bluuebunny/crossref_metadata_2025 huggingface dataset:
A structural approach to the study of viral immune evasion mechanisms (TECH1P.850)
Authors: Sgourakis Nikolaos , Natarajan Kannan , Margulies David , Bax Ad
Published: January 2023

Mouse cytomegalovirus encodes immunoevasin proteins that interfere with MHC-I antigen presentation to CD8+ T cells and natural killer (NK) cells. Members of the m02-m16 family contribute to viral survival and infectivity. One member of this family, m04/gp34, binds MHC-I molecules in the endoplasmic reticulum and accompanies them to the cell surface, while the related m06 binds and directs MHC-I to endosomes. Biochemical evidence suggests that m04 counters MHC-retaining functions of other viral proteins. Despite many genetic and functional studies, the structural basis of immunoevasin specificities and functions is poorly understood, due largely to difficulty in cocrystallization of these proteins with their ligands. There is no identifiable amino acid homology of any member of this family to any protein structure available. To bypass this structural bottleneck, we have developed a new technology that combines sparse datasets recorded from Nuclear Magnetic Resonance spectroscopy with sophisticated Rosetta macromolecular modeling methods. We apply this approach to determine the structure of the m04 core domain and its interactions with the H2-Dd MHC-I molecule. m04 has a novel Ig-like fold, with long loops providing a flexible scaffold for MHC-I binding. This study provides insight into a key site on MHC-I “discovered” by the virus and helps us to understand the function of m04 with respect to T and NK cell MHC recognition.
Analysis of mass cytometry data with viSNE reveals phenotypic heterogeneity within human Tregs (TECH1P.854)
Authors: Konrad Mark , Simonds Erin , Bendall Sean , George Thaddeus , Delucchi Pam
Published: January 2023

Regulatory T (Treg) cells play a fundamental role in the development and maintenance of immunological tolerance. Tregs are characterized by constitutive expression of the transcription factor Foxp3, in addition to a surface phenotype of CD4+CD25hiCD127lo. While this pattern of expression has proven highly useful for studying Tregs, the use of additional markers should provide greater resolution of minor subsets within the Treg compartment. Moreover, identification of a concise surface marker profile will facilitate isolation of viable Tregs by FACS. Mass cytometry is a new platform that couples flow cytometry with mass spectrometry, and enables single-cell analysis of at least 30 parameters simultaneously without the requirement for compensation. To profile Treg phenotypes in human peripheral blood, we employed a panel of metal-conjugated antibodies, against both surface and intracellular targets. High-dimensional analysis with the viSNE algorithm, a cytometry analysis tool based on the t-distributed stochastic neighbor embedding algorithm, revealed heterogeneity within the Treg compartment with respect to expression of the markers CD39, CTLA-4 and HLA-DR. Furthermore, we found these markers to be co-expressed primarily in Tregs with the highest level of Foxp3 expression. We propose a gating strategy for identification of Tregs by mass cytometry that can be used to further phenotype human regulatory T cells.
Defining accuracy and sensitivity of T-cell receptor repertoire profiling for monitoring recurrent/persistent disease in patients with mature T-cell neoplasms (TECH1P.857)
Authors: Sherwood Anna , Robins Harlan , Fromm Jonathan , Greisman Harvey , Sabath Daniel , Chung Moon-Wook , Bertucci Caterina , Wilcox Cara , Emerson Ryan , Rieder Mark , Wood Brent , Wu David
Published: January 2023

Accurate and sensitive identification of recurrent or persistent disease in T-cell neoplasms is important for proper patient care. We developed a method that amplifies rearranged TCR CDR3 sequences and uses high-throughput sequencing (HTS) to sequence tens of thousands of chains simultaneously. Because the technology utilizes gDNA the frequency of sequenced CDR3 chains is representative of the relative frequency of each CDR3 sequence in the sample population of T cells. Thus these assays can describe both the breadth of the T-cell receptor repertoire and quantify individual clones; enabling tracking the presence and frequency of neoplastic clones. To demonstrate the potential of this technology, we first test the assay’s sensitivity and accuracy (1 lymphocyte in 1M total cells). Then, to show utility, we apply the technology to monitor persistent/recurrent disease in a set of mature T-cell lymphoma samples. To identify the neoplasm’s CDR3 chain, we sequence the TRB and TRG repertoire of 35 index samples. Then, to diagnose persistent disease, we sequence the TCR repertoire of follow-up samples and search for the neoplastic sequence. We then define persistent disease based on the sensitivity and accuracy of the assay. Concurrently, as part of standard clinical practice, a number of other methods were used to identify persistent disease. We compare these two methods to demonstrate that HTS technology is a viable alternative to traditional methods to detect and monitor lymphoma.
Intracellular signaling as measured by single cell network profiling (SCNP) is stable in longtitudinal samples of peripheral blood mononuclear cells (PBMC) from healthy donors (HD) across multiple immune cell subsets (TECH1P.839)
Authors: Ptacek Jason , Hawtin Rachael , Louie Brent , Evensen Erik , Cordeiro James , Mittleman Barbara , Atallah Michelle , Cesano Alessandra , Gregersen Peter , Bridges S.
Published: January 2023

