bluuebunny/crossref_metadata_2025 huggingface dataset:
Restoring CD8+ T cell anti-HIV activity using combinatorial immune checkpoint blockade
Authors: Jones Amber D , Van Duyne Rachel , Khakhina Svetlana , Smith Stephen M , Klase Zachary A
Published: February 2023

Antiretroviral therapy (ART) effectively reduces viral load and improves life expectancy but does not fully restore host immune function. During chronic infection, T cells upregulate multiple inhibitory immune checkpoint receptors (ICR) which results in the loss of T cell functionality; a state called T cell exhaustion. In this study, we utilized HIV-1 controllers (HC) who have an inherent ability to control viral replication in the absence of ART but eventually gain viral susceptibility due to unknown mechanisms. We hypothesize that efficient control of viral replication becomes compromised by CD8+ T cell exhaustion. Using our cohort, we demonstrate that CD8+ T cells of ART naïve HCs are both necessary and sufficient to suppress viral replication. We also demonstrate that during times of clinical viral control, ART naïve HCs have low expression of exhaustion markers and sustain in vitro viral control. Upon loss of control, our HCs upregulate exhaustion markers and are susceptible to in vitro infection. This study evaluates the synergistic effects of immune checkpoint blockade (ICB) to determine if blockade of ICRs can restore anti-viral functionality of exhausted CD8+ T cells. Using our in vitro spreading infection assay, we demonstrate that treatment with single ICB results in a modest improvement of anti-viral functionality in CD8+ T cells from HCs who have lost viral control. However, former HCs who support in vitro viral replication demonstrate restored suppression of viral replication in tissue culture in the presence of combinatorial anti-PD1 and anti-TIM3 ICB. Our data suggests that combinatorial ICB can restore CD8+ T cells anti-viral functionality and has the potential to be a novel HIV therapeutic intervention.
A single immunization with CAF08 provides newborns with Th1-mediated protection against Respiratory Syncytial Virus infection
Authors: van Haren Simon D. , Pedersen Gabriel , Kumar Azad , Ruckwardt Tracy , Moin Syed , Moore Ian , Minai Mahnaz , Liu Mark , Pak Jensen , Borriello Francesco , Ahmed Saima , Helmel Michaela , Andersen Peter , Graham Barney S , Steen Hanno , Christensen Dennis , Levy Ofer
Published: February 2023

Respiratory Syncytial Virus (RSV) is a leading cause of morbidity and mortality in children, due in part to their distinct immune system, characterized by impaired induction of T-helper 1 (Th1)□immunity. Here we describe cationic adjuvant formulation (CAF)-08, a liposomal vaccine formulation tailored to induce Th1 immunity in early life via synergistic engagement of Toll-like Receptor 7/8 and the C-type lectin receptor Mincle. Quantitative phosphoproteomics applied to human dendritic cells revealed a key role for Protein Kinase C-d for enhanced Th1 cytokine production in neonatal dendritic cells and identified signaling events resulting in antigen cross-presentation. In vivo, a single immunization at birth with CAF08-adjuvanted RSV pre-fusion antigen protected newborn mice from RSV infection through induction of antigen-specific CD8+ and Th1 cells. Overall, we describe a novel pediatric adjuvant formulation and characterize its mechanism of action providing a promising avenue for development of early life vaccines against RSV and other intracellular pathogens.
Influence of vaccine adjuvant physical properties on antigen processing and presentation in the lymph node
Authors: Ball Alexander , Pompano Rebecca R
Published: February 2023

Despite the efficacy of vaccines, there is a growing need for novel vaccine adjuvants that can drive type-1 immunity, a response governed by antigen presenting cells (APCs). Current methods used to evaluate efficacy of novel adjuvants fail to consider the spatiotemporal events of antigen presentation that dictate the development of cellular immunity. Understanding how adjuvants affect the initial development of type-1 immunity would result in the design of more effective adjuvants. We hypothesized that adjuvants induce unique spatiotemporal patterns within lymph nodes that differ based on the extent to which an adjuvant drains to the LN. These patterns are then responsible for shaping the adaptive immune response. We tested this hypothesis by converting alum, a depot-forming adjuvant that remained at the site of inoculation, into nanoparticles (np) that enter the lymphatics and drained to the LN after vaccination. The size and charge of the np alum was measured through dynamic light scattering and zeta potential. The immunostimulatory effects of both alum and np alum were measured through inflammasome activation of bone marrow-derived macrophages. The two adjuvant formulations were then used to examine the influence of depot-forming alum versus lymph-node (LN) draining alum on skin-resident dendritic cell migration to the LN. The number of migratory DCs in the LN after vaccination was measured by flow cytometry. Next, we made use of live ex vivo lymph node slices to measure chemokine secretion in the LN by ELISA and to observe patterns of DC migration through widefield microscopy. This study illustrates the impact of the physical properties of an adjuvant in shaping the early stages of the adaptive immune response.
Pre-existing cellular immunity to SARS-CoV-2 through an immunodominant epitope
Authors: Grant Emma J , Lineburg Katie E , Swaminathan Srividhya , Chatzileontiadou Demetra S.M. , Szeto Christopher , Sloane Hannah , Panikkar Archana , Raju Jyothy , Crooks Pauline , Rehan Sweera , Nguyen Andrea T , Lekieffre Lea , Neller Michelle , Tong Zhen Wei Marcus , Jayasinghe Dhilshan , Chew Keng Yih , Lobos Christian A , Halim Hanim , Burrows Jacqueline M , Riboldi-Tunnicliffe Alan , Chen Weisan , D’Orsogna Lloyd , Khanna Rajiv , Short Kirsty R , Smith Corey , Gras Stephanie
Published: February 2023

