Analysis of T follicular and T peripheral helper lymphocytes in autoimmune thyroid disease.

PURPOSE: Peripheral helper T (Tph) cells have an important role in the induction of humoral immune responses and autoantibody production. Accordingly, it is feasible that this lymphocyte subset has a relevant role in the pathogenesis of autoimmune thyroid diseases (AITD). In this study we aim to analyze the levels and function of Tph cells in blood samples from patients with AITD. METHODS: We performed an observational study with cases and controls. Blood samples were obtained from nineteen patients with Hashimoto's thyroiditis (HT), twenty-four with Graves' disease (GD), and fifteen healthy controls. In addition, the levels of follicular T helper (Tfh) cells and Tph cells, the release of interleukin-21 (IL-21) by these lymphocytes and the number of plasmablasts were analyzed by multi-parametric flow cytometry analyses. RESULTS: Increased percentages of Tfh and Tph lymphocytes were detected in patients with HT and GD. Furthermore, an enhanced synthesis of the cytokine IL-21 by these cells was observed. Accordingly, we detected significant higher percentages of plasmablasts in patients with GD, and these values tended to be also higher in HT patients. Moreover, significant positive associations were observed between the levels of Tfh or Tph and the number of plasmablast or anti-TSHR Ab titers in patients with AITD. CONCLUSION: Our data suggest that Tph lymphocytes may have a relevant role in the pathogenesis of AITD.

B-cells depletion after treatment with rituximab predicts relapse of IgG4-related disease.

OBJECTIVES: B cell depletion therapy with rituximab is effective in most patients with IgG4-related disease (IgG4-RD) but requires repeated cycles to prevent disease flares. We here aimed to assess B cells after rituximab to predict relapse of IgG4-RD and guide retreatment. METHODS: Patients with active IgG4-RD included in this retrospective study fulfilled the ACR/EULAR Classification Criteria. Total CD19+ B cells, plasmablasts, naïve and memory B cells were measured on peripheral blood by flow-cytometry at baseline and six months after rituximab. All patients were treated with two 1 g infusions of rituximab 15 days apart and monitored for 48 months. Disease response was assessed using the IgG4-RD Responder Index. RESULTS: Thirty-three patients were included. Six months after rituximab, disease response was observed in all patients. Complete depletion of CD19+ B cells, plasmablasts, naïve and memory B cell depletion was achieved in 30%, 55%, 39%, and 42% of cases, respectively. Twenty-three relapses (70%) were observed at a median time of 24 months after rituximab. Relapse rate was significantly higher in patients who failed to achieve complete depletion of CD19+ cells (60% vs 17%, p= 0.02), naïve B cells (54% vs 15%, p= 0.01), or memory B cells (50% vs 16%, p= 0.03) six months after rituximab. The median relapse free survival time was shorter in patients who failed to achieve complete depletion of CD19+ cells (19 vs 38 months, p= 0.02), naïve B cells (16 vs 38 months, p= 0.01), or memory B cells (19 vs 38 months, p= 0.03) six months after rituximab. CONCLUSIONS: The degree of B cell depletion six months after rituximab may predict disease flare and may instruct on the pacing of B cell depletion therapy in IgG4-RD.

Non-cross-reactive epitopes dominate the humoral immune response to COVID-19 vaccination - kinetics of plasma antibodies, plasmablasts and memory B cells.

