Suppressive lymphocte subset in COVID-19
It has been known that CD3+CD4-CD8-double-negative T cells are suppressive T cells (21) The percentage of CD3+CD4-CD8- was 3.6%±3.4 in the mild group; 4.5%±3.6 in the moderate group;5.4%±4.5 in the severe group and 2.1%±1 in controls. No difference has been found between controls and mild group, however a significant difference was observed between controls and both moderate and severe groups (p<0.01; Fig 7a, b, c).
As an activation related suppression marker, HLA-DR on T lymphocytes was examined. CD3 +HLA-DR+ cells was elevated in all Covid-19 patients in comparison to controls in general (16%±11 vs 5.9%±4.6 respectively; p<0.01) This was 13%±8 in the mild; 19%±11 in the moderate and, 17%±14 in the severe group. (p<0.01; Fig 7d, e, f). No difference has been found among patients’ groups.
CD8+CD28- T cells are a subset of regulatory T cells which has suppressive effect on CD4+ T cells (22, 23). In our study, the percentage ofCD8+CD28- , suppressor T cells were found to be significantly increased in all groups of COVID-19 patients [17% ±8.9 ( 17%±7.6 in the mild, 16%±9 in the moderate 18.5%±10 in the severe group) vs 11.5% ±6.7 in the control group; p<0.03; Figure 7g, h, i)
Lymphocyte apoptosis in the periphery depends on spontaneous activation of caspase-3 in patients with Covid-19:  A special attention was focused on the lymphocyte population. Lymphocyte early apoptosis/activation rate (PhiPhilux only) following PMA stimulation was 36%±14.8 in patients with Covid-19 while that was 16.6%±6.2 in the control group (p=0.02; Fig 8a). Lymphocyte late apoptosis (PhiPhulux+PI stained cells) rate was 19.8%±11 in patients’ group; 9.6%±7.7 in the control group. Although Covid-19 patients showed higher late apoptosis on average, this difference did not reach statistical significance (Fig 8a). There was no difference between the two groups in the measurement of cell death, which was evaluated with PI stained only cells (20.5%±8.5 vs 16.8%±15.6; p>0.05). However, unlike controls, we observed that the relative number of PMA-induced lymphocytes in Covid-19 patients decreased significantly compared to their initial samples indicating spontaneous cell death. However, due to the small number of samples, we did not perform statistical analysis for this.
Total cell death was calculated by summing percentages of late apoptotic cells (PhiPhilux+PI) and cell death (PI only). This was significantly high in lymphocytes of Covid-19 patients (42%±14.5 vs 22.6%±12.3 in controls; p<0.05; Fig 8a). In addition to percentage increases, folds increases have been calculated by dividing PMA stimulated MFI value to the MFI value of the unstimulated sample for each sample. Interestingly, COVID-19 patients showed significantly lower fold increase than controls (2.3 ± 1.8 in Covid-19 patients, 6.7 ± 4.2 in controls; p <0.01; Fig 8b). It was an intriguing question of how come they have significantly low stimulation response in folds increase assessments while the percentage of lymphocyte early apoptosis following PMA stimulation was significantly higher in COVID-19 patients. To find an answer, we compared initial activation levels in unstimulated samples from both control and patient groups. In patients with COVID-19, initial fluorochrome conversion value for caspase-3 was significantly higher that indicated spontaneous activation of caspase-3 (17%±8 versus 6.3%±4.6; p=0.01; Fig 8c). Therefore, we thought that spontaneous caspase 3 activation could make COVID-19 lymphocytes more vulnerable to cell death. Thus, one of the causes of lymphopenia in COVID-19 patients might be elevated basal caspase-3 activation of peripheral lymphocytes for an unknown reason.