key: cord-0744158-sxegniol
authors: Högelin, Klara Asplund; Ruffin, Nicolas; Pin, Elisa; Månberg, Anna; Hober, Sophia; Gafvelin, Guro; Grönlund, Hans; Nilsson, Peter; Khademi, Mohsen; Olsson, Tomas; Piehl, Fredrik; Al Nimer, Faiez
title: Development of humoral and cellular immunological memory against SARS-CoV-2 despite B-cell depleting treatment in multiple sclerosis.
date: 2021-09-02
journal: iScience
DOI: 10.1016/j.isci.2021.103078
sha: af21170b34738fde6a5a7e34e2bda0d64d8d3bea
doc_id: 744158
cord_uid: sxegniol

B-cell depleting therapies (BCDTs) are widely used as immunomodulating agents for autoimmune diseases such as multiple sclerosis. Their possible impact on development of immunity to SARS-CoV-2 has raised concerns with the COVID-19 pandemic. We here evaluated the frequency of COVID-19-like symptoms and determined immunological responses in participants of an observational trial comprising several multiple sclerosis disease modulatory drugs, (COMBAT-MS; NCT03193866) and in eleven patients after vaccination, with a focus on BCDT. Almost all seropositive and 17.9% of seronegative patients on BCDT, enriched for a history of COVID-19-like symptoms, developed anti-SARS-CoV-2 T-cell memory and T-cells displayed functional similarity to controls producing IFN-γ and TNF. Following vaccination, vaccine-specific humoral memory was impaired, while all patients developed a specific T-cell response. These results indicate that BCDTs do not abrogate SARS-CoV-2 cellular memory and provide a possible explanation as to why the majority of patients on BCDTs recover from COVID-19.

In December 2019, the first cases of infection with a new zoonotic pathogen were detected.

Severe acute respiratory syndrome virus-2 (SARS-CoV-2) causes a variety of clinical symptoms and syndromes, ranging from asymptomatic to lethal infections, that are altogether known as coronavirus disease 2019 . The ongoing COVID-19 global pandemic has sparked intense efforts to identify risk factors and characterize the pathophysiology of severe disease courses in order to reduce disease morbidity and mortality while waiting for vaccination. Demographic factors such as advanced age, male sex and comorbidities such as obesity, chronic obstructive pulmonary disease, cardiovascular and kidney diseases have all been found to associate to increased risk for severe disease or mortality (Izcovich et al., 2020) .

The immunologic host response to SARS-CoV-2 is complex with different components of the innate and adaptive systems synergistically interacting in the defense against the virus.

Importantly, both too little and too much immune activation can be detrimental, as severe immunosuppression as well as cytokine storm syndrome have been implicated as risk factors for severe COVID-19 disease Remy et al., 2020; Vabret et al., 2020) . Therefore, immunosuppressant therapies for autoimmune diseases on the one hand have been considered risk factors for a more severe disease course, while certain immunomodulators on the other hand have been clinically tested for possible attenuation of hyperinflammatory responses. Such trials have mainly involved modulators of cytokine and chemokine signaling, such as interleukin-6 blockers (Meyerowitz et al., 2020; Stone et al., 2020; Vabret et al., 2020) .

In parallel, epidemiological studies have assessed if immunosuppressive therapies are associated with more severe COVID-19 (Sadeghinia and Daneshpazhooh, 2020; Schoot et al., 2020) .

In particular, B-cell depleting anti-CD20 monoclonals, e.g. ocrelizumab (OCR), ofatumumab and rituximab (RTX), have raised concerns due to their potential to abrogate development of J o u r n a l P r e -p r o o f humoral responses to infectious agents including SARS-CoV-2 (Baker et al., 2020; Bar-Or et al., 2020; Houot et al., 2020; Meca-Lallana et al., 2020; Zabalza et al., 2020) . B-cell depleting therapies (BCDTs) are widely used in hematologic malignancies as well as in a variety of autoimmune diseases, such as rheumatoid arthritis and multiple sclerosis (MS). Emerging epidemiological evidence suggests that anti-CD20 therapies might be associated with a higher risk for a more severe COVID-19 disease course, but not increased mortality (Peeters et al., 2020; Safavi et al., 2020; Sormani et al., 2021; Zabalza et al., 2020) . However, caution should be exerted when interpreting this type of data since it relies on spontaneous reporting with difficulties in completely controlling for confounders. In addition, in persons with MS (pwMS), established risk factors seem to outweigh the impact of disease-modulatory therapies (DMTs) on COVID-19 outcomes (Bsteh et al., 2021; Louapre et al., 2020; Zabalza et al., 2020) .

