CD8+ T cells from a novel T cell receptor transgenic mouse induce liver-stage immunity that can be boosted by blood-stage infection in rodent malaria - PubMed (original) (raw)

. 2014 May 22;10(5):e1004135.

doi: 10.1371/journal.ppat.1004135. eCollection 2014 May.

Daniel Fernandez-Ruiz 1, Vanessa Mollard 2, Angelika Sturm 2, Michelle A Neller 3, Anton Cozijnsen 2, Julia L Gregory 1, Gayle M Davey 1, Claerwen M Jones 1, Yi-Hsuan Lin 1, Ashraful Haque 3, Christian R Engwerda 3, Catherine Q Nie 4, Diana S Hansen 5, Kenneth M Murphy 6, Anthony T Papenfuss 5, John J Miles 7, Scott R Burrows 3, Tania de Koning-Ward 8, Geoffrey I McFadden 2, Francis R Carbone 1, Brendan S Crabb 9, William R Heath 10

Affiliations

CD8+ T cells from a novel T cell receptor transgenic mouse induce liver-stage immunity that can be boosted by blood-stage infection in rodent malaria

Lei Shong Lau et al. PLoS Pathog. 2014.

Abstract

To follow the fate of CD8+ T cells responsive to Plasmodium berghei ANKA (PbA) infection, we generated an MHC I-restricted TCR transgenic mouse line against this pathogen. T cells from this line, termed PbT-I T cells, were able to respond to blood-stage infection by PbA and two other rodent malaria species, P. yoelii XNL and P. chabaudi AS. These PbT-I T cells were also able to respond to sporozoites and to protect mice from liver-stage infection. Examination of the requirements for priming after intravenous administration of irradiated sporozoites, an effective vaccination approach, showed that the spleen rather than the liver was the main site of priming and that responses depended on CD8α+ dendritic cells. Importantly, sequential exposure to irradiated sporozoites followed two days later by blood-stage infection led to augmented PbT-I T cell expansion. These findings indicate that PbT-I T cells are a highly versatile tool for studying multiple stages and species of rodent malaria and suggest that cross-stage reactive CD8+ T cells may be utilized in liver-stage vaccine design to enable boosting by blood-stage infections.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Characterization of T cells from the spleen and lymph node of PbT-I mice.

Cells were harvested from the spleen and the lymph nodes of PbT-I transgenic or littermate control mice (WT). FACS analysis was performed to characterize the expression of CD8, CD4 and the transgenic TCR alpha (Vα8.3) and beta (Vβ10) chains. (A) Representative dot-plots showing the proportions of CD8 versus CD4 cells in the spleen and lymph node of PbT-I and WT mice. (B) Representative histograms showing the expression of the transgenic TCR Vα8.3 and Vβ10 chains on the CD8 or CD4 single-positive cells from the spleen. This experiment was repeated three times with two mice per experiment.

Figure 2

Figure 2. PbT-I cells respond in the spleen to i.v. blood-stage PbA.

B6 mice were adoptively transferred with 2×106 Ly5.1+ PbT-I cells and the next day infected i.v. with 106 blood-stage PbA. Spleens were harvested three or five days later and the proliferation of PbT-I cells was analyzed. The gating strategy to identify PbT-I cells was similar to that shown in Figure S13. (A) Representative histograms showing the proliferation of PbT-I cells on day three or five post-infection. (B) Number of proliferating PbT-I cells in the spleen and blood of mice infected with PbA for three or five days. Data are pooled from three experiments. Each data point represents a mouse and the lines represent the mean. Data were compared using student t test (**, p<0.01; ***, p<0.001). (C) B6 mice were adoptively transferred with 106 CFSE-labeled Ly5.1+ PbT-I cells. The next day, mice were injected i.v. with 106 blood-stage PbA. Various tissues (spleen, blood, celiac lymph node (ceLN), portal LN (pLN), inguinal LN (iLN), mesenteric LN (mLN)) were harvested after 2 days and PbT-I cells examined for CD69 and CFSE expression. Profiles are gated on PbT-I cells. This experiment was performed three times (two-three mice per group) with similar results. Typical profiles are shown. (D) The mean percentage of CD69+ PbT-I cells for the analysis shown in (C). Histograms represent values from infected animals minus mean values from uninfected animals. Error bars represent standard error of the mean.

