bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of membrane bound IL-21 expanded natural killer cells for chronic lymphocytic leukemia therapy
Authors: Yano Max , Lance J. Rachel , Zapolnik Kevan , Mo Xiaokui , Lee Dean A , Byrd John , Muthusamy Natarajan
Published: February 2023

Chronic lymphocytic leukemia (CLL) is the most prevalent adult leukemia and remains incurable despite recent therapeutic advances. Natural killer (NK) cells have potent anti-cancer effects, but efforts to use NK cell therapy in CLL have been limited by poor NK cell growth and anti-leukemia activity. We sought to determine if stimulating NK cells ex vivo with K562-based feeder cells expressing membrane-bound IL-21 (mbIL-21) (Denman et al., 2012) could overcome the limitations of prior techniques to create an effective therapy for CLL.

Our data demonstrate that healthy donor-derived mbIL-21 expanded NK cells have increased antibody-dependent cellular cytotoxicity (ADCC) and direct cytotoxicity (DC; without targeting antibody) against CLL cell lines. This technique can also overcome CLL-induced NK suppression – CLL patient-derived NK cells expanded equally and were as potent as normal donor-derived NKs, including against both allogeneic and autologous CLL tumor cells. CLL tumor cells are not susceptible to DC but are very vulnerable to ADCC with anti-CD20 antibodies (rituximab or obinutuzumab). The lack of direct cytotoxicity of tumor cells was correlated to low NK:CLL quantitative conjugate formation, suggesting a lack of tumor target recognition.

Blocking antibodies to FasL, TNFα, and TRAIL show that only TRAIL contributes modestly to anti-CLL tumor cytotoxicity, while application of folimycin demonstrates that perforin/granzyme release is the major mechanism of cytotoxicity. In vivo, treatment with expanded NK cells plus obinutuzumab significantly prolongs survival in the OSU-CLL human xenograft model. Based on these data, we are planning a clinical trial of mbIL-21 expanded NK cells plus obinutuzumab in CLL patients.
TetTCRII-SeqHD: High-dimensional analysis of antigen-specific ex vivo CD4+ T cells
Authors: Schonnesen Alexandra A , Xia Amanda , Parker Patrik , Jiang Ning
Published: February 2023

Objective:

We present a method for high-throughput and inexpensive generation of DRB1 tetramers to assess CD4+ T cell specificity with special applications in oncoimmunology.

Methods:

Peptides were generated by in vitro transcription and translation (IVTT) of dsDNA. These peptides were then loaded onto DRB1 MHC molecules via chemical exchange and tetramerized using DNA barcoded streptavidin as previously described. As a proof of concept, we generated 250 DNA-tetramers bearing peptide segments from a variety of naïve and experienced antigens. These tetramers were pooled and used to stain primary CD4+ T cells from healthy donors. These cells were then labeled with Abseq antibodies (BD Biosciences), sorted and loaded onto the BD Rhapsody.

Results:

We analyzed ~5000 CD4+ T cells from healthy peripheral blood and assessed their phenotype, specificity and TCR sequence. We demonstrated that this technique robustly identifies and characterizes antigen-specific CD4+ T cells. It replicates previous findings in low-throughput systems in terms of clonality and phenotype distribution across memory and naïve T cell phenotypes. Given these results, we then analyzed tumor infiltrating lymphocytes and identified foreign specific cells within this compartment. We also found explicit differences in the phenotypes of antigen specific cells within the tumor and a paired healthy tissue sample.

Conclusions:

In conclusion, we validate the use of a high-throughput technology for assessing the specificity, phenotype, TCR, and gene expression patterns in antigen-specific CD4+ T cells in peripheral blood and tumor infiltrating lymphocytes.
Accelerating ADCP screening using the iQue® Advanced Flow Cytometry Platform
Authors: McBain Kirsty A , Weldon Caroline , Szybut Clare , Dale Tim , Carter Mark
Published: February 2023

Antibody-dependent cellular phagocytosis (ADCP) is an immune process stimulated by monoclonal antibodies (mAbs) that promotes specific recruitment of phagocytes to eliminate targeted cancer cells. In vitro functional assays are used extensively in drug development to characterize the effects of mAbs on a range of mechanisms of action, including ADCP.

We describe a high throughput assay for ADCP quantification via co-localization analysis of labeled target cells with CD14 positive effector cells. Target cells were incubated in 96 or 384 well plates with clinically relevant mAbs prior to addition of PBMCs or isolated immune cells (e.g. monocytes or macrophages). Cells were analyzed using the iQue®3 Advanced Flow Cytometer and inbuilt software. Various effector:target (E:T) cell ratios were used to validate the assay.

Maximal ADCP of non-adherent Ramos target cells by PBMCs in the presence of Truxima was 43 ± 2% with an E:T ratio of 20:1, reducing to 19 ± 6% with a 5:1 ratio. Further pharmacological data was obtained for adherent AU565 cells, comparing the response between anti-HER2 mAb isotypes. The potency of response to anti-HER2-IgG1 mAb (Trastuzumab) was five times greater than to another native human isotype, anti-HER2-IgA2, with EC50 values of 6.6 ng/mL and 35 ng/mL, respectively. These data are in line with previous characterizations of isotype effector functions which showed increased ADCP functionality with IgG1 compared to IgA2 mAbs.

