Figure 1.
Decreased number of antigen-specific CD8+ T cells in Btla−/− mice after LM-OVA infection.
A- WT and Btla−/− mice were orally infected with 1×109 LM-OVA and 7 days p.i., OVA-specific CD8+ T cells were detected by staining spleen cells with OVA-tetramer (Kb/OVA257–264). The percentage and absolute number of OVA-tetramer+ CD8+ T cells found in WT (circles and open bars) and Btla−/− (squares and filled bars) mice are shown. Each symbol represents an individual mouse from a total of 11–14 mice per group, pooled from three independent experiments. B- IFNγ production by CD8+ T cells isolated from WT and Btla−/− mice, 7 days p.i., was calculated by intracellular staining after ex vivo stimulation of the cells with OVA257–264 peptide. The percentage and absolute number of IFNγ+CD8+ T cells is shown. Data shown correspond to the average of 4–6 mice per group and are representative of three independent experiments. C- OVA-tetramer staining in MLN cells and IEL isolated from LM-OVA infected animals, 7 days p.i. Data pooled from three independent experiments are shown. D- Reduced number of memory CD8+ T cells in Btla−/− mice. The percentage and absolute number of OVA-specific memory CD8+ T cells in the spleen of LM-OVA-infected animals was calculated by staining cells with OVA-tetramers, 70 days p.i. E- IFNγ intracellular staining performed on CD8+ T cells isolated from infected mice 70 days p.i., restimulated ex vivo with OVA257–264 peptide. Data shown correspond to the average of 4 mice per group and are representative of three independent experiments.
Figure 2.
BTLA expression on OT1 cells is not required for their accumulation after LM-OVA infection.
Purified naïve WT or Btla−/− OT1 cells were adoptively transferred into CD45.1+ recipient mice and one day post transfer, mice were orally infected with LM-OVA as indicated in Material and Methods. A- The percentage (top panel) and absolute number (bottom panel) of donor WT and Btla−/− OT1 cells in the peripheral blood of infected mice was tracked at different time points after infection. OT1 cell numbers in the peripheral blood were calculated as numbers of OT1 cells per 3×104 peripheral blood lymphocytes (PBL). Data shown correspond to the average of 6–8 mice per group. B- Percentages of CD45.2+ WT and Btla−/− OT1 cells in the spleen, MLN and IEL of infected mice, 7 days p.i. Each symbol represents an individual mouse from a total of 7–9 mice per group, pooled from two independent experiments. C- Absolute number of WT (open bars) and Btla−/− (filled bars) OT1 cells in the spleen and MLN of infected animals at 7 days p.i. Data shown correspond to the average of the number of animal indicated in B. D- Percentage of OT1 memory cells in the spleen and IEL of WT (open bars) and Btla−/− (filled bars) mice, 70 days p.i. Absolute number of OT1 cells in the spleen of these animals is also represented. Differences between percentage and absolute numbers of WT and Btla−/− OT1 are not statistically significant. Data shown was pooled from two independent experiments with total of 7–9 mice per group, and E, F- Co-transfer experiment with 5×104 CD45.1+CD45.2+ WT and CD45.2+ Btla−/− naïve OT1 cells transferred into CD45.1+ recipient mice that were subsequently infected with LM-OVA. Data shown correspond to the percentage of WT (circles) and Btla−/− (squares) cells within the total OT1 cells found in the spleen and IEL of infected mice, 7 days (E) and 70 days (F) p.i. Each symbol represents an individual mouse from a total of 9 mice per group, pooled from two independent experiments.
Figure 3.
BTLA expression in the host environment is required for expansion/survival of CD8 T cells.
5×104 purified naïve CD45.1+ WT CD8+ OT-1 cells were adoptively transferred into WT or Btla−/− recipient mice. One day after transfer, mice were orally infected with 1×109 LM-OVA. A- The percentage and absolute number of donor CD45.1+ OT1 cells in the peripheral blood of WT (circles and open bars) and Btla−/− (squares and filled bars) hosts was tracked at different time points following infection. OT1 numbers in the peripheral blood were calculated as indicated in figure 2A. Percentages (B) and absolute numbers (C) of CD45.1+ OT1 cells in WT and Btla−/− recipient mice, 7 days p.i. Data pooled from three independent experiments is shown. D- Percentage and absolute number of memory OT1 cells in the spleen and MLN of WT and Btla−/− recipient mice, 70 days p.i. Data pooled from two independent experiments is shown. E- Co-transfer experiments with WT (CD45.1+CD45.2+) and Btla−/− (CD45.2+) naïve OT1 cells transferred into congenic CD45.1+ WT or Btla−/− recipients. The percentage and absolute number of CD45.2+ WT or Btla−/− cells within total OT1 cells found in the spleen of transferred recipients measured 70 days p.i., is shown. F- Percentage (left) and absolute number (right) of total OT1 cells (CD45.2+) in the spleen, MLN and IEL of co-transferred WT and BTLA-deficient recipient mice, 70 days after LM-OVA infection.
Figure 4.
Reduced accumulation of antigen-specific CD8+ T cells in Hvem−/− mice.
