The chemokines CCL5, CCL2 and CXCL12 play significant roles in the migration of Th1 cells into rheumatoid synovial tissue.

As the T-cell population in the synovial tissue (ST) in rheumatoid arthritis (RA) is dominated by T helper (Th) 1 cells, this study was designed to examine whether there is a preferential migration of polarized T cells to ST, and to identify the chemokines responsible for the migration. This was done by developing 10 T-cell clones specific for an arbitrary antigen (mouse immunoglobulin G (IgG)) from the peripheral blood (PB) of a healthy donor sensitized to mouse IgG. The Th polarizations of the clones were determined by measuring secreted interferon-gamma and interleukin-4, following anti-CD3 stimulation. Migration to pools of RA ST cell-derived supernatants was analysed. Expression of the chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CXCR3 and CXCR4 were analysed by flow cytometry. Th1 clones showed significantly higher migration to RA ST cell-derived supernatant compared with Th2 clones. Blocking of either of the chemokines, CCL5 or CCL2, strongly inhibited migration of the Th1 cells between 56 and 77%, while blocking of CXCL12 inhibited migration between 44 and 61%. Blocking of CXCL10 had only a minor inhibitory effect. Our results demonstrate a selective migration of Th1 cells to RA ST supernatant and that blocking either CCL5, CCL2 or CXCL12 significantly inhibits T-cell migration. This indicates that CCL5, CCL2 and CXCL12 play significant roles in attracting Th1 cells towards the RA ST, and may prove potent targets for obstructing T-cell migration to the synovium.

Clonal analysis of a human antimouse antibody (HAMA) response.

Circulating human antimouse antibodies (HAMAs) directed to mouse immunoglobulin G (IgG) are clinically significant, compromising mouse antibody therapy and imaging, and interfering in immunological assays. To investigate the HAMA response, 20 stable cell lines secreting human monoclonal antibodies reactive with mouse IgG were established from a donor with a history of exposure to mice. Their subclass and domain specificities were established by solid-phase binding, indirect haemagglutination assays and immunoblotting, using Igs of known subclass and Ig fragments. The heavy-chain variable region gene usage was determined for 12 HAMAs. Eight HAMAs were IgM, 11 HAMAs were IgG4 and one HAMA was IgG1, indicating an IgG4-dominated response. All of the IgG HAMAs reacted with epitopes present on the Fc portion; one was subclass-specific, nine were subclass-restricted and two were pan-IgG-reactive. Measurement of their affinities gave dissociation constants typically in the nanomolar range. Seven and five HAMAs were derived from variable heavy-chain 3 (VH3) and VH1 gene segments, respectively. The IgG HAMAs used different VH segments to the IgM HAMAs. JH regions were coded by JH4 in eight HAMAs. DH segment usage appeared to be restricted in the IgM HAMAs. Two IgG HAMAs were clonally related. These monoclonal HAMAs are potentially useful as reagents for detecting mouse IgG and as reference reagents for the investigation of the HAMA response in patients undergoing mouse monoclonal antibody therapy and for the investigation of the influence of HAMAs on immunodiagnostic tests.

Association of antigen specificity and migratory capacity of memory T cells in rheumatoid arthritis.

