bluuebunny/crossref_metadata_2025 huggingface dataset:
Memory CD8 T cells in RSV infection: friend or foe?
Authors: Stoley Megan E , Knudson Cory J , Pewe Lecia L , Meyerholz David K , Langlois Ryan A , tenOever Benjamin R , Harty John T , Varga Steven M
Published: January 2023

Respiratory syncytial virus (RSV) is the leading cause of severe respiratory tract infection in infants and young children. CD8 T cells play a critical role in mediating viral clearance following an acute RSV infection. However the role of memory CD8 T cells in providing protection against RSV remains understudied. To generate high magnitude CD8 T cell memory in the absence of CD4 T cell memory and antibodies, we immunized naïve mice with dendritic cells pulsed with an RSV-derived peptide followed by a boost with a recombinant Listeria monocytogenes expressing the same RSV-derived epitope. Memory CD8 T cells significantly reduced viral titers following RSV challenge, but did so at the expense of increased airway dysfunction, weight loss, and mortality compared to controls. Importantly, the severe immunopathology and mortality observed was specific to the context of an RSV infection, as prime-boosted mice challenged with a recombinant influenza virus expressing the same RSV-derived epitope did not exhibit enhanced disease. The induction of a pro-inflammatory cytokine storm mediated by TNF-α and IFN-γ was observed in the serum of prime-boosted mice following RSV challenge. Additionally, RSV-specific memory CD8 T cells produced large amounts of IFN-γ locally within the lung, and adoptive transfer of wild-type but not IFN-γ-deficient memory CD8 T cells resulted in enhanced airway dysfunction and weight loss. Our results indicate that memory CD8 T cells are able to mediate protection against RSV infection. However, memory CD8 T cells acting alone in the absence of antibodies and memory CD4 T cells induce significant immunopathology and mortality through the induction of a systemic pro-inflammatory cytokine storm and local IFN-γ production.
Differential antigen presentation and transport by neonatal and adult dendritic cell subsets during Respiratory Syncytial Virus (RSV) infection
Authors: Lau-Kilby Annie W , Kehl Margaret , Malloy Allison MW
Published: January 2023

Dendritic cells (DCs) are critical for the induction of anti-viral CD8+ T cells. Young infants mount poor antiviral responses contributing to increased disease susceptibility and severity compared with acquisition later in life. Using a mouse model of respiratory syncytial virus (RSV), we have previously shown differences in the number of DCs that can carry antigen from the lung to the lung-draining lymph node (dLN) during infection between adults and neonates, and this is associated with age-dependent differences in RSV-specific CD8+ T cell responses. However whether adult or neonatal DCs more readily present antigen for T cell engagement is unknown.

In this study, we used an ovalbumin-expressing RSV (RSV-OVA) to track antigen presentation through early infection. In the lung, expression of MHCI-bound OVA (MHCI-OVA) was highest on adult CD103+ DCs; while in the dLN a greater number of neonatal CD103+ DCs expressed MHCI-OVA. Quantification of the migration-associated chemokine receptor 7 identified increased expression on neonatal MHCI-OVA-expressing CD103+ DCs compared to adults, suggesting adults may more readily present antigen but neonatal CD103+ DCs were more apt to migrate upon antigen acquisition. However, neonates failed to induce an OVA-specific T cell response in the lung in contrast to adults.

Together our data indicate that in neonates MHCI-OVA+ CD103+ DCs were more proficient at migration resulting in a greater number of MHCI-OVA-presenting CD103+ DCs in the dLN;- however, they failed to induce an OVA-specific T cell response. Thus antigen abundance was not the primary determinant of CD8+ T cell induction during RSV infection in neonates, suggesting that alternate mechanism(s) limit their DC-mediated T cell responses.
Tetherin controls T cell proliferation and exhaustion by shaping the early distribution of a persistent viral infection
Authors: Kenyon Elizabeth A , Nayak Debasis , McGavern Dorian B
Published: January 2023

Type I interferons participate in the host defense against most viral infections through induction of innate antiviral proteins like tetherin (BST-2). In vitro studies have shown that tetherin can physically sequester viruses by linking them to infected cells, which in turn reduces virion release. It is not currently understood how this antiviral protein functions in vivo to control an immune response mounted against a persistent viral infection. We sought novel mechanistic insights into the function of tetherin by studying its role in the establishment of a persistent lymphocytic choriomeningitis virus (LCMV) infection. Following viral infection, tetherin was significantly upregulated in B cells, plasmacytoid dendritic cells (DCs), conventional DCs, and brain-resident microglia. Genetic deletion of tetherin resulted in an early failure to contain a persistence-prone strain of LCMV in the splenic marginal zone. This resulted in a rapid re-distribution of LCMV into the red and white pulp at early time points post-infection. This also interfered with the early priming of antiviral T cells by causing them to localize preferentially to the splenic red pulp where they proliferated less and acquired an impaired phenotype characterized by upregulation of exhaustion markers (PD-1, LAG-3, TIGIT), loss of cytokine production, and decreased motility. Importantly, these early alterations impeded the ability of the host to control persistence-prone LCMV. Viral control was delayed in peripheral tissues, and lifelong persistence was established in the brain. These data demonstrate that tetherin plays a critical in vivo role in shaping the anatomical distribution and adaptive immune response against a persistent viral infection.
Transposon mutagenesis screen identifies host-encoded anti-viral factors
Authors: Bruchez Anna M , Sha Ky , Chen Li , Lacy-Hulbert Adam , Stuart Lynda
Published: January 2023

Ebola virus causes sporadic outbreaks of severe hemorrhagic fever. Owing to the high mortality rate, it poses a significant health risk and is a potential bioterrorism agent. However, our knowledge of how it usurps host cells to complete its life cycle, and more importantly, how this can be combated, is limited.

