Project description:IL-6 is known to contribute to the differentiation of CD4<sup>+</sup> T cells into different subsets of effector T helper cells. Less is known about the potential of IL-6 in regulating CD8<sup>+</sup> T cell effector function. Here, we identify IL-6 as a master regulator of IL-21 in effector CD8<sup>+</sup> T cells. IL-6 promotes the differentiation of a subset of naive CD8<sup>+</sup> T cells that express IL-6R into a unique population of effector CD8<sup>+</sup> T cells characterized by the production of high levels of IL-21 and low levels of IFN-?. Similar to CD4<sup>+</sup> T follicular helper (Tfh) cells, IL-21-producing CD8<sup>+</sup> T cells generated in the presence of IL-6 directly provide help to B cells to induce isotype switching. CD8<sup>+</sup> T cell-derived IL-21 contributes to the production of protective virus-specific IgG antibodies during influenza virus infection. Thus, this study reveals the presence of a new mechanism by which IL-6 regulates antibody production during viral infection, and a novel function of effector CD8<sup>+</sup> T cells in the protection against viruses.
Project description:Avian influenza virus (AIV) infection is a continuing threat to both humans and poultry. Influenza virus specific CD8+ T cells are associated with protection against homologous and heterologous influenza strains. In contrast to what has been described for humans and mice, knowledge on epitope-specific CD8+ T cells in chickens is limited. Therefore, we set out to identify AIV-specific CD8+ T-cell epitopes. Epitope predictions based on anchor residues resulted in 33 candidate epitopes. MHC I inbred chickens were infected with a low pathogenic AIV strain and sacrificed at 5, 7, 10 and 14 days post infection (dpi). Lymphocytes isolated from lung, spleen and blood were stimulated ex vivo with AIV-specific pooled or individual peptides and the production of IFN? was determined by ELIspot. This resulted in the identification of 12 MHC B12-restricted, 3 B4-restricted and 1 B19-restricted AIV- specific CD8+ T-cell epitopes. In conclusion, we have identified novel AIV-derived CD8+ T-cell epitopes for several inbred chicken strains. This knowledge can be used to study the role of CD8+ T cells against AIV infection in a natural host for influenza, and may be important for vaccine development.
Project description:The development of novel human vaccines would be greatly facilitated by the development of in vivo models that permit preclinical analysis of human immune responses. Here, we show that nonobese diabetic severe combined immunodeficiency (NOD/SCID) beta(2) microglobulin(-/-) mice, engrafted with human CD34+ hematopoietic progenitors and further reconstituted with T cells, can mount specific immune responses against influenza virus vaccines. Live attenuated trivalent influenza virus vaccine induces expansion of CD8+ T cells specific to influenza matrix protein (FluM1) and nonstructural protein 1 in blood, spleen, and lungs. On ex vivo exposure to influenza antigens, antigen-specific CD8+ T cells produce IFN-gamma and express cell-surface CD107a. FluM1-specific CD8+ T cells can be also expanded in mice vaccinated with inactivated trivalent influenza virus vaccine. Expansion of antigen-specific CD8+ T cells is dependent on reconstitution of the human myeloid compartment. Thus, this humanized mouse model permits preclinical testing of vaccines designed to induce cellular immunity, including those against influenza virus. Furthermore, this work sets the stage for systematic analysis of the in vivo functions of human DCs. This, in turn, will allow a new approach to the rational design and preclinical testing of vaccines that cannot be tested in human volunteers.
Project description:MicroRNA microarray expression dataset evaluating relative changes in microRNA expression levels between naïve and effector OT-I CD8+ T cells during influenza virus infection in mice. Overall design: Naïve OT-I CD8+ T cells were FACS sorted (based on CD44- and CD62L+ expression) from the spleens on uninfected female OT-I mice. Naïve cells were kept for analysis of microRNA expression in naïve OT-I CD8+ T cells. Additionally, 1x10e4 cells were adoptively transferred into uninfected female C57BL/6 mice (via tail vein injection) followed by intranasal infection with WSN-OVA influenza virus. At day 9, donor OT-I cells were FACS sorted from the lungs of influenza virus-infected mice for analysis of microRNA expression in effector OT-I CD8+ T cells. Total RNA was isolated from sorted cells and miRNA gene expression was assessed using an Affymetrix murine miRNA 4.0 gene chip. CEL files were normalized and analyzed using Affymetrix Expression Console, and miRNA annotation is from miRBASE version 21.