Among the T cell pool of multiple specificities in the rheumatoid synovial tissues (ST) we have previously shown a lack of proliferative response of T cells to Acanthamoeba polyphaga [1]. In contrast, peripheral blood (PB) derived T cells proliferate to the antigen. The aim of the present study was to establish whether there is a preferential migration of some T cell specificities to the joint in rheumatoid arthritis (RA) patients dependent on the chemokine system, and to identify which chemokine receptors are involved in the migratory process. For this purpose, PB-derived T cell lines and clones from RA patients specific for A. polyphaga, herpes simplex virus (HSV) and Campylobacter jejuni were developed. Their migratory capacities towards ST-derived chemokine supernatants were analysed. Expression of CCR1, CCR2, CCR5, CCR6, CCR7, CXCR3 and CXCR4 were analysed by FACS, and attracting chemokines were identified by blocking studies. We found that the migratory capacities of T cells specific for C. jejuni and HSV were markedly higher against synovial chemokines than T cells specific for A. polyphaga. CCR5 and CXCR3 were expressed by all high-migrating T cell lines and clones. CCR2 was expressed at higher levels on the high-migrating T cell lines compared with the low-migrating A. polyphaga lines. Neutralization of RANTES (regulated upon activation normal T cell expressed and secreted) in the ST cell-derived supernatant reduced T cell migration of all T cell lines and clones by 60-90%, while neutralization of MCP-1 reduced the migratory capacity of CCR2-expressing T cells by 45-80%. In conclusion, the ability of T cells to migrate towards chemokines produced by ST cells is associated with the T cell specificity. Blocking of single chemokines substantially reduced the migratory capacity of memory T cells to ST cell-derived supernatant indicating unique roles for each chemokine receptor in the process of T cell migration.

T-cell responses to viral, bacterial and protozoan antigens in rheumatoid inflammation. Selective migration of T cells to synovial tissue.

OBJECTIVE: To identify any preferential or selective migration of T-cell specificities to inflamed tissues of rheumatoid arthritis (RA) patients. METHODS: Lymphocytes from peripheral blood (PB) and synovial tissue (ST) were isolated from RA patients and stimulated with a panel of crude antigen preparations from 18 bacterial, protozoan and viral sources. Proliferative responses of the T lymphocytes to each antigen and group of antigens were compared in PB and ST. Antigen-specific T-cell clones were developed and their migratory capacities towards synovial chemokines were compared. RESULTS: ST-derived T cells showed a small but significantly higher stimulation index (SI) to the group of intestinal bacteria compared with PB T cells. Conversely, responses of ST-derived T cells to Acanthamoeba polyphaga (AP) were both profoundly and significantly lower compared with PB-derived T cells. The viral antigens as a whole gave comparable reactivities in blood and ST. The migratory capacity of AP-specific T-cell clones towards chemokines produced by ST was profoundly poorer compared with Campylobacter jejuni- and herpes simplex virus-specific T-cell clones. CONCLUSIONS: The results indicate a selective migration of T cells of given specificities to the inflamed rheumatoid synovium.

T-cell receptor repertoire in human germinal centres.

A search for an antigen-driven expansion of T lymphocytes in the inflamed joints in rheumatoid arthritis (RA) patients have been going on for decades. We here analyzed the human germinal centre T-cell receptor (TCR) Vbeta gene usage with polymerase chain reaction (PCR) combined with sequence analysis, to address the question of clonality in tonsils and synovial tissue from RA patients. Our data show a large degree of TCR heterogeneity in both these histological structures. Furthermore, clonally related T cells were found within different closely located germinal centres indicating either an active T-cell migration between germinal centres (GC) or that a T-cell clone may seed more than one GC.

Significantly depressed percentage of CD27+ (memory) B cells among peripheral blood B cells in patients with primary Sjögren's syndrome.

CD27 has been found to be expressed on somatically mutated B cells and is thus a positive marker for memory B cells in peripheral blood (PB). Since abnormal immunogloblin (Ig) production is characteristic of the autoimmune diseases primary Sjögren's syndrome (pSS) and rheumatoid arthritis (RA), we have analyzed in detail the CD27 expression on PB B cell from these patient groups. Staining of PB B cells with monoclonal antibodies (MoAb) specific for CD19 and CD27 revealed a significantly depressed percentage of CD27+ PB B cells in patients with pSS (14.8 +/- 1.6%) compared to both healthy donors (31.3 +/- 4.7%, P = 0.005) and patients with RA (40.8 +/- 4.1%, P = 0.0001). In addition, the percentages of both the IgD+CD27+ and the IgD-CD27+ B-cell subpopulations were significantly lower in pSS patients compared to RA patients and healthy donors. However, the relative proportion of IgD- and IgD+ cells among the CD27+B cells were almost the same for the three groups. Our data suggest a disturbance in the differentiation of peripheral B cells and possibly a bias towards plasma cell differentiation, resulting in a depressed percentage of CD27+ memory PB B cells in pSS. These results are potentially of pathological significance and of diagnostic value.

