SARS-CoV-2 is a highly transmissible and pathogenic coronavirus, emerged at the end of 2019, causing the global COVID-19 pandemic. The disease presents with a wide spectrum of symptoms, ranging from mild flu-like symptoms to severe, life-threatening conditions including cytokine storms. Initial clinical symptoms such as: fever, general weakness, and/or muscle pain (1), are often accompanied by shortness of breath, fatigue, and/or loss of taste and smell (2). Direct contact, including transmission through secretions and droplets from the nasal and oral mucosa during coughing or sneezing is widely recognized as a primary route of infection (3).

Coronaviruses are pleomorphic particles with a spherical envelope composed of a two-layered lipid structure embedded with structural membrane (M), envelope (E), and spike (S) proteins, and containing a positive-sense RNA genome. The spike glycoprotein, composed of subunits S1 and S2, includes a receptor-binding domain (RBD) responsible for interacting with the host cell receptor (1, 4). The SARS-CoV-2 spike glycoprotein binds the angiotensin-converting enzyme 2 (ACE-2), a membrane bound carboxypeptidase expressed in various human tissues (5). Following viral entry, the innate immune response acts as the first line of defense, employing multiple strategies to detect and eliminate the virus. Natural Killer cells (NK), macrophages, monocytes, dendritic cells, and neutrophils are rapidly activated and play a critical role in generating inflammatory cytokines, such as type I interferons (6).

SARS-CoV-2 primarily replicates in the epithelium of the upper respiratory tract, leading to the activation of antigen-presenting cells (APCs), and subsequently, the activation of naïve CD4⁺ and CD8+ T as well as memory T cells. Activated effector T lymphocytes migrate to the infection sites and play a crucial role in the antiviral immune response. CD4⁺ T cells promote macrophage activation, stimulate antibody production by B cells and assist CD8+ T cells in developing effective cytotoxicity response (7). CD8⁺ T cells, upon recognition of viral peptides via presented via Major Histocompatibility Complex Class I (MHC I), contribute to the formation of SARS-CoV-2-specific memory T cells and induce apoptosis in infected cells through the activation of perforin and granzymes. Extensive activation of macrophages, monocytes, neutrophils, and T lymphocytes, along with elevated levels of cytokines and chemokines, such as IFNα, IFNγ, IL-1β, IL-6, IL-12, IL-18, IL-33, TNFα, TGFβ, CCL2, CCL3, CCL5, CXCL8, CXCL9, and/or CXCL10 leads to a strong inflammatory response, which can culminate in a cytokine storm, acute respiratory distress syndrome (ARDS) and multiorgan failure (8). B lymphocyte independently recognize viral antigens and subsequently proliferate and differentiate into antibody-producing plasma cells and memory B cells (9).

Although knowledge and experience related to COVID-19 grown exponentially in recent years, a deeper understanding of the immunopathology associated with the SARS-CoV-2 infection remains desirable and is recognized as a global health challenge (10). Effective management and treatment of SARS-Cov2 still require improvement, particularly in predicting potentially severe conditions in infected patients at the time of admission. A significant decrease in overall lymphocyte count, especially T cells has been widely observed in the blood of COVID-19 patients (11). In severe cases, a marked deficiency of eosinophils and lymphocytes, significantly reducing the number of T, B, and NK cells, has also been reported (12). This profile is specific to the blood of affected patients and is, at least in part, related to the migration of these immune cells to the lungs (13).

Our study examines changes in T lymphocytes, specific T lymphocytes subsets, including: CD4^+/-CD8^+/-, CD4⁺CD25^+/-, CD8⁺CD25^+/-, CD4⁺CD45RA^+/-, CD8⁺CD45RA^+/-, CD4⁺CD25^HiCD127^Lo, CD4⁺CD25^HiCD127^LoFoxP3⁺, as well as selected cytokines/chemokines in COVID-19 patients and healthy controls. In this study, samples were collected from COVID-19 patients hospitalized at the Military Institute of Medicine – National Research Institute (MIM-NRI) in Warsaw between February and May 2021. Samples from healthy individuals recruited between February and November 2021 were also used. The aim of the study was to identify a panel of potential biomarkers associated with COVID-19 severity in order to enhance the understanding of its immunopathology and to propose future diagnostics tools capable of predicting the severity of the disease course.

