Influenza-specific CD8 Research Articles

SARS-CoV-2-specific CD8+ T cells from people with long COVID establish and maintain effector phenotype and key TCR signatures over 2 years

Long COVID occurs in a small but important minority of patients following COVID-19, reducing quality of life and contributing to healthcare burden. Although research into underlying mechanisms is evolving, immunity is understudied. SARS-CoV-2-specific T cell responses are of key importance for viral clearance and COVID-19 recovery. However, in long COVID, the establishment and persistence of SARS-CoV-2-specific T cells are far from clear, especially beyond 12 mo postinfection and postvaccination. We defined ex vivo antigen-specific B cell and T cell responses and their T cell receptors (TCR) repertoires across 2 y postinfection in people with long COVID. Using 13 SARS-CoV-2 peptide-HLA tetramers, spanning 11 HLA allotypes, as well as spike and nucleocapsid probes, we tracked SARS-CoV-2-specific CD8+ and CD4+ T cells and B-cells in individuals from their first SARS-CoV-2 infection through primary vaccination over 24 mo. The frequencies of ORF1a- and nucleocapsid-specific T cells and B cells remained stable over 24 mo. Spike-specific CD8+ and CD4+ T cells and B cells were boosted by SARS-CoV-2 vaccination, indicating immunization, in fully recovered and people with long COVID, altered the immunodominance hierarchy of SARS-CoV-2 T cell epitopes. Meanwhile, influenza-specific CD8+ T cells were stable across 24 mo, suggesting no bystander-activation. Compared to total T cell populations, SARS-CoV-2-specific T cells were enriched for central memory phenotype, although the proportion of central memory T cells decreased following acute illness. Importantly, TCR repertoire composition was maintained throughout long COVID, including postvaccination, to 2 y postinfection. Overall, we defined ex vivo SARS-CoV-2-specific B cells and T cells to understand primary and recall responses, providing key insights into antigen-specific responses in people with long COVID.

Development of Influenza-Specific CD4 T Cell-Mediated Immunity in Children Following Inactivated Influenza Vaccination.

While both cellular and humoral immunity are important in immunologic protection against influenza, how the influenza-specific CD4 T cell response is established in response to early vaccination remains inadequately understood. In this study, we sought to understand how the CD4 T cell response to IIV is established and develops throughout early childhood. Influenza-specific CD4 T cell responses were quantified following IIV over two influenza seasons in 47 vaccinated children between 6 months and 8 years of age who had no documented history of natural influenza infection during the study. PBMCs were stimulated with peptide pools encompassing the translated regions of the pH1, H3, HAB, and NP proteins and CD4 T cell responses were assessed via multiparameter flow cytometry. There was boosting of H3- and HAB-specific CD4 T cells but not cells specific for the pH1 HA protein post-vaccination. A positive correlation between age and the magnitude of the influenza-specific CD4 T cell response was seen, with an overall greater magnitude of IFNγ-producing cells in subjects ≥3 years of age. Changes in CD4 T cell functionality were also noted in older compared to younger children, with increases in CD4 T cells producing IFNγ and TNF or IL-2 as well as IFNγ alone. IIV elicits a CD4 T cell response to H3 and HAB, with increases in the magnitude of the CD4 T cell response and changes in cellular functionality throughout childhood. This suggests that repeated influenza vaccination contributes to the development of anti-influenza CD4 T cell memory in children.

Safety and Immunogenicity of a Carbohydrate Fatty Acid Monosulphate Ester Adjuvant Combined with a Low-Dose Quadrivalent Split-Virion Inactivated Influenza Vaccine: A Randomised, Observer-Blind, Active-Controlled, First-in-Human, Phase 1 Study.

Seasonal influenza vaccine effectiveness is low. Carbohydrate fatty acid monosulphate ester (CMS), a new oil-in-water adjuvant, has proven potency in animal models with suggested capacity for dose-sparing. The objective was to evaluate safety and immunogenicity of CMS when added to a low-dose influenza vaccine (QIV) in humans. In a randomised, double-blind, active-controlled, first-in-human study, sixty participants (18-50 years) received either 0.5 mg CMS or 2 mg CMS with 1/5th dose QIV, or a full dose QIV without CMS. Adverse events (AE) were monitored until 7 days post-vaccination. Haemagglutinin inhibition (HI) titres in serum and CD4+ T cells in PBMCs were determined at day 0, 7, 28, and 180. Mean age was 37.6 (±10.1) years and 42/60 (70.0%) were female. Pain at injection site (42/60, 86.7%) and headache (34/60, 56.7%) were reported most and more frequently in the 2 mg CMS group. HI titres and the frequency of influenza specific CD4+ T cells were equal across strains for the three cohorts on all visits, increased until day 28 and decreased at day 180 to values higher than baseline. CMS was safe in humans. Humoral and cell-mediated immunogenicity was similar across vaccines, even with 1/5th antigen dose. CMS can have beneficial implications in low-resource settings or in a pandemic context.

