bluuebunny/crossref_metadata_2025 huggingface dataset:
Proteogenomics and de novo sequencing based approach for neoantigen discovery from the immunopeptidomes of patient CRC liver metastases using Mass Spectrometry
Authors: shah Darshit , Rye-Weller Anna , Dhanik Ankur , Cygan Kamil , Olsen Olav , Olson William , Salzler Robert
Published: January 2023

Introduction

Using High-resolution Mass Spectrometry (MS), we developed a novel immunopeptidomics workflow using de novo sequencing. This method does not rely on a reference database to deduce full-length peptide sequences. As a consequence, peptide discovery is not limited to the reference database and allows for the discovery of novel peptides directly from MS.

Goal

Identify HLA-associated peptides that appear in high frequency in tumors through de novo sequencing and missed by reference database searches.

Method

We employed a highly selective anti-HLA Class I monoclonal antibody, W6/32, to enrich HLA-complexes from 21 CRC liver metastases and identified HLA associated peptides using a Thermo Orbitrap Fusion Lumos on-line with a nanoLC 1200. RNA-Seq was also performed on each tumor. MS raw spectra were analyzed by de novo assisted PEAKS-X (BSI)

Results

Approximately 1600 unique de novo sequenced HLA-associated peptides were identified in a single LC-MS analysis with high confidence (95% ALC cut-off) from each tumor. Approximately 45% of the 1600 unique de novo sequenced peptides matched to reference databases (e.g Uniprot.), leaving a large percentage of peptides with no match. Interestingly, some of these unmatched peptides have been validated as neoantigens that arise from gene mutation, spliced peptides generated by the proteasomes, and non-coding region derived peptides.

Conclusion

These de novo sequenced peptides without matches in reference databases may provide a unique source of disease-specific HLA class I-presented peptides and trigger specific immune responses. Some of the higher frequency unmatched de novo sequenced peptides may thus serve as tumor-specific targets for next-generation immunotherapies.
Destabilization of exotoxin A diminishes serum antibody titer and affinity maturation by reducing peptide-MHCII abundance
Authors: Moss Daniel L , Song Kejing , Wanek Alanna G , Shu Qingbo , Hu Tony , Kolls Jay K , Mettu Ramgopal R , Landry Samuel J
Published: January 2023

Potent antibody responses depend on the proteolytic processing of protein antigens and the presentation of MHC Class II bound peptides to CD4+ T cells. We have previously reported a single amino acid substitution (R494A) made to pseudomonas exotoxin A domain III (PE-III) that alters processing and antibody responses with only minor changes to CD4+ T-cell priming. These findings could not explain how the R494A substitution causes a substantial reduction in antibody titer. Previous work by our laboratory and others has shown that the stability of protein antigens affects T-cell priming through effects on antigen processing. However, there is no direct mechanism for how antigen processing alters antibody immunogenicity. We hypothesized that destabilization of PE-III by the R494A substitution reduces the abundance of PE-III peptide-containing MHCII complexes on B cells and thereby reduces their capacity to solicit T cell help for affinity maturation. Here we have observed that antibodies raised against PE-III R494A exhibit lower avidity than those raised against wild type PE-III. Furthermore, R494A PE-III was a poorer source of T-cell epitopes when provided to splenocytes from mice immunized with either wild type or R494A. Currently, PE-III peptide abundance is being analyzed by mass spectrometry, and antigen specific B cells are being characterized by single cell RNA sequencing.
Molecular requirements for B cell receptor engagement and activation
Authors: Ferapontov Alexey , Omer Marjan , Nielsen Jesper , Thiel Steffen , Kjems Jørgen , Degn Søren Egedal
Published: January 2023

B cells are a central component of the adaptive branch of the immune system, responsible for humoral immunity through production of antibodies. Antigen engagement of the B cell receptor (BCR) is the first step in B cell activation; yet despite its recognized importance, the exact mechanism of BCR activation remains controversial. A central point of contention has been whether monovalent antigen is capable of activating the BCR or not. To address this question, we leveraged the B1-8hi knock-in BCR model, in which λ light-chain positive B cells display intermediate and high affinity binding to the haptens NP ((4-hydroxy-3-nitrophenyl)acetyl) and NIP ((4-hydroxy-3-iodo-5-nitrophenyl)acetyl), respectively, and combined this with a recently developed nucleic acid based scaffolding technology (Holliday junctions [HJs]) to prepare defined mono-, di- and trivalent displays of NP and NIP. Using confocal imaging, flow and imaging cytometry, as well as calcium-flux analyses, we find that HJs carrying 2 or 3 copies of either NP or NIP can bind and activate B1-8hi B cells across a wide range of concentrations, whereas HJs carrying a single NP require high concentrations for any measurable activity. Most interestingly, HJs carrying a single NIP are able to bind B1-8hi B cells at low concentrations, approaching equimolar stoichiometry, but despite measurable BCR engagement do not elicit activation. Our results may reconcile previous contradictory reports regarding monovalent antigen activation, by providing a framework for understanding complex interaction effects of antigen affinity, valency and concentration.
Pertussis toxin treatment promotes a B cell driven disease during TH17 EAE
Authors: Maria Zahra , Axtell Robert C.
Published: January 2023