Nevertheless, a recent pre-pandemic study showed an increased rate of severe infections with B-cell depletion compared to other DMTs in pwMS (Luna et al., 2020) . Therefore, suggested guidelines have included increased social distancing, choice of another medication than anti-CD20 or extending dosing intervals (Korsukewitz et al., 2020) . While epidemiological data to some degree are at hand, it remains unknown if and to what degree individuals with anti-CD20 treatment develop immunity to SARS-CoV-2. The objective of this study was to provide a detailed characterization of humoral and cellular immunity to SARS-CoV-2 in a populationbased cohort of patients with relapsing-remitting MS exposed to a number of different DMTs.

Our findings suggest that most patients develop cellular immunity to SARS-CoV-2 that does not significantly differ between different DMTs, including anti-CD20 treated patients with suppressed B-cell levels.

J o u r n a l P r e -p r o o f with the multiplex bead array and IFN-γ responses in FluoroSpot correlated well between SARS-CoV-2 spike and nucleocapsid domains in each method respectively, while instead there was no evident correlation between the strength of humoral and T-cell immunity (Figures S1C and S1D). Based on this and previous studies, we defined SARS-CoV-2 T-cell positive samples as those that had ≥ 12.5 ΔIFN-γ SFU/ 2.5 x 10 5 cells for S1 and/or N (Sekine et al., 2020) .

Eighteen out of 24 pwMS and 6 out of 8 HC that were SARS-CoV-2 seropositive with either ECLIA or multiplex bead array for spike (S1S2) and nucleocapsid also displayed SARS-CoV-2 T-cell reactivity for S1 or N peptides in FluoroSpot (Figure 2A ). Seven pwMS were positive in the multiplex bead array for only one of the two peptide domains (spike S1S2 foldon or nucleocapsid C) and hence were considered as seronegative in the final assessment. Of those seven, three displayed SARS-CoV-2 T-cell reactivity for S1 or N peptides in the FluoroSpot assay. Four patients with T-cell reactivity were negative with ECLIA but tested seropositive with the multiplex bead array, while no individual displayed positivity with ECLIA and was seronegative with the multiplex bead array. These four patients displayed a lower MFI for nucleocapsid C (p = 0.02), but otherwise did not differ in other factors including time from infection (p = 0.13) compared to those being positive in both assays. Of note, only 3 out of 19 (16%) of seropositive anti-CD20-treated pwMS lacked detectable T-cell responses ( Figure 2B ).

Although this could depend on the few samples tested, it may also be affected by the HLA-type of these patients and/ or the limited number of SARS-CoV-2 peptides tested (Nguyen et al., 2020) . On the contrary, 16.4% (9/55), 13.5% (5/37) and 28.6% (2/7) of pwMS treated with anti-was at least as good in the anti-CD20 treated group (RTX and OCR) as in patients with other treatments and HC ( Figure 2B ).

There was a tendency that donors with fever, anosmia and symptoms more than 2 weeks displays a higher frequencies of SARS-CoV-2 memory T-cell responses as compared to donors only reporting cough, suggesting a lower specificity of the latter for COVID-19 ( Figure 2C ).

There was, however, no apparent difference in the levels of anti-SARS-CoV-2 antibodies between treatment types ( Figure 2D ) or reported symptoms (data not shown). A tendency for a decrease over time in antibody levels to N (r = -0.333, p = 0.067), but not to S, or T-cell memory responses to S1 or N was observed, partly explaining the observation that antibodies and T-cell reactivity did not correlate (Figures 2E and 2F) . No apparent effect of age or sex was seen (data not shown). Unfortunately, few subjects had been tested with PCR, since in Sweden such testing during the first phase of the pandemic was mainly restricted to hospital admissions (Table S1) .

