SARS-CoV-2 Cause Complement Activation Induces Excessive CD16+ T Cells With Increased Cytotoxic Functions

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Researchers from Charité – Universitätsmedizin Berlin-Germany and the University of Bonn-Germany have in a new study discovered that SARS-CoV-2 caused complement activation induces excessive T Cell cytotoxicity especially in severe disease.

The study team identified highly activated, CD16+ T cells with increased cytotoxic functions in severe COVID-19. CD16 expression enabled immune complex-mediated, T cell receptor-independent degranulation and cytotoxicity not found in other diseases.
 
It was found that the CD16+ T cells from COVID-19 patients promoted microvascular endothelial cell injury and release of neutrophil and monocyte chemoattractants.
 
These CD16+ T cell clones persisted beyond acute disease maintaining their cytotoxic phenotype.
 
The increased generation of C3a in severe COVID-19 induced activated CD16+ cytotoxic T cells.
 
Importantly the study findings showed that the proportions of activated CD16+ T cells and plasma levels of complement proteins upstream of C3a were associated with fatal outcome of COVID-19, supporting a pathological role of exacerbated cytotoxicity and complement activation in COVID-19.
 
The study findings were published in the peer reviewed journal:
https://www.sciencedirect.com/science/article/pii/S0092867421015622

Severe acute respiratory distress syndrome Coronavirus 2 (SARS-CoV-2) infection in humans causes a diverse spectrum of clinical manifestations, ranging from asymptomatic disease to acute respiratory distress syndrome (ARDS) and multi-organ failure (Miyazawa, 2020).

In addition to direct virus-induced injury to the respiratory system and other organs, increasing evidence suggests that the immune response evoked by SARS-CoV-2 infection contributes to the pathophysiology of Coronavirus disease (COVID-19), particularly during severe disease courses (Gustine and Jones, 2021; McKechnie and Blish, 2020; Vabret et al., 2020).

Both, CD4+ T helper cells and CD8+ cytotoxic T lymphocytes (CTL), contribute to the control of respiratory viral infections. Consequently, SARS-COV-2-specific CD4+ and CD8+ T cells have been associated with milder COVID-19 (Jacob, 2020; Tan et al., 2021). While this had been interpreted as a predominantly protective role of T cell responses (Rydyznski Moderbacher et al., 2020), complementary data do not unequivocally support this idea (Feng et al., 2020; Mathew et al., 2020; Peng et al., 2020; Thieme et al., 2020).

The extent of SARS- CoV-2-specific T cell responses could not be directly tied to disease severity, with high T cell numbers not necessarily translating into mild COVID-19 (Le Bert et al., 2021). In fact, the number of SARS-CoV-2-specific CD4+ and CD8+ T cells were found to be comparable or even higher in COVID-19 patients displaying severe versus mild disease (Feng et al., 2020; Le Bert et al., 2021; Mathew et al., 2020; Peng et al., 2020; Thieme et al., 2020).

A higher state of T cell activation in all T cell compartments (CD4+, CD8+, double-negative (DN)) in patients progressing to severe COVID-19 was reported (Zenarruzabeitia et al., 2021).

Interstitial T cell infiltration is observed in pathological specimens of COVID-19 pneumonia along with macrophage accumulation in the alveolar space, and it has been hypothesized that infiltrating T cells also contribute to alveolar wall damage and endothelial cell injury known as lymphocytic endotheliitis (Miyazawa, 2020; Varga et al., 2020).

All this argues for a complex relationship between T cell immune responses and disease outcome during COVID-19 beyond a mere quantitative influence. It is likely that additional factors present in the microenvironment will shape the quality of T cell responses and consequently impact pathology. It is therefore important to identify whether and which T cell subsets have a pathogenic role. Also, mechanisms by which potentially pathogenic T cells are induced need to be revealed, as studies on this matter are currently lacking (Yan et al., 2021).

Here, we combined single-cell proteomics and transcriptomics with mechanistic studies to reveal alterations in the T cell compartment, their upstream signals, and functional relevance, which explain important immunopathological features observed in severe COVID-19. Mass cytometry (CyTOF) and single-cell RNA-seq (scRNA-seq) combined with VDJ-seq-based T cell clonotype identification were used to determine COVID-19- and severity-specific alterations in the T cell compartment. In addition to the severity-independent formation of highly activated HLA-DRhiCD38hiCD137+Ki67+ T follicular helper (TFH) -like cells and CD8+

CTLs in COVID-19, we describe a C3a-driven induction of activated CD16 expressing cells in patients with severe COVID-19. These T cells display increased immune complex-mediated, TCR-independent cytotoxicity causing activation and release of chemokines by lung endothelial cells. This mechanism may contribute to the profound lung damage and endotheliitis observed in patients with severe COVID-19.

