Ig Family Research Articles

MEC1 and MEC2: two new cell lines derived from B-chronic lymphocytic leukaemia in prolymphocytoid transformation

We report the establishment and characterization of two cell lines, MEC1 and MEC2, that grew spontaneously on two subsequent occasions from the peripheral blood (PB) of a patient with B-chronic lymphocytic leukemia (B-CLL) in prolymphocytoid transformation. The patient was EBV-seropositive, his leukemic cells were EBNA negative, but the spontaneously grown cell lines are EBNA-2 positive. In liquid culture MEC1 cells grow adherent to the vessel wall and as tiny clumps; MEC2 cells do not adhere and form large clumps. The doubling time of MEC1 is 40h and of MEC2 is 31h. Both cell lines express the same light ( κ) and heavy chains ( μ, δ) as the fresh parental B-CLL cells at the same high intensity, share the expression of mature B cell markers (CD19,CD20, CD21, CD22), differ in the expression of CD23 and FMC7, are CD11a+, CD18+, CD44+, CD49d+, CD54+ and express at high levels both CD80 and CD86. CD5 is negative on MEC1 cells (as on the vast majority of parental cells) and it has been lost by MEC2 cells after several months of culture. The cells have a complex karyotype. The tumour origin of MEC1 and MEC2 has been demonstrated by Southern blot analysis of the IgH loci and by Ig gene DNA sequencing. They use the VH4 Ig family and have not undergone somatic mutations (94.8% homology with germline Ig gene 4-59). Cytofluorographic analysis and RT-PCR reveal that MEC1 and MEC2 overexpress Bc1-2 together with Bax, express large amounts of Bcl-xL and trace amounts of Bcl-xS.

The Role of Adhesion Molecules in Human Leukocyte Attachment to Porcine Vascular Endothelium: Implications for Xenotransplantation

Many obstacles still prevent successful xenotransplantation of porcine donor organs. When hyperacute rejection is averted, transplanted pig organs are subject to acute vascular and cellular rejection. In autologous systems, leukocyte recruitment into inflamed tissues involves selectins, integrins, and Ig family members. To determine whether these mechanisms allow human leukocytes to effectively enter porcine grafts, the pathways by which human leukocytes adhere to TNF-alpha-stimulated porcine aortic endothelium were examined under static and physiologic flow conditions. L-selectin and E-selectin had overlapping functions in neutrophil capture and rolling, whereas Ab blockade of E-selectin and the beta2 integrins inhibited firm arrest of rolling neutrophils. Combined blockade of selectins and beta2 integrins resulted in negligible human neutrophil attachment to pig endothelium. Lymphocyte attachment to porcine endothelium was primarily L-selectin mediated, whereas beta2 integrin and VCAM-1/very late Ag-4 (VLA-4) interactions promoted static adhesion. Concurrent beta2 integrin, VLA-4, VCAM-1, and L-selectin blockade completely inhibited lymphocyte attachment. Thus, interactions between leukocyte-endothelial cell adhesion receptor pairs remained remarkably intact across the human-porcine species barrier. Moreover, disrupting the adhesion cascade may impair the ability of human leukocytes to infiltrate a transplanted porcine organ during rejection.

Optimal selectin-mediated rolling of leukocytes during inflammation in vivo requires intercellular adhesion molecule-1 expression.