Cytomegalovirus vaccine vector-induced effector memory CD4 + T cells protect cynomolgus macaques from lethal aerosolized heterologous avian influenza challenge

An influenza vaccine approach that overcomes the problem of viral sequence diversity and provides long-lived heterosubtypic protection is urgently needed to protect against pandemic influenza viruses. Here, to determine if lung-resident effector memory T cells induced by cytomegalovirus (CMV)-vectored vaccines expressing conserved internal influenza antigens could protect against lethal influenza challenge, we immunize Mauritian cynomolgus macaques (MCM) with cynomolgus CMV (CyCMV) vaccines expressing H1N1 1918 influenza M1, NP, and PB1 antigens (CyCMV/Flu), and challenge with heterologous, aerosolized avian H5N1 influenza. All six unvaccinated MCM died by seven days post infection with acute respiratory distress, while 54.5% (6/11) CyCMV/Flu-vaccinated MCM survived. Survival correlates with the magnitude of lung-resident influenza-specific CD4 + T cells prior to challenge. These data demonstrate that CD4 + T cells targeting conserved internal influenza proteins can protect against highly pathogenic heterologous influenza challenge and support further exploration of effector memory T cell-based vaccines for universal influenza vaccine development.

Mucosal signatures of pathogenic T cells in HLA-B*27+ anterior uveitis and axial spondyloarthritis.

HLA-B*27 was one of the first HLA alleles associated with an autoimmune disease, i.e., axial spondyloarthritis (axSpA) and acute anterior uveitis (B27AAU), which cause joint and eye inflammation, respectively. Gastrointestinal inflammation has been suggested as a trigger of axSpA. We recently identified a bacterial peptide (YeiH) that can be presented by HLA-B*27 to expanded public T cell receptors (TCRs) in the joint in axSpA and the eye in B27AAU. While YeiH is present in enteric microbiota and pathogens, additional evidence that pathogenic T cells in HLA-B*27-associated autoimmunity may have had a prior antigenic encounter within the gastrointestinal tract remains lacking. Here, we analyze ocular, synovial, and blood T cells in B27AAU and axSpA, showing that YeiH-specific CD8 T cells express a mucosal gene set and surface proteins consistent with intestinal differentiation, including CD161, integrin α4β7, and CCR6. In addition, we find an expansion of YeiH-specific CD8 T cells in the blood of axSpA and B27AAU over healthy controls, whereas influenza-specific CD8 T cells were equivalent across groups. Lastly, we demonstrate the dispensability of TRBV9 for antigen recognition. Collectively, our data suggest that, in HLA-B27-associated autoimmunity, early antigen exposure and differentiation of pathogenic CD8 T cells may occur in enteric organs.

A combination of TLR9 and STING agonists favors antigen cross-presentation and promotes Th1 immune response against influenza virus

Abstract Adjuvants can boost the type, magnitude, breadth, and durability of vaccine responses. We have previously demonstrated that combining adjuvants through rational design can overcome age-specific impairments in inducing Th1 immunity, trigger antigen cross-presentation, and enable CD8-driven protective immunity of protein-based vaccine formulations. Here, we screened 78 adjuvant combinations to explore their molecular mechanism of action through human in vitro modeling and identified combinations that synergistically promote the expansion of antigen-specific CD4+ cells, induce cross-presentation on MHC class I resulting in activation of antigen-specific CD8+ cells, and redirect the type of immune response to favor the production of Th1-promoting cytokines. The combination of CpG (TLR9 agonist) and 2,3-cGAMP (STING agonist) promoted influenza-specific CD4+ and CD8+ T cell activation and selectively favored the secretion of Th1-polarizing cytokines TNF and IL-12p70. Activation of T-bet-expressing Th1 cells was also observed with this adjuvant combination. We also demonstrate the phenotypic reprogramming towards conventional type 1 dendritic cells by CpG + 2,3-cGAMP, and the ability of this adjuvant combination to induce similar immune responses in immune cells from newborns and older adults. Identification of the adjuvant combination CpG + 2,3-cGAMP may prove key to the future development of vaccines against intracellular pathogens such as respiratory viruses.

