CD4+ T cells display a spectrum of recall dynamics during re-infection with malaria parasites

Abstract

Children in malaria-endemic regions can experience repeated Plasmodium infections over short periods of time. Effects of re-infection on multiple co-existing CD4⁺ T cell subsets remain unresolved. Here, we examine antigen-experienced CD4⁺ T cells during re-infection in mice, using scRNA-seq/TCR-seq and spatial transcriptomics. TCR transgenic T_EM cells initiate rapid Th1/Tr1 recall responses prior to proliferating, while GC Tfh counterparts are refractory, with T_CM/Tfh-like cells exhibiting modest non-proliferative responses. Th1-recall is a partial facsimile of primary Th1-responses, with no upregulated effector-associated genes being unique to recall. Polyclonal, TCR-diverse, CD4⁺ T cells exhibit similar recall dynamics, with individual clones giving rise to multiple effectors including highly proliferative Th1/Tr1 cells, as well as GC Tfh and Tfh-like cells lacking proliferative capacity. Thus, we show substantial diversity in recall responses mounted by multiple co-existing CD4⁺ T cell subsets in the spleen, and present graphical user interfaces for studying gene expression dynamics and clonal relationships during re-infection.

Fig. 1. PbTII cells exhibit a spectrum of transcriptional states in the spleen after primary infection and antimalarial drug treatment.

A Schematic of scRNA-seq experiment to study PbTII cells at day 28 post-infection. B UMAPs of PbTII cell-derived scRNA-seq data before and after integration of our two independent experiments, this study and Soon et al.—cells coloured by experimental origin. C UMAPs of PbTII cell gene expression patterns for Th1 (Cxcr6, Ifng), Tfh (Cxcr5, Bcl6), T_CM (Ccr7, Sell, Tcf7, Klf2), proliferation (Mki67), and IFN-high (Ifit1, Ifit3, Irf7) genes. D UMAP of PbTII cell states depicted by colours and dotted boundaries. E Estimated cell-type abundance for GC Tfh, T_CM, and Th1-memory (red), compared with the same cDC2, GC B cells and follicular B cells in each panel, assessed across a Slide-seqV2 spatial transcriptomic map of mouse spleen at day 30 p.i.—scale bar: 500 µm: white boxes shown at higher magnification within each inset panel, with each dot representing a 10 µm bead. F Box plots showing % positive occurrence of GC Tfh, T_CM, or Th1-memory cells in each GC (each open circle denoting 1 GC). Cell abundance (computed using cell2location) >0.2 is considered a positive occurrence. Centre line indicates median and box limits upper and lower quartiles; two-sided Wilcoxon signed rank test: ****P < 0.0002 (exact values: P = 0.00019 for GC Tfh vs. Tcm; P = 1.134 × 10⁻⁵ for GC Tfh vs. Th1-memory).

Fig. 2. Re-infection triggers an early transcriptional response from Th1-memory PbTIIs.

A Schematic of scRNA-seq experiment to study in vivo responses of antigen-experienced versus naïve PbTII cells during re-infection. B–D Representative FACS plots showing B IFN-γ, C CXCR6, and D Ki67 expression in antigen-experienced PbTII cells at day 28 p.i. and 1 and 3 days post re-infection, compared to co-transferred naïve comparator PbTIIs. Data combined from two independent experiments showing similar results (n = 9 mice for day 28 p.i, 15 mice each for 1 and 3 days post-re-infection); two-way ANOVA with Šídák’s multiple comparison testing; ****P < 0.0001. E UMAP of antigen-experienced PbTII cells and naïve comparators, prior to and 1 day after re-infection. F UMAPs of PbTII expression of various genes associated with Th1 (Cxcr6, Ifng), Tfh (Cxcr5, Bcl6), T_CM (Ccr7, Sell, Tcf7, Klf2), proliferation (Mki67), Sostdc1⁺ cells, and early activation (Cd69).

Fig. 3. PbTII cells exhibit a spectrum of recall dynamics during re-infection.

A (Left) UMAP representation of antigen-experienced PbTII cells prior to and 1, 2, and 3 days post-re-infection; two apparent trajectories indicated by arrows; GC Tfh cells and Sostdc1⁺ Tfh cells marked with dotted boundaries. (Right) Th1, Tcm, and Tfh signature scores. (Bottom) Expression patterns for various genes associated with Th1 (Cxcr6, Ifng), Tfh (Cxcr5, Bcl6), Sostdc1⁺ cells, T_CM (Ccr7, Sell, Tcf7, Klf2), and proliferation (Mki67) states. B Volcano plots depicting the number and LogFC (lfc mean) of differentially expressed genes (genes with Bayes factor > 3) from comparing Th1 cells (Left), T_CM/Tfh cells (Right), and GC Tfh cells (Bottom) prior to and post re-infection. Number of significantly upregulated/downregulated genes and the top 10 upregulated/downregulated genes annotated on volcano plots.

