Association of follicular helper T and follicular regulatory T cells with severity and hyperglycemia in hospitalized COVID-19 patients

Figures & data

Figure 1. A schematic flow chart of the study protocol.

Figure 2. Flow cytometry gating strategy of follicular helper T cells (Tfh) and follicular regulatory T cells (Tfr) using FACSDiva software (Becton Dickinson Biosciences, USA).

(a) Lymphocytes were gated (R1) according to their forward and side scatter properties. (b, c) CD4+ T cells were then selected by drawing (R2), then (R3) was set to identify the CD4+ T cells expressing CXCR5 (Tfh). (d) A dot plot showing the activated Tfh cells (CD4+CXCR5+ICOS+Foxp3-), resting Tfh (CD4+CXCR5+ICOS-Foxp3-), and Tfr cells (CD4+CXCR5+ICOS+Foxp3+).

Table 1. Demographic, clinical and laboratory features of the COVID-19 patients

Parameter	Patients (n = 34)
Demographic data
Age*	61.7 ± 2
Sex Male	13 (38.2%)
Female	21 (61.8%)
Clinical findings
Dyspnea	26 (76.5%)
Fever	25 (73.5%)
Cough	26 (76.5%)
Diarrhea	23 (67.6%)
Headache	7 (20.6%)
Myalgia	13 (38.2%)
Fatigue	18 (52.9%)
Anorexia	8 (23.5%)
Sore throat	11 (32.4%)
Severity
Non-severe	20 (59%)
Severe	14 (41%)
Diabetic patients	23 (67.6%)
FBG (70–99 mg/dl)	222.8 ± 17
−diabetic patients	273 ± 20
-non-diabetic patients	129.5 ± 8
HA1c (4–5.6%)	6.7 ± .2
CBC findings*
TLC (4-11X10^9/L)	11.6 ± 1
Neutrophil count (2–7 ×10^9/L)	10.6 ± 1
Lymphocyte count (1–4.8 ×10^9/L)	2 ± .4
COVID-19 related tests*
CRP (up to 1 mg/dl)	63.5 ± 9
Ferritin (22–322 ng/ml)	843.4 ± 156
D dimer (up to .55 mg/L)	2.8 ± .5
LDH (100–190 U/L)	427.2 ± 34
Renal functions*
Urea (2.5–7.1 µmol/L)	12.2 ± 2
Creatinine (44.2–97 µmol/L)	138.6 ± 25
Liver functions*
ALT (0–45 U/L)	33.1 ± 3.4
AST (0–34 U/L)	38.5 ± 3
Total protein (64–83 g/L)	57 ± 2
Albumin (34–50 g/L)	29.5 ± 1
Total bilirubin (0–34 µmol/L)	19.9 ± 4
Direct bilirubin (0–3.4 µmol/L)	8.2 ± 2
Prothrombin time (11–14 sec)	13.4 ± .4
Prothrombin concentration (70–130%)	84.6 ± 3
Blood gases*
SpO2 (>95%)	84.5 ± 2
PaCO2 (35–45 mmHg)	33.2 ± 2
Respiratory rate (13–28/min) *	32.4 ± 2

Data were presented as number (percentage) or *mean ± SE (normal reference ranges.

FBG fasting blood glucose, CBC complete blood count, TLC total leukocyte count, CRP C-reactive protein, LDH lactate dehydrogenase, ALT alanine aminotransferase, AST aspartate aminotransferase, SpO₂ Oxygen saturation, PaCO₂ partial pressure of carbon dioxide.

Figure 3. Comparison of the frequencies of follicular helper (Tfh) and follicular regulatory T (Tfr) cells between COVID-19 patients and controls (p-value*** <.0001, ** <.01, ns=not significant). Percentages of ICOS+ and ICOS- cells were calculated from CD4+CXCR5+ Tfh cells.

Figure 4. Comparison of the percentages of follicular helper (Tfh) and follicular regulatory T (Tfr) cells between severe and non-severe cases of COVID-19 (p-value: *** <.0001, ** <.01, ns=not significant). Percentages of ICOS+ and ICOS- cells were calculated from CD4+CXCR5+ Tfh cells.

Figure 5. A heat map showing the levels of activated Tfh cells (Cd4+cxcr5+icos+foxp3-), resting Tfh (Cd4+cxcr5+icos-Foxp3-), and Tfr cells (Cd4+cxcr5+icos+foxp3+) in relation with clinical and laboratory findings in COVID-19 patients. as shown, most of the CD4+CXCR5+ Tfh cells were activated (Icos+) in severe cases and the increase in its level was associated with higher LDH, D-dimer, and ferritin, as well as respiratory rate, and lower PaCo₂. Resting Tfh and Tfr cells showed opposite relations with the laboratory indices of severity.

Figure 6. Comparison of the percentages of follicular helper (Tfh) and follicular regulatory T (Tfr) cells between diabetic and non-diabetic cases of COVID-19 and healthy controls (p-value: *** <.0001, ** <.01, ns=not significant). Percentages of ICOS+ and ICOS- cells were calculated from CD4+CXCR5+ Tfh cells.

Data availability statements

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request after taking a permission from our ethical committee.