bluuebunny/crossref_metadata_2025 huggingface dataset:
Caveolin-1 is critical for Lymphocyte Trafficking into Central Nervous system during Experimental Autoimmune Encephalomyelitis
Authors: WU HAO , Deng Rui-xia , Chen Xing-miao , Wu Wu-tian , Shen Jian-gang
Published: January 2023

Multiple Sclerosis (MS) is a progressive autoimmune disease in central nervous system (CNS) with its pathogenesis not fully understood. In the present study, we demonstrated that caveolin-1, a major membrane scaffolding protein, plays a critical role in experimental autoimmune encephalomyelitis (EAE), a laboratory murine model of multiple sclerosis (MS). Immunized WT mice have increases of caveolin-1 expressions in serum and CNS tissues associated with disease development. After immunization, Cav-1knockout mice showed remarkable resistance with alleviated incidences and symptoms. With caveolin-1 deficiency, encephalitogenic T lymphocytes processed less efficient trafficking into CNS parenchyma, probably a sequence of declined expressions of adhesion molecule ICAM-1 and VCAM-1 in the white matter of CNS tissues. In agreement with in vivo studies, in vitro silencing of caveolin-1 in endothelial cells prevented the up-regulation of ICAM-1 expression, leading to less trans-endothelial migration of encephalitogenic TH1 and TH17 cells. Taken together, the results indicate caveolin-1 as an active modulator of lymphocyte trafficking into CNS and may be a potential therapeutic target for neuroinflammatory diseases.
Neutrophil recruitment limited by high affinity bent β2 integrin binding ligand in cis
Authors: Fan Zhichao , McArdle Sara , Mikulski Zbigniew , Gutierrez Edgar , Ginsberg Mark , Groisman Alex , Ley Klaus
Published: January 2023

Neutrophils are essential in innate immunity and inflammation. Many neutrophil functions are β2 integrin-dependent. Integrins can extend (E+) and acquire a high affinity conformation with an “open” headpiece (H+). The canonical switchblade model of integrin activation proposes that the E+ conformation precedes H+, and the two are believed to be structurally linked. Here, by using high-resolution quantitative dynamic foot printing (qDF) microscopy combined with a homogenous conformation-reporter binding assay in a microfluidic device, we show that a substantial fraction of β2 integrins on human neutrophils acquires an unexpected E−H+ conformation. Beside the observations consistent with the canonical switchblade model, we discovered a new alternative model that some β2 integrins transition from E−H− to E−H+ to E+H+ during activation. Using flow cytometry based Förster resonance energy transfer (FRET) assay, we find out that E−H+ β2 integrin binds its ligand intercellular adhesion molecule 1 (ICAM-1) in cis. And this cis binding significantly inhibits the conformational transitioning of β2 integrin from E−H+ to E+H+ and the neutrophil adhesion under flow, which indicates a novel endogenous anti-inflammatory mechanism inhibits neutrophil accumulation and inflammation.
Induction of adipose-resident NKT cells can be instructed by non ligand-binding motifs of the TCR β chain.
Authors: Vieth Joshua A , Das Joy , Ranaivoson Fanomezana , Comoletti Davide , Denzin Lisa K , Sant’Angelo Derek B
Published: January 2023

Invariant Natural Killer T (NKT) cells, a CD1d-restricted subset of αβ T cells, have been increasingly recognized for their role in a wide variety of functions associated with autoimmunity, cancer, infection, tolerance, and obesity. The semi-invariant TCR expressed by NKT cells is unique in its recognition of glycolipids presented by CD1d, heterodimer rigidity, and limited binding footprint compared to conventional TCR:MHC interactions. This distinctive interaction has been studied extensively in vitro, though the role of the rigid α:β heterodimer in the selection and development of NKT cells remains elusive. We investigated a hydrophobic patch on the β chain of the NKT TCR, suggested to play a role in heterodimer stability. Partial disruption of this patch, while permissive of ligand binding, resulted in decreased populations of NKT cells, diminished development, and effector function. Complete disruption of the patch resulted in the ablation of the NKT cell compartment entirely, while still accommodating conventional T cell development. NKT cells expressing the mutant NKT TCR acquired an adipose-resident NKT phenotype while in the thymus, and accumulated in the adipose tissue in mice. Hence, we show that in vivo, non-ligand binding regions of the NKT TCR are critical for the development of these important immune cells, and mediate the selection of adipose-resident NKT cells.
Development of a scurfy-like phenotype in Foxp3-YFP-cre NKAP cKO mice
Authors: Dash Barsha , Constans Megan , Chung Ji Young , Arocha Sinibaldo Romero , Shapiro Virginia
Published: January 2023

NKAP is a transcriptional repressor required at various stages of conventional αβ T cell development. Here, we asked if NKAP is also required in regulatory T cells. To answer this, we created Foxp3-YFP-cre NKAP conditional knock-out (cKO) mice in which NKAP deletion occurs upon the expression of Foxp3 during Treg development in the thymus. Deletion of NKAP results in lethal autoimmunity necessitating euthanasia at 3–4 weeks of age that is reminiscent of Foxp3-deficient scurfy mice. Foxp3-YFP-cre NKAP cKO mice present uncontrolled conventional T cell activation, Treg abnormalities and a reduction of total number of Tregs in the periphery. Therefore, NKAP is required for Treg dependent peripheral tolerance.
Src-Like Adaptor Protein 2 regulates NKT lineage diversification
Authors: Tuttle Kathryn , Peterson Lisa K. , Bedel Romain , Zhang Jingjing , Dragone Leonard L. , Gapin Laurent
Published: January 2023

