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.