Unique transcriptional profile of liver-resident memory CD8+ T cells induced by immunization with malaria sporozoites

Abstract

Sterile immunity against live Plasmodium infection can be achieved by immunization with radiation-attenuated sporozoites. This protection is known to be mediated in part by antigen-specific memory CD8(+) T cells, presumably those residing in the liver. We characterized and compared the transcriptional profile of parasite-specific memory CD8(+) T cells residing in the liver and spleen after immunization of mice with irradiated sporozoites. Microarray-based expression analysis of these memory CD8(+) T cells indicated that liver-resident memory cells display a distinct gene expression profile. We found major differences in the expression of immune function genes as well as genes involved in the cell cycle, cell trafficking, transcription and intracellular signaling. Importantly, the malaria parasite-induced liver-resident CD8(+) T cells display a transcriptional profile different to that described for CD8(+) T cells following other microbial challenges.

Figure 1. Experimental design and cell isolations

(A) Naive CD8Py transgenic cells (Thy1.1⁺) were adoptively transferred into BALB/c mice (Thy1.2⁺) which were then immunized with irradiated P. yoelli sporozoites. 30-45 days later, Thy1.1⁺ CD8⁺ T cells were isolated by FACS. (B) Expression of CD44 on Thy1.1⁺ CD8⁺ T cells day 45 after immunization. (C) FACS plots showing the T cell populations prior to and after cell sorting. Number indicates percentage among total lymphocytes. Data are representative of three independent cell isolations.

Figure 2. Overview of microarray analysis

(A) Total number of genes that were differentially expressed compared to naive control. (B) Principal component analysis (PCA) to all nine arrays (3 in each group) showing distinct global gene expression in naive, spleen and liver resident memory cells sorted in Figure 1.

Figure 3. Differential gene expression is observed between liver and spleen resident memory CD8⁺ T cells

(A) Differentially expressed genes were selected based on a cutoff of absolute fold change > 1.8 and FDR (q-value) <0.1. Cluster analysis was performed on the differentially expressed genes using hierarchical clustering (B) validation of microarray results assessed by RT-PCR from independently sorted samples.

Figure 4. GO analysis of liver and spleen memory CD8⁺ T cells

(A) Overexpression of GO terms. Dotted line represented a enrichment score of 1.3 (p-value of 0.05). Numbers in brackets represented total number of genes in that specific GO term. (B) Fold-changes between liver and spleen memory CD8⁺ T cells of indicated genes. (C) Expression of Ki67 and (D) IL7R/CD127 between naive, spleen- and liver-memory CD8Py cells one months and five months post immunization. Data are representative of three independent experiments.

Figure 5. Fold changes of genes in specific GO terms in liver and spleen memory CD8⁺ T cells

Fold-change of genes of (A) chemokine and cytokine receptors and (B) carbohydrate binding. (C) Protein expression of indicated chemokine receptor genes. Data are representative of two independent experiments. (D) Fold-change of genes in transcription and (E) in intracellular signaling. (F) Protein expression of Eomes and T-bet among naive-, spleen- and liver-memory CD8Py cells. Data are representative of three experiments.

Figure 6. GSEA analysis of liver-resident memory CD8Py T cells

Protein expression of (A) GZMB and (B) CD69 among spleen and liver memory CD8Py. (C-E) GSEA was performed to determine whether up-regulated effector genes set [19] (C), repeated immunization gene set [21] (D) and exhaustion associated gene set [22] (E) show specific enrichment to liver memory CD8Py induced by irradiated sporozoite immunization. (F) Fold changes (as liver/spleen) of selected exhaustion markers in sorted Thy1.1⁺CD8⁺ memory cells from mice immunized with irradiated P. yoelii sporozoites. Dotted lines represent fold-change cutoff = 1.8, * indicates FDR < 0.1 (G) Histograms showing expression of CTLA-4, LAG3, CD160 and PD1. Plots are gated on Thy1.1⁺ CD8Py. Data are representative of three experiments.