Limits...
Skin-draining lymph node priming is sufficient to induce sterile immunity against pre-erythrocytic malaria.

Obeid M, Franetich JF, Lorthiois A, Gego A, Grüner AC, Tefit M, Boucheix C, Snounou G, Mazier D - EMBO Mol Med (2012)

Bottom Line: However, it has recently been demonstrated that priming also occurs in the skin.We wished to establish if sterile protection could be obtained in the absence of priming by infected hepatocytes.We then compared and contrasted the patterns of priming with those obtained by intradermal immunization, where priming occurs in the liver.

View Article: PubMed Central - PubMed

Affiliation: Inserm, UMR-S 945, Paris, France. michel.obeid@inserm.fr

Show MeSH

Related in: MedlinePlus

Investigations on the priming site in immunized animalsResults expressed as mean ± standard deviation (SD). All experiments were repeated three times (n = 3) with five mice per group at each time. *p < 0.0001 for (A) and (B), 0.0017 for (C) or 0.0002 for (D) by Kruskal–Wallis test with Dunn's post-test. In (B) and (D) the * refer to statistical significance between data in the upper and lower panels.The level of T-cell priming following s.c. or i.d. γ-spz immunization (three 100 × 103 doses at weekly intervals) of BALB/c, Cd81+/+, or Cd81−/− mice (s.c. or i.d.), was assessed by measuring IFN-γ production subsequent to stimulation with a CS T-cell epitope, by cells isolated 4–5 days following the last immunizing dose from the right iLN draining the immunization site. Mice infected with PyWT sporozoites served as controls.In a separate experiment where the mice were immunized as above, the level of T-cell priming was similarly measured in cells isolated from iLN, their nLNs, the cLN, or from the spleen (spln). This was conducted for cells collected from animals that were untreated (upper panel) or FTY720-treated (lower panel) throughout the experiment.Mice were immunized s.c. or i.d. (two doses of 200 × 103 γ-spz at 1 weekly interval, or PBS for controls) while under FTY720 treatment or not. Protection (undetectable blood stage parasites between days 4 and 14 post-challenge) was assessed after an i.v. challenge with 5 × 103 PyWT.30 × 106 cells isolated from the iLN, nLN, cLN or spln of BALB/c, Cd81+/+, or Cd81−/− mice 5 days after immunization s.c. or i.d. with two doses of 200 × 103 γ-spz at 1-weekly interval, were adoptively transferred into each of the naïve recipient mice, which were challenged i.d. (5 × 103 PyWT sporozoites) 2 days later. The experiment was conducted for mice that were untreated (upper panel) or treated with FTY720 (lower panel) throughout the experiment. All recipient mice adoptively transferred with control mice splenocytes became patent by days 4 or 5 after PyWT challenge. Protection from challenge in the experimental animals was measured as above.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3569641&req=5

fig02: Investigations on the priming site in immunized animalsResults expressed as mean ± standard deviation (SD). All experiments were repeated three times (n = 3) with five mice per group at each time. *p < 0.0001 for (A) and (B), 0.0017 for (C) or 0.0002 for (D) by Kruskal–Wallis test with Dunn's post-test. In (B) and (D) the * refer to statistical significance between data in the upper and lower panels.The level of T-cell priming following s.c. or i.d. γ-spz immunization (three 100 × 103 doses at weekly intervals) of BALB/c, Cd81+/+, or Cd81−/− mice (s.c. or i.d.), was assessed by measuring IFN-γ production subsequent to stimulation with a CS T-cell epitope, by cells isolated 4–5 days following the last immunizing dose from the right iLN draining the immunization site. Mice infected with PyWT sporozoites served as controls.In a separate experiment where the mice were immunized as above, the level of T-cell priming was similarly measured in cells isolated from iLN, their nLNs, the cLN, or from the spleen (spln). This was conducted for cells collected from animals that were untreated (upper panel) or FTY720-treated (lower panel) throughout the experiment.Mice were immunized s.c. or i.d. (two doses of 200 × 103 γ-spz at 1 weekly interval, or PBS for controls) while under FTY720 treatment or not. Protection (undetectable blood stage parasites between days 4 and 14 post-challenge) was assessed after an i.v. challenge with 5 × 103 PyWT.30 × 106 cells isolated from the iLN, nLN, cLN or spln of BALB/c, Cd81+/+, or Cd81−/− mice 5 days after immunization s.c. or i.d. with two doses of 200 × 103 γ-spz at 1-weekly interval, were adoptively transferred into each of the naïve recipient mice, which were challenged i.d. (5 × 103 PyWT sporozoites) 2 days later. The experiment was conducted for mice that were untreated (upper panel) or treated with FTY720 (lower panel) throughout the experiment. All recipient mice adoptively transferred with control mice splenocytes became patent by days 4 or 5 after PyWT challenge. Protection from challenge in the experimental animals was measured as above.

