bluuebunny/crossref_metadata_2025 huggingface dataset:
Interferon gamma supresses T follicular helper cell and antibody responses upon viral infection
Authors: Kuka Mirela , Sala Eleonora , Mangione Marta , Laura Chiara , Nelli Maria , Consolo Eleonora , Beccaria Cristian Gabriel , Amit Ido , Iannacone Matteo
Published: August 2023

Although humoral and cellular immunity upon viral infections usually co-exist, sometimes one of the two responses emerges as dominant and is responsible for most of the antiviral activity. For example, vesicular stomatitis virus (VSV) infection induces early and potent neutralizing antibody (nAb) responses, whereas lymphocytic choriomeningitis virus (LCMV) infection induces strong cellular responses, but weak nAb responses. Recent work from our laboratory has shown that unbalance is observed also at the level of CD4 T cells responses, with subcutaneous VSV infection inducing strong TFH polarization that supports nAb responses, and subcutaneous LCMV in contrast promoting an extreme TH1 differentiation with no TFH. Single-cell RNA sequencing revealed that LCMV-specific TH1 cells comprise at least to distinct subsets, one of which expresses TCF1, which is usually expressed by TFH. We found that blocking of IFN gamma results in the transition of this atypical T-bet+TCF1+ cells into TFH cells, and in an increased number of germinal center B cells and antibodies. Our results identify IFN gamma as a molecular switch that suppresses TFH and antibody responses while supporting TH1 differentiation upon viral infection. This might be an additional mechanism whereby viruses like LCMV interfere with the generation of efficient antibody responses.

Research was supported by the Italian Ministry of Education, University and Research grants SIR-RBSI14BAO5, PRIN-2017ZXT5WR and PRIN-20209Y5YFZ (to M.K.) and by the European Research Council (ERC) Consolidator Grant 725038 (to M.I.)
The complement protein C1q modulates the activities of anti-influenza HA-stem and anti-SARS-Cov2-nonRBD antibodies and alters viral evolution
Authors: Kosik Ivan , Santos Jefferson J J , Angel Mathew , Hu Zhe , Holly Jaroslav , Gibbs James S , Gill Tanner U , Andrews Sarah , Gillespie Rebecca , Kanekiyo Masaru , Mcdermott Adrian , Yewdell Jonathan W
Published: August 2023

The broadly neutralizing hemagglutinin (BN-HA) stem antibodies represent an universal vaccine prospect. However, our understanding of how these antibodies interact with the influenza virus is limited by excluding other immune elements, such as complement, in our experimental systems.

Here, we show that the Fc binding protein of the complement system, C1q, grants BN-HA stem Abs a novel attachment inhibition (AI) and boosts virus neutralization (VN) activity. C1q promotes AI even if added to preformed BN-HA Ab-virion-RBC complexes, pointing at the steric nature of the phenomenon. Most probably by the same mechanism of steric hindrance, C1q also contributes to fusion inhibition. By employing a panel of engineered avidity-reverted anti-BNHA Abs we show that the C1q-mediated VN enhancement is independent of Ab avidity. We also show that C1q’s presence expands the escape viral repertoire beyond the HA stem domain. Among selected viral variants, near-RBS mutations (E156K-Sb, R224I-Ca, S145N-Ca) modulate HA avidity while causing antigenic drift. Expanding our discoveries to SARS-CoV-2, we identified non-RBD anti-SARS-CoV-2 spike Abs which exhibit C1q-VN enhancement. With an increased SARS-CoV-2 spike per viral particle amount, C1q-VN increased concomitantly. Altogether, our results emphasize the general role of C1q in Ab-mediated antiviral activity and viral evolution.

NIH/NIAID intramural funding
Evaluation of cutaneous immune response in a controlled human in vivo model of mosquito bites
Authors: Cantaert Tineke , Guerrero David , Vo Hoa Thi My , Lon Chanthap , Bohl Jennifer A , Nhik Sreynik , Chea Sophana , Man Somnang , Sreng Sokunthea , Pacheco Andrea R , Ly Sokna , Sath Rathanak , Lay Sokchea , Missé Dorothée , Huy Rekol , Leang Rithea , Kry Hok , Valenzuela Jesus G , Oliveira Fabiano , Manning Jessica E
Published: August 2023

Mosquito-borne viruses are a growing global threat. Initial viral inoculation occurs in the skin via the mosquito ‘bite’, eliciting immune responses that shape the establishment of infection and pathogenesis. Here we assess the cutaneous innate and adaptive immune responses to controlled Aedes aegypti feedings in humans living in Aedes-endemic areas. In this single-arm, cross- sectional interventional study (trial registration #NCT04350905), we enroll 30 healthy adult participants aged 18 to 45 years of age from Cambodia between October 2020 and January 2021. We perform 3-mm skin biopsies at baseline as well as 30 min, 4 h, and 48 h after a controlled feeding by uninfected Aedes aegypti mosquitos. The primary endpoints are measurement of changes in early and late innate responses in bitten vs unbitten skin by gene expression profiling, immunophenotyping, and cytokine profiling. The results reveal induction of neutrophil degranulation and recruitment of skin-resident den- dritic cells and M2 macrophages. As the immune reaction progresses T cell priming and regulatory pathways are upregulated along with a shift to Th2-driven responses and CD8+ T cell activation. Stimulation of participants’ bitten skin cells with Aedes aegypti salivary gland extract results in reduced pro-inflammatory cytokine production. These results identify key immune genes, cell types, and pathways in the human response to mosquito bites and can be leveraged to inform and develop novel therapeutics and vector- targeted vaccine candidates to interfere with vector-mediated disease.

