A humanized mouse that mounts mature class-switched, hypermutated and neutralizing antibody responses.

Humanized mice are limited in terms of modeling human immunity, particularly with regards to antibody responses. Here we constructed a humanized (THX) mouse by grafting non-γ-irradiated, genetically myeloablated KitW-41J mutant immunodeficient pups with human cord blood CD34+ cells, followed by 17ß-estradiol conditioning to promote immune cell differentiation. THX mice reconstitute a human lymphoid and myeloid immune system, including marginal zone B cells, germinal center B cells, follicular helper T cells and neutrophils, and develop well-formed lymph nodes and intestinal lymphoid tissue, including Peyer's patches, and human thymic epithelial cells. These mice have diverse human B cell and T cell antigen receptor repertoires and can mount mature T cell-dependent and T cell-independent antibody responses, entailing somatic hypermutation, class-switch recombination, and plasma cell and memory B cell differentiation. Upon flagellin or a Pfizer-BioNTech coronavirus disease 2019 (COVID-19) mRNA vaccination, THX mice mount neutralizing antibody responses to Salmonella or severe acute respiratory syndrome coronavirus 2 Spike S1 receptor-binding domain, with blood incretion of human cytokines, including APRIL, BAFF, TGF-ß, IL-4 and IFN-γ, all at physiological levels. These mice can also develop lupus autoimmunity after pristane injection. By leveraging estrogen activity to support human immune cell differentiation and maturation of antibody responses, THX mice provide a platform to study the human immune system and to develop human vaccines and therapeutics.

Rad52 mediates class-switch DNA recombination to IgD.

In B cells, IgD is expressed together with IgM through alternative splicing of primary VHDJH-Cµ-s-m-Cδ-s-m RNAs, and also through IgD class switch DNA recombination (CSR) via double-strand DNA breaks (DSB) and synapse of Sµ with σδ. How such DSBs are resolved is still unknown, despite our previous report showing that Rad52 effects the 'short-range' microhomology-mediated synapsis of intra-Sµ region DSBs. Here we find that induction of IgD CSR downregulates Zfp318, and promotes Rad52 phosphorylation and recruitment to Sµ and σδ, thereby leading to alternative end-joining (A-EJ)-mediated Sµ-σδ recombination with extensive microhomologies, VHDJH-Cδs transcription and sustained IgD secretion. Rad52 ablation in mouse Rad52-/- B cells aborts IgD CSR in vitro and in vivo and dampens the specific IgD antibody response to OVA. Rad52 knockdown in human B cells also abrogates IgD CSR. Finally, Rad52 phosphorylation is associated with high levels of IgD CSR and anti-nuclear IgD autoantibodies in patients with systemic lupus erythematosus and in lupus-prone mice. Our findings thus show that Rad52 mediates IgD CSR through microhomology-mediated A-EJ in concert with Zfp318 downregulation.

Lnc-GULP1-2:1 affects granulosa cell proliferation by regulating COL3A1 expression and localization.

BACKGROUNDS: Long non-coding RNA is a novel group of non-protein coding transcripts over 200 nt in length. Recent studies have found that they are widely involved in many pathological and physiological processes. In our previous study, we found that lnc-GULP1-2:1 was significantly down-regulated in the ovarian cortical tissue of patients with primary ovarian insufficiency and predicted that lnc-GULP1-2:1 has a regulatory effect on COL3A1. RESULTS: In this study, we found that lnc-GULP1-2:1 was mainly localized in the cytoplasm of luteinized granulosa cells. The expression of lnc-GULP1-2:1 was lower in patients with diminished ovarian reserve but substantially elevated in patients with polycystic ovary syndrome. Overexpression of lnc-GULP1-2:1 in KGN cells significantly inhibited cell proliferation, likely through cell cycle related genes CCND2 and p16. Moreover, lnc-GULP1-2:1 expression was positively correlated with the level of COL3A in luteinized granulosa cells from patients with different ovarian functions as well as in multiple cell lines. Overexpression of lnc-GULP1-2:1 in KGN cells promoted the expression of COL3A1 and its translocation into the nucleus. Consistently, silencing COL3A1 in KGN cells also significantly inhibited cell proliferation. CONCLUSIONS: Lnc-GULP1-2:1 affects the proliferation of granulosa cells by regulating the expression and localization of COL3A1 protein, and may participate in the regulation of ovarian follicle development. This study will provide new insight into molecular mechanisms underlying ovarian follicular development, which will help generate novel diagnostic and therapeutic strategies for diseases related to ovarian follicular development disorders.