Natural Killer T (NKT) cells are a class of αβ T cells that express a semi-invariant T Cell Receptor (TCR) and recognize glycolipids presented by the non-polymorphic MHC molecule, CD1d. NKT cells are considered to be innate-like lymphocytes because they produce a robust cytokine response within hours of stimulation. This ‘poised effector’ state is acquired during development in the thymus, where NKT precursors differentiate into one of three distinct subsets and acquire TH1, TH2, or TH17 effector functions. It is not known which signals control the divergence of NKT1, NKT2 and NKT17 subsets. We have identified a novel role for Src-like adaptor protein 2 (SLAP2) in regulation of NKT cell development. We have found that SLAP2-deficiency causes accumulation of thymic NKT cells and alters lineage diversification. In the absence of SLAP2, the frequency and number of thymic NKT2 and NKT17 cells are increased with a concomitant decrease in NKT1 cells. SLAP2−/− NKT cells express elevated levels of PLZF and higher levels of CD5, both of which are downstream of TCR signals. Although SLAP2 has been shown to negatively regulate TCR signaling in vitro, analysis of SLAP2−/− thymocytes revealed that TCR levels and signaling in CD4+CD8+ double positive thymocytes are normal. These data suggest that SLAP2 does not directly regulate TCR during development, in vivo. The numbers and proportions of conventional αβ T cells are largely unaffected by SLAP2-deficiency. In light of these data, we predict that SLAP2 regulates signals that are important for NKT lineage diversification but are dispensable for conventional T cell development.
In utero induction of mixed chimerism results in deletion, receptor editing, and functional inactivation of antigen specific alloreactive B cells
Authors: McClain Lauren E , Kim Aimee G , Flake Alan W , Luning Prak Eline T , Peranteau William H
Published: January 2023

Purpose

B cell tolerance is critical for donor cell engraftment across immune barriers after in utero hematopoietic cell transplantation (IUHCT). Using our fetal transplant model we studied how B cells achieve tolerance to allogeneic MHC antigens.

Methods

10×106 C57Bl/6-GFP (H2Kb) bone marrow (BM) cells were injected intravenously into gestational day 14 Balb/c (H2Kd) fetuses. Mice were sacrificed at 3 days (P3), 1 and 4 months of age and donor-specific B cell tolerance including deletion, receptor editing and functional inactivation were assessed. Age matched naïve C57Bl/6 and Balb/c mice served as controls.

Results

The % of alloantigen specific splenic B cells was significantly decreased in allogenic chimeras as compared to controls (p=0.016) consistent with clonal deletion. Alloreactive B cells that escape deletion can undergo receptor editing. There was a significant increase in the % of B cells undergoing editing (lambda light chain expression) at P3 in recipients of IUHCT compared to controls (10.8% vs. 8.4%; p=0.02). Alloreactive B cells can be functionally inactivated such that they no longer make antibodies or express IgM. In contrast to naïve Balb/c mice immunized with C57Bl/6 splenocytes, immunization of recipients of IUHCT failed to elicit an anti-donor antibody response (MFI fold change: IUHCT-0.89 naïve-2.77; p<0.05). Furthermore, IgM expression on alloantigen specific splenocytes in chimeric animals was lower than controls (MFI: chimeric-1206, control-1472 ; p=0.03).

Conclusion

B cell tolerance after IUHCT is achieved by partial deletion in alloreactive splenic B cells, receptor editing in early mature splenocytes, and the remainder of alloreactive B cells demonstrate a functionally inactive phenotype.
X chromosome gene Ddx3x is involved in B-cell development
Authors: Liu Ke , Karmele Erik P , Perlot Thomas , Penninger Josef M , Waggoner Stephen N , Harley John B
Published: January 2023

Ddx3x, located on the X chromosome, belongs to the DEAD box RNA helicase family. Ddx3x is previously implicated in RNA metabolism, cell cycle control, apoptosis, tumorigeneses and viral infections.

In the process of exploring the role of ddx3x in the female predominance of systemic lupus erythematosus, we found abnormalities in B-cell development within conditional ddx3x-deficient mice.

Breeding of ddx3x-floxed mice with Vav1-Cre mice led to deletion of ddx3x in hematopoietic cells (Vav1ddx3x). Ddx3x expression was reduced 80% in the bone marrow (BM) of Vav1ddx3x mice. Hematopoietic deficiency of ddx3x is associated with a reduced proportion and absolute number of B cells, starting at the small pre-B cell stage, and then subsequently immature B cells and mature B cells in bone marrow, relative to wild-type mice. In spleen, the frequency and number of follicular B cells (FOB) were significantly decreased whereas the numbers of marginal zone, germinal center and plasma-blast B cells were sustained. Mixed Vav1ddx3x and wild-type bone marrow chimera mice demonstrated a B-cell intrinsic effect. Surprisingly, despite fewer B cells in the BM and spleen, serum IgM, IgA and IgG titers were elevated in Vav1ddx3x mice, suggesting ddx3x may affect B-cell function as well.

