Analysis Shows Diverse Cytokine, Chemokine, and Growth Factor Responses upon CD4 and CD8 Activation, Demonstrating Robust Regulation and Stability in Early Cytokine Responses to SARS-CoV-2 Infection
Upon stimulating CD4 or CD8 cells with targeted peptide pools (Fig. 1), we consistently observed minimal median concentrations (0.01 pg/ml) of cytokines, chemokines, and growth factors such as FGF-2, IFN-alpha-2, IFN-gamma, IL-7, IL-12-p40, IL-12-p70, IL-13, IL-15, IL-17A, IL-17F, and IL-22 across all time points studied. This persistent low-level presence suggests a limited impact on immune signaling processes, underscoring the unclear role of these factors. Stimulation using CD4- or CD8-directed peptides resulted in modest FLT-3L, IL-18, and TGF-alpha (0.71 pg/mL). Simultaneously, a range of other molecules, such as sCD40L, EGF, Eotaxin, G-CSF, GRO-alpha, IL-1-alpha, IL-1-beta, IL-1RA, IL-2, IL-6, IL-10, IL-27, MCP-3, MDC, MIG/CXCL9, PDGF-AA, PDGF-AB/BB, RANTES, TNF-alpha, and VEGF-A, exhibited substantial concentrations (86.49 pg/mL) consistently. Diverse immune activations were observed, accompanied by the release of a spectrum of cytokines and growth factors, suggesting the engagement of both pro-inflammatory and anti-inflammatory processes. Immune cell mobilization towards the site of stimulation was evident, with a harmonious interplay of these mediators crucial for immune response regulation and preservation of equilibrium, marked by sustained and moderate concentrations spanning an extended duration, indicative of a prolonged rather than transient response. The prolonged release of these molecules indicates their enduring role in the immune cascade triggered by CD4 or CD8 peptides. Their persistent presence highlights the essential need for ongoing immune regulation and coordination to effectively enhance the immune response. Significantly, robust elevation in IL-8, MCP-1, MIP-1-alpha, and MIP-beta occurred consistently upon CD4 and CD8 cell stimulation, highlighting their pivotal role in attracting and activating monocytes, macrophages, and neutrophils. These heightened secretion levels denoted an ongoing immune response characterised by mobilising and stimulating immune cells at the infection site, vital for combatting pathogens and addressing tissue damage.
Stimulation of CD4 and CD8 cells induced dynamic alterations in cytokine concentrations, yielding important implications for the immune response.
We comprehensively analysed 48 biomolecules, comparing their levels before and after stimulation with CD4- and CD8-directed peptide pools, revealing significant concentration changes in 19 factors (Fig. 2A). Enhanced post-stimulation levels of 13 factors signified a vigorous immune activation response, while six factors exhibited elevated pre-stimulation levels, implying their potential regulatory functions in the baseline state. Stimulation of the CD4-directed peptides resulted in a notable increase in five essential cytokines - Eotaxin, IL-1RA, IL-7, MCP-3, and M-CSF, compared to their baseline levels. The significant elevation of these cytokines highlights a robust immune response and emphasizes the crucial impact of targeting CD4 cells in enhancing immune activation. Furthermore, the swift and immediate cytokine response on Day 0 underscores their direct involvement in stimulating CD4 cells.
After CD8-directed peptide stimulation on day 0, a distinct cytokine response emerged, with significant increases observed in EGF, Eotaxin, IL-1RA, IL-4, IL-12-p40, IL-18, M-CSF, PDGF-AA, PDGF-AB/BB, RANTES, and VEGF-A levels compared to the baseline. These altered cytokine profiles underscore their potential influence on immune modulation, encompassing vital roles in cell growth, chemotaxis, inflammation regulation, and angiogenesis promotion. Notably, the persistence of heightened Eotaxin and VEGF-A concentrations, even on the seventh day, surpassing baseline levels, underscores their enduring significance in CD8-mediated immune responses. This sustained elevation implies a continuous role in immune cell recruitment and angiogenesis, crucial for establishing a robust antiviral immune defence. By Day 14, only IL-12-p40 showed a noticeable rise (7.5 pg/ml) following CD8 stimulation, suggesting its potential involvement in modulating Th1 immune responses and supporting cytotoxic functions. These findings hint at the sustained engagement of CD8-mediated immunity during the subsequent phases.
