whole wells that no important event is missed. This can be done in order to relate different treated cells and to make results between different experiments comparable.
Immunophenotypic profiling reveals novel T cell subset associated with “tolerance” in pediatric transplant recipients
Authors: Zhang Wenming , Rao Mahil , Amouzgar Meelad , Reitsma Andrea , Pena Josselyn , Nova Patricia , Shah Anika , Bendall Sean , Esquivel Carlos , Martinez Olivia , Krams Sheri
Published: November 2024

Achieving allograft health remains a challenge in pediatric organ transplantation. Recently. we performed multilineage, single-cell analysis of the immune cell composition in pediatric solid-organ transplant recipients who received a heart, liver, or kidney allograft (n=52); 24 children had stable graft function while 28 children had a rejection episode. Hierarchical clustering of the mean cell type proportion across allograft types and health states demonstrated a distinct subpopulation of CD4 T cells (CD45RA-,CD25+,CD5+,CD38-) was significantly (p<0.05) associated with stable graft function and were termed TOT cells. The cosine similarity analysis of proteomics data showed that TOT cells are phenotypically most like Tregs and least like CD4+ TEMRA. To characterize the TOT cells, we developed an 8-marker flow cytometry panel to specifically sort Treg and TOT populations and performed 10x single-cell RNA-seq and TCR-seq with subsequent analysis in R and Loupe Browser. Differentially expressed genes that were specifically associated with TOT cells included ANXA1, IL7R, TCF7, THEMIS, NELL2, KLRB1 and CD40LG while Treg expressed FOXP3, IKZF2, CTLA4, IL2RA, and RTKN2, consistent with their known function in immune suppression. TCR analysis indicates unique expansions of T cell clonotypes in TOT cells not seen in Treg. Further investigation into the immunoregulatory capacity of TOT cells could be instrumental in developing new strategies to prolong graft survival.
KCa3.1 channels regulate NK cell tumor infiltration in head and neck cancer
Authors: Alshwimi Abdulaziz , Chimote Ameet , Weatherford Layne , Wulff Heike , Khodoun Marat , Wise-Draper Trisha , Conforti Laura
Published: November 2024

NK cells are important mediators of antitumor immunity in head and neck squamous cell carcinoma (HNSCC) where they need to infiltrate the tumor and make contact with the cancer cells in order to kill them. However, low NK cell tumor infiltration has been associated with poor outcomes and immunotherapy resistance. Previously we have shown that KCa3.1 channels are important regulators of Ca2+ fluxes and chemotaxis in T cells (Chimote et al., Science Signaling 2018). However, little is known about the role of KCa3.1 in NK cell chemotaxis. In vitro studies with human NK cells activated with IL2/IL15 showed that the KCa3.1 selective blocker TRAM-34 inhibited chemotaxis and random migration, while pharmacological KCa3.1 activation increased chemotaxis. In vivo experiments with a humanized HNSCC mouse model generated by implantation of Cal27 cells (a human HNSCC cell line) in immune-deficient NSG mice, followed by injection of healthy donor PBMCs, showed that SKA-31, a selective KCa3.1 activator, significantly reduced tumor burden compared to vehicle. This effect was not due to an effect of SKA-31 on cancer cells. HNSCC cell lines and patient tumor biopsies showed low to null KCa3.1 expression, and KCa3.1 activation did not increase their proliferation. However, quantitation of immunohistochemistry images of tumors from the HNSCC mice revealed a 2-fold increase in infiltrated NK cells in the SKA-31 treated group, thus supporting the important role of KCa3.1 in anti-tumor immunity.
KRAS inhibition triggers coordinated neoplastic and immune remodeling in the tumor microenvironment
Authors: Lu Daniel , Osgood Tao , Ma Shining , Estrada Juan , Arias Vanessa , Yamawaki Tracy , Hoang Uyen , Rex Karen , DeVoss Jason , Canon Jude , Lipford Rusty , Wang Songli , Coxon Angela , Li Chi-Ming
Published: November 2024