In conclusion, we showed that ddx3x deficiency affects B-cell development resulting in a loss of B cells in bone marrow and the periphery and an increase in serum immunoglobulin levels.
EZH2 regulates cell cycle and survival in B and T lymphocyte progenitors
Authors: Jacobsen Jennifer Andrene , Woodard Jennifer , Kee Barbara L
Published: January 2023

The histone methyltransferase EZH2 catalyzes the H3K27me3 histone modification, which is broadly associated with repressed and poised chromatin. EZH2 is required for both B and T cell development, but its specific role in these lineages is not clear. Here we used an Il7racre to delete Ezh2 in all lymphocytes. We confirmed that EZH2 is required for B and T cell development, but found that it was dispensable during NK cell development. EZH2-deficient pro-B cells failed to expand in vitro, and showed increased apoptosis and cell cycle defects. These cells expressed increased mRNA from the cell cycle regulators, p16/INK4a and p19/ARF, which are encoded at the Cdkn2a locus and are known targets of EZH2 in other tissues. ARF prevents ubiquitin-mediated degradation of the tumor suppressor p53 leading to cell cycle defects and apoptosis. p53 was increased in EZH2-deficient pro-B cells and canonical p53 target genes were also increased. We crossed EZH2-deficient mice to the Cdkn2a−/− background to determine whether Cdkn2a upregulation affected B and T cell development in EZH2-deficient mice. Abrogation of Cdkn2a partially rescued B cell development and robustly restored T cell development in EZH2-deficient mice. These results indicate that EZH2 is required for survival and cell cycle regulation in B and T cell progenitors.
Regulation of B cell responses in allergy by histamine receptors
Authors: Velez Tania Estefania , Swartzendruber Julie A , Hulse Kathryn E , Bryce Paul J.
Published: January 2023

Histamine is a bioactive amine stored in mast cells and basophils and released upon antigen-specific IgE-dependent activation, mediating responses during allergic reactivity, including vasodilation, smooth muscle constriction and inflammatory cell recruitment. These are regulated through four receptors (H1R-H4R) that are differentially expressed on various cell types. Despite this, increasing evidence suggests that histamine can be produced during the priming phase of allergen responses, with H4R on dendritic cells being important for initiating Type 2 immunity. Here, we sought to determine the roles of these receptors in supporting antigen-specific B cell responses. Interestingly, our data demonstrates that both murine and human B cells only expressed H1R and H2R; these also showed differential expression levels on specific B cell subsets and upon activation. Using an Ovalbumin-alum (OVA-alum) model of allergic immunization, mice lacking H2R produced significantly lower levels of OVA-IgE, whereas mice lacking H1R were unaffected. Surprisingly, absence of both H1R and H2R (H1R/H2R DKO) exhibited undetectable levels of OVA-IgE. This was not due to T cell-intrinsic influences since restimulation of splenocytes from immunized H1R/H2R DKO mice showed that Th2 cytokine production was unaltered compared to WT. Instead, in vitro stimulation of B220+ B cells by αCD40 and IL-4 yielded significantly lower IgE production from H1R/H2R DKO mice than WT and defines a key requirement for these receptors in permitting B cells from generating IgE antibodies. Our data establishes a novel requirement for histamine, a molecule typically considered in regulating allergic responses, in supporting the generation of allergic sensitivity.
A novel mouse model of hypersensitivity to salt-soluble wheat protein: comparison of soft vs. durum wheat varieties
Authors: Gangur Venugopal , Jin Yining , Ebaugh Sarah , Secord James , Ng Perry KW , Olson Eric
Published: January 2023

Immune-mediated adverse reactions to wheat are rising at an alarming rate on a global scale for reasons that are not completely understood. Wheat is identified as a major red-flag allergenic food in many countries including the USA. Although mouse model of alcohol-soluble gluten hypersensitivity is available, a mouse model of hypersensitivity to salt-soluble wheat proteins (SWP)--the most common of the wheat allergens—is unavailable. Here we tested the hypothesis that SWP from both soft and durum wheat will elicit allergic response in BALB/c mice. High quality SWP from soft (Ambassador) and durum (Carpio) wheat were prepared, characterized and used in the study. Pups were weaned onto and maintained on a wheat-free diet. Group of mice (n=5/group) received three i.p. injections with SWPs plus alum. Blood was collected and used in the specific (S) IgE, IgG1/G2a antibody analysis. Upon i.p. challenge, mice were monitored for hypothermia shock response (HSR) and mast cell protease (mMCP)-1 response. Although both soft and durum wheat SWPs elicited robust SIgE Ab responses, durum wheat was superior (IgE titers: 905 vs. 2560). Both proteins elicited marked but comparable SIgG1/IgG2a Ab responses. Upon challenge, both proteins elicited HSR; however, durum-SWP elicited 6.7 times higher mMCP-1 response than soft-SWP. In summary, we report a novel mouse model of hypersensitivity to salt-soluble wheat proteins for the first time. These data also demonstrate that the durum (Carpio) wheat is more allergenic than the soft (Ambassador) wheat variety that we studied.
Efficacy of Curcuma longa in osteoarthritis: Association of IL-1β, IL-10 and MMP-9 with severity of disease
Authors: Saksena Anil Kumar , Srivastava Shobhit , Khattri Sanjay , Kumar Santosh
Published: January 2023

Osteoarthritis (OA) is a progressively debilitating immune mediated inflammatory disorder in which various biomarkers have been reportedly increased. This study was carried out to establish a correlation between IL-1β, IL-10 and MMP-9 in various grades of OA of knee and to observe the effect of Curcuma longa (CL-an indigenous drug) in such patients through a randomized, placebo controlled, double blind trial.

After obtaining clearance from Institutional Ethics Committee, 160 patients of either sex aged between 40 to 80 years, meeting the inclusion and exclusion criteria, were enrolled and randomly divided in two groups. The grading of the disease was done according to Kellgren-Lawrence (KL) grading scale. Placebo and extract of CL were given along with the standard treatment to the two groups in blind manner. The assessment of the patients was done after two and four months.

