Association of ferritin autoantibodies with giant cell arteritis/polymyalgia rheumatica.

OBJECTIVES: Polymyalgia rheumatica (PMR) and giant cell arteritis (GCA) are relatively common inflammatory disorders. Establishing the diagnosis however may be difficult, since so far no specific biomarkers of the disorders are available. METHODS: As a screening procedure, the authors used protein arrays for the detection of new autoantigens in GCA and PMR. The results of the protein array were confirmed by different ELISAs detecting IgG antibodies against the human ferritin heavy chain, N-terminal 27 amino acids of the human ferritin heavy chain or the homologous peptide of Staphylococcus epidermidis. Sera of patients with only GCA (n=64), only PMR (n=47) and both PMR and GCA (n=31) were used. RESULTS: In the ELISA using the human ferritin peptide, the sensitivity of IgG antibodies against ferritin was 92% in 36 GCA and/or PMR patients before initiation of treatment, 22/32 (69%) in patients with disease flares and 64/117 (55%) in the total cohort including treated and inactive patients. In controls, the false positive rate was 11/38 (29%) in systemic lupus erythematosus, 1/36 (3%) in rheumatoid arthritis, 0/31 (0%) in late onset rheumatoid arthritis, 3/46 (6.5%) in B-non-Hodgkin's lymphoma and 1/100 (1%) in blood donors. In the ELISA using the ferritin peptide of S epidermidis, 89% of 27 patients with untreated GCA and PMR were positive. CONCLUSION: Antibodies against the ferritin peptide were present in up to 92% of untreated, active GCA and PMR patients. They can be useful as a diagnostic marker of PMR and GCA.

Evidence of the contribution of the X chromosome to systemic sclerosis susceptibility: association with the functional IRAK1 196Phe/532Ser haplotype.

OBJECTIVE: Several autoimmune disorders, including systemic sclerosis (SSc), are characterized by a strong sex bias. To date, it is not known whether genes on the sex chromosomes influence SSc susceptibility. Recently, an IRAK1 haplotype that contains the 196Phe functional variant (rs1059702), located on Xq28, was found to confer susceptibility to systemic lupus erythematosus (SLE). This study was undertaken to test for an association between SSc and the IRAK1 SLE risk haplotype. METHODS: We tested for an association with the IRAK1 SLE risk haplotype in a discovery set of 849 SSc patients and 625 controls. IRAK1 rs1059702 was further genotyped in a replication set, which included Caucasian women from Italy (493 SSc patients and 509 controls) and Germany (466 SSc patients and 1,083 controls). RESULTS: An association between the IRAK1 haplotype and SSc was detected in the discovery set. In both the discovery and replication sets, the rs1059702 TT genotype was found to be associated with specific SSc subsets, highlighting a potential contribution to disease severity. A meta-analysis provided evidence of an association of both the T allele and TT genotype with the overall disease, with an odds ratio (OR) of 1.20 and 95% confidence interval (95% CI) of 1.06-1.35 for the T allele (P = 0.003) and an OR of 1.49 and 95% CI of 1.06-2.10 for the TT genotype (P = 0.023). However, the most notable associations were observed with the diffuse cutaneous, anti-topoisomerase I antibody positive, and SSc-related fibrosing alveolitis subsets (OR 2.35 [95% CI 1.51-3.66], P = 1.56 × 10(-4), OR 2.84 [95% CI 1.87-4.32], P = 1.07 × 10(-6), and OR 2.09 [95% CI 1.35-3.24], P = 9.05 × 10(-4), respectively). CONCLUSION: Our study provides the first evidence of an association between IRAK1 and SSc, demonstrating that a sex chromosome gene directly influences SSc susceptibility and its phenotypic heterogeneity.

Association of the CD226 Ser(307) variant with systemic sclerosis: evidence of a contribution of costimulation pathways in systemic sclerosis pathogenesis.