Introduction: COVID-19 vaccines are highly effective in inducing protective immunity. While the serum antibody response to COVID-19 vaccination has been studied in depth, our knowledge of the underlying plasmablast and memory B cell (Bmem) responses is still incomplete. Here, we determined the antibody and B cell response to COVID-19 vaccination in a naïve population and contrasted it with the response to a single influenza vaccination in a primed cohort. In addition, we analyzed the antibody and B cell responses against the four endemic human coronaviruses (HCoVs). Methods: Measurement of specific plasma IgG antibodies was combined with functional analyses of antibody-secreting plasmablasts and Bmems. SARS-CoV-2- and HCoV-specific IgG antibodies were quantified with an in-house bead-based multiplexed immunoassay. Results: The antibody and B cell responses to COVID-19 vaccination reflected the kinetics of a prime-boost immunization, characterized by a slow and moderate primary response and a faster and stronger secondary response. In contrast, the influenza vaccinees possessed robust immune memory for the vaccine antigens prior to vaccination, and the recall vaccination moderately boosted antibody production and Bmem responses. Antibody levels and Bmem responses waned several months after the 2nd COVID-19 vaccination, but were restored upon the 3rd vaccination. The COVID-19 vaccine-induced antibodies mainly targeted novel, non-cross-reactive S1 epitopes of the viral spike protein, while cross-reactive S2 epitopes were less immunogenic. Booster vaccination not only strongly enhanced neutralizing antibodies against an original SARS-CoV-2 strain, but also induced neutralizing antibodies against the Omicron BA.2 variant. We observed a 100% plasma antibody prevalence against the S1 subunits of HCoVs, which was not affected by vaccination. Discussion: Overall, by complementing classical serology with a functional evaluation of plasmablasts and memory B cells we provide new insights into the specificity of COVID-19 vaccine-induced antibody and B cell responses.

Estradiol mediates greater germinal center responses to influenza vaccination in female than male mice.

Adult females of reproductive age develop greater antibody responses to inactivated influenza vaccines (IIV) than males. How sex, age, and sex steroid concentrations impact B cells and durability of IIV-induced immunity and protection over 4 months post-vaccination (mpv) was analyzed. Vaccinated adult females had greater germinal center B cell and plasmablast frequencies in lymphoid tissues, higher neutralizing antibody responses 1-4 mpv, and better protection against live H1N1 challenge than adult males. Aged mice, regardless of sex, had reduced B cell frequencies, less durable antibody responses, and inferior protection after challenge than adult mice, which correlated with diminished estradiol among aged females. To confirm that greater IIV-induced immunity was caused by sex hormones, four core genotype (FCG) mice were used, in which the testes-determining gene, Sry, was deleted from chromosome Y (ChrY) and transferred to Chr3 to separate gonadal sex (i.e., ovaries or testes) from sex chromosome complement (i.e., XX or XY complement). Vaccinated, gonadal female FCG mice (XXF and XYF) had greater numbers of B cells, higher antiviral antibody titers, and reduced pulmonary virus titers following live H1N1 challenge than gonadal FCG males (XYM and XXM). To establish that lower estradiol concentrations cause diminished immunity, adult and aged females received either a placebo or estradiol replacement therapy prior to IIV. Estradiol replacement significantly increased IIV-induced antibody responses and reduced morbidity after the H1N1 challenge among aged females. These data highlight that estradiol is a targetable mechanism mediating greater humoral immunity following vaccination among adult females.IMPORTANCEFemales of reproductive ages develop greater antibody responses to influenza vaccines than males. We hypothesized that female-biased immunity and protection against influenza were mediated by estradiol signaling in B cells. Using diverse mouse models ranging from advanced-age mice to transgenic mice that separate sex steroids from sex chromosome complement, those mice with greater concentrations of estradiol consistently had greater numbers of antibody-producing B cells in lymphoid tissue, higher antiviral antibody titers, and greater protection against live influenza virus challenge. Treatment of aged female mice with estradiol enhanced vaccine-induced immunity and protection against disease, suggesting that estradiol signaling in B cells is critical for improved vaccine outcomes in females.

Ultrahigh frequencies of peripherally matured LGI1- and CASPR2-reactive B cells characterize the cerebrospinal fluid in autoimmune encephalitis.

