bluuebunny/crossref_metadata_2025 huggingface dataset:
No change in HIV cellular burden and lower ADCC despite increased immune activation after combined immunotherapy with peg-IFN-α2b and two broadly neutralizing antibodies in suppressed HIV+ subjects
Authors: Papasavvas Emmanouil , Morris Jessicamarie , Ross Brian , Fair Matthew , Giron Leila , Azzoni Livio , Abdel-Mohsen Mohamed , Mounzer Karam , Kostman Jay , Tebas Pablo , Montaner Luis
Published: November 2024

Background: Pegylated interferon α2b (peg-IFN-α2b) and the broadly neutralizing antibodies (bNabs) 3BNC117 and 10-1074 were individually shown to increase HIV suppression during antiretroviral therapy (ART) interruption (ATI). We evaluated the anti-HIV and immune effect of combined immunotherapy with peg-IFN-α2b+bNAbs in persons with HIV infection (PWH).

Methods: Peripheral blood mononuclear cells and plasma from 14 ART-suppressed PWH were assessed at baseline (ART), ART+4 weeks of peg-IFN-α2b, and ATI+26 weeks of peg-IFN-α2b+bNAbs immunotherapy for: 1) intact HIV DNA, 2) NK constitutive, and in vitro gp120 or IFN-α-induced antibody-dependent cell-mediated cytotoxicity (ADCC) and direct cytotoxicity, and 3) immune activation.

Results: Immunotherapy did not alter cell-associated intact HIV DNA levels nor IFN-α-induced NK direct cytotoxicity. Yet, it decreased gp120-induced ADDC, and the percentage (%) of CD56lo/+CD16+ on CD56+ while increasing the % of CD56hi and CD56lo/+CD16- on CD56+ suggesting that decreased expression of CD16 (Fc receptor) on NK could result to lower ADCC. Immunotherapy also decreased CD57 expression on CD56lo/+CD16+, while increased Tetherin, CD38, HLA-DR, NKp46, and CD169 on immune cells, and pro-inflammatory bisected GlcNAc glycans on bulk IgG.

Conclusion: Decreased ADCC and circulating CD16+ NK cell subsets despite increased immune activation may explain the lack of change in HIV cell burden after Peg-IFN-α2b+bNAbs administration in PWH.
Longitudinal analysis of immunological response to vaccination
Authors: Chenchik Alex , Kobzik Lester , Liu Tianbing , Hu Dongfang , Makhanov Mikhail , Paraiso Kitt , Ghias Khadija , Diehl Paul
Published: November 2024

This study explores the dynamic nature of the immune responses to vaccination with Td (Tetanus-diphtheria), PPSV23 (pneumococcal), and Shingrix (herpes zoster) vaccines. We performed detailed transcriptomic analysis, including genome-wide expression profiling, adaptive immune receptor (AIR) profiling, and single-cell transcriptome analysis of sorted T & B cells. We collected whole blood samples from a single donor at multiple time points before and after vaccination. RNA and DNA were extracted, and the DriverMap™ genome-wide targeted expression profiling (EXP) assay and AIR profiling were run for both T & B cell receptors from RNA and DNA. NGS results were analyzed using the MiXCR pipeline (AIR panel) and Salmon analysis (EXP panel). Whole transcriptome pathway enrichment analysis showed innate immune response with significant activation of interferon and cytokine signaling on Day 2 after immunization, followed by activation of adaptive immunity on Days 7-10, and return to baseline levels by Days 15-22. Response to vaccinations involved both TCR and BCR signaling with significant activation of immunoglobulin clonotypes specific for vaccine antigens. We characterized specific cell types responsible for adaptive immune responses through T/B cell sorting experiments and 10x analysis. This study highlights the importance of integrated transcriptome, immune repertoire, and single-cell profiling analysis to understand the complex interplay of innate and adaptive immune mechanisms.
Yersinia pestis NLP subunit vaccine platform: Protective efficacy and immunological profile
Authors: Davies Michael , Dankmeyer Jennifer , Rill Nathaniel , Klimko Christopher , Shoe Jennifer , Hunter Melissa , Talyansky Yuli , Qiu Ju , Rasley Amy , Fischer Nicholas , Cote Christopher , Biryukov Sergei
Published: November 2024

Yersinia pestis (Yp) is the etiological agent of plague, a disease that remains a concern as demonstrated by recent outbreaks in Madagascar. Infection with Yp results in a rapidly progressing illness that can only be treated with antibiotics. Live attenuated or whole-cell inactivated vaccines confer short-lived protection against bubonic but not pneumonic plague. Subunit vaccine formulations may circumvent some of these shortfalls. Here we compare the immunogenicity generated by the most advanced subunit vaccine (rF1V) against a nanolipoprotein particle (NLP)-based vaccine. BALB/c mice were immunized twice, four weeks apart. Four weeks later, splenocytes and sera were collected for immune profiling and mice were aerosol challenged with Yp CO92. Both formulations induced a strong IgG response against F1 and V proteins, along with a robust memory B cell response and cell-mediated response with Th1- and Th2-related cytokines. NLP-based vaccine induced a stronger cytokine response against F1, V, and F1V proteins relative to the rF1V vaccine. As with rF1V, the inclusion of Alhydrogel in NLP vaccine formulations was critical for enhanced immunogenicity and protective efficacy. Addition of Francisella tularensis antigens to the Yp NLP vaccine platform did not impair vaccine efficacy, based on protection and antibody response. The modularity and lipid bilayer structure of NLPs allows for incorporation of many protective antigens with variable ratios against multiple pathogens.
A non-canonical mechanism of antiviral activity of Oligoadenylate Synthetase 1
Authors: Harioudh Munesh , Perez Joseph , Chong Zhenlu , Nair Sharmila , So Lomon , McCormick Kevin , Ghosh Arundhati , Shao Lulu , Srivastava Rashmi , Soveg Frank , Ebert Thomas , Atianand Maninjay , Hornung Veit , Savan Ram , Diamond Michael , Sarkar Saumendra
Published: November 2024