Here we describe a genome-wide forward genetic approach to identify host genes, known as restriction factors, which confer resistance to viral infection. In this system cells are mutagenized with a transposon that activates genes close to its insertion site. Libraries of mutagenized cells are screened with a cytotoxic virus and resistant cell populations are selected. Illumina sequencing is used to characterize transposon insertion sites and identify candidate resistance genes.

We have performed this screen in five human cell lines to identify genes conferring resistance to either vesicular stomatitis virus (VSV) or recombinant VSV bearing the Ebola virus surface glycoprotein. We have identified genes associated with resistance to only Ebola-VSV, or to both viruses, demonstrating that resistance occurs by targeting either viral entry or viral replication. One of the genes our screen identified is NPC1, the Ebola virus entry receptor, thus confirming that this is a valid method of identifying virus-host interactions. We have also identified a number of novel candidate Ebola restriction factors, which we are characterizing further, and are extending the screen to additional cell lines and viruses. This work provides new insights into host anti-viral defense mechanisms and may identify novel therapeutic targets that can be utilized to promote host-encoded anti-viral immunity.
Mutations in the 5′ NTR and the non-structural protein 3A of the coxsackievirus B3 selectively attenuate myocarditogenicity
Authors: Massilamany Chandirasegaran , Gangaplara Arunakumar , Basavalingappa Rakesh Halekote , Rajasekaran Rajkumar A , Steffen David , Pattnaik Asit K , Reddy Jay
Published: January 2023

The 5′ non-translated region (NTR) is an important molecular determinant that controls replication and virulence of coxsackievirus B (CVB)3. In our studies of CVB3-induced myocarditis, we sought to generate an infectious clone of the virus for routine in vivo experimentation. By determining the viral nucleotide sequence, we identified three new nucleotide substitutions in the clone that differed from the parental virus strain: C97U in the 5′ NTR; a silent mutation, A4327G, in non-structural protein 2C; and C5088U (resulting in P1449L amino acid change) in non-structural protein 3A of the virus leading us to evaluate the role of these changes in the virulence properties of the virus. We noted that the disease-inducing ability of the infectious clone-derived virus in three mouse strains was restricted to pancreatitis alone, and the incidence and severity of myocarditis were significantly reduced. We then reversed the mutations by creating three new clones, representing 1) U97C; 2) G4327A and U5088C; and 3) their combination together in the third clone. The viral titers obtained from all the clones were comparable, but the virions derived from the third clone induced myocarditis comparable to that induced by wild type virus; however, the pancreatitis-inducing ability remained unaltered, suggesting that the mutations described above selectively influence myocarditogenicity. Our data point to a possibility that, CVB3 viruses containing such altered nucleotides may evolve naturally leading to their survival.
Tracheobronchial papillomatosis.
Authors: Somerville Lindsay Ayers Lucas
Published: January 2023

Recurrent respiratory papillomatosis (RRP) is a rare clinical entity characterized by squamous cell papillomas in the tracheobronchial tree. RRP is caused by human papillomavirus (HPV) 6 and 11. HPV-specific memory T cells in adults suggest most infections are asymptomatic or self-limited. However, a small subset of patients develop persistent HPV infection and invasive malignancies. RRP is observed in infants following vertical transmission and in adults in the second and third decade following sexual transmission. There is low malignant potential, although patients with smoking or radiation history, p53 mutations, or HPV-11 subtype have increased risk of transformation into squamous cell cancer.

We present a case of tracheobronchial papillomatosis in a 73-year-old man who presented with exertional dyspnea. History was notable for a laryngeal polyp resected 30 years prior. Pulmonary function testing suggested variable intrathoracic obstruction (Figure A). Computerized tomography of the chest revealed a 1.4 × 1.1 × 2.0 soft tissue mass adherent to the mid thoracic trachea (Figure B). We performed rigid bronchoscopy with laser devascularization and mechanical debridement. Pathology demonstrated well-differentiated squamous epithelium overlying fibrovascular cores (rete ridges) with elongated nuclei characteristic of squamous papillomatosis (Figure C).

The quadrivalent HPV vaccine is approved for prevention of genital warts and gynecologic malignancies caused by HPV 6, 11, 16 and 18. Vaccination could potentially reduce the prevalence of RRP by decreasing vertical and sexual transmission of HPV 6 and 11. Ongoing investigations are currently evaluating the role of vaccination in patients with preexisting RRP.
Crucial role of FOXO1 in inflationary CD8+ T-cell responses during persistent viral infection
Authors: Delpoux Arnaud , Michelini Rodrigo Hess , Verma Shilpi , Wellisch Brittney , Lai ChenYen , Benedict Chris A , Hedrick Stephen M
Published: January 2023

