Peripheral deletion of Ova-specific CD8⁺ can be evidenced in double transgenic [OT-I×SM-Ova]F1 mice after thymectomy.

<p>(<b>A</b>) Double transgenic [OT-I×SM-Ova]F1 and control OT-I mice were thymectomized at day 0. The total numbers of Vα2⁺Vβ5⁺CD8⁺ cells were determined from blood samples one day before thymectomy and 60 or 90 days after thymectomy. (<b>B</b>) Representative flow cytometry overlay profile illustrating the level of CD44 expression in peripheral blood cells from unmanipulated [OT-I×SM-Ova]F1 or OT-I control mice in the gated Vα2⁺Vβ5⁺CD8⁺ population. (<b>C</b>) Representative flow cytometry profiles and a scatter plot graph illustrating the percentage of CD44^highCD62L^high cells found in blood samples from unmanipulated [OT-I×SM-Ova]F1 or OT-I control mice in the gated Vα2⁺Vβ5⁺CD8⁺ population. Data are representative of 2 independent experiments with 8–9 mice per group.</p

Lack of detectable Ova-specific CD8⁺ T cell responses in immunized SM-Ova mice.

<p>(<b>A</b>) B6 or SM-Ova mice were immunized with rAAV-Ova, replicative VSV-Ova or live Lm-Ova. Seven days after infection with VSV-Ova or Lm-Ova, or 14 days after injection of rAAV-Ova, mice were inoculated with 1×10⁶ Ova-bearing EG-7 tumor cells and monitored during 30–40 days for tumor development. (<b>B</b>) In other groups of mice, animals were immunized with the same vaccines and splenocytes were analyzed 7 or 14 days after by flow cytometry to evaluate the percentage of CD8⁺ T cell recognizing the immunodominant peptides of Ova or VSV-associated nucleoprotein using H-2K^b/Ova_257–264 or H-2K^b/VSV pentamers staining, respectively. Representative flow cytometry profiles are shown and numbers indicate percentage of pentamer-positive cells in the CD8⁺-gated population. Background staining using H-2K^b/VSV pentamers were always below 0.3% of CD8⁺ cells as also shown for the staining using H-2K^b/Ova_257–264 pentamers (<b>C</b>) Bar graphs represent mean percentages of CD8⁺ T cells positively stained with the indicated H-2K^b/Ova_257–264 or H-2K^b/VSV pentamers in B6 (black bars) or SM-Ova (open bars). Data are representative of 3 independent experiments, each one performed with 5–7 mice per group.</p

Adoptively transferred OT-I CD8⁺ T cells are tolerized in SM-Ova recipient mice.

<p>Indicated numbers of purified CD8⁺ T cells from CD45.1⁺ OT-I mice were adoptively transferred into B6 or SM-Ova mice. Recipients were immunized one day later (<b>A</b>) or 3 weeks later (<b>B</b>) with Lm-Ova and splenocytes were collected, enumerated and analyzed 7 days after immunization by flow cytometry. Bar graphs show the total numbers of CD8⁺CD45.1⁺ cells per spleen and the mean percentage of cells expressing PD-1 in the CD8⁺CD45.1⁺ gated population. Representative flow cytometry overlay profiles are shown in (<b>B</b>), illustrating the intensity of PD-1 staining in the CD8⁺CD45.1⁺ gated cells recovered from B6 (black histograms) or SM-Ova mice (open histograms) when 10⁵ or 10⁴ OT-I cells were adoptively transferred. Negative staining controls obtained with an isotype-matched irrelevant antibody are also shown (dashed grey lines). Data are representative of 2 independent experiments each one performed with 5–8 mice per group.</p

Treg depletion or adoptive cell transfer to CD3^−/− mice is not sufficient to restore an Ova-specific CD8⁺ T cell response.