OBJECTIVE: The nonsynonymous polymorphism rs763361 of the CD226 gene, which encodes DNAX accessory molecule 1, which is involved in T cell costimulation pathways, has recently been identified as a genetic risk factor for autoimmunity. The purpose of this study was to test for association of the CD226 rs763361 polymorphism with systemic sclerosis (SSc) in European Caucasian populations. METHODS: CD226 rs763361 was genotyped in 3,632 individuals, consisting of a discovery sample (991 SSc patients and 1,008 controls) and a replication sample (999 SSc patients and 634 controls). All study subjects were of European Caucasian origin. Expression of CD226 was assessed on peripheral blood mononuclear cells obtained from 21 healthy donors genotyped for CD226 rs763361. RESULTS: The CD226 rs763361 T allele was found to be associated with SSc in both the discovery and the replication samples, showing the following results in the combined populations: odds ratio (OR) 1.22 (95% confidence interval [95% CI] 1.10-1.34), P = 5.69 × 10(-5) . The CD226 T allele was also associated with various SSc subsets, highlighting a potential contribution to disease severity. The most remarkable associations of the CD226 TT risk genotype were observed with the diffuse cutaneous SSc subtype, the anti-topoisomerase I antibody-positive, and SSc-related fibrosing alveolitis subsets: OR 1.86 (95% CI 1.42-2.43), P = 5.15 × 10(-6) , OR 1.82 (95% CI 1.38-2.40), P = 2.16 × 10(-5) , and OR 1.61 (95% CI 1.25-2.08), P = 2.73 × 10(-4) , respectively. CD226 expression was not significantly influenced by CD226 rs763361 genotypes whatever the T cell subtype investigated. CONCLUSION: Our results establish CD226 as a new SSc genetic susceptibility factor underlying the contribution of costimulation pathways in the pathogenesis of SSc. Further work is nevertheless needed to define the causal variant at the CD226 locus as well as the functional consequences.

NLRP1 influences the systemic sclerosis phenotype: a new clue for the contribution of innate immunity in systemic sclerosis-related fibrosing alveolitis pathogenesis.

BACKGROUND: Recent evidence has highlighted a potential role of interleukin 1ß (IL-1ß) in systemic sclerosis (SSc). NLRP1 provides a scaffold for the assembly of the inflammasome that promotes the processing and maturation of pro-IL-1ß. In addition, NLRP1 variants were found to confer susceptibility to autoimmune disorders. OBJECTIVE: /st> To study a possible association of the NLRP1 rs6502867, rs2670660 and rs8182352, rs12150220 and rs4790797 with SSc in the European Caucasian population. METHODS: NLRP1 single nucleotide polymorphisms were genotyped in 3227 individuals comprising a discovery set (870 SSc patients and 962 controls) and a replication set including individuals from Germany (532 SSc patients and 324 controls) and Italy (527 SSc patients and 301 controls), all individuals being of European Caucasian origin. RESULTS: Conditional analyses revealed a significant association for the NLRP1 rs8182352 variant with both anti-topoisomerase-positive and SSc-related fibrosing alveolitis (FA) subsets under an additive model: p=0.0042, OR 1.23 (95% CI 1.07 to 1.41) and p=0.0065 OR 1.19 (95% CI 1.05 to 1.36), respectively. Logistic regression analysis showed an additive effect of IRF5 rs2004640, STAT4 rs7574865 and NLRP1 rs8182352 risk alleles on SSc-related FA. CONCLUSIONS: Our results establish NLRP1 as a new genetic susceptibility factor for SSc-related pulmonary fibrosis and anti-topoisomerase-positive SSc phenotypes. This provides new insights into the pathogenesis of SSc, underlining the potential role of innate immunity in particular in the FA-positive SSc subphenotype, which represents a severe subset of the disease.

Association of a KCNA5 gene polymorphism with systemic sclerosis-associated pulmonary arterial hypertension in the European Caucasian population.

