Defining terminally differentiating B cell populations in rainbow trout immune tissues using the transcription factor XbpI

Abstract

The nature of antibody-secreting cells in the rainbow trout is poorly defined. Here we describe a flow cytometric approach to help differentiate between four major trout B cell subsets present during terminal B cell differentiation: resting B cells, activated B cells, plasmablasts, and plasma cells. To aid in the identification of B cell subsets, the LPS-inducible transcription factor XbpI-S was used as a marker. An antibody specific to the stable form of inducible transcription factor X-box protein I (XbpI) was generated, which detects XbpI-S protein expression for species within the Oncorhyncus genus, including rainbow trout. Combinatorial expression patterns, or B cell signatures, were established using antibodies to XbpI-S, Pax5, and IgM in combination with a proliferation marker. We show that XbpI-S induction in trout splenic B cells increases throughout a 10-day in vitro LPS-induction period and that increased XbpI-S expression correlates with increased HCmu expression in the cell. PBLs displayed a lower level of XbpI-S induction during this incubation period, compared to spleen. We conclude that trout B cells follow a highly conserved B cell activation pathway, albeit slower than what has been observed in mammalian species. The use of XbpI-S as an activation marker for trout humoral immune activation promises to be useful for future in vivo studies, and can be applied to a broad range of teleost species.

Figure 1. Trout XbpI-S transcripts and XbpI-S antibody design

A. Photograph of ethidium bromide-stained, 2.5% agarose gel containing 10 µl PCR-product from D0 (0), D1 (1), and D7 (7). Molecular weight markers (MW), in bps, are indicated on the right. Locations of the expected 121 nt (XbpI-S) and predicted 147 nt (XbpI-U) fragments are indicated by lined and dotted arrows respectively. The asterix indicates the position of PCR primers. B. Schematic for the Unspliced form of XbpI (XbpI-U; light grey box) and the Spliced form (XbpI-S), which differ at their C-terminal protein sequence (dark grey area) [13]. Position of the tXbpI peptide used for immunization is indicated. C. Comparison XbpI-S protein sequence in the region corresponding to aa 334–364 of mouse XbpI-S (Genbank AF4431921). For trout, predicted protein sequence based on Genbank cDNA sequence (BX075133) and genomic trout XbpI DNA sequence were used (** Dr. Palti, National Center for Cold and Cool Water Aquaculture; unpublished data).

Figure 2. Western blot analysis on Oncorhyncus immune tissues using tXbpI-S antibody

A. XbpI-S expression in freshly isolated rainbow trout PBLs and SPL (spleen) cells (5 × (10)⁵ cells/lane). MWM are shown on the right in kD. B. Staining of spleen tissue (30 µg protein/lane) from 3 other Oncorhynchus species: Oncorhynchus nerka (ON), Oncorhynchus kisutch (OK), and Oncorhynchus gorbuscha (OG). C. Rainbow trout spleen immune cells cultured in the presence of LPS and collected on day 0 (D0) – day 6 (D6). Cell lysates of 5 × (10)⁵ splenic cells/lane. MWM (in kD) are indicated on the right.

Figure 3. One-color flow cytometry and detection of XbpI-S expression in in vitro LPS-activated spleen and PBL cells

A. Left: histogram showing increased intensity of XbpI-S staining (using tXbpI-S conjugated to Alexa 555) upon LPS-stimulation. Insert: dot-plot to show the gated lymphocyte population. Grey solid line: isotype control; black solid line, D0; black dotted line, D2; black dot-striped line, D4; black bold line, D8. Arrows indicate the intensity peak of the XbpI-S stained populations for each day. Cell populations that did not stain with tXbpI-S mark the 0% intensity peak. 100% indicates the maximum fluorescent intensity for XbpI-S (mean intensity value of Population 1 in Figure 4A). Blue dotted lines indicate gates for determining cell frequency of Peak 1 (XbpI-S−), Peak 2(XbpI-S+/−), and Peak 3 (XbpI-S+ cells. Right panel: list of cell frequencies (in percent) for each peak and for each day. B. Relative fluorescent intensity (in percent) in spleen B cells; results from 4 independent experiments with 3 fish pooled in each. C. As B. but for PBL, 4 independent experiments using 3 fish each.

Figure 4. Use of two-color flow cytometry to identify populations of terminally differentiating trout B cells

Spleen cultures were activated in vitro with LPS. A. Contour graph of Day 4 cells after staining with anti-IgM (total HCmu) and tXbpI-S antibodies; three distinct staining (Population 1–3) and one non-staining (double-negative; DN) cell population. B. Control (Day 4) sample stained with C555 and C647 isotype control antibodies. C. Detection of resting B cells and MPCs in Day 0 cells using anti-IgM (total HCmu) in combination with anti-Pax5 antibody. D. Detection of activated B cells, TPCs and MPCs in Day 4 cells using the same antibodies as in C. E. FSC/SSC profiles for each of the gated Populations 1–3 and DN cells.

Figure 5. Detecting trout plasmablasts (PB)

Trout splenic in vitro LPS-activated cultures (day 4). T/M-PCs are also indicated. A. Using anti-IgM (HCmu) with Edu. B. Using anti-Pax5 with Edu C. Using anti-tXbpI-S with Edu, and D. Using IgM pre-staining to detect membrane-IgM-positive proliferating cells, with Edu. E. FSC/Edu profiles for LPS-activated splenic B cells on days 2, 4, and 8 (D2, D4, and D8). D0 cells did not receive the Edu reagent.

Figure 6. Relationship between HCmu and XbpI-S expression during LPS-activation of trout B cells

Activation period: Day 0–10 after LPS activation. Mean intensity fluorescent value for each sample was determined using FACSArray software (see text). A. Relative HCmu intensity for days 0, 2, 4, 6, and 10 post LPS activation. Means and SE (N=4). B. Scatter plot showing the relationship between total HCmu intensity and XbpI-S intensity, using days 0, 2, 4, 6, 8, and 10 fluorescent intensity values for both spleen and PBL. Values on X- and Y-axes indicate percent fluorescent intensity relative to their maximum value.

Figure 7. Dynamics of trout terminal B cell differentiation in spleen

Cell frequencies were measured on specific days after LPS-activation in vitro. Days after LPS activation shown on the X-axis. A. Cell staining with tXbpI-S and anti-IgM (HCmu). Mean percent of each cell type and SE (N=4). Diamonds: frequencies of resting mature B cells (Population 3; XbpI-S+/−/HCmu+), squares: activated B cells and plasmablasts (Population 2; XbpI+/HCmu++) and triangles: frequency of (M/T)-plasma cells (Population 1; XbpI-S+/HCmu+++). B. Trout spleen, frequency of plasmablasts (Edu+/Pax5+ cells). Mean and SE (N=4). C. Staining as in B, but for mouse spleen, mean and SE (N=4).