bluuebunny/crossref_metadata_2025 huggingface dataset:
Phenotype of CD39/CD73 expressed on T cells in a mouse model of IPEX syndrome
Authors: Liu Yuying , Armbrister Shabba A , Okeugo Beanna , Daniel Rhea C , Rhoads J Marc
Published: January 2023

The combination of CD39 and CD73 degrade ATP to adenosine that shifts ATP-driven pro-inflammatory immune cell activity toward an anti-inflammatory state. This immunological switch is a major mechanism for regulatory T cells (Tregs) to control inflammation. However, in Treg-deficiency-induced autoimmune pathologies, exemplified by IPEX syndrome (immune dysregulation, polyendocrinopathy, enteropathy, with X-linked inheritance, as modeled by the scurfy (SF) mouse), the expression of these molecules on T cells is unknown. We analyzed homeostatic markers CD39 and CD73 on T cells in spleen and peripheral blood by flow cytometry in SF males and their sibling WT male mice at d21. Among Foxp3+ Tregs in WT mice, ~68% cells had CD39 and 85% cells had CD73 expression. In the spleens of SF mice, the % of CD39+ CD4+ T cells was higher than the % in WT mice, while no difference was observed in the CD73+ CD4+ T cells. In addition, the data showed ~70% of NonTregs expressed CD39, 2-fold higher than both % of CD73+ NonTregs in SF, and % of CD39+ NonTregs in WT. Only ~10% of these CD39+ and CD73+ NonTregs had the CD25 expression. The proportion of CD8+ T cells in splenocytes and blood of SF mice were significantly higher than those in WT mice. Amongst CD8+ T cells, the % of CD39+ T cells was significantly increased, conversely, the % of CD73+ T cells was significantly decreased in SF compared to WT mice. We conclude that normally, CD73 is a more highly expressed Treg surface marker than CD39; however, in Treg-deficiency, there is a higher expression by CD4+ T and CD8+ T cells of CD39 than CD73; and CD39 and CD73 expression may not be due to cell activation. This study provides data to aid further discovery of potential biomarkers and therapeutic targets in Treg deficiency diseases.

Supported by grants from NIH/NIAID (R03AI153725)
EGR2 deletion displays similar and differential effects on immunological development and function in autoinflammation-prone B6/lpr and normal B6 mice
Authors: Dai Rujuan , Wang Zhuang , Heid Bettina , Eden Kristin , Reilly Christopher M , Ahmed S Ansar
Published: January 2023

Transcription factor early growth response protein 2 (EGR2) prevents the development of lupus-like autoimmune disease in C57BL/6 (B6) mice by negatively regulating T cell activation, inflammatory cytokine IFNγ and IL-17 production and by suppressing humoral responses. However, increased EGR2 expression has been observed in lupus and also other autoimmune disorders, which contradicts the autoimmune suppressive role of EGR2. Here, we derived conditional EGR2−/− B6/lpr and EGR2−/− B6 mice to investigate and compare the immune regulatory role of EGR2 in autoinflammation (B6/lpr) versus normal physiological (B6) conditions. Significantly, we found that in B6/lpr mice, but not in B6 mice, EGR2 deletion dramatically suppressed serum levels of anti-dsDNA autoantibodies, total IgG, IgG1, IgG2a, and IgM. We further demonstrated that while EGR2 deletion promoted germinal center B (GCB) cell development in both B6 and B6/lpr mice, it significantly reduced the differentiation of plasma and plasmablasts cells in B6/lpr mice. EGR2 deletion promoted splenomegaly and T cell activation in both B6 and B6/lpr mice, but it differentially regulated the profiles of immune cell subpopulations in the spleens of B6 and B6/lpr mice. Especially, EGR2 deletion dramatically suppressed the development of double negative T cells in B6/lpr mice. Moreover, we found that EGR2 had an opposite role in regulating IL-17 production in in vitro activated splenocytes from B6/lpr and B6 mice, although EGR2 had a similar suppressive role for IFNγ production. Together, our data strongly suggest that the immune and autoimmune regulatory roles of EGR2 are context dependent, and should not be generalized in normal physiology and different pathological conditions.

Supported by VMCVM-VCOM Center for One Heath Research Seed Grant Program
ABCB7 is required for B cell development, proliferation, and class switch recombination
Authors: Lehrke Michael Jonathan , Shapiro Michael J , Rajcula Matthew , Theodore Keith , Lee Hyun Se Kim , Shapiro Virginia Smith
Published: January 2023

Iron-sulfur (Fe-S) clusters are cofactors essential for the activity of numerous enzymes including DNA polymerases, helicases, and glycosylases. They are synthesized in the mitochondria as Fe-S intermediates and are exported to the cytoplasm for maturation by the mitochondrial transporter ABCB7. Here, we demonstrate that ABCB7 is required for bone marrow B cell development, proliferation, and class switch recombination, but is dispensable for peripheral B cell homeostasis in mice. Conditional deletion of ABCB7 using Mb1-cre resulted in a severe block in bone marrow B cell development at the pro-B cell stage. The loss of ABCB7 did not alter expression of transcription factors required for B cell specification or commitment. While increased intracellular iron was observed in ABCB7-deficient pro-B cells, this did not lead to increased cellular or mitochondrial reactive oxygen species, ferroptosis, or apoptosis. Interestingly, loss of ABCB7 led to replication-induced DNA damage in pro-B cells, independent of VDJ recombination, and these cells had evidence of slowed DNA replication. Interestingly, ABCB7 appeared to be dispensable for splenic B cell homeostasis, as CD23-cre ABCB7 cKO mice had normal splenic B cell populations. However, stimulated ABCB7-deficient splenic B cells from CD23-cre ABCB7 cKO mice had a striking loss of proliferation and a defect in class switching. Thus, ABCB7 is essential for early B cell development, proliferation, and class switch recombination.