The translational arrest of protein synthesis is often a cellular response to the virus infection. However, some antiviral proteins continue to be translated through unknown mechanisms during this translational shutdown. Here, we report a mechanism by which antiviral effectors are upregulated during this translational shutdown. We found that interferon (IFN) stimulated gene, Oligoadenylate Synthetase 1 (OAS1), binds AU-rich elements (ARE) of specific mRNA, including IFNβ, prolonging the half-life and continued expression. This increased IFN expression protects from WNV infection in vitro and in vivo via downstream IFNAR signaling. This mechanism is common between human OAS1 and mouse Oas1b, independent of OAS enzyme activity and RNase L. However, human OAS1 inhibits SARS-CoV-2 replication through its canonical enzyme activity via RNase L, thus establishing two different mechanisms of OAS1 antiviral activity. These results establish OAS1 as an ARE-binding protein with a broader non-canonical function that protects IFN expression from translational shutdown.
Macrophage galactose-type lectin modulates host immunometabolism during Mycobacterium tuberculosis infection
Authors: Paez Reina , Chauhan Sadhana , Naqvi Kubra , Puebla-Clark Lucinda , Gelman Benjamin , Martinez-Martinez Yazmin , Prideaux Brendan , Endsley Janice
Published: November 2024

Macrophages (Mφ) are the primary replicative niche for Mycobacterium tuberculosis (Mtb), a prominent bacterial pathogen responsible for nearly 1.5 million deaths annually. Appropriate metabolic reprogramming and activation of these cells is a major determinant of host-pathogen interactions and control of infection. Macrophage galactose-type lectin (MGL) is an immunomodulatory CLR primarily expressed on myeloid cells. We previously demonstrated a host-protective role for MGL in Mtb infection, with MGL-1 deficient (MGLKO) mice exhibiting increased bacterial burden and production of pro-inflammatory cytokines. Additionally, more lipid-laden cells were observed in the vicinity of granulomatous TB lesions in MGLKO mice, as compared to WT littermates. Extracellular flux analysis further revealed baseline differences in Mφ metabolism, with MGLKO BMDM exhibiting a glycolytic bias, consistent with the pro-inflammatory bias in vivo. LC-MS/MS lipidomic analysis of Mtb-infected whole-lung homogenate and BMDM revealed significant differences in lipid species between MGLKO and WT, with greater accumulation of DHA and other LCPUFA species observed in MGLKO samples. Importantly, these lipids were also observed to be concentrated within TB lesions of MGLKO and WT mice via mass spectrometry imaging (MSI), highlighting the relevance of these findings to disease outcomes. Taken together, we identify a novel role for MGL as a determinant of host immunometabolic response to Mtb infection.
Two immunodominant antigens from Madurella mycetomatis were identified by IgG antibodies in infected mice
Authors: Espinoza Martínez Pablo , Salinas Carmona Mario , Vázquez Marmolejo Anna , Vera Cabrera Lucio , Solís Marfil Pedro
Published: November 2024

Madurella mycetomatis is the most frequent etiological agent of eumycetoma, a chronic infectious disease that is acquired by traumatic inoculation of the microbe, mainly in the lower limbs. It usually affects young people between the ages of 15 and 30 and its treatment is complicated, involving surgical approach and chronic use of antifungals. To date there is lack of accessible immunological test for the diagnosis of this disease. In this work we obtained protein antigens from the cellular extract of M. mycetomatis, which can be used in the design of an immunodiagnostic test. 8-12 week old female BALB/C mice were infected with a suspension of M. mycetomatis using 3 different inoculation routes: intraperitoneal, dorsal subcutaneous and foot pad. Crude cellular extract of M. mycetomatis, protein antigens were analyzed on an SDS-PAGE gel, which were recognized by serum of mice infected with M. mycetomatis using the Western blot technique. The IgG anti-M. mycetomatis identified 2 immunodominant antigens were recognized by antibodies in all infected mice with M. mycetomatis. The identification of these antigens in sera from patients with eumycetoma has diagnostic potential.
Ifit2 interacts with murine-β-coronavirus RSA59 nucleocapsid protein to restrict its replication and spread
Authors: Sharma Madhav , Sarma Jayasri
Published: November 2024

The antiviral efficacy of Ifit2, an interferon-induced protein with tetratricopeptide repeats 2, is known for its ability to restrict the neurotropic murine-β-coronavirus, RSA59 infection. Intracranial injection of RSA59 in Ifit2 deficient (-/-) compared to wild-type (WT) mice results in impaired microglial activation and peripheral lymphocyte infiltration into the brain with severe morbidity and mortality in the acute phase of the disease. RSA59-infected Ifit2-/- mice also showed extensive viral replication, spreading throughout the spinal cord with impaired microglial activation and T-cell infiltration. Cervical-lymph-nodes of RSA59-infected Ifit2-/- mice exhibited reduced activation of CD4+ T cells and impaired IFNγ expression while preserving the blood-brain barrier integrity. To understand Ifit2's impact on the chronic demyelination phase, Ifit2-/- and Ifit2+/+ (WT) mice were observed for neuropathological outcomes upon RSA59 infection. The chronic disease phase in RSA59-infected Ifit2-/- mice demonstrated severe demyelination and persistent viral load. Furthermore, understanding molecular interactions in a reductionist approach using co-immunoprecipitation showed that upregulated Ifit2 in RSA59-infected primary astrocytes can interact with viral nucleocapsid protein. Thus, this study highlights that Ifit2's direct interaction with viral nucleocapsid protein can restrict RSA59 spread throughout the CNS in the acute phase, thus limiting severe chronic demyelination.
THE ROLE OF THE VIRAL PROTEIN VPR IN THE EPIGENETIC REGULATION OF HIV-1 LATENCY
Authors: Lewis Catherine , Browne Edward
Published: November 2024

