key: cord-0980261-brqe8k73
authors: Pérez-Gómez, Alberto; Gasca-Capote, M Carmen; Vitallé, Joana; Ostos, Francisco J.; Serna-Gallego, Ana; Trujillo-Rodríguez, María; Muñoz-Muela, Esperanza; Giráldez-Pérez, Teresa; Praena-Segovia, Julia; Navarro-Amuedo, María D.; Paniagua-García, María; García-Gutiérrez, Manuel; Aguilar-Guisado, Manuela; Rivas-Jeremias, Inmaculada; Jimenez-Leon, María R.; Bachiller, Sara; Fernández-Villar, Alberto; Pérez-González, Alexandre; Gutiérrez-Valencia, Alicia; Benhnia, Mohammed Rafii-El-Idrissi; Weiskopf, Daniela; Sette, Alessandro; López-Cortes, Luis F.; Poveda, Eva; Ruiz-Mateos, Ezequiel
title: Deciphering the quality of SARS-CoV-2 specific T-cell response associated with disease severity, immune memory and heterologous response
date: 2021-12-28
journal: bioRxiv
DOI: 10.1101/2021.12.28.474325
sha: 15cbe8206f39a039487e9cb971e5add59473c279
doc_id: 980261
cord_uid: brqe8k73

SARS-CoV-2 specific T-cell response has been associated with disease severity, immune memory and heterologous response to endemic coronaviruses. However, an integrative approach combining a comprehensive analysis of the quality of SARS-CoV-2 specific T-cell response with antibody levels in these three scenarios is needed. In the present study we found that, in acute infection, while mild disease was associated with high T-cell polyfunctionality biased to IL-2 production and inversely correlated with anti-S IgG levels, combinations only including IFN-γ with absence of perforin production predominated in severe disease. Seven months after infection, both non-hospitalized and previously hospitalized patients presented robust anti-S IgG levels and SARS-CoV-2 specific T-cell response. In addition, only previously hospitalized patients showed a T-cell exhaustion profile. Finally, combinations including IL-2 in response to S protein of endemic coronaviruses, were the ones associated with SARS-CoV-2 S-specific T-cell response in pre-COVID-19 healthy donors’ samples. These results have implications for protective immunity against SARS-CoV-2 and recurrent COVID-19 and may help for the design of new prototypes and boosting vaccine strategies.

Host immune response against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is a key factor in the progression of Coronavirus Disease 2019 (1) and its deregulation results in fatal disease in hospitalized COVID-19 patients (2, 3) . The coordination of different branches of adaptive immunity, such as CD4+, CD8+ T-cell and antibody responses, is essential for the resolution of COVID-19 (4) . Despite the already known role of T-cell response against SARS-CoV-2 infection, there are still gaps that need to be clarify in relation to the quality of this response and its association with: i) disease severity in acute infection, ii) long-lasting immune memory and iii) the heterologous response found in healthy donors (5) .

Seminal studies in SARS-CoV-1 infection models showed that both CD4+ (6) and CD8+ (7) T-cell response were involved in protection and virus clearance in acute infection. In SARS-CoV-2 infection, the predominant CD4+ T-cell response has been associated with mild disease and enhanced early virus clearance in acute infection while its absence was associated with fatal COVID-19 outcome (4, 8, 9) . Although at a lower level of magnitude, Laboratory, Baltimore, MD, #HP6043-HRP) was used at a 1: 2,000 dilutions in 1% milk containing 0.05% Tween-20 in PBS. Plates were washed 4 times with 0.05% PBS-Tween-20. The plates were developed using fast o-phenylenediamine Peroxidase Substrate (Merck, #P9187), the reaction was stopped using 1M HCl, and the optical density at 490 nm (OD490) was read on a Multiskan GO Microplate Spectrophotometer (ThermoFisher Scientific) within 2 hrs.

