The COVID-19 outbreak presents tremendous challenges for the human race. In general, the causative organism, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causes a spectrum of disease, ranging from asymptomatic to fatal. A new study published on the preprint server medRxiv* in April 2020 suggests that an overactive immune response is responsible for the more severe manifestations of the illness.
Novel Coronavirus SARS-CoV-2 Colorized scanning electron micrograph of a VERO E6 cell (blue-green) exhibiting elongated cell projections and signs of apoptosis, after infection with SARS-COV-2 virus particles (yellow), which were isolated from a patient sample. Image captured at the NIAID Integrated Research Facility (IRF) in Fort Detrick, Maryland. Credit: NIAID
Immunity is generally linked to a better disease course and outcome. However, it is also known that a hyperimmune response can occur after viral infections, causing widespread damage and organ failure.
Recently, it has been found that severe illness is associated with high cytokine expression, including interleukin 6 (il-6). This has been called a cytokine storm and suggests an abnormal or imbalanced immune function. On the other hand, critically ill patients with COVID-19 have very low numbers of immune cells and reduced immune activation.
The current study aims to provide a detailed picture of how both specific and nonspecific immune responses take place in COVID-19 to produce disease of varying severity.
The researchers included 53 patients, 21 with moderate, 18 with severe and 14 with critical COVID-19 illness. Those with moderate and severe illness joined the study once they became symptomatic, and were tested for the virus using reverse transcription-polymerase chain reaction (RT-PCR). In those with a critical illness, the diagnosis had been made about 14 days before (median), and the patients were in the intensive care unit (ICU) at the time of joining the study.
Only one blood sample could be collected from 10 of 14 patients in the critical group.
Thus, there was a significant difference in the total duration of the illness between these three groups of patients. To account for this potential confounding factor, blood samples of patients in the moderate and severe groups were also collected over a period of 8 days after they joined the study.
The blood samples were used to isolate peripheral blood mononuclear cells (PBMCs). The researchers analyzed the counts of white cells in the blood, such as lymphocytes, granulocytes, and monocytes.
They also analyzed the changes in the severity of disease over the follow-up period of 8 days.
Reduced lymphocytes with cytotoxic phenotypes in critical or severe illness
They found that for most patients, the PBMC counts were subnormal, both at the initial visit and on follow-up. The number of natural killer cells (NK) was markedly reduced in severe and critical cases, but eosinophil numbers were higher in critical patients.
They used relative PBMC counts to compensate for individual variations caused by the subnormal levels. They found significantly lower levels of lymphocytes as a proportion of PBMCs in critical cases compared to moderate cases.
The ratio of T cells with CD4 to CD8 was higher in severe and critical cases relative to moderate cases because of a drop in CD4+ cells.
T cells with the memory phenotype that are long-lasting and preserve the memory of the virus were lower in both CD4 and CD8 subgroups in critical cases. Terminally differentiated T cells form a subtype of CD8 cells, which were also lower in critical and severe cases.
Activated CD4 cells with HLA-DR expression at its lowest level were found to be markedly reduced among critical cases, with the decline slowing from severe to moderate cases. A similar gradient was seen with CD57+CD4+ or CD57+CD8subtypes. The CD57 T cell is a highly migratory memory or effector T cell that is terminally differentiated and functionally competent.
CD11a-positive CD4 and CD8 T-cells showed a steep drop, as did CD28+CD4 T-cells, in critical cases compared to severe or moderate cases.
Among the B cells, transitional and marginal zone CD19 cells were found to show a progressively reducing trend in patients with severe and critical symptoms.
All these findings reflect a loss of activated and terminally differentiated cytotoxic T cells in severe and critical patients, remaining observable even at the follow-up visit.
They found that 10 of 18 patients improved sufficiently to move from the severe to the moderate disease cohort during their follow up. In these ten patients, the trend showed a reversal, with increased numbers of T cells in those subsets which had registered significantly falling numbers at the initial visit. At follow up, those who switched to moderate disease had a T cell profile similar to those who were classified as moderate at the first visit.
The researchers comment, “The improvement in the clinical course from severe to moderate in our study patients accompanied by the increase of affected T cells might point out an inflammation-triggered lymphocyte migration rather than apoptosis.” T cells carrying the key integrin molecule CD11a are drastically fewer in critically ill cases. Integrins are essential for the T cell to be activated and to migrate.
The next step was to examine how this affected specific T cell immunity to the virus.
Increased number and function of specific T cells
The investigators found that moderately ill patients had a slightly lower chance of having detectable specific T cells directed against the virus than severe or critical cases, at 44% vs 75% and 80%, respectively. At follow-up, all groups showed an increase to 77% in the moderate cases to 100% in the severe cases.
Less than 45% of patients had detectable CD8+CD137+ T-cells in moderate disease, but this percentage increased in severely ill cases from 50% to 75%. In critically ill patients, the percentage was 80%.
The number of patients who had T cells specific to the viral antigen and producing granzyme B or cytokines like TNF-α, INFγ or IL-2, at detectable levels was higher in severely and critically ill patients compared to moderate cases.
S-protein reactive and IL-2 producing T cell subsets were more often found and at higher counts in critical relative to moderate cases, with only a slight difference in cytokine levels between severe or critical vs. moderate patients. However, S-reactive T cells were generally very low in number.
At the initial visit, all groups of patients had the same differentiation profile for the T cells that were reactive with the S protein. The number and frequency of the S-protein reactive CD4+CD154 T cell subset, but not CD8+CD137 cells, was correlated with the antibody titer at all levels of disease severity.
What do these results imply?
The researchers conclude: there are much fewer circulating T-, NK- and B-cell subsets in patients with severe and critical disease relative to moderate illness. Secondly, critical cases had the lowest numbers of specialized, activated, and functional T cells, with the gradient increasing from critical to moderate illness.
The subsets most affected are those with cytotoxic effector response to foreign antigens, though the reason is unknown. However, it could be that the sudden and drastic decline is due to a migration of these activated nonspecific lymphocytes in response to inflammation, rather than lymphocyte death.
This is supported by the correlation with the level of severity of illness and the reversal of the trend as patients recover and switch from severe to moderate illness. The increased expression of IL-6 in severe and critical patients recruits activated T cells and macrophage, as shown in another study.
On the other hand, virus-specific T cells are present in higher numbers in critical illness, perhaps due to abnormal migration of the antigen-specific cells to the infected tissue.
An alternative expression is that this reflects the situation of the inflamed tissue. Even though these virus-specific effector cells may be protective against the virus, there is a clear association between the number of such cytokine-producing cells and the severity of illness.
“The large number of antigen-specific effector T cells leads to injury,” they hypothesize. This study provides initial evidence that the immune response is part of the problem in severe and critical COVID-19 illness, and this could mean that immunomodulation plays an important part in future clinical trials for the management of such patients.
medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.