Leukocyte interactions with vascular endothelium during inflammation occur through discrete steps involving selectin-mediated leukocyte rolling and subsequent firm adhesion mediated by members of the integrin and Ig families of adhesion molecules. To identify functional synergy between selectin and Ig family members, mice deficient in both L-selectin and intercellular adhesion molecule 1 (ICAM-1) were generated. Leukocyte rolling velocities in cremaster muscle venules were increased significantly in ICAM-1-deficient mice during both trauma- and tumor necrosis factor alpha-induced inflammation, but rolling leukocyte flux was not reduced. Elimination of ICAM-1 expression in L-selectin-deficient mice resulted in a sharp reduction in the flux of rolling leukocytes during tumor necrosis factor alpha-induced inflammation. The observed differences in leukocyte rolling behavior demonstrated that ICAM-1 expression was required for optimal P- and L-selectin-mediated rolling. Increased leukocyte rolling velocities presumably translated into decreased tissue emigration because circulating neutrophil, monocyte, and lymphocyte numbers were increased markedly in L-selectin/ICAM-1-deficient mice. Furthermore, neutrophil emigration during acute peritonitis was reduced by 80% in the double-deficient mice compared with either L-selectin or ICAM-1-deficient mice. Thus, members of the selectin and Ig families function synergistically to mediate optimal leukocyte rolling in vivo, which is essential for the generation of effective inflammatory responses.

Predicting amino acid sequences of the antibody human VH chains from its first several residues.

A new method for classification of Ig sequences is suggested. The defining characteristic of a class is presence of particular residues at several class-determining positions. Sequences within a class follow the same amino acid pattern, i.e., residues at identical positions are, in an overwhelming majority of sequences of that class, identical or chemically related. Thus, once the class of a sequence is determined, one can predict the residue(s) at almost any position in the sequence. In this paper, results of analysis of 1,172 human heavy chains are presented. It was shown that a sequence can be assigned to one of six classes depending on which residues are found at its positions 1, 3, 5, 6, 7, 9, 10, 12, and 13. It is important to note that it is possible to achieve same six-class classification of the human heavy chains on the basis of a different set of positions found not at the beginning but near the end of the sequence (around position 80). For every class, an amino acid pattern of an entire sequence (complementarity determining regions excepting) has been determined. Our approach allowed us to reconstruct the incomplete human heavy chains in which residues at certain positions at the beginning or end of the chain are known. We developed a software tool for analysis, classification, and prediction of residues in sequences of the Ig family.

Expression of Two Ig Family Adhesion Molecules in the Murine Hair Cycle: DCC in the Bulge Epithelia and NCAM in the Follicular Papilla

The hair cycle involves remodeling of cells and of cell groups into a complex follicular structure. During skin appendage development, adhesion molecules such as neural cell adhesion molecule (NCAM) and deleted in colon carcinoma (DC) participate in the formation of cell groups. NCAM has been found to be expressed in the mesenchyme during mouse hair follicle induction. DCC expression has been observed in the epithelial cells of the developing feather. We postulate that these two molecules may also define cell groups in the cycling hair follicle. Here we report their spatio-temporal expression patterns during the depilation-induced murine hair cycle. NCAM expression was also examined in positive and negative hair-inductive follicular papilla cell lines. Throughout the hair cycle, DCC expression was confined to the basal keratinocytes of the epidermis and the epithelial portion of the hair follicle. During mid-anagen, two types of deleted in colon carcinoma staining were observed. One was a cell surface pattern seen in the epithelial cells in the bulge region where the follicular stem cells reside. The other was a diffuse cytoplasmic staining pattern in the transient hair follicle epithelia located below the bulge region. Prominent NCAM staining was observed in the follicular papilla throughout the hair cycle and was accompanied by weak staining of the matrix epithelia. NCAM expression correlated with hair induction by a follicular papilla cell line. The results suggest that DCC and NCAM define the permanent cell groups of the hair follicle and that NCAM is important for hair induction.

Investigation of myelin/oligodendrocyte glycoprotein membrane topology.