Cytomegalovirus (CMV) establish persistent infection that induce the accumulation of virus-specific T cells over time in a process called memory inflation. Although persistence of antigen (Ag) is considered essential, the molecular pathway for the inflationary T-cell development is poorly understood. Mainly, inflationary CD8 T-cells have an effector-memory phenotype (KLRG1hi CD27−), but a low percentage of them express a central-memory phenotype (KLRG1lo CD27+), which are considered as their memory precursors. To study the role of FOXO1 in CD8 T cells during a persistent viral infection, we analyzed mouse bone marrow chimeras in which FOXO1 was specifically deleted in CD8 T cells. These mice were infected with MCMV-Δm157 strain, and the expansion and phenotype of inflationary and acute-contracting T cells was examined. The results revealed that the IE3 and M38 CD8 T cell responses do not inflate in absence of FOXO1. Furthermore, with an absence of FOXO1, there were fewer Ag-specific CD8 T cells that produced both IFNγ and TNFα. Consistent with this lack of CD8 effector cells the mice were less able to control the virus in the spleen and the liver at day 6 after infection. Moreover, we found that FOXO1 KO Ag-specific CD8 T cells fail to up-regulate the memory associated transcription factors, TCF-1 and EOMES, correlating with a lower percentage of memory precursors (KLRG1lo CD27+). Finally, we found that FOXO1 KO Ag-specific CD8 T cells cycle normally (Ki67+), but display an anergic state, as measured by calcium mobilization activation, and are prone to apoptosis. Collectively, these results demonstrate an intrinsic role for FOXO1 in establishing the inflationary memory program that is essential to forming long-lived effector memory cells
Masking of antigenic epitopes by antibodies shapes the humoral immune response to influenza
Authors: Zarnitsyna Veronika I , Ellebedy Ali H , Davis Carl , Jacob Joshy , Ahmed Rafi , Antia Rustom
Published: January 2023

The immune responses to influenza, a virus that exhibits strain variation, show complex dynamics where prior immunity shapes the response to the subsequent infecting strains. Original antigenic sin (OAS) describes the observation that antibodies to the first encountered influenza strain, specifically antibodies to the epitopes on the head of influenza’s main surface glycoprotein, haemagglutinin (HA), dominate following infection with new drifted strains. OAS suggests that responses to the original strain are preferentially boosted. Recent studies also show limited boosting of the antibodies to conserved epitopes on the stem of HA, which are attractive targets for a ‘universal vaccine’. We develop multi-epitope models to explore how pre-existing immunity modulates the immune response to new strains following immunization. Our models suggest that the masking of antigenic epitopes by antibodies may play an important role in describing the complex dynamics of OAS and limited boosting of antibodies to the stem of HA. Analysis of recently published data confirms model predictions for how pre-existing antibodies to an epitope on HA decrease the magnitude of boosting of the antibody response to this epitope following immunization. We explore strategies for boosting of antibodies to conserved epitopes and generating broadly protective immunity to multiple strains.
Novel antitumor complexes of bispecific antibodies using ALT-803 as a scaffold demonstrate Tetra-specific binding activities
Authors: Liu Bai , Kong Lin , Noel Terra , Han Kaiping , You Lijing , Dee Michael , Rhode Peter R , Jeng Emily K , Alter Sarah , Wong Hing C
Published: January 2023

IL-15 and its receptor α (IL-15Ra) are co-expressed in antigen presenting cells allowing trans-presentation of IL-15 to immune cells bearing IL-2Rβγc and stimulation of effector immune responses. We have demonstrated that an IL-15 superagonist:IL-15Rα-Fc complex (ALT-803) is capable of stimulating T cell and NK cell responses. We have reported that ALT-803 could be exploited to create a functional scaffold for the design of multivalent disease-targeted complexes (Liu et al, J. Biol. Chem. 2016 291: 23869). The lead molecule based on anti-CD20 rituximab is currently in pre-clinical development. In this study, this approach was further optimized by generating an IL-15 superagonist:IL-15Rα-Fc complex (2B8T3M) comprising two anti-human CD20 scFv domains and two anti-human CD3 scFv domains. 2B8T3M molecules exhibit CD3, CD20, Fc-receptor and IL-15Rbgc tetra-specific binding activities and IL-15 bioactivity. In contrast to 2B8T2M which redirects NK cells to lyse CD20+ B-lymphoma cells (ADCC), 2B8T3M was capable of redirecting both CD8+ T cells via the anti-CD3 scFv domains and NK cells via the Fc domain against CD20+ Daudi cells. In order to investigate antitumor activities in vivo of the tetra-specific molecules in immunocompetent mouse, we have also constructed and generated mouse CD3/CD19-binding T3M complexes. Antitumor efficacy of these molecules is being assessed in mouse A20 B cell lymphoma model. These findings suggest that tetra-specific T3M complexes may serve as novel, T-cell based targeted immunotherapeutics for treating cancer.
Protective function of Calcium permeable cation channel TRPM2 during pneumoseptic Klebsiella pneumoniae infection
Authors: Sharma Jyotika , Tripathi Jitendra , Sharma Atul , Jondle Christopher Ned , Mishra Bibhuti B.
Published: January 2023

Calcium influx mediated by plasma membrane calcium permeable cation channels play essential role in immune homeostasis. Transient receptor potential melastatin 2 (Trpm2) is a trp superfamily member cationic channel which is reported to shape several effector functions of myeloid cells. Because of its role in neutrophil chemotaxis, we examined a yet undetermined function of Trpm2 in pathogenesis of Klebsiella pneumoniae (KPn), a causative agent of pneumonic sepsis with a significant neutrophil component. Here we show that Trpm2−/− mice exhibit significantly increased susceptibility to pulmonary KPn infection corresponding with an increased bacterial burden and exacerbated tissue pathology. This correlated with a modulation of local and systemic inflammatory response in-vivo in KPn infected Trpm2−/− mice as well as altered calcium influx, ROS generation and MAP kinase activity in Trpm2 deficient neutrophils compared with their WT counterparts. Our data suggest that Trpm2 regulates KPn pathogenesis by controlling neutrophil mediated inflammation and oxidative stress.
Combined inhibition of JAK/STAT and BCL2 during ex vivo NK cell expansion enriches a highly cytotoxic NK cell subset
Authors: Walker Kirsti Lorraine , Gavcovich Tara B , Ea Dennis , Capitini Christian M.
Published: January 2023