KRAS inhibitors are a novel, recently approved class of small molecules that target cancers with KRAS mutations, and understanding molecular mechanisms by which these inhibitors stimulate anti-tumor immunity will improve patient outcomes. In this study, we utilized a multi-omic approach combining single-cell RNA-seq, spatial transcriptomics, and flow cytometry to define cell signatures and signaling events associated with response in a murine model expressing KRAS-G12C tumors. Treatment with KRAS inhibitors but not MEK inhibitors drove neoplastic differentiation toward cell states with increased oxidative stress and extracellular matrix remodeling capacity. These changes were accompanied by spatially-defined Notch and Integrin signaling patterns between neoplastic, stromal, and classical dendritic cell (cDC) niches, leading to accelerated cDC maturation and CD8 effector T-cell activation. Our multi-omic design distills the complex cellular consequences and intercellular communication patterns in the tumor microenvironment following KRAS inhibition and provides a framework for contextualizing and validating disease-relevant signatures to guide therapeutic strategy.
Host genetic mechanisms regulate gammaherpesvirus mediated exacerbation of CNS autoimmunity.
Authors: Sessions Katherine , Holt Emily , Waytashek Courtney , Usherwood Edward , Krementsov Dimitry
Published: November 2024

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). Chronic infection with the gammaherpesvirus Epstein Barr virus (EBV) is the leading environmental risk factor, and the mechanisms behind this risk, including the role of host genetics, are still unclear. We have previously shown that host genetics play an essential role in the infection outcomes of a rodent pathogen homologous to EBV, murine gammaherpesvirus 68 (MHV68), where poor control of MHV-68 replication was associated with an expansion of cytotoxic CX3CR1+KLRG1+ T helper cells (ThCTL). To explore how genetically-determined infection outcomes modulate CNS autoimmunity, C57BL/6J (B6) and 129S1/SvImJ (129S) mice were first chronically infected with MHV-68 and then immunized for induction of experimental autoimmune encephalomyelitis (EAE), a rodent model of MS. Viral load was higher in 129S mice, and EAE clinical scores were more exacerbated by MHV-68 infection compared with B6. MHV-68 infection induced higher levels of CD8 T cells and IFNg production in CNS in both strains. In contrast, MHV-68 infection only promoted ThCTL infiltration into the CNS of B6 mice. Ongoing experiments will address the functional role of CD8 T cells and ThCTL. Because poor control of EBV and enhanced anti-EBV responses are associated with greater MS risk, our findings suggest that this could result from genetically determined control of viral load and augmented ThCTL and/or CD8 T cell responses in the CNS.
Mouse models advancing immunology research at The Jackson Laboratory
Authors: Beckwith Jason , Merriam Jennifer , Valenzuela Alicia , Pomerleau Darcy , Davis Crystal , Kelmenson Jennifer
Published: November 2024

The Jackson Laboratory (JAX) provides genetically-defined mice to the scientific community. This poster will highlight recently added novel models with immunology research application.

The portfolio of strains useful for transplantation/PDX and “humanized” mouse research continues to expand and complement the NSG (NOD scid IL2rgc-/-) model as ideal tools for engraftment of human or mouse tissues.

Novel fluorescent strains include Traffic Light Reporters for tracking DNA repair pathways, Gene Editing Reporters for detecting homology directed repair (HDR) and non-homologous end joining (NHEJ) repair events, as well as several CRISPR Editing, Bxb1 Integrase Editing, Base Editor and Prime Editor tools.

Recently added CRISPR Array Repair Lineage Tracing (CARLIN and DARLIN) component strains allow researchers to achieve high barcode diversity in targeted cell populations.

In addition to cryopreserving each strain, our genetic quality control program confirms mutation identity and genetic background, and screens for unwanted alleles (such as stray GFP, Cre, neo, etc.).

Search the comprehensive collection using the improved JAX Mouse Search site (mice.jax.org).

This resource includes models created by many generous donating institutions. Researchers are encouraged to donate their mouse lines via a very short Strain Submission form (jax.org/donate-a-mouse).

