key: cord-0783485-3f0pgo4z
authors: Kaneko, Naoki; Kuo, Hsiao-Hsuan; Boucau, Julie; Farmer, Jocelyn R.; Allard-Chamard, Hugues; Mahajan, Vinay S.; Piechocka-Trocha, Alicja; Lefteri, Kristina; Osborn, Matthew; Bals, Julia; Bartsch, Yannic C.; Bonheur, Nathalie; Caradonna, Timothy M.; Chevalier, Josh; Chowdhury, Fatema; Diefenbach, Thomas J.; Einkauf, Kevin; Fallon, Jon; Feldman, Jared; Finn, Kelsey K.; Garcia-Broncano, Pilar; Hartana, Ciputra Adijaya; Hauser, Blake M.; Jiang, Chenyang; Kaplonek, Paulina; Karpell, Marshall; Koscher, Eric C.; Lian, Xiaodong; Liu, Hang; Liu, Jinqing; Ly, Ngoc L.; Michell, Ashlin R.; Rassadkina, Yelizaveta; Seiger, Kyra; Sessa, Libera; Shin, Sally; Singh, Nishant; Sun, Weiwei; Sun, Xiaoming; Ticheli, Hannah J.; Waring, Michael T.; Zhu, Alex L.; Alter, Galit; Li, Jonathan Z.; Lingwood, Daniel; Schmidt, Aaron G.; Lichterfeld, Matthias; Walker, Bruce D.; Yu, Xu; Padera, Robert F.; Pillai, Shiv; Abayneh, Betelihem A.; Allen, Patrick; Antille, Diane; Armstrong, Katrina; Balazs, Alejandro; Barbash, Max; Boyce, Siobhan; Braley, Joan; Branch, Karen; Broderick, Katherine; Daley, George; Ellman, Ashley; Fedirko, Liz; Flaherty, Keith; Flannery, Jeanne; Forde, Pamela; Gettings, Elise; Golan, David; Griffin, Amanda; Grimmel, Sheila; Grinke, Kathleen; Hall, Kathryn; Healey, Meg; Heller, Howard; Henault, Deborah; Holland, Grace; Kayitesi, Chantal; Lam, Evan C.; LaValle, Vlasta; Lu, Yuting; Luthern, Sara; Marchewska, Jordan; Martino, Brittni; Millstrom, Ilan; Miranda, Noah; Nambu, Christian; Nelson, Susan; Noone, Marjorie; O’Callaghan, Claire; Ommerborn, Christine; Pacheco, Lois Chris; Phan, Nicole; Porto, Falisha A.; Reissis, Alexandra; Ruzicka, Francis; Ryan, Edward; Selleck, Katheleen; Sharpe, Arlene; Sharr, Christianne; Slaugenhaupt, Sue; Sheppard, Kimberly Smith; Suschana, Elizabeth; Wilson, Vivine; Worrall, Daniel
title: Loss of Bcl-6-expressing T follicular helper cells and germinal centers in COVID-19
date: 2020-08-19
journal: Cell
DOI: 10.1016/j.cell.2020.08.025
sha: fe2bf3d956389395fe193746cf3efec204e19779
doc_id: 783485
cord_uid: 3f0pgo4z

Summary Humoral responses in COVID-19 disease are often of limited durability, as seen with other human coronavirus epidemics. To address the underlying etiology, we examined postmortem thoracic lymph nodes and spleens in acute SARS-CoV-2 infection and observed the absence of germinal centers, a striking reduction in Bcl-6+ germinal center B cells but preservation of AID+ B cells. Absence of germinal centers correlated with an early specific block in Bcl-6+ TFH cell differentiation together with an increase in T-bet+ TH1 cells and aberrant extra-follicular TNF-α accumulation. Parallel peripheral blood studies revealed loss of transitional and follicular B cells in severe disease and accumulation of SARS-CoV-2-specific “disease-related” B cell populations. These data identify defective Bcl-6+ TFH cell generation and dysregulated humoral immune induction early in COVID-19 disease, providing a mechanistic explanation for the limited durability of antibody responses in coronavirus infections and suggest that achieving herd immunity through natural infection may be difficult.