Biomarkers of immune function from viable cells would be of high value for many clinical applications. The immune system responds rapidly to a variety of stimuli, so identifying immune measures that are stable, robust, and relevant to clinical features/outcomes can be challenging. SCNP is a multiparametric flow cytometry assay measuring induced changes in intracellular signaling, providing a functional measure of pathway capacity in multiple cell subsets without physical separation. To assess the longitudinal stability of signaling capacity, PBMCs from 11 healthy donors (7 female, age 29 - 60) were collected at 2 time points and characterized by SCNP of 32 nodes (combinations of a modulator and an intracellular signaling readout) in 21 cell subsets. Signaling stability was assessed for each node:subset combination, calculating the ratio of the signaling variability between time points to the mean signaling level. Of 258 node:subset combinations measured: 160 (62%) had a ratio <0.1; 227 (88%) were <0.2 (i.e. the variability between time points is <20% of the average signaling). Memory B cells had the greatest, though modest, variability in signaling between sample times (median ratio 0.15, range 0.04 - 0.43); naive CD4- T cells had the least variation (median ratio 0.066, range 0.03 - 0.13). This study serves as reference for biomarker development for immune monitoring, demonstrating that signaling capacity is a stable, robust phenotype over time and within an individual.
Self-education via Ly49 receptor engagement of donor and host iNKT cells following in utero hematopoietic cellular transplantation (TRAN1P.874)
Authors: Strong Beverly , Rotzler Tess , Wadhwani Ram , Turner Lucas , Shaaban Aimen
Published: January 2023

Invariant NKT (iNKT) cells recognize glycolipid-CD1d complexes via their TCR but also express inhibitory Ly49 receptors (iLy49s) specific for MHC I ligands. However, little is known regarding the importance of iLy49s in the self-recognition of MHC I by iNKT cells during development. We hypothesized that self-engagement of iLy49s during development alters the phenotype and function of self-responsive iNKT cells. We examined this hypothesis in prenatal Balb/c to B6 and B6 to Balb/c allogeneic chimeras that were established by in utero hematopoietic cellular transplantation (IUHCT) of E14 fetal liver light density cells into age-matched fetal recipients. After birth, we compared iLy49 expression and the functional response of iNKT cells in the allogeneic chimeras to age-matched controls. The levels of allospecific iLy49s on host iNKT and donor iNKT were significantly reduced in all organs tested suggesting developmental recognition of both donor and host MHC class I had occurred. In contrast, the expression of irrelevant receptors was not altered. Lastly, iNKT cells from chimeric mice that expressed allospecific iLy49s exhibited significantly increased production of IL-4 in response to TCR stimulation suggesting that a shift toward a regulatory phenotype had occurred. Future studies will directly evaluate the impact of self-recognition by iNKT cells in the developmental pathogenesis of autoimmunity, allergy and allospecific tolerance.
Lectin-like transcript-1 (LLT1) and exosomal PCNA inhibit NK cell effector function against prostate cancer cells (TUM2P.892)
Authors: Mathew Porunelloor , Horton Nathan , Bowen Kelly , Mathew Stephen
Published: January 2023

NK cell functions are regulated by inhibitory and activating receptors binding corresponding ligands on the surface of target cells, providing vital first line defenses against infections and cancer. In order to establish and grow, cancer cells must escape NK cell-mediated killing. By altering the expression of ligands for NK cell receptors (down-regulating activating ligands and up-regulating inhibitory ligands) cancer cells may evade NK mediated killing. Here we have examined the expression of LLT1 (Lectin-like transcript-1 or CLEC2D), a ligand for the NK cell inhibitory receptor NKRP1A (CD161) as well as proliferating cell nuclear antigen (PCNA), a ligand for NKp44 on surface of prostate cancer cell DU145. Expression of LLT1 on DU145 cell inhibited NK cell cytolytic activity though NKRP1A (CD161). Similarly, expression of exosomal PCNA by DU145 inhibited NK cell cytotoxicity through NKp44. The association of HLA I and PCNA on DU145 forms an inhibitory ligand for NKp44. Blocking the inhibitory signals by anti-LLT1 and anti-NKp44 monoclonal antibodies enhanced the killing of prostate cancer cells by NK cells. Thus tumor cells may use multiple pathways to inhibit NK effector function and combinatorial targeting of these pathways could lead to efficient elimination of cancer cells.
Biomarker: long term study of breast cancer specific CDR3s in peripheral blood (TUM2P.894)
Authors: Alleyne Jessica , Wang Chunlin , Yang Qunying , Byrne-Steele Miranda , Ng Enders , Kwong Ava , Shin Vivian , Lu Chiung-Mei , Han Jian
Published: January 2023

We have identified a group of T cell receptors, represented by their unique CDR3 sequences, that are closely associated with breast cancer. Multiplex PCR was used to amplify the T cell receptor beta (TCRβ) inclusively and semi-quantitatively; the amplicons were sequenced with the Illumina HiSeq platform. Here, we report a longitudinal study where one patient’s peripheral blood samples were evaluated for repertoire changes before and after the treatment. At regular intervals over four years, we collected five peripheral blood samples from the patient. The blood samples were sorted into Tc, Tr, Th, and NKT cell subsets, and the TCRβ genes were amplified and sequenced. We have also sequenced the TCRβ from the patient’s surgically removed breast cancer tissue. We have identified two kinds of biomarkers for breast cancer: public and private biomarkers. The public biomarkers are those CDR3s that are shared with other patients who were also diagnosed with breast cancer; the private biomarkers are expressed exclusively within a particular patient and are also found in the disease tissue. The study has shown that a group of CDR3s can be used as breast cancer-specific biomarkers because they are tightly associated with breast cancer patients, and their expression level can be quantitatively measured from peripheral blood. The discovery of this new class of cancer specific biomarkers will facilitate the development of new drugs and also improve the utility of existing therapies.
Natural killer (NK) cell high-mobility group box 1 (HMGB1) is required for anti-tumor function (TUM2P.895)
Authors: LI Guanqiao , Liang Xiaoyan , Kaus James , Basse Per , Zeh Herbert , Lotze Michael
Published: January 2023