JAX is supported by the NIH, The Howard Hughes Medical Institute and other private charitable foundations.
Generation of type I interferon autoantibody tests for at-risk populations
Authors: Premsrirut Prem , Garcia Reino Eduardo , Gao Nicholas , Ayoluwa Nana , Sosa Elie , Brereton Racquel , Patera Andrew , Desikan Shreya , Davis Evan , Cheng Hui-Ting , Mendoza Rachelle
Published: November 2024

A plethora of evidence has shown that 10-20% of cases of severe COVID-19 occur in individuals harboring autoantibodies that neutralize type I interferons (IFNs) and thus block their ability to initiate innate defenses and dampen viral replication. These autoantibodies are, therefore, currently the strongest and most common determinants of a growing range of severe viral diseases, especially in the elderly; yet, no routine test is available to the general population to gain knowledge of their immune insufficiency and potential risk from future infections. Here, we developed a set of assays to establish the levels of anti-IFN autoantibodies and their neutralizing activity and performed validation studies in a cohort of hospitalized COVID patients (N=388) to assess their sensitivity and specificity. Consistent with previous findings, our tests developed in a CLIA-certified laboratory were able to detect anti-IFN autoantibodies in a sizable fraction of patients with severe and critical COVID-19 pneumonia. Taken together, our results suggest that anti-IFN autoantibody testing should be considered for standard clinical screening.
VIP deficiency disrupts immune ovarian homeostasis
Authors: Gori Soledad , Castagnola Lara , Gallino Lucila , Vota Daiana , Grasso Esteban , Parborell Fernanda , Pérez Leirós Claudia , Hauk Vanesa , Ramhorst Rosanna
Published: November 2024

Ovarian aging is associated with inflammation, contributing to the decline in oocyte count and quality. Since the vasoactive intestinal peptide (VIP) is an immunopeptide found in the reproductive tract with anti inflammatory effects inducing a tolerogenic response, we focus on VIP contribution to ovarian immune homeostasis preventing premature aging. We observed that young VIP KO mice (3 months old) display an altered ovarian histology with an increase in atretic follicles and less mature follicles as in aged WT mice (8 months old). This microenvironment was accompanied by an increased fibrosis deposition, ROS and IL-1β production. Interestingly, we found a significant decrease in ovarian reserve and the presence of foamy macrophages in young VIP KO ovary associated with premature aging. Getting insight into the mechanism involved in foam cell generation, we pre-conditioned young WT or VIP KO macrophages with WT or VIP KO ovarian media for 48h to mimic the ovarian microenvironment. Macrophages from VIP KO mice display a differential phagocytic ability of apoptotic bodies after preconditioning with WT or VIP KO ovarian media. However, only in VIP KO macrophages conditioned with VIP KO ovarian media we found an increase in lipid droplet accumulation, consistent with the formation of foamy macrophages. These findings highlight the role of VIP in regulating ovarian immune microenvironment, thereby preserving ovarian health and preventing premature aging.
V-set immunoglobulin domain-containing protein 4 (VSIG4) as a potential non-invasive biomarker of lupus nephritis
Authors: Teymur Aygun , Zhang Shu , Saxena Ramesh , Cai Qi , Wu Tianfu
Published: November 2024

Objective: This study aimed to identify a non-invasive biomarker of lupus nephritis (LN) for diagnosis, disease monitoring or flare prediction of LN.

Methods: Both the VSIG4 free form and immune complex (ICx) bound form in the urine of LN patients (N = 97) were examined using ELISA, to compare with chronic kidney diseases (CKD) controls and healthy controls. Immunohistochemistry was used to confirm the elevated tissue expression of VSIG4 in the renal biopsy of LN. Immunofluorescence was used to examine the VSIG4-mediated immune complex deposition in the kidney of LN.

Results: The urinary levels of both VSIG4 free form (P = 0.01) and VSIG4-ICx (P = 6.3 x 10-14) and were significantly elevated in LN patients, particularly in renal active LN (P = 4.8 x 10-16) compared to healthy controls. VSIG4-ICx effectively discriminated HC from LN (AUC = 0.84, P< 0.01) and active LN from inactive LN (AUC = 0.91, P< 0.01). The urinary immune complex form of VSIG4 was positively correlated with renal pathology activity index (AI). Immunohistochemistry highlighted the significantly elevated VSIG4 expression in both glomeruli and tubules in LN patients, compared to healthy controls.

