B-cell receptor reactivity against Rothia mucilaginosa in nodular lymphocyte-predominant Hodgkin lymphoma

Abstract

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a Hodgkin lymphoma expressing functional B-cell receptors (BCR). Recently, we described a dual stimulation model of IgD+ lymphocyte-predominant cells by Moraxella catarrhalis antigen RpoC and its superantigen MID/hag, associated with extralong CDR3 and HLA-DRB1*04 or HLADRB1* 07 haplotype. The aim of the present study was to extend the antigen screening to further bacteria and viruses. The fragment antibody-binding (Fab) regions of seven new and 15 previously reported cases were analyzed. The reactivity of non-Moraxella spp.-reactive Fab regions against lysates of Rothia mucilaginosa was observed in 5/22 (22.7%) cases. Galactofuranosyl transferase (Gltf) and 2,3-butanediol dehydrogenase (Bdh) of R. mucilaginosa were identified by comparative silver- and immuno-staining in two-dimensional gels, with subsequent mass spectrometry and validation by western blots and enzyme-linked immunosorbent assay. Both R. mucilaginosa Gltf and Bdh induced BCR pathway activation and proliferation in vitro. Apoptosis was induced by recombinant Gltf/ETA'-immunotoxin conjugates in DEV cells expressing recombinant R. mucilaginosa-reactive BCR. Reactivity against M. catarrhalis RpoC was confirmed in 3/7 newly expressed BCR (total 10/22 reactive to Moraxella spp.), resulting in 15/22 (68.2%) cases with BCR reactivity against defined bacterial antigens. These findings strengthen the hypothesis of bacterial trigger contributing to subsets of NLPHL.

Figure 1.

Reactivity of recombinant IgD⁺ lymphocyte-predominant cell-derived Fab regions with bacterial lysates. (A) Representative immuno-dot blots of bacterial lysates, showing reactivity against lysate of M. catarrhalis from previously reported Fab regions, and from Fab regions of new cases as validation. In addition there are nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL)-derived recombinant Fab regions of five cases (#2, #12, #15, #16, #19) bound to lysates of R. mucilaginosa. (B) Representative western blot of polyacrylamide gel electrophoresis of lysate of R. mucilaginosa using pooled and individual recombinant Fab regions of NLPHL, which had shown reactivity against R. mucilaginosa as primary antibody. Two bands of approximately 35-40 kDa and 75 kDa were detected. (C) Silver staining of lysate of R. mucilaginosa for 45 min of blotting gel (Proteome factory AG, Berlin, Germany), and immunostain of antigens as dots in a two-dimensional gel blot using the recombinant pooled NLPHL Fab region as primary antibody. (D) Enzyme-linked immunosorbent assay with recombinant C-terminally FLAG-tagged potential antigens of R. mucilaginosa identified by mass spectrometry. The respective Fab region at a concentration of 10 μg/mL (positive control = rec. the Fab region of patient #11) showed a reactivity against galactofuranosyl transferase and 2,3-butanediol dehydrogenase of R. mucilaginosa.

Figure 2.

Serological responses against Gltf and Bdh of R. mucilaginosa. (A) Sera of patients with nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) and classical Hodgkin lymphoma (cHL) (diluted 1:100) were tested for antibodies against galactofuranosyl transferase (Gltf) and 2,3-butanediol dehydrogenase (Bdh) of R. mucilaginosa. (B) Titers of anti-Gltf antibodies and anti-2,3-Bdh antibodies in seropositive patients with NLPHL (left) or cHL (right). The curves represent the optical density (OD) at 490 nm of different serum dilutions. Patients with NLPHL had titers between 1:200 and 1:800. (C) Analysis of Ig classes, and IgG subclasses of serum-antibodies against Gltf and 2,3-Bdh of R. mucilaginosa in patients with NLPHL. The antiGltf antibodies and anti-2,3-Bdh antibodies were exclusively of the IgG class, mostly IgG1. (D) Light chains of serum-antibodies against Gltf and 2,3-Bdh of R. mucilaginosa in patients with NLPHL.

Figure 3.

Stimulation of different types of lymphocyte-predominant cells by Gltf of R. mucilaginosa. (A) Activation of the B-cell receptor (BCR) signaling pathway. Western blot analysis of components of the BCR signaling pathway shows activation by R. mucilaginosa galactofuranosyl transferase (Gltf) and by M. catarrhalis RpoC in DEV cells transfected and expressing R. mucilaginosa-Gltf-reactive recombinant BCR of case #2 with stronger bands of the activated isoforms pTyr525/526 SYK, pTyr96 BLNK, pTyr759 PLCγ2 and pTyr223 BTK and of MYC after incubation with R. mucilaginosa Gltf. Similarly, reaction to the BCR pathway after incubation with M. catarrhalis RpoC to DEV cells with RpoC-reactive BCR of case #3. (B) Tetrazolium proliferation assay with the transfected DEV cells expressing recombinant nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL)-derived BCR with reactivity against Gltf of R. mucilaginosa or RpoC of M. catarrhalis. Stimulation with Gltf of R. mucilaginosa increased proliferation in DEV cells transfected to express Gltf-reactive BCR but not in DEV cells expressing RpoC-reactive BCR. Incubation by ETA’ toxin-conjugated to Gltf of R. mucilaginosa or to RpoC/ETA’ of M. catarrhalis on DEV cells expressing either recombinant NLPHL-derived BCR (#2) with reactivity against R. mucilaginosa Gltf or against M. catarrhalis RpoC-reactive (#3) led to a decrease in proliferation. *P<0.05; **P≤0.01, ***P≤0.001, ****P≤0.0001. (C) Apoptosis induced by ETA’ toxin-conjugated to Gltf of R. mucilaginosa or RpoC of M. catarrhalis is dependent on doxycycline-induced expression of Gltf- or RpoC-reactive BCR on lymphocyte-predominant cells. Characterization of DEV cells transfected to express doxycycline-inducible IgD⁺ recombinant BCR with (from case #2) reactivity against Gltf of R. mucilaginosa or RpoC of M. catarrhalis (from case #3) by annexin-V/FITC and propidium iodide staining after 24 h culture in the presence of RpoC/ETA’, Gltf/ETA’ or staurosporin. Dox: doxycycline.