Inflammation durably imprints memory CD4+ T cells

Abstract

Adaptive immune responses are induced by vaccination and infection, yet little is known about how CD4+ T cell memory differs when primed in these two contexts. Notably, viral infection is generally associated with higher levels of systemic inflammation than is vaccination. To assess whether the inflammatory milieu at the time of CD4+ T cell priming has long-term effects on memory, we compared Spike-specific memory CD4+ T cells in 22 individuals around the time of the participants' third SARS-CoV-2 mRNA vaccination, with stratification by whether the participants' first exposure to Spike was via virus or mRNA vaccine. Multimodal single-cell profiling of Spike-specific CD4+ T cells revealed 755 differentially expressed genes that distinguished infection- and vaccine-primed memory CD4+ T cells. Spike-specific CD4+ T cells from infection-primed individuals had strong enrichment for cytotoxicity and interferon signaling genes, whereas Spike-specific CD4+ T cells from vaccine-primed individuals were enriched for proliferative pathways by gene set enrichment analysis. Moreover, Spike-specific memory CD4+ T cells established by infection had distinct epigenetic landscapes driven by enrichment of IRF-family transcription factors, relative to T cells established by mRNA vaccination. This transcriptional imprint was minimally altered following subsequent mRNA vaccination or breakthrough infection, reflecting the strong bias induced by the inflammatory environment during initial memory differentiation. Together, these data suggest that the inflammatory context during CD4+ T cell priming is durably imprinted in the memory state at transcriptional and epigenetic levels, which has implications for personalization of vaccination based on prior infection history.

Figure 1.. Spike-specific CD4+ T cells form a distinct cluster in the activation-induced marker assay

A. Study schematic. B. Representative flow cytometry plots for vaccine- and infection-primed participants 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. P value by Wilcoxon test. Frequency shown in red. E. 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. P value by Wilcoxon test. Frequency shown in red. G. Schematic of experimental design. 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 genes expressed at adjusted P < 0.05 for AIM-Reactive CD4+ T cell compared to CD4+ T cell cluster. J. Top 20 clonotypes identified among the AIM-Reactive CD4+ T cluster. K. Per-participant TCR overlap between AIM-Reactive CD4+ TCRs and the Adaptive Biotechnologies SARS-CoV-2-reactive TCR database. L. Distribution of epitope specificity of AIM-Reactive CD4+ T cells as inferred from the reference SARS-CoV-2 TCRβ dataset. See also Figure S1 and Tables S1–S2.

Figure 2.. Infection-primed Spike-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. Spike-specific CD4+ T cells projected onto UMAP and clustered for gene expression of 27 select 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 Spike-specific CD4+ T cell cluster distribution at pre-and post-booster time points (unpaired two-way ANOVA with Sidak posttest). See also Figure S2.

Figure 3.. Inflammation during priming results in durable transcriptional effects in Spike-specific CD4+ T cells

A. PCA of post-booster vaccine-primed (orange) and infection-primed (purple) Spike-specific CD4+ T cells. Each symbol indicates one participant. B. Heatmap of differentially expressed genes between post-booster vaccine- and infection-primed cohorts. C-D. Gene ontology analysis for differentially genes expressed at adjusted P < 0.05 for vaccine-primed (C) and infection-primed (D) Spike-specific CD4+ T cells at post-booster time point. E-H. GSEA for Mitotic Spindle (E), G2M Checkpoint (F), Interferon Alpha (G), and Interferon Gamma Response (H) gene sets for Spike-specific CD4+ T cells. Light gray line indicates pre-booster time point and dark gray line indicates post-booster time point. I. GSEA results for Hallmark gene sets enriched at FDR < 0.05 post-booster. Positive enrichment scores denote enrichment towards the infection-primed cohort in (E-I). See also Figure S3 and Tables S4–S5.

Figure 4.. Vaccine-primed and infection-primed Spike-specific CD4+ T cells have distinct epigenetic landscapes.

A. Study schematic. B. UMAP of stimulated CD4+ T cells pooled across samples and clustered for gene expression. C. Difference in scaled expression for IFI44L and HLA-C between post-infection and post-vaccination samples. Nominal P values shown and determined by DESeq2. D. GSEA for Interferon Gamma Response and Interferon Alpha Response gene sets for transcriptional profiling of Spike-specific memory CD4+ T cells. Positive enrichment scores indicate enrichment for the post-infection cohort. E. Heatmap open chromatin regions (OCRs) in the Spike-specific CD4+ T cells post-vaccination (yellow) and post-infection (purple). F. Number of differentially accessible regions (DARs) shown in (E) at a nominal P value < 0.05. G. Representative ATAC-seq tracts shown at the FRP1 and LGALS3BP loci. H-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 post-vaccination Spike-specific memory CD4+ T cells (H) and post-infection Spike-specific memory CD4+ T cells (I). J. TFs shown for the −log₁₀ adjusted P value from post-infection OCR motif analysis against the TF predicted by ChEA3 using the infection-primed gene expression data. See also Figure S4 and Tables S6–S7.

Figure 5.. Breakthrough infection minimally alters the transcriptional profile of Spike-specific CD4+ T cells

A. Study schematic. B. Example 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+ co-expression 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) Spike-specific CD4+ T cells. Each symbol indicates one participant. E. Spike-specific CD4+ T cells projected onto UMAP and clustered as described in Fig 2D. Bar graphs of vaccine-versus infection- primed Spike-specific CD4+ T cell cluster distribution at post-booster and post-breakthrough time points (two-way ANOVA). F. Volcano plot showing differentially expressed genes at adjusted P < 0.05. Genes in purple denote enrichment in infection-primed Spike-specific CD4+ T cells, orange for vaccine-primed post-booster Spike-specific CD4+ T cells, and red for post-breakthrough Spike-specific CD4+ T cells. G. Summary bar graph of DEGs in (F). H. GSEA for Interferon Gamma Response and Interferon Alpha Responses gene sets in infection-primed samples. I-J. GSEA for Hallmark gene sets at FDR < 0.05 when comparing infection-primed, post-booster samples to post-breakthrough (I) and vaccine-primed, post-booster samples to post-breakthrough (J). Positive enrichment scores denote enrichment for the post-breakthrough samples and negative enrichment scores signify enrichment for post-booster samples. See also Figure S5 and Tables S3, S8–S9.

Figure 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 Spike-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-D. Summary data at each time point. P values by Wilcoxon test. E-F. Enrichment scores for the ‘vaccine’ and ‘infection’ signatures for the four clonotypes from infection-primed participants (E) and vaccine-primed participants (F) that repeated in all time points shown. P values by Wilcoxon test. See also Figure S6.