Measurement of IFN-γ and IL-2 for the assessment of the cellular immunity against SARS-CoV-2

Abstract

The study of specific T-cell responses against SARS-CoV-2 is important for understanding long-term immunity and infection management. The aim of this study was to assess the dual IFN-γ and IL-2 detection, using a SARS-CoV-2 specific fluorescence ELISPOT, in patients undergoing acute disease, during convalescence, and after vaccination. We also evaluated humoral response and compared with T-cells with the aim of correlating both types of responses, and increase the number of specific response detection. Blood samples were drawn from acute COVID-19 patients and convalescent individuals classified according to disease severity; and from unvaccinated and vaccinated uninfected individuals. IgGs against Spike and nucleocapsid, IgMs against nucleocapsid, and neutralizing antibodies were also analyzed. Our results show that IFN-γ in combination with IL-2 increases response detection in acute and convalescent individuals (p = 0.023). In addition, IFN-γ detection can be a useful biomarker for monitoring severe acute patients, as our results indicate that those individuals with a poor outcome have lower levels of this cytokine. In some cases, the lack of cellular immunity is compensated by antibodies, confirming the role of both types of immune responses in infection, and confirming that their dual detection can increase the number of specific response detections. In summary, IFN-γ/IL-2 dual detection is promising for characterizing and assessing the immunization status, and helping in the patient management.

Figure 1

IFN-γ (a) and IL-2 (b) T-cell responses against the SARS-CoV-2 antigens. The specific response for each cytokine is represented using the SI (stimulation Index). Differences between two group conditions were calculated using the two-tailed Mann–Whitney U test. P is considered significant when < 0.05 (* < 0.05, ** < 0.01, *** < 0.001, and *** < 0.0001). SI: Stimulation index; Non-Invasive Ventilation (NIV); Invasive Mechanical Ventilation (IMV).

Figure 2

SI ratios between both cytokine responses (SI IL-2/SI IFN-γ) for the different study groups. Values over 1 meant higher IL-2 response whilst values below 1 meant higher IFN-γ response. Samples with a 0 value for IFN-γ SI and IL-2 higher than 0 were excluded for avoiding indeterminate results in the ratio calculation. Differences between conditions were calculated using the two-tailed Mann–Whitney U test. P is considered significant when < 0.05 (* < 0.05, ** < 0.01, *** < 0.001, and *** < 0.0001). SI: Stimulation index; Non-Invasive Ventilation (NIV); Invasive Mechanical Ventilation (IMV).

Figure 3

Correlations between IFN-γ (green) and IL-2 (orange) T-cell response with time since the first dose of the vaccine administration (a); time since diagnosis in acute patients (b) and convalescent individuals (c); and time since symptomatology appearance in acute patients (d). For figure (b), some samples were collected 35 days post-diagnosis during patient hospitalization in semi-critical or in intensive care units with severe symptomatology. For (c), some samples were collected 10 days after diagnosis but are considered convalescent as they correspond to healthcare workers with mild symptoms or without symptoms during the infection and have either a posterior negative PCR during their routine examinations or a finalization of the symptomatology. The specific response for each cytokine is represented using the SI. Correlations were calculated using the two-tailed non-parametric Spearman test. SI: Stimulation index.

Figure 4

Correlations between IFN-γ (green) and IL-2 (orange) T-cell response with IgGs against Spike (a), IgGs against NCP (excluding the uninfected vaccinated individuals) (b), IgMs in acute patients (c), and per-centage of neutralization (d). The specific response for each cytokine is represented using the SI. Correlations were calculated using the two-tailed non-parametric Spearman test. In (a), results equal to or over 384 BAU/mL were excluded for statistical analysis (the results including the ones over > 384 BAU/ml maintained significance and moderate correlations: SR IFN-γ = 0.317; SR IL-2 = 0.429). SI: Stimulation index. BAU: Binding antibody units.