Clonal expansion and somatic hypermutation of V(H) genes of B cells from cerebrospinal fluid in multiple sclerosis.

The cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients is characterized by increased concentrations of immunoglobulin (Ig), which on electrophoretic analysis shows restricted heterogeneity (oligoclonal bands). CSF Ig is composed of both serum and intrathecally produced components. To examine the properties of intrathecal antibody-producing B cells, we analyzed Ig heavy-chain variable (V(H)) region genes of B cells recovered from the CSF of 12 MS patients and 15 patients with other neurological diseases (OND). Using a PCR technique, we could detect rearrangements of Ig V(H) genes in all samples. Sequence analysis of complementarity-determining region 3 (CDR3) of rearranged VDJ genes revealed expansion of a dominant clone or clones in 10 of the 12 MS patients. B cell clonal expansion was identified in 3 of 15 OND. The nucleotide sequences of V(H) genes from clonally expanded CSF B cells in MS patients demonstrated the preferential usage of the V(H) IV family. There were numerous somatic mutations, mainly in the CDRs, with a high replacement-to-silent ratio; the mutations were distributed in a way suggesting that these B cells had been positively selected through their antigen receptor. Our results demonstrate that in MS CSF, there is a high frequency of clonally expanded B cells that have properties of postgerminal center memory or antibody-forming lymphocytes.     

Partial characterization of the shift from IgG to IgA synthesis in the clonal differentiation of human leukemic bone marrow-derived lymphocytes

An unusual B-cell proliferation was noted in an individual (Tun) which was characterized by the presence of two separate populations of chronic lymphocytic leukemia (CLL) cell staining on the surface and in the cytoplasm for either IgG(k) or IgA(k). Utilizing an idiotypic antiserum prepared from the associated serum monoclonal IgG(k) protein the idiotype was detected on the surface and in the cytoplasm of both the IgG- and IgA-bearing cell populations. These observations are consistent with a common clonal origin and a switch mechanism involving IgG and IgA synthesis. Sequential-labeling of Surface Ig and intracellular Ig with antisera conjugated to opposite fluorochromes documented the progressive maturation of the terminal differentiation of the IgA-bearing cell population at a level before morphologically distinct plasma cells. The distribution and pattern of surface and cytoplasmic IgG and IgA staining in individual cells suggest that the direction of switching is from IgG to IgA synthesis. The demonstration of shared idiotypic specificity between the IgG- and IgA-bearing populations is consistent with a transition in Ig heavy chain synthesis resulting from an alternation in the CH gene. It is concluded that certain CLL clones may manifest a switch from IgG to IgA synthesis at a level of B-cell differentiation which encompasses both the B lymphocyte and the Ig-synthesizing plasma cell.     

Both IgM and IgG anti-DNA antibodies are the products of clonally selective B cell stimulation in (NZB x NZW)F1 mice

Disease activity in systemic lupus erythematosus is closely associated with the appearance of immunoglobulin (Ig)G antibody to native DNA in both humans and mice. Like normal antibody responses, the anti-DNA autoantibody first appears as IgM and then switches to IgG. Structural studies of IgG anti-DNA suggest that these antibodies are the products of clonally selected, specifically stimulated B cells. The origins of the IgM anti-DNA have been less clear. To determine whether the earlier appearing IgM anti-DNA antibody in autoimmune mice also derives from clonally selected, specifically stimulated B cells or B cells activated by nonselective, polyclonal stimuli, we have analyzed the molecular and serological characteristics of a large number of monoclonal IgM anti-DNA antibodies from autoimmune (NZB x NZW)F1 mice. We have also analyzed IgM and IgG anti-DNA hybridomas obtained from the same individual mice to determine how the later-appearing IgG autoantibody may be related to the earlier-appearing IgM autoantibody within an individual mouse. The results demonstrate that: (a) IgM anti-DNA, like IgG, has the characteristics of a specifically stimulated antibody; (b) IgM and IgG anti-DNA antibodies have similar variable region structures and within individual mice may be produced by B cells derived from the same clonal precursors; (c) recurrent germline and somatically derived VH and VL structures may influence the specificity of anti-DNA monoclonal antibody for denatured vs. native DNA; and (d) the results provide a structural explanation for the selective development of IgG antibody to native DNA as autoimmunity to DNA progresses in (NZB x NZW)F1 mice.     