Non-parametric statistical analyses were performed using Statistical Package for the method was utilized to identify and discard outliers. Differences between different groups were analyzed by two-tailed Mann-Whitney U test. The Wilcoxon test was used to analyze paired samples. Categorical variables were compared using the χ2 test or the Fisher's exact test. The Spearman test was used to analyze correlations between variables.

All differences with a P value of < 0.05 were considered statistically significant.

Patients hospitalized with acute SARS-CoV-2 infection showed higher CD4+ and lower CD8+ T-cells levels compared with sex-and age-matched pre-COVID-19 healthy donors (HD), which resulted in higher CD4:CD8 T-cell ratio (Fig. 1A, left panel) . SARS-CoV-2 infection was also associated with lower effector memory (EM) and terminally differentiated effector memory (TEMRA) CD4+ T-cell levels ( 

We assayed SARS-CoV-2 specific T-cell response by intracellular cytokine staining (ICS), this technique is a well-stablished method for evaluating virus specific T-cell response (22, 26) . ICS, despite of using a high number of cells, allowed us to have higher sensitivity to assay the magnitude and quality of the T-cell response. We performed CD4+ and CD8+ T-cell response specific to spike (S) and nucleocapsid (N) peptide pools. The specific T-cell response to each stimuli was determined by the sum of the expression of each assayed cytokine (IFN-γ, IL-2 and TNF-α). To classify an individual as a responder, we consider a threshold higher than 0.05%, as previously published (22) . First, as expected, we observed a higher magnitude of the response in most of T-cell subsets for both peptide pools (N and S) in hospitalized acute SARS-CoV-2 infected patients (Acute) compared to HD samples ( Fig. 2A-B , top panels). However, there were differences neither in the magnitude of the response nor in the proportion of responders in the TEMRA subset for both CD4+ and CD8+ T-cells and for S and N stimuli ( Fig. 2A-B ). In fact, there were no differences in the levels of responders for all CD8+ T-cell subsets for N peptides (Fig. 2B , bottom panels). Overall, 75% and 82% of HD had SARS-CoV-2 specific CD4+ and CD8+ T-cell response, respectively, considering S+N peptides and all T-cell subsets ( Fig. 2C-D) .

Second, comparing the response to S and N peptide pools, there was a higher magnitude of response to S compared to N stimulus in central memory (CM) CD4+ T-cell (p=0.042) and a trend in total memory (MEM) CD4+ T-cells (p=0.126) ( Fig. 2A , top panels), however there were no differences for CD8+ T-cell subsets (Fig. 2B , top panels).

Additionally, a cumulative SARS-CoV-2-specific T-cell measurement was calculated as the sum of the S and N responses (Fig. S2 ). Our data show that, all the patients had detectable SARS-CoV-2 specific T-cell response considering together the response against S and N peptide pools and to all the CD4+ and CD8+ T-cell subsets ( Fig.2C-D,   Fig. S2 ).

Finally, when comparing CD4+ and CD8+ T-cell response in hospitalized patients (Acute), the magnitude of SARS-CoV-2 specific total memory (MEM) CD4+ T-cell response ( Fig. 2A, top panel) was higher compared MEM CD8+ T-cell response ( Fig. 2B , top panel) for protein S (p=0.048), but not different for the rest of subsets and for protein N ( Fig. 2A-B , top panels). In the same way, there was a higher percentage of responders for MEM CD4+ T-cells compared to MEM CD8+ T-cells in S protein (p=0.025), there were no differences in the proportion of responders for the rest of subsets for S and N proteins ( Fig. 2A-B , bottom panels).

associated with disease severity while IL-2 production in S-specific CD8+ T-cells is associated with mild disease.

We next analyzed in acute infection the association of S-specific CD4+ T-cell response with disease severity in hospitalized patients segregated as mild and severe patients. The S-specific CD4+ T cell response was significantly higher in the TEMRA CD4+ T-cell subset in severe compared to mild patients (Fig. 3A) . When individual cytokine production was analyzed, these higher levels were attributed to IFN-γ production in Sspecific TEMRA CD4+ subset ( Fig. 3B; Fig S3A) , but not for IL2 or TNF-α (Fig. S3B ).

