Memory B Cells Research Articles

Durability and cross-reactive immune memory to SARS-CoV-2 in individuals 2 years after recovery from COVID-19: a longitudinal cohort study

SARS-CoV-2-specific adaptive immunity more than 1 year after initial infection has not been well characterised. The aim of this study was to investigate the durability and cross-reactivity of immunological memory acquired from natural infection against SARS-CoV-2 in individuals recovered from COVID-19 2 years after infection. In this longitudinal cohort study, we recruited patients who had recovered from laboratory-confirmed COVID-19 and were discharged from Jinyintan Hospital (Wuhan, China) between Jan 7 and May 29, 2020. We carried out three successive follow-ups between June 16 and Sept 3, 2020 (6 months), Dec 16, 2020, and Feb 7, 2021 (1 year), and Nov 16, 2021, and Jan 10, 2022 (2 years), in which blood samples were taken. We included participants who did not have re-infection or receive a SARS-CoV-2 vaccination (infected-unvaccinated), and participants who received one to three doses of inactivated vaccine 1-2 years after infection (infected-vaccinated). We evaluated the presence of IgG antibodies, neutralising antibodies, and memory B-cell and memory T-cell responses against the prototype strain and delta and omicron variants. In infected-unvaccinated participants, neutralising antibody titres continually declined from 6-month to 2-year follow-up visits, with a half-life of about 141·2 days. Neutralising antibody responses to omicron sublineages (BA.1, BA.1.1, BA.2, BA.4/5, BF.7, BQ.1, and XBB) were poor. Memory B-cell responses to the prototype strain were retained at 2 years and presented cross-reactivity to the delta and omicron BA.1 variants. The magnitude of interferon γ and T-cell responses to SARS-CoV-2 were not significantly different between 1 year and 2 years after infection. Multifunctional T-cell responses against SARS-CoV-2 spike protein and nucleoprotein were detected in most participants. Recognition of the BA.1 variant by memory T cells was not affected in most individuals. The antibody titres and the frequencies of memory B cells, but not memory T cells, increased in infected-vaccinated participants after they received the inactivated vaccine. This study improves the understanding of the duration of SARS-CoV-2-specific immunity without boosting, which has implications for the design of vaccination regimens and programmes. Our data suggest that memory T-cell responses primed by initial viral infection remain highly cross-reactive after 2 years. With the increasing emergence of variants, effective vaccines should be introduced to boost neutralising antibody and overall T-cell responses to newly emerged SARS-CoV-2 variants. Chinese Academy of Medical Sciences, National Natural Science Foundation of China, Fundamental Research Funds for the Central Universities for Peking Union Medical College, Beijing Natural Science Foundation, UK Medical Research Council.

Alteration in innate immune cues assessed by analyzing peripheral blood immune system in post-covid patients

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has led to global morbidity and mortality. Some patients fully recover from COVID-19, whereas around 45% subjects suffer from various persistent symptoms (fatigue, cognitive impairment, impaired thermoregulation, skin diseases, etc.) for at least four months after SARS-CoV-2 infection regardless of disease severity. Such persistent post-infection effects are known as long-COVID, post-acute effects of COVID-19, or post-COVID state. SARS-CoV-2 infection is accompanied by damage to the innate immune system. Considering the role of natural killer cells and the activation of the complement system in COVID-19 as well as the regulatory properties related to CD46 and its potential involvement in cell virus entry, we found necessary to study immune system parameters associated with impairment of these innate immune cues on various leukocyte subpopulations in post-COVID patients. We studied 92 immune system parameters, including: pan-leukocyte markers for gated lymphocytes, phenotyping of T cells, T-helper inducers, cytotoxic T-lymphocytes, NK- and TNK-cells, T-regulatory cells/suppressors, B-lymphocytes, including B-memory cells, activated helpers, early activation of lymphocytes, activated T-lymphocytes, and late lymphocyte activation markers. Levels of total IgM, IgG, IgA, specific IgM, IgG to coronavirus COVID-19, C1-inhibitor, C3a, and C5a complement components were measured by enzyme immunoassay using Multiscan FC Thermoscientific enzyme immunoassay analyzer (China) and Vector Best reagent kits (Russia). A complete blood count was conducted to study 25 parameters: leukocyte, erythrocyte, and platelet hematopoietic lineages as well as the quantitative and qualitative composition of hematopoietic lineages. Our study results showed that in some patients, six months after suffering from COVID-19, there was a decrease in the level of NK cells (48%) and CD46+pan-leukocyte marker cells (64.5%). A decrease in NK cell levels was accompanied by increased level of total T- and B-lymphocytes, and altered platelet and erythroid hematopoietic lineages. In patients with reduced CD46 expression on T-lymphocytes, both their total count and NK cell count were significantly reduced. Our data also suggest that CD46 might be potentially involved in development of SARS-CoV-2 infection and the post-COVID state. Thus, in 50–65% of patients who have experienced SARS-CoV-2 infection, damage to the innate immune system persists after six months being accompanied by impaired erythroid and platelet hematopoietic lineages. The data obtained indicate a need for using immunocorrective therapy in such patients.

Integrative Single-Cell RNA-Seq and Single-Cell ATAC-Seq Analysis of Human Plasma Cell Differentiation

