Potential of the immune complex transfer enzyme immunoassay for antigens and antibodies to improve the sensitivity and its limitations

To better characterize the cellular immune response taking place in the MS central nervous system, we investigated the blood and CSF T cell receptor (TCR) V beta 5 and V beta 17 repertoire in HLA-typed patients with recently diagnosed MS or other neurological diseases (OND). Using a RT-PCR based technique, we analysed directly ex vivo the CDR3 size of TCR beta chains utilizing V beta 5 (eight patients with MS and one with OND) or V beta 17 (eight patients with MS and six with OND) gene segments on paired blood-CSF samples. Globally, the analysis of V beta 5-J beta and V beta 17-J beta repertoire showed a less diverse pattern in the CSF samples than in the corresponding peripheral blood lymphocytes both in MS and in OND patients. However, we did not detect any recurrent clonal expansion within the V beta 5+ T cells in MS patients, underlining the potential limits of V beta 5-based immunotherapy in MS. We found an expanded T cell population using the same V beta 17-J beta 1.6 combination with identical CDR3 length in the CSF of three MS patients and none of the control patients. These results suggest selective expansion of T cells expressing this segment gene in the MS central nervous system.     

In vitro triggering of somatic mutation in human naive B cells

During T cell-dependent immune response, germinal center B cells accumulate somatic mutations in their Ig V(D)J genes and give rise to affinity-selected B cells. We tested several culture conditions for triggering somatic mutation in human tonsillar naive slgD+CD23+ cells after cross-linking their membrane Igs. CD40 activation, in the presence of exogenous cytokines (IL-2, IL-4, and IL-10), induced proliferation and isotype switch without somatic mutation. In contrast, after coculture with anti-CD3-activated cloned T cells, somatic mutation accumulated in a fraction of naive B cells. Mutations included shared as well as independent events in clonally related sequences, allowing reconstitution of genealogic trees generated in vitro. Naive tonsillar B cells sorted for slgD expression can be induced to mutate their Ig V(H) gene upon coculture with activated T cells, thereby providing a model to study somatic hypermutation in vitro.     

Pairing of variable heavy and variable kappa chains in individual naive and memory B cells

A functional Ig consists of two heterodimers each of which is composed of a heavy and a light chain. Although there is increasing knowledge about the events that govern the rearrangement of the genes encoding each individual chain, only very limited information is available about the mechanisms governing the pairing of variable heavy (V(H)) and variable light (V(L)) chains. Using a single cell PCR, we were able to obtain V(H) and Vkappa chains from 144 individual human CD19+/IgM+ B cells. Pairing of specific V(H) or Vkappa families was not observed, nor was the length or the amino acid composition of the CDR3s of V(H) and Vkappa chains in individual B cells similar. Comparison of V(H) and Vkappa genes in B cells in which one or both contained evidence of somatic hypermutation with those with no mutations revealed a significant decrease in the mean length of the V(H) CDR3. Moreover, there was a significant correlation between the frequencies of mutations in V(H) and Vkappa gene pairs in individual B cells. These results indicate that Ag-mediated selection as opposed to V(H)DJ(H) recombination or subsequent Ig chain pairing tended to approximate the CDR3 lengths and the frequency of mutations of V(H) and Vkappa in individual B cells.     

Human Herpesvirus-6 (HHV-6) infection in multiple sclerosis: a preliminary report

