The humoral response to xenografts is controlled by a restricted repertoire of immunoglobulin VH genes

BACKGROUND: The early phases of the host immune response to xenografts are dominated by anti-donor antibodies. The immunological pathways responsible for mediating the host humoral responses to xenografts are largely unknown, and this report addresses the nature of the immunoglobulin genes controlling the host antibody response to xenografts. METHODS: cDNA libraries established from rat anti-hamster monoclonal antibodies and splenic lymphocytes from LEW rats rejecting hamster heart xenografts were used to clone, sequence, and identify the immunoglobulin genes responsible for encoding rat xenoantibodies to hamster heart grafts. Libraries for germline variable region heavy chain (VH) genes encoding the anti-hamster xenograft antibodies were established by genomic DNA cloning and analyzed by nucleotide sequencing. The frequency of Ig VH gene usage for controlling the antibody responses to hamster xenografts was examined by colony-filter dot hybridization. The nucleic acid structure of these genes was then compared to their genomic progenitors to identify the number and structural diversity expressed by the Ig VH genes used to mediate the response. RESULTS: Rat monoclonal antibodies selected for their ability to precipitate the rejection of hamster xenografts exclusively use a closely related group of VH genes. The VH genes used by these antibodies are restricted to a single family of germline genes (VHHAR) for which 15 family members have been identified. The frequency of VHHAR gene usage in splenic IgM-producing B cells from LEW rats rapidly expands from 0.8% in naive animals to 13% in recipients 4 days after xenotransplantation. cDNA libraries expressing VHHAR genes were established from splenic lymphocytes derived from naive or xenograft recipients at 4 and 21 days after transplantation. Examination of 20 cDNA clones revealed that the majority (75%) of these clones express VHHAR genes displaying limited somatic mutation. CONCLUSIONS: The use of a closely related group of Ig VH genes in a germline configuration to control the early humoral response to xenografts suggests that this response may represent the utilization of a primitive, T cell-independent pathway of antibody production by the graft recipients.     

Germline structure and differential utilization of Igha and Ighb VH10 genes

Ab heavy chains encoded by mouse VH10 genes have been of particular interest due to their frequent association with DNA binding. We reported previously that VH10 sequences are over-represented in the preimmune repertoire considering the apparent number of germline-encoded VH10 gene segments. In this report, we show that the VH10 family consists of three and two germline genes in the Igha and Ighb haplotypes, respectively. The complete nucleotide sequences of these five genes, including promoters and recombination signal sequences, were determined and allow unambiguous assignment of allelic relationships. The usage of individual VH10 genes varied significantly and ranged from 0.2% to an extraordinary 7.2% of the VH genes expressed by splenic B cells. Since the promoter and recombination signal sequence elements of all five VH10 genes are identical, we suggest that the few amino acid differences encoded by these five germline VH10 genes determine their representation in the preimmune repertoire. Rearrangements of the most frequently used VH10 gene have an apparent bias for histidine at position 95 of complementarity-determining region-3 (CDR3). These CDR3s are also biased for asparagine, an amino acid associated with the CDRs of DNA binding Abs. Together, these results suggest that high VH10 gene use is the result of B cell receptor-mediated selection and may involve DNA and/or ligands that share antigenic features with DNA.     

Preferential use of the VH4 Ig gene family by diffuse large-cell lymphoma

Ig heavy chain variable region (VH) genes expressed by human diffuse large-cell lymphoma (DLC) and follicular lymphoma (FL) were identified and analyzed with respect to germline gene families. In 67 cases of FL, VH region genes were expressed in a pattern similar to that of normal B cells, with a predominance of the large VH3 gene family being used. In contrast, of the 17 cases of DLC, there was an extremely biased use of VH genes. Of these DLC tumors, 88% expressed genes from the small VH4 gene family; and even among these tumors, there was a limited use of genes, with 11 cases producing Igs derived from the VH4.21 germline gene. Although most of the VH genes expressed by DLC tumor cells contained mutations with respect to their germline counterparts, almost all of these mutations occurred before the clonal expansion of the tumor. This contrasts with our previous findings of ongoing mutations in FL and represents a fundamental difference between these two malignancies. This preferential gene use implies an important role for the VH4 gene family, and specifically for VH4.21, in the genesis of DLC.     

