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Prolonged antigen presentation is required for optimal CD8+ T cell responses against malaria liver stage parasites.

Cockburn IA, Chen YC, Overstreet MG, Lees JR, van Rooijen N, Farber DL, Zavala F - PLoS Pathog. (2010)

Bottom Line: Firstly, reducing the time primed CD8+ T cells were exposed to antigen in vivo severely reduced the final size of the developing memory population.Secondly, fully developed memory cells expanded in previously immunized mice but not when transferred to naïve animals.Finally, persisting antigen was able to prime naïve cells, including recent thymic emigrants, to become functional effector cells capable of eliminating parasites in the liver.

View Article: PubMed Central - PubMed

Affiliation: Johns Hopkins Malaria Research Institute and Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Baltimore, Maryland, USA.

ABSTRACT
Immunization with irradiated sporozoites is currently the most effective vaccination strategy against liver stages of malaria parasites, yet the mechanisms underpinning the success of this approach are unknown. Here we show that the complete development of protective CD8+ T cell responses requires prolonged antigen presentation. Using TCR transgenic cells specific for the malaria circumsporozoite protein, a leading vaccine candidate, we found that sporozoite antigen persists for over 8 weeks after immunization--a remarkable finding since irradiated sporozoites are incapable of replication and do not differentiate beyond early liver stages. Persisting antigen was detected in lymphoid organs and depends on the presence of CD11c+ cells. Prolonged antigen presentation enhanced the magnitude of the CD8+ T cell response in a number of ways. Firstly, reducing the time primed CD8+ T cells were exposed to antigen in vivo severely reduced the final size of the developing memory population. Secondly, fully developed memory cells expanded in previously immunized mice but not when transferred to naïve animals. Finally, persisting antigen was able to prime naïve cells, including recent thymic emigrants, to become functional effector cells capable of eliminating parasites in the liver. Together these data show that the optimal development of protective CD8+ T cell immunity against malaria liver stages is dependent upon the prolonged presentation of sporozoite-derived antigen.

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Antigen persists in the draining lymph nodes.A. Mice were immunized i.v. or i.d. with 5×104 irradiated sporozoites. Fourteen days later 1×104 CFSE labeled transgenic cells were transferred to the mice, some of the mice that had been immunized i.d. received daily doses of 30 µg FTY720 after cell transfer to prevent egress of primed T cells. Eight days after cell transfer transgenic cells were enriched from various tissues by positive selection. Flow cytometry plots show representative data from each group. Values are the mean ± standard deviation of the number of divided transgenic cells recovered per tissue of three mice per group. B. Histograms of the data presented in A. (n = 3, mean ± SE, P>0.05, * = P<0.05, *** = P<0.001). Data in A and B are representative of four similar experiments.
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ppat-1000877-g004: Antigen persists in the draining lymph nodes.A. Mice were immunized i.v. or i.d. with 5×104 irradiated sporozoites. Fourteen days later 1×104 CFSE labeled transgenic cells were transferred to the mice, some of the mice that had been immunized i.d. received daily doses of 30 µg FTY720 after cell transfer to prevent egress of primed T cells. Eight days after cell transfer transgenic cells were enriched from various tissues by positive selection. Flow cytometry plots show representative data from each group. Values are the mean ± standard deviation of the number of divided transgenic cells recovered per tissue of three mice per group. B. Histograms of the data presented in A. (n = 3, mean ± SE, P>0.05, * = P<0.05, *** = P<0.001). Data in A and B are representative of four similar experiments.

Mentions: Given that antigen does not appear to persist as exo-erythrocytic forms in the liver, we hypothesized that it may be retained in lymphoid organs, particularly those draining the site of immunization. To test this, mice were immunized i.v. or intra-dermally (i.d.) in the right ear. Fourteen days later labeled transgenic cells were transferred to the mice. When sporozoites were injected i.v. significant numbers of dividing T cells were observed in the spleen but not in the lymph nodes of mice (Figure 4A). In contrast, following i.d. immunization, dividing cells were seen in the draining auricular lymph nodes as well as the spleen. Significantly, 10-fold more dividing cells were seen in the draining right auricular lymph nodes compared to the non-draining left auricular lymph nodes (Figure 4A and B), indicating that antigen is retained in the lymphoid tissues where sporozoite antigen is first trapped and presented to naïve cells. In addition we also saw cells dividing in the spleen and liver, the divided cells found in these organs following i.d. immunization could have migrated from the ear draining lymph node or they could have been primed in-situ. To distinguish these possibilities another group of mice that received parasites i.d. was given daily injections of 30 µg FTY720 after T cell transfer to prevent the egress of activated T cells from the lymph nodes in which they are primed [22]. In drug treated animals we saw an increased accumulation of activated cells in the draining lymph node, the spleen and the liver (Figure 4A and B) suggesting that the spleen and liver might also be sites of antigen retention. Previously we determined that lymphoid organs, chiefly the skin-draining lymph nodes, but also the spleen, are the principal sites of T cell priming [23] – here we show that antigen is mainly retained in these sites. We also find some evidence of antigen persistence in the liver, which cannot be excluded as a site of T cell priming.


