In this study we describe main changes in the T cell compartment of patients with Covid–19 pneumonia who had been admitted to the Infectious Diseases Clinics (University Hospital of Modena, Northern Italy) because of the severity of their symptoms. Most were lymphopenic, and most did not required non invasive ventilation, indicating that at the time of blood collection the disease was not too advanced. Our data thus indicate that relevant immune alterations, both phenotypic and functional, are present from the earliest stages of the infection, when they become clinically relevant. Studies on asymptomatic, infected individuals are now crucial to better understand the immunopathogenesis of the Covid–19.
This study evidences that Covid–19 patients with pneumonia display important phenotypic changes that could be used as possible prognostic markers. Moreover, the production of IL–17 by both CD4+ and CD8+ T cells, along with the high plasma level of this crucial cytokine, able to strengthen inflammation and activate neutrophils, is a main finding that could pave the way to novel therapeutic approaches based upon IL–17 blockage.
Very few data exist on the changes in the T cell compartment in patients affected by SARS-CoV–2 infection, and some have been published in a non-peer reviewed manner, so being difficult to discuss. Previous studies have shown changes in the T cell family, which is characterized by relevant signs of exhaustion (18,19). It has also been reported that in the peripheral blood of most Covid–19 patients the proportions of T cells subsets can remain within the normal range, but a decrease can exist in CD4+ and CD8+ T cell counts or in CD4+/CD8+ ratio (20,21). Our data are in agreement with these observations, which are deepened with detailed analysis of different subpopulations of both CD4+ and CD8+ T lymphocytes. Indeed, if on the one side no gross changes can be detected simply searching for markers related to naive, memory or effector cells, on the other a more sophisticated and detailed analysis have revealed significant differences and truly important immune alterations.
Regulatory T cells (Treg) are crucial for regulating immune homeostasis and self‐tolerance (22,23). They express the expression of the forkhead box transcription factor Foxp3, but can also be identified by detecting the high expression of the IL‐2 receptor α‐chain (CD25) and low/null expression of IL‐7 receptor α‐chain (CD127) (23), along with other surface molecules such as CD39 (24.) and CD73 (25). Treg are considered as gate‐keeper of a huge variety of immune reactions. They are able to suppress autoimmune phenomena, dampen allergic reactions or block transplant rejection, but they can also inhibit a protective immune response against invading pathogens or tumors (26). This is clearly an unproductive immunosuppression that can either cause unwanted reactions or promote the progression of a disease or an infection. Here we show that, on the one hand, different types of Tregs are increased in peripheral blood from Covid–19 patients and that their plasma contains high amounts of IL–10, a typical inhibitory cytokine produced by these cells. On the other hand, we have found that several markers of T cell activation are present in these subjects, whose plasma, rich of proinflammatory molecules, is definitely a mirror of the cytokine storm that characterize this disease.
Patients with Covid–19 had increased amounts of CD8+ T cells expressing CD57, which has been considered for decades the key marker of in vitro replicative senescence and is associated either to human aging or prolonged chronic infections (27). CD57 can be used to detect functional immune deficiency in patients with autoimmune diseases, infectious diseases, and cancers. It has been suggested that CD57+ cells display a high susceptibility to activation‐induced cell death, and are not able to undergo cell proliferation despite preserved ability to secrete cytokines after activation (28). Thus, activating cells with this type of exhaustion not only results in the lack of clonal expansion but also in the production of molecules that can cause inflammation.
Then, we have observed that cells from patients had high expression of PD1, a crucial inhibitory immune mediator that, after activation by its ligands PD-L1 and PD-L2, plays an important role in the induction and maintenance of peripheral tolerance, and for keeping the stability and integrity among T cells (29). The PD–1/PD-L1 axis however mediates also potent inhibitory signals to block proliferation and function of T effector cells, causing inimical effects on antiviral immunity. It is to note that significantly high plasma levels of PD-L1 were also found in our patients, suggesting that an inhibition of the immune response could occur that follows this pathway.
An excessive inflammatory response evidenced by elevated levels of proinflammatory cytokines and chemokines has been described in patients affected by the SARS-Cov that caused the epidemic in 2003 (30,31). Moreover, in vitro experiments revealed that several different cell types from those patients were able to produce high amounts of cytokines (32,33). High plasma levels of pro-inflammatory molecules indicating a TH1/TH17 response were reported in patients with MERS-CoV infection, with increased concentrations of IFN-γ, TNF-α, IL–15 and IL–17 (34). Similar data, along with skewed in vitro cytokine production by T cells, have been described in patients affected by the Middle East Respiratory Syndrome caused by the MERS coronavirus (35). In our Covid–19 patients, plasma concentrations of many pro-inflammatory cytokines and chemokines, including IL–6, IL–17, TNF-α, IL–8, IL–1a, CCL2, CCL3, CCL4, MIP–2 and CXCL6, were strongly increased, according to all the aforementioned reports that have described the so-called cytokine storm.
