key: cord-0791131-7dy82ax3 authors: de Campos-Mata, L.; Tejedor Vaquero, S.; Tacho-Pinot, R.; Pinero, J.; Grasset, E. K.; Arrieta Aldea, I.; Rodrigo Melero, N.; Carolis, C.; Horcajada, J. P.; Cerutti, A.; Villar-Garcia, J.; Magri, G. title: SARS-CoV-2 sculpts the immune system to induce sustained virus-specific naïve-like and memory B cell responses date: 2021-04-30 journal: nan DOI: 10.1101/2021.04.29.21256002 sha: d63aafd86eb5db869ded224363a8f6a584024e8a doc_id: 791131 cord_uid: 7dy82ax3 SARS-CoV-2 infection induces virus-reactive memory B cells expressing unmutated antibodies, which hints at their emergence from naive B cells. Yet, the dynamics of virus-specific naive B cells and their impact on immunity and immunopathology remain unclear. Here, we longitudinally studied moderate to severe COVID-19 patients to dissect SARS-CoV-2-specific B cell responses overtime. We found a broad virus-specific antibody response during acute infection, which evolved into an IgG1-dominated response during convalescence. Acute infection was associated with increased mature B cell progenitors in the circulation and the unexpected expansion of virus-targeting naive-like B cells that further augmented during convalescence together with virus-specific memory B cells. In addition to a transitory increase in tissue-homing CXCR3+ plasmablasts and extrafollicular memory B cells, most COVID-19 patients showed persistent activation of CD4+ and CD8+ T cells along with transient or long-lasting changes of key innate immune cells. Remarkably, virus-specific antibodies and the frequency of naive B cells were among the major variables defining distinct immune signatures associated with disease severity and inflammation. Aside from providing new insights into the complexity of the immune response to SARS-CoV-2, our findings indicate that the de novo recruitment of mature B cell precursors into the periphery may be central to the induction of antiviral immunity. To date, the rapidly spreading severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected around 142 million people, resulting in more than three million deaths worldwide 1 . Infection with SARS-CoV-2 causes the coronavirus disease 2019 , which is characterized by a wide variety of clinical manifestations that range from asymptomatic to acute respiratory distress syndrome (ARDS), multi organ failure and death 2 . Although such diversity in disease pathogenesis is partially explained by the patient's comorbidities as well as genetic and socio-demographic factors, severe manifestations of the disease are strongly associated with dysregulated immune responses 3, 4 . Immune dysregulation in severe COVID-19 patients is characterized by delayed and impaired type I interferon responses that associate with failure to control primary infection 5, 6 . The resulting aberrant activation of innate immune cells leads to an exacerbated release of pro-inflammatory cytokines, causing systemic inflammation and tissue damage 7 . Interestingly, interferon signaling and hyper inflammation may associate with autoimmunity. Indeed, severe COVID-19 patients develop autoantibodies against immunomodulatory proteins, including antibodies against type I interferon [8] [9] [10] . Besides playing a role in immunopathogenesis, the host immune response is a major determinant of recovery and immune protection through the development of durable SARS-CoV-2-specific T and B cell responses. Several studies have documented the early activation of CD4 and CD8 T cells following SARS-CoV-2 infection as well as the generation of durable virus-specific T cell responses required for immune protection [11] [12] [13] [14] . In the early response to SARS-CoV-2, infected individuals generate antibodies against the viral nucleocapsid (NP) and spike (S) proteins. Around 90% of COVID-19 patients produce detectable neutralizing antibody responses against the receptor binding domain (RBD) of the viral S protein, which persist for up to 8 months [15] [16] [17] . Early humoral responses are driven by the transient expansion of antibody-secreting plasmablasts. During convalescence, humoral memory is sustained by somatically-mutated memory switched B cells and long-lived plasma cells 14, 16, 18, 19 . Of note, recent studies identified convergent antibody responses to SARS-CoV-2 by B cells with preferential immunoglobulin heavy chain variable-joining (IGHV-J) gene usage and minimal somatic hypermutation 18, [20] [21] [22] [23] [24] [25] . These findings suggest that humoral protection involves SARS-CoV-2 recognition by naïve B cells with little or no antigen-driven affinity maturation required 21, 26 . In spite of our growing understanding of SARS-CoV-2 infection, both kinetics and composition of virus-specific B cell responses remain poorly understood. In particular, the dynamics of virus-reactive naïve B cells and their role in immune protection and immunopathology are unclear. In addition, the temporal trajectories of innate and adaptive immune responses to SARS-CoV-2 and their functional relationship remain elusive. A better understanding of these facets of SARS-CoV-2 infection may help in the evaluation of the protective effects afforded by individual vaccination programs. Here, we longitudinally profiled global and virus-specific B cell responses from a cohort of moderate to severe COVID-19 patients at different stages of SARS-CoV-2 infection. We also explored the relationship of B cell responses to SARS-CoV-2 with the activation of effector and regulatory cells from the innate or adaptive immune system. We identified specific properties of immune response dynamics from COVID-19 patients as well as unique immune trajectories that associate with disease severity and inflammation. We also found some evidence of a novel mechanism adopted by the host immune system to fight SARS-CoV-2. This mechanism may involve the continuous peripheral recruitment of early mature B cell precursors to enhance viral recognition by the naïve B cell repertoire. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 30, 2021. ; https://doi.org/10.1101/2021.04.29.21256002 doi: medRxiv preprint To dissect the dynamics of humoral immune responses to SARS-CoV-2, we collected blood and serum samples from a cohort of 25 hospitalized COVID-19 patients in the acute phase of infection (COVT1). Of these patients, 24% (n = 6; darker symbols in Figures) were admitted to intensive care unit (ICU). On average, COVT1 samples were collected 11 days post-symptom onset (PSO). A follow-up blood sample was drawn from 20 of 25 patients (COVT2), including 4 ICU patients, averaging 70 days PSO (Fig. 1A) . Blood and serum samples were also collected from healthy controls (n = 21). Aside from being a major risk factor for fatal outcome in COVID-19 patients, age profoundly influences the immune system of healthy individuals. To minimize these age-related effects, we mostly selected patients younger than 65 (median age = 51) and included age-matched healthy controls (median age = 50). A summary of demographic and clinical data of all the individuals included in this study is provided (Table S1) . To elucidate whether COVID-19 patients can mount a broad and long-lasting humoral response to SARS-CoV-2, we performed isotype-specific (IgM, IgA and IgG) and subtype-specific (IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4) enzyme-linked immunosorbent assays (ELISAs) that quantified antibody responses to the receptor-binding domain (RBD) of the spike protein or nucleocapsid protein (NP) from SARS-CoV-2. Compared to healthy controls, sera from COVID-19 patients in acute infection (COVT1) and convalescence (COVT2) showed significantly higher titers of all RBD-and NP-specific antibody classes and subclasses analyzed (Fig. S1 ). According to our longitudinal study and as previously reported 18, 27 , RBD-and NP-specific IgM and IgA titers significantly decreased two months PSO (Fig. 1B, C) , presumably because they mainly originated from short-lived plasmablasts (PBs). Interestingly, both IgA1 and IgA2 subclasses contributed to the decline of virus-specific IgA, suggesting a limited involvement of mucosal immunity during convalescence. Unlike RBD-and NP-specific IgM and IgA, virus-specific IgG titers were maintained over the first two months PSO (Fig. 1B, C) . Remarkably, RBD-specific IgG1 was the only IgG subclass that significantly increased overtime (Fig. 1B) . We further studied the dynamics of the humoral response in COVID-19 throughout the study's time course and found that the magnitude of relative changes in virus-specific IgM or IgG titers directly correlated with their initial levels at COVT1. Indeed, patients with initial higher IgM or lower IgG titers showed greater relative decrease or increase, respectively (Fig. 1D) , similarly to what has been previously reported for convalescent individuals at later time points 18 . Thus, SARS-CoV-2 infection triggers a broad antibody response in terms of antigen-specificity and antibody isotypes in early stages and an expansion of an RBD-specific and IgG1-dominated response during convalescence. To further explore the dynamics of the B cell response to SARS-CoV-2, we analyzed circulating B cells from healthy controls and COVID-19 patients in the acute and convalescent phase of infection by high-dimensional spectral flow cytometry ( Table S2) . As reported by others 28 , the frequency of CD19 + B cells within live peripheral blood mononuclear cells (PBMCs) was significantly higher in COVT1 compared to healthy controls or COVT2 ( Fig. 2A) . T-distributed stochastic neighbour embedding (tSNE) defined the major B cell populations (Fig. 2B) , the contribution of each group of samples to these populations (Fig. 2C) , and the differential expression of multiple surface B cell proteins within these populations (Fig. 2D) . In parallel, we queried the data by traditional gating (Fig. S2A, C) . This analysis revealed that, for most COVID-19 patients, the acute response to SARS-CoV-2 was dominated by a transitory expansion of CD38 ++ CD10 − CD27 high plasma cells (PCs; Fig. 2B-F) , the majority of which expressed HLA-DR + (Fig. 2G) , an antigen-presenting protein typically expressed by newly generated short-lived PBs 29 . Next, the analysis of antibody classes and subclasses confirmed the induction of unswitched as well as IgA1 or IgG class-switched PCs soon after infection ( Fig. 2H and Fig. S2B ). We then assessed the homing potential of these PCs through the analysis of CXCR3 and CXCR4 chemokine receptors, which guide PCs to inflamed tissues or bone marrow, respectively 30 . We found that the majority of PCs from COVT1 had a CXCR3 + CXCR4phenotype, suggesting their targeted migration into inflamed tissues (Fig. 