key: cord-328853-0iqdqcp6
authors: Neidleman, Jason; Luo, Xiaoyu; Frouard, Julie; Xie, Guorui; Gill, Gurjot; Stein, Ellen S.; McGregor, Matthew; Ma, Tongcui; George, Ashley F.; Kosters, Astrid; Greene, Warner C.; Vasquez, Joshua; Ghosn, Eliver; Lee, Sulggi; Roan, Nadia R.
title: SARS-CoV-2-specific T cells exhibit phenotypic features of robust helper function, lack of terminal differentiation, and high proliferative potential
date: 2020-08-19
journal: Cell Rep Med
DOI: 10.1016/j.xcrm.2020.100081
sha: 
doc_id: 328853
cord_uid: 0iqdqcp6

SUMMARY Convalescing COVID-19 patients mount robust T cell responses against SARS-CoV-2, suggesting an important role for T cells in viral clearance. To date, the phenotypes of SARS-CoV-2-specific T cells remain poorly defined. Using 38-parameter CyTOF, we phenotyped longitudinal specimens of SARS-CoV-2-specific CD4+ and CD8+ T cells from nine individuals who recovered from mild COVID-19. SARS-CoV-2-specific CD4+ T cells were exclusively Th1 cells, and predominantly Tcm with phenotypic features of robust helper function. SARS-CoV-2-specific CD8+ T cells were predominantly Temra cells in a state of less terminal differentiation than most Temra cells. Subsets of SARS-CoV-2-specific T cells express CD127, can homeostatically proliferate, and can persist for over two months. Our results suggest that long-lived and robust T cell immunity is generated following natural SARS-CoV-2 infection, and support an important role for SARS-CoV-2-specific T cells in host control of COVID-19.

The first cases of COVID-19 were reported in December of 2019 in Wuhan, China, and 36 soon thereafter its causative agent was identified as SARS-CoV-2, a beta-coronavirus with 79% 37 sequence identity to the SARS-CoV that had emerged in 2003 1 . SARS-CoV-2 has proven to be 38 much more transmissible than its SARS-CoV counterpart, quickly spreading around the world 

Many individuals exposed to SARS-CoV-2 are asymptomatic or exhibit only a mild 48 course of disease, suggesting that natural immunity can effectively combat this virus. While 49 most studies on SARS-CoV-2 immunity have focused on the humoral immune response, 50 emerging data suggest T cell-mediated immunity is also likely to play an important role in 51 eliminating the virus. Lymphopenia, as characterized by reduced numbers of CD4+ and CD8+ T 52 cells, is predictive of disease severity 3 . In addition, levels of activated T cells increase at the 53 time of SARS-CoV-2 clearance 4 . Furthermore, the clonality of T cell receptor (TCR) sequences 54 5 is higher in patients with mild rather than severe COVID-19, suggesting a role for antigen-55 specific T cell responses in symptom resolution. A beneficial role for T cells in combating 56 COVID-19 would be in line with observations that both CD4+ and CD8+ T cells are protective 57 against the closely-related SARS-CoV 6-8 . However, the nature of the response is also 58 important, as Th1 responses appear to be protective against SARS-CoV while Th2 responses 59 J o u r n a l P r e -p r o o f were associated with immunopathology 9-11 . Th17 responses have also been implicated in 60 immunopathology during coronavirus infections 12 .

Only a limited number of studies have characterized the SARS-CoV-2-specific T cell 62 response. These studies have focused on the breadth of the T cell response, and detected T 63 cells recognizing spike and non-spike epitopes [13] [14] [15] [16] [17] . In some 13, 16 but not other 17 studies, 64 responses were also detected in individuals who had not been infected with SARS-CoV-2, 65 presumably reflecting recognition of cross-reactive epitopes from other coronaviruses. These 66 responses primarily involved non-spike ORFs 16 . ELISA revealed that peptide-treated PBMCs 67 upregulated IFNγ but not IL4 or IL17, suggesting a Th1 response 14, 16 . Limited phenotyping 68 based on CD45RA and CCR7 suggested SARS-CoV-2-specific CD4+ T cells to be more of the 

In this study, we conducted an in-depth phenotypic analysis of SARS-CoV-2-specific 78 CD4+ and CD8+ T cells circulating in the bloodstream of individuals who had recently recovered 79 from COVID-19. This was achieved by combining detection of SARS-CoV-2-specific T cells 80 together with CyTOF, a mass spectrometry-based single-cell phenotyping method that uses 81 antibodies conjugated to metal lanthanides to quantify expression levels of both surface and 82 intracellular proteins 19 . As spectral overlap is not a limitation with CyTOF, large phenotyping 83 panels of nearly 40 parameters can be implemented, allowing for a high-resolution view of . We report here that SARS-CoV-2-specific CD4+ and CD8+ T cells from convalescent 86 individuals are diverse, exhibit features different from antigen-specific T cells against CMV, 87 include cells with both lymphoid and tissue homing potential, harbor phenotypic features of 88 functional effector cells, and are long-lived and capable of homeostatic proliferation.