Supported by 1R21 AI157328-01 to V.S.S., T32AI007425 to M.J.L.
Regulation of metabolism by protein modifications in autoimmunity
Authors: Yang Mei-Ling , James Eddie A , Overbergh Lut , Herold Kevan C , Mamula Mark J
Published: January 2023

In type 1 diabetes (T1D), inflammation and oxidative stress in pancreatic islets amplifies various post-translational modifications (PTMs) of self-proteins. The loss of immune tolerance to islet PTMs triggers an autoreactive T cell response and contributes to the destruction of insulin-producing beta cells in T1D. The major function of beta cells is to secret insulin in response to glucose uptake in order to maintain glucose homeostasis. In this study, we identify autoantibodies against to beta subunit of prolyl-4-hydroxylase (P4Hb; native and carbonylated form) and glucokinase (GK; native and citrullinated form) in both human T1D and murine models. Glucokinase serves as a glucose sensor to initiate glycolysis and insulin signaling in beta cells. P4Hb is required for the accurate folding of insulin. By mass spectrometry, six carbonyl residues and eleven citrulline residues were mapped in oxidative rhP4Hb and PAD-treated rhGK, respectively. Of interest, autoreactive CD4+ T cells to citrullinated GK epitopes are present in the circulation of T1D patients. In regards to glucose metabolism, the carbonylated-P4Hb is amplified in stressed human islets coincident with decreased glucose-stimulated insulin secretion and disturb (increase) proinsulin to insulin ratios. The citrullination alters GK biologic activity (Km) and suppresses glucose-stimulated insulin secretion. Moreover, PAD2/4 inhibitors can partially restore IFNg +IL-1b suppressed glucose stimulated insulin secretion in INS-1E beta cells. Our studies implicate the crucial enzymes, glucokinase and P4Hb, as the biomarkers, providing new insights into creating autoantigens and define the impact of metabolic PTMs on the aberrant beta cell functions of T1D.

This research was supported by the Juvenile Diabetes Research Foundation (Innovative Grant to M.J.M., 2-SRA-2018-551-S-B to E.A.J.), and National Institutes of Health (DK104205-01 to K.H. and M.J.M.).
Blocking the Migration of Circulatory T cells Mitigates the Accumulation of Allergen-induced Tissue-Resident Memory T cells
Authors: Sethi Gurupreet Singh , Croft Michael
Published: January 2023

Targeting lymphocyte egress from lymph nodes with S1PR agonists has been approved for multiple sclerosis and colitis, but their potential in asthma is unclear. Tissue-resident memory CD4 T cells (Trm) are thought to be a major contributor to asthma relapse but the role of circulatory T cells is not fully understood. We used a murine model of house dust mite allergen-driven asthma exacerbation and transient i.v. injection of antibody and the Trm markers CD44hi CD62Llo and CD69+ to distinguish lung-localized CD4 T cells from circulatory T cells. Trm were retained in lungs after the initial allergen encounter and expanded upon repeated allergen exposures. To determine the role of circulatory T cells, mice were given FTY720, a S1PR agonist. Blocking the primary migration of circulatory T cells alleviated the formation and accumulation of lung Trm. Even so, subsequent allergen challenges still resulted in strong Trm accumulation. This was blocked if FTY720 was also given during allergen re-exposure showing that new allergen-driven circulatory T cells formed this lung Trm pool at times well after the initial sensitization. In contrast, once lung Trm cells developed at high frequency, they no longer relied on circulatory T cells with their expansion and further survival of the Trm pool being unaffected by FTY720. Overall, this suggests that targeting circulatory T cells during allergen insults might be employed to inhibit the accumulation of lung Trm in individuals with recent-onset asthma and may promote long-lasting airway tolerance. However, in individuals that have had asthma for some time and developed a stable population of lung Trm, targeting circulatory T cells may be ineffective in reducing the pathogenic T cell pool that maintains disease
Systemic bile acids potentiate airway hyperresponsiveness and modulate Th17 cell function in obesity-associated asthma
Authors: Manni Michelle L , Heinrich Victoria A , Uvalle Crystal E , Manuel Allison , Ellgass Madeline R , Mullett Steven J , Normann Marigny C , Koziel Corrine , Fajt Merritt L , Wenzel Sally E , Holguin Fernando , Freeman Bruce A , Wendell Stacy L
Published: January 2023

Obesity-associated asthma is phenotypically characterized by low grade inflammation and resistance to standard therapies. Like many other asthma endotypes, the molecular mechanisms contributing to obesity-associated asthma are diverse and ambiguous. A panoply of metabolites that act as signaling mediators are altered by obesity and may also promote lung dysfunction and a pro-asthma phenotype. Recent metabolomics studies indicate that bile acid profiles are altered in individuals with asthma, thus these metabolites were investigated in a murine model of obesity-associated asthma and clinically. In obese mice with allergic airway disease (AAD), the levels of β-muricholic acid (βMCA) and tauro-β-muricholic (tβMCA) were significantly increased in serum and positively correlated with impaired lung function. Two conjugated bile acids, glycocholic acid (GCA) and glycoursodeoxycholic acid (GUDCA), were elevated in the plasma of high body mass index (BMI) individuals and correlated with both BMI and airway hyperreactivity as measured by FEV1, forced expiratory volume in one second. In vitro differentiated T helper (Th) cells were treated with tβMCA and βMCA, which resulted in changes in T cell metabolism that were specific to Th17 cells. In addition, tβMCA treatment increased IL-17 production from Th17 cells upon stimulation. Finally, the anti-inflammatory and metabolic regulatory activities of nitro-oleic acid (NO2-OA) were evaluated in the murine model of obesity-associated asthma. NO2-OA reduced levels of βMCA and tβMCA, with a concomitant reduction in tissue resistance and elastance, providing a potential approach to improving the current standard of care for obese individuals with asthma.