Intrathecal synthesis of central nervous system (CNS)-reactive autoantibodies is observed across patients with autoimmune encephalitis (AE), who show multiple residual neurobehavioral deficits and relapses despite immunotherapies. We leveraged two common forms of AE, mediated by leucine-rich glioma inactivated-1 (LGI1) and contactin-associated protein-like 2 (CASPR2) antibodies, as human models to comprehensively reconstruct and profile cerebrospinal fluid (CSF) B cell receptor (BCR) characteristics. We hypothesized that the resultant observations would both inform the observed therapeutic gap and determine the contribution of intrathecal maturation to pathogenic B cell lineages. From the CSF of three patients, 381 cognate-paired IgG BCRs were isolated by cell sorting and scRNA-seq, and 166 expressed as monoclonal antibodies (mAbs). Sixty-two percent of mAbs from singleton BCRs reacted with either LGI1 or CASPR2 and, strikingly, this rose to 100% of cells in clonal groups with ≥4 members. These autoantigen-reactivities were more concentrated within antibody-secreting cells (ASCs) versus B cells (P < 0.0001), and both these cell types were more differentiated than LGI1- and CASPR2-unreactive counterparts. Despite greater differentiation, autoantigen-reactive cells had acquired few mutations intrathecally and showed minimal variation in autoantigen affinities within clonal expansions. Also, limited CSF T cell receptor clonality was observed. In contrast, a comparison of germline-encoded BCRs versus the founder intrathecal clone revealed marked gains in both affinity and mutational distances (P = 0.004 and P < 0.0001, respectively). Taken together, in patients with LGI1 and CASPR2 antibody encephalitis, our results identify CSF as a compartment with a remarkably high frequency of clonally expanded autoantigen-reactive ASCs whose BCR maturity appears dominantly acquired outside the CNS.

Prior dengue virus serotype 3 infection modulates subsequent plasmablast responses to Zika virus infection in rhesus macaques.

Immunodominant and highly conserved flavivirus envelope proteins can trigger cross-reactive IgG antibodies against related flaviviruses, which shapes subsequent protection or disease severity. This study examined how prior dengue serotype 3 (DENV-3) infection affects subsequent Zika virus (ZIKV) plasmablast responses in rhesus macaques (n = 4). We found that prior DENV-3 infection was not associated with diminished ZIKV-neutralizing antibodies or magnitude of plasmablast activation. Rather, characterization of 363 plasmablasts and their derivative 177 monoclonal antibody supernatants from acute ZIKV infection revealed that prior DENV-3 infection was associated with a differential isotype distribution toward IgG, lower somatic hypermutation, and lesser B cell receptor variable gene diversity as compared with repeat ZIKV challenge. We did not find long-lasting DENV-3 cross-reactive IgG after a ZIKV infection but did find persistent ZIKV-binding cross-reactive IgG after a DENV-3 infection, suggesting non-reciprocal cross-reactive immunity. Infection with ZIKV after DENV-3 boosted pre-existing DENV-3-neutralizing antibodies by two- to threefold, demonstrating immune imprinting. These findings suggest that the order of DENV and ZIKV infections has impact on the quality of early B cell immunity which has implications for optimal immunization strategies. IMPORTANCE: The Zika virus epidemic of 2015-2016 in the Americas revealed that this mosquito-transmitted virus could be congenitally transmitted during pregnancy and cause birth defects in newborns. Currently, there are no interventions to mitigate this disease and Zika virus is likely to re-emerge. Understanding how protective antibody responses are generated against Zika virus can help in the development of a safe and effective vaccine. One main challenge is that Zika virus co-circulates with related viruses like dengue, such that prior exposure to one can generate cross-reactive antibodies against the other which may enhance infection and disease from the second virus. In this study, we sought to understand how prior dengue virus infection impacts subsequent immunity to Zika virus by single-cell sequencing of antibody producing cells in a second Zika virus infection. Identifying specific qualities of Zika virus immunity that are modulated by prior dengue virus immunity will enable optimal immunization strategies.

GLUT1-mediated glucose import in B cells is critical for anaplerotic balance and humoral immunity.

Glucose uptake increases during B cell activation and antibody-secreting cell (ASC) differentiation, but conflicting findings prevent a clear metabolic profile at different stages of B cell activation. Deletion of the glucose transporter type 1 (GLUT1) gene in mature B cells (GLUT1-cKO) results in normal B cell development, but it reduces germinal center B cells and ASCs. GLUT1-cKO mice show decreased antigen-specific antibody titers after vaccination. In vitro, GLUT1-deficient B cells show impaired activation, whereas established plasmablasts abolish glycolysis, relying on mitochondrial activity and fatty acids. Transcriptomics and metabolomics reveal an altered anaplerotic balance in GLUT1-deficient ASCs. Despite attempts to compensate for glucose deprivation by increasing mitochondrial mass and gene expression associated with glycolysis, the tricarboxylic acid cycle, and hexosamine synthesis, GLUT1-deficient ASCs lack the metabolites for energy production and mitochondrial respiration, limiting protein synthesis. We identify GLUT1 as a critical metabolic player defining the germinal center response and humoral immunity.

