What is Cytokine Release Syndrome (CRS)?
Cytokine-associated toxicity, also known as cytokine release syndrome (CRS), is a non–antigen-specific toxicity that occurs as a result of high-level immune activation and can be triggered by a variety of factors such as certain drugs and infections. As the name implies, several cytokines are released and elevated during CRS. The main cytokines implicated during the pathogenesis of CRS include interleukin-6 (IL-6), tumor necrosis factor (TNF-α), interferon-γ (IFN-γ) and interleukin-10 (IL-10) (Fig.1).
The term “cytokine release syndrome” was first described in the 1990s, when the anti- CD3 T-cell antibody (OKT3) was introduced into the clinic as an immunosuppressive treatment for organ transplantation.. Cytokine release syndrome (CRS) represents potentially life-threatening toxicity recently observed following infusion of several antibody-based therapies with natural and bi-specific antibodies, several adoptive T-cell therapies for cancer, severe viral infections such as influenza  and most recently in those patients presenting with severe COVID-19 infections.
The proposed mechanism of how CRS is induced comes from the activation of mainly T cells or by the lysis of immune cells which induces a release of IFN-γ and TNF-α as observed in severe viral infections or by activation of T cells from treatment with various adoptive T-cell therapeutic agents. Release of these cytokines triggers a chain reaction due to the activation of innate immune cells like macrophages and endothelial cells, which release additional cytokines, hence the term "cytokine storm or cytokine release syndrome." IL-6 is a key component of CRS and it appears that using an anti-IL-6 antibody, like tocilizumab, may help to immunosuppress the release of this cytokine helping to save patients’ lives.
Figure 1. Cytokine Release Syndrome can be triggered by activation of T cells due to a severe viral infection like influenza or by the administration of therapeutic stimuli such as an adoptive T-cell therapy or by direct target cell lysis with the subsequent release of cytokines like INF-γ and TNF-α. These cytokines can trigger a cascade reaction due to the activation of innate immune cells, hence the term "cytokine storm."
What is the role of Cytokine Release Syndrome (CRS) in Adoptive T-cell Therapies?
Over the past several years, immune-based therapies for treating cancer have become more potent, effective, and widely utilized for treatment and subsequently, unique toxicity profiles identified in treated patients. Cytokine release syndrome (CRS) is a life-threatening toxicity associated with numerous immunotherapeutic techniques involving monoclonal antibodies, bispecific antibodies, and adoptive T cell therapies that results in the release of a large number of cytokines such as, IL-6, IFN-γ, and TNF-α from the target cells.(Fig. 2). Cytokines, when released in excessive amounts into the circulation, produce many adverse side-effects like nausea, chills, fever, rashes, headache, hypotension, etc.
Most patients present mild to moderate symptoms which can easily be managed, but some patients show life-threatening symptoms, and as such CRS has become increasingly recognized as one of the most severe and frequent adverse effects of these therapies. 
CRS represents a potentially serious complication of CAR-T therapy. Cytokines are released when the interaction between tumor and immune effector cells occurs. This reaction can originate not only from the CAR-T itself but also from host immune cells such as macrophages, which respond in part to CAR-T activation. The CRS cytokine associated toxicity that results comes from excessive immune activation of these cell types.
Immune activation is the primary underlying mechanism of immune-therapeutic treatments and is used to attain the desired clinical benefit. However, the activation of the immune system by these immune-therapeutic agents beyond normal expectations results in the massive release of cytokines and hence the adverse toxic effects in the patient. It appears that the management of these unique toxicities may be controlled by the administration of an anti-IL-6 receptor antibody, Tocilizumab, and may help reverse the syndrome. 
Figure 2. Cytokine profile of CRS and potential mechanism of action in Adoptive T-cell therapies. Profound activation of T cells or lysis of other immune cells induces the release of IFN-γ and/or TNF-α, which leads to the activation of macrophages, dendritic cells, other immune cells, and endothelial cells. These cells, in turn, release pro-inflammatory cytokines that produce larger amounts of IL-6. IL-6 then acts in a positive feedback manner that further activates T cells and other immune cells to release additional cytokines.
What is the role of Cytokine Release Syndrome (CRS) in COVID-19?
Cytokines and chemokines play an important role in immunity, but when the immune response becomes dysregulated and exasperated by these factors they've been shown in many cases to cause lung damage and diminish patient survival. When beta coronaviruses, like SARS-CoV-2 (COVID-19), infect monocytes, macrophages, and dendritic cells, they can become activated and secrete IL-6 along with other inflammatory cytokines(Fig. 3).
