Supplementary MaterialsS1 Fig: M2-expressing self-employed B cell line shows upregulation of CD80, CD86 and ICAM-1
Supplementary MaterialsS1 Fig: M2-expressing self-employed B cell line shows upregulation of CD80, CD86 and ICAM-1. error of the mean. Statistical significance was assessed having a one-tailed College students t-test.(TIF) pone.0142540.s001.tif (458K) GUID:?1DE91643-BF3C-4447-A1FD-B5737D636741 S2 Fig: M2 expression in an self-employed B cell line promotes conjugation with TH cells. eGFP self-employed B cell lines were pulsed over night, or not, with different concentrations of OVAp and incubated with OVAp-specific CD4+ T cells at a 2:1 percentage. (A) Percentage of conjugates after 30min of incubation upon variance of the OVAp concentration. T cell populations were loaded with DDAO, to allow their discrimination. Results shown correspond to imply of three self-employed experiments. Statistical significance refers to assessment between M2 and M2Y conditions. (B) Percentage of conjugates per image after 30min of incubation, determined by confocal microscopy, upon variance of the OVAp concentration. Conjugate count was blind and based on B-TH contact and pTyr polarization to the contact zone. 15 to 35 images were taken per sample, for an equal number of analyzed T Cspg2 cells within each OVAp concentration. Only images with a minimum of three T cells were considered for analysis. Results are from one experiment. (C) Fold increase of the number of conjugates created with eGFP-M2- (open bars) or eGFP-M2Y- (packed bars) expressing B cells relative to M2Y condition. eGFP-M2-expressing B cells, eGFP-M2Y-expressing B cells and CD4+ T cells were combined at a 1:1:1 percentage and incubated for 30min. Prior to conjugation M2Y-expressing B and T cell populations were labeled with the Alda 1 live dyes CMTMR and DDAO, respectively, to allow their discrimination. Conjugate formation was analyzed on a LSR Fortessa circulation cytometer as the percentage of eGFP+DDAO+ (M2) or eGFP+CMTMR+DDAO+ (M2Y) events in the total DDAO+ human population. (D) Representative FACS plots for each OVAp concentration. Percentage of T Alda 1 cells conjugating with M2- or M2Y-expressing B cells is definitely indicated in the respective quadrant. In circulation cytometry experiments, error bars represent standard error of the mean. Statistical significance between organizations was evaluated by a one-tailed unpaired College students t test. In confocal microscopy experiments, statistical significance of the difference between organizations was evaluated by a Mann-Whitney U test.(TIF) pone.0142540.s002.tif (1.0M) GUID:?72003574-5562-4337-A0C1-FE043FA9EBA5 S3 Fig: An independent M2-expressing B cell line requires specific peptide to promote TH cell activation. (A) Average of the percentage of CD4+ T cells mobilizing calcium when conjugated with eGFP-M2-expressing (black bars), eGFP-M2Y-expressing (white bars) or eGFP-expressing (grey bars) B cells. eGFP self-employed B cell lines were pulsed over night, or not, with different concentrations of OVAp and incubated with OVAp-specific CD4+ T cells for 5 min. Prior to conjugation T cells were loaded with Indo-I, a calcium indication. Ionomycin was used like a positive control. Calcium fluxes were measured on a MoFlow cytometer for 21 moments and were based on the 405/530 emission percentage over time. Graph shows results from one experiment. (B) Quantification of conjugates showing IFN- polarization to the contact zone per field. Prior to incubation B and T cells were labelled with CMFDA and CMAC live dyes, respectively. Cells were incubated for 2.5h, fixed and stained for IFN- and pTyr. Conjugates were evaluated by confocal microscopy based on B-TH contact and IFN- polarization. Only images with a minimum of three T cells were considered for analysis. Statistical significance of the difference between organizations was evaluated by a Mann-Whitney U test.(TIF) pone.0142540.s003.tif (249K) GUID:?432530C4-7F86-4F3C-B51B-89C00D894F4F Data Availability StatementAll relevant data are within the paper and its Supporting Information documents. Abstract Establishment of prolonged infection in memory space B cells by murid herpesvirus-4 (MuHV-4) depends on the proliferation of latently infected germinal center B cells, for which T cell help is essential. Whether the disease is capable of modulating B-T helper cell connection for its personal benefit is still unknown. Here, we investigate if the MuHV-4 latency connected M2 protein, which assembles multiprotein complexes with B cell signaling proteins, plays a role. We observed that M2 led to the upregulation of adhesion and co-stimulatory molecules in transduced B cell lines. In an MHC-II restricted OVA peptide-specific system, M2 polarized to the B-T helper contact zone. Furthermore, it advertised B cell polarization, as shown by the Alda 1 improved proximity of the Alda 1 B cell microtubule organizing center to the interface. Consistent with these data, M2 advertised the formation of B-T helper cell conjugates..