Pigs are natural hosts for influenza A viruses and play a critical role in influenza epidemiology. However, little is known about their influenza-evoked T-cell response. We performed a thorough analysis of both the local and systemic T-cell response in influenza virus-infected pigs, addressing kinetics and phenotype as well as multifunctionality (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], and interleukin-2 [IL-2]) and cross-reactivity. A total of 31 pigs were intratracheally infected with an H1N2 swine influenza A virus (FLUAVsw) and consecutively euthanized. Lungs, tracheobronchial lymph nodes, and blood were sampled during the first 15 days postinfection (p.i.) and at 6 weeks p.i. Ex vivo flow cytometry of lung lymphocytes revealed an increase in proliferating (Ki-67+) CD8+ T cells with an early effector phenotype (perforin+ CD27+) at day 6 p.i. Low frequencies of influenza virus-specific IFN-γ-producing CD4+ and CD8+ T cells could be detected in the lung as early as 4 days p.i. On consecutive days, influenza virus-specific CD4+ and CD8+ T cells produced mainly IFN-γ and/or TNF-α, reaching peak frequencies around day 9 p.i., which were up to 30-fold higher in the lung than in tracheobronchial lymph nodes or blood. At 6 weeks p.i., CD4+ and CD8+ memory T cells had accumulated in lung tissue. These cells showed diverse cytokine profiles and in vitro reactivity against heterologous influenza virus strains, all of which supports their potential to combat heterologous influenza virus infections in pigs.IMPORTANCE Pigs not only are a suitable large-animal model for human influenza virus infection and vaccine development but also play a central role in the emergence of new pandemic strains. Although promising candidate universal vaccines are tested in pigs and local T cells are the major correlate of heterologous control, detailed and targeted analyses of T-cell responses at the site of infection are scarce. With the present study, we provide the first detailed characterization of magnitude, kinetics, and phenotype of specific T cells recruited to the lungs of influenza virus-infected pigs, and we could demonstrate multifunctionality, cross-reactivity, and memory formation of these cells. This, and ensuing work in the pig, will strengthen the position of this species as a large-animal model for human influenza virus infection and will immediately benefit vaccine development for improved control of influenza virus infections in pigs.

Background: Antibody tests are essential tools to investigate humoral immunity following SARS-CoV-2 infection or vaccination. While first-generation antibody tests have primarily provided qualitative results, accurate seroprevalence studies and tracking of antibody levels over time require highly specific, sensitive and quantitative test setups. Methods: We have developed two quantitative, easy-to-implement SARS-CoV-2 antibody tests, based on the spike receptor binding domain and the nucleocapsid protein. Comprehensive evaluation of antigens from several biotechnological platforms enabled the identification of superior antigen designs for reliable serodiagnostic. Cut-off modelling based on unprecedented large and heterogeneous multicentric validation cohorts allowed us to define optimal thresholds for the tests' broad applications in different aspects of clinical use, such as seroprevalence studies and convalescent plasma donor qualification. Findings: Both developed serotests individually performed similarly-well as fully-automated CE-marked test systems. Our described sensitivity-improved orthogonal test approach assures highest specificity (99.8%); thereby enabling robust serodiagnosis in low-prevalence settings with simple test formats. The inclusion of a calibrator permits accurate quantitative monitoring of antibody concentrations in samples collected at different time points during the acute and convalescent phase of COVID-19 and disclosed antibody level thresholds that correlate well with robust neutralization of authentic SARS-CoV-2 virus. Interpretation: We demonstrate that antigen source and purity strongly impact serotest performance. Comprehensive biotechnology-assisted selection of antigens and in-depth characterisation of the assays allowed us to overcome limitations of simple ELISA-based antibody test formats based on chromometric reporters, to yield comparable assay performance as fully-automated platforms.