Figure 3

Figure 3. PbT-I cells infiltrate the brain and accelerate ECM.

B6 mice were adoptively transferred with 2×106 or 2×104 Ly5.1+ PbT-I cells or 2×106 herpes simplex virus-specific gBT-I cells or left uninjected. The next day mice were infected i.v. with 106 blood-stage PbA. (A) Mice were monitored for the development of ECM. Data are pooled from three independent experiments. The differences between 2×106 PbT-I and the group that did not receive any transgenic cells or the group that received gBT-I cells are statistically significant (p<0.0001) as determined by a Log-rank test. (B) Mice were adoptively transferred with PbT-I cells (filled circle) or gBT-I cells (filled square) or no cells (open circle) and were sacrificed on days 4, 5 or 6 post-infection. Their brains were then analysed for the infiltration of PbT-I cells (left) or gBT-I cells (right). Data are pooled from 2–4 experiments. Data were compared using student t test (*, p<0.05).

Figure 4

Figure 4. PbT-I cells induce ECM in mice lacking endogenous CD8+ T cells.

B6 mice were either left undepleted (closed triangles) or were depleted of endogenous CD8+ T cells (-CD8) seven days before the adoptive transfer of 2×106 naïve PbT-I (filled square) or gBT-I cells (filled circle) or no cells (open triangles). The next day, mice were infected i.v. with 106 blood-stage PbA and monitored for the development of cerebral malaria. Data are pooled from three experiments. The difference in survival between the following groups was statistically significant (p<0.0001) as determined by the Log-Rank test: i. B6, -CD8, + PbT-I and B6, -CD8; ii. B6, -CD8, + PbT-I and B6, -CD8, + gBT-I.

Figure 5

Figure 5. PbT-I cells respond to P. chabaudi AS.

B6 mice were adoptively transferred i.v. with 2×106 CFSE-labeled PbT-I cells. The next day, mice were injected i.v. with 105 P. chabaudi AS. Six or seven days later, spleens were harvested and the proliferation of PbT-I was analyzed. (A) Representative histograms of CFSE-labeled PbT-I cells in the spleen of uninfected or P. chabaudi infected mice. (B) Number of PbT-I cells in the spleens of mice on days 6–7. The lines represent the mean and each data point represents a mouse. Data are pooled from three experiments. Data were compared using student t test (***, p<0.001).

Figure 6

Figure 6. PbT-I cells proliferate in response to irradiated sporozoites.

B6 mice were adoptively transferred with 2×106 CFSE-labeled PbT-I cells. The next day, mice were injected i.v. with 5×104–105 radiation attenuated sporozoites (RAS) or equivalent salivary extract. Four days later spleens were harvested and the proliferation of PbT-I cells was analyzed. (A) Representative histograms showing the proliferation of PbT-I cells in response to irradiated sporozoites. (B) Pooled data showing the number of divided PbT-I cells from four experiments. The lines represent the mean and each data point represents a mouse. Mice similarly treated but left until day 7 post-challenge showed no breakthrough in blood-stage infection indicating full attenuation of sporozoites. Data were compared by one-way ANOVA and Tukey's multiple comparison test (*, p<0.05).

Figure 7

Figure 7. PbT-I cells are primed mainly in the spleen when irradiated sporozoites are delivered by the i.v. route.

B6 mice were adoptively transferred with 106 CFSE-labeled PbT-I cells. The next day, mice were injected i.v. with 105 irradiated PbA sporozoites. Various organs (spleen, blood, liver, different lymph nodes) were harvested on days 1–4 post-vaccination and the activation and proliferation of PbT-I cells was analyzed by flow cytometry. For gating strategy to identify PbT-I cells see Figure S13. (A) Representative histograms showing the proliferation of PbT-I cells versus the upregulation of CD69 in the various organs. (B) Number of divided PbT-I cells in the different organs. The insert shows a more sensitive scale to identify the few divided PbT-I cells in the liver and lymph nodes on days three and four post-infection. (C) Percentage of CD69+ PbT-I cells in each organ on days 1-2. Error bars represent standard error of the mean. Data are pooled from three independent experiments.