The primary obstacle to HIV-1 cure is the persistence of a long-lived latent reservoir, best characterized in memory CD4+ T cells. New research supports latency prevention as a therapeutic strategy. However, such an approach will require a better understanding of how HIV-1 establishes latency. We recently showed that histone deacetylases (HDACs) are critical for entry into latency. Whether HDACs interact with specific viral factors, particularly the HIV-1 accessory protein and coactivator Vpr, to regulate latency establishment is unknown. We therefore sought to determine how Vpr, both alone and in combination with the HDAC inhibitor vorinostat (VOR), regulates HIV-1 latency establishment. In a primary CD4+ T-cell model, we observed a p300-dependent synergy between Vpr and VOR in maintaining viral gene expression. We next assessed whether Vpr delivery during latent infection, a stage when viral proteins are not normally expressed, could enhance the ability of VOR alone to induce viral gene expression. We found that Vpr delivery reactivated latently infected CD4+ T cells to a greater extent than VOR treatment alone and that the combination of Vpr delivery and VOR treatment led to the highest level of reactivation. An improved understanding of how host and viral factors interact during HIV-1 latency establishment in CD4+ T cells will aid in the development of new therapies that prevent HIV-1 entry into the latent reservoir or that reverse latency in latently infected cells.
ZBP1 regulates cytokine production and cell death in lung fibroblasts following influenza virus infection
Authors: Jordan Summer , Boyd David , Gautam Avishekh , Balachandran Siddharth
Published: November 2024

Regulated cell death following influenza viral infection is an important determinant of disease severity and is required for effective viral clearance. However, extensive cell death can lead to severe tissue damage and compromised lung function. Z-DNA binding protein-1 (ZBP1) is a critical sensor of influenza A virus (IAV) that can activate both apoptotic (non-inflammatory) or necrotic (inflammatory) cell death pathways. The cell type-specific effects of ZBP1 activation during influenza virus infection have not been clearly defined. Here, our single-cell gene expression studies identified lung fibroblasts as a cell type expressing high levels of Zbp1 following IAV infection in vivo. Following infection in vitro, we observed that ~20% of primary lung fibroblasts were readily infected by IAV but did not support high levels of spreading infection. Lung fibroblasts from wildtype mice underwent cell death with ~50% reduction in cell survival at 24 hours while lung fibroblasts from Zbp1 knockout mice were almost completely resistant to cell death. Analysis of cytokine production prior to cell death demonstrated that IAV infection in lung fibroblasts triggered an early ZBP1-dependent production of inflammatory proteins. Knockout of ZBP1 reduced inflammatory cytokine/chemokine production, but left interferon pathways intact. These studies identify lung fibroblasts as a key cell type activating Zbp1-mediated cytokine production and cell death during influenza virus infection.
Distinct roles for interferon-α and interferon-β in recovery from Sindbis virus encephalomyelitis
Authors: Nguyen Benjamin , Stanley Elise , Chen Emily , Griffin Diane
Published: November 2024

Alphaviruses are viruses that can cause encephalomyelitis, which is inflammation of the brain and spinal cord due to the immune response to viral infection. Neurons are the primary target cells of Sindbis virus (SINV), the prototypic alphavirus. Interferon (IFN) regulatory factor (IRF) 7 is a transcription factor important for amplification of type I IFN and is required for survival from SINV infection. Irf7-/- mice do not produce IFNα and rapidly succumb to infection – despite local production of IFNβ – while wild type (WT) mice recover. Treatment of Irf7-/- mice with IFNα increases survival and promotes viral clearance, suggesting that IFNα is required for recovery, and further, myeloid-specific expression of IRF7 is necessary for survival. Additionally, Ifnb-/- mice do not phenocopy Irf7-/- mice, further suggesting that IFNα is critical for survival while IFNβ is dispensable despite usage of the same receptor. Interestingly, Ifnb-/- mice have similar peak viral titers to Irf7-/- mice but clear virus with kinetics similar to WT mice, implying that the protective effects of IFNα go beyond restriction of viral replication. Irf7-/-Ifnb-/- double knock-out mice have disease similar to Irf7-/- mice but with earlier onset, suggesting an immunomodulatory role for IFNβ in delaying clinical disease. These findings show subtype-specific differences in protection by type I IFN and can inform treatment options for more severe forms of alphavirus-induced encephalomyelitis.
Co-evolution of antibody responses against human immunodeficiency virus (HIV-1) envelope
Authors: Arora Juhi , Birungi Kevina , Schramm Chaim , Johnston Timothy , Henry Amy , Nortelus Sarah , Jacob Sari , Zemil Michelle , Kavusak Erin , Rao Mekhala , Smith Gabriel , Lewitus Eric , Bose Meera , Sanders-Buell Eric , Rolland Morgane , Polonis Victoria , Douek Daniel
Published: November 2024