The iQue® platform provides a solution for rapid profiling of ADCP effector function in both adherent and suspension target cell models. This accelerates antibody discovery by highlighting potential efficacy and toxicity of antibody drug candidates, leading to greater productivity and insight.
Characterizing tumor-infiltrated immune cells with spatial context using an integrated RNAscope-immunohistochemistry workflow in FFPE tissues
Authors: Dikshit Anushka , Phatak Jyoti , Hernandez Lydia , Doolittle Emerald , Murlidhar Vasudha , Zhang Bingqing , Ma Xiao-Jun
Published: February 2023

Characterizing heterogenous populations of tumor-infiltrating immune cells requires a multi-omics approach. Here we demonstrate a newly developed integrated in situ hybridization (ISH) and immunohistochemistry (IHC/IF) workflow that can substantially improve RNA-protein co-detection, enabling the visualization and characterization of tumor immune infiltrates at single-cell and spatial resolution.

To characterize tumor-infiltrating immune cells in a tumor TMA (tumor microarray), we utilized the RNAscope Multiplex Fluorescence assay in combination with the RNA-Protein Co-detection Kit to detect multiple immune cell populations. Immune cells such as macrophages, T cells and NK cells were detected using antibodies against CD68, CD8, CD4 and CD56 in combination with probes targeting CCL5, IFNG, GNZB, IL-12, NCR1 etc.

We identified CD4+ regulatory T cells and CD8+ cytotoxic T lymphocytes. Additionally, we determine the activation states of CD8+ T cells by visualizing IFNG and GZMB expression. Furthermore, infiltrating macrophages were detected by CD68 protein expression while the M1 and M2 subsets were differentiated by using the M2-specific marker, CD163. NK cells were identified by detecting CD56 protein in combination with CCL5 and NCR1 RNA expression. The degree of immune cell infiltration varied based on the tumor type.

In conclusion, the new RNAscope-ISH-IHC co-detection workflow and reagents enable optimized simultaneous visualization of RNA and protein targets by enhancing the compatibility of antibodies, including many previously incompatible antibodies with RNAscope . This new workflow provides a powerful approach to identifying and characterizing tumor infiltrating immune cells.
Immunogenomic determinants of tumor microenvironment correlate with survival in high-risk neuroblastoma
Authors: Bao Riyue , Spranger Stefani , Hernandez Kyle M. , Zha Yuanyuan , Pytel Peter , Luke Jason J , Gajewski Thomas , Volchenboum Samuel L , Cohn Susan L , Desai Ami V
Published: February 2023

Background:

Tumor-infiltrating CD8+ T cells and neoantigens are predictors of a favorable prognosis and response to immunotherapy in adult cancer, but little is known about their role in pediatric malignancies. Here, we analyzed the prognostic strength of T cell-inflamed (Tinfl) expression and neoantigen load (neo) in high-risk neuroblastoma (NBL). We investigated transcriptional programs associated with the non-T cell-inflamed (non-Tinfl) phenotype for mechanisms of immune exclusion.

Methods:

Using a defined Tinfl gene expression signature, we categorized high-risk NBL from the TARGET (Therapeutically Applicable Research to Generate Effective Treatments, n=123) and the GMKF (Gabriella Miller Kids First, n=48) programs into non-Tinfl, Tinfl, and intermediate groups. Cox proportional hazard models were used to test the association between survival and tumor group, neo, MYCN amplification. Pathways activated in non-Tinfl vs. Tinfl tumors were identified.

Results:

Patients with high-risk Tinfl tumors showed improved overall survival (OS) compared to those with non-Tinfl tumors (P<0.05), independent of MYCN amplification, in both TARGET and GMKF. Higher neo was associated with better event-free survival and OS (P<0.005) and was independent of Tinfl. Activation of MYCN, ASCL1, SOX11, and KMT2A was inversely correlated with the Tinfl signature in both cohorts.

Conclusions:

Our results indicate that tumors from children with high-risk NBL harboring a strong Tinfl signature have a favorable clinical outcome, and neo is a prognosis predictor, independent of Tinfl. Strategies to target SOX11 and other signaling pathways associated with non-Tinfl tumors should be pursued as potential immune-potentiating interventions.
Ag-dependent Engagement of CD8+ T Cells with H-2Kb on Brain Endothelium Required for CD8+ T Cell entry and Cerebral Manifestation of Malaria
Authors: Fain Cori E. , Zheng Jiaying , Jin Fang , Ayasoufi Kathy , Tritz Zachariah Peter , Khadka Roman , Hansen Michael , Johnson Aaron J
Published: February 2023

Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection with high mortality. The immune system is a key participant as the cerebral manifestation is heavily reliant on CD8+ T cell infiltration and neuroinflammation. In this disease etiology, elevated blood cytokine levels lead to increased adhesion and antigen (Ag)-presenting molecules on brain endothelium prior to CD8+ T cell entry and onset of neurological symptoms. Using the animal model of experimental cerebral malaria (ECM) we sought to further define these interactions in vivo at the blood-brain barrier. While direct interaction with MHC class I on cerebrovascular endothelial cells (CECs) has been put forward as a putative mechanism for ECM, it has not been directly confirmed in vivo. We therefore hypothesized that Ag presentation by discrete MHC class I molecules on CECs would be required for CD8+ T cells to enter the brain and establish ECM. To test this, we crossed CDH5-Cre mice with novel H-2Kb LoxP and H-2Db LoxP mice to create endothelium-specific, individual class I cKO mice. In CDH5-Cre H-2Kb mice, we show that with T cell development fully intact, CD8+ T cell infiltration into brain is decreased in frequency and number at baseline and during the infection. T cells that do enter are increasingly naive with effector responses attenuated. Importantly, mice with loss of H-2Kb on CECs show extended survival and protection from cerebral complications; as assessed by 2-photon imaging, small-animal MRI and immunofluorescence. Together, these data demonstrate that parasite-specific CD8+ T cells must undergo Ag-dependent engagement with H-2Kb class I molecule on CECs in order to fully activate CD8+ T cells to enter brain parenchyma and mediate ECM development.
Immunopeptidomics analysis of Lyme arthritis: insights into infection and autoimmunity
Authors: Danner Rebecca , Pereckas Michael , Rouse Joseph , Wahhab Amanda , Lochhead Robert
Published: February 2023

Background:

Lyme arthritis (LA) is caused by infection with the Lyme disease spirochete Borrelia burgdorferi (Bb) and in humans is often accompanied by autoimmune T and B cell responses. In this study, we used an immunopeptidomics approach to gain further insight into mechanisms of infection-induced autoimmunity.