WT or Hvem−/− mice were orally infected with 1×109 LM-OVA and the amount of antigen-specific CD8+ T cells was measured. A- Percentage and absolute number of OVA-specific CD8+ T cells in splenocytes isolated from WT or Hvem−/− mice, 7 days p.i. B- IFNγ production by WT or Hvem−/− splenic CD8+ T cells after ex vivo restimulation with OVA257–264 peptide. The percentage of OVA-specific CD8+ T cells in the MLN (C) and IEL (D) of WT and Hvem−/− mice was also calculated, 7 days after infection. Data shown correspond to 6–9 mice per group, pooled from two independent experiments. E- Percentage and absolute number of memory OVA-specific CD8+ T cells in the spleen of WT and Hvem−/− mice, 70 days p.i. F- IFNγ production by OVA-specific memory CD8+ T cells isolated from the spleen of WT and Hvem−/− mice 70 days p.i. and restimulated ex vivo with OVA-peptide. Data were pooled from at least two independent experiments, each one performed with 5–7 mice per group.
Figure 5.
Hvem expression in OT1 cells is required for their accumulation after LM-OVA infection.
5×104 purified naïve WT or Hvem−/− OT1 cells were adoptively transferred into CD45.1+ recipient mice. One day after the transfer, mice were orally infected with 1×109 LM-OVA and the amount of transferred OT1 cells was monitored at 7 or 70 days p.i. The percentage of CD45.2+ WT and Hvem−/− effector OT1 cells in the spleen, MLN and IEL of the recipient animals (A), as well as the absolute number of these cells in the spleen and MLN of infected mice (B), were measured 7 days p.i. Data shown correspond to 7 mice per group, pooled from two independent experiments. The percentage (C) and absolute number (D) of WT and Hvem−/− memory OT1 cells in the recipient mice were also calculated, 70 days after LM-OVA infection. Data shown are representative of two independent experiments. E-F- Co transfer experiments with equal numbers of naïve WT (CD45.1+CD45.2+) and Hvem−/− (CD45.2+) OT1 cells injected into CD45.1+ recipient mice. One day after co-transfer, mice were orally infected with LM-OVA and the percentage of WT and Hvem−/− cells gated on total OT1 cells, as well as the absolute number of both OT1 cell populations was calculated at 7 days (E) and 70 days (F) following infection. Data shown are representative of at least two independent experiments.
Figure 6.
HVEM promotes the survival of OT1 cells after LM-OVA infection.
Purified naïve WT or Hvem−/− CD8+ OT1 cells were adoptively transferred into CD45.1+ recipient mice and one day later, mice were orally infected with LM-OVA. A- HVEM expression on naïve OT1 cells and OT1 cells isolated from peripheral blood of LM-OVA infected mice was assessed at the indicated times after infection. To analyze the early expansion of WT and Hvem−/− CD8+ T cells in mice infected with LM-OVA, 5×105 CFSE-labeled WT or Hvem−/− OT1 cells were transferred into CD45.1+ recipients and the percentage of OVA-specific T cells in the MLN of the hosts, 4 days p.i., was analyzed (B). Proliferation of WT and Hvem−/− OT1 cells in the MLN of infected mice was assessed by CFSE dilution profiles (C). Histogram shows the percentage of WT (open bar) and Hvem−/− (filled bar) OT1 cells undergoing more than two cell divisions. The accumulation of activated CD8+ effector T cells was also monitored at day 5 p.i., by measuring the percentage and absolute number of WT and Hvem−/− OT1 cells in the spleen and MLN of LM-OVA-infected animals (D). The ability of WT and Hvem−/− OT1 cells to expand and contract following LM-OVA infection was determined by tracking the percentage and absolute number of the CD45.2+ OT1 cells in the spleen of infected mice at different time points after infection (E). The proliferation of WT and Hvem−/− OT1 cells in the spleen of infected mice at the peak of the immune response was evaluated by measuring the intracellular levels of the proliferation marker Ki-67 (F).
Figure 7.
BTLA promotes the long-term survival of activated CD8+ T cells through its interaction with HVEM on T cells.
WT mice were injected intraperitoneally with an anti-BTLA blocking antibody (clone 6A6) or isotype control (IgG1), one day before and 2 days after oral infection with 1×109 LM-OVA. A,B- 7 days p.i., endogenous OVA-specific CD8+ effector T cell response was assessed by OVA-tetramer staining (A) and IFNγ intracellular staining after ex vivo stimulation with OVA257–264 peptide (B). C- The percentage and absolute number of endogenous OVA-specific memory CD8+ T cells in the spleen of mice treated with the anti-BTLA mAb as indicated above, were monitored 70 days post LM-OVA infection. Data shown are representative of two independent experiments. D- To assess the effect of blocking BTLA-HVEM interaction at the memory phase of the immune response, WT mice were orally infected with LM-OVA were treated with the anti-BTLA mAb at days 20, 27, 30 and 34 p.i. Endogenous OVA-specific CD8+ memory T cells were measured in the spleen and IEL of the animals, 40 days p.i. Data shown correspond to the average of 8 mice per group pooled from two independent experiments. E,F- To rule out that the anti-BTLA antibody works by engaging BTLA expressed on T cells, WT or Btla−/− OT1 cells were adoptively transferred into CD45.1+ WT mice that were treated with the anti-BTLA mAb as described in A. 7 days after LM-OVA infection, the percentage (E) and absolute number (F) of CD45.2+ OT1 cells in the recipients were measured. Data shown was pooled from two independent experiments. G,H- Co-transfer experiment with equal numbers of CD45.1+CD45.2+ WT and CD45.2+ Hvem−/− OT1 cells adoptively transferred into CD45.1+ WT or CD45.1+ Btla−/− recipient mice. The percentage of memory WT and Hvem−/− cells within total OT1 cells in the spleen of the recipients was measured 70 days p.i. (G). The ratio of WT vs. Hvem−/− memory OT1 cells in the different recipients was calculated (H). Data shown correspond to 7 mice per group, pooled from two independent experiments.