Journal: Nature Communications

Article Title: MHC class II functions as a host-specific entry receptor for representative human and swine H3N2 influenza A viruses

doi: 10.1038/s41467-026-69267-6

Figure Lengend Snippet: a HEK-293T cells were transfected with either HLA-DRA + HLA-DRB1 or SLA-DRA + SLA-DRB1. MHCII-expressing cells and cells transfected with an empty plasmid were desialylated at 24 h post-transfection for 24 h. Cells were infected with either sOH/04 or hVIC/11 and infections were incubated for 24 h before processing. Subsequently, cells were stained for MHCII (pink), sialic acid (red), and FLUAV (green). Co-localization between MHCII and FLUAV can be seen as white. Scale bar= 30 μm. Images are representative of three independent experiments. Transient expression of MHCII allows FLUAV entry into desialylated HEK-293T cells. Cells were transfected with either HLA-DR ( b ) or SLA-DR ( c ) and further desialylated prior to infection with hVIC/11 or sOH/04. At 24 hpi, cells were fixed and stained for MHCII and FLUAV. The percentage of FLUAV-positive cells was determined by flow cytometry ( n = 3 independent experiments). d Percentage of FLUAV-positive cells among desialylated MHCII-expressing cells determined by flow cytometry represented as the mean ± SEM of three independent experiments. sOH/04 is shown in blue while hVIC/11 can be seen in orange. e Percentage of infected HEK-293T cells (regardless of MHCII expression) determined by flow cytometry. Data are presented as the mean ± SEM of three independent experiments. f MHCII-expressing cells were incubated with 20 mM NH 4 Cl for 1 h prior to infection with either sOH/04 (blue) or hVIC/11 (orange) at an MOI of 1. Infections were maintained at 37 °C for 24 h in presence of NH 4 Cl and intracellular vRNA was determined at 24 hpi by RT-qPCR. Data are presented as the mean ± SEM of n = 3 independent experiments. g Transfected HEK-293T cells were incubated with 10 nM Bafilomycin A1 before infection and were subsequently infected at an MOI of 1 with sOH/04 (blue) or hVIC/11 (orange). Intracellular FLUAV vRNA was quantified at 24 hpi by RT-qPCR. Data are presented as the mean ± SEM of three independent experiments. L.D. limit of detection. Untreated: non-transfected, non-desialylated cells. Empty vector: desialylated cells transfected with an empty pCAGGS plasmid. HLA-DR: desialylated cells expressing HLA-DR. SLA-DR: desialylated cells expressing SLA-DR.

Article Snippet: Purified RNA was used as template for RT-qPCR using the Quantabio qScript XLT One-Step RT-qPCR ToughMix kit (Quantabio, Beverly, MA) and specific primers targeting the M segment of the virus (Supplementary Table ).

Techniques: Transfection, Expressing, Plasmid Preparation, Infection, Incubation, Staining, Flow Cytometry, Quantitative RT-PCR

Journal: Nature Communications

Article Title: MHC class II functions as a host-specific entry receptor for representative human and swine H3N2 influenza A viruses