We examined cerebral spinal fluid (CSF) from multiple sclerosis (MS) patients and patients with other neurological diseases (OND) for antibody specific for Human Herpesvirus-6 (HHV-6) and for HHV-6 DNA detectable by PCR. CSF from MS patients had a higher frequency of IgG antibody to HHV-6 late antigens (39.4%) compared with CSF from OND (7.4%). In contrast, the frequency of detectable IgG antibody in CSF from MS patients specific for Epstein-Barr Virus (EBV) (12.1%) and Human Cytomegalovirus (HCMV) (6.1%) was much lower. Two of 12 MS CSFs (16.7%) also contained HHV-6 DNA detected by PCR. None of four OND CSF were positive for HHV-6 DNA. Plasma from 16 patients with MS, eight with OND and 72 healthy donors were tested for antibodies by ELISA to HHV-6 early (p41/38) and late (gp110) proteins. Although no differences in anti-gp110 IgG antibody were detected between MS patients, patients with other neurological diseases, and normals, IgG antibody to early protein p41/38 was detected in > 68% of the plasma from MS patients, 12.5% from OND patients and 27.8% of the controls. IgM antibody to p41/38 was present in > 56% of MS patients, 12.5% of OND patients, and 19% of controls. These data suggest that more than half of the MS patients had active, ongoing HHV-6 infections. HHV-6 was also isolated from peripheral blood mononuclear cells (PBMC) from 3/5 MS patients who were in relapse or had progressive disease and was identified as HHV-6 Variant B. These preliminary results support the hypothesis that HHV-6 may be a co-factor in the pathogenesis of some cases of MS.     

Somatic diversification and affinity maturation of IgM and IgG anti-DNA antibodies in murine lupus

Molecular events occurring during the process of generation of pathogenic immunoglobulin (Ig)G anti-DNA antibodies in systemic lupus erythematosus (SLE) were studied using a newly established method. We analyzed the Ig variable (V) region gene sequence and DNA-binding activity of IgM and IgG anti-DNA monoclonal antibodies (mAb) from individual SLE-prone (NZB x NZW) F1 mice. The first event appeared to be clonal selection and expansion of IgM anti-DNA clones, in which several clones had intraclonal V gene mutations. Although the number of mutations was small, the mutated IgM clones were associated with an increase in DNA-binding activity. The somatic mutations located in complementarity-determining regions (CDR) and in framework regions (FR) of V genes were apparently related to changes in DNA-binding activity. IgG anti-DNA clones that progressively increased in number with aging had numerous somatic mutations in the V region genes and there was a pair of clones which showed an intraclonal accumulation of mutations, in association with increase in the DNA-binding activity. All these findings show that somatic mutations associated with affinity maturation of the V region begin immediately before isotype-switching from IgM to IgG of the clones that have been selected and expanded, in an antigen-driven manner and/or by other forces. We propose that further accumulations of intraclonal somatic hypermutation, in association with selection and expansion of high affinity IgG clones, may lead to formation of highly pathogenic anti-DNA antibodies.     

Identification of functional human splenic memory B cells by expression of CD148 and CD27

Memory B cells isolated from human tonsils are characterized by an activated cell surface phenotype, localization to mucosal epithelium, expression of somatically mutated immunoglobulin (Ig) variable (V) region genes, and a preferential differentiation into plasma cells in vitro. In spleens of both humans and rodents, a subset of memory B cells is believed to reside in the marginal zone of the white pulp. Similar to tonsil-derived memory B cells, splenic marginal zone B cells can be distinguished from naive follicular B cells by a distinct cell surface phenotype and by the presence of somatic mutations in their Ig V region genes. Although differences exist between human naive and memory B cells, no cell surface molecules have been identified that positively identify all memory B cells. In this study, we have examined the expression of the receptor-type protein tyrosine phosphatase CD148 on human B cells. CD148(+) B cells present in human spleen exhibited characteristics typical of memory B cells. These included an activated phenotype, localization to the marginal zone, the expression of somatically mutated Ig V region genes, and the preferential differentiation into plasma cells. In contrast, CD148(-) B cells appeared to be naive B cells due to localization to the mantle zone, the expression of surface antigens typical of unstimulated B cells, and the expression of unmutated Ig V region genes. Interestingly, CD148(+) B cells also coexpressed CD27, whereas CD148(-) B cells were CD27(-). These results identify CD148 and CD27 as markers which positively identify memory B cells present in human spleen. Thus, assessing expression of these molecules may be a convenient way to monitor the development of memory B cell responses in immunocompromised individuals or in vaccine trials.     