Intranasal Epitope-Polymer Vaccine Lodges Resident Memory T Cells Protecting Against Influenza Virus.

Intranasal vaccines, unlike injectable vaccines, boost immunity along the respiratory tract; this can significantly limit respiratory virus replication and shedding. There remains a need to develop mucosal adjuvants and vaccine delivery systems that are both safe and effective following intranasal administration. Here, biopolymer particles (BP) densely coated with repeats of MHC class I restricted immunodominant epitopes derived from influenza A virus namely NP366, a nucleoprotein-derived epitope and PA224, a polymerase acidic subunit derived epitope, are bioengineered. These BP-NP366/PA224 can be manufactured at a high yield and are obtained at ≈93% purity, exhibiting ambient-temperature stability. Immunological characterization includes comparing systemic and mucosal immune responses mounted following intramuscular or intranasal immunization. Immunization with BP-NP366/PA224 without adjuvant triggers influenza-specific CD8+ T cell priming and memory CD8+ T cell development. Co-delivery with the adjuvant poly(I:C) significantly boosts the size and functionality of the influenza-specific pulmonary resident memory CD8+ T cell pool. Intranasal, but not intramuscular delivery of BP-NP366/PA224 with poly(I:C), provides protection against influenza virus challenge. Overall, the BP approach demonstrates as a suitable antigen formulation for intranasal delivery toward induction of systemic protective T cell responses against influenza virus.

Human In vitro Modeling Identifies Adjuvant Combinations that Unlock Antigen Cross-presentation and Promote T-helper 1 Development in Newborns, Adults and Elders

Adjuvants are vaccine components that can boost the type, magnitude, breadth, and durability of an immune response. We have previously demonstrated that certain adjuvant combinations can act synergistically to enhance and shape immunogenicity including promotion of Th1 and cytotoxic T-cell development. These combinations also promoted protective immunity in vulnerable populations such as newborns. In this study, we employed combined antigen-specific human in vitro models to identify adjuvant combinations that could synergistically promote the expansion of vaccine-specific CD4+ cells, induce cross-presentation on MHC class I, resulting in antigen-specific activation of CD8+ cells, and direct the balance of immune response to favor the production of Th1-promoting cytokines. A screen of 78 adjuvant combinations identified several T cell-potentiating adjuvant combinations. Remarkably, a combination of TLR9 and STING agonists (CpG + 2,3-cGAMP) promoted influenza-specific CD4+ and CD8+ T cell activation and selectively favored production of Th1-polarizing cytokines TNF and IL-12p70 over co-regulated cytokines IL-6 and IL-12p40, respectively. Phenotypic reprogramming towards cDC1-type dendritic cells by CpG + 2,3-cGAMP was also observed. Finally, we characterized the molecular mechanism of this adjuvant combination including the ability of 2,3-cGAMP to enhance DC expression of TLR9 and the dependency of antigen-presenting cell activation on the Sec22b protein important to endoplasmic reticulum-Golgi vesicle trafficking. The identification of the adjuvant combination CpG + 2,3-cGAMP may therefore prove key to the future development of vaccines against respiratory viral infections tailored for the functionally distinct immune systems of vulnerable populations such as older adults and newborns.

Genetically engineered CD80-pMHC-harboring extracellular vesicles for antigen-specific CD4+ T-cell engagement.

The identification of low-frequency antigen-specific CD4+ T cells is crucial for effective immunomonitoring across various diseases. However, this task still encounters experimental challenges necessitating the implementation of enrichment procedures. While existing antigen-specific expansion technologies predominantly concentrate on the enrichment of CD8+ T cells, advancements in methods targeting CD4+ T cells have been limited. In this study, we report a technique that harnesses antigen-presenting extracellular vesicles (EVs) for stimulation and expansion of antigen-specific CD4+ T cells. EVs are derived from a genetically modified HeLa cell line designed to emulate professional antigen-presenting cells (APCs) by expressing key costimulatory molecules CD80 and specific peptide-MHC-II complexes (pMHCs). Our results demonstrate the beneficial potent stimulatory capacity of EVs in activating both immortalized and isolated human CD4+ T cells from peripheral blood mononuclear cells (PBMCs). Our technique successfully expands low-frequency influenza-specific CD4+ T cells from healthy individuals. In summary, the elaborated methodology represents a streamlined and efficient approach for the detection and expansion of antigen-specific CD4+ T cells, presenting a valuable alternative to existing antigen-specific T-cell expansion protocols.