2I, J) . Compared to healthy controls and COVT2, COVT1 samples also showed a significantly increased proportion of CXCR3 + CXCR4 + PCs (Fig. 2I, K) . The tSNE projections also highlighted prominent differences in the transitional and naïve B cell compartments among healthy controls, COVT1 and COVT2 (Fig. 2B-D) . Using conventional gating strategies (Fig. S2C) and pairwise longitudinal comparisons, we identified an increased proportion of immature CD21 low transitional B cells (Fig. 2L ) as well as a highly significant transient expansion of naïve B cells during the acute phase of infection (Fig. 2M, N) . Furthermore, our data revealed a positive correlation between the frequency of naïve B cells and the frequency of CD19 + B cells (Fig. S2D) , which implies that naïve B cell expansion is responsible for the increased frequency of CD19 + B cells. These changes likely reflect an increased inflammation-dependent mobilization All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 30, 2021. ; https://doi.org/10.1101/2021.04.29.21256002 doi: medRxiv preprint of developing B cells from the bone marrow into the periphery, a phenomenon also observed in other infections 31 . Compared to similar cells from healthy controls, naïve B cells from COVT1 exhibited significantly lower expression of CD21, HLA-DR, IgD and the chemokine receptors CCR6, CXCR5 and CXCR3 but not CCR7, presumably due to their activation by signals from the inflammatory environment ( Fig. S2E-K) . Thus, aside from promoting a transient induction of CXCR3 + unswitched and class-switched PBs, SARS-CoV-2 may induce the mobilization of precursors of mature B cells together with the transient expansion and activation of naïve B cells. To gain insight into the dynamics of B cell memory (ME) responses to SARS-CoV-2, we analyzed the frequency of different antigen-experienced B cell populations in healthy controls and COVID-19 patients during acute infection and convalescence. First, we found that the frequency of ME B cells within total CD19 + B cells was significantly lower in acute COVT1 compared to COVT2 and healthy controls ( Fig. S3A and Fig. 3A) , presumably due to the transient expansion of newly formed PBs and naïve B cells. Unlike PCs but similar to naïve B cells, the frequency of CXCR3 + ME B cells diminished during the acute phase of infection (Fig. 3B) , suggesting a possible impairment in their recruitment into inflamed tissues and lymphoid follicles 32 . Within the ME B cell compartment, we observed a significant and persistent reduction in the proportion of IgM + IgD + CD27 + ME unswitched B cells in COVID-19 patients ( Fig. S3A and Fig. 3C) and a progressive expansion of IgG1-expressing ME B cells (Fig. 3D) within the memory compartment. Interestingly, the frequency of unswitched ME B cells positively correlated with the serum concentration of inflammatory marker ferritin (Fig. 3E ) as well as with NPspecific IgM titers (Fig. 3F) . Moreover, COVID-19 patients with higher plasma concentration of ferritin and other markers of inflammation (data not shown) also showed a lower frequency of IgG1 ME B cells during acute infection (Fig. 3G) . Remarkably, ME B cells expressing other IgG subclasses did not increase after infection (Fig. S3B) . These results may reflect the consolidation of efficient anti-viral IgG1-mediated immunity in less severe patients. In agreement with findings published previously 29 , we categorized IgD -CD27double negative (DN) ME B cells as early activated CD27 -CD21 + CD11c -ME or DN1, extrafollicular CD27 -CD21 -CD11c + PB precursors or DN2, and DN3 CD27 -CD21 -CD11c -B cells (Fig. S3C) . The analysis of these ME B cell subsets revealed a significant and transient increase in the frequency of CD27 -IgD -DN cells in COVT1 compared to COVT2 and healthy controls (Fig. 3H) , as reported by others 28 . Among DN B cells, DN2 cells are All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 30, 2021. ; https://doi.org/10.1101/2021.04.29.21256002 doi: medRxiv preprint recognized as primed precursors of antibody-secreting cells that differentiate from newly activated naïve B cells through an extra-follicular pathway. In contrast, DN3 has unknown origin and function 28 . First, we quantified the frequency of DN2 cells within different ME B cells subsets distinguished on the basis of their expressed isotype or subclass. We found a significantly increased proportion of DN2 in ME B cells expressing IgG3, IgG1 and, to lesser extent, IgA1 (Fig. S3D) . The frequency of extrafollicular ME DN2 B cells and DN3 cells were higher in COVT1 compared to COVT2 and healthy controls (Fig. 3I, J) . Interestingly, the most significant increase in DN2 was observed within the IgG1 + ME B cell subset (Fig. 3K) . Thus, SARS-CoV-2 infection induces the transient expansion of extrafollicular IgG1 + ME B cells, which is followed by a long-lasting IgG1 + ME B cell response. Next, a fluorescently labeled recombinant RBD probe was used to identify SARS-CoV-2-specific B cells capable of producing potentially neutralizing antibodies (Fig. S4A) . We confirmed the specificity of our assay by comparing our results to those obtained using a double discrimination strategy via the inclusion of two fluorescently labeled RBD probes (Fig. S4B) . Antigen-binding CD19 + B cells were further characterized according to the expression of