Methods:

C57BL/6 (B6) mice, which develop mild, self-limiting LA, and B6 Il10−/− mice, which develop chronic, autoimmune-like LA, were inoculated with 2×104 Bb. Inguinal and popliteal lymph nodes (LN) were harvested from infected mice at 4 weeks and 16 weeks post-inoculation. MHC class II molecules were isolated by immunoaffinity capture and MHC-bound peptides were identified by LC/MS/MS.

Results:

Nearly 10,000 MHCII-bound peptides were identified. At 4 weeks post-inoculation, representing the peak of LA, proteins involved in leukocyte trans-endothelial migration, tissue repair, and immune activation, including a known Lyme autoantigen ApoB-100, were over-represented in both B6 and Il10−/− mice. Peptides from these proteins returned to near baseline levels in LN from B6 mice at 16 weeks post-inoculation, when arthritis resolves, but were further enriched in Il10−/− mice at 16 weeks, during the chronic, autoimmune-like phase. Surprisingly, only 27 peptides derived from Bb proteins were identified, all but one of which were from proteins found in the inner membrane, periplasm, or cytosol.

Conclusions:

This study identified immune-relevant proteins presented by APCs in draining LN that are associated with LA development, which included ApoB-100, a known Lyme autoantigen in humans. Further studies are underway to assess T cell responses to identified Bb and self-peptides, including ApoB-100, during Bb infection.
INF-λ enhances expression of MHC class I molecules on thymic epithelial
Authors: Benhammadi Mohamed , Dumont-Lagacé Maude , Mathé Justine , Kobayashi Koichi S. , Gaboury Louis , Brochu Sylvie , Perreault Claude
Published: February 2023

Introduction: The major histocompatibility complex class I (MHC-I) molecules is a cell surface glycoprotein involved in the presentation of endogenously derived peptides, to the TCR of CD8 T cells. Moreover, several vital processes specifically depend on the interaction between TCR and the MHC-I presented by the epithelial cells (ECs). However, the regulation of the MHC-I expression has been studied almost exclusively in hematolymphoid cells and very little is known about this process in ECs. In the present work, we performed a deep analysis of MHC-I expression in primary ECs freshly harvested from the thymus, skin, gut, and lung.

Methods and Results: Our flow cytometry analysis showed that the abundance of cell surface MHC-I was overall 10- to 100-fold greater in thymic ECs (TECs) than in extrathymic EC types. Our transcriptomic analysis revealed a dominant role of IFN signalling in the differential MHC-I expression found in TECs vs. extrathymic ECs. In fact, superior MHC I expression in TECs is unaffected by deletion of Ifnar1 or Ifngr1, but is lessened by deletion of Aire, Ifnlr1, Stat1 or Nlrc5, and is driven mainly by type IFN-λ produced by TECs. Moreover, Ifnlr1−/− mice showed impaired negative selection of CD8 thymocytes, and at 9 months of age, present autoimmune manifestations.

Conclusion and Relevance: In conclusion, our study opens the door to a new level of understanding of MHC-I regulation in ECs and will pave the way for a more detailed exploration of the impact of IFN-λ signaling in thymic functions.
TSLP rather than IL-33 signaling drives intestinal mucosal mast accumulation and allergen-induced diarrhea in mice following epicutaneous sensitization to food allergen.
Authors: Brandt Eric B , Bolcas Paige E , Shik Dana , Ruff Brandy P , Hershey Gurjit K Khurana
Published: February 2023

Background:

A major route of sensitization to food allergen is through an impaired skin barrier. IL33 and TSLP have both been implicated in epicutaneous sensitization and development of food allergy.

Method:

We assessed the respective contributions of TSLP and IL33 to the development of food allergy in TSLP and IL33 receptor deficient mice following epicutaneous food allergen sensitization, where mice were exposed to thrice weekly skin patches of either saline, OVA or a combination of OVA and Aspergillus fumigatus extract (ASP).

Results:

ASP+OVA-patched but not OVA-patched mice developed an atopic dermatitis (AD)-like skin phenotype, while sensitization to OVA occurred in both OVA and OVA+ASP patched mice. OVA-specific IgE levels were significantly lower in OVA(±ASP)-patched TSLPR−/− mice compared to wild type mice and ST2−/− mice. Repeated intragastric challenges with 50mg of OVA, induced intestinal accumulation of mast cells but not ILC2s in OVA(±ASP)-patched WT and ST2−/− mice, and to a much lesser degree in TSLPR−/− mice. While OVA-induced mast cell degranulation assessed by measuring MCPT1 blood levels and the development of food allergy (diarrhea occurrences) was observed in almost all WT mice and two thirds of OVA+ASP patched ST2−/− mice, all TSLPR−/− mice were protected from developing food allergy and had significantly lower MCPT1 levels.