Ex vivo expansion of human NK cells with CD137L and IL-15 is being explored both preclinically and clinically as a means of increasing the number and activation of NK cells for a variety of cancers, but results to date have not shown consistent efficacy. JAK1/2 inhibition impairs NK cell maturation, activation and cytotoxicity. However, the absence or mutation of STAT1/3 molecules which are typically phosphorylated by JAK1/2 enhance NK cytotoxicity. We hypothesize that inhibition of the JAK/STAT pathway in CD137L/IL-15 activated NK cells stimulate NK cell activation and increase more potent effectors if combined with a BCL2 inhibitor. NKs were ex vivo expanded with CD137L/IL-15 for 12 days, then exposed to increasing concentrations of Ruxolitinib (0.313 – 10μM) and/or Venetoclax (6.25 – 200nM) for 24 hours before analysis. Ruxolitinib and Venetoclax enriched a CD56dim cytotoxic subset and decreased a CD56 bright cytokine-producing subset in a dose-dependent manner. Ruxolitinib alone increased CD16 and CD69 expression which indicates NK cell activation. Importantly, both drugs did not induce markers associated with senescence (CD57) or exhaustion (PD-1/Tim-3) or decrease NK cell viability. Treatment of two human T cell acute lymphoblastic leukemia (T-ALL) cell lines with 1.25μM Ruxolitinib and 25nM Venetoclax decreased proliferation and increased apoptosis of T-ALL cells in a synergistic manner (CI<1; p<0.001). Usage of Ruxolitinib and Venetoclax has implications on both the biology of ex vivo activated NK cells and T-ALL, and may be a novel pharmacologic combination for improving adoptive cell therapy of leukemia.
Mouse Models for Immunology Research available from The Jackson Laboratory Repository
Authors: Valenzuela Alicia , Fancher Karen , Lutz Cat , Rockwood Stephen
Published: January 2023

The Jackson Laboratory Repository serves as a centralized facility for the development, distribution and cryopreservation of mouse models of human biology and disease. Hundreds of new strains are added annually to one of the largest collections of characterized mouse strains available.

Our newest strains carry genes associated with T lymphocyte regulation and development. T cells from the Fyb (ADAP) knockout (KO) exhibit impaired proliferation and TCR-mediated adhesion. T cells from the Cd27 KO have a diminished viral response and a delayed memory cell response. A conditional KO of Kdm6b (Jmjd3), a histone demethylase, results in altered T cell differentiation. Finally, conditional expression of an HA-tagged mutant human transcription factor, FOXO, is associated with regulatory T cell function.

Mice carrying the Ifnar1 (interferon αβ receptor 1) KO allele have a reduced immune response and increased susceptibility to viral infection. The Ifnar1 KO, combined with the Ifngr1 (interferon γ receptor 1) KO allele provide a model for Zika virus (and other viruses) pathogenesis.

New tool strains include two conditional GFP knockin reporters, one in Spic, a transcription factor expressed in macrophages, and the second in Ebf1, a transcription factor involved in B cell development. A knockin to Zbtb46, a zinc finger transcription factor, expresses Cre recombinase in classical dendritic cells.

Donating a strain to the Repository fulfills the NIH’s requirements for sharing mice. Researchers wishing to have strains considered for inclusion in the Repository are encouraged to submit their strains: www.jax.org/donate-a-mouse.

This work is supported by NIH, HHMI, and several private charitable foundations.
MicroRNA-155 regulates T-bet expression in anti-viral CD8+ T cells
Authors: Hope Jennifer L , Stairiker Christopher , Spantidea Panagiota , Gracias Donald Tom , Carey Alison J , van Meurs Marjan , Brouwers-Haspels Inge , Fike Adam J , Rijsbergen Laurine , Mueller Yvonne M , Fraietta Joseph A , Klop Roos , Erkeland Stefan , Katsikis Peter D
Published: January 2023

MicroRNAs (miRNAs) are small, single-stranded non-coding RNAs that play essential roles in regulating key cellular processes, and are highly conserved across species. miRNA are known to regulate immune cells and play an important role in effective CD8+ T cell (CTL) responses to viral infection and tumors. To understand the role of miRNA in CTL responses we performed microarray miRNA expression profiling and identified unique in vivo expression patterns of miRNAs in naïve and effector anti-viral CTL. In particular, miR-155, which we and others previously have demonstrated to regulate CTL responses, was significantly upregulated in effector CTL. We show that further increasing the levels of miR-155 by retroviral overexpression significantly augments anti-viral effector CTL and skews memory CD8+ T cells towards an effector memory phenotype. MiR-155 overexpression resulted in enhanced T-bet expression, which is known to be required for effector CTL and to promote effector memory cell formation. MiR-155 overexpression-induced T-bet expression was required for miR-155 mediated increases in effector CTL. Our studies have revealed an important and unexpected link between miR-155 and T-bet transcription factor in the context of in vivo CTL responses.
Combating Antibody-dependent Enhancement of Dengue Viral infection in Peripheral Blood Monocytes by NK Cell-mediated Antibody Dependent Cell Cytotoxicity
Authors: Sun P , Williams Maya , Nagabhushana Nishith , Morrison Brian J
Published: January 2023

Background

The dengue virus (DENV) group is composed of 4 serotypes, DENV-1, -2, -3 and -4. The 4 serotypes of DENV share70% sequence homology, therefore cross-reactivity among 4 serotypes is common. Cross-reactive antibodies (Ab) can cause Ab-dependent enhancement (ADE) of viral infection. ADE is thought to be an underlying mechanism of elevated immune-pathology associated with severe dengue diseases. Human monocytes and macrophages are target cells for ADE. This study investigates NK cell-mediated Ab-dependent cell cytotoxicity (ADCC) as a critical control mechanism following an established ADE intracellularly.