Fig. 4. Transcriptome dynamics of Th1-recall predicts suggest early RNA processing associated with rapid effector function and subsequent proliferation.

A UMAP of antigen-experienced PbTII cells prior to and 1, 2, and 3 days post-re-infection, with only Th1-like cells coloured according to inferred pseudotime values (from 1-dimensional BGPLVM, GPfates), split into early, mid and late-pseudotime. B Expression dynamics for pseudo-temporally variable genes in Th1-like cells along pseudotime, genes grouped according to similar dynamics, represented as signature scores. C Summary of gene ontology enrichment analysis of biological processes associated with genes for selected groups from (B). X-axis represents negative log-transformed P-values that indicate the extent of enrichment of biological processes; Fisher’s exact test is used to identify over-represented GO terms. D Expression dynamics for gene groups 2–5 for Th1-like PbTII cells during primary infection; BP denotes Th1/Tfh bifurcation point. E Co-expression network analysis of genes (represented as nodes) in Dynamics 2 and 4. Edge weight corresponds to Spearman’s rho values (only showing rho > 0.2). Gene labels in the inset coloured according to the dynamic of origin. F Schematics of enriched biological pathways associated with genes from Dynamic 4. Genes found in Dynamic 4 are highlighted in red.

Fig. 5. Th1-recall is characterised by the upregulation of a select few genes.

A UMAP of PbTII cells after scVI integration of naïve and primary Th1 scRNA-seq data from Soon et al.. with Th1-recall scRNA-seq in this study (open circles) coloured according to pseudotime values (after 1-dimensional BGPLVM)—apparent Th1-recall trajectory indicated with an arrow. B (Left) Venn diagrams showing overlap in lists of differentially expressed genes (Bayes factor > 3) from peak Th1-primary (relative to naïve PbTIIs), compared to peak Th1-recall (relative to naïve PbTIIs). (Right) Summary of gene ontology enrichment analysis of biological processes associated with genes common or unique to each comparison; Fisher’s exact test used to identify over-represented GO terms. C Sankey plot schematic summarising dynamics of 1319 genes initially upregulated in Th1-primary cells (compared to naïve PbTIIs), as cells proceed to memory and then to peak Th1-recall (Up: upregulated, Down: downregulated, Stable: no significant change; at each stage in reference to the preceding stage). Full list of all 60 genes upregulated from Th1-memory to recall is shown on the right.

Fig. 6. TCR-diverse, antigen-experienced CD4⁺ T cells exhibit varied recall responses during re-infection.

A Representative FACS plot showing GFP⁺ PbTII cells, CD11a and CXCR3 expression, and CXCR6 and CXCR5 expression (orange: gated on CD11a^hiCXCR3⁺, grey: gated on CD11a^loCXCR3⁻) in CD4⁺ T cells prior to and 3 days after re-infection. Graphs show the percentage of CXCR6⁺ and CXCR5⁺ cells from CD11a^hiCXCR3⁺ versus CD11a^loCXCR3⁻ CD4⁺ T cells (n = 5 mice). Statistical analysis was performed using a Wilcoxon signed-ranks test. ****P < 0.0001. B Schematic of scRNA-seq and TCR-seq experiment to study CD11a^hiCXCR3⁺ polyclonal CD4⁺ T cells prior to and 3 days after re-infection. C UMAP of CD11a^hiCXCR3⁺ and inferred naïve (CD11a^loCXCR3^-) polyclonal CD4⁺ T cells. Cells sharing the same TCR chains connected with edges. Only families with four or more cells sharing the same TCR chains are shown in the plot. Naïve cells and iNKT cells are marked with dotted boundaries. D UMAP of CD11a^hiCXCR3⁺ cells expressing Th1, Tcm, and Tfh signature scores. E UMAP of CD11a^hiCXCR3⁺ cells expressing genes associated with Th1 (Cxcr6, Ifng), Tfh (Cxcr5, Bcl6, Pdcd1), T_CM (Ccr7, Sell, Tcf7, Klf2), proliferation (Mki67), Sostdc1⁺ cells, and Treg (Foxp3) cells. F UMAP of CD11a^hiCXCR3⁺ polyclonal CD4⁺ T cells prior to and 3 days post-re-infection. Differentiation trajectories are indicated with arrows. GC Tfh cells and Sostdc1⁺ Tfh cells are marked with dotted boundaries. G Volcano plots showing the number of differentially expressed genes (genes with Bayes factor > 3) comparing prior to and after re-infection for each inferred state: Th1 cells (Top-Left), T_CM/Tfh cells (Top-Right), GC Tfh cells (Bottom-Left), and Treg cells (Bottom-Right). Number of significantly upregulated/downregulated genes and the top 10 upregulated/downregulated genes annotated on volcano plots.