Evidence that multiple myeloma Ig heavy chain VDJ genes contain somatic mutations but show no intraclonal variation

To investigate whether somatic hypermutation occurs in multiple myeloma (MM) Ig VH region genes, we have cloned and sequenced the expressed VH genes from five cases of MM. The sequences were obtained after polymerase chain reaction (PCR) on total RNA isolated from the bone marrow, using 5' VH family-specific leader and 3' C gamma- or C alpha-specific primers. MM-specific CDR3 oligonucleotides were produced to isolate VH genes expressed by the malignant plasma cells. In all five cases, the productive Ig gene used the VH3 family. Extensive sequence analysis of multiple independent M13 clones showed no intraclonal variation with no evidence for ongoing somatic hypermutation in MM VH region genes. We were able to identify possible germline counterparts of the expressed VH genes in two cases. Comparison of these genes shows that the MM VH region genes have somatic mutations characteristic for an antigen-driven process. In the other three cases, no close homology could be found with published VH3 sequences. These findings implicate that, in MM, clonal proliferation takes place in a cell type that has already passed through the phase of somatic hypermutation.     

Xenoantibodies to pig endothelium are expressed in germline configuration and share a conserved immunoglobulin VH gene structure with antibodies to common infectious agents

BACKGROUND: The rejection of pig xenografts in humans is initiated by preformed antibodies that may be related to the natural antibodies that formulate a first line of defense against infectious agents. Immunoglobulin gene variable domains encoding the antibodies that react with similar epitopes expressed on xenoantigens and bacteria may share structurally similar antigen-binding site configurations. METHODS: We sequenced the VH immunoglobulin genes and germline progenitors of two rat monoclonal antibodies that recognize pig xenoantigens. Nucleic and amino acid sequences of these xenoantibodies were compared with immunoglobulin genes encoding antibodies that react with bacteria or viruses. RESULTS AND CONCLUSIONS: VH genes encoding rat anti-pig xenoantibodies are expressed in germline configuration and share structural similarities, including identical amino acids in key antigenic contact sites that define antibody canonical structural groups, with antibodies to infectious agents.     

Expression of monovalent fragments derived from a human IgM autoantibody in E. coli. The input of the somatically mutated CDR1/CDR2 and of the CDR3 into antigen binding specificity

A hybridoma producing a polyspecific human monoclonal IgM antibody (named CB03) has been derived from a fusion of mouse myeloma cells with human spleen lymphocytes obtained from an autoimmune patient suffering from chronic idiopathic thrombocytopenia. The antibody was found to be encoded by somatically mutated VHI and VlambdaIII genes. To study the input of mutated complementarity regions (CDRs) into antibody specificity, the antigen binding features of the purified complete IgM antibody were compared with (i) a Fab fragment by hot tryptic digestion and (ii) recombinant monovalent fragments expressed in E. coli. In detail, vectors were constructed encoding for (i) rFab03 and single chain Fv03 fragments containing the VH and VL genes connected by a linker sequence, (ii) scFc1.1. fragments containing the VH germline equivalent and the CB03 wild-type CDR3 region, and (iii) scFv fragments containing the CDR1 and CDR2 in germline configuration and the CDR3 expressed in the CB253 human fetal B cell hybridoma producing a polyspecific IgM antibody. The expression vectors contained at the 3' end either a (His)6 motif allowing purification on Ni(2+)-agarose or a c-myc tag for specifically detecting the expression products by a murine monoclonal antibody. Western blotting and ELISA analyses of the expression products indicate: (i) recombinant Fab fragments were found in the bacterial periplasm in extremely low amounts (1-10 micrograms from 1 litre bacterial culture), (ii) scFv fragments were obtained in suitable amounts from bacterial periplasm (800-1000 micrograms/l), (iii) the monovalent recombinant fragments as well as the Fab obtained by tryptic digestion reflected the polyspecific antigen binding features of the complete IgM antibody, but did bind to the antigens with much lower affinity, and (iv) the CDR3 was found to be of critical importance for the antigen binding pattern of this particular IgM. We discuss the expression of recombinant scFv fragments in E. coli as a suitable method in studying the role of the somatic mutation in autoantibody generation.     

