key: cord-0789337-cmc4ocay
authors: Welch, Jennifer L; Xiang, Jinhua; Chang, Qing; Houtman, Jon C D; Stapleton, Jack T
title: Human T cells express Angiotensin Converting Enzyme 2 at levels sufficient to interact with the SARS-CoV-2 Spike protein
date: 2021-12-16
journal: J Infect Dis
DOI: 10.1093/infdis/jiab595
sha: f86537123123812d3abf7bfc578fdc2048f232be
doc_id: 789337
cord_uid: cmc4ocay

The pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is not completely understood. SARS-CoV-2 infection frequently causes significant immune function consequences including reduced T cell numbers and enhanced T cell exhaustion that contribute to disease severity. The extent to which T cell effects are directly mediated through infection or indirectly result from infection of respiratory-associated cells is unclear. We show that primary human T cells express sufficient levels of angiotensin converting enzyme 2 (ACE-2), the SARS-CoV-2 receptor, to mediate viral binding and entry into T cells. We further show that T cells exposed to SARS-CoV-2 particles demonstrate reduced proliferation and apoptosis compared to uninfected controls, indicating that direct interaction of SARS-CoV-2 with T cells may alter T cell growth, activation, and survival. Regulation of T cell activation and/or turnover by SARS-CoV-2 may contribute to impaired T cell function observed in patients with severe disease.

A c c e p t e d M a n u s c r i p t 6 ug/million cells) to block Fc receptors on B cells. Data were acquired on an LSR II flow cytometer using single stained AbC bead kit (ThermoFisher) for compensation [17] . Viable cells were gated based on forward and side scatter, doublets excluded and fluorescence minus one controls were assessed. PEquantitation beads (BD Quantibrite) were analyzed according to manufacturer's instruction to evaluate ACE-2 molecules/cell. At least 10,000 total events were collected in each experiment and the FlowJo program (Tree Star Inc.) was used for data analysis.

Recombinant trimeric SARS-CoV-2 spike recombinant protein (AcroBiosystems) was labeled with FITC Conjugation Kit (Fast)-Lightning-Link (Abcam) according to manufacturer's instructions. Unlabeled protein was detected by fixation/permeabilization (BD Biosciences) and anti-Spike S1 (Genetex) and anti-Rabbit APC (Biolegend). ACE-2 binding was blocked with 2µg/mL anti-ACE-2 (R&D Systems) for 3 h prior to spike recombinant protein addition [7] . Cells were treated with 2.5 µg/mL recombinant protein, and cell viability was verified by MTT (Sigma-Aldrich) assay (not shown). Statistics: Statistics were performed using GraphPad software V 9.0.0 (GraphPad Software Inc.). Two-sided Student's t test was used to compare results between test and controls. P values less than 0.05 were considered statistically significant.

To address the conflicting descriptions of ACE-2 expression and T cells [10] [11] [12] [13] , multiple approaches were used to detect ACE-2 expression in resting and activated Jurkat-T and primary human T cells . Using qRT-PCR, Jurkat-T, PBMCs, and purified CD3+ T cells contained detectable ACE-2 RNA ( Fig 1A) . As expected, positive control VeroE6 cells with high expression of ACE-2 contained significantly (~ 1000-fold) more ACE-2 RNA than the Jurkat-T cell line or primary PBMCs and enriched CD3+ T cells [25] [26] [27] . ACE-2 RNA was not detected in immortalized Ramos-B cells (Fig 1A) , thus we utilized Ramos-B as negative controls thereafter.

Unexpectedly, ACE-2 RNA levels were significantly increased 10 to 100-fold in Jurkat-T and purified CD3+ T cells following activation through the T cell receptor (TCR) with anti-CD3/CD28 or following stimulation with PMA and ionomycin ( Fig 1A) .

T cell activation was confirmed by measuring cellular IL-2 release (Fig 1B) . In addition to mRNA expression, ACE-2 protein was detected in PBMCs, purified CD3+ A c c e p t e d M a n u s c r i p t 8 (Fig 1C) . Similar to mRNA results, the Vero cells had considerably more ACE-2 protein and Ramos-B cells had negligible ACE-2 ( Fig 1C) . ACE-2 protein levels also increased with cell activation ( Fig. 1C includes the fold-change ACE-2 increase relative to actin).