Project description:Purpose: A growing body of evidence highlights the association of exuberant CD8+ T cell responses with influenza acute lung injury. Given their indispensable role in viral clearance, we studied the double-edged function of CD8+ T cells as a bridge between infection resolution and lung pathology. Methods: we performed scRNA-Seq to study the cellular heterogeneity and regulation of cellular response in CD8+ T cells during peak viral (day 7) and post-viral resolution phase (day 14). Results: Our single-cell RNA-Seq data reveal significant differences in CD8+ T cell heterogeneity during different stages of influenza infection (peak viral load vs. infection resolved state). While CXCR3hi CD8+ T effector memory (Tem) was associated with a more robust cytotoxic response (compared to CXCR3low CD8+ T central memory, Tcm), both CD8+ Tem and Tcm exhibited equally potent effector function. The ablation of CXCR3 mitigates lung injury without affecting viral clearance. IFN-gamma was dispensable to the recruitment and regulation of cytotoxic response of CXCR3+ CD8+ T cells. Using scRNAseq, we identified unique regulons associated with regulating cytotoxic response in CXCR3hi CD8+ T cells. Conclusions: Collectively, our data identify CXCR3 as a potential therapeutic target to curb lung injury during influenza pathogenesis. Overall design: scRNAseq profiles of mock, 7 days and 14 days post-IAV infection
Project description:<h4>Purpose</h4>Human interleukin-21 (IL-21) is a class I cytokine previously reported in clinical studies on immune responsive cancers. Here we report the effects of systemic IL-21 therapy on the immune system in two phase 1 trials with this novel cytokine.<h4>Experimental design</h4>Recombinant IL-21 was administered by intravenous bolus injection at dose levels from 1 to 100 microg/kg using two planned treatment regimens: thrice weekly for 6 weeks (3/week); or once daily for five consecutive days followed by nine dose-free days (5 + 9). The following biomarkers were studied in peripheral blood mononuclear cells (PBMC) during treatment: phosphorylation of STAT3, alterations in the composition of leukocyte subsets, ex vivo cytotoxicity, expression of effector molecules in enriched CD8(+) T cells and CD56(+) NK cells by quantitative RT-PCR, and gene array profiling of CD8(+) T cells.<h4>Results</h4>Effects of IL-21 were observed at all dose levels. In the 5 + 9 regimen IL-21 induced a dose dependent decrease in circulating NK cells and T cells followed by a return to baseline in resting periods. In both CD8(+) T cells and CD56(+) NK cells we found up-regulation of perforin and granzyme B mRNA. In addition, full transcriptome analysis of CD8(+) T cells displayed changes in several transcripts associated with increased cell cycle progression, cellular motility, and immune activation. Finally, cytotoxicity assays showed that IL-21 enhanced the ability of NK cells to kill sensitive targets ex vivo.<h4>Conclusions</h4>IL-21 was biologically active at all dose levels administered with evidence of in vivo NK cell and CD8(+) T cell activation.