V region diversity in human anti-insulin antibodies. Preferential use of a VHIII gene subset

Data on structures used by human antibody repertoires are derived principally from lymphoid malignancies and from autoantibodies that often express VH genes from the developmentally regulated fetal repertoire. To determine whether human immune responses generated by exogenous Ag use a pool of VH genes distinct from the fetal repertoire, nucleotide and predicted amino acid sequences were determined for five anti-insulin B cell clones from a type I diabetic patient treated with human insulin. The data show that a set of VHIII genes is preferentially used by human anti-insulin B cells. Structural features indicate that these expressed VH are derived from germ-line genes that are not frequent in fetal repertoires and these genes have undergone Ag-driven somatic mutation. The preferential use of related VH segments contrasts with the BALB/c anti-insulin response, which uses multiple V genes elements largely unmutated from germ-line sequences. In addition, long CDRH3 structures in human anti-insulin mAb are generated by complex gene interaction mechanisms that are not seen in murine anti-insulin mAb. Interestingly, similar potential insulin-binding structures are used by antibodies from both species. These findings suggest that human responses to exogenous insulin may express a limited number of VH genes and depend upon somatic mutation and complex D gene interactions in CDRH3 to expand the repertoire. Although these antibodies bind autologous insulin, VH gene usage and structural features that predominate in the response are not characteristic of the fetal repertoire.     

B cell superantigens: potential modifiers of the normal human B cell repertoire

Staphylococcal protein A (SPA), HIV gp120, and staphylococcal enterotoxins (SE) are B cell superantigens that induce VH specific B cell responses. In addition, the red blood cell antigens, i/I, have some features of a B cell superantigen. Binding of SPA, SE and HIV gp120 are VH family specific, whereas binding of i/I is VH gene specific. SPA and HIV gp120 function by stimulating VH3-expressing B cells, whereas SE appear to function by enhancing survival of the appropriate VH-expressing B cells. Moreover, HIV gp120 has been shown to delete VH3-expressing B cells. In this review, we describe evidence that shows how these superantigens may play a role in shaping the normal B cell repertoire.     

VH usage and somatic hypermutation in peripheral blood B cells of patients with rheumatoid arthritis (RA)

The human antibody repertoire has been demonstrated to have a marked V-gene-dependent bias that is conserved between individuals. In RA patients, certain heavy chain V genes (VH) have been found to be preferentially used for encoding autoantibodies. To determine if such preferential use of VH genes in autoantibodies is associated with a general distortion of the V gene repertoire in RA patients, the VH composition of peripheral blood B cells was analysed among four RA patients and four age- and sex-matched healthy controls. Usage of individual VH genes (eight VH3 and three VH4 genes tested by hybridization with a set of gene-specific oligonucleotide probes) was highly biased among RA patients, but no evidence of a distortion in the bias was observed compared with healthy controls. However, the occurrence of somatic mutations in these VH genes (estimated by differential hybridization with motif-specific oligonucleotide probes targeted to CDR and FR of the tested genes, and by DNA sequence analysis) was strikingly different between patients and healthy subjects. The number of VH3 rearrangements that had accumulated somatic mutations and the number of mutations per rearrangement were significantly elevated in three of the four RA patients. A slight but not significant elevation in mutations among rearranged VH4 genes was also observed in these patients. These data suggest that although usage of individual VH genes among peripheral blood B cells is not affected by the disease, the autoimmune process may involve a significant fraction of the B cell compartment.     

Autoreactivity of human VH domains from cDNA libraries: analysis with a bacterial expression system