Myelin/oligodendrocyte glycoprotein (MOG) is a CNS-specific integral membrane protein that is an atypical member of the immunoglobulin (Ig) superfamily with two potential transmembrane domains based upon hydropathy analysis. With only one other exception, all Ig family members possess a single or no membrane spanning region. In order to analyze MOG membrane topology, we prepared stably transfected cells that express mouse MOG and used three domain-specific antisera to ascertain the localization of these hydrophobic domains. As expected, MOG's glycosylated N-terminal Ig-like domain was identified as extracellular, because membrane permeabilization was not required for immunoreactivity with the MOG1-125 antiserum. In contrast, both MOG154-169 and MOG198-218 antisera stained cells only upon permeabilization. These data indicate that only MOG's N-terminal hydrophobic domain spans the lipid bilayer, and we propose that MOG's C-terminal hydrophobic domain associates with the cytoplasmic face of the plasma membrane. As for MOG's second hydrophobic domain, it is clear that either orientation (transmembrane versus membrane-associated) would be unique among Ig-like proteins, and the implications of our proposed topology for MOG in oligodendroglial plasma membrane are discussed.

Identification of novel K562 membrane proteins that adhere to bone marrow fibroblasts

Adhesion of myeloid leukemia cells to the bone marrow (BM) microenvironment is mediated in part by Beta 1 and Beta 2 integrins. Cells of the erythroleukemia line K562, derived from a patient with chronic myeloid leukemia, bind to BM fibroblasts (BMFs) but the adhesion cannot be accounted for by integrins or other known adhesion proteins including CD44 or members of the Ig or selectin families. Membrane fragments from K562 cells were radioiodinated and allowed to adhere to BMF monolayers. Adherent proteins were solubilized together with the fibroblasts, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and visualized by autoradiography. Four adherent proteins were consistently observed. Two of these, with reduced molecular weights of 52 kD and 35 to 37 kD, were prominent. Addition of soluble thrombospondin and heparin but not fibronectin inhibited binding of K562 membrane proteins to adherent BMFs and immobilized thrombospondin- and heparin-bound K562 proteins. The 52-kD protein has a multimeric structure nonreduced and has characteristics of a glycoprotein. Its adhesion to fibroblasts is divalent cation and temperature sensitive. The 35- to 37-kD protein, whose function is divalent cation but not temperature sensitive, is phosphoinositol-linked and has characteristics identical to an adherent 35- to 37-kD protein identified on murine progenitor cells. Membrane preparations from two cases of acute myeloid leukemia showed an adherent 35- to 37-kD protein and in one case an adherent 52-kD protein without other adherent bands. A K562 subclone with reduced adherence to BMFs showed reduced amounts of adherent 52-kD and 35- to 37-kD proteins. These proteins may be responsible for the adhesion of malignant and normal hematopoietic progenitor cells to the BM microenvironment.

Identification of novel K562 membrane proteins that adhere to bone marrow fibroblasts

Abstract Adhesion of myeloid leukemia cells to the bone marrow (BM) microenvironment is mediated in part by Beta 1 and Beta 2 integrins. Cells of the erythroleukemia line K562, derived from a patient with chronic myeloid leukemia, bind to BM fibroblasts (BMFs) but the adhesion cannot be accounted for by integrins or other known adhesion proteins including CD44 or members of the Ig or selectin families. Membrane fragments from K562 cells were radioiodinated and allowed to adhere to BMF monolayers. Adherent proteins were solubilized together with the fibroblasts, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and visualized by autoradiography. Four adherent proteins were consistently observed. Two of these, with reduced molecular weights of 52 kD and 35 to 37 kD, were prominent. Addition of soluble thrombospondin and heparin but not fibronectin inhibited binding of K562 membrane proteins to adherent BMFs and immobilized thrombospondin- and heparin-bound K562 proteins. The 52-kD protein has a multimeric structure nonreduced and has characteristics of a glycoprotein. Its adhesion to fibroblasts is divalent cation and temperature sensitive. The 35- to 37-kD protein, whose function is divalent cation but not temperature sensitive, is phosphoinositol- linked and has characteristics identical to an adherent 35- to 37-kD protein identified on murine progenitor cells. Membrane preparations from two cases of acute myeloid leukemia showed an adherent 35- to 37- kD protein and in one case an adherent 52-kD protein without other adherent bands. A K562 subclone with reduced adherence to BMFs showed reduced amounts of adherent 52-kD and 35- to 37-kD proteins. These proteins may be responsible for the adhesion of malignant and normal hematopoietic progenitor cells to the BM microenvironment.