On day 0 the level of IL-1β, MMP-9 and IL-10 was found to be significantly higher in grade 3 and 4 patients as compared to grade 1 and 2 patients. After oral administration of CL extract there was significant clinical improvement (as compared to placebo) in all grades of patients as adjudged by WOMAC score and VAS. There was also significant decrease in levels of IL-1β and MMP-9 in parallel with the clinical improvement. Moreover, decline in the level of IL-10, after treatment with CL was not significant.

It is concluded that IL-1β, MMP-9 and IL-10 have strong parallel association with severity of OA. Further, CL produced significant relief in the patients of OA of knee, when given along with standard treatment.
High Monocyte Turnover Predicts Progression to AIDS in SIV Infected Aging Rhesus Macaques
Authors: Fahlberg Marissa , Walker Edith , Penney Toni , Cai Yanhui , Kuroda Marcelo J , Didier Elizabeth S
Published: January 2023

Incidence of newly-acquired HIV infections in persons over age 50 is increasing, from 13% in 2006 to 21% in 2013. Advanced age corresponds with lower 12-month survival and ART is contraindicated with the simultaneous use of many chronic disease medications. To address the need for age-specific interventions, we are studying aged rhesus macaques (RM) infected with SIV. In a pilot study, four young (6.27–7.37 years) and six older (17.71–23.82 years) RM were injected i.v. with SIVmac239. The older RM exhibited lower survival times compared to younger macaques (112.5 ± 76.8 days v. 353 ± 74.9 days, respectively). In young adult RM with SIV, we reported that increasing monocyte turnover (via BrdU uptake) as well as declining CD4+ cell counts, predicted disease progression. Among the aged RM, we observed that 3 animals developed increased or high monocyte turnover (HMT) after the first month of infection (35.2% ± 2.36) while the other 3 maintained steady state or low monocyte turnover (LMT) (5.74% ± 4.92) similar to that in the young animals (9.97% ± 2.34). Older RM with LMT survived longer (151 ± 85.6 days) than those with HMT (74 ± 54.5 days). The young adult and aged RM with LMT exhibited a dramatic decline of CD4+ T cells during peak viral load (−46% and −41%) 2 wks post infection compared to only a 22% decrease in aged RM with HMT. Also, the numbers of naïve T cells (CD4 and CD8) were lower in the HMT aged animals than in the LMT and young adults, at baseline and 2 wks post infection. These results suggest that there may be an additional or alternative preferential host cell target of SIV early in infection of older susceptible RM that we speculate to be macrophages. Work is continuing to examine the role of macrophages in pathogenesis of HIV infection in the elderly.
Role of TWEAK/Fn14 in chronic inflammatory disorders of the skin
Authors: Sidler Daniel , Herro Rana , Kawakami Yuko , Kawakami Toshi , Burkly Linda C , Croft Michael
Published: January 2023

Atopic dermatitis and Psoriasis are inflammatory disorders of the skin. Despite particular differences in respect of etiology, pathogenesis and clinical manifestations, these diseases share histological features such as epidermal hyperplasia and dermal fibrosis. Discovering molecules that contribute to the development of both diseases could provide opportunities for therapeutic intervention. It is accepted that keratinocytes and dermal fibroblasts play a pivotal role in both diseases by amplifying and maintaining inflammatory and fibrotic responses. TNF superfamily members, including TWEAK (TNFSF12), have been implicated in control of these cell types, but experimental insights are scarce. Employing TWEAK-deficient mice, we demonstrated that the TWEAK/Fn14 axis is indispensable for experimental atopic dermatitis and psoriasis. Furthermore, we found that subcutaneous injection of recombinant TWEAK into naïve mice is sufficient to induce robust inflammation with histological and molecular signatures of both atopic dermatitis and psoriasis. Moreover, in vitro experiments revealed that keratinocytes and dermal fibroblasts produced key chemokines and cytokines in response to TWEAK that are characteristic of both atopic dermatitis and psoriasis, and these molecules were amplified when TWEAK was combined with the signature cytokines IL-13 and IL-17. Similarly, subcutaneous injection of TWEAK with IL-13 or IL-17 synergized with respect to infiltration and intra cutaneous expression of proinflammatory molecules. These studies help to understand the network of factors active in the pathogenesis of skin inflammatory disorders, and highlight TWEAK as a central regulator of both atopic dermatitis and psoriasis.
The auto-reactive profile of B cells from early MS patients
Authors: Monson Nancy , Rivas Jacqueline , Ireland Sara , Rounds William , Ligocki Ann , Ma Lisha , Frohman Elliot , Greenberg Benjamin , Cowell Lindsay G , Stowe Ann Marie
Published: January 2023

My lab’s recent work has led to the discovery that antibodies expressed by memory B cells in the cerebrospinal fluid (CSF) of patients with Multiple Sclerosis recognize neurons and astrocytes in the brain. Genetic analysis further revealed that only certain heavy chain genes accommodate binding to these brain targets, and that memory B cells from control cohorts rarely express these antibody genes. In fact, in a recent clinical trial, we demonstrated that the prevalence of these antibody genes expressed by memory B cells in the CSF support identification of patients who either have MS or will develop MS in the future with 89–94% accuracy. This Antibody Gene Signature (AGS) is a novel biomarker that heralds the advent of antibody genetics as a means to support categorization of patients early in their disease course. Our goal for this project was to determine whether antibodies expressed by activated memory B cells that have initiated antibody production (called “plasmablasts”) in the peripheral blood of MS patients also recognize neurons and astrocytes. We cloned and expressed more than 50 antibodies expressed by peripheral plasmablasts of early MS patients and control cohorts. We found that antibodies expressed by peripheral plasmablasts from patients who develop MS bind to neurons and astrocytes in the brain. Those plasmablasts that did not express antibody heavy chains associated with the AGS did not bind to brain targets. Interestingly, only those patients in our cohort who have an elevated frequency of peripheral plasmablasts continue to accumulate brain MRI activity. It is possible that brain-reactive peripheral plasmablasts contribute to the brain damage associated with MS.
ER stress generates immunogenicity in human pancreatic β cells
Authors: Marre Meghan L. , James Eddie A , Piganelli Jon D
Published: January 2023