Supported by R01HL146445 (MLM), S10OD023402 (SGW), R61HL157069 (SGW, BAF, FH), R01HL132550 (BAF)
Androgen receptor signaling decreases glutaminolysis in Th17 cells to reduce airway inflammation in asthma
Authors: Chowdhury Nowrin U , Cephus Jacqueline-Yvonne , Voss Kelsey , Gandhi Vivek D , Peebles Ray S , Rathmell Jeffrey C , Newcomb Dawn C
Published: January 2023

Women have higher rates of severe asthma compared to men. Severe asthma has increased Th2-mediated eosinophilic and/or Th17-mediated neutrophilic airway inflammation. We showed androgens, signaling through the androgen receptor (AR), decreased Th2- and Th17-mediated airway inflammation, and that Th17 and Th2 rely on glutaminolysis during allergic airway inflammation. Therefore, we hypothesized that AR signaling decreases glutaminolysis in Th2 and Th17 cells, resulting in decreased airway inflammation. Th2 and Th17 were differentiated in vitro from wild-type male and ArTfm male mice, with a nonfunctional AR. Mitochondrial metabolism and glycolysis were determined by measuring oxygen consumption rate (OCR), reactive oxygen species (ROS), and the extracellular acidification rate (ECAR). In Th17 cells, AR signaling decreased OCR, while increasing mitochondrial ROS, suggesting decreased glutaminolysis. Further, both 5α-DHT (0.1nM) and CB839 (a glutaminase inhibitor, 0.5μM) reduced OCR in Th17 cells from male mice. AR signaling had no effect on ECAR in Th17 cells or OCR and ECAR in Th2 cells. To induce eosinophilic and neutrophilic airway inflammation, house dust mite (HDM) was intranasally administered to Arfloxed CD4-Cre and Arfloxed male mice and markers of glycolysis and glutaminolysis were determined by flow cytometry. AR signaling decreased airway neutrophils, the number of Th17 lung cells, and GLUD1, a glutaminolysis marker, expression in T cells, but had no effect on eosinophils or Th2 cells. Combined, these data showed that AR signaling decreased glutaminolysis in Th17, but not Th2, cells. Understanding these pathways may provide new potential therapeutic targets in women with difficult-to-treat asthma.

Supported by grants from NIH (R01 HL12254, R01 HL136664, T32 GM007347)
Mouse model of late-onset neutrophilic asthma reveals an epithelium-lymphocyte-neutrophil communication circuit underlying destructive airway neutrophilia.
Authors: Shenoy Anukul T , Korkmaz Filiz , De Ana Carolina Lyon , Odom Christine , Barker Kimberly , Goltry Wesley , Ramanujan Aditya , Martin Ian , Shao Feng-Zhi , Jones Matthew , Quinton Lee , Fine Alan , Chen Felicia , Belkina Anna , Mizgerd Joseph
Published: January 2023

While childhood-onset asthma is extensively modeled in labs and includes TH2 cell driven-, steroid-responsive, eosinophilic etiology, little is understood about late-onset asthma which presents as severe, steroid-resistant, and destructive neutrophilic disease with poor outcomes. Lack of appropriate disease models widens this knowledge gap leading to fewer therapies and poorer quality of life for neutrophilic asthma patients. Here, we discover that while conventional models of allergic asthma (relying on naïve sensitized mice acutely challenged with aerosolized ovalbumin) induces eosinophilic asthma, transient and recurrent aeroallergenic exposure over extended durations (as would occur in humans progressing into adulthood) reprograms the lung myeloid and lymphoid landscape to instigate neutrophilic asthma. Mice with such lung history harbor diverse clusters of lung-resident CD4+ TRM cells including a novel RORγt-negative-IL17A+ TH17 subset; the latter detectable in asthmatic adult human lungs. On allergen reencounter, these RORγt-negative TH17 cells, rapidly secrete IL-17A which signals into lung epithelial and stromal cells to express CXCL5 and induce neutrophilic asthma including peribronchial neutrophilia, vascular leakage and lung damage. We find that lung epithelial antigen presentation is crucial to regulate disease severity by skewing CD4+ TRM phenotypes in asthmatic lungs. Specifically, epithelial MHC-II supports TH1 TRM cells and IFNγ secretion; the latter being a potent suppressor of IL-17A-induced CXCL5 and airway neutrophilia. Thus, using a relevant model of the disease, we identify an ‘epithelium-lymphocyte-neutrophil’ circuitry as a critical regulator of late-onset neutrophilic asthma.

Supported by NIH grants including HL147461 to F.T.K., HL142199 to K.A.B., HL136725 to M.R.J., GM120060 and HL111449 to L.J.Q., AI115053, HL135756, and HL137081 to J.P.M. and T32 HL007035 for support of trainees.
Periopathogens Modulate Inflammation by Repressing miR-142-3p and Augmenting miR-155/NFκB axis
Authors: Valverde Araceli Maria , Schaller Samantha , Brandini Daniela Atili , Brambila Maria F , Martinez Gloria , Chapa Gabriela , Li Wei , Wu Christine , Rahat Rani , Siddiqui Hasan , Nares Salvador , Naqvi Afsar
Published: January 2023

MicroRNA (miR) play a critical role in the pathogenesis of immune-mediated diseases. We hypothesize that periopathogens (P. gingivalis [Pg] and A. actinomycemtemcomitans [Aa]) impair host immune response by perturbing miR expression. Periopathogen burden, cytokine and miR expression was quantified by qPCR in healthy and inflamed human gingival biopsies. Human primary CD14+ monocytes were differentiated to M1 or M2 macrophages (MΦ) and challenged with live Pg or Aa to monitor expression profiles of pro- and anti-inflammatory miRs by qPCR. Supernatant levels of cytokines were assessed by multiplex bead assay while, protein levels were quantified by western blot. Murine gingival miRNA and mRNA profiles were validated during disease progression in a liguture-induced periodontitis model and bone loss measured by microCT. Higher periopathogen burden and inflammatory cytokine expression was observed in inflamed gingiva with a higher expression of pro-inflammatory miR-155 and reduced levels of anti-inflammatory miR-142-3p. This corroborates with NFκB activation and downregulation of PU.1 suggesting a distinct role of a miR and transcription factor (TF) axis. miR-155-mediated downregulation of PU.1, a TF of miR-142, suppressed transcription of miR-142, thereby alleviating silencing of IL-6 mediated by miR-142-3p. Our murine ligature model showed progressive bone loss in the presence of Pg and higher expression of cytokine profiles. Local delivery of miR-142-3p showed reduction in IL-6. Periopathogen-mediated activation of miR-155/NFκB axis augments inflammatory signaling by decreasing levels of PU.1 and expression of its target miR-142. This mechanism may be critical in the pathogenesis of periodontal disease.

