Fig 1.
WT but not H84T, induces human T cell expansion by interacting with dendritic cells (DCs) in an HLA-DR and CD86-dependent manner.
(A, B) CFSE labelled PBMCs or isolated T cells from the same donor were exposed to nothing (black) or to 2 μg/mL WT (blue), H84T (red) or D133G (grey) BanLec for 72 hours. T cell proliferation was detected by CFSE dilution using flow cytometry. Graphs show frequencies of live proliferating CFSElow (A) CD4 T cells and (B) CD8 T cells from five individual donors with mean ± SEM. (C–E) DCs were exposed to nothing or 2 μg/mL WT, H84T or D133G for 1 hour, and then co-cultured with CFSE labelled autologous CD4 or CD8 T cells (1:10 ratio MDDC:T cells) for 5 days. T cell proliferation was detected by CFSE dilution using flow cytometry. (C) Dot plots show live CD4 or CD8 T cells and numbers indicate frequency of proliferating CFSElow T cells. One representative donor of four is shown. (D, E) Graphs show frequencies of live proliferating CFSElow (D) CD4 T cells and (E) CD8 T cells in autologous DC-T cell co-culture from four individual donors. (F, G) DCs were treated with 3 μg/mL isotype control (black), anti-HLA-ABC (orange), anti-HLA-DR (green) or anti-CD86 (maroon) blocking antibodies for 1 hour, and then exposed to 2 μg/mL WT, H84T or D133G and co-cultured with CFSE labelled autologous CD4 or CD8 T cells (1:10 ratio DC:T cells) for 5 days. Bar graphs show frequency of live proliferating CFSElow (F) CD4 T cells and (G) CD8 T cells from four individual donors with mean ± SEM. (H-I) DCs were exposed to nothing or 2 μg/mL WT, H84T or D133G and cultured for 24 hours, then the supernatants were collected and added to CFSE labelled autologous CD4 or CD8 T cells and cultured for 3 days. Bar graphs show frequency of live proliferating CFSElow (H) CD4 T cells and (I) CD8 T cells from four individual donors with mean ± SEM. Friedman test with Dunn’s multiple comparisons test was used to assess statistically significant differences at * p < 0.05 (** p < 0.01).
Fig 2.
H84T treatment of DCs results in higher frequencies of IAV-specific CD8 T cells in response to replicating IAV.
(A) DCs were exposed to nothing or 2 μg/mL WT, H84T and D133G for 1 hour and then exposed to no virus or 0.6 MOI IAV for 4 hours. Subsequently, CFSE labelled autologous CD4 or CD8 T cells were added (1:10 ratio MDDC:T cells) and co-cultured for 7 days. T cell proliferation was detected by CFSE dilution using flow cytometry. (B, C) Bar graphs show frequency of live proliferating CFSElow (B) CD4 T cells and (C) CD8 T cells from four individual donors with mean ± SEM. (D, E) Bar graphs show the fold change of proliferated (D) CD4 T cells and (E) CD8 T cells with mean ± SEM, in which the frequencies of IAV induced CD4 and CD8 T cell proliferation were divided by no virus conditions. (F, G) DCs from HLA-A2 positive donors with detectable IAV-specific CD8 T cell memory responses were generated and exposed to nothing or to 2 μg/mL WT, H84T or D133G for 1 hour, and then exposed to no IAV or 0.6 MOI IAV for 4 hours. Subsequently, CFSE labelled autologous CD8 T cells were added (1:10 ratio DC:T cells) and co-cultured for 7 days. IAV M1-specific CD8 T cells were identified with an HLA-A2 Influenza A M1 dextramer and analysed using flow cytometry. (F) Dot plots show live CD8 T cells from one representative donor. The CFSElow M1 Dextramer+ population (upper left quarter of the plots) represents proliferated IAV-specific CD8 T cells detected by IAV M1 Dextramer. Frequencies of CFSElow M1 Dextramer+ CD8 T cells out of total CD8 T cells are displayed. (G) Bar graphs show frequency of live CFSElow M1 Dextramer+ CD8 T cells from six individual donors with mean ± SEM. Statistical differences were assessed using RM one-way ANOVA with Tukey’s multiple comparisons test and considered significant at * p < 0.05 (** p < 0.01).
Fig 3.
H84T effectively limits IAV infection in human DCs, and consequently preserves DC viability but allows expression of viral antigen for presentation to T cells.