Pertussis toxin (PTX) is an enigmatic co-adjuvant that is required for disease induction in active Experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. The objective of this study was to investigate the functional role of PTX during adoptive transfer TH17-EAE. TH17-EAE was induced by transferring myelin-specific TH17 cells, harvested and cultured from MOG35–55 immunized wild type (WT) donor mice, into WT or B cell deficient (μMT) recipient mice. Recipient mice were treated with PTX or PBS on days 0 and 2 and monitored for paralysis. In WT mice, PTX delayed disease onset but did not alter disease severity or incidence. We found that PTX reduced trafficking of myeloid cells and inflammatory T helper cells in the CNS. Importantly, PTX treatment increased the number of B cells in the brain with increased antigen presenting function, suggesting an altered disease toward a B cell-dependent pathology. When disease progression was compared between WT and μMT mice treated with PTX or PBS, there was no significant difference in TH17-EAE in the PBS treated groups. However, PTX treatment significantly delayed disease onset, severity and incidence in the μMT mice vs. WT. In conclusion, our current data suggests that during TH17-EAE, PTX restricts the trafficking of pathogenic T helper cells and myeloid cells into the CNS and thereby delays the disease onset. However, not only PTX does not alter trafficking of B cells into the CNS it also stimulates a more inflammatory B cell phenotype and therefore promotes a B cell driven disease. This study provides novel insights to interactions between TH17 and B cells in mice and has defined a new model to study B cell activity during multiple sclerosis.
Expansion of polyreactive and autoreactive IgG B cells in NOD.H-2h4 mice, a model of Sjögren’s Syndrome
Authors: Engel Pablo , Gutiérrez-Cózar Rebeca , Puñet-Ortiz Joan , Carrillo Jorge , Blanco Julìa , Moya Manuel Sáez
Published: January 2023

In genetically prone individuals, chronic immune activation may lead to expansion of autoreactive lymphocyte clones that can induce organ damage developing autoimmune disorders. Sjögren’s Syndrome (SjS) is one of the most common systemic chronic autoimmune diseases that primarily affects exocrine glands with progressive dryness of mouth and eyes. Despite the accumulated evidences of profound B-cell alterations of humoral immunity, the repertoire and onset of B-cell autoreactivity in SjS remains to be determined. We hypothesize that SjS mice will have an increased frequency of self-reactive B cells with a progressive evolution to antigen-driven oligoclonality. Here, we study the B cell repertoire of NOD.H-2h4 mice, a mouse model of spontaneous autoimmunity mimicking SjS without developing diabetes. All female mice presented salivary gland infiltrates and autoantibodies such as anti-dsDNA and anti-Ro52, both characteristic of SjS, at the age of 24 weeks. A library of 456 hybridomas from NOD.H-2h4 mice splenocytes at three different ages: 28, 47 and 66 weeks old was created. The presence of mono or polyreactive autoantibodies against dsDNA, Ro52, LPS, insulin, histones and OVA was evaluated by ELISA, and their staining patterns and cellular reactivity were tested by IFA and FACS. We observed a high percentage of autoreactivity among B-cell clones from NOD.H-2h4 mice. IgM autoreactive B cells were maintained, while polyreactive and autoreactive IgG clones increased with mice age. Interestingly, all anti-Ro52 autoantibodies were polyreactive. Our results indicate that in the NOD.H-2h4 mouse model of SjS, IgG+ B cells are mainly polyreactive and might expand following an unknown antigen-driven positive selection process.
Single Cell RNA Sequencing of Human Liver Infiltrating B Cells in Primary Biliary Cholangitis (PBC)
Authors: Zhang Weici , Wu Xunyao , Sun Ying , Duan Weijia , Dhaliwal Sandeep , Leung Patrick S.C. , Bowlus Christopher , Zhang Xuan , Gershwin M Eric
Published: January 2023

Increasing evidence supports the concept that dysregulated B cell differentiation and activation are pivotal in the initiation and progression of autoimmune disorders, in both antibody (Ab)-dependent and Ab-independent mechanisms. Antimitochondrial antibodies (AMA), the serologic hallmark of PBC, are present in greater than 95% of patients with PBC, with a long latency period between the detection of autoantibodies and the onset of clinical disease. However, the mechanisms involved in the role of B cells in the pathogenesis of PBC have remained enigmatic. Herein, we have applied scRNA sequencing to simultaneously characterize B cell repertoire and transcriptome status in blood-liver paired samples from patients with PBC. Our data reveal that there is a dramatic increase in the heterogeneity of the blood CD27− B-cell population in PBC as compared to control. Strikingly, this overwhelmingly expanded naïve-like population were IgM+/IgD+, and encoded by germline sequences. In contrast with blood B cells, the dominant liver CD27− B-cell population is comprised of a high frequency of class-switched clonal B cells, which strongly express MHC class II genes. Upon stimulation with CpG (a surrogate TI-antigen), PBC CD27− B-cell populations markedly expanded and were accompanied by a significantly reduced expression of MARCKS, a newly identified molecule that suppresses active BCR signaling, as compared to healthy controls. Our data indicate that PBC CD27− B-cells exhibit abnormalities and heterogeneity, which may arise from a consistent exposure to TI-antigens. Our findings underscore the significance of B cells in PBC and suggest that more efforts should be placed on PBC B cell immunobiology and as potential therapeutic targets.
Defects in lysosome acidification are evident in active, but not inactive, systemic lupus erythematosus (SLE)
Authors: Kang SunAh , Rogers Jennifer L. , Clowse Megan E.B. , Sheikh Saira Z. , Vilen Barbara J.
Published: January 2023

