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Visualizing early splenic memory CD8+ T cells reactivation against intracellular bacteria in the mouse.

Bajénoff M, Narni-Mancinelli E, Brau F, Lauvau G - PLoS ONE (2010)

Bottom Line: Memory CD8(+) T cells are endowed with enhanced antimicrobial effector functions that perfectly tail them to rapidly eradicate invading pathogens.Within these clusters that only last few hours, memory CD8(+) T produce inflammatory cytokines such as IFN-gamma and CCL3 nearby infected myeloid cells known to be crucial for L.m killing.Altogether, we describe how memory CD8(+) T cells trafficking properties and the splenic micro-anatomy conjugate to create a spatio-temporal window during which memory CD8(+) T cells provide a local response by secreting effector molecules around infected cells.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale Unité 924, Groupe Avenir, Valbonne, France. bajenoff@ciml.univ-mrs.fr

ABSTRACT
Memory CD8(+) T cells represent an important effector arm of the immune response in maintaining long-lived protective immunity against viruses and some intracellular bacteria such as Listeria monocytogenes (L.m). Memory CD8(+) T cells are endowed with enhanced antimicrobial effector functions that perfectly tail them to rapidly eradicate invading pathogens. It is largely accepted that these functions are sufficient to explain how memory CD8(+) T cells can mediate rapid protection. However, it is important to point out that such improved functional features would be useless if memory cells were unable to rapidly find the pathogen loaded/infected cells within the infected organ. Growing evidences suggest that the anatomy of secondary lymphoid organs (SLOs) fosters the cellular interactions required to initiate naive adaptive immune responses. However, very little is known on how the SLOs structures regulate memory immune responses. Using Listeria monocytogenes (L.m) as a murine infection model and imaging techniques, we have investigated if and how the architecture of the spleen plays a role in the reactivation of memory CD8(+) T cells and the subsequent control of L.m growth. We observed that in the mouse, memory CD8(+) T cells start to control L.m burden 6 hours after the challenge infection. At this very early time point, L.m-specific and non-specific memory CD8(+) T cells localize in the splenic red pulp and form clusters around L.m infected cells while naïve CD8(+) T cells remain in the white pulp. Within these clusters that only last few hours, memory CD8(+) T produce inflammatory cytokines such as IFN-gamma and CCL3 nearby infected myeloid cells known to be crucial for L.m killing. Altogether, we describe how memory CD8(+) T cells trafficking properties and the splenic micro-anatomy conjugate to create a spatio-temporal window during which memory CD8(+) T cells provide a local response by secreting effector molecules around infected cells.

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Memory ‘CD8 clusters’ concentrate anti-listeria effector cells.(A,C,D,E) 200 naïve GFP+ OT-I cells were transferred in C57BL/6 mice. One day after, recipient mice as well as naive GFP+ lys-M Tg mice (B) were injected i.v. with 0.1×LD50 Wt L.m-OVA. One month later, all mice were injected i.v. with 10×LD50 Wt L.m-OVA. Spleens were harvested 6 (A,B,C,D) or 12 hrs later (E), sectioned, stained with anti-B220, -CD4, -CD8 (A); anti-CD3, -collagen IV (B) anti-CD11c, -listeria (C); anti-IFN-γ, and hoescht (D), anti-iNOS (E) specific Abs and analyzed by confocal microscopy. In (D), IFN-γ secretion by endogenous CD8+ T cells and GFP+ OT-I memory cells was also assessed by flow cytometry. Data are representative of 3 independent experiments.
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pone-0011524-g006: Memory ‘CD8 clusters’ concentrate anti-listeria effector cells.(A,C,D,E) 200 naïve GFP+ OT-I cells were transferred in C57BL/6 mice. One day after, recipient mice as well as naive GFP+ lys-M Tg mice (B) were injected i.v. with 0.1×LD50 Wt L.m-OVA. One month later, all mice were injected i.v. with 10×LD50 Wt L.m-OVA. Spleens were harvested 6 (A,B,C,D) or 12 hrs later (E), sectioned, stained with anti-B220, -CD4, -CD8 (A); anti-CD3, -collagen IV (B) anti-CD11c, -listeria (C); anti-IFN-γ, and hoescht (D), anti-iNOS (E) specific Abs and analyzed by confocal microscopy. In (D), IFN-γ secretion by endogenous CD8+ T cells and GFP+ OT-I memory cells was also assessed by flow cytometry. Data are representative of 3 independent experiments.

