Table 1.
Main cell populations fractions within non activated or PHA-activated human PBMCs at 24, 48 and 72 h after H-1 PV infection.
Figure 1.
PHA-activated PBMCs are affected by H-1 PV infection.
PBMCs were inoculated with increasing amounts of purified H-1 PV or mock-treated. MOI : multiplicity of infection, expressed as the number of plate-forming unit/cell; p.i : post-infection. A. CD8+ effector T cell, B lymphocyte and NK cell populations are affected by H-1 PV infection within PHA-activated PBMCs, whereas CD4+ helper T cells are not. Cells were collected 72 h p.i and labelled for appropriate cell surface antigens and the fraction of main lymphocyte populations within PBMCs was analyzed using flow cytometry. Results are expressed as percentages of total cells within PBMCs. B. PHA-activated PBMC proliferation ability is decreased upon H-1 PV infection. Cell proliferation was assessed by metabolic incorporation of tritiated thymidine into cellular DNA. Assays were performed at different times after H-1 PV inoculation but only 48 and 72 h p.i conditions are shown. Results from one representative experiment are represented as means of triplicate wells with ± standard deviation bars and expressed in count per minute (cpm). Statistical analysis for 4 independent experiments was performed using a Mann-Whitney test (* p<0,05; ** p<0,01 relative to Mock condition). C. PBMCs viability is decreased upon H-1 PV infection. Cell viability was evaluated using a test based on a bioluminescent reaction measuring the amount of ATP present in living cells. Assays were performed at different times after H-1 PV inoculation but only 48 and 72 h conditions are shown. Results from one representative experiment are represented as means of trilplicate wells with ± standard deviation bars and expressed in relative light unit (RLU). Statistical analysis for 4 independent experiments was performed using a Mann-Whitney test (* p<0,05 relative to Mock condition). Representative data from 4 independent experiments.
Table 2.
Main cell populations fractions within non activated or PHA-activated human PBMCs at 72 h after H-1 PV infection.
Figure 2.
H-1 PV exerts its effect on PHA-activated PBMCs in an NS1-dependent manner.
PBMCs were inoculated with increasing amounts of purified H-1 PV or mock-treated. MOI : multiplicity of infection, expressed as the number of plate-forming unit/cell; p.i : post-infection. A. H-1 PV alters PHA-activated PBMC morphology. For each condition, pictures of cells were taken in the course of infection but only those taken 48 h post-infection are shown. Scale bar = 100 µm. B. PHA-activated PBMCs are lysed upon H-1 PV infection. H-1 PV cytotoxicity was assessed using a test based on a bioluminescent reaction measuring the leak of a cellular marker from cytoplasm to culture medium, which reveals the loss of plasma membrane integrity. Assays were performed at different times after H-1 PV inoculation but only 48 and 72 h conditions are shown. Results are represented as means of triplicate wells with ± standard deviation bars and expressed in relative light unit (RLU). C. PHA-activated PBMC death upon H-1 PV infection is related to NS1 protein expression but does not depend on caspase 3 activation. Non activated (NA) and activated (A) PBMC total protein extracts were prepared in the course of infection. Equal amounts of proteins were separated by 4–12% SDS-PAGE and analyzed by Western blot for the presence of NS1 protein, cleaved caspase 3 and both full-length and cleaved PARP. β-actin was used as a loading control. D. PBMCs do not support an H-1 PV productive infection. Both PBMCs and culture supernatants were collected and tested for the presence of infectious viral particles using the Tissue Culture Infectious Dose 50 method. The graph represents the time course of both released and non-released virions amounts which were added and expressed in plaque forming unit (pfu). Representative data from 3 independent experiments.
Figure 3.
PHA-activated PBMCs are overactivated during H-1 PV infection and secrete Th1 and Th2 type cytokines.
