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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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CD11c+ and phagocytic cells are required for continued antigen presentation.A. CD11c-DTR and WT mice were immunized i.v. with 5×104 irradiated sporozoites at day 0. Groups of CD11c-DTR and wild-type mice were treated with 100ng of DT on the indicated days. On day 14, 2×105 CFSE labeled transgenic cells were transferred to the mice. Ten days later, the number of divided transgenic cells was determined by FACs (n = 3, mean ± SE, * = P<0.05, ** = P<0.01, data representative of four experiments). B. Groups of mice were immunized on day 0 as in A and treated with clodronate liposomes (200 µl i.v./mouse) at the indicated days to deplete splenic macrophage populations. On day 14, 2×105 CFSE labeled transgenic cells were transferred to the mice. Ten days later, the number of divided transgenic cells was determined by FACs (n = 3, mean ± SE, * = P<0.05 data representative of two experiments). C. Groups of mice were immunized on day 0 with irradiated sporozoites and treated with 200 µg of the anti-plasmacytoid DC mAb 120G8 or control IgG at the indicated days. On day 14, 2×105 transgenic cells were transferred to the mice. Ten days later the number of divided transgenic cells was determined by FACs. (n = 3, mean ± SE, data representative of two experiments). D. Jh (B cell deficient) and WT mice were immunized with irradiated sporozoites. 14 days later 2×105 transgenic cells were transferred to the mice. Ten days after cell transfer the number of divided transgenic cells in immune mice was determined by FACs and compared to background proliferation of cells transferred to naïve mice (n = 3, mean ± SE, ** = P<0.01, data representative of two experiments).
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ppat-1000877-g005: CD11c+ and phagocytic cells are required for continued antigen presentation.A. CD11c-DTR and WT mice were immunized i.v. with 5×104 irradiated sporozoites at day 0. Groups of CD11c-DTR and wild-type mice were treated with 100ng of DT on the indicated days. On day 14, 2×105 CFSE labeled transgenic cells were transferred to the mice. Ten days later, the number of divided transgenic cells was determined by FACs (n = 3, mean ± SE, * = P<0.05, ** = P<0.01, data representative of four experiments). B. Groups of mice were immunized on day 0 as in A and treated with clodronate liposomes (200 µl i.v./mouse) at the indicated days to deplete splenic macrophage populations. On day 14, 2×105 CFSE labeled transgenic cells were transferred to the mice. Ten days later, the number of divided transgenic cells was determined by FACs (n = 3, mean ± SE, * = P<0.05 data representative of two experiments). C. Groups of mice were immunized on day 0 with irradiated sporozoites and treated with 200 µg of the anti-plasmacytoid DC mAb 120G8 or control IgG at the indicated days. On day 14, 2×105 transgenic cells were transferred to the mice. Ten days later the number of divided transgenic cells was determined by FACs. (n = 3, mean ± SE, data representative of two experiments). D. Jh (B cell deficient) and WT mice were immunized with irradiated sporozoites. 14 days later 2×105 transgenic cells were transferred to the mice. Ten days after cell transfer the number of divided transgenic cells in immune mice was determined by FACs and compared to background proliferation of cells transferred to naïve mice (n = 3, mean ± SE, ** = P<0.01, data representative of two experiments).

Mentions: Since antigen persists in both lymphoid and non-lymphoid tissues we investigated the roles that professional APCs such as myeloid DCs, macrophages or plasmacytoid DCs may play a role maintaining and presenting antigen over the long term. Mice were depleted of APC subsets at one of two time-points – either before immunization on d0 or after immunization and prior to the transfer of transgenic cells on d14 (ie. at the time we detect persistent antigen presentation). To assess the role of DCs we used mice which express the diphtheria toxin receptor under the control of a CD11c promoter; DCs can be depleted in these animals with low doses of diphtheria toxin (DT) [24]. Macrophages were depleted from WT mice using clodronate liposomes (CL) [25] while plasmacytoid DCs were depleted using the mAb 120G8 [26]. DT treatment of CD11c-DTR transgenic mice resulted in ∼90% depletion of CD11c+ cells, while clodronate lipsome depletion induced ∼90% depletion of CD11bint F4/80-, CD11bintF4/80+ and CD11bhiF4/80+ macrophage populations (Figure S3). Interestingly we found that depletion of CD11c+ cells in DTR mice and treatment with CL had similar effects. In both cases, depletion at the time of immunization eliminated persisting antigen (Figure 5A and B). It may be that DCs and macrophages may be acting co-operatively to trap and retain antigen. Alternatively since the treatments are not mutually exclusive (Figure S3) a single cell type that is depleted by both treatments, for example immature DCs, may be important for antigen persistence.