doi: 10.1038/s41467-026-69267-6

Figure Lengend Snippet: a A549 cells were transduced with a lentivirus encoding a shRNA targeting CMAS (shRNA-CMAS) or a shRNA-scrambled negative control. α2,6-SA and α2,6-SA levels on the cell surface were evaluated by lectin staining and flow cytometry. b MHCII expression in A549 cells was evaluated by flow cytometry. Cells were transfected with equimolar quantities of either HLA-DRA + HLA-DRB1 or SLA-DRA + SLA-DRB1 and MHCII expression was assessed at 48 h post-transfection. c MHCII expression levels on shRNA-CMAS A549 cells were quantified at 48 h post transfection by flow cytometry. shRNA-CMAS cells transfected with an empty plasmid (blue), HLA-DR-expressing plasmids (black), SLA-DR-expressing plasmids (yellow), or reconstituted with a shRNA-resistant CMAS cDNA (white) were infected with either hVIC/11 ( d ) or sOH/04 ( e ). Viral titers were measured in the supernatant at 0, 12, 24, 48, and 72 hpi by RT-qPCR. Infection of shRNA-scrambled A549 cells was included as both shRNA and transduction controls (red). L.D= limit of detection. Data are presented as the mean ± SEM of three independent experiments. Similarly, shRNA-scrambled or shRNA-CMAS cells were transfected with either HLA-DR or SLA-DR, were incubated with 10 μg/mL of an anti-HLA-DR, anti-SLA-DR, or a control antibody (anti-GFP) for 1 h prior to infection with hVIC/11 ( f ) or sOH/04 ( g ). At 48 hpi viral titers in the supernatant were quantified by RT-qPCR. Data are presented as the mean ± SEM of three independent experiments. The effect of FLUAV replication in either shRNA-scrambled or shRNA-CMAS MHCII-expressing cells on the cell viability was evaluated for hVIC/11 ( h ) and sOH/04 ( i ). Cells were transfected and infected for further determination of cell viability at 48 hpi. For figures panels h and i, data is presented as the mean ± SEM of n = 3 independent experiments. Mock infection: non-infected cells. Empty vector: Cells transfected with an empty pCAGGS plasmid. HLA-DR: Cells expressing HLA-DR. SLA-DR: Cells expressing SLA-DR. CMAS: Cells transfected with a shRNA-resistant CMAS cDNA.

Article Snippet: Purified RNA was used as template for RT-qPCR using the Quantabio qScript XLT One-Step RT-qPCR ToughMix kit (Quantabio, Beverly, MA) and specific primers targeting the M segment of the virus (Supplementary Table ).

Techniques: Transduction, shRNA, Negative Control, Staining, Flow Cytometry, Expressing, Transfection, Plasmid Preparation, Infection, Quantitative RT-PCR, Incubation, Control

Journal: Nature Communications

Article Title: MHC class II functions as a host-specific entry receptor for representative human and swine H3N2 influenza A viruses

doi: 10.1038/s41467-026-69267-6

Figure Lengend Snippet: a hVIC/11 HA trimer model made using PyMOL based on previously published crystal structure (PDB: 4WE8 ). Sialic acid RBS is highlighted in pink while residues 138, 186, and 193 (H3 numbering) are shown in red. HEK-293T cells transfected with HLA-DR- ( b ) or SLA-DR-expressing plasmids ( c ) and then desialylated with 100 mU/mL of neuraminidase. Subsequently, cells were infected at an MOI of 0.01 with hVIC/11-A138S, hVIC/11-V186G, or hVIC/11-F193Y. At 24 hpi cells were stained for MHCII (APC) and FLUAV (Alexa488). d FLUAV-positive cells among MHCII-expressing HEK-293T cells was determined by gating live, MHCII + cells only. Percentages were obtained by flow cytometry. Data is presented as the mean ± SEM of n = 3 independent experiments. e Total amount of FLUAV-infected HEK-293T cells was determined by multi-color flow cytometry. Data are presented as the mean ± SEM of three independent experiments. To evaluate virus replication in A549 cells, shRNA-scrambled (red), shRNA-CMAS cells transfected with an empty plasmid (blue), shRNA-CMAS cells transfected with HLA-DR-encoding plasmids (black), shRNA-CMAS cells transfected with SLA-DR-expressing plasmids (yellow), and shRNA-CMAS cells transfected with a shRNA-resistant CMAS cDNA (white) were infected at an MOI of 0.01 with either hVIC/11-A138S ( f ), hVIC/11-V186G ( g ), or hVIC/11-F193Y ( h ). Viral titers in the supernatant were quantified at 0, 12, 24, 48, and 72 hpi by RT-qPCR. Data are presented as the mean ± SEM of three independent experiments. L.D. limit of detection.

Article Snippet: Purified RNA was used as template for RT-qPCR using the Quantabio qScript XLT One-Step RT-qPCR ToughMix kit (Quantabio, Beverly, MA) and specific primers targeting the M segment of the virus (Supplementary Table ).

Techniques: Transfection, Expressing, Infection, Staining, Flow Cytometry, Virus, shRNA, Plasmid Preparation, Quantitative RT-PCR