Prolonged antigen presentation is required for optimal CD8+ T cell responses against malaria liver stage parasites.

Cockburn IA, Chen YC, Overstreet MG, Lees JR, van Rooijen N, Farber DL, Zavala F - PLoS Pathog. (2010)

Antigen persists in the draining lymph nodes.A. Mice were immunized i.v. or i.d. with 5×104 irradiated sporozoites. Fourteen days later 1×104 CFSE labeled transgenic cells were transferred to the mice, some of the mice that had been immunized i.d. received daily doses of 30 µg FTY720 after cell transfer to prevent egress of primed T cells. Eight days after cell transfer transgenic cells were enriched from various tissues by positive selection. Flow cytometry plots show representative data from each group. Values are the mean ± standard deviation of the number of divided transgenic cells recovered per tissue of three mice per group. B. Histograms of the data presented in A. (n = 3, mean ± SE, P>0.05, * = P<0.05, *** = P<0.001). Data in A and B are representative of four similar experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2865532&req=5

ppat-1000877-g004: Antigen persists in the draining lymph nodes.A. Mice were immunized i.v. or i.d. with 5×104 irradiated sporozoites. Fourteen days later 1×104 CFSE labeled transgenic cells were transferred to the mice, some of the mice that had been immunized i.d. received daily doses of 30 µg FTY720 after cell transfer to prevent egress of primed T cells. Eight days after cell transfer transgenic cells were enriched from various tissues by positive selection. Flow cytometry plots show representative data from each group. Values are the mean ± standard deviation of the number of divided transgenic cells recovered per tissue of three mice per group. B. Histograms of the data presented in A. (n = 3, mean ± SE, P>0.05, * = P<0.05, *** = P<0.001). Data in A and B are representative of four similar experiments.
Mentions: Given that antigen does not appear to persist as exo-erythrocytic forms in the liver, we hypothesized that it may be retained in lymphoid organs, particularly those draining the site of immunization. To test this, mice were immunized i.v. or intra-dermally (i.d.) in the right ear. Fourteen days later labeled transgenic cells were transferred to the mice. When sporozoites were injected i.v. significant numbers of dividing T cells were observed in the spleen but not in the lymph nodes of mice (Figure 4A). In contrast, following i.d. immunization, dividing cells were seen in the draining auricular lymph nodes as well as the spleen. Significantly, 10-fold more dividing cells were seen in the draining right auricular lymph nodes compared to the non-draining left auricular lymph nodes (Figure 4A and B), indicating that antigen is retained in the lymphoid tissues where sporozoite antigen is first trapped and presented to naïve cells. In addition we also saw cells dividing in the spleen and liver, the divided cells found in these organs following i.d. immunization could have migrated from the ear draining lymph node or they could have been primed in-situ. To distinguish these possibilities another group of mice that received parasites i.d. was given daily injections of 30 µg FTY720 after T cell transfer to prevent the egress of activated T cells from the lymph nodes in which they are primed [22]. In drug treated animals we saw an increased accumulation of activated cells in the draining lymph node, the spleen and the liver (Figure 4A and B) suggesting that the spleen and liver might also be sites of antigen retention. Previously we determined that lymphoid organs, chiefly the skin-draining lymph nodes, but also the spleen, are the principal sites of T cell priming [23] – here we show that antigen is mainly retained in these sites. We also find some evidence of antigen persistence in the liver, which cannot be excluded as a site of T cell priming.

Bottom Line: Firstly, reducing the time primed CD8+ T cells were exposed to antigen in vivo severely reduced the final size of the developing memory population.Secondly, fully developed memory cells expanded in previously immunized mice but not when transferred to naïve animals.Finally, persisting antigen was able to prime naïve cells, including recent thymic emigrants, to become functional effector cells capable of eliminating parasites in the liver.

View Article: PubMed Central - PubMed

Affiliation: Johns Hopkins Malaria Research Institute and Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Baltimore, Maryland, USA.

ABSTRACT
Immunization with irradiated sporozoites is currently the most effective vaccination strategy against liver stages of malaria parasites, yet the mechanisms underpinning the success of this approach are unknown. Here we show that the complete development of protective CD8+ T cell responses requires prolonged antigen presentation. Using TCR transgenic cells specific for the malaria circumsporozoite protein, a leading vaccine candidate, we found that sporozoite antigen persists for over 8 weeks after immunization--a remarkable finding since irradiated sporozoites are incapable of replication and do not differentiate beyond early liver stages. Persisting antigen was detected in lymphoid organs and depends on the presence of CD11c+ cells. Prolonged antigen presentation enhanced the magnitude of the CD8+ T cell response in a number of ways. Firstly, reducing the time primed CD8+ T cells were exposed to antigen in vivo severely reduced the final size of the developing memory population. Secondly, fully developed memory cells expanded in previously immunized mice but not when transferred to naïve animals. Finally, persisting antigen was able to prime naïve cells, including recent thymic emigrants, to become functional effector cells capable of eliminating parasites in the liver. Together these data show that the optimal development of protective CD8+ T cell immunity against malaria liver stages is dependent upon the prolonged presentation of sporozoite-derived antigen.

Show MeSH
Related in: MedlinePlus