Activation and Exhaustion Status of T Cells During COVID-19 Infection
The activation, exhaustion, and proliferation response of T and B cells are considered an integral determinant of the disease severity. Unequivocally, studies have shown lymphocytopenia as a predictive marker which may also determine the disease severity in COVID-19 patients (Liu J. et al., 2020; Tan L. et al., 2020b; Wang et al., 2020b; Yang A.P. et al., 2020; Yang X. et al., 2020; Zhang et al., 2020a). However, contradictory reports exist regarding the functional and exhaustion status of these cells during COVID-19. Further, understanding these changes throughout the disease has remained a challenge, considering the complexity in the underlying immune response, comorbid condition, and previous exposure to the infections.
Peripheral blood study of a single patient (50-year male) revealed robust activation of CD4+ and CD8+ T cells marked by HLA-DR expression (Xu Z. et al., 2020). However, the major limitation of this study was that only a single patient was studied. Using multiparameter flow cytometry approach Kuri-Cervantes et al. (2020) studied 35 COVID-19 patients (n = 7 moderate and n = 28 severe). The study revealed that a subset of severe cases displayed T cell activation as revealed by CD38 and HLA-DR expression in both CD4+ and CD8+ T cells (Kuri-Cervantes et al., 2020). By analyzing, PBMCs derived from healthy (n = 5) and severe cases (n = 16), the authors found an increase in the percentage of cytotoxic CD8+ memory cells as revealed by perforin and granzyme B.
Similarly, a subset of severe cases had increased Ki-67 expressing CD4+ and CD8+ T cells, displaying proliferation. At the same time, these findings revealed heterogeneous T cell response but overall suggested a skew towards the activation and proliferation status of these cells in a subset of severe cases. The limitation of this finding is again the small sample size which may be the reason for the inconclusive findings of the T cell status concerning the disease severity.
Similar multiparameter flow cytometry approach was used by De Biasi et al. (2020) to study T cell response in healthy (n = 12) and COVID-19 patients (n = 21). The study found activated status of CD4+ and CD8+ T cells as revealed by an increase in CD38+HLA-D population. Activated status of the CD4+ T and CD8+ T cells was further confirmed by production of IFN-γ, TNF-α, IL-17, and IL-2 when stimulated in vitro. The major limitation of this study was that the sample size was small, which restricted the comparison between the T cell responses across patients with various disease severity. In another study, Song et al. (2020) showed the activated status of CD8+ T but not CD4+ T cells in severe (n = 9) than mild (n = 20) patients. The activated status of CD8+ T cells reflected by the increased population of CD38+HLA-DR+, HLA-DR+, and CD38+HLA-DR+ marker expression (Song et al., 2020). Further, CD8+ T cells were associated with increased cytolytic markers like granzyme B, perforin, and granulysin with more pronounced activation in severe than mild.
While across studies, it has become apparent that T cells show robust activation status in severe cases than mild and moderate. These cells also exhibit exhaustion status, which may occur concomitantly with their activation status. Deep immune profiling of 125 patients by Mathew et al. (2020) demonstrated that both CD4+ and CD8+ T cells exhibit activation status as revealed by coexpression of CD38 and HLA-DR which corresponded to the disease severity. Further, these cells were also associated with concomitant expression of proliferation (Ki-67) and exhaustion (PD-1) markers. This study thus suggests that hyperactivated status of T cells may eventually lead to their exhaustion, and thus these functional and exhaustion features of T cells may reflect the disease severity.
A study by Zheng M. et al. (2020) in a cohort of 68 COVID-19 patients revealed extensive CD8+ T cell exhaustion as shown by increased expression of NKG2A. Intracellular cytokine staining (IFN-γ, IL-2, and granzyme B) further confirmed a decrease in the activation profile of these cells, which was more pronounced in severe (n = 55) than mild (n = 13) cases (Zheng M. et al., 2020). As mentioned earlier in the study by Song et al. (2020) and De Biasi et al. (2020) T cells showed activation status that was also concomitantly seen with express of exhaustion markers PD-1 and TIM-3 on CD8+ T cells and TIM-3 on CD4+ cells. The exhaustion was more pronounced in severe cases (n = 9) than mild (n = 20). However, both these studies did not consider the age of the patients when comparing the disease severity. Further, the study did not consider the temporal dynamics of these cells while measuring their functional properties.