Multiple combination of cytokines, together with CD107a and perforin expression revealed that combinations only including IFN-γ+ CM (Fig. 3C, Fig. S3C ) and TEMRA cells (Fig. S3D ) were increased in severe compared with mild patients. The same occurred for combinations including IFN-γ+ and TNF-α+ CM cells (Fig. S3E) . However, combinations including IL-2, such as IL2+TNF-α+ MEM cells were increased in mild compared to severe patients (Fig. 3D ). In fact, S-specific MEM CD4+ T-cell polyfunctionality was higher in mild patients, mainly because of increased bi-functional combinations including IL-2 (IL2/TNF-α, IL2/IFN-γ and IL-2/perforin) that were not present in severe patients, where IFN-γ/TNF-α combination was predominant (Fig. 3E ).

It is also important to highlight that perforin expression was higher in MEM and EM subsets of mild patients in comparison with severe patients (Fig. S3F ). This was reflected in a higher S-specific MEM polyfunctional index in mild compared to severe patients ( Fig. 3F) . Additionally, we observed that the bulk of S-specific CD4+ T-cell response in the different subsets was inversely associated with different inflammatory markers, while specific combinations including IFN-γ were directly associated with plasmatic IP-10 levels (Fig. S4) . Overall, a high polyfunctional S-specific CD4+ T-cell response biased to IL-2 production was associated with mild disease, while combinations only including IFN-γ were associated with severe disease outcomes.

In relation to S-specific CD8+ T-cells, the bulk of CM CD8+ T-cell response was higher in mild compared to severe patients (Fig. 3G ). We observed that the cytokine responsible of these differences was IL-2, which presented higher levels in MEM, CM and EM Sspecific CD8+ T-cells in mild subjects ( Fig. 3H; Fig. S5A ) while very low levels and no differences were observed in IFN-γ+ and TNF-α+ production (Fig. S5B) . These results were confirmed by combinations only including IL-2 without the expression of the rest of the cytokines, CD107a and perforin, in the same subsets: MEM, CM and EM (Fig. 3I ).

Similar results were observed for three and four functions (Fig. S5C ). In summary, IL-2 production in not terminally differentiated S-specific CD8+ T-cells was associated with mild disease progression in hospitalized acute SARS-CoV-2 infected patients.

We also analyzed in detail the quality of N-specific T-cell response. MEM and CM IL2+ and EM TNF-α+ N-specific CD4+ T cell levels were higher in mild compared to severe patients ( Fig. 4A-B) . We did not observe differences for the bulk of IFN-γ+ N-specific CD4+ T-cell response ( Fig. S6A-B ). Following the same profile of S-specific CD4+ Tcell response, combinations including only IL2+ and TNF-α+ in MEM, CM and EM N-specific CD4+ T-cells were associated with mild disease progression (Fig. 4C) . Besides, we observed higher levels of combinations with triple cytokine positive MEM, CM and EM CD4+ T-cells in mild compared to severe patients (Fig. 4D ). In fact, MEM N-specific response showed a higher proportion of triple and a variety of double combinations (Fig.   4E ), likewise a higher MEM polyfunctional index in mild compared to severe patients (Fig. 4F) . These results were reproduced in polyfunctionality of CM and EM subsets with three and four functions that were also associated with mild disease progression (Fig.   S6C ). We did not observe great differences in N-specific CD8+ T-cell response according with disease severity, only higher levels of combinations with only IFN-γ+ T-cells in severe compared to mild patients (Fig. S6D ).