Division of Intramural Research at National Institute of Allergy and Infectious Diseases at the National Institutes of Health #1ZIAAI001066-12: J.M., F.O.; Howard Hughes Medical Institute-Wellcome #208710/Z/17/Z: T.C.; French National Research Agency #ANR-17-CE15-00029: D.M.; Calmette-Yersin PhD Scholarship: D.G.
SARS-CoV-2 seroreversion is associated with a decrease in T cell functionality in children
Authors: Files Megan A , Kehoe Leanne , Adler Amanda , Englund Janet , Seshadri Chetan
Published: August 2023

The kinetics and durability of T cell and antibody responses to SARS-CoV-2 in children have not been well-characterized. We leveraged a longitudinal convalescent cohort of children (N = 30) aged 6 months to 20 years with PCR-confirmed (28/30) or serology-confirmed (2/30) COVID-19 in whom peripheral blood mononuclear cells (PBMC) and sera were archived at approximately 1 month, 6 months, and 12 months post-symptom onset. Nucleocapsid-specific (N) T cell functionality was evaluated utilizing intracellular flow cytometric analysis and combinatorial polyfunctionality analysis of antigen-specific T-cell subsets (COMPASS). CD4+ T cell responses to N declined over time with low cytokine production in children less than 5 years. These data suggest that cell-mediated immunity to natural SARS-CoV-2 infection in children wanes quickly, with the greatest decreases seen in children less than 5 years of age. Conversely, spike-specific (S) CD4+ T cell functionality scores remained stable up to one year after initial infection, and included signals from Th1 cytokines (IFN-γ, IL-2, TNF-α). Consistent with this, anti-S neutralizing antibody titers increased over the course of the study in children greater than 5 years, likely as a result of vaccination. Because children experience more mild disease compared to adults but can carry similar viral loads, our data suggest children may be susceptible to re-infection in the absence of vaccine-induced protection.
The proviral role of B cell-intrinsic IL-17RA signaling in the establishment of chronic gammaherpesvirus infection
Authors: Jondle Christopher Ned
Published: August 2023

Epstein-Barr virus (EBV) is a human specific gammaherpesvirus that establishes lifelong infection in >95% of all adults and is associated with multiple cancers, including B cell lymphomas. Murine gammaherpesvirus 68 (MHV68) is a natural rodent pathogen that is genetically and biologically related to EBV and importantly provides a tractable animal model to study viral and host factors that impact chronic infection. Both of these viruses have a natural tropism for B cells and usurp B cell differentiation to drive a robust polyclonal germinal center response, which is needed to establish the latent viral reservoir in memory B cells. The factors which these viruses use to usurp the host germinal center response to establish chronic infection are not clearly defined.

Using MHV68 we discovered that global IL-17RA signaling as well as T-cell intrinsic IL-17RA signaling is proviral and supports the gammaherpesvirus-driven germinal center response needed to establish chronic infection. Loss of global and T-cell intrinsic IL-17RA signaling resulted in a significant attenuation in the germinal center response as well as viral latency and reactivation.

Given the B cell tropism of MHV68 we generated a B cell specific model of IL-17RA deficiency to understand the significance of IL-17RA signaling in virally infected cells. Loss of IL-17RA signaling in B cells during MHV68 infection resulted in an attenuated germinal center response as well as reduced viral reactivation and latency, similar to what was observed in the loss of global and T-cell intrinsic IL-17RA signaling. This indicates that IL-17RA signaling in both virally infected and uninfected cells play an important proviral role in the establishment of chronic gammaherpesvirus infection.

WMed Start up funds and an American Cancer Society Postdoctoral Award (134165-PF-19-176-01-MPC) which was used to generate the B cell specific IL-17RA deficient mice.
PD-L1 blockade enhances brain resident memory T cell function and stimulates brain to lymph node crosstalk
Authors: Musial Shawn C , Kleist Sierra A , Searles Tyler G , Degefu Hanna N , Isaacs Jordan F , Skorput Alexander G J , Rosato Pamela C
Published: August 2023

Resident memory T cells (TRM) are a unique subset of memory T cells that persist within non-lymphoid tissues. Previous studies have identified TRM within the brain (bTRM) after peripheral infection and vaccination in mice, with more recent appreciation in human brain tissue. While bTRM are important for protection of the CNS against reinfection, their heterogeneity at the single cell level and overall involvement in shaping the neuroimmune landscape remains unknown. Here, we defined viral-specific bTRM heterogeneity by single cell RNA sequencing and identified unique subsets dependent on local antigen encounter. These data also confirmed previous findings that bTRM express markers associated with T cell exhaustion, such as PD-1. Despite this, intracranial delivery of cognate viral peptide led to robust bTRM reactivation and initiated a cascade of immune activation and accumulation within the brain, including rapid activation of microglia, NK cells and T cells, DC maturation, and infiltration of macrophages and monocyte derived DCs. In the presence of PD-L1 blockade, despite observing higher effector molecule production from reactivated bTRM, we found no apparent difference in downstream immune activation in the brain. Interestingly, however, we found a significant increase in DC and B cell maturation in the CNS draining deep cervical lymph nodes (dcLN) only in the presence of PD-L1 blockade. These data indicate a potential unique crosstalk between bTRM and APCs in the dcLN normally suppressed by PD-1/PD-L1 signaling. These studies provide insight into brain TRM functions and potential mechanisms for strengthening immune activation that can guide immunotherapies for immunologically cold brain tumors.