Better strategies are needed in SLE to identify the receptors and molecular events that promote inflammation. We identified a defect in lysosomal acidification in multiple hematopoietic cell types in the MRL/lpr, NZM2410, and sle123 murine models of lupus. On myeloid cells, lysosome dysfunction is induced by chronic Fcgamma receptor (FcγR) signal transduction and results in the recycling of internalized IgG-immune complexes, and their accumulation on the cell surface. This perpetuates FcγR activation, promoting BAFF and IFNα/β secretion upstream of autoantibody production, lupus nephritis, and tertiary lymphoid organogenesis. Here, we define whether lysosomal defects and accumulation of nuclear antigens (Ags) are evident on human blood cells in SLE. Our data show that diminished lysosome function and the accumulation of nuclear Ags on patients’ blood cells are more evident in active, compared to inactive disease. On monocytes, 78% of active (n=18), but only 38% of inactive (n=16) patients showed diminished lysosomal acidification. On B cells, 47% of active, while only 9% of inactive patients, showed defects in lysosomal acidification. To assess whether lysosomal defects were coincident with the accumulation of nuclear Ags, we measured surface DNA levels (n = 57 active, 51 inactive patients, 109 HCs). In active patients, surface DNA levels were also elevated on blood B cells (7-fold; p = <0.002), DCs (3.7 fold; p = 0.06), T cells (2.4-fold; p = 0.03), and monocytes (1.8-fold; p = 0.09). Because monocytes exhibited lysosome defects, the low levels of surface DNA on monocytes was unexpected. However, it raises the possibility that monocytes activated by lysosome dysfunction and chronic FcγR signaling leave the blood.
PU.1 mediates hematopoietic stem cell self-renewal during cellular stress by non-canonical Wnt signaling
Authors: Welner Robert S , Bararia Deepak , Levantini Elena , Camacho Virginia , Patel Sweta , Matkins Victoria R. , Tenen Daniel G
Published: January 2023

Hematopoietic stem cells (HSCs) reside in a dormant state during tissue homeostasis. However, secreted niche factors and cellular programs that maintain HSC dormancy following hematopoietic stress remain unknown. HSC are capable of re-entering quiescence following proliferation, suggesting local factors control HSC exhaustion. In the present study, we found that the myeloid-associated transcription factor PU.1 alters cell cycle activity and differentiation during cellular stress. PU.1 is typically associated with myeloid differentiation and lineage commitment, but during HSC perturbation, PU.1 regulates HSC maintenance and self-renewal potential. Taken together, PU.1 is required to limit HSC exhaustion; furthermore, limiting PU.1 expression during stress leads to impaired HSC function. Using cell-specific reporter models and reductionist culture assays, we find that a non-canonical Wnt-PU.1 axis constrains HSC differentiation and activation to maintain tissue homeostasis. Moreover, using scRNAseq and ChIPseq, we found unique cellular programs associated with PU.1 localization that mediate cell cycling and metabolism of proliferative HSC are mediated by Wnt. In the context of cellular stress, exposure to non-canonical Wnt signaling transitions HSCs into PU.1 expressing cells with increased engraftment potential and multilineage reconstitution. Our results suggest Wnt-induced PU.1 expression limit proliferation and differentiation programs to promote HSC function. Altogether, our data identify PU.1 as a key mediator of HSC maintenance in response to cellular stress.
Exploring the transcriptional impact of ERα in the B cell response of systemic lupus erythematosus
Authors: Richard Mara Lennard , Tsao Betty P. , Chung Dongjun , Gilkeson Gary S , Cunningham Melissa A
Published: January 2023

Systemic lupus erythematosus (SLE) is a heterogeneous, autoimmune disease. Being female is a significant, baseline risk factor for developing lupus. The mechanisms responsible for enhanced risk remain unknown. The nuclear hormone receptor and key transcription factor, estrogen receptor alpha (ERα), has a number of immune gene targets; yet, understanding of its regulation of the immune response is incomplete. Lupus prone mice, expressing a truncated functional ERα knockout, survived longer and had reduced renal disease. However, a complete knockout of ERα was not protective. Our goal is to identify the molecular mechanisms utilized by ERα in regulating the immune response in lupus B cells. We identified ERα associated genes and determined expression differences in B cells isolated from female African American (AA) lupus patients. RNA-seq analysis indicates that 60% of ERα target genes were deferentially expressed and 82% of these genes were upregulated in patients compared to controls. To understand ERα function, full-length ERα and an isoform, ERα46 (lacking the AF-1 activation domain), were transfected into an EBV transformed SLE B cell line and the expression of ERα gene targets, including inflammatory cytokines (IL1β and IL6) examined. Transfection results varied by gene and suggest the interaction between ERα isoforms is complex. The effect of epigenetic modifying agents on ERα protein expression in transfected SLE B cells was also studied. Evidence suggests that an increase in DNA methylation leads to increased protein expression of ERα isoforms. The regulatory role of ERα in lupus B cells is complex, involves epigenetic changes and interplay between isoforms. Results support a role for ERα in the pathogenesis of SLE.
TNIP1/ABIN1 mutation contributes to lupus nephritis via chemokine IP-10 induction
Authors: Powell David W , Brady Makayla P , Barati Michelle T , Caster Dawn J
Published: January 2023