Plasma cells (PC) are highly specialized cells representing the end stage of B cell differentiation. They play an important role in humoral immunity by synthesizing and secreting antibodies protecting the host against infections. We have shown that PC differentiation (PCD) can be reproduced in vitrofrom memory B cells (MBC). MBC differentiate into CD20 low/-CD38 - pre-plasmablasts (prePB), CD20 -CD38 +CD138 - plasmablasts (PB) and CD20 -CD38 +CD138 + PC. The molecular changes occurring during PCD are recapitulated in this in vitrodifferentiation model. However, a major challenge exists to decipher the spatiotemporal epigenetic and transcriptional programs that drives the early stages of PCD. We combined single-cell (sc) RNA-seq and scATAC-seq to decipher the trajectories involved in PCD. scRNA-seq experiments on the four populations (MBC, prePB, PB and PC) generated during normal PCD revealed a highly specific transcriptomic profile for MBC and PC, and a strong heterogeneity of the prePB and PB. The prePB stage presented the most differentially expressed genes (DEG) with almost 2000 DEG showing that the most important changes take place during this stage. Epigenetic analysis using scATAC-seq technology showed that prePB, PB and PC stages were clearly separated from the MBC highlighting chromatin remodeling induced by B cell activation and differentiation. As observed on the transcriptomic level, the number of differentially accessible peaks was higher in prePB (4660) than in other stages (MBC: 641; PB: 44; PC: 105). Among genes that were commonly identified using scATAC-seq and RNA-seq, we identified several TF such as BATF and BATF3 in prePB. Interestingly, differentially accessible peaks characterizing the prePB stage were enriched in motifs of BATF3, FOS and BATF belonging to the TF AP-1 family and among these differentially accessible peaks, 38% of peaks presented BATF3 motif. Since BATF3 TF was previously identified operating in short impulse manner at prePB stage, BATF3 target genes may represent a key transcriptional node involved in PCD. The integration of the chromatin accessibility and transcriptomic data revealed a more mature population of prePB cells characterized by open chromatin in PC genes without significant expression suggesting that a population of prePB were already committed to become antibody-secreting cells. To focus on the understanding of the processes occurring during the transition from prePB to PB which remains largely unknown, we computationally clustered and ordered cells. Pseudotemporal analyses underlined maturation trajectories in prePB with early prePB characterized by downregulation of B cell markers ( CD19, CD22, CD83, CCR7, CCL17 and CCL22) and B cell TF (SPIB, PAX5, STAT5A, RUNX3 and BATF3) together with upregulation of PC markers, adhesion molecules and growth factor receptors ( CD27, CD38, SLAMF7, BCMA, ITGA4, IL-6R, IL-6ST and INSR). The transition from early prePB to more mature prePB is associated with downregulation of AICDA and PRC2 complex ( EZH2, EED). In the transitional prePB, a first wave of UPR activation is observed, associated with mTORC1 pathway activation. In these prePB, HSAP5, also called binding immunoglobulin protein (BiP) is overexpressed together with ERN1, leading to the activation of the Ire1 pathway, known to splice XBP-1 (sXBP-1) producing a highly active TF. Then, prePB overexpress EGR1, FOS, CXCR4 and TFRC. Mature prePB overexpress KLF2 that participate in bone marrow PC homing. Then, PB overexpress gene involved in conventional UPR and protein secretion associated to immunoglobulin gene expression. Altogether, these data underlined that prePB present already UPR priming through mTORC1 pathway activation to prepare for PC function. XBP1 driven UPR activation will be coordinated in PB related to antibody synthesis increase. Integration of transcriptomic and epigenetic data at the single cell level revealed that a population of prePB already undergone epigenetic remodeling related to PC profile together with UPR activation and are committed to differentiate in PC. These results and the supporting data generated with our PCD model provide a resource for the identification of molecular circuits that are crucial for early and mature PC biological function and survival. These data thus provide critical insights into epigenetic- and transcriptional-mediated reprogramming events that sustain PCD.

Novel Isoforms Identified By Isoseq Analysis Drive Expression Differences in Key Genes That Delineate the Subtypes of Waldenstrom's Macroglobulinemia

Background: Alternative isoforms can alter regulatory motifs, binding partners, localization, and function of gene or even inhibit its function. While isoform regulation in humans is generally well described, there are still many tissue specific isoforms that remain to be fully documented. Moreover, mutations and broad dysregulation of transcriptional and RNA processing machinery is common in cancer resulting in additional novel isoforms. Analysis of 249 MYD88 mutated patients from our multi-omic data set of 253 treatment-naive patients with Waldenstrom's Macroglobulinemia (WM) identified three subtypes of WM: B-cell like (BCL), Plasma cell like (PCL), and an intermediate clone enriched for early/smoldering WM from which the other two evolved. (Hunter et al, ASH 2022) The BCL and PCL subtypes have distinct clinical as well as mutational and transcriptional characteristics. To investigate isoform usage, including novel disease related isoforms, we utilized PacBio single molecule real-time (SMRT) sequencing which can produce reads up to 10Kb allowing for end-to-end single transcript sequences for most genes. Methods: PacBio Isoseq analysis was performed on RNA isolated from CD19+ bone marrow (BM) samples from 11 WM patients; CD19+CD27+ memory B-cells (MB) from two healthy donors; and CD138+ plasma cell (PC) from two healthy donors. SMRT analysis data was aligned and processed using minimap2 with Cupcake. Observed isoforms were filtered, categorized, and annotated using Squanti. High confidence novel isoforms were then added into hg38 Gencode annotation which was used to realign our existing high depth Illumina 150bp paired end RNASeq data set of 249 untreated MYD88 mutated WM patients using STAR and Salmon. DTU analysis and functional annotation was performed using the isoformSwitchAnalyzeR package from Bioconductor in R. Results: The 11 patients in PacBio novel isoform discovery cohort had a median age of 63 (range 48-78 years) at time of sample acquisition; serum IgM of 2,150 (598-5,830 mg/dL); and BM involvement of 75% (range 20-95%). Six (55%) were female; 4 (36%) had a familial history of WM; 4 (36%) had a familial history of other B-cell malignancies; and 3 (27%) were sporadic. MYD88 and CXCR4 mutations were present in 10/11 (91%) and 6/11 (55%) patients, respectively. In the MYD88 mutated RNASeq cohort, 102/249 (41%) were female, the median age at biopsy was 66 (range: 31-95 years), BM involvement was 50% (5-95%), and the median serum IgM was 3,162 (104-10,321 mg/dL). PacBio analysis added 194,997 novel isoforms to the gencode annotation which ranged in function from minor alterations in untranslated regions (UTRs) to the introduction of novel coding sequences. Of the 3,134 genes differentially expressed between BCL and PCL, 1,175 (37.5%) and 1,149 (36.6%) had greater that 50% of their expression assigned to novel isoforms for BCL and PCL, respectively (p=NS), which highlights the impact of the improved annotation. The top result from the DTU analysis was DUSP22, a negative regulator of MAPK and ERK1/2 signaling ( Figure 1). DUSP22 was overexpressed in early/smoldering WM and PCL, with overexpression driven by a novel internal start with a partial intron inclusion. These novel isoforms express only half of DUSP22's functional domain but also contain novel 5' signal peptide sequences targeting the transcript to the endoplasmic reticulum for translation. Another top result was FCRLB, an FC receptor that can promote survival in B-lymphocytes. In HD and BCL, the minimal expression corresponded to a canonical non-coding isoform, while in PCL and early/smoldering WM most of the expression resulted from novel coding isoforms that contained the core protein coding regions of the gene. Other genes with significant DTU included HDAC4, HDAC9, and ARID5B all of which are critical modulators of chromatin accessibility. Functional validation of novel isoforms and DTU analysis of additional genomic, and clinical factors is ongoing. Conclusions: Novel transcripts identified by PacBio IsoSeq analysis drive expression of key genes including the important MAPK/ERK regulator DUSP22, and FC receptor FCRLB that promotes survival of B-lymphocytes. Other genes of interest included modulators of chromatic accessibility. Our findings demonstrate important new insights into isoform usage associated with WM subtype in WM and provide novel insights into the underlying biology of the disease.