WHO’s declaration states that SARS-CoV-2 outbreaks continue to occur globally, still causing life-threatening conditions—particularly in unvaccinated individuals. In 2024, new outbreaks were reported, though they were not widespread across multiple continents (15–17). On March 28, 2025, the European Centre for Disease Prevention and Control updated its list of SARS-CoV-2 variants of concern, including Omicron, KP.3 and BA.2.86, while Omicron XEC and LP.8.1 remain under consistent monitoring (18). Since the emergence of SARS-CoV-2, numerous studies have investigated immune response parameters, particularly immune cells, cytokines and chemokines, in relation to the clinical progression of COVID-19 (19–23).

In our study, we analyzed the immunological profiles of hospitalized COVID-19 patients during the 2021 pandemic in Warsaw, Poland, to identify potential biomarkers associated with immune dysregulation and prospective disease severity. Such biomarkers should be an early prognostic indicators of COVID-19 progression. Identifying them is important for predicting disease course and enabling timely, individualized therapeutic strategies.

We screened patients with severe and non-severe COVID-19 for selected subsets of T lymphocytes, cytokines specific to Th1/Th2/Th17 responses, and chemokines from the CC and CXC families, and compared the results with those of healthy volunteers. Samples were collected within 24 h of hospital admission, prior to the onset of symptoms used for stratifying patients into non-severe or severe groups.

Changes in lymphocytes percentages in the blood are often among the first signs of inflammation, with disease progression reflected by shifts in the distribution of various lymphocyte populations. In our study, we observed that the population of total lymphocytes and CD3⁺ T lymphocytes decreased in a severity-dependent manner, consistent with previously reported findings (24–28). However, we did not observe any significant changes in the percentage of helper T cells (CD4⁺) or cytotoxic T cells (CD8⁺), in contrast to other studies (24, 25, 27, 29). These discrepancies are unlikely to be explained by methodological differences, as standard and widely accepted techniques were used. Instead, population-specific factors and/or the influence of group size in the non-severe/severe cohorts may have contributed to the observed differences.

Recent studies have shown that double-positive lymphocytes may be involved in the pathogenesis of HIV (30), Dengue virus (31), Hantaan virus (32) and Hepatitis C virus (33). In our study, the double-positive lymphocytes population was significantly decreased in all COVID-19 patients compared to healthy individuals. These findings are consistent with the study by Kalpakci et al. (24) but not with Zahran et al. results (26). The role of double-positive lymphocytes in SARS-CoV-2 infection therefore remains unclear and requires further investigation. Moreover, double-positive lymphocytes are not considered as suitable biomarkers for COVID-19 severity, as their population is relatively small, and consequently difficult to identify.

Naïve T lymphocytes, precursors to memory and effector T cells, circulate in the periphery until activated by new antigens-presenting cells (APCs) (34). In our study, we observed a modest yet statistically significant decrease in the naïve CD4⁺CD45RA⁺/CD8⁺CD45RA⁺ T lymphocyte population in COVID-19 patients. Previous studies have reported an increase in the naïve CD4⁺CD45RA⁺ subpopulation in patients with non-severe and severe COVID-19, which contrasts with our findings (35–37). Since naïve T lymphocytes are crucial for maintaining a diverse T cell receptor (TCR) repertoire to recognize a broad range of pathogens (36), even a slight reduction in COVID-19 cases could suggest early signs of impaired immune responsiveness. However, given the relatively small effect size, this finding should be interpreted cautiously and warrants confirmation in larger cohorts.