Adaptive immunity is initiated in secondary lymphoid organs and is influenced by the milieu generated by the initial activation of the innate immune system. Longitudinal studies on humoral immunity in COVID-19 as well as studies in convalescent subjects indicate that humoral immunity is often short lived and that most SARS-CoV-2 antibodies exhibit limited somatic hypermutation (Brouwer et al., 2020; Long et al., 2020; Robbiani et al., 2020) .

Understanding how the adaptive immune system is modulated in severe COVID-19 disease thus requires interrogation of secondary lymphoid organs in the acute phase of infection, where these responses are generated, but most studies to date have largely focused on peripheral blood samples.

SARS-CoV-2 infection results in a broad spectrum of clinical manifestations from asymptomatic to rapidly fatal, but the reasons for this heterogeneity are not known. Severely ill patients experience a life-threatening acute respiratory distress syndrome, and, even in an advanced care setting, some patients sustain severe lung damage and succumb early Zhou et al., 2020) . Virus is found in the lungs and airways early in infection but not as the disease progresses (Schaefer et al., 2020) . Damage-associated molecular patterns (DAMPs) released by infected pneumocytes likely combine with viral pathogen-associated molecular patterns (PAMPs) to activate innate immunity (Vardhana and Wolchok, 2020) . The cytokine milieu thus generated would be predicted to influence the induction of lymphocyte activation by antigen conveyed directly in the lymph or by dendritic cells to draining lymph nodes. Viremia likely also leads to the initiation of immune responses in the spleen.

Many of the features of severe human coronavirus disease in COVID-19 and in SARS are strikingly similar. Progressive lymphopenia has been described in SARS-CoV-2 infection (Guan J o u r n a l P r e -p r o o f et al., 2020) and the degree of lymphopenia has been correlated with increases in circulating IL-6 and IL-8 . Lymphopenia was also observed in SARS at the peak of active disease which was also characterized by cytokine storm and acute respiratory distress (Perlman and Dandekar, 2005) . Autopsy studies in SARS showed atrophy of lymphoid organs including lymph nodes, spleen and Peyer's patches and loss of germinal centers (Gu et al., 2005) . Autopsy studies in COVID-19 have also identified splenic white pulp atrophy , Buja et al., 2020 and lymphocyte depletion in spleen and lymph nodes (Lax et al., 2020) . However, numerous viral and non-viral infections do give rise to cytokine storm, acute respiratory distress and lymphopenia (Tisoncik et al., 2012) . Splenic white pulp atrophy has also been histopathologically demonstrated in Ebola and Marburg disease (Martines et al., 2016; Rippey et. al., 1984) and in H5N1 influenza (Gao et al. 2010; Lu et al., 2008) . These data, taken together, suggest that many different viral and infectious triggers can contribute to a similar constellation of immunological phenomena that may drive pathology.

In persons with COVID-19, the magnitude and durability of antibody responses are greater in those with more severe disease (Ju et al., 2020; Amanat et al., 2020) but are often of low magnitude (Robbiani et al., 2020) and appear to lack durability (Long et al., 2020) . This may be similar to SARS and MERS where humoral responses were generally not durable except in a subset of individuals (Mo et al., 2006; Zumla et al., 2015) . Impaired infection-induced protective immunity has also been documented by repeated infections with the human coronaviruses CoV 229E, NL63, OC43 and HKU1 in patients with less severe respiratory tract infections (Galanti et al., 2018) . Reinfection could be possibly attributed to viral strain subtypes, but the reason/s for the general lack of durable humoral immune responses to coronaviruses has never been established.

