No evidence of immune exhaustion after repeated SARS-CoV-2 vaccination in vulnerable and healthy populations

Abstract

Frequent SARS-CoV-2 vaccination in vulnerable populations has raised concerns that this may contribute to T cell exhaustion, which could negatively affect the quality of immune protection. Herein, we examined the impact of repeated SARS-CoV-2 vaccination on T cell phenotypic and functional exhaustion in frail older adults in long-term care (n = 23), individuals on immunosuppressive drugs (n = 10), and healthy adults (n = 43), in Canada. Spike-specific CD4⁺ and CD8⁺ T cell levels did not decline in any cohort following repeated SARS-CoV-2 vaccination, nor did the expression of exhaustion markers on spike-specific or total T cells increase. T cell production of multiple cytokines (i.e. polyfunctionality) in response to the spike protein of SARS-CoV-2 did not decline in any cohort following repeated vaccination. None of the cohorts displayed elevated levels of terminally differentiated T cells following multiple SARS-CoV-2 vaccinations. Thus, repeated SARS-CoV-2 vaccination was not associated with increased T cell exhaustion in older frail adults, immunosuppressed individuals, or healthy adults.

Fig. 1. Spike-specific CD4⁺ and CD8⁺ T cell levels and expression of PD-1, TIM-3, and LAG-3 following subsequent SARS-CoV-2 vaccinations.

Spike-specific T cell levels and surface phenotypes were assessed by AIM assay and flow cytometry in the LTC, RA, and HA cohorts. a Frequencies of spike-specific CD4⁺ T cells. p = 0.0420*. b Frequencies of spike-specific CD8⁺ T cells. p = 0.0048**, p = 0.0002***. The solid red lines indicate the median of each group. c Frequencies of spike-specific CD4⁺ T cells expressing each combination of PD-1, TIM-3, and LAG-3, displayed as the mean of each combination as a proportion of all combinations. d Frequencies of spike-specific CD8⁺ T cells expressing each combination of PD-1, TIM-3, LAG-3, displayed as the mean of each combination as a proportion of all combinations. LTC: 3mo2 n = 14, 3mo3 n = 23, 3mo4 n = 18. RA: 3mo2 n = 7, 3mo3 n = 9, 3mo4 n = 7. HA: 3mo2 n = 42, 3mo3 n = 20, 3mo4 n = 7. For a, b, participant vaccination history is indicated by circles (BNT162b2 only), squares (mRNA-1273 only), triangles (mixed BNT162b2 and mRNA-1273), or diamonds (mRNA only but one vaccine unknown). 3moX denotes 3 months post dose X. Multivariable linear mixed models accounting for age and sex were used to assess changes in spike-specific T cells levels within each cohort, and changes in the frequencies of PD-1, TIM-3, and LAG-3, within each cohort (a–d). FDR adjusted p values were obtained within each cohort for c and d, to account for multiple testing on cells from the same parent population. If p values are not indicated, the result was not significant.

Fig. 2. T cell functionality and polyfunctionality following repeated SARS-CoV-2 vaccination.

PBMCs were stimulated for 24 h using a peptide pool derived from the spike protein of SARS-CoV-2, and intracellular cytokine stains were conducted to evaluate cytokine production using flow cytometry. COMPASS heatmap of posterior probabilities of a spike-specific response in a CD4⁺ T cells b and CD8⁺ T cells for each cytokine combination. COMPASS functionality score (FS) for c CD4⁺ T cells and d CD8⁺ T cells in each cohort following repeated SARS-CoV-2 vaccinations. COMPASS polyfunctionality score (PFS) for e CD4⁺ T cells and f CD8⁺ T cells in each cohort following repeated SARS-CoV-2 vaccinations. For a, b, each column represents a different cytokine combination as indicated by the shaded legend beneath the heatmap, while each row is a unique participant. The blue scaling within the heatmaps indicates the posterior probabilities. Colored bars on the left side denote the cohorts associated with the rows, and timepoint within each cohort. For c–f, participant vaccination history is indicated by circles (BNT162b2 only), squares (mRNA-1273 only), triangles (mixed BNT162b2 and mRNA-1273), or diamonds (mRNA only but one vaccine unknown). The solid red lines indicate the median of each group. 2 indicates 3mo2, 3 is 3mo3, and 4 is 3mo4. LTC: 3mo2 n = 14, 3mo3 n = 23, 3mo4 n = 18. RA: 3mo2 n = 7, 3mo3 n = 9, 3mo4 n = 8. HA: 3mo2 n = 41, 3mo3 n = 21, 3mo4 n = 7. Multivariable linear mixed models accounting for age and sex were used to assess changes in the CD4⁺ and CD8⁺ T cell FS and PFS within a given cohort following additional SARS-CoV-2 vaccinations. For heatmaps, columns of cytokine combination subsets with posterior probabilities less than 0.005 for all participants are not displayed. If p values are not indicated, the result was not significant.

Fig. 3. Immunophenotyping of circulating CD4⁺ T cell compartment following repeated SARS-CoV-2 vaccination.

