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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 naïve CD8 T+ cells are reactivated in the WP.200 naïve GFP+ OT-I cells were transferred in C57BL/6 mice that were injected i.v. the following day with 0.1×LD50 Wt L.m-OVA. One month later, recipient mice were injected i.v. with 3×106 naïve CMTPX labelled OT-I cells. One day after, mice were injected i.v. with 10×LD50 Wt L.m or Wt L.m-OVA. Spleens were harvested 24 hrs later, sectioned, stained for collagen IV expression and analyzed by confocal microscopy. Inserts show the clusterization of naïve OT-I cells in the WP of Wt L.m-OVA challenged animals. Data are representative of 2 different experiments.
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pone-0011524-g010: L.m-specific naïve CD8 T+ cells are reactivated in the WP.200 naïve GFP+ OT-I cells were transferred in C57BL/6 mice that were injected i.v. the following day with 0.1×LD50 Wt L.m-OVA. One month later, recipient mice were injected i.v. with 3×106 naïve CMTPX labelled OT-I cells. One day after, mice were injected i.v. with 10×LD50 Wt L.m or Wt L.m-OVA. Spleens were harvested 24 hrs later, sectioned, stained for collagen IV expression and analyzed by confocal microscopy. Inserts show the clusterization of naïve OT-I cells in the WP of Wt L.m-OVA challenged animals. Data are representative of 2 different experiments.

Mentions: In vitro, memory CD8+ T cells are able to rapidly secrete IFN-γ in response to inflammatory cytokines such as IL-12 and IL-18 in absence of any TCR-triggering. Interestingly, Forman et al. have demonstrated that this non-specific secretion contributes to the control of L.m growth in the spleen of secondary infected mice [29]. Therefore, we sought to determine if L.m-specific and non-specific memory CD8+ T cells were equally able to join the effector clusters and express effector functions during secondary L.m infection. To this aim, we adoptively transferred 200 GFP+ OT-I cells in recipient mice that were injected the day after with 0.1×LD50 Wt L.m-OVA. One month later, mice were challenged for 6 hours with 10×LD50 Wt L.m or Wt L.m-OVA bacteria and their spleens sectioned and stained for collagen IV expression (Figure 9A). In order to compare if OT-I memory cells were able to join effector clusters in absence of a specific TCR trigger, we also co-stained these sections for CD8 expression and delineated 3 regions of interest: the entire splenic WP, the clusters of endogenous CD8+ T cells in the RP and the rest of the RP. Densities of OT-I cells present in these 3 regions were calculated (Figure 9B). Data show that memory OT-I cells were equally capable to join effector clusters and secrete IFN-γ to the same proportion (∼20% of all OT-I cells in both conditions) in mice challenged with Wt L.m and Wt L.m-OVA, indicating that the capacity of memory CD8+ T cells to cluster and provide a rapid response does not require antigen-specific re-activation of the cells (Figure 9A, B and C).Interestingly, we detected CCL3 secretion by memory OT-I cells only in the spleens isolated from animals injected with Wt L.m-OVA, suggesting that memory CD8+ T cells need antigen-specific stimuli to secrete CCL3 in contrast to IFN-γ (Figure 7). Finally, we also investigated whether naïve OT-I cells were able to join the effector clusters of secondary infected mice or if this capacity was restricted to memory OT-I cells. For this, we used the same experimental setup but adoptively transferred the immunized mice with 3×106 CMTPX-labelled naïve OT-I cells before re-infection. Spleens were harvested 6 hours later, and the densities of memory and naïve OT-I cells were calculated in the 3 regions of interest delineated above (Figure 9A and B). Data show that naïve OT-I cells failed to integrate the effector clusters in both groups of mice. However, as anticipated, naïve OT-I cells aggregated in typical “activation clusters” only in the splenic WP of mice challenged for 24 hours with Wt L.m-OVA (Figure 10 and [15]), indicating that naive CD8+ T cell activation during a secondary response takes place in the WP yet much after memory CD8+ T cells reactivation had occurred in the RP.