Supported by grants from the NIH (K22AI148508-02, T32 AI007363)
CD8 T cell exhaustion is dynamically controlled by the chromatin regulatory factor BRD4
Authors: Mullins Genevieve N , Green Jarred M , Green William D , Milner J Justin
Published: August 2023

In the context of chronic viral infections and cancer, the protective potential of CD8 T lymphocytes is often dramatically blunted and characterized by a functionally exhausted cell state. T cell exhaustion contributes to pathogen persistence and can limit responsiveness to promising immunotherapy strategies. Therefore, defining the signals controlling the development and maintenance of an exhausted T cell state has critical implications for global public health. While it has become apparent that T cell exhaustion is a transcriptionally and epigenetically driven process, critical epigenetic mechanisms mediating T cell exhaustion remain unresolved. To this end, we sought to define how the ‘chromatin reader’ BRD4 controls an exhausted state in a CD8 T cell-intrinsic manner during chronic viral infection. Utilizing inducible deletion mouse models and RNAi, we found that BRD4 is a critical regulator of T cell differentiation and function. BRD4 was essential for driving a terminally exhausted CD8 T cell state. Mechanistically, BRD4 was found to bind near and modulate expression of key genes that influence T cell differentiation and exhaustion. Finally, small molecule inhibition of BRD4 was seen to reverse a terminally exhausted cell state. These findings provide insights into the epigenetic underpinnings of T cell exhaustion and have important implications for enhancing immunotherapy efficacy.

Supported by a CGIBD pilot award and grant from NCI (R00CA234430).
Brain-resident memory cells (bTRM) and virus reactivation
Authors: Gupta Priyanka , Hu Shuxian , Nomura Wen Sheng , Lokensgard James R
Published: August 2023

Background

Neuroimmune responses terminate MCMV acute infection, however, a subset of neurons still harbor latent viral genomes. Factors regulating viral reactivation remain to be elucidated.

Methods

Using RNAscope, IE1 nucleic acid was detected within brains at D5 and D30 p.i. Using real-time RT-PCR, we assessed expression of IE1, E1, and gB transcripts. Using multi-color flow cytometry, we assessed depletion of bTRM following icv injection of either α-CD8 Ab or α-CD103-sap. Using luciferase-expressing transgenic mice, we longitudinally assessed reactivation of cre-MCMV after T-cell depletion using live small animal imaging. Explant assay was employed to study the role of bTRM in reactivation. Using Nanostring, we identified changes following bTRM-depletion.

Results

Brain infection was demonstrated by X-gal staining as well as IE1 staining using RNAscope at 5 d p.i. After 30 d p.i., the virus established latency, as indicated by absence of transcripts (IE1, E1, and gB). After establishment, we injected either α-CD8 Ab or α-CD103-sap into the brain and showed 90–95% T-cell depletion. We infected mice with cre-MCMV, which expresses Cre recombinase. We observed enhanced imaging signals indicative of IE promoter activity in depleted animals. Surprisingly, we efficiently recovered virus from untreated mice, but not from those depleted of bTRM. Furthermore, on Nanostring analysis, we identified gpnmband hmox1, upregulated 11.48- and 6.7-fold following bTRM depletion.

Conclusion

When we depleted bTRM, there was transient IE expression, sufficient to activate surveying microglia. This eventually resulted in a tissue-wide anti-viral state. These data provide new insights into the role of bTRM in controlling reactivation.

This project was supported by award numbers NS-038836 from the National Institute of Neurological Disorders and Stroke
TLR3 Signaling is Required for Robust Extrafollicular B Cell Response (EF) in Influenza A/PR/8 Infection
Authors: Keller Emma J , Lam Jonathan , Baumgarth Nicole
Published: August 2023

Following influenza A virus (IAV) infection B cells rapidly proliferate and differentiate into plasma cells (PCs) in extrafollicular foci (EF) within mediastinal lymph nodes (medLN), resulting in protective antibody secretion. We had shown that co-expression of both TLR adaptor proteins, TRIF and MyD88, is required for robust EF responses. Only two TLRs, TLR3 and 4, signal using TRIF. As shown previously TLR3 and 7, but not TLR4, expression were upregulated by medLN B cells within 2 days post infection (dpi) in a Type-I IFN-dependent manner, suggesting that TLR3 might utilize TRIF in B cells during IAV infection. To test this we generated MyD88 × TLR3 double-deficient (DKO) mice. Flow cytometry on MedLN at 7 dpi showed significantly reduced EF B cells (CD19lo B220lo CD38lo CD24hi) and CD138+plasma cells (PCs), including IAV-specific PCs, as well as reduced serum IAV-specific IgG and IgG2C in DKO compared to C57BL/6 or single- MyD88 and TLR3 deficient mice. IAV infection of mixed bone marrow chimeras indicated that the TLR3-TRIF axis contributes to EF formation at least in part in a B cell-intrinsic manner. In vitro stimulation of B cells with the TLR3 agonist, Poly: IC, caused expression of several activation/costimulatory molecules but did not induce proliferation. Together, the data suggest that TLR3-mediated B cell is a non-redundant driver of rapid protective antibody responses.