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)

CD11c+ and phagocytic cells are required for continued antigen presentation.A. CD11c-DTR and WT mice were immunized i.v. with 5×104 irradiated sporozoites at day 0. Groups of CD11c-DTR and wild-type mice were treated with 100ng of DT on the indicated days. On day 14, 2×105 CFSE labeled transgenic cells were transferred to the mice. Ten days later, the number of divided transgenic cells was determined by FACs (n = 3, mean ± SE, * = P<0.05, ** = P<0.01, data representative of four experiments). B. Groups of mice were immunized on day 0 as in A and treated with clodronate liposomes (200 µl i.v./mouse) at the indicated days to deplete splenic macrophage populations. On day 14, 2×105 CFSE labeled transgenic cells were transferred to the mice. Ten days later, the number of divided transgenic cells was determined by FACs (n = 3, mean ± SE, * = P<0.05 data representative of two experiments). C. Groups of mice were immunized on day 0 with irradiated sporozoites and treated with 200 µg of the anti-plasmacytoid DC mAb 120G8 or control IgG at the indicated days. On day 14, 2×105 transgenic cells were transferred to the mice. Ten days later the number of divided transgenic cells was determined by FACs. (n = 3, mean ± SE, data representative of two experiments). D. Jh (B cell deficient) and WT mice were immunized with irradiated sporozoites. 14 days later 2×105 transgenic cells were transferred to the mice. Ten days after cell transfer the number of divided transgenic cells in immune mice was determined by FACs and compared to background proliferation of cells transferred to naïve mice (n = 3, mean ± SE, ** = P<0.01, data representative of two experiments).
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Related In: Results  -  Collection

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ppat-1000877-g005: CD11c+ and phagocytic cells are required for continued antigen presentation.A. CD11c-DTR and WT mice were immunized i.v. with 5×104 irradiated sporozoites at day 0. Groups of CD11c-DTR and wild-type mice were treated with 100ng of DT on the indicated days. On day 14, 2×105 CFSE labeled transgenic cells were transferred to the mice. Ten days later, the number of divided transgenic cells was determined by FACs (n = 3, mean ± SE, * = P<0.05, ** = P<0.01, data representative of four experiments). B. Groups of mice were immunized on day 0 as in A and treated with clodronate liposomes (200 µl i.v./mouse) at the indicated days to deplete splenic macrophage populations. On day 14, 2×105 CFSE labeled transgenic cells were transferred to the mice. Ten days later, the number of divided transgenic cells was determined by FACs (n = 3, mean ± SE, * = P<0.05 data representative of two experiments). C. Groups of mice were immunized on day 0 with irradiated sporozoites and treated with 200 µg of the anti-plasmacytoid DC mAb 120G8 or control IgG at the indicated days. On day 14, 2×105 transgenic cells were transferred to the mice. Ten days later the number of divided transgenic cells was determined by FACs. (n = 3, mean ± SE, data representative of two experiments). D. Jh (B cell deficient) and WT mice were immunized with irradiated sporozoites. 14 days later 2×105 transgenic cells were transferred to the mice. Ten days after cell transfer the number of divided transgenic cells in immune mice was determined by FACs and compared to background proliferation of cells transferred to naïve mice (n = 3, mean ± SE, ** = P<0.01, data representative of two experiments).
Mentions: Since antigen persists in both lymphoid and non-lymphoid tissues we investigated the roles that professional APCs such as myeloid DCs, macrophages or plasmacytoid DCs may play a role maintaining and presenting antigen over the long term. Mice were depleted of APC subsets at one of two time-points – either before immunization on d0 or after immunization and prior to the transfer of transgenic cells on d14 (ie. at the time we detect persistent antigen presentation). To assess the role of DCs we used mice which express the diphtheria toxin receptor under the control of a CD11c promoter; DCs can be depleted in these animals with low doses of diphtheria toxin (DT) [24]. Macrophages were depleted from WT mice using clodronate liposomes (CL) [25] while plasmacytoid DCs were depleted using the mAb 120G8 [26]. DT treatment of CD11c-DTR transgenic mice resulted in ∼90% depletion of CD11c+ cells, while clodronate lipsome depletion induced ∼90% depletion of CD11bint F4/80-, CD11bintF4/80+ and CD11bhiF4/80+ macrophage populations (Figure S3). Interestingly we found that depletion of CD11c+ cells in DTR mice and treatment with CL had similar effects. In both cases, depletion at the time of immunization eliminated persisting antigen (Figure 5A and B). It may be that DCs and macrophages may be acting co-operatively to trap and retain antigen. Alternatively since the treatments are not mutually exclusive (Figure S3) a single cell type that is depleted by both treatments, for example immature DCs, may be important for antigen persistence.

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