Discussion

Excessive T cell activation and altered phenotypes can contribute to infection-associated organ damage. Early after the first reports on immune profiles of COVID-19 patients (Sette and Crotty, 2021) discussions on their putative role in immune protection versus pathology started.

In our study, we provide evidence that SARS-CoV-2 infection – in contrast to other acute and chronic infections – promotes the formation of highly activated and proliferating HLA- DR+CD38hiCD137+CD69+ T helper cells and CD8+ T cells independent of disease severity, although this response occurred faster in severe COVID-19 patients. More importantly, in severe COVID-19 patients, we detected differentiation of activated CD16+ T cells, which showed an increased immune complex-mediated cytotoxic potential and a potential to activate lung microvascular endothelial cells.

Expanded clones within the CD16+ T cell compartment persisted and maintained their high cytotoxic potential. We identified C3a as an upstream signal for the differentiation of the altered activated T cell phenotype. Proportions of activated CD16+ T cells and plasma complement protein abundance levels were associated with worse outcomes of patients with severe COVID-19. Thus, SARS-CoV-2-triggered complement activation creates an inflammatory milieu that drives differentiation of T cells with high immunopathogenic potential.

A balanced T cell activation is decisive for the course of infection. The formation of CD8+ tissue-resident memory T cells (Trm) during primary infection is known to restrain viral spread upon secondary influenza infections. Yet, enhanced accumulation of Trm cells in an imbalanced environment such as during aging can support excessive inflammation leading to organ damage and impaired repair (Goplen et al., 2020). In COVID-19, large numbers of such Trm-like CD8+ T cells have been identified in the airways (Liao et al., 2020).

Blood samples acquired during the acute phase of severe COVID-19 contain high numbers of HLA-DR+CD38hiKi67+ in both CD4+ and CD8+ T cell compartments (Mathew et al., 2020; Rydyznski Moderbacher et al., 2020; Stephenson et al., 2021), a finding that we corroborated by CyTOF analysis.

Severe COVID-19 patients showed a faster increase of the CD38hiHLA- DR+Ki67+ICOS+ TFH-like CyTOF cluster 7 proportions accompanied by an earlier antibody response (Figure S1B&5A). We identified an elevated and activated T cell population expressing CD16 across the three major T cell compartments. Activated CD16+ T cells showed increased TCR-independent pathogenic potential.

The activated CD16+ CD4+ and CD8+ T cells enriched in severe COVID-19 expressed high levels of chemokine receptors such as CXCR3 and CCR6 (Figure 1D). This clearly distinguished them from the other CD16lo T cell clusters e.g., cluster 31. CXCR3 and CCR6 might promote the migration of activated CD16+ T cells into the inflamed lungs (Oja et al., 2018; Shanmugasundaram et al., 2020).

Pronounced CCR6 expression on T cells has been described in severe COVID-19 (Fenoglio et al., 2021; Tiwari-Heckler et al., 2021), which we can link here to unexpected phenotypic and functional properties. Immunofluorescence co- staining of CD3 and CD16 in lung samples of an autopsy cohort showed enrichment of CD3+CD16+ T cells in COVID-19 compared to influenza pneumonia or other causes of ARDS (Figure 3I+J). Although strong T cell activation is a feature of both severe COVID-19 and

influenza pneumonia, specific differences have been described between both diseases (Youngs et al., 2021).

We found that approximately 5% of the activated CD16+ CD8+ T cells respond to stimulation with SARS-CoV-2 peptides. This is in line with the previously described positive correlation between ex vivo-determined HLA-DR+CD38hiKi67+CD8+ T cells and SARS-CoV-2-specific CD8+ T cells (Rydyznski Moderbacher et al., 2020). Activated CD16+ T cells show significant enrichment of SARS-CoV-2 specific T cells compared to activated CD16- T cells (Figure 5F).

The remaining, non-responding activated CD16+ T cells may recognize other SARS-CoV-2 epitopes or may be driven by bystander activation and/or homeostatic proliferation (Bergamaschi et al., 2021; Mathew et al., 2020). This indicates that cognate T cell activation plays an important role in the generation of activated CD16+ T cells along with environmental signals in a complement split product-rich inflammatory milieu.

