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Studies on the humoral response to homologous BNT162b2 mRNA-vaccination focus mainly on IgG antibody dynamics, while long-term IgA kinetics are understudied. Herein, kinetics of IgG and IgA levels against trimeric-Spike (S) and Receptor-Binding-Domain (RBD) were evaluated by in-house ELISAs in 146 two-dose vaccinated Greek healthcare workers (HCWs) in a 9-month period at six time points (up to 270 days after the first dose). The effect of a homologous booster third dose was also studied and evaluated. The peak of immune response was observed 21 days after the second dose; 100% seroconversion rate for anti-S and anti-RBD IgG, and 99.7% and 96.3% respectively for IgA. IgG antibody levels displayed higher increase compared to IgA. Declining but persistent anti-SARS-CoV-2 antibody levels were detected 9 months after vaccination; IgG and IgA anti-S levels approached those after the first dose, while a more rapid reduction rate for anti-RBD antibodies led to significantly lower levels for both classes, supporting the need for a booster dose. Indeed, a homologous booster third dose resulted in enhanced levels of anti-S of both classes, whereas anti-RBD didn’t exceed the peak levels after the second dose. Previous SARS-CoV-2 infection, flu vaccination, BMI<35 and the occurrence of an adverse event upon vaccination, were associated with higher IgG antibody levels over time, which however were negatively affected by age increase and the presence of chronic diseases. Overall, after concurrently using the S and RBD target-antigens in in-house ELISAs, we report in addition to IgG, long-term persistence of IgA antibodies. Regarding antibody levels, homologous mRNA vaccination gives rise to an effective anti-viral protection up to 9 months negatively correlated to age. Considering that COVID-19 is still a matter of public concern, booster vaccine doses remain critical to vulnerable individuals.

The IGHV4-34 gene is intrinsically autoreactive due to carrying a germline(GL)-encoded (super)antigenic motif binding various self (and exogenous) antigens, while it is one of the few IGHV genes that contain a GL-encoded N-glycosylation (N-glyc) site. IGHV4-34 is overrepresented in chronic lymphocytic leukemia (CLL), particularly in cases expressing B cell receptor immunoglobulin (BcR IG) with a significant load of somatic hypermutation (SHM; 'mutated' CLL, M-CLL). Moreover, a large fraction of IGHV4-34 M-CLL cases are clustered in different stereotyped subsets, of which the best studied is subset #4, the largest within M-CLL, defined by the expression of IgG-switched IGHV4-34/IGKV2-30 BcR IG with a distinctive SHM imprint. Considerably smaller than subset #4 is subset #201, defined by the expression of IGHV4-34/IGLV1-44 BcR IG of the IgMD isotype. Subset #201 is noteworthy owing to recurrent replacement SHMs that frequently lead to the creation of novel N- glyc motifs within the VH domain. This may be functionally relevant, considering that N-linked glycosylation is a widespread post-translational modification that is largely SHM-induced during antigen-specific immune responses and can modulate antibody (Ab) affinity towards antigen. That said, nothing is yet known about the antigen reactivity of subset #201 BcR IG and whether/how it could be affected through SHM-induced changes of N-linked glycosylation. In order to obtain insight into this issue, 4 subset #201 clonotypic IGs were expressed as recombinant monoclonal Abs (mAbs) of the mu isotype in HEK293 human cells, in either the authentic SHM state ('wildtype', WT-mAbs) or after reverting specific SHMs that altered N-glyc sites (R-mAbs) by site-directed mutagenesis. Since not all N-glyc motifs are eventually glycosylated, we used the NetNglyc 1.0 Server (http://www.cbs.dtu.dk/services/NetNGlyc/) for the prediction of N-glycan occupancy. Binding to MEC1 B CLL, Jurkat T and HEK293 cells was assessed by flow cytometry. Reactivity against nuclear Hep-2 cell extract, nDNA, actin, myosin, thyroglobulin (TG), β-amyloid, carbonic anhydrase, F(ab')2 and the non-self hapten trinitrophenyl was tested by ELISA. Non-subset #201 M-CLL mAbs (n=14, including 3 subset #4 mAbs), were used as controls. None of the subset #201 WT-mAbs displayed reactivity in any of the ELISAs. However, unlike most CLL mAbs, all subset #201 WT-mAbs bound to live MEC1 cells, while also exhibiting