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease. Kidney inflammation or lupus nephritis (LN) is the most common and severe complication of SLE and mechanisms of pathogenesis are not clearly defined and current therapies are toxic and ineffective. We previously reported TNIP1 gene variants as risks for LN. TNIP1 encodes the protein ABIN1, which is a polyubiquitin binding protein that negatively regulates the prominent immune regulatory transcription factor NF-κB. We reported that transgenic mice with impaired ABIN1 ubiquitin binding function (ABIN1[D485N]) spontaneously develop SLE-like autoimmunity and LN and that ABIN1 determines the severity of LN via activation of kidney and immune cell inflammation. Interferon gamma-inducible protein -10 (IP-10) is a pro-inflammatory chemokine and NF-κB target that has been implicated in the pathogenesis and as a diagnostic marker of LN. The current project tested a hypothesis that LN development is mediated by induction of P-10 expression due to loss of cellular ABIN1 ubiquitin binding activity. We found that serum, urine, and kidney cell IP-10 expression is enhanced in ABIN1[D485N] mice. We also found that urinary IP-10 levels are higher in LN patients with TINIP1/ABIN1 variant rs4958881 when compare to LN patients without the TNIP1 variant and healthy controls. Our findings indicate that TNIIP/ABIN1 mutation contributes to the pathogenesis of LN via kidney and immune cell induction of IP-10 secretion and that serum and urinary IP-10 are promising diagnostic markers for LN especially in patients with TNIP1 variants. Further, successful Phase 2 clinical trials with IP-10 mAb for ulcerative colitis indicate its potential for effective LN treatment.
Role of 2B4 on dermal γδ T cells in the development of psoriasis-like skin inflammation
Authors: Kim Jiyoung , Kim Tae-Jin , Lee Kyung-mi
Published: January 2023

Psoriasis is a chronic inflammatory skin disease that has comorbid with systemic inflammatory disease, however, their precise pathologic mechanisms remain poorly understood. Transcriptome analysis of dermis in the psoriasis patient has revealed that the signaling lymphocyte activation molecule (SLAM) family receptor, 2B4 (CD244), was significantly down-regulated in the skin of psoriasis patients, suggesting that 2B4 might have served as an important regulator in the process of psoriatic inflammation. To test this hypothesis, we induced psoriatic skin inflammation with topical application of Imiquimod (IMQ), a toll-like receptor 7 ligand, on the back skin of WT or 2B4KO mice and examined their clinical disease score up to 6 days. As expected, IMQ-treated WT mice exhibited the signs of erythema, scales and skin thickness, however all these clinical manifestations were significantly worsened in 2B4KO mice. Furthermore, 2B4 deficiency caused elevated expression of antimicrobial peptides, along with increased IL-17A, a key cytokine driving the pathogenesis of psoriasis. Upon further analysis, we found that increased IL-17A production in the psoriatic skin of 2B4KO mice was attributed to the presence of dermal γδ T cells, not DCs or monocytes, within inflamed skin. Taken together, we present a previously unidentified role of 2B4 as a key coreceptor regulating the pathogenesis of psoriasis via controlling the dermal γδ T cells.
A novel mouse model of proteasome associated autoinflammatory syndromes
Authors: Brown Ivy Khristine , O’Brien Tommy , Jin Jingxiao , Jiang Simon , Reinhardt R. Lee
Published: January 2023

Proteasome associated autoinflammatory syndromes (PRAAS) represent a rare class of early-onset autoinflammatory diseases, including chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE syndrome). CANDLE is an inherited autosomal recessive disease associated with recurrent fevers, skin lesions, lipodystrophy and other comorbidities. Point mutations in the proteasome subunit beta type-8 (psmb8) gene, which encodes the beta-5i (ß5i) catalytic subunit of the 20S immunoproteasome, have been linked to CANDLE. The immunoproteasome is induced in cells in response to interferon (IFN) signaling. In cells lacking functional immunoproteasomes, oxidant-damaged proteins aggregate and likely create a feed-forward loop resulting in greater cytokine-mediated stress. We have developed a novel mouse model via targeted mutation within the psmb8 gene, which harbors a specific point mutation (224C>T) present in CANDLE patients. Our data shows that this mutation leads to marked differences in the expression of the immunoproteasome subunit ß5i within these mice. Thus, this mouse model is likely to exhibit changes in the immunoproteasome consistent with human disease upon interferon signaling. As mice with the targeted mutation do not directly yield a CANDLE-like phenotype at rest, it suggests that induction of disease-associated morbidity requires additional drivers. To test this, we are investigating interferons as initiators of disease pathogenesis. Future research will entail subjecting these mice to viral infection in order to identify if such triggers can initiate PRAAS-like symptoms in CANDLE patients. These mice may prove to an essential tool in understanding such disorders.
Prominent antibody mediated auto-immune response in idiopathic CD4 lymphopenic patients
Authors: Perez-Diez Ainhoa , Wong Chun-Shu , Liu Xiangdong , Mystakelis Harry , Song Jian , Sheikh Virginia , Anderson Megan , Lisco Andrea , Siegel Richard M , Tsang John , Sereti Irini
Published: January 2023

Patients with Idiopathic CD4 Lymphocytopenia (ICL) have low circulating CD4 T cell numbers, accompanied sometimes by low numbers of CD8, B and/or NK cells, making them susceptible to opportunistic infections. The etiology is unknown and there is no familiar linkage. In spite of their immune deficiency 32% of ICL patients have clinically defined auto-immune disease. To analyze the presence of auto-antibodies in these patients we hybridized sera from 34 patients and 15 healthy control (HC) to a 9,000 proteins human array. When identifying positive targets as the ones with Z score ≥4 we found that all 34 ICL patients, unrelated to their autoimmune disease manifestations, had IgG antibodies against tens of proteins while the HC had none. There was considerable variability in both numbers and identities of targets. We next evaluated whether some of these antigens might be displayed on the T cell membrane. Using a flow-based assay we tested sera from 72 ICL patients and found that 50% of them had either IgG, mostly but not exclusively IgG1 and/or IgG4 isotypes, or IgM antibodies against T cell membrane proteins. Functionally, 50% of the plasma with IgG1 or IgG3 antibodies induced antibody dependent cell mediated cytotoxicity against healthy T-lymphocytes. In addition, sera from 20% of the patients with anti-lymphocyte antibodies induced complement deposition on healthy CD3 T cells and some of them induced complement dependent cytotoxicity. Furthermore, we observed complement deposition (C1q, C4c and C3b) on CD4 T cells in 14% of the 72 ICL patients when analyzed directly ex vivo. Our data support a high prevalence and pathogenicity of autoantibodies in ICL, which offers a potential therapeutic target.
Effect of combined low dose IL-2 and polyclonal Tregs infusion in Type 1 Diabetes patients
Authors: Dong Shen , Mowery Cody , Hiam Kamir , Herold Kevan C , Gitelman Stephen , Esensten Jonathan , Liu Weihong , Lares Angela , Nguyen Vinh , Lee Michael , Leinbach Ashley , Ye Jimmie , Tang Qizhi , Bluestone Jeffrey A
Published: January 2023