Human mucosal addressin cell adhesion molecule-1 (MAdCAM-1) demonstrates structural and functional similarities to the alpha 4 beta 7-integrin binding domains of murine MAdCAM-1, but extreme divergence of mucin-like sequences.

The mucosal vascular addressin, mucosal addressin cell adhesion molecule-1 (MAdCAM-1), is an Ig family adhesion receptor preferentially expressed by venular endothelial cells at sites of lymphocyte extravasation in murine mucosal lymphoid tissues and lamina propria. MAdCAM-1 specifically binds both human and mouse lymphocytes that express the homing receptor for Peyer's patches, the integrin alpha 4 beta 7. Functional expression cloning was used to isolate a cDNA from a macaque mesenteric lymph node library that encodes the homologue to murine MAdCAM-1. The macaque cDNA was subsequently used to clone the human homologue as well. Expression of human MAdCAM-1 RNA is restricted to mucosal tissues, gut-associated lymphoid tissues and spleen. Human MAdCAM-1 selectively binds both murine and human lymphocyte cell lines expressing alpha 4 beta 7. Human and macaque MAdCAM-1 have two Ig-like domains that are similar to the two amino-terminal integrin binding domains of murine MAdCAM-1. The conservation of sequences within the region homologous to the mucin/IgA domain of murine MAdCAM-1 is, however, much less apparent. These receptors exhibit considerable variation from murine MAdCAM-1 with respect to the length of the mucin-like sequence and the lack of a membrane proximal Ig (IgA-like) domain. The isolation of these different species of MAdCAM-1 demonstrates greater seleoffctive pressure for maintenance of amino acids involved in alpha 4 beta 7 binding than those sequences presumably involved in the presentation of carbohydrates for selectin binding.

Structural requirements for mucosal vascular addressin binding to its lymphocyte receptor alpha 4 beta 7. Common themes among integrin-Ig family interactions.

The mucosal vascular addressin, MAdCAM-1, is an Ig family adhesion receptor preferentially expressed by venular endothelial cells at sites of lymphocyte extravasation in mucosal lymphoid tissues and lamina propria. MAdCAM-1 binds the lymphocyte homing receptor for Peyer's patches, the integrin alpha 4 beta 7. We describe a point mutation within the first Ig domain of MAdCAM-1 that abolishes activation-independent alpha 4 beta 7 binding. This point mutation resides within an eight-amino acid motif with homology to sequences important for the integrin binding ability of the related vascular Ig family members, ICAM-1 and VCAM-1. To understand the contributions of the individual MAdCAM-1 domains, chimeric exchanges with the beta 1 integrin ligand, ICAM-1, were made. The two N-terminal domains of MAdCAM-1 are sufficient to confer alpha 4 beta 7 binding comparable to that of native MAdCAM-1. A chimera containing only the N-terminal Ig domain (domain 1) of MAdCAM-1 can also bind (to a lesser extent) alpha 4 beta 7, but only after integrin (to a lesser extent) alpha 4 beta 7, but only after integrin activation. Conversely, the first domain of ICAM-1 appears sufficient to bind activated LFA-1. These data suggest that the first domain of MAdCAM-1 is sufficient for interaction with alpha 4 beta 7, but that sequences within the second domain support this interaction, either by providing additional contact points for integrin binding or by contributing to the conformation or presentation of the N-terminal domain. The second domain of MAdCAM-1 can also support activation-dependent LFA-1 binding to domain 1 of ICAM-1. The findings parallel studies of VCAM-1 binding to alpha 4 beta 1 and suggest that structural differences exist between vascular Ig-like ligands for alpha 4 vs beta 2 integrins.

