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. 2024 May:103:105114.
doi: 10.1016/j.ebiom.2024.105114. Epub 2024 Apr 18.

IL-1 receptor 1 signaling shapes the development of viral antigen-specific CD4+ T cell responses following COVID-19 mRNA vaccination

Hong-Jai Park  1 , Min Sun Shin  1 , Junghee J Shin  1 , Hyoungsu Kim  2 , Byunghyun Kang  3 , Jennefer Par-Young  1 , Serhan Unlu  1 , Yuliya Afinogenova  1 , Jason Catanzaro  4 , Juan Young  5 , Minhyung Kim  6 , Sang Jin Lee  7 , Sangchoon Jeon  8 , Sungyong You  6 , Michael K Racke  9 , Richard Bucala  1 , Insoo Kang  10
Affiliations

IL-1 receptor 1 signaling shapes the development of viral antigen-specific CD4+ T cell responses following COVID-19 mRNA vaccination

Hong-Jai Park et al. EBioMedicine. 2024 May.

Abstract

Background: The innate immune cytokine interleukin (IL)-1 can affect T cell immunity, a critical factor in host defense. In a previous study, we identified a subset of human CD4+ T cells which express IL-1 receptor 1 (IL-1R1). However, the expression of such receptor by viral antigen-specific CD4+ T cells and its biological implication remain largely unexplored. This led us to investigate the implication of IL-1R1 in the development of viral antigen-specific CD4+ T cell responses in humans, including healthy individuals and patients with primary antibody deficiency (PAD), and animals.

Methods: We characterized CD4+ T cells specific for SARS-CoV-2 spike (S) protein, influenza virus, and cytomegalovirus utilizing multiplexed single cell RNA-seq, mass cytometry and flow cytometry followed by an animal study.

Findings: In healthy individuals, CD4+ T cells specific for viral antigens, including S protein, highly expressed IL-1R1. IL-1β promoted interferon (IFN)-γ expression by S protein-stimulated CD4+ T cells, supporting the functional implication of IL-1R1. Following the 2nd dose of COVID-19 mRNA vaccines, S protein-specific CD4+ T cells with high levels of IL-1R1 increased, likely reflecting repetitive antigenic stimulation. The expression levels of IL-1R1 by such cells correlated with the development of serum anti-S protein IgG antibody. A similar finding of increased expression of IL-1R1 by S protein-specific CD4+ T cells was also observed in patients with PAD following COVID-19 mRNA vaccination although the expression levels of IL-1R1 by such cells did not correlate with the levels of serum anti-S protein IgG antibody. In mice immunized with COVID-19 mRNA vaccine, neutralizing IL-1R1 decreased IFN-γ expression by S protein-specific CD4+ T cells and the development of anti-S protein IgG antibody.

Interpretation: Our results demonstrate the significance of IL-1R1 expression in CD4+ T cells for the development of viral antigen-specific CD4+ T cell responses, contributing to humoral immunity. This provides an insight into the regulation of adaptive immune responses to viruses via the IL-1 and IL-1R1 interface.

Funding: Moderna to HJP, National Institutes of Health (NIH) 1R01AG056728 and R01AG055362 to IK and KL2 TR001862 to JJS, Quest Diagnostics to IK and RB, and the Mathers Foundation to RB.

Keywords: Antigen-specific T cells; COVID-19; IL-1 receptors; IL-1β; Primary antibody deficiency; mRNA vaccines.

PubMed Disclaimer

Conflict of interest statement

Declaration of interests MKR is an employee of Quest Diagnostics. IK and RB received research funding from Quest Diagnostics.