Background

IL-2 is a well characterized cytokine that drives the expansion of activated human T cells. Its mechanism of action involving the high affinity receptor CD25 confers to IL-2 a crucial role in the homeostasis and function of regulatory T cell (Treg) basally expressing CD25. It has been suggested that low doses of IL-2 treatment in vivo, selectively expands Tregs without altering effector T cell or NK populations. This preferential targeting is key to cell-based immunotherapeutics that aim to prevent autoimmunity such as Type 1 Diabetes.

Methods

A phase I protocol was initiated to treat recently diagnosed T1D patients with a single infusion of ex-vivo expanded autologous polyclonal Tregs followed by two 5-days courses of low-dose IL-2. Patients were monitored for safety and diabetes-related metabolic profiles. PBMCs were analyzed at different time points by CyToF and 10X genomic single-cell RNAseq to monitor T cell activation.

Results

The overall safety profile was excellent but all 9 patients treated with the combined therapy failed to maintain c-peptide production at pre-treatment levels. We observed an expansion of Tregs and granzyme B (GZMB)+ NK subsets. More interestingly, single cell T cell receptor and gene expression sequencing allowed us to detected in all the patients a population of (GZMB)-producing CD8+ T that expanded clonally after IL-2 treatment.

Conclusion

These data suggest that the usage of low-dose IL-2 together with polyclonal Tregs infusion exacerbate the expansion of Tregs but also of a GZMB+ CD8+ T cells and NK population already present in blood. Thus, the failure for all patients to maintain C-peptide level may be due to a shift of the immune balance toward activation rather than tolerance.
Lack of Polyfunctional Cytomegalovirus-specific T cells in Hemodialysis Patients
Authors: Lay Fang-Yun , Chen I-Yu , Shu Kai-Hsiang , Chia Jean-San , Chiu Yen-Ling
Published: January 2023

Polyfunctional T cells are critical for maintaining protection against pathogens. Patients with end-stage renal disease (ESRD) are at increased risks for infectious complications and their T cell immunity against viruses may be impaired. Our current study intends to investigate the effects of renal disease on T cell immunity by analyzing T cell differentiation and polyfunctionality response against cytomegalovirus (CMV), an ubiquitous pathogen. Based on our research, when compared to same-age healthy individuals, ESRD patients are characterized by a significant loss of polyfunctional CMV specific CD4+ and CD8+ T cells, upregulation of effector phenotypic markers and increased surface PD-1 and TIM-3 expression. Furthermore, we performed transcriptome experiment and identified the functional impairment pathway that is distinct from T cell exhaustion and cannot be reversed by checkpoint blockade. These results give the new insight of designing future immunomodulation therapies.
Increased NKG2D expressing NK cells in patients with chronic HBV infection
Authors: Gao Y , He Wenjing , Lang Dou
Published: January 2023

Introductions

Hepatitis B virus (HBV) infection remains a significant health issue worldwide. It is a major cause of cirrhosis, HCC and liver failure. A large number of studies showed that host immune responses are the key factor for HBV clearance and persistence. In here, we investigated the role of NK cells in chronic HBV infections.

Methods

We compared the expression frequency, and functionality of NKGs and KIRs repertoire on NK cells in 170 samples by multi-colour flow cytometry. To further investigate the functionality of the NK cells, we performed K562 target cell killing assay and cytokine driven perforin released assay.

Results

The percentage of circulation and liver resident NK cells did not significantly alter in HBV patients compared to the healthy controls (HCs). Enrichment of NK cells was observed in the healthy controls but not in the HBV infected patients. The intracellular expression of Granzyme B was significantly reduced in NK cells derived from HBV-infected patients as compared to HCs. No differences were observed in the expression of NKG2A and NKG2C. Interestingly, the levels of NKG2D was significantly increased in the circulating NK cells of HBV infection but not in the liver resident NK cells. The K562 target cell killing ability and IFN-gamma production correlated with the NKG2D expression, particularly in the HBV patients as compared to HCs.