HMGB1 is an evolutionarily ancient protein, serving as genome guardian, autophagy promoter and DAMP molecule, essential in immunity and cancer development. Its role in NK cell activitity(lysis, DC maturation and cytokine secretion) remains unknown. To study the role of HMGB1 in NK cell functionality, we generated transgenic NKH mice with targeted genomic ablation of HMGB1 in NK cells. HMGB1- NK cells are less responsive to IL-2/IL-18 in vivo and vitro(p=.0001). Consistent with a less mature phenotype(CD27+/CD11b-), they expand more effectively with Flt3 ligand(p=.03). Reserve capacities of OXPHOS and glycolysis decrease with HMGB1 deletion(p<.001). Activated NK cells secrete HMGB1 via vesicular exocytosis and up-regulate cognate receptors such as RAGE. This fails to occur in NKH model. Deletion of HMGB1 impairs NK ability to mature DCs. This can be rescued with addition of exogenous HMGB1. Serum IFN-γ levels and cytotoxicity are reduced in NKH mice receiving high dose IL-2(p<.001). Additionally, tumors engraft more readily in NKH mice(p <.0001), but grow at comparable rates with respect to WT, suggesting NK cells are crucial in early tumor surveillance, but not overall growth kinetics. Thus, accelerated tumor engraftment results in diminished survival in NKH cohorts (p <.05). In conclusion, endogenous HMGB1 promotes NK cell development and IL-2-induced proliferation. Besides, HMGB1 sustains NK cell bioenergetics and promotes diverse NK functions including early tumor control.
Functional consequences of TCR transgenic β-chain expression (TUM2P.908)
Authors: Kazansky Dmitry , Silaeva Yulia , Vagida Murad , Kalinina Anastasia , Khromykh Ludmila
Published: January 2023

We have generated mouse strain expressing transgenic β-chain of TCR 1D1 (belonging to the Vβ6 family) on the genetic background B10.D2(R101) (KdIdDb). The repertoire of peripheral T lymphocytes in these mice contains 70-80% of T cells expressing transgenic β-chain and 20-30% of T cells expressing endogenous β-chains. Expression of transgenic β-chain resulted in T cells with phenotype CD44-CD62L+ characteristic of “naïve” T lymphocytes. On the contrary, CD8+ T lymphocytes expressing endogenous β-chains had surface phenotype of activated effectors CD44+CD62L-. In wild-type B10.D2(R101) mice, immunization with lymphoma EL4 (H-2b) cells induced a vigorous CTL response targeted to the H-2Kb molecule and resulted in full rejection of the tumor cells. In contrast, transgenic mice developed a compromised proliferative response in MLR and were unable to reject transplanted allogeneic EL4 cells. During the immune response to EL4 cells, mostly CD8+ T-lymphocytes with endogenous β-chains became CD44+CD62L- effectors in transgenic mice, whereas only a small part of the T-lymphocytes expressing transgenic β-chains became effectors. Effectors CD8+CD44+CD62L- expressing endogenous β-chains showed enhanced expression of PD-1 and contained increased percent of KLRG1-positive cells. The acquisition of these characteristic features was associated with immune selection of H-2Kb-negative EL4 cells and subsequent tumor escape.
A common gut commensal as a cross-reactive candidate in antiphospholipid syndrome (HUM3P.250)
Authors: Dehner Carina , Ruff William , VIEIRA SILVIO , Goodman Andrew , Kriegel Martin
Published: January 2023

Antiphospholipid syndrome (APS) is an autoimmune clotting disease characterized by autoantibodies interfering with coagulation. Given the vast antigenic potential of the gut microbiota, we hypothesized that adaptive immune responses to gut commensals may sustain chronic autoreactivity via cross-reactivity. We identified in in silico searches the common, gram-positive gut commensal Roseburia intestinalis as a prime candidate for molecular mimicry, containing peptide sequences highly homologous to both T and B cell immunodominant epitopes of the major autoantigen in APS, the plasma protein b2-glycoprotein I (b2GPI). PBMCs from APS patients proliferated significantly more to R. intestinalis protein extracts compared to phylogenetically closely related Eubacterium rectale, which lacks homologous peptides. We therefore cloned b2GPI-specific CD4 memory T cells and tested cross-reactivity with peptides in vitro. Preliminary data supports that IL-2 but not IL-4, IL-10 or IL-17 secretion can be elicited from an autoantigen-specific Th1 clone in response to a R. intestinalis peptide. Furthermore, Western blotting using patient plasma samples revealed specific bands for R. intestinalis including a potential cross-reactive protein at 24 kDa. Pre-adsorption using recombinant b2GPI prior to Western blotting showed a decrease in this candidate band. These data support a working model whereby autoreactive T and B cell responses are sustained by cross-reactivity with a common gut commensal.
A novel subset of fibrocyte-like cells in synovial fluid communicate with CD8 T cells and shape the local inflammatory milieu in Juvenile Idiopathic Arthritis (JIA). (HUM3P.253)
Authors: Ferguson Ian , Yano Hiroshi , Griffin Patricia , Michel Joshua , Kietz Daniel , de Vallejo Abbe
Published: January 2023