Our research reveals that cytokine levels vary when CD4 and CD8 cells are activated, suggesting diverse immune responses to stimuli. These changes help us understand the unique and precise ways our immune system operates. Our results provide insight into the initial cellular responses in people with mild or symptom-free COVID-19.
Stimulation targeted specifically at CD4 and CD8 cells elicits distinct cytokine and growth factor profiles.
The Wilcoxon signed-rank test revealed distinct differences in cytokine concentrations between CD4 and CD8 stimulation across various time points (Day 0, Day 7, and Day 14), comprising ten cytokines on Day 0, twelve cytokines on Day 7 (including EGF, Eotaxin, IL-1-beta, IL-8, and IL-18), and nine cytokines on Day 14 (such as EGF, Eotaxin, Fractalkine, IL-2, MCP-1, MCP-3, PDGF-AA, PDGF-AB/BB, and VEGF-A), underscoring the dynamic immunomodulatory responses elicited by these peptide stimulations (Fig. 2B). Distinct cytokine patterns emerged from the use of CD4 and CD8 peptides for stimulation. Notably, CD4-directed peptides prompted elevated levels of Fractalkine, IL-2, IL-17A, MCP-1, MCP-3, and PDGF-AB/BB, while CD8-directed peptides induced a significant increase in 13 cytokines including EGF, Eotaxin, FLT-3L, IL-1-beta, IL-3, IL-8, IL-18, IL-27, PDGF-AA, PDGF-AB/BB, RANTES, TNF-alpha, and VEGF-A. Notable differences in cytokine levels affirm that the activation of CD4 and CD8 cells elicits different immune responses, possibly exerting distinct influences on immune modulation and disease progression.
Elevated cytokine levels in symptomatic individuals reveal the significant involvement of TGF-alpha, GRO-alpha, IL-6, and IL-10 in viral infection symptoms.
In a cohort of 15 participants, with 53.3% presenting symptoms and 46.7% asymptomatic, an unpaired Wilcoxon rank sum test revealed significant cytokine level discrepancies. Notably, TGF-alpha, linked to CD8-directed responses, exhibited notably higher median levels (median: 0.175 pg/ml; IQR: 0.00–0.74 pg/ml) in symptomatic individuals compared to asymptomatic counterparts (median: 0.00 pg/ml; IQR: 0.00–0.00 pg/ml), as shown in Fig. 2C. Similarly, symptomatic subjects exhibited distinct and significantly elevated distributions of CD4-directed GRO-alpha (714.34 pg/ml; IQR: 520.48-1629.48 pg/ml), IL-6 (1941.39 pg/ml; IQR: 713.14-3261.55 pg/ml), IL-10 (27.97 pg/ml; IQR: 20.09–92.74 pg/ml), and TGF-alpha (0.35 pg/ml; IQR: 0.00-0.98 pg/ml) than the asymptomatic group. Elevated levels of TGF-alpha, GRO-alpha, IL-6, and IL-10 assume pivotal roles within the immune system. TGF-alpha governs the immune response to viral infections by modulating cellular growth and differentiation, while GRO-alpha attracts immune cells to infection sites, bolstering viral clearance. IL-6 modulates immune responses and potentially contributes to symptom manifestation. Simultaneously, IL-10, an anti-inflammatory cytokine, is heightened to counter excessive inflammation triggered by the virus. The initial symptoms directly shape the concentrations of these cytokines, underscoring their potential in both symptom development and expression during viral infections.
Utilising Random Forest Analysis for Machine Learning-Based Prediction of Key Cytokines in the Dynamic Immune Response over Time.
