bluuebunny/crossref_metadata_2025 huggingface dataset:
Natural CD4+ T regulatory cell down-regulation of monocytic myeloid derived suppressor cell phenotypic and functional subpopulations in LP-BM5 murine retrovirus-induced immunodeficiency (VIR1P.1149)
Authors: OConnor Megan , Vella Jennifer , Green William
Published: January 2023

Immunomodulatory cellular subsets, including myeloid derived suppressor cells (MDSCs) and T regulatory cells (Tregs), contribute to the immunosuppressive tumor microenvironment and have been targets of immunotherapy, but their role in retroviral-associated immunosuppression is less understood. Due to known crosstalk between Tregs and MDSCs in the tumor microenvironment, it is of interest to look at the interplay between these immune cells during LP-BM5-induced murine AIDS. Following in vivo natural Treg (nTreg) depletion, and subsequent LP-BM5 retroviral infection, enriched MDSCs from infected nTreg-depleted mice exhibit increased suppression of T cell, but not B cell, responses, as compared to MDSCs derived from non-depleted LP-BM5-infected controls. MDSCs from LP-BM5-infected nTreg-depleted mice also display altered phenotypic subsets, which correlate with functional differences. In addition, LP-BM5-induced MDSCs modulate in vitro Treg phenotype and function. These collective data highlight an antagonistic relationship between MDSCs and Tregs, and may provide insight into the immunotherapeutic targeting of these cells during retroviral infection.
High avidity and functionality of virus-specific CD8 T cells associated with protection during HCV reinfection (VIR2P.1168)
Authors: Boisvert Maude , Abdel-Hakeem Mohamed , Bruneau Julie , Soudeyns Hugo , Shoukry Naglaa
Published: January 2023

The majority of acute hepatitis C virus (HCV) infection (75%) leads to chronicity and liver diseases. While 25% of infected individuals can naturally clear the infection, long-term protection is elusive and reinfection can occur. We have previously showed that protection from viral persistence upon HCV reinfection correlates with expansion of HCV specific T cells with increased breadth. Viral persistence was associated with limited expansion of virus specific T cells. CD8 T cell receptor (TCR) repertoire analysis showed that clonotypes associated with protection upon reinfection are recruited from the memory population, with focusing of the repertoire. We hypothesized that protective immune memory response is associated with selection of T cell clonotypes with the highest TCR avidity and a polyfunctional profile. We FACS sorted HCV-specific CD8 T cells to generate individual clones representative of clonotypes of high, medium or low frequency in the TCR repertoire. Tetramer titration assay showed that the TCR avidity correlated with the clonotype expansion in vivo upon reinfection. All clones showed a polyfunctional profile but the main function varied between clones. In conclusion, our results suggest that the main determinant for the expansion of memory HCV specific CD8+ T cells during HCV reinfection was the TCR avidity. The results of this study have implications for the development of vaccination regimens to boost generation and selection of such clonotypes.
HLA-F and MHC open conformers in a novel HIV-1 immunization strategy
Authors: Geraghty Daniel , Gooodridge Jodie P , Wang Kevin , Burian Aura , McNevin John , McElrath Margaret J , Lee Ni
Published: January 2023

HLA-F is expressed as a protein independent of bound peptide or b2-microglobulin and surface expression is upregulated in dendritic cells, monocytes and most lymphocyte subsets upon activation. Classical MHC class I (MHCI) is also expressed on proliferating lymphoid cells as so-called ‘open conformers (OCs)’, in addition to the ubiquitously expressed form complexed with peptide and b2-microglobulin. Previous studies showed that HLA-F binds most MHCI proteins as open conformers without peptide but not as peptide bound complex. These studies were extended to show that both HLA-F and MHCI OC are ligands for a specific subset of killer Ig-like receptors (KIRs). The HLA-F/MHCI physical interaction was further implicated in the function of HLA-F and MHCI open conformers in a general mode of exogenous MHCI antigen uptake and antigen presentation by activated immune cells that differs from the canonical MHCI endogenous antigen presentation. We are currently testing the hypothesis that antigen entry is governed by a synergy between specific structural characteristics of the exogenous antigen and the MHC-I allele types of target cells. New evidence is presented using HIV-1 p24 gag derived long polypeptides suggesting a requirement for HLA class I peptide epitope specificity in antigen uptake, upstream of antigen presentation, which subsequently can be presented by either or both of MHCI and MHCII. These experiments suggested that the physical proximity of HIV-1 class I and class II peptide epitopes within a p24 polypeptide can govern antigen presentation of epitopes through either MHCI or MHCII or both. A goal is to manipulate these features in designing effective immunogens for directed stimulation of antigen-specific host responses.
Proteasomal degradation by irreversible binding of Nedd8.
Authors: Vijayasimha Kartikeya , Palmer Amy , Dolan Brian P
Published: January 2023

A large fraction of endogenous antigenic peptides presented by the major histocompatibility complex-1 (MHC1) is thought to be generated by rapid proteasomal degradation of newly synthesized defective ribosomal products (DRiPs). Poly-ubiquitination is responsible for the proteasomal degradation of a majority of intracellular proteins. However, recent data demonstrate that the inhibition of poly-ubiquitin chain assembly itself does not fully inhibit DRiP presentation, thus indicating the involvement of other players. We therefore suspected that other ubiquitin-like (UBL) molecules may be involved in antigen presentation. A recently described chemical inhibitor of the UBL termed Neural precursor cell expressed, developmentally down-regulated 8 (Nedd8) conjugation was used in conjunction with a cell-based assay to measure direct antigen presentation. Inhibition of Nedd8 conjugation led to a striking decrease in presentation of peptides derived from DRiPs, but not from peptides derived from retired forms of the same model antigen. To determine if Nedd8 conjugation to a substrate led to proteasomal-mediated degradation, we created a mutant form of Nedd8, which substituted the three of the four C-terminal glycine residues to alanine and thus inhibited the processing of Nedd8 into its active form. When fused to GFP, we found the mutant Nedd8 led to a rapid, proteasomal mediated destruction of the fusion protein. Wild-type Nedd8-GFP fusion protein was processed as expected and protein detection was not detected. Here we show a mechanism of proteasomal degradation mediated directly by Nedd8 conjugation, and a possible role for Nedd8 in the MHC1 presentation pathway.
TLR9 is indispensable for MR1-mediated bacterial antigen presentation
Authors: Liu Jianyun , Brutkiewicz Randy Richard
Published: January 2023