Bm1-Bm5 classification of peripheral blood B cells reveals circulating germinal center founder cells in healthy individuals and disturbance in the B cell subpopulations in patients with primary Sjögren's syndrome.

Analyses of B cells in the bone marrow and secondary lymphoid tissues have revealed a broad range of cell surface markers defining B cell subpopulations, but only a few of these have been used to analyze B cell subpopulations in peripheral blood (PB). We report here the delineation of circulating PB B cell subpopulations by staining for CD19, CD38, and IgD in combination with CD10, CD44, CD77, CD95, CD23, IgM, and the B cell memory marker CD27. The utility of this approach is shown by the demonstration of disturbances of circulating B cell subpopulations in patients with autoimmune disease. Five mature B cell (Bm) subpopulations were identified in normal PB that were comparable with the tonsillar Bm1, Bm2, early Bm5, Bm5 subpopulations and, surprisingly, to the germinal center (GC) founder cell subpopulation (Bm2' and Bm3delta-4delta), suggesting that some GC founder cells are circulating. No PB B cells resembled the Bm3 and Bm4 GC cells. Remarkably, some cells with the CD38-IgD+ phenotype, previously known as naive Bm1 cells, expressed CD27. The CD38-IgD+ subpopulation therefore includes both naive Bm1 cells and IgD+ memory B cells. This new classification of B cell developmental stages reveals disturbances in the proportions of B cell subpopulations in primary Sjögren's syndrome (pSS) patients compared with healthy donors and rheumatoid arthritis patients. Patients with pSS contained a significantly higher percentage of B cells in two activated stages, which might reflect a disturbance in B cell trafficking and/or alteration in B cell differentiation. These findings could be of diagnostic significance for pSS.

Switch in chemokine receptor phenotype on memory T cells without a change in the cytokine phenotype.

Th1 and Th2 cells as defined by their cytokine profile are associated with the expression of the chemokine receptors CCR5 and CCR3, respectively. In committed human memory Th1 cells the cytokine profile is irreversibly expressed. However, it is not known if the chemokine receptor phenotypes of Th1 and Th2 cells are permanently associated to the cytokine profile or if it can be changed. To analyze the possibility of inducing a switch in chemokine receptor phenotype on memory Th cells we used differentiated memory Th cells isolated from synovial tissue (ST) samples of patients with rheumatoid arthritis (RA). Freshly isolated T cells, T-cell lines and T-cell clones from these tissues were manipulated with Th1 (interleukin (IL)-12 + anti IL-4) or Th2 (IL-4 + anti IL-12) inducing conditions. The surface expression of CCR5 and CCR3 was analyzed by flowcytometry and interferon (IFN)-gamma and IL-4 production by ELISA. A Th1-inducing cytokine environment increased the expression of CCR5 in Th1 cells and induced the expression of CCR5 in Th2 cells as compared to culture condition with only IL-2. Induction of CCR5 expression on Th2 clones was associated with secretion of some IFN-gamma. Moreover, the Th2-associated chemokine receptor CCR3 could be expressed on both Th1-dominant cell lines, and clones of Th1 and Th0 type after culture conditions with IL-4. This expression of CCR3 was associated with a reduced IFN-gamma production, but no IL-4 production could be induced. The IL-4-treated Th1 clones had a reduced migratory capacity against chemokines produced by ST cells compared to nonmanipulated T-cell clones. In contrast, the same IL-12-treated Th1 clones showed an increased migratory potential. Induction of the Th2-associated marker CCR3 on memory Th1 cells demonstrates that a change in chemokine receptor phenotype related to the Th2 type can be induced on terminally differentiated Th1 cells, without a change in the cytokine profile.