Broadly neutralizing antibodies (bNAbs) target epitopes on the Envelope (Env) glycoprotein of HIV-1 and neutralize diverse strains with high breadth and potency. Studying the ontogeny of bNAb development will help identify better immunogens for vaccine design. RV217 is an acute infection cohort with samples collected prior to seroconversion and continued 2+ years following detection of viral RNA (RNA+). From participant 40512, living with CRF01_AE infection, we isolated several bNAbs at day 646 post-RNA+ that targeted the membrane proximal external region (MPER) on the HIV Env. These included the VRC42 lineage, with neutralization breadth of 97% on multi-subtype pseudovirus panels. Sequence analysis revealed sequential mutations in the HIV-1 Env, indicating virus evolution. The participant acquired another CRF01_AE HIV-1 infection (i.e., superinfection) between days 330-401 post-RNA+, increasing antigenic diversity, and continued to develop breadth to 98% at day 1142 post-RNA+. Using rapid amplification, transfection, and production of immunoglobulins (RATP-Ig), we sorted MPER+ B cells and isolated an additional 22 VRC42 lineage mAbs at day 1142 post-RNA+. Interestingly, origins of the 4 sublineages were traced back to day 154 post-RNA+. These mAbs continued to evolve out to day 1142 and showed greater activity against late autologous viruses, suggesting that co-evolving HIV-1 Env and antibodies led to development of neutralization breadth.
First lung infection permanently enlarges LN and enhances new T cell responses
Authors: Stolley Michael , Masopust David
Published: November 2024

Humans experience about four respiratory infections per year. Immunology and vaccinology studies in mice are typically performed in naïve specific pathogen free (SPF) animals responding to their very first respiratory challenge. We found that the first respiratory infection induces lifelong enlargement of lung-draining mediastinal LNs. Furthermore, infection-experienced medLNs supported better naïve T cell surveillance and effector responses to new unrelated infections and engendered more biased accumulation and memory establishment within the lung. Moreover, we observed that weight loss induced by influenza infection was substantially reduced in mice that had recovered from a previous unrelated respiratory viral challenge. These data show that the lack of infectious history in SPF mice alters their immune response to lung infections. Preclinical vaccination and immunology studies should consider the previous infectious experience of the model organism.
Glycolysis and HIF-1α signaling are important for granulocytic myeloid-derived suppressor cell activity during Staphylococcus aureus biofilm infection
Authors: Arumugam Prabhakar , Horn Christopher , Roy Zachary , Heim Cortney , Fallet Rachel , Bertrand Blake , Shinde Dhananjay , Thomas Vinai , Romanova Svetlana , Bronich Tatiana , Hartman Curtis , Garvin Kevin , Kielian Tammy
Published: November 2024

Prosthetic joint infection (PJI) is a severe complication following hip and knee arthroplasty and is reported to affect 1-2% of total arthroplasties. Staphylococcus aureus (S. aureus) contributes to most PJIs, with a higher treatment failure rate partly due to biofilm formation. Granulocytic myeloid-derived suppressor cell (G-MDSC) expansion and IL-10 secretion are critical for inducing an immunosuppressive environment that supports S. aureus persistence during PJI. Recently, S. aureus-derived lactate was shown to promote IL-10 production by G-MDSCs. However, the importance of G-MDSC metabolism in programming their anti-inflammatory activity is unknown. Single-cell RNA sequencing (scRNA-seq) of infected tissues from a mouse model of S. aureus PJI revealed enrichment of glycolysis and hypoxia pathway genes in G-MDSCs. Increased glycolytic activity in G-MDSCs was confirmed following S. aureus biofilm co-culture and attenuating glycolysis with either 2-deoxyglucose or granulocyte-specific HIF-1a deletion reduced bacterial growth during S. aureus PJI. These changes coincided with less G-MDSC suppressive activity and a transformation towards a proinflammatory phenotype. scRNA-seq of samples from PJI patients revealed heightened glycolysis and hypoxia signatures, supporting findings with the mouse PJI model. Together, these results suggest that glycolysis and HIF-1a pathways are critical for the anti-inflammatory activity of G-MDSCs during S. aureus biofilm infection.
Natural IgM Maintains Ocular Homeostasis into Old Age
Authors: Webster Sarah , Les Sydney , Heck Daken , Tsuji Naomi , Clemente Michael , Jones Prentiss , Holodick Nichol
Published: November 2024

The eye is designed to properly focus light through optically clear structures, the cornea and lens, at the anterior onto photosensitive nervous tissue, retina, at the posterior for vision. Maintaining these tissues is critical, but the integrity of eye tissues is often lost with age, leading to disease. Although the eye is immune privileged, lymphocytes may enter retinal tissue under certain circumstances, usually in disease. Natural antibodies, such as natural IgM, are produced by B cells without infection or immunization and maintain tissue homeostasis. We found that vitreous humor contains IgM and IgG. We examined mice deficient in secreted IgM (ms-/-) and found young mice developed cataracts and mild retinal degeneration, which was enhanced with age. Examination of aged ms-/- mice revealed significant reactive gliosis by the resident Müller glia and increased microglia infiltration into retinal layers. The retina of aged ms-/- mice displayed an increased pro-inflammatory cytokine profile and increased retinal death resulting from apoptosis. We found B cells localized to the retinal pigment epithelium responsive to LPS stimulation and display a diverse repertoire influenced by age and lack of IgM. These data demonstrate a critical role for natural IgM in maintaining ocular homeostasis, which preserves anterior clarity and posterior retinal health. A better understanding of intraocular maintenance will enable the development of therapeutics to support vision into old age.
Characterizing the mechanism of catecholamines transport in the Mesenteric lymph Node (MLN) using Fast Scan Cyclic Voltammetry (FSCV).
Authors: Ojha Sarbeshwar
Published: November 2024

Neuroimmune communication is one of the key components in regulating the body’s response to physiological challenges and maintaining homeostasis while facilitating immune stress response. Within the gastrointestinal tract, the mesenteric lymph nodes play a key role in governing the immune response, and this regulation involves communication between immune cells and neural components.Sympathetic neurons are thought to co-release norepinephrine and dopamine to modulate cytokine signaling during inflammation and therefore measurements of these neurochemicals in the lymph node are critical for understanding immunity. Here, we have expanded our electrochemical waveform and way of performing the experiment in ex vivo lymph node slices to study how these neurochemicals communicate in the lymph node during different conditions. We have also tried blocking the different neurotransmitter transporter with different antagonist drugs for different catecholamines to see the differential change and came to know that these transport blockers have significant effect on holding the neurochemical in the extracellular space when we block their transporter.

This work presents the first-time detection on role of different transport blocker and their impact in the mechanism of catecholamine release in the MLN on a millisecond timescale, which will provide significant insight into the mechanisms of neuron-immune communication and different drug related studies.​
A20 is a negative regulator of both NF-κB and JNK signaling following T cell receptor stimulation in humans
Authors: Dabbah-Krancher Gina , Ruchinskas Allison , Snow Andrew
Published: November 2024

A20 is a ubiquitously expressed, inducible ubiquitin-editing enzyme that restrains NF-kB-dependent inflammatory signaling throughout the body. In T cell receptor (TCR) signaling, the molecular mechanisms utilized and their ultimate impact on human T cell function remain unclear. TCR engagement triggers the assembly of the CARD11-BCL10-MALT1 (CBM) protein complex, a signaling platform that governs the activation of downstream transcription factors including NF-kB and c-Jun partner AP-1. Utilizing WT and A20KO Jurkat T cells, we demonstrate A20 is required to negatively regulate both NF-kB and JNK/AP-1 signaling. By employing a novel set of A20 mutant constructs with NF-kB and AP-1 driven reporter systems in Jurkat transfectants, we discovered the ZnF7 domain is essential for CBM localization and signal modulation. We confirmed these findings in CRISPR-mediated A20 knockout human primary effector T cells, demonstrating sustained TCR-induced NF-kB and JNK signaling, enhanced upregulation of activation markers, and increased cytokine secretion associated with a skewed Th9/Th17 phenotype. Moreover, loss of A20 altered apoptosis sensitivity in primary human T cells, resulting in decreased restimulation induced cell death (RICD) and increased cytokine withdrawal induced death (CWID). These findings further elucidate the mechanism of A20-dependent regulation of both NF-kB and c-Jun in T cells, and highlight the functional importance of A20 in maintaining adaptive immune homeostasis.
Sequential regulation of Lck K63-ubiquitination and autophosphorylation by the E3 ligase UBR2 and the phosphatase DUSP22 in TCR signaling
Authors: Tan Tse-Hua , Chuang Huai-Chia
Published: November 2024

Dual-specificity phosphatase 22 (DUSP22, also named JKAP) downregulation in peripheral blood T cells is correlated with human SLE nephritis and T-cell leukemia. DUSP22 also dephosphorylates and inactivates Lck in the turn-off stage of TCR signaling, leading to suppression of autoimmune disease.

We found that DUSP22 dephosphorylated the E3 ligase UBR2, leading to SCF E3 ubiquitin complex-mediated UBR2 ubiquitination and degradation. Remarkably, UBR2 induced Lck K63-linked ubiquitination, leading to subsequent Lck Tyr394 autophosphorylation and activation upon TCR signaling. Thus, DUSP22 also can inactivate Lck indirectly by inducing UBR2 degradation. Moreover, the proinflammatory cytokine induction and inflammatory responses in DUSP22 KO mice was blocked by knocking out UBR2 in DUSP22/UBR2 double KO mice. The UBR2-Lck interaction and Lck Lys63-linked ubiquitination were indeed induced in T cells of human SLE patients. Taken together, DUSP22 dephosphorylates UBR2 and induces its degradation, leading to inactivation of Lck and suppression of inflammation. Overall, our results reveal a novel activation mechanism of Lck by K63-linked ubiquitination in TCR signaling.
Mechanism of Treg conversion to exTreg in atherosclerosis
Authors: Lyu Qingkang
Published: November 2024

Foxp3+ regulatory T cells arise from precursors in the thymus and can be induced from naïve CD4 T cells. They are indispensable for maintaining self-tolerance and limiting collateral damage during an immune response. Under inflammatory conditions, Treg cells acquire plasticity and instability, ultimately leading to a loss of CD25 and FoxP3. These exTregs lose suppressive function. exTregs have been reported to express lineage-defining transcription factors of helper CD4 T cell like Th1, Th2, Th17 or Tfh cells. In mouse models of atherosclerosis, conversion of Tregs to exTregs is accelerated. The molecular mechanism of Tregs conversion to exTregs is unknown. Previous studies have shown that plasma IL-6 was increased in atherosclerosis. The IL6 receptor heterotrimer IL6R (CD126) and gp130 (CD130) is expressed in Treg cells. We hypothesized that IL-6 may play a role during the conversion of Treg to exTreg. Using Treg and exTreg cell lineage-tracker mice (FoxP3eGFP−Cre-ERT2 ROSA26CAG-fl-stop-fl-tdTomato) on the Apoe-/- background, we are able to distinguish Treg from exTreg that lost Foxp3 expression in mice model of atherosclerosis. We established an in vitro culture system for Treg cells based on bead-bound anti-CD3 and anti-CD28. Preliminary data showed that IL-6 dramatically increased the proportion of exTreg cells in vitro. These data suggest that IL-6 may promote Treg to exTreg conversion.
3D extension of spatial patterning analysis of cellular ensembles (SPACE) to extract parameters for dynamic tumor modeling
Authors: Sreejithkumar Vivek , Schrom Edward , Germain Ronald
Published: November 2024

Investigating the cellular interactions within three-dimensional (3D) tumor microenvironments is essential for understanding tumor growth and progression. Building upon the success of our previous computational framework, SPACE, designed for spatial analysis of high-plex tissue images, we are developing 3D-SPACE. This innovation extends spatial pattern analysis to 3D volumetric tissue images. Volumetric images more accurately capture nuances of cellular arrangements and tissue structures, offering a more realistic representation of the tumor microenvironment.

Applying 3D-SPACE to analyze full 3D tumor datasets has the potential to reveal crucial parameters for understanding tumor outcomes. This approach not only elucidates previously undiscovered spatial patterns but also allows for the quantification of parameters such as contact probabilities between different cell types or the killing efficiencies of key effector cells in situ. These spatially-derived parameters can then be used in predictive equations that model and forecast tumor growth based on cellular interactions.

Using a 3D tumor model and 3D-SPACE, we can quantify cell interactions and develop predictive equations for tumor growth dynamics, with the aim of deriving a predictive reproduction number (R₀) for tumor growth. Working towards a predictive R₀ for tumor growth offers a valuable approach for predicting tumor growth outcomes.

This work was supported in part by the Intramural Research Program of NIAID, NIH.
Label-free morphological profiling and isolation of immune cell subsets using VisionSort, a novel, AI-based flow cytometry platform.
Authors: Schneider Greg , Wagatsuma Keisuke , Tsubouchi Asako , Packiasamy Juliet , Uematsu Mika , Nomaru Hiroko , Teranishi Kazuki , Akai Satoru , An Yuri , Komoriya Kaoru , Kawamura Yoko , Ota Sadao
Published: November 2024

Identification, characterization, and minimally invasive isolation of specific populations of human immune cells are critical for understanding and treating disease. Here we present data on label-free identification of three immune cell subsets by morphological profiling using VisionSort; a label-free, artificial intelligence (AI)-driven cellular analysis and sorting platform. By capturing single-cell digital phenotypes, we characterized mouse T-cells and generated ‘ground truth’ functional profiles for activated and non-activated T cells. A set of machine-learning derived classifiers was generated to identify these phenotypic classes in unlabeled T-cell subsets. The classifier showed an area under the curve (AUC) performance for differentiating between phenotypically defined T cell populations of 0.917. In addition, by using unsupervised machine learning, we were able to resolve activated and non-activated T cell populations label free, using morphological data alone. Using a similar approach, we show label-free differentiation/classification of B cells from plasma cells with an AUC score of 0.941 and M1 and M2 polarized macrophages with an AUC score of 0.878 +/- 0.002 (n=6). Here we report results on the use of a novel, label-free cytometry platform to characterize and isolate human immune cell subsets using morphological profiling and AI with applications for investigators in basic life sciences and drug developers in small molecule, antibody, and cell therapy R&D.
Single-cell full-length TCR/BCR profiling assay with high sensitivity and CITE-seq compatibility enables deeper immune cell characterization
Authors: Genero Gracia , Han Zhiyuan , Chen Lin , Gordon Chelsea , Vadrevu Samatha , Sakofsky Cynthia , Li Ruifang , Wang Larry , Padmanabhan Raghavendra , Jensen Devon , Ayer Aruna
Published: November 2024

Single-cell full-length TCR/BCR assays provide the ability to profile the highly diverse T cell receptor (TCR) and B cell receptor (BCR) repertoires of the adaptive immune response. The identification of T cell clonotype can be challenging due to insufficient sensitivity to detect low abundance TCR transcripts. Furthermore, cell type identification can be limited using only transcriptomic profiles.

Here, we present data with high resolution of TCR clonotype using a new highly sensitive TCR/BCR assay combined with CITE-seq using the BD™ AbSeq Assay. We profiled human and mouse T cells alongside whole transcriptome analyses with and without CITE-seq. The enhanced assay showed high clonotype detection with transcriptome-only profiling and with CITE-seq. Results also showed high gene expression correlation (R2>0.97), consistent library quality and mapping, and better resolution of cell subtype clustering with CITE-seq. Compared to transcriptome-only profiling, the coupling of surface protein expression with transcriptomic and clonal information of each cell provides deeper insights of the immune repertoire and enhances detection of cell subsets within a complex population. The results demonstrate the utility of our improved immune profiling assay with highly sensitive TCR/BCR detection for in-depth multiomics analyses of single cells.

For Research Use Only. Not for use in diagnostic or therapeutic procedures. © 2023 BD. All rights reserved.
Direct cloning of novel and specific human neutralizing anti-interferon-alpha or anti-interferon-beta antibodies
Authors: Papasavvas Emmanouil , Lu Lily , Fair Matthew , Oliva Isabela , Cassel Joel , Majumdar Sonali , Mounzer Karam , Kostman Jay , Tebas Pablo , Bar-Or Amit , Muthumani Kar , Montaner Luis
Published: November 2024

Background: Type I interferons (IFNs) play a pivotal role in the development of immune responses and have significant clinical importance. However, dysregulated activation of type-I IFN response has been associated with a wide range of disorders. Therefore, it is crucial to develop tools that facilitate the understanding of their mechanism of action, enabling the development of more effective therapeutic strategies.

Methods: We identified, cloned, and characterized by enzyme-linked immunosorbent assay (ELISA), IFN receptor dimerization assay, signal transducer and activator of transcription 1 (STAT-1) phosphorylation, surface plasmon resonance, and western blot the binding/neutralizing/specificity properties of novel human anti-IFN-α and IFN-β monoclonal antibodies (Abs). The Abs were sourced from individuals with multiple sclerosis with a history or ongoing IFN-β therapy, and from antiretroviral therapy (ART)-suppressed individuals with chronic HIV-1 infection who had been treated with IFN-α.

Results: We observed that clones AH07856, and AH07857 effectively neutralize IFN-α-mediated IFN receptor dimerization and signaling but not IFN-β. Likewise, clones AH07859, and AH07866, neutralize IFN-β-mediated IFN receptor dimerization and signaling but not IFN-α.

