Innate stimulation of B cells ex vivo enhances antibody secretion and identifies tumour-reactive antibodies from cancer patients

Human B cells and their expressed antibodies are crucial in conferring immune protection. Identifying pathogen-specific antibodies following infection is possible due to enhanced humoral immunity against well-described molecules on the pathogen surface. However, screening for cancer-reactive antibodies remains challenging since target antigens are often not identified a priori and the frequency of circulating B cells recognising cancer cells is likely very low. We investigated whether combined ex vivo culture of human B cells with three innate stimuli, interleukin-17 (IL-17), B cell activation factor (BAFF) and the toll like receptor 9 (TLR-9) agonist DNA motif CpG ODN 2006 (CpG), each known to activate B cells through different signalling pathways, promote cell activation, proliferation and antibody production. Combined IL-17+BAFF+CpG prolonged B cell survival and increased proliferation compared with single stimuli. IL-17+BAFF+CpG triggered higher IgG secretion, likely by activating differentiated, memory and class-switched CD19 +CD20 +CD27 +IgD - B cells. Regardless of anti-FOLR antibody seropositive status, IL-17+BAFF+CpG combined with a monovalent tumour-associated antigen (folate receptor alpha (FOLR)) led to secreted antibodies recognising the antigen and the antigen-expressing IGROV1 cancer cells. In a seropositive individual, FOLR stimulation favoured class-switched memory B cell precursors (CD27 -CD38 -IgD -), class-switched memory B cells and anti-FOLR antibody production, while IL-17+BAFF+CpG combined with FOLR, promoted class-switched memory B cell precursors and antibody-secreting (CD138+IgD-) plasma cells. Furthermore, IL-17+BAFF+CpG stimulation of peripheral blood B cells from patients with melanoma revealed tumour cell-reactive antibodies in culture supernatants. These findings suggest that innate signals stimulate B cell survival and antibody production and may help identify low-frequency antigen-reactive humoral responses.

DYRK1a mediates BAFF-induced noncanonical NF-kB activation to promote autoimmunity and B cell leukemogenesis

B cell-activating factor (BAFF) mediates B cell survival and, when deregulated, also contributes to autoimmune diseases and B cell malignancies. The mechanism connecting BAFF receptor (BAFFR) signal to downstream pathways and pathophysiological functions is not well understood. Here we identified DYRK1a as a kinase that responds to BAFF stimulation and mediates BAFF-induced B cell survival. B cell-specific DYRK1a deficiency causes peripheral B cell reduction and ameliorates autoimmunity in a mouse model of lupus. An unbiased screen identified DYRK1a as a protein that interacts with TRAF3, a ubiquitin ligase component mediating degradation of the noncanonical NF-kB-inducing kinase (NIK). DYRK1a phosphorylates TRAF3 at serine-29 to interfere with its function in mediating NIK degradation, thereby facilitating BAFF-induced NIK accumulation and noncanonical NF-kB activation. Interestingly, B cell acute lymphoblastic leukemia (B-ALL) cells express high levels of BAFFR and respond to BAFF for noncanonical NF-kB activation and survival in a DYRK1a-dependent manner. Furthermore, DYRK1a promotes a mouse model of B-ALL through activation of the noncanonical NF-kB pathway. These results establish DYRK1a as a critical BAFFR signaling mediator and provide novel insight into B-ALL pathogenesis.

Circulating CD138 (syndecan-1) enhances APRIL-mediated autoreactive B cell survival and differentiation in MRL/Lpr mice

High levels of serum CD138, a heparan sulfate-bearing proteoglycan, correlates with increased disease activity in systemic lupus erythematosus (SLE) patients. Mechanisms responsible for serum CD138 production and its biological function in SLE disease remain poorly understood. In this study, corroborating patient data, we detected an increase in serum CD138 in MRL/Lpr mice parallel to disease activity. Although TCR+CD138+ T cells expand in MRL/Lpr mice as the disease progresses, surprisingly, TCR+CD138- cells were the primary source of circulating CD138 as the transfer of TCR+CD138- cells to young MRL/Lpr mice, but not TCR+CD138+ cells, resulted with higher serum CD138 in the recipient mice. We found that elevated trypsin, expressed by TCR+CD138- cells, was able to cleave CD138 from T cells. Moreover, suggesting the contribution of cleaved CD138 to the increase in blood CD138, trypsin inhibitors defined trypsin inhibitor (DTI) or leupeptin increased CD138 expression on TCR+CD138- cells. Furthermore, soluble CD138 was able to bind a proliferation inducing ligand (APRIL) and enhanced APRIL-mediated plasma cell generation and autoreactive antibody production through the phosphorylation of extracellular-signal-regulated kinase (ERK) in B cells. APRIL receptor, transmembrane activator, calcium modulator, and cyclophilin ligand interactor (TACI) was involved in the enhancement of APRIL activity by CD138, as the synergistic effect of APRIL and CD138 was ablated on TACI deficient B cells. These findings indicate a regulatory role for soluble CD138 in B cell differentiation and autoreactive antibody secretion in SLE disease.