Supported by R01 DE027980, R21 DE026259, R03 DE027147
Treg donation of sIL-6R to promote intestinal integrity and repair
Authors: Klatt Emily Eileen , Sagstetter Mary , Bamidele Adebowale , Gonzalez Michelle M , Ramos Guilherme Piovezani , Gibbons Hunter R , Wixom Alexander , Hamdan Feda H , Neto Manuel Braga , Faubion William A
Published: January 2023

IBD affects roughly 1.6 million US adults, with its prevalence increasing since the late 1990s. Characterized by unchecked intestinal inflammation, multiple proinflammatory cytokines including IL6 have been implicated as critical factors in this pathology. It has been established that EZH2 is a gene involved in the production of specific histone methyltransferase enzymes, as well as in the differentiation of Treg cells. The association between the Treg master transcription factor, Foxp3 and EZH2 results in the suppressive function of Tregs, with our previous work revealing impairment of this interaction under the influence of IL6 signaling in an inflamed intestinal setting. We investigated this further in a murine preventative T cell transfer model of colitis that involved injecting donor Treg cells that lacked IL6Ra, into immunocompromised mice. Histological scoring of the mice receiving the IL6Rko Tregs revealed a resolved inflammatory milieu and flow cytometric analysis of cells isolated from the spleens and MLNs showed increased Foxp3+ expression when compared to mice that received WT Tregs. Foxp3IL6Rako mice were subjected to a DSS model of colitis to further characterize the absence of the IL6R on Treg function in the intestinal milieu. Exacerbated signs of colitis were observed through histology and colon shortening of the conditional knockout mice when compared to their WT counterparts. We postulate that Tregs could be acting as a donor of sILR to promote intestinal integrity and healing. Further experiments will be conducted to define a role for the IL6R in the development of Foxp3+ cells, along with outlining the instrumental process of sIL6R donation in the intestinal milieu.

Supported by Mayo Clinic Graduate School for Biomedical Sciences
Gut microbiota modulates the development of murine Kawasaki disease vasculitis
Authors: Jena Prasant K , Wakita Daiko , Gomez Angela C , Carvalho Thacyana T , Narayanan Meena , Lee Youngho , Cani Patrice D , de Vos Willem M , Devkota Suzanne , Underhill David M , Chen Shuang , Shimada Kenichi , Crother Timothy R , Arditi Moshe , Rivas Magali Noval
Published: January 2023

Alterations of gut microbiota composition and function influence the development of cardiovascular disease, but the role of these aberrations remains poorly understood in Kawasaki Disease (KD), an acute pediatric vasculitis that targets coronary arteries. Using a murine model of KD vasculitis, we found that germ-free and antibiotic-treated mice displayed reduced inflammation and cardiovascular lesions. Development of KD vasculitis in mice was associated with alterations in the composition of the intestinal microbiota, specifically decreased abundance of Akkermansia muciniphila and Faecalibacterium prausnitzii. Supplementation with live or pasteurized A. muciniphila or F. prausnitzii attenuated the severity of KD cardiovascular inflammation. Oral administration of the short-chain fatty acids acetate or butyrate, which are produced by A. muciniphila and F. prausnitzii respectively, or treatment with Amuc_1100, a purified protein isolated from the outer membrane of A. muciniphila, ameliorated the severity of KD cardiovascular lesions. Reduced development of KD vasculitis in mice receiving either pasteurized A. muciniphila or Amuc_1100 was associated with improvements of gut barrier function. These results reveal an underappreciated gut microbiota-cardiovascular inflammation axis during murine KD vasculitis. Our findings may stimulate the development of novel diagnostic tools and therapeutic strategies that modulate the intestinal microbiota composition and function for KD patients.

Research is supported by the NIH grants R01AI072726 to M.A. and R01HL139766 to M.N.R.
A human ectopic-lymphoid-follicle-on-a-chip for testing vaccines and adjuvants
Authors: Zhai Yunhao , Goyal Girija , Prabhala Pranav , Patil Aditya , Kim Min Sun , Junaid Abidemi , Ku Min Wen , Mahajan Gautam , Bausk Bruce , Gilboa Tal , Lazarovits Roey , Sharma Sanjay , Cohen Limor , Ferrante Thomas , Walt David R , Ingber Donald E
Published: January 2023

New vaccine candidates evaluated in animals can lead to unpredicted toxicities or poor efficacy in human clinical trials due to species-specific differences in immune responses. To address this problem, we developed the human ectopic lymphoid follicle (LF) chip containing primary human immune cells, including autologous blood B and T lymphocytes and antigen presenting cells, cultured in a three-dimensional extracellular matrix (ECM) gel within an organ-on-a-chip microfluidic device. Superfusion via a parallel channel separated by a microporous membrane is required for LF formation and prevents lymphocyte autoactivation. These germinal center-like LFs contain B cells expressing activation-induced cytidine deaminase and exhibit plasma cell differentiation upon activation. Increased basal lymphocyte proliferation is observed in the human LF chip as compared to conventional 2D cultures lacking ECM or perfusion. The human LF chips demonstrated improved antibody responses to split virion influenza vaccination compared to conventional planar 2D cultures, which were enhanced by the addition of a squalene-in-water emulsion (SWE) adjuvant, and this was accompanied by increases in LF size and number. To extend this work, we modelled intramuscular vaccination by co-culturing monocytes with muscle cells in the presence of different vaccines and adjuvants. We found that presence of muscle cells can promote cytokine production, antigen uptake and monocyte activation. Seeding these monocytes into the LF chips with autologous T and B cells could promote cytokine production and LF formation, which extends the LF chips usage for testing intramuscular vaccination.

