SARS-CoV-2 inflammation durably imprints memory CD4 T cells

Abstract

Memory CD4 T cells are critical to human immunity, yet it is unclear whether viral inflammation during memory formation has long-term consequences. Here, we compared transcriptional and epigenetic landscapes of Spike (S)-specific memory CD4 T cells in 24 individuals whose first exposure to S was via SARS-CoV-2 infection or mRNA vaccination. Nearly 2 years after memory formation, S-specific CD4 T cells established by infection remained enriched for transcripts related to cytotoxicity and for interferon-stimulated genes, likely because of a chromatin accessibility landscape altered by inflammation. Moreover, S-specific CD4 T cells primed by infection had reduced proliferative capacity in vitro relative to vaccine-primed cells. Furthermore, the transcriptional state of S-specific memory CD4 T cells was minimally altered by booster immunization and/or breakthrough infection. Thus, infection-associated inflammation durably imprints CD4 T cell memory, which affects the function of these cells and may have consequences for long-term immunity.

Fig. 1.. S-specific CD4 T cells form a distinct cluster in the activation-induced marker assay.

(A) Cohort design of COVID-19 vaccine-primed and SARS-CoV-2 infection-primed adults receiving third dose of BNT162b2 vaccine. Pre-booster and post-booster sampling events are indicated. (B) Representative flow cytometry plots for vaccine- and infection-primed participants’ CD4 T cells at pre-booster and post-booster time points for expression of CD69 and CD137 after the activation-induced markers (AIM) assay. Frequency shown in red. (C) Summary data for vaccine-primed (orange, n=8) and infection-primed (purple, n=8) participants’ PBMC after AIM assay. P-values by Wilcoxon matched-pairs signed rank test. (D) S_167-180 HLA class II tetramer and CD45RA expression plots for vaccine- and infection-primed participants at the post-booster time point. Summary data shown for vaccine-primed (orange, n=8) and infection-primed (purple, n=8) participants. Statistical significance was determined by Wilcoxon test. Frequency shown in red. (E) Spearman correlation between frequency of CD69⁺CD137⁺ CD4 T cells after AIM and the frequency of S_167-180⁺ CD4 T cells at the post-booster time point (n=16). (F) Representative plots for CD45RA and CCR7 expression by S_167-180⁺ CD4 T cells from vaccine- and infection-primed participants. Data from the same experiment in (D). P-value by Wilcoxon test. Frequency shown in red. (G) Experimental design schematic. UMAP projection of CD4 T cells from the AIM assay, pooled across samples and clustered using gene expression. (H) Scaled expression of TNFRSF4 (OX40), TNFRSF9 (CD137), TFRC (CD71), LTA. (I) Gene ontology for differentially expressed genes at adjusted P<0.05 for S-reactive CD4 T cell compared to CD4 T cell cluster. (J) Top 20 clonotypes by frequency in the S-reactive CD4 T cluster. (K) Per-participant TCR overlap between S-reactive CD4 TCRs and the Adaptive Biotechnologies SARS-CoV-2-reactive TCR database. (L) Distribution of epitope specificity of S-reactive CD4 T cells as inferred from the reference SARS-CoV-2 TCRβ dataset. See also fig. S1.

Fig. 2.. Infection-primed S-specific CD4 T cells exhibit a cytotoxic profile.

(A) Scaled expression of IFNG, IL2, TNF, and GZMB. (B) Scaled expression for IL2 and GZMB between cohorts at pre- and post-booster timepoints. Nominal P-values reported. (C) Polyfunctionality analysis. (D) S-specific CD4 T cells projected onto UMAP and clustered for gene expression of 27 selected parameters. UMAP colored by density and split by cohort. (E) Scaled expression of differentially expressed genes at adjusted P<0.01 for each cluster. (F) Vaccine-primed versus infection-primed S-specific CD4 T cell cluster distribution at pre- and post-booster time points (unpaired two-way ANOVA with Sidak posttest). See also fig. S2.

Fig. 3.. Inflammation during priming results in durable transcriptional effects in S-specific CD4 T cells.

(A) PCA of post-booster vaccine-primed (orange) and infection-primed (purple) S-specific CD4 T cells. Each symbol indicates one participant. (B) Heatmap of differentially-expressed genes between post-booster vaccine- and infection-primed cohorts. (C) Gene ontology analysis for differentially-expressed genes at adjusted P<0.05 for infection-primed S-specific CD4 T cells at post-booster time point. (D and E) GSEA for (D) “interferon alpha response” and (E) “interferon gamma response” gene sets for S-specific CD4 T cells. Light gray line indicates pre-booster time point and dark gray line indicates post-booster time point. (F) GSEA results for hallmark gene sets enriched at FDR<0.05 post-booster. Positive enrichment scores denote enrichment towards the infection-primed cohort in (D to F). See also fig. S3.

Fig. 4.. Vaccine-primed and infection-primed S-specific CD4 T cells have distinct epigenetic landscapes.