Significant efforts are being made worldwide to understand the immune response to SARS-CoV-2, responsible for the COVID-19 pandemic, including the role of preexisting T cell immunity. Understanding the mechanisms that promote crossrecognition by T cells induced by seasonal coronaviruses will be critical for future predictions on the role of pre-existing immunity in protection against severe disease. We demonstrate that the SARS-CoV-2 nucleocapsid (N) protein induces an immunodominant response in HLA-B7+ COVID-19-recovered individuals that is also readily detectable in unexposed donors. This immunodominant response is driven by a single N-encoded epitope that displays a high degree of conservation with the homologous region in circulating coronaviruses. We show that T cell-mediated crossreactivity can be detected towards the circulating OC43/HKU-1 coronaviruses, but not the 229E or NL63 coronaviruses, due to different peptide conformations. This cross-reactivity is driven by private T cell receptor repertoires with a bias for TRBV27 and a long CDR3b loop in unexposed and COVID-19-recovered individuals. Together, our findings demonstrate the basis of pre-existing immunity to a conserved and highly immunogenic SARS-CoV-2 epitope driven by cross-reactive memory T cells, suggesting long-lived protective immunity.
Immunodominant, Qa-1-restricted CD8 T cell response elicited against a non-canonical translation product in influenza A virus
Authors: Hogan Michael J , Maheshwari Nikita , Nicastri Annalisa , Ternette Nicola , Eisenlohr Laurence C.
Published: February 2023

The extent to which non-canonical translation products drive antiviral T cell responses is incompletely understood. We explored this question using an immunopeptidomic approach in an influenza virus model. C57Bl/6 mouse-derived cells were infected with influenza virus A/Puerto Rico/8/1934 (PR8), and MHC-bound peptides were eluted and analyzed by tandem mass spectrometry. Mass spectra were searched against a database of all viral gene segments translated in all possible reading frames, irrespective of the presence of start codons. We identified a 9-mer peptide, here termed M-SL9, that maps to an alternative reading frame of the sequence encoding matrix protein 1 (M1). To validate M-SL9 as a T cell epitope, we infected C57Bl/6 mice with PR8, collected tissues, and measured T cell reactivation by synthetic M-SL9 peptide. Remarkably, 10% of all CD8 T cells in the lung were specifically reactivated by M-SL9 peptide, and the vast majority of these were polyfunctional, producing two or more Th1 cytokines. An analysis of M-SL9 processing and presentation revealed that M-SL9 is restricted to the non-classical MHC class Ib molecule, Qa-1, and that presentation is dependent on the second 5′-proximal AUG codon within the M1-coding sequence, suggesting that its translation results from leaky ribosomal scanning. These data contribute to a growing body of work showing the importance of non-canonical MHC presentation in protective immune responses.
T cell insufficiency enables emergence of polyomavirus antibody escape variants
Authors: Lauver Matthew D , Goetschius Daniel J , Hafenstein Susan L , Lukacher Aron E
Published: February 2023

Deficient adaptive immunity underlies CNS diseases caused by JC polyomavirus (JCPyV), a ubiquitous member of the human virome. The best characterized of these is progressive multifocal leukoencephalopathy (PML), a frequently fatal brain demyelinating disease. Risk factors for PML are poorly defined but mainly involve conditions of T cell insufficiency or dysfunction, including AIDS and particular immunomodulatory therapies (natalizumab, rituximab). JCPyV isolates from PML patients’ brains frequently contain point mutations in the major capsid protein, VP1. However, these variants are not present in the patients’ kidneys, the major site of JCPyV persistence. How/if these mutations confer neurovirulence is actively debated. Mapping a common JCPyV-PML VP1 mutation into mouse polyomavirus (MuPyV), we found that this variant virus was resistant to a neutralizing monoclonal antibody (mAb) and poorly replicated in the kidney. Cryo EM analysis unambiguously defined the antibody epitope and identified the mechanism of antibody escape. Passaging MuPyV in the presence of VP1 mAb allowed emergence of antibody escape variants in vitro. To investigate the connection between T cell impairment and antibody escape in vivo, we infected antibody-deficient μMT mice with MuPyV, treated them with VP1 mAb, then subjected them to CD4 and CD8 antibody-mediated T cell depletion during persistent infection. In contrast to non-T cell depleted mice, which maintained long term control over MuPyV infection, T cell depletion led to lethal MuPyV disease resulting from the outgrowth of antibody-escape variants. These data show that a narrow PyV antibody response compounded by T cell insufficiency promotes emergence of nAb escape neurotropic variants.
Characterization of cross-reactive immunity following coronavirus vaccination or natural infection
Authors: Dangi Tanushree , Palacio Nicole M , Sanchez Sarah , Penaloza-MacMaster Pablo
Published: February 2023