Conclusions

Our study demonstrated that NK cells activity and status might play an important role in controlling HBV infection.
Longitudinal immunomonitoring across the virome using a highly-multiplexed serological assay and citizen science
Authors: Engelbrektson Anna Lynn , Fink Zane , Ladner Jason , Altin John
Published: January 2023

Hundreds of viral species are known to infect humans. During a typical year, the average adult is naturally infected with ~10 new viruses, many asymptomatically, but most leaving a humoral footprint. The detection of viral exposures and the associated immune responses is useful for various purposes – clinical diagnostics, epidemiological surveillance, immunological profiling, and potentially as biomarkers or risk factors for complex, immune-related diseases –however, current assays are incommensurate with the diversity of the human virome. Here, we present a new approach to chart the diversity and dynamics of humoral anti-viral responses across 3 axes – pathogens, population and time. In the pathogen dimension, we used a novel platform for highly-multiplexed DNA-barcoded peptide-based serology (‘PepSeq’) to comprehensively assay blood samples for reactivity across a set of 244,000 30mer peptides designed to represent the proteomes and known diversity of all viruses known to infect humans. To survey the population and time dimensions, we used Citizen Science to recruit a cohort of participants for self-collected longitudinal blood samples. In a pilot study of 48 volunteers, each giving 10 weekly samples, we detect humoral reactivity against ~5–20 commonly-encountered viruses in each individual, and resolve viruses encountered during the sampling period from historical infections with high confidence. We expect that cost-effective, longitudinal and deep immunomonitoring across the virome will provide a powerful new view into immunological dynamics and the contribution of viral exposures to disease.
Defective macrophage mediated immune responses in HIV negative Mycobacterium avium complex (MAC) patients with pulmonary diseases
Authors: Jung Bock-Gie , Samten Buka , Brown-Elliott Barbara A , Wallace Richard J , Idell Steven , Wadle Carly , Philley Julie V , Vankayalapati Ramakrishna
Published: January 2023

Mycobacterium avium complex (MAC), which includes M. avium and M. intracellulare, is widely distributed in the environment, and exposure to these organisms is inevitable. Despite widespread exposure, the disease is unusual in immunocompetent hosts. During the past two decades, MAC represents a common cause of progressive pulmonary disease in the United States. To gain insight into the immunologic factors that predispose persons to MAC infections, we evaluated the immune responses of HIV negative (-) MAC patients with pulmonary diseases in comparison to their healthy HIV− household contacts (HHCs). Whole blood from 20 pairs of HIV− MAC patients and healthy HHCs was immunolabeled, and the percentages of various immune cell populations were determined by flow cytometry. Among various cell types, MAC patients have significantly lower ratio of monocytes compared to healthy HHCs. In 11 pairs of HIV− MAC patients and healthy HHCs, peripheral blood mononuclear cells (PBMC) of MAC patients produced lower levels of interleukin (IL)-10 and IL-1 family (IL-1β, IL-18, IL-1α) out of 34 cytokines and chemokines measured by multiplex ELISA upon MAC stimulation, compared with that of healthy HHCs. In two MAC patients, PBMC restricted MAC growth less efficiently compared with their healthy HHCs despite there was no deficiency in restricting MAC growth by monocyte derived macrophages of six MAC patients. Our findings suggest that the interactions among various immune cells are required for efficient clearance of MAC by macrophages in MAC patients. Studies are underway to pinpoint the nature of defective immune responses that prevents efficient clearance of MAC by macrophages of MAC patients.
Phenotypic changes of mucosal-associated invariant T (MAIT) cells during and after acute human sepsis
Authors: Labuz Daniel , Trivedi Shubhanshi , Anderson Cole , Araujo Claudia , Blair Toni , Middleton Elizabeth , Campbell Robert A. , Hale Scott , Rondina Matthew T. , Leung Daniel T.
Published: January 2023

Sepsis is a devastating syndrome that is characterized by organ dysfunction caused by a hyperinflammatory environment following a dysregulated immune response to an infection. Aside from the initial insult of sepsis, patients who survive are at risk of immunosuppression, likely due to the impact of sepsis on both innate and adaptive immunity. There remains a lack of effective interventions for sepsis, possibly due to incomplete understanding of its pathobiology. MAIT cells are innate-like T cells that possess features of both innate and adaptive immunity and are activated by microbial-derived compounds of the riboflavin synthesis pathway, restricted by the evolutionarily conserved receptor MHC class-1 related protein (MR1). We aim to study the role that MAIT cells play during and after acute sepsis. We obtained blood and isolated peripheral blood mononuclear cells from 12 paired septic patients during acute sepsis and 90 days after diagnosis of sepsis, and age and sex-matched healthy donor controls. Using flow cytometry, we see that acute septic patients’ MAIT cells are highly activated and show higher inhibitory receptor expression compared to healthy donors and convalescent patients’ MAIT cells. We show that acute septic patient’s MAIT cells produced significantly less interferon gamma, and lower IFNG expression compared to paired convalescent patients after ex vivo stimulation with E. coli. Further investigation into other effectors of MAIT cells is ongoing. Our data suggest that acute septic patients’ MAIT cells are highly activated and are dysfunctional upon stimulation with E. coli. The understanding of the biological role of MAIT cells during and after acute sepsis can provide a novel therapeutic strategy for septic patients.
Fc-bridged immune cell activation and its role in infectious diseases and autoimmune diseases
Authors: Dai Hongsheng , Zhang Tongwen , Caligiuri Michael A
Published: January 2023