Mucosal-associated innate T (MAIT) cells are conserved T cells that express a semi-invariant T cell receptor (TCR; Vα7.2 in human and Vα19 in mice). The development of MAIT cells requires the antigen (Ag) presenting major histocompatibility complex (MHC)-related molecule, MR1, as well as commensal bacteria, because germ-free or MR1-deficient animals have few MAIT cells. B cells were the first identified (and the most prominent) antigen presenting cells (APCs) for MAIT cells. Previous studies have shown that MR1 is ubiquitously expressed, but is mostly localized in the cytosol. B cells normally express very few MR1 molecules on the cell surface. Upon a bacterial infection, MR1 translocates to cell surface with loaded Ag, interacts with the invariant TCRα and activates MAIT cells. However, it remains unknown how MR1 molecules are processed and loaded with bacterial Ag in APCs. We have found that treating B cells with a TLR9 agonist CpG increases MR1 surface expression. The upregulation of surface MR1 was blocked by protein transport inhibitor Brefeldin A, suggesting that endocytic trafficking stimulated by TLR9 activation is important for the surface expression of MR1 in B cells. Further, knocking-down TLR9 expression by shRNA reduces MR1-mediated activation of MAIT cells. Overall, our results indicate that TLR9 is important for MR1-mediated bacterial Ag presentation. Further studies are needed to elucidate how TLR9 controls MR1 endocytic trafficking and bacterial Ag processing and/or loading onto MR1.
Rab43 facilitates cross-presentation of cell-associated antigens by CD8a+ dendritic cells
Authors: Theisen Derek J , Kretzer Nicole , Tussiwand Roxane , Briseno Carlos , Grajales-Reyes Gary , Wu Xiaodi , Durai Vivek , Albring Jorn , Bagadia Prachi , Murphy Theresa , Murphy Kenneth M
Published: January 2023

Batf3-dependent classical dendritic cells (cDCs) are essential for cross-presentation and clearance of intracellular pathogens through priming of CD8 T cells. By analyzing differences in gene expression between cDC subsets we identified vesicular transport molecules unique to cross-presenting dendritic cell subsets. RAB43, a GTPase that is localized to the golgi, is selectively expressed in Batf3-dependent CD8a+ and CD103+ cDCs and was highly conserved between mouse and human. Mice with germline and conditional Rab43 deletions are viable and fertile with normal hematopoiesis. However, mice lacking Rab43 showed reduced cross-presentation of cell-associated, but not soluble, antigens both in vitro and in vivo. Bone-marrow derived DCs (BMDCs) from Rab43 deficient mice show normal cross-presentation efficiency, suggesting that Rab43 is involved in optimizing cross-presentation in vivo through a pathway unique to CD8a+ DCs.
A Sec22b knockout mouse offers novel insights into the molecular mechanisms of crosspresentation
Authors: Wu Shin-Rong Julia , Johnson Claire , Zajac Cynthia , Sun Yaping , Mathewson Nathan , Toubai Tomomi , Oravecz-Wilson Katherine , Khoriaty Rami , Ginsburg David , Reddy Pavan
Published: January 2023

Crosspresentation critically contributes to the adaptive immune response to tumors and viral infection by presenting extracellular antigen on MHC I to activate CD8 T cells. However, its molecular regulation is poorly understood. Using in vitro shRNA-mediated knockdown (KD) methods, another group had shown that Sec22b, an ER-SNARE, specifically regulates crosspresentation. Thus, we tested the hypothesis that Sec22b would mediate crosspresentation in vivo. We generated DC-specific Sec22b knockout (KO) mice, which have an intact, functional immune system. After adoptively transferring OT-I T cells into these mice, then injecting with soluble OVA i.p., we analyzed OT-I T cell proliferation to measure crosspresentation. Surprisingly, we saw no difference in OVA crosspresentation by KO versus FL mice. This observation was verified in vitro using KO DCs from BM and spleen crosspresenting soluble and insoluble OVA. We concluded Sec22b is not necessary for crosspresentation, invalidating our hypothesis. However, we were able to reproduce the reduced crosspresentation by shRNA-mediated KD of Sec22b. Intriguingly, when we treated KO BMDCs with the same Sec22b-targeting shRNA, we again observed reduced crosspresentation comparable to that seen in shRNA-treated WT BMDCs. This suggested the shRNA mediates crosspresentation through its effects on an off-target gene. RNAseq data on shRNA-treated BMDCs provided a candidate list of potential crosspresentation mediators. Our data demonstrates that (a) Sec22b is not necessary for crosspresentation, (b) there may be a novel critical mediator of crosspresentation targeted by the shRNA, and (c) extrapolating mechanisms or phenotypes based on KD studies alone must be done with caution.
γδ T cells can play a role in provoking an inflammatory attack on the cornea
Authors: O’Brien Rebecca L , Yang Zhifang , Huang Yafei , Loomis Josh , Owen Amie L , Born Willi K H
Published: January 2023