Cloning and expression of a murine Fab fragment recognizing a defined linear epitope of Chironomus thummi thummi major allergen Chi t 1-9.

We have cloned and expressed in bacteria the genes coding for the Fab fragment of the monoclonal antibody (MoAb) M3. M3 is a murine IgG1 antibody reactive with the major allergen Chi t 1-9 of Chironomus thummi thummi. The major allergen Chi t 1-9 is known to be an aggressive inhalant allergen and causes type-I allergy. The immunoglobulin (Ig) fragment genes were cloned as a synthetic dicistronic operon. In this operon each gene is preceded by a bacterial signal sequence to direct the recombinant protein to the periplasmic space of the bacteria. The cloned genes were expressed in Escherichia coli using the strong T7-RNA-polymerase-based system. Sequence analysis revealed that the light and heavy chains of MoAb M3 belong to the V kappa II and V kappa IIC group of the Ig family, respectively. Genes of the V kappa II and V kappa IIC group are known to be used in response to haptens. The apparent affinity constants of the parent antibody M3 and the recombinant Fab fragment are nearly equivalent.

Functional constraints against variations on molecules from the tissue level: slowly evolving brain-specific genes demonstrated by protein kinase and immunoglobulin supergene families.

In the protein kinase family, the basic function of kinase domain is similar among members. According to the standard view of functional constraint, the molecular evolutionary rate depends on functional and structural features characteristic of individual molecules (local constraint). Thus the evolutionary rate of the kinase domain is expected to be similar for different members. Contrary to this expectation, a comparison of the evolutionary rates revealed a wide difference among members; it amounts to about 100 times difference between the maximum and minimum rates. A similar result was also found in members of the immunoglobulin (Ig) family. In addition, significant correlations in evolutionary rate were observed between the kinase domain and the Ig-like domain in the receptor protein tyrosine kinases and between the kinase domain and the SH domain in the nonreceptor-type kinases. Furthermore, the evolutionary rates of family members that are expressed tissue specifically differ widely, depending on their tissue distribution: members expressed in the brain evolve with significantly slower rates than those expressed in the immune system. These results strongly suggest the presence of an alternative constraint (global constraint) against changes on molecules derived from higher levels like tissues or organs.

Interaction between human CD2 and CD58 involves the major beta sheet surface of each of their respective adhesion domains.

The CD58 binding site on human CD2 was recently shown by nuclear magnetic resonance structural data in conjunction with site-directed mutagenesis to be a highly charged surface area covering approximately 770A2 on the major AGFCC'C" face of the CD2 immunoglobulin-like (Ig-like) NH2-terminal domain. Here we have identified the other binding surface of the CD2-CD58 adhesion pair by mutating charged residues shared among CD2 ligands (human CD58, sheep CD58, and human CD48) that are predicted to be solvent exposed on a molecular model of the Ig-like adhesion domain of human CD58. This site includes beta strand residues along the C strand (E25, K29, and K30), in the middle of the C' strand (E37) and in the G strand (K87). In addition, several residues on the CC' loop (K32, D33, and K34) form this site. Thus, the interaction between CD2 and CD58 involves the major beta sheet surface of each adhesion domain. Possible docking orientations for the CD2-CD58 molecular complex are offered. Strict conservation of human and sheep CD58 residues within the involved C and C' strands and CC' loop suggests that this region is particularly important for stable formation of the CD2-CD58 complex. The analysis of this complex offers molecular insight into the nature of a receptor-ligand pair involving two Ig family members.