J o u r n a l P r e -p r o o f 5 A better understanding of alterations to components of the humoral immune system, especially in secondary lymphoid organs, provides an opportunity to decipher why natural infections with coronaviruses often do not provide durable immunity. A granular analysis of B and T lymphocytes in draining lymph nodes and spleens of SARS or MERS patients was never reported, leaving the underlying basis for the lymphopenia and the general lack of durability of antibody responses in those diseases unresolved. Since COVID-19 disease most significantly affects the lungs, we undertook an analysis of thoracic lymph nodes examining lymphoid architecture and lymphocyte populations using multi-color immunofluorescence, multispectral imaging and cell-cell interaction analyses from the time of disease onset in persons with diverse disease outcomes. Given that viremia has been observed in this illness Lescure et al., 2020) we also interrogated spleens both in the acute and late disease settings and complemented these studies with examination of peripheral blood samples in a separate cohort wherein convalescence could also be studied. Our results identify a striking absence of lymph node and splenic germinal centers and Bcl-6 expressing B cells, defective Bcl-6 + T follicular helper cell generation and differentiation and dysregulated SARS-CoV-2 specific humoral immunity early in COVID-19 disease, providing a mechanistic explanation for the limited durability of humoral immunity and the less robust somatic hypermutation seen in this disease following natural infection.

We have used a human tissue imaging platform with quantitative high-resolution automated slide-scanning microscopy, exploiting both regular and confocal approaches and multispectral imaging, in order to interrogate human lymphoid and non-lymphoid organs at the single cell level. These approaches crucially preserve architecture over broad swaths of tissue.

Thoracic lymph nodes in severely ill COVID-19 patients who succumbed in less than eight days after admission (the group designated "Early"; less than 10 days from the onset of respiratory symptoms; Table S1 ), displayed a lack of germinal centers and these were also absent in those who succumbed later (15-36 days after admission, categorized as "Late") ( Fig. 1A, B) . Controls were thoracic lymph nodes from age matched individuals who succumbed from non-COVID-19 causes ( Table S2) . Quantitation revealed dramatic early loss of both B and T cells, absolute numbers declining to about a third of their non-COVID-19 controls, and this persisted in late disease ( Fig.1 D, E) , though distinct T and B cell zones could always be clearly discerned.

Human control lymph nodes contain germinal centers possibly because of ongoing adaptive immunity initiated by commensal antigens. The absence of germinal centers in the thoracic lymph nodes of acutely ill COVID-19 patients in whose lungs we have already described very high viral loads (Schaefer et al., 2020) was particularly surprising.

Bcl-6 expressing germinal center B cells were also markedly reduced in COVID-19 but there was a preservation of AID expressing B cells, although these were diffusely distributed compared to controls (Fig. 1C, F, G) .

J o u r n a l P r e -p r o o f 7

Analysis of spleens in this same group of COVID-19 patients also revealed a preponderance of red pulp and paucity of white pulp ( Fig. 2A, B) , a marked reduction in B and T cell numbers (Fig. 2B, D, E) and a marked reduction in Bcl-6 + germinal center B cells (Fig. 2C,   F ). There was, however, very clear and quantitative preservation of AID + B cells in both early and late splenic tissue (Fig. 2C, G) . Importantly follicular dendritic cells (FDC) were present in both lymph nodes and spleen in these patients, indicating that the lack of these cells was not contributing to the lack of germinal center B cells (Fig. S1 ). Together these data indicate that early in severe COVID-19 disease, even within ten days of the onset of respiratory symptoms, there is severe attrition in B and T cell numbers and a striking reduction in Bcl-6 + B cells in lymph nodes and the spleen and the loss of germinal centers. Interestingly AID + B cells are preserved indicating that activated helper T cells are still likely to be in frequent contact with antigen specific B cells.

