Figure 1.
Langerhans cells and langerin+ dermal DCs are dispensable for anti-CS CD8+ T cell responses.
MuLangerin-DTR/EGFP and C57BL/6 mice received a single IP injection of DT (1 μg). 24 hours after DT treatment, 5×103 naive OT-1 cells were adoptively transferred to recipient mice. Mice were immunized through the bites of 20 irradiated P. berghei CS5M-infected mosquitoes 1 day after cell transfer and 2 days after DT treatment. A. Percentage of OT-1 cells of total CD8+ T cells recovered 10 days after sporozoite inoculation. B. Percentage of OT-1 cells producing IFN-γ as determined by intracellular staining and flow cytometry 10 days after inoculation by irradiated P. berghei CS5M–infected mosquito bites (mean ± SEM; n = 4–5/group). Data representative of 2 similar experiments.
Figure 2.
Sporozoite migration to the DLN is required for robust CD8+ T cell priming.
A. Naïve mice were injected ID with 2×104 P. berghei CS5M sporozoites treated for 20 minutes with 25 ug/ml of mAb, 2G3 (isotype control) or anti-P. berghei CS (3D11), or 16 ug/ml of Fab fragments prepared from the 3D11 antibody. Total RNA was isolated from DLNs at the indicated times. Parasite burdens were quantified by RT-PCR and pooled from 2 similar experiments; mean ± SEM, n = 10/group. B. 5×103 naïve OT-1 cells were transferred to mice. 24 hours later, mice were injected ID with 2×104 irradiated P. berghei CS5M sporozoites treated with 25 ug/ml 2G3 or 3D11 mAb or 16 ug/ml 3D11:Fab. Proportion of CD8+ T cells of OT-1 origin recovered 10 days post-inoculation; n = 5/group, mean ± SEM. Data representative of 2 similar experiments. C. 5×103 naïve OT-1 cells were transferred to mice. Mice were injected ID with 2×104 irradiated P. berghei CS5M or P. berghei CS5MΔN sporozoites 1 day after cell transfer. Proportion of CD8+ T cells of OT-1 origin recovered 10 days post-inoculation; n = 5/group, mean ± SEM. Data are representative of 4 similar experiments. D. 5×103 naive OT-1 cells were transferred to mice. 1 day after transfer mice were immunized IV with 2×104 irradiated P. berghei CS5M sporozoites or P. berghei CS5MΔN sporozoites. Expansion of OT-1 cells was measured 10 days after inoculation; mean ± SEM, n = 4–6/group. Data representative of 3 similar experiments.
Figure 3.
Localization, quantification, and dynamics of sporozoites in the DLN.
Immunofluorescence (IF) images depicting sporozoites and CS protein in the DLNs at various time points after ID inoculation with 1×105 P. berghei CS5M sporozoites. A. IF images of a DLN 30 minutes post-inoculation. Box 1 and 2 indicate enlarged areas of a DLN montage. Arrowhead denotes a sporozoite in the subcapsular sinus. Subcapsular sinus abbreviated as SCS. Colors of the word labels correspond to the colors of the stains here and throughout. White dotted line demarcates the paracortex. B. IF images of a DLN 1 hour post-inoculation. Box 3 indicates a maximum intensity projection of a 30 μm z stack. Box 4 and 5 are higher magnification images of sporozoites in the DLN 1 hour after ID inoculation. C. Number of sporozoites in whole DLNs at various time points after ID inoculation. The mean from one experiment is shown and is representative of 8 similar experiments. D. IF image of whole parasites interacting with DCs 5 and 8 hours post-inoculation. White dotted line marks boundary between the medullary sinus and LN parenchyma. Box 6 is an enlarged image of a DC-associated sporozoite. E. IF images of CS-positive events in a DLN 8 hours post-inoculation. White dotted line indicates boundary between the medullary sinus and LN parenchyma or the B cell follicle and cortex. Box 7 and 8 are higher magnification images of CS-positive events associated with DCs. F. Number of CS-positive events associated with DCs in the DLNs at 5 and 8 hours post-inoculation. Mean from one experiment is shown and is representative of 2 similar experiments. G. Maximum intensity projection of a 2-photon image from a CD11c-EYFP DLN 5 hours after ID inoculation with 1×105 P. berghei GFP sporozoites (pseudo-colored red). Second-harmonic generation (SHG, capsule); z stack of 30 μm. H. Time course of sporozoite uptake by a DC taken 3 hours after injection by infectious mosquito bites. Time, min:sec. See also S1 and S2 Movies.