In addition to the analyses in the acute phase, we analyzed the magnitude of SARS-CoV- We also compared the magnitude of S-versus N-specific CD4+ T-cell response in both groups. We found that the magnitude of MEM and CM response was higher in S compared to N in both, previously hospitalized (p=0.008; p=0.008, respectively) and nonhospitalized patients (p=0.014; p=0.009, respectively) seven months after SARS-CoV-2 infection (Fig. 5A, top panel) . Secondly, we analyzed the magnitude of SARS-CoV-2 specific CD8+ T-cell response and we found that previously hospitalized patients presented higher S-specific EM T-cells compared to non-hospitalized patients ( Furthermore, we found that the magnitude of MEM and EM response was higher in N compared to S peptides in non-hospitalized patients (p=0.026; p=0.024, respectively) while no differences were found in previously hospitalized patients seven months after SARS-CoV-2 infection (Fig. 5C ). Finally, we compared the magnitude of response between CD4+ and CD8+ T-cells. We found that in both groups, MEM and CM Sspecific CD4+ T-cell response was higher than in CD8+ T-cells (p=0.005 and p=0.036 for previously hospitalized; p=0.004 and p=0.013 for non-hospitalized, respectively), while no differences were found for N stimulus ( Fig. 5A -C).

After finding a similar magnitude of SARS-CoV-2 specific T-cell response in both groups of individuals seven months after infection, we assayed the quality of T-cell response and exhaustion markers in previously hospitalized and non-hospitalized patients. The TIGIT expression in all the CD4+ T-cell subsets were higher in previously hospitalized than in non-hospitalized patients (Fig. 6A) . We did not find differences between groups in TIGIT+ CD8+ T-cells ( 

Next, we analyzed antibody levels against S protein and the association of this humoral response with disease severity and T-cell immunity. In acute infection, we observed a trend to increased antibody levels in severe compared to mild patients (Fig. 7A ). Seven months after SARS-CoV-2 infection anti-S IgG levels remained high, similar to severe patients in acute infection and at higher levels compared to mild patients in both previously hospitalized and in non-hospitalized patients (Fig. 7A) . As expected, all the groups had higher antibody levels compared to HD (Fig. 7A) . In relation to T-cell response, in general, SARS-CoV-2 specific T-cell response was inversely associated with anti-S IgG levels in acute infection (Fig. S9A) , while a direct correlation was observed seven months after infection (Fig. S9B) . A representative example was the inverse correlation of S-specific EM CD4+ T-cell producing IL2 in acute infection (Fig. 7B) compared to the direct correlation found seven months after infection (Fig. 7C) .

Similar to previously reported (21), we found that a high percentage of HD (pre-COVID-19 samples) presented detectable CD4+ and CD8+ SARS-CoV-2 specific T-cell response (75% and 82%, respectively) (Fig. 2) . In order to characterize this immune response, we performed anti-S IgG levels and specific T-cell response by ICS using an optimized peptide pool for the four human endemic coronaviruses (21) . In acute SARS-CoV-2 infected participants we observed a direct correlation of anti-S SARS-CoV-2 IgG levels with those of three out of the four endemic coronaviruses (HCoV-NL63, -OC43 and -HKU1) (Fig. S10 ). When we split this group, we only found a positive correlation of anti-S SARS-CoV-2 IgG and anti-S HCoV-NL63 and -OC43 levels in severe (Fig. S10 ) but no correlation was found in mild patients (Fig. S10) . In HD we also found a positive correlation of anti-S SARS-CoV-2 IgG and anti-S HCoV-OC43, -229E and HKU-1 levels ( Fig. S10) . Finally, in all the groups together, anti-S SARS-CoV-2 IgG levels were directly associated with anti-S IgG levels of the beta-coronaviruses HCoV-OC43 and -HKU1 (Fig. S10 ). After that, we performed S-specific T-cell response to the optimized peptide pool of endemic coronaviruses (SE) in HD. We found detectable SE T-cell response in all the CD4+ and CD8+ T-cell subsets (Fig. 8A) . Analyzing the bulk of SE endemic coronaviruses correlated to S-specific for SARS-CoV-2 in CM and EM CD4+subsets ( Fig. 8B -C) and in CM CD8+ subset (Fig. 8D) . Attending to the quality of this response, it was mainly monofunctional and IL-2 production prevailed in CD4+ and CD8+ T-cells respect to other cytokines ( Fig. 8E; Fig. S11A ). Interestingly, combinations including IL-2, but not IFN-γ, in response to human endemic coronaviruses correlated with S-specific SARS-CoV-2 response, for CD4+ MEM (Fig. 8F ), CM and EM subsets (Fig. S11B-C) and CD8+ CM (Fig. 8G) .