(A) Study schematic of post-vaccine and post-infection sampling events for mRNA vaccine-primed and SARS-CoV-2 infection-primed adults, respectively. (B) UMAP of stimulated CD4 T cells pooled across samples and clustered for gene expression. (C) Difference in scaled expression for IFI44L and HLAC between post-infection and post-vaccination samples. Nominal P-values shown as determined by DESeq2. (D) GSEA for “interferon alpha response” and “interferon gamma response” gene sets for transcriptional profiling of S-specific memory CD4 T cells. Positive enrichment scores indicate enrichment for the post-infection cohort. (E) Heatmap open chromatin regions (OCRs) in the S-specific CD4 T cells post-vaccination (yellow) and post-infection (purple). (F) Number of differentially accessible regions (DARs) shown (E) at a nominal P-value<0.05. (G) Representative ATAC-seq tracts shown at the FRP1 and LGALS3BP loci. (H and I). Enrichment of transcription factor (TF) binding motifs over background in the top differentially-accessible OCRs based on nominal P-value <5×10⁻³) in (H) post-vaccination S-specific memory CD4 T cells and (I) post-infection S-specific memory CD4 T cells. (J) TFs shown for the -log₁₀ Padj value from post-infection OCR motif analysis against the TF predicted by ChEA3 using the infection-primed gene expression data. See also fig. S4.

Fig. 5.. Breakthrough infection minimally alters the transcriptional profile of S-specific CD4 T cells.

(A) Breakthrough SARS-CoV-2 infection in vaccine-primed adults after third dose of BNT162b2 vaccine. (B) Representative flow plots for post-booster vaccine- and infection-primed participants and post-breakthrough samples for expression of CD69 and CD137 after AIM assay. Frequency shown in red. (C) Summary plots of CD69⁺CD137⁺ coexpression in CD4 T cells at post-booster and post-breakthrough time points. P-values by Wilcoxon matched-pairs signed rank test (top) and Wilcoxon unpaired test (bottom). (D) PCA of transcriptional profiling of post-booster vaccine-primed (orange) and infection-primed (purple) and post-breakthrough (red) S-specific CD4 T cells. Each symbol indicates one participant. (E) S-specific CD4 T cells projected onto UMAP and clustered as described in Fig. 2D. Bar graphs of vaccine- versus infection- primed S-specific CD4 T cell cluster distribution at post-booster and post-breakthrough time points (two-way ANOVA with Sidak’s posttest). (F) Volcano plot showing differentially expressed genes at adjusted P<0.05. Genes in purple denote enrichment in infection-primed S-specific CD4 T cells, orange for vaccine-primed post-booster S-specific CD4 T cells, and red for post-breakthrough S-specific CD4 T cells. (G) Summary bar graph of DEGs in (F). (H) GSEA “interferon alpha response” and “interferon gamma response” gene sets in infection-primed samples. (I and J) GSEA for Hallmark gene sets at FDR<0.05 when comparing (I) infection-primed, post-booster samples to post-breakthrough and (J) vaccine-primed, post-booster samples to post-breakthrough. Positive enrichment scores denote enrichment for the post-breakthrough samples and negative enrichment scores signify enrichment for post-booster samples. See also fig. S5.

Fig. 6.. Clonotypes enriched for the vaccine-primed gene signature can undergo inflammatory imprinting by breakthrough infection.

(A) Gene set variation analysis (GSVA)-derived enrichment scores for the “vaccine” and “infection” signatures plotted in 2-D Cartesian space for S-specific CD4 T cells pooled from all individuals and split by time point and cohort. Median frequencies of first and third quadrants in black. Summary plots for each individual shown. (B to D). Summary data at each time point. P-values by Wilcoxon test. (E and F). Enrichment scores for the “vaccine” and “infection” signatures for (E) the four clonotypes from infection-primed participants and (F) vaccine-primed participants that repeated in all time points shown. P-values by Wilcoxon test. See also fig. S6.

Fig. 7.. Infection-primed transcriptional signature confers a proliferative disadvantage.

(A) Gene ontology analysis for differentially expressed genes at Padj<0.05 for vaccine-primed S-specific CD4 T cells relative to infection-primed cells at post-booster time point. (B and C) GSEA for (B) “mitotic spindle” and (C) “G2M checkpoint” gene sets for S-specific CD4 T cells. Light gray line indicates pre-booster time point and dark gray line indicates post-booster time point. Negative enrichment scores denote enrichment towards the vaccine-primed cohort. (D) Study schematic of in vitro proliferation experiments with vaccine-primed and infection-primed post-booster samples. PBMCs were stained with CTR and stimulated with S peptides and IL2 for 5 days. (E and F) Representative flow cytometry plots and summary data for vaccine- (n=9) and infection-primed (n=10) participants’ (E) CD4 T cells and (F) B cells post-booster for CD71 and CellTrace Red (CTR) after PBMC stimulation with S peptides. (G) Spearman correlation between frequency of CTR^loCD71⁺ CD4 T cells and CTR^loCD71⁺ CD19⁺ cells. (H) Representative flow cytometry plots and summary data for vaccine- (n=9) and infection-primed (n=10) participants’ B cells expression of CD38 and CD27 after PBMC stimulation with S peptides. (I) Spearman correlation between frequency of CTR^loCD71⁺ CD4 T cells and CD38⁺CD27⁺ CD19⁺ cells. Frequency shown in red and P-values by Wilcoxon test in (E, F, and I). Vaccine-primed cohort points and correlation lines shown in orange and infection-primed cohort in purple for (G and I).