In vivo role of interleukin 4 in T cell tolerance induced by aqueous protein antigen

High doses of aqueous protein antigens induce a form of immunological tolerance in which interleukin 2 (IL-2)- and interferon gamma (IFN- gamma)-secreting T helper type 1 (Th1) cells are inhibited, but IL-4- secreting (Th2) cells are not. This is manifested by reduced proliferation of antigen-specific T cells upon in vitro restimulation, and marked suppression of specific antibody responses of the immunoglobulin (Ig)G2a, IgG2b, and IgG3 isotypes, but not of IgG1 and IgE. The role of the immunomodulatory cytokine IL-4 in this model of unresponsiveness to protein antigens has been examined. Administration of tolerizing antigen itself primes splenic CD4+ T cells for secretion of lymphokines, both IL-2 and IL-4. Neutralization of IL-4 in vivo with the anti-IL-4 antibody 11B11 during tolerance induction augments IFN- gamma production by T cells of tolerant mice, and reverses the suppression of IgG2a, IgG2b, and IgG3. This blockade of IL-4 function does not, however, restore the proliferative responses of T cells, suggesting that reduced T cell proliferation is due to direct T cell inactivation or anergy. Inhibiting the activity of IL-4 in vivo also inhibits the expansion of antigen-specific Th2-like cells, which are resistant to the induction of unresponsiveness. Thus, the immunologic consequences of high-dose tolerance are due to a combination of clonal T cell anergy and IL-4-mediated immune regulation.     

Evidence for locally synthesized and clonally restricted immunoglobulin in the synovial fluid from rheumatoid arthritis patients

In this study, we examined the immunoglobulin (Ig) present in synovial fluid (SF) from patients with rheumatoid arthritis (RA) to determine if it was locally produced and to assess the presence of clonally restricted (oligoclonal) immunoglobulin. We studied SF/serum pairs from 55 RA patients and 23 patients with degenerative joint disease (DJD). We found increases in total protein, IgG, IgA, and IgM in RA vs DJD SF (P less than 0.01). The immunoglobulin present in RA appeared to be locally produced as evidenced by significant increases (P less than 0.01) in the immunoglobulin indices. Regression analysis among the levels of IgG, IgA, and IgM RF and the Ig indices suggested that only a minority of the locally synthesized Ig present was specific for RF. To provide evidence of clonal restriction, we further analyzed the SF specimens by isoelectric focusing and assessed the presence of oligoclonal bands present only in RA SF. In 7/55 RA specimens (13%) we found unique SF IgG bands. All bands were of similar isoelectric point (pI), being quite cathodic with pI greater than 7.5. Our evidence supports synthesis of Ig within RA synovium, with a minority of patients showing prominent and unique SF Ig bands. This suggests an oligoclonal response in SF of some patients, but polyclonal Ig synthesis in most.     

Identification of Epstein-Barr virus proteins as putative targets of the immune response in multiple sclerosis

MS is a chronic inflammatory and demyelinating disease of the CNS with as yet unknown etiology. A hallmark of this disease is the occurrence of oligoclonal IgG antibodies in the cerebrospinal fluid (CSF). To assess the specificity of these antibodies, we screened protein expression arrays containing 37,000 tagged proteins. The 2 most frequent MS-specific reactivities were further mapped to identify the underlying high-affinity epitopes. In both cases, we identified peptide sequences derived from EBV proteins expressed in latently infected cells. Immunoreactivities to these EBV proteins, BRRF2 and EBNA-1, were significantly higher in the serum and CSF of MS patients than in those of control donors. Oligoclonal CSF IgG from MS patients specifically bound both EBV proteins. Also, CD8(+) T cell responses to latent EBV proteins were higher in MS patients than in controls. In summary, these findings demonstrate an increased immune response to EBV in MS patients, which suggests that the virus plays an important role in the pathogenesis of disease.     