Conclusion: Our findings underscore the diagnostic potential of VSIG4 as a non-invasive urinary biomarker for LN. With its correlation to disease activity and renal pathology, VSIG4 presents a promising avenue for enhanced diagnosis and monitoring of lupus nephritis, paving the way for improved patient care.
Amyotrophic lateral sclerosis is associated with changes in peripheral neutrophil heterogeneity and function
Authors: Baird Lillia , Teener Samuel , Webber-Davis Ian , Feldman Eva , Goutman Stephen , Murdock Benjamin
Published: November 2024

There is overwhelming evidence that the immune system plays a significant role in amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease characterized by motor neuron degeneration. Expected survival is 2-4 years from disease onset and treatment options are limited. There is significant clinical and pathogenic heterogeneity and an incomplete understanding of the underlying pathogenesis, including mechanisms of immune contribution. Neutrophils are consistently implicated in ALS, with increased count in the blood and infiltration into the spinal cord. Increased neutrophil count and activation are associated with shortened survival and neutrophil levels are the single best predictor of future ALS progression. Despite this, little is known about which neutrophil subpopulations are dysregulated or the underlying mechanisms contributing to disease progression. Primary neutrophils were assessed via flow and ex vivo stimulation. NET remnants (MPO:DNA, elastase, extracellular DNA) were quantitated in plasma. Distinct populations were observed in controls and ALS subjects, and ex vivo analysis of primary neutrophils and analysis of NET remnants demonstrates changes in effector functions that may contribute to BBB disruption and neuronal damage in ALS. These differences in neutrophil populations and function provide compelling evidence that neutrophils play a role in ALS progression, as well as provide novel targets for therapeutics.
Comparison of the immunological response in mice after bronchoscopic and surgical methods for delivery of Pseudomonas aeruginosa to murine airways
Authors: Schelde Karen , Sonneborn Claire , Buczkowski Natalia , Bobinsky Annemarie , Host Derek , Jafri Anjum , Barbato Elisha , Brumbaugh Sarah , Rietsch Arne , Hodges Craig , Bonfield Tracey , Drumm Mitchell
Published: November 2024

We have established improved methods to access the mouse airways and lungs for pulmonary research. We have applied a non-surgical, bronchoscopic system with a camera to bacterial infection studies and found it reduced mortality and weight change in infected mice compared to our previously used procedure, a tracheostomy involving surgery. The two procedures were compared with and without bacteria to identify the inflammatory responses to the surgery and to the bacteria involved. We found the mouse tolerated the bronchoscope much better than the tracheostomy which allowed delineation of the effects of the bacteria from those of the surgical wound. The bronchoscope appeared to reduce stress to the mice, compared to the tracheostomy, and provided a significant benefit of reducing morbidity and mortality, thus reducing the number of mice needed for experiments. We found that mice receiving sterile beads instilled in the lung by tracheostomy lost more weight compared to mice undergoing bronchoscopy. Adding bacteria gave rise to even greater weight loss displaying significantly higher in surgery compared to bronchoscopy groups. Furthermore for surgery, immune cells (especially neutrophils) were increased in broncho-alveolar lavage fluid, as were pro-inflammatory cytokines TNF-α, IL-1β, and IL-6. The effects from the surgical wound are significant and should be recognized and incorporated into experimental designs to best reflect the condition the mouse is intended to model.
γδ T cells are crucial effectors of antibacterial response in T2D complicated with diabetic foot
Authors: Lehman Natalia , Suchodolski Wojciech , Jakubik Karol , Huang Tsan-Shin , Zarobkiewicz Michał , Bojarska-Junak Agnieszka , Roliński Jacek
Published: November 2024