[Mechanism about LMP1 of EB Virus Promoting Plasma Blast Differentiation of DLBCL Cell via mTORC1].

OBJECTIVE: To investigate possible mechanism on protien LMP1 expressed by EBV inducing plasmablast differentiation of DLBCL cell via the mTORC1 pathway. METHODS: The expression levels of LMP1 protein, CD38 and the phosphorylation levels of p70S6K in EBV+ and EBV- DLBCL cell lines were detected by Western blot. Cell lines overexpressing LMP1 gene stablely were constructed and LMP1 gene was silenced by RNAi. The expression of LMP1 gene was verified by RT-qPCR. The expression levels of LMP1 and CD38 and the phosphorylation levels of p70S6K in each group were detected by Western blot. RESULTS: Compared with EBV-DLBCL cells, the expression of LMP1 was detected on EBV +DLBCL cells (P =0.0008), EBV +DLBCL cells had higher phosphorylation levels of p70S6K (P =0.0072) and expression levels of CD38(P =0.0091). Compared with vector group, the cells of LMP1OE group had higher expression levels of LMP1 and CD38 (P =0.0353; P <0.0001), meanwhile molecular p70S6K was phosphorylated much more(P =0.0065); expression of LMP1 mRNA was verified(P <0.0001). Compared with si-NC group, expression level of LMP1 protein(P =0.0129) was not detected and phosphorylated p70S6K disappeared of LMP1KO group (P =0.0228); meanwhile, expression of CD38 decreased,although there was no significant difference (P =0.2377). CONCLUSION: LMP1 promotes DLBCL cells plasmablast differentiation via activating mTORC1 signal pathway.

Case Report: Gene expression profiling of COVID-19 vaccination-related lymphadenopathies reveals evidence of a dominantly extrafollicular immune response.

mRNA-based vaccines against SARS-CoV-2 have been proven to be very efficient in preventing severe COVID-19. Temporary lymphadenopathy (LA) has been observed as a common adverse event following immunization. Here we describe a case series of three female patients with prominent local to generalized LA after SARS-CoV-2 mRNA-1273 vaccination, which led to lymph node biopsy due to the suspicion of lymphoma or metastasis. All three patients morphologically showed similar patterns of follicular hyperplasia and especially extrafollicular blast activation. Two of the three patients only had short-lasting humoral immune responses to the vaccination. Gene expression profiling (GEP) using the HTG Immune response panel revealed that all three patients clustered together and clearly differed from the GEP-patterns of COVID-19, infectious mononucleosis and non-specific follicular hyperplasia. The closest similarities were seen with lymph nodes showing extrafollicular activation of B-blasts as well as hemophagocytosis. The GEP of the vaccination-induced LA was reminiscent of an immune response with little potential of immunologic memory. mRNA-1273 vaccination-induced LA may to a certain extend reflect disordered immune response with potentially poor immunologic memory in affected individuals.

The spectrum of B cells in the pathogenesis, diagnosis and therapeutic applications of immunoglobulin G4-related disease.

Immunoglobulin G4 (IgG4)-related disease is a chronic fibroinflammatory disease mediated by immune disorders. Given the challenging clinical diagnosis and treatment, knowledge of the pathogenesis of IgG4-related disease is important. The typical elevation of serum IgG4 concentrations and infiltration of IgG4-positive plasma cells in the involved tissues indicate the involvement of B lymphocytes in the pathogenesis of IgG4-related disease. Mass production of autoantibodies reflects abnormal activation of B cells, which causes tissue damage. Circulating plasmablasts are recently discovered markers that correlate with serum IgG4 concentration, the extent of organ involvement and disease activity. B-cell depletion therapy is an emerging curative strategy that can significantly alleviate clinical manifestations and achieve remission in patients with IgG4-related disease. These findings highlight the potential role of B cells in IgG4-related disease. In this review, we discuss the pathogenic impact of B lymphocytes on IgG4-related disease and describe novel therapies targeting B cells.

Early Cytomegalovirus Reactivation in Renal Recipients Is Associated with High Levels of B Cell Maturation Antigen Transcript Expression Prior to Transplantation.