Rhythmic profile of memory T and B-cells along childhood and adolescence

Immunobiography describes the life-long effects of exogenous or endogenous stimuli on remodeling of immune cell biology, including the development of memory T and B-cells. The present study aimed at investigating the rhythms of changes in phenotypic features of memory T and B-cells along childhood and adolescence. A descriptive-observational investigation was conducted including 812 healthy volunteers, clustered into six consecutive age groups (9Mths–1Yr; 2Yrs; 3–4Yrs; 5–7Yrs; 8–10Yrs; 11–18Yrs). Immunophenotypic analysis of memory T-cell (CD4+ and CD8+) and B-cell subsets were performed by flow cytometry. The results pointed out that memory-related biomarkers of T and B-cells displayed a bimodal profile along healthy childhood and adolescence, regardless of sex. The first stage of changes occurs around 2Yrs, with predominance of naive cells, while the second and more prominent wave occurs around the age 8–10Yrs, with the prevalence of memory phenotypes. The neighborhood connectivity profile analysis demonstrated that the number of correlations reaches a peak at 11–18Yrs and lower values along the childhood. Males presented higher and conserved number of correlations when compared to females. Altogether, our results provide new insights into immunobiography and a better understanding of interactions among the cellular subsets studied here during childhood and adolescence.

Early Subclones Showing Aberrant Co-Expression of Stem Cell, T Cell and Myeloid Genes Can be Detected By Flow Cytometry in MYD88 Mutated Waldenström's Macroglobulinemia Patients

Background: Our RNA-Seq analysis of 249 untreated MYD88 mutated WM patients showed aberrant expression of stem cell, pre-B-cell, T-cell and myeloid genes in early WM B clones. This expression pattern was associated with inferior progression free survival with BTK inhibitor therapy and diminished with advancing disease. We therefore sought to validate this early signature at the protein level using flow cytometry and evaluate its potential as a clinical assay to monitor WM disease evolution. Methods: RNA-Seq predictions were validated on an independent cohort of treatment naïve MYD88 mutated WM patients and healthy (HD) samples. We designed a 13-color panel for flow cytometry detection of surface markers CD19, CD138, k and l immunoglobulin (Ig) light chains, CD34, CD117 (KIT), CD4, CD8A, CD33, FCGR3A (CD16), CEACAM8 (CD66b), pre-BCR (using an antibody that recognizes a unique conformational epitope of the BCR complex formed by the association of IGLL1 and VPREB1); Zombie Yellow was used to label dead cells. Peripheral B (PBMC) and/or BM mononuclear cells (BMMC) were isolated from WM and HD samples by density gradient centrifugation using Ficoll-Paque medium. Data acquisition was performed for at least 10^6 nucleated cells per tube in BDLSR Fortessa flow cytometer using the FACSDiva software. Results: RNA-Seq analysis showed high expression of genes atypical of mature B-cells that significantly diminished with increasing disease burden. For validation purposes, we selected genes reaching at least a 10-fold drop in their expression levels between the earliest and the most advanced disease cohort. In particular, the T-cell and myeloid genes including CD4, CD8A, FCGR3A, CD33, CEACAM8 as well as the prePCR surrogate light chain genes IGLL1 and VREPB1 respectively decreased from 67.6, 7.1, 80.5, 23.2, 8.3, 114, 38.3 to 2.4, 0.9, 1.7, 0.7, 0.4, 12.2 and 4.3 transcripts per million (TpM). A subclonal stem cell signature was detected, including CD34 and KIT, which dropped from 1.67 and 0.64 to 0.15 and 0.06 TpM, respectively. It is well established that WM represents a mature, post germinal center clone. Consistent with this notion, expression of memory B-cell and plasma blast genes in our RNASeq data set, including PRDM1, XBP1 and SPIB, was high in all patients regardless of disease progression and expression of the studied atypical genes. Our independent patient validation cohort had a median age of 68 (range 50-88 years), BM involvement by immunohistochemistry of 40% (range 10-95%), hemoglobin of 13.2 (range 8.1-13.4 g/dL) and serum IgM of 686 (range 536-1,151 mg/dL). An IgMk monoclonal spike was detected in all cases. All patients were mutated for MYD88. Within the CD19+k+-selected cells, we detected a subpopulation of cells co-expressing CD34, KIT, CD4, CD8A, FCGR3A, CD33, CEACAM8 and preBCR in all the patients, including in PBMC. As predicted, these markers were relatively enriched in samples with lower BM disease burden ( Figure 1, 2). Even in those with the highest BM involvement, the subclone was still detectable in both cellular compartments. Conversely, HD controls showed normal B cell populations and no evidence of the aberrant subclone. Conclusions: In WM primary cells, we observed the existence of a clonal B cell population that appears to re-activate stem cell programming early on and progressively is replaced by clones bearing more typical B cell markers with disease expansion. This study lays the foundation to develop a widely accessible methodology to accurately diagnose IgM MGUS/early-stage WM and monitor disease evolution. Flow-based sorting of these subclones will allow targeted multi-omic studies with insights into disease pathogenesis and targeted therapeutic applications.

Atypical Stem Cell, Pre-B-Cell, T-Cell and Myeloid Gene Expression Characterizes Early Waldenstrom's Macroglobulinemia Clones Which Diminishes with Advancing Disease and Has Therapeutic Implications

