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. 2013 Jan 14;210(1):59-70.
doi: 10.1084/jem.20121801. Epub 2013 Jan 7.

A Mutated B Cell Chronic Lymphocytic Leukemia Subset That Recognizes and Responds to Fungi

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Free PMC article

A Mutated B Cell Chronic Lymphocytic Leukemia Subset That Recognizes and Responds to Fungi

Robbert Hoogeboom et al. J Exp Med. .
Free PMC article

Abstract

B cell chronic lymphocytic leukemia (CLL), the most common leukemia in adults, is a clonal expansion of CD5(+)CD19(+) B lymphocytes. Two types of CLLs are being distinguished as carrying either unmutated or somatically mutated immunoglobulins (Igs), which are associated with unfavorable and favorable prognoses, respectively. More than 30% of CLLs can be grouped based on their expression of stereotypic B cell receptors (BCRs), strongly suggesting that distinctive antigens are involved in the development of CLL. Unmutated CLLs, carrying Ig heavy chain variable (IGHV) genes in germline configuration, express low-affinity, poly-, and self-reactive BCRs. However, the antigenic specificity of CLLs with mutated IGHV-genes (M-CLL) remained elusive. In this study, we describe a new subset of M-CLL, expressing stereotypic BCRs highly specific for β-(1,6)-glucan, a major antigenic determinant of yeasts and filamentous fungi. β-(1,6)-glucan binding depended on both the stereotypic Ig heavy and light chains, as well as on a distinct amino acid in the IGHV-CDR3. Reversion of IGHV mutations to germline configuration reduced the affinity for β-(1,6)-glucan, indicating that these BCRs are indeed affinity-selected for their cognate antigen. Moreover, CLL cells expressing these stereotypic receptors proliferate in response to β-(1,6)-glucan. This study establishes a class of common pathogens as functional ligands for a subset of somatically mutated human B cell lymphomas.

