IntroductionSeveral studies have demonstrated that leukemic B cells of patients with chronic lymphocytic leukemia (CLL) express a restricted immunoglobulin (Ig) heavy chain repertoire and that expression of Ig variable heavy chain (IGHV) genes in CLL is not random. [1][2][3][4][5] One particular IGHV gene, the 51p1 allele of IGHV1-69, generally is expressed with little or no somatic mutation in 10 to 20% of all cases of this disease. 6 Furthermore, CLL B cells that express 51p1 have a preferential use of certain diversity (IGHD) and junctional (IGHJ) gene segments with restricted reading frames (RF) that encode relatively long third complementarity determining regions (CDR3), which have conserved amino acid motifs that are not characteristic of the CDR3s of the Ig expressed by nonneoplastic tonsilar or blood B cells that use 51p1. 3,7,8 The CDR3 is typically the most variable region of the Ig heavy chain and is generally directly involved in binding to conventional antigens. As such, the expression of such conserved Ig with restricted CDR3 argues that the Ig repertoire expressed in this disease is highly selected, suggesting a potential role for antigen in the development and/or progression of CLL.More recently we identified 15 unrelated CLL cases that expressed nearly identical Ig, with Ig heavy and light chain variable regions encoded by 51p1 and IGKV3-20 (A27), respectively. 9 The heavy and light chains of these 15 samples were virtually identical to SMI, a previously characterized CLL that expressed a polyreactive IgM/ autoantibody with low-affinity-binding activity for a variety of self-antigens. 10 The CDR3 regions of both the heavy and light chains together comprise the Ig binding site for antigen. The prevalent use of the 51p1 allele of IGHV1-69 in CLL with little or no somatic mutation allowed us to examine the effect of different Ig heavy chain CDR3 (HCDR3) motifs on Ig light chain pairing in CLL cases that otherwise expressed nearly identical Ig heavy chain variable regions. For this, we examined the IGKV and IGLV gene usage and CDR3 structure of 258 CLL cases that expressed unmutated Ig heavy chains encoded by 51p1 that were identified in a large cohort of 1846 nonselected CLL cases followed by the CLL Research Consortium (CRC). Methods Patient materialBlood was collected from consenting patients who satisfied the diagnostic and immunophenotypic criteria for B-cell CLL 11 and who presented for evaluation at the referral centers of the CRC. Institutional review board approval from each participating institution and informed consent were obtained in all cases, in accordance with the Declaration of Helsinki. Peripheral blood mononuclear cells were isolated by density gradient centrifugation using Ficoll-Hypaque 1077 (Sigma-Aldrich, St Louis, MO), washed twice, and analyzed directly or suspended in fetal calf serum containing 10% dimethylsulfoxide (DMSO; Sigma-Aldrich) for storage in liquid nitrogen. All samples contained more than 90% CLL B cells as Submitted February 8, 2007; accepted June 18, 2007. Pr...

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Analysis of the immunoglobulin (Ig) heavy chains expressed by the leukemic B cells of patients with chronic lymphocytic leukemia (CLL) has demonstrated that expression of Ig variable heavy chain (VH) genes in CLL is not random. Certain VH genes are more frequently expressed in CLL than in the normal adult B cell repertoire, and some, such as the 51p1 allele of VH1-69, also use of certain diversity (D) and junctional (JH) gene segments that encode third complementarity determining regions (CDR3) with conserved molecular structures. We identified 15 CLL cases among 1,220 examined that express nearly identical Ig heavy and light chains, encoded by 51p1/D3-16/JH3 and VKA27, respectively (Blood, 104:2499, 2004). The highly restricted and virtually identical structure of these B cell receptors strongly suggests selection for Ig in CLL that have a particular binding activity. However, little information is currently available about the light chains expressed by CLL B cells that have 51p1-encoded Ig heavy chains that use other D and JH segments encoding CDR3 that also are repeatedly observed in this disease. We analyzed the VL genes used by 235 CLL cases found to express 51p1-encoded Ig heavy chains among 1,605 CLL patients examined. First, we find restricted light chain isotype expression, as 72% of samples express kappa and 28% express lambda light chains, compared to 65% kappa and 35% lambda within the cohort of all 1,605 CRC CLL samples, and about 60% kappa and 40% lambda expression in normal blood B cells. Nucleotide sequence analysis of the Ig light chain V gene used by these 235 cases revealed that each had greater than 98% homology to an identified germline VK or Vl gene. Additionally, we identified non-stochastic pairing of particular VK and Vl genes with 51p1-encoded heavy chains that have highly-conserved CDR3. Twenty of the 235 cases (8.5%) were found to have Ig light chains encoded by VKO2. Seventeen (85%) of such cases had 51p1-encoded Ig heavy chains that used D2-2 and JH6, 15 of which had nearly identical CDR3 using the amino acid motif DIVVVPAAI. The VKO2-encoded light chains paired with these Ig heavy chains all had nearly identical CDR3 with the amino acid sequence motif QQSYSTPRT. Similarly, seven of the 235 cases (3%) were found to have Ig light chains encoded by Vl3-9. Six (86%) of such cases had 51p1-encoded Ig heavy chains that used D3-3 and JH6, and all had highly conserved heavy chain CDR3 with the amino acid motif YDFWSGYYPNYYYYGMDV. The Vl3-9-encoded light chains paired with these Ig heavy chains all had nearly identical CDR3 with the amino acid sequence motif QVWDSSTXV. Finally, we identified seven additional samples that express a heavy chain using D3-16 and JH3 that have nearly identical CDR3 amino acid sequences GGGYDYIWGSYRPNDAFDI, and also express light chains encoded by VKA27. These seven samples combined with the previous 15 represent all of the 51p1-encoded heavy chains that utilize D3-16 and JH3, as well as 52% (22 of 42) of all 51p1-encoded CLL samples that express VKA27-encoded light chains. These studies reveal for the first time that CLL cases using the same unmutated Ig heavy chain have non-stochastic pairing with disparate Ig light chains that is predicated upon the Ig heavy chain CDR3 structure. Because the CDR3 typically forms a major part of antibody binding site(s) for antigen, these data provide compelling evidence for antigen selection of the antibodies expressed in CLL.