Tissue-resident, memory CD8+ T cells are effective in clearing intestinal Eimeria falciformis reinfection in mice

Abstract

Eimeria, a cousin of malarial parasites, causes coccidiosis that results in huge losses in the poultry industry. Although live coccidiosis vaccines have been developed and used widely for the successful control of the disease, the mechanism underlying protective immunity remains largely unknown. Using Eimeria falciformis as a model parasite, we observed that tissue-resident memory CD8⁺ T (Trm) cells accumulated in cecal lamina propria following E. falciformis infection in mice, especially after reinfection. In convalescent mice challenged with a second infection, E. falciformis burden diminished within 48-72 h. Deep-sequencing revealed that CD8⁺ Trm cells were characterized by rapid up-regulation of effector genes encoding pro-inflammatory cytokines and cytotoxic effector molecules. While FTY720 (Fingolimod) treatment prevented the trafficking of CD8⁺ T cells in peripheral circulation and exacerbated primary E. falciformis infection, such treatment had no impact on the expansion of CD8⁺ Trm cells in convalescent mice receiving secondary infection. Adoptive transfer of cecal CD8⁺ Trm cells conferred immune protection in naïve mice, indicating that these cells provide direct and effective protection against infection. Overall, our findings not only explain a protective mechanism of live oocyst-based anti-Eimeria vaccines but also provide a valuable correlate for assessing vaccines against other protozoan diseases.

Keywords: Apicomplexa pathogen; Eimeria falciformis; intestinal immunity; protective immunity; resident memory T cell.

Figure 1

Protective immunity induced by primary E. falciformis (E.f.) infection targeted early schizonts. (A) Experimental design for E. falciformis primary infection and reinfection. Naïve mice remained uninfected for the duration of study. For the infected mice, each infection doses is indicated. (B) Survival curves after primary infection and secondary infection with 5000 oocysts of E. falciformis. n=9 per treatment group. (C) Kinetics of oocyst output at 6-13 days post primary infection and reinfection with 5000 oocysts of E. falciformis, # denotes the mice with severe diarrhea, to the extent that faeces collection was not possible. n=6 per treatment group. (D) Body weight change at 8 days post primary infection and secondary infection with 5000 E. falciformis. n=4 per treatment group. (E) H&E staining of cecum from mice at 5 days after primary infection and reinfection with 5000 E. falciformis. n=3 per treatment group, magnification ×200. (F) Histology scoring of cecum at 5 days after primary infection and reinfection with 5000 E. falciformis. (G) Frozen sections for E. falciformis (green) and DAPI (blue) in cecum from single infected and reinfected mice (to visualize parasites in the cecum at 8 and 24 h post infection, mice were infected twice with E. falciformis expressing GFP and reinfected after 3 weeks. n=3 per group, magnification ×1000. (H) Quantification the E. falciformis in cecum at 8, 24, 48 and 72 hours post primary infection and reinfection. n=3 per group, HPF, high power field (×1000). Bar graphs capture mean ± SD from three independent replicates, **p ≤ 0.01, PI, primary infection; SI, secondary infection.

Figure 2

CD8⁺ T cells rose rapidly in the lamina propria of cecum after reinfection with E. falciformis. (A) H&E staining of cecum at 24 hours post primary infection and secondary infection with 5000 E. falciformis. n=3 per treatment group, magnification ×200. (B) Multi-parameter immunohistochemical staining of cecal CD4⁺ and CD8⁺ T-cells in three groups after 24 h primary infection and reinfection. n=3 per treatment group, magnification ×600. (C) Flow cytometric for expression of CD4 ⁺ and CD8 ⁺ on CD3 ⁺ T cells in IEL of cecum at 24 hours after primary infection and reinfection with 5000 E. falciformis. n=6 per treatment group. (D) Flow cytometric for expression of CD4 ⁺ and CD8 ⁺ on CD3 ⁺ T cells in LPL of cecum at 24 hours after primary infection and reinfection. n=6 per treatment group. Results are mean ± SD from three independent experiments, ns, no statistical significance, **p ≤ 0.01, PI, primary infection; SI, secondary infection.

Figure 3

Cecal CD8⁺ Trm cells (in blue color) rose rapidly in reinfected mice. (A) Multi-parameter immunohistochemical staining for CD8⁺, CD69⁺ and CD103⁺ Trm cells enriched in the cecum at 24 hours post primary infection and reinfection with 5000 E. falciformis. n=3 per treatment group, scale bar =50 μm. (B) Representative flow cytometric plots for expression of CD69 ⁺ and CD103 ⁺ on CD8⁺ T cells in LP of cecum at 24 hours post primary infection and reinfection with 5000 E. falciformis. n=6 per treatment group. (C) Summary bar graph of CD8⁺Trm, gated on live CD8⁺ cells. (D) t-Distributed Stochastic Neighbor Embedding (tSNE) plots of LP-infiltrating single CD3⁺ cells. Results are mean ± SD from three independent experiments, ns, no statistical significance, **p ≤ 0.01.

Figure 4

Cecal CD8⁺ Trm cells produced high levels of pro-inflammatory cytokines and cytotoxic effectors molecules after E. falciformis reinfection. (A) Schematic of SMART-seq experiments. CD8⁺ T-cells were obtained from cecal lamina propria of naïve and reinfected mice (24 hours post reinfection) for comparison. (B) Heatmap of selected differentially expressed genes in two groups (>2 fold; p < 0.05). (C) Volcano plots selected differentially expressed genes between naïve CD8⁺ T and CD8⁺ Trm cells. (D) Flow cytometry plots representing IFN-γ and/or TNF-α production by cecal CD8⁺ Trm cells. n=6 per treatment group. (E) Summary bar graph of IFN-γ and/or TNF-α producing CD8⁺ Trm cells in LP, **p ≤ 0.01, PI, primary infection; SI, secondary infection.

Figure 5

CD8⁺ Trm cells were responsible for protection against E. falciformis infection. (A) Experimental design for treatment with FTY720 scheme. Mice were given FTY720 by intraperitoneal injection for 5 d before primary or secondary infection and during infection with E. falciformis. (B) Kinetics of oocyst output of mice treated or untreated with FTY720 at 6-13 days post reinfection with 5000 E. falciformis. n=6 per treatment group. (C) Body weight change of mice treated or untreated with FTY720 at 8 days post reinfection with 5000 E. falciformis. n=6 per treatment group. (D) Representative flow cytometric plots for expression of CD69 and CD103 on CD8⁺ T-cells in LPL from mice treated or untreated with FTY720 at 24 hours post reinfection with 5000 E. falciformis. n=6 per treatment group. (E) Summary bar graph of CD8⁺ Trm, gated on live CD8⁺ cells. (F) Kinetics of oocyst output of mice received CD8⁺ Trm or PBS at 6-13 days post infection with 100 E.falciformis. n=5 per treatment group. (G) Total oocyst output of mice received CD8⁺ Trm or PBS. n=5 per treatment group. Results are mean ± SD from three independent experiments, ns, no statistical significance between treatment groups, **p ≤ 0.01, PI, primary infection; SI, secondary infection.