Treg lymphocytes play an important role in modulating the immune response. Their increased activity can attenuate immune effectiveness, while they also prevent excessive immune activation by limiting effector lymphocyte activation and regulating cytokine production (38, 39). Patients in both the non-severe and severe groups showed a reduced percentage of Treg lymphocytes CD4⁺CD25^HiCD127^Lo and nTreg lymphocytes with FoxP3 expression CD4⁺CD25^HiCD127^LoFoxP3⁺, which are widely recognized as subpopulations with regulatory functions (40). The available literature presents a heterogeneous depiction of the dynamic changes in Treg populations, as COVID-19 patients have been described with increased (41–44), decreased (23, 25, 36) or no change (27, 45–47) in this lymphocyte populations. Similarly to our results, Jiménez-Cortegana et al. (29) observed a reduced percentage of Treg lymphocytes with the CD4⁺CD25^HiCD127^Lo phenotype in COVID-19 patients compared to healthy controls. In contrast, Dai et al. (28) and Fentoligo et al. (27) reported no differences in percentage of the Treg lymphocyte with CD4⁺CD25⁺FoxP3⁺ phenotype between patients with different COVID-19 severity (28) and between patients with severe COVID-19 compared to healthy individuals (27). Additionally, Wang et al. (48) showed that, the percentage of Treg lymphocyte with a CD4⁺CD25⁺CD127^- phenotype increased during progression from mild to severe conditions, then decreased during progression from severe to critical conditions. These discrepancies may result from the different criteria used for identifying Treg lymphocytes and the varying sampling timelines during the infection course. Moreover, differences may also arise from the use of whole blood cells (25, 28, 49)) versus peripheral blood mononuclear cells (23, 27, 47). Our findings suggest that impaired Treg lymphocyte function may contribute to disease progression and severity, but this requires confirmation in larger cohorts.

Hyperactivation of T lymphocytes and the excessive release of pro-inflammatory cytokines increase vascular permeability and plasma leakage, ultimately leading to lung injury, severe respiratory distress syndrome, and multi-organ failure (50–53). This process is recognized as a major contributor to severe COVID-19 and the associated mortality. In our study, we demonstrated that SARS-CoV-2 infection caused no significant differences in concentrations of IL2, IL4, TNF and CXCL8 in the sera of COVID-19 patients compared to healthy volunteers. However, a significant increase was observed in the anti-inflammatory IL10, as well as the pro-inflammatory cytokines IL6, IFNγ and IL17A, and the pro-inflammatory chemokines CCL2, CCL5, CXCL9, CXCL10 in patients with both non-severe and severe COVID-19. Notably, CXCL10 exhibited a severity-dependent pattern, progressively increasing over the course of the disease. Overall, the observed cytokine and chemokine increases appeared to be associated with disease severity, although the relatively small cohort size call for cautious interpretation and confirmation in larger studies.

The available literature extensively describes changes in cytokine and chemokine concentrations in serum (19, 54, 55) and plasma (21, 56, 57) in patients with varying severity of COVID-19. Bourhis et al. (55) reported increased serum concentrations of IL4, IL6, IL10 in SARS-CoV-2 infected patients compared to healthy controls, with the latter cytokines being higher in severe and critical cases. They also showed increased concentrations of the CCL2 and CXCL10 in accordance with disease progression, consistent with our data (55). Similarly, Ravindran et al. (21) demonstrated that plasma concentrations of IL6 and CXCL10 were significantly elevated in patients with severe COVID-19. Huang et al. (20), identified IL6, IL10 and CXCL10 as factors strongly associated with progression from mild to severe disease. Trifonova et al. (19), reported that CXCL8 and CXCL10 distinguished mild from moderate/severe cases, although not at statistically significant levels, while IL6 and IL10 concentrations were significantly higher in severe compared to mild and moderate cases. Zuñiga et al. (58), described dynamic changes in CXCL10 concentrations in the sera of Zika virus patients: levels were significantly elevated from enrollment (day 0) until day 28 compared to healthy controls, peaking on days 0 and 3, and decreasing by days 7 and 28. CXCL10 also appears to be an important factor in other viral diseases, such as SARS and measles, where the concentration of this chemokine is elevated and increases with disease progression, while a decreases are associated with recovery (59–61). Taken together, these observations suggest that CXCL10 may be a broadly relevant marker of immune activation in viral infections. However, its potential role as a diagnostic or prognostic biomarker in COVID-19 requires further clarification through studies using sequentially collected samples and larger, representative patient cohorts.