Supported by grants from the Defense Advanced Research Projects Agency under Cooperative Agreement Number W911NF-12-2-0036, the National Institutes of Health grant UG3HL141797, Bill and Melinda Gates Foundation and the Wyss Institute for Biologically Inspired Engineering.
Anti-CD3 Bi-Fab crosslinking of T cells enables their fratricide by activation induced cell death (AICD) and cytotoxicity
Authors: Pages Diana Gil , Nelson Alfreda , Wang Liangyu , White Tommi , Schrum Adam , Cannon John
Published: January 2023

We showed in the past anti-CD3 Fabs can form stable bivalent non-covalent dimers we called Bi-Fabs, which appear stimulatory to T cells according to the up-regulation of the early activation marker CD69. Now we show that incubation of naïve T cells with anti-CD3 Bi-Fabs in the presence of proper co-stimulation results in their robust stimulation in comparison to mAb and F(ab’)2 species with same specificity. Nonetheless, anti-CD3 Bi-Fab treatment leads to abortive T cell proliferation with impaired accumulation of divided T cells. After detailed studies to dissect the mechanisms of T cell death enabled by Bi-Fab molecules in comparison with anti-CD3 mAb and F(ab′)2 recognizing the same epitope, we observed that anti-CD3 Bi-Fabs promote T cell fratricidal killing via activation induced cell death (AICD) and CD8 cytolytic activity (CTL) more efficiently than the mAb and F(ab′)2 counterparts. While low resolution structural studies indicate that Bi-Fab and F(ab’)2 adopt similar shapes, molecular dynamic simulation shows that F(ab’)2 is a highly flexible molecule, whereas Bi-Fabs are more rigid and of linear conformation, sampling a smaller range of Fab-Fab angles than F(ab’)2. We hypothesize anti-CD3 Bi-Fab is superior as T cell killer stimulatory modality over mAb and F(ab’)2 of equal specificity due to Bi-Fab’s greater rigidity when crosslinking in trans TCR/CD3 molecules located in neighboring T cells. These findings may be applicable into the design of Ig-based immunotherapeutic drugs targeting the TCR/CD3 complex.

Supported by MU start-up funds
CD16 and NKG2D co-clustering facilitates quality of primary NK cell responses
Authors: Sykulev Yuri , Tsygankova Oxana , Anikeeva Nadia , Maskalenko Nicholas , Campbell Kerry
Published: January 2023

Cooperation between CD16 and NKG2D receptors has been demonstrated both in animal and NK cell line models leading to an enhanced cytotoxicity and tumor elimination. However, the mechanisms of the receptor cooperation are not known, and whether the receptors form co-clusters has not been determined either. We exposed human primary polyclonal NK cells with CD16 158V/V-genotype to planar lipid bilayers (PLB) presenting Herceptin and anti-NKG2D Ab in the presence of ICAM-1. NK cells interacting with Herceptin-containing PLBs + ICAM-1 formed mostly unstable synapse, i.e., kinapses, while stimulation with anti-NKG2D alone or Herceptin+anti-NKG2D bilayers + ICAM-1 led to formation of mature synapses showing complete segregation of the receptors from ICAM-1, increased synapse stability, and efficient NK cell cytotoxicity. The cells exposed to PLBs with both anti-NKG2D and Herceptin ligands were mostly confined within the initial contact area while the cells responding to Herceptin alone were significantly more motile. Regardless of stimulatory ligands displayed on PLB, the frequency of adhered cells was very similar, but the degranulation was observed within 40% of kinapses and 80% of mature synapses, suggesting that degranulation efficiency is linked to synapse stability. Importantly, more efficient degranulation was associated with rapid and sustained Ca2+ signaling as opposed to oscillating Ca2+ signaling observed for NK cells showing less efficient degranulation. Our data demonstrated that Herceptin alone functions as a weak agonist, while co-activation of CD16 and NKG2D results in rapid and sustained Ca2+ signaling that mediates stable synaptic interface and efficient cytolytic activity by human NK cells.

Supported by Grant from NIH U01AI148117
Gene therapy increases MHC class-I and T cell Infiltration to promote anti-tumor immune response in 4T1 breast cancer model
Authors: Shi Guilan , Heller Richard
Published: January 2023

Background

Immune checkpoint inhibition (ICI) has led to impressive gains in survival for many patients. However, many cancer patients treated with ICI experience intrinsic or acquired resistance which directly affects efficacy. Decreased levels of MHC class-I molecules on cancer cells and tumor infiltrating lymphocytes (TILs) have been associated with poor clinical outcomes.

Methods

Treatment of breast cancer cell lines with a plasmid encoding IL-12 (pIL-12) and vector pUMVC3 were analyzed using flow cytometry, western blotting and then validated functionally. The 4T1 breast cancer animal model was used to study T cell infiltration into the tumor as well as response following local delivery of pIL-12 with electrotransfer.

Results

Enhanced MHC class-I expression in 4T1 tumor cells, and elevated CD8+ T-cell infiltration into 4T1 tumor tissue were observed after pIL-12 gene electrotransfer. Furthermore, increased capacity to lyse 4T1 cells was observed with cultured cytotoxic T lymphocytes (CTLs) from long-term surviving mice compared to control group. A direct translation was observed in tumor-bearing mice, including attenuating tumor growth and improved tumor-free survival in mice. An immune memory response which protected against rechallenge was induced also.