Figure 8

Figure 8. CD8α+ DC are required for presentation of irradiated sporozoites delivered by the i.v. route.

B6 or Batf3-/- mice were adoptively transferred with 106 CellTracker Violet-labeled PbT-I cells then infected with irradiated PbA sporozoites. (A) Representative histograms show the proliferation of PbT-I cells in B6 and Batf3-/- mice on day 5 after injection with irradiated sporozoites. (B) Pooled data showing the proliferation of PbT-I cells from three experiments. The lines represent the mean and each data point represents a mouse. Data were compared by one-way ANOVA and Tukey's multiple comparison test (***, p<0.001).

Figure 9

Figure 9. PbT-I cells proliferate to greater numbers when sequentially primed with liver-stage then blood-stage PbA parasites.

B6 mice were adoptively transferred with 5×104 GFP-expressing PbT-I cells then on day 0 they were injected with 5×104 live sporozoites alone, or 5×104 radiation-attenuated sporozoites alone (RAS), or RAS followed 2 days later by 104 iRBC (RAS + iRBC), or nothing followed 2 days later by 104 iRBC (iRBC) or left uninfected (naïve). On day 7 spleens were harvested and the number of PbT-I cells enumerated. Data are pooled from four experiments. At the time of sacrifice, blood parasitemias were equivalent in the two groups of mice given iRBC (RAS + iRBC = 5.6±1.8; iRBC alone = 5.1±0.7). Data were compared by one-way ANOVA and Tukey's multiple comparison test (n.s., non-significant; **, p<0.01; ***, p<0.001).

Figure 10

Figure 10. Activated PbT-I cells confer protection against a sporozoite challenge.

(A) B6 mice were infected i.v. with 300 (n = 25), 520 (n = 22) or 900 (n = 15) PbA sporozoites and the % of mice that developed a blood-stage infection (black bars) by day 14 is shown. Data are pooled from three to five experiments. Data were analyzed using Fisher's exact test with no significant difference between groups (p>0.05). (B) B6 mice were adoptively transferred with 7×106 in vitro activated PbT-I cells (n = 20) or activated virus-specific gBT-I cells (n = 16) or Nil (n = 17). Two hours later, mice were infected i.v. with 520 sporozoites. Blood-stage parasitemia was monitored to day 14 post-infection. Shown is the percentage of mice that develop a blood-stage infection (black bars) by day 14. Data are pooled from three experiments. Data were analyzed using Fisher's exact test with the PbT-I treated group significantly different from the other two groups (p<0.0001) and no significant difference between these two groups (p>0.05).

Figure 11

Figure 11. Identification of a peptide antigen (NCYDFNNI) recognized by endogenous T cells and PbT-I cells from PbA-infected mice.

(A) B6 mice were infected with 106 blood-stage PbA then cured by treatment with chloroquine from days 4 to 6. On day 7 mice were adoptively transferred with peptide-labeled target cells and a day later spleens were recovered and examined for target cell killing. Data are from two experiments, each of which used a test peptide, NCYDFNNI, and control peptides (NNFDFNNL or NIYDFNFI; pooled). (B, C) IFNγ production by T cells responding to PbA. B6 mice were either left untreated (B) or were adoptively transferred with 5×104 PbT-I cells (C). All mice were then infected with 106 blood-stage PbA and cured of infection by chloroquine treatment from days 4 to 6. On day 7, spleens were removed and an intracellular cytokine assay performed to detect IFNγ expression by endogenous CD8 T cells (B) or PbT-I cells (C). Data are from two experiments, each of which used a test peptide, NCYDFNNI, and a control peptide (SIINFEKL; OVA). Data were compared using student t test (***, p<0.001; ****, p<0.0001).

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