Immunoglobulin G (IgG) is critical to both humoral and cellular immunity. Conventionally, protective functions of IgG against infection and cancer require utilization of both its Fab and Fc domains. IgG Fab binds specific antigen and Fc engages complement or various Fcγ receptors (FcγRs) expressed on immune cells. We recently discovered that the IgG Fc domain alone can play a critical role in the immune cell’s recognition and elimination of certain pathogens, in vitro and in vivo, without the need for the Fab domain of IgG. Many human pathogens, such as herpes simplex virus 1 (HSV1), human cytomegalovirus (HCMV), and Staphylococcus aureus (S.A), express proteins capable of binding IgG Fc (called FcBP hereafter to differentiate from FcγR). FcBPs bind the IgG Fc domain at a site that is distinct and non-overlapping with the site bound by FcγRs. We have demonstrated that a trimeric FcBP-Fc-FcγR complex is formed when the FcγR-bearing immune cell with its bound IgG recognizes, binds to and is therefore activated by the pathogen expressing FcBP. The IgG Fc domain serves as a bridge between the FcγR positive immune cells and the FcBP-bearing pathogens. We have named this process “Fc-bridged cell-mediated cytotoxicity”, or FcBCC. We have demonstrated that FcBCC (1) results in the in vivo elimination of HSV1; (2) prevents fatal HSV1 infection; (3) depends on IgGFc and the FcγR+ NK cell, but (4) does not depend on IgG Fab. Thus IgG Fc alone is necessary and sufficient for engaging both the pathogens and the FcγR-bearing immune effector cells in FcBCC. Other than infectious diseases, results showing FcBCC in some autoimmune diseases will also be presented. Research in my lab is supported by Southern Medical University startup fund.
Studying platelet-HIV interactions and potential to promote viral spread
Authors: Simpson Sydney , Singh Meera V. , Dewhurst Stephen , Maggirwar Sanjay
Published: January 2023

HIV-infected individuals experience persistent inflammation, which is associated with increased platelet activation. There is growing evidence that platelets contribute to immune responses, through the release of inflammatory granules and complex formation with other cells including monocytes and CD4+ T-cells, both of which are susceptible to infection. Previous studies have found evidence that HIV is engulfed by platelets. However, such studies were primarily conducted in vitro. It is important to study consequences of HIV-platelet interactions, and to ascertain whether or not they occur in patients.

We confirmed previous in vitro findings through co-culturing platelets from healthy donors, with HIV virions, and using electron microscopy identified virus-like particles inside platelets. Utilizing RNAscope in tandem with RT-PCR we created a timeline of virus-platelet associations and found viral genomic RNA remained associated with platelets for a prolonged period of time. Importantly, platelet-associated virus remained infectious, as platelets were able to spread infection to activated CD4+ T-cells in coculture experiments. Furthermore, we established a system to screen for HIV-positive platelets. We isolated platelets from cART-naïve viremic patients and performed super resolution microscopy and imagestream flow cytometry. This analysis revealed evidence of HIV-positive platelets in these infected individuals. In contrast, the amount of HIV-positive platelets was drastically, although not entirely, reduced following 3 months of cART treatment. Our findings suggest that platelet-virus interactions may be important in contributing to viral spread, and the development and maintenance of viral reservoirs.
Expression of the Src-family kinase LynA tunes myeloid-cell signaling and function
Authors: Brian Ben F , Nunez Myra , Freedman Tanya S
Published: January 2023

The activity of Src-family kinases (SFKs), which phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs) upon receptor ligation, is a critical factor regulating myeloid cell activation. The SFK Lyn has roles in activating myeloid cells, by phosphorylating ITAMs, and also in inducing suppressive signaling via phosphatase activation. Alternative splicing of lyn mRNA produces two proteins, LynA and LynB, and emerging evidence from our lab and others suggests that LynA and LynB interact with different proteins and preferentially activate different signal cascades. In macrophages LynA is uniquely susceptible to rapid ubiquitin-mediated degradation and functions as a rheostat that regulates ITAM signal strength. Phosphorylation of tyrosine 32 in the unique region of LynA preferentially targets LynA for polyubiquitination by the E3 ligase c-Cbl. In contrast to macrophages, mast cells express little c-Cbl. Upon activation, LynA is not rapidly degraded in mast cells, and SFK-mediated signaling is amplified relative to macrophages. Previous studies of LynA have been hampered by knockout models that lack both LynA and LynB. To understand the role LynA plays in immune development and activation, we used CRISPR-Cas9 gene editing to generate novel mouse strains that express either LynA or LynB. Preliminary studies in these mice have demonstrated novel roles for LynA. Notably, LynA−/− mice have an enrichment in splenic dendritic cells and alveolar macrophages. Thus, LynA is not only an important regulator of myeloid cell activation, tuning cell-specific signaling thresholds, but also helps regulate the function of these cells. Further studies will investigate the impact of LynA deletion in myeloid inflammation sensing.
6-Methyladenine may enhance immunostimulatory activity of bacterial genomic DNA
Authors: Tsukamoto Yumiko , Tamura Toshiki , Maeda Yumi , Miyake Kensuke , Ato Manabu
Published: January 2023

Background and Aim

CpG motifs in DNA sequences are recognized by TLR9 and activate immune cells. Bacterial genomic DNA (gDNA) has modified cytosine bases (5-methylcytosine (5mC)) and modified adenine bases (6-methyladenine (6mA)). It is well-known that 5mC inhibits immune activation by CpG DNA; however, it is still unclear whether 6mA inhibits immune activation by CpG DNA. In this study, we tried to examine whether the existence of 6mA affected the immunostimulatory activity of gDNA.

Materials and Methods

We focused on adenine methyltransferases (MTases) expressed by Mycobacterium bovis BCG (BCG). Each adenine MTases has specific target sites. Therefore, we used oligodeoxynucleotides (ODNs) with 6mA on the target sequences of MTases from BCG. We stimulated murine bone-marrow derived macrophages (BMDMs) with ODNs harboring 6mA or ODNs without 6mA. We then compared the immunostimulatory activity of ODNs with 6mA to ODNs without 6mA using. We also developed adenine MTase-deficient BCG. We analyzed whether 6mA on gDNA from BCG is involved in the immunostimulatory activity of BCG gDNA.

Results

Our results showed that 6mA located at the target sequence of mamA, an adenine MTase from BCG, enhanced IL-12p40 production from BMDMs stimulated with CpG DNA.