Background: In previous studies using multi-omic data for 253 treatment-naive patients with Waldenstrom's Macroglobulinemia (WM), we identified three subtypes of WM: B-cell like (BCL), Plasma cell like (PCL), and an intermediate clone enriched for early/smoldering WM from which the other two evolved (Hunter et al, ASH 2022). The BCL and PCL subtypes show distinct mutation and transcriptional signatures, as well as clinical characteristics. Diffusion pseudo-time (DPT) analysis suggests all patient tumors, regardless of subtype have a shared evolutionary path and can be divided into Early and Late DPT determined stages that mirror disease progression. Additional analysis of DPT suggests it represents a continuous process akin to clonal evolution. We therefore sought to further characterize the DPT signal in MYD88 mutated WM. Methods: RNASeq was performed on CD19-selected bone marrow (BM) samples from 249 of the 253 treatment-naive WM patients who were MYD88 mutated, as well as 13 paired CD19+CD27- and CD19+CD27+ selected healthy donor (HD) peripheral blood samples. Whole exome sequencing of CD19-selected BM cells along with CD19-depleted peripheral blood mononuclear controls was also performed for 215 of the WM samples. To assess BM histology, slides from 66 BM paraffin blocks from these WM patients were analyzed. Results: WM patients had median age of 66 (range 31-95 years), BM involvement of 50% (5-95%) and serum IgM level of 3,162 (104-10,321 mg/dL). The median follow-up for the study cohort was 8.8 (range 0.2 - 33.8 years). Patients were stratified into 5 cohorts by DPT values. Analysis of BM involvement by DPT cohorts revealed a stepwise increase with the median involvement rising from 20% in the lowest to 80% in the highest cohort (p<0.0001). Increasing DPT predicted for shorter time from biopsy to first therapy (p=0.0003) but was not predictive of time from diagnosis to first therapy consistent with DPT alignment by stage of WM development. RNASeq analysis revealed high expression of genes atypical of mature B-cells that diminished with increasing DPT ( Figure 1). Among these were the myeloid chemo-attractants CXCL1, CXCL8 and CXCL12 which fell from a median of 4.4, 67.2 and 41.1 to 0.1, 3.6 and 0.8 transcripts per million (TpM) in the lowest and highest DPT cohorts, respectively (p<0.0001). Accordingly, all the top 20 gene ontology (GO) terms associated with DPT were related to activation, degranulation, and migration of myeloid cells and leukocytes. Pathological review of the 66 BM slides ranked by DPT, showed increased early myeloid cells and eosinophils clustering near small lymphoid aggregates, and as DPT increased, increased lymphocytes and mast cells with decreased myeloid cell involvement was observed. By RNASeq, pre-B-cell genes including MYB, the VDJ recombination genes RAG1, RAG2 and DNTT as well as the surrogate light chain genes IGLL1 and VPREB1 fell from 22.5, 10, 13.5, 61.1, 144 and 38.3 to 2.2, 1, 1.1, 9.1, 12.2 and 4.3 TpM, respectively (p<0.0001). T-cell and myeloid genes including CD4, CD8A, FCGR3A (CD16), CD14 and CD33 also decreased with increased DPT from 67.6, 7.1, 80.5, 200, and 23.2 to 2.4, 0.9, 1.7, 3.6 and 0.7 TpM, respectively (p<0.0005). A subclonal stem cell signature was also detected, including CD34 and KIT (CD117), which fell from 1.67 and 0.64 to 0.15 and 0.06 TpM, respectively (0<0.0001). CD34, KIT, preBCR, CD4, CD8A, FCGR3A, CEACAM8 (CD66b), and CD33 expression was confirmed by CD19+ light chain restricted flow cytometry in WM but not in HD samples. In addition, the MB and plasma blast genes PRDM1 and XBP1 were elevated in all samples regardless of DPT, consistent with a putative memory B-cell origin. Progression free survival (PFS) in response to proteasome inhibitors revealed strong stratification by subtype (p=0.001), as well as inferior PFS with BTK-inhibitors in low DPT patients (p= 0.044; Figure 2), suggesting less BTK dependence in the early DPT clones. Consistent with this finding, gene set enrichment identified increased BCR signaling with increased DPT. Conclusions: This analysis suggests that stem cell program reactivation and atypical marker expression characterize early WM clones that are ultimately replaced by clones exhibiting more typical B-cell markers with disease progression. The findings may be relevant to treatment selection and provide a framework for investigating subtype and early/late evolutionary staging in the treatment approach to WM.

Self-Assembled Multiepitope Nanovaccine Provides Long-Lasting Cross-Protection against Influenza Virus.

Seasonal influenza vaccines typically provide strain-specific protection and are reformulated annually, which is a complex and time-consuming process. Multiepitope vaccines, combining multiple conserved antigenic epitopes from a pathogen, can trigger more robust, diverse, and effective immune responses, providing a potential solution. However, their practical application is hindered by low immunogenicity and short-term effectiveness. In this study, multiple linear epitopes from the conserved stem domain of hemagglutinin and the ectodomain of matrix protein 2 are combined with the Helicobacter pylori ferritin, a stable self-assembled nanoplatform, to develop an influenza multiepitope nanovaccine, named MHF. MHF is prokaryotically expressed in a soluble form and self-assembles into uniform nanoparticles. The subcutaneous immunization of mice with adjuvanted MHF induces cross-reactive neutralizing antibodies, antibody-dependent cell-mediated cytotoxicity, and cellular immunity, offering complete protection against H3N2 as well as partial protection against H1N1. Importantly, the vaccine cargo delivered by ferritin triggers epitope-specific memory B-cell responses, with antibody level persisting for over 6 months post-immunization. These findings indicate that self-assembled multiepitope nanovaccines elicit potent and long-lasting immune responses while significantly reducing the risk of vaccine escape mutants, and offer greater practicality in terms of scalable manufacturing and genetic manipulability, presenting a promising and effective strategy for future vaccine development.

B-2 lymphocytes and the balance of pro and anti-inflammatory cytokines in infectious and autoimmune phenotypes of common variable immune deficiency

Objective: comparative characteristics of the subpopulation composition of B- lymphocytes and the cytokine spectrum of peripheral blood in infectious and non-infectious manifestations of CVID.Materials and methods: the results of observation of 10 people diagnosed with CVID have been presented. Six patients the clinical manifestation had an infectious phenotype of the disease of synopulmonary localization. Four patients were diagnosed with Crohn's disease, hemolytic anemia, and autoimmune hepatitis. The level of cytokines IL-4, IL-10, IL-17, TNF-α, IFN-γ in blood serum was determined by the ELISA, the phenotypic characteristic of B cells of peripheral blood cells was carried out by flow cytofluorimetry.Results: the proportion of memory B cells in the healthy donors is 30% of all B-lymphocytes, on infectious manifestation of CVID – 12%, with autoimmune – 14%. The number of switched memory B-lymphocytes relative to the total pool of B-2 cells in infectious manifestation is greater (2.3%) than in autoimmune ones (1.4%). In patients with infectious manifestations the content of IFN-γ and TNF-α was increased inrelation to healthy donors of the comparison group, there were no differences in the serum amount of IL-17, with autoimmune manifestations, the content of all these cytokines was increased, IFN-γ and TNF-α being to a greater extent.Conclusion: impaired formation of memory B-lymphocytes and cytokine dysregulation of immune processes are detected regardless of the variant of clinical manifestation of CVID. The nature and degree of changes differ depending on the clinical phenotype of the disease.

The trajectory of human B-cell function, immune deficiency, and allergy revealed by inborn errors of immunity.

The essential role of B cells is to produce protective immunoglobulins (Ig) that recognize, neutralize, and clear invading pathogens. This results from the integration of signals provided by pathogens or vaccines and the stimulatory microenvironment within sites of immune activation, such as secondary lymphoid tissues, that drive mature B cells to differentiate into memory B cells and antibody (Ab)-secreting plasma cells. In this context, B cells undergo several molecular events including Ig class switching and somatic hypermutation that results in the production of high-affinity Ag-specific Abs of different classes, enabling effective pathogen neutralization and long-lived humoral immunity. However, perturbations to these key signaling pathways underpin immune dyscrasias including immune deficiency and autoimmunity or allergy. Inborn errors of immunity that disrupt critical immune pathways have identified non-redundant requirements for eliciting and maintaining humoral immune memory but concomitantly prevent immune dysregulation. Here, we will discuss our studies on human B cells, and how our investigation of cytokine signaling in B cells have identified fundamental requirements for memory B-cell formation, Ab production as well as regulating Ig class switching in the context of protective versus allergic immune responses.

Antibody-Mediated Suppression Regulates the Humoral Immune Response to Influenza Vaccination in Humans.

