Cross-reactive CD4+ T cells enhance SARS-CoV-2 immune responses upon infection and vaccination

Abstract

The functional relevance of preexisting cross-immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a subject of intense debate. Here, we show that human endemic coronavirus (HCoV)–reactive and SARS-CoV-2–cross-reactive CD4⁺ T cells are ubiquitous but decrease with age. We identified a universal immunodominant coronavirus-specific spike peptide (S816-830) and demonstrate that preexisting spike- and S816-830–reactive T cells were recruited into immune responses to SARS-CoV-2 infection and their frequency correlated with anti–SARS-CoV-2-S1-IgG antibodies. Spike–cross-reactive T cells were also activated after primary BNT162b2 COVID-19 messenger RNA vaccination and displayed kinetics similar to those of secondary immune responses. Our results highlight the functional contribution of preexisting spike–cross-reactive T cells in SARS-CoV-2 infection and vaccination. Cross-reactive immunity may account for the unexpectedly rapid induction of immunity after primary SARS-CoV-2 immunization and the high rate of asymptomatic or mild COVID-19 disease courses.

Cross-reactive CD4⁺ T cells enhance SARS-CoV-2 immune responses upon infection and vaccination.

(A) Preexisting SARS-CoV-2 S-II– and spike (S)816-830–cross-reactive T cells show booster response characteristics upon SARS-CoV-2 infection and vaccination and their abundance correlates with higher protective anti–SARS-CoV-2 S1-IgG titers and higher functional T cell avidity. (B) The frequency of HCoV-reactive and SARS-CoV-2–cross-reactive T cells decreases with age.

Fig. 1.. CD4⁺ T cell cross-reactivity against the SARS-CoV-2 orfeome.

(A) Ex vivo stimulation of PBMCs from COVID-19 convalescent patients (top panel, n = 59) and unexposed individuals (bottom panel, n = 60). The percentage of CD40L⁺4-1BB⁺ CD4⁺ T cells among stimulated PBMCs was divided by the percentage of these cells among unstimulated PBMCs to determine the SI shown on the y-axis. The SARS-CoV-2 orfeome peptide pools used for stimulation are shown below the bottom panel. Gray labels highlight proteins exclusive for SARS-CoV-2 (i.e., those not shared with HCoVs). Gray circles (COVID-19) or red circles (unexposed) identify donors with an SI ≥ 3. Dotted lines indicate an SI of 1.5 and 3. Statistically significant differences between COVID-19 convalescents and unexposed groups (with respect to each peptide pool) are indicated above the bottom panel. *P < 0.05, **P < 0.01, and ***P < 0.001; ns, not significant at P > 0.05 by unpaired Student’s t test. (B) Bars show the proportions of individuals with the indicated number of SARS-CoV-2 orfeome peptide pool stimulations with an SI ≥ 3. (C) Proportions of individuals with an SI ≥ 3 for each stimulation in each indicated stimulation combination.

Fig. 2.. The magnitude of SARS-CoV-2 cross-reactivity decreases with age.

(A) Scatter plots showing the SI (CD40L⁺41BB⁺ CD4⁺ T cells) among PBMCs stimulated with SARS-CoV-2 S-I, SARS-CoV-2 S-II, or CEFX (known T cell–stimulating peptides from CMV, EBV, influenza virus, and other common pathogens) plotted against age in n = 568 unexposed donors and n = 174 COVID-19 convalescents. Dotted lines indicate an SI of 1.5 and 3. (B) Frequencies of CD3^lo cells among S-I–, S-II–, or CEFX-reactive CD40L⁺4-1BB⁺ CD4⁺ T cells over age. CD3^lo frequencies are shown for T cell responses with an SI ≥ 1.5. Regression lines denote linear regression on age in each group; the corresponding Pearson correlation coefficients are shown.

Fig. 3.. Frequencies of high-functional-avidity T cells specific for spike S-II from HCoVs decrease with age.