Conclusion:

Epicutaneous sensitization to food allergen and subsequent development of food allergy does not require atopic dermatitis skin lesions and is dependent on TSLP, suggesting that prophylactic targeting of TSLP may be useful in mitigating the development of food allergy early in life.
Analysis of transdermal sensitization and oral anaphylaxis using salt-soluble protein extracts from diploid wheats in an adjuvant-free mouse model
Authors: Gao Haoran , Jorgensen Rick , Raghunath Rajsri , Chandra Shivam , Wiersma Andrew , Nagisetty Siddharth , Olson Eric , Ng Perry KW , Gangur Venugopal
Published: February 2023

Wheat allergies are growing at an alarming rate for reasons that are not well understood. The commonly consumed wheats belong to the AABB and AABBDD genotypes. The AA wheat is less commonly consumed, and the DD wheat is not commercially available. It is unknown at present whether the diploid wheats elicit allergic reactions in mice. Here we tested the hypothesis that the salt-soluble protein extracts (SSPEs) from AA and DD wheats will induce food allergy in Balb/cJ male mice in an adjuvant-free model. We grew the DD wheat at our university and used it in this study. The AA wheat was obtained from www.einkorn.com. Balb/cJ male mice were bred and maintained on a plant protein-free diet. Mice were exposed to SSPEs or saline via skin once a week for nine weeks. SSPE-specific IgE (sIgE) was measured using an ELISA. After sensitization, mice were orally challenged with SSPEs or saline to elicit a hypothermia shock response (HSR). We found that both AA- and DD-SSPEs elicited significant and comparable levels of sIgE responses upon skin exposures. Oral challenge with DD-SSPE but not saline in DD-SSPE sensitized elicited a mild HSR lasting up to only 15 minutes. In contrast, oral challenge with AA-SSPE but not saline in AA-SSPE sensitized mice elicited a severe HSR lasting for 30 minutes. These data together suggest that the diploid wheats induce comparable sensitization, but DD wheat is less potent in inducing oral anaphylaxis compared to the AA wheat.
PD-1 regulates affinity maturation of allergen-specific antibodies and severity of peanut allergy in mice
Authors: Lama Jyoti K , Iijima Koji , Kobayashi Takao , Kita Hirohito
Published: February 2023

Programmed cell death-1 (PD-1), a member of the CD28 family, is critical for regulation of immune cell activation and maintenance of peripheral tolerance. Blocking antibodies against PD-1 have been used to sustain activation of T cells in cancer immunotherapy. T follicular helper (Tfh) cells play important role in the differentiation and maturation of antigen-specific B cells. Tfh cells highly and constitutively express PD-1; however, our knowledge is limited regarding the role of PD-1 in Tfh cell function and antibody response. We addressed this question by using a Tfh-dependent mouse model of peanut allergy. When naïve BALB/c mice were exposed to peanut flour by inhalation for 4 weeks, they developed peanut allergy as demonstrated by increased plasma levels of peanut-specific IgE and IgG antibodies and manifestation of acute anaphylaxis upon challenge with peanut extract. To examine the roles of PD-1, we administered a PD-1 blocking antibody to the animals during their exposure to peanut flour. Mice administered anti-PD-1 did not develop anaphylaxis even when they were challenged with peanut extract. Anti-PD1 treatment increased the number of Tfh cells and germinal center B cells in draining lymph nodes, and it increased plasma levels of peanut-specific IgG antibodies. Importantly, PD-1 blockade promoted the accumulation of low-affinity antibodies while it maintained the levels of high-affinity antibodies. These findings suggest that quality of allergen-specific antibodies affects the severity of allergic immune responses. Understanding the mechanisms that explain how PD-1 regulates the Tfh-B interaction and affinity maturation of antibodies likely leads to a better understanding of pathophysiology of allergic diseases.
Functions of an Environmental Allergen in Induction of Food Allergy.
Authors: Buelow Lauren M , Tat Kiet , Schroeder-Carter Lindsay , Carroll Daniela , Hoji Aki , Cook-Mills Joan M
Published: February 2023

Food allergy can be induced in neonatal mice with heterozygous skin barrier mutations and skin co-exposure to detergent, food allergen and a ubiquitous environmental allergen Alternaria Alternata (Alt) at doses in dust. In this study it was determined whether cytokines are induced by Alt in development of allergy to peanut extract (PNE). In preliminary RNAseq data, Alt induced expression of IL33, oncostatin M (OSM) and amphiregulin (Areg) in the skin of neonatal flakey tail (FT+/−) mice with heterozygous skin barrier mutations. In FT+/−, but not WT neonates, qPCR analyses revealed skin expression of IL33 and OSM following Alt or Alt+PNE exposure compared to neonates with saline skin exposure, while IL33 and OSM were not induced by skin exposure to PNE alone. The expression of Areg was induced by Alt, PNE or Alt+PNE in FT+/− neonates. It was determined whether intradermal injection of recombinant IL33 (rIL33), rAreg or rOSM could substitute for Alt during skin sensitization to PNE. PNE skin sensitization with intradermal rIL33 was sufficient for oral PNE induced anaphylaxis, whereas skin sensitization with intradermal rAreg or rOSM during skin exposure to PNE was not. Injection of neutralizing antibodies for Areg and OSM blocked anaphylaxis in Alt+PNE exposed neonates, indicating that Areg and OSM are required for PNE-induced anaphylaxis prior to skin sensitization. Analysis of skin IL33, Areg and OSM gene expression and plasma protein for IL33, Areg and OSM defined a pathway for IL33, Areg and OSM in Alt+PNE skin. Thus, Alt stimulated expression of IL33 and a pathway of OSM and Areg in concert with PNE to induce food allergy in FT+/− pups. These data have important implications for studies of detection of risks for sensitization to food allergens.
Dysregulated TGF-β Signaling Leads to Cell-Intrinsic Changes in T cell Development that Promote Allergic Inflammation
Authors: Haque Tamara , Weissler Kate , Schmiechen Zoe , Leak Lashawna , Laky Karen , Frischmeyer-Guerrerio Pamela A
Published: February 2023

