Immunological Tolerance to Muscle Autoantigens Involves Peripheral Deletion of Autoreactive CD8+ T Cells

Muscle potentially represents the most abundant source of autoantigens of the body and can be targeted by a variety of severe autoimmune diseases. Yet, the mechanisms of immunological tolerance toward muscle autoantigens remain mostly unknown. We investigated this issue in transgenic SM-Ova mice that express an ovalbumin (Ova) neo-autoantigen specifically in skeletal muscle. We previously reported that antigen specific CD4+ T cell are immunologically ignorant to endogenous Ova in this model but can be stimulated upon immunization. In contrast, Ova-specific CD8+ T cells were suspected to be either unresponsive to Ova challenge or functionally defective. We now extend our investigations on the mechanisms governing CD8+ tolerance in SM-Ova mice. We show herein that Ova-specific CD8+ T cells are not detected upon challenge with strongly immunogenic Ova vaccines even after depletion of regulatory T cells. Ova-specific CD8+ T cells from OT-I mice adoptively transferred to SM-Ova mice started to proliferate in vivo, acquired CD69 and PD-1 but subsequently down-regulated Bcl-2 and disappeared from the periphery, suggesting a mechanism of peripheral deletion. Peripheral deletion of endogenous Ova-specific cells was formally demonstrated in chimeric SM-Ova mice engrafted with bone marrow cells containing T cell precursors from OT-I TCR-transgenic mice. Thus, the present findings demonstrate that immunological tolerance to muscle autoantigens involves peripheral deletion of autoreactive CD8+ T cells

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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