Integrins as dynamic regulators of vascular function

The vascular endothelium lines the entire cardiovascular system and serves as a nonthrombogenic and selectively permeable boundary between the blood-stream and extravascular space. Endothelial cells are polar cells that are continuously subjected to fluid-generated forces on their luminal surface whereas their abluminal surface resides on basement membranes/extracellular matrix. The integrin family of cell-surface heterodimeric glycoproteins is located along both of these surfaces and participates in maintaining the normal endothelium and in the dynamic changes associated with the pathophysiology of the endothelium. Endothelial cell beta 1 and beta 3 integrins function together with other families of adhesion molecules during vasculogenesis, angiogenesis, inflammation, and wound healing. Leukocyte beta 1 and beta 2 integrins, in conjunction with members of the Ig and selectin gene families expressed on endothelium, mediate leukocyte recruitment to sites of inflammation. The structural and functional properties of integrins make them uniquely suited to mediate these essential and complex processes in the vasculature.

Cell adhesion in a dynamic flow system as compared to static system. Glycosphingolipid-glycosphingolipid interaction in the dynamic system predominates over lectin- or integrin-based mechanisms in adhesion of B16 melanoma cells to non-activated endothelial cells.

Initial adhesion of B16 melanoma variants to non-activated endothelial cells is mediated through specific interaction between GM3 (NeuAc alpha 2----3Gal beta 1----4Glc beta 1----Cer) expressed on melanoma cells and lactosylceramide (LacCer, Gal beta 1----4Glc beta 1----Cer) expressed on endothelial cells. This adhesion is predominant over integrin- or lectin-mediated adhesion in a dynamic flow experimental system employing a parallel plate laminar flow chamber (Lawrence, M. B., Smith, C. W., Eskin, S. G., and McIntire, L. V. (1990) Blood 75, 227-237). In this system, a tumor cell suspension flows over a glass plate coated with glycosphingolipid, lectin, or fibronectin, and adhesion is recorded on videotape. These conditions were designed to mimic the microvascular environment in which tumor metastatic deposition takes place. In contrast, lectin- and fibronectin-based mechanisms are predominant in previously used static adhesion systems. Under static conditions, the relative degree of adhesion of the four B16 variants to endothelial cells or to LacCer-coated plates was the same as their relative degree of GM3 expression (i.e. BL6 approximately F10 greater than F1 greater than WA4), and adhesion was inhibited in the presence of methyl-beta-lactoside, or liposomes containing LacCer or GM3. Adhesion was also inhibited by pretreatment of B16 cells with anti-GM3 antibody DH2 or sialidase and by pretreatment of endothelial cells with anti-LacCer antibody T5A7. Under dynamic flow conditions, WA4 cells did not adhere to mouse endothelial cells at high shear stress (greater than 2.5 dynes/cm2) but did adhere at lower shear stress. In contrast, BL6 and F10 cells adhered strongly at both low and high shear stress. BL6 cell adhesion to endothelial cells at both low and high shear stress was inhibited in the presence of antibody DH2, ethyl-beta-lactoside, or lactose, as well as by pretreatment of BL6 cells with sialidase. Thus, some clear differences, as well as similarities, in cell adhesion under static versus dynamic conditions are demonstrated. These findings suggest that melanoma cell adhesion to endothelial cells, based on GM3/LacCer interaction, initiates metastatic deposition, which may trigger a series of "cascade" reactions leading to activation of endothelial cells and expression of Ig family or selectin receptors, thereby promoting adhesion and migration of tumor cells.

The scid mutation disrupts gene rearrangement at the rejoining of coding strands.

The gene rearrangement mechanism in lymphocytes is a highly ordered and regulated process, containing distinct stages and tissue specificity for most of the V(D)J rearrangement events. Several lines of evidence indicate that a common “recombinase” function mediates the rearrangement process for all the immunoglobulin (Ig) and T cell receptor (TCR) gene elements. Whereas significant progress has been acheived in the molecular mapping of the organization of the Ig and TCR gene families and the structure of rearrangement products, very little progress has been made in identifying the proteins important to the recombination pathway.

