Regulation of IgM+ B Cell Activities by Rainbow Trout APRIL Reveals Specific Effects of This Cytokine in Lower Vertebrates

Abstract

Tumor necrosis factor ligand superfamily members such as B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) have been identified in mammals as key regulators of B cell homeostasis and activation. However, the immune functions of APRIL are not as well defined as those of BAFF. Furthermore, while BAFF is present in all vertebrates, APRIL is missing in some animal groups, suggesting that BAFF has compensated the functions of APRIL in these species. In this context, we thought of great interest to explore the effects of APRIL on teleost B cells, given that APRIL appears for the first time in evolution in bony fish. Thus, in this study, we have performed an extensive analysis of the effect of APRIL on B cells using rainbow trout (Oncorhynchus mykiss) as a model species. Our results demonstrate that APRIL induces a specific proliferation of IgM+ B cells by itself and increases IgM secretion without promoting a terminal differentiation to plasma cells. APRIL also increased the levels of surface MHC II and augmented the capacity of these cells to process antigen, effects that were exclusively exerted on IgM+ B cells. Although our results point to a highly conserved role of APRIL on B cell homeostasis and activation throughout evolution, some specific differential effects have been observed in fish in comparison to the effects of APRIL previously described in mammals. Finally, the effects that APRIL induces on rainbow trout IgM+ B cells described in this paper have been compared with those previously reported in response to BAFF.

Rainbow Trout IgM+ B Cells Preferentially Respond to Thymus-Independent Antigens but Are Activated by CD40L

“…Lipopolysaccharide (LPS) from Escherichia coli O26:B6, polyinosinic-polycytidylic acid (poly I:C), concanavalin A (ConA) from Canavalia ensiformis, collagenase type IV from Clostridium histolyticum, cytochalasin B from Drechslera dematioidea (cyto B), and benzocaine were purchased from Merck Millipore and used at concentrations previously described (22,23). 2,4,6-Trinitrophenyl hapten conjugated to keyhole limpet hemocyanin (TNP-KLH), TNP-LPS, and TNP-Ficoll were acquired from LGC Biosearch Technologies.…”

“…Single-cell suspensions from spleen, skin, head kidney, gills, and intestine were prepared by pushing the tissues through 100 µm nylon cell strainers (BD Biosciences) with L-15 medium containing 100 IU/ml penicillin, 100 µg/ml streptomycin (P/S), 10 U/ml heparin, and 5% fetal calf serum (FCS) (Thermo Fischer Scientific). In the case of intestine and skin, before cell extraction, tissue samples were digested as previously described (23). All cell suspensions were then placed onto 30/51% Percoll (GE Healthcare) density gradients and centrifuged at 500 × g for 30 min at 4 • C. Cells at the interface were collected, washed twice in L-15 medium containing P/S and 5% FCS, and adjusted to 2 × 10 6 cells in the same media.…”

“…The anti-trout IgM [1.14 mAb mouse IgG1 coupled to fluorescein isothiocyanate or to allophycocyanin (APC), 1 µg/ml], the anti-trout major histocompatibility complex (MHC) II β-chain (mAb mouse IgG1 coupled to APC, 2 µg/ml), and the antitrout CD8 (mAb rat IgG coupled to phycoerythrin, 7 µg/ml) used in this study have been previously characterized (23). Leukocytes were incubated with specific antibodies for 30 min on ice, washed three times with staining buffer (L-15 without phenol red containing 1% FCS), counterstained with 0.2 µg/ml 4 ′ ,6-diamidino-2-phenylindole (Sigma), to remove dead cells, and analyzed.…”

“…In all cases, 20,000 events (live single cells) were acquired per sample. FACS sorting of IgM + B cells, splenic T cells, or skin CD8 + dendritic cells (DCs) was performed on a FACSAria TM III flow cytometer (BD Biosciences) equipped with BD FACSDiva TM software as previously described (23,24). The purity of the sorted samples was verified by flow cytometry as described in Figure S1B.…”

“…To evaluate the levels of transcription of different immune genes in sorted leukocyte populations, DNase I-treated total RNA was reverse transcribed directly from FACS sorted populations using the Power SYBR Green Cells-to-Ct Kit (Invitrogen) following the manufacturer's instructions. Real-time PCR was performed using SYBR Green PCR core Reagents (Applied Biosystems) using specific primers (Table S1) and following the manufacturer's instructions as previously described (23).…”