Ocular immune privilege results from a complex set of mechanisms that together prevent immunogenic inflammation, but still allow the eye to deal with potentially infectious agents in a localized way that does not compromise vision. Failure of immune privilege can unleash a damaging autoimmune attack on the eye. γδ T cells have been found to be important for these processes in a number of different systems. In mice of the C57BL/10 background (B10), we have previously shown that γδ. T cells help prevent the spontaneous development of keratitis, an inflammation of the cornea that B10.TCRδ −/− mice are highly prone to develop, particularly the females. Paradoxically, B10 mice that can produce γδ T cells but not αβ T cells also develop keratitis at a high rate. We recently found that Vγ4+ cells from the spleens of keratitic B10.TCRβ−/− mice, but not Vγ1+ cells, can adoptively transfer the disease to normally keratitis-resistant B10.TCRβ−/−δ−/− hosts. Immunofluorescence staining of corneal whole mounts from B10.TCRβ−/− mice revealed that Vγ4+ cells infiltrate the keratitic corneas, and show a strong bias to secrete IL-17. In contrast, Vγ1+ cells were more rare in the keratitic corneas and did not produce IL-17. The majority of the γδ T cells in keratitic corneas were Vγ1-negative, Vγ4-negative, and Vγ7-negative, however, and did not produce IL-17. We hypothesize that although some γδ T cell subsets protect against autoimmune attack on the cornea, certain Vγ4+ γδ T cells instead promote keratitis by infiltrating the corneas and secreting IL-17, which attracts and mobilizes neutrophils, resulting in tissue damage.
The TAM receptors Axl and Mer play a joint-specific protective role in experimental arthritis
Authors: Waterborg Claire Elisabeth Joyce , Través Paqui González , Zagórska Anna , Lemke Greg , Beermann Silke , van de Loo Fons Adrianus
Published: January 2023

Introduction

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by an unrestrained inflammatory response in synovial joints. One family of tyrosine kinase receptors involved in anti-inflammatory feedback mechanism are Tyro3, Axl and Mer (TAM). The plasma level of the TAM receptor ligand Gas6 is significantly reduced in RA patients suggesting this feedback mechanism is impaired in RA. We investigated the individual role of each TAM receptor in experimental arthritis.

Methods

The KRN serum transfer model of arthritis was induced by two intraperitoneal injections of arthritic K/BxN serum in TAM receptor single gene knock-out mice (Tyro3−/−, Axl−/−, Mer−/−) and wild-type (WT) mice. Ankle joints were macroscopically scored for 7 days and knee and ankle joints were taken at the end of the experiment for histology and immunohistochemistry.

Results

Mer−/− mice had an increased macroscopic ankle score until day 4 whereas Axl−/− mice had an enhanced macroscopic score from day 4 until the end of the experiment. Histology of the ankle joints showed significantly increased arthritis pathology in Axl−/− mice compared to WT. In contrast, enhanced macroscopic score and arthritis pathology in the knee joints of Mer−/− mice was observed. Both in ankle and knee joints, no protective or aggravating role of Tyro3 was seen. To explain the discrepancy of Axl involvement between ankle and knee, we looked for Axl expression in synovium of naïve mice. The lining layer of ankle synovium was completely Axl-positive whereas only mild expression in the knee joints was observed.

Conclusion

These findings identify the TAM receptors Axl and Mer as important protective players in arthritis in addition to being expressed in a joint-specific manner.
Plasmacytoid dendritic cells in late-stage lupus mice defective in producing IFN-α
Authors: Liao Xiaofeng , Li Song , Settlage Robert E , Sun Sha , Ren Jingjing , Reihl Alec , Zhang Husen , Karyala Saikumar , Reilly Christopher , Ahmed S Ansar , Luo Xin M
Published: January 2023

Plasmacytoid dendritic cells (pDCs) are professional type I IFN producers believed to promote lupus. However, questions exist about whether they function at the same level throughout the course of lupus disease. We analyzed high-purity pDCs sorted from lupus mice. Although pDCs produced a large amount of IFN-α during disease initiation, those sorted from late-stage lupus mice were found to be defective in producing IFN-α. These pDCs expressed an increased level of MHC, suggesting a functional drift to Ag presentation. We examined the potential mechanism behind the defect and identified a novel transcriptional factor, Foxj2, which repressed the expression of several genes in pDCs, but not IFN-α. Dysregulation in pDCs appears to be predisposed, because they exhibited an altered transcriptional profile before the onset of clinical signs. Our results suggest that pDCs do not function the same throughout the disease course and lose the ability to produce IFN-α in late-stage lupus mice.
The gut microbiome influences arthritis development in IIJ mice
Authors: Paras Tyler M , Percival Frank , McQuade Eileen
Published: January 2023

Inherited Inflamed Joint (IIJ) mice spontaneously develop inflammatory arthritis at an early age. The gut microbiome has been implicated in several murine models for inflammatory arthritis, however the role of the microbiome in IIJ arthritis is unknown. The purpose of this study was to determine whether the gut microbiome affects arthritis development in IIJ mice and characterize the microbiome of IIJ mice in various age and disease states using next generation sequencing techniques. Pre-arthritic (pAR) IIJ mice were treated with water containing neomycin, ampicillin, vancomycin, and metronidazole for 5 weeks. Disease onset and severity was scored based on swelling of the large joints throughout the treatment period. Antibiotic treatment delayed disease onset and reduced disease severity in IIJ mice. Additionally, the IIJ gut microbiome in pAR, 5 week old arthritic mice (AR), and age matched controls (pN and NAR, respectively) were characterized using MiSeq sequencing of 16S rRNA amplicons. pAR mice were enriched with an unclassified member of the Clostridiales, whereas pN mice were dominated with species from the S24-7 family and Bacteroides, both members of the Bacteroidetes. Differences between the bacterial communities of experimental groups were apparent from the non-metric multidimensional scaling (NMDS) plots, but were not significant by PERMANOVA. Together these results suggest that the gut microflora of IIJ mice increase host susceptibility to inflammatory arthritis, which may be mediated by a unclassified member of the Clostridiales. Classical triggers of the innate immune system are also being investigated for effects on arthritis incidence and severity.
LCMV infection prevents MOG-specific T cell trafficking to the CNS and delays EAE
Authors: Cosby Jennifer M , Evavold Brian D
Published: January 2023