Previous studies showed that VH domains of several anti-DNA Abs can bind DNA in the absence of VL. In the current work, we tested the VH autoreactive potential more generally, examining VH domains that did not come from known autoantibodies. Using a bacterial expression system, we produced 11 fusion proteins, each containing a VH domain and a B domain of staphylococcal protein A. The VH domains were coded in cDNA libraries from circulating B cells of healthy young adult humans. Thus, binding properties of the Ig molecules from which they came were unknown. The B cells had not been stimulated in vitro. Seven cDNA clones combined the frequently expressed VH3-23 gene segment with varied DH and JH segments. The other clones contained unmutated VH3-7, VH3-9, VH3-53, and VH4-39 segments. We compared these bacterial expression products with single-chain Fv, VH and VL domains of IgM mAb 18/2, a VH3-23-encoded, DNA-binding autoantibody. Submicromolar concentrations of 5 of the 11 VH domains bound to ssDNA. Those and one more also bound to immobilized poly(dT), and two bound to circular plasmid dsDNA. Soluble poly(dT) was the most potent inhibitor in competitive ELISA. Seven of the VH domains also bound to immobilized nuclear ribonucleoprotein, four to histone and none to thyroglobulin. Two interacted with the matrix of a Sephacryl S-100 column. The polyreactive autoantigen-binding properties of these VH domains raise the question of whether these properties may play a role in the formation of the VH repertoire of circulating B cells.     

Molecular cloning of multiple cDNAs encoding human enzymes structurally related to 3 alpha-hydroxysteroid dehydrogenase

Leukaemic B cells from patients with chronic lymphocytic leukaemia (B-CLL) are known to express the pan T-cell marker CD5 and a restricted set of immunoglobulin (Ig) variable region heavy (VH) and light (VL) chains encoded by germline or minimally mutated germline genes. We have studied surface expression of certain VH and VK gene products on peripheral blood B lymphocytes from 23 patients with B-CLL, using a panel of monoclonal antibodies (MoAbs) recognizing germline encoded cross-reactive idiotypes (CRI) associated with VHI (G6, G8), VHIII (B6, D12), VKIIIb (17-109) and an epitope linked to the VKIII light chain subgroup (C7). While only 1.7-3.2% of peripheral blood B lymphocytes from normal individuals expressed the VHI-associated CRI (VHI-CRI), these CRI were expressed on virtually all the leukaemic B cells from 17-22% of the CLL patients. The VHIII-associated CRI (VHIII-CRI), however, were found in 8.5-13% of the CLL B cells. Fifty per cent of the IgMK-expressing CLL cells (7/14) expressed the VKIII light chain subgroup of which only one expressed the VKIIIb-associated CRI (VKIIIb-CRI), 17-109. The anti-VHI-associated CRI antibodies were used to study their regulatory effect on in vitro Ig synthesis by the leukaemic cells. A significant suppression of spontaneous and mitogen-driven Ig production was observed in all cases studied. These results demonstrate an over-expression of VHI and VKIII gene products in B-CLL and suggest that B cells expressing these CRI are particularly susceptible to lymphoproliferative stimuli. The anti-CRI antibodies can be used to modulate Ig production by the leukaemic cells and may be of potential value for selective immunotherapy.     

Variant effects of non-native kissing-loop hairpin palindromes on HIV replication and HIV RNA dimerization: role of stem-loop B in HIV replication and HIV RNA dimerization

Splenic marginal-zone B cells, marginal-zone B cells of Peyer's patches in the gut, and nodal marginal-zone B cells (also identified as monocytoid B cells) share a similar morphology and immunophenotype. These cells likely represent a distinct subset of B cells in humans and rodents, but their precise ontogenetic relationship as well as their origin from B cells of the germinal center is still debated. To study this, we performed a mutation analysis of the rearranged immunoglobulin variable genes (VH) of microdissected single nodal and splenic marginal-zone cells. In addition, we investigated the presence of proliferating cells and B-cell clones in the human splenic and nodal marginal zone as well as adjacent germinal centers. This was performed by immunohistochemical staining for the Ki-67 antigen and denaturing gradient gel analysis of amplified immunoglobulin heavy chain genes' complementarity determining region 3 of microdissected cell clusters. A variable subset of nodal and splenic marginal-zone B cells showed somatic mutations in their rearranged VH genes, indicating that both virgin and memory B cells are present in the nodal and splenic marginal zone. Nodal and splenic marginal-zone B cells preferentially rearranged VH3 family genes such as DP47, DP49, DP54, and DP58. A preferential rearrangement of the same VH genes has been shown by others in the peripheral CD5(-) IgM+ B cells. These data suggest that the splenic and nodal marginal-zone B cells are closely related B-cell subsets. We also showed that marginal-zone B cells may cycle and that clones of B cells are frequently detected in the nodal as well as the splenic marginal zone. These clones are not related to those present in adjacent germinal centers. These data favor the hypothesis that clonal expansion occurs in the marginal zone. Whether the somatic hypermutation mechanism is activated during the clonal expansion in the marginal zone and which type of immune response triggers the clonal expansion need to be elucidated.     