Mentions: First, we assessed T-cell priming through the production of IFN-γ after stimulation by a specific parasite antigen, at different sites in i.d.-immunized mice, where priming is expected to occur concomitantly in the skin, the liver and the spleen, and in s.c.-immunized mice, where priming is expected to occur principally, or entirely, in the skin. Groups of BALB/c, Cd81−/− or Cd81+/+ mice were immunized s.c. or i.d. with γ-spz. Cells were harvested from different sites for the IFN-γ assays. iLN cells from either s.c.- or i.d.-immunized mice showed strong IFN-γ production, following stimulation with a CS epitope, which contrasted with the weak IFN-γ production by iLN cells isolated from PyWT infected mice (Fig 2A). However, activated T cells could leave the priming site and circulate to other lymph nodes and organs within few days (4–5 days) after i.d. vaccination (Chakravarty et al, 2007). Consequently, we additionally sought the presence of activated T cells in the contra-lateral non-draining inguinal-lymph node (nLN), the celiac-lymph node (cLN) and the spleen. Five days after s.c. or i.d. vaccination with γ-spz, IFN-γ-producing T cells were equally present in the iLN, nLN, cLN and the spleen (Fig 2B). This could either be due to migration of activated T cells from the priming site in the skin (iLN) to the nLN, cLN and the spleen, or due to the presence of multiple priming sites. In order to distinguish between the two possibilities, migration of T lymphocytes out of the priming site was inhibited. This was achieved by treating mice with the immunosuppressive drug FTY720, which downregulates the expression of sphingosine-1-phosphate receptors (S1PRs) on the T lymphocyte surface, thereby preventing lymphocytes from migrating along a S1P gradient and reducing the T-cell egress from draining-lymph nodes (Brinkmann et al, 2010). In the i.d.- or s.c.-vaccinated and FTY720-treated mice, the level of activated T cells in the iLN increased about twofolds in contrast to the nLN, where activated T cells were no longer found (Fig 2B). Furthermore, treatment with FTY720 also reduced the level of activated T cells in the cLN and the spleen to negligible levels in the s.c.-immunized mice but not in the i.d.-immunized mice, where their levels increased about 1.5-fold (Fig 2B). These data suggest that whereas the iLN, CLN and the spleen are all priming sites in i.d.-immunized mice, in s.c.-immunized mice priming occurs mainly, if not exclusively, in the skin-draining lymph nodes (iLN).


Skin-draining lymph node priming is sufficient to induce sterile immunity against pre-erythrocytic malaria.

Obeid M, Franetich JF, Lorthiois A, Gego A, Grüner AC, Tefit M, Boucheix C, Snounou G, Mazier D - EMBO Mol Med (2012)

Investigations on the priming site in immunized animalsResults expressed as mean ± standard deviation (SD). All experiments were repeated three times (n = 3) with five mice per group at each time. *p < 0.0001 for (A) and (B), 0.0017 for (C) or 0.0002 for (D) by Kruskal–Wallis test with Dunn's post-test. In (B) and (D) the * refer to statistical significance between data in the upper and lower panels.The level of T-cell priming following s.c. or i.d. γ-spz immunization (three 100 × 103 doses at weekly intervals) of BALB/c, Cd81+/+, or Cd81−/− mice (s.c. or i.d.), was assessed by measuring IFN-γ production subsequent to stimulation with a CS T-cell epitope, by cells isolated 4–5 days following the last immunizing dose from the right iLN draining the immunization site. Mice infected with PyWT sporozoites served as controls.In a separate experiment where the mice were immunized as above, the level of T-cell priming was similarly measured in cells isolated from iLN, their nLNs, the cLN, or from the spleen (spln). This was conducted for cells collected from animals that were untreated (upper panel) or FTY720-treated (lower panel) throughout the experiment.Mice were immunized s.c. or i.d. (two doses of 200 × 103 γ-spz at 1 weekly interval, or PBS for controls) while under FTY720 treatment or not. Protection (undetectable blood stage parasites between days 4 and 14 post-challenge) was assessed after an i.v. challenge with 5 × 103 PyWT.30 × 106 cells isolated from the iLN, nLN, cLN or spln of BALB/c, Cd81+/+, or Cd81−/− mice 5 days after immunization s.c. or i.d. with two doses of 200 × 103 γ-spz at 1-weekly interval, were adoptively transferred into each of the naïve recipient mice, which were challenged i.d. (5 × 103 PyWT sporozoites) 2 days later. The experiment was conducted for mice that were untreated (upper panel) or treated with FTY720 (lower panel) throughout the experiment. All recipient mice adoptively transferred with control mice splenocytes became patent by days 4 or 5 after PyWT challenge. Protection from challenge in the experimental animals was measured as above.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3569641&req=5