Cell-cell interactions in synovitis: antigen presenting cells and T cell interaction in rheumatoid arthritis.

The synovial tissue in rheumatoid arthritis (RA) patients is enriched with mature antigen presenting cells (APCs) and many T lymphocytes. Interactions between APCs and T cells are essential for the initiation and amplification of T-cell-dependent immune responses, and may therefore play an important role in the chronic inflammatory processes in the synovium. The nature of the antigen(s) involved in RA still remains elusive. However, interactions and signaling through the costimulatory molecules CD28-CD80/86 and CD40-CD40L are critical during APC-T cell interaction for optimal cell activation. This review discusses how such costimulatory signals can be involved in the initiation and amplification of the inflammatory reactions in the synovium. Blocking of the signaling pathways involved in APC-T cell interactions might provide a specific immuno-therapeutic approach for the treatment of RA.

Characterization and V(H) sequences of human monoclonal anti-F(ab')(2) autoantibodies from normals and Sjögren's syndrome patients.

To investigate the genetic background of anti-F(ab')(2) autoantibodies and the mechanism behind their production we have analyzed 10 human monoclonal antibodies directed against IgG F(ab')(2) and IgG Fab. They were all derived from peripheral blood by the EBV/hybridoma technique. Eight were from three healthy individuals and two from two patients with primary Sjögren's syndrome (pSS). They react with epitopes on distinct regions of IgG, including epitopes present on or near the hinge of IgG, epitopes on the Fd gamma, and an antigenic determinant(s) present on lambda light chains. These determinants are either exposed on the intact IgG molecule or revealed following pepsin or papain digestion. The V(H) germline gene repertoire used is diverse and with considerable overlap with that used by rheumatoid factors (RF). The two IgG antibodies from normals are extensively mutated (13 and 24 mutations/V(H)), but with a replacement to silent mutation ratio in the CDR(H)1 + 2 of only 3.7. The IgM antibodies from normals are also heavily mutated (mean 10 mutations/V(H)). This suggests that anti-F(ab')(2) from normals are generated by an antigen-driven somatic hypermutation mechanism. In contrast, the two IgM antibodies from pSS are virtually unmutated in both V(H) and V(L). Together with published data of pSS RF and anti-Ro 52-kDa sequences (1-3), this suggests that there is an expanded population of naïve B cells with autoantibody specificities in the peripheral blood of pSS patients.

Potential contribution of IL-17-producing Th(1)cells to defective repair activity in joint inflammation: partial correction with Th(2)-promoting conditions.

To assess the contribution of cell interactions to the production of cytokines and type I collagen, fixed synovium T cell clones were cocultured on synoviocytes and levels of IL-6, LIF and PICP, a marker of type I collagen synthesis measured. Levels of IL-6 and LIF were higher with Th(1)than with Th(0)and Th(2)clones. Levels of PICP were decreased with Th(1)clones and increased with Th(2)clones. IL-17-producing T cells, all Th(1), were among the highest inducers of cytokine and inhibitors of collagen synthesis. Preincubation of clones in Th(1)conditions (IL-12 plus anti-IL-4) increased IL-6 production, whereas Th(2)conditions (IL-4 plus anti-IL-12) strongly inhibited IL-6 production and restored repair activity. As rheumatoid synovium is infiltrated by Th(1)cells, local cell interactions result in a pro-inflammatory pattern with defective repair, which can be reversed at least in part, by a Th(2)pattern.

Molecular analysis of human immunoglobulin heavy chain variable region associated determinants recognized by anti-VH3 antibodies 7B4, B6 and D12.