Characterization of a non-T, non-B human lymphocyte (L cell) with use of monoclonal antibodies. Its regulatory role in B lymphocyte function.

These studies investigate the role of L lymphocytes in regulating terminal B lymphocyte differentiation. L cells have abundant Fc IgG receptors and comprise 10--15% of human peripheral blood mononuclear cells (PBMC). L cells lack the conventional markers of B and T lymphocytes and in culture, do not develop into B cells, T cells, or macrophages. Additionally, use of monoclonal antibodies failed to detect on L cells, surface antigens specific for B cells, T cells, and macrophages. In these studies, purified L cell subpopulations depleted of macrophages were co-cultured with autologous PBMC in the presence of pokeweed mitogen and at the end of 8 d, development of intracytoplasmic immunoglobulin (Ig) was determined. L cells were depleted of B and T cells by rosetting techniques and, in addition, by cytotoxicity techniques using monoclonal-specific antisera to T cells. In 14 individuals, L cells when co-cultured with PBMC, enhanced Ig synthesis by 83% +/- 62 SD, and also enhanced cell proliferation. Radiated L cells lost enhancing properties. To study the role of their high density Fc IgG receptors, L cells pretreated with IgG antibody-sensitized erythrocytes were used (i.e., after lysis of rosettes). Such L cells significantly inhibited Ig synthesis (by greater than 50%) despite promoting cell proliferation. Antibody-sensitized erythrocyte-rosetted macrophages did not inhibited Ig synthesis. Thus, positive and negative influences can be mediated by the same cell, depending on the state of Fc-receptor stimulation. Such cells may play a more prominent role in "feed-back" regulation of Ig synthesis by virtue of having abundant Fc IgG receptors.     

Molecular analysis of original antigenic sin. I. Clonal selection, somatic mutation, and isotype switching during a memory B cell response

To determine how the memory B cell population elicited to one epitope might be used in immune responses to other, structurally related epitopes, we explored the phenomenon of original antigenic sin. Strain A/J mice reproducibly respond to immunization with p-azophenylarsonate (Ars) by production of anti-Ars antibodies encoded predominantly by a single VH gene segment (VHIdCR). The structural analogue of Ars p-azophenylsulfonate (Sulf) fails alone to elicit such V regions, but can do so in A/J mice previously immunized with Ars, providing a means to specifically examine B cells capable of responding secondarily to a crossreactive antigen (i.e., memory cells). VHIdCR-expressing hybridomas were derived from the Ars-primed, Sulf-boosted original antigenic sin response of A/J mice at various times after Ars priming, and the properties of the antibodies they express and the structure of the genes encoding these antibodies were characterized. The data obtained support the following conclusions: (a) The Ars-induced memory B cell population capable of being crossreactively stimulated by Sulf is largely formed from a small fraction of all B cells participating in the anti-Ars primary response that express somatically mutated V regions; (b) the antibody repertoire and clonal composition of this population are stable over long periods of time; (c) memory B cells are capable of clonal expansion in the absence of a high rate of V gene somatic mutation; (d) the activation requirements for clonal selection of memory, versus naive B cells appear to differ; and (e) a major fraction of Ars-induced memory B cells express either IgM or IgG3 prior to and during the initial stages of the sin response.     

Rituximab induces sustained reduction of pathogenic B cells in patients with peripheral nervous system autoimmunity

