γδ T Cells Are Required for the Induction of Sterile Immunity during Irradiated Sporozoite Vaccinations

Abstract

Whole-sporozoite vaccines confer sterilizing immunity to malaria-naive individuals by unknown mechanisms. In the first PfSPZ Vaccine trial ever in a malaria-endemic population, Vδ2 γδ T cells were significantly elevated and Vγ9/Vδ2 transcripts ranked as the most upregulated in vaccinees who were protected from Plasmodium falciparum infection. In a mouse model, absence of γδ T cells during vaccination impaired protective CD8 T cell responses and ablated sterile protection. γδ T cells were not required for circumsporozoite protein-specific Ab responses, and γδ T cell depletion before infectious challenge did not ablate protection. γδ T cells alone were insufficient to induce protection and required the presence of CD8α⁺ dendritic cells. In the absence of γδ T cells, CD8α⁺ dendritic cells did not accumulate in the livers of vaccinated mice. Altogether, our results show that γδ T cells were essential for the induction of sterile immunity during whole-organism vaccination.

Figure 1

Vδ2 T cell levels after the fifth vaccination were highest in vaccinees who remained uninfected throughout follow-up a) Representative histogram showing two populations of γδ T cells identified by differential levels of γδ TCR expression (top panel) and an overlay of Vδ2 population on total γδ T cells (bottom panel). Comparison of the percentage of (b) Vδ2⁺γδTCR⁺ T cells and (c) Vδ2⁻γδTCR⁺ T cells in vaccinees who remained uninfected throughout follow-up (blue dots) compared to vaccinees who developed parasitemia (red dots) or to unvaccinated individuals (black dots). Green dots are the unvaccinated individuals who remained uninfected. (d) Heatmap with hierarchical clustering of RNAseq data showing the top 15 genes that were differentially expressed between vaccinated/uninfected (VU), vaccinated/infected (VI) and unvaccinated (UV) study volunteers 3 days after the fifth vaccination. Upregulated genes are denoted in green and downregulated genes are in red.

Figure 2

Comparison of sterile protection after PbSPZ challenge in a) vaccinated C57Bl/6, γδ KO and BATF3KO mice. b) Representative flow cytometry plots showing the depletion of total γδ T cells and Vγ4⁺ T cells after administration of the GL3 and UC310A6 mAb respectively. c) Comparison of sterile protection in mice depleted of total γδ T cells using the GL3 mAb prior to each vaccination or before challenge. d) Comparison of sterile protection in mice depleted of the Vγ4⁺ γδ T cells using the UC310A6 mAb prior to each vaccination or before challenge. e) Representative flow cytometry plots of CD8 depletions in protected mice. f) Effect of CD8 depletion on protection after rechallenge. Sterile protection was defined as absence of blood stage parasitemia after challenge with 10³ P. berghei ANKA sporozoites. All vaccination studies were done twice.

Figure 3. γδ T cells were required during irradiated PbSPZ vaccination to induce effector αβ T cells

Representative flow plot of (a) CD8⁺CD11a^hiKLRG1⁺ cells and (c) CD4⁺CD11a^hiKLRG1⁺ cells in the γδKO mice and C57Bl/6 mice in whole blood at Day 31 (3 days after last vaccination). Comparison of the percentage of (b) CD8⁺CD11a^hiKLRG1⁺ and (d) CD4⁺CD11a^hiKLRG1⁺ in C57Bl/6 mice treated with GL3 mAb (red dashed line), C57Bl/6 mice treated with UC310A6 mAb (purple solid line), γδKO (red solid line), BATF3KO (black line) control C57Bl/6 mice that did (blue solid line) or did not receive anti-KLH (“isotype” blue dashed line) mAb. N=10 in each group. Data are presented as medians with interquartile ranges measured 3 days after each vaccination (days 3,17 and 31) and the day of challenge (day 66). (e) Comparison of antibody titers (expressed as OD) to the repeat region of the P. berghei CSP in sera from γδKO and C57Bl/6 mice at day 66.

Figure 4. Absence of γδ T cells impaired antigen-specific immune responses and protection from infectious challenge

(a) Representative flow plots of liver cells gated on CD8 showing the percentage of IFNγ⁺ events in the unstimulated and PfTRAP₁₃₀ peptide-stimulated cells in C57Bl/6 and γδKO mice. (b) Comparison of IFNγ⁺ CD8 T cells in γδKO (red) and C57Bl/6 mice (blue) in the liver and spleen. The values shown were determined by subtracting the control unstimulated from the PbTRAP₁₃₀ stimulated samples. Data are presented as medians with interquartile ranges.

Figure 5. CD8α⁺ DC and B220⁺ γδ T cells in SPZ vaccinated mice

(a) Representative flow cytometry plot of CD8α DC in the liver identified as CD8⁺B220⁻ in γδKO and C57Bl/6 mice (D66). Events were gated on CD3⁻CD11c⁺. (b) Comparison of CD8⁺B220⁻ cell percentage in C57Bl/6, γδKO, BATF3KO and C57Bl/6 treated with GL3 mAb.