We also observed that gDNA from mamA-deficient BCG induced less IL-12p40 from BMDMs than gDNA from mamA-intact BCG.

Conclusion

We concluded from these results that adenine methylation on ODNs may enhance immune activity induced by bacterial gDNA.
MAP Kinase Phosphatase (Mkp)-1 Controls Type I Interferon (IFN) and PD-L1 Expression during Sepsis
Authors: Liu Yusen , Kirk Sean G. , Cash Charles J. , Batty Abel J. , Wang Xiantao , Murphy Parker R. , Li Jinhui , Ackerman Williams E. , Hafner Markus
Published: January 2023

Mkp-1 is a critical negative regulator of the inflammatory response and plays a crucial role for bacterial clearance in an animal model of sepsis. Mkp-1−/− mice exhibit augmented inflammation, increased bacterial burden, elevated organ damage, and increased mortality relative to Mkp-1+/+ mice following systemic E. coli infection. To understand the immunological functions of Mkp-1, we performed RNA-seq analysis with mouse livers. Under normal conditions, 357 genes were differentially expressed between Mkp-1+/+ and Mkp-1−/− mice. However, following E. coli infection > 5,400 genes displayed differential expression between the two groups. Among genes upregulated by E. coli infection and exacerbated by Mkp-1 knockout are IL-6, IL-10, as well as a large number of IFN-inducible genes including Mx1/2, Gbp2/3, Isg15, Usp18, Ifitm1-3, STAT3, and immune checkpoint protein PD-L1. qRT-PCR and Western blot analyses verified E. coli infection-induced PD-L1 expression and further exacerbation in Mkp-1−/− mice. Examination of the RNA-seq dataset also revealed a clear interferon signature. ELISA analyses on the serum revealed low IFN-β levels in uninfected Mkp-1+/+ and Mkp-1−/− mice, increased IFN-β levels in E. coli-infected Mkp-1+/+ mice, and further augmented IFN-β levels in E. coli-infected Mkp-1−/− mice. Similarly, Mkp-1−/− bone marrow-derived macrophages also produced more IFN-β than Mkp-1+/+ macrophages following stimulation with either LPS or heat-killed E. coli. Analysis of the signaling pathways critical for IFN-β expression indicates a marked increase in TBK1 activity. Our studies suggest that exaggerated expression of type I interferons and PD-L1 contribute to the phenotypes of Mkp-1−/− mice in the model of sepsis.
The study of a dendritic cells-based standardized evaluation system for immunity-boosting functional foods
Authors: Kwon Jaeyul , Kim Ye-Eun , Sun Pureum , Go Eunbyeol , Heo Bu Yeon , Choi In-Wook , Han Bok Kyung , Kwon Bo-In , Lee Young-Ha , Kang Seong Wook
Published: January 2023

Dendritic cells (DC) are specialized antigen-presenting cells that initiate and orchestrate the body’s immune response to bridge the innate and adaptive systems. Here we report developing a standardized evaluation system for immunity-boosting functional foodstuffs using bone marrow-derived dendritic cells. A DC-based evaluation system can indicate the type, magnitude, and specificity of immune responses elicited by an immune-boosting food item. We monitored the expression profile of DC maturation markers, including CD11b, CD11c, CD40, PD-L1/L2, CD80, CD86, and MHC, by flow cytometry to define immunity indexes. These indexes are then consolidated to a 4-point scale to describe the immunogenic and tolerogenic capabilities of the immunity-boosting food candidate. Among well-known immunoregulatory materials, red ginseng extracts, fucoidan, and polysaccharide-K were found to belong to the highest grade of immunogenicity, while extract of the Ostericum root was among the most tolerogenic foods. Extracts of Peucedanum japonicum Thunberg root and Psidium guajava L. leaves also showed high tolerogenic capabilities. Our results suggest that this assay is an effective method to characterize and screen the immune boosting effects of food and pharmaceutical materials in a standardized manner.
Integrin αv limits TLR signaling in plasmacytoid dendritic cells
Authors: Lorant Alina K , Lacy-Hulbert Adam
Published: January 2023

SLE is a chronic, systemic autoimmune disease affecting millions of people worldwide. SLE is caused by a breakdown of tolerance to self-derived nucleic acids (NA) and characterized by the presence of autoantibodies to DNA/RNA. Plasmacytoid dendritic cells (pDCs) are highly tuned to respond to NA and express a range of receptors for RNA and DNA, including TLRs 7 and 9. Following TLR engagement, pDCs produce high levels of type I interferon (IFN-I), which promotes loss of tolerance, implicating pDCs in development of autoimmunity. However, relatively little is known about how innate immune signaling is regulated in pDCs, and particularly how they distinguish self-derived from foreign NA.

TLRs 7 and 9 are both localized in endosomal compartments, and their NA ligands must be internalized and processed before they can bind. One source of these NA is dying cells and loss of their receptors is associated with SLE. We studied the role of an apoptotic cell receptor, Integrin αv, in TLR signaling of pDCs and found that αv limits IFN-I production. In mice without αv, there is a systemic increase in IFN-I and inflammatory cytokines after TLR7 stimulation. This corresponds with an increase in pDC activation and the number of IFN-I-producing pDCs in the spleen. αv-knockout pDCs show increased TLR signaling and produce more IFN-I and inflammatory cytokines when stimulated in culture.

These data indicate αv has a pDC-intrinsic role in limiting TLR signaling, which we propose is through activation of non-canonical autophagy. Based on these data, we hypothesize that co-receptors involved in capture and internalization of nucleic acids provide contextual ‘cues’ to regulate TLR signaling and prevent autoimmunity.