To better understand the absence of germinal centers in COVID-19, we explored the possibility that the tissue milieu might contribute to defective T follicular helper cell differentiation. In both the lymph nodes and spleen, in early as well as late disease, CD4 + ICOS + T FH cells were diminished (Fig. 3A, C, E, G) , and CD4 + CXCR5 + T FH cells were present but reduced in numbers ( Fig. S2 ) but the decrease in CD4 + Bcl-6 + germinal center type T FH (GC-T FH ) cells was striking (Fig. 3B, D, F and H) . Tissue quantitation confirmed significant differences for both early and late disease compared to controls. Because these changes were J o u r n a l P r e -p r o o f 8 seen both in thoracic lymph nodes and in the spleen these data are consistent with the view that circulating factors in severely ill COVID-19 patients may impair GC-T FH cell differentiation and thus abrogate the generation of germinal centers. Although in principle phenotypically defined CD4 + Bcl-6 + T cells could include both T FH cells and T follicular regulatory cells, we stained cells simultaneously with CD4, CXCR5, FOXP3 and Bcl-6 among other markers and used multispectral imaging to establish that while there were FOXP3 + T regs present, there was no overlap in Bcl-6 and FoxP3 expression in COVID-19 secondary lymphoid organs, indicating that there are very few if any T follicular regulatory cells in COVID-19 (Fig. S3A) . Although there is a developmental role for TNF-α in primary lymphoid follicular development (Pasparakis et al., 1996; Korner et al., 1997) , germinal center loss has been described in the context of cytokine storm in mouse models, has been reversed by TNF-α blockade (Ryg-Cornejo et al., 2016 , Popescu et al., 2019 and also linked genetically to an abundance of TNF-α (Popescu et al., 2019) . We therefore also examined activated secondary lymphoid tissues from controls and COVID-19 lymph nodes for TNF-α expression. In this case we used tonsils from non-COVID infected patients as a control for activated lymphoid tissue. While TNF-α is expressed at low levels in the follicle in controls, in COVID-19 it is expressed very abundantly both inside and outside the follicle ( Fig. S3C and D) . These data indicate that the differentiation of activated CD4 + T cells into GC-type Bcl-6 + T FH cells is specifically blocked in COVID-19. Given the information obtained from the above animal models (Popescu et al., 2019; Ryg-Cornejo et al., 2016) , it is possible that the aberrant and exuberant synthesis of TNF-α at the site of T FH differentiation in COVID-19 lymph nodes may contribute to the lack of germinal centers and the impaired quality and durability of the antibody response to SARS-CoV-2 in this disease.

J o u r n a l P r e -p r o o f

We hypothesized that the reduction in GC-T FH cell numbers likely reflects a block in differentiation, and next sought to determine if this reduction was specific to this particular CD4 + T cell subset. We quantitated CD4 + T cell subsets in the lymph nodes and spleens using the well-established transcription factors T-bet, GATA-3, RORγt and FOXP3 as key markers. In contrast to the reduced GC-T FH cell numbers, T H1 cells were consistently increased early and late in both the lymph nodes and spleen, whereas an increase in T H17 cells was more variable (Fig. 4) .

In contrast, a consistent reduction in T H2 cells was observed (Fig. 4) . Late in the disease FOXP3 + Tregs made up a large part of the population of CD4 + T cells. Overall there was an increase in secondary lymphoid organ CD4 + T cells relative to CD8 + T cells in COVID-19 secondary lymphoid organs though this was variable (Fig. S4) . These data indicate that the defect in GCtype T FH cell differentiation is specific and suggest that this defect may be indirectly linked to the strong T H1 response seen in this disease.

The preservation in COVID-19 of AID + B cells and the relatively large proportions of CD4 + CXCR5 + T FH cells (that do not express Bcl-6) and T H1 cells, both known to express CD40L, led us to hypothesize that even though there were no germinal centers, there may be frequent T-B conjugates in COVID-19 within follicles as well as in extra-follicular locations.