Conclusion

Our findings suggest that the capacity of an induced endogenous immune response with pIL12 via the recruitment of CD8+ T cells and increased MHC class-I expression in tumor cells results in attenuating the resistance of anti-PD1 and leads to eradication of established, aggressive tumors and generates systemic tumor-specific protective immunity. Combinations of anti-PD-1 with pIL-12 gene therapy may be an effective future therapeutic strategy.

Supported by NCI R01 CA186730
BET inhibitors synergize with anti-PD1 by rescuing TCF1+ progenitor exhausted T cells in Acute Myeloid Leukemia
Authors: Romine Kyle Alexander , Cho Hyun-jun , Kosaka Yoko , Flynn Patrick , Byrd Kaelan H , Coy Jesse L , Newman Matthew T , Scott Jaime , Loo Christopher , Lind Evan F
Published: January 2023

Acute Myeloid Leukemia (AML) is the most common adult leukemia and has a 5-year survival of under 30%. AML is caused by uncontrolled proliferation of myeloid cells resulting from a combination of mutations that affect proliferation, differentiation and epigenetic state. For this reason, drugs targeting epigenetic modifications are being studied in AML. AML cells avoid immune recognition though inhibiting the function of multiple cell types, especially T cells and therefore immune checkpoint blockade presents a promising therapy; however, clinical trials to date have shown very modest efficacy. T cell exhaustion has been shown to be a regulated process involving transcriptional and epigenetic changes. BET proteins, which are chromatin readers, have been implicated in maintaining this exhaustion state. In these studies, we investigated the effects of the BET inhibitor (BETi) JQ1 on T cell exhaustion and checkpoint responsiveness in a murine model of AML and AML patient samples. The AML mouse model bears FLT3-ITD and deletion of TET2 restricted to myeloid lineages and is resistant to anti-PD1 therapy. This mouse model of AML expanded terminally exhausted T cells and impaired proliferative capacity after TCR stimulation. Ex vivo treatment with BETi and anti-PD1 reverses CD8+ T cell exhaustion via rescue of proliferative dysfunction and expansion of more functional precursor exhausted T cells in patient samples and AML splenocytes. Finally, we show that BETi rescues anti-PD1 resistance in vivo and reduces tumor burden in multiple organ sites and enriches CD8+ T cells in the bone marrow. In total, we demonstrate that combining BETi and anti-PD1 therapy in the treatment of AML is a rational strategy to overcome anti-PD1 resistance.
Dissecting molecular mechanisms underlying T cell co-potentiation when targeting the TCR/CD3 complex with anti-CD3 Fab fragments
Authors: Huynh Hien T , Puthanapura Chandrashekara Mahadevappa , Nelson Alfreda D , Becher Laura R E , Abergel Megan , Hu Shuang , Alarcon Balbino , Schrum Adam , Pages Diana Gil
Published: January 2023

A cognate antigen engagement of TCR induces a conformational change in the CD3 complex (CD3Δc) that is required for productive T cell activation. CD3Δc fails when TCRs interact with poorly immunogenic antigens that do not active T cells. Anti-CD3 monovalent Fab fragments bound to the CD3 complex mimic CD3Δc, producing T cell “co-potentiation” when associated with TCRs bound to weak antigens that enhances T cell activation, neither stimulating non-engaged T cells, nor interrupting T cell responses to strong antigens. Previously, we showed T cell co-potentiation can be exploited as a novel immunotherapeutic principle by reducing melanoma burden in B6 mice treated with anti-CD3 Fabs. TCR signaling initiation requires CD3Δc for the exposure of a conserved sequence in the cytosolic domain of CD3ɛ rich in prolines (PRS) that enables the recruitment of adaptor protein Nck. The mutation of cysteines in the CXXC motif at the extracellular domain of CD3ɛ inhibits the conformational change across the plasma membrane that results on PRS accessibility. Here, using two strains of B6 mice that express mutations in either the CXXC or the PRS motifs in CD3ɛ, we study the potential contribution of each motif in T cell co-potentiation. Our results from these studies favor the involvement of the CXXC and PRS motifs in the molecular mechanism supporting the co-potentiation of T cell activation by anti-CD3 Fab fragments.

Supported by MU Start-up funds and NIH grants: NCI, U01 CA244314; NIAID, R01 AI097187
Tumor Adjacent IL2 Cytokine Factories Eradicates Ovarian Cancer in Mice Through Cytotoxic T-cell Activation with Safe and Predictable Dosing in Non-human Primates
Authors: Carmona Guillaume , Nash Amanda , Jarvis Maria , Newman Ryan , Schladenhauffen Jake , Aghlara-Fotovat Samira , Hernandez Andrea , Nouraein Shirin , Mukherjee Sudip , Hecht Andrew , Cui Yufei , Lee Jared , Rios Peter , Zhang David , Xu Chunyu , Sheth Rahul Sheth , Peng Weiyi , Oberholzer Jose , Igoshin Oleg , Jazaeri Amir , Veiseh Omid
Published: January 2023