SARS-CoV-2 has infected more than 100 million people worldwide. Several vaccine candidates have been deployed under emergency use authorization, but it is unclear whether a coronavirus (CoV) vaccine can protect against other CoV. To investigate this proof-of-concept, we evaluated cross-reactive immunity following vaccination with a modified vaccinia Ankara expressing SARS-CoV-1 spike protein. We first vaccinated C57BL/6 mice intramuscularly and then measured heterologous antibodies (SARS-CoV-2, OC43 and mouse hepatitis virus, MHV) by ELISA. Interestingly, the SARS-CoV-1 vaccine elicited cross-reactive antibodies that recognize these other CoV. Sera from mice immunized with the SARS-CoV-1 vaccine neutralized SARS-CoV-2 pseudovirus in vitro (5-fold greater than control naïve sera; p=0.007), and transfer of these immune sera into naïve mice provided partial protection after heterologous challenges. Similar cross-reactivity was observed following immunization with a SARS-CoV-2 vaccine, and we mapped a conserved CD8 T cell epitope in both SARS-CoV-1 and CoV-2 spike protein, allowing us to develop an MHC tetramer to track this cross-reactive response. Finally, we interrogated whether a CoV infection could elicit cross-reactive immunity. We show that an OC43 infection generated cross-reactive antibodies against SARS-CoV-2, OC43 and MHV, and conferred partial protection against MHV challenge. In summary, our findings demonstrate that cross-reactive immunity can be elicited by vaccination, providing a framework for the rational design of universal CoV vaccines. Moreover, these data suggest that prior infection with endemic CoV may provide partial protection against other CoVs.
CD8 co-receptor links T cell avidity and metabolism
Authors: Clutton Genevieve Tyndale , Weideman Ann Marie , Kallon Sallay , Xu Yinyan , Warren Joanna , Lenarcic Erik , Lin Lin , Council Olivia , Muehlbauer Michael , Mincey Adam , Hill Demitrius , Moorman Nathaniel , RolTisch  , Newgard Christopher , Bain James , Armistead Paul , Hudgens Michael , Goonetilleke Nilu
Published: February 2023

This study investigated the mechanistic basis behind the reported superior efficacy of high-avidity CD8 T cells. CMV and HIV are both chronic viral infections, but while CMV-specific CD8 T cells can mediate lifelong viral control, in untreated HIV infection HIV-specific CD8 T cells progressively lose function. Using in vitro studies of human cells, we show that avidity-dependent downregulation of the CD8 co-receptor directly programs metabolism, due to a novel association between CD8 and the glucose transporter GLUT1. We used flow cytometry to profile ex vivo and virus-specific CD8 T cells from HIV-infected individuals on antiretroviral therapy. Ex vivo, cells expressing low levels of CD8 (CD8dim) expressed more CD69 but less cell surface GLUT1, and took up less glucose (2-NBDG) than CD8bright T cells. Following antigen stimulation, CD3, CD8, and GLUT1 were downregulated from the cell surface in an avidity-dependent manner. CMV-specific CD8 T cells, which were of higher avidity, downregulated these proteins to a greater extent than lower-avidity HIV-specific CD8 T cells. GLUT1 downregulation strongly correlated with CD8 but not CD3 downregulation. Antibody-mediated downregulation of CD8 from the cell surface resulted in reduced glucose uptake and increased fatty acid (Bodipy) uptake, independent of CD3. Finally, CD3, CD8, and GLUT1 downregulation by HIV-specific CD8 T cells was impaired following viral escape mutations that reduced CD8 T cell avidity. We confirmed this finding in a transduction setting with a single clonal TCR. Our data reveal a novel function of the CD8 co-receptor, linking the avidity and metabolism of CD8 T cells.
Characterization of hepatitis C virus-specific Th1 and Th1-biased circulating T follicular helper CD4+ T cells that arise following pregnancy
Authors: Phelps Christopher , Walker Christopher M. , Honegger Jonathan R.
Published: February 2023

Chronic hepatitis C virus (HCV) infection is marked by a loss of HCV-specific CD4+ T cells and CD8+ T cell exhaustion. However, recovery of antiviral CD4+ T cells and decreased viremia occurs in some women after pregnancy. We hypothesize that recovery of HCV-specific Th1 and Tfh biased CD4+ T cell populations contributes to viral control postpartum. Here we undertook a detailed phenotypic analysis of lineage-defining chemokine receptor expression on recovering HCV-specific CD4+ T cells using tetramer staining and flow cytometry of PBMCs from 14 women with chronic HCV infection. Seven women had >10-fold decreases in viremia by 3 months postpartum (3PP) (controllers) and the remaining half did not (non-controllers). At 3PP, tetramer+CD4+T cells were predominantly CXCR5+/CXCR3+/CCR6− (cTfh1) with most of the remaining HCV-specific CD4+ T cells being CXCR5−/CXCR3+/CCR6−(Th1). The proportions of cTfh1 cells within tetramer+CD4+ T cell populations were higher in controllers compared to non-controllers (median 74.3% vs. 52.8%, p=0.0262, Mann-Whitney), while the proportions of Th1 cells were higher in non-controllers (p=0.007, Mann-Whitney). Higher cTfh1 frequencies correlated with viral control (p=0.0072, Spearman). In all mothers, over 85% of HCV-specific cTfh1 cells were PD-1+. ICOS expression correlated with reduced viral control. Together, these data suggest that a shift of HCV-specific CD4+ T cell responses towards a cTfh1 phenotype contributes to the unique viral control that occurs in some women after pregnancy.
Viral and tumor immunity is enhanced in mice expressing the pro-autoimmune allele of tyrosine phosphatase Ptpn22
Authors: Orozco Robin C , Marquardt Kristi , Mowen Kerri A , Pratumchai Isaraphorn , Teijaro John R , Sherman Linda A
Published: February 2023