Variants in genes in the Transforming Growth Factor Beta (TGFβ) signaling pathway are associated with allergic diseases suggesting that this cytokine may be a central player in the allergic diathesis. The role of TGFβ in preserving T cell mediated self-tolerance is well appreciated but how disruption in this pathway promotes allergic inflammation is unclear. We previously demonstrated that patients with Loeys-Dietz Syndrome (LDS), an autosomal dominant disorder caused by mutations in TGFBR1 and TGFBR2, are highly predisposed to allergic disease. LDS patients and mice harboring a knock in allele (Tgfbr1mut) of an LDS mutation known to cause severe disease in humans exhibit higher levels of total and food specific IgE. These serologic changes were associated with higher frequencies of T follicular helper (Tfh) cells and lower T follicular regulatory (Tfr) cells. Using a mixed bone marrow and fetal liver chimera model, we showed that these alterations in T cell development are cell intrinsic. Chimera models also demonstrated that the TGFβ mutation drives higher IgE production despite the presence of wildtype Tregs. Furthermore OVA specific Tgfbr1mutOTII cells were more likely to accumulate in the peyers patch and differentiate into Tfh cells in response to orally ingested OVA. These findings correlated with increased AKT/pS6 signaling downstream of TCR in Tgfbr1mut T cells, and mTOR inhibition decreased Tgfbr1mut OTII Tfh cells in response to fed ova. Additionally, RNA sequence analysis of murine Tgfbr1mut Tfh cells revealed enhanced PI3K/AKT pathway activity. These findings suggest that TGFβ signaling plays an important role in T cell development and function via the PI3K/AKT pathway that when disrupted promotes allergic inflammation.
The role of the stress-activated transcription factor Nrf2 in OVA-induced food allergy
Authors: Jin Yining , Boss Allison P. , Bursley Jenna K. , Gangur Venugopal , Rockwell Cheryl Elizabeth
Published: February 2023

Food allergies are rising at a striking rate, but the underlying cause is unclear. The purpose of the studies was to determine the effect of the food preservative tert-butylhydroquinone (tBHQ) in a mouse model of food allergy. Our preliminary studies show that skin-sensitization with OVA induced an elevated OVA-IgE level in mice on a tBHQ diet. Likewise, in response to oral challenge, a greater decrease in body temperature during anaphylaxis was observed in the tBHQ group. Previously published in vitro studies suggest that tBHQ promotes Th2 polarization through activation of the stress-activated transcription factor, Nrf2. Therefore, we proposed the hypothesis that tBHQ exacerbates allergic response in OVA sensitized mice through activation of Nrf2. We implemented adoptive transfers of CD4 T cells from wild-type or Nrf2-null mice into immunodeficient mice (all mice received wild-type B cells). The study revealed marked genotype differences. Notably, dietary tBHQ had little effect on food allergy in recipients of Nrf2-deficient CD4 T cells. These mice showed attenuated sensitization as compared to wild-type CD4 T cells recipients. Strikingly, mice receiving Nrf2-deficient T cells did not become anaphylactic following challenge. In contrast, tBHQ caused an increase in OVA-IgE levels and a greater drop in body temperature in mice receiving wild-type CD4 T cells, suggesting these effects are Nrf2 dependent. In addition, recipients of Nrf2-deficient CD4 T cells showed an attenuated percentage of Th2 cells and mast cell degranulation response, following challenge. Taken together, these data suggest a critical role for Nrf2 during food allergy and demonstrate that tBHQ exacerbates food allergy in a CD4 T cell-Nrf2 dependent manner.
Mixed species live biotherapeutic product improves immunological tolerance in food allergy
Authors: Qayum Amina Abdul , Valladares Ricardo , Gehrig Jeanette
Published: February 2023

The human gut microbiota is central to early-life immune system education, including the establishment of immune tolerance, and thus represents an important target for the development of preventative interventions against food allergy (FA).

The composition of the gut microbiota, and specific bacterial members within the microbiota, have been shown to impact allergic disease development in animal models by limiting basophil hematopoiesis, influencing macrophage polarization, and enhancing regulatory T cell activity. With a growing body of evidence showing the gut microbiota is an important environmental factor contributing to FA development comes an opportunity to translate this knowledge into novel approaches for treating and preventing FA.

Using a proprietary human gut microbiota consortium, we have developed a mixed species live biotherapeutic product (LBP) to alleviate FA development and severity. We show that this LBP can directly interact with human immune cell populations modulating immune signaling pathways including TGF-beta, FFARs, chemotaxis, and NFkB inhibition. We also demonstrate that supplementation with this LBP can reduce OVA induced oral anaphylactic severity in mice with humanized gut microbiota. This protective effect may be attributed to increased fecal antigen-specific IgA levels and enhanced intestinal TGFb levels. These data describe immunological mechanism of a mixed species LBP that is protective in mouse FA model.
Dendritic cell metabolism regulates allergic sensitization in asthma
Authors: Tharakan Anuj , Martin Rebecca
Published: February 2023