We reported previously that children with JIA carry senescent CD31+CD8+ T cells disproportionate with age. Such cells are inflammatory effectors via CD31-driven TCR-independent activation pathway. Here, we hypothesized that these T cells interact with non-hematopoietic cells in the synovial space to perpetuate joint inflammation. JIA patients medically indicated to undergo arthrocentesis were recruited. Synovial aspirates were screened by flow cytometry, and short-term cultures were established. Results showed two subsets of plastic-adherent fibrocyte-like cells (FLC). About 75% were procollagen-CD45-CD14- cells that variably expressed CD34. The other ~25% were procollagen+CD45+CD14+IL17RA+ CD34- FLC; a finding unlike similar cells reported for adult-onset rheumatoid arthritis. FLC exposed to rIL-17 and/or rTNFα induced production of MMPs that were reversed with corticosteroid, TNFi, anti-IL6R, or experimental NFkB inhibitors. Co-culture of FLC with autologous CD8 T cells resulted in production of the same array of MMPs and inflammatory cytokines that were partially blocked by neutralizing anti-CD31 antibody. These results suggest a FLC-CD8 T cell communication circuit in the maintenance of joint inflammation in JIA. Further understanding of the regulation and/or the consequences of this local cell-cell interaction could shed light into the systemic complications of this childhood disease that impose lifelong health burden. [Supported by Nancy E Taylor Foundation and NIH].
Functional evaluation of PTPN22 modulation in human T cells (HUM3P.260)
Authors: Perry Daniel , Saikumar Lakshmi Priya , Zhang Lin , Brusko Todd
Published: January 2023

A major event in type 1 diabetes (T1D) pathogenesis occurs when T cells aberrantly target and destroy insulin-producing beta cells. A SNP variant of protein tyrosine phosphatase, non-receptor type 22 (PTPN22) has been associated with T1D by GWAS. This gene encodes for lymphoid phosphatase (LYP), which functions to dephosphorylate proximal TCR signaling molecules, and is thus a major determinant for T cell function. The susceptible variant produces an R620W amino acid change in the LYP protein. As yet, there is no consensus as to how this variant affects T cell signaling and function, and ultimately, contributes to T1D pathogenesis. We hypothesized that the utilization of lentiviruses to stably transduce primary human T cells would elucidate the mechanisms of the LYP-620W variant in T1D pathogenesis. We found that TCR signaling and proliferation were blunted by overexpressing the LYP-620R variant (-25.39±4.15% Ca++ flux, 0.616±0.041 fold expansion), but were only modestly affected by the LYP620W variant (-5.66±2.11% Ca++ flux, 0.823±0.041 fold expansion). In addition, LYP modulation only affected proliferation when APCs were used for activation, as opposed to antibody-coated beads. Finally, endogenous PTPN22 expression was found to be dynamic throughout an activation time course. Our results implicate the T1D-associated LYP-620W variant to be a hypomorph, and have important implications when considering interventions to restore defective pathways for proper immune regulation.
Forced miR-146a expression causes autoimmune lymphoproliferative syndrome in mice via downregulation of Fas in germinal center B cells (HUM3P.249)
Authors: Li Jingyi , Guo Qiuye , Wan Ying , Wu Yuzhang
Published: January 2023

MicroRNAs, a class of small non-coding RNAs, play a major role in various cellular activities during psychological and pathological processes through inhibiting the direct targets expression. MiR-146a is remarkable induced after LPS stimulation and virus infection and highly expressed in patients with immune disorders such as rheumatoid arthritis, Sjögren's syndrome and psoriasis. To date, whether the high level of miR-146a contributes the pathogenesis of immune disorder disease s remains unknown. In order to elucidate precious functions of miR-146a in vivo, we generated a miR-146a over-expression transgenic mouse. The level of miR-146a has been increased 3 to 8 folds higher than WT mice, which is similar to the levels of patients with immune disorders. miR-146a transgenic mice spontaneously developed a ALPS feathered disease with the distinct manifests including elevated double negative T cells, enlarged spleens and lymph nodes, higher serum IgG, and inflammatory infiltrations in livers and lungs at early age. Further analysis revealed that Fas was the direct target of miR-146a and was specifically down-regulated in germinal center B cells. In addition, B cells of miR-146a TG mice showed hyper-homeostasis proliferation after adoptive transferred into SCID mice. Collectively, our results clearly establish the notion that miR-146a may get involved in the pathogenesis of ALPS through targeting Fas in GC B cells and might be a potential therapeutic target.
Myeloid-derived cells reside in subcutaneous adipose tissue but are not found in circulation: a DC-expressing BDCA-1 CD206 and hematopoetic progenitor cells (HUM4P.273)
Authors: Gonzalez Juana , Walker Jeanne , Stanwix Jenny , Correa da Rosa Joel , Krueger James
Published: January 2023

Obesity is an insulin resistance-associated metabolic disorder which is a hallmark of and risk factor for type 2 diabetes and the metabolic syndrome. It is also often linked to cardiovascular disease, certain cancers and inflammatory diseases. Different immune cells have been identified in visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) which are indicators of inflammation associated with insulin resistance and systemic endothelial dysfunction in the peripheral vasculature. However, detailed studies of infiltrating leukocytes and associated cytokines are largely absent in human adipose tissue. One hypothesis that may link obesity with inflammatory immune response is that Th17 T-cells may be involved with pathological processes. Since past studies have shown increased production of CCL20 by adipocytes in obese humans , this chemokine would be expected to chemoattract DCs and Th17 T-cells into adipose tissue; associated Th17 activation could be the nidus for inflammatory diseases. In this study, we have found that obese SAT samples have a higher percentage of DCs than in the lean SAT samples of a DC which is BDCA-1 positive. Half of these BDCA-1 cells express CD206, this population is not found in blood. We also found a population unique to SAT, a hematopoietic progenitor cell. We additionally found an increase of Th17 cells in blood of obese subjects in comparison to lean subjects.
FcRγ- and IL-33-dependent house dust mite extract antigen-uptake in lung antigen presenting cells (HYP4P.311)
Authors: Camacho Daniel , Tjota Melissa , Sperling Anne
Published: January 2023