The 1858C>T allele of the tyrosine phosphatase PTPN22 (causing amino acid substitution R620W) is present in 5–10% of the North American population and is strongly associated with numerous autoimmune diseases. Specifically, this alternative allele affects lymphocyte activation, toll-like receptor signaling, and cytokine production in various autoimmune contexts. Despite the importance of these inflammatory networks in pathogen and tumor clearance, the influence of PTPN22 and its pro-autoimmune allele have on viral and tumorigenic conditions is poorly defined. To interrogate these roles, we used CRISPR/Cas9 to generate mice expressing the Ptpn22 R619W molecule, which is the ortholog of R620W in humans. Using LCMV-cl13 infection and B16 tumor models, we tested the hypothesis that pleiotropic effects of Ptpn22 R619W enhances anti-viral and anti-tumor immunity. Importantly, R619W bearing mice have less tumor burden and clear chronic viral infection faster than wildtype mice. Consistent with these results, R619W mice have enhanced antigen-specific T cell activation during viral infection and more tumor infiltrating activated T cells. Also, R619W bearing mice have a more immunostimulatory cDC phenotype during disease. As this gene is expressed in all immune cells, we interrogated the role that the R619W molecule has on T cell intrinsic versus extrinsic mechanisms of T cell activation. We show an additive mechanism in which both T cell and non-T cell compartments expressing the R619W molecule cause the most T cell activation. Our results suggest that a pro-autoimmune allele can be beneficial by promoting anti-viral and anti-tumor immune responses through multiple immune cell types, and may have a protective effect in these diseases.
Age-dependent differences in the immune response to human metapneumovirus
Authors: Parks Olivia Bartlett , Lan Jie , Zhang Yu , Williams John V
Published: February 2023

Objective:

Human metapneumovirus (HMPV) is a leading cause of serious respiratory infection in older adults, but pathophysiology in this population is incompletely understood.

Methods:

We developed an elderly mouse model of HMPV using 72-week-old mice. Aged mice and 6-week-old mice were infected intratracheally with HMPV and euthanized at different time points to measure viral titer and cellular immune response. We also infected young v. aged macrophages in vitro to determine cytokine response.

Results:

Aged HMPV-infected mice demonstrated increased weight loss, higher clinical disease scores, increased lung histopathology, and delayed viral clearance with virus persisting in the lung through day 9 post-infection while 6-week young mice cleared infection completely by day 7. Aged mice showed a significant increase in absolute number of CD8+ T cells in bronchoalveolar lavage (BAL) and lung; however, CD8+ T cells in aged mice exhibited fewer HMPV-specific tetramer+ CD8+ cells in both BAL and lung. Most aged cells expressed two or more impairment receptors (PD1, LAG3, or TIM3). Luminex analysis of lung homogenate and BAL revealed increased Th2 and inflammatory cytokine expression in aged mice. Aged HMPV-infected peritoneal macrophages in vitro had significantly increased expression of IFNb and decreased IFNg, while bone marrow-derived macrophages had decreased IFNl expression.

Conclusions:

These findings suggest that aged mice have a dysregulated CD8+ T cell response to HMPV, increasing disease but delaying viral clearance. Altered cytokine production by aged macrophages may skew CD8+ T cell response. Future studies will investigate the specific mechanisms surrounding impaired immune response in the aged host.
Female obese mice have a higher mortality rate and altered immune responses following viral infection in comparison to wild type mice
Authors: Geerling Elizabeth , Hassert Mariah , Stone E Taylor , Steffen Tara L , Pinto Amelia K
Published: February 2023

A rise in adiposity in the United States has resulted in >70% of adults being overweight or obese. Globally, areas that have seen a rise in obesity also have an increase in viral pathogens, resulting in a double disease burden. Studies done using influenza, hepatitis C, West Nile and dengue viruses have shown that excessive adiposity alters immune responses to vaccination. Similarly, obese individuals have a greater risk for developing COVID-19 following SARS-CoV-2 infection and a higher mortality rate than non-obese individuals. However, limited studies have been done to identify mechanisms responsible for obesity-induced impaired immune function. Based on published studies and our preliminary data, we hypothesized that obesity-associated chronic inflammation alters the immune system, enhancing susceptibility to viral diseases and hindering vaccine efficacy. To study this, we are using West Nile virus and SARS-CoV-2 in murine models of obesity. By infecting regular chow-fed (wt) and high fat diet-induced obese (ob) mice, we compared virus-specific immune responses at varying time points post infection. Results from these studies indicate that female ob mice have a higher mortality rate, dysfunctional virus-specific T cell responses and lowered efficacy of neutralizing antibodies. However, ob males do not show these differences when compared to wt males. These results demonstrate that obesity can enhance disease severity following viral infection which may have long term consequences for the generation of protective immune responses to vaccination, and sex appears to play a role in this phenomenon. Our ongoing studies are focused on determining how sex and chronic inflammation alter immune responses to these viral pathogens.
Reducing the priming dose of a SARS CoV-2 vaccine improves vaccine-elicited immunity
Authors: Sanchez Sarah , Palacio-Betancur Nicole , Dangi Tanushree , Penaloza-MacMaster Pablo
Published: February 2023

SARS-CoV-2 caused a global pandemic that has killed over 2 million people. While several vaccine candidates have received emergency use authorization, there is still limited vaccine availability and lack of knowledge on optimal vaccine dosing. A recent AstraZeneca vaccine study with an adenovirus vector suggested that priming with a low dose (LD) is superior than priming with a standard dose (SD). We extended these results to a murine vaccination model to further understand the mechanism of how limiting the priming dose affects vaccine-elicited immunity. We first primed C57BL/6 mice intramuscularly with an adenovirus-based vaccine expressing SARS CoV-2 spike (Ad5-spike), either with a LD (106 PFU) or a SD (109 PFU), followed by a SD boost three weeks later. An initial priming with a SD resulted in a higher magnitude of adaptive immune responses relative to an initial priming with a LD, consistent with the notion that adaptive responses are proportional to the priming antigen dose. However, T cell responses generated by a LD prime exhibited more rapid central memory differentiation, suggesting that they could display improved recall expansion following subsequent booster immunization. Interestingly, mice that were primed with a LD exhibited significantly more potent anamnestic T cell responses upon boosting, relative to mice that were primed and boosted with a SD. Antibody responses were also significantly improved in the LD/SD vaccine regimen. Overall, our data suggest that limiting the priming dose may offer a substantial long-term benefit for SARS CoV-2 vaccines. These findings may be useful for improving vaccine availability and also for the rational design of prime-boost vaccine regimens for SARS CoV-2 and other diseases.
Immune profiling of COVID-19 infection in pediatric solid organ transplantation
Authors: Ekong Udeme D , Paul Saikat , Royal Scott , Chahine Joeffrey , Yazigi Nada , Kaufman Stuart , Khan Khalid , Matsumoto Cal , Kroemer Alexander , Fishbein Thomas
Published: February 2023

Background:

We describe the clinical presentation & immune response to COVID-19 infection in pediatric SOT.

