CLL Stereotyped Subset #4 Igs Acquire Binding to Viable B Lymphocyte Surfaces By Somatic Mutations, Isotype Class Switching, and with the Prerequisite of IG Self-Association

The selection and transformation of a normal B cell into the leukemic state in chronic lymphocytic leukemia (CLL) is mediated at least in part by the structure of the B cell receptor (BCR) immunoglobulin (IG) antigen binding site. A dramatic indication of the role played by BCR is demonstrated by those CLL cases expressing "stereotyped" BCR IGs, characterized by very similar VH CDR3 amino acid sequences often encoded by specific IGHV, IGHD, and IGHJ gene segments. CLL stereotyped subset #4 BCR IGs expressing IGHV4-34/IGHD5-18/IGHJ6 and IGKV2-30/IGKJ2 rearrangements are prime examples of this. Moreover, these clones are always IgG isotype class switched, and contain IGHV and IGLVκ somatic mutations. Moreover, some of these mutations are shared among patients, and are almost invariably similar physicochemically and located at specific positions in the antigen binding site. In the following study, we evaluated the effects of these unique, acquired structural changes on the (auto)antigenic specificities of subset #4, using 3 mAbs belonging to the subset.

Gamma-irradiated Ramos B and Jurkat T cell line cells were incubated with subset #4 IGs under non-permeabilizing conditions, and binding to the B and T cell surfaces was detected by flow cytometry. Unlike most other CLL BCR IGs, the average binding percentage to apoptotic Ramos B cells of subset #4 mAbs was low. Conversely, each subset #4 IG bound live B cells well (CLL 183 - 44.8%; CLL 240 - 55.6%; CLL 342 - 52.0%). Moreover, all 3 IGs exhibited minimum binding to live and apoptotic Jurkat T cells. Next we exposed the subset #4 IGs to viable human B lymphocytes from tonsillectomy samples to confirm that binding was not limited to transformed B cells, and indeed the IGs bound well to CD19⁺ while minimally to CD3⁺ tonsil cells. When murine mAbs reactive with appropriate B-cell surface antigens were used to define subpopulations of normal B cells with which the subset #4 IGs interacted, we found that the subset #4 IGs and a non-subset #4 IGHV4-34-expressing CLL case (CLL 141) bound viable, naïve human B cells as expected from other studies. However, it became clear that the subset #4 IGs also interacted with viable memory B cells (CLL 183 - 28.1%; CLL 240 - 34.0%; CLL 342 - 42.7%; CLL 141 - 11.8%), a subpopulation that CLL 141 do not bind; the latter similar to IGHV4-34-expressing IGs in other disease states. Furthermore, although DNA and the "i" and "I" carbohydrate antigens are common targets of IGHV4-34 utilizing antibodies in systemic lupus erythematosus and cold agglutinin disease, respectively, the subset #4 Igs did not bind to either of these autoantigens when tested by ELISA or glycan binding arrays, respectively.

Strikingly, subset #4 IG binding to viable Ramos B cells and memory B lymphocytes depended on an asparagine (N) to aspartic acid (D) mutation at position 66 of FR3 in the rearranged IGKV2-30 gene (N vs. D: 5.26% vs. 55.6%); this amino acid residue is shared by many other clones in this subset and comes about by somatic hypermutation (SHM). Interestingly and consistent with the previous finding, another focused mutation of this subset, glutamic acid (E) at position 28 of FR1 in the rearranged IGHV4-34 (G28E), was responsible for the loss of DNA binding and for the diminished I/i glycan binding.

Finally, binding to viable Ramos B cells required the presence of the IgG isotype characteristic of this subset, since reactivity with Ramos B lymphocytes disappeared when the stereotyped antigen-binding site was linked to IgM (183, 240, 342 IgG vs. IgM: 44.8%, 55.6%, 51.2% vs. 24.5%, 1.85%, 7.02%, respectively). This requirement for IgG isotype binding to live Ramos B cells is likely due to the documented subset #4 self-association which requires the gamma isotype, since the change of a single amino acid at VH FR1 (E^16HA), which disrupts self-association, eliminated binding to Ramos B lymphocytes.

Together our findings indicate that distinct autoantigens mediate the positive and negative selection of the structural elements acquired by SHM and isotype class switching that are characteristic of this unique subset of stereotyped BCR IGs. Moreover, our data show that the reactivity of subset #4 IGs with viable B-cell surface requires the self-association of these IGs and, therefore, strongly suggests that viable B-cell surface binding is mediated by a non-conventional binding site created by self-associated surface membrane bound IGs.