The absence of germinal centers (most germinal center B cells are IgD -CD27 -Bcl-6 + AID + CXCR5 + CD19 + cells) offered the opportunity to directly ask whether many of the scattered activated AID + B cells inside and outside follicles in COVID-19 secondary lymphoid organs were also IgD -CD27 -"double negative" B cells. These cells are often observed in chronic infectious contexts including in viral infections as well as in autoimmunity (Portugal et al., 2017; Jenks et al., 2019) . They are considered to be "disease-related" cells and are generally described as "extra-follicular", implying they are not derived from the germinal center reaction, but are frequently class switched and have the hallmarks of having been induced in a T-dependent manner without germinal center based selection. At extra-follicular and follicular sites these double negative B cells may be less capable of inducing the differentiation of the Bcl-6 + GC-T FH cells that are required to induce germinal centers.

By applying computational tools to systematically quantitate the area of cytoplasmic overlap between sets of two cells of two different cell types, and using pre-determined cut-offs to assess true cell-cell interaction, we quantified the degree and intimacy of plasma membrane contacts between cells and observed the presence of numerous T-B conjugates in COVID-19 lymph nodes and spleens (Fig. S5) . The presence of IgD -CD27 double negative cells both within and outside follicles in COVID-19 lymph nodes and spleens was clearly evident (Fig. S5) .

IgG expressing class-switched plasmablasts were prominent both within the follicular and extrafollicular areas (Fig. S3B) . These data indicate that in the absence of germinal center formation in COVID-19 an "extra-follicular" type of class-switched B cell response, more typical of disease rather than of long-lasting protection, predominates in secondary lymphoid organs. as compared to convalescent COVID-19 patients and healthy controls (Fig. 6A, B) , though in terms of absolute numbers these relative increases were blunted by the greater degree of B cell lymphopenia (Fig. S7) . Within the IgD + CD27 compartment, severely ill patients with COVID-19 and high CRP levels, specifically, showed an increase in a number of disease-related presumed non-germinal center derived activated B cells as compared to convalescent patients.

These include activated naïve B cells (IgD + CD27 -CD21 lo CD11c hi ) (Kaminski et al., 2012) as 6C) .

Additionally, IgD -CD27 -CXCR5 -B cells that include populations described by Kaminski et al. based on the expression of CXCR5 and CD11c were expanded in severely ill COVID-19 patients with high CRP levels, specifically, as compared to convalescent patients and healthy controls J o u r n a l P r e -p r o o f 13 ( Fig. 6D) . The sum accumulation of these activated B cells was markedly higher in the severely ill COVID-19 patients with high versus intermediate CRP levels and also in comparison to convalescent patients and healthy controls (Fig. 6E) . Paralleling the trends observed with the loss of early transitional B cells, the gain in circulating activated B cells correlated with higher CRP levels and increased patient morbidity as measured by symptom duration and length of inpatient hospitalization (Fig. 6F) .

Although recent studies have also documented circulating activated B cell populations in COVID-19 patients Kuri-Cervantes et al., 2020; Woodruff et al., 2020) the antigen specificity of these cells was not evaluated. We tested whether the activated B cell populations we had identified were specific for the SARS-CoV-2 Spike receptor binding domain (RBD) using recombinant RBD labeled separately with APC and PE fluorophores. Cells that stained with both labeled probes were considered authentic RBD-specific B cells (Fig. 7A) . This probe contains a very small fraction of all the potential antibody epitopes in SARS-CoV-2 but is highly specific for this particular virus (Premkumar et al., 2020) . All the activated and mainly disease-related populations of relevance in COVID-19 contain SARS-CoV-2 specific cells ( Fig.   7A , B), establishing that they were expanded as a result of an adaptive immune response to this virus. In particular the presence of antigen-specific double negative B cells, switched memory B cells, and plasmablasts are all consistent with an extra-germinal center/extra-follicular type classswitched antibody response to SARS-CoV-2. These data establish that the aberrant non-germinal center type activated B cells that accumulate in tissues also accumulate in the blood of severely ill and convalescent COVID-19 patients and these include virus-specific B cells. Given that they bear the hallmarks of not being from germinal centers, they are therefore unlikely to provide optimal or durable humoral immunity.