Method:

Medical records of COVID-19 PCR+ & seropositive patients (SP) were reviewed for details of their disease course. Blood was obtained during PCR or seropositivity for immunophenotyping. Controls were pediatric solid organ transplant (SOT) patients with negative COVID-19 (NCs) status. As induction of immunological memory is central to anti-pathogen adaptive immunity induced by infection, immunohistochemistry of liver tissue for tissue resident memory cells (TRM) was performed.

Results:

15 patients had COVID positive status (7 PCR+ & 8 SP) between March 2020 & January 2021. 4 symptomatic PCR+ patients were hospitalized for 24–72-hours & 1 symptomatic SP patient had a prolonged PICU stay. Infrequent TRM were seen in liver biopsies of PCR+ & SP patients vs. biopsies from same patients that pre-date a COVID-19+ status. Patients with COVID + status appeared to segregate into 2 groups: 1 group with activated CD4+ response, & the 2nd group lacking obvious activated CD4+ response, similar to NC patients (n=16). The PCR+ patients expressed significantly less CD4+ & CD8+ IFN-γ compared to the SP patients (p=0.009; p=0.01 respectively). Similarly, activated CD8 “EMRAs” was significantly lower in PCR+ patients compared to SP patients (p=0.01).

Conclusion:

COVID-19 infection in pediatric SOT patients is generally mild with notable increased frequency of CD8+ “EMRAs” & IFN-γ in SP patients, presumably a result of antigen-experienced cells.
Role of IL-17-EGFR axis in alleviated lung inflammation and recovery during the late phase of acute influenza virus infection
Authors: Huang William , Huang Ching-Tai , Hung Chen-Yiu , Chen Tse-Ching , Chang Chia-Shiang , Huang Yu-Lin , Chen Ting-An , Lin Yung-Chang , Lin Chun-Yen , Dutta Avijit
Published: February 2023

Influenza hemagglutinin (HA)-specific T-Cell-Receptor (TCR) transgenic mice sustain the influenza virus infection of an inoculum size which is fatal in wild type mice. There is a prominent IL-17 response of the HA-specific TCR transgenic CD4+ T cells in late phase of infection in the transgenic mice. We have demonstrated that the late phase IL-17 response is a de novo response of naïve T cells under guidance of the Th1 cells with influenza viral neuraminidase-activated TGF-β. There is also a late phase up-regulation of epidermal growth factor receptor (EGFR) on the transgenic CD4+ T cells in the lungs. EGFR inhibition with Gefitinib resulted in impaired sustainability of the infection of the transgenic mice. They suffered from profound body weight loss and severe disease with significant mortality. IL-17-induced EGFR-mediated signaling cascade has been documented in skin stem cells that are involved in wound healing. We will further dissect the role of this cascade in alleviation of lung inflammation and recovery from the disease during the late phase of acute influenza virus infection.
ILC1 and TRM occupy the same mucosal barrier niche and provide non-overlapping protection against infection
Authors: Vrba Sophia , Wynne-Jones Erica , Reynoso Glennys , Malo Courtney , Shannon John , Hickman Heather
Published: February 2023

Tissue-resident memory T cells (TRM) occupy front-line niches in barrier tissues where they are poised to rapidly respond to secondary infection. Intriguingly, even though new infections result in new populations of TRM, there appears to be enough space in the tissue to accommodate new TRM without displacing older cells. We have recently been investigating how the oral mucosa is protected against infection using a newly developed model of viral infection of the inner lip with vaccinia virus (VACV). Many orthopoxviruses including variola (the causative agent of smallpox), are thought to have invaded through the oropharyngeal mucosa. We have previously shown that group 1 innate lymphoid cells (ILCs) are the major force protecting the oral mucosa during VACV infection. ILC1s serve as the primary source for IFN-γ, which is critical to restrict viral replication. In a tissue heavily populated with ILC1s, however, it is unclear what, if any, role other tissue resident memory cells (including TRM) play during infection. We have used a combination of static and intravital imaging of Rag−/− Il12rg−/− mice and Ncr1-gfp+ reporter mice to spatially map ILC1 and TRM locations in the oral mucosa. Using Rag−/−Il12rg−/− mice and Rag− /− T-bet-ZsGreen reporter mice and adoptive transfer of virus-specific T cells, we plan to examine the function of ILC1s and TRM in the absence of the other cell population as well as when both are present. We are further exploring whether competition for local cytokines could be regulating niche occupancy. Together, these data will expand our understanding on the complex interplay between innate and adaptive immune cells during viral infection of a critical but often overlooked barrier tissue.
Activated peripheral TFH cells and MAIT cells together with increased plasma TNF-α levels mark the pathogenesis of acute dengue virus infection
Authors: Murugesan Amudhan , Anbalagan Annai Meena , Sathappan Usha Preeyaa , manickan Elanchezhiyan , Larsson Marie , Yong Yean K , Shankar Esaki M , Velu Vijayakumar
Published: February 2023