Thus, presence of high amounts of molecules that are produced by several cell types and act on innate immune cells has always to be taken into consideration. Among these molecules, IL–8 might have a particular importance, being able to recruit neutrophils from the blood to infected or injured tissue. IL–8 production can be induced by a wide range of stimuli such as TNF-α, IL–1, bacteria, virus, and cellular stress, and it can be synthetized by a several cell types, including monocytes, macrophages, endothelial and epithelial cells, fibroblasts, T lymphocytes, hepatocytes, synovial cells and keratinocytes (36). Its receptors, CXCR1 and CXCR2, are expressed on neutrophils, monocytes, CD8+ T and NK cells, mast cells, basophils and myeloid-derived suppressor cells. In neutrophils, receptor activation stimulates the release of granule enzymes and the production of reactive oxygen species (37). When a respiratory virus like SARS-CoV–2 enters the alveolus, alveolar epithelial cells are the first cells the virus encounters and infect, and can produce IL–8 that in turn attracts and activates neutrophils and macrophages, which start damaging the organ and eventually trigger a much more complex series of pathogenic events, including, among others, endothelial damage, platelet activation, intravascular thrombosis. Even if in our patients with pneumonia it was not possible to ascertain the site of IL–8 production, it is possible to hypothesize that alveolar cells were deeply involved in this phenomenon.
Surprisingly, we also observed a marked increase of immunosuppressive cytokines, including IL–10 and TH2 cytokines, like IL–4 and IL–13. This could indicate that the activation of immune system is really massive, indiscriminately involving all cells, and depleting their residual resources.
Galectin–1, –3 and –9 were increased in patients if compared to controls. Galectins represent a family of soluble β-galactoside-binding proteins widely expressed at sites of inflammation and infection that have emerged as a new class of damage-associated molecular patterns (DAMPs) or resolution-associated molecular patterns, serving to magnify or to fix inflammatory responses (38,39). In particular, galectin–1 acts typically as a pro-resolving mediator by repressing a number of innate and adaptive immune programs. Conversely, galectin–3 and –9 have been proposed to act as alarmins (or DAMPs) that amplify inflammatory responses during sepsis and several types of infection. This means that the condition of hyperinflammation, which is observed in patients with Covid–19, is driven by a plethora of molecules, including those belonging to the galectin family, that act against each other and that could likely become pharmacological targets. To our knowledge, these are the first data describing the presence of these types of soluble mediators in patients with Covid–19, and further studies are needed to investigate their importance in the immunopathogenesis of the disease.
A large number of mediators that we have measured are produced by T lymphocytes, and such a massive cytokine release is very similar to what happens during a polyclonal, superantigen-driven, T-cell activation. Superantigens like SEB can bind directly either the Vβ portion of the T-cell receptor or to MHC class II molecules, thus rapidly and non-specifically activating T cells to produce IFN- and several other cytokines (40). A four-fold increase in the level of circulating IFN- was indeed observed in plasma from Covid–19 patients if compared to controls. IFN- in turn activates macrophages, which produce pro-inflammatory cytokines, which then overwhelm the system. When patients’ cells were stimulated anti-CD3/28 or SEB, an increased number of CD4+ T cells producing IFN-, TNF-, IL–17A and IL–2 was observed if compared to healthy controls, indicating that T cells from Covid–19 patients have a higher ability than those from healthy individuals to respond in vitro to stimulatory challenges. Studies are in course to investigate the functional phenotype of specific T cells, i.e., those responding to peptide antigens derived from SARS-CoV–2.
In conclusion, in Covid–19 patients with pneumonia, we have found the presence of: i) increased markers of T cell exhaustions, activation, senescence; ii) an altered differentiation of different T cell subtypes; iii) high plasma level of a variety of cytokine, from those with proinflammatory action to those that are able to inhibit the immune response, from those that indicate a skewing towards TH1 to those that reveal a skewing towards TH2; iv) massive in vitro production of several cytokines, with a potential skewing of activated cells towards TH17 phenotype.
Thus, Covid–19 looks very similar to a catastrophic sepsis, with concomitant aspects of immune inhibition, activation, exhaustion and complex alterations within cells at different stage of differentiation, not to mention the fact that almost all kind of cytokines are produced and released in a senseless and uncoordinated manner. The overall picture that emerges underlines the ability of SARS-CoV–2 to provoke, in a very fast time, a dramatically confused immune response. However, and thanks to the enormous efforts that the scientific community is carrying out studying relentlessly the immunopathogenesis of Covid–19, the progression of the viral infection starts to show some weaknesses. A therapeutic approach now exists based on the administration of drugs that block IL–6 pathway, and is now significantly ameliorating the course of the disease (41). IL–17 is crucial in recruiting and activating neutrophils, cells that can migrate to the lung and are heavily involved in the pathogenesis of Covid–19. We show here that a significant skewing of T cell activation towards TH17 functional phenotype exists in Covid–19 patients, and we suggest that blocking IL–17 pathway by the biological drugs that are already available and used to treat different pathologies could be a novel, additional strategy to improve the health of patients infected by SARS-CoV–2.