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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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Duration of antigen presentation, and antigen persistence after mosquito biting.A. Mice were immunized with 5×104 irradiated sporozoites i.v. and 14 days later 2×103–2×106 CFSE labeled transgenic cells were transferred to the mice and to unimmunized controls. Ten days later transgenic cells were enriched from the spleen and their CFSE profile determined by FACs. Representative plots from one of three mice per group are shown. Values given are the total number of divided transgenic (CD8+ Thy1.1+ CFSElo) cells isolated from the spleen and in parentheses the % of total recovered transgenic cells that have divided (data representative of numerous similar experiments). B. Mice were immunized as in A. and 1×104 transgenic cells were transferred to mice at different time points after immunization. Ten days after transfer the cells were recovered and the number of divided transgenic cells recovered was determined (n = 3, mean ± SE; data representative of two similar experiments). C. Three mice were bitten by 8–10 infected P. yoelii infected mosquitoes. Fourteen days later 1×104 transgenic cells were transferred to the mice and 10 days later the number of divided transgenic cells was determined. Bars represent values for individual mice; data shown from two independent experiments (i and ii).
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ppat-1000877-g002: Duration of antigen presentation, and antigen persistence after mosquito biting.A. Mice were immunized with 5×104 irradiated sporozoites i.v. and 14 days later 2×103–2×106 CFSE labeled transgenic cells were transferred to the mice and to unimmunized controls. Ten days later transgenic cells were enriched from the spleen and their CFSE profile determined by FACs. Representative plots from one of three mice per group are shown. Values given are the total number of divided transgenic (CD8+ Thy1.1+ CFSElo) cells isolated from the spleen and in parentheses the % of total recovered transgenic cells that have divided (data representative of numerous similar experiments). B. Mice were immunized as in A. and 1×104 transgenic cells were transferred to mice at different time points after immunization. Ten days after transfer the cells were recovered and the number of divided transgenic cells recovered was determined (n = 3, mean ± SE; data representative of two similar experiments). C. Three mice were bitten by 8–10 infected P. yoelii infected mosquitoes. Fourteen days later 1×104 transgenic cells were transferred to the mice and 10 days later the number of divided transgenic cells was determined. Bars represent values for individual mice; data shown from two independent experiments (i and ii).

Mentions: In the previous experiment, the proliferation of transgenic cells, transferred weeks after initial immunization, suggested the presence of persisting antigen. However, since T cell proliferation was limited we initially hypothesized that the amount of antigen that persists may be small and the phenomenon of marginal significance. Alternatively, we reasoned that the transfer of high numbers of transgenic cells might be inhibiting a more robust proliferative response that might be seen with more physiological numbers of cells [21]. Accordingly, we titrated different numbers of labeled transgenic cells into previously immunized mice and later isolated the transgenic cells from the spleen by magnetic bead separation to determine the degree of proliferation in the antigen specific cell population (Figure S2). Strikingly the extent of transgenic cell proliferation substantially increased if fewer precursor cells were used (Figure 2A): in mice that received 2×106 cells only ∼50% of the recovered cells had proliferated - most of them fewer than 6 times; however, in mice that received more physiological numbers of transgenic cells essentially all the transgenic cells had divided and diluted out the CFSE label completely. Similarly to earlier experiments, P. berghei immunization did not induce transgenic cell proliferation even if low numbers of cells were transferred (Figure S1B). In subsequent experiments where we wished to analyze the differentiation of cells primed by persisting antigen, a low number of cells (2×103) was transferred; where we were concerned with detecting only the presence or absence of persisting antigen higher numbers of cells were used (1×104–2×105) which removed the need for technically challenging T cell purification.


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)

Duration of antigen presentation, and antigen persistence after mosquito biting.A. Mice were immunized with 5×104 irradiated sporozoites i.v. and 14 days later 2×103–2×106 CFSE labeled transgenic cells were transferred to the mice and to unimmunized controls. Ten days later transgenic cells were enriched from the spleen and their CFSE profile determined by FACs. Representative plots from one of three mice per group are shown. Values given are the total number of divided transgenic (CD8+ Thy1.1+ CFSElo) cells isolated from the spleen and in parentheses the % of total recovered transgenic cells that have divided (data representative of numerous similar experiments). B. Mice were immunized as in A. and 1×104 transgenic cells were transferred to mice at different time points after immunization. Ten days after transfer the cells were recovered and the number of divided transgenic cells recovered was determined (n = 3, mean ± SE; data representative of two similar experiments). C. Three mice were bitten by 8–10 infected P. yoelii infected mosquitoes. Fourteen days later 1×104 transgenic cells were transferred to the mice and 10 days later the number of divided transgenic cells was determined. Bars represent values for individual mice; data shown from two independent experiments (i and ii).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2865532&req=5

ppat-1000877-g002: Duration of antigen presentation, and antigen persistence after mosquito biting.A. Mice were immunized with 5×104 irradiated sporozoites i.v. and 14 days later 2×103–2×106 CFSE labeled transgenic cells were transferred to the mice and to unimmunized controls. Ten days later transgenic cells were enriched from the spleen and their CFSE profile determined by FACs. Representative plots from one of three mice per group are shown. Values given are the total number of divided transgenic (CD8+ Thy1.1+ CFSElo) cells isolated from the spleen and in parentheses the % of total recovered transgenic cells that have divided (data representative of numerous similar experiments). B. Mice were immunized as in A. and 1×104 transgenic cells were transferred to mice at different time points after immunization. Ten days after transfer the cells were recovered and the number of divided transgenic cells recovered was determined (n = 3, mean ± SE; data representative of two similar experiments). C. Three mice were bitten by 8–10 infected P. yoelii infected mosquitoes. Fourteen days later 1×104 transgenic cells were transferred to the mice and 10 days later the number of divided transgenic cells was determined. Bars represent values for individual mice; data shown from two independent experiments (i and ii).
Mentions: In the previous experiment, the proliferation of transgenic cells, transferred weeks after initial immunization, suggested the presence of persisting antigen. However, since T cell proliferation was limited we initially hypothesized that the amount of antigen that persists may be small and the phenomenon of marginal significance. Alternatively, we reasoned that the transfer of high numbers of transgenic cells might be inhibiting a more robust proliferative response that might be seen with more physiological numbers of cells [21]. Accordingly, we titrated different numbers of labeled transgenic cells into previously immunized mice and later isolated the transgenic cells from the spleen by magnetic bead separation to determine the degree of proliferation in the antigen specific cell population (Figure S2). Strikingly the extent of transgenic cell proliferation substantially increased if fewer precursor cells were used (Figure 2A): in mice that received 2×106 cells only ∼50% of the recovered cells had proliferated - most of them fewer than 6 times; however, in mice that received more physiological numbers of transgenic cells essentially all the transgenic cells had divided and diluted out the CFSE label completely. Similarly to earlier experiments, P. berghei immunization did not induce transgenic cell proliferation even if low numbers of cells were transferred (Figure S1B). In subsequent experiments where we wished to analyze the differentiation of cells primed by persisting antigen, a low number of cells (2×103) was transferred; where we were concerned with detecting only the presence or absence of persisting antigen higher numbers of cells were used (1×104–2×105) which removed the need for technically challenging T cell purification.

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