reactivity to HEK293 cells that was significantly higher when compared to non-subset #201 M-CLL (p=0.0095) or subset #4 (p=0.05); additionally, 1/4 subset #201 mAb displayed weak binding to Jurkat T cells. Three of 4 subset #201 mAbs bore a novel N-glyc site introduced by SHM in codons VL CDR1 36-38 of the clonotypic lambda light chains. Reversion to the GL in one such mAb resulted in enhanced binding to all 3 cell lines [fold change (FC) of binding of the R- vs WT-mAb to MEC1, Jurkat and HEK293: 1.3, 7.9 and 3.3, respectively) and in strong anti-TG activity. The GL-encoded N-glyc site in VH CDR2 57-59, that has been reported to be mostly unoccupied, was targeted by SHM in 2/4 subset #201 mAbs: reversion to GL decreased binding to both MEC1 and HEK293 cells (FC: -8 and -1.4 respectively). Finally, in 2/4 cases, SHM at codons VH FR3 67-68 inserted an N-glyc site that, however, is not predicted to acquire N-glycans. Reversion to GL enhanced the binding of one of these mAbs to MEC1 and HEK293 cells (FC: 2.1 and 5.6, respectively). The same mAb bore an additional predicted N-glyc site introduced by SHM at VH FR3 90-92; reversion of this change to GL augmented binding to both MEC1 and HEK293 cells (FC: 4.1 and 9.7, respectively). Double reversion of both aforementioned SHMs conferred further increased binding than any of the single reversions, implying a synergistic effect. Acquisition of novel N-glyc sites is not an intrinsic characteristic of either M-CLL in general or IGHV4-34 M-CLL in particular and its high incidence in subset #201 implies a selective process likely due to distinct (auto)antigenic pressure. Indeed, subset #201 mAbs exhibit an antigen reactivity profile that differs from that of typical polyreactive mAbs, including natural autoantibodies and other CLL mAbs, binding selectively to viable lymphoblastoid cell line cells and human HEK293 epithelial cells. These results further emphasize the importance of SHM in shaping the distinct (auto)antigenic recognition profile of CLL mAbs. Disclosures Chatzidimitriou: Janssen: Honoraria. Stamatopoulos:Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding.

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Background Oligoarticular juvenile idiopathic arthritis (oligo-JIA) is considered as an antigen-driven lymphocyte-mediated autoimmune disease. Natural antibodies (NAbs) are pre-immune antibodies produced in the absence of exogenous antigen stimulation, participating in both, innate and adaptive immunity. Considering their major immunoregulatory role in homeostasis and autoimmune pathogenesis, we designed this study to further elucidate their role in oligo-JIA pathogenesis. Methods Seventy children with persistent oligo-JIA and 20 healthy matched controls were enrolled in the study. Serum IgM and IgA antibodies against human G-actin, human IgG F(ab΄)2 fragments and the hapten TriNitroPhenol (TNP) as well as the total concentration of serum IgM and IgA were measured by in-house enzyme-immunoassays. Kolmogorov–Smirnov normality test, Kruskal–Wallis H and Mann–Whitney tests were used to assess data distribution, and significant differences of non-parametric data between groups of the study. Backward regression analysis was used to analyze the effect of multiple factors (age, gender, disease activity, anti-nuclear antibody positivity, presence of uveitis) on continuous dependent variables (activities and activity/ concentration ratios of IgM and IgA NAbs). Results The ratios of IgA anti-TNP, anti-actin and anti-F(ab΄)2 levels to total serum IgA concentration were found to be significantly increased in patients with oligo-JIA compared to healthy subjects. Significantly elevated levels of IgM anti-TNP antibodies were also found in children with inactive oligo-JIA compared to those of children with active disease and of healthy controls. In the presence of anterior uveitis, IgM anti-TNP levels were significantly higher than in patients without uveitis or in healthy controls. Backward regression analysis revealed that the disease activity and the presence of anterior uveitis independently affect IgM anti-TNP levels. Conclusuions Our findings are in accordance with the hypothesis that NAbs contribute to the pathogenesis of autoimmune diseases and provide additional evidence that disturbances in natural autoimmunity may contribute to the as yet unclarified pathogenesis of oligo-JIA.