Type 1 diabetes (T1D) is an autoimmune disease in which autoreactive T cells target and destroy pancreatic islet β cells. The key events that break peripheral tolerance in patients genetically predisposed to autoimmunity remain poorly understood. Many physiological and environmental triggers associated with T1D cause endoplasmic reticulum (ER) stress, which may increase abnormal protein post-translational modification (PTM). We hypothesized that β cell ER stress generates neo-antigens that can activate autoreactive T cells in T1D. Chemical (Thapsigargin) induction of ER stress in human islets or insulinomas increased their recognition (135–360 fold) by human T cells known to preferentially recognize deamidated GAD65, as measured by IFNγ secretion. This increased immunogenicity was accompanied by increased activation of the PTM enzyme tissue transglutaminase 2 (Tgase2; 2 fold). We will confirm the role of this enzyme in ER stress-induced human β cell immunogenicity with shRNA. We are also working to validate this pathway as a therapeutic target as follows: First, we are measuring calcium dynamics to determine if cytosolic calcium is crucial to ER stress-dependent immunogenicity. Second, we are assessing whether other triggers of ER stress, such as cytokines and glucose, also generate GAD65 PTM in human β cells. β cell ER stress generated through a variety of sources may be an opportunity for therapeutic intervention to prevent the destruction of β cells. Our on-going work will define the ER stress-induced pathways and PTM that contribute to recognition of GAD65. These findings will also have implications for the recognition of other autoantigens and for the development of T1D.
Immune regulatory receptors expressed on NK cells in type 1 diabetes.
Authors: Suwannasaen Duangchan , Thrope Jerill , Greenbaum Carla , Long Sarah Alice
Published: January 2023

Auto-reactive T cells specific to islet antigens contribute to the onset of type 1 diabetes (T1D), yet NK cells may also play a role in disease onset and progression. Mouse models suggest a dual role for NK cells in disease onset and progression while human studies have found deficiencies in NK number and function. However, human studies are confounded by variability in cohorts (pre-T1D, recent onset, long-standing T1D) and more phenotypic and functional analysis as has been performed in murine studies. In our initial studies, we characterized activation and inhibitory markers on NK cells of longstanding T1D (n=5) as compared to healthy controls (n=6). NK cells from the same sample were then compared to known T cell phenotypes. PBMC were analyzed by flow cytometry for markers of T and NK cells (CD45RA, CCR7, CD56, CD4 and CD8), immune regulation (PD1, KLRG1, TIGIT and CD57) and activation (CD69 and CD25). Results showed that there was a significant elevation of T cells but a decrease of NK subsets (CD56dim and bright) in T1D compared to healthy controls. Both CD57+ and CD57+TIGIT+ NK cells were significantly reduced in T1D, implying a more immature and less regulatory phenotype or more activated phenotype. In the same subjects, both CD4 and CD8 memory T cell populations expressing PD1 were significantly lower in T1D than in healthy controls. Moreover, the CD8 EMRA (CCR7− CD45RA+) T cells exhibited decreased KLRG1, TIGIT and CD57 expression. Taken together, these findings revealed a skewing of NK cell subsets in T1D. Further functional characterization of both NK and T cells in the same sample may lead to identification of biomarkers for T1D.
Increased expression of IRF-5 in the adipose tissue in obesity: Implication in metabolic inflammation
Authors: THOMAS REEBY SARA , Kochumon Shihab , Ahmad Rasheed , Sindhu Sardar
Published: January 2023

Obesity is marked by a state of chronic low-grade inflammation called metabolic inflammation which plays a role in the development of insulin resistance and type-2 diabetes (T2D). We and others have shown that the innate immune toll-like receptors (TLRs) are involved in metabolic inflammation/insulin resistance. The transcription factor called interferon regulatory factor (IRF)-5 is a major regulator of proinflammatory macrophage polarization; however, its role in obesity/T2D remains unclear. Therefore, we determined modulations in the IRF-5 expression and analyzed their relationship with signature metabolic inflammatory markers in the subcutaneous adipose tissues (AT) obtained from adult persons classified as lean, overweight, and obese on the basis of BMI. The data show that IRF-5 expression was upregulated (P<0.05) in obese as compared with lean individuals at both mRNA and protein levels; and these changes correlated with BMI (r=0.3; P= 0.03) and body fat percentage (r=0.48; P=0.0005). The elevated IRF-5 expression also correlated positively (P<0.05) with various inflammatory markers including TNF-α (r=0.47), IL-6 (r=0.31), IL-1β (0.36), IL-18 (r=0.27), CXCL-10 (r=0.30); IL-8 (r= 0.33), CCL-5 (r=0.41); CCR-5 (r=0.42); CCR-2 (0.54) as well as TLR adapter proteins MyD88 (r=0.61) and IRAK-1 (r=0.50). Interestingly, IRF-5 expression was found to associate with fasting blood glucose (r=0.40, P = 0.008) and glycated hemoglobin (r=0.45, P=0.009). As expected, IRF-5 expression correlated negatively with plasma adiponectin levels (r= −0.43; P=0.009). Collectively, our data show that increased AT- IRF-5 expression is concordant with signature inflammatory markers which may have significance in metabolic inflammation.
The role of monoclonal paraproteins in Systemic Capillary Leak Syndrome (SCLS)
Authors: Hakeem Reem M , Druey Kirk , Zhihui Xie
Published: January 2023