Both Humoral and Cellular Immunity Limit the Ability of Live Attenuated Influenza Vaccines to Promote T Cell Responses.

One potential advantage of live attenuated influenza vaccines (LAIVs) is their ability to establish both virus-specific Ab and tissue-resident memory T cells (TRM) in the respiratory mucosa. However, it is hypothesized that pre-existing immunity from past infections and/or immunizations prevents LAIV from boosting or generating de novo CD8+ T cell responses. To determine whether we can overcome this limitation, we generated a series of drifted influenza A/PR8 LAIVs with successive mutations in the hemagglutinin protein, allowing for increasing levels of escape from pre-existing Ab. We also inserted a CD8+ T cell epitope from the Sendai virus nucleoprotein (NP) to assess both generation of a de novo T cell response and boosting of pre-existing influenza-specific CD8+ T cells following LAIV immunization. Increasing the level of escape from Ab enabled boosting of pre-existing TRM, but we were unable to generate de novo Sendai virus NP+ CD8+ TRM following LAIV immunization in PR8 influenza-immune mice, even with LAIV strains that can fully escape pre-existing Ab. As these data suggested a role for cell-mediated immunity in limiting LAIV efficacy, we investigated several scenarios to assess the impact of pre-existing LAIV-specific TRM in the upper and lower respiratory tract. Ultimately, we found that deletion of the immunodominant influenza NP366-374 epitope allowed for sufficient escape from cellular immunity to establish de novo CD8+ TRM. When combined, these studies demonstrate that both pre-existing humoral and cellular immunity can limit the effectiveness of LAIV, which is an important consideration for future design of vaccine vectors against respiratory pathogens.

Allergen exposure functionally alters influenza-specific CD4+ Th1 memory cells in the lung.

CD4+ lung-resident memory T cells (TRM) generated in response to influenza infection confer effective protection against subsequent viral exposures. Whether these cells can be altered by environmental antigens and cytokines released during heterologous, antigen-independent immune responses is currently unclear. We therefore investigated how influenza-specific CD4+ Th1 TRM in the lung are impacted by a subsequent Th2-inducing respiratory house dust mite (HDM) exposure. Although naïve influenza-specific CD4+ T cells in the lymph nodes do not respond to HDM, influenza-specific CD4+ TRM in the lungs do respond to a subsequent allergen exposure by decreasing expression of the transcription factor T-bet. This functional alteration is associated with decreased IFN-γ production upon restimulation and improved disease outcomes following heterosubtypic influenza challenge. Further investigation revealed that ST2 signaling in CD4+ T cells during allergic challenge is necessary to induce these changes in lung-resident influenza-specific CD4+ TRM. Thus, heterologous antigen exposure or ST2-signaling can drive persistent changes in CD4+ Th1 TRM populations and impact protection upon reinfection.

Modulation of mucosal immunity and tissue resident memory formation by NS1-deficient influenza A virus

Abstract Recurrent influenza A virus (IAV) epidemics and occasional pandemics as a result of viral genome reassortment and human adaptation pose significant health burdens globally. The non-structural protein 1 (NS1) mediates attenuation of host response and facilitates viral replication in the nucleus. While intranasal vaccination using NS1-deleted live attenuated virus has been shown to induce pulmonary mucosal immunity in animal models, the immunological details have not been fully studied. In vivo mouse infection using wild type (WSN/WT) and NS1-deleted WSN (WSN/Del) showed WSN/Del induced massive type I interferon response in murine lungs, whereas pulmonary inflammation was minimal. Skewed migration of CD103+ dendritic cells (DC) to mediastinal lymph nodes upon WSN/Del infection was observed with enhanced formation and persistence of tissue resident influenza-specific memory CD8 T cells (T RM). After rechallenge, while influenza-specific T RMin both groups expanded comparably, more polyfunctionality was seen in T RMfrom WSN/Del immunized mice than WSN/WT recovered mice. Single cell RNA-seq of migratory DC in initial immunization showed differential gene programs associated with WSN/Del infection. Ongoing work is undertaken to identify the signals provided by DC that may lead to preferential priming of T cells to tissue resident lineage. Since T RMhave been illustrated to confer heterosubtypic protection, knowledge on the specific physiological cues that facilitate T RMformation and retention will be useful to vaccine development.