Nine convalescent and three uninfected participants (Table S1) (Table S2) .

CD4+ and CD8+ T cells were identified by sequential gating on live, singlet CD3+ cells 104 expressing the corresponding co-receptor (Fig. S1 ). Spike-specific CD4+ and CD8+ T cells 105 producing IFNγ were detected in convalescent but not uninfected individuals (Fig. 1, Table S3 ),

suggesting a robust spike-specific Th1 response. In contrast, spike-specific CD4+ T cells did not 107 include cells of the Th2 and Th17 lineages, as no IL4-or IL17-producing cells were detected 108 following spike peptide stimulation, although such cells were detected following PMA/ionomycin 

To obtain a global view of the phenotypic features of SARS-CoV-2-specific T cells, we 114 visualized the data by t-distributed stochastic neighbor embedding (t-SNE) 23 . We gated on the 115 IFNγ-producing spike-specific cells (Fig. 1 ) and overlaid these on total T cells treated with co-116 stimulation alone. As most of the convalescent donors were CMV+ (Table S1, Table S3 in that they occupied multiple regions of the t-SNE. However, these cells were concentrated 120 within one or two major regions of the t-SNE, suggesting that their phenotypes are biased 121 towards particular subsets. The phenotypes of spike-specific T cells were not identical to those 122 of CMV-specific T cells, as in every donor there were regions of the t-SNE occupied by CMV-123 specific T cells that were devoid of spike-specific cells (Fig. 2 , purple ovals), although overall the 124 CD8+ T cells against the two viruses were more similar to each another than as for the CD4+ T 125 cells. This was further confirmed by demonstrating that the distributions of subset clusters, as 126 defined using the clustering algorithm DensVM 24 , were different among T cells specific for 127 SARS-CoV-2 as compared to those specific for CMV, particularly for CD4+ T cells (Fig. S4 ).

These results suggest that spike-specific T cells are not randomly distributed among T cell 129 subsets and differ from CMV-specific T cells, a finding that is perhaps expected as CMV differs Tfh function 26 , but is also an activation marker; therefore one concern was its levels were high 154 on SARS-CoV-2-specific cells simply because these cells were responding to antigen 155 stimulation. We believe that to not be the case because 1) ICOS-CXCR5-cells do not 

The predicted original states of SARS-CoV-2 had high levels of ICOS, supporting the notion that 165 these cells exhibit phenotypic features of cells with robust helper function (Fig. 3D ).

We next assessed whether SARS-CoV-2-specific CD4+ T cells exhibit features denoting 167 longevity and an ability to proliferate. CD127, the alpha chain of the IL7 receptor, is involved in 168 cell survival and is required for IL7-driven homeostatic proliferation 28 . We found that among the 

To directly assess whether SARS-CoV-2-specific T cells were capable of homeostatic acting on previously-generated cross-reactive memory B cells. We speculate that the 301 convalescent individuals we analyzed had previously been exposed to endemic coronaviruses 302 and that this prior exposure, along with conditions favoring a Th1 response in these individuals,

provided the necessary conditions for eliciting a robust and effective response against SARS-

CoV-2. Testing this intriguing hypothesis will require serological and immune cell analyses of a 305 large collection of specimens collected before and after exposure to SARS-CoV-2.

In contrast to their CD4+ counterparts, SARS-CoV-2-specific CD8+ T cells were 307 predominantly Temra cells, antigen-experienced cells that re-express the naïve cell marker 

Whether such treatment will boost the numbers of SARS-CoV-2-specific T cells remains to be 343 determined, but is conceivable given that CD127 is expressed on these cells.

In summary, our phenotypic analysis reveals long-lived lymph-node homing cTfh CD4+

T cells and CD27+CD28+ CD8+ Temra as the major subsets of SARS-CoV-2-specific T cells 346 that persist following recovery from mild COVID-19. The system we developed here to conduct (Table S1 ). Results are gated on live, singlet CD4+ or CD8+ T cells. ** p < 0.01, **** 

See also Fig. S3, Fig. S4, Fig. S5 

These fixed cells were stored at -80°C until analysis by CyTOF.

For identification of antigen-specific T cells, unless otherwise indicated, 6 million freshly- 

The cells were then incubated at 4°C for an additional 5 minutes. After 3 washes in RP10, the 583 cells were cultured for 5 days in RP10 in the absence or presence of 10 ng/ml human IL-7 (R&D 584 System). Cells were then stimulated for 6 hours with 0.5 µg/ml anti-CD49d clone L25 and 0.5 The CyTOF data were exported as FCS files, and samples were de-barcoded according 599 to manufacturer's instructions (Fluidigm) . Events corresponding to antigen-specific cells were 600 identified by gating on live, singlet intact CD3+CD19-CD4+ or CD8+ T cells expressing IFNγ,

Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus 625 origins and receptor binding

Johns Hopkins Coronavirus Resource Center

Viral and 628 host factors related to the clinical outcome of COVID-19

Breadth of concomitant immune responses prior 631 to patient recovery: a case report of non-severe COVID-19

Blood single cell immune profiling reveals the interferon-MAPK pathway mediated adaptive 634 immune response for COVID-19

A mouse-adapted SARS-coronavirus causes disease and 637 mortality in BALB/c mice

T cell responses to whole SARS coronavirus in humans

T cell responses are required for protection from 642 clinical disease and for virus clearance in severe acute respiratory syndrome coronavirus-643 infected mice

Understanding the T cell 645 immune response in SARS coronavirus infection

Vaccine efficacy in senescent mice challenged with 648 recombinant SARS-CoV bearing epidemic and zoonotic spike variants

Prior immunization with severe acute respiratory syndrome (SARS)-652 associated coronavirus (SARS-CoV) nucleocapsid protein causes severe pneumonia in mice 653 infected with SARS-CoV

Presence of SARS-CoV-2-reactive T cells in COVID-19 patients and 659 healthy donors

Phenotype of SARS-CoV-2-662 specific T-cells in COVID-19 patients with acute respiratory distress syndrome

Detection of SARS-CoV-2-Specific Humoral and Cellular Immunity in COVID-19 Convalescent 666 Individuals. Immunity. 2020/05/16

Targets of T Cell Responses to SARS-CoV

Coronavirus in Humans with COVID-19 Disease and Unexposed Individuals

Broad and strong memory CD4 (+) and CD8 (+) T cells induced by SARS-CoV-2 in 672 UK convalescent COVID-19 patients

Longitudinal high-throughput TCR repertoire proling 675 reveals the dynamics of T cell memory formation after mild COVID-19 infection

Single-cell mass cytometry of differential immune and drug 679 responses across a human hematopoietic continuum

Mass Cytometric Analysis of HIV Entry

Replication, and Remodeling in Tissue CD4+ T Cells

HIV efficiently infects T cells from the endometrium and remodels them to 685 promote systemic viral spread

Broadly targeted human cytomegalovirus-specific CD4+ 688 and CD8+ T cells dominate the memory compartments of exposed subjects

High-dimensional analysis of the murine myeloid cell 694 system

Induction of ICOS+CXCR3+CXCR5+ TH cells correlates 697 with antibody responses to influenza vaccination

ICOS is an inducible T-cell co-stimulator structurally and functionally related 700 to CD28

Human blood CXCR5(+)CD4(+) T cells are 703 counterparts of T follicular cells and contain specific subsets that differentially support antibody 704 secretion

Interleukin 7 regulates the survival and generation of memory CD4 cells

Infection with cytomegalovirus but not herpes simplex 710 virus induces the accumulation of late-differentiated CD4+ and CD8+ T-cells in humans

Multiparameter flow cytometric analysis of CD4 and CD8 T cell subsets in young and old people

COVID-19: a case for inhibiting IL-17? Nat 716 Rev Immunol

Pathogenic T cells and inflammatory monocytes incite inflammatory storm in severe COVID-19 719 patients

Follicular B helper T cells express CXC chemokine receptor 5, localize to B cell follicles, and 722 support immunoglobulin production

CXC 724 chemokine receptor 5 expression defines follicular homing T cells with B cell helper function

CXCR5 expressing human central memory CD4 T cells and their relevance for 728 humoral immune responses

SARS-CoV-2 infection induces germinal center responses with robust 731 stimulation of CD4 T follicular helper cells in rhesus macaques

Differential expression of chemokine receptors 737 and chemotactic responsiveness of type 1 T helper cells (Th1s) and Th2s

Spatial map of human T cell compartmentalization and 741 maintenance over decades of life

Human cytomegalovirus-specific 743 memory CD8+ and CD4+ T cell differentiation after primary infection

Cellular immune correlates of protection against 747 symptomatic pandemic influenza

Epstein-Barr virus-specific CD8(+) T cells that re-750 express CD45RA are apoptosis-resistant memory cells that retain replicative potential

Human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T(EMRA) 754 cells in early infection are linked to control of HIV-1 viremia and predict the subsequent viral 755 load set point

The hallmarks of CMV-specific CD8 T-cell differentiation

Dengue-specific CD8+ T cell subsets display 761 specialized transcriptomic and TCR profiles

Large-Scale HLA Tetramer Tracking of T Cells during Dengue Infection Reveals 764 Broad Acute Activation and Differentiation into Two Memory Cell Fates

Lack of peripheral memory B cell responses in recovered patients with severe 771 acute respiratory syndrome: a six-year follow-up study

Memory T cell 773 responses targeting the SARS coronavirus persist up to 11 years post-infection

Convergent Antibody Responses to SARS-CoV-2 Infection in 777 Convalescent Individuals

Coronavirus: Immune clue sparks treatment hope. BBC 779 News

An Optimized and Validated Method for Isolation and 782 Characterization of Lymphocytes from HIV+ Human Gut Biopsies

Mass cytometric analysis of HIV-infected genital T cells unveils viral strategies to 786 promote dissemination

Automatic Classification of 788

Cellular Expression by Nonlinear Stochastic Embedding (ACCENSE)