Dengue virus (DENV) infection has become an increasingly common public health concern in tropical and subtropical regions. Dengue infection appears to cause immune activation in a wide array of immune cells. Here, we estimated Th1 and Th2 cytokines and correlated their levels with primary and secondary dengue. We also investigated the signatures of immune activation of circulating Tfh cells and mucosal-associated invariant T (MAIT) cells in adults with confirmed acute clinical dengue virus infection. We found that IL-2, TNF-α, IL-4, and IL-10 levels were significantly elevated in patients with secondary DENV infection, whereas the level of IL-13 remained unaltered during both primary and secondary infections. No statistically significant difference was noticed with IL-12p70, IL-5, IFN-γ, and GM-CSF between the healthy controls and the primary and secondary dengue. Increase of 1 unit of TNF-α was associated with a decrease of 160 units of blood platelets. We found a higher frequency of activated Tfh cells and pTfh cells in clinical DENV infection. We also found similar activated and expanding phenotypes of MAIT cells. Our study suggests that TNF-α could play a key role in the pathogenesis of dengue along with activated pTfh cells and circulating MAIT cells in acute DENV infection.
HLA class II binding analysis for SARS-CoV-2 Spike through cell-surface MHC density assay
Authors: Miyadera Hiroko , Jiang Nian
Published: February 2023

Introduction: We developed MHC-peptide interaction assay that measures cell-surface expression of covalently linked peptide-MHC II complex. The utility of the assay has been validated for known CD4+ T-cell epitopes of Mycobacterium tuberculosis antigens (Miyadera et al. submitted). Using this assay (named “delta MHC (ΔMHC) assay”), we screened HLA II-binding regions in SARS-CoV-2 Spike protein to identify potential T-cell epitopes.

Method:

ΔMHC assay uses engineered fibroblast cell line that stably expresses DPA1. The cell line is transduced with pMXs-IG (Kitamura et al. 2003 Exp Hematol) that carries DPB1*05:01-peptide fusion. The assay measures cell-surface HLA expression through flow cytometry and calculates the expression levels normalized to the internal control GFP and control peptide. The peptides (15-mer) that covered the entire Spike protein were analyzed in this study.

Results:

We performed the assay for HLA-DP5 (DPA1*02:02-DPB1*05:01), which is present at high frequency in East Asian populations. Among the 211 Spike peptides analyzed, we found > 8 peptides that showed strong binding to DP5, indicating that these regions might act as T-cell epitopes. Additional >10 regions showed intermediate binding. Part of binding regions were overlapped with DR-restricted T-cell epitopes reported for SARS-CoV (Yang et al. 2009, Int Imm). Some of the DP5-binding regions were also overlapped with viral mutation sites of contagious variants (VOC 2020 12/01 (UK), 501Y.V2 (South Africa), B1.1.24B (Brazil)). The binding analyses of the variants for DP5 and DP0401 are underway.
Novel potent IFN-γ-inducing CD8+ T cell epitopes conserved among diverse Bovine Viral Diarrhea Virus strains
Authors: SANGEWAR NEHA Sangewar , Waghela Suryakant D , Yao Jianxiu , Sang Huldah , Bray Jocelyne M , Mwangi Waithaka
Published: February 2023

Studies of immune responses elicited by Bovine Viral Diarrhea Virus (BVDV) vaccines have primarily focused on the characterization of neutralizing B cell and CD4+ T cell epitopes. Despite availability of vaccines for decades, BVDV prevalence in cattle remains largely unaffected. There is limited knowledge regarding the role of BVDV-specific CD8+ T cells in immune protection and indirect evidence suggests that they play a crucial role during BVDV infection. In this study, presence of BVDV-specific CD8+ T cells that are highly cross-reactive in cattle was demonstrated. Most importantly, novel potent IFN-γ-inducing CD8+ T cell epitopes were identified from different regions of BVDV polyprotein. Eight CD8+ T cell epitopes were identified from structural BVDV antigens: Erns, E1, and E2 glycoproteins. In addition, from nonstructural BVDV antigens: Npro, NS2-3, NS4A-B, and NS5A-B, twenty CD8+ T cell epitopes were identified. Majority of these IFN-γ-inducing CD8+ T cell epitopes were found to be highly conserved among more than two hundred strains from BVDV-1 and -2 genotypes. These conserved epitopes were also validated as cross-reactive since they induced high recall IFN-γ+ CD8+ T cell responses, ex vivo, in purified CD8+ T cells from BVDV-1- and -2-immunized cattle. Altogether, twenty-eight bovine MHC I-binding epitopes were identified from key BVDV antigens that can elicit broadly reactive CD8+ T cells against diverse BVDV strains. The data presented here will lay the groundwork for the development of a contemporary CD8+ T cell-based BVDV vaccine capable of addressing BVDV heterogeneity more effectively than current vaccines.
Fam49b, the source of antigenic peptide for ERAAP surveillance, controls thymocyte development.
Authors: Park Chansu , Guan Jian , Rhee Peter , Sadegh-Nasseri Scheherazade , Shastri Nilabh
Published: February 2023

The aminopeptidase ERAAP, the ER aminopeptidase associated with antigen processing, is essential for trimming of peptides presented by classical and non-classical MHC class I molecules. The function of ERAAP is monitored by semi-invariant CD8+ T cells that recognize the QFL ligand. The QFL ligand consists of a 9-mer peptide encoded by the highly conserved Fam49a/b genes and is presented by non-classical Qa-1bmolecules expressed on surface of ERAAP-deficient cells only. Recently, Fam49b is identified as a novel negative regulator of TCR signaling, that functions by binding to active rac-1 and modulates reorganization of cytoskeleton. However, the function of Fam49b in T cells development has remained unclear. We show that Fam49b-deficient thymocytes receive higher TCR signaling and are defective in T cell development. Our studies reveal that Fam49b plays an important role in regulating thymocytes development and lineage commitment of conventional T cells as well as agonist selected T cells such as CD8αα αβ T cells. [This research was supported by NIH grants R01 AI130210 and R37 AI060040]
Unusual distribution of a non-classical MHC I restricted CD8+ T population
Authors: Guan Jian , Sadegh-Nasseri Scheherazade , Shastri Nilabh
Published: February 2023