Harmonization and qualification of intracellular cytokine staining to measure influenza-specific CD4+ T cell immunity within the FLUCOP consortium.

Despite the knowledge that cell-mediated immunity (CMI) contributes to the reduction of severe influenza infection, transmission, and disease outcome, the correlates of protection for cell-mediated immunity remain still unclear. Therefore, measuring the magnitude and quality of influenza-specific T cell responses in a harmonized way is of utmost importance to improve characterisation of vaccine-induced immunity across different clinical trials. The present study, conducted as part of the FLUCOP project, describes the development of a consensus protocol for the intracellular cytokine staining (ICS) assay, in order to reduce inter-laboratory variability, and its qualification. In order to develop a consensus protocol, the study was divided into different stages. Firstly, two pilot studies evaluated critical parameters in the analytical (read-outs) and post-analytical (gating strategies and data analysis) methods applied by eight different laboratories within the FLUCOP consortium. The methods were then harmonized by fixing the critical parameters and the subsequent consensus protocol was then qualified by one FLUCOP member. The antigen-specific cell population was defined as polypositive CD4+ T cells (i.e. positive for at least two markers among CD40L/IFNγ/IL2/TNFα), which was shown to be the most sensitive and specific read-out. The qualification of this consensus protocol showed that the quantification of polypositive CD4+ T cells was precise, linear and accurate, and sensitive with a lower limit of quantification of 0.0335% antigen-specific polypositive CD4+ T cells. In conclusion, we provide the description of a harmonized ICS assay, which permits quantitative and qualitative evaluation of influenza vaccine-induced T cell responses. Application of this harmonized assay may allow for future comparisons of T cell responses to different influenza vaccines. It may facilitate future assessments of potential correlates of protection with the promise of application across other pathogens.

Influenza-Induced CD103+ T Resident Memory Cells Exhibit Enhanced Functional Avidity over CD103- Memory T Cells in the Mediastinal Lymph Node.

Influenza virus–specific tissue-resident memory CD8 T cells (Trms) targeting conserved viral proteins provide strain-transcending heterosubtypic immunity to infection. Trms in the lung combat reinfection through rapid cytolytic function and production of inflammatory cytokines to recruit other immune cells. Influenza-specific Trms are also generated in the lung draining mediastinal lymph node (mLN) and can provide immunity to heterologous virus infection in this tissue, although their role in combating influenza infection is less well defined. Functional avidity, a measure of T cell sensitivity to Ag stimulation, correlates with control of viral infection and may be important for immune detection of recently infected cells, when low numbers of surface peptide–MHC complexes are displayed. However, the functional avidity of influenza-specific Trms has not been previously compared with that of other memory CD8 T cell subsets. In this article, a methodology is presented to compare the functional avidity of CD8 T cell subsets across murine tissues, with a focus on influenza-specific mLNs compared with splenic CD8 T cells, by stimulating both populations in the same well to account for CD8 T cell–extrinsic variables. The functional avidity of influenza-specific mLN effector CD8 T cells is slightly increased relative to splenic effector CD8 T cells. However, CD103+ mLN Trms display increased functional avidity compared with splenic memory CD8 T cells and CD103− memory CD8 T cells within the mLN. In contrast, lung-derived CD103+ Trms did not exhibit enhanced functional avidity. mLN CD103+ Trms also exhibit increased TCR expression, providing a potential mechanism for their enhanced functional avidity.

In Vitro Stimulation with Live SARS-CoV-2 Suggests Th17 Dominance In Virus-Specific CD4+ T Cell Response after COVID-19.