PBMCs were inoculated with increasing amounts of purified H-1 PV or mock-treated. MOI : multiplicity of infection, expressed as the number of plate-forming unit/cell. A. Early and late activation marker expression is increased at the surface of PHA-activated PBMCs upon H-1 PV infection. In the course of infection, cells were collected, labelled for the expression of early (CD69) and late (CD30) activation markers and analyzed using flow cytometry. Results are expressed as percentages of activated cells within T lymphocytes (CD3+ cells). B. H-1 PV enhances the ability of PHA-activated PBMCs to secrete Th1 and Th2 type cytokines. In the course of infection, culture supernatants were collected and tested for cytokine detection by enzyme-linked immunosorbent assay (ELISA). Results are expressed in pg/ml as means of duplicate wells and after subtraction of the background value. Statistical analysis for 3 independent experiments was performed using a Mann-Whitney test (* p<0,05; ** p<0,01 relative to Mock condition). Representative data from 3 independent experiments.
Figure 4.
Unlike PBMCs, CD4+ T cells do not undergo any major impairment upon H-1 PV infection.
CD4+ T cells were inoculated with increasing amounts of H-1 PV or mock-treated. MOI : multiplicity of infection, expressed as the number of plate-forming unit/cell; p.i : post-infection. A. CD4+ T cell proliferation ability is not altered upon H-1 PV infection. Cell proliferation was assessed by metabolic incorporation of tritiated thymidine into cellular DNA. Only 48 and 72 h p.i conditions are shown. Results are represented as means of triplicate wells with ± standard deviation bars and expressed in count per minute (cpm). B. H-1 PV does not affect CD4+ T cell viability. Cell viability was evaluated using a test based on a bioluminescent reaction measuring the amount of ATP present in living cells. Only 48 and 72 h p.i conditions are shown. Results are represented as means of triplicate wells with ± standard deviation bars and expressed in relative light unit (RLU). C. Infection of CD4 T cells by H-1 PV is not related to any morphological changes. For each condition, pictures of cells were taken in the course of infection but only those taken 48 h after H-1 PV inoculation are shown. Scale bar = 100 µm. D. H-1 PV does not modify CD4+ T cell basal lysis. H-1 PV cytotoxicity was assessed using a test based on a bioluminescent reaction measuring the leak of a cellular marker from cytoplasm to culture medium, which reveals the loss of plasma membrane integrity. Results are represented as means of triplicate wells with ± standard deviation bars and expressed in relative light unit (RLU). E. CD4+ T cells transiently express a slight amount of NS1 protein. Equal amounts of total CD4+ T cell proteins were separated by 4–12% SDS-PAGE and analyzed by Western blot for the presence of NS1 protein. Erk2 was used as a loading control. Representative data from 2 independent experiments.
Figure 5.
H-1 PV preserves or even enhances non-activated and activated CD4+ T cell secretion ability.
CD4+ T cells were inoculated with increasing amounts of purified H-1 PV or mock-treated as described in Materials and Methods section. MOI : multiplicity of infection, expressed as the number of plate-forming unit/cell; p.i : post-infection. A. Early and late activation marker expression is increased at the surface of CD4+ T cells upon H-1 PV infection. In the course of infection, cells were collected, labelled for the expression of early (CD69) and late (CD30) activation markers and analyzed using flow cytometry. Results are expressed as percentages of activated cells within CD4+ T cell population. B. H-1 PV enhances CD4+ T cell ability to secrete Th1 and Th2 type cytokines. In the course of infection, culture supernatants were collected and tested for cytokine detection by enzyme-linked immunosorbent assay (ELISA). Results are expressed in pg/ml as the mean of duplicate wells and after subtraction of the background value. Statistical analysis for 2 independent experiments was performed using a Mann-Whitney test (* p<0,05 relative to Mock condition). Representative data from 2 independent experiments.
Figure 6.
H-1 PV does not affect neutrophils viability.