However, all 7 out of 71 pwMS on RTX treatment with a positive SARS-CoV-2 PCR test also had antibodies and/or T-cell immunological memory, while all 8 pwMS that reported negative PCR did not.

These findings indicate that cellular and humoral immune response against SARS-CoV-2 may arise or persist independently and that BCDTs do not prevent development of SARS-CoV-2 immunity.

LFA-1 conformation in response to T-cell activation in combination with intracellular cytokine staining using flow cytometry (Dimitrov et al., 2018; Schollhorn et al., 2021) . The observation that the percentage of LFA-1 + IFN-γ + T-cells correlated well with FluoroSpot IFN-γ SFU for anti-CD3 (r = 0.438, p = 0.01), N, S, S1 and M (r = 0.672, p < 0.0001) and N and S1 (r = 0.447, p = 0.013) peptide stimulation ( Figures 3A-D) confirmed the specificity of this approach.

Analyses of LFA-1 + IFN-γ + T-cells revealed that nearly two third of activated T-cells were CD4 + for SARS-CoV-2 peptides, and approximately 15% were CD8 + . Notably, these proportions were reversed following anti-CD3 and EBV peptide stimulation, with a vast majority of activated T-cells being CD8 + ( Figure 3E ). Further investigation is necessary to establish if the observed CD4 + T-cell skewing with SARS-CoV-2 peptides is a consequence of peptide-length or if it reflects the biological characteristics of the response to this virus. Importantly, the coexpression of LFA-1 with either CD40L, IFN-γ, TNF or GM-CSF in CD4 + and CD8 + T-cells were similar in pwMS on anti-CD20 treatment and in HC and was at least as high in patients on anti-CD20 treatment as in HC and pwMS on other treatments (Figures 3F-J; Figure S2 ). Of note, the highest T-cells responses were observed in 2 patients that had been hospitalized, in accordance with the reported association between immune response and symptoms (Sekine et al., 2020) . Our results thus point to CD4 + T-cells as IFN-γ producing cells upon SARS-CoV-2 peptide stimulation in this FluoroSpot setting. Importantly, the T-cell response of pwMS following SARS-CoV-2 is functional and similar to that of healthy controls, even after BCDT. 9 responses. The percentages of B and memory B lymphocytes displayed an inverse correlation with the strength of T-cell memory when checked in all Covid + patients. This possibly reflects the fact that patients on anti-CD20 treatment with low B-cells develop a robust cellular immunological memory to SARS-CoV-2 ( Figures 4A and 4B ). The percentage of T regulatory cells correlated with levels of S1S2 antibodies, perhaps indicating that both are affected by the severity of COVID-19 ( Figure 4A ) (Wang et al., 2020) . Circulating T follicular helper cells (Tfh) reflect the Tfh activity in lymph nodes that help antigen-specific B-cells to produce antibodies, which has been found to correlate with SARS-CoV-2 antibody levels (Crotty, 2019; Juno et al., 2020) . Accordingly, we here found a correlation between circulating Tfh cells and nucleocapsid C antibody levels, both in the whole Covid + cohort (Figures 4A and 4B) and when separately analyzing the Covid + anti-CD20 treated patient group (Figures 4C and 4D) . We therefore confirm the association between Tfh cells with antibody responses.

To better understand how RTX affects humoral and cellular immunological memory after COVID-19, we stratified anti-CD20 treated patients into those that at the time of COVID-19like symptom were; a) completely B-cell depleted (<0.01; B-cell count x 10 9 / L), b) partially B-cell repleted (0.01-0.08; B-cell count x 10 9 / L) and c) completely B-cell repleted (>0.08; Bcell count x 10 9 / L). Among the 26 Covid + patients, 7 out of 14 with complete B-cell depletion status, 5/7 with partial repletion and 3/3 with complete repletion status developed antibodies.