To examine ACE-2 cell surface expression, we utilized flow cytometric analysis and confirmed that Jurkat-T cells express low levels of ACE-2 and that levels increase with cell activation ( Fig. 2A) . Based on the shift of the entire curve compared to > 5% of the unstained negative control cells, it appears that the entire cell population expresses at least some ACE-2 ( Fig Examination of healthy blood donor's primary PBMCs found that CD3+ T cells contained ~353 ACE-2 surface molecules per cell, and expression also significantly increased to 579 or 765 molecules per cells following stimulation via TCR or PMA and ionomycin respectively ( Fig 2D) .

Since the SARS-CoV-2 spike protein mediates binding to ACE-2 in permissive cells, we evaluated if the level of ACE-2 present on T cells was sufficient to mediate A c c e p t e d M a n u s c r i p t 9 binding to SARS-CoV-2 spike protein. To address this, we determined the amount of FITC-labeled SARS-CoV-2 trimeric spike protein bound to Jurkat-T cells compared to nonspecific FITC binding by flow cytometry (Fig. 3A-B ). This interaction was partially dependent on ACE-2, as blocking of ACE-2 with anti-ACE-2 antibody [7] moderately, but significantly, reduced spike protein binding levels in Jurkat-T cells ( Fig 3C) . Detection of bound spike protein after anti-ACE-2 blocking may indicate incomplete antibody blocking or interaction between spike protein and alternative surface molecules as suggested by others [14] . The binding of the FITC-labeled spike trimers to Jurkat-T cells was not due to protein alterations during fluorescent labeling, as SARS-CoV-2 specific antibodies detected unlabeled SARS-CoV-2 trimeric protein incubated with T cells (Fig 3D) . 

To determine if replication competent SARS-CoV-2 binds to T lymphocytes, SARS-CoV-2 particles were incubated with Jurkat-T, PBMCs, purified CD4+, and purified CD3+ primary lymphocytes at 4 o C for 3 h to allow virus binding while preventing internalization events [21] . Subsequent washing and evaluation of SARS-CoV-2 RNA present in cells found significant SARS-CoV-2 bound to Jurkat-T cells, PBMCs, purified CD4+ T, and purified CD3+ T cells compared to Ramos-B cells (Fig 5A) .

Surprisingly, virus binding levels in primary cell populations were comparable to levels in VeroE6 cells (Fig 5A) containing significantly more ACE-2 (Fig 1) . These data strongly suggest that low levels of ACE-2 are sufficient to mediate SARS-CoV-2 binding to cells.

Significant levels of intracellular SARS-CoV-2 RNA was detected in Jurkat-T, PBMCs, purified CD4+ T, and purified CD3+ T cells compared to Ramos-B cells ( Fig   5B) ; however, intracellular virus levels were orders of magnitude lower than that detected in VeroE6 cells 3 days post-infection (Fig 5B) . To evaluate internalization of SARS-CoV-2, cells were incubated with SARS-CoV-2 particles at 37 o C for 3 h. Cells were then treated with 0.25% trypsin-EDTA and washed to remove surfaceassociated virus. Complete removal of input virus was verified by SARS-CoV-2 extracellular RNA evaluation remaining in the final wash (Fig 5C) . Release of SARS-CoV-2 RNA from these cells was measured in cell culture supernatants 5 d postinfection. Extracellular or supernatant viral RNA levels in PBMC and T cell models were not different than levels in Ramos-B cells (Fig 5D) . Furthermore, these PBMC and T cell supernatants were not able to infect VeroE6 cells (not shown) while VeroE6 supernatants contained infectious virus (TCID 50 = 10 6 infectious A c c e p t e d M a n u s c r i p t 11 particles/mL) (not shown). Together, these data indicate that SARS-CoV-2 particles bind and enter into T cells, but are not able to replicate or produce detectable infectious particles.

Previous reports identified reduced T cell numbers in patients with severe COVID-19, and this has been hypothesized to be related to enhanced apoptosis [5, 6, 29] .

To examine potential functional consequences of SARS-CoV-2 internalization on the T cell growth cycle, we evaluated T cell proliferation and apoptosis in T cells following incubation with SARS-CoV-2. Ki67 surface staining was used as an indicator of cell proliferation, and SARS-CoV-2 infected PBMC and purified CD3+ T cells had significantly reduced proliferation compared to uninfected controls (Fig 6A) .