Mentions: We next asked whether such early clustering of memory OT-I cells may be related to the fast control of bacterial growth. We previously demonstrated that reactivated L.m-specific CD8+ T cells rapidly secrete CCL3, a chemokine that promotes the antimicrobial oxidative burst production in innate immune cells, an effector function required for secondary protection against L.m [28]. Others have also shown that memory CD8+ T cells rapidly secrete IFN-γ and that such secretion partially contributes to the protective recall response [29], [30]. Finally, several studies have demonstrated that innate cells such as neutrophils and inflammatory monocytes expressing the myeloid marker Ly-6C -also defined as TNF/NO-producing DCs (Tip-DCs)- produce antimicrobial mediators known to be critical for pathogens clearance [31], [32]. Therefore, we characterized the composition of the OT-I clusters induced 6 hours after the challenge infection and looked for the presence of the innate immune cell types and effector functions described above. Spleens sections from Wt L.m-OVA challenged mice (6 h) were stained for the expression of B220, CD8, CD4 (Figure 6A); CD11c, L.m antigens (Figure 6C) and CD8, IFN-γ (Figure 6D). Attention was focused on the clusters of memory OT-I cells. Data show that these clusters were massive (>100 µm), composed of both CD4+ and CD8+ T cells (but not B cells) and were usually found around L.m-infected CD11c+ cells, similar to what was recently described during the secondary challenge infection against Toxoplasma gondii in LNs [18]. Most interestingly, we observed both on tissue sections and by flow cytometry an intense IFN-γ secretion by memory OT-I cells in these clusters that we therefore named “effector clusters” (Figure 6D). Although we were unable to detect CCL3 on sections, likely due to a low expression of this chemokine, we did observe CCL3 secretion in memory OT-I cells by flow cytometry. Interestingly, CCL3 expression was always observed in IFN-γ secreting OT-I cells (Figure 7). As IFN-γ secretion by memory OT-I cells was only observed in these effector clusters, we concluded that OT-I cells secreting CCL-3 were also localized in these clusters. To determine if neutrophils were also present there, we took advantage of the GFP-expressing lys-M knock-in mouse in which neutrophils express high amounts of GFP [33]. Massive and local accumulations of neutrophils were indeed found in these clusters (Figure 6B), in agreement with our previous study demonstrating that these cells underwent activation at such early time following the secondary infection [28]. Inflammatory monocytes produce radical oxygen intermediates (ROI) critical for secondary protection against L.m infections ([28] and unpublished data). As these intermediates cannot be detected by immunofluorescence, we used the ability of activated inflammatory monocytes to express the inducible nitric oxide synthase (iNOS) to locate these cells on tissue sections [34]. Spleen sections of memory mice challenged for 3, 6, 12 and 24 hours with wt L.m-OVA were stained for iNOS and collagen IV expression (Figure 6E and Figure 8). At 6 hours, the peak of the effector cluster formation, we failed to detect iNOS expression in agreement with previous studies [34]. At 12 hours, however, iNOS expression was detected in the OT-I remaining clusters localized in the RP, suggesting that inflammatory monocytes activation and/or recruitment also occurs in these clusters. At 24 hours, when the majority of OT-I cells had relocalized to the WP, we were still able to visualize patches of iNOS-expressing cells in the RP nearby OT-I cells. Thus altogether, these results demonstrate that, very early after a secondary infection, L.m-specific memory CD8+ T cells aggregate in clusters in the splenic RP where they release effector molecules nearby L.m-infected innate immune cells. At the same time and later on, neutrophils as well as inflammatory monocytes are also found in these effector clusters producing antimicrobial mediators such as ROI that are essential for the control of the secondary bacterial burden [28].