Methods

To model the physiological situation, whole human PBMCs were infected with DENV in the presence of autologous serially diluted sera. ADE in monocytes was detected by intracellular staining of viral antigens and by RT-PCR. NK cell activation was monitored in the same cultures by a degranulation marker CD107a. Other NK cell markers, cell proliferation and cytokine/chemokines were investigated.

Results

ADE was only detected in monocytes with the serially diluted immune sera. NK cell activation occurred concomitantly with the detection of ADE. The peak of the ADCC response was 48 hours post infection, but the response was still detectable at day 7. Minimal activation of T cells was observed. The power of ADE in monocytes ranged between 3 to 10.2 folds when NK cells were depleted as compared to 0.6 to 1.8 folds under normal condition. Activated NK cells had elevated expression of CD69, CD335, CD336, and CD96, but had downregulated CD337 and CD314.

Conclusion

ADCC is activated when DENV escapes from Ab neutralization and establishes infection in host cells, and it can act as the first line of defense to combat ADE.
Anti-spike IgG exacerbates lung injury by skewing monocyte-macrophage responses during SARS-CoV infection
Authors: Liu Li , Wang Haibo , Wei Qiang , Tang Hangying , Nishiura Kenji , Kwok Hauyee , Peng Jie , Alvarez Xavier , Lackner Andrew , Chen Zhiwei
Published: January 2023

Deceased SARS patients displayed severe acute lung injury (ALI) with exuberant inflammatory responses yet faster neutralizing IgG development. The mechanism underlying this discrepancy remains elusive. Here, we combine vaccination and passive immunization strategies to determine whether anti-spike (S) IgG modulates SARS-CoV-mediated lung injury in Chinese rhesus macaques. We found that macaques pre-vaccinated with MVA-S displayed more severe lung injury compared with MVA-vaccinated controls after SARS-CoV challenge. Moreover, passive immunization of purified anti-spike IgG but not control IgG resulted dose-dependently in enhanced lung infection and severer lung injury, associated with prolonged responses of pro-inflammatory alveolar MAC387+ and CD163+TGF-b− macrophages, IL-6 production and delayed wound-healing CD163+TGF-b+ macrophages response. Consistently, accumulated pro-inflammatory CD163+TGF-b− macrophages were found in lungs of deceased SARS patients. Our results demonstrated that anti-spike IgG exacerbates lung injury likely by enhancing infection and skewed alveolar monocyte-macrophage responses, which may have significant implications to CoV-mediated pathogenesis and immunotherapy.
Efficient control of chronic LCMV infection by CD4 T cell epitope-based heterologous prime-boost vaccination in a murine model
Authors: He Ran
Published: January 2023

CD4+ T cells are essential for sustaining CD8+ T cell responses during chronic infection. The adoptive transfer of virus-specific CD4+ T cells has been shown to efficiently rescue exhausted CD8+ T cells. However, the question of whether endogenous virus-specific CD4+ T cell responses can be enhanced by certain vaccination strategies and subsequently reinvigorate exhausted CD8+ T cells remains unexplored. In this study, we developed a CD4+ T cell epitope-based heterologous prime-boost immunization strategy and examined the efficacy of this strategy using a mouse model of chronic lymphocytic choriomeningitis virus (LCMV) infection. We primed chronically LCMV-infected mice with the Listeria monocytogenes vector expressing the LCMV glycoprotein-specific I-Ab-restricted CD4+ T cell epitope GP61-80 (LM-GP61) and subsequently boosted the primed mice with an influenza virus A (PR8 strain) vector expressing the same CD4+ T cell epitope (IAV-GP61). This heterologous prime-boost vaccination strategy elicited strong anti-viral CD4+ T cell responses, which further improved both the quantity and quality of the virus-specific CD8+ T cells and led to better control of viral loads. The combination of this strategy and the blockade of the programmed cell death-1 (PD-1) inhibitory pathway further enhanced anti-viral CD8+ T cell responses and viral clearance. Therefore, heterologous prime-boost immunization that selectively induces virus-specific CD4+ T cell responses in conjunction with inhibitory pathway blockade may represent a promising therapeutic approach to treating patients with chronic viral infection.
A subset of NK1.1+ follicular helper CD4+ T cells promotes early extrafollicular plasmablast production during Plasmodium yoelii infection
Authors: Stumhofer Jason Scott , Wikenheiser Daniel J
Published: January 2023