<p>(<b>A</b>) B6 (n = 3) and SM-Ova (n = 3) mice were injected i.p. at day −4 and day −1 with 250 µg of anti-CD25 (PC-61) antibody to inactivate endogenous Treg, or with PBS, followed by the injection of rAAV-Ova at day 0. Splenocytes from treated mice were collected 14 days after immunization and analyzed as before by flow cytometry for the presence of Ova-specific CD8⁺ cells using H-2K^b/Ova_257–264 pentamer staining. Representative FACS profiles and bar graphs are shown and numbers correspond to percentages of cells positively stained with the H-2K^b/Ova_257–264 pentamer in the gated CD8⁺ population. (<b>B</b>) 2×10⁷ purified CD8⁺ T cells originating from B6 or SM-Ova donor mice were adoptively transferred into syngenic CD3^−/− recipient mice (n = 6) together with 3×10⁷ purified CD4⁺ T cells originating exclusively from B6 donors. Recipient CD3^−/− mice were immunized with rAAV-Ova one day later and splenocytes were collected, enumerated and analyzed 14 days after by flow cytometry to evaluate the percentages and absolute numbers of Ova-specific CD8⁺ cells. Representative profiles gated on the CD8⁺ population are shown in the left panel and the total numbers of Ova-specific CD8⁺ per spleen are shown in the right bar graph.</p

Adoptively transferred OT-I CD8⁺ T cells are deleted in SM-Ova mice.

<p>(<b>A</b>) 5×10⁶ purified CD8⁺CD45.1⁺ OT-I cells were adoptively transferred into SM-Ova or control B6 mice. Recipients were euthanized 2 days or 11 weeks after to determine the percentages (indicated in the representative flow cytometry panels on top) and total numbers (represented below in the bar graphs) of CD8⁺CD45.1⁺ OT-I cells recovered from the LNs and spleen of recipient mice. (<b>B</b>) 5×10⁶ CFSE-labeled CD8⁺CD45.1⁺ OT-I cells were injected i.v. into SM-Ova or B6 control mice. Proliferation of gated CD8⁺CD45.1⁺ cells was evaluated in the LN and spleen of recipient mice by flow cytometry 7 or 15 days after their adoptive transfer. Representative profiles are shown and the indicated numbers correspond to the percentage of gated cells that have diluted CFSE upon cell division. Data are representative of 2 independent experiments performed with 10 mice per group.</p

Peripheral deletion of endogenously produced Ova-specific CD8⁺ T cells in chimeric SM-Ova mice.

<p>B6 (n = 8) and SM-Ova (n = 13) recipient mice were lethally irradiated and reconstituted one day after with bone marrow cells collected from B6 donor mice mixed with 10% bone marrow cells collected from CD45.1⁺ OT-I donor mice. (<b>A</b>) Left bar graph illustrates the percentages of cells in the CD4⁺, CD19⁺ or CD8⁺ gated populations expressing the CD45.1 marker in blood samples from B6 (black bars) or SM-Ova (open bars) recipient mice 6 weeks after bone marrow transplantation. Right bar graph illustrates the percentage of CD8⁺ PBL expressing the CD45.1 marker 6, 8 or 10 weeks after bone marrow transplantation. (<b>B</b>) Absolute numbers of CD8⁺ CD45.1⁺ OT-I T cells found in LN, spleen, blood and thymus of chimeric mice were enumerated and analyzed by flow cytometry 10 weeks after bone marrow engraftment. (<b>C, D</b>) Bar graphs show the percentages of cells expressing CD69, PD-1, CD44 or Ki-67 marker in the gated CD8⁺CD45.1⁺ population. (<b>E</b>) Representative flow cytometry overlay profiles illustrate the level of Bcl-2 expression in the gated CD8⁺CD45.1⁺ T cells population collected from the LN and spleen of B6 (dark histograms) or SM-Ova (open histograms) recipient mice 10 weeks after bone marrow transplantation. Isotype-matched negative staining controls are also shown (dashed grey lines). The corresponding bar graph illustrating the mean fluorescence intensities (MFI) of Bcl-2 expression is shown in the right panel.</p