Neural Protein 1B236/Myelin‐Associated Glycoprotein (MAG) Defines a Subgroup of the Immunoglobulin Superfamily

We have reviewed the structure and properties of the neural protein 1B236/MAG. This molecule consists largely of five Ig-like domains separated from its carboxyl terminal tail by a single membrane-spanning region. Two forms of the protein differ in the length and sequence of the carboxyl terminus: these are encoded by alternatively spliced mRNAs that are differentially expressed during postnatal neural development. The Ig-like domains of 1B236/MAG are unusual in having structural similarities to Ig V domains but with short Cys-Cys distances characteristic of C domains. Several other Ig-like molecules exhibit this structural feature, including the cell adhesion molecule N-CAM, which is most closely related in sequence to 1B236/MAG. We have proposed 1B236/MAG as the prototype for this subgroup of the Ig family and offer a model for this type of Ig domain structure. 1B236/MAG probably acts as a cell adhesion molecule to mediate interactions between cells in a fashion similar to that proposed for N-CAM. In particular, 1B236/MAG may be involved in interactions between myelinating oligodendrocytes or Schwann cells and axons or between adjacent layers of myelin membrane during the process of myelin compaction. It is most likely that the homophilic or heterophilic interactions of 1B236/MAG occur through binding to the Ig-like domains. The structure of 1B236/MAG is therefore quite consistent with its proposed function and may serve as the model for this class of cell-cell interaction molecules. One would predict, for example, that the neuron-glia cell adhesion molecule Ng-CAM, also known as NILE or L1 (Bock et al. 1985, Friedlander et al. 1985), which mediates interactions between neurons and glial cells, would have a very similar structure to those of N-CAM and 1B236/MAG. In addition, the carboxyl terminal tails of the 1B236/MAG proteins may also be involved in interactions with cytoskeletal components, during membrane vesicle transport through the glial cytoplasm during myelination or through neuronal axoplasm or cytoplasm. The availability of full-length cDNA clones of 1B236/MAG mRNAs with the ability to express these products in vitro will enable the structure and interactions of 1B236/MAG to be tested in detail.

Variable region sequences of murine IgM anti-IgG monoclonal autoantibodies (rheumatoid factors). A structural explanation for the high frequency of IgM anti-IgG B cells.

The nucleotide sequences of heavy and light chains from 10 monoclonal IgM anti-IgG1 (RF) antibodies were determined and reported here as translated amino acid sequences. Only three families of VK light chains were used in these antibodies: VK1 (two examples), VK8 (three examples), and VK19 (four examples). This represents a significant nonrandom selection of light chains. In contrast, all other variable region gene segments (i.e., VH, DH, JH, and JK) were used in a pattern consistent with random selection from the available pool of germline genes. In two cases, the same anti-IgG1 specificity was generated by a combination of very homologous light chains with unrelated heavy chains. We infer from this that the light chain is the segment used by these antibodies to bind IgG1. The nature of these sequences provides an explanation for the curious observation that as many as 15% of splenic B cells in normal mice may be expressing IgM anti-IgG; if, as our data suggest, certain light chains in combination with many different heavy chains can be used in assembling the anti-IgG specificity, then, because of combinatorial association in which the heavy chain is not relevant for specificity, the fraction of IgM-producing B cells expressing these light chains should approximate the fraction of B cells making IgM anti-IgG. We calculate, based on data presented in several other studies, that 5-17% of B cells express one of the VK types observed in monoclonal RF. This agrees well with estimates for the number of B cells making IgM anti-IgG. In addition, our findings could rule out other explanations of the high percentage of B cells making RF, such as constant stimulation by antigen or presence of numerous antigenic epitopes since it was shown that IgM anti-IgG1 antibodies are not somatically mutated and that they are structurally homogeneous. We aligned the VK sequences of the RF in hopes of finding some primary sequence homology between the represented VK families which might point to residues involved in the binding interaction. Although we found no such homology in the hypervariable regions, we did find significant and unexpected homology in the FR2 and FR3 of these light chains. We noted that these regions are exposed in the Ig structure and postulate that they may be involved in a unique type of binding interaction between two Ig family domains, i.e., VK binding to a constant region domain of IgG.