7B4, B6 and D12 are murine monoclonal antibodies (MoAb) that bind to some human immunoglobulin heavy chain products of the closely related V3-30, V3-30.3 and V3-33 genes from the VH3 family. B6 and D12 have additional reactivities with some immunoglobulins (Ig) encoded by the V3-11 and V3-7 genes; D12 also reacts with some V3-43 gene Ig. We show here, by site-directed mutagensis, that the lysine at position 57 in the complementarity-determining region 2 (CDR-2) of the V3-30 gene product is crucial for epitope recognition by all three anti-VH3 MoAbs. Further analysis of the amino-acid sequences of a large panel of Ig reactive, or nonreactive, with MoAb 7B4 indicates that the determinant recognized by 7B4 is dependent on the presence of the tetrapeptide sequence NKYY between positions 56 and 59 in the CDR-2. Comparing the efficiency of 7B4 reactivity with VH3 gene-encoded human Ig indicates that amino-acid position 4 in the frame region 1 (FR-1) may also influence the binding of 7B4 to Ig encoded by three very closely related germline genes, V3-30, V3-30.3 and V3-33. NKYY is also found on the gp120 V3 region of human immunodeficiency virus (HIV)-2, SIV and HTLV-4. We also report that other tetrapeptide sequences found on the 56-59 motif of heavy chain variable regions encoded by germline genes are expressed on the solvent exposed V2 region of gp120 of HIV-1 isolates. The possible significance of these observations is discussed.

Analysis of 'natural' and vaccine-induced haemophilus influenzae type B capsular polysaccharide serum antibodies for 3H1, a V3-23-associated idiotope.

The variable (V-) region repertoire of antibodies (Abs) to Haemophilus influenzae capsular polysaccharide (Hib PS) has been extensively studied in individuals vaccinated against the microbe, but to a lesser extent in subjects who generated such Abs in response to a 'natural' encounter with this microbe or its antigenic mimics. To gain an insight into the repertoire of Hib PS-reactive Abs in vaccinated and non-vaccinated individuals, we used a monoclonal Ab, 3H1, which detects an idiotypic marker associated with an Ab V-region gene, V3-23. We show here that Hib PS-reactive Abs with detectable 3H1 idiotope can be quantified by an indirect inimunoezymatic assay in serum samples of non-vaccinated healthy adults as well as of recently vaccinated healthy infants. The percentage of Abs that was simultaneously Hib PS-reactive and 3H1-positive ranged widely (from 0 to 68%) among individual serum samples from both groups of subjects. No dramatic differences in the expression of 3H1 idiotope on Hib PS-reactive Abs were found between vaccinated and non-vaccinated individuals. Our results are consistent with the hypothesis that the utilization of V-region genes in Hib PS-reactive Abs that individuals generate after a 'natural' encounter with Hib PS or its mimics is similar to that in these Abs elicited by Hib PS conjugate vaccines.

Changes in the Th1 or Th2 cytokine dominance in the synovium of rheumatoid arthritis (RA): a kinetic study of the Th subsets in one unusual RA patient.

OBJECTIVE: To perform a kinetic study of the Th1/Th2 balance in the rheumatoid arthritis (RA) synovium. METHODS: Three different synovial tissue (ST) samples were obtained from one patient with erosive RA. The characterization of Th1 and Th2 responses was performed by interferon-gamma and interleukin-4 measurements and by expression of the chemokine receptors CCR5 and CCR3. Measurements of secreted and surface immunoglobulin determined the types of B cells. RESULTS: The first ST sample yielded 31 CD4+ T cell clones which showed an unusual Th2 dominant pattern in the inflamed synovium. The Th2 response was associated with predominantly synovial IgG B cells, and a predominantly Th1 profile in the peripheral blood. In contrast, ST samples obtained 2 and 2.5 yr later displayed first a Th0 and thereafter a Th1 profile, and the synovial B cell response was predominantly of IgM type. The T cell lines from the Th1/Th0 tissues expressed the Th1 marker CCR5 but not CCR3, while the T cells from the Th2 tissue expressed the Th2 marker CCR3 and no CCR5. CONCLUSION: These results demonstrate that a predominantly Th2 response can be associated with active erosive RA. However, the Th2 profile was not permanent and changed into a Th0 and thereafter a Th1 profile.