OBJECTIVE: Pulmonary arterial hypertension (PAH) has emerged as a leading cause of death in systemic sclerosis (SSc). The genetic basis of PAH has been unraveled in recent years, with a major role played by transforming growth factor ß receptors; however, some other candidate genes have also been advocated, including potassium voltage-gated channel, shaker-related subfamily, member 5 (KCNA5). We undertook this study to determine whether KCNA5 polymorphisms confer susceptibility to SSc and its vascular phenotype, including PAH. METHODS: Four KCNA5 single-nucleotide polymorphisms (SNPs), rs10744676, rs1860420, rs3741930, and rs2284136, were genotyped in a discovery set of 638 SSc patients and 469 controls. In addition, rs10744676 was genotyped in an independent replication sample (938 SSc patients and 564 controls) and in a cohort of 168 patients with different PAH subtypes. RESULTS: The KCNA5 rs10744676 variant was found to be associated with SSc in the discovery sample, with an odds ratio (OR) of 0.62 (95% confidence interval [95% CI] 0.48-0.79, adjusted P = 0.0003) in comparison with controls (C allele frequency 11.4% versus 17.2%). When subphenotypes were investigated, an association was found solely for PAH associated with SSc (OR 0.31 [95% CI 0.13-0.71], adjusted P = 0.04). The other KCNA5 SNPs tested were not associated with any SSc subset. The above association with PAH associated with SSc was replicated in the second set. In the combined population, rs10744676 was strongly associated with PAH associated with SSc in comparison with controls (OR 0.36 [95% CI 0.21-0.63], P = 0.0002). In the independent cohort of patients with PAH, after investigating PAH subtypes, only rs10744676 showed an association with PAH associated with SSc. CONCLUSION: Our results provide the first evidence for an association between the KCNA5 rs10744676 variant and PAH associated with SSc.

[Genetic analysis in collagen vascular diseases]. / Genetik bei Kollagenosen.

Genetic risk factors are known to exist for all collagen vascular diseases. They are most important for systemic lupus erythematosus (SLE) and systemic scleroderma (SSc), as shown by the systematic analysis of family data. Both diseases to date share most of the validated risk factors (PTPN22, STAT4, BANK1, TNFAIP3, IRF5, BLK) underlining once again their relationship. Moreover, most of these factors were also shown to be associated with other autoimmune diseases. Many additional risk factors exist, but need further analysis. The HLA complex is of special interest, as many loci within this region, some highly polymorphic, may contribute to the total genetic risk.

Quantitative studies on T cell diversity. IV. Mathematical analysis of multiple limiting populations of effector and suppressor T cells.

Limiting dilution (LD) analyses of polyclonally activated T cells yielded results suggesting the existence of multiple paired populations of effector and suppressor precursors for a number of different T cell functions and specificities analyzed. These populations occur at graded frequencies and suppression occurs within a pair but not between pairs. In this paper, we establish the mathematical basis for the interpretation of these multi-component limiting dilution results. First, we derive equations for a number of mathematical models and identify one model that both makes biological sense and can be used to reproduce experimental data. Second, within this model, we identify parameters such as the frequency of suppressive cells and the number of suppressive cells required for suppression. The results suggest that within each paired population, suppressor precursors are 20 times more frequent that effector precursors. Furthermore, a similar but variable excess of suppressor cells is required for suppression to become effective. Together with the high frequency (1/50-1/500) of most effector T cell precursors previously reported, the results suggest that up to 40% of the T cells can become involved in suppression of an antigen-specific effector T cell population. These studies may provide exact estimates for predictions to be tested in experiments on immune regulation.

Quantitative studies on T cell diversity. III. Limiting dilution analysis of precursor cells for T helper cells reactive to xenogeneic erythrocytes.