CXCL10 is a chemokine secreted by various cell types e.g.: fibroblasts, endothelial cells, T lymphocytes and monocytes/macrophages. It binds to the CXCR3 receptor, expressed on immune cells such as T and B lymphocytes, and its activation promotes chemotaxis, proliferation, and recruitment of macrophages, Th1 lymphocytes, and NK cells (22). This increased leukocyte activity may contribute to systemic inflammation and tissue damage (22). In our study, CXCL10 differentiated patients with a severe disease course from those with non-severe disease and from healthy controls. CXCL10 levels significantly correlated with total lymphocyte count and with IL6, IFNγ, CCL2, and CXCL9. Rydyznski Moderbacher et al. (62) also showed that CXCL10 strongly correlated with disease severity and the number of CD4⁺ and CD8⁺ T cells, while Lore et al. (63) demonstrated positive correlation between CXCL10 and CCL2, IFNγ, IL1Ra, CCL5, CCL11, IL6. Additionally, Fabris et al. (64) reported additive correlations between IL6 and IL10, IL10 and CXCL10, TNFα and CXCL8.

In our cohort, CXCL10 showed consistent associations with disease severity and with several other immune parameters, suggesting its involvement in the broader dysregulation of the immune response in COVID-19. While these findings highlight CXCL10 as a potentially informative biomarker, its role should be interpreted with caution given the limited sample size and the complexity of the cytokine network. Validation in larger and more diverse patient populations will be necessary to establish whether CXCL10, alone or as part of a biomarker panel, can reliably predict COVID-19 severity.

In our study CXCL10 was identified as a crucial chemokine in the severe course of COVID-19, serving as an early marker of diseases progression. Additionally, the correlation between CXCL10 and the percentage of total lymphocytes, as well as the levels of IL6, IFNγ, CCL2 and CXCL9 suggests a complex immune dysregulation occurring in COVID-19. This may serve as an associated panel for predicting the severity of disease.

Our single-center study was conducted during the COVID-19 pandemic in 2021 among hospitalized, unvaccinated patients. At that time in Poland, infections were mainly caused by the Alpha, Beta, and Delta variants, and successive age and occupational groups of the population were gradually included in vaccination programs. Since the study was conducted, the immunological landscape has continued to evolve with the emergence of new SARS-CoV-2 variants, such as Omicron, as well as due to widespread vaccination. The predictive value of biomarkers identified on the basis of 2021 data may not be the same for infections caused by currently circulating SARS-CoV-2 variants and in vaccinated individuals. However, some of the biomarkers we identified may remain relevant regardless of vaccination status and viral variant. Kawasuji et al. (65) demonstrated no statistically significant differences in serum levels of CXCL10, IL6, and IFNγ when comparing vaccinated and unvaccinated patients. Moreover, other studies indicate that some of the biomarkers we analyzed also show significantly higher levels in patients with severe SARS (CXCL10, CCL2, IFNγ) and MERS (CXCL10, CCL2, IL6) compared to patients with mild forms of these diseases (66–70). These observations may point to the stability of certain biomarkers irrespective of vaccination and SARS-CoV-2 variants. Furthermore, the repeated involvement of CXCL10 and CCL2 in severe SARS, MERS, and COVID-19 may indicate shared mechanisms of inflammatory responses in severe coronavirus infections. In summary, further studies in representative patient cohorts are needed to confirm whether the biomarkers identified in the present work remain useful for risk stratification in today’s predominantly vaccinated populations and with respect to currently circulating variants. The identification of universal biomarkers could facilitate the assessment of the risk of severe COVID-19 and the implementation of potential interventions aimed at restoring immunological balance in affected patients.