Immunoglobulin VH gene mutational analysis suggests that primary effusion lymphomas derive from different stages of B cell maturation

Primary effusion lymphoma (PEL) is a recently described distinct subtype of non-Hodgkin's lymphoma associated with infection by the Kaposi's sarcoma-associated herpesvirus, also called human herpesvirus-8. Most cases of PEL are also associated with the Epstein-Barr virus (EBV). In order to better characterize the cellular origin of PEL, we investigated the immunoglobulin (Ig) heavy chain variable region (VH,) genes expressed by tumor cells of the BC-1 and BC-3 cell lines derived from PELs and five original PEL specimens. In the six EBV-positive PELs examined, including the BC-1 cell line, the expressed VH gene sequences showed numerous point mutations relative to the putative germline VH gene sequences. In addition, the VH, segment of one of these cases showed intraclonal sequence heterogeneity, indicating ongoing somatic mutation. In five cases, the distribution and type of mutations indicated that tumor cells had been selected by antigen. Because somatically mutated Ig genes are expressed by B cells that have reached a germinal center/post-germinal center stage of development, these findings suggest that the PEL cell of origin is a germinal center or post-germinal center B cell in most cases. In contrast, the VH gene segment expressed by tumor cells of the BC-3 cell line, which was originated from an EBV-negative PEL obtained from an HIV-negative patient, was unmutated, suggesting a pre-germinal center B cell origin for tumor cells of this particular PEL cell line. Taken together, these findings suggest that development of PELs may not be restricted to one stage of B cell differentiation and may represent transformation of B cells at different stages of ontogeny.     

Isolated U-fiber involvement in MS: preliminary observations

Mantle cell lymphoma is a distinct clinicopathological entity associated with t(11;14) and cyclin D1 overexpression. The majority of cases show uniform morphological and phenotypic features characterized by a monotonous proliferation of small-to-medium-sized irregular B cells that express CD5 and bright surface immunoglobulin IgM and IgD. By sequence analysis of the rearranged immunoglobulin heavy chain variable genes (VH), it has been shown that these lymphoma cells carry little if no somatic mutations, as described for the fetal CD5+ cells or B1 cells. Besides mantle cell lymphoma with classic histological features, a morphological variant of mantle cell lymphoma with blastic features and a more aggressive clinical course has been described. To investigate whether this variant is closely related, by the cell of origin, to typical cases, we analysed the presence and the pattern of somatic mutations of the VH genes in a series of nine cases diagnosed as such. Our cases of blastic mantle cell lymphomas rearrange most frequently VH4 and VH3 family genes. In three cases there was a complete homology to published germline genes, and a near complete homology was documented in another three. In contrast, the remaining three cases showed somatic mutations in their rearranged VH genes. Mutation analysis revealed evidence for antigen selection in one of these three cases. Taken together, these data are similar to those of normal adult-type B1 cells and those described for chronic lymphocytic leukaemia (CLL) but slightly different to those reported for classic mantle cell lymphoma. It is likely that blastic mantle cell lymphoma as well as CLL originates from adult-type B1 cells. More cases will need to be studied to determine whether classic mantle cell lymphoma is different from the blastic subtype and if it arises from fetal-type B1 cells.     

Mutation analysis of the rearranged immunoglobulin heavy chain genes of marginal zone cell lymphomas indicates an origin from different marginal zone B lymphocyte subsets