(A, B) DCs were exposed to nothing or 2 μg/mL WT, H84T or D133G for 1 hour, and then exposed to no IAV, or 0.6 MOI IAV with or without 20 mM NH4Cl for 24 hours to prevent viral fusion. (A) Dot plots show live DCs and numbers depict the frequency of IAV nucleoprotein (NP)+ DCs from one representative donor. (B) Graphs show frequency of IAV NP + DCs from five individual donors with mean ± SEM. (C–E) DCs were exposed to nothing or 2 μg/mL WT, H84T or D133G for 1 hour, then exposed to no IAV or 0.6 MOI IAV for 24, 48 or 72 hours. Viability of DCs was detected by Annexin V and PI staining using flow cytometry. (C) Dot plots show DCs and numbers depict the frequency of PI or Annexin V positive DCs from one representative donor out of four. (D) Graphs show frequency of necrotic (orange) and apoptotic (maroon) cells out of total cells after 24 hours of exposure to IAV in four donors with mean ± SEM. Paired t test was used to analyze the data and data considered significant at * p < 0.05 (** p < 0.01, *** p < 0.001). (E) Line graphs show the frequency of necrotic and apoptotic cells with mean ± SEM in four individuals. (F, G) IAV M1 protein in cell lysates from DCs exposed to nothing, 2ug/mL BanLec (WT, H84T or D133G) with or without 0.6 MOI IAV for 24 hours were determined by Western blot. Tubulin was used as the loading control. (F) One representative donor from four is shown. An overexposed blot is shown to highlight that, unlike in the BanLec WT treated cells, there is indeed a detectable band in H84T treated cells corresponding to IAV M1 protein. (G) Graph shows the analysis of IAV-M1 expression level by gray value in DCs exposed to replicating IAV using blots that were not overexposed. (H, I) DCs from HLA-A2 positive donors with detectable IAV-specific CD8 T cell memory responses were generated and exposed to nothing or to 2 μg/mL H84T or D133G BanLec for 1 hour, and then exposed to heat-inactivated IAV (HI IAV) or IAV M1 peptide (GILGFVFTL) for 4 hours. Subsequently, CFSE labelled autologous CD8 T cells were added and co-cultured for 7 days. IAV M1-specific CD8 T cells were identified with an HLA-A2 Influenza A M1 dextramer and analysed using flow cytometry. T cell proliferation was detected by CFSE dilution. Graph shows frequencies of live CFSElow M1 Dextramer+ CD8 T cells with (H) HI IAV and (I) IAV M1 peptide stimulation with mean ± SEM values from five individual donors. Dotted lines show the mean frequencies of live CFSElow M1 Dextramer+ CD8 T cells in DC-CD8 T cell co-culture without HI IAV or IAV M1 peptide stimulation. Statistical differences were assessed using the Friedman test with the Dunn’s multiple comparisons test and considered significant at * p < 0.05.
Fig 4.
H84T restores the capacity of DCs to initiate pathogen-specific CD8 T cell expansion against CMV antigen post replicating IAV infection.
(A) DCs differentiated from HLA-A2 + donors were pre-treated with nothing or H84T or D133G for 1h, and then exposed to IAV (0.6 MOI) or HI IAV (0.6 MOI) for 2h. Then the cells were washed and the recombinant cytomegalovirus (CMV) pp65 protein were added as a second source of antigen. After 4h incubation, DCs were washed and co-cultured with autologous CFSE labelled CD8 T cells for 10 days. CMV-specific CD8 T cells were identified with an HLA-A2-CMV pp65-dextramer and analysed using flow cytometry. (B) Dot plots show live CD8 T cells from one representative donor. The CFSElow CMV pp65 Dextramer+ population (upper left quarter of the plots) represents proliferated CMV-specific CD8 T cells. Frequencies of CFSElow CMV pp65 Dextramer+ CD8 T cells out of total CD8 T cells are displayed. Bar graphs show frequency of live CFSElow CMV pp65 Dextramer+ CD8 T cells in conditions (C) without BanLec, or in the presence of (D) D133G or (F) H84T from four individual donors with mean ± SEM. (E) Bar graphs show frequency of live CFSElow CMV pp65 Dextramer+ CD8 T cells in DC-CD8 T cell co-culture with replicating IAV exposure from four individual donors with mean ± SEM. (C-F) Dotted lines show the mean frequency of live CFSElow CMV pp65 Dextramer+ CD8 T cells in DC-CD8 T cell co-culture without CMV pp65 protein stimulation. (G) DCs differentiated from HLA-A2 + donors were pre-treated with nothing or H84T for 1h, and then exposed to nothing or IAV (0.6 MOI) for 2h. Then the cells were washed and the recombinant CMV pp65 protein added as a second source of antigen. The viability of the DCs was detected by Annexin V and PI staining using flow cytometry at 0, 2, 4, 6, 8, 10,12,16 and 24h post IAV infection. Line graph shows the frequency of live DCs out of total DCs from four individual donors with mean ± SEM. Statistical differences were assessed using RM one-way ANOVA with Tukey’s multiple comparisons test and considered significant at * p < 0.05 (** p < 0.01).