Supported by NIH/NIAID R01AI117890 (NB) and NIH T32 HL007013-40 (JHL and EJK).
LPAR5-Mediated Regulation of CD8 T Cell Immunity During Chronic Viral Infections
Authors: D’Antonio Marc , Ware Brian C , Turner Jacqueline A , Davenport Bennett J , Morrison Thomas E , Torres Raul M
Published: August 2023

Persistent viral infections such as Human Immunodeficiency Virus (HIV), Hepatitis B and C Viruses (HBV & HCV), and Epstein Barr Virus (EBV) result in high mortality rates, tremendous healthcare costs, and limit global life expectancies. Paramount to establishing and maintaining chronic infections, viruses must evade or suppress T cell-mediated immunity, often through promoting the generation of dysfunctional or “exhausted” CD8 T cells. Although there are considerable research efforts investigating mechanisms of T cell exhaustion, further work is necessary to identify and characterize therapeutically targetable regulators of exhaustion programming. Work from our lab has focused on the ability of the bioactive lipid, lysophosphatidic acid (LPA), to regulate CD8 T cell function through LPA receptor 5 (LPAR5) signaling. Importantly, significantly elevated levels of LPA have been identified in chronic viral infections and cancers. We previously demonstrated that activation of LPAR5 on CD8 T cells during TCR engagement suppresses downstream signaling events. Since TCR-induced transcriptional networks regulate T cell exhaustion programming, we hypothesize that LPA-mediated changes in gene expression during chronic infections promotes the generation of dysfunctional CD8 T cells. To investigate the role of LPA in the differentiation of exhausted CD8 T cells, we utilized the LCMV Clone 13 infection model. We postulate that the elevated levels of LPA identified in persistent infections and our initial findings investigating LPAR5 signaling in CD8 T cells provide strong a rationale to evaluate LPA signaling as a previously unexplored pathway by which chronic viruses promote T cell exhaustion to establish and maintain persistence.

RO1AI143261-02 T32-AI07405
CD4 T cell deficiency narrows virus-neutralizing IgG coverage for Polyomavirus and selects for antibody-escape variants
Authors: Ayers Katelyn Nicole , Lauver Matthew D , Lukacher Aron E
Published: August 2023

CD4 T cell deficiencies predispose patients to progressive multifocal leukoencephalopathy (PML), a brain disease caused by JC polyomavirus (JCPyV). PML is linked to immunomodulatory therapies that preferentially lower CD4 T cells in the CSF (natalizumab); but how CD4 T cells control JCPyV is undefined. “Blind spots” in the ability to neutralize JCPyV variants with mutations in the VP1 capsid protein were described in PML patients. We recently reported that a VP1 mAb selects for VP1-mutant escape variants both in vitro and in vivo; some of these variant viruses are neurovirulent. We hypothesized that alterations in the Ab response due to CD4 T cell insufficiency underlie emergence of Ab-escape mouse polyomavirus (MuPyV) variants. T-cell independent (TI) VP1-specific IgG (via anti-CD4 mAb depletion, CD40L blockade, MHCII KO mice, IL21R KO mice) neutralizes MuPyV but has reduced titer and avidity than anti-VP1 IgGs in CD4 T cell-sufficient mice. Serial passaging of MuPyV in vitro with sera from TI mice selected an Ab-escape VP1 mutant containing amino acid deletions at residue 145–146 in an external loop of VP1 (MuPyV.145–146). In contrast to sera from healthy MuPyV-infected mice, TI sera failed to neutralize MuPyV.145–146. Depletion of CD4 T cells during persistent MuPyV infection, as a model of acquired CD4 T cell insufficiency (i.e. onset of immunomodulatory mAbs) via anti-CD4 mAb depletion, CD40L blockade, and anti-CD20 mAb B cell depletion also reduced anti-VP1 IgG titer and avidity. Ongoing studies seek to determine if a similar loss in recognition of the 145–146 VP1 epitope occurs. Our data shows that a narrowed antiviral IgG response due to CD4 T cell deficiency is a critical antecedent for outgrowth of Ab-escape PyV VP1 mutant viruses.

Supported by grants from the NIH (5R01NS088367, 5R01NS092662, and R35NS127217) (AEL) Judy S. Finkelstein Memorial Award (KNA) T32 CA06395 (KNA)
Intermediate levels of preexisting protective antibody allow priming of protective T cell immunity against Influenza
Authors: Mansoor Mishfak Abdullah Mohamed , Malavet Valeria Flores , Ng Terry , Arvelo Andrea C , Strutt Tara M
Published: August 2023

Overcoming interfering impacts of preexisting immunity to generate universally protective influenza A virus (IAV)-specific T cell immunity through vaccination is a high priority. Here, we passively transfer varied amounts of H1N1-IAV-specific immune serum prior to H1N1-IAV infection to determine how different levels of preexisting Ab influence the generation and protective potential of heterosubtypic T cell responses in a murine model. Surprisingly, IAV nucleoprotein (NP)-specific CD4 and CD8 T cell responses are readily detected in infected recipients of IAV-specific immune serum regardless of the amount transferred. When compared to responses in control groups and recipients of low and intermediate levels of convalescent serum, NP-specific T cell responses in recipients of high levels of IAV-specific serum, which prevent overt weight loss and reduce peak viral titers in the lungs, are, however, markedly reduced. While detectable at priming, this response recalls poorly and is unable to mediate protection against a lethal heterotypic (H3N2) virus challenge at later memory timepoints. A similar failure to generate protective heterosubtypic T cell immunity during IAV priming is seen in offspring of IAV-primed mothers that naturally receive high titers of IAV-specific Ab through maternal transfer. Our findings support that priming of protective heterosubtypic T cell responses can occur in the presence of intermediate levels of preexisting Ab. These results have high relevance to vaccine approaches aiming to incorporate and evaluate cellular and humoral immunity against IAV and other viral pathogens against which T cells can protect against variants escaping Ab-mediated protection.