Preexisting immunity, including memory B cells and preexisting antibodies, can modulate antibody responses to influenza in vivo to antigenically related antigens. We investigated whether preexisting hemagglutination inhibition (HAI) antibodies targeting the K163 epitope on the hemagglutinin (K163 antibodies) could affect antibody responses following vaccination with A/California/07/2009-like A(H1N1)pdm09 influenza viruses in humans. Pre- and postvaccination sera collected from 300 adults (birth years, 1961-1998) in 6 seasons (2010-2016) were analyzed by HAI assays with 2 reverse genetics viruses and A(H1N1) viruses circulated from 1977 to 2018. Antibody adsorption assays were used to verify the preexisting K163 antibody-mediated suppression effect. Preexisting K163 antibody titers ≥80 affected HAI antibody responses following influenza vaccination containing A/California/07/2009-like antigens. At high K163 antibody concentrations (HAI antibody titers ≥160), all HAI antibody responses were suppressed. However, at moderate K163 antibody concentrations (HAI antibody titer, 80), only K163 epitope-specific antibody responses were suppressed, and novel HAI antibody responses targeting the non-K163 epitopes were induced by vaccination. Novel antibodies targeting non-K163 epitopes cross-reacted with newly emerging A(H1N1)pdm09 strains with a K163Q mutation rather than historic 1977-2007 A(H1N1) viruses. K163 antibody-mediated suppression shapes antibody responses to A(H1N1)pdm09 vaccination. Understanding how preexisting antibodies suppress and redirect vaccine-induced antibody responses is of great importance to improve vaccine effectiveness.

BIOM-42. LONGITUDINAL IMMUNE PROFILES OF LOWER GRADE ADULT GLIOMA PATIENTS

Abstract Immune dysregulation is a known characteristic of glioma, however there is little known about the peripheral immune status of lower-grade (LrGG) glioma patients across the course of disease. Using immunomethylomic (deconvoluted blood DNA methylation) data from the UCSF Immune Profiles Study, we explore changes in patient peripheral blood immune cell profiles in newly diagnosed and recurrent LrGG patients. For patients receiving radiation (RT) and temozolomide (TMZ) (n = 75), blood is collected pre-RT/TMZ, post-RT/TMZ, before, during, and after adjuvant TMZ (if applicable). For patients followed only by MRI (n = 53) and those with other treatment plans (n = 67), blood is collected at MRIs, and if applicable, before radiation. For all patients, blood is also collected before new treatment intervention. For patients (LrGG IDH mutated) with paired pre- and post-surgery samples (n = 139), we observed a significant post-surgery increase in the count of memory B-cells (p = 1.7e-5), neutrophils (p = 0.0001), basophils (p = 8.7e-5), monocytes (p = 0.0001), and T-regulatory cells (p = 0.0001) along with a significant decrease in natural killer cells (p = 0.0003). Treatment was also associated with altered patient immune profiles. The use of dexamethasone was associated with an increased count of B-memory (p = 0.0001) and B-naïve cells (p = 0.0001), monocytes (p = 2.1e-5), and neutrophils (p = 3.7e-6) post-surgery. Patients receiving chemoradiation (n = 12) exhibited significant and temporally sustained decreases in B-memory (p < 0.0001), B-naïve (p = 0.005), CD4 naïve cells (p = 0.002), and in neutrophils (p = 0.001). Patient follow-up, data collection, and further analyses are ongoing. These results shed light on the dynamic and variable immune changes in LrGG patients and provide a guide for future prognostic and therapeutic studies.

PSIX-7 Attenuation of Immune Allergic Response Pathways in Canines with Dermatological Problems Fed a Polyphenol-Rich Food

Abstract Pathogenesis of canine dermatological problems is mediated by multiple effector cells including lymphocytes, eosinophils, and mast cells. We have previously reported a significant increase in the gene expression of interleukin-5 receptor alpha (IL-5RA) in dogs with dermatological problemscompared with controls, as well as a modest increase in C-C chemokine receptor 3 (CCR3), IL-4 and IL-5. We now investigated whether 2 tests foods (one rich in polyphenols) would provide beneficial effects in canines with dermatological problems (n = 8) compared with controls (n = 8). Dogs were pre-fed control food for 28 days and then divided into two groups based on their age, gender and phenotype (Healthy, Derm) followed by a cross-over design. At each phase, dogs were fed for 42 days with test food 1 (TF1) and the second group was fed with a greater polyphenol content Test food 2 (TF2) for 42 days followed by a crossover. Blood samples were collected at the end of pre-feed, end of phase 1, and end of phase 2. The study was approved by IACUC and AWC, Hill’s PNC. RNA was extracted from blood collected in PAXgene tubes and gene expression was investigated using the NanoString nCounter platform. To assess the food effect, a mixed model analysis was performed using treatment (food) as a fixed effect and animal as random effect using JMPPro. TF2 fed dogs decreased pruritus scores, increased the response rate (improvement %) to 30.77% compared with dogs fed TF1. There was a significant decrease in the interleukin-5 receptor alpha (IL-5RA) in dogs fed TF2 when compared with baseline (P < 0.05). IL-5RA is a receptor expressed on eosinophils as well as committed eosinophil precursors in the bone marrow. There was also a decrease in FCRL2 in dogs fed TF2 (P < 0.05). This gene is preferentially expressed in memory B-cells and plasma cells and also involved in B-cell receptor signaling. The generation of plasma cells requires naïve B cell activation to differentiate into antibody secreting cells or memory B cells. Given the lower levels of FCRL2 with TF2, it is possible that the production of subsequent IgE is attenuated. Another signaling molecule that was significantly decreased with TF2 was the innate immune response molecule C3AR1, a receptor for Complement C3a that induces chemotaxis, granule release, as well as superoxide production. TF1 and TF2 significantly decreased CCR3, a receptor on eosinophils and basophils. CCR3 antagonists are effective in reducing allergic inflammation. Our results indicate the important role of eosinophilic allergic response in dermatological disorders in dogs. TF2 appears to have a beneficial effect in attenuating allergy-inducing signaling pathways of innate and adaptive immune responses. Targeting these molecules may be important as part of newer therapeutic approaches, including nutritional strategies, to manage dermatological problems in dogs.