Early plasmablast induction is a hallmark of Plasmodium infection, including P. yoelii, and is thought to be essential for control of acute parasite burden. Although long understood to be a T-cell dependent phenomenon, control of early plasmablast differentiation in response to Plasmodium is poorly understood. Here, we identify a subset of CD4+ T cells that express the innate NK cell marker NK1.1 as being critical for early plasmablast and parasite-specific Ab production. Interestingly, NK1.1+ CD4+ T cells arise from conventional, naive NK1.1− CD4+ T cells and their induction is independent of CD1d but critically reliant on MHC-II. Early NK1.1+ CD4+ T cells express more ICOS than conventional (NK1.1−) CD4+ T cells and subsequently expand more rapidly and give rise to PD-1high follicular helper T (Tfh) cells more frequently than conventional CD4+ T cells. Further study of this population revealed that NK1.1+ Tfh cells are more likely to be complexed with plasmablasts than conventional Tfh cells. Moreover, NK1.1+ Tfh cells interact almost exclusively with PD-L1high plasmablasts and more readily express markers indicative of apoptosis than conventional Tfh cells. Ultimately, depletion of NK1.1+ cells significantly impairs class-switched parasite-specific antibody production during early P. yoelii infection. Together, these data suggest NK1.1+ Tfh cells are essential for driving early plasmablast induction during Plasmodium infection and may represent a unique Tfh cell subset whose modulation could promote effective vaccine design.
IL-15 complex-stimulated NK cells protect mice from cerebral malaria
Authors: Burrack Kristina Stoermer , Huggins Matthew A , Hart Geoffrey T , Johnson Aaron J , Jameson Stephen C , Hamilton Sara E
Published: January 2023

Cerebral malaria (CM) is one of the most lethal complications of Plasmodium falciparum infection, responsible for a large fraction of the nearly 500,000 malaria-related deaths annually. In the experimental mouse model of CM (ECM), mice are inoculated with red blood cells infected with Plasmodium berghei ANKA (PbA) parasites and develop a CM-like disease within 6–10 days post-infection (dpi) resulting from CD8 T cell-mediated damage to the CNS. We found that treatment of C57Bl/6 mice with interleukin (IL)-15 complexes (IL-15C; IL-15 bound to an IL-15Rα-Fc fusion protein) prevented the development of PbA-induced ECM. IL-15C treatment stimulates natural killer (NK) and CD8 T cells. Interestingly, adoptive transfer of IL-15C-stimulated NK cells, but not CD8 T cells, prevented ECM. Similar complexes formed with IL-2 (IL-2C; IL-2 bound to the anti-IL-2 S4B6 antibody) also induce robust expansion and activation of NK cells, but fail to protect against ECM. Rescue from ECM was not associated with reduced parasitemia at 6 dpi. Instead, IL-15C treatment resulted in reduced CD8 T cell activation in the brain at 6 dpi and reduced blood brain barrier breakdown. Moreover, we found that in vivo IL-15C, but not IL-2C, treatment of mice induces IL-10 expression in NK cells, and that IL-10 is required for IL-15C-mediated protection from ECM. These data indicate that NK cells – which are typically involved in promoting inflammatory responses – can also restrain damaging immune responses, and that differences between the activation of NK cells by IL-2C and IL-15C regulates their capacity to control the inflammatory response to blood stage malaria infection.
Th1 differentiation drives the accumulation of intravascular, non-protective CD4 T cells during tuberculosis
Authors: Sallin Michelle A , Sakai Shunsuke , Kauffman Keith , Young Howard A , Zhu Jinfang (Jeff) , Barber Daniel L
Published: January 2023

Mycobacterium tuberculosis (Mtb) is the leading cause of death by infectious disease worldwide. The development of novel TB vaccination strategies would have dramatic global public health benefits, but have been hindered by an incomplete understanding of the mechanisms of protective immunity against Mtb infection. Recent data indicate that the differentiation state of Th1 cells determines their protective capacity against tuberculosis. We have shown that less-differentiated CXCR3+T-betdim Th1 cells migrate into the lung, expand and control Mtb infection after transfer into infected recipients, while KLRG1+CX3CR1+T-bethigh terminal effectors poorly migrate into the lung, fail to expand and cannot transfer protection against Mtb infection. IL-12/23p40 and T-bet have been shown to promote terminal differentiation, leading to the hypothesis that these host protective factors may have unrecognized detrimental effects on the quality of the Mtb-specific CD4 T cell response. We find IL-12/23p40 promotes Th1 cell expansion and maturation beyond the CD73+CXCR3+T-betdim stage, and T-bet prevents deviation of Th1 cells into Th17 cells. Nevertheless, IL-12/23p40 and T-bet are also essential for the production of a subset of intravascular CX3CR1+KLRG1+Th1 cells. Furthermore, T-bet potently suppresses the development of CD69+CD103+ tissue resident phenotype effectors in the lung. In contrast, CD4 T cell-derived IFNγ inhibits the accumulation of intravascular CX3CR1+KLRG1+Th1 cells. Thus, although IL-12 and T-bet are essential host survival factors, they simultaneously oppose lung CD4 T cell responses at several levels, demonstrating a functional dichotomy of Th1 polarization in tuberculosis.
Alternative polyadenylation signals regulate HLA-A surface expression
Authors: kulkarni smita , Ramsuran Veron , Rucevic Marijana , Lied Alexandra , kulkarni Viraj , Le Gall Sylvie , Carrington Mary
Published: January 2023

Genomic variations in the 5′ and 3′ untranslated region (UTR) have been shown to influence Human leukocyte antigen (HLA) class I expression, the strength of immune responses and disease outcomes beyond peptide binding. Sequencing of the 3′UTR of common HLA-A alleles indicated a presence of three polyadenylation signals (PAS). The proximal and the middle PAS are conserved whereas the distal PAS is disrupted in some alleles. Using 3′ rapid amplification of cDNA ends (3′RACE), we confirmed expression of two distinct forms of HLA-A 3′UTR that use the proximal or the distal PAS and differ in length by 100 base pairs. HLA-A alleles varied in the usage of PAS, with some alleles using only the proximal, and others using both the PAS to differing degrees. The short and long 3′UTRs cloned in luciferase vectors and expressed in the human T cell line-Jurkat showed similar mRNA expression levels. However, the long 3′UTR conferred lower luciferase expression as compared to the short form, indicating translation inhibition of the long 3′UTR. RNA affinity pulldown followed by mass-spectrometric analysis indicated enrichment of an RNA-binding protein (RBP), Syncrip, among proteins bound to the long 3′UTR. Only the long form of cellular HLA-A 3′UTR co-precipitated with Syncrip. The siRNA depletion of Syncrip increased surface expression of HLA-A alleles with the long 3′UTR, whereas an allele expressing only the short form was unaffected. Further, specific blocking of the proximal 3′UTR reduced surface expression without a decrease in mRNA expression. These data demonstrate allele-specific variation in the PAS usage by HLA-A modulated its expression, which adds another layer of regulation and variation to the expression of HLA-A.
JAK/STAT Signaling of Natural Killer (NK) Cells Following Cytokine Stimulation
Authors: Rodriguez-Nieves Jennifer , Tuck Ryan , De Paris Kristina
Published: January 2023