The Systemic Capillary Leak Syndrome (SCLS) is a rare, orphan disease characterized by bouts of acute, reversible plasma extravasation into tissues, which leads to hemoconcentration, hypoalbuminemia, hypotension, and vascular collapse. Although the cause of SCLS is unknown, 80–95 % of SCLS patients have a monoclonal gammopathy of unknown significance (MGUS), in which clonal plasma cells secretes large amounts of monoclonal immunoglobulins or “paraproteins”. MGUS is a premalignant precursor to multiple myeloma (MM) and is notably detected in most SCLS patient sera. Another marked distinction is that acute, but not basal SCLS sera contain elevated concentrations of inflammatory mediators known to promote endothelial permeability, notably Vascular Endothelial Growth Factor (VEGF) and Angiopoietin 2 (Angpt2). To address these findings experimentally, we purified whole IgG from patient and control sera to examine whether these immunoglobulins have an impact on endothelial cell barrier function and performed functional assays to characterize the observed changes in morphology. Preliminary data suggest that patient IgG binds to endothelial cells and elicits significant F-actin stress fiber formation and VE-cadherin internalization. SCLS IgG seems to be critical in triggering endothelial dysfunction and dissecting the underlying signaling mechanisms involved will help us better understand disease pathogenesis and potentially pave the way for therapeutic target development.
SMAC mimetic in combination with HDACi effectively release brakes on cell death of chronic myeloid leukemia K562 cells accelerating ligand-mediated apoptosis.
Authors: Hontz Adrianne E , Vrazo Alexandra C , Figueira Sarah K , Kieser Pearce C , Risma Kimberly A
Published: January 2023

Enthusiasm for immunotherapy has reached an all-time high within the last year due to remarkable clinical outcomes using strategies directed at activating T cells. The mechanisms by which newly activated T cells execute tumor target cells have not been described; it is assumed that CD4 and CD8 T cells may utilize ligation of TNF receptor family members, Fas, TNF, and TRAIL. However, not all tumors are susceptible to T cell killing by death receptor ligands. Chronic myeloid leukemia K562 cells are notably resistant to pro-apoptotic, ligand-induced cell death; whereas, T cell leukemia Jurkat cells undergo rapid caspase 3/7 activation and cell death (within 4–6 hours) in response to pro-apoptotic death ligands Fas and TRAIL. We hypothesized that K562 cells may be sensitized to respond similarly by targeting the regulatory proteins controlling activation of executioner and/or initiator caspases, thus accelerating cell death. SMAC mimetic compounds (SMC) are a novel class of drugs that target pro-survival signaling molecules. Single agent treatment with SMC enhanced TRAIL- and TNF-induced caspase activation, but had only modest effects on cell death after 24 hours. Addition of histone deacetylase inhibitors (HDACi) in combination with SMC, accelerated TNF- and TRAIL- mediated caspase activation and led to rapid cell death in K562 cells. By relieving multiple levels of inhibition in the tumor targets, we achieved rapid and efficient killing of previously resistant tumor cells using recombinant death receptor ligands. Combination therapy with SMC and HDACi may enhance current immunotherapy approaches designed to 1) activate tumor specific T cells expressing death receptor ligands or 2) administer recombinant ligands such as TRAIL.
Mouse models for immunology research available from The Jackson Laboratory Repository.
Authors: Valenzuela Alicia , Lutz Cathleen M , Rockwood Stephen , Sasner Michael
Published: January 2023

The Jackson Laboratory Repository serves as a centralized facility for the development, distribution and cryopreservation of mouse models of human biology and disease. Hundreds of new strains are added annually to one of the largest collections of characterized mouse strains available.

This poster presents our newest strains involving genes associated with adaptive immunity. The Pdcdl (programmed cell death 1 - PD1) knockout (KO) mice allow increased infiltration of inflammatory cells in several disease models. CC10-OVA transgenic mice direct OVA expression to lung epithelium; and a KO/KI mouse expresses tamoxifen-inducible cre under the control of the Cd19 promoter in B lymphocytes.

New mouse models with mutations in genes involved in the innate immune response include: Dectin-1 (Clec7a) KO mice, which exhibit increased sensitivity to fungal infection and a dysregulated chemokine response; GM-CSF (Csf2) KO mice with impaired phagocytosis, a lupus-like disorder and glomerulonephritis; mice with a KO in MPYS (Tmem173), a pattern recognition receptor that activates type 1 interferon; Ccr2gfp KI/KO mice used in tracking monocyte chemotaxis; a transgenic mouse model of hyperimmunoglobulin E syndrome that expresses modified Stat3; and a new addition to our extensive collection of interleukin mutants, Il1a, a new conditional KO/reporter allele that is produced by monocytes and macrophages.

Additionally, several Cas9-expressing lines, requiring only a specific single guide RNA to generate single or multiple simultaneous mutations, are available. These lines overcome the burden of packaging size limits.