High Dimensional Detection of Non-Malignant B-Cells and Its Clinical Implications in Patients with Chronic Lymphocytic Leukaemia (CLL) Undergoing Frontline Therapy

Introduction: The analysis of non-malignant B-cells in chronic lymphocytic leukaemia (CLL) is challenging due to their scarcity and paucity of reliable identification methods. However, given the well-defined immunophenotypic characteristics of CLL cells, it is feasible to distinguish and quantify non-malignant B-cells by incorporating an extensive panel of B-cell markers into unsupervised high-dimensional analysis. In this study, we performed high dimensional mass cytometry analysis of circulating B-cells in patients enrolled in the NCRI phase III RIAltO trial, which compared frontline chemoimmunotherapy (CIT) regimens in CLL. Methods: Mass cytometry was performed on samples obtained from CLL patients enrolled in the RIAltO trial (NCT01678430). Patients were randomly assigned to receive bendamustine or chlorambucil plus ofatumumab, with or without idelalisib. Samples were collected before treatment and at early (median 11.2 [IQR 11 - 11.7] months) and late (18.5 [16.1 - 22.6] months) post-treatment timepoints, as well as at disease relapse. Healthy controls (HC) were included for comparison. CD19 + B cells were analysed using a customised antibody panel with 29 surface-antigen markers. Results: A total of 198 samples from 79 patients and three HC were analysed. Unsupervised analysis using uniform manifold approximation and projection (UMAP) and FlowSOM identified two distinct groups of CD19 + B-cell clusters. Malignant clusters were identified based on established immunophenotypic markers of CLL, while non-malignant clusters were identified using the inverse co-expression of these markers (CD5 -CD43 -ROR1 -CD20 +CD79b +CD81 +). In keeping with the assumption that non-malignant clusters comprised physiological B-cells, they were the majority cell type among HC, rare before treatment or at relapse, and variably detected following therapy. When relating non-malignant clusters with clinical outcomes, their re-emergence following therapy correlated with an increase in markers of haematopoietic recovery, including haemoglobin concentration (R 2 = 0.4, p = 0.002), neutrophil count (R 2 = 0.39, p = 0.002) and platelet count (R 2 = 0.41, p = 0.002). Furthermore, re-emergence of non-malignant, CD27 + memory B-cells the at late post-treatment timepoint positively correlated with serum immunoglobulins (Ig): IgA (R 2 = 0.41, p = 0.012), IgM (R 2 = 0.37, p = 0.027) and IgG (R 2 = 0.42, p = 0.01) levels. Notably, stratification of patients based on post-treatment median cluster size showed that a higher frequency of non-malignant B-cells correlated with significantly longer time to progression (TTP; median 49.9 [95% CI: 28.5 - NR] versus 9.7 [7.2 - 26.8] months, p < 0.001). This correlation remained significant after adjusting for baseline characteristics, CLL prognostic factors, treatment allocation, minimal residual disease (MRD) status, and haematopoietic recovery. Subsequently, a Boolean gating strategy which prospectively defines non-malignant B-cells (CD5 -CD43 -ROR1 -CD20 +CD79b +CD81 +) was devised. Despite a weak correlation with MRD (R 2 = -0.27, p = 0.084), the proportion of non-malignant B cells at the end of treatment was found to be an independent determinant of TTP and overall survival (OS) among patients with ≥ 0.1% residual CLL cells. Conclusion: To our knowledge, this is the first study to identify correlations between the re-emergence of non-malignant B-cells following anti-CLL therapy and specific clinical endpoints such as haematopoietic recovery, serum Ig levels, TTP and OS. Although further validation is required in the context of targeted agents, our findings suggest that therapy-induced re-emergence of non-malignancy B-cells may have important biological and clinical implications in CLL.

Changes in Methylation and Chromatin Accessibility Underlie Subtype Classification and Disease Evolution in Waldenström's Macroglobulinemia

Background: We previously identified three subtypes of Waldenstrom's Macroglobulinemia (WM): B-cell like (BCL), Plasma cell like (PCL), and an intermediate group enriched for early/smoldering WM from which the other two subtypes evolved (Hunter et al, ASH 2022). Diffusion pseudo-time (DPT) analysis suggested all WM samples could be placed on a shared evolutionary path independent of subtype, and when samples were separated into Early and Late DPT values, it mirrored disease progression. The role of epigenetic dysregulation that underlies subtype classification and disease evolution remains poorly understood in WM. Methods: We therefore performed chromatin availability analysis using ATACSeq and bisulfite sequencing of WM patients. ATAC-Seq libraries were constructed from 37 CD19+ BM cells from treatment-naive WM patients along with 6 paired CD19+CD27- and CD19+CD27+ selected healthy donor (HD) peripheral blood (PB) controls and 3 HD CD138+ plasma cell (PC) samples. Enhanced reduced representational bisulfite sequencing (ERRBS) was also performed on 65 MYD88 mutated and 13 MYD88 wild type (WT) WM patients using CD19-selected BM lymphoplasmacytic cells. For 11 WM MYD88 mutated patients, CD138+ cells were also selected. In addition ERRBS was also performed on paired CD19+ and CD138+ selected samples from 5 IgM multiple myeloma (MM) patients, 6 paired CD19+CD27- and CD19+CD27+ selected healthy donor (HD) peripheral blood cells; 8 HD CD138+ selected PC; as well as MYD88 mutated BCWM.1, BCWM.2, MWCL-1, RPCI-WM1, OCI-Ly3, TMD-8, HBL-1 and SU-DHL2 cells; MYD88 wild-type OCI-Ly7, OCI-Ly19, Ramos B-cells. To infer 5-hydroxymethylcytosine (5HMC) levels, oxidative bisulfite sequencing was also performed for 73 samples including the HD, MM, WT and WM CD19/138 pairs. All ATAC-Seq and 37 of the WM ERRBS samples were part of our multi-omic study that included 253 treatment naïve patients who underwent whole exome and RNASeq sequencing. Results: A UMAP projection of the CpG ERRBS data clustered WM patients into two distinct groups ( Figure 1). One group clustered directly with HD Memory B-cells (MB) and was greatly enriched for CXCR4 mutated WM patients. The other WM group clustered with IgM MM patients and was largely CXCR4 WT. The findings are consistent with the BCL and PCL WM subtypes, which were confirmed by a perfect assignment correlation with our larger multi-omic data set wherein these subtypes were defined. Notably, BCWM.1, BCWM.2 and MWCL-1 WM cell lines clustered with the WM samples. The 5HMC data displayed no clear clustering of the patient samples but clearly separated all HD samples from those of WM and IgM MM samples, consistent with a role for 5HMC dysregulation in oncogenesis. Differential methylation analysis showed differences by MYD88 and CXCR4 mutation status, deletions in Chr6q, familial history for WM, WM subtype, and stratification by early and late DPT values. Notably, the only comparison that did not yield any differences was WM CD19+ to WM CD138+, suggesting that this may not be a meaningful epigenetic distinction in WM. Similar comparisons were performed on the ATAC peaks. Differential chromatin accessibility was noted near many of the corresponding differentially expressed genes. ATAC peak coverage significantly correlated with the expression of top genes. This was particularly true for WM subtype and DPT comparisons ( Table 1). DNA sequences from differentially accessible peaks between BCL and PCL subtypes were also evaluated for relative TF motif enrichment in open vs. closed chromatin revealing PAX5, FUBP1, and ZNF121 enrichment in BCL subtypes using PCL subtype peaks as a background control. TFs REL, SP1, SPIB, BC11A and JUN were enriched PCL subtype compared with BCL subtype. Correlation between DPT associated genes revealed two clear groups, one associated with stem cell and pre-B-cell genes (Group 1) and the other with inflammatory, myeloid, and T-cell genes (Group 2). Motif analysis of differential peaks correlating with genes in each group demonstrated comparative enrichment for MYC, MXI1, and E2F2 in group 1 while RUNX1, NFKB1, and ATF1 were in enriched in group 2. Conclusions: This is the first independent validation of our previously reported multi-omic driven WM subtype classification. The studies underscore that epigenetic differences underlie the biology of WM subclassification and support a strong role for epigenetic changes driving WM evolution.