Fig. 7. TCR diverse polyclonal GC Tfh cells lack the capacity to proliferate upon re-infection.

A UMAP of CD11a^hiCXCR3⁺ polyclonal CD4⁺ T cells 3 days post-re-infection with cell states depicted by colours and dotted boundaries. Proliferating cells show Tfh/Th1 signature scores. B UMAP of proliferating Tfh-like cells and GC Tfh cells 3 days post-re-infection (Top) and UMAP of proliferating Th1-like cells and Th1 cells (Bottom) after regressing out G2M phase and S phase signature scores. C Violin plots showing expression of genes associated with proliferation (Mki67), GC Tfh (Pdcd1, Thy1—expressed at a low level in GC Tfh cells), Tfh (Cxcr5, Bcl6, and Tox2), and T_CM (Ccr7). D UMAP of CD11a^hi CXCR3⁺ polyclonal CD4⁺ T cells (with 5% spike-in of CD11a^loCXCR3^- naïve cells) showing GC Tfh cells highlighted in pink and TCR sharing (straight edges) from those proliferating Tfh-like cells (coloured by mouse of origin; n = 5 mice). E Gating strategy to identify proliferating and non-proliferating Tfh-like cells (Top). FACS plots and scatter plot of PD-1 expression on Ki67⁺ and Ki67^- Tfh-like cells (Bottom). FACS plots are representative of three independent experiments, with at least four mice per experiment (n = 13 total). Wilcoxon matched-pairs signed rank test was used to assess difference in PD-1 expression between Ki67⁺ and Ki67⁻ populations pooled from three independent experiments. F Sorting strategy for isolated T cell subsets from anti-malarial treated mice following infection with PcAS (Top). Representative histograms (from two independent experiments) showing CellTrace^TM Far Red intensity in naïve (no stimulation), naïve (anti-CD3/anti-CD28 stimulation), non-Tfh (anti-CD3/anti-CD28 stimulation), and GC Tfh (anti-CD3/anti-CD28 stimulation) populations prior to and 3 days post-stimulation (Bottom). G Experimental design: antigen-experienced T_CM/Tfh-like or non-Tfh cells (harbouring Th1-like Tem cells) were sorted from infected/drug-treated mice at 28 days post-infection. 360,000 cells were transferred into separate groups of naïve, congenically marked recipient mice, which were infected the following day. Spleens were assessed 5 days later. H Representative FACS plots showing gating strategy to detect CD4⁺ CD45.2⁺ cells, including a “no cell transfer control”, and scatter plots of numbers of splenic CD4⁺ CD45.2⁺ cells; one-way ANOVA with Šídák’s multiple comparison testing; ***P = 0.0008; **P = 0.0051. I Representative FACS plots and gating for assessment of CXCR5 and CXCR6 expression by CD4⁺ CD45.2⁺ cells, and corresponding scatter plots of numbers of CXCR5⁺ or CXCR6⁺ CD4⁺ CD45.2⁺ cells. Scatter plots show data from 2 independent experiments (n = 2/experiment for “no-cell transfer”; n = 5/experiment for other groups). Bars represent mean ± SEM; one-way ANOVA with Šídák’s multiple comparison testing, CXCR5 plot: *P = 0.0137, **P = 0.0068; CXCR6 plot **P = 0.0020, ***P = 0.0001.

Fig. 8. Th1-memory clones exhibit a robust proliferative Tr1 recall response during re-infection.

A UCSC genome browser tracks displaying genomic accessibility signals around the Il10 gene locus. (Top) Mean bulk ATAC-seq genome coverage. Boxes at the top of each coverage represent peaks called using MACS2. Data representative of two independent experiments showing similar results. (Middle) Accessibility profiles of 200 randomly selected single cells from plate-based single-cell ATAC-seq data. (Bottom) Percentage of cells with open accessibility at each genomic region B UMAP of PbTII cells and polyclonal CD4⁺ T cells showing mRNA expression of Ifng, Il10, Il2, Tnf, Ctla4, Lag3, Havcr2, and Tigit. C UMAP of PbTII cells (Left) and polyclonal CD4⁺ T cells (Right) with Il10 and Ifng co-expressing cells highlighted in red. D UMAP of CD11a^hiCXCR3⁺ polyclonal CD4⁺ T cells (with 5% spike-in of CD11a^loCXCR3⁻ naïve cells) showing TCR sharing (straight edges) from those cells co-expressing Il10 and Ifng (highlighted in open circles and coloured by mouse of origin; n = 5 mice).