Blocking of PDL-1 interaction enhances primary and secondary CD8 T cell response to herpes simplex virus-1 infection.

The blocking of programmed death ligand-1 (PDL-1) has been shown to enhance virus-specific CD8 T cell function during chronic viral infections. Though, how PDL-1 blocking at the time of priming affects the quality of CD8 T cell response to acute infections is not well understood and remains controversial. This report demonstrates that the magnitude of the primary and secondary CD8 T cell responses to herpes simplex virus-1 (HSV-1) infection is subject to control by PDL-1. Our results showed that after footpad HSV-1 infection, PD-1 expression increases on immunodominant SSIEFARL peptide specific CD8 T cells. Additionally, post-infection, the level of PDL-1 expression also increases on CD11c+ dendritic cells. Intraperitoneal administration of anti-PDL-1 monoclonal antibody given one day prior to and three days after cutaneous HSV-1 infection, resulted in a marked increase in effector and memory CD8 T cell response to SSIEFARL peptide. This was shown by measuring the quantity and quality of SSIEFARL-specific CD8 T cells by making use of ex-vivo assays that determine antigen specific CD8 T cell function, such as intracellular cytokine assay, degranulation assay to measure cytotoxicity and viral clearance. Our results are discussed in terms of the beneficial effects of blocking PDL-1 interactions, while giving prophylactic vaccines, to generate a more effective CD8 T cell response to viral infection

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Anti-PDL-1 antibody treatment at the time of priming enhances secondary SSIEFARL specific CD8 T cell response.

<p>C57BL/6 mice were intra-peritoneally administered with either anti-PDL-1 or Isotype matched antibody on -1 and 3day post footpad HSV-1 infection. Thirty-two days post-infection both groups of mice were intra-peritoneally challenged with 1.5×10⁶ p.f.u of vaccinia virus encoding gB_498–505 peptide. After 4 days of challenge, mice were euthanized and single cell suspension obtained from the spleen and peripheral blood of control and anti-PDL-1 antibody treated mice were used for flow-cytometeric studies. A, Representative FACS plots and bar graphs demonstrate the percentage of SSIEFARL Tetramer+ CD8 T cells in the spleen and peripheral blood of control and anti-PDL-1 treated groups of mice. B, FACS plots and bar graphs represent SSIEFARL specific IFN-γ producing memory CD8 T cells as determined by <i>ex-vivo</i> stimulation of the splenocyte and peripheral blood mononuclear cells (PBMC) obtained from control and anti-PDL-1 treated groups with gB_498–505 peptide (10 µg/ml) for 6 h in presence of Brefeldin A. C, Representative FACS plots and bar graphs demonstrate the percentage of granzyme B expressing memory CD8 T cells in spleen and PBMC of control and anti-PDL-1 treated mice post-challenge. D, Histogram FACS plots, derived from gated CD8 T cell, and bar diagram represent the proportion of CD107a/b expressing memory CD8 T cells in spleen and peripheral blood of control and anti-PDL-1 treated groups of mice. Data shown is derived from two similar experiments with a total of five mice per group in each experiment. Statistical significance was determined by unpaired student’s t test where *p<0.05 and **p<0.01 are considered statistically significant.</p

Kinetics of PDL-1 expression on CD11c+ dendritic cells derived from popliteal lymph node (PLN) of HSV-1 infected mice.

<p>A, A representative histogram overlay, obtained from gating on CD11c+ cells, denotes the expression of PDL-1 on CD11c+ dendritic cells in PLN at different time-points post-infection. Naïve represents level of PDL-1 expression on CD11c+ DCs in the PLN of uninfected mice. B, Bar graph shows mean ± S.D. of PDL-1 mean fluorescence intensity (MFI) on CD11c+ DCs in naive and infected mice at different time-points post-infection. Data shown is derived from four mice at each time-point.</p

Anti-PDL-1 antibody treatment increases the frequency and absolute numbers of SSIEFARL specific CD8 T cell in PLN and spleen tissue of HSV-1 infected mice.

<p>Anti-PDL-1 or Isotype matched antibody was intra-peritoneally administered on -1 and 3 days post footpad HSV-1 infection. Both groups of mice were euthanized on day 6 post-infection and single cell suspensions from PLN and spleen tissue were cell surface stained with gB_498–505 tetramer to detect immunodominant SSIEFARL peptide specific CD8 T cells. The representative FACS plots are gated on CD8 T cells and demonstrate the proportion of SSIEFARL specific CD8 T cells in isotype and anti-PDL-1 treated mice in PLN and spleen, respectively. The scatter plots represent average percentage and the bar diagram shows the average absolute numbers of SSIEFARL specific CD8 T cells in PLN and spleen tissue of isotype and anti-PDL-1 antibody treated groups of mice. Data shown is the representative of two independent experiments including 4mice/experiment. Statistical significance was determined by unpaired students t test with *P<0.05, **P<0.01 and ***P<0.0001 were considered statistically significant.</p

Granzyme B secreting potential of PD-1^high and PD-1^medium CD8 T cells in spleen during lytic phase of HSV-1 infection.

