Peripheral lymphocyte subset alteration in patients with COVID-19 having differential clinical manifestations

Abstract

Background & objectives: The COVID-19 disease profile in Indian patients has been found to be different from the Western world. Changes in lymphocyte compartment have been correlated with disease course, illness severity and clinical outcome. This study was aimed to assess the peripheral lymphocyte phenotype and subset distribution in patients with COVID-19 disease from India with differential clinical manifestations.

Methods: Percentages of peripheral lymphocyte subsets were measured by flow cytometry in hospitalized asymptomatic (n=53), mild symptomatic (n=36), moderate and severe (n=30) patients with SARS-CoV-2 infection, recovered individuals (n=40) and uninfected controls (n=56) from Pune, Maharashtra, India.

Results: Percentages of CD4+Th cells were significantly high in asymptomatic, mild symptomatic, moderate and severe patients and recovered individuals compared to controls. Percentages of Th memory (CD3+CD4+CD45RO+), Tc memory (CD3+CD8+CD45RO+) and B memory (CD19+CD27+) cells were significantly higher in the recovered group compared to both asymptomatic, mild symptomatic patient and uninfected control groups. NK cell (CD56+CD3-) percentages were comparable among moderate +severe patient and uninfected control groups.

Interpretation & conclusions: The observed lower CD4+Th cells in moderate+severe group requiring oxygen support compared to asymptomatic+mild symptomatic group not requiring oxygen support could be indicative of poor prognosis. Higher Th memory, Tc memory and B memory cells in the recovered group compared to mild symptomatic patient groups might be markers of recovery from mild infection; however, it remains to be established if the persistence of any of these cells could be considered as a correlate of protection.

Keywords: Asymptomatic; SARS-CoV-2; lymphocyte subset; mild symptomatic; moderate +severe patients; recovered individuals.

Fig. 1

(A) Gating strategy for the enumeration of natural killer (NK) and natural killer T-like (NKT-like) cells in different study groups. Lymphocyte gate was recognized by forward scatter (FSC) & side scatter (SSC). Further, the CD3 negative cells from this population were screened for CD56+ expression to identify the NK cells and were identified as CD3-CD56+ and NKT-like (NKT-like) cells were CD3+CD56+. (B) Gating strategy for the enumeration of B and memory B cells in different study groups. B cells from lymphocytes were identified with the help of CD19 expression. For further differentiation, expression of CD27 was considered as memory B cells.

Fig. 1

(C) Gating strategy for the enumeration of T helper and T cytotoxic cells in different study groups. Lymphocyte populations were identified based on forward and side scatter properties of PBMCs (peripheral blood mononuclear cells). The CD3 positive population was further discriminated to identify CD4 and CD8 T cells. T cell subsets differentiate into T helper cells (CD3+CD4+CD8-) and T cytotoxic cells (CD3+CD4-CD8+). (D) Gating strategy for the enumeration of memory Th and memory Tc cells in different study groups. T cell subsets differentiation into T helper (CD3+CD4+CD8-) and T cytotoxic (CD3+CD4-CD8+) cells from CD3+ cells. Further screening of memory Th cells (CD3+CD4+CD8-CD45RO+) and memory Tc cells (CD3+CD4-CD8+CD45RO+) were done.

Fig. 1

(E) Gating strategy for the enumeration of T regulatory (Treg) cells (CD4+CD25+ CD127-) in different study groups. Lymphocyte gate was recognized by FSC & SSC. Further, the CD4 positive cells from this population were screened for CD25+CD127- expression to identify (Treg) cells.

Fig. 2

Flowcytometric analysis of NK/NKT-like, B and memory B cells, T cell subsets. PBMCs from 53 asymptomatic, 36 mild symptomatic, 30 (Moderate+Severe) COVID-19 patients, 40 recovered individuals and 56 uninfected controls were stained and acquired on flowcytometer. Vertical scatter plots denote the comparisons of frequencies of immune cells and their subpopulation among different study groups: (A) NK cells and NKT-like cells profile, (B) B and memory B cells profile, (C) T-helper and T-cytotoxic cells profile, (D) Memory Th and Tc cells, Tregs profile. Data are presented as percentage of immune cells out of lymphocytes. The dots represent individual values and bars represent Mean+SD values (P *** <0.0001). ns, not significant

Fig. 3

Flow cytometric analysis of NK/NKT-like, T cell subsets. PBMCs from 89 patients not requiring O2 support (Asymptomatic+ Mild symptomatic), 30 patients requiring O2 support (Moderate+ Severe), 36 mild symptomatic & 15 severe patients were stained and acquired on flowcytometer. Vertical scatter plots denote the comparisons of frequencies of immune cells and their subpopulation among different study groups: (A) CD3-CD56+ NK cells (B) CD3+CD56+ NKT-like cells (C) CD3+CD4+Th (D) CD3+CD8+Tc cells Data are presented as percentage of immune cells out of lymphocytes. The dots represent individual values and bars represent Mean+SD values. (P ***<0.0001).