Cytomegalovirus (CMV) infection is the most frequent infection episode in kidney transplant (KT) recipients. Reactivation usually occurs in the first three months after transplantation and is associated with higher cellular and/or antibody-mediated rejection rates and poorer graft performance. CMV induces the expression of BAFF (B-cell-activating factor, a cytokine involved in the homeostasis of B cells), which communicates signals for survival and growth to B cells and virus-specific plasma cells via the R-BAFF (BAFF receptor), TACI (the calcium modulator, the cyclophilin ligand interactor), and BCMA (B cell maturation antigen) receptors. These molecules of the BAFF system have also been suggested as biomarkers for the development of alloantibodies and graft dysfunction. This prospective study included 30 CMV-IgG seropositive KT recipients. The expression levels of the genes BAFF-R, transmembrane activator and CAML interactor (TACI), and B cell maturation antigen (BCMA) in peripheral blood leukocytes (PBL) pre-KT were determined using qPCR. qPCR was also used to monitor CMV reactivation in the first three months following KT. The remainder of the KT recipients were classified as CMV- reactivation, and those with more than 500 copies/mL in at least one sample were classified as CMV+ reactivation. There were no discernible variations in the BAFF-R and TACI transcript expression levels. In the CMV+ group, we examined the relationship between the transcript levels and peak viremia. Peak viremia levels and BCMA transcript levels showed a strong correlation. BAFF-R and TACI expressions showed no measurable differences. In patients with early CMV reactivation, high BCMA receptor expression was associated with increased plasmablast, lymphocyte B cell class-switched levels (LBCS), and viral load. Our findings demonstrate that pre-KT BCMA transcript levels increased in KT recipients with early CMV reactivation. These transcript levels positively correlate with peak viremia and weakly with plasmablast and LBCS levels in PBLs.

Plasma cells are not restricted to the CD27+ phenotype: characterization of CD27-CD43+ antibody-secreting cells.

Circulating antibody-secreting cells are present in the peripheral blood of healthy individuals reflecting the continued activity of the humoral immune system. Antibody-secreting cells typically express CD27. Here we describe and characterize a small population of antibody-secreting class switched CD19+CD43+ B cells that lack expression of CD27 in the peripheral blood of healthy subjects. In this study, we characterized CD27-CD43+ cells. We demonstrate that class-switched CD27-CD43+ B cells possess characteristics of conventional plasmablasts as they spontaneously secrete antibodies, are morphologically similar to antibody-secreting cells, show downregulation of B cell differentiation markers, and have a gene expression profile related to conventional plasmablasts. Despite these similarities, we observed differences in IgA and IgG subclass distribution, expression of homing markers, replication history, frequency of somatic hypermutation, immunoglobulin repertoire, gene expression related to Toll-like receptors, cytokines, and cytokine receptors, and antibody response to vaccination. Their frequency is altered in immune-mediated disorders. Conclusion: we characterized CD27-CD43+ cells as antibody-secreting cells with differences in function and homing potential as compared to conventional CD27+ antibody-secreting cells.

Antibody-Secreting Cell Isolation from Different Species for Microfluidic Antibody Hit Discovery.

The recent advent of microfluidic-assisted antibody hit discovery as standard methodology accelerated pharmaceutical research. While work on compatible recombinant antibody library approaches is ongoing, the major source of antibody-secreting cells (ASCs) remains to be primary B cells of mostly rodent origin. As fainting viability and secretion rates can lead to false-negative screening results, careful preparation of these cells is an essential prerequisite for successful hit discovery. We here describe procedures to enrich plasma cells from relevant tissues of mice and rats and plasmablasts from human blood donations. Although freshly prepared ASCs yield the most robust results, suitable freezing and thawing protocols to preserve the viability and antibody secretory function can circumvent extensive process time and allow transferring of samples between laboratories. An optimized procedure is described to yield similar secretion rates after prolonged storage when compared to freshly prepared cells. Finally, the identification of ASC-containing samples can increase the probability of success of droplet-based microfluidics-two methods for pre- or in-droplet staining are described. In summary, the preparative methods described herein can facilitate robust and successful microfluidic antibody hit discovery.