Two patients with complete depletion were seropositive with the multiplex bead array but not with ECLIA and displayed more than 5 but less than 11 IFN-γ + spots after stimulation with SARS-CoV-2 peptides (Figures 5A and 5B; Table S3 ). All but 2 patients with complete depletion displayed SARS-CoV-2 T-cell memory without any apparent effect of depletion status on the strength of T-cell reactivity measured by FluoroSpot or on SARS-CoV-2 T-cell memory functionality measured by LFA-1 and intracellular cytokine expression, respectively J o u r n a l P r e -p r o o f ( Figures 5C and 5D ). In addition, one patient with complete depletion and one patient with partial depletion not being tested with the multiplex bead array displayed T-cell memory (data not shown).

Recent epidemiological studies have indicated a reduced ability of the immune system of pwMS on anti-CD20 to produce antibodies (Sormani et al., 2021; Zabalza et al., 2020) . We therefore investigated what factors might affect the SARS-CoV-2 antibody production in pwMS on RTX treatment. The number of previous RTX infusions did not correlate with SARS-CoV-2 antibody levels or the strength of T-cell memory ( Figures 5E and 5F ). When separating seropositive and seronegative patients, they did not differ with regards to strength of T-cell reactivity. However, seronegative pwMS displayed less time from last RTX dose to COVID-19 symptoms (p = 0.009) and higher number of previous RTX doses (p = 0.048) (Figures 5G-J). We cannot exclude a partial effect of waning of antibodies in seronegative pwMS since we saw a correlation (p = 0.029) between time from last RTX dose to symptoms and time from symptoms to sampling ( Figure S3 ), and thus those effects were lost in multivariate analyses (data not shown). Of note, two patients that developed COVID-19 within two weeks of last RTX infusion remained seronegative, while displaying a robust memory T-cell response. While short time since RTX treatment potentially impairs B-cell responses, the lack of SARS-CoV-2 antibody response may also be the result of waning antibody titers, which were tested 205 and 234 days after COVID-19, respectively ( Figure S3 ) (Ibarrondo et al., 2020) . Interestingly, a patient that had received a RTX infusion more than two years ago while still being completely depleted on repeated testing also did not develop antibodies, but displayed T-cell reactivity to SARS-CoV-2.

These results demonstrate that absence of measurable B-cell levels in peripheral blood does not prevent development and strength of a T-cell immune or strength of humoral response against SARS-CoV-2.

Although the number of pwMS treated with DMTs other than RTX was low, we nevertheless investigated how these affected humoral and cellular SARS-CoV-2 immune responses in order to provide a preliminary indication and a basis to compare with RTX. Three pwMS on OCR (days since last infusion; 202, 214, 222), another anti-CD20 therapy, tested seropositive with either ECLIA and/or multiplex bead array, suggesting that OCR, as for RTX, does not prevent SARS-CoV-2 specific antibody production. One pwMS on interferon-beta, two pwMS on dimethyl fumarate, a treatment that increases oxidative burst and dampening of MS-associated adaptive immune responses (Carlstrom et al., 2019; Luckel et al., 2019) , two patients on fingolimod, a drug that blocks the egress of CCR7 + naïve and central memory T-cells from the lymph nodes (Pappu et al., 2007) , one pwMS on cladribine, a drug that depletes B-and T-cells, and one pwMS on natalizumab, a VLA-4 blocker that impedes the transmigration of lymphocytes into the brain (Piehl, 2020) , tested positive in ECLIA and/or multiplex bead array suggesting that these immunotherapies used in pwMS do not impede a humoral anti-viral immune response against SARS-CoV-2. The IFN-γ T-cell memory response after stimulation with the SARS-CoV-2 domain peptides is shown in Figure S4 . Although treatment-induced lymphopenia does not abrogate the development of T-cell immunity, 3 out of 5 seropositive pwMS treated with other DMTs displayed low or undetectable SARS-CoV-2 specific T-cell response in both assays. Further investigations are needed to corroborate these observations in larger cohorts.