Supported grants from NIH (RO1 AI165406) Startup funds from the University of Central Florida foundation
Antigen-specific reactivation of brain tissue resident memory CD8 T cells directly induces severe neuroinflammation and blood-brain-barrier disruption
Authors: Ayasoufi Katayoun , Wolf Delaney M , Jin Fang , Bamkole Michael A , Tritz Zachariah , Maynes Mark A , Pfaller Christian K , Hansen Michael , Fain Cori E , Johnson Aaron J
Published: August 2023

Intracranial infection with neurotropic Theiler’s Murine Encephalomyelitis Virus induces robust generation of brain TRMs against the immunodominant antigen, VP2121–130, following viral clearance in C57BL/6 mice. While the presence of TRMs in the brain is documented, neurological consequences of TRM reactivation and their causal role in neuropathology remain understudied. We disabled peripheral CD8 T cells via anti CD8 depleting strategies and FTY720 treatment to determine the causal link between brain TRM reactivation and neuropathology. We depleted peripheral T cells and then administrated VP2 peptide IV to reactivate TRMs 60–200 days post infection as this elegant technique isolates responses of brain TRMs away from other branches of memory. We used state-of-the-art flow cytometry and MRI analysis to evaluate neurological consequences of TRM reactivation. TRM reactivation induced severe neuroinflammation in the brain as evident by infiltration of myeloid cells and blasting of T cells. MRI detected gadolinium leakage marking severe blood-brain-barrier (BBB) disruption when brain TRMs were reactivated. Depletion of peripheral CD8 T cells with low-dose anti CD8 antibodies or their sequestration via FTY720 did not alter neuroinflammation indicating brain TRM reactivation causally initiates neuropathology. Confirming this, tissue depletion of all TRMs in the brain using high-dose anti CD8 antibodies prevented both neuroinflammation and neuropathology. Hence, TRM reactivation causes severe neuroinflammation and BBB disruption from within the brain independently of peripheral T cells. Targeting brain TRM reactivation is a novel potential therapeutic for patients with many acute and chronic neurological diseases.

Supported by grants from NIH (K99NS117799-01A1 (KA), R01NS103212 (AJJ), and RF1NS122174(AJJ)
Persistent CD4 T cell functional deficits in individuals recovered from prolonged symptomatic SARS-CoV-2 infection
Authors: Kovats Susan , Lawrence Christina , Turner Sean , Pezant Nathan , Redinger Nancy , Guthridge Carla , Smith Ken , Guthridge Joel M , James Judith A , Farris A Darise
Published: August 2023

We assessed phenotypic changes in CD4+ T cells in 17 individuals (male and female, age 26–71, not hospitalized) after recovery from moderate SARS-CoV-2 infection. A subset of 5 individuals with protracted symptom duration showed marked downregulation of CD4 on CD3+CD8− T cells and a poor response to TCR stimulation with SEB, as judged by failure to upregulate activation markers CD134, CD25 and CD69. This defective response (DR) group included 3 subjects sampled <30 days and 2 sampled >150 days post-recovery. In longitudinal samples obtained 3–12 months later, 4 of 5 individuals harbored T cells showing a recovered response (RR), with CD4 levels comparable to normal response (NR) healthy controls. One individual did not show a recovered response until 10 mo later. Levels of anti-S1 IgG did not correlate with the CD4 defect. Selected plasma cytokines (IL-1RA, APRIL, VEGF) elevated in the DR group correlated with low CD4 levels. The SEB activation program, defined as genes differentially expressed (DE) between paired unstimulated and stimulated CD3+CD8− T cells, showed a >50% reduction in the number of DE genes in the DR group compared to the RR and NR groups. Upstream driver analysis of genes uniquely DE in the NR and RR groups, representing a recovered/normal activation program, showed strongly reduced influence of VEGF. Conversely, similar analysis of genes uniquely DE in the DR group showed reduced influence of TCR signaling. In sum, recovery from prolonged symptomatic Covid-19 is often associated with a global CD4+ T cell response defect, defined by low surface CD4 and defective T cell activation at the transcriptional level, and correlating with elevated cytokines such as VEGF associated with severe or long Covid-19.

Supported by NIH U19 AI062629
HIV-1 Tat impairs the establishment and maintenance of latency in a quiescent T cell line model of HIV infection
Authors: Gomez-Rivera Francisco , Terry Valerie H , Virgilio Maria C , Collins Kathleen L
Published: August 2023

Human Immunodeficiency Virus (HIV) persists as a transcriptionally silent, latent provirus despite optimal therapy. Latently infected cells avoid immune detection and viral cytopathic effects becoming a long-lived reservoir of HIV. To elucidate factors responsible for the establishment of latency, our lab developed two novel HIV-1 reporter constructs (89.6 VT1 and 454 VT2). Studies using these reporters demonstrated that the multiplicity of infection (MOI) (the ratio of viral particles to cells) strongly correlated with the proportion of active versus latent infection in a “quiescent” T cell line. This observation suggested the hypothesis that at low viral inoculum, the availability of a viral factor, such as the HIV transactivator of transcription (Tat), was limiting and affected the likelihood that active versus latent infection was established. To test this, we (1) examined relative tat levels in reporter viruses that established different proportions of latent versus active infection (2) measured active versus latent infection in a cell line that constitutively overexpressed tat and (3) modified the dual reporter 89.6 VT1 to overexpress tat after an IRES element. In every case, higher tat levels correlated with increased active infection and a reduction in dependency of active infection on the amount of inoculum. Ongoing studies will test the extent to which Tat levels vary in native HIV proviral genomes. These studies provide insight into the molecular mechanisms that govern latency establishment at high versus low viral infection conditions and may shed light on new approaches to eradicate latently infected cells.