CD27, CD21, CD11c, HLADR, IgM, IgD, IgA and the Ig light chain  ( Fig. S4C and Table S3 ). In agreement with published studies 14, 17, 33 , SARS-CoV-2 infection induced a rapid increase in the frequency of RBD-specific CD19 + B cells at T1 that persisted 2 months after viral exposure (Fig. 4A) . Interestingly, the phenotypic characterization of these antigen-binding B cells revealed significant changes in virusspecific B cell populations between the acute and convalescent phase of infection ( Fig. 4B and Fig. S4D ). Consistent with the observed expansion of PCs, COVT1 showed an increased frequency of RBD-specific CD27 high CD21 -PCs compared to healthy controls and COVT2 ( Fig. 4C and data not shown). Remarkably, virtually 100% of these RBDspecific cells were newly generated HLADR + PBs (Fig. 4D ). In addition, RBD-targeting IgM + IgD + CD27naïve B cells increased progressively in COVID-19 patients (Fig. 4E) . These cells were enriched in CD11c + early activated B cells in COVT1 and even more in COVT2 (Fig. 4F-G) . Of note, the frequency of RBD-specific naïve B cells during convalescence strongly correlated with the frequency of total naïve B cells in the acute phase of infection (Fig. 4H) , suggesting that the early mobilization of developing B cells to the periphery and the expansion of naïve B cells could contribute to humoral defense by increasing germline RBD-specific B cells. To elucidate the All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 30, 2021. ; https://doi.org/10.1101/2021.04.29.21256002 doi: medRxiv preprint mechanisms leading to the sustained enrichment of RBD-targeting naïve B cells, we measured the plasma concentration of IL-7, a cytokine involved in B and T lymphopoiesis 34 . Compared to healthy controls, COVID-19 patients showed more IL-7 during acute infection (Fig. S4E ) and its concentration correlated with the frequency of RBD-specific naïve B cells in COVT2 (Fig. S4F) , supporting a direct role of this cytokine in enhancing the expansion and recruitment of relatively immature transitional and naïve B cells with useful specificities. Additionally, RBD-specific naïve B cells expressed more Ig compared to total naïve B cells (Fig. 4I) . Given that Ig light chain has a more varied structural repertoire than Ig 35 , Ig enrichment may provide the host with the advantage of developing a broader humoral response to SARS-CoV-2. We then analyzed whether RBD-specific ME B cells were induced and maintained throughout the study's time course. The number of RBD + ME B cells in COVT1 was significantly greater than in healthy controls and increased even further in COVT2 (Fig. 4J ). Similarly, there was a sustained increase in the number of RBD-specific IgG (IgM -IgD -IgA -) class-switched ME and, to a lesser extent, in RBD-specific IgM + IgD + CD27 + unswitched ME B cells (Fig. 4K-L) . The RBD-specific ME compartment included only a few IgA + and unswitched IgM + IgD -ME cells and these subsets did not increase overtime ( Fig. S4G) . Consistent with our characterization of B cell subsets, RBD-specific extrafollicular ME B cells, or DN2, expanded during the acute phase of infection but their frequency dramatically decreased in convalescence (Fig. 4M) . To assess the persistency of virus-specific ME B cell responses, RBD-targeting B cell subsets were analyzed by flow cytometry in a small cohort of non-hospitalized convalescent individuals three months (COVT3) and six months (COVT6) PSO (Fig. S5 ). In agreement with previous reports 14, 17, 18 , RBD-specific B cells persisted up to 6 months after infection (Fig. S5A ) and mainly consisted of IgG + ME B cells (Fig. S5B) . Accordingly, RBD-specific IgG and, to a lesser extent, IgA were significantly higher in sera from COVT3 and COVT6 compared to healthy controls (Fig. S5C) . Thus, SARS-CoV-2 infection promotes a rapid and transient induction of RBD-specific PBs and extrafollicular ME B cells as well as a long-lasting expansion of RBD-targeting activated naïve B cells and IgG class-switched ME B cells. To further characterize the dynamics of immune responses over time in COVID-19 patients, we simultaneously analyzed by multicolor spectral flow cytometry the phenotypic landscape of other circulating lymphoid and myeloid populations using an inhouse developed 23-color antibody panel ( Table S4 ). The projection of the data for All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 30, 2021. ; https://doi.org/10.1101/2021.04.29.21256002 doi: medRxiv preprint CD19 -CD3 + cells in tSNE space allowed the definition of the main T cell subsets, the contribution of each group of samples to T cell populations, and the expression patterns of relevant T cell surface proteins ( Fig. 5A-C) . In parallel, we queried the data by traditional gating (Fig. S6) . As reported previously 12, 36 , the acute phase of infection was characterized by a striking reduction in the frequency of CD3 + T cells (Fig. 5D) , which was driven by a decrease in the frequency of CD4 + and, to an even larger extent, CD8 + T cells (Fig. S7A, B) . Compared to healthy controls, COVT2 convalescent individuals still had a significantly decreased frequency of CD3 + cells (Fig. 5D ) and in particular of CD4 + T helper cells (Fig. S7A) , which suggested their persistent loss from the periphery. We then evaluated how SARS-CoV-2 infection associated with temporary or long-lasting perturbations in both CD4 and CD8 T cell compartments. Consistent with findings published earlier 12 , the frequency of naïve T cells within total CD4 + T cells in acutely infected patients was significantly lower compared to age-matched healthy controls and further declined during convalescence (Fig. 5E ). This reduction was associated with a prominent expansion of activated CD38 + HLA-DR + CD4 + T cells during the acute and, to a lesser extent, convalescent phase of infection (Fig. 5F) , as observed in other viral infections 37, 38 . We then analyzed the frequency of circulating T follicular helper (cTFH) cells. Tfh cells are a specialized subset of CD4 + T cells that provide cognate help to antigen-specific B cells in the germinal center (GC) to initiate and maintain humoral immune responses 39 . These cells include a circulating counterpart, cTfh cells, which co-express PD-1 and CXCR5 as their GC-based equivalents do (Fig. S6) . In general, cTfh cells can be used as a surrogate to evaluate the Tfh cell activity in lymphoid tissues 40 . Of note, activated CD38 + ICOS + cTfh cells likely reflect a recent exit from the GC immediately after antigen encounter 41 . The frequency of cTfh cells was significantly higher in COVT2 patients compared to healthy controls and COVT1 patients, which is consistent with a model of antigen persistency and long-lasting GC reaction previously suggested by others 22 (Fig. 5G ). Interestingly, recently activated CD38 + ICOS + cTfh cells were more profoundly increased during the acute phase, which probably reflects the peak of GC responses in the early phase of the infection (Fig. 5H) . (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 30, 2021. in COVT2 compared to COVT1 upon pairwise comparison, whereas Th2 cells showed a decreased frequency over time (Fig. S7C) . Given the role of CD8 + T cells in viral infection and consistent with previous reports 12,36 , COVID-19 was associated with several changes in the CD8 + T cell compartment. We observed a significantly reduced frequency of naïve CD8 + T cells during convalescence as well as an expansion of CM and EM3 CD8 + T cells (Fig. 5I-K) . On the other hand, our analysis showed no differences in the frequency of EM1, EM2 and EMRA CD8 + T cells between groups (Fig. S7D) . We also found that activated CD38 + HLA-DR + CD8 + T cells strongly expanded during the acute phase of infection and still persisted at a higher frequency compared to healthy controls during convalescence (Fig. 5L) . As expected, patients with a higher increase in activated CD4 + T cells showed a higher frequency of activated CD8 + T cells as well (Fig. 5M) . Thus, SARS-CoV-2 causes a marked T cell loss during the acute phase of infection, which is associated with long-lasting and coordinated activation of CD4 + and CD8 + T cells as well as expansion and activation of professional B cell-helping T cells. Acute SARS-CoV-2 infection triggers alterations in circulating innate immune cell subsets, some of which have been associated with COVID-19 severity 42 . Nevertheless, how these perturbations persist during convalescence after viral clearance is unclear. We combined global high-dimensional mapping via tSNE of CD19 -CD3cells with traditional gating analysis to study significant alterations in the phenotype and frequency of peripheral blood innate immune cell subsets in COVT1 and COVT2 and healthy controls ( Fig. 6A-C and Fig. S8A ). Although the frequency of total circulating CD11c + HLA-DR + myeloid cells among live PBMCs in COVT1 and COVT2 was comparable to that of healthy controls (Fig. S8B) , we observed significant differences in the phenotype as well as subset composition of these myeloid cells. We initially explored the expression of the activation-induced molecule CD38 and HLA-DR on circulating myeloid cells. We found a striking induction of CD38 and a reduction in the expression of HLA-DR during acute infection ( Fig. 6D-E) . Of note, the decrease in HLA-DR may interfere with proper antigen presentation and has been directly linked to an immunosuppressive phenotype of monocytes during sepsis 5 . Indeed, HLA-DR levels strongly correlated with disease severity defined according to the patient's oxygen requirement (Fig. S8C) . However, these changes were only transient, as CD38 and HLA-DR returned to their homeostatic expression levels in the convalescent phase ( Fig. 6D- All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. We then analysed the frequency of CD14 + CD16conventional monocytes, CD14 + CD16 + intermediate monocytes and CD14 dim CD16 + non-conventional monocytes ( Fig. 6A-C and Fig. S8A ). Classical CD14 + CD16monocytes remained unaltered, whereas intermediate CD14 + CD16 + monocytes showed a higher frequency in COVT2 compared to COVT1 and healthy controls ( Fig. S8D-E) . Furthermore, we observed a robust but transient decrease in the frequency of non-conventional CD14 dim CD16 + monocytes (Fig. 6B , C and F), which appeared to be strongly activated during acute infection (Fig. 6G) . Non-conventional monocytes have a patrolling function and contribute to the antiviral response 43 , which may point to their selective recruitment to the inflamed lung upon FcRIIIA-mediated activation by virus-IgG immune complexes. Accordingly, nonconventional monocytes have been shown to be enriched in the lungs of critical COVID-19 patients 44 . The