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Splenic marginal-zone lymphoma (SMZL) ontogeny and evolution is a complex process, involving, amongst others, (super)antigenic stimulation, molecular deregulation of genes involved in the physiological differentiation of splenic marginal zone B cells (e.g. NOTCH2 and KLF2), and epigenetic alterations leading to silencing of different tumor suppressor genes and overexpression of oncogenes, including the PRC2-complex (EZH2, EED, and SUZ12). However, despite important recent progress, advances in the fundamental understanding of SMZL have not yet been translated in improved patient management, highlighting the need for further molecular investigations. In this context, here we aimed at detailed characterization of: (i) epigenetic modifiers, particularly the histone methyltransferase EZH2, a marker of clinical aggressiveness and druggable target in lymphoma; (ii) immune signaling capacity of SMZL B cells; and, (iii) antigen reactivity profile of the SMZL clonotypic B Cell Receptor immunoglobulin (BcR IG). Quantification of EZH2 mRNA levels using quantitative Real-time PCR (n=26 SMZL patients) and protein levels using western blotting (WB) (n=14) revealed that EZH2 was expressed in all cases yet in a heterogeneous manner (2^-ΔCt range: 0.079-1.38; ΕΖΗ2+ cells: 3.11%-44.8%). Interestingly, higher proliferation rates (Ki67+ cells) were observed in cases with high EZH2 protein levels (n=4) compared to cases with low EZH2 (n=6) (FD=2.6; p&lt;0.05). Stimulation of SMZL CD19+ B cells (n=8) through i) the BcR with anti-IgM ii) Toll-Like receptor 9 (TLR9) with CpG and iii) BcR/TLR for 60 minutes affected pBTK, pERK and pNF-kB levels as revealed by WB and flow cytometry (FCM). Although, great heterogeneity was observed in the responses between different cases, categorization of SMZL cases based on EZH2 levels showed that, at basal level, EZH2high cases expressed higher pBTK, pERK and pNF-κB compared to EZH2low cases (FC=2.4, FC=6,1, FC=12.7, respectively; p&lt;0.05 for all comparisons). BcR and TLR9 activation and, especially, co-stimulation with anti-IgM/CpG induced the expression of pBTK, pERK and pNF-κB in EZH2low cases, while the opposite was observed in EZH2high cases. Moreover, stimulation through both the BcR and BcR/TLR9 for 24 hours could also induce EZH2 expression compared to the unstimulated control cells (FC=1.6; p&lt;0.05 for both comparisons). To gain insight into the type of antigenic stimuli that could activate BcR signaling through the BcR we investigated the antigen binding profile of 10 SMZL clonotypic BcR IGs expressed as recombinant human IgM monoclonal antibodies (mAbs). Cases were representative of the most common IGHV genes in SMZL (IGHV1-2*04, IGHV4-34 and IGHV3-23). Reactivity (at 15 μg/ml) against various autoantigens was evaluated by ELISA. Five of 9 (56%), 4/7 (57%), 5/10 (50%), 2/9(22%), 2/9 (22%), 3/9 (33%), 3/8 (38%), 4/8 (50%) and 4 of 9 (44%) SMZL IgM mAbs displayed reactivity against native DNA, nuclear Hep-2 cell extract, actin, myosin, thyroglobulin, β-amyloid, carbonic anhydrase, F(ab')2 and the hapten trinitrophenyl respectively, with optical density (OD) values greater than the cut-off point. Moreover, 5/10 (50%) SMZL IgM mAbs recognized more than 2 antigens and were characterized as polyreactive. Each of these SMZL mAbs exhibited a distinct antigen polyreactivity profile, while heterogeneity was observed even amongst cases expressing the same gene or cases with similar IGHV gene mutational load. Recognition of epitopes on the surface of viable MEC1 B and HEK293 cells was assessed by FCΜ. Two of 3 and 5/11 mAbs bound to viable MEC1 B cells and HEK293 cells, respectively: binding to HEK293 cells was more pronounced for minimally mutated/unmutated versus mutated mAbs (p&lt;0.05). Importantly, stimulation of SMZL CD19+ B cells with the SMZL BcR cognate antigens actin and myosin resulted in regulation of pERK/pPLCγ2 in 3/6 and 1/2 cases, respectively, as revealed by WB. Overall, SMZL cells can be activated by microenvironmental signals albeit in a different manner depending on EZH2 levels, highlighting links between the epigenetic and the signaling machinery with potential therapeutic implications. Moreover, the recognition of a wide range of autoantigens by the SMZL mAbs indicates that SMZL B cell clones may arise from polyreactive B cells, likely resident in the normal splenic marginal zone. Disclosures Stamatopoulos: Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding.