key: cord-0789006-8s1dpwmv
authors: Neidleman, Jason; Luo, Xiaoyu; George, Ashley F.; McGregor, Matthew; Yang, Junkai; Yun, Cassandra; Murray, Victoria; Gill, Gurjot; Greene, Warner C.; Vasquez, Joshua; Lee, Sulggi A.; Ghosn, Eliver; Lynch, Kara; Roan, Nadia R.
title: Distinctive features of SARS-CoV-2-specific T cells predict recovery from severe COVID-19
date: 2021-06-29
journal: Cell Rep
DOI: 10.1016/j.celrep.2021.109414
sha: 3040da46e56ef62e1779d5d900510ae96e984797
doc_id: 789006
cord_uid: 8s1dpwmv

Although T cells are likely players in SARS-CoV-2 immunity, little is known about the phenotypic features of SARS-CoV-2-specific T cells associated with recovery from severe COVID-19. We analyze T cells from 34 COVID-19 patients with severities ranging from mild (outpatient) to critical culminating in death. Relative to patients that succumbed, individuals that recovered from severe COVID-19 harbor elevated and increasing numbers of SARS-CoV-2-specific T cells capable of homeostatic proliferation. In contrast, fatal COVID-19 displays elevated numbers of SARS-CoV-2-specific regulatory T cells and a time-dependent escalation in activated bystander CXCR4+ T cells as assessed by longitudinal sampling. Together with the demonstration of increased proportions of inflammatory CXCR4+ T cells in the lungs of severe COVID-19 patients, these results support a model whereby lung-homing T cells activated through bystander effects contribute to immunopathology, while a robust, non-suppressive SARS-CoV-2-specific T cell response limits pathogenesis and promotes recovery from severe COVID-19.

The COVID-19 pandemic caused by SARS-CoV-2 has taken an unprecedented toll on the world's healthcare systems and economy, and a year since its emergence had already 38 claimed over 2 million lives with fatality rates reaching as high as 20% in some countries (Sorci Because activation markers but not IFN were expressed in the baseline specimens ( 

Their frequencies ranged from undetectable to almost 2% of T cells, were not significantly 108 different between the patient groups (Fig. 1B) , and did not correlate with time since initial PCR+ group harbored significantly higher proportions of CD8+ Tem and CD4+ Tscm cells, and lower 151 proportions of CD8+ Ttm cells.

We then examined the percentages of CD4+ Tregs and circulating T follicular helper 153 (cTfh) cells, subsets important in immunoregulation and helping antibody responses, 154 respectively. The frequencies of Tregs were similar between the three patient groups, although 155 there was a trend for higher levels in the severe group. By contrast, the moderate and severe 156 groups had significantly higher frequencies of SARS-CoV-2-specific cTfh than the mild group Among total CD4+ T cells, the most noticeable difference was a single cluster (B4.9), composed 175 of activated memory CD4+ T cells expressing high levels of the lung homing receptor CCR6 (Ito 176 et al., 2011) , which was present at higher frequencies in the severe group (Fig. 3E ). Analysis of 177 total CD8+ T cells revealed four clusters that were significantly enriched in the severe group 178 (Fig. 4E) . These clusters all expressed elevated levels of HLADR, but only some expressed 179 CD38 (Fig. 4F ), suggesting that HLADR may be a more universal marker of SARS-CoV-2-180 induced global T cell activation than CD38. One cluster (B8.1) was enriched in the mild group 181 (Fig. 4E ), albeit insignificantly, and this cluster expressed low levels of both HLADR and CD38 182 (Fig. 4F ).

Examination of SARS-CoV-2-specific T cell clusters revealed one CD4+ and one CD8+ 184 T cell cluster enriched in the severe group, and one CD4+ T cell cluster enriched in the mild 185 group (Fig. 3G, 4G ). The two clusters enriched in the severe group shared phenotypic features, 186 including increased expression levels of the activation markers HLADR and CD38 and 187 exhaustion markers PD1 and CTLA4, and decreased expression of the IL7 receptor alpha chain enriched in the mild group (Fig. 3H) . To determine whether a subset of the markers identified by expressing the two activation/exhaustion markers PD1 and CTLA4. PD1+CTLA4+ cells were (Fig. 3I, 4I ). These findings point to an overabundance of exhausted T cells recognizing CoV-2 spike during severe COVID-19, consistent with increased severity-associated T cell in the ICU, 32% of whom subsequently died from COVID-19. We next focused on these 201 specimens to try to identify T cell signatures associated with survival from severe disease. Age 202 did not account for the differential survival outcomes, as the ages of survivors and non-survivors 203 were not significantly different. Although male sex is a risk factor for severe COVID-19, we in 204 fact found among our ICU patients a lower proportion of male patients that died (22%, 205 compared to 40% for females). Furthermore, we found similar frequencies of SARS-CoV-2-206 specific T cells among our hospitalized male and female patients, and no marked phenotypic 207 differences although there was a trend for higher activation of SARS-CoV-2-specific T cells in 208 females (Fig. S3) , consistent with previous reports of total HLADR+CD38+ T cells being higher 209 in female COVID-19 patients (Takahashi et al., 2020) . Because prior studies found high levels 210 of SARS-CoV-2 antibodies to be associated with more severe disease (Garcia-Beltran et al., 211 2020; Liu et al., 2019; Woodruff et al., 2020) , we assessed whether IgG and IgM against the than mild cases (Fig. S3) .

We then examined whether T cells from survivors and non-survivors differed in their 216 response to spike peptide stimulation. Non-survivors more frequently failed to mount a robust specific CD8+ T cell response trended higher in the survivors, but this did not reach statistical 219 significance because of lower overall responses. Patients that mounted detectable SARS-CoV-220 2-specific T cell responses harbored significantly lower frequencies of total HLADR+CD69+ 221 CD4+ and CD8+ T cells (Fig. S4) , suggesting that an overall heightened state of T cell 222 activation may hinder the development of T responses directed specifically against SARS-CoV-223 2.

Interestingly, two individuals mounted very robust CD8+ T cell responses to spike 225 peptides, and one of these survived while the other did not (highlighted in blue and red, 226 respectively, in Fig. 5A ). After comparing all the antigens on these two patients' CD8+ T cells,

we found that IL6 levels within the SARS-CoV-2-specific CD8+ T cells were lower in the survivor 228 than in the non-survivor, and this phenomenon was not observed among total CD8+ T cells 229 (Fig. 5B ). Of note, the subtleness of the increase in IL6 in SARS-CoV-2-specific CD8+ T cells 230 from the non-survivor was not due to poor antibody quality, as a positive control of LPS-231 stimulated monocytes led to robust detection of IL6 (Fig. 5C ). This observation prompted us to 232 examine IL6 expression among all the ICU individuals that did elicit SARS-CoV-2-specific CD8+ 233 T cell responses. Indeed, we found a highly significant increase in IL6-producing SARS-CoV-2-234 specific CD8+ T cells in non-survivors, but not among total CD8+ T cells (Fig. 5B ). These results

suggest the possibility that SARS-CoV-2-specific CD8+ T cells producing both IFN and IL6 236 may contribute to lethal immunopathogenesis, although follow-up studies will be needed to 237 confirm this hypothesis.

We next assessed whether the major T cell subsets were differentially distributed among 239 the survivors and non-survivors. Memory CD4+ T cells were statistically more frequent in the significantly more frequent among non-survivors, though total Tregs were not (Fig. 5D ). In 243 contrast, Tfh frequencies were equivalent between survivors and non-survivors (Fig. 5D ).

Together, these observations are consistent with a model whereby Tregs recognizing SARS-

CoV-2 spike can hinder the elicitation of a robust SARS-CoV-2-specific T cell response, and 246 thus prevent recovery from severe disease. 