Figures

Fig. 1
Fig. 1
Multiplexed single-cell RNA sequencing reveals transciptomic features of SARS-CoV-2 spike (S) protein, influenza virus (Flu) and cytomegalorvirus (CMV)-specific memory CD4+T cells in healthy subjects. (a) Representative flow cytometry plots of CD4+ T cell subsets (indicated by #1–7) identified based on OX40 and 4-1BB expression following overnight stimulation of PBMCs of COVID-19 mRNA vaccinated healthy individuals with or without S protein overlapping peptides, influenza virus (Flu), or cytomegalovirus (CMV) lysates. scRNA-seq analysis of FACS-purified subsets #1–7 in (A) (n = 2) was done. (b) UMAP projection of indicated CD4+ T cell subsets. (c) Heatmap showing differentially expressed genes (DEGs) in indicated CD4+ T cell subsets. (d) Volcano plot showing DEGs between OX40+4-1BB+ and OX40-4-1BB- CD4+ T cells in S protein-stimulated PBMCs. (e) Plots showing IL1R1 and IL1R2 expression in indicated CD4+ T cell subsets.
Fig. 2
Fig. 2
SARS-CoV-2 Spike (S) protein-specific memory CD4+T cells in healthy subjects immunized with COVID-19 mRNA vaccine have a distinct expression pattern of molecules as determined by CyTOF. (a) Flow cytometric analysis of IL-1R1 and IL-1R2 expression by indicated cell subsets showing expression levels of IL-1R1 and IL-1R2 (mean fluorescence intensity or MFI) in 5 healthy subjects immunized with COVID-19 mRNA vaccine. Left pannels, representative histograms. (b–d) CyTOF analysis showing distinct metaclusters in CD4+ T cells specific for S protein, Flu and CMV. PBMCs of COVID-19 mRNA vaccinated healthy individuals were incubated overnight with or without S protein overlapping peptides, Flu or CMV lysates followed by CyTOF analysis. CD4+ T cell populations indicated above the t-SNE plots in Fig. 1b were identified in 35 samples from 5 subjects according to the gating strategy as in Fig. 1a and further analyzed using using PhenoGraph and metaclustering. (b)t-SNE plots showing distinct metaclusters in indicated CD4+ T cells. Numbers 1 and 2 in the t-SNE plot of all cells indicate metaclusers 1 and 2, respectively. Unstim, unstimulated. (c) Heatmap showing expression levels of indicated molecules by individual metaclusters. (d) Frequency of metaclusters 1 and 2 in S protein-, Flu- and CMV-specific OX40+4-1BB+ CD4+ T cells. Bars and error bars indicate mean and 95% CI. P-values by one-way ANOVA with Dunnett's post hoc analysis.
Fig. 3
Fig. 3
IL-1β with or without anti-IL-1R2 neutralizing antibody enhances IFN-γ+expression by human CD4+T cells in response to SARS-CoV2 spike (S) protein. (a–d) Flow cytometric analysis of IFN-γ+ CD4+ T cells in PBMCs (n = 7 COVID-19 mRNA vaccinated healthy subjects) which were incubated for 3 days in the presence or absence of S protein overlapping peptides (Spike) with or without recombinant human IL-1β (5 or 10 ng/ml) (a–d) or a combination of human IL-1β and anti-IL-1R2 neutralizing antibody (20 μg/ml, c-d). Representive dot plots (a, c). Each dot in scatter graphs (b, d) represents data from indicated conditions. Bars and error bars (Box-and-whisker) indicate median ± interquartile range (b) and (d). P-values by the Wilcoxon matched-pairs signed rank test (b) or the Sign test (b, d).
Fig. 4
Fig. 4
IL-1R1 expessing memory CD4+T cells specific for SARS-CoV-2 spike (S) protein increase in healthy subjects following the 2nd dose of COVID-19 mRNA vaccine, correlating with anti-S protein IgG production. (a–f) PBMCs of healthy subjects (n = 7) were obtained before and 3–4 weeks after the 1st and 2nd doses of Pfizer-BioNTech or Moderna COVID-19 mRNA vaccine. Cells were incubated overnight with or without S protein overlapping peptides and analyzed by CyTOF. CD4+ T cell populations indicated above the t-SNE plots in (b) were identified according to the gating strategy as in Fig. 1a and further analyzed using using PhenoGraph and metaclustering. (a) Blood collection time points (T). (b)t-SNE plots showing distinct metaclusters in indicated CD4+ T cell populations at T1 and T2. Numbers 1 and 2 in the t-SNE plot of all cells indicate metaclusers 2 and 4, respectively. (c) Heatmap showing expression levels of indicated molecules by individual metaclusters. (d) Graph showing the frequency of metaclusters 2 and 4 at T1 and T2. Bars and error bars indicate mean and 95% CI. P-values were obtained by the paired t-test. (e–f) The relationship of geometric mean metal intensities (GMMI) of IL-1R1 (e) and IL-1R2 (f) expressed on spike protein-specific OX40+4-1BB+ CD4+ T cells with serum anti-s protein IgG levels. 95% CI for correlation coefficient are 0.0015–0.96 (e, 1st dose), 0.039–0.96 (e, 2nd dose), −0.98 to −0.39 (f, 1st dose), −0.52 to 0.88 (f, 2nd dose). Sera were obtained at 10–14 days after the 1st and 2nd doses of COVID-19 mRNA vaccine. P and r values were obtained by Pearson correlation.
Fig. 5
Fig. 5
Administrating anti-IL-1R1 neutralizing antibody decreases spike protein (S) -specific CD4+T cells expressing IFN-γ in mice immunized with COVID-19 mRNA vaccine. (a) Schematic diagrm showing IL-1R1 neutralizing antibody administration schedule in C57BL/6 mice immunized with BNT162b2 COVID-19 mRNA vaccine. (b–c) Flow cytometric analysis of IFN-γ+, TNF-α+, and IL-2+ CD4+ T cells in splenocytes from unvaccinated, vaccinated, and vaccinated mice treated with anti-IL-1R1 neutralizing antibody (Ab) (n = 13). For intracelluar cytokine analysis, splenocytes were incubated overnight with or without S protein overlapping peptides. (d) Anti-S protein IgG levels were determined in sera from the same mice (n = 13). Bars and error bars indicate mean and 95% CI. P-values were obtained by the unpaired t-test.
Fig. 6
Fig. 6
SARS-CoV-2 spike (S) protein-specific memroy CD4+T cells in patients with primary antibody deficiency (PAD) express IL-1 receptor 1 after COVID-19 vaccination, without showing correlation with serum anti-S protein IgG levels. PBMCs were obtained from patients with PADs (4 CVID and 8 other PADs) following the 1st and 2nd doses of COVID-19 mRNA vaccine. Cells were incubated overnight with or without S protein overlapping peptides followed by flow cytometric analysis. (a) Flow cytometric analysis of IL-1R1 expression by indicated CD4+ T cell subsets. Representative histograms and scatter graphs showing mean fluorescence intensity (MFI) of IL-1R1. (b) Heatmap illustrating the expression levels of indicated molecules (z-scores of MFI) on CD4+ T cell subsets defined by the expression of OX40 and 4-1BB. Bars and error bars indicate mean and 95% CI (a). P-values were obtained by ANOVA (a).
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