Conclusion: We describe novel neutralizing Abs that selectively block human IFN-α or IFN-β with potential applications in research, and in therapeutics and other medical interventions.
Use of a Near Infrared Probe to Assess Intracellular Hydrogen Sulfide Production
Authors: Liu Jianyun , Mesfin Fikir , Zeng Lifan , Manohar Krishna , Hunter Chelsea , Shelley W , Brokaw John , Markel Troy
Published: November 2024

Hydrogen sulfide (H2S) is a ubiquitous gas and has been recently identified as the third biological gasotransmitter, along with the more well studied nitric oxide (NO) and carbon monoxide (CO). Intensive studies on its potential as a therapeutic agent for cardiovascular, inflammatory, infectious, and neuropathological diseases have been undertaken. Numerous studies have shown that H2S can regulate PI3K/AKT, NF-κB and MAPK signaling pathways, suppressing LPS-induced inflammation. However, its use in the clinical setting and mechanistic studies are limited in part due to the lack of effective H2S detection methods. In the current study, we investigated a compound as a potential near infrared (NIR-HS) probe to measure intracellular H2S. After being synthesized and purified using chromatography, NIR-HS probe was added to a cell-free environments in the presence of hydrogen sulfide salts (Na2S and NaHS) or a H2S-slow-releasing donor (GYY4137) to determine its ability in detecting H2S. We further demonstrated that the NIR-HS probe can detect endogenous H2S in different cell lines. Detected by using the NIR-HS probe, knocking down and overexpressing the endogenous H2S synthases (CBS, CTH, and MPST) altered the production of H2S, respectively. Further studies are needed to demonstrate its use in large scale and in vivo environment to further demonstrate its effectiveness in detecting H2S.
Finding the needle in the haystack- detection of circulating endothelial cells with a new EpiSpot
Authors: Preyer Rosemarie , Strecker Kevin , Ettischer-Schmid Nicole
Published: November 2024

Revascularisation in the setting of critical limb ischema (CLI) is the treatment objective. For the evaluation of an angiogenic effect, peripheral circulating endothelial cells (CECs) and endothelial progenitor cells in the blood stream should be detected in an EpiSpot system. The development of the assay and proof of principle with human umbilical vein endothelial cells (HUVEC) are part of this work.

Therefore, vascular endothelial growth factor (VEGF), the coagulation factor VIII (FVIII), E-Selectin, von Willebrand Faktor (vWF) and Interleukin-6 (IL-6) antibody pairs were tested and concentration and incubation times were evaluated with HUVECs in combination with PBMCs. Flow-cytometric analysis was done in parallel to determine the applicability of HUVECs as a model for circulating endothelial cells. Beside this, isolated and enriched endothelial cells (ECs) from peripheral blood were characterised as CECs by using FACS.

E-Selectin has been selected to identify ECs unambiguously in the established assay system. This was proven by titration of HUVEC in PBMCs, and recovery rates of 10 HUVEC / 200.000 PBMCs were achieved.

The developed assay-system can detect low numbers of endothelial cells in general. The amount of circulating endothelial cells in healthy volunteers is relatively low, therefore additional experiments will reveal the usefulness of the assay for CLI treatments and will allow to define the sensitivity in patient samples.

Funding EU Horizon 2020(n° 733006 PACE)
Simultaneous profiling of single-cell BCR clonal diversity and whole transcriptomes from diseased and healthy PBMCs in a single experiment using split-pool combinatorial barcoding
Authors: Sapre Ajay , Papalexi Efthymia , Salvino Alec , Marrujo Simone , Gallareta-Olivares Guillermo , Roco Charles , Rosenberg Alexander
Published: November 2024

Many autoimmune diseases are characterized by aberrant B cells which play a central role in pathogenesis. Each B cell expresses a B cell receptor (BCR), which determines its specificity in binding to different epitopes. For autoimmune diseases such as Rheumatoid Arthritis, Lupus, and Type 1 Diabetes our current understanding of the role of unique BCR repertoires is poorly understood; however, recent advances in single-cell RNA sequencing has allowed the field to simultaneously profile both the BCR and whole transcriptome leading to the characterization of clonal diversity of B cell subtypes.

Traditional methods using microfluidic barcoding are low throughput (1,000s-10,000s of cells) and costly. To overcome these limitations, we developed a method to simultaneously characterize the BCR repertoire and full transcriptomes of up to 1 million cells from up to 96 samples in one experiment.

For proof of concept, we applied this method to simultaneously profile the BCR and whole transcriptome on multiple healthy and diseased samples. Using the whole transcriptome data, we detected all major subtypes of B cells in peripheral blood. From the BCR data, we defined BCR isotypes, captured a majority of cells with a paired chain, detected full length chains, and differentiated dominant & rare clones. This technology paves the way for high-throughput and sensitive screening of BCR repertoires to further the understanding of B cell biology and disease.
Defining healthy and breast tumor-associated neutrophils at a whole-organism, single-cell resolution
Authors: Hsu Alex , Alshetaiwi Hamad , Gong Yanwen
Published: November 2024

Neutrophils accumulate systemically in tumor-bearing organisms, and have been shown to promote primary tumor progression and metastasis through suppression of the anti-tumor immune response. However, neutrophils have specialized phenotypes in different organs, and their full transcriptional heterogeneity and maturation trajectory has yet to be defined across healthy and cancer states. Here, we performed single-cell RNA sequencing on isolated neutrophils from bone marrow, spleen, lymph nodes, blood, mammary fat pad/tumor, and lungs from wild-type and breast tumor-bearing MMTV-PyMT mice. Our analysis reveals distinct transcriptional programs which distinguish systemic neutrophil function in normal homeostasis and cancer. In wild-type mice, we describe a neutrophil maturation trajectory that begins with granulopoiesis in the bone marrow, continues development in the spleen and blood, and reaches a fully mature state in the lymph nodes, mammary fat pad, and lungs. This is captured by five maturation stages, N0-N4, each with its own characteristic gene signature. In MMTV-PyMT mice, we observe a systemic upregulation of genes including Ifitm1, Wfdc17, Clec4e, Igfbp6, and Saac, which have been implicated in cancer-induced immunosuppression. Taken altogether, this work provides a multi-organ perspective into neutrophil development across healthy and cancer states and identifies markers specific to breast tumor-associated neutrophils which may be potential immunotherapeutic targets.
IL3-driven crosstalk of T cell and basophil enhances antitumor immunity
Authors: Mayberry Colleen , Wei Jian , Chang Chih-Hao
Published: November 2024