To compare T cells from survivors and non-survivors in an unbiased fashion, we 251 subjected them to clustering via FlowSOM, and found phenotypic differences between the two 252 groups for both CD4+ (Fig. 6A , B) and CD8+ (Fig. S6A , B) T cells. Among total CD4+ T cells, 253 two clusters (B4.1 and B4.9) were enriched in the survivor group, although the former did not 254 meet statistical significance (Fig. 6C ). Both clusters consisted of activated (HLADR+) memory 255 (CD45RO+) cells. While B4.1 expressed the additional activation markers CD38, CD69, and 256 CCR5, B4.9 did not, consistent with earlier observations of HLADR being a more universal 257 marker of T cell activation. Similar analyses among total CD8+ T cells revealed cluster B8.6 to 258 be more frequent among survivors, albeit insignificantly after multiple correction (Fig. S6C ). This 259 cluster also harbored activated cells, and were predominantly Tem cells as suggested by low features of exhausted T cells (expressing high levels of PD1 and CTLA4), S4.9 exhibited 270 features of cTfh cells with high proliferative potential (expressing high levels of CXCR5 and 271 CD127) (Fig. 6E) . Manual gating confirmed a significant increase in CD127-expressing SARSalso expressed high levels of activation marker CD69 (Fig. 6E) , we assessed whether activated 274 CD127+ cells were increased in survivors, and found this to indeed be the case (Fig. 6G ).

Further analysis of cluster S4.9 confirmed it to be highly activated (expressing activation 276 markers HLADR, CD38, CCR5, and Ox40 in addition to CD69), exhibit features of Tcm

(expressing high levels of CCR7 and CD62L), and have mucosal tissue-homing potential 278 (expressing high levels of CCR6 and CD49d) (Fig. S7 ). By contrast, cluster S4.5 did not harbor 279 most of these features (Fig. S7) . A cluster of SARS-CoV-2-specific CD8+ T cells significantly 280 enriched in survivors was also identified (S8.2), and this cluster harbored phenotypic features 281 distinct from the cluster of survivor-associated SARS-CoV-2-specific CD4+ T cells (Fig. S6F ).

S8.2 was not activated (expressing low levels of HLADR and CD38) and, unlike S4.9, 283 expressed low levels of CD127. Accordingly, unlike what was observed among SARS-CoV-2-284 specific CD4+ T cells, the percentages of CD127-expressing cells among SARS-CoV-2-specific 285 CD8+ T cells were not increased among survivors (Fig. S6G ). This suggests that relative to their 286 CD4+ counterparts, SARS-CoV-2-specific CD8+ T cells in survivors may be less long-lived, 287 although the observation that these cells express low levels of the terminal differentiation 288 marker CD57 (Fig. S6F ) suggests that they may be able to differentiate through non- 

To provide further support for this model, we mined a public single-cell RNAseq 307 (scRNAseq) dataset (Liao et al., 2020) of bronchoalveolar lavage (BAL) specimens from 308 severe/critical COVID-19 patients, and compared it to BAL from moderate COVID-19 which 309 harbored lower lung viral loads (Fig. 7D ). Elevated expression of CXCR4 was observed in T but 310 not epithelial cells of severe patients (Fig. 7E ). Visualization of the T cells by UMAP (Fig. 7F , 311 S11A) revealed co-expression of CXCR4 and CD69 in the same subsets (Fig. 7G ). These cells 312 likely infiltrated from the periphery as they express low levels of the T resident memory (Trm) 313 marker CD103 (Fig. 7H ). To assess whether pulmonary T cells may themselves recruit more 314 CXCR4+ T cells, we assessed for expression of the CXCR4 ligands CXCL12 and HMGB1 315 (Schiraldi et al., 2012) . While CXCL12 was not detected (Fig. S8B) , HMGB1 was, particularly in 316 a subset of CD8+ T cells over-represented in severe patients (Fig. 7I ). These CD8+ T cells 317 comprise a unique cluster (Fig. S8C ) and express relatively low levels of CXCR4 but high levels lung by HMGB1 produced by pulmonary cells particularly CD8+ Trm cells, which could 321 contribute to COVID-19-associated mortality.