Infections have been implicated in playing a role in autoimmune diseases. Most reports discuss infectious insults as an initiator of autoimmune disease. Despite the idea that microbes act as triggers for autoimmune disease, their direct role in multiple sclerosis for example is still a topic of debate. Instead of assessing the role of infection in initiating disease, we address the converse hypothesis and test whether some infections can have protective effects. We investigated the role of LCMV infection during the well-established C57Bl/6 EAE model of chronic-progressive disease. In our model, mice infected with LCMV had a delay in EAE disease course compared to uninfected controls with significantly fewer myelin tetramer positive CD4 T cells in the CNS. However, roughly equal numbers of myelin-specific T cells were seen in the periphery suggesting that absence in the CNS was not due to lack of antigen-specific expansion. The CNS was instead populated with LCMV specific CD8 T cells. Therefore, EAE disease course is delayed during LCMV infection as a result of a lack of myelin-specific T cell homing into the CNS and the characteristic neuroinflammation mediated by this T cell population.
Dysregulation of intestinal IL17 signaling & the microbiome exacerbate autoimmune neuroinflammation.
Authors: Castillo Patricia , Kumar Pawan , Kolls Jay K
Published: January 2023

Multiple sclerosis (MS) is a chronic autoimmune disorder of the central nervous system (CNS). Studies in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, strongly indicate that both Th17 cells and the commensal microbiome modulate disease pathogenesis. The commensal, segmented filamentous bacteria (SFB), regulates Th17 responses. Given the relationship between commensals and Th17 cells and the role of each of these factors in EAE, we investigated the significance of enteric IL-17R signaling in EAE using intestinal specific IL-17 receptor knockouts (Il17ra/rcfl/flx villin cre+ mice).

We hypothesized that disruption of the reciprocal regulatory relationship between enteric IL-17 signaling and the gut microbiota leads to dysbiosis, expansion of Th17 cells, and increased predisposition to autoimmune neuroinflammation. Our data suggested that Il17ra/rcfl/fl x villin cre+ mice, which have higher SFB levels, exhibit earlier EAE onset and increased EAE severity and incidence as compared to littermate cre− controls. Treatment with vancomycin ameliorated disease, further supporting our hypothesis. In addition, preliminary data indicated that cre+ mice have increased CCR2 and CCR6 gut expression at baseline. At day 9-post immunization in the same mice, there was increased expression of CCR2 and Nox2 in the spinal cord and Csf2 in the gut. Together, this suggested that there could be increased migration into the CNS of cre+ mice, contributing to exacerbated disease.

These studies will make a significant conceptual advance by elucidating how dysregulation of enteric IL-17R signaling and the commensal microbiota contribute to the pathogenesis of autoimmune conditions such as Multiple Sclerosis.
Intravital imaging of T and B cell migration across high endothelial venules in lymph node
Authors: Choe Kibaek , Song Eunjoo , Lee Soo Yun , Ahn Soyeon , Kim Pilhan , Moon Jieun
Published: January 2023

High endothelial venules (HEVs) in lymph node (LN) facilitate an effective recruitment of circulating lymphocytes from the blood. HEVs have distinctive cuboidal-shaped endothelial cells and prominent perivascular sheaths consisting of fibroblastic reticular cells (FRCs). Yet, detailed dynamic behaviors of lymphocytes during trans-endothelial migration, intra-perivascular channel crawling and trans-FRC migration in HEV are still poorly understood. In this work, we adapted a custom-design confocal microscope to visualize T and B cell migration across HEV in real time in vivo. Actin-DsRed transgenic mice were used to visualize HEV-endothelial cells and T or B cells obtained from actin-GFP mice were adoptively transferred. At the same time, FRCs were labeled in vivo by injecting anti-ER-TR7 antibody conjugated with far-red fluorophore to the footpad. T and B cells squeezed in between endothelial cells (ECs) and then migrated along the perivascular channel, narrow space between ECs and FRCs, for searching a proper site to exit by trans-FRC migration. Interestingly, B cells spent twice longer time in the perivascular channel than T cells although their total moving distances in the perivascular channel were similar. In addition, we observed that there existed preferred exit sites (“exit ramps”) from the perivascular channel for both of T and B cell. To observe whether T and B cell exit through the same exit ramp, we simultaneously transferred DsRed+ T and GFP+ B cells into wild type mouse. Indeed, T and B cells followed each other though the same exit ramp from the perivascular channel. There also existed “entrance ramps” into the perivascular channel; preferred sites for trans-endothelial migration for both of T and B cells.
Chemokine independent T cell migration
Authors: Walling Brandon Lee , Capece Tara , Kim Minsoo
Published: January 2023

