Unmutated and mutated chronic lymphocytic leukemias derive from self-reactive B cell precursors despite expressing different antibody reactivity

Abstract

B cell chronic lymphocytic leukemia (CLL) is a disease of expanding monoclonal B cells whose B cell receptor (BCR) mutational status defines 2 subgroups; patients with mutated BCRs have a more favorable prognosis than those with unmutated BCRs. CLL B cells express a restricted BCR repertoire including antibodies with quasi-identical complementarity-determining region 3 (CDR3), which suggests specific antigen recognition. The antigens recognized by CLL antibodies may include autoantigens since about half of CLL B cells produce autoreactive antibodies. However, the distribution of autoreactive antibodies between Ig heavy-chain variable-unmutated (IgV-unmutated) CLL (UM-CLL) and IgV-mutated CLL (M-CLL) is unknown. To determine the role of antibody reactivity and the impact of somatic hypermutation (SHM) on CLL antibody specificity, we cloned and expressed in vitro recombinant antibodies from M- and UM-CLL B cells and tested their reactivity by ELISA. We found that UM-CLL B cells expressed highly polyreactive antibodies whereas most M-CLL B cells did not. When mutated nonautoreactive CLL antibody sequences were reverted in vitro to their germline counterparts, they encoded polyreactive and autoreactive antibodies. We concluded that both UM-CLLs and M-CLLs originate from self-reactive B cell precursors and that SHM plays an important role in the development of the disease by altering original BCR autoreactivity.

Figure 1

UM-CLL B cells express antibodies with long heavy- and light-chain CDR3s. IgH (A) and Igκ (B) CDR3 length in amino acids from control CD5⁺ (open bars), M-CLL (gray bars), and UM-CLL (black bars) B cells is indicated below the x axes. The average CDR3 length for control CD5⁺, M-CLL, and UM-CLL B cells was 15.5, 15.6, and 18.6 amino acids respectively. Significant P values were obtained by Student’s t test when UM-CLL IgH CDR3 length was compared with that of control CD5⁺ (P = 0.004) and M-CLL (P = 0.008) B cells. The y axes indicate the frequency of IgH and Igκ CDR3 lengths in each group.

Figure 2

A majority of CLL B cells express HEp-2 reactive antibodies. (A) Data shown are from ELISAs for anti–HEp-2 cell reactivity of recombinant antibodies cloned from control CD5⁺ (left), UM-CLL (middle), and M-CLL (right) B cells. Dotted lines show ED38-positive control antibody (18, 19). The percentage of autoreactive clones for each fraction and their P values are indicated. (B) CLL B cells mostly express anticytoplasmic antibodies. HEp-2–reactive antibodies from CLL B cells show various IFA patterns including rare nucleolar (CLL 261) and nuclear and cytoplasmic patterns (CLLs 266 and RF22) whereas most CLL antibodies recognized different structures in the cytoplasm (CLLs 249, 109, 403, 355, and GO13). (C) Frequency of self-reactive antibodies in UM-CLL (left) and M-CLL (right) with nuclear, nuclear/cytoplasmic, or cytoplasmic staining patterns and frequency of nonreactive antibodies.

Figure 3

CLL antibodies with quasi-identical Ig CDR3s recognize similar antigens. Antibodies belonging to 3 sets (sets I, IV, and V) of highly similar heavy and light chains as well as antibodies encoded by V_H1-69/D3-3/ J_H6 show quasi-identical staining cytoplasmic patterns specific to each group.

Figure 4

UM-CLL B cells express polyreactive antibodies. Antibodies cloned from control CD5⁺ (left), UM-CLL (middle), and M-CLL (right) B cells were tested by ELISAs for reactivity with ssDNA, dsDNA, insulin, and LPS. Dotted lines show ED38-positive controls (18, 19). Percentages represent frequency of polyreactive antibodies in each fraction. Significant P values obtained when UM-CLL polyreactive frequency was compared with that of control CD5⁺ and M-CLL B cells are indicated.

Figure 5

IgH CDR3 sequences of unmutated revertant antibodies engineered from M-CLL antibodies. (A) The 183 mutated sequence (183) to be reverted according to germline V_H, D, and J_H counterparts was selected based on the identification of mutations in bold in the IgH CDR3 sequences. Homologous nucleotides to the original germline segment sequences are represented by dots whereas nucleotides to be substituted in the reverted sequence are shown in bold. Deducted amino acids are shown in upper-case letters. 183R, the resulting sequence of the 183 unmutated revertant. (B) IgH amino acid CDR3 sequences of mutated-CLL and engineered unmutated revertants (R). Homologous amino acids to the original mutated CLL sequences (top) are represented by dots whereas substituted amino acids in the reverted sequence (bottom) are shown. IgH CDR3 D and J_H gene segments are indicated.

Figure 6

Most unmutated revertant antibodies engineered from M-CLL antibodies are HEp-2 reactive and/or polyreactive. (A) HEp-2 reactivity is increased in revertant CLL antibodies. Data shown are from ELISAs for anti–HEp-2 cell reactivity of revertant (filled squares) and M-CLL antibodies (open squares). (B) A majority of revertant CLL antibodies acquire polyreactivity. Revertant (filled symbols) and M-CLL antibodies (open symbols) were tested by ELISAs for reactivity with ssDNA (circles), dsDNA (triangles), insulin (diamonds), and LPS (squares). (C) Revertant CLL antibodies show IFA staining patterns that are different from those of their M-CLL counterparts. Mutated CLL HEp-2 staining patterns (top) were compared with unmutated revertant patterns (bottom). CLL 261 reverts from nucleolar (CLL 261) to cytoplasmic (CLL 261R) whereas others modify (CLLs 109R, 183R, 240R, 342R, 374R, and 215R) or acquire (CLL 189R) cytoplasmic staining patterns.

Figure 7

Strategy to revert mutated CLL antibodies to unmutated original counterpart. (1) Using a multi-template PCR strategy, germline V_H and J_H templates were first amplified with overlapping reverted CDR3 primers combined with primers annealing with either upstream germline V_H genes or downstream J_H segments. (2) A second PCR reaction fused the 2 overlapping V_H and J_H PCR fragments generating germline revertant antibody seqence.