frequency of circulating myeloid dendritic cells (mDCs) and basophils were also strongly reduced in COVT1 compared to healthy controls, but both returned to homeostatic levels two months PSO ( Fig. 6H-I) . Interestingly, we detected increased CD25 expression on basophils from COVT1 compared to COVT2, which suggests basophil activation during the acute phase of infection (Fig. S8F) . Finally, we analyzed the frequency of circulating plasmacytoid DCs (pDCs) and natural killer (NK) cells, two subsets of the innate immune system that play crucial protective roles in viral infections. As reported previously 45 , the frequency of pDCs significantly decreased during acute infection (Fig. 6J) . Remarkably, convalescent individuals maintained significantly lower frequency of pDCs compared to healthy controls, even after viral clearance (Fig. 6J) , suggesting persistent impaired type I IFN responses in infected individuals. Although our data did not reveal any significant changes in the frequencies of natural killer (NK) cells within live cells, the analysis of CD38 expression indicated an activated phenotype during the acute phase of the infection (Fig. 6K, L) . Moreover, the characterization of NK cell subsets based on the relative expression of CD56 and CD16 showed a persistent decrease in the frequency of circulating immature CD56 bright CD16cells and an increase in the proportion of naturally cytotoxic CD56 dim CD16 high cells within total NK cells ( Fig. 6M and Fig. S8G) . Thus, SARS-CoV-2 infection drives profound changes in the circulating innate immune compartment, some of which persist after viral clearance during convalescence. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 30, 2021. ; https://doi.org/10.1101/2021.04.29.21256002 doi: medRxiv preprint The immune system is composed of layered defense mechanisms of increasing specificity that protect the host from infection. To investigate the putative associations between innate and adaptive immune trajectories developed upon SARS-CoV-2 infection, we performed pairwise correlations across 41 variables that were significantly different among the groups analyzed, including virus-specific antibody titers measured by ELISA and selected immune parameters identified by high-dimensional flow cytometry (Data File S1). We found that the global antibody response to SARS-CoV-2 positively correlated with the frequency of circulating PCs, extrafollicular DN2 ME B cells, activated CD4 + T cells, cTfh cells and CD8 + T cells. It also positively correlated with CD38 expression on circulating myeloid cells, which suggests a close coordination between innate and adaptive immune responses. On the other hand, the level of virus-specific antibody titers, the percentage of circulating PCs and the frequency of CD19 + cells inversely correlated with the frequency of CD3 + T cells, non-conventional monocytes and pDCs, which suggests virus-induced interference with different components of both adaptive and innate immune responses in a fraction of COVID-19 patients (Fig. 7A) . Next, principal component analysis (PCA) was performed to examine the general distribution of healthy controls and infected individuals during acute and convalescent stage according to these immune parameters. The analysis revealed that patients during acute infection were notably more distant from healthy controls than same patients during convalescence in PCA space (Fig. 7B) . Virus-specific IgA titers, as well as the frequency of IgG and IgM PCs, activated CD4 T cells and pDCs, were among the variables that mostly contributed to the observed clustering in PC1 space. The frequency of naïve B cells and, to a lesser extent, IgM + IgD + ME B cells, IgA1 PCs and NP-specific IgM titers were the immune features that mostly contributed to the clustering in PC2 space (Fig. 7C, D) . Remarkably, when we computed the Euclidean distance from each COVID-19 patient at T1 to the centroid of healthy controls, we observed a positive correlation between Euclidean distance and disease severity defined according to the patient's oxygen requirement and serum concentration of ferritin and lactate dehydrogenase (Fig. 7E) . Of note, the same analysis performed on samples from COVT2 did not show equivalent correlation patterns (data not shown), which suggests that immunological recovery trajectories occur independently of disease severity status. Thus, cross-dataset correlation and principal component analysis reveals coordinated immune responses in COVID-19 patients as well as immune parameters associated with disease severity and inflammation. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Another possible explanation for the reduction in virus-specific IgA1 and IgA2 two months PSO is the relocation of IgA-secreting PCs to mucosal inflamed tissues, including the upper respiratory tract. Indeed, acutely infected patients showed increased proportion of CXCR3 + CXCR4 -PCs that are programmed to migrate to inflamed tissues rather than All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 30, 2021. ; https://doi.org/10.1101/2021.04.29.21256002 doi: medRxiv preprint bone marrow niches. Consistently, two earlier reports described elevated SARS-CoV-2specific IgA antibodies in nasal fluids, tears and saliva of infected individuals 27, 48 . However, a recent study failed to detect PBs in the lung of deceased COVID-19 patients 49 . Overall, our data are in agreement with recent reports showing detectable S-specific IgG and neutralizing antibodies at 6-8 months PSO and rapid decline of circulating IgA titers 14, 15, 17, 18, 33 The increased frequency in CD21 low transitional and naïve B cells in COVT1 patients compared to age-matched healthy controls and COVT2 patients was consistent with the All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 30, 2021. ; https://doi.org/10.1101/2021.04.29.21256002 doi: medRxiv preprint mobilization of B cell precursors to the periphery following virus-induced inflammation 51 . In the presence of massive immune sensing of viral particles, the immune system may be evolutionary programmed to recruit mature B cell precursors from the bone marrow into the periphery as a "last-ditch" defense against invading virions. Multiple inflammation-induced cytokines, including IL-7, may contribute to this process 34 . In agreement with this hypothesis, we documented a significant expansion of RBDspecific naïve B cells enriched in Ig that were largely activated and more numerous in (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. For all COVID-19 patients and healthy volunteers, sera were collected from whole blood in silica-treated tubes (BD Biosciences) where the blood was incubated for 30 min without movement to trigger coagulation. Next, samples were centrifuged for 10 min at 1300 g at room temperature (RT), heat-inactivated at 56ºC for 1 hour and stored at -80ºC prior to use. PBMCs were isolated from whole blood collected with EDTA anticoagulant via Ficoll-Paque Premium (Cytiva) following manufacturer's instructions. Briefly, each sample was diluted with an equal volume of phosphate buffered saline (PBS). The diluted blood was carefully layered over an equal volume of Ficoll solution in a Falcon tube, followed by centrifugation at 400 g for 30 min at 20℃ without brake. The upper layer containing plasma was collected and stored at -80ºC. The layer of mononuclear cells was gently removed and washed twice with PBS. Pelleted cells were counted using Turk solution, resuspended in fetal bovine serum (FBS, Gibco) with 10% Dimethyl sulfoxide (DMSO, Sigma) and stored in liquid nitrogen prior to use. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The pCAGGS RBD construct, encoding for the receptor-binding domain of the SARS- ELISAs performed in this study were adapted from previously established protocols 46 . Serum samples were heat-inactivated at 56ºC for 1 hour and stored at -80ºC prior to use. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Spearman's rank correlation coefficient (ρ) was indicated by heat scale; significance was indicated by *P < 0.05, **P < 0.01, and ***P < 0.001; FDR correction was performed using the Benjamini-Hochberg procedure at the FDR < 0.05 significance threshold. Principal component analysis (PCA) was used to identify the most important features from 41 variables (including antibody titers and immune parameters; Data File S1) using COVT1 (n = 25), COVT2 (n = 20) and healthy controls (n = 16). The PCA was conducted using the "prcomp" function in R and visualized using the "factoextra" package (Kassambara A, and Mundt F. factoextra: Extract and Visualize the Results of Multivariate Data Analyses. 2020). All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Bars represent mean ± SEM. Dark-colored dots show ICU patients. Two-tailed Mann-Whitney U test was performed to compare HCs with COVT1 and HCs with COVT2. Wilcoxon matched pairs test was performed to compare COVT1 with COVT2 (*P < 0.05, **P < 0.01, and ***P < 0.001). Unless mentioned otherwise, HCs, n=19; COVT1, n=25; COVT2, n=20. test was performed to compare HCs with COVT1 and HCs with COVT2. Wilcoxon matched pairs test was performed to compare COVT1 with COVT2 (*P < 0.05, **P < All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 30, 2021. ; https://doi.org/10.1101/2021.04.29.21256002 doi: medRxiv preprint 0.01, and ***P < 0.001). Unless mentioned otherwise, HCs, n=19; COVT1, n=25; COVT2, n=20. Wilcoxon matched pairs test was performed to compare COVT1 with COVT2 (*P < 0.05, All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. test was performed to compare COVT1 with COVT2 (*P < 0.05, **P < 0.01, and ***P < 0.001). HCs, n=21; COVT1, n=25; COVT2, n=20. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. r stands for Spearman's rank-order correlation. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. IgM NP (log 10 AUC) IgM + IgD + ME (% ME) All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Distance to HC centroid LDH (UI/L) All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 30, 2021. ; https://doi.org/10.1101/2021.04.29.21256002 doi: medRxiv preprint WHO Coronavirus Disease (COVID-19) Dashboard Clinical Characteristics of Coronavirus Disease 2019 in China COVID-19: consider cytokine storm syndromes and immunosuppression. The Lancet vol The trinity of COVID-19: immunity, inflammation and intervention Complex Immune Dysregulation in COVID-19 Patients with Severe Respiratory Failure Impaired type I interferon activity and inflammatory responses in severe COVID-19 patients. Science (80-. ) Cytokine Storms: Understanding COVID-19. Immunity Autoantibodies against type I IFNs in patients with lifethreatening COVID-19. Science (80-. ) Prothrombotic autoantibodies in