Multiple tolerance checkpoints restrain affinity maturation of B cells expressing the germline precursor of a lupus patient-derived anti-dsDNA antibody in knock-in mice.

Anti-dsDNA antibodies are a hallmark of systemic lupus erythematosus and are highly associated with its exacerbation. Cumulative evidence has suggested that somatic hypermutation contributes to the high-affinity reactivity of anti-dsDNA antibodies. Our previous study demonstrated that these antibodies are generated from germline precursors with low-affinity ssDNA reactivity through affinity maturation and clonal expansion in patients with acute lupus. This raised the question of whether such precursors could be subjected to immune tolerance. To address this, we generated a site-directed knock-in (KI) mouse line, G9gl, which carries germline-reverted sequences of the VH-DH-JH and Vκ-Jκ regions of patient-derived, high-affinity anti-dsDNA antibodies. G9gl heterozygous mice had a reduced number of peripheral B cells, only 27% of which expressed G9gl B-cell receptor (BCR). The remaining B cells harbored non-KI allele-derived immunoglobulin heavy (IgH) chains or fusion products of upstream mouse VH and the KI gene, suggesting that receptor editing through VH replacement occurred in a large proportion of B cells in the KI mice. G9gl BCR-expressing B cells responded to ssDNA but not dsDNA, and exhibited several anergic phenotypes, including reduced surface BCR and shortened life span. Furthermore, G9gl B cells were excluded from germinal centers (GCs) induced by several conditions. In particular, following immunization with methylated bovine serum albumin-conjugated bacterial DNA, G9gl B cells occurred at a high frequency in memory B cells but not GC B cells or plasmablasts. Collectively, multiple tolerance checkpoints prevented low-affinity precursors of pathogenic anti-dsDNA B cells from undergoing clonal expansion and affinity maturation in GCs.

Self-reactive and polyreactive B cells are generated and selected in the germinal center during γ-herpesvirus infection.

Immune responses against certain viruses are accompanied by auto-antibody production although the origin of these infection-associated auto-antibodies is unclear. Here, we report that murine γ-herpesvirus 68 (MHV68)-induced auto-antibodies are derived from polyreactive B cells in the germinal center (GC) through the activity of short-lived plasmablasts. The analysis of recombinant antibodies from MHV68-infected mice revealed that about 40% of IgG+ GC B cells were self-reactive, with about half of them being polyreactive. On the other hand, virion-reactive clones accounted for only a minor proportion of IgG+ GC B cells, half of which also reacted with self-antigens. The self-reactivity of most polyreactive clones was dependent on somatic hypermutation (SHM), but this was dispensable for the reactivity of virus mono-specific clones. Furthermore, both virus-mono-specific and polyreactive clones were selected to differentiate to B220lo CD138+ plasma cells (PCs). However, the representation of GC-derived polyreactive clones was reduced and that of virus-mono-specific clones was markedly increased in terminally differentiated PCs as compared to transient plasmablasts. Collectively, our findings demonstrate that, during acute MHV68 infection, self-reactive B cells are generated through SHM and selected for further differentiation to short-lived plasmablasts but not terminally differentiated PCs.

Mouse model of Epstein-Barr virus LMP1- and LMP2A-driven germinal center B-cell lymphoproliferative disease.

Epstein-Barr virus (EBV) is a major cause of immunosuppression-related B-cell lymphomas and Hodgkin lymphoma (HL). In these malignancies, EBV latent membrane protein 1 (LMP1) and LMP2A provide infected B cells with surrogate CD40 and B-cell receptor growth and survival signals. To gain insights into their synergistic in vivo roles in germinal center (GC) B cells, from which most EBV-driven lymphomas arise, we generated a mouse model with conditional GC B-cell LMP1 and LMP2A coexpression. LMP1 and LMP2A had limited effects in immunocompetent mice. However, upon T- and NK-cell depletion, LMP1/2A caused massive plasmablast outgrowth, organ damage, and death. RNA-sequencing analyses identified EBV oncoprotein effects on GC B-cell target genes, including up-regulation of multiple proinflammatory chemokines and master regulators of plasma cell differentiation. LMP1/2A coexpression also up-regulated key HL markers, including CD30 and mixed hematopoietic lineage markers. Collectively, our results highlight synergistic EBV membrane oncoprotein effects on GC B cells and provide a model for studies of their roles in immunosuppression-related lymphoproliferative diseases.

Bystander inhibition of humoral immune responses by Epstein-Barr virus LMP1.

Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1), which mimics a constitutively active receptor, is required for viral transformation of primary B cells. LMP1 is expressed in EBV-infected germinal center (GC) B cells of immunocompetent individuals, suggesting that it may contribute to persistent EBV infection. In this study, we generated and analyzed mice that expressed LMP1 under the control of the CD19 or activation-induced cytidine deaminase (AID) promoter. Expression of LMP1 induced activation of B cells but severely inhibited their differentiation into antibody-secreting cells (ASCs) in vitro and GC B cells in vivo. LMP1-expressing (LMP1+) B cells not only suppressed the functions of wild-type (WT) B cells in in vitro co-culture, but also blocked differentiation of WT B cells into GC B cells and ASCs in immunized bone marrow chimeric mice. Microarray analysis revealed that the gene encoding indoleamine 2,3-dioxygenase 1 (IDO1), a major enzyme involved in the tryptophan metabolic process, was highly induced by LMP1. Either inhibition of IDO1 activity by methyl-l-tryptophan or knockout of Ido1 in LMP1+ B cells could rescue WT B cells from such suppression. IDO1-induced tryptophan consumption and production of tryptophan metabolites appeared to be responsible for inhibition of B-cell function. We conclude that LMP1 expression in antigen-committed B cells not only directly impairs GC B-cell differentiation, but also indirectly inhibits the functions of neighboring B cells, resulting in suppression of humoral immune responses. Such bystander inhibition by LMP1+ B cells may contribute to immune evasion by EBV.