The SARS-CoV-2 and influenza viruses are the main causes of human respiratory tract infections with similar disease manifestation but distinct mechanisms of immunopathology and host response to the infection. In this study, we investigated the SARS-CoV-2-specific CD4+ T cell phenotype in comparison with H1N1 influenza-specific CD4+ T cells. We determined the levels of SARS-CoV-2- and H1N1-specific CD4+ T cell responses in subjects recovered from COVID-19 one to 15 months ago by stimulating PBMCs with live SARS-CoV-2 or H1N1 influenza viruses. We investigated phenotypes and frequencies of main CD4+ T cell subsets specific for SARS-CoV-2 using an activation induced cell marker assay and multicolor flow cytometry, and compared the magnitude of SARS-CoV-2- and H1N1-specific CD4+ T cells. SARS-CoV-2-specific CD4+ T cells were detected 1–15 months post infection and the frequency of SARS-CoV-2-specific central memory CD4+ T cells was increased with the time post-symptom onset. Next, SARS-CoV-2-specific CD4+ T cells predominantly expressed the Th17 phenotype, but the level of Th17 cells in this group was lower than in H1N1-specific CD4+ T cells. Finally, we found that the lower level of total Th17 subset within total SARS-CoV-2-specific CD4+ T cells was linked with the low level of CCR4+CXCR3– ‘classical’ Th17 cells if compared with H1N1-specific Th17 cells. Taken together, our data suggest the involvement of Th17 cells and their separate subsets in the pathogenesis of SARS-CoV-2- and influenza-induced pneumonia; and a better understanding of Th17 mediated antiviral immune responses may lead to the development of new therapeutic strategies.

Immunization with live influenza virus induces greater polyfunctional CD4+ T cell response compared to inactivated virus

Previous work has shown that infection of dendritic cells with influenza virus results in more diverse and efficient antigen presentation on MHC-II compared to treatment with excess doses of killed influenza virus. Accordingly, immunization of mice with live influenza virus elicits a greater frequency and breadth of CD4+ T cells than killed virus, as determined by interferon (IFN)-gamma ELISpot. Here, we sought to more fully characterize the differences in CD4+ T cell functionality induced by live vs. killed virus vaccines. C57Bl/6 mice were immunized with live influenza virus A/Puerto Rico/8/1934 (PR8) or a large excess dose of inactivated PR8. Efforts were made to control for the antigen load and adjuvant effects. Influenza-specific CD4+ T cells were evaluated by peptide/MHC-II tetramer staining, intracellular cytokine staining after peptide restimulation, and flow cytometry. Greater frequencies of PR8-specific CD4+ T cells were elicited by live vs. killed virus, as measured by tetramer staining or expression of the cytokines IFN-gamma, TNF, or IL-2. Of particular note, live virus induced a significantly higher frequency of polyfunctional CD4+ T cells, producing two or more cytokines when stimulated with peptide. This result was evident when polyfunctional CD4+ T cells were expressed as a fraction of all CD4+ T cells or PR8 epitope-specific CD4+ T cells. The difference in polyfunctional CD4+ T cells was maintained when antigenically distinct influenza virus B/Lee/1940 was mixed with killed PR8 as an adjuvant or when killed PR8 was administered in a prime/boost series. The data are consistent with a model whereby endogenous antigen presentation on MHC-II transmits more potent activating signals to CD4+ T cells than exogenous presentation, leading to a greater level of type 1 helper cytokine production and potentially greater antiviral effects.

T Cell Epitope Discovery in the Context of Distinct and Unique Indigenous HLA Profiles.

CD8+ T cells are a pivotal part of the immune response to viruses, playing a key role in disease outcome and providing long-lasting immunity to conserved pathogen epitopes. Understanding CD8+ T cell immunity in humans is complex due to CD8+ T cell restriction by highly polymorphic Human Leukocyte Antigen (HLA) proteins, requiring T cell epitopes to be defined for different HLA allotypes across different ethnicities. Here we evaluate strategies that have been developed to facilitate epitope identification and study immunogenic T cell responses. We describe an immunopeptidomics approach to sequence HLA-bound peptides presented on virus-infected cells by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Using antigen presenting cell lines that stably express the HLA alleles characteristic of Indigenous Australians, this approach has been successfully used to comprehensively identify influenza-specific CD8+ T cell epitopes restricted by HLA allotypes predominant in Indigenous Australians, including HLA-A*24:02 and HLA-A*11:01. This is an essential step in ensuring high vaccine coverage and efficacy in Indigenous populations globally, known to be at high risk from influenza disease and other respiratory infections.

Examining effector functions of lung CD8+ tissue resident memory T cells in humans.