Our current understanding of T cell migration is focused on chemokine-mediated integrin activation, which dictates that chemokines provide critical directional signals to guide T cell migration to target tissues within the body. Emerging evidence, however, supports that activated T cells can migrate to certain vascularized tissues, despite chemokine receptor deficiency or with the inhibition of chemokine receptors. This suggests the presence of a chemokine-independent mechanism through which T cells can activate integrins, induce adhesion and subsequent cell migration. We found with OT-I T cell receptor transgenic mice that integrin LFA-1 mediated migration of naïve T cells was strictly dependent on chemokine stimulation. In contrast, activated CD8+ T cells migrated in the absence of chemokines both in vitro and in vivo. Additionally, activated CD8+ T cells migrate in the presence of the chemokine inhibitors pertussis toxin and Gallein (a Gβγ inhibitor), further supporting chemokine-independent T cell migration. Using mass spectrometry, we discovered three cytoskeletal molecules, non-muscle myosin heavy chain IIA (MyH9), αII-spectrin, and βII-spectrin, associate with LFA-1 in T cells. Western blot analysis of mouse T cells revealed expression levels of MyH9 and αII-spectrin remained stable after T cell activation, but surprisingly, βII-spectrin expression was dramatically down-regulated during CD8+ T cell activation. Over-expression of βII-spectrin in activated CD8+ T cells abolished chemokine-independent migration on integrin ligand ICAM-1. Therefore, we hypothesize that βII-spectrin provides a mechanical link between LFA-1 and the cytoskeleton to regulate T cell migration without chemokine stimulation.
Differential regulation of human and mouse IL33 expression in the lungs of human IL33 BAC transgenic mice.
Authors: Pivniouk Vadim I , Rosenbaum David , Herrell Amanda , Pivniouk Oksana , Rafaels Nicholas , Mathias Rasika , Barnes Kathleen , Vercelli Donata
Published: January 2023

Interleukin-33 (IL33) is a member of the IL-1 cytokine family. IL33 polymorphisms are strongly associated with asthma susceptibility and IL33 deletion attenuates asthma in mice. However, the use of mouse models to elucidate the role of IL-33 in asthma is hampered by notably different tissue-specific patterns of IL33 expression in humans and mice.

To bypass this limitation, we generated mice carrying a 157 kB human BAC transgene (TG) that includes IL33 and its flanking sequences on chromosome 9. This TG contains protective alleles for a number of asthma-associated polymorphisms. Five founders with 1, 2, or 6 BAC copies were obtained. All were healthy, fertile, and transmitted the transgene at the expected Mendelian ratio.

To compare expression of transgenic human (h) and endogenous mouse (m)IL33, mice were challenged intranasally with Alternaria, which induces rapid release of mIL-33 in the lungs. Bronchoalveolar lavage fluid analysis showed that hIL-33 was also induced by Alternaria. Analysis of lung RNA by real-time PCR showed that Alternaria-induced hIL33 transcription was TG copy number-dependent and comparable with that of mIL33.

IL33 is known to be expressed by human but not mouse endothelial cells. Indeed, we found high levels of hIL33 RNA in endothelial cell-enriched mouse CD31+ lung cells while mIL33 was barely detectable.

These results suggest that tissue-specific hIL33 expression is controlled by cis-acting element(s) within the IL33 locus. More generally, our BAC TG appears to include all cis-regulatory elements necessary for faithful tissue-specific and copy number-dependent regulation of hIL33 expression, suggesting our mice are an ideal tool to study IL33 and its role in asthma.
HEB plays a critical role in the generation of IL-17 producing Vγ2+ γδ T cells
Authors: In Tracy , Trotman-Grant Ashton , Anderson Michele K
Published: January 2023

IL-17 producing Vγ2+ γδ T cells (Vγ2 T cells) form an integral part of the immune system of various lymphoid and mucosal tissues in mice. The γδ T cells acquire the ability to produce IL-17 during their thymic development but the molecular basis of this programming is not well understood. Here, we aim to characterized the role of HEB in the development of IL-17 producing Vγ2 T cells by utilizing HEB conditional knockout mice on a vav-Cre background (HEB conKO). First, we observed that there was a block in the development of Vγ2 T cells in the fetal thymic organ culture (FTOC) from HEB conKO embryos at E14. However, in contrast to the near absence of fetal thymic Vγ2 T cells in FTOC, neonatal thymus of HEB conKO mice supported the development of Vγ2 T cells, albeit at a significantly lower frequency than the wildtype thymus. Further, the frequency of Vγ2 T cells in the thymus of adult mice was the equivalent to that in the wildtype thymus, showing a gradual increase in the development of Vγ2 T cells in the HEB conKO thymus with age. As IL-17 producing γδ T cells have been shown to be generated preferentially during fetal development, we characterized the functional subsets of Vγ2 T cells found in adult HEB conKO mice. In fact, there was a significant reduction in the frequency of RORγt+ Vγ2+ T cells in the thymus of adult HEB conKO mice, as well as in the lungs, spleen and lymph nodes. Furthermore, the HEB-deficient Vγ2 T cells in the lungs, spleen and lymph nodes exhibited a profound deficiency in their ability to produce IL-17 in response to stimulation with IL-1β, IL-23 and IL-21 or PMA/Ionomycin. Collectively, our work shows for the first time that HEB is required for the generation of IL-17 producing Vγ2 T cells for various lymphoid and mucosal tissues.
Hematopoietic stem cell niches control multipotent progenitor differentiation
Authors: Pereira Joao P , Lim Vivian , Gomes Ana , Hara Takahiro , Herndler-Brandstetter Dietmar , Flavell Richard A , Nagasawa Takashi , Ikuta Koichi
Published: January 2023