How common is cytokine release syndrome in COVID-19 patients?
CRS is common in patients with COVID-19, and elevated levels of serum IL-6 correlates with respiratory failure and adverse clinical outcomes.  In COVID-19 infected individuals, IL-6, IL-10, and TNF-α surge during infection and decline during recovery.
Patients with severe disease have significantly higher levels of IL-6, IL-10 and TNF-α, decreased IFN-γ expression in CD4+ T cells and fewer CD4+ and CD8+ T cells indicating that cytokine release syndrome may dampen adaptive immunity against the virus. . Flow cytometry analysis of COVID-19 patients found a decrease in CD4 and CD8 T cells but an increase in Th17 populations.
Th17 cells are T helper cells that differentiate from To when stimulated with IL-6 and IL-23. Severe cases of the disease seem to show a sustained decrease in the proportion of lymphocytes compared to those found in mild cases. Also, CD8 T cells decrease, and inflammatory cytokines like IL-6, IL-10, IL-2, and IFN-γ increase in severe cases in the peripheral blood. .
A recent article  has indicated that mild or severe cytokine storms, accompanied by high levels of IL-6, occurs in patients with severe COVID-19 and is a leading cause of death in these patients. (Fig. 4). It appears that blocking the IL-6 signaling pathway with the IL-6 inhibitor Tocilizumab may be used as a method of treatment of patients with severe symptoms from a COVID-19 infection.
These results show that COVID-19 induced cytokine release syndrome (CRS) is associated with disease severity and outcome. Understanding immune dysregulation in patients with COVID-19 will provide not only a greater understanding of the viruses’ pathogenesis but also identifies potential targets for immune-therapeutics and provide insights into effective vaccine designs. Targeted immunomodulation that reduces CRS may effectively reduce severe pulmonary inflammation and hopefully reduce patient mortality.
Figure 3. Cytokine Release Syndrome in COVID-19 Patients.
Figure 4. Evolution of Cytokine Response Syndrome During COVD-19 Infection. Compared with uninfected individuals (left panel), moderate COVID-19 cases exhibit an increase in IL-6 and a decrease in total T lymphocyte counts, particularly CD4+ T cells and CD8+ T cells (middle panel). Severe COVID-19 cases have further increased production of IL-6, IL-2R, IL-10, and TNF-α, while total T lymphocytes, particularly CD4+ T cells and CD8+ T cells, and IFN-γ–expressing CD4+ T cells markedly decrease (right panel). The level of cytokine storm and T cell lymphopenia is associated with pulmonary damage, respiratory distress, and unfavorable outcome.
Expertise in flow cytometry is critical in advancing our understanding of the role that cytokine release syndrome (CRS) plays in both the development of cellular therapies like CAR-T and in the monitoring of patients with infectious diseases, like COVID-19. In particular, flow cytometry-based measurements for the quantification of cellular subsets and inflammatory cytokines in the blood during and immediately following treatment have been early indicators for predicting the severity of CRS before onset.
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1. Transplantation. 1990 Apr;49(4):697-702. In Vivo Cell Activation Following OKT3 Administration. Systemic Cytokine Release and Modulation by Corticosteroids. L. Chatenoud et.al. https://www.ncbi.nlm.nih.gov/pubmed/2109379
2. Microbiol Mol Biol Rev. 2012 Mar;76(1): 16-32. Into The Eye of the Cytokine Storm. JR. Tisoncik et.al. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3294426/
3. Int J Antimicrob Agents. 2020 Mar 29:105954. The Cytokine Release Syndrome (CRS) of Severe COVID-19 and IL-6 Receptor (IL-6R) Antagonist Tocilizumab may be key to Reduced Mortality. C. Zhang et. al.
|Authored by: Susan Reynolds|
Susan Reynolds has worked at FlowMetric for over four years. She has over three decades of experience in the areas of flow cytometry and assay development. Her work has focused on the therapeutic areas of immunology, immune-oncology, cell therapy, and gene therapy. Sue holds a graduate degree in Immunology from the University of Maine and an executive education MBA from MIT Sloan School of Management. Sue's career includes drug development research working within Pfizer and Sanofi and as a flow cytometry technical application specialist within Thermo Fisher Scientific and BD Biosciences. Sue was part of Becton-Dickinson’s Global HIV Health initiative working in Africa with the World Health Organization to advance training in flow cytometric applications for advancing vaccine development in the areas of HIV and malaria.