The B cell-depleting IgG1 monoclonal antibody rituximab can persistently suppress disease progression in some patients with autoimmune diseases. However, the mechanism underlying these long-term beneficial effects has remained unclear. Here, we evaluated Ig gene usage in patients with anti-myelin-associated glycoprotein (anti-MAG) neuropathy, an autoimmune disease of the peripheral nervous system that is mediated by IgM autoantibodies binding to MAG antigen. Patients with anti-MAG neuropathy showed substantial clonal expansions of blood IgM memory B cells that recognized MAG antigen. The group of patients showing no clinical improvement after rituximab therapy were distinguished from clinical responders by a higher load of clonal IgM memory B cell expansions before and after therapy, by persistence of clonal expansions despite efficient peripheral B cell depletion, and by a lack of substantial changes in somatic hypermutation frequencies of IgM memory B cells. We infer from these data that the effectiveness of rituximab therapy depends on efficient depletion of noncirculating B cells and is associated with qualitative immunological changes that indicate reconfiguration of B cell memory through sustained reduction of autoreactive clonal expansions. These findings support the continued development of B cell-depleting therapies for autoimmune diseases.     

Evidence for a bone marrow B cell transcribing malignant plasma cell VDJ joined to C mu sequence in immunoglobulin (IgG)- and IgA-secreting multiple myelomas

Multiple myeloma is a B cell malignancy characterized by the expansion of plasma cells producing monoclonal immunoglobulins (Ig). It has been regarded as a tumor arising at the B, pre-B lymphocyte, or even stem cell level. Precursor cells are presumed to proliferate and differentiate giving rise to the plasma cell clonal expansion. Antigenic features and specific Ig gene rearrangement shared by B lymphocytes and myeloma cells have supported this hypothesis. However, the existence of such a precursor is based upon indirect evidence and is still an open question. During differentiation, B cells rearrange variable (V) regions of Ig heavy chain genes, providing a specific marker of clonality. Using an anchor polymerase chain reaction assay, these rearranged regions from five patients with multiple myeloma were cloned and sequenced. The switch of the Ig constant (C) region was used to define the B cell differentiation stage: V regions are linked to C mu genes in pre-B and B lymphocytes (pre-switch B cells), but to C gamma or C alpha in post-switch B lymphocytes and plasma cells (post- switch B cells). Analysis of bone marrow cells at diagnosis revealed the presence of pre-switch B cells bearing plasma cell V regions still joined to the C mu gene. These cells were not identified in peripheral blood, where tumor post-switch B cells were detected. These pre-switch B cells may be regarded as potential myeloma cell precursors.     

Survival and clonal expansion of mutating "forbidden" (immunoglobulin receptor-deficient) epstein-barr virus-infected b cells in angioimmunoblastic t cell lymphoma

Angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) is a peculiar T cell lymphoma, as expanding B cell clones are often present besides the malignant T cell clones. In addition, large numbers of Epstein-Barr virus (EBV)-infected B cells are frequently observed. To analyze the differentiation status and clonal composition of EBV-harboring B cells in AILD, single EBV-infected cells were micromanipulated from lymph nodes of six patients with frequent EBV(+) cells and their rearranged immunoglobulin (Ig) genes analyzed. Most EBV-infected B cells carried mutated Ig genes, indicating that in AILD, EBV preferentially resides in memory and/or germinal center B cells. EBV(+) B cell clones observed in all six cases ranged from small polyclonal to large monoclonal expansions and often showed ongoing somatic hypermutation while EBV(-) B cells showed little tendency for clonal expansion. Surprisingly, many members of expanding B cell clones had acquired destructive mutations in originally functional V gene rearrangements and showed an unfavorable high load of replacement mutations in the framework regions, indicating that they accumulated mutations over repeated rounds of mutation and division while not being selected through their antigen receptor. This sustained selection-free accumulation of somatic mutations is unique to AILD. Moreover, the survival and clonal expansion of "forbidden" (i.e., Ig-deficient) B cells has not been observed before in vivo and thus represents a novel type of viral latency in the B cell compartment. It is likely the interplay between the microenvironment in AILD lymph nodes and the viral transformation that leads to the survival and clonal expansion of Ig-less B cells.     