14

Long-lasting B cell memory and the highest affinity pathogen-specific antibodies are derived within germinal centers in secondary lymphoid organs. Germinal centers are anatomically structured to facilitate the selection of high affinity B cell with long life spans (Victora and Nussenzweig, 2012) . Longevity of such responses exceeds decades for some infectious diseases and when a substantial portion of a population is infected, can contribute to herd immunity. In contrast, antibody responses to SARS-CoV-2 appear to be similar to other human coronaviruses in being short-lived in a large fraction of individuals (Long et al., 2020) .

Understanding the reasons for this decline in responses requires architectural studies of lymphoid organs from patients coupled with cell-cell interaction analyses, as well as approaches to reliably identify, quantify and physically locate the diverse immune cell types that contribute to antibody induction. Using quantitative multicolor immunofluorescence combined with multispectral imaging and cell-cell interaction analyses of autopsy specimens as well as analyses of peripheral blood samples in parallel cohorts with acute SARS-CoV-2 infection, we show evidence for dysregulated humoral immune induction early in COVID-19 including a striking absence of germinal centers in the earliest stages of infection, defective Bcl6 + T FH cell generation and aberrant lymphoid TNF-α production.

The absence of Bcl-6 + T follicular helper cells (and the consequent absence of germinal centers) in COVID-19 secondary lymphoid organs provides an explanation for a phenomenon anecdotally observed in autopsies of many different severe viral infections. These findings also provide a mechanistic basis for the recent descriptions of non-durable humoral immune responses, impaired humoral immunity and the low levels of somatic hypermutation in antibodies from convalescent COVID-19 patients (Long et al., 2020; Robbiani et al., 2020; Brouwer et al., 2020) . The alteration of the cytokine milieu in secondary lymphoid organs in this disease likely reflects a continuum across the spectrum of disease. While T-dependent B cell activation, class switching and some low level somatic hypermutation do occur in COVID-19, the germinal center reaction is sub-optimal or totally absent and this will likely, in due course of time, be reflected in less durable class-switched antibody responses similar to those seen in SARS and MERS. These findings have some bearing on concepts such as herd immunity and immunity passports following natural infection with SARS-CoV-2. They strongly underscore the need and relevance of vaccination approaches to the prevention of COVID-19.

Severe infections by many different human viruses result in high levels of circulating cytokines and peripheral lymphopenia, but few studies have examined secondary lymphoid organs where immune responses are generated. Autopsy studies have revealed lymphoid depletion of the spleen and lymph nodes in Ebola, Marburg, and in H5N1 (Martines et al., 2015; Rippey et al., 1984; Lu et al., 2008; Gao et al., 2010) . In SARS, lymphoid depletion and loss of germinal centers was also reported (Gu et al., 2005) . Autopsy studies in these severe viral Because of our focus on the loss of germinal centers, we have concentrated on TNF-α because of its known ability, when produced in excess, to contribute to impaired T FH cell differentiation and germinal center loss. Many other cytokines are induced in COVID-19 and probably contribute to some aspects of the phenotypes that we describe here. IL-6, for example, though it has pleiotropic effects, is known to suppress lymphopoiesis and induce myelopoiesis (Maeda et al., 2009) , and it might thus contribute to the B lymphopenia that we document here. 