To further examine the effect of BCDT on SARS-CoV-2 immunity, the humoral and cellular immune responses were determined in pwMS four to twelve weeks after the second dose of SARS-CoV-2 mRNA vaccine. The T-cell response was analyzed both before and four weeks J o u r n a l P r e -p r o o f after vaccination for one patient and was negative for both the N and S1 peptides at baseline but became positive only for S1 after vaccination ( Figure 6A ). After four weeks, 7 out of 10 (70%) patients had antibodies to the spike protein and 10 out of 10 (100%) had a positive Tcell response exclusively to the S1 peptides. After twelve weeks, one patient had seroconverted from positive to negative but all patients (3 out of 3) still had a specific T-cell response ( Figure   6B ). To be noted, for the seroconverted patient, the twelve-week sample was analyzed using a different antibody-detection method with a higher cutoff level for seropositivity. To examine which factors that may influence the development of humoral immunity in anti-CD20 treated pwMS, the time from last infusion to first vaccine dose for each MS patient was evaluated.

Interestingly, for the patients who had received an infusion within four months before their first dose, only two out of five (40%) had detectable SARS-CoV-2 specific antibodies four weeks after vaccination. The remaining patients with more than 4 months since last infusion, all five out of five (100%) had detectable antibodies four weeks after vaccination. However, regardless of time since last infusion or serology status, all pwMS developed a robust and specific T-cell response ( Figure 6C ). When analyzing the percentages of B-cells, all seronegative samples displayed less than 0.2% B-cells of live lymphocytes. Interestingly, among the seropositive pwMS, there were also two samples with less than 0.2% B-cells ( Figure 6D ), indicating that a virtual absence of B-cells in peripheral blood is not a good predictor of not producing detectable antibodies after vaccination. These data also support the observation that SARS-CoV-2 specific T-cells can arise and persist even in the absence of detectable SARS-CoV-2 antibodies in pwMS that are being treated with anti-CD20.

We here determined humoral and cellular immune responses to SARS-CoV-2 in a large population-based cohort of pwMS being treated with different DMTs. Since the start of the COVID-19 pandemic, concerns have been raised regarding a worsened COVID-19 disease course, increased risk of re-infection and dampened vaccine responses in patients treated with immunomodulators, in particular anti-CD20 therapies (Luna et al., 2020; Montalban et al., 2017) . Indeed, pwMS treated with anti-CD20 display increased susceptibility to severe infections compared to those treated with interferons, even if this increased risk mainly regards bacterial rather than viral infections (Luna et al., 2020) . Our study did not aim to provide data on a possible clinical impact of DMT on susceptibility to COVID-19 or its resulting severity.

However, in our population-based COMBAT-MS cohort of 620 patients, out of which at least 4.5% had had COVID-19 as verified with ECLIA, 2 patients (both on rituximab) required hospitalization, arguing against a major clinical impact in this cohort.

In order to extend these epidemiological observations to an immunological context, also prompted by the ongoing pandemic, we determined immunological responses to SARS-CoV-2 in a population-based cohort of patients with MS participating in the ongoing prospective COMBAT-MS study. Interestingly, we find that the immune system of all patients except two treated with RTX reacts to acute COVID-19 infection by producing a functional T-cell response irrespective of B-cell depletion status. The two patients with no T-cell response displayed antibodies, which may relate to the combination of HLA-type and the peptides used in this study rather than being biological. Thus, we did not observe any apparent effect of B-cell Our results further suggest that lymphopenia induced by other MS DMTs in general does not noticeably affect the quantitative or qualitative aspects of the resulting SARS-CoV-2 adaptive immune response, even though SARS-CoV-2-induced lymphopenia has been shown to correlate with severe COVID-19 (Weiskopf et al., 2020) . Indeed, two lymphopenic pwMS, one on dimethyl fumarate and one on cladribine developed both antibodies and T-cell response against SARS-CoV-2. It is currently unclear to what degree the cellular and humoral immune responses contribute together or separately towards long lasting protection against re-infection, which needs to be addressed in further studies (Jarjour et al., 2021) .

