IgT, a primitive immunoglobulin class specialized in mucosal immunity

Abstract

Teleost fish are the most primitive bony vertebrates that contain immunoglobulins. In contrast to mammals and birds, these species are devoid of immunoglobulin A (IgA) or a functional equivalent. This observation suggests that specialization of immunoglobulin isotypes into mucosal and systemic responses took place during tetrapod evolution. Challenging that paradigm, here we show that IgT, an immunoglobulin isotype of unknown function, acts like a mucosal antibody. We detected responses of rainbow trout IgT to an intestinal parasite only in the gut, whereas IgM responses were confined to the serum. IgT coated most intestinal bacteria. As IgT and IgA are phylogenetically distant immunoglobulins, their specialization into mucosal responses probably occurred independently by a process of convergent evolution.

Figure 1

Purification and structural characterization of IgT. (a) Coomassie blue staining of purified serum IgM and IgT (2 µg) resolved by SDS-PAGE. Red arrowheads indicate heavy and light chains of IgT. Left margin, molecular size in kilodaltons (kDa). (b,c) Immunodetection of IgM and IgT (~0.2 µg) with a polyclonal antibody (b) and mAb (c, right) to trout IgT or an IgM-specific mAb (c, left). (d,e) Fractionation of serum (0.5 ml; d) or gut mucus (0.5 ml; e) by gel filtration (top left), followed by immunoblot analysis of the fractions with IgM- and IgT-specific mAbs (below). Right, 4–15% SDS-PAGE of gel-filtration fractions corresponding to elution volumes of 8.5 ml and 11.5 ml under nonreducing conditions, followed by immunoblot analysis with mAb to trout IgM or IgT. Red arrowheads indicate monomers. A₂₈₀, absorbance at 280 nm. (f,g) Immunoblot and densitometric analysis of the concentration of IgM and IgT in serum (f) and gut mucus (g); n = 10–15 fish. (h) Ratio of IgT to IgM in serum and gut mucus, calculated from the values in f and g. Numbers above bars (f–h) indicate mean. Data are representative of at least three independent experiments (mean and s.e.m. in f,g).

Figure 2

Identification of a previously unknown B cell lineage that expresses only surface IgT. (a) Flow cytometry (left) of blood leukocytes double-stained with IgM- and IgT-specific mAbs (n = 5 fish), and transmission electron microscopy (top right) and Giemsa staining (bottom right) of sorted IgT⁺ B cells. Scale bar, 2 µm (top right); original magnification (bottom right), ×40. (b) Gene-expression profiles of sorted IgM⁺ cells, IgM⁻ IgT⁻ double-negative cells (DN), and IgT⁺ cells among blood leukocytes (n = 5 fish). β-actin, loading control; IgM(mem), IgD(mem) and IgT (mem), membrane forms of IgM, IgD and gT heavy chains; TCRα and TCRβ, T cell antigen receptorα- and β-chains; CSF-1R, receptor for colony-stimulating factor 1. (c) Flow cytometry of leukocytes from peripheral blood (PBL), head kidney (HKL), spleen (SPL), peritoneal cavity (PCL) and intestine (INTL). Numbers adjacent to outlined areas indicate percent IgM⁺ cells (top left) or IgT⁺ cells (bottom right). (d) Frequency of IgM⁺ and IgT⁺ cells among total B cells (n = 15 fish). Red dashed line indicates 50%. (e) Immunofluorescence staining for IgM (red) and IgT (green) in a cryosection of rainbow trout spleen; nuclei (Nucl; blue) are stained with the DNA-intercalating dye DAPI (isotype-matched control antibodies, Supplementary Fig. 3a). Original magnification, ×20. Data are representative of at least three independent experiments (mean and s.e.m. in d).

Figure 3

Phagocytic and intracellular killing capacities of IgT⁺ B cells. (a) Flow cytometry of peripheral blood leukocytes incubated with 1-µm fluorescent latex beads (labeled with fluorescein isothiocyanate (FITC)) and then stained with mAb to trout IgT or IgM (n = 9 fish). Phag⁻, nonphagocytic; Phag⁺, phagocytic. (b) Phagocytic and nonphagocytic cells in IgT⁺ or IgM⁺ B cell subsets of peripheral blood leukocytes (n = 9 fish). Numbers in bars indicate mean percent phagocytic cells. (c) Transmission electron microscopy of various stages of ingestion of 1-µm beads (red ‘x’) by phagocytic IgT⁺ B cells from peripheral blood leukocytes. Scale bar, 2 µm. (d) Inhibitory effect of cytochalasin B on the phagocytic capacity of IgT⁺ B cells, presented as the percentage of phagocytic cells relative to that of PBS-treated control cells. (e) Intracellular bacterial killing by sorted IgM⁺ and IgT⁺ B cells and total peripheral blood leukocytes incubated with live E. coli and lysed; lysates were inoculated onto Luria-Bertani agar plates and surviving intracellular bacteria were counted. Results are presented as percent of live bacteria at time 0, set as 100%. Data are representative of at least three independent experiments (mean and s.e.m. in b).