Ia-mediated signal transduction leads to proliferation of primed B lymphocytes

One of the most controversial questions in immunology is the molecular basis by which Th lymphocytes deliver activating signals to quiescent B lymphocytes during T cell-dependent immune responses. Recent studies suggest that T cell-dependent activation of quiescent B lymphocytes may involve signaling mediated by direct T helper cell-B cell contact. Since B cell membrane-associated MHC-encoded class II molecules (Ia) must be recognized by Th lymphocytes for generation of T cell-dependent humoral immune responses, they are obvious candidates for receptors of this signal. Here we report that stimulation of quiescent murine B cells with IL-4 and antibodies against the B cell antigen receptor for 12-16 h primes cells to proliferate in response to immobilized mIa binding ligands. In the presence of additional lymphokines, these B cells differentiate to secrete Ig of IgM and IgG classes. These results suggest that Ia molecules are receptors for direct, T helper cell-B cell contact mediated signaling that results in B cell proliferation.     

B cell dependence on and response to accessory signals in murine lupus strains

B cell hyperactivity, a feature common to all lupus-prone murine strains, may be caused by hyperresponsiveness to, overproduction of, or bypassing of certain signals required for B cell activation, proliferation, and differentiation. In this study, we have compared the responses of B cells from three lupus-prone strains of mice (BXSB males, MRL and NZB/W females) and normal strains in a number of assays for which two or more signals are required to obtain a response. In medium to low density cultures of B cells from BXSB and NZB/W but not MRL/l lupus mice, the cells' proliferation induced by bacterial lipopolysaccharide (LPS) or anti-mu antibody was much higher than that of B cells from normal controls. At low B cell density, polyclonal activation by these substances and subsequent Ig secretion were dependent on accessory signals present in supernatants of concanavalin A-treated normal lymphocytes (CAS) or on the MRL/l proliferating T cell-derived B cell differentiation factor (L-BCDF) in both lupus-prone and immunologically normal mice. However, the responses of B cells from BXSB and NZB/W, but not MRL/l, mice to these accessory signals were higher than those of normal mice. Ig synthesis by fresh B cells of BXSB and NZB/W mice cultured in the absence of mitogens but in the presence of CAS or L-BCDF was higher than by similar cells from other strains, suggesting an increased frequency of B cells activated in vivo in these two autoimmune strains of mice. The patterns of IgG subclass secretion in response to LPS (without added CAS or L-BCDF) were abnormal in all lupus strains, with a predominance of IgG2b and/or IgG2a and low levels of IgG3, contrary to normal B cells for which IgG3 synthesis predominated. However, IgG1 synthesis in vitro by autoimmune and normal B cells alike was highly dependent on T cell-derived soluble mediators. Antigen-specific responses to SRBC in vitro of B cells from all lupus strains, like those of B cells from normal strains, required a minimum of three signals (antigen, LPS, T cell-derived antigen nonspecific helper factors). Yet, once triggered, B cells of BXSB and NZB/W mice gave higher responses than those of the other strains. We conclude that B cells of lupus mice have signal requirements similar to those of normal mice. Nevertheless, B cells of BXSB and NZB/W, but not MRL/l, lupus mice hyperrespond or process some accessory signals abnormally.     

Oligoclonal T lymphocytes in the cerebrospinal fluid of patients with multiple sclerosis [published erratum appears in J Exp Med 1988 Jul 1;168(1):459]

We have investigated the T cell populations in the cerebrospinal fluid (CSF) of chronic progressive multiple sclerosis (MS) patients. Individual T cells from the CSF and blood were cloned before expansion and their clonotypes were defined by analysis of rearranged T cell receptor beta chain and gamma chain genes. 87 T cell clones from blood and CSF of two patients with chronic progressive MS were examined for common TCR gene rearrangement patterns. In one patient, 18 of 28 CSF-derived T cell clones demonstrated common TCR gene rearrangements indicating oligoclonal T cell populations; in the blood, two patterns were found twice among 26 T cell clones. In another patient, 5 of 27 CSF-derived clones had common TCR gene rearrangement patterns. In contrast, no common beta chain rearrangement pattern was found among 67 T cell clones derived from the blood or CSF of a patient with subacute sclerosing panencephalitis, among 20 clones from the CSF of a patient with herpes zoster meningoencephalitis, or among 66 clones from a normal subject. A subject with atypical, fatal MS of 8-mo duration was also studied and did not have oligoclonal T cells in the CSF or blood. These results demonstrate that distinct oligoclonal T cell populations can be found in the CSF immune compartment of subjects with nonmalignant inflammatory disease and they can create a new avenue for the investigation of the specificity of the T cell response within the central nervous system.     

