Pathological changes of the spleen of the infected mice
To examine the pathological changes in the spleens of Plasmodium yoelii-infected mice, the mice were euthanized 12 days post-infection and the spleens were removed. As shown in Fig. 1A, the size and colour of the spleens in the infected group is bigger and darker compared with the normal control group. The weight of the spleen and the number of spleen cells from infected mice were significant higher than that from the normal control mice (***P<0.001,**P<0.01). Paraffin sections were made and stained with hematoxylin and eosin to observe the effects of infection on spleen microstructure. Splenic sinusoid hyperemia, germinal center hyperplasia and cellular infiltration of T cells were noted in the spleen of the infected mice(Fig. 1D).
The induced γδTCR+ cells and CD11b+γδTCR+ cells in the spleen of the infected mice
In order to examine the role of γδTCR+ cells, we compared scRNA-seq data from normal control spleen cells, to infected cells. After filtering cells, a total of 11656 cells in normal spleen and 9998 cells in infected spleen remained for analysis. After UMAP projection of cell clusters, there was a total of 12 distinct clusters representing major spleen groups. Feature plots showing the expression of Tcrg-C2 gene was cell-type specific, with predominance for expression in CD4+T cell and CD8+T cell from normal group (Fig. 2A). In contrast, Tcrg-C2 gene was mainly enriched in NKT cells/ γδT cells after infection. The frequency of cells expressing Tcrg-C2 among all cells from infected group was higher than normal control group. We observed the expression of CD3, CD19, as well as CD11b in Tcrg-C2-expressing cells (Fig. 2C). It is clear that the infected group has a much greater proportion of CD11b-expressing cells amongst Tcrg-C2-expressing cells than is the case with normal group. We then explored the expression γδTCR+CD3+ cells, γδTCR+CD19+ cells, γδTCR+CD11b+ cells by incubating with different fuorescence labeled markers. The γδTCR+cells were gated first, and then expression levels of CD3, CD19 and CD11b were examined by FACS (Fig. 2D). The proportions of γδTCR+ cells and γδTCR+ CD3+cells were higher (P < 0.05) compared with that in the normal mice. There was no obvious different in the γδTCR+CD3+ cells and γδTCR+CD19+ cells between the normal and infected hosts.
The differentially expressed genes (DEGs) analysis
Regarding the large amount of data obtained by RNAseq, we conducted a screen of the genes in 12dpi vs. control groups and obtained 4759 genes for further analysis. We utilized the ballgown package of R to analyze the DEGs of the String Tie-assembled and-quantified genes (P<0.05 or Q<0.05), and the differential expression multiple was screened as >1.5. Then, we obtained 330 DEGs: 212 were up-regulated and 118 were down-regulated (Fig. 3), where Gzmb, Gzma, Ccl5, Ccl4 and Ccl3 were significantly up-regulated and Il1b, Ccr7 and Il7r were significantly down-regulated.
The state of the Splenetic γδTCR+CD11b+cells
The states of the infected Itgam and Tcrg-C2 co-expressing cells (γδTCR+CD11b+ cells ) were determined by measuring the Klrk1, Klrc1, Sell, Cd44, Cd69, Il2ra, Fcgr3, Cd40, Pdcd1, Cd274, Btla, and Havcr2 gene expressions. Violin plots showed expression level of genes. The black whiskers extended from the bars represent the upper (max) and lower (min) adjacent values in the data. The black lines in the middle of the bars showed the median values (Fig. 4A). Klrc1 (NKG2A), Cd40 (CD40), Pdcd 1(PD-1), Havcr2 (CTLA-4) highly expressed in infected γδTCR+CD11b+ cells compared to normal cells. Comparing to normal γδTCR+CD11b+ cells, infected cells expressed lower levels of Klrk1, Sell, Cd274, Btla genes, which was validated by single-cell RNA sequencing. Similar phenomenon was observed in results of proportion of cells that expressed those genes within the whole γδTCR+CD11b+ population(Fig. 4B).
Cytokine Gene Expressions of γδTCR+ CD11b+Cells
Since the cytokine–cytokine receptor interaction was enriched in γδTCR+CD11b+ cells, we investigated the expression level of cytokine expression profiles in γδTCR+ CD11b+ cells. Although many cytokine genes are known to be involved in the infection, we filtered for some differentially expressed genes in two groups(Fig. 5). Notably, violin plots confirmed that Ifng, Il10, Fas and Csf1 were highly expressed in infected groups and the expressions of Il1b and Tnf were decreased. In line with the above findings, the proportion of cells that expressed those genes within the whole γδTCR+CD11b+ population showed the same trend (Fig. 5B).
The functional analyses of DEGs
After identifying DEGs, we analyzed the DEGs using Gene Ontology enrichment analysis to explore the potential functions of these genes. The up-regulated genes and down-regulated genes in the 12dpi vs. control cells were evaluated by GO analysis, respectively(Fig. 6). In comparison with the normal cells, the infected cells expressed high levels of genes were enriched in the items related to energy metabolism, including oxidative phosphorylation and respiratory electron transport chain. It revealed that in addition to the items related to energy metabolism, infammatory-related pathways, especially leukocyte activation involved in immune response, was also enriched among the up-regulated DEGs (Fig. 6B). The genes, including Ndufs8, Cox5b, Uqcrb, Lgals1, Pycard, and Ifng, were highly expressed in the 12dpi vs. control cells. Moreover, down-regulated genes related, such as Ccr7, Itch, Foxp1, Tcf7 and Cd44, were mainly enriched in alpha-beta T cell activation pathway. The analysis of the down-regulated genes showed that the functions of a series of genes were also related to myeloid leukocyte migration, such as Dusp1, Il1b, Ccr7, Fcer1g and Il17ra(Fig. 6D).
Phenotypic Changes in Splenetic γδTCR+ CD11b+ Cells
To study the characteristics of γδTCR+ CD11b+ cells during Plasmodium yoelii
infection, the expressions of Ly6g and Ly6c2 genes were detected. The scRNA-seq analysis showed that the γδTCR+ CD11b+ cells marked by high levels of Ly6c2 in both infected and normal groups(Fig. 7A,B). We then looked for evidence of different subsets expression γδTCR+ CD11b+ cells by incubating with different fuorescence labeled markers: Ly6G and Ly6C(Fig. 7C). The γδTCR+CD11b+ cells were gated first, and then expression levels of Ly6G and Ly6C were examined by FACS. The proportion of Ly6C+ Ly6G-γδTCR+ CD11b+ cells was higher than that of Ly6G+ Ly6C-γδTCR+ CD11b+ cells in the normal and infected spleen (P<0.05). There was no obvious different in the Ly6C+ Ly6G-δTCR+ CD11b+ cells between the normal and infected hosts.