Unlike mice and humans, porcine γδ T cells represent a prominent subset of T cells in blood and secondary lymphatic organs. GATA-3, T-bet and Eomesodermin (Eomes) are transcription factors with crucial functions in T-cell development and functional differentiation, but their expression has not been investigated in porcine γδ T cells so far. We analyzed the expression of these transcription factors in γδ thymocytes, mature γδ T cells from blood, spleen, lymph nodes, and lung tissue as well as in vitro stimulated γδ T cells on the protein level by flow cytometry. GATA-3 was present in more than 80% of all γδ-thymocytes. Extra-thymic CD2− γδ T cells expressed high levels of GATA-3 in all investigated organs and had a CD8α−/dimCD27+perforin− phenotype. T-bet expression was mainly found in a subset of CD2+ γδ T cells with an opposing CD8αhighCD27dim/−perforin+ phenotype. Eomes+ γδ T cells were also found within CD2+ γδ T cells but were heterogeneous in regard to expression of CD8α, CD27, and perforin. Eomes+ γδ T cells frequently co-expressed T-bet and dominated in the spleen. During aging, CD2−GATA-3+ γδ T cells strongly prevailed in young pigs up to an age of about 2 years but declined in older animals where CD2+T-bet+ γδ T cells became more prominent. Despite high GATA-3 expression levels, IL-4 production could not be found in γδ T cells by intracellular cytokine staining. Experiments with sorted and ConA + IL-2 + IL-12 + IL-18-stimulated CD2− γδ T cells showed that proliferating cells start expressing CD2 and T-bet, produce IFN-γ, but retain GATA-3 expression. In summary, our data suggest a role for GATA-3 in the development of γδ-thymocytes and in the function of peripheral CD2−CD8α−/dimCD27+perforin− γδ T cells. In contrast, T-bet expression appears to be restricted to terminal differentiation stages of CD2+ γδ T cells, frequently coinciding with perforin expression. The functional relevance of high GATA-3 expression levels in extra-thymic CD2− γδ T cells awaits further clarification. However, their unique phenotype suggests that they represent a thymus-derived separate lineage of γδ T cells in the pig for which currently no direct counterpart in rodents or humans has been described.

Although swine are natural hosts for influenza A viruses, the porcine T-cell response to swine influenza A virus (FLUAVsw) infection has been poorly characterized so far. We have studied Ki-67 expression and FLUAVsw-specific production of IFN-γ, TNF-α and IL-2 in CD4+ and CD8β+ T cells isolated from piglets that had been intratracheally infected with a H1N2 FLUAVsw isolate. IFN-γ+TNF-α+IL-2+ multifunctional CD4+ T cells were present in the blood of all infected animals at one or two weeks after primary infection and their frequency increased in four out of six animals after homologous secondary infection. These cells produced higher amounts of IFN-γ, TNF-α and IL-2 than did CD4+ T cells that only produced a single cytokine. The vast majority of cytokine-producing CD4+ T cells expressed CD8α, a marker associated with activation and memory formation in porcine CD4+ T cells. Analysis of CD27 expression suggested that FLUAVsw-specific CD4+ T cells included both central memory and effector memory populations. Three out of six animals showed a strong increase of Ki-67+perforin+ CD8β+ T cells in blood one week post infection. Blood-derived FLUAVsw-specific CD8β+ T cells could be identified after an in vitro expansion phase and were multifunctional in terms of CD107a expression and co-production of IFN-γ and TNF-α. These data show that multifunctional T cells are generated in response to FLUAVsw infection of pigs, supporting the idea that T cells contribute to the efficient control of infection.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-015-0182-3) contains supplementary material, which is available to authorized users.

The CD3−NKp46+ phenotype is frequently used for the identification of natural killer (NK) cells in various mammalian species. Recently, NKp46 expression was analyzed in more detail in swine. It could be shown that besides CD3−NKp46+ lymphocytes, a small but distinct population of CD3+NKp46+ cells exists. In this study, we report low frequencies of CD3+NKp46+ lymphocytes in blood, lymph nodes, and spleen, but increased frequencies in non-lymphatic organs, like liver and lung. Phenotypic analyses showed that the majority of CD3+NKp46+ cells coexpressed the CD8αβ heterodimer, while a minor subset expressed the TCR-γδ, which was associated with a CD8αα+ phenotype. Despite these T-cell associated receptors, the majority of CD3+NKp46+ lymphocytes displayed a NK-related phenotype (CD2+CD5−CD6−CD16+perforin+) and expressed mRNA of NKp30, NKp44, and NKG2D at similar levels as NK cells. Functional tests showed that CD3+NKp46+ lymphocytes produced IFN-γ and proliferated upon cytokine stimulation to a similar extent as NK cells, but did not respond to the T-cell mitogen, ConA. Likewise, CD3+NKp46+ cells killed K562 cells with an efficiency comparable to NK cells. Cross-linking of NKp46 and CD3 led to degranulation of CD3+NKp46+ cells, indicating functional signaling pathways for both receptors. Additionally, influenza A(H1N1)pdm09-infected pigs had reduced frequencies of CD3+NKp46+ lymphocytes in blood, but increased frequencies in the lung in the early phase of infection. Thus, CD3+NKp46+ cells appear to be involved in the early phase of influenza infections. In summary, we describe a lymphocyte population in swine with a mixed phenotype of NK and T cells, with results so far indicating that this cell population functionally resembles NK cells.