Somatic mutations in the variable regions of a human IgG anti-double- stranded DNA autoantibody suggest a role for antigen in the induction of systemic lupus erythematosus

The processes that govern the generation of pathogenic anti-DNA autoantibodies in human systemic lupus erythematosus (SLE) are largely unknown. Autoantibodies may arise as a consequence of polyclonal B cell activation and/or antigen-driven B cell activation and selection. The role of these processes in humoral autoimmunity may be studied by molecular genetic analysis of immunoglobulin (Ig) variable (V) regions of antibodies that are characteristic of SLE. We have analyzed the gene elements that encode a high affinity, IgG anti-double-stranded DNA autoantibody secreted by a monoclonal Epstein-Barr virus (EBV)-transformed cell line derived from a patient with active SLE. In addition, we have identified, cloned, and sequenced the germline counterparts of the VH and VL genes expressed in this autoantibody. The comparison of both sets of gene elements shows that the autoantibody VH and VL regions harbor numerous somatic mutations characteristic of an antigen-driven immune response. The light chain expressed in this autoantibody is a somatically mutated variant of the kv325 germline gene that is frequently associated with paraproteins having autoantibody activity and with Ig molecules produced by malignant B cells that express the CD5 antigen. Furthermore, the utilized DH segment has been repeatedly found in multireactive, low affinity IgM anti-DNA autoantibodies from SLE patients and healthy individuals. These results suggest that pathogenic IgG anti-DNA autoantibodies in human SLE may arise through antigen-driven selection of somatic mutations in the gene elements that frequently encode multireactive IgM autoantibodies.     

Interleukin 4 causes isotype switching to IgE in T cell-stimulated clonal B cell cultures

Although it has been established that IL-4 enhances both IgG1 and IgE secretion in LPS-stimulated B cell cultures, these studies failed to determine whether IL-4 preferentially induces isotype switching or preferentially allows for the maturation of precommitted precursor cells. To distinguish between these possibilities, it is necessary to ascertain the effect of IL-4 on the isotypes secreted by individual precursor cells during clonal expansion. Therefore, clonal cultures of B cells stimulated with a Th2 helper cell line specific for rabbit Ig and rabbit anti-mouse IgM were established. The majority of B cells are capable of undergoing clonal expansion under these conditions. To vary the level of IL-4 present, either IL-4 or anti-IL-4 was added to cultures. In the presence of IL-4 there was an increase in the proportion of clones that secreted IgE and a decrease in the proportion of clones that secreted IgM. The addition of IL-4 to cultures also increased the amount of IgE secreted by individual clones. Thus, these experiments definitively prove that IL-4 causes specific heavy chain class switching to IgE in Th2-stimulated B cell cultures. In contrast, IL-4 does not affect the proportion of clones secreting IgG1, suggesting that other consequences of Th cell-B cell interactions play a role in the generation of an IgG1 response.     

脑脊液免疫固定电泳酶标抗体染色及154例结果分析

目的评价脑脊液免疫固定电泳结合酶标抗体染色技术的使用和154例检测结果应用价值。方法对脑脊液和血清标本同时进行琼脂糖凝胶电泳,使用酶标抗人免疫球蛋白IgG,采用免疫固定技术后,用TTF3显色。显色后使脑脊液标本呈现出寡克隆条带。结果154例脑脊液和血清标本,经电泳免疫固定酶标显色后,出现血脑屏障受损病例8例,寡克隆条带病例9例。其中18例多发性硬化病人中,有7例出现寡克隆条带,2例出现血脑屏障受损。结论脑脊液免疫固定电泳酶标抗体染色技术,脑脊液无须浓缩,检测灵敏度高,是检测脑脊液中内源性合成免疫球蛋白的直观和可靠的方法。