(A) Scatter plots show the SI of CD40L⁺4-1BB⁺ CD4⁺ T cells in unexposed individuals (n = 568) and COVID-19 convalescents (n = 174) after PBMC stimulation with HCoV (229E, NL63, OC43, and HKU1) spike S-I or S-II peptide pools plotted against age. Dotted lines indicate an SI of 1.5 and 3. (B) Frequencies of CD3^lo cells in CD40L⁺4-1BB⁺ CD4⁺ T cells from unexposed and COVID-19 convalescents plotted against age. CD3^lo frequencies are shown for T cell responses with an SI ≥ 1.5. Regression lines denote linear regression on age in each group; the corresponding Pearson correlation coefficients are shown.

Fig. 4.. Peptide S816-830 constitutes an immunodominant epitope of SARS-CoV-2 T cell cross-reactivity.

(A) Bars show the proportions of unexposed individuals <65 years of age (n = 491) and COVID-19 convalescents (n = 174, 18–79 years) with S-I– or S-II–specific T cell responses to HCoV and/or SARS-CoV-2 with an SI ≥ 3. (B) Plots showing the SI (CD40L⁺4-1BB⁺ CD4⁺ T cells) of short-term T cell lines derived from OC43 S-I– and S-II–reactive primary T cells after restimulation with autologous antigen-presenting cells in the presence of OC43 or SARS-CoV-2 spike glycoprotein pools S-I and S-II. The dotted line indicates an SI of 3. (C) The SIs of CD40L⁺4-1BB⁺ CD4⁺ T cells from unexposed (n = 48) or COVID-19 convalescents (n = 22) after stimulation with the single peptide 204_3 (S816-830), the control single peptide 284 (S1133-1147), or the S-II peptide pool. (D) Levels (optical density, OD) of anti–S809-826 peptide IgG (ELISA) in unexposed young (<65 years) and elderly (>65 years) individuals as well as COVID-19 convalescents. ELISA plates were coated with an 18-aa peptide overlapping by 11 aa with S816-30. Serum was diluted 1:100. (E) Bars show the frequencies of common class-II HLA alleles in definite S816-830 responders (SI ≥ 3) and definite nonresponders (SI < 1.5) (n = 308). +/+, homozygous; +/–, heterozygous. *P < 0.05, **P < 0.01, ***P < 0.001; ns at P > 0.05 by Student’s t test.

Fig. 5.. HCoV-specific SARS-CoV-2–cross-reactive T cells are recruited into the primary SARS-CoV-2 infection response.

(A to C) SI of CD40L⁺4-1BB⁺ CD4⁺ T cells (A), frequencies of HLADR⁺CD38⁺ cells (B), and frequencies of CD3^lo cells (C) among CD40L⁺4-1BB⁺ CD4⁺ T cells after stimulation with SARS-CoV-2 S-I, S-II, and CEFX peptide pools of donors before infection (baseline) and at four different follow-up time points (table S2) after symptom onset. CD3^lo frequencies are shown only for T cell responses with an SI ≥ 1.5. (D) Changes to CD3^lo frequencies among CD40L⁺4-1BB⁺ CD4⁺ T cells between baseline, follow-up 2 (10 to 16 days after symptom onset), and follow-up 4 (29 to 71 days after symptom onset) (left plot), and statistics (right plot) for baseline and follow-up measurement time point 2 in cross-reactive donors (baseline SI ≥ 3, red circles) and non–cross-reactive donors (baseline SI < 3, white circles). (E and F) SI (E) and frequency (F) of CD3^lo of CD40L⁺4-1BB⁺ CD4⁺ T cells after stimulation with peptide S816-830 or control peptide S1133-1147. CD3^lo frequencies are shown for T cell responses with an SI ≥ 1.5. (G) Levels (OD) of anti–S809-826 peptide IgG (ELISA) at baseline and follow-up time point 1 (3 to 9 days after symptom onset). ELISA plates were coated with an 18-aa peptide overlapping by 11 aa with S816-830. (H) Anti–S1-IgG binding antibody units (BAUs) in cross-reactive (baseline SI ≥ 3, red circles) and non–cross-reactive donors (baseline SI < 3, white circles) were plotted against time (left) and compared between baseline and follow-up 3 (right). (I) Scatter plots showing the relationship between anti–SARS-CoV-2 S1 IgG antibody levels (OD) at follow-up 4 and the SI of CD40L⁺4-1BB⁺ CD4⁺ T cells upon S-II stimulation at baseline (left), the relationship between neutralizing antibody titers (PRNT50) at follow-up 4, and the SI of CD40L⁺4-1BB⁺ CD4⁺ T cells upon S-II stimulation (left) or S-I stimulation (right) at baseline. (J) Heatmap showing the change in SI of CD40L⁺4-1BB⁺ CD4⁺ T cells after stimulation with S-II pools of the indicated HCoVs. Δ represents the change of the parameter at the given time point relative to baseline (i.e., white depicts no increase). Asterisks indicate S816-830 peptide responders. For (A), (B), (E), and (F), *P < 0.05, **P < 0.01, ***P < 0.001, and ns at P > 0.05 by repeated-measures one-way-ANOVA with Dunnett’s correction. For (C) and (G), *P < 0.05, **P < 0.01, ***P < 0.001, and ns at P > 0.05 by paired Student’s t test. For (D) and (H), *P < 0.05, **P < 0.01, ***P < 0.001, and ns at P > 0.05 by Student’s t test. For (E), ns at P > 0.05 by paired Student’s t test. For (I) ns at P > 0.05 by Pearson correlation.