Visualizing early splenic memory CD8+ T cells reactivation against intracellular bacteria in the mouse.

Bajénoff M, Narni-Mancinelli E, Brau F, Lauvau G - PLoS ONE (2010)

Memory ‘CD8 clusters’ concentrate anti-listeria effector cells.(A,C,D,E) 200 naïve GFP+ OT-I cells were transferred in C57BL/6 mice. One day after, recipient mice as well as naive GFP+ lys-M Tg mice (B) were injected i.v. with 0.1×LD50 Wt L.m-OVA. One month later, all mice were injected i.v. with 10×LD50 Wt L.m-OVA. Spleens were harvested 6 (A,B,C,D) or 12 hrs later (E), sectioned, stained with anti-B220, -CD4, -CD8 (A); anti-CD3, -collagen IV (B) anti-CD11c, -listeria (C); anti-IFN-γ, and hoescht (D), anti-iNOS (E) specific Abs and analyzed by confocal microscopy. In (D), IFN-γ secretion by endogenous CD8+ T cells and GFP+ OT-I memory cells was also assessed by flow cytometry. Data are representative of 3 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2902518&req=5

pone-0011524-g006: Memory ‘CD8 clusters’ concentrate anti-listeria effector cells.(A,C,D,E) 200 naïve GFP+ OT-I cells were transferred in C57BL/6 mice. One day after, recipient mice as well as naive GFP+ lys-M Tg mice (B) were injected i.v. with 0.1×LD50 Wt L.m-OVA. One month later, all mice were injected i.v. with 10×LD50 Wt L.m-OVA. Spleens were harvested 6 (A,B,C,D) or 12 hrs later (E), sectioned, stained with anti-B220, -CD4, -CD8 (A); anti-CD3, -collagen IV (B) anti-CD11c, -listeria (C); anti-IFN-γ, and hoescht (D), anti-iNOS (E) specific Abs and analyzed by confocal microscopy. In (D), IFN-γ secretion by endogenous CD8+ T cells and GFP+ OT-I memory cells was also assessed by flow cytometry. Data are representative of 3 independent experiments.
Mentions: We next asked whether such early clustering of memory OT-I cells may be related to the fast control of bacterial growth. We previously demonstrated that reactivated L.m-specific CD8+ T cells rapidly secrete CCL3, a chemokine that promotes the antimicrobial oxidative burst production in innate immune cells, an effector function required for secondary protection against L.m [28]. Others have also shown that memory CD8+ T cells rapidly secrete IFN-γ and that such secretion partially contributes to the protective recall response [29], [30]. Finally, several studies have demonstrated that innate cells such as neutrophils and inflammatory monocytes expressing the myeloid marker Ly-6C -also defined as TNF/NO-producing DCs (Tip-DCs)- produce antimicrobial mediators known to be critical for pathogens clearance [31], [32]. Therefore, we characterized the composition of the OT-I clusters induced 6 hours after the challenge infection and looked for the presence of the innate immune cell types and effector functions described above. Spleens sections from Wt L.m-OVA challenged mice (6 h) were stained for the expression of B220, CD8, CD4 (Figure 6A); CD11c, L.m antigens (Figure 6C) and CD8, IFN-γ (Figure 6D). Attention was focused on the clusters of memory OT-I cells. Data show that these clusters were massive (>100 µm), composed of both CD4+ and CD8+ T cells (but not B cells) and were usually found around L.m-infected CD11c+ cells, similar to what was recently described during the secondary challenge infection against Toxoplasma gondii in LNs [18]. Most interestingly, we observed both on tissue sections and by flow cytometry an intense IFN-γ secretion by memory OT-I cells in these clusters that we therefore named “effector clusters” (Figure 6D). Although we were unable to detect CCL3 on sections, likely due to a low expression of this chemokine, we did observe CCL3 secretion in memory OT-I cells by flow cytometry. Interestingly, CCL3 expression was always observed in IFN-γ secreting OT-I cells (Figure 7). As IFN-γ secretion by memory OT-I cells was only observed in these effector clusters, we concluded that OT-I cells secreting CCL-3 were also localized in these clusters. To determine if neutrophils were also present there, we took advantage of the GFP-expressing lys-M knock-in mouse in which neutrophils express high amounts of GFP [33]. Massive and local accumulations of neutrophils were indeed found in these clusters (Figure 6B), in agreement with our previous study demonstrating that these cells underwent activation at such early time following the secondary infection [28]. Inflammatory monocytes produce radical oxygen intermediates (ROI) critical for secondary protection against L.m infections ([28] and unpublished data). As these intermediates cannot be detected by immunofluorescence, we used the ability of activated inflammatory monocytes to express the inducible nitric oxide synthase (iNOS) to locate these cells on tissue sections [34]. Spleen sections of memory mice challenged for 3, 6, 12 and 24 hours with wt L.m-OVA were stained for iNOS and collagen IV expression (Figure 6E and Figure 8). At 6 hours, the peak of the effector cluster formation, we failed to detect iNOS expression in agreement with previous studies [34]. At 12 hours, however, iNOS expression was detected in the OT-I remaining clusters localized in the RP, suggesting that inflammatory monocytes activation and/or recruitment also occurs in these clusters. At 24 hours, when the majority of OT-I cells had relocalized to the WP, we were still able to visualize patches of iNOS-expressing cells in the RP nearby OT-I cells. Thus altogether, these results demonstrate that, very early after a secondary infection, L.m-specific memory CD8+ T cells aggregate in clusters in the splenic RP where they release effector molecules nearby L.m-infected innate immune cells. At the same time and later on, neutrophils as well as inflammatory monocytes are also found in these effector clusters producing antimicrobial mediators such as ROI that are essential for the control of the secondary bacterial burden [28].