Molecular characterization of IgA- and/or IgG-switched chronic lymphocytic leukemia B cells

The immunoglobulin (Ig) variable region (V) genes expressed by IgM chronic lymphocytic leukemia (CLL) B cells display little or no somatic mutations. However, preliminary findings have shown that Ig V genes of IgA and IgG CLLs may be somatically mutated, suggesting that isotype-switched CLLs may represent a "subtype" of the disease. To investigate the degree and nature of somatic mutations and the role of antigen (Ag) in the clonal selection and expansion of isotype-switched CLLs, and to determine whether specific oncogene or tumor suppressor gene mutations are associated with isotype-switched CLLs, we analyzed the expressed Ig VH gene, bcl-1 and bcl-2 proto-oncogene, and p53 tumor suppressor gene configurations of 3 IgA-, 1 IgG-, and 1 IgA/ IgG-expressing CLLs. These isotype-switched CLL B cells expressed surface HLA-DR, CD19, CD23, and CD5, and displayed no alterations of the bcl-1 and bcl-2 oncogenes and the p53 tumor-suppressor gene. The cDNA VH-D-JH gene sequence was joined with that of the C alpha gene in the B cells of the three IgA CLLs, and with that of the C gamma gene in the IgG CLL B cells. In the IgA/IgG-coexpressing CLL B cells, identical VH-D-JH cDNA sequences were spliced to either C alpha or C gamma genes. In all five CLLs, the pattern of C mu DNA probe hybridization to the digested genomic DNAs was consistent with deletion of the C mu exon from the rearranged Ig gene locus, suggesting that these CLL B cells had undergone DNA switch recombination. In one IgA CLL, the expressed VH gene was unmutated. In all other class-switched CLLs, the Ig VH segment gene was mutated, but the point mutations were not associated with intraclonal diversification. In one IgA and in the IgA/IgG-coexpressing CLL, the nature and distribution of the mutations were consistent with Ag selection. These findings suggest that IgA- and/or IgG-expressing CLLs represent, in their VH gene structure, transformants of B cells at different stages of ontogeny. They also suggest that Ag may play a role in the clonal selection of some of these isotype-switched leukemic cells, but bcl-1 and bcl-2 oncogene rearrangements and p53 tumor suppressor gene mutation are not associated with the pathogenesis of isotype-switched CLLs.     

Dominant clonotypes in the repertoire of peripheral CD4+ T cells in rheumatoid arthritis

Clonal expansion of T cell specificities in the synovial fluid of patients has been taken as evidence for a local stimulation of T cells. By studying the T cell receptor (TCR) repertoire of CD4+ T cells in the synovial and peripheral blood compartments of patients with early rheumatoid arthritis (RA), we have identified clonally expanded CD4+ populations. Expanded clonotypes were present in the peripheral blood and the synovial fluid but were not preferentially accumulated in the joint. Dominant single clonotypes could not be isolated from CD4+ cells of HLA-DRB1*04+ normal individuals. Clonal expansion involved several distinct clonotypes with a preference for V beta 3+, V beta 14+, and V beta 17+CD4+ T cells. A fraction of clonally related T cells expressed IL-2 receptors, indicating recent activation. The frequencies of clonally expanded V beta 17+CD4+ T cells fluctuated widely over a period of one year. Independent variations in the frequencies of two distinct clonotypes in the same patient indicated that different mechanisms, and not stimulation by a single arthritogenic antigen, were involved in clonal proliferation. These data support the concept that RA patients have a grossly imbalanced TCR repertoire. Clonal expansion may result from intrinsic defects in T cell generation and regulation. The dominance of expanded clonotypes in the periphery emphasizes the systemic nature of RA and suggests that T cell proliferation occurs outside of the joint.     