In agreement, Zheng H.Y. et al. (2020) showed reduced functional activation of CD4+ T cells in severe (n = 6) than mild (n = 10) group as revealed by a lower proportion of IFN-γ and IL-2 expressing CD4+ T cells. While IL-2 expressing CD4+ T cell population was also significantly lower in healthy vs mild group. Further, CD8+ T cells displayed exhaustion as revealed by an increase in CTLA-4 in severe cases than mild and TGIT in severe than healthy, while PD-1 was more in mild than healthy. Exhaustive states of both CD4+ and CD8+ T cells were also present in patients requiring ICU (Diao et al., 2020). The exhaustive state was apparent by an increase in PD-1 and Tim-3 expression, which was more pronounced in CD8+ than CD4+ T cells. These studies along with others thus suggest that robust activation followed by the exhaustion of CD4+ and CD8+ T cells may be responsible for the disease progression, while therapies like checkpoint inhibitors (anti-PD-1 antibody; NCT04268537) which may prevent T cell exhaustion and restore their functional state may benefit some patients. More studies are necessary before using such an approach can be used for therapeutic intervention.
A post-mortem study of deceased COVID-19 patients conducted to find the status of these cells at the site of infection. T cell profiling and their activation status in the lungs revealed an increase in the presence of CD4+ and CD8+ T cells exhibiting activation status (Song et al., 2020). This increase in infiltration of these cells was concomitantly associated with their decline in peripheral blood. Others presented a similar activation profile of CD8+ T cells (Kuri-Cervantes et al., 2020; Mathew et al., 2020). This activated state of CD8+ T cells was consistently present across studies, with reports of immune profiling in BALF samples from COVID-19 patients, which showed increased CD4+ and CD8+ T cells in the lungs in both mild and severe cases along with the increased expression of CD8+ T cell cytolytic genes like GZMA and GZMK (Liao et al., 2020). Thus, these studies point towards heterogeneous activation and exhaustion status of T cells in peripheral blood, while a more consistent activated status at the site of infection (lungs) (Figure 4).
FIGURE 4  T and B cell immune response during SARS-CoV-2 infection. (A) The activation status of CD4+ and CD8+ T in the circulation is indicated by CD38+ HLA-DR+. These activated T cells are further recruited at the sites of infection (initially lungs) in the presence of their respective chemokines. The activated CD4+ T cells are marked by the presence of cytokines like IFN-γ, IL-2, IL-12, IL-6, and GM-CSF, whereas activated CD8+T (cytotoxic T cells) are marked by the secretion of granzymes, perforins, and IFN-γ. During SARS-CoV-2 infection, activated CD8+T cells exhibiting increased expression of granzyme A, B, and K (GZM-B, GZM-A, and GZM-K) were found in the lungs (Liao et al., 2020; Song et al., 2020; Zheng M. et al., 2020). (B) T cells were also found to exhibit exhausted state as marked by the expression of PD-1, Tim3, and NKG2A. However, most studies showing exhausted T cells were confined to the peripheral blood, while lungs were mostly shown to have activated T cells but with concomitant expression of some exhaustive markers, suggesting that the activation state is followed by exhaustion. The exhaustive T cells are marked by the reduced expression of respective chemokines and cytolytic granules. (C) Similarly, antibody-producing B cells (plasmablasts; PB) were shown to exhibit activation status as reflected by the expression of IL4R, TNFSF13B, and XBP1, while at the same time, the exhausted status of these cells was also reported in the peripheral blood. Exhaustive state of B cells is reflected by a decrease in antibody production. Further, it appears that unlike CD4+ T cells, the activation status of CD8+ T cells is more pronounced, which may account for their relatively faster exhaustion state (Wherry et al., 2007). Interestingly, by studying the CD8+T cell response in convalescent patients, Habel et al. (2020) found that these cells skewed toward naïve, stem cell and central memory phenotypes, with low effector T cells. While comparing the response with Influenza A viruses, SARS-CoV-2 directed CD8+ T exhibit relatively lower response. Others have also shown a significant decline in CD8+ T cell subsets (naïve, effector, and memory) in COVID-19 patients, with a more pronounced decline in critical (n = 3) than severe (n = 5), and mild (n = 4), suggesting their robust activation during early disease followed by exhaustion during the critical condition (Wang W. et al., 2020). On the contrary, CD4+ T cells were higher in the mild and critical cases than severe cases and healthy control (n = 12). These results imply that the overall T cell response is heterogenous, while CD8+ response, though robust during infection and correlates with the disease severity; but the response may not be long-lasting, at least in some cases.
Both CD4+ and CD8+ T cells also exhibit dysregulated response (Qin et al., 2020). Decreased levels of CD4+ regulatory cells as marked by CD3+ CD4+ CD25+ CD127low+ population was found in severe cases. Similarly, the study found decreased CD8+ suppressor T cells (CD3+, CD8+, CD28+) in severe cases. Overall, more comprehensive studies are warranted with larger cohort size, to profile local vs systemic T cell response and persistence simultaneously, and correlate these responses with disease severity in age-matched patients.