Hematopoietic stem cells (HSC) self-renew in bone marrow niches formed by CXCL12+ mesenchymal progenitor and endothelial cells. Here, we show that hematopoietic multipotent progenitors (MPPs) encounter lineage-instructive differentiation signals in HSC niches. Conditional deletion of CXCR4 in MPPs profoundly reduced differentiation into common lymphoid progenitors (CLPs), which significantly decreased lymphopoiesis. CXCR4 was required for CLP positioning near IL-7+ cells and, consequently, for optimal IL-7R signaling. IL-7+ cells form a large subset of CXCL12-abundant reticular mesenchymal progenitor cells capable of differentiation into osteoblasts and adipocytes, and a minor subset of BM sinusoidal endothelial cells. Conditional Il7 deletion in Lepr+ mesenchymal progenitor cells dramatically reduced B-lineage committed CLPs, developing B cell subsets, and peripheral mature B cells. Importantly, more than 50% of HSCs and MPPs were found in direct contact with IL-7+ cells, and more than 75% of HSCs and MPPs resided at <15 micrometers away from IL-7+ cells. Our studies demonstrate that MPPs and CLPs rely on positional cues to encounter short-range lymphopoietic signals provided by CXCL12+ mesenchymal progenitors. We conclude that HSC maintenance and multilineage differentiation are distinct cell lineage decisions controlled by HSC niches.
Embryonic stem cell-derived B-1 cells engraft in immunodeficient mice, recapitulating YS B lymphopoiesis.
Authors: Yoshimoto Momoko , Lin Yang , Yoder Mervin C.
Published: January 2023

It has been reported that B cells are produced from embryonic stem cells (ESCs) when co-cultured with OP9 stromal cells. However, these ESC-derived B cells have not been well characterized; it is unknown whether they belong to the B-1 or B-2 lineage and whether they are transplantable into immunodeficient mice.

We previously reported that the first wave of B lymphopoiesis occurs in the extra-embryonic yolk sac (YS) at the time prior to hematopoietic stem cell (HSC) emergence and these cells belong to the B-1 cell lineage. YS-derived B progenitor cells display a AA4.1+CD19+B220dim B-1 specific progenitor phenotype, are transplantable into immunodeficient mice, and secrete natural antibodies; all key features of B-1 cells. Since differentiation of ESC into blood cells recapitulates YS hematopoiesis, we hypothesized that B progenitor cells produced from mouse ESCs in vitro belong to the B-1 cell lineage. We co-cultured ESCs with OP9 stromal cells and generated Flk1+ cells. Isolated Flk1+ cells were replated onto OP9 stromal cells, and after 9–12 days of co-culture, AA4.1+CD19+B220+ cells were detected. We transplanted these cells into sublethally irradiated immunodeficient neonates. Donor-derived B-1 cells were detected in the recipient peritoneal cavity up to 6 months after transplantation. Furthermore, ESC-derived B-1 cells produced anti-phosphorylcholine antibodies detected by Elispot assay, showing the ability of these innate B-1 cells to produce natural antibodies.

Thus, we have demonstrated that ESC-derived B cells engraft in vivo as functional B -1 cells secreting natural antibodies. These B cell developmental processes recapitulate B lymphopoiesis in the YS and might be utilized for innate immune cell therapy in the future.
Bone marrow basophils enhance immature B cell survival by recruitment and contact dependent signals
Authors: Moreau Joshua Michael , Paige Christopher J
Published: January 2023

B cell development in the bone marrow relies upon an organized tissue structure. Maturing progenitors migrate between several distinct niches and towards the sinusoidal vessels before entering circulation. Microenvironments specific for early developmental stages are provided by stroma expressing CXCL12 (pre-pro-B), IL-7 (pro-B), or Galactin-1 (pre-B); however, the identity of a defined niche for immature B cells remains unclear. A DX5+Thy1lo cell has been shown to provide IgM+ progenitors survival signals through direct cell-cell contact; but the cell type responsible, factors provided, and the influence this help may have on B cell function remains unknown. Here, utilizing in vitro B cell selection assays and in vivo depletion experiments, we identify the DX5+Thy1lo cells as basophils. We demonstrate that this population produces chemotactic factors, which act to recruit immature B cells and therefore enable provision of pro-survival signals directly via cell-cell contact. Furthermore, the bone marrow basophil population expanded during the first 24 hours following induction of inflammation by injection of Freund’s incomplete adjuvant. This response correlated with an increase in the number of immature B cells within the marrow, as well as, a reduction of lambda light chain usage in the immature but not mature B cell population. These results reveal that bone marrow basophils actively provide a supportive microenvironment for immature B cells and that the sensitivity of basophils to inflammatory stimuli may modulate BCR repertoire development.
Effects of neonatal antigen exposure on natural antibody repertoire development
Authors: New James Stewart , Dizon Brian L.P. , King R. Glenn , Kearney John F.
Published: January 2023

B-1 B cells produce natural antibodies that contribute significantly to maintenance of immunological homeostasis, however the role exogenous antigens play in the development of the B-1 B cell repertoire is unclear. N-acetyl-D-glucosamine (GlcNAc)-reactive antibodies are universally represented in mammals, and some GlcNAc-reactive B cell clonotypes are autoreactive due to the conservation of orthologous GlcNAc epitopes in mammals and microorganisms. We report here that neonatal immunization with GlcNAc-bearing Group A Streptococcus significantly expands CD5-bearing GlcNAc-reactive B-1 B cell populations, alters the GlcNAc- reactive clonal repertoire, and suppresses class-switch antibody responses upon secondary immunization in adulthood. We further find that in the absence of exogenous antigen stimulation, germ-free mice exhibit log-reductions in the number of GlcNAc-reactive B-1 B cells and the levels of circulating antibodies. Importantly, clonal analyses of B cells from germ-free mice reveals a lack of GlcNAc-reactive innate-like B cell clonotypes that are highly conserved between mice raised under SPF conditions. Collectively, these data indicate that exposure to exogenous antigen during perinatal development can directly influence clonal representation within certain B-1 B cell specificities. These observations suggest that integration of signals derived from exogenous antigen is required for the development of certain clonal specificities that are negatively selected in the absence of exogenous stimuli. Given the functions of natural antibodies, these observations may hold major implications for the roles of environmental factors in the development of autoimmune disease.
Neurokinin A reduces FcɛRI-triggered inflammation mediated by cutaneous mast cells.
Authors: Sumpter Tina L , Tkacheva Olga , Falo Louis D , Larregina Adriana T
Published: January 2023