A very high proportion of T cells from acute COVID-19 and especially severe patients express cytotoxic molecules such as Perforin and Granzyme B (Shuwa et al., 2021). The increased cytotoxic profile persisted for up to six months and was associated with poorer recovery. The CD16+ T cells identified in severe COVID-19 did not only express higher levels of PRF1 and GZMB but also LAMP1 and STX11 (Figure 2H+S2B), which explains their increased general degranulation potential (Figure 3D+E) (Spessott et al., 2017).

So far, CD16+ T cells have been described mainly in patients with chronic infections or inflammation (Björkström et al., 2008; Clémenceau et al., 2011; Jacquemont et al., 2020). In these conditions, CD16+ T cells displayed a more differentiated phenotype like the one adopted during COVID-19 convalescence (Figure 4C, D & F). In acute COVID-19, we also identified elevated transcription of various granzyme genes including Granzyme K (Figure S2B). Interestingly, increased numbers of Granzyme K expressing effector memory T cells have been observed in blood samples of older individuals and these T cells were shown to augment cytokine and chemokine production by fibroblasts (Mogilenko et al., 2021).

It was shown that extracellular Granzyme K proteolytically activates Protease-activated receptor-1 leading to increased release of IL-6 and CCL2 (MCP-1) by endothelial cells (Sharma et al., 2016). Particularly in severe COVID-19, we demonstrated that T cells also induce CCL2 and XCL8 by co-cultured primary lung endothelial cells upon anti-CD16 mediated degranulation (Figure 3G).

This establishes a general link between the immune complex triggering of local CD16+ T cells and endothelial cell-mediated release of monocyte and neutrophil chemoattractants, a hallmark of severe COVID-19 (Rendeiro et al., 2021). Altogether, activated CD8+ T cells adopt antibody- dependent cellular cytotoxicity (ADCC) properties known for NK cells (Lee et al., 2021). ADCC can have protective but also disease exaggerating roles (Yu et al., 2021).

The binding of antigen-antibody complexes to CD16 on activated T cells might therefore counteract anti- inflammatory immune complex clearing systems via complement receptor 1 (Fernandez-Arias et al., 2013; Kavai, 2008). Notably, patients suffering from severe COVID-19 have been reported to display high levels of spike-reactive IgG with significantly reduced Fc fucosylation.

This change in the Fc glycosylation increases binding affinity to CD16, leading to increased CD16-mediated effector function (Ferrara et al., 2011; Hoepel et al., 2021; Vivier et al., 2008). As such, the distinct serological profile observed in severe COVID-19 with afucosylated, spike-directed IgG, and an inherently increased inflammatory capacity could further enhance the pathogenic potential of CD16+ T cells.

In a search for important environmental signals driving differentiation of activated CD16+ T cells, we detected a positive correlation between high serum C3a levels and proportions of CD16+ T cell clusters (Figure 5E). It has been reported that serum C3 hyperactivation is a risk factor for COVID-19 mortality (Sinkovits et al., 2021) and widespread complement activation by all three pathways and thus generation of C3a has also been described in patients with severe COVID-19 (Chouaki Benmansour et al., 2021; Defendi et al., 2021; Satyam et al., 2021).

The disease-promoting activity of the complement system was observed for other coronaviruses, as SARS-CoV infection caused less systemic inflammatory response and lung injury in C3 knock-out as compared to wild type mice (Gralinski et al., 2018). Furthermore, it has been shown that SARS-CoV-2 infection of lung epithelial cells induces transcription of complement genes leading to the generation of activated C3a (Yan et al., 2021). Signaling via complement receptors such as C3AR1 and cell-autonomous complosome in human T cells enhances induction of CD4+ Th1 responses and cytotoxic function of CD8+ T cells (Arbore et al., 2018).

Here, we show that increased C3a generation in severe COVID-19 patients promotes differentiation of CD16+, highly cytotoxic CD4+ and CD8+ T cells (Figure 5F-H). Interestingly, targeting distal complement effects by receptor blockade in a humanized preclinical model of SARS-CoV-2 infection prevented acute lung injury (Carvelli et al., 2020).

Results from the first clinical trials on complement inhibition in COVID-19 also showed promising effects resulting in reduced inflammation and faster normalization of neutrophil and lymphocyte counts (Mastaglio et al., 2020; Mastellos et al., 2020; Polycarpou et al., 2020). In this context, C3 inhibition enabled a broader and better therapeutic potential as compared to C5 neutralisation (Mastellos et al., 2020).

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