In the present study, analyzing 103 subjects, we describe features of SARS-CoV-2 specific humoral and T-cell response differentially associated with disease severity in hospitalized patients during acute infection. This response is long-lasting seven months after infection independently whether patients were previously hospitalized or not, although previous hospitalization was associated with exhausting T-cell features present in acute infection. Finally, we comprehensively analyzed the features of the high levels of cross-reactive response between SARS-CoV-2 and human endemic coronaviruses in healthy donors.

We used ICS for the systematic analysis of SARS-CoV-2 specific T-cell response. ICS is a technique commonly used for analyzing T-cell response against viral infections (22, 26) and can be complementary to other strategies as T-cell receptor dependent activation induced marker (AIM) (16, 27, 28) . Although a high amount of cells is needed, a comprehensive cytokine dependent functional characterization of virus specific T-cell response can be achieved (5) . We analyzed the response against protein S and N, because these are the main targets, in terms of magnitude, of SARS-CoV-2 specific T-cell response (16) . Next, we sought to analyze the immune memory to SARS-CoV-2 seven months after infection in two groups of subjects with different course of the disease: patients that overcame the disease without the need of hospitalization and previously hospitalized patients. We observed that in both groups, all subjects displayed detectable T-cell response, considering S+N response and all CD4+ and CD8+ T-cell subsets. This is in accordance with immune memory found to SARS-CoV infection, which have been shown to last for years (36) and agreed with the magnitude of T-cell response found eight months after SARS-CoV-2 infection in previously non-hospitalized subjects (14). Although in that study using AIM they found only SARS-CoV-2 Specific CD8+ T-cell response in 50% while we observed 91% of responders but 75% in previously hospitalized patients (14). Despite the general absence of difference in the magnitude of T-cell response between both groups, in terms of quality of this response, previously hospitalized patients showed higher T-cell exhaustion levels (TIGIT and PD-1 expression) and higher S and N-specific T-cell levels of combinations of only including IFN-γ and TNF-α production compared to non-hospitalized patients. Additionally, non-hospitalized patients presented higher IL-2 and perforin production in N-specific CD8+ T-cells compatible with a preserved antiviral activity. This profile is reminiscent of the one found in severe compared to mild patients in acute infection. However, on the contrary to what happened in acute disease, seven months after infection in previously hospitalized subjects, anti-S IgG levels were directly, not inversely, associated with SARS-CoV-2 specific T-cell response, especially that enriched in IL-2 production which was associated with a good prognosis in acute infection. These results demonstrate that anti-SARS-CoV-2 humoral and cellular response are long-lasting and robust at least seven months after infection in both non-hospitalized and previously hospitalized patients. However, previously hospitalized patients showed T-cell exhaustion and some signs of SARS-CoV-2-specific T-cell response associated with disease progression in acute infection, although this response was more IL-2 biased, which was associated with good prognosis. This defects found in previously hospitalized patients seven months after infection may be selectively associated with long-COVID symptoms, as has been recently reported four months after infection (11); however, we cannot confirm it because information about long-lasting symptoms were not recorded in this cohort as this was not the aim of the present study.