Figure 4

Proliferative and immunoglobulin-secreting capacities of IgT⁺ and IgM⁺ B cells in response to microbial stimulation. (a) Proliferation of IgT⁺ and IgM⁺ B cells among trout head kidney leukocytes left unstimulated (Control) or stimulated for 1, 3 or 7 d with V. anguillarum bacterin (Vibrio), presented as frequency of cells positive for the thymidine analog EdU (5-ethynyl-2′-deoxyuridine) in the IgT⁺ or IgM⁺ B cell subset (n = 10–12 fish). (b) IgM and IgT in supernatants of cells treated as in a, assessed by immunoblot and densitometric analysis and presented relative to values on day 0 (n = 6 fish). (c) Immunoblot analysis of supernatants of cells treated as in a, probed with IgT- and IgM-specific antibodies (n = 6 fish). (d) Flow cytometry of IgT⁺ and IgM⁺ head kidney leukocytes on day 7 in the presence or absence of V. anguillarum bacterin; circles outline the predominant small or large B cell populations. FSC, forward scatter; SSC, side scatter. (e) IgM and IgT in supernatants of head kidney leukocytes collected 7 d after intraperitoneal injection of a mixture of lipopolysaccharide and V. anguillarum bacterin (n = 6 fish), sorted into large and small IgT⁺ and IgM⁺ cells and cultured for 2 d, assessed by immunoblot and densitometric analysis and presented relative to values at day 0. P values, unpaired Student’s t-test. Data are representative of three independent experiments (mean and s.e.m. in a,b,e).

Figure 5

Accumulation of IgT⁺ B cells in the GALT of fish that survived infection with C. shasta. Differential interference contrast images of immunofluorescence staining of trout gut cryosections from uninfected control fish (a) and fish that survived infection with C. shasta (b–d; n = 5 per group), stained for IgM (red), IgT (green) and C. shasta (Cer; magenta); nuclei are stained with DAPI (isotype-matched control antibody staining, Supplementary Fig. 3b). Blue dotted lines in a,b outline the border of the lamina propria (LP); magenta arrows point to the parasite located in the gut lumen (Lu). (c) Enlarged images of the areas outlined in b, without differential interference contrast, showing infiltrating IgT⁺ B cells in the gut lamina propria (left) or epithelium (white arrowheads, right). (d) Localization of a parasite in the gut epithelium (left) or lumen (right); green arrows indicate IgT⁺ B cells that seem to be secreting IgT. Scale bars, 20 µm. Data are representative of at least three independent experiments.

Figure 6

Immune responses in the gut of trout infected with C. shasta are mediated by the IgT system. (a) IgT⁺ and IgM⁺ B cells in gut cryosections of uninfected control fish and fish that survived infection with C. shasta (n = 5 per group), counted in 25 fields (original magnification, ×20). (b) Gut mucus IgT and IgM (n = 9–12 fish per group). (c) Real-time PCR analysis of IgT and IgM transcripts from the gut, presented relative to expression in control fish, set as 1 (n = 7 fish per group). (d,e) ELISA of specific anti–C. shasta IgT and IgM in the serum (d) and gut mucus (e), presented as end-point titers (n = 9 fish per group). P values, unpaired Student’s t-test. Data are representative of three independent experiments (mean and s.e.m.).

Figure 7

Most trout gut luminal bacteria are predominantly coated with IgT. (a,b) Staining of propidium iodide–positive trout gut lumen bacteria (3 × 10⁴) with anti-IgT (a; solid lines) or anti-IgM (b; solid lines) or their respective isotype-matched control antibodies (shaded histograms). (c) Coating of bacteria with IgT or IgM. Each symbol represents an individual fish (n = 17); red horizontal lines indicate the median. P value, nonparametric Mann–Whitney test. (d–g) Differential interference contrast images of luminal bacteria stained with a DAPI-Hoeschst solution (blue; d), anti-IgM (red; e) or anti-IgT (green; f), or merged IgT and IgM staining (orange; g). Isotype-matched control antibody staining, Supplementary Figure 5f,g. Orange arrows indicate bacteria stained for both IgT and IgM. Scale bar, 5 µm. (h) Immunoblot analysis of IgT and IgM on luminal bacteria: lane 1, 0.2 µg purified IgM or IgT; lanes 2–6, luminal bacteria (n = 5 fish). Data are representative of at least five independent experiments.

Figure 8

Gut mucus IgT and IgM associate with a trout pIgR. (a) Phylogenetic analysis of trout pIgR, constructed on the basis of the amino acid sequences of domain 5 from all pIgR sequences used. Numbers in diagram (percent) represent 1,000 bootstrap replications. GenBank accession numbers: human (Homo sapiens), NM_002644); mouse (Mus musculus), NM_011082; rabbit (Oryctolagus cuniculus), NM_001171045; pig (Sus scrofa), NM_214159; chicken (Gallus gallus) NM_001044644; African clawed frog (Xenopus laevis), EF079076; rainbow trout (Oncorhynchus mykiss), FJ940682; fugu (Takifugu rubripes), AB176853; common carp (Cyprinus carpio), GU338410; and orange-spotted grouper (Epinephelus coioides), FJ803367. (b) Immuno-fluorescence microscopy of trout pIgR expressed on MDCK cells transfected with a plasmid encoding Flag-tagged trout pIgR and stained with rabbit antibody to trout pIgR (α-pIgR; green), mouse anti-Flag (α-Flag; red) and DAPI (blue). Original magnification, ×20. (c) Immunoblot analysis of trout pIgR in cell lysates (5 µg) from MDCK cells transfected with empty plasmid (Vector) or plasmid encoding Flag-tagged trout pIgR (TpIgR) and in serum (~2 µg) and gut mucus (~2 µg), separated by SDS-PAGE under reducing conditions. (d) Coimmunoprecipitation (CoIP) of tSC from gut mucus with rabbit anti-IgT or mouse anti-IgM, followed by immunoblot analysis (IB) under reducing conditions (tSC detection) or nonreducing conditions (IgT and IgM detection) with anti-pIgR, anti-IgT or anti-IgM; IgG purified from serum obtained from rabbits before immunization (Prebleed) and mouse IgG1 (Isotype control) serve as negative controls for rabbit anti-IgT and mouse anti-IgM, respectively. Data are representative of at least three independent experiments.