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 naïve CD8 T+ cells are reactivated in the WP.200 naïve GFP+ OT-I cells were transferred in C57BL/6 mice that were injected i.v. the following day with 0.1×LD50 Wt L.m-OVA. One month later, recipient mice were injected i.v. with 3×106 naïve CMTPX labelled OT-I cells. One day after, mice were injected i.v. with 10×LD50 Wt L.m or Wt L.m-OVA. Spleens were harvested 24 hrs later, sectioned, stained for collagen IV expression and analyzed by confocal microscopy. Inserts show the clusterization of naïve OT-I cells in the WP of Wt L.m-OVA challenged animals. Data are representative of 2 different experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0011524-g010: L.m-specific naïve CD8 T+ cells are reactivated in the WP.200 naïve GFP+ OT-I cells were transferred in C57BL/6 mice that were injected i.v. the following day with 0.1×LD50 Wt L.m-OVA. One month later, recipient mice were injected i.v. with 3×106 naïve CMTPX labelled OT-I cells. One day after, mice were injected i.v. with 10×LD50 Wt L.m or Wt L.m-OVA. Spleens were harvested 24 hrs later, sectioned, stained for collagen IV expression and analyzed by confocal microscopy. Inserts show the clusterization of naïve OT-I cells in the WP of Wt L.m-OVA challenged animals. Data are representative of 2 different experiments.
Mentions: In vitro, memory CD8+ T cells are able to rapidly secrete IFN-γ in response to inflammatory cytokines such as IL-12 and IL-18 in absence of any TCR-triggering. Interestingly, Forman et al. have demonstrated that this non-specific secretion contributes to the control of L.m growth in the spleen of secondary infected mice [29]. Therefore, we sought to determine if L.m-specific and non-specific memory CD8+ T cells were equally able to join the effector clusters and express effector functions during secondary L.m infection. To this aim, we adoptively transferred 200 GFP+ OT-I cells in recipient mice that were injected the day after with 0.1×LD50 Wt L.m-OVA. One month later, mice were challenged for 6 hours with 10×LD50 Wt L.m or Wt L.m-OVA bacteria and their spleens sectioned and stained for collagen IV expression (Figure 9A). In order to compare if OT-I memory cells were able to join effector clusters in absence of a specific TCR trigger, we also co-stained these sections for CD8 expression and delineated 3 regions of interest: the entire splenic WP, the clusters of endogenous CD8+ T cells in the RP and the rest of the RP. Densities of OT-I cells present in these 3 regions were calculated (Figure 9B). Data show that memory OT-I cells were equally capable to join effector clusters and secrete IFN-γ to the same proportion (∼20% of all OT-I cells in both conditions) in mice challenged with Wt L.m and Wt L.m-OVA, indicating that the capacity of memory CD8+ T cells to cluster and provide a rapid response does not require antigen-specific re-activation of the cells (Figure 9A, B and C).Interestingly, we detected CCL3 secretion by memory OT-I cells only in the spleens isolated from animals injected with Wt L.m-OVA, suggesting that memory CD8+ T cells need antigen-specific stimuli to secrete CCL3 in contrast to IFN-γ (Figure 7). Finally, we also investigated whether naïve OT-I cells were able to join the effector clusters of secondary infected mice or if this capacity was restricted to memory OT-I cells. For this, we used the same experimental setup but adoptively transferred the immunized mice with 3×106 CMTPX-labelled naïve OT-I cells before re-infection. Spleens were harvested 6 hours later, and the densities of memory and naïve OT-I cells were calculated in the 3 regions of interest delineated above (Figure 9A and B). Data show that naïve OT-I cells failed to integrate the effector clusters in both groups of mice. However, as anticipated, naïve OT-I cells aggregated in typical “activation clusters” only in the splenic WP of mice challenged for 24 hours with Wt L.m-OVA (Figure 10 and [15]), indicating that naive CD8+ T cell activation during a secondary response takes place in the WP yet much after memory CD8+ T cells reactivation had occurred in the RP.

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