LUBAC Suppresses IL-21-Induced Apoptosis in CD40-Activated Murine B Cells and Promotes Germinal Center B Cell Survival and the T-Dependent Antibody Response

B cell activation by Tfh cells, i.e., through CD154 engagement of CD40 and IL-21, and survival within GCs are crucial for the T-dependent Ab response. LUBAC, composed of HOIP, SHARPIN, and HOIL-1, catalyzes linear ubiquitination (Linear M1-Ub) to mediate NF-κB activation and cell survival induced by TNF receptor superfamily members, which include CD40. As shown in this study, B cells expressing the Sharpin null mutation cpdm (Sharpincpdm) could undergo proliferation, CSR, and SHM in response to immunization by a T-dependent Ag, but were defective in survival within GCs, enrichment of a mutation enhancing the BCR affinity, and production of specific Abs. Sharpincpdm B cells stimulated in vitro with CD154 displayed normal proliferation and differentiation, marginally impaired NF-κB activation and survival, but markedly exacerbated death triggered by IL-21. While activating the mitochondria-dependent apoptosis pathway in both Sharpin+/+ and Sharpincpdm B cells, IL-21 induced Sharpincpdm B cells to undergo sustained activation of caspase 9 and caspase 8 of the mitochondria-dependent and independent pathway, respectively, and ultimately caspase 3 in effecting apoptosis. These were associated with loss of the caspase 8 inhibitor cFLIP and reduction in cFLIP Linear M1-Ub, which interferes with cFLIP poly-ubiquitination at Lys48 and degradation. Finally, the viability of Sharpincpdm B cells was rescued by caspase inhibitors but virtually abrogated – together with Linear M1-Ub and cFLIP levels – by a small molecule HOIP inhibitor. Thus, LUBAC controls the cFLIP expression and inhibits the effects of caspase 8 and IL-21-activated caspase 9, thereby suppressing apoptosis of CD40 and IL-21-activated B cells and promoting GC B cell survival.

Extrafollicular CD4 T cell-derived IL-10 functions rapidly and transiently to support anti-Plasmodium humoral immunity

Immunity against malaria depends on germinal center (GC)-derived antibody responses that are orchestrated by T follicular helper (TFH) cells. Emerging data show that the regulatory cytokine IL-10 plays an essential role in promoting GC B cell responses during both experimental malaria and virus infections. Here we investigated the cellular source and temporal role of IL-10, and whether IL-10 additionally signals to CD4 T-cells to support anti-Plasmodium humoral immunity. Distinct from reports of virus infection, we found that IL-10 was expressed by conventional, Foxp3-negative effector CD4 T cells and functioned in a B cell-intrinsic manner only during the first 96 hours of Plasmodium infection to support humoral immunity. The critical functions of IL-10 manifested only before the orchestration of GC responses and were primarily localized outside of B cell follicles. Mechanistically, our studies showed that the rapid and transient provision of IL-10 promoted B cell expression of anti-apoptotic factors, MHC class II, CD83, and cell-cell adhesion proteins that are essential for B cell survival and interaction with CD4 T cells. Together, our data reveal temporal features and mechanisms by which IL-10 critically supports humoral immunity during blood-stage Plasmodium infection, information that may be useful for developing new strategies designed to lessen the burden of malaria.

Monogenic autoimmunity caused by TLR7 gain-of-function

While circumstantial evidence supports enhanced TLR7 signaling as a mechanism of human systemic autoimmune disease, we have lacked the proof afforded by lupus-causing TLR7 gene variants. Here we describe monogenic human systemic lupus erythematosus (SLE) caused by TLR7 gain-of-function. We identified a de novo, novel, missense TLR7 Y264H variant in a child with severe lupus and additional novel or rare variants in probands with interferonopathies or systemic autoimmunity (Aicardi Goutieres Sd, SLE, Sjogren’s Sd, and juvenile idiopathic arthritis). The variants increased NF-κB and IFN-β activity and the de novo TLR7 Y264H variant was sufficient to cause lupus when introduced in mice. We show that constitutive TLR7 signaling drives aberrant survival of BCR-activated B cells that would otherwise die, and accumulation of CD11c+ age-associated B cells and germinal center (GC) B cells in a B cell-intrinsic manner. Follicular and extrafollicular helper T-cells were also increased but these phenotypes were cell-extrinsic. MyD88-deficiency rescued autoimmunity, aberrant B cell survival, and all cellular and serological phenotypes. Despite prominent spontaneous GC formation in mice carrying the TLR7 Y264H variant, we show that TLR7-driven lupus was not ameliorated when the TLR7 Y264H mice were made GC-deficient suggesting extrafollicular origin of pathogenic B cells. We establish the importance of TLR7 for human SLE pathogenesis, which paves the way for therapeutic TLR7 or MyD88 inhibition.

B Cell–Activating Factor Promotes B Cell Survival in Ectopic Lymphoid Tissues in Nasal Polyps

Ectopic lymphoid tissues (eLTs) characterized by B cell aggregation contribute to the local immunoglobulin production in nasal polyps (NPs). B cell-activating factor (BAFF) is vital for B cell survival, proliferation, and maturation. The purpose of this study is to investigate whether BAFF is involved in the B cell survival and eLT formation in NPs. The mRNA and protein levels of BAFF in NP tissues with and without eLTs were detected by PCR and ELISA assay, respectively. The cellular sources of BAFF and active caspase-3-positive B cells in NPs were studied by immunofluorescence staining. B cells purified from NP tissues were stimulated with BAFF and were analyzed by flow cytometry. Stromal cells purified from NP tissues were stimulated with lymphotoxin (LT) α1β2, and BAFF levels in culture supernatants were analyzed by ELISA. Compared with those in control tissues and NPs without eLTs, the BAFF levels were elevated in NPs with eLTs. Abundant BAFF-positive cells and few active caspase-3-positive apoptotic B cells were found in NPs with eLTs, in contrast to those in NPs without eLTs. There was a negative correlation between the numbers of BAFF-positive cells and frequencies of apoptotic B cells in total B cells in NP tissues. BAFF protected nasal polyp B cells from apoptosis in vitro. Stromal cells were an important cellular source of BAFF in NPs with eLTs. LTα1β2 induced BAFF production from nasal stromal cells in vitro. We propose that BAFF contribute to eLT formation in NPs by promoting B cell survival.