Increasing human monoclonal antibody cloning efficiency with a whole-cell modified immunoglobulin-capture assay (mICA).

Expression cloning of fully human monoclonal antibodies (hmAbs) is seeing powerful utility in the field of vaccinology, especially for elucidating vaccine-induced B-cell responses and novel vaccine candidate antigen discovery. Precision of the hmAb cloning process relies on efficient isolation of hmAb-producing plasmablasts of interest. Previously, a novel immunoglobulin-capture assay (ICA) was developed, using single protein vaccine antigens, to enhance the pathogen-specific hmAb cloning output. Here, we report a novel modification of this single-antigen ICA using formalin-treated, fluorescently stained whole cell suspensions of the human bacterial invasive pathogens, Streptococcus pneumoniae and Neisseria meningitidis. Sequestration of IgG secreted by individual vaccine antigen-specific plasmablasts was achieved by the formation of an anti-CD45-streptavidin and biotin anti-IgG scaffold. Suspensions containing heterologous pneumococcal and meningococcal strains were then used to enrich for polysaccharide- and protein antigen-specific plasmablasts, respectively, during single cell sorting. Following application of the modified whole-cell ICA (mICA), ~61% (19/31) of anti-pneumococcal polysaccharide hmAbs were cloned compared to 14% (8/59) obtained using standard (non-mICA) methods - representing a ~4.4-fold increase in hmAb cloning precision. A more modest ~1.7-fold difference was obtained for anti-meningococcal vaccine hmAb cloning; ~88% of hmAbs cloned via mICA versus ~53% cloned via the standard method were specific for a meningococcal surface protein. VDJ sequencing revealed that cloned hmAbs reflected an anamnestic response to both pneumococcal and meningococcal vaccines; diversification within hmAb clones occurred by positive selection for replacement mutations. Thus, we have shown successful utilization of whole bacterial cells in the ICA protocol enabling isolation of hmAbs targeting multiple disparate epitopes, thereby increasing the power of approaches such as reverse vaccinology 2.0 (RV 2.0) for bacterial vaccine antigen discovery.

Thymus antibody-secreting cells: once forgotten but not lost.

Antibody-secreting cells are essential contributors to the humoral response. This is due to multiple factors which include: 1) the ability to secrete thousands of antibodies per second, 2) the ability to regulate the immune response and 3) the potential to be long-lived. Not surprisingly, these cells can be found in numerous sites within the body which include organs that directly interface with potential pathogens (e.g., gut) and others that provide long-term survival niches (e.g., bone marrow). Even though antibody-secreting cells were first identified in the thymus of both humans and rodents in the 1960s, if not earlier, only recently has this population begun to be extensively investigated. In this article, we provide an update regarding the current breath of knowledge pertaining to thymus antibody-secreting cells and discuss the potential roles of these cells and their impact on health.

Metabolic Reprogramming During B-Cell Differentiation.

B cells engaging with antigen and secondary signals provided by T cell help, or ligands for Toll-like receptors, undergo a step-wise process of differentiation to eventually produce antibody-secreting plasma cells. During the course of this conversion, the cells transition from a resting, non-growing state to an activated B-cell state engaged in DNA synthesis and mitosis to a terminally differentiated, quiescent cell state with expanded organelles necessary for high levels of secretion. Each of these phases is accompanied by considerable changes in metabolic requirements. To facilitate evaluation of this metabolic reprogramming, methods for the in vitro differentiation of human B cells that incorporates each of the transitional stages are described.

Intrinsically determined turnover underlies broad heterogeneity in plasma-cell lifespan.

Antibodies produced by antibody-secreting plasma cells (ASCs) underlie multiple forms of long-lasting immunity. Here we examined the mechanisms regulating ASC turnover and persistence using a genetic reporter to time-stamp ASCs. This approach revealed ASC lifespans as heterogeneous and falling on a continuum, with only a small fraction surviving for >60 days. ASC longevity past 60 days was independent of isotype but correlated with a phenotype that developed progressively and ultimately associated with an underlying "long-lived" ASC (LL ASC)-enriched transcriptional program. While some of the differences between LL ASCs and other ASCs appeared to be acquired with age, other features were shared with some younger ASCs, such as high CD138 and CD93. Turnover was unaffected by altered ASC production, arguing against competition for niches as a major driver of turnover. Thus, ASC turnover is set by intrinsic lifespan limits, with steady-state population dynamics governed by niche vacancy rather than displacement.