Splenic T cells exposed to concanavalin A (Con A), and subsequently to factors produced by rat spleen cells in response to Con A (Con A sup), acquire the ability to function as helper T (TH) cells in response to xenogeneic erythrocytes (RBC). Help is measured as the reconstitution of the plaque-forming cell response of a spleen cell population depleted of T cells by treatment with anti-Thy-1 serum and complement. We propose that precursor TH cells differentiate during the in vitro treatment into mature TH cells. As differentiation occurs under limiting dilution conditions, an estimation of the precursor frequency should in principle be possible. However, a single-hit Poisson distribution does not fit our data. Instead, we observe, dependent on the T cell concentration, three separate "peaks" of response. In many experiments, using sheep, horse, and chicken RBC as antigens, we reproducibly find these "peaks" at 40-190, 600-3,000, and 20,000-100,000 T cells, placed into limiting dilution cultures, respectively. By various experiments we can show that the helper activity is not due to passively transferred rat factors, but to the titrated cells themselves. The active cell is a T cell that appears to function in an antigen-specific way and to require direct cell contact to do so. It thus resembles the classical helper T cell. As we find precursor TH cells already at very low concentrations of T cells, we titrated the range between 0 and 100 T cells/well carefully. The bent shape of the titration curves does not always allow a statistically satisfying regression analysis, and we therefore cannot estimate precise precursor frequencies from every experiment. However, a common sense argument can be made that these frequencies must be on the order of 1/10-1/100 T cells. We propose that the limiting dilution curves obtained in this system most likely reflect fundamentally important cellular interactions that regulate immunological effector functions. We favor a concept of independently interacting sets of helper and suppressor T cells of various frequencies, but other models are possible.

Quantitative studies on T cell diversity. I. Determination of the precursor frequencies for two types of streptococcus A-specific helper cells in nonimmune, polyclonally activated splenic T cells.

A limiting-dilution system is described that makes use of T cell growth factor T cell expansion and allows the determination of precursor frequencies for various regulatory and effector T cells in nonimmune, polyclonally, or specifically activated T cell populations. Two different sets, a frequent and a rare set, of T helper cell precursors with specificity for trinitrophenyl-group A streptococcal vaccine, could be identified: the frequent set is of the Lyt-123 phenotype, and is present at frequencies of from 1/1,000 to 1/6,000 splenic T cells. It is only active at low cell numbers, whereas it is completely inactivated at greater cell numbers, presumably by suppressor T cells of lower frequency but greater potency. The rare set is of the Lyt-1 phenotype, is present at frequencies of from 1/10,000 to 1/70,000, and is not sensitive to suppressor cells present within the tested cell numbers. We suggest that the frequent set contains primiary helper cell precursors, whereas the rare set contains helper T memory cells preselected by previous exposure to other antigens. The results are discussed with respect to other reports on the involvement of more than one set of helper cells in antibody production.

Isolated hapten-binding receptors of sensitized lymphocytes. III. Evidence for idiotypic restriction of T-cell receptors.

The primary antibody response of C57BL/6 mice to the 4-hydroxy-3-nitrophenylacetyl (NP) hapten is restricted to antibody molecules expressing the NPb idiotype. This idiotype is a genetic marker for V genes in the heavy chain linkage group. In the secondary response, the frequency of NPb idiotype-positive molecules within the antibody population drops to very low values. Accordingly, isolated NP binding receptors from NP-sensitized B lymphocytes are largely devoid of this idiotype. In contrast, the NPb idiotype is expressed on the majority of the receptor fractions which we consider T-cell derived. This finding suggests that the antigen receptors of T lymphocytes may be restricted to the expression of major (germ-line encoded?) heavy chain idiotypes.

Involvement of the T cell antigen receptor and of Lyt-2 in the cytotoxic function of aged killer (AK) T cells.

Aged killer (AK) T cells are antigen-independent, IL-2-requiring variants of antigen-dependent CTL clones that have lost their original antigen specificity and have acquired, instead, specific cytotoxicity for P815 target cells. In this report we study whether AK cells use a similar or a different target cell recognition system than that of bona fide CTL. To this end, we selected from a cloned AK line variants that are partially or completely deficient in specific target recognition and/or in cytotoxic function, and analyzed these variants for expression of the T cell antigen receptor and of Lyt-2. Variants were selected from the prototype AK line (Cl 96) with specific, as well as lectin-facilitated, cytotoxicity for P815 tumor cells. Variants could be grouped into four types with increasing degrees of functional deficiency, which correlated with loss of T cell receptor and/or loss of Lyt-2. In short, loss of Lyt-2 was reflected in loss of specific target recognition, and loss of the T cell antigen receptor was reflected in loss of all cytotoxic activity. We conclude from these results that both Lyt-2 and the T cell antigen receptor are required for specific target cell recognition and the T cell antigen receptor is, in addition, required for cytotoxic function. Moreover, since AK cells express a somatically acquired specificity that differs from that of their clonal precursors, it appears that cytotoxic T cells may change their antigen receptor from one specificity to another during tissue culture.