Evidence that human immunoglobulin M rheumatoid factors can Be derived from the natural autoantibody pool and undergo an antigen driven immune response in which somatically mutated rheumatoid factors have lower affinities for immunoglobulin G Fc than their germline counterparts.

The question of whether immunoglobulin (Ig)M rheumatoid factors (RF) arise as the result of an abnormal expansion of already existing clones producing natural autoantibodies or emerge as new clones that are somatically mutated owing to an antigen driven immune response has never been conclusively answered. In this study, an inhibition ELISA was utilized to measure the affinities of recombinant antibodies using VH segments reverted back to their closest germline counterparts (germline revertants). In all cases, the somatically mutated parental RFs had a decreased affinity for immunoglobulin (Ig)G Fc compared to the germline revertant, indicating that the antibodies in the germline configuration had the higher affinities. This demonstrates that somatic mutation is not a prerequisite to generate disease associated antibodies. The presence of mutations in the parental IgM RFS suggests that these cells had been involved in a germinal centre reaction. As the germinal centre is the conventional site of the acquisition of mutations during an antigen driven response, these data suggest a role for germinal centres in the generation of the antibody diversity in addition to the selection of higher affinity antibodies. Assuming that only antigen selected cells survive deletion, these data support the hypothesis that IgM RFS can be derived from the natural autoantibody repertoire and result from an antigen driven response. Mechanisms controlling the survival of B cells based on the affinity/avidity of the immunoglobulin receptor are shown to be functional in patients with rheumatoid arthritis.

Immunoglobulin variable genes and epitope recognition of human monoclonal anti-Ro 52-kd in primary Sjögren's syndrome.

OBJECTIVE: To clone and characterize human anti-Ro/SSA autoantibodies from a patient with primary Sjögren's syndrome (pSS). METHODS: Monoclonal antibodies (mAb) were raised from the peripheral blood of a patient with pSS using Epstein-Barr virus transformation and a hybridoma technique. Specificity was determined using cell extracts, recombinant Ro 52-kd, Ro 60-kd, and La proteins as well as Ro 52-kd peptides in enzyme-linked immunosorbent assay (ELISA) and Western blot. The immunofluorescence pattern was analyzed using cultured human and mouse cell lines. Complementary DNA was amplified by polymerase chain reaction, and Ig variable (V)-region genes were directly sequenced. RESULTS: Two human anti-Ro 52-kd mAb of IgM isotype, denoted SG1 and SG3, were cloned from the peripheral blood of a patient with pSS. The 2 mAb reacted with the Ro 52-kd antigen in cell extracts of human cell lines and mouse cell lines, and with purified human recombinant Ro 52-kd protein in ELISA and Western blot. SG1 reacted specifically with 1 peptide, amino acids 136-156, of the Ro 52-kd protein, and SG3 was mapped to react with a recombinant fragment representing amino acids 136-292. Immunofluorescence studies revealed cytoplasmic staining with both mAb. Both were encoded by V(H)3-family genes. SG1 was highly homologous to the DP-77 germ-line gene, with 2 replacement mutations and 1 silent. It utilized the DPL-11 germ-line gene from the Vlambda2-family gene, with 1 silent mutation. SG3 was 100% homologous to the DP-47 germ-line gene, combined with a Vkappa1-family gene that was 100% homologous to the A30 germ-line gene. CONCLUSION: Two human mAb were demonstrated to be specific for the Ro 52-kd protein and to be directed against 2 different epitopes, 1 linear and 1 conformation-dependent, within a region previously described to be immunodominant. Somatic hypermutation appeared to be of minor importance in generating these 2 specificities.

Light chain variable (VL) sequences of rheumatoid factors (RF) in patients with primary Sjögren's syndrome (pSS): moderate contribution of somatic hypermutation.