Similarly, caspase-3 activity was measured as a marker of T cell apoptosis.

Caspase-3 levels were significantly reduced in infected cells (Fig 6B) , demonstrating that SARS-CoV-2 infected T cells are proliferating at a reduced rate and have reduced apoptosis in vitro. Thus, T cell turnover may be reduced during SARS-CoV-2 infection contributing to reduced cell numbers. This contribution is likely small; however, because the cells are resistant to apoptosis, the surviving cells likely contribute to the massive inflammatory milieu observed in patients with severe disease.

SARS-CoV-2 infected individuals, and this is generally attributed to an indirect regulation of T cell homeostasis following SARS-CoV-2 infection [30] [31] [32] . However, few studies have evaluated direct effects of SARS-CoV-2 in T cells, and there are A c c e p t e d M a n u s c r i p t 12 conflicting reports on the expression of ACE-2 receptor levels on human T cells [10] [11] [12] [13] [14] . Our data supports findings of others that both immortalized and ex vivo human T cells contain detectable levels of ACE-2 RNA and protein (Fig 1) [12, 13] . The level of ACE-2 measured was sufficient to mediate SARS-CoV-2 spike protein trimer binding (Fig. 3-4) and virus binding and entry (Fig 5) . Of note, T cells did not produce infectious virus, thus replication appears to be restricted at a post-entry replication cycle step (Fig 5) . Similar results identified SARS-CoV-2 binding and uptake in CD4+

T cells [14] , and a separate study reported low but detectable levels of SARS-CoV-1specific genomic RNA [33] . Consistent with our finding that T cells do not produce infectious virus, SARS-CoV-1 replicating (minus strand) RNA was not found in PBMCs [33] . A potential corollary is that influenza A virus may cause abortive infection in macrophages and dendritic cells; however, some strains appear to lead to productive infection (34) . It has been suggested that this may contribute to viral amplification and dissemination, and understanding strain differences may provide insights into pathogenicity and immunogenicity.

Although few studies have evaluated T cell turnover in coronavirus infections, a

Jurkat-T and PBMCs [33] . In contrast to our results with SARS-CoV-2, no significant differences in apoptosis measurements in SARS-CoV-1 infected cells were observed when compared to mock-infected controls [33] . However, similar to our in vitro results, a COVID-19 clinical study did not identify significant increases in the apoptosis-associated indicator, caspase-3 in T cells [34, 35] . Thus, T cell apoptosis, as measured by caspase-3 activation, does not appear to be increased during SARS-CoV-2 infection (Fig 6B) , and enhanced apoptosis may not be responsible for the reduced T cell numbers observed in infected patients.

A c c e p t e d M a n u s c r i p t 13 Bulk PBMC and purified CD3+ T cells infected with SARS-CoV-2 showed reduced proliferation (Fig 6) . These data are consistent with findings by others that showed healthy PBMC treated with a cocktail of SARS-CoV-2 spike, nucleoprotein, and protease recombinant proteins demonstrated significantly reduced CD4+ and CD8+ cell proliferation [36] . In addition, proliferation of circulating CD4+ and CD8+ T cells obtained from COVID-19 patients was significantly reduced in patients with prolonged hospitalization (≥5 days) [36] . Interestingly, even patients in the most prolonged hospitalization group (>20 days) who tested negative for virus by PCR at the time of lymphocyte collection had reduced T cell proliferation [36] . This is consistent with our finding that productive viral replication was not required to alter normal T cell function, and that T cell interaction with viral proteins appears to be sufficient to interfere with proliferation. Our data suggest that longitudinal assessment of T cell ACE-2 expression in patients with different COVID-19 disease severity is warranted. If higher levels of ACE-2 correlate with T cell activation in subjects with severe disease, this may suggest a link between ACE-2 and inflammation, contributing to immunopathogenesis underlying severe SARS-CoV-2 infection. This may be true in other infections as well, as studies of T cells obtained from HIV-suppressed patients found reduced proliferation rates compared to uninfected controls [37] . The authors in this study hypothesize that low but persistent HIV infection in these cells may prolong the half-life of T cells through reduced proliferation which may in turn maintain the infected cell population [37] . 

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We thank Dr. James McLinden for critical discussions and review of the manuscript.We thank Thomas Kaufman for assistance with flow cytometry, and Dr. Wendy Maury for reagents.

All authors report no conflicts of interest.

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