Opposing roles of RUBCN isoforms in autophagy and memory B cell generation.

RUBCN (also known as Rubicon) was originally identified as a negative regulator of autophagy, a process by which cells degrade and recycle damaged components or organelles and that requires the activity of the class III PI3K VPS34 and the mTORC1 protein complex. Here, we characterized the role of a shorter isoform, RUBCN100, as an autophagy-promoting factor in B cells. RUBCN100 was translated from alternative translation initiation sites and lacked the RUN domain of the longer, previously characterized RUBCN130 isoform. Specific deficiency of RUBCN130 in B cells enhanced autophagy, which promoted memory B cell generation. In contrast to RUBCN130, which is localized in late endosomes and lysosomes and suppresses the enzymatic activity of VPS34, an effect thought to mediated by its RUN domain, RUBCN100 was preferentially located in early endosomes and enhanced VPS34 activity, presumably because of the absence of the RUN domain. Furthermore, RUBCN100, but not RUBCN130, enhanced autophagy and suppressed mTORC1 activation. Our findings reveal that the opposing roles of two RUBCN isoforms are critical for autophagy regulation and memory B cell differentiation.

Steric recognition of T-cell receptor contact residues is required to map mutant epitopes by immunoinformatical programmes.

MHC class I-restricted CD8 T-lymphocyte epitopes comprise anchor motifs, T-cell receptor (TCR) contact residues and the peptide backbone. Serial variant epitopes with substitution of amino acids at either anchor motifs or TCR contact residues have been synthesized for specific interferon-γ responses to clarify the TCR recognition mechanism as well as to assess the epitope prediction capacity of immunoinformatical programmes. CD8 T lymphocytes recognise the steric configuration of functional groups at the TCR contact side chain with a parallel observation that peptide backbones of various epitopes adapt to the conserved conformation upon binding to the same MHC class I molecule. Variant epitopes with amino acid substitutions at the TCR contact site are not recognised by specific CD8 T lymphocytes without compromising their binding capacity to MHC class I molecules, which demonstrates two discrete antigen presentation events for the binding of peptides to MHC class I molecules and for TCR recognition. The predicted outcome of immunoinformatical programmes is not consistent with the results of epitope identification by laboratory experiments in the absence of information on the interaction with TCR contact residues. Immunoinformatical programmes based on the binding affinity to MHC class I molecules are not sufficient for the accurate prediction of CD8 T-lymphocyte epitopes. The predictive capacity is further improved to distinguish mutant epitopes from the non-mutated epitopes if the peptide-TCR interface is integrated into the computing simulation programme.

Starvation-induced autophagy via calcium-dependent TFEB dephosphorylation is suppressed by Shigyakusan.

Kampo, a system of traditional Japanese therapy utilizing mixtures of herbal medicine, is widely accepted in the Japanese medical system. Kampo originated from traditional Chinese medicine, and was gradually adopted into a Japanese style. Although its effects on a variety of diseases are appreciated, the underlying mechanisms remain mostly unclear. Using a quantitative tf-LC3 system, we conducted a high-throughput screen of 128 kinds of Kampo to evaluate the effects on autophagy. The results revealed a suppressive effect of Shigyakusan/TJ-35 on autophagic activity. TJ-35 specifically suppressed dephosphorylation of ULK1 and TFEB, among several TORC1 substrates, in response to nutrient deprivation. TFEB was dephosphorylated by calcineurin in a Ca2+ dependent manner. Cytosolic Ca2+ concentration was increased in response to nutrient starvation, and TJ-35 suppressed this increase. Thus, TJ-35 prevents the starvation-induced Ca2+ increase, thereby suppressing induction of autophagy.

Effects of mass retention of dissolved organic matter and membrane pore size on membrane fouling and flux decline.

Ultrafiltration (UF) fouling has been attributed to concentration polarization, gel layer formation as well as outer and inner membrane pore clogging. It is believed that mass of humic materials either retained on membrane surface or associated with membrane inner pore surface is the primary cause for permeate flux decline and filtration resistance build-up in water supply industries. While biofilm/biofouling and inorganic matter could also be contributing factors for permeability decline in wastewater treatment practices. The present study relates UF fouling to mass of dissolved organic matter (DOM) retained on membrane and quantifies the effect of retained DOM mass on filtration flux decline. The results demonstrate that larger pore membranes exhibit significant flux decline in comparison with the smaller ones. During a 24-h period, dissolved organic carbon mass retained in 10 kDa membranes was about 1.0 gm(-2) and that in 100 kDa membranes was more than 3 times higher (3.6 gm(-2)). The accumulation of retained DOM mass significantly affects permeate flux. It is highly likely that some DOMs bind or aggregate together to form surface gel layer in the smaller 10 kDa UF system; those DOMs largely present in inner pore and serving as pore blockage on a loose membrane (100 kDa) are responsible for severe flux decline.