Natural Killer (NK) cells are an important component of the innate immune system, capable of providing a fast and effective response against virally infected cells. NK cells are mainly characterized by their cytotoxic function and their ability to secrete cytokines. It has been shown that infant NK cells have decreased cytotoxicity and cytokine-secreting function, suggesting hyporesponsiveness of NK cells during the first year of life. Because NK cell activation is dependent on cytokine stimulation, we hypothesized that infant NK cells are hyporesponsive due to deficiencies in signaling following cytokine stimulation. We examined the activation of human infant and adult NK cells in response to IL2, IL12 or IFNα stimulation, cytokines important in NK survival and function. Using Phosflow analysis, we measured the phosphorylation of transcription factors, STATs, specific for the distinct cytokines. Cord blood NK cells showed significantly lower pSTAT activation when compared to adult NK cells when treated with IL2 or IFNα but not IL12. However, despite pSTAT4 activation in response to IL12, no nuclear translocation was observed by ImageStreamX Mark II analysis. NK cells in 6 and 12-month-old infants showed similar frequencies of pSTAT NK cells compared to adults when treated with IL2, IL12 or IFNα, suggesting that cytokine signaling in NK cells is age-dependent. Surprisingly, pSTAT activation was restored in cord blood NK cells when treated with a cocktail of IL2, IL12 and IL15. These results suggest that JAK/STAT signaling in infant NK cells is impaired at different steps in the pathway in response to specific cytokines.
Cereblon deficiency increases polyamine synthesis in activated T-cells through the amplification of c-MYC and the arginine and proline metabolic pathway
Authors: Beatty Matthew S , Hesterberg Rebecca S , Han Ying , Epling-Burnette Pearlie
Published: January 2023

Thalidomide, and its immunomodulatory drug analogs (IMiD) including lenalidomide and pomalidomide, activate T-cells and reverse T-cell anergy in cancer patients. Previously we have shown that cereblon, the molecular target of IMiD compounds, is a negative regulator of T-cell metabolism and signaling following suboptimal antigen stimulation. To explore this mechanistically, differentially-regulated genes were subjected to gene set enrichment analysis (GSEA) for transcriptional regulators and global metabolomic profiling was conducted by mass spectrometry in activated Crbn+/+ versus Crbn−/− T-cells. With results confirmed by 1H-NMR spectra analysis, we show that Crbn deficiency leads to an increase in intermediates of the “Arginine/Proline” pathway and enhanced polyamine (putrescine, spermidine, and spermine) biosynthesis. Wang et al showed previously that c-MYC controls polyamine synthesis downstream of mTORC1 during T-cell activation. In activated Crbn−/− CD8+ T-cells, the kinetics and magnitude of c-Myc mRNA were similar to Crbn+/+ T-cells. However, c-MYC is largely regulated through post-transcriptional mechanisms including ubiquitin proteasome-mediated degradation and regulation of c-Myc mRNA translation. Interestingly, polyamines directly bind U- and AU-rich elements (AREs) in the 3′-untranslated region (3′-UTR) of c-Myc mRNA and spermidine is the substrate for a unique post-translational modification required for the efficient translation of c-MYC. Western blot analysis revealed more rapid and sustained expression of c-MYC in activated Crbn−/− T-cells compared to Crbn+/+ T-cells suggesting that CRBN is a regulator of the MYC-polyamine circuit controlling T-cell metabolic fate.
STAT5b Controls IgE-mediated Mast Cell Functions In Vitro and In Vivo
Authors: Kiwanuka Kasalina N , Ryan John J
Published: January 2023

Signal Transducers and Activators of Transcription (STATs) are latent transcription factors that mediate a number of cellular responses including but not limited to cell survival, proliferation and differentiation. This protein family consists of seven members, STATs 1 – 6 including two closely related molecules, STAT5a and STAT5b, that show 96% amino acid sequence homology and are critical for lymphoid, myeloid, and erythroid cell development and function. We recently identified Stat5b as the critical regulator of IgE-mediated cytokine production in mast cells. STAT5b knockout (KO) cells show decreased cytokine production in vitro. In vivo STAT5b KO mice demonstrate decreased sensitivity to IgE-mediated passive systemic anaphylaxis accompanied with decreased production of IL-6 and IL-13 compared to wildtype counterparts. There was no difference in MAR-1-mediated passive systemic anaphylaxis or mast cell number and distribution. Interestingly STAT5b KO mice demonstrate elevated levels of serum IgE and an exacerbated response to histamine-mediated passive systemic anaphylaxis. We previously found that Fyn-mediated Stat5 tyrosine phosphorylation is critical for Stat5-dependent effects. The current work demonstrates that Stat5b is also phosphorylated on serine residues during IgE signaling in mast cells, via a Src-independent pathway requiring PI3-kinase function. Preliminary data show that STAT5b tyrosine and serine phosphorylation either independently or in tandem regulate IgE-induced cytokine production. Elucidating the role of STAT5b in the inflammatory response as well as the key players in STAT5b serine phosphorylation will yield new targets for mast cell-associated diseases.
Establishing the HIV reservoir: HIV-susceptible cells and the signals that recruit them
Authors: Packard Thomas Alexander , Herzig Eytan , Roan Nadia R , Greene Warner C
Published: January 2023