Abstract Due to their position in the lung tissue, CD8+ tissue resident memory T cells (TRM) act as sentinels of the respiratory tract that rapidly respond to, and mediate protection against, respiratory viruses. In mice, TRM have been shown to mediate protection at barrier sites by producing cytokines, chemokines, and performing cell lysis. In the lungs specifically, our lab has shown that influenza-specific CD8+ TRM rapidly produce IFNγ, but airway TRM are poorly cytolytic in mice. In humans, less is known about the effector functions of virus-specific CD8+ TRM in the lungs, and thus this study seeks to fill that gap in knowledge. Using cells from healthy human lungs, we first identified and quantified the frequency of antigen-specific cells in our lung donors by performing intracellular cytokine staining (IFNγ+) and activation induced marker assays (CD137+ CD25+). Then, by performing a series of in vitro peptide stimulation and cytokine neutralization experiments, we investigated which cytokines are produced by lung CD8+ TRM, and how those cytokines impact local innate and epithelial cells. Initial results show that, when stimulated with their cognate antigen, lung CD8+ TRM produce cytokines that directly activate innate immune cells. Results of this study suggests that human CD8+ TRM in the lungs act to rapidly reprogram local immune cells, and these data will ultimately help us understand how CD8+ TRM fit into the overall immune response to respiratory viruses. Supported by grants from the NIH/NHLBI (R35 HL150803) and by NIH/NIAD through the Centers of Excellence for Influenza Research and Response (75N93019R00028).

Pneumonia Severity and Phase Linked to Virus-Specific T Cell Responses with Distinct Immune Checkpoints during pH1N1 Infection.

The detailed features and the longitudinal variation of influenza-specific T cell responses within naturally infected patients and the relationship with disease severity remain uncertain. In this study, we characterized the longitudinal influenza-specific CD4+ and CD8+ T cell responses, T cell activation, and migration-related cytokine/chemokine secretion in pH1N1-infected patients with or without viral pneumonia with human PBMCs. Both the influenza-specific CD4+ and CD8+ T cells presented higher responses in patients with severe infection than in mild ones, but with distinct longitudinal variations, phenotypes of memory markers, and immune checkpoints. At 7 ± 3 d after onset of illness, effector CD8+ T cells (CD45RA+CCR7-) with high expression of inhibitory immune receptor CD200R dominated the specific T cell responses. However, at 21 ± 3 d after onset of illness, effector memory CD4+ T cells (CD45RA-CCR7-) with high expression of PD1, CTLA4, and LAG3 were higher among the patients with severe disease. The specific T cell magnitude, T cell activation, and migration-related cytokines/chemokines possessed a strong connection with disease severity. Our findings illuminate the distinct characteristics of immune system activation during dynamic disease phases and its correlation with lung injury of pH1N1 patients.

Establishment of influenza-specific lung tissue-resident helper memory CD4+ T cells relies on expression the eATP sensor P2RX7

Abstract Influenza remains an infection of high medical relevance. Development of universal anti-influenza vaccines will require a better knowledge of lung immune responses. That includes influenza-specific CD4+ T cells, which establish long-term lung residency and induce a diversified local protective response to subsequent infection. Lung-resident memory CD4+ T cells are heterogeneous, including Th1-like cells, and a subset that shares phenotypic and functional features with follicular helper (Tfh) cells – called tissue-resident helper (Trh). Notably, both Tfh and lung Trh cells express the extracellular ATP (eATP) sensor P2RX7 at high levels. Given its known deleterious role in Tfh cells, we investigated if P2RX7 could also limit the Trh pool in influenza-infected lungs. Contrary to our hypothesis, T cell-specific P2RX7 ablation led to a significant decrease in the Trh cell subset. Consequently, lungs of T cell-P2RX7 knockout mice harbor reduced B cell and CD8+ T cell responses. Initial Trh cell establishment is not affected by P2RX7 knockout; rather, P2RX7-deficient Trh cells gradually decay over time. This is associated with increased apoptosis and decreased in situ proliferation of P2RX7-knockout Trh cells. Our bulk RNA-seq analysis of lung CD4+ T cells revealed many cell cycle-associated genes poorly expressed in P2RX7-knockout cells. Some (but not all) Trh-fate genes are also decreased in P2RX7 deficiency. We are currently performing single-cell RNA-seq to define whether P2RX7 promotes the Trh fate in a particular subset of lung precursor cells. Our data shows that, in response to influenza, P2RX7 promotes lung-resident Trh cells, suggesting this eATP sensor has opposite roles in Tfh-like CD4+ T cells in a tissue-specific manner. Supported by grant from NIH (R00 AI139381)