Somatic Hypermutation in Enhancer Regions Shapes Non-Coding Myeloma Genome, Generating DNA Breaks and Driving Etiology through Mutation and Structural Variation

INTRODUCTION: Little is known about the pattern and function of mutations within the 98% of the genome which is non-coding (nc). Whole-genome sequencing (WGS) can identify the full range of single nucleotide variants (SNVs), insertions/deletions (InDels), copy-number variants (CNVs), and structural variants (SVs), which are critical to disease progression. Here, we characterize the non-coding genome to gain significant insight into the role of mutations in gene regulatory elements in the etiology of multiple myeloma (MM) and to models of how it develops. METHODS: We studied 302 of MM precursor and newly diagnosed MM (NDMM) patients with high-coverage WGS, where each SNV/InDel was confirmed by two or more algorithms. Results were validated on an independent cohort of 256 NDMM with 80X WGS data. A pipeline employing a consensus mechanism for determining the final set of somatic events was used, including Mutect2, Strelka2, and VarScan2 for SNVs; Mutect2, Strelka2, VarScan2, and SvABA for InDels; Battenberg and FACETS for CNVs; Manta, SvABA, DELLY2, and IgCaller for SVs (https://github.com/pblaney/mgp1000). The R package fishHook was used toidentify statistically significant enrichment of mutations. To identify nc-variants we partitioned the genome into 10 kb tiles that were iteratively shifted by 500 bp and tested each tile against a regression model built into fishHook. The model includes a series of covariates that inform replication timing, sequencing context, and chromatin states. RESULTS: We identified 2,039,841 SNVs and 492,746 InDels in total. The tumor mutational burden (TMB) varies between molecular subgroups with the t(4;14) being significantly higher at 3.23 (somatic mutations per Mb) in comparison to the t(11;14) at 2.57 (FDR adj. P=0.035), which was closer to patients without a subtype translocation at 2.78. For ncSNVs and ncInDels, we identified 4,374 and 272 tiles respectively with significant mutation enrichment genome-wide (FDR adj. P<0.05). As tiles may overlap, we collapsed contiguous segments into consensus regions assigning the nearest coding gene as an identifier and termed these “mutation-enriched regions” or MERs. We identified 282 MERs associated with 203 genes for ncSNVs and 26 MERs associated with 25 genes for ncInDels. The two types of regions overlap at six loci ( TENT5C, OR2T2, FOXD4L1, BCL6, BLOC1SS- TXNDC5, PLD5P1). Thus, we identified 302 MERs associated with 221 genes, with some of the most highly mutated MERs included BCL6 (76.2% of patients), BLOC1S5- TXNDC5 (28.1%), ZFP36L1 (22.2%), BTG2 (21.2%), IRF8 (16.2%), TENT5C (13.6%), and CCND1 (12.3%). In total 19,743 of the 2,532,587 mutations fall into MERs with 1.3-65.6% of patients having one of these mutations. We evaluated the MERs for functional relevance by intersecting the regions with a list of 8,357 genome-wide enhancer (E) and super-enhancer (SE) elements derived from germinal-center B cells (GCB), DLBCL (Bal et al. Nature 2022) and MM (Lovén et al. Cell 2013). In total, 17.9% (54/302) of the MERs were identified, involving 45 genes. These MERs intersected with 28 Es and 20 SEs, with a non-random distribution of mutations within them. Of the total MER mutations, 21.9% (4,317/19,743) fell into some form of enhancer element. Breaking these down further 41.6% (1,798/4,317) are in Es, and 58.4% (2,519/4,317) are in SEs. All the E mutations were MM specific; of the SE mutations, 6.0% (18/302) of patients had a mutation in an ABC-DLBCL specific SE, 16.6% (50/302) in a GCB specific SE and 64.2% (194/302) in a MM specific SE. We examined the distribution of mutations within the SE regions and found they are non-random suggesting a selective mechanism. We intersected the SE regions with SV and found an excess at TENT5C, BTG2, BLOC1S5-TXNDC5 and ZFP36L1. A focused analysis of chr1p, chr1q, chr6q and chr14 revealed the importance of mutationally induced breaks within the SE and its translocation to a receptor site often 8q the site of MYC. CONCLUSIONS: We provide evidence for an important contribution of mutations within E and SE regions to the etiology of MM. This may involve either direct selection of mutations within the GC or by the re-entry of a memory B-cell carrying a pattern of mutations it acquired in a pre-MM phase, which then acquires a MM-specific driver. FIGURE: Distribution of mutations across MM genomes. A) Tumor mutational burden across MM subtypes. B) Q-Q plots of fishHook model for SNVs

Multi-Omic Insights into the Mutational Landscape and Dysregulated Transcriptional Programs of Autoimmune B-Cells in Systemic Lupus Erythematosus