Cytotoxic T lymphocytes (CTLs) are vital to combat cancer yet become exhausted in the immunosuppressive tumor microenvironment. Here we probed the role of CTL-derived IL-3 in enhancing CTL anti-tumor response and preventing exhaustion. Transwell culture of CTLs, genetic manipulation of T-cell IL-3 expression, and T cell and Il-4 deficient mice were used to evaluate factors in T-cell exhaustion, proliferation, and survival. We found reduced IL-3 production in intratumoral CTLs, correlating with exhaustion, and revealed that CTL-derived IL-3 influences basophils, synergistically amplifying CTL anti-tumor immunity. Introducing IL-3-activated basophils slows tumor growth and basophil depletion lessens IL-3 efficacy. Ingenuity Pathway Analysis of transcriptomic data revealed that IL-3-treated basophil-conditioned medium enhances T-cell function via IL-4 stimulation, resulting in altered cytokine signaling, inflammation, and T-cell activation. Augmenting IL-3 confers increased CTL activity, reduced exhaustion, and lowered tumor burden. Furthermore, IL-3 increases basophil-derived IL-4, elevating CTL IFN-γ production and viability. CTLs are vital as IL-3 has no impact on tumor growth in RAG1-/- mice or after CTL-depletion; moreover, IL-4-/- tumor mice do not benefit from IL-3 supplementation. This research unveils IL-3-mediated CTL-basophil crosstalk regulates anti-tumor immunity and offers the prospect of harnessing IL-3 as a promising approach for enhancing cancer immunotherapy.
The role of interleukin 27 in donor specific alloantibody responses
Authors: Valenzuela Jose , Fan Ran , Gorbacheva Victoria , Chuluyan Eduardo , Fairchild Robert , Valujskikh Anna
Published: November 2024

There is a great clinical need for new approaches inhibiting pathogenic donor specific alloantibodies (DSA) and antibody-mediated rejection (AMR) in transplantation. Targeting interleukin-6 (IL-6) showed modest promise for AMR treatment. Interleukin-27 (IL-27) also belongs to IL-6 cytokine family, utilizes the same receptor signaling chain, gp130, and has many overlapping functions with IL-6. The current study tested the role of IL-27 in humoral immune responses using mouse transplant models. First, B6.WT and B6.IL-27R-/- recipients (H-2b) were transplanted with BALB/c heart allografts (H-2d). By d. 21 posttransplant, B6.WT recipients developed serum DSA against donor class I and class II MHC. In contrast, DSA responses were markedly reduced in B6.IL-27R-/- recipients. We next tested how IL-27 neutralization affects DSA using mAbs against p28 IL-27 subunit. B6.WT recipients transplanted with BALB/c skin allografts and treated with mAb against either p28, IL-6, or IL-6R or control IgG for the first two weeks posttransplant. While control recipients developed anti-H-Dd DSA, none of anti-p28 mAb treated recipients generated DSA, comparable to the effects of anti-IL-6 or anti-IL-6R therapies. Anti-IL-27 mAb resulted in a more profound decrease in the numbers of antibody secreting spleen plasma cells compared to anti-IL6 and anti-IL-6R treatments. Our data identify IL-27 as a novel therapeutic target to inhibit pathogenic DSA responses in transplant recipients.
Sustained cross-talk between donor T cells and IL-33+ stromal cells maintains CD4 T cell differentiation and determines GVHD outcomes
Authors: Dwyer Gaelen , Mathews Lisa , Chalmers Bailey , Naaz Afsana , Poholek Amanda , Sacirbegovic Faruk , Shlomchik Warren , Turnquist Heth
Published: November 2024

Conditioning before allogeneic hematopoietic stem cell transplantation (AlloHSCT) increases the tissue injury signal IL-33 in fibroblastic reticular cells (FRC). Released IL-33 directly stimulates donor CD4 T cells to prime IL-12-independent Type 1 T helper cell (Th1) differentiation and expansion. Tissue stroma upregulates IL-33, but a role for IL-33 in sustaining the pathogenic donor Th1 responses causing GVHD is unclear. We compared B6 mice with inducible ST2 deletion (R26-CreERT2xSt2fl/fl) to wildtype (WT) R26-CreERT2 as T cell donors in a lethal GVHD model (B6 to BALB/c). Donor ST2 deletion at days 10-14 post AlloHSCT increased CD4 T cells Foxp3 expression with reciprocal decreases in Tbet expression in both the lymphoid organs and target tissues. Sustained IL-33 signaling also maintained donor T cell TCF1 expression. Ablating ST2 after GVHD development improved clinical scores and promoted recipient weight gain. How bioactive IL-33 is released from nuclear sequestration remains undefined. RNAseq analysis suggested that IL-33 stimulates T cell granzyme B (GzmB) expression and B6 GzmB deficient (Gzmb-/-) donor T cells displayed reduced activation and expansion similar to ST2 deficient CD4 T cells. In contrast to GzmBWT, anti-IL-33 antibodies had no impact on GzmBKO T cell responses. Thus, cross-talk between donor T cells and IL-33+ stroma orchestrates the T cell identities that are critical to sustain the pathogenic CD4 T cell responses causing GVHD.