Plasmablasts in previously immunologically naïve COVID-19 patients express markers indicating mucosal homing and secrete antibodies cross-reacting with SARS-CoV-2 variants and other beta-coronaviruses.

Antigen-specific class-switched antibodies are detected at the same time or even before IgM in serum of non-vaccinated individuals infected with SARS-CoV-2. These derive from the first wave of plasmablasts formed. Hence, the phenotype and specificity of plasmablasts can reveal information about early B-cell activation. Here we have analyzed B cells and plasmablasts circulating in blood of COVID-19 patients not previously exposed to SARS-CoV-2 during and after disease. We find that during infection with the original Wuhan strain, plasmablasts in blood produce IgA1, IgG1, and IgM, and that most express CCR10 and integrin ß1, only some integrin ß7, while the majority lack CCR9. Plasmablast-secreted antibodies are reactive to the spike (S) and nucleocapsid (N) proteins of the Wuhan strain as well as later variants of concern, but also bind S proteins from endemic and non-circulating betacoronaviruses. In contrast, after recovery, antibodies produced from memory B cells target variants of SARS-CoV-2 and SARS-CoV-1 but compared to previously non-infected individuals do not show increased binding to endemic coronaviruses. This suggests that the early antibody response to a large extent stems from pre-existing cross-reactive class-switched memory B cells, and that although newly formed memory cells target the novel SARS-CoV-2 virus the numbers of broadly cross-reactive memory B cells do not increase extensively. The observations give insight into the role of pre-existing memory B cells in early antibody responses to novel pathogens and may explain why class-switched antibodies are detected early in the serum of COVID-19 patients.

Case report: Immune profiling links neutrophil and plasmablast dysregulation to microvascular damage in post-COVID-19 Multisystem Inflammatory Syndrome in Adults (MIS-A).

Despite surviving a SARS-CoV-2 infection, some individuals experience an intense post-infectious Multisystem Inflammatory Syndrome (MIS) of uncertain etiology. Children with this syndrome (MIS-C) can experience a Kawasaki-like disease, but mechanisms in adults (MIS-A) are not clearly defined. Here we utilize a deep phenotyping approach to examine immunologic responses in an individual with MIS-A. Results are contextualized to healthy, convalescent, and acute COVID-19 patients. The findings reveal systemic inflammatory changes involving novel neutrophil and B-cell subsets, autoantibodies, complement, and hypercoagulability that are linked to systemic vascular dysfunction. This deep patient profiling generates new mechanistic insight into this rare clinical entity and provides potential insight into other post-infectious syndromes.

Analysis of Plasmablasts From Children With Kawasaki Disease Reveals Evidence of a Convergent Antibody Response to a Specific Protein Epitope.

BACKGROUND: Kawasaki disease (KD) is a febrile illness of young childhood that can result in coronary artery aneurysms and death. Coronavirus disease 2019 (COVID-19) mitigation strategies resulted in a marked decrease in KD cases worldwide, supporting a transmissible respiratory agent as the cause. We previously reported a peptide epitope recognized by monoclonal antibodies (MAbs) derived from clonally expanded peripheral blood plasmablasts from 3 of 11 KD children, suggesting a common disease trigger in a subset of patients with KD. METHODS: We performed amino acid substitution scans to develop modified peptides with improved recognition by KD MAbs. We prepared additional MAbs from KD peripheral blood plasmablasts and assessed MAb characteristics that were associated with binding to the modified peptides. RESULTS: We report a modified peptide epitope that is recognized by 20 MAbs from 11 of 12 KD patients. These MAbs predominantly use heavy chain VH3-74; two-thirds of VH3-74 plasmablasts from these patients recognize the epitope. The MAbs were nonidentical between patients but share a common complementarity-determining region 3 (CDR3) motif. CONCLUSIONS: These results demonstrate a convergent VH3-74 plasmablast response to a specific protein antigen in children with KD, supporting one predominant causative agent in the etiopathogenesis of the illness.