Project description:Seasonal influenza virus infections cause 290,000-650,000 deaths annually and severe morbidity in 3-5 million people. CD8+ T-cell responses towards virus-derived peptide/human leukocyte antigen (HLA) complexes provide the broadest cross-reactive immunity against human influenza viruses. Several universally-conserved CD8+ T-cell specificities that elicit prominent responses against human influenza A viruses (IAVs) have been identified. These include HLA-A*02:01-M158-66 (A2/M158), HLA-A*03:01-NP265-273, HLA-B*08:01-NP225-233, HLA-B*18:01-NP219-226, HLA-B*27:05-NP383-391 and HLA-B*57:01-NP199-207. The immunodominance hierarchies across these universal CD8+ T-cell epitopes were however unknown. Here, we probed immunodominance status of influenza-specific universal CD8+ T-cells in HLA-I heterozygote individuals expressing two or more universal HLAs for IAV. We found that while CD8+ T-cell responses directed towards A2/M158 were generally immunodominant, A2/M158+CD8+ T-cells were markedly diminished (subdominant) in HLA-A*02:01/B*27:05-expressing donors following ex vivo and in vitro analyses. A2/M158+CD8+ T-cells in non-HLA-B*27:05 individuals were immunodominant, contained optimal public TRBV19/TRAV27 TCR?? clonotypes and displayed highly polyfunctional and proliferative capacity, while A2/M158+CD8+ T cells in HLA-B*27:05-expressing donors were subdominant, with largely distinct TCR?? clonotypes and consequently markedly reduced avidity, proliferative and polyfunctional efficacy. Our data illustrate altered immunodominance patterns and immunodomination within human influenza-specific CD8+ T-cells. Accordingly, our work highlights the importance of understanding immunodominance hierarchies within individual donors across a spectrum of prominent virus-specific CD8+ T-cell specificities prior to designing T cell-directed vaccines and immunotherapies, for influenza and other infectious diseases.
Project description:During a primary influenza infection, cytotoxic CD8+ T cells need to infiltrate the infected airways and engage virus-infected epithelial cells. The factors that regulate T cell motility in the infected airway tissue are not well known. To more precisely study T cell infiltration of the airways, we developed an experimental model system using the trachea as a site where live imaging can be performed. CD8+ T cell motility was dynamic with marked changes in motility on different days of the infection. In particular, significant changes in average cell velocity and confinement were evident on days 8-10 during which the T cells abruptly but transiently increase velocity on day 9. Experiments to distinguish whether infection itself or antigen affect motility revealed that it is antigen, not active infection per se that likely affects these changes as blockade of peptide/MHC resulted in increased velocity. These observations demonstrate that influenza tracheitis provides a robust experimental foundation to study molecular regulation of T cell motility during acute virus infection.
Project description:Type 2 effector production of IL-13, a demonstrated requirement in models of fibrosis, is routinely ascribed to CD4(+) Th2 cells. We now demonstrate a major role for CD8(+) T cells in a murine model of sterile lung injury. These pulmonary CD8(+) T cells differentiate into IL-13-producing Tc2 cells and play a major role in a bleomycin-induced model of fibrosis. Differentiation of these Tc2 cells in the lung requires IL-21, and bleomycin treated IL-21- and IL-21R-deficient mice develop inflammation but not fibrosis. Moreover, IL-21R-expressing CD8(+) cells are sufficient to reconstitute the fibrotic response in IL-21R-deficient mice. We further show that the combination of IL-4 and IL-21 skews naive CD8(+) T cells to produce IL-21, which, in turn, acts in an autocrine manner to support robust IL-13 production. Our data reveal a novel pathway involved in the onset and regulation of pulmonary fibrosis and identify Tc2 cells as key mediators of fibrogenesis.
Project description:Aged individuals have increased morbidity and mortality following influenza and other viral infections, despite previous exposure or vaccination. Mouse and human studies suggest increased senescence and/or exhaustion of influenza virus-specific CD8 T cells with advanced age. However, neither the relationship between senescence and exhaustion nor the underlying transcriptional pathways leading to decreased function of influenza virus-specific cellular immunity in elderly humans are well-defined. Here, we demonstrate that increased percentages of CD8 T cells from aged individuals express CD57 and KLRG1, along with PD-1 and other inhibitory receptors, markers of senescence, or exhaustion, respectively. Expression of T-box transcription factors, T-bet and Eomes, were also increased in CD8 T cells from aged subjects and correlated closely with expression of CD57 and KLRG1. Influenza virus-specific CD8 T cells from aged individuals exhibited decreased functionality with corresponding increases in CD57, KLRG1, and T-bet, a molecular regulator of terminal differentiation. However, in contrast to total CD8 T cells, influenza virus-specific CD8 T cells had altered expression of inhibitory receptors, including lower PD-1, in aged compared with young subjects. Thus, our data suggest a prominent role for senescence and/or terminal differentiation for influenza virus-specific CD8 T cells in elderly subjects.