We used a machine learning-driven Random Forest analysis to determine the predicted key cytokines steering the immune response at distinct time points. Our approach unveiled a dynamic cytokine pattern marked by fluctuating importance across diverse CD4 (Fig. 3A) and CD8 (Fig. 3B) peptide pool stimulation scenarios, accentuating the intricate nature of their influence. During the initial infection phase (Day 0), IL-8 and G-CSF were identified as pivotal cytokines, orchestrating the immune cascade in response to CD4-directed peptides. Transitioning to Day 7, a distinct cluster of cytokines, including MCP-1, EGF, IL-6, IL-3, and MIP-1-beta, emerged, pivotal in harmonizing cellular activities. By Day 14, the pronounced emergence of IL-1-beta, MCP-3, IL-2, and IL-6 had assumed central roles in modulating later-stage immune processes. Notably, G-CSF initiated CD8-directed immune responses on Day 0, while Day 7 revealed the prominence of cytokines like MCP-3, VEGF-A, MDC, GRO-alpha, and MIG/CXCL9 in shaping the intermediate immune phase. By Day 14, GRO-alpha, IL-10, and EGF ultimately assumed significance in driving CD8 stimulation. The study revealed significant predictive capabilities of crucial cytokines, including IL-8, G-CSF, MCP-1, EGF, IL-6, IL-3, MIP-1-beta, IL-1-beta, MCP-3, IL-2, GRO-alpha, IL-10, VEGF-A, MDC, and MIG/CXCL9 - when exposed to CD4- or CD8-directed spike peptides on specific days, emphasizing their potential as effective targets for therapeutic interventions. These findings underscore the intricate role of time-dependent cytokine variations in immune responses, providing a foundational understanding of tailored immunological treatments to modulate CD4 and CD8 stimulation responses.
Three Distinct Cytokine Clusters Reveal MCP-1 and IL-8 as Critical Regulators with Persistent and Specialized Functions in CD4 T-Cell Stimulation
Principal Component Analysis (PCA) was used to reduce the dimensionality of 48 cytokines, preserving crucial information while condensing it. Subsequently, k-means clustering facilitated the grouping of cytokines into distinct clusters based on their similarities in the reduced space. Analysis of CD4-directed spike antigens on Days 0, 7, and 14 revealed three distinct groups represented by blue, red, and green clusters, where cytokines of the same colour exhibited high similarity while different-coloured cytokines demonstrated significant differences (Fig. 4). The cytokines IL-6, MCP-1, MIP-1-alpha, and MIP-1-beta formed a tightly interconnected red cluster, suggesting collaborative immune responses or shared regulatory mechanisms. In contrast, IL-8 stood apart in a distinct blue cluster, indicating unique characteristics and regulatory mechanisms, implying divergent functions. Meanwhile, a green cluster encompassing diverse cytokines, including sCD40L, EGF, Eotaxin, and IL-10, exhibited overlapping expression patterns, unveiling potential shared biological pathways and functional associations. Within the red cluster, MCP-1, a protein recognised for its involvement in monocyte recruitment during inflammation, stood out. Its unique placement in the red group suggests a unique function, making it a promising candidate as a specific marker for monitoring monocyte-mediated immune responses. The diverse array of cytokines within the green group, including sCD40L, EGF, Eotaxin, FGF-2, FLT-3L, Fractalkine, G-CSF, GM-CSF, GRO-alpha, IFN-alpha-2, IFN-gamma, IL-1-alpha, IL-1-beta, IL-1RA, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12-p40, IL-12-p70, IL-15, IL-17A, IL-17E/IL-25, IL-17F, IL-18, IL-22, IL-27, IP-10, MCP-3, M-CSF, MDC, MIG/CXCL9, MIP-1-alpha, MIP-1-beta, PDGF-AA, PDGF-AB/BB, RANTES, TGF-alpha, TNF-beta, and VEGF-A, collectively holding functional importance. These cytokines are vital in immune responses, cell growth, inflammation, and blood vessel formation. They help determine how immune cells develop, grow, and produce other cytokines. They also have a role in attracting and activating immune cells. Among them, IL-8 is noteworthy as it helps bring neutrophils, a type of immune cell, to areas with infection or inflammation. By day 14, the red cluster exclusively featured MCP-1, highlighting its specialised function in recruiting monocytes. The green cluster cytokines included IL-17A, IL-17E/IL-25, IL-17F, IL-18, IL-27, IP-10, MCP-3, M-CSF, MDC, MIG/CXCL9, MIP-1-alpha, MIP-1-beta, PDGF-AA, PDGF-AB/BB, RANTES, TGF-alpha, TNF-beta, and VEGF-A, these synergistically regulate the cellular immune response. The blue cluster featured IL-8 by the same day, underscoring its specific role in coordinating neutrophil responses and fostering inflammation.
