SARS-CoV-2 infection and mRNA vaccination prime transcriptionally distinct CD4+ T cell memory landscapes

In a recent study posted to bioRxiv*, researchers reported that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and mRNA vaccination induce distinct memory T cells.

Study: Molecularly distinct memory CD4+ T cells are induced by SARS-CoV-2 infection and mRNA vaccination. Image Credit: Design_Cells/Shutterstock


T-cell memory is critical for long-term protection against viruses and correlates with immune protection. Many individuals develop T-cell memory against the SARS-CoV-2 spike as a result of natural infection or vaccination. Notably, the cluster of differentiation 4-positive (CD4+) T cell responses target epitopes conserved across variants.

During initial priming, factors such as the site and antigen exposure, inflammatory signals, cytokine milieu, and interactions between cells imprint the resultant memory pools and influence T-cell responses upon re-exposure to the antigen. Viral infection induces a highly inflammatory state that is not seen with vaccination. How inflammation associated with infection affects memory CD4+ T cells remains to be studied.

The study and findings

In the present study, researchers explored the transcriptional landscapes of CD4+ T cells specific to the SARS-CoV-2 spike protein before and after the third vaccine dose (booster dose). Peripheral blood mononuclear cells (PBMCs) were collected from 14 adults approximately eight months after the second vaccine dose (pre-boost) and one month after booster vaccination (post-boost) to examine immune memory longitudinally.

Seven adults had coronavirus disease 2019 (COVID-19) in the Spring of 2020 and were designated infection-primed, i.e., their first exposure to the SARS-CoV-2 spike was through infection. The remaining seven were infection-naïve and were deemed vaccine-primed (the first spike exposure was the BNT162b2 mRNA vaccine). Moreover, five vaccine-primed subjects later experienced breakthrough infections during the Omicron wave.

The activation-induced markers (AIM) assay was used to identify memory CD4+ T cells. PBMCs were stimulated with spike peptide pools, and AIM-reactive CD4+ T cells were detected by flow cytometry. After the AIM assay, the authors performed multimodal single-cell RNA sequencing to examine transcription-level differences in spike-specific memory CD4+ T cells.

Seven major clusters were identified in stimulated PBMCs, two of which were almost absent in unstimulated controls. Activation-associated genes, such as interferon-γ (IFNG), interleukin 2 (IL2), and lymphotoxin-α (LTA), were highly enriched in these two clusters. Dimensionality reduction applied with 27 parameters to identify AIM-reactive CD4+ T cell clusters revealed 11 distinct clusters.

The distribution of cells across these 11 clusters was similar between the two cohorts at pre- and post-boost time points. Most CD4+ T cells in both cohorts were identified in cluster 0 expressing CD27, selectin L (SELL), and transcription factor 7 (TCF7), suggestive of a central memory state. Post-booster vaccination, infection-primed CD4+ T cells were more in cluster 1 that expressed cytotoxic genes than in other clusters. The polyfunctionality of CD4+ T cells was similar between the two cohorts and was not markedly altered upon booster vaccination.

Sixty-nine and 220 genes were differentially expressed in CD4+ T cells before and after booster vaccination, respectively, between the two cohorts. CD4+ T cells from infection-primed adults differentially expressed IFN-stimulated genes. In contrast, CD4+ T cells from vaccine-primed individuals showed differential expression of genes involved in nuclear factor kappa B (NF-kB) signaling. Gene set enrichment analysis (GSEA) was performed to test for differences at a pathway level.

It revealed significant enrichment for IFN α and γ response gene sets in CD4+ T cells for infection-primed subjects at the pre-boost time point. Booster vaccination did not substantially alter gene set enrichment, suggesting that CD4+ T cells were differentially imprinted at priming and the transcriptional profile was minimally changed upon re-exposure to the spike during mRNA vaccination.

In contrast, GSEA revealed significant enrichment of the NF-kB signaling gene set in vaccine-primed individuals at the post-boost time point. Moreover, the mitotic spindle and G2M checkpoint gene sets were enriched at pre- and post-boost time points, suggesting a robust expression of proliferation-associated genes in vaccine-primed CD4+ T cells.

As such, CD4+ T-cell frequency was two-fold higher in vaccine-primed adults than infection-primed individuals one month after the second mRNA vaccination, indicating that imprinting at the time of priming of memory CD4+ T cells can result in durable effects on cellular activation and may also influence the proliferative potential.

Lastly, SARS-CoV-2 vaccine breakthrough infections in five vaccine-primed participants caused subtle changes in the transcriptional profile of the CD4+ T cells. However, it did not recapitulate the transcriptional landscape of infection-primed CD4+ T cells.


In summary, the findings indicated that the imprint of inflammation during the formation of SARS-CoV-2 spike-specific memory CD4+ T cells resulted in persistent transcriptional changes sustained even after mRNA vaccination relative to CD4+ T cells primed during mRNA vaccination. SARS-CoV-2 vaccine breakthrough infection did not cause dramatic changes to the transcriptional landscape of vaccine-primed CD4+ T cells. Taken together, the study provided valuable insights into factors that impact functionality and quality of memory CD4+ T-cell responses, which will help inform future vaccine designs.

*Important notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
  • Gray-Gaillard, S. et al. (2022) "Molecularly distinct memory CD4+ T cells are induced by SARS-CoV-2 infection and mRNA vaccination". bioRxiv. doi: 10.1101/2022.11.15.516351.

Posted in: Medical Science News | Medical Research News | Disease/Infection News

Tags: Antigen, Assay, Blood, CD4, Cell, Coronavirus, Coronavirus Disease COVID-19, covid-19, Cytokine, Cytometry, Flow Cytometry, Frequency, Gene, Genes, Inflammation, Interferon, Interleukin, Omicron, Proliferation, Protein, Respiratory, RNA, RNA Sequencing, SARS, SARS-CoV-2, Severe Acute Respiratory, Severe Acute Respiratory Syndrome, Spike Protein, Syndrome, T-Cell, Transcription, Vaccine

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Tarun Sai Lomte

Tarun is a writer based in Hyderabad, India. He has a Master’s degree in Biotechnology from the University of Hyderabad and is enthusiastic about scientific research. He enjoys reading research papers and literature reviews and is passionate about writing.

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