HIV-1 infected individuals require lifelong antiretroviral therapy (ART). If ART is discontinued, HIV rebounds from a reservoir of cells containing latent provirus—persistence of this reservoir is the most significant roadblock to curing people infected with HIV. The HIV reservoir is formed rapidly following infection and remains relatively stable throughout the lifetime of an infected individual on ART: this reservoir persistence likely stems from a central memory phenotype of host T cells.

Transmitted HIV binds and enters cellular targets via the co-receptor, CCR5, which is primarily expressed on effector memory CD4 T cells. How does HIV rapidly establish a central memory reservoir? Abortive HIV infection of lymphoid CD4 T cells activates IFI16, which can form inflammasomes, triggering pyroptosis. IFI16 also can activate STING to produce type 1 interferon and the CCL2 chemokine. Thus, an early innate immune response to HIV sets up a zone of inflammation.

CCL2 attracts cells expressing the receptor CCR2. Strikingly, CCR2 delineates a population of lymphoid CD4 T which co-express CCR5 and markers of central memory (CCR7 & CD62L)—precisely the same type of cells that make up the viral reservoir. We found that these lymphoid CCR2+ T cells migrate in response to CCL2 and are susceptible to infection with R5- or X4-tropic strains of HIV. We propose that CCL2 produced early in HIV infection lures CCR2+ central memory T cells to the site of inflammation. Here these cells can become productively infected and produce virus, or become latently infected and contribute to the stable reservoir. These findings raise the possibility that inflammation contributes to seeding of the latent reservoir, and might be reduced by antagonists of CCL2/CCR2.
The effect of HIV-1 infection on S1P-R1 expression and function in thymocytes and their progenitors during entry into and egress from the human thymus
Authors: Resop Rachel Samantha , Craft Joshua , Vatakis Dimitrios N , Uittenbogaart Christel H
Published: January 2023

Lack of adequate T cell regeneration in HIV infected individuals is likely due to a defect in the entry of hematopoietic stem cells (HSC) into and egress of naïve T cells from the thymus to the periphery. We studied the effect of HIV-1 infection on the receptors to Sphingosine-1-phosphate (S1P), a chemotactic sphingolipid mediator, during thymocyte trafficking. Our novel findings show that HIV infection changes the expression patterns of S1P-R1 on thymocytes about to exit the thymus.

To examine the dynamics of S1P-R1 expression on HSC progenitors and thymocytes during HIV infection, mice implanted with human fetal thymus/liver infected with CXCR4- or CCR5-tropic HIV-1 were injected intravenously with CFSE-labeled HSC. In infected mice CFSE labeled CD34+ progenitors developed into mature thymocytes in the thymic implant and a subset expressed S1P-R1, indicating that entry into the thymus and development are functional during early HIV infection. Surprisingly, we found that S1P-R1 was significantly upregulated in mature thymocytes post-HIV infection. Intriguingly, S1P-R1 was also upregulated by HIV within the CD3+CD69+ population, which normally does not express S1P-R1. S1P-R1 signaling as measured by phosphorylated Akt (pAkt) was not impaired in infected thymocytes, which is interesting in the context of published data demonstrating that S1P-R1 response in HIV infection may be impaired in other cell types.

Further results show that the mechanism of increased S1P-R1 in the thymus by HIV may be due to cytokines including Interferon-beta (IFN-β), which significantly increased S1P-R1 expression in both CD3hiCD69- and CD3+CD69+ thymocyte subsets treated in vitro with exogenous cytokine.
Monitoring the response to TB treatment using T cells biomarkers in TB/HIV patients undergoing ART
Authors: Adekambi Toidi , Ibegbu Chris , Cagle Stephanie , Ray Susan , Rengarajan Jyothi
Published: January 2023

TB is the leading cause of death in HIV-infected persons. Anti-TB treatment in TB/HIV co-infected persons is therefore a public health priority. In patients undergoing anti-TB treatment and ART, ART initiation is often deferred by 2–8 weeks at the physician’s discretion in patients presenting CD4 counts >350 cells/ml to sufficiently reduce the Mtb burden in order to minimize potential drug interactions and complications related to the immune reconstitution inflammatory syndrome (IRIS). However, we currently lack reliable “real time” diagnostic methods to assess Mtb clearance during TB treatment. Isolation of Mtb from sputum can take 4–6 weeks and the rate of sputum smear-negative/active TB cases is upto 66% higher in HIV(+) than in HIV (−) patients. PCR-based technology while sensitive, does not differentiate between live and dead Mtb. We have recently identified specific host immune activation markers (CD38 and HLA-DR) on Mtb-specific CD4+ T cells that correlate with Mtb clearance during and after TB treatment of HIV (−) pulmonary active TB using flow cytometry. We have extended these studies to assess the performance of these biomarkers in diagnosing ATB in HIV(+) patients. Further, we are exploring the value of these biomarker profiles on T cells from TB/HIV co-infected patients undergoing ART and TB treatment, to evaluate their potential to serve as clinical correlates of Mtb clearance in ART treated TB/HIV+ patients.