Supported by grants from NIH/ NIAID (R01 AI148084 S1)
Influenza virus infection modulates expression of the anti-inflammatory mucin 1 in both epithelial and immune cells
Authors: Abdelhamid Leila , Scull Margaret Adele
Published: August 2023

Cell-associated mucin 1(MUC1/Muc1) is a component of the mucus barrier in the respiratory tract that acts as a physical barrier and signaling molecule. Our prior work shows that MUC1 expression is increased during influenza virus (IAV) infection in human airway epithelial (HAE) cultures in vitro. Here we further detail MUC1 expression patterns and report that IAV-induced cytokines, including type I interferons (IFNs), trigger the expression of a low-molecular-weight form of MUC1 which localizes to the cytoplasm and nucleus, and is expressed in different epithelial cell types. Further, IFN-driven changes in the cellular composition of HAE toward a more secretory phenotype require MUC1, suggesting that MUC1 plays an important role in shaping the epithelial response during viral infection. Using a mouse model of IAV infection, we confirmed the upregulation of Muc1 throughout the epithelium in vivoand observed altered Muc1 levels in lung immune cells. Specifically, Muc1 was upregulated in interstitial macrophages but downregulated in antigen-presenting cells (APCs) including alveolar macrophages and dendritic cells as early as 2 days post-infection (dpi). By 5 dpi, Muc1 was significantly upregulated in various T cell subsets and their effector counterparts including CD4+, CD8+, and double-negative effector T cells. We also found a negative correlation between Muc1 levels in APCs and the expansion of effector T cell populations, supporting the putative immunoregulatory roles of MUC1/Muc1. Our ongoing work further explores the impact of Muc1 expression in specific cellular subsets on the immune response and IAV pathogenesis.

This work was supported by the National Heart, Lung, and Blood Institute (R01HL151840, to MAS)
CD40-dependent mechanism of virus control in a mouse model of HBV infection
Authors: Bailey Jacob T , Rexhouse Catherine , Moshkani Safiehkhatoon , Robek Michael D
Published: August 2023

An estimated 296 million people worldwide currently live with chronic hepatitis B virus (HBV) infection. Up to 90% of newborns and 30% of children with acute hepatitis B develop chronic infections, causing lifelong complications including liver cirrhosis and cancer. HBV persistence is a consequence of T and B cell dysfunction, so immunotherapy has been proposed as a means of HBV control. TNF receptor superfamily member CD40 has shown therapeutic value in overriding T cell exhaustion and inducing lymphocyte responses in solid tumors and viral infections. To determine the efficacy of CD40 targeting in generating HBV-resolving immune responses, mice were simultaneously transduced with adeno-associated virus encoding the HBV genome (AAV-HBV) and treated with the agonistic CD40 antibody FGK4.5 to activate CD40 signaling. AAV-HBV mice receiving FGK4.5 saw a transient reduction in HBV as determined by serum antigen analysis, while antigen levels in CD4-depleted mice treated with FGK4.5 became undetectable over seven weeks. HBV control was strongly associated with the generation of HBV-specific CD8+ T cells, increased serum ALT, and elevated expression of intrahepatic CD8+ T cell-related genes. Histological analysis of the liver revealed loss of hepatitis B core antigen-positive hepatocytes and increased inflammatory foci. Neither Treg cell depletion nor checkpoint inhibition in CD4-replete mice affected HBV control. Lastly, CD40 activation in CD4-depleted chronically infected mice also reduced both serum antigenemia and liver HBV RNA. Taken together, these results implicate a critical role of CD4+ T cells in maintaining HBV immune tolerance and further suggest CD40 as a potential therapeutic target for chronic HBV infection.

Supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award numbers R01AI124006 and R01AI148354. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
NAC1 constrains CD4+ T cell memory formation through the ROCK1-mediated pathway
Authors: Wang Liqing , Song Jianxun
Published: August 2023

Nucleus accumbens-associated protein 1 (NAC1) is a transcriptional co-factor that has been extensively investigated in cancer biology. Our recent studies indicate that NAC1 can modulate the functional activity of regulatory T cells and tumoral expression of NAC1 restrains antitumor immunity. However, little is known about its regulation of memory T cells. In the current study, we analyzed CD4+ T cells from wild type (WT) and NAC1-decificient (−/−) mice and used the vaccinia virus (VACV) as the tool to investigate the precise role and mechanism of NAC1 in controlling CD4+ T cell memory. In vitro, we identified that NAC1 is essential for T cell metabolism, including both glycolysis and oxidative phosphorylation, which positively modulates CD4+ T cell survival. We also revealed that NAC1 deficiency has an interruption of the AMPK-mTOR pathway, resulting in decreased glycolysis and defective autophagy of CD4+ T cells. Mechanically, loss of NAC1 downregulates ROCK1 expression and reduces the phosphorylation and stabilization of BECLIN1. When overexpressing ROCK1 in NAC1−/− CD4+ T cells, both the defective autophagy and the interrupted AMPK-mTOR pathway were restored. In vivo, when i.p. challenged with VACV, NAC1−/− mice had improved VACV-specific CD4+ memory T cell formation at 35 days compared with WT mice. Furthermore, in adoptively transferred experiments when overexpressing ROCK1 of NAC1−/− CD4+ T cells (Thy1.2+) in congenic hosts (Thy1.1+), the increased memory T cell formation of NAC1−/− CD4+ T cells was reverted to a normal WT level. Conclusively, we define that NAC1 acts as a suppressor of CD4+ T cell memory formation through the ROCK1-mediated pathway, and targeting NAC1 may be adapted to promote memory CD4+ T cell development.
Asymmetric Cross-Protection is Elicited by Distinct Influenza B Lineages
Authors: Page Caroline Katherine , Shepard Justin , Ray Sean , Lorton Ana T , Rowe Dawne , Tompkins Stephen Mark
Published: August 2023

