Persistent High Percentage of HLA-DR+CD38high CD8+ T Cells Associated With Immune Disorder and Disease Severity of COVID-19

Abstract

Background: The global outbreak of coronavirus disease 2019 (COVID-19) has turned into a worldwide public health crisis and caused more than 100,000,000 severe cases. Progressive lymphopenia, especially in T cells, was a prominent clinical feature of severe COVID-19. Activated HLA-DR⁺CD38⁺ CD8⁺ T cells were enriched over a prolonged period from the lymphopenia patients who died from Ebola and influenza infection and in severe patients infected with SARS-CoV-2. However, the CD38⁺HLA-DR⁺ CD8⁺ T population was reported to play contradictory roles in SARS-CoV-2 infection.

Methods: A total of 42 COVID-19 patients, including 32 mild or moderate and 10 severe or critical cases, who received care at Beijing Ditan Hospital were recruited into this retrospective study. Blood samples were first collected within 3 days of the hospital admission and once every 3-7 days during hospitalization. The longitudinal flow cytometric data were examined during hospitalization. Moreover, we evaluated serum levels of 45 cytokines/chemokines/growth factors and 14 soluble checkpoints using Luminex multiplex assay longitudinally.

Results: We revealed that the HLA-DR⁺CD38⁺ CD8⁺ T population was heterogeneous, and could be divided into two subsets with distinct characteristics: HLA-DR⁺CD38^dim and HLA-DR⁺CD38^hi. We observed a persistent accumulation of HLA-DR⁺CD38hi CD8⁺ T cells in severe COVID-19 patients. These HLA-DR⁺CD38^hi CD8⁺ T cells were in a state of overactivation and consequent dysregulation manifested by expression of multiple inhibitory and stimulatory checkpoints, higher apoptotic sensitivity, impaired killing potential, and more exhausted transcriptional regulation compared to HLA-DR⁺CD38^dim CD8⁺ T cells. Moreover, the clinical and laboratory data supported that only HLA-DR⁺CD38^hi CD8⁺ T cells were associated with systemic inflammation, tissue injury, and immune disorders of severe COVID-19 patients.

Conclusions: Our findings indicated that HLA-DR⁺CD38^hi CD8⁺ T cells were correlated with disease severity of COVID-19 rather than HLA-DR⁺CD38^dim population.

Keywords: CD38; COVID-19; HLA-DR; immune disorder; severity.

Figure 1

Elevated HLA-DR⁺CD38^hi CD8⁺ T cells during acute infection of COVID-19. Flow cytometry analysis of HLA-DR and CD38 expression was performed on PBMCs collected from healthy donors, M/M and S/C patients with COVID-19 infection. (A) Representative FACS contour plots showed three subpopulations of HLA-DR⁺ CD8⁺ T cells from healthy donor and COVID-19 patients: HLA-DR⁺CD38⁻ (I), HLA-DR⁺CD38^dim (II), HLA-DR⁺CD38^hi (III). (B) Scatter dot plots of the three percentages of HLA-DR⁺ CD8⁺ T cells from healthy donors and COVID-19 patients within 2–3 weeks post onset (n = 9–20 each group). P Values were obtained by unpaired two-tailed Student’s t tests and Mann–Whitney U test and repeated measures by one-way ANOVA or Kruskal-Wallis test followed by Tukey’s or Dunn’s multiple comparisons test. *P < .05, **P < .01, ***P < .001, ****P < .0001. (C) Longitudinal data of three subpopulations were graphed for eight S/C and seven M/M patients with three time points at least. (D) Temporal changes of three subpopulations in M/M (n =32) and S/C (n = 10) groups during hospitalization were shown. The 95% confidence interval indicated by colored areas. The normal range of each population was gray shaded region.

Figure 2

HLA-DR⁺CD38^hi CD8⁺ T cells consisted of enhanced percentage of T_TM and decreased T_N and T_E. Flow cytometry analysis of T_N, T_CM, T_TM, T_EM, and T_E frequency was performed on PBMCs collected from patients with infection of COVID-19 (n = 20). (A) Gating strategy for T_N, T_CM, T_TM, T_EM, and T_E in three CD8⁺ T populations. (B) The percentage of T_N, T_CM, T_TM, T_EM, and T_E on each CD8⁺ T population (I, II, III). P Values were obtained by paired two-tailed Student’s t tests and Wilcoxon matched-pairs signed-rank test and repeated measures by one-way ANOVA or Friedman test followed by Holm-Sidak’s multiple comparisons or Dunn’s multiple comparisons test. **P < .01, ***P < .001, ****P < .0001.

Figure 3

HLA-DR⁺CD38^hi CD8⁺ T cells exhibited the phenotype of overactivation. Flow cytometry analysis of expression of CD69 (A), ICOS (B), OX40 (C), 4-1BB (D), and GITR (E) on three CD8⁺ T populations (I, II, III) from COVID-19 patients (n=20). Representative histograms (left) and plots (right) were shown. P Values were obtained by paired two-tailed Student’s t tests and repeated measures by one-way ANOVA test followed by Holm-Sidak’s multiple comparisons test. *P < .05, **P < .01, ****P < .0001.

Figure 4

HLA-DR⁺CD38^hi CD8⁺ T cells displayed phenotypic and transcriptional state of exhaustion. (A–D) Flow cytometry analysis of expression of PD-1 (A), TIM3 (B), LAG3 (C), and TIGIT (D) on the three CD8⁺ T population (I, II, III) from COVID-19 patients (n = 20). Representative histograms (left) and plots (right) were shown. (E) Representative flow data (left) and dot plots (right) of percentage of T-bet^dimEomes^hi and T-bet^hiEomes^dim cells among I, II, III from COVID-19 patients (n = 20). P Values were obtained by paired two-tailed Student’s t tests and repeated measures by one-way ANOVA test followed by Holm-Sidak’s multiple comparisons test. **p < .01, ***p < .001, ****p < .0001.

Figure 5

HLA-DR⁺CD38^hi CD8⁺ T cells exhibited enhanced susceptibility to apoptosis and highly proliferative potential. Flow cytometry analysis of the expression of Bcl-2 (A), BAX (B), Granzyme B (C), perforin (D), ki67 (E), and CD71 (F) on the three CD8⁺ T population (I, II, III) from patients with infection of COVID-19 (n = 20). Representative histograms (left) and plots (right) display the expression of the above receptors on I, II, III. P Values were obtained by paired two-tailed Student’s t tests and repeated measures by one-way ANOVA test followed by Holm-Sidak’s multiple comparisons test. *p < .05, **p < .01, ***p < .001, ****p < .0001.