T cell lymphopenia was identified early on during the COVID-19 pandemic as a hallmark 325 of severe disease, implying an important role for T cells in the control of SARS-CoV-2. Yet, the 326 T cell subsets that may contribute to recovery and the role of T cells directly recognizing SARS-

CoV-2 epitopes has not been investigated in depth. In this study, we compared total and SARS-

CoV-2-specific T cells from mild, moderate, and severe cases of COVID-19, and within the 329 severe cases conducted in-depth analyses of longitudinal specimens to identify features 330 predicting who will survive severe COVID-19. As discussed below, we discovered T cell 331 features associated with recovery from disease, but also some implicated in disease 332 pathogenesis. As part of our study, we provide as a resource the raw CyTOF single-cell 333 datasets for all the total and SARS-CoV-2-specific T cells (see Methods).

The phenotypes of total T cells from COVID-19 patients from our study are largely 337 consistent with a recent study that conducted deep-profiling of immune cells (including T cells) 338 using high-parameter flow cytometry (Mathew et al., 2020) . We, like the prior study, had 339 observed higher frequencies of CD8+ Ttm cells in mild patients, and a higher frequency of 340 activated, PD1-expressing T cells in severe patients. We also, similar to multiple other studies 341 (Mathew et al., 2020; Rydyznski Moderbacher et al., 2020) , found memory T cells to be 342 significantly more abundant in severe patients, although this was likely due to the older age of 343 the severe group, which affects memory cell frequencies (Rydyznski Moderbacher et al., 2020) . CD4+ Temra cells were markedly depleted. The biological significance of this difference is this paralleled a corresponding increase in total cTfh cells in more severe cases, the latter of 

To our knowledge, this is the first study to directly compare via in-depth immune survive disease. We discovered in a cross-sectional analysis that survivors mounted a higher SARS-CoV-2-specific T cell response, and longitudinal analyses revealed that this response 376 increases in survivors prior to recovery, whereas it does not in non-survivors. These data 377 suggest that SARS-CoV-2-specific T cells are protective during severe COVID-19, and are in 378 line with a number of other reports, including: a recent report of greater expansion of SARS-

CoV-2-specific T cells during moderate than severe COVID-19 (Liao et al., 2020) ; the finding 380 that antigen-specific T cells against SARS-CoV-1, a close relative of SARS-CoV-2, are 381 protective in mouse infection models (Zhao et al., 2016) ; and a recent study demonstrating 382 SARS-CoV-2-specific T cell responses, as defined by AIM markers, to be associated with less 383 severe disease (Rydyznski Moderbacher et al., 2020) . Although that latter study did not focus 384 on fatal vs. non-fatal outcomes, it included one fatal case with longitudinal sampling, and 385 observed that patient to have had no detectable SARS-CoV-2-specific T cell responses 16 and 386 11 days prior to death, in line with our findings. Together, the data are consistent with the notion 387 SARS-CoV-2-specific T cells being beneficial rather than detrimental for surviving severe 388 COVID-19. That being said, we did find that SARS-CoV-2-specific CD8+ T cells co-producing 389 IFN and IL6 were elevated in individuals that did not survive severe disease. Plasma IL6 levels 390 associate with COVID-19 severity (Huang et al., 2020; Mathew et al., 2020; Zhou et al., 2020a) 391 and predict COVID-19-associated death (Del Valle et al., 2020) . However, save a study that 392 reported elevated IL6 production in total CD4+ T cells from COVID-19 patients in the ICU (Zhou 393 et al., 2020b), IL6 is not thought to be produced by T cells during SARS-CoV-2 infection.