Recombinant IL-2 has been approved for the treatment of melanoma and renal cancer. However, IL-2 therapy is limited by its short half-life in circulation and the associated toxicities that emerge as a result of high systemic exposure. To overcome these limitations, we developed a localized cytokine LOCOcyteTM platform composed of allogeneic and engineered cells which produce potent immune effector molecules for local delivery with temporal regulation of activity. Administration of AVB-001 in mice and non-human primates demonstrated that IL2 local concentration (IP space) was 100x higher than the systemic concentration. Additional studies in mice demonstrated dose-dependent levels of IL2 in the intraperitoneal cavity in mice. Treatment of peritoneal tumors in mice using a single administration of AVB-001 provided sustained eradication of peritoneal tumors in an ovarian cancer mouse model. Our data in mice confirmed local increases in activation (CD25+CD8+) (8.6 fold) and proliferation (Ki67+CD8+)(3.4 fold) of cytotoxic T cells within the IP space in comparison to sham mice. Finally, a single administration of AVB-001 led to therapeutic levels of IL2 in the IP space in NHP and produced local and systemic T cell biomarker profiles that predict efficacy, AVB-001 was well tolerated by all animals with no abnormal clinical observations during the study. Our findings demonstrate that the LOCOcyteTM platform has a dose-dependent PK/PD effect, is safe and efficacious in animal models and enables itself to deliver a diverse set of cytokines alone or in combination which is presently explored. Combined, our pre-clinical results provide a rationale for testing AVB-001 in ovarian cancer patients.
Novel low-avidity glypican-3 specific CARTs resist exhaustion and mediate durable antitumor effects against hepatocellular carcinoma
Authors: He Yukai , Caraballo-Galva Leidy D , Jiang Xiaotao , Hussein Mohamed S , Zhang Huajun , Mao Rui , Brody Pierce , Peng Yibing , He Aiwu Ruth , Kehinde-Ige Mercy , Sadek Ramses , Qiu Xiangguo , Shi Huidong
Published: January 2023

Background & Aims

Chimeric antigen receptor engineered T cells (CARTs) for hepatocellular carcinoma (HCC) and other solid tumors are not as effective as they are for blood cancers. CARTs may lose function inside tumors due to persistent antigen engagement. The aims of this study are to develop low-affinity monoclonal antibodies (mAb) and low-avidity CARTs for HCC and to test the hypothesis that low-avidity CARTs can resist exhaustion and maintain functions in solid tumors, generating durable antitumor effects.

Methods and results

New human glypican-3 (hGPC3) mAbs were developed from immunized mice. We obtained 3 hGPC3-specific mAbs that stained HCC tumors, but not the adjacent normal liver tissues. One of them, 8F8, bound an epitope close to that of GC33, the frequently used high-affinity mAb, but with ~17 fold lower affinity. We then compared the 8F8 CARTs to GC33 CARTs for their in vitro function and in vivo antitumor effects. In vitro, low-avidity 8F8 CARTs killed both hGPC3high and hGPC3low HCC tumor cells to the same extent as high-avidity GC33 CARTs. 8F8 CARTs expanded and persisted to a greater extent than GC33 CARTs, resulting in durable responses against HCC xenografts. Importantly, compared to GC33 CARTs, there were 5 folds more of 8F8-BBz CARTs in the tumor mass for a longer period of time. Remarkably, the tumor infiltrating 8F8 CARTs were less exhausted and apoptotic, and more functional than GC33 CARTs.

Conclusion

The novel low-avidity 8F8-BBz CART resists exhaustion and apoptosis inside tumor lesions, demonstrating a greater therapeutic potential than high-avidity CARTs.
Development of swine immune reagents for analysis of immune correlates for vaccines, infection, and in biomedical research
Authors: Gourapura Renukaradhya J , Loving Crystal , Kenney Scott , LaBresh Joanna , Patil Veerupaxagouda , Byrne Kristen A , Manirarora Jean , Walker Kristen , Jovan Aquino , Chick Shannon , Dai Chai , Hailstock Taylor , Lunney Joan K
Published: January 2023

The goal of the USDA-NIFA Swine Immune Toolkit Initiative has been to generate priority immune reagents based on inputs from veterinary immunology researchers worldwide, and pipeline them for marketing. So far in our efforts we express soluble proteins in yeast system and then produce panels of monoclonal antibodies (mAbs) using collaborations with commercial partners for protein expression and mAb production. So far we generated several new panels of mAbs reactive to porcine IL-6, IL-13, IFN-γ, IL-17A, IL-28B, CXCL10, and BAFF and screened their reactivity in multiple immune assays. Reactivity tests of labeled anti-IL-6 and anti-IL-13 mAbs for intracellular staining of porcine immune cells using flow cytometry assay are in progress. Our results have confirmed the reactivity of porcine IL-17A, IFN-γ and CXCL10 mAbs. A sensitive sandwich ELISA is now available for IL-17A, IL-13 and CXCL10; other targets are being screened for best mAb pairs for ELISA. Planning has been initiated for the generation of IL-5 and IL-21 mAbs; and use of SLA-I & -II tetramers to identify swine CD4 and CD8 T cells specific for influenza virus peptides. For each target, our goal is to provide the veterinary community with new commercial reagents and standardized assay techniques for their research efforts. Tools and reagents generated by this project will undoubtedly advance our understanding of swine immune responses to disease, vaccine and biomedical research efforts.

Supported by USDA-NIFA AFRI grant # 2019-67015-29815
The SARS-CoV-2 encoded ORF8 protein Stimulates Human Monocytes to Produce Pro-Inflammatory Cytokines
Authors: Ruan Gordon J , Wu Xiaosheng , Manske Michelle K , Nowakowski Kevin E , Abeykoon Jithma P , Tang Xingyi , Yu Yue , Witter Taylor L , Taupin Vanessa , Paludo Jonas , Ansell Stephen M , Badley Andrew D , Schellenberg Matthew J , Witzig Thomas E
Published: January 2023