fig02: Investigations on the priming site in immunized animalsResults expressed as mean ± standard deviation (SD). All experiments were repeated three times (n = 3) with five mice per group at each time. *p < 0.0001 for (A) and (B), 0.0017 for (C) or 0.0002 for (D) by Kruskal–Wallis test with Dunn's post-test. In (B) and (D) the * refer to statistical significance between data in the upper and lower panels.The level of T-cell priming following s.c. or i.d. γ-spz immunization (three 100 × 103 doses at weekly intervals) of BALB/c, Cd81+/+, or Cd81−/− mice (s.c. or i.d.), was assessed by measuring IFN-γ production subsequent to stimulation with a CS T-cell epitope, by cells isolated 4–5 days following the last immunizing dose from the right iLN draining the immunization site. Mice infected with PyWT sporozoites served as controls.In a separate experiment where the mice were immunized as above, the level of T-cell priming was similarly measured in cells isolated from iLN, their nLNs, the cLN, or from the spleen (spln). This was conducted for cells collected from animals that were untreated (upper panel) or FTY720-treated (lower panel) throughout the experiment.Mice were immunized s.c. or i.d. (two doses of 200 × 103 γ-spz at 1 weekly interval, or PBS for controls) while under FTY720 treatment or not. Protection (undetectable blood stage parasites between days 4 and 14 post-challenge) was assessed after an i.v. challenge with 5 × 103 PyWT.30 × 106 cells isolated from the iLN, nLN, cLN or spln of BALB/c, Cd81+/+, or Cd81−/− mice 5 days after immunization s.c. or i.d. with two doses of 200 × 103 γ-spz at 1-weekly interval, were adoptively transferred into each of the naïve recipient mice, which were challenged i.d. (5 × 103 PyWT sporozoites) 2 days later. The experiment was conducted for mice that were untreated (upper panel) or treated with FTY720 (lower panel) throughout the experiment. All recipient mice adoptively transferred with control mice splenocytes became patent by days 4 or 5 after PyWT challenge. Protection from challenge in the experimental animals was measured as above.
Mentions: First, we assessed T-cell priming through the production of IFN-γ after stimulation by a specific parasite antigen, at different sites in i.d.-immunized mice, where priming is expected to occur concomitantly in the skin, the liver and the spleen, and in s.c.-immunized mice, where priming is expected to occur principally, or entirely, in the skin. Groups of BALB/c, Cd81−/− or Cd81+/+ mice were immunized s.c. or i.d. with γ-spz. Cells were harvested from different sites for the IFN-γ assays. iLN cells from either s.c.- or i.d.-immunized mice showed strong IFN-γ production, following stimulation with a CS epitope, which contrasted with the weak IFN-γ production by iLN cells isolated from PyWT infected mice (Fig 2A). However, activated T cells could leave the priming site and circulate to other lymph nodes and organs within few days (4–5 days) after i.d. vaccination (Chakravarty et al, 2007). Consequently, we additionally sought the presence of activated T cells in the contra-lateral non-draining inguinal-lymph node (nLN), the celiac-lymph node (cLN) and the spleen. Five days after s.c. or i.d. vaccination with γ-spz, IFN-γ-producing T cells were equally present in the iLN, nLN, cLN and the spleen (Fig 2B). This could either be due to migration of activated T cells from the priming site in the skin (iLN) to the nLN, cLN and the spleen, or due to the presence of multiple priming sites. In order to distinguish between the two possibilities, migration of T lymphocytes out of the priming site was inhibited. This was achieved by treating mice with the immunosuppressive drug FTY720, which downregulates the expression of sphingosine-1-phosphate receptors (S1PRs) on the T lymphocyte surface, thereby preventing lymphocytes from migrating along a S1P gradient and reducing the T-cell egress from draining-lymph nodes (Brinkmann et al, 2010). In the i.d.- or s.c.-vaccinated and FTY720-treated mice, the level of activated T cells in the iLN increased about twofolds in contrast to the nLN, where activated T cells were no longer found (Fig 2B). Furthermore, treatment with FTY720 also reduced the level of activated T cells in the cLN and the spleen to negligible levels in the s.c.-immunized mice but not in the i.d.-immunized mice, where their levels increased about 1.5-fold (Fig 2B). These data suggest that whereas the iLN, CLN and the spleen are all priming sites in i.d.-immunized mice, in s.c.-immunized mice priming occurs mainly, if not exclusively, in the skin-draining lymph nodes (iLN).

Bottom Line: However, it has recently been demonstrated that priming also occurs in the skin.We wished to establish if sterile protection could be obtained in the absence of priming by infected hepatocytes.We then compared and contrasted the patterns of priming with those obtained by intradermal immunization, where priming occurs in the liver.

View Article: PubMed Central - PubMed

Affiliation: Inserm, UMR-S 945, Paris, France. michel.obeid@inserm.fr

Show MeSH
Related in: MedlinePlus