Notably, we here studied a low dose RTX (500 mg) protocol, which is lower than the approved dose for rheumatoid arthritis, and is based on the notion that memory B-cells rather than B-cells in general are implicated in MS immunopathogenesis, and which also translates in a long lasting effect after interrupting anti-CD20 therapy (Baker et al., 2017; Jelcic et al., 2018; Juto et al., 2020; Novi et al., 2020) . In addition, dosing intervals had been extended already before the start of the pandemic, explaining why a proportion of patients had dosing intervals in the range of 9-24 months or more. However, the observations that cellular immunological responses were not affected by depletion status or time since last dose and that pwMS on the more potent Bcell depleting OCR dosing regimen also displayed an adaptive response suggest that the results J o u r n a l P r e -p r o o f of the current study can be extrapolated to other doses of RTX and other anti-CD20 therapies (Evertsson et al., 2020) . A further finding of our study is that the proportion of pwMS that tested positive for SARS-CoV-2 in the routine antibody test (ECLIA; 4.5%) is lower than the 11.6% seropositive prevalence reported in healthy blood donors by the Public Health Agency of Sweden for the Stockholm population already in June 2020 2020). Furthermore, the post-vaccination data are in accordance with previous studies reporting an attenuated antibody response to SARS-CoV-2 mRNA and also other types of vaccines for patients on anti-CD20 treatment (Achiron et al., 2021; Baker et al., 2020; Bar-Or et al., 2020; Bigaut et al., 2021; Gallo et al., 2021; Guerrieri et al, 2021) and also demonstrate that measurement of B-cells in blood is a poor predictor of capacity to develop a humoral response.

In combination with recent literature that demonstrates a contribution of T-cell immunity in COVID-19 (McMahan et al., 2021; Soresina et al., 2020) , even in the absence of detectable antibodies, they help explain why most of the patients on anti-CD20 treatment recover, and suggest a potential effect of vaccination even in the absence of B-cells.

A limitation of this study is that most individuals were not confirmed with PCR testing for SARS-CoV-2 during the acute phase due to the lack of testing capacity early in the pandemic.

Thus, since patients on anti-CD20 treatment are generally more prone to respiratory tract infections (Luna et al., 2020) , it is likely that early in the pandemic and while PCR verification 

Patients that were SARS-CoV-2 positive in either serological and/or T-cell assays are included.

(A) Correlation of SARS-CoV-2 antibody levels for S1S2 or N with ΔIFN-γ SFUs for S1 (n = 22) or N (n = 22) peptides respectively.

(B-C) Anti-SARS-CoV-2 antibody levels for S1S2 and N and number of ΔIFN-γ SFUs for S1

or N peptides in patients with B-cell repletion, partial repletion, depletion. No significant differences were seen.

(D) Percentages of LFA-1 + IFN-γ + in patients with B-cell repletion (n = 1), partial repletion (n = 4), depletion (n = 5).

(E) Correlation of antibody levels for S1S2 and N with number of RTX infusions.

(F) Correlation of ΔIFN-γ SFUs for S1 and N peptides with number of RTX infusions.

(G-J) Comparison of seropositive (n = 14) and seronegative (n = 8) patients regarding number of ΔIFN-γ SFUs for S1 (p = 0.803) or N (p = 0.815) or days from symptom onset to sampling (p = 0.140) or days from last RTX dose to symptom onset (p = 0.009) or number of RTX infusions (p =0.048).

(A-C, E-J) B-cell depletion status; n = 3 repletion, n = 6 partial repletion, n = 13 depletion.

Dots represent individual data points. Box plots represent median and 95% CI. See also Table   S3 and Figure S3 . negative control (medium only) and SARS-CoV-2 specific peptides (N and S1) in one pwMS on anti-CD20 before and four weeks after SARS-CoV-2 vaccination.

(B) Serology status and number of ΔIFN-γ SFUs after stimulation with N or S1 peptides four (n = 10) and twelve (n = 4) weeks after SARS-CoV-2 vaccination in pwMS on anti-CD20.

(C) Serology status and number of ΔIFN-γ SFUs after stimulation with S1 peptides four weeks after vaccination (n = 10) and days from last anti-CD20 infusion to first vaccine dose. J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Faiez Al Nimer (faiez.al.nimer@ki.se).

This study did not generate new unique reagents. 