Revealing the Key Dynamics of Cytokine Profiles from PCA Analysis of CD8-Stimulated Immune Response
The integration of PCA and K-means clustering analysis of CD-8-elicited responses on days 0, 7, and 14 also delineated the three distinct red, green, and blue clusters (Fig. 5). The cytokines MCP-1, MIP-1-alpha, and MIP-1-beta showed similar expression patterns in the red cluster. This similarity hints at a possible coordinated immune response or a shared mechanism directing the movement of immune cells to areas of inflammation. On day 0, the green cluster exhibited a diverse array of cytokines, including sCD40L, EGF, Eotaxin, FLT-3L, Fractalkine, G-CSF, GM-CSF, GRO-alpha, IFN-alpha-2, IFN-gamma, IL-1-alpha, IL-1-beta, IL-1RA, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12-p40, IL-12-p70, IL-13, IL-17A, IL-17E/IL-25, IL-17F, IL-18, IL-22, IL-27, IP-10, MCP-3, M-CSF, MDC, MIG/CXCL9, PDGF-AA, PDGF-AB/BB, RANTES, TGF-alpha, TNF-alpha, TNF-beta, and VEGF-A. These are critical in guiding immune functions, managing cellular activities, controlling inflammation, and supporting growth and differentiation. They also facilitate communication between cells, maintaining a balance in body responses. Notably, the presence of IL-8 in the blue cluster is significant. IL-8 is a crucial chemokine that attracts and activates neutrophils, essential for the early stages of immune responses against infections. This highlights IL-8's unique role in the beginning stages of disease and its importance in overall immune system functionality.
By the seventh day, a noticeable cytokine pattern was observed. IL-6, MCP-1, MIP-1-alpha, MIP-1-beta, and RANTES grouped closely together, forming a distinct cluster, suggesting a synchronised immune response. These cytokines typically play a role in drawing and activating immune cells during inflammation. A wide variety of cytokines, such as sCD40L, EGF, Eotaxin, FLT-3L, Fractalkine, G-CSF, GM-CSF, GRO-alpha, IFN-alpha-2, IFN-gamma, IL-1-alpha, IL-1-beta, IL-1RA, IL-2, IL-3, IL-4, IL-5, IL-7, IL-9, IL-10, IL-12-p40, IL-12-p70, IL-17A, IL-17E/IL-25, IL-18, IL-22, IL-27, IP-10, MCP-3, M-CSF, MDC, MIG/CXCL9, PDGF-AA, PDGF-AB/BB, TGF-alpha, TNF-alpha, TNF-beta, and VEGF-A, clustered in green. This grouping indicates that these molecules likely cooperate to initiate and maintain immune responses.
Fourteen days after CD8-targeted stimulation, we observed a notable clustering of specific cytokines: GRO-alpha, IL-6, MCP-1, MIP-1-alpha, MIP-1-beta, and RANTES. These cytokines are essential for guiding the movement of immune cells, moderating inflammation, and supporting the function of T cells and monocytes in the immune system. Simultaneously, a distinct cluster was observed, which included a broad range of cytokines such as sCD40L, EGF, Eotaxin, FLT-3L, and several interleukins (e.g., IL-1-alpha, IL-2, IL-4) and growth factors (e.g., PDGF-AA, TGF-alpha). Notably, this group also comprised cytokines like TNF-alpha, TNF-beta, and VEGF-A, among others. This diverse grouping suggests a complex interplay of immune markers.