Antibodies to the axolemma-enriched fraction in the cerebrospinal fluid and serum of patients with multiple sclerosis and other neurological diseases

Antibodies to an axolemma-enriched fraction (AEF) antigen have been detected in the cerebrospinal fluid (CSF) and serum of patients with Multiple Sclerosis (MS) using an enzyme-linked immunosorbent assay (ELISA). A marginal elevation (P < 0.08) of anti-AEF IgG was found in MS CSF when compared with OND samples. When CSF was diluted to a standardized IgG concentration, the anti-AEF IgG level in MS CSF was significantly elevated (P=0.007) when compared to OND CSF. MS serum was also found to contain a significantly higher level (P < 0.001) of anti-AEF IgG when compared to OND serum using the ELISA technique.     

Expansion of a recurrent V beta 5.3+ T-cell population in newly diagnosed and untreated HLA-DR2 multiple sclerosis patients

We have used a PCR-based technology to study the V beta 5 and V beta 17 repertoire of T-cell populations in HLA-DR2 multiple sclerosis (MS) patients. We have found that the five MS DR2 patients studied present, at the moment of diagnosis and prior to any treatment, a marked expansion of a CD4+ T-cell population bearing V beta 5-J beta 1.4 beta chains. The sequences of the complementarity-determining region 3 of the expanded T cells are highly homologous. One shares structural features with that of the T cells infiltrating the central nervous system and of myelin basic protein-reactive T cells found in HLA-DR2 MS patients. An homologous sequence was not detectable in MS patients expressing DR alleles other than DR2. However, it is detectable but not expanded in healthy DR2 individuals. The possible mechanisms leading to its in vivo proliferation at the onset of MS are discussed.     

Human immunoglobulin (Ig)M+IgD+ peripheral blood B cells expressing the CD27 cell surface antigen carry somatically mutated variable region genes: CD27 as a general marker for somatically mutated (memory) B cells

Immunoglobulin (Ig)M+IgD+ B cells are generally assumed to represent antigen-inexperienced, naive B cells expressing variable (V) region genes without somatic mutations. We report here that human IgM+IgD+ peripheral blood (PB) B cells expressing the CD27 cell surface antigen carry mutated V genes, in contrast to CD27-negative IgM+IgD+ B cells. IgM+IgD+CD27(+) B cells resemble class-switched and IgM-only memory cells in terms of cell phenotype, and comprise approximately 15% of PB B lymphocytes in healthy adults. Moreover, a very small population (<1% of PB B cells) of highly mutated IgD-only B cells was detected, which likely represent the PB counterpart of IgD-only tonsillar germinal center and plasma cells. Overall, the B cell pool in the PB of adults consists of approximately 40% mutated memory B cells and 60% unmutated, naive IgD+CD27(-) B cells (including CD5(+) B cells). In the somatically mutated B cells, VH region genes carry a two- to threefold higher load of somatic mutation than rearranged Vkappa genes. This might be due to an intrinsically lower mutation rate in kappa light chain genes compared with heavy chain genes and/or result from kappa light chain gene rearrangements in GC B cells. A common feature of the somatically mutated B cell subsets is the expression of the CD27 cell surface antigen which therefore may represent a general marker for memory B cells in humans.     

The arts and humanities: a creative approach to developing nurse leaders

There is still much controversy about the precursor cell type in multiple myeloma (MM). Some authors claim that it is a pre-B cell, others state that it is a memory B cell or plasmablast. We have recently shown that the VDJ region of the MM immunoglobulin heavy chain gene is somatically hypermutated and antigen selected, without intraclonal variation or evolution in time. By using a patient-specific PCR approach we have now obtained evidence that the premyeloma cell can be situated in the pre-switched B-cell compartment and that heavy chain switching can occur without further somatic mutation. Based on the MM immunoglobulin sequences derived from the bone marrow, patient-specific CDR2 and CDR3 oligonucleotides were designed. B lymphocytes were separated from plasma cells based on the expression of CD19 and HLA class II or surface bound IgM using immunomagnetic beads. The expressed Ig sequences were amplified by RT-PCR using patient specific CDR2 primers and isotype specific primers (C mu, C gamma, and C alpha). Myeloma-specific Ig sequences were detected by a myeloma-specific CDR3 probe and sequenced. In one out of five cases we found in the peripheral blood clonally related IgM and IgA sequences with the same somatic mutations as the MM-IgG sequence. In another case of an IgG MM we found in the bone marrow clonally related IgA sequences with the same somatic mutations. These findings, together with the fact that myeloma-Ig genes contain somatic mutations without intraclonal variation, suggest that the clonogenic cell in multiple myeloma can originate from a pre-switched but somatically mutated B cell.     

