In a recent study published on Research Square* preprint server, researchers explored the underlying mechanism leading to thymic atrophy and subsequent lymphopenia in patients with coronavirus disease 2019 (COVID-19).
Previous studies have reported dysregulated T cell function and lymphopenia in COVID-19 patients. However, the literature does not shed light on the immunological and pathological alterations of the thymus after infection with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).
The thymus is the primary site of T cell development; aging, pathogenic infections, nutritional deficiencies, cancer and hormonal changes impact its health, which is defined by its performance. Peripheral thymocyte escape, developmental pathway arrest, or increased apoptosis are all responsible for thymic atrophy due to pathogenic infection.
About the study
In the current study, researchers intranasally infected human angiotensin-converting enzyme 2 (hACE2)-Tg transgenic hamsters or mice with 105 PFU of live SARS-CoV-2. The viral inoculum for hamsters and mice was 100 μl for hamsters and 50 μl for hACE2-Tg mice. The control group received Dulbecco’s Modified Eagle’s Medium (DMEM) instead of live virus, and untested animals received injections of phosphate buffered saline (PBS).
From the day before viral challenge to day three after infection, animals in the test group received a subcutaneous (sc) injection of remdesivir (RDV) at 25 mg/kg body, and animals in the control group received injections of PBS.
They used an anti-mouse interferon-gamma (IFNγ) antibody for IFN-γ neutralization at a dose of 10 mg/kg body mass at two time points one day before the SARS-CoV-2 challenge and two days after the challenge by intraperitoneal injections. The control group received only the immunoglobulin G antibody.
The team recorded the animals’ body mass daily post-challenge. Similarly, they sacrificed six animals from the test and control groups, and their thymuses were extracted and examined for any gross morphological changes.
The researchers analyzed thymus sections using immunofluorescence microscopy for the nucleocapsid (N) protein. A trained pathologist scored the hematoxylin and eosin (H&E) stained sections on a scale of 0 to 5, where a score of 5 indicated the highest pathologic feature. They also performed immunophenotyping of human peripheral blood mononuclear cells (PBMC).
The team also isolated ribonucleic acid (RNA) from homogenized thymus cells, transcribed it into complementary deoxyribonucleic acid (cDNA), and performed quantitative polymerase chain reaction (qPCR). They used the cloned transcript (as a template) to generate a standard curve to estimate the RNA copy number of the SARS-CoV-2 N gene. The researchers used the trypan blue exclusion method to determine the number of living cells of the thymus and lymph nodes.
Researchers compared and analyzed thymus and body mass, gene expression, and enzyme immunoassay (ELISA) results using either one-way ANOVA or two-way ANOVA.
hACE2-Tg mice infected with Wuhan-Hu strain 1 SARS-CoV-2 developed profound thymic atrophy with seven- to eight-fold reduced size, primarily arresting developing thymocytes at double-negative stage 1 (DN1). Additionally, intriguingly, SARS-CoV-2-induced thymocyte apoptosis resulted in increased cell death.
qPCR data showed the presence of SARS-CoV-2 N gene RNA in all thymocyte cell subpopulations. Immunofluorescence microscopy also detected the presence of virus in the cellular compartments of thymocytes. However, the investigation could not identify the exact mechanism of SARS-CoV-2 entry into thymocytes.
Therefore, the authors hypothesized that either infected progenitor T cells were migrating to the thymus, thus disseminating the infection, or the virus was migrating to the thymus by direct impoundment.
Remarkably, IFN-γ adequately induced thymic atrophy because neutralizing IFN-γ by neutralizing monoclonal antibodies rescued thymic atrophy. In contrast, interleukin 17 (IL-17), IL-4, granzyme B (GzB), and perforin-1 (Prf-1) had limited roles in SARS-CoV-induced thymic atrophy -2. Interestingly, RDV antiviral therapy effectively rescued mice from SARS-CoV-2-related thymic atrophy.
Also, not Omicron but severe Delta-induced thymic atrophy worse than the ancestral strain in Delta-infected mice, with profoundly impaired T-cell development.
Anti-SARS-CoV-2 P4A2 monoclonal neutralizing antibody therapy initiated at an early stage of infection helped inhibit thymic pathology and rescued the infected animal by restoring the T cell developmental pathway.
Overall, the study demonstrated that thymic dysregulation and thymic atrophy cause SARS-CoV-2-related lymphopenia and changes in peripheral T cell receptor (TCR) repertoire. Given that thymic atrophy leads to a loss of the peripheral TCR repertoire, study results could improve understanding of how T cell response was reduced during COVID-19 and help develop new vaccine candidates. .
Research Square publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be considered conclusive, guide clinical practice/health-related behaviors, or treated as established information.