Figure 4.
CD8α+ DCs present sporozoite antigens to CD8+ T cells in the LN cortex.
A. 2×106 naïve OT-1 cells were transferred to mice 1 day before ID inoculation with 1×105 irradiated P. berghei CS5M sporozoites. Popliteal LNs were harvested at the indicated time points and confocal images of popliteal LNs were prepared from 30 μm thick sections. White dotted line demarcates the cortex. B stands for B cell follicle. White arrowheads indicate examples of CD8+ T cell clusters. Representative images from 4 independent experiments with 2 mice per time point. B. Representative OT-1 cluster 8 hours after ID inoculation of P. berghei CS5M sporozoites. The phenotype of the DC within the cluster (depicted with a white dotted circle) was determined by histo-cytometry; CD8α+ DC signal (top panel) and CD11b+ DC signal (bottom panel) within the cluster. C. Representative histo-cytometry scatter plots depicting the percentage of all imaged DCs (black dots) and DCs associated with OT-1 clusters (red dots) in DLNs 8 and 16 hours after ID inoculation of sporozoites. D. The percentages of OT-1 cluster-associated CD8α+ or CD11b+ DCs (red bars) vs. all imaged DCs (black bars) were quantified from 3 independent experiments with 4 DLNs/time point (mean ± SEM).
Figure 5.
CD8+ T cell activation correlates with durable interactions between CD8+ T cells and DCs.
A-C. TdTomato-expressing polyclonal CD8+ T cells and GFP-expressing OT-1 cells were transferred to CD11c-YFP mice 1 day before ID inoculation with 1×105 irradiated P. berghei CS5M sporozoites. A. Tracks of polyclonal and OT-1 cells in the popliteal LN acquired by MP-IVM 12 hours after ID inoculation of sporozoites. Mean speed analysis (B) and confinement ratio (C) of TdTomato polyclonal CD8+ T cells and GFP OT-1 cells in situ, mean ± SEM. Data points represent individual cells from one experiment, representative of four similar experiments with mean value indicated. D and E. 1×106 OT-1 cells were transferred to naïve mice before ID inoculation with 1×105 irradiated P. berghei CS5M or P. berghei ANKA (WT) sporozoites. Single cell suspensions were prepared from popliteal LNs at the indicated time points. D. Proportion of OT-1 cells expressing CD69 based on a gate drawn on naïve OT-1 cells as controls. E. Percentage of OT-1 cells expressing IFN-γ at the indicated time points after ID inoculation. Data are representative of 2 similar experiments; mean ± SEM, n = 6/group. See also S3 Movie.
Figure 6.
CD8α+ DCs are necessary for the presentation of sporozoite antigens to CD8+ T cells.
A and B. WT and Batf3−/− mice received 1×106 CFSE-labeled OT-1 cells 1 day prior to ID injection of 2×104 irradiated P. berghei CS5M sporozoites. DLNs were collected 3 days post-inoculation. A. Representative CFSE profiles of OT-1 cells from 1 of 3 similar experiments. B. Expansion of OT-1 cells in the DLN; mean ± SEM, n = 7/group. Data are representative of 3 similar experiments. C. 0.5–1×104 naive OT-1 cells were transferred to WT and Batf3−/− mice 1 day before immunization via 20 irradiated P. berghei CS5M-infected mosquito bites. Responses were examined 8, 14, and 17 days post-inoculation. Proportion of CD8+ T cells of OT-1 origin after P. berghei CS5M-infected mosquito bites. Data are pooled from 2 similar experiments mean ± SEM; n = 9/group (day 8) and n = 6/group (day 14 and 17).
Figure 7.
Summarizes Figs. 1–6 as a model.
Sporozoite-mediated delivery of malaria antigens to the DLNs is required for CD8+ T cell priming. Anopheline mosquitoes inject sporozoites into the skin of their mammalian host, a tissue populated by epidermal Langerhans cells, langerin+ dermal DCs (CD207+), and langerin- dermal DCs (CD207-). Following sporozoite inoculation into the dermis, a proportion of sporozoites enter the DLNs via the afferent lymphatic vessel and are found in close association with CD169+ macrophages lining the subcapsular and medullary sinuses. LN-resident DCs directly sample and phagocytose sporozoites and sporozoite-derived CS particles within the LN parenchyma. Sporozoite-specific CD8+ T cells are primed by LN-resident CD8α+ DCs residing along the cortical ridge and the T cell zone of the DLN as early as 8 hours after immunization. Printed with permission from Heidi Sinsel, 2014.