The human heavy chain Ig V region gene repertoire is biased at all stages of B cell ontogeny, including early pre-B cells

The expressed human Ig repertoire is not an equal representation of all V(H) segments present in genomic DNA. Studies have shown that a restricted set of V(H) gene segments are over-represented in Ab repertoires of fetal/neonatal and adult B cells. Additionally, this restricted set of V(H) genes is frequently expressed by autoimmune and tumor B cells. To investigate at which developmental stage a bias in the repertoire begins, we compared the V(H)3 and V(H)4 family repertoires of pre-B and immature B cells from bone marrow and mature B cells from peripheral blood of two adults. We found that the V4-34 and V4-59 gene segments of the V(H)4 family and the V3-23 gene segment of the V(H)3 family dominate the repertoires of the surface Ig-negative early pre-B as well as immature and mature B cells. Furthermore, the pattern of utilization of other V(H)3 family members suggests that certain genes that are frequently rearranged during early stages of B cell development are subsequently disfavored during later stages of B cell maturation. We conclude that the over-representation of certain V genes could arise from sequential mechanisms operating at both early and later stages of B cell development. These V(H)-mediated mechanisms might include preferential rearrangement and/or efficiency of pairing with the surrogate light chain at the surface Ig-negative, early pre-B cell stage and ligand selection at more mature, surface Ig-positive, B cell stages.     

Similar characteristics of the CDR3 of V(H)1-69/DP-10 rearrangements in normal human peripheral blood and chronic lymphocytic leukaemia B cells

The variable heavy chain (V(H)) gene segment V(H)1-69/DP-10 has been shown to be over-represented in B-cell chronic lymphocytic leukaemia (CLL). Because of certain similar characteristics of their complementarity determining region 3 (CDR3), including preferential utilization of J(H)6 elements and an extended length, it has been suggested that antigenic stimulation might be involved in leukaemogenesis. Utilizing single-cell PCR to amplify and sequence genomic DNA from individual normal human peripheral blood B cells, we have obtained 7/421 productively and 1/69 nonproductively rearranged V(H) genes that used V(H)1-69/DP-10. All productive rearrangements were unmutated, used J(H)6 and had an average CDR3 length similar to that previously found in V(H)1-69/DP-10-expressing CLL cells. These results suggest that CLL may arise from B cells commonly found in the peripheral B-cell repertoire and do not represent expansion of a unique subset of specific antigen-reactive B cells.     

Cadmium inhibits proteoglycan and procollagen production by cultured human lung fibroblasts

OBJECTIVE: Previously, we showed that 15-20% of patients with rheumatoid arthritis (RA) have oligoclonal expansions of peripheral blood CD8+ T cells expressing T cell receptors encoded by the V(alpha)12 (AV12S1) gene. To better understand the significance of these expansions, the present study was undertaken to determine their specificity. METHODS: We cloned and characterized V(alpha)12+,CD8+ T cells from the peripheral blood of 1 RA patient with a clonal expansion of these T cells. RESULTS: The T cell clones were autoreactive since they recognized autologous, but not allogeneic, antigen-presenting cells. Upon activation, these T cells secreted interleukin-4 and interleukin-10. The autoreactive T cell clones were class I major histocompatibility complex (MHC) restricted, by either HLA-B60 or HLA-Cw3. CONCLUSION: A large population of class I MHC-restricted CD8+ T cells in a patient with RA is clonally expanded and autoreactive. These cells define a novel immune aberration in RA and provide a tool for defining the autoantigens that activate expanded T cell populations in vivo.     

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.