CD4 + Bcl6 + GC-type T FH (H) in spleens from COVID-19 patients (purple, n = 10) and controls (blue, n = 7). COVID-19 samples include early (purple, n = 4) and late (red, n = 6) COVID-19

patients. Mann-Whitney U test used to calculate p-value. Error bars represent mean ±SEM. *p < 0.05; **p < 0.01; ***p < 0.001. See also Fig. S2 , S3 and S4 and Tables S1, S2 and S3. Table S4 and compared to healthy controls (n=4). Quantitation shown by B cell J o u r n a l P r e -p r o o f 26 level for each individual patient with mean, standard deviation, and significance by one-way ANOVA of log % B cell value indicated (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Representative COVID-19 contour plots shown with healthy control contour plots and full B cell flow cytometry gating strategy outlined (Fig. S6) 

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Dr. Shiv Pillai pillai@helix.mgh.harvard.edu

The SARS-CoV-2 RBD protein is made freely available through Dr. Aaron Schmidt who may be reached through the Lead Contact or directly.

The published article includes all data generated or analyzed during this study, and summarized in the accompanying tables, figures and Supplemental materials. Table S1and S2) including 11 with laboratory confirmed COVID-19 who underwent autopsy in 2020.

Sample allocation to experimental groups: All patients had tested positive for SARS-CoV-2 by RT-PCR of nasopharyngeal swabs in a laboratory during hospital admission. All cases were divided into two groups; early (less than ten days from respiratory symptoms onsets to death, hospitalization of up to 8 days), and late (hospitalized for 15-36 days prior to death). Thoracic lymph nodes were obtained from six age-matched non-COVID-19 patients who underwent autopsies at the Brigham and Women's Hospital in the same time window. In addition, seven spleens from automobile accident victims were obtained from the Ragon Institute Tissue Core.

These were histologically normal. Information on age and gender is in Table S3 . Ten discarded control tonsils from Massachusetts General Hospital not matched for age and gender were also used for initial titration and validation of certain antibodies and as controls for TNF-α and CD8 staining studies,

Peripheral blood samples were drawn from both Outpatients and Inpatients with COVID-19 at Massachusetts General Hospital and fresh blood was analyzed for flow cytometry using a multicolor panel.

Sample Size estimation: Power calculations were not undertaken pior to the initiation of these studies. However, the primary end-point was the sum of activated B cells (as a % CD19+ B cells in peripheral blood). Using a two sided Student's t-test to compare log values of convalescent and severe (CRP hi) COVID-19 patient samples, with groups of 10, we had more than 90% power to detect an effect of size of 1.20 between groups based on simulation studies using 10,000 Monte Carlo samples with a type 1 error rate of 5%.

Allocation to Experimental Groups: Data is presented on B cell populations from 68 patients, including moderately ill, severely ill and convalescent patients. Convalescence was defined as a clinically asymptomatic state on the date of blood draw, either from a baseline asymptomatic state or recuperated from moderate clinical symptoms of COVID-19. Moderate disease was defined as active clinical symptoms of COVID-19 on the date of blood draw that did not necessitate a hospital admission. Severe disease was defined as active clinical symptoms of COVID-19 on the date of blood draw that did necessitate a hospital admission. Severe 

Tissue samples were fixed in formalin, embedded in paraffin, and sectioned. These specimens were incubated with the following antibodies: anti-CD3 (clone: A045229-2; DAKO), anti-CD4 

Images of the tissue specimens were acquired using the TissueFAXS platform (TissueGnostics). For quantitative analysis, the entire area of the tissue was acquired as a digital grayscale image in five channels with filter settings for FITC, Cy3, Cy5 and AF75 in addition to DAPI. Cells of a given phenotype were identified and quantitated using the TissueQuest software J o u r n a l P r e -p r o o f 36 (TissueGnostics), with cut-off values determined relative to the positive controls. This microscopy-based multicolor tissue cytometry software permits multicolor analysis of single cells within tissue sections similar to flow cytometry. In addition, multispectral images (sevencolors staining) were unmixed using spectral libraries built from images of single stained tissues for each reagent using the StrataQuest (TissueGnostics) software. StrataQuest software was also used to quantify cell-to-cell contact. In the StrataQuest cell-to-cell contact application, masks of the nuclei based on DAPI staining establish the inner boundary of the cytoplasm and the software "looks" outwards towards the plasma membrane boundary. Overlap of at least 3 pixels of adjacent cell markers is required to establish a "contact" criterion. Although the software has been developed and validated more recently, the principle of the method and the algorithms used have been described in detail elsewhere (Ecker et al., 2004) . For the RBD-specific characterization, 6-7 million fresh PBMCs were stained using fluorescently labeled SARS-CoV-2 RBD in addition to the B cell panel antibodies listed above.