Nearly half a billion people worldwide have type 2 diabetes (T2D), which poorly controlled may lead to severe consequences, including diabetic foot. Growing evidence highlights the role of γδ T subsets in the antibacterial response. A total of 70 patients were divided into three groups: T2D, T2D with diabetic foot (DF), and individuals without T2D. Flow cytometry was used to assess the frequency of peripheral blood γδ T along with activation (CD69, CD56) and senescence (CD57) markers. Intracellular expression of cytokines was tested on peripheral γδ T and Vδ2 in vitro cultures. PBMCs were stimulated with S. aureus and P. aeruginosa lysates and the expression of CD25, HLA-DR, along with CD137, CD154 on Vδ1 and Vδ2 cells was determined. The predominance of the Vδ2 was observed in DF group. γδ T cells in DF patients were significantly activated, as evidenced by CD69 and CD56 expression. Moreover, upon phosphoantigen stimulation, both peripheral and zoledronate-derived Vδ2 exhibited increased levels of intracellular IL-17A and TNF compared to patients without T2D. The γδ T cell responses to bacterial lysates were more pronounced in Vδ2 than in Vδ1 cells within the DF group. Among Vδ1 cells more of them were CD137 positive than Vδ2. Overall, the data show differentiated antibacterial properties of Vδ1 and Vδ2 cells in T2D patients. Understanding of the role of γδ T cell subpopulations may improve the prevention of T2D complications.
IM156, a new AMPK activator, ameliorates polymicrobial sepsis by regulating inflammatory responses
Authors: Kang Ji Hyeon , Lee Sung Kyun
Published: November 2024

IM156, a biguanide with a higher potency of AMP-activated protein kinase (AMPK) activation than metformin, exhibits inhibitory activity against angiogenesis and cancer. In this study, we explored the anti-inflammatory effects of IM156 against polymicrobial sepsis. Administration of IM156 significantly improved the survival rate and tissue damage in cecal ligation and puncture (CLP)-induced sepsis. Mechanistically, IM156 markedly reduced the number of bacterial cells in the peritoneal fluid and peripheral blood in a CLP-induced sepsis model. IM156 also inhibited splenocyte apoptosis and the production of inflammatory cytokines, including IL-1β, IL-6, and IL-10, in CLP mice. Furthermore, IM156 strongly inhibited the reactive oxygen species (ROS) generation by enhancing antioxidative effects, which can lead to reduced neutrophil extracellular trap (NET) formation in response to lipopolysaccharide. In conclusion, these findings collectively indicate that IM156 can suppress the inflammatory response and provide protection against polymicrobial sepsis. These results suggest the potential of IM156 as a novel therapeutic agent for sepsis control.
Macrophage GPR18 signaling activation by Resolvin D2 mediates metabolic switch and cardiac repair after myocardial infarction.
Authors: Thakkar Abhishek , Thorp Edward , DeBerge Matthew , Fredman Gabrielle
Published: November 2024

Higher inflammatory response is a major cause of heart failure and mortality post myocardial infarction (MI), a major health problem worldwide. Resolution of inflammation, well established as an actively orchestrated programmed response, is mediated by specialized pro-resolving lipid mediators (SPM) and cellular processes like efferocytosis. Resolvin D2 (RvD2), one such potent SPM, is expressed in leukocytes and stimulates the resolution of inflammation through signaling by its receptor GPR18. RvD2 signaling attenuates inflammation in immune-related conditions through macrophage (Mφ) cell-intrinsic and extrinsic mechanisms. The therapeutic potential of SPM has been demonstrated in treatment of sepsis and arthritis. However, the role of Mφ RvD2-GPR18 signaling in heart post-MI has not been elucidated. Here, we demonstrate (i) induction of GPR18 receptor on cardiac Mφ, (ii) >50% reduction in survival of myeloid- and Mφ-specific GPR18-/- mice, (iii) reduced heart function in GPR18-/- mice post-MI, (iv) a GPR18-dependent suppression of TLR4 signaling cytokines, and (v) suppression of TLR4-induced glycolytic metabolism by RvD2-GPR18 signaling in vitro in Mφ from human GPR18 chimeric adult mice. To summarize, our data suggest GPR18-dependent metabolic and phenotype switch to anti-inflammatory Mφ and cardiac repair post-MI. Further interrogation will delineate the intracellular pathways downstream of GPR18 and demonstrate the potential of RvD2 in reducing mortality post-MI.