Researchers are encouraged to deposit mice to the Repository: www.jax.org/donate-a-mouse.
Helios expression defines two distinct populations of Foxp3+ regulatory T cells
Authors: Thornton Angela M , Kilaru Gokhul , Burr Patrick , Rieder Sadiye , Muljo Stefan Adi , Shevach Ethan M
Published: January 2023

The transcription factor Helios is expressed in a subset of Foxp3+ Tregs and we have proposed that Helios is a marker of thymic derived Treg (tTreg). Other studies have suggested that Helios is primarily a marker of T cell activation. In order to study the two Treg subpopulations, we have generated Helios-GFP reporter mice and crossed them to Foxp3-RFP reporter mice. The expression pattern of Helios in the reporter mice is similar to that detected by intracellular staining. When Foxp3+ Helios+ and Foxp3+ Helios− were isolated by FACS and transferred to normal mice, both phenotypes were stable. Both populations expressed a highly demethylated TSDR. A comparison of the two populations using RNA-Seq demonstrated 600 differentially expressed genes (500 higher in Helios+, 100 higher in Helios−). The Helios+ population expressed a more activated (Ly-6C−) phenotype and had slightly higher suppressive capability in vitro. Both subsets were equivalent in their ability to suppress IBD in vivo, but the Helios+ subset was more effective in the protection of RAG−/− mice from disease induced by transfer of splenocytes from scurfy mice. Thus, Helios expression can differentiate two distinct populations of Treg and may differentiate peripherally derived Treg (pTreg) from tTreg.

This research was supported by the Intramural Research Program of the NIH, NIAID.
Early TNFα signaling results in pulmonary DC1 polarization and programing of murine myeloid precursor cells in the bone marrow towards DC1 polarization throughout Cryptococcus neoformans infection
Authors: Eastman Alison J , Potchen Nicole , Zhou Guolei , Xu Jintao , Neal Lori , Malachowski Antoni , Kunkel Steven L , Kryczek Ilona , Osterholzer John J , Olszewski Michal A
Published: January 2023

TNFα is required for protective Th1/Th17 immunity to Cryptococcus neoformans (CN), an opportunistic pulmonary fungal pathogen. The effects of TNFα are linked to the stable, early classical activation of dendritic cells (DC1), preventing alternative (DC2) activation. We hypothesized that TNFα signaling facilitates epigenetic modification of key DC genes during CN infection. We tested this hypothesis using CBA/J mice infected intratracheally with CN and injected once on day 0 intraperitoneally with control or a TNFα-depleting antibody (αTNFα). αTNFα mice had no difference in pulmonary CFU at 7 days post-infection (dpi), but had significantly higher fungal burden and extrapulmonary dissemination at 14 and 28 dpi. DCs from the lungs of control mice at 7 dpi had increased association of iNOS and IL-12b promoters with the activating modification histone 3 lysine 4 trimethylation (H3K4me3), while DCs from αTNFα mice did not. Because DCs have a relatively short half-life during infection, we assessed the bone marrow (BM) myeloid precursor cells (MPCs) from infected animals with and without TNFα. Intranuclear flow cytometry for H3K4me3 showed distinct patterns of global trimethylation between infected control and αTNFα mice as early as 7 dpi. We next tested whether BM-derived DCs (BMDCs) matured ex vivo from infected animals would yield DC1 or DC2 cells. BMDCs from control animals became DC1, and resisted changes to polarization when challenged with the DC2-skewing IL-4, while BMDCs matured from TNFα-depleted mice maintained DC2 polarization when challenged with IFNγ. We conclude that TNFα epigenetically modifies key DC1 genes in MPCs, thereby sustaining lasting DC1 programing of DCs required for clearance of CN from the infected lungs.
The mechanisms of Gamma delta T cells in the occurrence and development of hepatocellular carcinoma
Authors: Ye Yanshuo , Wang Zhanpeng , Li Zhuonan , Liu Hongyu , Li Wei
Published: January 2023

γδ T cells are unique population of T cells in which their function still remain unclear. We have demonstrated that γδ T cell mainly located in the liver in mice. Our previous data based on liver transplantation model suggests that γδT cells are involved in the processes of liver transplant tolerance and rejection, and may play a role in the induction of liver graft rejection. In contrast to Foxp3+ CD4+ CD25+ Treg, the majority of γδT cells were mainly located in the liver and did not express TCRαβ and NK receptors, but expressed moderate levels of PD-1 and PD-L1 molecules. It has recently been shown that γδT cells play an important role against tumors and infectious diseases.

Methods

In this study we employed mouse orthotopic hepatocellular carcinoma model, critically examine the role of γδ T cells in the liver and peripheral on liver cancer development.

Results

The ratio of CD3+ T cells were decreased, but γδ T cells were increased in the surrounding tissue of the tumor and the spleen. Anti-γδ T cells antibody plus IL-2 treatment to tumor barrier mice could further increase the γδ T cells in the liver and spleen and limited the growth of the tumor.

Conclusion

γδ T cell may plays an important role in immunologic defence against hepatocellular carcinoma. More studies underlying the mechanisms and interplay among liver immune cells are under investigation.
Dendritic cell subset positioning governs clonal diversity and magnitude of the CD8 T cell response
Authors: Gerner Michael , Kastenmuller Wolfgang , Germain Ronald N
Published: January 2023

Although the molecular requirements for CD8 T cell responses have been well characterized, subunit vaccine formulations struggle to generate robust CD8 T cell immunity. One possible basis for this may be the relationship of dendritic cell (DC) subset topography to micro-anatomically dictated antigen dispersal and subsequent down-stream innate and adaptive cellular engagement. To address this issue directly in situ we utilized advanced quantitative imaging approaches (histo-cytometry) and found that protein immunization led to non-linear concentration gradients across draining lymph nodes. This graded distribution in turn affected which DC subset had access to high vs. low concentrations of antigen. Because DC subsets are specialized in MHC-I vs. MHC-II antigen processing and presentation, the result of this intersection of antigen dispersal and cell positioning was the predominant residence of MHC-I presenting DC in regions with lower antigen bioavailability. Modulating the antigen dose directly influenced the fraction of MHC-I presenting DC with access to sufficient antigen to activate T cells, allowing for direct control over the clonal diversity and magnitude of the response. These findings indicate that tissue microanatomy related to DC subset distribution and antigen dissemination within the lymph node is a critical regulator of CD8 T cell immunity.