Estrogen Reverses HDAC Inhibitor-Mediated Repression of Aicda and Class-Switching in Antibody and Autoantibody Responses by Downregulation of miR-26a.

Estrogen contributes to females' strong antibody response to microbial vaccines and proneness to autoimmunity, particularly antibody-mediated systemic autoimmunity, in females. We have hypothesized that this is due to estrogen-mediated potentiation of class switch DNA recombination (CSR) and somatic hypermutation (SHM). As we have shown, estrogen boosts AID expression, which is critical for both CSR and SHM, through upregulation of HoxC4, which together with NF-κB critically mediates Aicda (AID gene) promoter activation. We contend here that additional regulation of Aicda expression by estrogen occurs through epigenetic mechanisms. As we have shown, histone deacetylase inhibitors (HDIs) short-chain fatty acid (SCFA) butyrate and propionate as well as the pharmacologic HDI valproic acid upregulate miRNAs that silence AID expression, thereby modulating specific antibody responses in C57BL/6 mice and autoantibody responses in lupus-prone MRL/Faslpr/lpr mice. Here, using constitutive knockout Esr1-/- mice and B cells as well as conditional knockout Aicdacre/creEsr1flox/flox mice and B cells, we showed that the HDI-mediated downregulation of Aicda expression as well as the maturation of antibody and autoantibody responses is reversed by estrogen and enhanced by deletion of ERα or E2 inhibition. Estrogen's reversion of HDI-mediated inhibition of Aicda and CSR in antibody and autoantibody responses occurred through downregulation of B cell miR-26a, which, as we showed, targets Aicda mRNA 3'UTR. miR-26a was significantly upregulated by HDIs. Accordingly, enforced expression of miR-26a reduced Aicda expression and CSR, while miR-26a-sponges (competitive inhibitors of miR-26a) increased Aicda expression and CSR. Thus, our findings show that estrogen reverses the HDI-mediated downregulation of AID expression and CSR through selective modulation of miR-26a. They also provide mechanistic insights into the immunomodulatory activity of this hormone and a proof-of-principle for using combined ER inhibitor-HDI as a potential therapeutic approach.

Transcriptional profiling of long noncoding RNAs and their target transcripts in ovarian cortical tissues from women with normal menstrual cycles and primary ovarian insufficiency.

Previous studies have shown that long noncoding RNAs (lncRNAs) show a highly tissue- and disease-specific expression pattern and that they regulate the expression of neighboring genes. Because lncRNAs have been shown to be secreted into the general circulation, they may be used as diagnostic tools for some diseases. Primary ovarian insufficiency (POI) is a disease in which women have menstrual cessation before the age of 40, accompanied by elevated follicle stimulating hormone and decreased estrogen levels. In this study, ovarian cortical tissues from five women with normal menstrual cycles and from five POI patients were used for next-generation RNA sequencing. We found 20 differentially expressed lncRNAs with 12 upregulated and eight downregulated lncRNAs in cortical tissues of POI ovaries, compared with normal controls (fold change ≥ 2 and false discovery rate[FDR] ≤ 0.05). We also found 52 differentially expressed messenger RNA transcripts, with 33 upregulated and 19 downregulated ones (foldchange ≥ 2 and FDR ≤ 0.05). Functional annotation showed that these differentially expressed transcripts were associated with follicular development and granulosa cell function. Thirteen differentially expressed lncRNAs and their targeted neighboring transcripts were coregulated in ovarian cortical tissues, including lnc-ADAMTS1-1:1/ADAMTS1, lnc-PHLDA3-3:2/CSRP1, lnc-COL1A1-5:1/COL1A1, lnc-SAMD14-5:3/COL1A1, and lnc-GULP1-2:1/COL3A1. Furthermore, serum levels of these lncRNAs in POI patients were significantly different from those in normal patients ( p < 0.05), and expression differences were consistent with those in ovarian cortical tissues. This study showed that key lncRNAs were differentially expressed in both ovarian cortical tissues and serum samples between women with normal menstrual cycles and POI patients. Further studies on the regulation of ovarian lncRNAs during follicular development are critical in understanding the etiologies of POI. Analyses of lncRNA expression in serum samples might provide a basis for early diagnosis and treatment of POI.