Finally, we found high levels of cross-reactive CD4+ and CD8+ T-cell response to SARS-CoV-2 (75% and 82%, respectively) in pre-COVID-19 HD samples. These levels were even higher than those found in previous cohorts showing 20-50% of cross-reactivity (8, 16, 36, 37) . Using an optimize peptide pool (21) for the four endemic coronaviruses: NL63, OC43, 229E and HKU1, we found that endemic S-specific CD4+ and CD8+ Tcell response was directly correlated with SARS-CoV-2 S-specific CD4+ and CD8+ Tcell response in EM and CM subsets. These results confirm cross-reactive SARS-CoV-2 specific T-cell response with endemic coronavirus. Comprehensively analyses of endemic S-specific T-cell response was mainly induced by TEMRA CD4+ T-cells and CM CD8+ T-cells and we found that the response was totally biased to IL-2 production, what may explain some previously published results using IFN-γ ELISPOT that did not find cross-reactive response (10). In fact, combinations including IL-2, but not IFN-γ, were the ones associated with SARS-CoV-2 S-specific CD4+ and CD8+ T-cell response.

These results suggest that this pre-existing T-cell memory may have the potential to induce low COVID-19 fatality. However, whether a different quality of endemic T-cell specific response, based mainly in IL-2 or IFN-γ production, may contribute to variations in COVID-19 progression is currently unknown.

One limitation of this study is that all patients included were recruited in the first wave of COVID-19 in Spain, in those dates, experimental therapies with very limited but transitory immunosuppressive effects were administered what may have affected the levels of immune parameters in acute infection. However, in those patients who were treated with IFN-β and corticosteroids, samples were collected time enough after these therapies to reverse the potential effects (24 days [7 -28] and 9 days [1 -21] , respectively) what may have not affected the results presented herein. Seven months postinfection, one potential bias may come from the group of hospitalized subjects that were composed by patients with previous mild (42%) or severe (58%) disease, however, as no differences were found in any parameter associated to T-cell response (data not shown) between this two subgroups, they formed part of the same group of previously hospitalized and were compared with non-hospitalized patients. ICS needs a notable amount of cells to be assayed, this avoid us to perform endemic virus-specific T-cell response in COVID-19 samples; however, it has allowed us to obtain comprehensive data about the quality of SARS-CoV-2 specific T-cell response. Finally, anti-S IgG levels were assayed against the whole S protein and not for RBD, cross-reactive reaction cannot be excluded and results have to be interpreted taking this into account. In the same way, further research is needed to confirm and correlate our T-cell response profile with neutralizing antibody levels and B-cell polyfunctionality. individual. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Mann-Whitney U test was used for groups' comparisons and Spearman test for non-parametric correlations. 

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TNF-α and IL-2 production (bottom panels). (B) and (D) Bar graphs describe the number and percentage of responders for S peptide pool, as the sum of any CD3+CD4+ or CD3+CD8+ T-cell subset (%S Responders); for N peptide pool, as the sum of any CD3+CD4+ or CD3+CD8+ T-cell subset (%N Responders) and the total responders as the sum of CD3+CD4+ or CD3+CD8+ S and N responses (% of total Responders). The medians with the interquartile ranges are shown. Each dot represents an individual

Representative dot plot showing IL-2 production in N-specific CM CD8+ T-cells (right panel). (F) N-specific EM CD8+ T-cell levels of cells producing PRF (left panel). N-specific EM CD8 T-cell polyfunctionality pie charts (right panel). For all the pie charts each sector represents the proportion of SARS-CoV-2-specific T-cells producing two (blue) and one (yellow) function. Arcs represents the type of function (IFN-γ, TNF-α, IL-2, CD107a and PRF) expressed in each sector

U test was used for groups' comparisons. shows the percentage of responders considering a responder subjects as those with the percentage of SE-specific T-cells higher than 0.05% considering the sum of IFN-γ, TNFα and IL-2 production. (B) Correlation between S-Specific and SE-specific CM CD4+ T cell levels. (C) Correlation between S-Specific and SE-specific EM CD4+ T cell levels and (D) Correlation between S-Specific and SE-specific CM CD8+ T-cell levels in healthy donors. (E) Pie graphs represent IFN-γ, IL-2 and TNF-α expression in each Tcell subset where median percentages of this expression are shown in right table. (F) Correlation between S-Specific and SE-specific MEM CD4+ IL-2+ T cells and (G) S-Specific and SE

*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Mann-Whitney U test was used for groups' comparisons and Spearman test for non-parametric correlations