Bottom Line: Memory CD8(+) T cells are endowed with enhanced antimicrobial effector functions that perfectly tail them to rapidly eradicate invading pathogens.Within these clusters that only last few hours, memory CD8(+) T produce inflammatory cytokines such as IFN-gamma and CCL3 nearby infected myeloid cells known to be crucial for L.m killing.Altogether, we describe how memory CD8(+) T cells trafficking properties and the splenic micro-anatomy conjugate to create a spatio-temporal window during which memory CD8(+) T cells provide a local response by secreting effector molecules around infected cells.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale Unité 924, Groupe Avenir, Valbonne, France. bajenoff@ciml.univ-mrs.fr

ABSTRACT
Memory CD8(+) T cells represent an important effector arm of the immune response in maintaining long-lived protective immunity against viruses and some intracellular bacteria such as Listeria monocytogenes (L.m). Memory CD8(+) T cells are endowed with enhanced antimicrobial effector functions that perfectly tail them to rapidly eradicate invading pathogens. It is largely accepted that these functions are sufficient to explain how memory CD8(+) T cells can mediate rapid protection. However, it is important to point out that such improved functional features would be useless if memory cells were unable to rapidly find the pathogen loaded/infected cells within the infected organ. Growing evidences suggest that the anatomy of secondary lymphoid organs (SLOs) fosters the cellular interactions required to initiate naive adaptive immune responses. However, very little is known on how the SLOs structures regulate memory immune responses. Using Listeria monocytogenes (L.m) as a murine infection model and imaging techniques, we have investigated if and how the architecture of the spleen plays a role in the reactivation of memory CD8(+) T cells and the subsequent control of L.m growth. We observed that in the mouse, memory CD8(+) T cells start to control L.m burden 6 hours after the challenge infection. At this very early time point, L.m-specific and non-specific memory CD8(+) T cells localize in the splenic red pulp and form clusters around L.m infected cells while naïve CD8(+) T cells remain in the white pulp. Within these clusters that only last few hours, memory CD8(+) T produce inflammatory cytokines such as IFN-gamma and CCL3 nearby infected myeloid cells known to be crucial for L.m killing. Altogether, we describe how memory CD8(+) T cells trafficking properties and the splenic micro-anatomy conjugate to create a spatio-temporal window during which memory CD8(+) T cells provide a local response by secreting effector molecules around infected cells.

Show MeSH
Related in: MedlinePlus