The COVID-19 pandemic, caused by SARS-CoV-2, has resulted in millions of cases and deaths worldwide. Clinical manifestations range from asymptomatic infection to acute respiratory distress syndrome (ARDS). Various risk factors and comorbidities influence disease progression. This study focuses on the role of the cytotoxic immune response in determining the outcome of critical COVID-19 in unvaccinated individuals admitted to the ICU. A strong cytotoxic response is crucial for viral clearance, and its impairment could contribute to severe disease and death. The researchers hypothesized that a sustained cytotoxic response by PBMCs is essential for survival in critically ill, unvaccinated COVID-19 patients. Understanding this immune response is crucial for developing effective immunomodulatory therapies to improve patient outcomes and combat future outbreaks. Prior research has highlighted the importance of cytotoxic T cells and NK cells in controlling viral infections, and the role of cytokines like IL-15 in regulating these cell populations. This study aims to investigate the longitudinal changes in PBMC cytotoxicity in critically ill COVID-19 patients, and the potential for IL-15 to restore or enhance this response.

The literature review extensively covers the existing knowledge on COVID-19 pathogenesis, immune responses, and the role of cytotoxic cells. Studies demonstrating the correlation between lymphopenia, immune exhaustion, and severe COVID-19 outcomes are discussed. The role of IL-15 in immune cell regulation and its potential in immunotherapy is also explored. Several key studies are cited to highlight the existing understanding of immune dysregulation in COVID-19, including the role of cytokine storms and the exhaustion of T cells and NK cells. The literature review lays the groundwork for the study’s rationale by emphasizing the need for research into potential immunomodulatory therapies.

This observational, longitudinal study enrolled 23 unvaccinated individuals with critical COVID-19 admitted to the ICU. Blood samples were collected at admission and bi-weekly for 13 weeks, or until death or discharge. PBMCs and plasma were isolated. PBMC cytotoxic activity against SARS-CoV-2 was assessed using Vero E6 cells infected with pseudotyped SARS-CoV-2. Caspase-3 activity was quantified to measure cytotoxicity. Flow cytometry was used to analyze immune cell populations and expression of exhaustion markers (PD-1, TIGIT). The effect of in vitro IL-15 treatment (13 ng/mL) on PBMC cytotoxicity was evaluated. SARS-CoV-2 RNAemia was detected by RT-qPCR. Statistical analysis included Mann-Whitney U-test, Wilcoxon matched-pairs signed rank test, one-way ANOVA, Tukey's multiple comparisons test, and Fisher's exact test. The study followed ethical guidelines with informed consent from all participants. The detailed methodology encompasses the study design, participant recruitment, sample collection and processing, experimental procedures for assessing cytotoxicity and immune cell analysis, and the statistical methods employed to analyze the data. This detailed description ensures reproducibility and transparency.

The study found no significant differences between the Exitus and Survival groups in sociodemographic, clinical, or biochemical data at baseline. However, PBMCs from the Exitus group showed significantly reduced cytotoxic activity against SARS-CoV-2-infected cells at admission and at 4 weeks, which did not improve during hospitalization. IL-15 treatment did not restore cytotoxicity in the Exitus group. In contrast, PBMCs from the Survival group showed significantly increased cytotoxicity at Week 4, and IL-15 treatment further enhanced this effect. The Exitus group exhibited increased expression of immune exhaustion markers (PD-1, TIGIT) in NKT, CD4+, and CD8+ T cells. While CD8+ T cell counts were initially higher in the Exitus group, their cytotoxic activity, indicated by CD107a expression, was lower compared to the Survival group. Lymphocyte counts were higher in the Survival group for most of the study duration. No SARS-CoV-2 RNAemia was detected at baseline in either group. The magnitude of effect size calculations supported the statistical significance of the findings.

The key finding of persistently low cytotoxic activity in the Exitus group, even with IL-15 stimulation, points to immune exhaustion as a significant factor in fatal COVID-19. This exhaustion, indicated by elevated PD-1 and TIGIT expression, likely impairs the ability of immune cells to respond to IL-15 and clear the virus. In contrast, the Survival group's ability to mount a sustained cytotoxic response, further enhanced by IL-15, suggests the critical role of this immune function in COVID-19 recovery. The initial higher CD8+ T cell count in the Exitus group might reflect a compensatory response overwhelmed by exhaustion. The study suggests that monitoring immune exhaustion markers and response to IL-15 could be valuable prognostic indicators. While the study highlights the therapeutic potential of IL-15, it also reveals the limitations of IL-15 monotherapy in overcoming exhaustion, indicating the need for combination therapies.

This study demonstrates the crucial role of a sustained cytotoxic PBMC response in determining survival in critical, unvaccinated COVID-19 patients. Immune exhaustion, characterized by increased expression of PD-1 and TIGIT, appears to be a key factor contributing to fatal outcomes. While IL-15 shows promise in boosting cytotoxic activity in responsive individuals, combination therapies are necessary to overcome immune exhaustion. Future research should focus on identifying and testing more effective immunomodulatory strategies, including combination therapies with IL-15 and other immunomodulators.

The primary limitation of this study is the relatively small sample size. While statistically significant differences were found, a larger study would provide greater power and more robust conclusions. Another limitation is the focus on unvaccinated individuals, which might limit the generalizability of the findings to vaccinated populations. The in vitro IL-15 treatment might not fully reflect the complex in vivo dynamics of the immune response.