The immune response to influenza B virus (IBV) infection is understudied compared to influenza A virus (IAV), despite IBV accounting for 20% of influenza-related hospitalizations annually. Influenza B was first isolated in the 1940s and subsequently diverged into two distinct lineages, Victoria (B/Vic) and Yamagata (B/Yam), defined by changes in the hemagglutinin (HA) and basic polymerase (PB1 and PB2) gene segments. The two IBV lineages are antigenically distinct, resulting in the development of influenza vaccines containing both B/Vic and B/Yam in addition to H1N1 and H3N2 IAVs. Differential epidemiologic dynamics of the IBV lineages and the recent proposed extinction of B/Yam suggests the lineages do not elicit equivalent immunity. Human studies have shown B/Yam-infected persons are susceptible to reinfection with the same lineage virus, suggesting B/Yam-elicited immunity is insufficient compared to B/Vic-elicited immunity, which prevents homotypic reinfection. Here we established a murine model of contemporary IBV infection that supports observational studies. We demonstrated immune responses elicited by B/Yam infection is insufficient to protect against a heterolineal infection (B/Vic challenge). Conversely, B/Vic-elicited immunity protected against homologous and heterolineal (B/Yam) challenge. We assessed virus-specific serum antibody (Ab) responses following primary infection and challenge. Serological data suggest B/Yam infection failed to elicit cross-reactive Ab, allowing B/Vic infection, while B/Vic elicited serum Ab against both lineages. Ongoing studies seek to define the virus-specific B-cell response to B/Yam and B/Vic infection and to confirm these results in a ferret model of IBV infection.

Supported by grants from NIH Contract 75N93021C00018
Profiling of the antibody response to viral and bacterial antigens and its correlations with time-to-hospital discharge: Covacta and Mariposa study
Authors: Shrivastava Divya , Budde Petra Ilse , Onabajo Olusegun , Bräutigam Manuel , Zucht Hans-Dieter , McBride Jacqueline , Bauer Rebecca , Chandler Scott , Kuebler Peter
Published: August 2023

In this study the role of the humoral response to viral and bacterial antigens in hospitalised patients with severe COVID-19 and its association with clinical endpoint time-to-hospital discharge (TTHD) was investigated. Here, we analysed the antibody (Ab) response to SARS-CoV-2 structural and non structural proteins, other influenza(s) and bacterial proteins using bead-based protein and peptide arrays comprising different viral epitopes. In total, 459 pre-treatment serum samples from hospitalised COVID-19 patients enrolled in the COVACTA and MARIPOSA clinical trials were analysed to identify Ab reactivity to protein antigens and linear epitopes associated with TTHD. Overall we found that most patients had IgG-Abs to SARS-CoV-2 S protein (89%), N protein (90%) and RBD domain (95%). Three major epitopes of the S protein were identified which map to the C-terminus of the RBD, TMPRSS2 cleavage site and fusion peptide 1, and close to the heptad repeat 2 domain. These epitopes could be further researched to understand the immunity to COVID-19 and how it affects outcomes.

Abs binding to several peptides mapping to the RBD C-terminus and N-terminal to heptad repeat 2 were associated with longer TTHD. Abs to an epitope mapping to the intracellular domain of S protein and to the RNA-binding domain of the N protein showed the strongest association with longer TTHD. Abs to seasonal corona viruses and influenza A & B, S. aureus, and rhinovirus were associated with shorter TTHD. To conclude, epitope-specific multiplexed Ab profiling of anti-SARS-CoV-2 response may potentially be used to predict clinical outcome. Pre-existing humoral immunity to seasonal corona viruses and influenza may also confer some protective effects against COVID-19
The metabolic role of gut dysbiosis in altered T cell responses to influenza virus infection
Authors: Cheetham Susana , Meza-Perez Selene , Sanchez Aaron Silva , Randall Troy
Published: August 2023

Commensal gut microbiota modulates protective immune responses to viral infections at extra-intestinal sites, including the lungs. Gut dysbiosis induced by broad-spectrum antibiotic treatment appears to alter adaptive immunity to influenza infection (IAV/PR8). However, the mechanism by which microbial metabolism influences immune responses across the gut-lung axis remains unclear. Here, we used flow cytometric analysis to investigate T cell responses to IAV in specific pathogen free (SPF), Altered Schaedler flora (ASF) and germ-free mice models. We show that, compared to SPF mice, ASF and germ-free mice have significantly reduced levels of regulatory T cells (Tregs) and virus specific cytotoxic CD4 and CD8 T cells at 10 days post infection. Further analysis of microbial mediators in the peripheral blood of these mice revealed significantly increased levels of L-arginine in the serum of ASF and germ-free mice compared to SPF mice. We noted that, germ-free and ASF mice lack bacteria that metabolize dietary amino acids, including L-arginine, regulating its increased availability in the peripheral blood of these mice. To investigate the role of L-arginine as a microbial mediator of reduced T cell responses to IAV, we supplemented L-arginine or administered neomycin (to induce selective disruption of arginine metabolizing bacteria) to separate cohorts of SPF mice. We observed altered cytotoxic and effector T cell responses at 10 days post infection in the experimental groups, however, Tregs were not significantly affected. Our data suggests that selective dysbiosis may increase L-arginine levels in the serum of SPF mice which may directly affect virus specific effector T cell responses in the lung mucosa.
RSV-specific T cell epitope presentation in the respiratory tract differs between adults and neonates as revealed by a novel T cell proliferation assay
Authors: Nhan Paula , DaMata Jarina P , Lu Zhongyan , Ennis Katherine H E , Malloy Allison M W
Published: August 2023