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can result in a cytokine storm that is associated with poor outcomes in patients. The activation of a NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome following SARS-CoV-2 infection has been shown to play a major role in inflammatory immune responses. In this study, we show that open reading frame 8 (ORF8) protein is abundantly secreted as a glycoprotein in vitro. The glycosylated ORF8 stimulates human CD14+/CD16+ monocytes to cause upregulation of proinflammatory cytokine production and cell surface marker expression within 24 hours. The data suggests that stimulation of human monocytes by ORF8 is not receptor mediated. Rather, ORF8 is likely phagocytosed by human monocytes. ORF8 then stimulates the monocytes by independently binding to the NACHT and LRR domains of the NLRP3 protein. Pharmacologic inhibition of NLRP3 significantly diminished the production of pro-inflammatory cytokines from ORF8-stimulated human monocytes. Finally, this study shows that the ORF8 protein is also secreted in patients with newly diagnosed coronavirus disease 2019 (COVID-19). Levels of ORF8 in the blood of patients diagnosed with COVID-19 correlated with disease mortality and trajectory of disease. ORF8 stimulation of monocytes causes pro-inflammatory cytokine production that leads to the development of severe COVID-19.
Exploration of shared antibody motifs in Kawasaki Disease (KD) and COVID-19 related Multisystem Inflammatory Syndrome of Childhood (MIS-C)
Authors: Rahman Zehra , Hicar Mark
Published: January 2023

Amidst the COVID-19 pandemic, there have been several studies elucidating the immune responses of individuals who have been infected with the SARS-CoV-2 virus. Antibodies (Abs) from different individuals that share the same variable gene and are highly similar in their third complementarity determining regions (CDR3) are termed public clonotypes. Eleven such public clonotypes for SARS-CoV-2 related neutralizing Abs have been proposed.

MIS-C was initially confused with KD as both conditions predominantly affect children and can have cardiac manifestations. KD has been previously associated with common cold coronaviruses and a shared immune response has been proposed.

Here we compare previously published neutralizing Abs in SARS-CoV-2 to Abs of pediatric patients with KD, acute COVID-19 and MIS-C in order to identify shared antibody motifs between the two immune responses. Meme analysis of B cell plasmablast derived heavy chain sequences were compared to published Sars-CoV-2 Ab datasets. Shared CDR3 sequence motifs were shown in seven instances in acute COVID-19, two instances in MISC, and none were shown in KD that appeared beyond shared D segment usage. We then compared Abs from MIS-C and KD with published public clonotypes of SARS-COV-2. We identified two of these in the total B cell sequences from MISC and none in the KD total B cell sequences. Data suggests one proposed SARS-CoV-2 public clonotype, that relies on a mid-CDR3 YDSSG motif, is not specific.

This study further supports that KD and MISC are distinct conditions not related by similar antigen driven immune response, which has implications for challenging the current therapeutic approach.

Supported by The Wildermuth Foundation and the Variety Club of Buffalo
Simultaneously targeting hyperinflammation and hyperglycemia following respiratory viral infection
Authors: Wei Xiaoqin , Zhu Bibo , Sun Jie
Published: January 2023

Individuals with hyperglycemia are at great risk of developing exaggerated pulmonary inflammation following infection with respiratory viruses including SARS-CoV-2. Conversely, SARS-CoV-2 infection has been linked to the development of new onset of hyperglycemia and diabetes. Despite global vaccination efforts, SARS-CoV-2 infection continues to be a great public health challenge, and corticosteroids targeting hyper-inflammation remain the mainstay treatment for severe COVID-19. However, steroidal medicine arises the risk of hyperglycemia, limiting its usage. Here, we showed that a second-generation thiazolidines drug MSDC-0602K (MSDC), a mitochondrial pyruvate carrier inhibitor, simultaneously decreased excessive pulmonary inflammation and hyperglycemia in normal and obese hosts during influenza infection, as such, reducing host morbidity and mortality. scRNA-seq analysis revealed that MSDC treatment reduced lung macrophage inflammation, and myeloid-specific ablation of MPC2 led to dampened lung inflammation. Further, MSDC inhibited human lung macrophage inflammatory responses following SARS-CoV-2 infection, and dampened inflammation in lung autopsies of COVID-19 patients. Mechanistically, MSDC treatment destabilized HIF-1a protein, a transcription factor promoting inflammatory responses of lung macrophages, by repressing cellular Acetyl-CoA levels and HIF-1a acetylation. Our results suggest that MSDC has the great potential to treat severe COVID-19 or other respiratory viral infection, particularly in patients with underlying metabolic conditions, by simultaneously repressing hyper-cytokinemia and hyperglycemia.

Supported by NIH AG069264, NIH AI150599
Development of SARS-CoV-2 specific neutralizing recombinant monoclonal antibodies
Authors: Desai Pavitra , Karuppuchamy Thangaraj , Syed Nazia , Unnikrishnan Sarath , Mathivanan Raghul , Priya Nancy , Balasubramanian Rajesh , Bhattiprolu Krishna , Sundarraj Swathi , Sridharan Haripriya
Published: January 2023

Development of effective therapeutic strategies to combat COVID-19 relies on understanding the cellular and molecular mechanisms of infection. Antibodies specific to SARS-CoV-2 aid in accelerating research to characterize viral protein function and immune response to infection. An urgent need for SARS-CoV-2 specific antibodies arose, as several antibodies already available in the market cross-reacted with other members of the coronavirus family.

To address this need for specificity and urgency, we collaborated with a leading pharmaceutical company to develop antibodies specific to SARS-CoV-2. We generated a single chain fragment variable (scFv) library for SARS-CoV-2 using computational expansion of published antibody structures against SARS-CoV-1. This enabled us to make recombinant antibodies in record time of 3 months. These synthetic sequences were grafted onto both human and human-rabbit chimeric Fc enabling use of these antibodies in a multitude of immunoassays. Our antibodies were targeted against the SARS-CoV-2 spike protein; the receptor binding domain in the spike protein plays a vital role in viral entry into host cells and subsequent infection, making it an important target for neutralizing antibodies and antiviral treatments. Some of our antibodies demonstrate neutralizing capabilities, making them a valuable tool to study neutralizing antibody response to infection. These antibodies are specific to SARS-CoV-2 and do not cross-react with other coronaviruses.

The specificity and versatility of our antibodies enables scientists to better understand SARS-CoV-2 and potentially aid in developing therapeutic strategies against COVID-19 which continues to be important as newer variants of the virus emerge.