We have characterized and sequenced the variable (V) region genes of the light (L) chains of 10 immunoglobulin (IgM) rheumatoid factor (RF) monoclonal antibodies (MoAb) derived by the hybridoma/Epstein-Barr virus (EBV) technique from the peripheral blood of patients with primary Sjögren's syndrome (pSS). Six out of 10 RFs used lambda (lambda) L chains, while four RFs used kappa (kappa) L chains. Five out of the six lambda RFs were encoded by Vlambda3 gene segments, the sixth one was encoded by a Vlambda1 gene segment. This preferential utilization of the Vlambda3 family genes suggests selective expansion of the B cell in pSS. Three of the kappa RFs used Vkappa3 gene segments, while the fourth used a Vkappa2 gene segment. Half of the RFs were found as unmutated copies of their closest germline (GL) gene. Interestingly these RFs were previously shown to use heavy (H) chains in GL gene configuration. Three RFs have very few mutations (2-3) and only two RFs have substantial numbers of mutations (6 and 11). This also correlated with the number of mutations in the respective H chains. In contrast to RFs in normal and RA these results further suggest the somatic mutation to be of moderate importance in the generation of RF from the peripheral blood of pSS patients.

Rheumatoid factors in primary Sjögren's syndrome (pSS) use diverse VH region genes, the majority of which show no evidence of somatic hypermutation.

Rheumatoid factor (RF) is the most common autoantibody found in patients with Sjögren's syndrome (SS). To study the genetic origin and the mechanisms acting behind its generation we have characterized and sequenced the immunoglobulin VH genes used by 10 IgM RF MoAbs derived from peripheral blood of six female patients with pSS. We compared the structure of the RF immunoglobulin VH genes with those obtained previously from rheumatoid arthritis (RA) patients and healthy immunized donors (HID). VH1 and VH4 were each used by four RF clones, one clone was encoded by VH3 family gene and one by VH2 family gene. This distribution frequency was different from that observed in RA, where VH3 was the dominant family, followed by VH1. Eight different germ-line (GL) genes encoded the clones and all of these genes were seen previously in RA and/or HID RF. Five clones rearranged to JH6, four rearranged to JH4 and one to JH5, in contrast to RF from RA and HID, where JH4 was most frequently used. D segment use and CDR3 structure were diverse. Interestingly, three out of four VH4 clones used the GL gene DP-79 that was seen frequently in RA RF. The degree of somatic mutation in the pSS RF was very much lower than seen in RA and HID RF. All the pSS RF clones except three were in or very close to GL configuration. This indicates that there is little role for somatic hypermutation and a germinal centre reaction in the generation of RF from peripheral blood in pSS.

Change in the Th1/Th2 phenotype of memory T-cell clones from rheumatoid arthritis synovium.

The stability of established memory T helper (Th)1/Th2 cells in chronic inflammatory diseases is not clear, and a shift of the cytokine balance could control chronic inflammation. In order to study the regulation of the Th phenotype of memory T cells, polyclonal T-cell lines and clones with a Th1, Th0 or Th2 phenotype were developed from rheumatoid synovial tissue. Th1 [interleukin (IL)-12 + anti-IL-4] and Th2 (IL-4 + anti-IL-12) promoting environments and IL-2 were used to manipulate the cytokine profile. Polyclonal T-cell lines of predominantly Th1 type could be shifted to produce Th2 cytokines, and polyclonal Th2/Th0 lines could be shifted to produce Th1 cytokines. However, this shift was due to an amplification of CD8+ T cells with a memory phenotype and a loss of the CD4+ T cells, giving Tc2 or Tc1 profiles, respectively. Th2 clones cultured repeatedly with IL-2 switched to either a Th0 or a Th1 phenotype, while both Th1 and Th0 memory clones kept a stable phenotype. Addition of Th2-promoting conditions strongly reduced the production of both interferon-gamma and IL-17, while Th1-promoting conditions increased the production of these cytokines. These results demonstrate that RA Th2 clones readily switch, while Th1 and Th0 clones are stable. However, induction of Th2 cytokines can be obtained in polyclonal polarized memory T cells due to amplification of Tc2 cells.