Human neutrophils cells were inoculated with increasing amounts of purified H-1 PV or mock-treated. MOI : multiplicity of infection, expressed as the number of plate-forming unit/cell; p.i : post-infection. C. Infection of Neutrophils by H-1 PV is not related to any morphological changes. For each condition, pictures of cells were taken in the course of infection but only those taken 48 h after H-1 PV inoculation are shown. Scale bar = 100 µm. B. H-1 PV does not affect Neutrophils viability. Cell viability was evaluated using a test based on a bioluminescent reaction measuring the amount of ATP present in living cells. 6 h, 18 h, 24 h, 48 h and 72 h p.i conditions are shown. Results are represented as means of triplicate wells with ± standard deviation bars and expressed in relative light unit (RLU). Representative data from 5 independent experiments.
Figure 7.
H-1 PV affects neither Treg cell phenotype nor viability while inhibiting their suppressive activity.
Treg cells were inoculated with increasing amounts of purified H-1 PV or mock-treated. MOI : multiplicity of infection, expressed as the number of plate-forming unit/cell; p.i : post-infection. A. Control of Treg cell purity. Cells were labelled for Treg cell markers and analyzed using flow cytometry. Results are expressed as percentages of CD4+CD25+, CD4+CD25+CD127- and CD4+CD25+FoxP3+ cells within the whole cell population. B. Treg cells do not undergo any major phenotypical changes upon infection. Cells were labelled for Treg cell markers and analyzed using flow cytometry 48 h p.i. Results are expressed as percentages of CD4+CD25+ and CD4+CD25+CD127- cells within the whole cell population. C. H-1 PV does not affect Treg cell viability. Cell viability was assessed 24 and 48 h p.i using a bioluminescent test measuring ATP amount in living cells. Results are represented as means of triplicate wells with ± standard deviation bars and expressed in relative light unit (RLU). D. Ex vivo Treg cells are able to suppress autologous activated PBMCs proliferation. Autologous PBMCs were activated (anti-CD3 and -CD28 antibodies) and cultured with increasing quantities of Treg cells. PBMCs proliferation was assessed by metabolic incorporation of tritiated thymidine into cellular DNA 48 h p.i. Results are represented as means of triplicate wells with ± standard deviation bars and expressed in count per minute (cpm). The black arrow indicates the ratio chosen for further experiments. E. H-1 PV is able to inhibit Treg cell suppressive activity. Autologous PBMCs were activated (anti-CD3 and -CD28 antibodies), cultured with Treg cells (4 PBMCs for 2 Treg cells) and mock- or H-1PV-treated. PBMC proliferation was assessed by metabolic incorporation of tritiated thymidine into cellular DNA 48 h p.i. Results are represented as means of triplicate wells with ± standard deviation bars and expressed in count per minute (cpm). Representative data from 3 independent experiments.
Figure 8.
H-1 PV is able to slow down and stabilize the growth of nasopharyngeal carcinoma in humanized SCID mice.
Immunodeficient SCID mice (Severe combined Immunodeficiency) were subcutaneously xenotransplanted with tumors of nasopharyngeal carcinoma (NPC) induced by C15 cells (human primary tumor cells of NPC). A. Timeline which summarizes the experimental protocol followed. After development of visible tumors, SCID mice were splenectomized and reconstituted (i.p) or not with 50.106 human PBMC. At the same moment they also received or not H-1PV directly injected within the tumor. SCID mice received only one injection of H-1 PV throughout the protocol. B. Results are presented in Tumor Volume Index versus days post-treatment. There is a progressive increase in tumor size for the four groups of mice. Only the groups, which did not receive H-1 PV, saw their tumors grow to 6–7 times the original size. H-1 PV stabilize the growth of nasopharyngeal carcinoma in humanized SCID mice. In two groups of mice that received H-1 PV, the growth of the tumor does not exceed a maximum of three times the size of the primary tumor. The size of these tumors stabilized to stay below the maximum threshold of 3. Representative data from 2 independent experiments.