A broad repertoire of peptides is constitutively presented through the MHC I molecules on the cell surface. ER aminopeptidase associated with Ag processing (ERAAP), a critical molecule in the antigen processing and presentation pathway, has profound effects on the generation of this peptide repertoire. Interestingly, the ERAAP deficient cells can be effectively detected by a unique population of the cytotoxic CD8+ T cell, named QFL-T cells which are specific for the ligand presented by the non-classical MHC Ib molecule, Qa-1b. The QFL-T cells showed similar characteristics with the other non-classical MHC Ib restricted unconventional T cells, such as the NKT or MAIT cells in their semi-invariant αβ TCRs, but their function(s) remains unknown. To study the potential function of the QFL-T cells, we assessed a variety of tissues for the ligand expression and the presence of the QFL-T cells. We observed high expression of this ERAAP dependent Qa-1b restricted ligand and relatively high frequency of the QFL-T cells in the small intestine. Remarkably, the number and the frequency of QFL-T cells among the intraepithelial lymphocytes (IELs) was tightly associated with the age and the gut microbiota of the mice. These findings suggest a role for gut microbiota in the homing/maintenance of the QFL-T cells in the small intestine.
Integration of affinity and abundance parameters for the identification of novel CD8+ T cell specificities in the CT26 tumor model
Authors: Arcia David , Boulanger Denise S. , James Edward N , Elliott Tim
Published: February 2023

The CT26 model is one of the most widely tested systems for cancer immunotherapy. Over 70% of the CD8+ CTL responses in CT26 tumors are directed against two peptides: SPSYVYHQF (AH1) presented by H-2Ld, or GGPESFYCASW (GSW11) presented by H-2Dd. Both peptides derive from the ecotropic murine leukaemia virus gp70 envelope glycoprotein, which is the highest expressed gene in CT26, and have relative low affinity values for their respective MHC-I. However, there is a lack of knowledge about other CTL specificities in CT26, the relationship between abundance and affinity of such peptides, and their roles in tumor rejection.

We identified 96 potential epitopes restricted by MHC-I molecules in BALB/c mice based on previously published immuno-transcriptomic data. We used a mathematical algorithm integrating the peptides relative affinity to MHC-I measured by NetMHC4.0, and their abundance in the proteome, which resulted in a prediction of the likelihood of peptide presentation.

Screening was performed in splenocytes from Treg-depleted CT26-challenged mice via IFNγ intracellular cytokine staining and T cell proliferation assays. Our approach led to the identification of a novel CTL specificity restricted by H-2Kd (Kd34), which shows moderate IFNγ responses in splenocytes from mice responding to the therapy. Moreover, using Kd34-specific dextramers, we were able to detect a small population of tumor-infiltrating lymphocytes (TILs) specific for this peptide, with similar phenotypic traits to AH1- and GSW1-specific TILs. The identification of such novel CD8+ T cell specificities will improve the understanding about the relationship between immunodominance and epitope abundance/affinity to better tailor vaccine strategies in cancer.
Dissociation of β2m from cell surface MHC class I triggers formation of noncovalent, transient heavy chain dimers
Authors: Springer Sebastian , Dirscherl Cindy , Löchte Sara , Hein Zeynep , Kopicki Janine-Denise , Harders Antonia , Linden Noemi , Weghuber Julian , Garcia-Alai Maria , Uetrecht Charlotte , Zacharias Martin , Lanzerstorfer Peter , Piehler Jacob
Published: February 2023

Major histocompatibility complex class I molecules (MHC I) that lose the antigenic peptide and the light chain beta-2 microglobulin (β2m) to become free heavy chains at the cell surface are known to associate, forming oligomers on the plasma membrane that are insufficiently understood. Here, we investigate the homotypic interaction of MHC I free heavy chains by combining a printed antibody micropattern assay with fluorescence recovery after photobleaching (FRAP) and with single molecule co-tracking in order to elucidate their molecular structure, abundance, and dynamics. We find that MHC I free heavy chain complexes are dimeric, transient, non-covalent, and mediated by the α3 domain. Free heavy chain interaction correlates with a decrease in the diffusion coefficient and an increase in the number of immobile molecules at the cell surface. Molecular docking and dynamics simulations suggest that in the complexes, the α3 domain of one FHC binds to another free heavy chain in a manner similar to the β2m light chain. We propose distinct functions of the MHC I free heavy chain dimers in signaling and in endocytic sorting.
Flow cytometry analysis of immune response to COVID-19 vaccine candidates using an in vitro stimulation model
Authors: Shi Xiaoshan , Bornheimer Scott J , Saksena Suraj , Widmann Stephanie , Tyznik Aaron
Published: February 2023

T cell immunity is vital for the control of viral infections. Cytotoxic CD8 T cells play a crucial role in eliminating virus-infected cells while CD4 helper T cells mediate host immune responses and promote B cells to secrete antibodies. For both, T cell recognition of viral antigens in the form of short peptides presented by HLA complexes is a prerequisite for T cell activation. Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has become a global pandemic that has disrupted economies and resulted in an enormous burden on health care systems. Despite the worldwide intensive efforts on development of COVID-19 therapeutics, a safe and effective COVID-19 treatment is not yet available. In this study, we established an in vitro stimulation system in which peripheral blood mononuclear cells were treated with putative SARS-CoV-2 immunogenic peptides. T cell immune responses were measured using different quantitative flow cytometry panels that were designed for evaluating cytokine production or identifying T cell subsets. Evaluation of host immune responses with a suite of flow cytometric panels may prove to be useful in the study of COVID-19.

