Impaired Cytotoxic CD8+ T Cell Response in Elderly COVID-19 Patients

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection induces a T cell response that most likely contributes to virus control in COVID-19 patients but may also induce immunopathology. Until now, the cytotoxic T cell response has not been very well characterized in COVID-19 patients. Here, we analyzed the differentiation and cytotoxic profile of T cells in 30 cases of mild COVID-19 during acute infection. SARS-CoV-2 infection induced a cytotoxic response of CD8⁺ T cells, but not CD4⁺ T cells, characterized by the simultaneous production of granzyme A and B as well as perforin within different effector CD8⁺ T cell subsets. PD-1-expressing CD8⁺ T cells also produced cytotoxic molecules during acute infection, indicating that they were not functionally exhausted. However, in COVID-19 patients over the age of 80 years, the cytotoxic T cell potential was diminished, especially in effector memory and terminally differentiated effector CD8⁺ cells, showing that elderly patients have impaired cellular immunity against SARS-CoV-2. Our data provide valuable information about T cell responses in COVID-19 patients that may also have important implications for vaccine development.IMPORTANCE Cytotoxic T cells are responsible for the elimination of infected cells and are key players in the control of viruses. CD8⁺ T cells with an effector phenotype express cytotoxic molecules and are able to perform target cell killing. COVID-19 patients with a mild disease course were analyzed for the differentiation status and cytotoxic profile of CD8⁺ T cells. SARS-CoV-2 infection induced a vigorous cytotoxic CD8⁺ T cell response. However, this cytotoxic profile of T cells was not detected in COVID-19 patients over the age of 80 years. Thus, the absence of a cytotoxic response in elderly patients might be a possible reason for the more frequent severity of COVID-19 in this age group than in younger patients.

Keywords: CD4+; CD8+; COVID-19; PD-1; SARS-CoV-2; aging; cytotoxic T cells; granzyme; perforin.

FIG 1

CD4⁺ T cells in COVID-19 patients. CD4⁺ T cells in blood of patients with mild COVID-19 and healthy donors were analyzed by flow cytometry. (A) Concentration of CD4⁺ T cells in peripheral blood of patients on the day of hospitalization and values for healthy donors of different ages. (B) The differentiation status of CD4⁺ T cells was determined by the expression of CD45RO, CCR7, and CD28. CD3⁺ CD4⁺ T cells were divided into naive (N; CD45RO⁻ CCR7⁺ CD28⁺), central memory (CM; CD45RO⁺ CCR7⁺ CD28⁺), transitional memory (TM; CD45RO⁺ CCR7⁻ CD28⁺), effector memory (EM; CD45RO⁺ CCR7⁻ CD28⁻), and terminally differentiated effector (E; CD45RO⁻ CCR7⁻ CD28⁻) subpopulations. (C) The production of GzmA, GzmB, and perforin in CD3⁺ CD4⁺ T cells is shown in representative dot plots. (D to F) Percentages of CD4⁺ T cells producing GzmA (D), GzmB (E), and perforin (F). Each dot represents an individual patient. Statistically significant differences are indicated by asterisks (*, P < 0.05; **, P < 0.01; unpaired t test with Welch’s correction).

FIG 2

Reduced numbers of circulating CD8⁺ T cells in elderly COVID-19 patients. CD8⁺ T cells in the blood of patients with mild COVID-19 were analyzed by flow cytometry. (A) Concentration of CD8⁺ T cells in peripheral blood of patients on the day of hospitalization and values for healthy donors of different ages. (B) Correlation of age with concentration of CD8⁺ T cells in the blood of acute COVID-18 patients. Each dot represents an individual patient. (C) The differentiation status of CD8⁺ T cells was determined by the expression of CD45RO, CCR7, and CD28. CD3⁺ CD8⁺ T cells were divided into naive (N; CD45RO⁻ CCR7⁺ CD28⁺), central memory (CM; CD45RO⁺ CCR7⁺ CD28⁺), transitional memory (TM; CD45RO⁺ CCR7⁻ CD28⁺), effector memory (EM; CD45RO⁺ CCR7⁻ CD28⁻), and terminally differentiated effector (E; CD45RO⁻ CCR7⁻ CD28⁻) subpopulations. Statistically significant differences are indicated by asterisks (*, P < 0.05; unpaired t test with Welch’s correction, Pearson’s correlation coefficient).

FIG 3

Production of cytotoxic molecules by CD8⁺ T cells. Production of cytotoxic molecules by CD8⁺ T cells in blood from patients with mild COVID-19 and healthy donors was characterized by flow cytometry. (A) Representative dot plots show the production of GzmA, GzmB, and perforin in CD8⁺ T cells. (B to D) Percentages of CD8⁺ T cells producing GzmA (B), GzmB (C), and perforin (D). Each dot represents an individual patient. Statistically significant differences are indicated by asterisks (*, P < 0.05; **, P < 0.001; nonparametric Mann-Whitney U test).

FIG 4

Production of cytotoxic molecules by PD-1⁺ CD8⁺ T cells. Expression of PD-1 and production of cytotoxic molecules in PD-1⁺ CD8⁺ T cells in the blood of patients with mild COVID-19 was characterized by flow cytometry. Percentages of CD8⁺ PD-1⁺ T cells (A) and of PD-1⁺CD8⁺ T cells producing GzmA (B), GzmB (C), and perforin (D) were calculated. Each dot represents an individual patient. (**, P < 0.01; nonparametric Mann-Whitney U test).

FIG 5

Production of cytotoxic molecules in different subpopulations of CD8⁺ T cells from COVID-19 patients. Differentiation of CD8⁺ T cells in the blood of patients with mild COVID-19 was analyzed by flow cytometry. (A) Representative histograms of production of GzmA, GzmB, and perforin in CD8⁺ T cells at different stages of differentiation. The frequencies of transitional memory (TM; CD45RO⁺ CCR7⁻ CD28⁺), effector memory (EM; CD45RO⁺ CCR7⁻ CD28⁻), and terminally differentiated effector (E; CD45RO⁻ CCR7⁻ CD28⁻) CD8⁺ T cells producing GzmA, GzmB, and perforin in the blood of patients with mild COVID-19 disease were detected by flow cytometry. (B to D) TM CD8⁺ T cells producing GzmA (B), GzmB (C), and perforin (D); (E to G) EM CD8⁺ T cells producing GzmA (E), GzmB (F), and perforin (G); (H to J) E CD8⁺ T cells producing GzmA (H), GzmB (I), and perforin (J). Each dot represents an individual patient. Statistically significant differences are indicated by asterisks (*, P < 0.05; Dunn test with the Benjamini-Hochberg correction for multiple testing).

FIG 6

Simultaneous production of GzmA, GzmB, and perforin by CD8⁺ T cells from COVID-19 patients. Differentiation of CD8⁺ T cells in the blood of patients with mild COVID-19 was characterized by flow cytometry. The frequencies of CD8⁺ T cells simultaneously producing GzmA, GzmB, and perforin from patients in the 29- to 66-year-old, 70- to 79-year-old, and 80- to 96-year-old age groups were calculated for transitional memory (TM; CD45RO⁺ CCR7⁻ CD28⁺) (A to C), effector memory (EM; CD45RO⁺ CCR7⁻ CD28⁻) (D to F), and terminally differentiated effector (E; CD45RO⁻ CCR7⁻ CD28⁻) (G to I) CD8⁺ T cells. Each dot represents an individual patient.