Systemic Lupus Erythematosus (SLE) is an autoimmune disease with diverse manifestations, from mild and sporadic to severe and life-threatening. SLE heterogeneity arises from various factors, including genetic, epigenetic, and immune niche-related features (Kaul et al., 2016; Jenks et al., 2018). However, the exact interactions and hierarchy between these features remain unclear. Recent studies have found somatic mutations in genes typically mutated in B-cell lymphomas, in subsets of B-cells that are associated with SLE pathogenesis (Piotrowski et al., 2015; Pullabhatla et al., 2018; Singh et al., 2020; Brown et al., 2022). These mutations may support B-cell clonal bursts, but direct information from clinical specimens on the impact of these mutations on B-cell lineages and their influence on SLE progression or malignant transformation is missing. To define the somatic mutational landscape of B-cells in SLE, we first profiled peripheral blood mononuclear cells obtained from a pilot cohort of four SLE patients using whole-exome sequencing (WES; 200X depth). Following GATK best practices (Van der Auwera and O'Connor, 2020), we identified somatic alterations in DDX11 [variant allele frequency (VAF): 10.2%], DNMT3A [VAF: 3.4%]; PMS2 [VAF: 47.9%], and BICD1 [VAF: 47.3%]; these genes have been linked to lymphoid malignancies and SLE pathogenesis (Piotrowski et al., 2015; Pullabhatla et al., 2018; Niroula et al., 2021; Saeed et al., 2021). We validated the presence of the top-identified mutations using droplet digital PCR technology on sorted B-cell subsets (naïve, memory, and double-negative [DN; CD19 hiIgD loCD27 lo]) from these SLE patients. DN cells are a heterogeneous subtype of autoreactive memory B-cells that play a crucial role in SLE pathogenesis being the precursors of autoantibody producing plasma cells (Wei et al., 2007; Jenks et al., 2018). To investigate the transcriptional and epigenetic alterations in the mutation-harboring B-cells, we then conducted single-cell multi-omic sequencing. We profiled sorted activated B-cells (CD19 hiIgD lo) from the four SLE cases, to largely exclude non-autoreactive naïve B-cells. To maintain a comparison with naïve B cells, we included a small proportion of these cells in the input. In total, we analyzed 35,339 cells, and identified different B-cell subsets with distinct gene expression patterns, including DN cells ( n = 1,437 cells; ZEB2 hiTRAF5 lo, Jenks et al., 2018). To uncover, in an unbiased way, transcriptional differences between B-cell subsets, we catalogued variation in transcriptomic signatures derived from the scRNA data using consensus non-negative matrix factorization. We found a unique gene expression program exclusively present in DN cells ( Panel A), which includes key genes implicated in immune regulation and the development of autoimmunity, such as PTPN22 (Chung & Criswell, 2007; Tizaoui et al., 2021) . Pathway analysis of the DN-specific program revealed an enrichment of complement system genes (FDR = 0.074; Hallmark), which are crucial for immune regulation and tolerance, such as CBLB (Tang et al., 2019). Notably, in DN cells from one of the SLE samples carrying lymphoma/SLE-associated mutations in BICD1, PMS2, DDX11, we observed an altered expression of this gene program compared to DN cells from other samples ( P-value = 3.6x10 -11; Panel A, inset), suggesting that a malfunctioning complement system in DN cells may lead to an overly active immune response (Weinstein et al., 2021). In line with this notion, using chromatin accessibility profiles as a measure of transcription factor (TF) regulatory activities, we showed increased activity of TFs involved in interferon-dependent innate immune response and in B-cell activation and differentiation (RFX and OCT-1, respectively; FDR < 0.05), in the DN B-cells of this sample ( Panel B). In summary, we uncovered a unique transcriptional program associated with autoimmune DN B-cells. This program showed enrichment in key genes and pathways involved in immune response regulation. The disruption of this program, accompanied by the somatic SLE-associated mutations, may lead to overactivation of DN B cells, ultimately contributing to autoimmunity. Further analyses involving a larger cohort of clinical specimens are needed to directly link B-cell genotypes with their transcriptional and epigenetic programs and to elucidate the role of the identified mutations in SLE pathobiology.

Diagnostic Values of METTL1-Related Genes and Immune Characteristics in Systemic Lupus Erythematosus.

Methyltransferase like 1 (METTL1) regulates epitranscriptomes via the m7G modification in mammalian mRNA and microRNA. Systemic lupus erythematosus (SLE) is caused by abnormal immune reactivity and has diverse clinical manifestations. RNA methylation as a mechanism to regulate gene expression is widely implicated in immune regulation. However, the role of m7G in immune response of SLE has not been extensively studied. Expression of METTL1 was identified in the public dataset GSE122459 and validated in an independent cohort of SLE patients. We investigated the association between METTL1-expression and clinical manifestations of SLE. Subsequently, differentially expressed genes (DEG) that were correlated with METTL1-expression in GSE122459 were used for functional enrichment analysis. The correlation between infiltrating immune cells and METTL1, as well as candidate biomarkers identified to be correlated with either METTL1 or immune cell infiltration were assessed by single-sample GSEA. Potential mechanisms were explored with Gene ontology and KEGG pathway enrichment. Diagnostic performances of candidate biomarkers in SLE were analyzed. The mRNA and protein expression of METTL1 in SLE patients were significantly decreased in both datasets. METTL1-coexpressed DEGs were enriched in several key immune-related pathways. Activated CD8 T cells, activated CD4 T cells, memory B cells and type 2 helper T cells were different between patients with high and low METTL1 expression. Further, activated CD8 T-cells, activated CD4 T-cells, memory B-cells were correlated with METTL1. The genes of LAMP3, CD83, PDCD1LG2, IGKVD3D-20, IGKV5-2, IGKV2D-30, IGLV3-19 and IGLV4-60 were identified as candidate targets that were correlated with immune cell proportion. Moreover, LAMP3, CD83, and PDCD1LG2 expression were of diagnostic value in SLE as indicated by ROC analysis. Our findings suggested that METTL1 and its candidate targets LAMP3, CD83, PDCD1LG2 may be used for diagnosing SLE and could be explored for developing targeted molecular therapy for SLE.

Mechanisms of Formation and Persistence of Ige Products and Potential Innovative Means of Therapy for Allergic Pathologies

The proposed review is devoted to the analysis of the main mechanisms of the formation of IgE-producing cells in the body and a brief review of the main, most striking candidate agents for use in innovative methods of therapy for IgE-dependent pathologies. Data are presented according to which the role of IgE+ plasma cells and various subpopulations of memory B-lymphocytes in the formation and persistence of the state of sensitization to a harmless allergen differs depending on the model system used or the clinical case under study. Therefore, drugs that target signaling pathways involved in the regulation of both plasma cells and memory B-lymphocytes are especially promising in the treatment of allergic diseases. The authors conclude that the components of the cellular response to oxidative stress and related genotoxic stress and ER stress are the most promising as such targets, since (a) all of them directly or indirectly affect the processes that regulate both of these subpopulations; b) are involved in the process of formation and maintenance of local allergic inflammation. The review presents data pointing to the particular promise of using nanoparticles of noble metals and complexes of rare earth metals of lanthanides in this regard, due to their ability to induce long-term effects in small doses due to changes in the properties of innate immunity cells and long-term accumulation in the body.

The Value of IgM Memory B-Cells in the Assessment of Splenic Function in Childhood Cancer Survivors at Risk for Splenic Dysfunction: A DCCSS-LATER Study.

Childhood cancer survivors (CCS) who received radiotherapy involving the spleen or total body irradiation (TBI) might be at risk for splenic dysfunction. A comprehensive screening test for examining splenic dysfunction is lacking. We investigated whether IgM memory B-cells could be used to assess splenic dysfunction in CCS who received a splenectomy, radiotherapy involving the spleen, or TBI. All CCS were enrolled from the DCCSS-LATER cohort. We analyzed differences in IgM memory B-cells and Howell-Jolly bodies (HJB) in CCS who had a splenectomy (n = 9), received radiotherapy involving the spleen (n = 36), or TBI (n = 15). IgM memory B-cells < 9 cells/µL was defined as abnormal. We observed a higher median number of IgM memory B-cells in CCS who received radiotherapy involving the spleen (31 cells/µL, p=0.06) or TBI (55 cells/µL, p = 0.03) compared to CCS who received splenectomy (20 cells/µL). However, only two CCS had IgM memory B-cells below the lower limit of normal. No difference in IgM memory B-cells was observed between CCS with HJB present and absent (35 cells/µL vs. 44 cells/µL). Although the number of IgM memory B-cells differed between splenectomized CCS and CCS who received radiotherapy involving the spleen or TBI, only two CCS showed abnormal values. Therefore, this assessment cannot be used to screen for splenic dysfunction.