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Male microbiota-associated metabolites restore macrophage efferocytosis in female lupus-prone mice via PPARγ and LXR signaling pathways
Authors: Harder James W , Ma Jing , Alard Pascale , Zhang Xiang , Yuan Fang , Kosiewicz Michele M
Published: February 2023

Sex and gut microbiota both influence systemic lupus erythematosus (SLE) development. We have found that male and lupus-prone female NZBxNZW F1 (BWF1) mice exhibit differences in gut microbiota and metabolomic profiles, and transfer of male microbiota suppresses disease in female mice. Here we determine how sex and microbiota may interact to affect SLE development. Transcriptomic analysis of female and male BWF1 spleens found phagocytosis-promoting genes were upregulated in males. Since defects in macrophage-mediated phagocytosis of apoptotic cells (efferocytosis) are found in SLE patients, we compared efferocytosis in vitro between male and lupus-prone female BWF1 mice. Macrophage-mediated efferocytosis was decreased in female compared to male BWF1 mice, but could be restored in female mice by transfer of male microbiota in vivo. Further transcriptomic analysis of spleens found that genes regulated by PPARγ and LXR receptor signaling were increased in male BWF1 mice. Our previous metabolomics analyses have found that two metabolites which can activate PPARγ and LXR signaling, phytol and its derivative phytanic acid, are higher in male BWF1 mice. We have also found that feeding phytanic acid to female mice delayed lupus onset. We show here that defective female BWF1 macrophage efferocytosis can be restored by phytanic acid treatment, and this requires both PPARγ and LXR signaling pathways. Furthermore, we found that phytanic acid may restore female BWF1 macrophage efferocytosis by upregulating the pro-efferocytic receptor, CD36. Taken together, our data indicate that BWF1 male microbiota produce metabolites that can enhance macrophage efferocytosis and suppress lupus.
The Contribution of Connexin 43 in Choroid Plexus to Experimental Autoimmune Encephalomyelitis
Authors: Reid Joshua , Manam Srikanth , Gadagkar Sudhindra R , Forsthuber Thomas , Nicholson Bruce J , Murthy Ashlesh K
Published: February 2023

Multiple Sclerosis (MS) is the most common progressive neurologic disease among young adults worldwide; yet the pathogenesis remains poorly understood. The myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalitis (EAE) in mice is a robust model and has been extensively used to understand the pathophysiology of MS. The expression of connexin 43 (CX43), a gap junction protein, has been shown to be enhanced in the choroid plexus during the development of acute EAE, with significantly increased CX43 expression at the peak of the disease. However, the contribution of such increased CX43 expression to the development of EAE has yet to be fully characterized. Using the Cre-Lox platform, we generated mice with conditional deletion of CX43 in the ciliated columnar epithelium of the choroid plexus (Foxj1Cre-CX43 KO mice; referred to as CX43 KO mice hereafter). We employed the MOG-induced EAE model in both male and female CX43 KO mice to understand the role of CX43 in the pathogenesis of EAE. Male CX43 and WT mice displayed comparable peak EAE clinical disease scores of 1.83±0.33 and 1.75±0.66, respectively. Female CX43 KO, however, displayed a significantly reduced peak EAE clinical disease score of 0.33±0.24 compared to 1.15±0.39 in female WT animals. These results suggest a role for CX43 in the ciliated columnar epithelium of choroid plexus to the development of EAE in female, not male, mice and underscore the need for further exploration of mechanism(s) underpinning the role of CX43 in the pathogenesis of EAE.
p38 MAP kinase signaling in microglia plays a sex-specific protective role in CNS autoimmunity and regulates microglial transcriptional states
Authors: Krementsov Dimitry N , McGill Mahalia , Richman Alyssa , Boyd Joseph , Frietze Seth
Published: February 2023

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system. MS is three times more common in women, yet more severe in men. The mechanisms underlying these sex differences remain largely unknown. MS is initiated by autoreactive T cells, but CNS-infiltrating myeloid cells are key effector cells driving pathology. The role of CNS-resident microglial cells is less clear. We have previously shown that genetic ablation of p38α MAP kinase broadly in the myeloid lineage is protective in the autoimmune model of MS, experimental autoimmune encephalomyelitis (EAE), but only in females, and not males. Here, we sought to define the mechanisms responsible, using genetic approaches and bone marrow chimeras to ablate p38α in microglial cells, peripheral myeloid cells, or both. Deletion of p38α in both cell types recapitulated the previous sex difference, with reduced EAE severity in females. Unexpectedly, deletion of p38α in the periphery was protective in both sexes. In contrast, deletion of p38α in microglia exacerbated EAE in males only, revealing opposing roles of p38α in microglia vs. periphery. Single-cell RNAseq revealed a diversity of microglial states, connected by distinct convergent transcriptional trajectories. Microglial p38α deficiency resulted in enhanced transition from homeostatic to disease-associated microglia, with downregulation of regulatory genes such as Atf3, Rgs1, and Btg2, and upregulation of inflammatory genes such as Cd74, Trem2, and MHC class I and II. These results reveal a p38α-dependent male-specific molecular pathway in microglia that is protective in CNS autoimmunity, suggesting that autoimmunity in males and females may be driven by distinct cellular and molecular pathways.