Allergic sensitization can be generated against various allergens, leading to Th2-mediated inflammation and subsequent asthmatic symptoms. However, the exact mechanisms behind allergic sensitization are not precisely understood. Our previous work demonstrated that house dust mite (HDM) signals through the FcRγ-associated receptor Dectin-2 and induces expression of IL-33 by DCs, leading to the differentiation of Th2 cells and asthma pathogenesis. Here, we sought to determine whether changes in antigen uptake by immune cells in the lung interstitium may be responsible for the diminished inflammation in response to HDM previously demonstrated in FcRγ- and IL-33-knockout mice. To this end, we evaluated antigen uptake in lung APC subsets 18 hours after intratracheal instillation of fluorescently labeled HDM in FcRγ- and IL-33-knockout mice. Our findings demonstrate the uptake of HDM by fewer lung-interstitial Ly6C+ monocytes in FcRγ-deficient mice, but equal uptake by DC subsets. Surprisingly, a defect in IL-33 alone resulted in reduced numbers of Ly6C+ monocytes, moDCs, and CD11b+ cDCs containing HDM in the lung interstitium. These data suggest a mechanism by which an FcRγ-dependent receptor is required for uptake of HDM by lung-interstitial Ly6C+ monocytes in order to upregulate IL-33 production in the lung, which may promote uptake by other APC subsets and induce Th2-mediated allergic airway inflammation.
Statins alter IL-33-mediated cytokine production in mouse mast cell (HYP4P.315)
Authors: Taruselli Marcela , Kolawole Elizabeth , Ryan John
Published: January 2023

Statins are HMG-CoA reductase inhibitors widely used to reduce hypercholesterolemia. Recent publications have shown that statins can act as immune suppressors by decreasing pro-inflammatory functions of APCs and T cells. We have found that the statin drug Fluvastatin suppresses IgE-mediated pro-inflammatory cytokine release from mast cells, likely by the decreasing prenylation of small G proteins. We extended these studies to the mast cell activator IL-33. Similar to previous studies, Atorvastatin decreased IL-33-mediated pro-inflammatory cytokine production. Unexpectedly, we have found that Fluvastatin increased TNF and IL-6 production upon IL-33-activation. This augmentation is dependent on stem cell factor (SCF), since in the absence of SCF Fluvastatin does not increase cytokine production. Suppressing G protein prenylation by treating mast cells with farnesyl protein transferase and geranylgeranyl transferase inhibitors did not mimic the effects of Fluvastatin. Furthermore, the product of HMG-CoA reductase, melvalonic acid, was unable to reverse Fluvastatin-mediated inhibition. These data suggest that the pro-inflammatory effects of Fluvastatin are an “off target” effect. Examining the effect of statins in IL-33-activated mast cells will elucidate how innate immunity may be altered by commonly prescribed medications.
Didox suppresses IgE-mediated mast cell response (HYP4P.317)
Authors: McLeod Jamie , Elford Howard , Ryan John
Published: January 2023

Mast cells play a central role in allergic reactions through high-affinity IgE receptor (FcεRI)-mediated responses. FcεRI cross-linking on mast cells activates multiple signaling pathways that lead to sustained inflammation through degranulation, prolonged generation of reactive oxygen species, and production of various cytokines and chemokines. These cellular responses are involved in inflammatory diseases including asthma, rheumatoid arthritis, multiple sclerosis and heart disease. As a synthetic anti-oxidant and a potent ribonucleotide reductase inhibitor capable of suppressing gene expression, Didox (3,4-Dihyroxybenzohydroxamic acid) has become an attractive therapeutic for treating inflammatory diseases. Here we investigated the anti-inflammatory abilities of Didox as a therapeutic approach to attenuate the allergic response. Our results show that Didox treatment yields a dose-dependent reduction in IgE-stimulated cytokine secretion among bone marrow-derived mast cell (BMMC). These effects are not directly linked to reduced receptor expression, or an increase in cell death and suggest a decrease in signaling associated with cytokine secretion. Together these data could indicate a role for Didox in the treatment of MC-associated inflammatory and autoimmune diseases.
Filaggrin deficiency in Stat6 transgenic mice exacerbates allergic skin inflammation (HYP4P.310)
Authors: Sehra Sarita , Akhtar Nahid , Nguyen Evelyn , Kaplan Mark
Published: January 2023

Atopic dermatitis (AD) is a chronic inflammatory skin disease induced by a complex interaction between susceptibility genes encoding skin barrier components and environmental antigen exposure. Although genome-wide association studies in AD have identified several susceptibility loci, loss of function mutations in the human filaggrin gene (FLG) is the major predisposing factor for AD. We have previously shown that 40% transgenic mice expressing constitutively active Stat6 (Stat6VT) in T cells spontaneously develop AD due to increased Th2 responses leading to decreased expression of barrier function genes including Flg. Flaky tail mice (FTM) have been widely used as a model of filaggrin deficiency-associated AD pathogenesis in patients. FTM have decreased barrier function, increased Th2 and Th17 responses following sensitization, but do not develop spontaneous allergic inflammation. In order to determine the effects of having both barrier dysfunction and increased Th2 responses, Stat6VT mice were mated to FTM. Our results demonstrate that over 90% of the STAT6VT mice carrying mutant Flg develop severe AD lesions by 3 months of age in comparison to 40% Stat6VT mice that develop disease between 4 to 5 months of age. Histopathological analysis of skin tissues revealed extensive thickening of the dermis with more severe infiltrates as compared to Stat6VT mice. Our studies suggest that skin barrier defects and altered Th2 responses separately contribute to the pathogenesis of AD.
Resistance of subacutely burned mice (SAB-mice) therapeutically treated with CCL1 antisense ODN against infections with MRSA or enterococci (INC4P.344)
Authors: Nishiguchi Tomoki , Lee Jong , Herndon David , Kobayashi Makiko , Suzuki Fujio
Published: January 2023

In previous studies, sepsis stemming for E. faecalis translocation and MRSA local infection has been greatly controlled in SAB-mice prophylactically treated with CCL1 antisense ODN. Effector cells (M1Mφ) in host antibacterial immunities are isolated from the infected SAB-mice after treatment with the ODN. In this study, the antibacterial resistance of SAB-mice therapeutically treated with the ODN was examined against infections with E. faecalis and MRSA. Mice 2 weeks after flame burn injury (3rd degree 25% TBSA) were infected with MRSA (106 CFU/mouse, i.v.). Decontaminated mice 2 weeks after burn injury were infected with E. faecalis (8 x 106 CFU/mouse, p.o.). These mice were therapeutically treated with the ODN (s.c., 10 mg/mouse) at various schedules starting 12 h after the infection. The antibacterial efficacies of the ODN were evaluated by reducing bacterial growth in blood and organs as compared to those of control mice. In the results, bacteria grew in the blood (>105 CFU/ml), liver (>106 CFU/gram), spleen (>105 CFU/gram), and kidneys (>107 CFU/gram) of control mice 3 to 5 days after MRSA infection. However, significant growth of the pathogen in their organs was not demonstrated in SAB-mice treated with the ODN 12, 24, and 36 h after the infection. Similar results were obtained in SAB-mice orally infected with E. faecalis. These results indicate that infections with MRSA and E. faecalis in SAB-mice are controllable by the therapeutic treatment with CCL1 antisense ODN.
Mycobacterial lipid antigen-specific innate T cell responses in TB-naïve macaques (INC4P.328)
Authors: Rout Namita , Tran Dollnovan , Exley Mark , Kaur Amitinder
Published: January 2023

Current tuberculosis (TB) vaccine strategies are targeted mainly towards conventional T cell responses to mycobacterial protein antigens. However, there is growing evidence for the role of mycobacterial lipid specific CD1-restricted T cell responses in anti-mycobacterial immunity. Understanding how the CD1-restricted T cells contribute to TB may provide critical insights into innate mechanisms of resistance to TB and aid the development of new therapies against TB. In this pilot study, we utilized cynomolgus macaques (CM) to characterize innate T cell responses to mycobacterial lipid antigens. PBMC from naïve CM were stimulated with purified mycobacterial lipid antigens, including Lipoarabinomannan (LAM), Phosphatidylinositol mannoside (PIM-1, 2, & 6) and total cellular lipids of MTb H37Rv, presented on CD1-a, -b, -c and -d transfectant C1R B cell lines. T cell responses were measured by IFN-γ, TNF-a and IL-2 ELISA in culture supernatants, and by intracellular cytokine staining for IFN-g, TNF-a, and IL-13. Robust TNF-a and IFN-g production was observed in response to CD1d/PIM and CD1b/LAM in all of the eight CM following 16 hours stimulation. Mainly CD8 positive and CD4/CD8 double negative T cells contributed to the dominant CD1-restricted Th1 cytokine response. The innate Th1-type response of T cells to MTb lipid antigens, particularly LAM and PIM, may be a significant component of the innate immune response in TB and thus promising targets for improved TB vaccines.
Cytosolic sensing of HIV by cGAS (INM2P.350)
Authors: Gao Daxing , Wu Jiaxi , Du Fenghe , Wu Youtong , Sun Lijun , aroh Chukwuemika , nan yan , Chen Zhijian
Published: January 2023

HIV infection abrogates adaptive immunity by the depletion of CD4 T cells. However, innate immune defense mechanisms against HIV is largely unknown. Here we show that pseudotyped HIV can infect human and mouse cell lines, leading to the production of interferons and other antiviral cytokines. Activation of innate immunity by HIV requires viral cDNA synthesis but not cDNA integration. We show that retrotranscribed HIV cDNA is sensed by the cytosolic DNA sensor cGAS, which then produces the second messenger 2'3'cGAMP to activate the adaptor STING. Importantly, wild type HIV also triggers cGAMP production in human primary macrophages, underscoring the key role of cGAS in HIV sensing. Furthermore, cytosolic sensing of other retroviruses such as murine leukemia virus and simian immunodeficiency virus also depends on cGAS. These results further our understanding of immune detection of HIV and can be potentially applied in HIV vaccine and adjuvant designs.
Inhibition of the STING pathway by the AIM2 inflammasome (INM2P.352)
Authors: Corrales Leticia , Woo Seng-Ryong , Gajewski Thomas
Published: January 2023

Tumor-derived DNA triggers the host STING pathway and the subsequent production of IFN-β by antigen-presenting cells, facilitating adaptive immunity against tumors. But cytosolic DNA also activates the AIM2 inflammasome, raising the question of functional impact of this pathway. We found that after DNA stimulation, macrophages or dendritic cells deficient in AIM2, ASC or caspase-1 produce substantially higher IFN-β. This observation suggests possible negative regulation of the STING pathway by the AIM2 inflammasome. We found no evidence for differential delivery of DNA into the cells, increased cell death triggered by activation of the inflammasome, or release of an inhibitory soluble factor. However, biochemical analysis revealed that the AIM2 inflammasome inhibits the early steps of STING pathway activation. Aggregation of STING and phosphorylation of TBK1 and IRF3 were highly enhanced in AIM2 inflammmasome-deficient cells. In addition, pre-treatment with a caspase-1 inhibitor increased the activation of TBK1/IRF3, suggesting that assembly and activation of the inflammasome complex is necessary for the inhibition of the STING pathway. Our data suggest the active AIM2 inflammasome inhibits the STING pathway at or upstream from STING. This crosstalk may have potential consequences for anti-tumor immunity which should be explored in vivo.
A mycobacterial lectin promotes bacilli adhesion to macrophages and influences pathogen growth during infection (INM3P.409)
Authors: Bafica Andre , Morales Stefanny , Eto Carol , Souza Nicole , Nogueira Lucas , Riley Lee , Mansur Daniel
Published: January 2023

Surface and secreted mycobacterial proteins play a major role on the infection process by mediating macrophage-bacteria interactions as well as the fate of cell’s death. We have previously described a secreted 13-kDa lectin (sMTL-13) in Mycobacterium tuberculosis (Mtb), encoded by Rv1419 gene, and although a possible importance for this protein as a major mycobacterial antigen was shown in tuberculosis patients, the importance of such lectin during infection has not been formally addressed. We have generated an Rv1419 knockout Mtb (ΔRv1419) to investigate a possible role of this gene during infection. In silico analysis of the protein as well as its detection on Mtb surface suggest that sMTL-13 may participate during bacteria entry in the host macrophage. This was confirmed by binding experiments performed in either murine or human derived macrophages. In addition, lower levels of TNF were detected in ΔRv1419-infected cells, indicating that this lectin may be important for pathogen recognition. Importantly, ΔRv1419 displays higher intracellular growth than the WT bacteria. Thus, although binding of Mtb to macrophages is decreased in the absence of Rv1419 gene, cells exposed to the knockout Mtb display higher bacterial replication rate and cell death. These data suggest that sMTL-13 expressed on Mtb regulates macrophage entry and perhaps cell fate, which could serve as a pathogen survival strategy, allowing bacterial growth without leading host cells to a premature death.
Population diversity in transcriptional responses of macrophages to TLR7/8 signaling (IRM12P.654)
Authors: SONG RAN , Dozmorov Igor , Liang Chaoying , Zhang Bo , wakeland Benjamin , Arana carlos , Viswanathan Kasthuribai , Riediger Linley , Wakeland Edward
Published: January 2023

The Toll-like receptors (TLR) of the innate immune system play a key role in the recognition of pathogens and the initiation of a robust innate immune response. Although an innate immune response is essential for resistance to pathogen infection, the magnitude and qualities of innate immune responses are quite variable in the human population. Here we performed transcriptome sequencing of monocyte-derived macrophages generated from 78 healthy individuals, including 10 replicates from repeat donors, to profile gene expression patterns in responses to TLR7/8 signaling. Extensive qualitative and quantitative diversity was apparent in the response of individuals in this panel. Cluster analysis discriminated nine distinct clusters of genes expressed in response to the TLR7/8 agonist (R848) with correlated expression variations within this panel. Interestingly, the induction of cytokine and chemokine transcription varied among donors and majority of these genes have very reproducible variability of their expression in 10 replicates. In addition, it was shown that donors with high expression of cytokine and chemokine genes after R848 stimulation were also high responder to Respiratory syncytial virus infection. Thus, there are the variations in gene expression among individuals with TLR7/8 stimulation, indicating that genetic polymorphisms in “master” regulators may correlate expression patterns of multiple genes during innate responses.
Role of interleukin-1 receptor antagonist in regulating specific Hepatitis B antibody production (IRM12P.646)
Authors: Chen Hairong , Zhao Yang , Tang Hong
Published: January 2023

While exogenous interleukin-1 beta was reported to facilitate antibody production, the function and mechanism of its conventional suppressor interleukin-1 receptor antagonist (IL-1Ra) in regulating specific antibody remain largely unknow. We showed that, upon aluminium-conjugated commercial hepatitis B surface antigen vaccination, hepatitis B antibody response was considerably amplified in IL-1Ra-/- mice, demonstrated by higher HBsAb level, increased germinal center and HBsAb secreting B cell numbers in dLN but not spleen. Mechanismly, IL-1 signalling pathway was, unexpectedly, found not to be involved in the process, evident by equivalent HBsAb titre in nalp3-/-, caspase-1-/- and IL-1R-/- mice as in wt. Surprisingly, the IL-17 production was amplified in IL-1Ra-/- mice, demonstrated by higher serum IL-17 level and higher number and frequency of IL-17 secreting cells in dLN of IL-1Ra-/- mice. Moreover, HBsAb level in IL1rn-/- x IL17-/- mice was comparable to that in wt mice, suggesting the essential role of IL-17 in regulating the elevated HBsAb level in IL-1Ra-/- mice. Furthermore, CD4 T cells was identified as the main source of IL-17 production in dLN of IL-1Ra-/- mice, indicating the essential role of IL-17 secreting CD4 T cells in upregulating HbsAb production. Therefore, the increased HBsAb production in IL-1Ra-/- was irrelevant to IL-1 pathway but dependend on evelated IL-17. We have thus proposed a novel mechanism of IL-1Ra in regulating specific antibody production.
Characterization of long-lived human memory CD8 T cells specific to yellow fever virus (LYM5P.707)
Authors: Yang Shu , Akondy Rama , Youngblood Ben , Edupuganti Srilatha , Mulligan Mark , Ahmed Rafi
Published: January 2023

The live yellow fever virus vaccine (YFV-17D) is a safe and effective vaccine that confers long-lasting protection after a single immunization. This vaccine results in the generation of virus memory T and B cells that persist for many years. We have used this vaccine to study the generation and maintenance of human memory CD8 T cells following an acute viral infection. Earlier studies using tetramers targeting an HLA-A2 restricted epitope have shown that YFV-specific CD8 T cells have polyfunctionality and recall potential - two hallmarks associated with effective T cell immunity. To better characterize these long-lived memory CD8 T cells we analyzed the peripheral blood mononuclear cells from HLA-A2+ individuals who had received a single dose of the vaccine 8 to 15 years previously. We will present data on the phenotype, function and epigenetic fingerprint of the YFV-specific memory CD8 T cells. Interestingly, these long-lived memory CD8 T cells had an epigenetic fingerprint that was consistent with their having passed through an effector stage during their differentiation.
OX40 signaling activates epigenetic mechanisms to repress Th17 cells and Th17-related autoimmune diseases (LYM5P.708)
Authors: Xiao Xiang , Shi Xiaomin , Fan Yihui , zhang xiaolong , wu minhao , liu wentao , Zhao Picheng , wu chenglin , Minze Laurie , Ghobrial Rafik , Li Xian
Published: January 2023

Th17 cells produce copious amount of IL-17, which mediates host antimicrobial responses and autoimmune diseases. The exact mechanisms that control Th17 homeostasis, especially those that inhibit Th17 differentiation, remain incompletely defined. Here, we found that the T cell costimulatory molecule OX40 is a potent repressor of Th17 cells and Th17-mediated autoimmune disease. Under Th17-polarizing conditions in vitro, engagement of OX40 strongly inhibited IL-17 production despite enhanced cell survival and proliferation. Mechanistically, OX40 engagement induced sustained activation of both canonical and non-canonical NF-kB pathways, and it is RelB that mediated dominant suppression of Th17 cells that is independent of p52 and p50. RelB directly recruited H3K9 methyltransferases G9a and Setdb1 upon binding to IL-17 locus, and this resulted in di- and tri-methylation of H3K9, which led to a closed epigenetic configuration of IL-17 locus that prevents RORγt binding to IL-17 locus. Conversely, inhibition of histone methyltransferase activity significantly reversed OX40-mediated suppression of Th17 cells. Importantly, in an EAE model, stimulation of OX40 in vivo inhibited the disease process, while RelB-deficient CD4+ T cells induced exacerbated disease. In conclusion, our study identified a new mechanism in the control of Th17 cells, and this mechanism relies on OX40 induced repressive epigenetic process. This finding may have important therapeutic implications in the clinic.
Preventing thymus involution in K5.Cyclin D1 transgenic mice sustains the naïve T cell compartment with age (LYM6P.716)
Authors: Bolner Michelle , Fink Pamela , Richie Ellen
Published: January 2023

The thymus maintains T cell receptor (TCR) repertoire diversity through perpetual release of self-MHC restricted naive T cells. However, thymus involution during the aging process reduces naïve T cell output, leading to defective immune responsiveness to newly encountered antigens. We have shown that age-related thymus involution is prevented in mice expressing a keratin 5 promoter-driven Cyclin D1 (K5.D1) transgene in thymic epithelial cells (TECs). Sustained T cell output from K5.D1 thymi prevents the age-associated decline of naïve T cells in the periphery. We find that K5.D1 recent thymic emigrants (RTEs) undergo typical phenotypic maturation. Competitive adoptive transfer studies with K5.D1 and nontransgenic RTEs indicate that K5.D1 RTEs are incorporated into the peripheral T cell pool comparably to nontransgenic controls. In addition, functional studies show that K5.D1 peripheral T cells are responsive to anti-CD3 and anti-CD28 stimulation in vitro. Collectively, these data show that preventing thymus involution not only sustains T cell output, but also provides functionally competent RTEs during aging.
Toxoplasma gondii gene products involved in the activation of the rat NLRP1 inflammasome (MPF4P.729)
Authors: Cirelli Kimberly , Butty Vincent , Saeij Jeroen
Published: January 2023

Toxoplasma gondii is an intracellular parasite, capable of infecting all warm-blooded animals. Upon infection of a host, Toxoplasma converts into cysts in brain and muscle tissue and establishes a life-long chronic infection. Rats, like humans, are resistant, surviving infection with high doses of mouse-virulent Toxoplasma strains. The Lewis rat is unique in its ability to completely clear infection and prevent cyst formation. We have previously shown that Toxoplasma-infected Lewis rat macrophages undergo rapid NLRP1-mediated pyroptosis. We hypothesize that this rapid cell death is a host mechanism to prevent parasite replication and dissemination to the brain and muscle tissues. Using a chemical mutagenesis screen, we have isolated several independent Toxoplasma mutants that fail to induce macrophage death and identified their mutations using whole genome sequencing. Using CRISPR/Cas9-mediated deletion of candidate genes, we have identified three Toxoplasma genes that contribute to macrophage pyroptosis. Parasites lacking these genes induce significantly less inflammasome activation, as measured by active IL-1β and pyroptosis, and are able to replicate within Lewis macrophages. We are determining if these mutants are able to form cysts in Lewis rats. Using biochemical techniques, we are probing the function of these individual parasite proteins and how they each contribute to NLRP1 activation.