Fig. 6.. HCoV-specific SARS-CoV-2–cross-reactive T cells are recruited into the BNT162b2 vaccine response.

(A) Serum anti-SARS-CoV-2 S1 IgG BAUs and IgA titer ratio were determined at baseline, day 7, and day 14 after primary vaccination with BNT162b, immediately before secondary vaccination (day 21) as well as 1 (day 28) and 2 weeks (day 35) after secondary vaccination. All values <1 were set to 1. The lower and upper cut-off levels for IgG were set at 32 and 3900, respectively; the corresponding IgA cut-offs were set at 0.6 and 10, respectively, indicated by dotted lines. (B) Plots showing the SI of CD40L⁺4-1BB⁺ CD4⁺ T cells after stimulation with S-I, S-II, and CEFX at baseline and at the indicated time points. (C) Difference in SI after stimulation with S-I and S-II at each time point relative to the previous time point. (D) Plots showing the frequencies of CD3^lo of CD40L⁺4-1BB⁺ CD4⁺ T cells after stimulation with S-I, S-II, and CEFX for responses with an SI ≥ 1.5. (E) Frequencies of CD3^lo of CD40L⁺4-1BB⁺ CD4⁺ T cells at days 0 and 7 in cross-reactive donors (baseline SI ≥ 3, red circles) and non–cross-reactive donors (baseline SI < 3, white circles). (F) Frequencies of HLADR⁺CD38⁺ cells among CD40L⁺4-1BB⁺ CD4⁺ T cells after stimulation with S-I, S-II, and CEFX at the indicated time points. (G and H) SI of CD40L⁺4-1BB⁺ CD4⁺ T cells (G) and frequencies of HLADR⁺CD38⁺ among these cells (H) after stimulation with HCoV S-II peptide pools at baseline and the indicated time points. (I and J) SI of CD40L⁺4-1BB⁺ CD4⁺ T cells (I) and frequencies of CD3^lo events (SI ≥ 1.5) (J) among these cells after stimulation with peptide S816-830 and control peptide S1133-1147 at baseline and the indicated time points. (K) Proportion of S816-830–reactive T cells over SARS-CoV-2 S-II–reactive T cells. (L) Relationship between responses to S816-830 and SARS-CoV-2 S-II peptide pool stimulation at day 0 (left) and day 7 (right). (M) OD of anti–S809-826-peptide IgG ELISA from sera before and 7 days after primary vaccination. For (A) and (F) to (J), *P < 0.05, **P < 0.01, ***P < 0.001, and ns at P > 0.05 by repeated-measures one-way-ANOVA with Dunnett’s correction. For (B) to (D) and (M), *P < 0.05, **P < 0.01, ***P < 0.001, and ns at P > 0.05 by paired Student’s t test. For (E), *P < 0.05, **P < 0.01, ***P < 0.001, and ns at P > 0.05 by Student’s t test. For (L), ns at P > 0.05 by Pearson correlation.