Our data suggest multiple mechanisms by which severe patients may control the 400 production of SARS-CoV-2-specific T cells. Hospitalized patients that produced few SARSof Tregs among total T cells has been reported to be both increased (De Biasi et al., 2020) and 

The authors declare no competing financial interests. 

Even in the uninfected specimen with the highest response to spike stimulation (red dot), the 539 proportion of IFN-producing cells was only 0.01% (inset), suggesting that the responses we 540 detect in COVID-19 specimens correspond to de novo SARS-CoV-2-specific T cells. *** p < 541 0.001 as determined by a Student's unpaired t-test. Each datapoint corresponds to a different 

 The raw CyTOF datasets generated from this study are available for download through 681 the public repository Dryad via the following link: https://doi.org/10.7272/Q67H1GTB 682  This paper does not report original code.

 Any additional information required to reanalyze the data reported in this paper is 684 available from the lead contact upon request. disease as defined by being in the ICU (designated "survivor" in this study), and 6 of these 691 individuals died from COVID-19 (designated "non-survivor" in this study). Moderate cases were 692 defined as non-ICU hospitalizations for COVID-19. Eight of the individuals that experienced 693 severe disease were sampled longitudinally at up to 4 timepoints. All specimens from 694 hospitalized patients were from a timepoint when the patients were still hospitalized, and this 695 ranged from 0-76 days after initial positive SARS-CoV-2 test. Mild cases consisted of those 696 never hospitalized for COVID-19, and were generally analyzed 20-154 after initial positive specimens from the mild cases were from outpatient individuals that were no longer 701 symptomatic at the time of sampling and therefore can be considered convalescent specimens.

Additional clinical features, including patient gender, age, race, and whether patients were given 703 convalescent plasma, dexamethasone, or remdesivir for COVID-19, are indicated in the table   704 below. Consistent with male sex, advanced age, and non-white race being risk factors for 705 severe disease, mild cases were predominantly female (81%, vs. 48% for hospitalized patients), 

The remaining cells were stimulated for 6 hours with the co-stimulatory agents 0.5 g/ml 741 anti-CD49d clone L25 and 0.5 g/ml anti-CD28 clone L293 (both from BD Biosciences) and 0.5 

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

CCR6 as a 924 mediator of immunity in the lung and gut

PD-1-Expressing SARS-CoV-2-Specific CD8(+) T Cells Are Not 973

Exhausted, but Functional in Patients with COVID-19

Antigen-Specific Adaptive Immunity to

SARS-CoV-2 in Acute COVID-19 and Associations with Age and Disease Severity

HMGB1 promotes recruitment of inflammatory 980 cells to damaged tissues by forming a complex with CXCL12 and signaling via CXCR4

Robust T cell immunity in convalescent 984 individuals with asymptomatic or mild COVID-19

Explaining among-country variation in COVID-19 986 case fatality rate

Two X-linked agammaglobulinemia patients develop 989 pneumonia as COVID-19 manifestation but recover

Sex differences in immune responses that underlie 998 COVID-19 disease outcomes

The SARS-CoV-2 T-cell immunity is 1001 directed against the spike, membrane, and nucleocapsid protein and associated with COVID 19 1002 severity

FlowSOM: Using self-organizing maps for visualization and interpretation 1008 of cytometry data

Dengue virus infection elicits highly polarized CX3CR1+

cytotoxic CD4+ T cells associated with protective immunity

Extrafollicular B cell responses correlate

Inborn errors of type I IFN immunity in patients with 1024 life-threatening COVID-19

Airway Memory CD4(+) T 1027

Longitudinal COVID-19 profiling associates IL-1RA and IL-10 with disease severity and 1034 RANTES with mild disease

Clinical course and risk factors for mortality of adult inpatients with

Pathogenic T cells and inflammatory monocytes incite inflammatory storm in severe 1040 COVID-19 patients

Single-Cell Sequencing of Peripheral Mononuclear Cells Reveals Distinct Immune

Finally, to better understand T cell features associated with survival of severe COVID-