serum from patients hospitalized with COVID-19 Diverse functional autoantibodies in patients with SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls Deep immune profiling of COVID-19 patients reveals distinct immunotypes with therapeutic implications Broad and strong memory CD4+ and CD8+ T cells induced by SARS-CoV-2 in UK convalescent individuals following COVID-19 Immunological memory to SARS-CoV-2 assessed for up to 8 months after infection. Science (80-) Robust neutralizing antibodies to SARS-CoV-2 infection persist for months Functional SARS-CoV-2-Specific Immune Memory Persists after Mild COVID-19 Rapid generation of durable B cell memory to SARS-CoV-2 spike and nucleocapsid proteins in COVID-19 and convalescence Evolution of antibody immunity to SARS-CoV-2 Maturation and persistence of the anti-SARS-CoV-2 memory B cell response Longitudinal Isolation of Potent Near-Germline SARS-CoV-2-Neutralizing Antibodies from COVID-19 Patients Stereotypic neutralizing VHantibodies against SARS-CoV-2 spike protein receptor binding domain in patients with COVID-19 and healthy individuals Convergent antibody responses to SARS-CoV-2 in convalescent individuals Structures of Human Antibodies Bound to SARS-CoV-2 Spike Reveal Common Epitopes and Recurrent Features of Antibodies Analysis of a SARS-CoV-2-Infected Individual Reveals Development of Potent Neutralizing Antibodies with Limited Somatic Mutation Next-Generation Sequencing of T and B Cell Receptor Repertoires from COVID-19 Patients Showed Signatures Associated with Severity of Disease Naive human B cells can neutralize SARS-CoV-2 through recognition of its receptor binding domain IgA dominates the early neutralizing antibody response to SARS-CoV-2 Extrafollicular B cell responses correlate with neutralizing antibodies and morbidity in COVID-19 Challenges and opportunities for consistent classification of human b cell and plasma cell populations Regulation of CXCR3 and CXCR4 expression during terminal differentiation of memory B cells into plasma cells Effects of acute and chronic inflammation on B-cell development and differentiation CXCR3 ligands: Redundant, collaborative and antagonistic functions Functional SARS-CoV-2-Specific Immune Memory Persists after Mild COVID-19 The key role of IL-7 in lymphopoiesis Structural repertoire of immunoglobulin λ light chains T cell responses in patients with COVID-19 Preexisting influenza-specific CD4 + T cells correlate with disease protection against influenza challenge in humans Human Effector and Memory CD8+ T Cell Responses to Smallpox and Yellow Fever Vaccines Helper Cell Biology: A Decade of Discovery and Diseases. Immunity Human Blood CXCR5+CD4+ T Cells Are Counterparts of T Follicular Cells and Contain Specific Subsets that Differentially Support Antibody Secretion T follicular helper cells in human efferent lymph retain lymphoid characteristics COVID-19 and the human innate immune system Nonclassical Monocytes in Health and Disease COVID-19 severity associates with pulmonary redistribution of CD1c+ DCs and inflammatory transitional and nonclassical monocytes Acute SARS-CoV-2 Infection Impairs Dendritic Cell and T Cell Responses SARS-CoV-2-specific antibody profiles distinguish patients with moderate from severe Identification of FcαRI as an inhibitory receptor that controls inflammation: Dual role of FcRγ ITAM Systemic and mucosal antibody responses specific to SARS-CoV-2 during mild versus severe COVID-19 SARS-CoV-2 in severe COVID-19 induces a TGF-βdominated chronic immune response that does not target itself Depletion of circulating IgM memory B cells predicts unfavourable outcome in COVID-19 Effects of acute and chronic inflammation on B-cell development and differentiation Predominant autoantibody production by early human B cell precursors. Science (80-. ) Phenotype and kinetics of SARS-CoV-2-specific T cells in COVID-19 patients with acute respiratory distress syndrome Immune determinants of COVID-19 disease presentation and severity Comprehensive mapping of immune perturbations associated with severe COVID-19 Type I interferon negatively controls plasmacytoid dendritic cell numbers in vivo Severe COVID-19 Is Marked by a Dysregulated Myeloid Cell Compartment This study was supported by the COVID-19 call grant from Generalitat de Catalunya, Department of Health Agència de Gestió d'Ajuts Universitaris i de Recerca Generalitat de Catalunya The DCEXS is a 'Unidad de Excelencia María de Maeztu designed and performed experiments, analyzed and discussed data and wrote the manuscript; J.P. performed data analysis produced recombinant SARS-CoV-2 antigens discussed data and wrote the manuscript A. selected COVID-19 patients, provided clinical data and discussed data designed and performed experiments, analyzed results, discussed data, and wrote the manuscript We want to particularly acknowledge the patients and the Parc de Salut Mar MARBiobanc (PT17/0015/0011) integrated in the Spanish National Biobanks Network from ISCIII for their collaboration. MARBiobanc's work was supported by grants from The authors declare that they have no competing financial interests. All data are available in the main text or the supplementary materials. Further information and requests for resources and reagents should be directed to and will be fulfilled by the corresponding author, Giuliana Magri (gmagri@imim.es).