Respiratory syncytial virus (RSV) invades the respiratory epithelium and provokes severe symptoms in young children leading to infant mortality. CD8 tissue resident memory (TRM) cells are associated with protection in adults against RSV, but are poorly established in neonatal mice consequent to primary infection. We hypothesized that antigen presentation contributes to age-dependent TRM establishment, thus we sought to define antigen kinetics in RSV-infected adult and neonatal mice.

CB6F1/J adult and neonatal mice were infected with RSV and lung tissue were isolated over the course of acute and convalescent infection. Single cell lung suspension was co-cultured in vitro with RSV Kd M282–90 (M2) or Db M187–195 (M) epitope-specific T cells isolated from CD8 T cell receptor (TCR) transgenic mice. T cells were stained with carboxyfluorescein succinimidyl ester (CFSE) to quantify proliferative response 48 hours post-incubation at 37°C via flow cytometry.

From lung tissue isolated at 7 days post infection (dpi), M and M2-specific CD8 T cells proliferated in response to co-culture; however, higher rates were induced by neonatal lung tissue primarily by M-specific T cells. By 8 and 9 dpi, proliferation induced by isolated neonatal lung continued while proliferation induced by adult lung tissue was not quantifiable by our assay.

Our findings suggest antigen availability in neonates exceeds that of adults and is age and epitope-dependent. These discoveries indicate that antigen accessibility does not independently correspond with the magnitude of the T cell response in early life. Exploring the inflammatory contributions to RSV-specific T cell defenses is required as this may aid in vaccine design and understanding disease pathogenesis.

Supported by grant from NIH R01-AI154619.
Single cell transcriptomics identify AHR and CD9 as markers of CD14+ atypical B cells
Authors: Blevins Lance K , Khan Isha O , Crawford Robert B , Kaminski Norbert E
Published: August 2023

B lymphocytes are adaptive immune cells responsible for the production of antigen specific antibodies and the release of pro- and anti-inflammatory cytokines. Pro-inflammatory B cells, often termed atypical B cells (at B cell) are a heterogenous population of B lymphocytes associated with aging, infection, and autoimmunity. Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor important for xenobiotic metabolism. However, AHR is heavily implicated in immune cell differentiation as a regulator of cell fate decisions. at B cells are believed to represent an alternative B cell lineage but the role of AHR in at B cells is not known. We have reported that human, primary CD5+ innate-like B cells (ILB) are preferentially sensitive to modulation by AHR-activation compared to CD5− B cells. CD5+ ILB are a heterogenous population of B cells enriched in IgM memory B cells, B1 B cells, marginal zone B cells, regulatory B cells, and at B cells. We used Single cell transcriptomics to elucidate AHR expression in human naïve, circulating CD19+ B cells isolated directly from PBMC and enriched for CD5 protein. We found that AHR expression correlated with expression of CD14 and were enriched in a myeloid gene signature. These results were validated in isolated CD9+ B cells and PBMC. Further, CD14 expressing B cells were enriched in CD9 isolated cells and expressed higher CD11c and Tbet and lower IgD, markers of atypical B cells, compared to CD9− B cells. These data suggest that CD9 and AHR are putative markers of CD14+ at B cells. (Supported by NIH grant P42ES004911)

Supported by NIH grant P42ES004911
Effects of IL-9 on TRAF3-deficient B cell activation
Authors: Jung Jaeyong , Gokhale Samantha , Victor Eton , Tsai Jemmie , Spirollari Eris , Matracz Brygida , Takatsuka Shogo , Kitamura Daisuke , Xie Ping
Published: August 2023

The pleiotropic cytokine interleukin-9 (IL-9) signals to target cells by binding to a heterodimeric receptor consisting of the IL-9 receptor (IL-9R) and the common subunit γ chain. In the present study, we found that the expression of IL-9R was strikingly up-regulated in mouse naïve follicular B cells genetically deficient in TRAF3, a critical regulator of B cell survival and function. The highly up-regulated IL-9R on Traf3−/− follicular B cells conferred responsiveness to IL-9, including IgM production and STAT3 phosphorylation. Interestingly, IL-9 significantly enhanced class switch recombination to IgG1 induced by B cell antigen receptor crosslinking plus IL-4 in Traf3−/− B cells but not in wild type B cells. Blocking the JAK-STAT3 pathway abrogated the effects of IL-9 on Traf3−/− B cells. Our study thus revealed a novel pathway that TRAF3 suppresses B cell activation and Ig isotype switching by inhibiting IL-9R-JAK-STAT3 signaling. Our findings provide new insights into the TRAF3-IL-9R axis in B cell function and have significant implications for the understanding and treatment of a variety of human diseases involving aberrant B cell activation.

This study was supported by the National Institutes of Health grants R01 CA158402 (P. Xie) and R21 AI128264 (P. Xie), the Department of Defense grant W81XWH-13-1-0242 (P. Xie), and a Busch Biomedical Grant (P. Xie). FACS analyses were supported by the Flow Cytometry Core Facility with funding from NCI-CCSG P30CA072720.