Mast cells (MCs) are potent effector cells in atopic diseases, like asthma and atopic dermatitis. In the skin, MCs reside in the dermis, in close proximity to sensory nerves. In this shared geographical space, MCs and neurons may have evolved common mechanisms for responding to noxious stimuli and for maintaining homeostasis, including responsiveness to neuropeptides. Sensory nerves release neuropeptides with immune functions including the tachykinin family member, neurokinin A (NKA). Recent studies highlight immune functions for NKA but a role for NKA in MC biology has not been explored. NKA signals with high affinity through the neurokinin 2 receptor (NK2R) that has been reported to be anti-inflammatory in the skin. In the current study, we hypothesized that NKA would dampen MC activation initiated by FcɛRI. Our data demonstrated that, in vitro, mouse bone marrow (BM) derived MCs expressed low levels of the NK2R, which was upregulated following FcɛRI activation and maintained by STAT3 signaling. The addition of NKA to MC cultures downregulated surface expression of the prototypical MC markers, FcɛRIα and c-Kit. This effect was overcome with IgE and cross-linking antigen (Ag). NKA alone did not affect degranulation or cytokine release. But, NKA increased the percentage of MCs releasing granules in response to IgE + Ag, while decreasing the secretion of IL-4, IL-13 and IL-6 in response to IgE + Ag. In vivo, intradermal administration of NKA reduced IgE-dependent MC functions in a murine model of passive cutaneous anaphylaxis. Our data suggest that NKA reduces the inflammatory capacity of MCs and that activation of the NKA/NK2R axis has a therapeutic potential for MC mediated atopic diseases.
Fluvastatin augments IL-33-mediated mouse mast cell function
Authors: Taruselli Marcela T , Kolawole Elizabeth Motunrayo , Ryan John J
Published: January 2023

Statins are HMG-CoA reductase (HMGCR) inhibitors that are the most commonly prescribed class of drugs for treatment of hypercholesterolemia. Recent studies have found that statins suppress some pro-inflammatory immune responses. Similarly, our lab has previously reported that fluvastatin suppresses pro-inflammatory IgE-mediated mast cell function in vitro and in vivo. However, other studies have shown statins can induce pro-inflammatory function in NK cells and monocytes. Due to the differing data on statins, we extended our studies to examine the effect of fluvastatin on IL-33 mediated mast cell function. Contrary to our IgE studies, fluvastatin augmented TNF and IL-6 production in IL-33-stimulated bone marrow derived mast cells. The increase in cytokine production was dependent on the presence of stem cell factor (SCF) during fluvastatin treatment. Mevalonic acid, the product of HMGCR, did not reverse the effect of fluvastatin, suggesting that fluvastatin action is not due to HMGCR targeting. Furthermore, inhibiting protein prenylation downstream of HMGCR with farnesyl transferase and geranylgeranyl transferase inhibitors did not emulate the effects of fluvastatin. The effects of fluvastatin were functionally important, since in vivo fluvastatin treatment enhanced IL-33-mediated neutrophil recruitment and increased plasma IL-6 and TNF. This study may elucidate the pro-inflammatory mechanisms of statins and reveal novel means by which mast cells are activated.
Helminth-induced B1 IgE is protective against allergic disease.
Authors: Martin Rebecca , Damle Sheela , Conrad Daniel H
Published: January 2023

Helminth infection is well known for its ability to generate large amounts of non-specific IgE. This hinders the parasite-specific IgE thereby reducing mast cell (MC) degranulation and anti-helminth immunity. This study shows that the innate-like B1 cells are responsible for the substantial amounts of IgE induced during the Th2 helminth infection, Nippostrongylus brasiliensis (Nb). We utilize a mouse model with defects in hematopoiesis that results in loss of B2 cells, but retains normal B1 development, the ADAM10 transgenic mouse. These mice have equivalent IgE production in Nb when compared to wild type (WT) controls that is T cell dependent. Utilizing the NP-KLH system, we demonstrate that these mice make no antigen-specific IgG1, either high or total affinity. They additionally make no increased overall antibody when boosted with the same antigen. In WT mice, we show a population of B1 plasma cells (CD11b+CD23−B220intCD138+) in vivo, that express secreted IgE message during Nb infection. We additionally show that B1 cells (CD11b+CD23−B220int) sorted from naïve and Nb infected mice make large amounts of IgE in in vitro culture with IL-4, anti-CD40, and IL-5 by ELISA and secreted IgE message by RT-PCR. These B1 cells become CD138+ after 5 days of culture. Finally and most importantly, we show that B1 IgE is first unable to induce MC degranulation in an active cutaneous anaphylaxis model and second, it can block MC degranulation in a model of passive cutaneous anaphylaxis. Overall, these data represents a protective response that has evolved for the helminth, where non-specific B1 IgE blocks MC degranulation and thereby protects the helminth from damage. Excitingly, this could be harnessed as a potential protection against allergic disease.
When signals get crossed: the consequences of dual allergen- and influenza-mediated signaling in mast cells
Authors: Temple Rachel Mary , Amiel Eyal , Obar Joshua J
Published: January 2023

