A flow cytometry-based proliferation assay for clinical evaluation of T-cell memory against SARS-CoV-2.

In general, the method of choice for evaluating immunity against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is detection of antibodies against the virus in patient sera. However, this is not feasible in patients who do not produce antibodies, either due to a primary immunodeficiency or secondary to treatment with immunosuppressive drugs. Assessment of the antiviral T cell response is an alternative to serological tests, but most T cell assays are labor-intensive and unsuitable for a clinical routine laboratory. We developed a flow cytometry-based assay for T cell proliferative responses against SARS-CoV-2, based on the detection of blast transformation of activated cells. The assay was validated on previously SARS-CoV-2 infected individuals and healthy seronegative blood donors, displaying 74% sensitivity and 96% specificity for previous infection with SARS-CoV-2. The usefulness of the assay was demonstrated in a patient with common variable immunodeficiency with a history of COVID-19. The described T-cell assay is a clinically relevant complement to serology in the evaluation of cellular immunity against SARS-CoV-2, which can be emulated by any routine lab with flow cytometric competence.

Long lasting local and systemic inflammation after cerebral hypoxic ischemia in newborn mice.

BACKGROUND: Hypoxic ischemia (HI) is an important cause of neonatal brain injury and subsequent inflammation affects neurological outcome. In this study we performed investigations of systemic and local activation states of inflammatory cells from innate and adaptive immunity at different time points after neonatal HI brain injury in mice. METHODOLOGY/PRINCIPAL FINDINGS: We developed a multiplex flow cytometry based method combined with immunohistochemistry to investigate cellular immune responses in the brain 24 h to 7 months after HI brain injury. In addition, functional studies of ex vivo splenocytes after cerebral hypoxic ischemia were performed. Both central and peripheral activation of CD11b(+) and CD11c(+) antigen presenting cells were seen with expression of the costimulatory molecule CD86 and MHC-II, indicating active antigen presentation in the damaged hemisphere and in the spleen. After one week, naïve CD45rb(+) T-lymphocytes were demonstrated in the damaged brain hemisphere. In a second phase after three months, pronounced activation of CD45rb(-) T-lymphocytes expressing CD69 and CD25 was seen in the damaged hemisphere. Brain homogenate induced proliferation in splenocytes after HI but not in controls. CONCLUSIONS/SIGNIFICANCE: Our findings demonstrate activation of both local and systemic immune responses months after hypoxic ischemic neonatal brain injury. The long term immune activation observed is of general importance for future studies of the inflammatory response after brain injury as most previous studies have focused on the first few weeks after damage, while the effects of the late inflammation phase may be missed. Furthermore, the self-reactive component raises the question if there is a correlation with development of autoimmune brain disease later in life.