Allergic asthma is a respiratory disease characterized by airway inflammation driven by T-helper 2 (Th2) cytokines and antigen-specific IgE (Ag-specific IgE). Ag-specific IgE production requires T follicular helper 13 cells (Tfh13s), which are a population of IL-4/13+ Tfh cells. Tfh13 polarization is induced by type 2 conventional dendritic cells (cDC2s), however, the mechanisms by which cDC2s regulate Tfh13 polarization are unclear. We hypothesized that cDC2s would exhibit distinct transcriptional responses following in vivo exposure to the Tfh13 inducing stimuli, Alternaria, compared to non-Tfh13 polarizing stimuli, such as LPS or the protease papain. Thus, we treated mice intranasally with LPS, papain, or Alternaria plus fluorescently tagged OVA and sorted OVA+ migratory cDCs from the mediastinal lymph node (mLN) for RNA-seq analysis. Principal component analysis revealed that these stimuli induced unique transcriptional signatures in migratory cDCs. Gene set enrichment analysis demonstrated significant downregulation of oxidative phosphorylation (OXPHOS) genes in Alternaria stimulated cDCs compared to LPS or papain. We therefore hypothesized that Alternaria induces metabolic perturbations in cDC2s, whereby they upregulate glycolytic metabolism and decrease OXPHOS, and this metabolic profile was involved in Tfh13 polarization. To test this hypothesis, mice were either fed ad libitum or fasted for 12 hours prior to Alternaria exposure. Fasted mice exhibited a significant reduction in mLN Tfh13s and Th2 cells. Thus, we demonstrate that glucose metabolism may regulate allergic sensitization in asthma. Future experiments will aim to elucidate the role of cDC2-intrinsic metabolism in Tfh13 polarization.
Resolvin D2 Promotes the Resolution of Lung Inflammation in Allergen-Induced Asthma
Authors: Peh Hong Yong , Bruggemann Thayse R , Wong WS Fred , Levy Bruce D
Published: February 2023

Background:

Asthma is a chronic inflammatory disease without complete resolution. The production of specialized pro-resolving mediators (SPMs) is defective in asthma, so they represent a potential therapeutic opportunity. We hypothesize that docosahexaenoic acid derived resolvin D2 (RvD2) can promote the resolution of asthma.

Methods:

BALB/c mice were challenged intra-tracheally with house dust mite (HDM). Bronchoalveolar lavage (BAL) fluid was collected at predefined protocol endpoints for peak inflammation and during resolution. To determine impact on resolution, some mice were given RvD2 (100 ng, intra-nasally) for 2 days after the last HDM challenge, and the resolution index for eosinophils and other markers were calculated.

Results:

Airway HDM challenge led to marked increases in BAL eosinophils and BAL fluid pro-inflammatory cytokine and chemokine levels. Histology demonstrated changes in airway inflammation and mucous cell metaplasia in HDM-challenged lungs. NF-κB expression was increased in the lungs of HDM allergic mice. Compared to vehicle, treatment with RvD2 significantly decreased BAL inflammatory macrophage and eosinophil counts, and accelerated the resolution of HDM-evoked inflammation with a marked decrease in the resolution index for BAL eosinophils from 5 days to 1 day. Interestingly, RvD2 attenuation of eosinophils specifically targeted a specific eosinophil population, but not the “resident” eosinophils. The impact of RvD2 on NF-kB activity and its GPR18 receptor-mediated actions is under current investigation to determine mechanisms for RvD2’s pro-resolving actions.

Conclusion:

RvD2 promotes resolution of allergen-induced inflammation in a murine model of allergic asthma.
G-protein coupled receptor kinase 2 (GRK2) regulates T cell response in a murine model of house dust mite-induced asthma
Authors: Subramanian Hariharan , Kammala Ananth Kumar , Yang Canchai , Panettieri Reynold A , Das Rupali
Published: February 2023

G protein-coupled receptor kinase 2 (GRK2) is an adapter protein that modulates G protein-coupled receptor (GPCRs) signaling. It also regulates the functions and activity of other intracellular proteins in many cell types. Accordingly, GRK2 is thought to contribute to disease progression by a variety of mechanisms related to its multifunctional roles. Indeed, GRK2 levels are enhanced in patient samples as well as in preclinical models of several diseases. We have previously shown that GRK2 regulates mast cell functions, and thereby contributes to exacerbated inflammation during allergic reactions. In the current study, we observed that GRK2 levels are enhanced in the lungs of human asthma patients and in mice sensitized to house dust mite extract (HDME) allergen. Consistent with these findings, interleukin (IL)-4 and IL-13 levels were reduced in the lungs of GRK2+/− mice in a HMDE mouse model of asthma. Because Th2 cells are the major source of these cytokines during asthma, we determined the role of GRK2 in regulating T cell-specific responses in our HMDE mouse model. We observed a significant reduction of airway hyperresponsiveness (AHR), lung eosinophil and lymphocyte count, serum IgE, Th2 cytokines (IL-4 and IL-13), goblet cell hyperplasia and mucus production in mice that had reduced GRK2 expression specifically in T cells. Collectively, our studies reveal an important role for GRK2 in regulating T cell response during asthma pathogenesis and further elucidation of the mechanisms through which GRK2 modulates airway inflammation will lead to the development of new therapeutic strategies for asthma.
Impact of oral probiotic treatment in murine model of atopic dermatitis
Authors: Jung Ariee , Swartzendruber Julie A , Incrocci Ryan , Toro Rosalind Del
Published: February 2023

The number of people with allergies has been on the rise over the last half-century; and the most common allergen afflicting humans, dogs and cats is the house dust mite (HDM). The rise in allergic disease correlates with an increasingly sterile living environment and reduced exposure to bacteria. We predict that exposure to non-pathogenic bacteria helps educate our immune system to become tolerant to innocuous material, such as allergens. Our previous studies have found that Bacillus subtilis, a gram-positive spore forming bacterium, can reduce the symptoms of HDM-induced allergic lung disease. Our current work is aimed at determining if B. subtilis can also provide protection in a murine model of atopic dermatitis. To test this, we administer B. subtilis by oral gavage 3 days before allergen exposure and weekly-post allergen exposure (total of 9 applications), while HDM extract in PBS is applied topically, after shaving the mouse back, 3 times a week for 8 weeks (total of 24 applications). After 8 weeks the serum is assessed for HDM-specific antibodies by ELISA. Additionally, we monitor symptoms of atopic dermatitis: reduced scratching, skin lesions and histological thickening of the skin. Our results, have identified HDM-specific IgM, IgG1 and IgE levels are unaffected by oral gavage of B. subtilis. This suggests orally administered probiotics are not sufficient to protect from topical allergen sensitization
ADAMTS4 promotes chronic airway inflammation and hyperresponsiveness in HDM-based mouse model of asthma: A potential therapeutic target for asthma traits
Authors: Ghonim Mohamed A. , Boyd David F. , Van de Velde Lee-Ann , Souquette Aisha , Vogel Peter , Thomas Paul G.
Published: February 2023