Association of the TNFAIP3 rs5029939 variant with systemic sclerosis in the European Caucasian population.

BACKGROUND: TNFAIP3 encodes the ubiquitin-modifying enzyme, a key regulator of inflammatory signalling pathways. Convincing associations between TNFAIP3 variants and autoimmune diseases have been reported. OBJECTIVE: To investigate the association of TNFAIP3 polymorphisms with systemic sclerosis (SSc). METHODS: Three single nucleotide polymorphisms (SNPs) in a set of 1018 patients with SSc and 1012 controls of French Caucasian origin were genotyped. Two intergenic SNPs, rs10499194 and rs6920220, and one located in TNFAIP3 intron 2, rs5029939, were selected. The TNFAIP3 rs5029939 found to be associated with SSc in this first set was then genotyped in a second set of 465 patients with SSc and 182 controls from Germany and 184 patients with SSc and 124 controls from Italy. Pooled odd ratios were calculated by Mantel-Haenszel meta-analysis. RESULTS: The rs5029939 G allele was found to be significantly associated with SSc susceptibility (pooled OR=2.08 (95% CI 1.59 to 2.72); p=1.16×10⁻7), whereas the rs10499194 and rs6920220 variants displayed no association. Only one of the predicted haplotypes investigated in the French sample was significantly associated with SSc (p=8.91×10⁻8), and this haplotype was discriminating only in the presence of the rs5029939 risk allele, suggesting that this SNP tags the association signal. The strongest associations of rs5029939 with subphenotypes, having large magnitudes for complex genetic disorders, were observed for diffuse cutaneous SSc (pooled OR=2.71 (1.94 to 3.79), p=5.2×10⁻9), fibrosing alveolitis (pooled OR=2.26 (1.61 to 3.17), p=2.5×10⁻6) and pulmonary arterial hypertension (pooled OR=3.11 (1.86 to 5.17), p=1.3×10⁻5). CONCLUSION: These results suggest that TNFAIP3 is a genetic susceptibility factor for SSc.

Skin sclerosis is only of limited value to identify SSc patients with severe manifestations--an analysis of a distinct patient subgroup of the German Systemic Sclerosis Network (DNSS) Register.

OBJECTIVES: In SSc, diagnosis and classification is based mainly on skin sclerosis. Herein, we investigated in a large multicentre cohort, to what extent skin sclerosis reflects organ involvement and additional clinical symptoms. METHODS: A total of 1200 SSc patients from the register of the German Systemic Sclerosis Network (DNSS), classified as either lcSSc or dcSSc, were analysed for their serological characteristics, clinical symptoms and organ manifestations in relation to skin involvement measured by the modified Rodnan skin score (mRSS). RESULTS: SSc patients with different mRSS did not differ significantly in their disease duration and in most of the clinical symptoms. They showed a similar distribution of most organ manifestations such as pulmonary arterial hypertension as well as cardiac, renal and nervous system involvement. More severe skin thickening was found to be associated with pulmonary fibrosis and gastrointestinal symptoms, as well as with digital ulcers and musculoskeletal involvement. CONCLUSIONS: In patients with SSc, potentially life-threatening complications and clinical symptoms with high impact on the quality of life occur independently from the extent of skin sclerosis. The diagnosis in SSc patients with a low mRSS could be missed or they could be insufficiently treated.

Comparison of patients with and without digital ulcers in systemic sclerosis: detection of possible risk factors.