This research was supported by the Intramural Research Program of the NIH, NIAID.
Dysfunctional expansion of immature granulocytes in sepsis survivors.
Authors: Fei Yurong , Chiu Pui Yan , Diamond Betty , Sherry Barbara
Published: January 2023

Sepsis is one of the leading causes of death worldwide. Surprisingly, increased mortality persists for years in sepsis survivors. This project investigated the contribution of myeloid cells to immune impairment in sepsis surviving mice. Sepsis was induced by cecal ligation and puncture (CLP). Sepsis survivors (2 weeks post-surgery) had significant increases in the CD11b+ Ly6Chigh and CD11b+ Ly6Clow myeloid populations in spleen, bone marrow and blood compared to controls. The expanded CD11b+ Ly6C populations in survivors were F4/80 negative. More CD11b+ Ly6C cells expressed the chemokine receptors CX3CR1 and CCR2, and the adhesion molecule CD62L. CD11b+ Ly6C populations in survivors also had higher levels of the mannose receptor CD206, Mer tyrosine kinase and the inhibitory collagen receptor LAIR-1; they appeared to be M2 like macrophages with presumed immune suppressive activity. Approximately 12% of the CD11b+Ly6Chigh population were CD115+ monocytes/macrophages, while the CD11b+ Ly6Clow comprised mainly immature granulocytes. CD11b+ Ly6Clow granulocytes in sepsis survivors had significantly reduced phagocytic activity compared to controls. A significant reduction in CD11b+ Ly6Clow granulocytes after LPS treatment was observed in controls but not survivors. Importantly, survivors had diminished serum production of TNF in response to endotoxin challenge, even though CD11b+ Ly6C granulocytes from the same mice produced comparable intracellular levels of TNF. In summary, our data indicate impaired cell exit and cytokine secretion, which is in part due to altered cell surface properties. This dysfunctional expansion of immature granulocytes may contribute to the impaired immune response in sepsis survivors.
Kupffer cell subsets differ between young and aged murine livers.
Authors: Stahl Elizabeth C , Brown Bryan N
Published: January 2023

The immune system, and in particular macrophages, are heavily implicated in wound healing, response to infection, and cancer progression, all of which are linked to mortality in the elderly population. Recently, it has been shown that tissue-resident macrophages from spleen and peritoneum become dysfunctional with aging, likely due to the aged microenvironment. The liver contains approximately 80% of total mammalian tissue-resident macrophages, known as Kupffer cells. Kupffer cells can be divided into two F4/80+ macrophage subsets: CD68+ and CD11b+ cells, which have different origins and functions. Currently, it is unclear how these macrophage subsets are affected by aging.

We sought to characterize differences in the Kupffer cell compartment from young (8–10 week) and aged (18–20 month) C57/Bl6 mice using immunofluorescent staining on tissue sections and flow cytometry. Preliminary histological analyses showed that the area of F4/80+ staining did not differ between young and aged mice. Interestingly the area of CD68+ staining was significantly higher in the aged murine livers. In addition, the area of CD32+ staining, which marks both endothelial cells and macrophage precursors, was significantly greater in aged mice, consistent with previous reports of blood vessel thickening in aged livers. Flow cytometry analysis confirmed differences in the macrophage subsets between young and aged murine livers. In addition, functional changes in phagocytosis were noted with aging, suggesting dysfunction of innate abilities. Characterizing the differences in the subsets of tissue-resident macrophages during aging will increase our understanding of age-related pathologies that will aid in the design of therapies in the future.
MiR-150 negatively regulates CD8+ T cell memory formation
Authors: Chen Zeyu , Stelekati Erietta , Kurachi Makoto , Yu Sixiang , Manne Sasikanth , Wherry E. John
Published: January 2023

MicroRNAs play an important role in CD8+ T cell differentiation and anti-viral immune responses. However, how microRNAs regulate CD8+ T cell memory formation remains poorly defined. Here, by microRNA profiling of CD8+ T cell during acute or chronic LCMV infection, we identified microRNAs that are specifically enriched in memory CD8+ T cells comparing to naïve, effector or exhausted CD8+ T cells. MiR-150, a microRNA that has been shown to be important in multiple biological processes, was identified as a potential memory CD8+ T cell biased microRNA. Functional interrogation of the role of miR-150 demonstrated that absence of miR-150 in CD8+ T cells enhanced memory CD8+ T cell differentiation, by both increasing KLRG-CD127+ memory precursors population during effector phase and enhancing CD127+CXCR3+ central memory phenotype long-term. Overexpression of miR-150 in CD8+ T cells, in contrast, negatively regulated CD8+ T cell memory formation. Furthermore, we found that miR-150 KO CD8+ T cells had higher expression of c-Myb comparing to miR-150 WT CD8+ T cells, a result observed using both in vitro CD8+ T cell differentiation and following infection with LCMV-Armstrong in vivo. Gene expression analysis implicated a relationship between the level of c-Myb expression and CD8+ T cell memory formation through potential anti-apoptotic molecules such as bcl-2. Thus, our studies identify a key role for miR-150 in CD8+ T cell memory and implicate a mechanism of CD8+ T cell memory effect by this microRNA through regulation of c-Myb.