Cells were then washed in PBS and fixed using 4% paraformaldehyde for 30 minutes at at 4 ℃.

Flow cytometry was performed on a BD Symphony (BD Biosciences, San Jose, CA) and rainbow tracking beads (8 peaks calibration beads, Fisher) were used to ensure consistent signals between flow cytometry batches. FCS files were analyzed, and B cell subsets were quantified using FlowJo software (version 10).

SARS-2 RBD (GenBank: MN975262.1) was cloned into pVRC vector containing an HRV 3Ccleavable C-terminal SBP-His 8X tag and sequence confirmed by Genwiz. The construct was transiently transfected into mammalian Expi293F suspension cells for recombinant expression. 5 days post-transfection, supernatants were harvested and clarified by low-speed centrifugation.

The RBD was purified by immobilized metal affinity chromatography (IMAC) using Cobalt-TALON resin (Takara) followed by size exclusion chromatography on Superdex 200 Increase 10/300 GL (GE Healthcare) in PBS. Purity was assessed by SDS-PAGE analysis. The fluorescent PE-SA (Invitrogen, Cat#12-4317-877) and APC-SA (Invitrogen; Cat#S32362) labels were added to the purified SBP-tagged RBD proteins through iterative complex formation, as previously described (Weaver et al. 2016) . The fluorescent SA conjugates were added to SBP-RBD in five increments to sequentially form the complexes. In this case the final molar ratio of probe to streptavidin valency was 1:1 (one SA-fluorophore can bind two SBP tags). After each stepwise addition of the fluorescent label, the mixture was incubated for 20 minutes and set rotating at 4C within an opaque 1.5 mL Eppendorf spin tube. Using this method, we generated fluorescent probes at a final concentration of 0.1ug/ul. An example of SBP-RBD (31,000g/mol) labeled with PE-SA (300,000g/mol) is described for a total 10 assays (0.5 ug labeled protein per assay): First, 5uL of SBP-RBD at 1ug/ul was diluted in 20.8uL PBS. Fluorescent PE-SA at 1ug/ul was then added in five increments, with an incremental volume of 4.8 uL for a final volume of 50uL.

Flow cytometry, clinical correlations and tissue studies. GraphPad Prism version 8 was used for statistical analysis, curve fitting and linear regression. A two-tailed Mann-Whitney U test was used to calculate p-values for continuous, non-parametric variables. For comparing more than one population, Kruskal-Wallis testing was used with Dunn's multiple comparison testing.

A p-value of < 0.05 was considered significant. CD4+ T-bet+ / CD4 (%)

CD4 

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Severe Malaria Infections Impair Germinal Center Responses by Inhibiting T Follicular Helper Cell Differentiation

In situ detection of SARS-CoV-2 in lungs and airways of patients with COVID-19

Costimulation modulation uncouples protection from immunopathology in memory T cell responses to influenza virus

Into the eye of the cytokine storm

Germinal centers

Follicular Helper T Cells

In vitro reconstitution of B cell receptor-antigen interactions to evaluate potential vaccine candidates

Critically ill SARS-CoV-2 patients display lupus-like hallmarks of extrafollicular B cell activation

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

Elevated exhaustion levels and reduced functional diversity of T cells in peripheral blood may predict severe progression in COVID-19 patients

Viral load dynamics and disease severity in patients infected with SARS-CoV-2 in Zhejiang province

A pneumonia outbreak associated with a new coronavirus of probable bat origin

A Novel Coronavirus from Patients with Pneumonia in China

Middle East respiratory syndrome