Despite the many advances in our understanding of viral infections, influenza A virus (IAV) continues to impose a significant heath burden and presents a serious threat for a worldwide pandemic. Severe IAV infection is characterized by a cytokine storm. Recent evidence indicates mast cell activation is a key initiator of this cytokine storm during severe IAV infection. During the 2009 H1N1 pandemic, asthma was the most common co-morbidity among hospitalized individuals suggesting a potential interplay between pre-existing mast cell disease and IAV infection. Experimental data examining the cross-talk that can occur during dual FcɛRI and IAV stimulation of mast cells is lacking. Here we show that signaling through FcɛRI concurrent with IAV infection (A/WSN/33) augments the mast cell response resulting in a highly synergist enhancement of cytokine synthesis and release by these cells in vitro. Surprisingly, while IAV induced cytokine production by A/WSN/33 is dependent on the MAVS pathway, using bone marrow cultured mast cells from MAVS and STING knockout mice, we determined this synergistic effect can occur independent of MAVS signaling and the STING adaptor. Furthermore, the synergy can be observed as early as 1 hour PI, prior to significant viral replication. These data suggest an early signaling event is responsible for the synergy. Our data supports a role for the lipid mediator sphingosine-1-phosphate, which is known to be important during allergen stimulation, and as our data demonstrates, is essential for proper IAV activation of mast cells. Taken together, our data suggest that IAV may incite asthmatic exacerbation through enhanced cytokine production from mast cells receiving two distinct stimuli simultaneously.
Dysregulation of ERK signaling in CD4-expressing cells induces osteochondromas
Authors: McGargill Maureen Ann , Wehenkel Marie , Corr Maripat , Guy Clifford S , Edwards Benjamin A , Calabrese Christopher , Pages Gilles , Pouyssegur Jacques , Vogel Peter
Published: January 2023

ERK1 and ERK2 are serine/threonine kinases critical for the proliferation and development of many cell types. Mice with a germline deletion of Erk1 bred to mice with a conditional deletion of Erk2 mediated by CD4cre (DKOCD4 mice) exhibit a profound block in T cell development in the thymus. Surprisingly, 80% of DKOCD4 mice spontaneously developed osteochondromas by 28 weeks of age. Histological analysis of these lesions revealed excessive accumulation of hypertrophic chondrocytes originating from the growth plates in the bone, but no mononuclear infiltrate indicative of inflammation. As the majority of CD4+ cells are T cells, we investigated whether these lesions were caused by deletion of Erk2 in T cells by breeding the DKOCD4 mice to Rag1−/− mice. Unexpectedly, osteochondromas still appeared in DKOCD4Rag1−/− mice. In fact, osteochondromas developed faster and were more severe in DKOCD4Rag1−/− mice compared to DKOCD4 mice, indicating that deletion of Erk2 in other cell types mediates excess cartilage accumulation. Furthermore, the addition of T cells to DKOCD4Rag1−/− mice delayed development of osteochondromas, showing that T cells play an important role in regulating cartilage homeotstasis. Furthermore, the development of the osteochondromas appears to be influenced by changes in the microbiota, as DKOCD4 mice treated with an antibiotic cocktail have a delay in osteochondroma development, while mice housed in non-specific pathogen free conditions had accelerated onset of tumors. Together these data suggest that Erk plays a critical role in a CD4+ cell type other than T cells, possibly an innate lymphoid cell, to alter cartilage growth. In addition, we present a novel role for T cells in regulating cartilage homeostasis.
Lupus T and B cell cross-reactivity between the human Ro60 autoantigen and Ro60 orthologs from the human microbiota
Authors: Greiling Teri , Dehner Carina Anja , Renfroe Stephen , Chen Xinguo , Vieira Silvio Manfredo , Ruff William , Goodman Andrew , Wolin Sandra , Kriegel Martin A
Published: January 2023

Human Ro60 is an RNA binding protein that is commonly targeted in systemic autoimmunity. We identified Ro60 orthologs that are conserved in a subset of skin, oral, and gut commensal species. Since anti-Ro60 antibodies are the earliest autoantibodies detected in lupus patients, we hypothesized that commensal Ro60 orthologs may trigger autoimmunity via autoepitope cross-reactivity in genetically susceptible individuals. While Ro60-producing gut commensals were common among both healthy controls and lupus patients, only antibodies from 4 Ro60-positive lupus patients, but not Ro60-negative subjects, co-immunoprecipitated Ro60 and its bound Y RNA from the Ro60-containing skin commensal P. propionicum. This suggests antibody cross-reactivity between human Ro60 antibodies and commensal Ro60. Next, Ro60-specific CCR6+ and CCR6- CD4 memory T cells clones from lupus patients were generated using a T cell library assay. Ro60 CCR6+ T cell clones proliferated in response to P. propionicum, demonstrating T cell cross-reactivity with commensal Ro60. Finally, germ-free mice monocolonized with B. thetaiotaomicron, a Ro60 ortholog-containing gut commensal, produced fecal anti-human Ro60 IgA antibodies (n=4, p=0.04), linking anti-Ro60 commensal responses in vivo with the generation of human Ro60 autoantibodies. In summary, Ro60 autoimmune T and B cells from human lupus patients cross-reacted with commensal Ro60 in vitro and commensal Ro60 triggered anti-Ro60 antibodies in vivo. Taken together, these data suggest that colonization with autoantigen ortholog-carrying species may sustain chronic autoimmunity in patients. This concept could lead to development of novel therapeutic approaches targeted at the human microbiota.