Figures

Figure 1.
Figure 1.
IGHV- and IGKV-sequences of V3-7Sh. (A) V3-7Sh CDR3 amino acid sequences and IGHV and IGKV rearrangements. (B) IGHV CDR3 lengths of 78 nonsubset IGHV3-7 and 4 V3-7Sh. (C) Frequencies of subset-biased replacement mutations in V3-7Sh and nonsubset IGHV3-7 sequences. n, number of mutated sequences; N, total number of sequences. Statistical significance analyzed by Fisher’s exact test. **, P < 0.01; ***, P < 0.001. (D) Schematic representation of V3-7Sh CLL IGHV. Lollipop-shaped symbols indicate somatic mutations as compared with the IGHV3-7 germline. Closed and open circles represent replacement and silent mutations, respectively. Boxes highlight subset-biased replacement mutations and the shared glutamic acid at position 106.
Figure 2.
Figure 2.
Recombinant V3-7Sh sIgM are specific for yeasts. (A) Staining of tissue microarrays with sIgM of V3-7Sh, two nonsubset M-CLLs, and two polyreactive U-CLLs. Displayed are stainings of kidney, duodenum, liver, and spleen tissues of healthy human donors. Stainings are representative of at least two independent experiments. (B) Flow cytometry staining of yeast species and zymosan with V3-7Sh sIgM (blue histograms) or CLL81, a nonsubset IGHV3-7–encoded control sIgM (red histograms). Displayed stainings are representative of three V3-7Sh sIgM. (C) ELISA for C. albicans with 23 CLL-derived recombinant sIgM. Data are representative of three independent assays. (D) Staining of hyphae in cytological specimens of cervical smears (top panel) and staining of yeast in hair follicles of paraffin-embedded skin biopsies (bottom panel). Displayed stainings are representative of all three V3-7Sh sIgM. (E) Flow cytometry staining of wild-type C. neoformans (red histogram) and a mutant strain that lacks a capsule (blue histograms) with V3-7Sh sIgM. Displayed staining is representative of all three V3-7Sh sIgM. (F) Flow cytometry staining of wild-type S. pombe cells (red histogram) and zymolyase-treated S. pombe cell walls (blue histogram) with V3-7Sh sIgM. Displayed staining is representative of all three V3-7Sh sIgM. (G) Flow cytometry staining of zymosan, a cell wall preparation of S. cerevisiae with V3-7Sh sIgM (blue histograms), or control sIgM (red histograms). Displayed staining is representative of all three V3-7Sh sIgM.
Figure 3.
Figure 3.
Recombinant V3-7Sh IgM stain filamentous fungi. Staining of Aspergillus in formalin-fixed, paraffin-embedded lung tissue (A), Aspergillus fumigates (B), Penicillium chrysogenum (C), F. pedrosoi (D), and R. oryzae (E) with sIgM of V3-7Sh or CLL81, a nonsubset IGHV3-7–encoded control sIgM. All displayed stainings are representative of all three V3-7Sh sIgM.
Figure 4.
Figure 4.
Recombinant V3-7Sh sIgM bind β-(1,6)-glucan. (A) Binding of V3-7Sh sIgM to serial dilutions of zymosan, pustulan (β-(1,6)-glucan), curdlan (β-[1,3]-glucan), mannan, and amylose (α-[1,4]-glucan) on dot blot. Blots are representative of three independent experiments. (B) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of pustulan (blue histograms). Red histogram represents an unstained control. Staining is representative of three independent experiments. (C) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of pustulan (•), mannan (▪), and laminarin (β-[1,3]-glucan; ▴). Displayed graph is representative of all three V3-7Sh sIgM. (D) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of gentiobiose, a β-(1,6)-glucose disaccharide (blue histograms). Red histogram represents an unstained control. Staining is representative of three independent experiments. (E) Binding of V3-7Sh sIgM to zymosan in the presence of indicated concentrations of gentiobiose (•), laminaribiose (β-[1,3]-glucose disaccharide; ▾), cellobiose (β-[1,4]-glucose disaccharide; ▴), isomaltose (α-[1,6]-glucose disaccharide; ▪), and salicin (◆). Displayed graph is representative of all three V3-7Sh sIgM.
Figure 5.
Figure 5.
V3-7Sh BCRs are selected for β-(1,6)-glucan specificity. (A) Binding of V3-7Sh sIgM to zymosan after exchanging the IGKV2-24 light chain with nonendogenous Ig light chains. Data are representative of three independent experiments. (B) Binding of V3-7Sh sIgM to zymosan after substitution of the glutamic acid at position 106 into an arginine (E106R) and after reversion of somatic mutations to IGHV3-7 germline (gl) combined with E106R. Data are representative of two independent experiments.
Figure 6.
Figure 6.
V3-7Sh BCR are selected for β-(1,6)-glucan affinity. (A) SPR curves of binding of pustulan (0, 0.3, 1, and 3 µg/ml) to V3-7Sh sIgM (left) and V3-7Sh sIgM after reversion of somatic mutations (V3-7Sh germline; right). The response curves were fitted to a 1:1 binding model (orange lines). Curves are representative of two independent experiments. (B) Kinetic constants for pustulan binding to V3-7Sh sIgM. ka in 104 sec−1M−1, kd in 10−5 sec−1, KD in pM. Kinetic constants were calculated with data from at least five different anti-IgM–coated spots. The error value is the deviation in the kinetic constants between different coated spots. For calculations, an estimated average molecular weight of 20 kD was used for pustulan. (C) Association constants (ka) of pustulan binding to V3-7Sh sIgM with somatic mutations (black bars) and after reversion of somatic mutations to IGHV3-7 germline determined by SPR (white bars). Data are representative of two independent experiments.
Figure 7.
Figure 7.
β-(1,6)-glucan induces proliferation of primary V3-7Sh CLL cells. (A) Zymosan-FITC staining of CD19+CD5+ cells of V3-7Sh CLL (filled histogram) or control M-CLL (open histogram). Plot is representative of three independent experiments. (B) Frequency of CD19+CD5+Zymosan+ cells of three V3-7Sh CLL and six control M-CLLs (including two nonsubset IGHV3-7–expressing CLLs). (C) CFSE staining after 8 d of culture of V3-7Sh CLL cells (top) or control M-CLLs (bottom) in the presence of pustulan (left) or anti-light chain antibodies (right). Representative data for cell cultures from all three V3-7Sh CLL patients. (D) Precursor frequency after 8 d of culture in the presence of pustulan (left) or anti-light chain antibodies (right). Values are precursor frequencies after correction for basal proliferation. **, P < 0.01; NS; not significant. Statistical significance was analyzed by two-tailed Student’s t test.

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