The study design is shown in Figure 1 SARS-CoV-2 PCR test, b) SARS-CoV-2 antibody test. Furthermore, a blood test was sent for routine SARS-CoV-2 antibody detection (see below).

In a subcohort (n = 122) of patients enriched for positive symptoms according to the questionnaire and ongoing anti-CD20 therapy, we collected blood for serological (plasma) and cellular analyses. Peripheral blood mononuclear cells (PBMCs) were freshly isolated from sodium citrate-containing cell preparation tubes (BD Biosciences). All isolated PBMCs were cryopreserved in freezing media containing 10% dimethyl sulfoxide (DMSO; Sigma-Aldrich) and stored at -180°C. PBMCs from 8 samples from MS patients before the start of COVID-19 pandemic were also included in the analysis. Lastly, for the vaccination cohort, PBMCs from MS patients on anti-CD20 were collected before (n = 1), four (n = 10) and/or twelve (n = 4) weeks after SARS-CoV-2 mRNA vaccination. Seroconversion against the spike protein was tested at the hospital at the same date as the PBMCs sampling date and the results were later obtained from the patient's journal. See Table S4 for cohort characteristics. 

Since there is a considerable discussion about the sensitivity and specificity of the different assays in detecting anti-SARS-CoV-2 antibodies, we also included an additional method in the cohort of patients with more advanced immunological analyses. The protocol for detection of SARS-CoV-2 IgG using a multiplex bead array has been recently described (Rudberg et al., 2020) . In brief, the assay measured IgG reactivity towards two different virus protein variants, the spike glycoprotein ectodomain produced in HEK cells (Spike S1S2 foldon), and the nucleocapsid protein C-terminal domain produced in Escherichia coli (Nucleocapsid C), using a multiplex antigen-bead array in a high throughput 384-well plates format using a FlexMap3D

(Luminex Corp). Each antigen was coated on the surface of uniquely color-coded magnetic beads (bead ID) (Luminex Corp), and the antigen-reacting plasma IgG was captured and detected by fluorescent goat anti-hIgG (Invitrogen). Median fluorescent intensity (MFI) and

bead-count were determined for each antigen (bead ID). As previously described, a cutoff of reactivity was defined for each antigen as the mean MFI + 6SD of 12 negative controls included in each analysis. Samples were regarded as positive when reactive to both viral antigens.

Patient baseline characteristics were extracted from the Swedish MS-registry Figure S5D ).

PBMCs were thawed as previously described and rested overnight at 37 °C with 5% CO2. For each stimulation and control, 1 x 10 6 cells were seeded in a total volume of 200 µL as single samples in a U-bottom 96-well plate. Anti-CD3 was used as positive control and prepared as previously described. As an additional control, EBV Consensus and EBNA-1 peptides were pooled together (0.5 µg/mL each, Miltenyi Biotec). Wells containing only medium were used as negative control. For the SARS-CoV-2 peptide stimulation, the peptides were divided into two pools, one containing all four proteins; Prot_N, Prot_M, Prot_S and Prot_S1 and one containing only Prot_N and Prot_S1 (each at 1 µg/mL). Monensin (0.6 µL) and brefeldin A

(1.0 µL) (BD Bioscience) were added to each well before seeding the cells. The plate was incubated for 4 h in a 37 °C humidified incubator with 5% CO2. LFA-1 staining was performed as previously described (Dimitrov et al., 2018; Schollhorn et al., 2021) , by adding LFA-1 m24 J o u r n a l P r e -p r o o f Ab to the culture and cells incubated for 15 min at room temperature. Then EDTA was added for an additional 10 min RT. Cells were washed, fixed and permeabilized as described above and thus antibody mix was added (Key Resources Table; Figure S5A ). Measurements were performed on an Aurora spectral cytometer (Cytek), and data were analyzed with FlowJo v.10 (Tree Star).

Statistical Spike protein MFI (Spike S1S2 foldon) Spike S1 domain (S1) ΔIFN-γ SFUs/2. Spike S1 domain (S1) N S1 Medium Anti-CD3 SARS-CoV-2 peptides 

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