<p><i>Ex-vivo</i> cytotoxicity of splenic PD-1^high and PD-1^medium CD8 T cells on day 6 post-infection was compared after stimulating with SSIEFARL peptide as described in the methods. A, The representative FACS plot denotes the ratio of PD-1^high (2.88), PD-1^medium (7.54) and PD-1^negative (88.6) splenic CD8 T cells on day 6 post-infection. The frequencies of cell surface expressed CD107a/b by PD-1^high and PD-1^medium CD8 T cells are shown in second column. B, The bar diagram shows the mean ± S.D. of the frequency of cell surface expressing CD107a/b PD-1^high and PD-1^medium CD8 T cells from five mice. Statistical significance was determined with unpaired student’s t test where ***p<0.0001 were considered statistically significant.</p

Cytokine producing ability of PD-1^high and PD-1^medium CD8 T cells during lytic phase of HSV-1 infection.

<p>Mice infected with HSV-1 were euthanized on day 6 post-infection. PLN and spleen tissue obtained were homogenized to obtain singe cell suspension. CD8 T cells were stimulated with immunodominant SSIEFARL peptide and pro-inflammatory cytokine producing PD-1^high and PD-1^medium CD8 T cells were determined by flow cytometery as described in the methods. A and B, Representative FACS plots gated on CD8 T cell and bar diagrams denote the proportion of IFN-γ, TNF-α and IL-2 producing PD-1^medium and PD-1^high CD8 T cells in PLN and spleen on day 6 post-infection, respectively. Data shown is representative of two similar experiments with five mice in each group/experiment. Statistical significance was determined with unpaired student’s t test where *p<0.05, **p<0.01 and ***p<0.0001 were considered significant.</p

Anti-PDL-1 treatment increases the cytotoxic potential of HSV-1 specific effector CD8 T cell in the PLN and spleen tissue during lytic phase of HSV-1 infection.

<p>Mice from isotype and anti-PDL1 treated groups were euthanized on day 6 post-infection. Single cell suspensions obtained from PLN and spleen tissue were <i>ex-vivo</i> stimulated with SSIEFARL peptide in the presence of fluorochrome conjugated anti-CD107a/b antibodies followed by intra-cellular staining for granzyme B molecule. A, Representative FACS plots and scatter plots denotes the proportion of granzyme B expressing CD8 T cells in PLN and spleen tissue of control and anti-PDL1 treated groups on day 6 post-infection. B, Histogram FACS plots, derived from gated CD8 T cell, and the bar diagram demonstrate the frequency of CD8 T cells expressing membrane bound CD107a/b in PLN and spleen tissue of control and anti-PDL1 treated groups of mice. Data shown is derived from two similar experiments with four mice per group in each experiment. Statistical significance was determined by unpaired two-tailed student’s t test where *p<0.05 and **p<0.01 are considered statistically significant.</p

Kinetics of PD-1 expression on gB_498–505 (SSIEFARL) peptide specific CD8 T cells in PLN and spleen tissue of HSV-1 infected mice.

<p>C57BL/6 mice were infected with 1×10⁶ p.f.u of HSV-1 (KOS) in the hind footpad. Mice were euthanized on day 4, 6, 8 and 10 post-infection. PD-1 expression on gB_498–505 tetramer+ CD8 T cells was determined in the PLN and spleen by flow cytometery at each time-point. A, The representative FACS plots gated on CD8 T cell demonstrate the frequency of tetramer+ PD-1+ cells in PLN and spleen tissue at different time-points post-infection. PD-1^high and PD-1^medium expression on SSIEFARL specific CD8 T cells are shown at day 6 post-infection. B and C, The bar graphs represent the average percentage and absolute numbers of SSIEFARL tetramer+ CD8 T cells expressing PD-1 in PLN and spleen tissue of HSV-infected mice on different days post-infection. Data shown is derived from four mice at each time-point post-infection.</p

Anti-PDL-1 antibody treatment increases the proportion of IL-2, IFN-γ and TNF-α producing SSIEFARL specific effector CD8 T cells.

<p>Mice from anti-PDL-1 or isotype treated groups were euthanized on day 6 post infection. Single cell suspensions obtained from PLN and spleen tissue were stained intra-cellularly for pro-inflammatory cytokines after brief <i>in vitro</i> stimulation with SSIEFARL peptide (10 µg/ml). The representative FACS plots in A, B and C demonstrates the proportion of IFN-γ, TNF-α and IL-2 secreting CD8 T cells, respectively, in PLN and spleen tissue derived from isotype and anti-PDL-1 treated mouse. Scatter plots in panel A, B and C show mouse to mouse variation in the frequency of IFN-γ, TNF-α and IL-2 producing CD8 T cell in PLN and spleen tissue of isotype and anti-PDL-1 treated groups of mice. Data shown is derived from two similar experiments with four mice per group in each experiment. Statistical significance was determined by unpaired student’s t test where *p<0.05 and **p<0.01 are considered statistically significant.</p