Asthma is a chronic airway inflammation characterized by combination of immune responses and structural remodeling that involves both large and small airways. The global prevalence of asthma is on the rise with a sizable portion of asthmatics not responding to existing medication, thus posing a substantial health and economic burden. Matrix proteases including A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS) contribute to lung damage and repair responses. Our lab has recently reported a critical role for ADAMTS4 in the immunopathology of IAV infection-induced acute lung inflammation through exuberant activity of lung fibroblasts and remodeling of the extracellular matrix. Given the divergent nature of immune responses in chronic airway inflammation compared to infection, we sought to investigate the sustaining role of ADAMTS4 upon persistent exposure to aeroallergen. Using HDM-based asthma model, ADAMTS4 knockout mice exhibited a robust reduction in lung eosinophils, mucus production, and airway resistance compared to their wildtype counterparts. This was associated with a marked decrease in IL-4, IL-5, and IL-13 cytokine production and IgE secretion. Interestingly, ADAMTS4 gene deletion differentially blocked the myeloid cell recruitment to the lungs without affecting their development. Such effects were also associated with significant reduction in alveolar macrophages and peribranchial inflammation. These results suggest that the modulating immune responses by ADAMTS-4 is sustainable beyond the acute phase of inflammation and may be extensively contributing to the immunopathology of chronic airway inflammation, thus harboring a therapeutic potential for the treatment of asthma traits.
Exosomes of MSCs target M2-type Macrophages to promote TGF-β-Dependent Microvascular Stabilization and Functional Recovery in Spinal Cord Injury
Authors: Askenase Philip W
Published: February 2023

Intravenous (IV) infusion of mesenchymal stromal cells (MSC) stabilizes the blood-spinal cord barrier and improves functional neurological recovery after spinal cord injury (SCI). However, the MSCs do not traffic to the injury site, but rather lodge primarily in the lungs for a few days. Here, groups of 8 young Sprague Dawley rats received 2 x 10>6 MSCs, their derived exosomes (MSC-exos, 2 x 10>10). in single dose or dividing that dose over 3 days starting on the 10th day following severe contusive SCI, or vehicle control. Locomotion, cell targeting, expression of TGF-β, TGF-β-R, vascular proteins and permeability were determined at 3, 7 and 14 days after treatments. We report that MSC-exos are released in vivo by infused MSCs, distribute systemically, and at the injury site specifically associate with M2-type macrophages. While a single MSC infusion resulted in significant improvement in locomotor recovery, fractionated dosing of MSC-exos over three days was required to achieve similar extent of therapeutic effects as infusion of the MSCs. Both MSCs and fractionated doses of MSC-exos increased numbers and differentiation of M2-type macs expressing TGF-β, with increased accompanying expression of TGF-β receptors and tight junction proteins in the neighboring microvascular, resulting in reduction in blood spinal cord barrier vascular permeability.

SUMMARY: In SCI, IV infused MSCs release MSC-exos in vivo over time that target lesional M2-macs to increase their TGF-β production and further induce TGF-β signaling pathways in the local vasculature to mediate therapeutic effects, likely by release of locally active secondary exosomes transferring miRNAs that induce TGF-β pathways in the neighboring micro vasculature to promote healing.
Lymphangiogenesis near the cribriform plate after cerebral ischemia
Authors: Choi Yun Hwa , Hsu Martin , Laaker Collin , Sandor Matyas , Fabry Zsuzsanna
Published: February 2023

The central nervous system (CNS) has been described as “immune privileged”, but it has been recently hypothesized that antigens, antigen-presenting cells (APCs), and cerebrospinal fluid (CSF) may drain from the CNS into lymphatics in order to provide antigen drainage and fluid homeostasis. This re-discovery of lymphatics ignited an intense debate about the mechanisms of how antigens or immune cells drain from the CNS parenchyma in both steady state and neuroinflammation. Here, we study how lymphangiogenesis near the cribriform plate contributes to recovery of brain tissues following transient ischemic stroke. We employed the 60-minute transient middle cerebral artery occlusion (tMCAO) model of ischemic stroke in mice. We showed that lymphangiogenesis occurs near the cribriform plate, which peaked at day 7 and decreases after 14 days following tMCAO. The lymphangiogenesis occurred through interaction of vascular endothelial growth factor receptor (VEGFR)-3 and VEGF-C which are produced by CD11b+, CD11c+ dendritic cells. These lymphangiogenic vessels could transport CSF and immune cells which were confirmed by injection of Evans Blue dye through cisterna magna and flow cytometry, respectively. Flow cytometry analysis showed robust leukocyte accumulation, which include macrophages (CD45hi, CD11b+, CD11clow), dendritic cells (CD45hi, CD11b+, CD11chi), CD8 T cells (CD45hi, CD11b−, CD11c−, CD8+), and CD4 T cells (CD45hi, CD11b−, CD11c−, CD4+) near the cribriform plate after 7 days of tMCAO, which also correlated with lymphangiogenesis (CD45−, Podoplanin+, CD31+). Understanding the biology and mechanisms of CNS lymphatic drainages may lead to novel therapeutics for neuroinflammatory diseases.
Examining the effect of a 10-week golf program on circulating markers of inflammation in healthy, older adults: a pilot study
Authors: Moore Jared , Kanwar Kiran , Salem George
Published: February 2023

Objective

Golf is a unique, multimodal recreational exercise that has been shown to provide physical and cognitive benefits to older adults; therefore, the purpose of this study was to investigate how golf also affects circulating inflammatory marker levels in healthy, older adults.