Marginal zone cell lymphoma is a recently described entity among the non-Hodgkin's lymphomas. It likely originates from the marginal zone B cells in the spleen and equivalent cells in the lymph node and extranodal tissues. Recent evidence indicates that marginal zone B cells are functionally heterogeneous and may differ with respect to the pattern of somatic hypermutation in their Ig variable genes. To test whether marginal zone lymphomas may originate from different subsets of marginal zone B cells, we performed a sequence and mutation analysis of the rearranged Ig heavy chain (IgH) variable genes (VH) of a series of 14 cases of marginal zone lymphoma, occurring in the spleen (4), the lymph node (4), the stomach (2), the orbit (2), the tongue (1), and the skin (1). Our data show that marginal zone cell lymphomas preferentially rearrange the VH4, VH3, and VH1 family genes, without preference for any particular VH gene. Somatic mutations are present in 13 cases; one case of marginal zone cell lymphoma of the skin showed a germline configuration of the rearranged VH gene. Mutation analysis shows evidence of antigen selection in three cases of marginal zone cell lymphoma, one of the spleen, stomach, and orbit, respectively. No evidence of antigen selection was present in the other cases. These data indicate that marginal zone cell lymphomas may arise from different subsets of marginal zone B cells. In addition, lymphomagenesis may not be triggered by antigen in all cases of marginal zone cell lymphoma.     

Evidence for immunoglobulin heavy chain variable region gene replacement in a patient with B cell chronic lymphocytic leukemia

Immunoglobulin heavy chain variable (V) gene replacement is an unusual recombinatorial event characterized by rearrangement of a germline V gene to a preformed VDJ gene complex. This phenomenon has occasionally been implicated in the emergence of clonal subpopulations during the course of acute lymphoblastic leukemia; it has also been found in murine precursor B cell lines. V gene replacement has never been described in lymphoproliferative disorders corresponding to more differentiated stages of B cell ontogeny. The present communication provides evidence for the operation of the same mechanism in B cell chronic lymphocytic leukemia (B-CLL). Genomic DNA and total cellular RNA extracted from peripheral blood mononuclear cells of a 48-year-old female patient diagnosed as having typical B-CLL were subjected to polymerase chain reaction (PCR) amplification aiming to detect rearranged clonal heavy and light chain variable genes (VH and VL, respectively). PCR consistently gave two VH amplification products, both at the DNA and the RNA level; similar analysis for the VL region revealed the presence of a single rearranged VK gene. Direct sequence analysis of the PCR products revealed that, except for a number of silent mutations, the single rearranged VK gene was identical to the germline A1-A17 VK gene. The two rearranged VH gene segments belong to the VHl and VHIII gene families and are closely homologous, respectively, to the germline gene segments V1-18 and V3-30, which have been shown to be used by autoantibodies. Both rearranged VH genes showed identical in-frame D-N-JH junctions and JH gene usage (JH5b), whereas the VH-N-D junctions were different. The above findings indicate that, during the course of the disease of our patient, VH gene replacement took place giving rise to two different clonally related subpopulations. This raises the intriguing possibility that the recombinase machinery, which governs Ig recombinatorial processes, might be operative even at more advanced stages in B cell ontogeny.     

Differential contribution of M(r) 120 kDa rasGTPase-activating protein and neurofibromatosis type 1 gene product during the transition from growth phase to arrested state in human fibroblasts accompanied by a unique rasGTPase-activating activity

Production of IgG3 in MRL/Mp-lpr/lpr (MRL/lpr) lupus mice is one of the major factors to develop glomerulonephritis (GN) in these mice. To examine molecular characteristics of IgG3 responsible for GN in these mice, hybridoma clones producing IgG3 antibodies were prepared from one unmanipulated MRL/lpr mouse. Two clones, 2B11.3 and 7B6.8, were nephritogenic; that is, they caused severe glomerular lesions when injected to normal mice, moreover with a different histopathological manifestation. The 2B11.3 clone generated diffuse cell-proliferative lesions, while those induced by the 7B6.8 clone resembled wire loop lesions in human lupus nephritis. The cDNA sequence analysis of 7B6.8 antibody and the other IgG3 antibody, 1G3, non-nephritogenic, revealed that the C regions of the heavy and light kappa chains were completely the same between them. Furthermore, they were identical in deduced amino acid sequences to those from non-autoimmune BALB/c mice, indicating no allelic difference of Igh-8 between these two strains. The V regions of 2B11.3 and 7B6.8 antibodies were composed of different sets of VH, D, JH, Vk and Jk. Although both of the VH belonged to the J558 family, they seemed to use a different VH germline gene. These findings suggest that GN in MRL/lpr mice is generated by the expansion of clonally different B cells producing particular antibodies possibly with a different pathogenetic potency.