Flow cytometry was used to assess the frequencies of naïve (N, CCR7⁺CD45RA⁺), central memory (CM, CCR7⁺CD45RA⁻), effector memory (EM, CCR7⁻CD45RA⁻) and EM re-expressing CD45RA (EMRA, CD45RA⁺CCR7⁻) CD4⁺ T cells. a The mean frequency of each T cell subset was determined and plotted in a stacked bar format. b The frequencies of terminally differentiated CD4⁺ T cells (EMRA⁺CD57⁺CD28⁻), out of all CD4⁺ T cells, within each cohort following the second, third, and fourth SARS-CoV-2 vaccinations. The solid red lines indicate the median of each group. c The frequency of CD4⁺ T cells expressing each combination of the exhaustion markers PD-1 and TIGIT, displayed as the mean value within each cohort at each timepoint. For b, participant vaccination history is indicated by circles (BNT162b2 only), squares (mRNA-1273 only), triangles (mixed BNT162b2 and mRNA-1273), or diamonds (mRNA only but one vaccine unknown). 3moX denotes 3 months post dose X. LTC: 3mo2 n = 14, 3mo3 n = 22, 3mo4 n = 15. RA: 3mo2 n = 7, 3mo3 n = 9, 3mo4 n = 8. HA: 3mo2 n = 41, 3mo3 n = 19, 3mo4 n = 5. Multivariable linear mixed models accounting for age and sex were used to assess changes in the frequencies of each T cell subset within a given cohort following repeated SARS-CoV-2 vaccinations. FDR adjusted p values were obtained within each cohort for a and c to account for multiple testing on cells from the same parent population. If p values are not indicated, the result was not significant. p < 0.05 *, p < 0.01 **.

Fig. 4. Immunophenotyping of circulating CD8⁺ T cell compartment following repeated SARS-CoV-2 vaccination.

Flow cytometry was used to assess CD8⁺ T cell phenotypes. a The mean frequencies of naïve (N), CM, EM, and EMRA CD8⁺ T cells were determined and plotted in a stacked bar format. b The frequencies of terminally differentiated CD8⁺ T cells (EMRA⁺CD57⁺CD28⁻), out of all CD8⁺ T cells, within each cohort following the second, third, and fourth SARS-CoV-2 vaccinations. The solid red lines indicate the median of each group. c The frequency of CD8⁺ T cells expressing each combination of the exhaustion markers PD-1 and TIGIT, displayed as the mean value within each cohort at each timepoint. For b, participant vaccination history is indicated by circles (BNT162b2 only), squares (mRNA-1273 only), triangles (mixed BNT162b2 and mRNA-1273), or diamonds (mRNA only but one vaccine unknown). 3moX denotes 3 months post dose X. LTC: 3mo2 n = 14, 3mo3 n = 22, 3mo4 n = 15. RA: 3mo2 n = 7, 3mo3 n = 9, 3mo4 n = 8. HA: 3mo2 n = 41, 3mo3 n = 19, 3mo4 n = 5. Multivariable linear mixed models accounting for age and sex were used to assess changes in the frequencies of each T cell subset within a given cohort following repeated SARS-CoV-2 vaccinations. FDR adjusted p values were obtained within each cohort for a and c to account for multiple testing on cells from the same parent population. If p values are not indicated, the result was not significant. p < 0.05 *.

Fig. 5. Immune phenotype and exhaustion in HA with different dose intervals between the first and second SARS-CoV-2 vaccination.

Flow cytometry was used to assess T cell phenotypes and functional capacity in HA at 3 months post dose 2. The HA ‘recommended’ (Rec) cohort had <35 days between dose 1 and 2, the ‘delayed’ (Del) cohort had 35–42 days between dose 1 and dose 2, and the ‘extended’ (Ext) cohort had >42 days between dose 1 and dose 2. a Frequencies of spike-specific CD4⁺ T cells in each HA cohort. b Frequencies of spike-specific CD4⁺ T cells expressing each combination of PD-1, TIM-3, and LAG-3, displayed as mean for each combination. c Frequencies of spike-specific CD8⁺ T cells in each HA cohort. d Frequencies of spike-specific CD8⁺ T cells expressing each combination of PD-1, TIM-3, LAG-3, displayed as mean for each combination. COMPASS functionality score (FS) and polyfunctionality score (PFS) for e CD4⁺ T cells and f CD8⁺ T cells in response to stimulation with peptides from the spike protein of SARS-CoV-2. g Frequencies of naïve, central memory (CM), effector memory (EM) and EM re-expressing CD45RA (EMRA) CD4⁺ and CD8⁺ T cells, displayed as mean of each combination as a proportion of all combinations, in each cohort. h The frequencies of terminally differentiated CD4⁺ and CD8⁺ T cells. The frequency of i CD4⁺ T cells and j CD8⁺ T cells expressing each combination of PD-1 and TIGIT, displayed as mean of each combination as a proportion of all combinations, in each cohort. For a, c, e, f, h, participant vaccination history is indicated by circles (BNT162b2 only), squares (mRNA-1273 only), triangles (mixed BNT162b2 and mRNA-1273), or diamonds (mRNA only but one vaccine unknown). The solid red lines indicate the median of each group. HA Rec n = 20 (21 for AIMs), HA Del n = 7, HA Ext n = 14. ANOVAs were used to assess differences between dosing interval cohorts. FDR adjusted p values were obtained for b, d, g, i, j to account for multiple testing on cells from the same parent population. If the ANOVA was significant, after FDR correction if applicable, post-hoc tests were used to determine which dosing intervals differed from one another. If p values are not indicated, the result was not significant. p < 0.05 *.