Methods

Fifteen older adults completed a 10-week golf program with blood draws before and after the program. Participants had blood drawn using EDTA collection tubes after a 10 hour overnight fast. Plasma was isolated and stored at −80°C for approximately 16 weeks until run using a Millipore human cytokine magnetic panel and hsCRP ELISA. Results are presented as percent change with mean±standard deviation. Hedge’s g effect sizes are also presented.

Results

12/15 participants completed pre and post blood draws. C-reactive protein (CRP) levels decreased by 49.80% (3.01±2.21 vs 1.51±1.50 mg/L; g = 0.73). Interferon gamma (IFNγ) decreased by 14.25% (11.28±7.04 vs 9.67±7.67 pg/mL; g = 0.20) and tumor necrosis factor alpha (TNFα) decreased by 10.64% (19.29±8.67 vs 17.24±8.15 pg/mL; g = 0.23). Interleukin-10 decreased by 5.51% (22.68±12.85 vs 21.43±10.91 pg/mL; g = 0.10).

Conclusions

This program was performed with healthy, older adults and demonstrated that a 10-week golf program is safe and feasible for this population, and likely influences markers of inflammation, particularly CRP. This pilot work may be used to inform future studies in older adults using recreational activities, such as golf, to examine not only fitness benefits but changes in levels of chronic inflammation.
The immunological profile of maternal obesity at 28 weeks gestation underpins common negative pregnancy outcomes
Authors: Rees April , Richards Oliver , Jones Nick , Thornton Catherine
Published: February 2023

Maternal obesity has been implicated in adverse pregnancy outcomes such as miscarriage, preeclampsia and gestational diabetes mellitus (GDM) and poses a risk to the neonate. There is a knowledge gap surrounding obesity and changes to the maternal immune system.

Hypothesis: Obesity exacerbates systemic inflammation in pregnant women at 28 weeks of gestation.

Samples: Peripheral blood was collected from fasted women at 26–28 weeks of gestation; all were GDM-negative

Results:

Insulin (p=<0.0001) and PAI-1 (p=0.0487) levels were increased in obese versus non-obese pregnant women. Obese women had a relative neutrophilia (p=0.0429) accompanied by diminished eosinophil (p=0.0178) and NKT cell (p=0.0124) numbers. The inflammatory cytokine profile of MNCs was mostly unchanged with obesity but the Th profile of pregnant women with obesity appears skewed towards Th17: on stimulation with CytoStim™, IL-6 (p=0.0183), TNFα (p=0.0478), IL-17F (p=0.0183) and IL-22 (p=0.0183) levels were increased; Th2 cells (p=0.0250) were also reduced with maternal obesity. Monocyte phenotypes showed some changes with obesity; on intermediate monocytes, CD86 (p=0.0068) was decreased yet CD163 (p=0.0421) was increased, and CCR2 expression was elevated on classical (p=0.0040) and intermediate (p=0.0340) monocytes.

There is a shift to Th1 and Th17 in obesity in the general populations, whereas pregnancy is associated with a shift to Th2 and Treg. Here, we show that this profile is compromised in maternal obesity, which might underpin detrimental effects on pregnancy. Further research should investigate homogenous populations of cells such as eosinophils, monocytes and T cells, to further understand the immunological consequences of maternal obesity.
Absorption After Neural Tube Teratogenesis is Dependent on Maternal Immune Cell Function
Authors: Ott Katherine C , Oria Marc , Redden Catherine R , Kang Hee Kap , Skital Veronica , Turner Lucas E , Alhajjat Amir A , Duru Soner , Peiro Jose L , Shaaban Aimen F
Published: February 2023

Introduction: Neural tube defects (NTDs), are severe malformations of the developing central nervous system. After having a pregnancy affected by NTD, the recurrence risk increases 20x. The predisposition to having children affected by NTDs has not been explained and may relate to surveillance by the maternal immune system as maternal immune cells such as NK and T cells have been shown to traffic in the fetal circulation. We hypothesized that maternal NK and T cells are necessary for selective fetal absorption after the development of an NTD.

Methods:

We challenged this hypothesis in an outbred murine model of valproic acid (VA)-induced fetal neural tube teratogenesis (NTT) performed of pregnant CD1 dams at E8. To evaluate protection from NTTs, maternal NK cells were selectively eliminated (mAb PK136) on E7. Maternal CD4+ and CD8+ T cells were selectively eliminated (GK1.5 and 2.43) on E6. All litters were harvested on E14 and prevalence of NTT compared between each treatment group and untreated controls.

Results:

The frequency of litters with VA-induced NTT was significantly higher when maternal NK cells or maternal T cells were selectively depleted compared to controls (100% and 88.2% vs 80.0%, p<0.05). Only 10% of NK depleted dams had greater than 3 late absorptions per litter and T cell depleted dams had 0% compared to controls that had 30%, p<0.05. This data shows that both cell types play a critical role in the late absorption process. Litter sizes were similar between the groups.

Conclusion:

These findings demonstrate that selective fetal absorption for NTT is dependent on intact maternal immune cell function. Further studies are needed to define the roles that maternal NK and T cells have in the absorption process during abnormal development.