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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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L.m specific memory CD8+ T cells preferentially traffic in the bloodstream.(A) 200 naïve GFP+ OT-I cells were transferred in C57BL/6 mice. One day later, recipient mice were injected i.v. with 0.1×LD50 Wt L.m-OVA. Thirty days after this immunization, recipient mice were injected i.v with 3×106 naïve CMTPX-labelled polyclonal CD8+ T cells. The following day, mice were injected i.p with PBS or 100 µg of FTY720. Animals were killed 24 hours later and their LNs, spleen and blood harvested, stained for CD8 expression and analyzed by flow cytometry. Numbers indicate the percentages of GFP+ OT-I memory cells and CMTPX naive polyclonal CD8+ T cells among total CD8+ T cells. (B) Wt BALB/c mice were injected i.v with 0.1×LD50 Wt L.m and further treated as in (A) with the exception that polyclonal naive CD8+ T cells were labelled with CFSE and that L.m specific endogenous memory CD8+ T cells were identified as LLO91–99/H-2Kd tetramers+ CD8+ cells. Data are representative of 3 independent experiments.
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pone-0011524-g004: L.m specific memory CD8+ T cells preferentially traffic in the bloodstream.(A) 200 naïve GFP+ OT-I cells were transferred in C57BL/6 mice. One day later, recipient mice were injected i.v. with 0.1×LD50 Wt L.m-OVA. Thirty days after this immunization, recipient mice were injected i.v with 3×106 naïve CMTPX-labelled polyclonal CD8+ T cells. The following day, mice were injected i.p with PBS or 100 µg of FTY720. Animals were killed 24 hours later and their LNs, spleen and blood harvested, stained for CD8 expression and analyzed by flow cytometry. Numbers indicate the percentages of GFP+ OT-I memory cells and CMTPX naive polyclonal CD8+ T cells among total CD8+ T cells. (B) Wt BALB/c mice were injected i.v with 0.1×LD50 Wt L.m and further treated as in (A) with the exception that polyclonal naive CD8+ T cells were labelled with CFSE and that L.m specific endogenous memory CD8+ T cells were identified as LLO91–99/H-2Kd tetramers+ CD8+ cells. Data are representative of 3 independent experiments.

Mentions: The RP filters the blood content and can be assimilated to a blood-filled sponge rather than a truly organised tissue like the WP [26]. Therefore, we hypothesized that the presence of memory OT-I cells in the splenic RP may solely be the result of their endless trafficking in the bloodstream. To test this hypothesis, we adoptively transferred 200 GFP+ OT-I cells in recipient mice that were injected i.v. 1 day later with 0.1×LD50 Wt or Wt-OVA-expressing bacteria. One month later, 3×106 polyclonal naïve CD8+ T cells were labelled with a Red tracker (CMTPX) and injected i.v. in the mice in order to generate an internal control of naïve T cell trafficking pattern. LNs, spleens, and blood cells were stained for CD8 expression and analyzed by flow cytometry (Figure 4A, upper panel). While the naive cells represented a stable proportion of total CD8+ T cells in the 3 compartments, memory OT-I cells were poorly present in the LNs of primed animals but more abundant in their blood and spleens, consistent with their CD62Llow CCR7low phenotype (not shown). Similar results were obtained when following the endogenous memory CD8 response generated against the naturally processed listeriolysin (LLO)-derived epitope LLO91–99 presented by H-2Kd using LLO91–99/H-2Kd tetramers (Figure 4B, upper panel). Neither OT-I nor LLO91–99/H-2Kd tet+ cells were detected in mice injected 30 days earlier with Wt bacteria or PBS respectively (not shown).


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)

L.m specific memory CD8+ T cells preferentially traffic in the bloodstream.(A) 200 naïve GFP+ OT-I cells were transferred in C57BL/6 mice. One day later, recipient mice were injected i.v. with 0.1×LD50 Wt L.m-OVA. Thirty days after this immunization, recipient mice were injected i.v with 3×106 naïve CMTPX-labelled polyclonal CD8+ T cells. The following day, mice were injected i.p with PBS or 100 µg of FTY720. Animals were killed 24 hours later and their LNs, spleen and blood harvested, stained for CD8 expression and analyzed by flow cytometry. Numbers indicate the percentages of GFP+ OT-I memory cells and CMTPX naive polyclonal CD8+ T cells among total CD8+ T cells. (B) Wt BALB/c mice were injected i.v with 0.1×LD50 Wt L.m and further treated as in (A) with the exception that polyclonal naive CD8+ T cells were labelled with CFSE and that L.m specific endogenous memory CD8+ T cells were identified as LLO91–99/H-2Kd tetramers+ CD8+ cells. Data are representative of 3 independent experiments.
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Related In: Results  -  Collection

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pone-0011524-g004: L.m specific memory CD8+ T cells preferentially traffic in the bloodstream.(A) 200 naïve GFP+ OT-I cells were transferred in C57BL/6 mice. One day later, recipient mice were injected i.v. with 0.1×LD50 Wt L.m-OVA. Thirty days after this immunization, recipient mice were injected i.v with 3×106 naïve CMTPX-labelled polyclonal CD8+ T cells. The following day, mice were injected i.p with PBS or 100 µg of FTY720. Animals were killed 24 hours later and their LNs, spleen and blood harvested, stained for CD8 expression and analyzed by flow cytometry. Numbers indicate the percentages of GFP+ OT-I memory cells and CMTPX naive polyclonal CD8+ T cells among total CD8+ T cells. (B) Wt BALB/c mice were injected i.v with 0.1×LD50 Wt L.m and further treated as in (A) with the exception that polyclonal naive CD8+ T cells were labelled with CFSE and that L.m specific endogenous memory CD8+ T cells were identified as LLO91–99/H-2Kd tetramers+ CD8+ cells. Data are representative of 3 independent experiments.
Mentions: The RP filters the blood content and can be assimilated to a blood-filled sponge rather than a truly organised tissue like the WP [26]. Therefore, we hypothesized that the presence of memory OT-I cells in the splenic RP may solely be the result of their endless trafficking in the bloodstream. To test this hypothesis, we adoptively transferred 200 GFP+ OT-I cells in recipient mice that were injected i.v. 1 day later with 0.1×LD50 Wt or Wt-OVA-expressing bacteria. One month later, 3×106 polyclonal naïve CD8+ T cells were labelled with a Red tracker (CMTPX) and injected i.v. in the mice in order to generate an internal control of naïve T cell trafficking pattern. LNs, spleens, and blood cells were stained for CD8 expression and analyzed by flow cytometry (Figure 4A, upper panel). While the naive cells represented a stable proportion of total CD8+ T cells in the 3 compartments, memory OT-I cells were poorly present in the LNs of primed animals but more abundant in their blood and spleens, consistent with their CD62Llow CCR7low phenotype (not shown). Similar results were obtained when following the endogenous memory CD8 response generated against the naturally processed listeriolysin (LLO)-derived epitope LLO91–99 presented by H-2Kd using LLO91–99/H-2Kd tetramers (Figure 4B, upper panel). Neither OT-I nor LLO91–99/H-2Kd tet+ cells were detected in mice injected 30 days earlier with Wt bacteria or PBS respectively (not shown).

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