BACKGROUND: Digital ulcers (DU) are a major complication in the course of systemic sclerosis (SSc). In recent years, efficacious, but expensive therapies (e.g. iloprost, sildenafil, bosentan) have been shown to improve healing or to reduce the recurrence of DU. For optimal management it would be useful to identify the risk factors for DU. Such statistical analyses have been rare because they require a high number of patients. OBJECTIVES: To identify potential risk factors for DU in patients with SSc. METHODS: We used the registry of the German Network for Systemic Scleroderma and evaluated the data of 1881 patients included by August 2007. We assessed potential risk factors for DU by comparing patients with (24.1%) and without active DU at time of entry (75.9%). RESULTS: Multivariate analysis revealed that male sex, presence of pulmonary arterial hypertension (PAH), involvement of the oesophagus, diffuse skin sclerosis (only when PAH was present), anti-Scl70 antibodies, young age at onset of Raynaud's phenomenon (RP), and elevated erythrocyte sedimentation rate (ESR) significantly impacted on the appearance of DU. Certain combinations increased the patients' probability of presenting with DU, with the highest probability (88%) for male patients with early onset of RP, ESR>30 mm h(-1), anti-Scl70 antibodies and PAH. Patients with DU developed RP, skin sclerosis and organ involvement approximately 2-3 years earlier than patients without DU. CONCLUSIONS: The results reveal possible risk factors for the occurrence of DU in SSc. As DU are prone to local complications, prophylactic vasoactive treatment for patients presenting with these factors may be justified.

Association of rheumatoid arthritis with Mdm2 SNP309 and genetic evidence for an allele-specific interaction between MDM2 and p53 P72R variants: a case control study.

OBJECTIVE: This study examines two common, functional, single nucleotide polymorphisms (SNP) in the genes coding the human homolog of murine-double-minute-2 (MDM2) and p53 in patients with rheumatoid arthritis (RA) based on the hypothesis that p53 may be an important negative regulator of the pro-inflammatory transcription factor nuclear factor kappa b (NFKappaB). METHODS: Genomic DNA was obtained from 221 patients with RA who fulfilled at least 4 ACR criteria and from 521 healthy controls. Mdm2 SNP309 and p53 P72R were genotyped by polymerase chain reaction and restriction enzyme analysis. RESULTS: In RA patients the frequencies of the mdm2 SNP309 G allele and both G-containing genotypes were significantly reduced (G allele: OR: 0.75, 95% CI: 0.59-0.95, p=0.016; genotype TG: OR: 0.71, 95% CI: 0.50-1.00; genotype GG: OR. 0.58, 95% CI: 0.34-0.99; both: p=0.049). Concerning p53 P72R, no differences in allele or genotype frequencies were detected. A combined analysis of both polymorphisms revealed a significant interaction between them (p=0.046). In individuals carrying >1 p53 72R allele, MDM2 had a protective effect, whereas in individuals homozygous for p53 72P, MDM2 had the opposite effect. CONCLUSION: The function of MDM2 depends on the p53 P72R genotype, resulting in either an increased or reduced risk for RA. We suggest that in most cases MDM2 stabilizes the conformation of p53, whereas in p53 PP-positive subjects MDM2 supports the degradation of p53.

The registry of the German Network for Systemic Scleroderma: frequency of disease subsets and patterns of organ involvement.

OBJECTIVE: Systemic sclerosis (SSc) is a rare, heterogeneous disease, which affects different organs and therefore requires interdisciplinary diagnostic and therapeutic management. To improve the detection and follow-up of patients presenting with different disease manifestations, an interdisciplinary registry was founded with contributions from different subspecialties involved in the care of patients with SSc. METHODS: A questionnaire was developed to collect a core set of clinical data to determine the current disease status. Patients were grouped into five descriptive disease subsets, i.e. lcSSc, dcSSc, SSc sine scleroderma, overlap-syndrome and UCTD with scleroderma features. RESULTS: Of the 1483 patients, 45.5% of patients had lcSSc and 32.7% dcSSc. Overlap syndrome was diagnosed in 10.9% of patients, while 8.8% had an undifferentiated form. SSc sine scleroderma was present in 1.5% of patients. Organ involvement was markedly different between subsets; pulmonary fibrosis for instance was significantly more frequent in dcSSc (56.1%) than in overlap syndrome (30.6%) or lcSSc (20.8%). Pulmonary hypertension was more common in dcSSc (18.5%) compared with lcSSc (14.9%), overlap syndrome (8.2%) and undifferentiated disease (4.1%). Musculoskeletal involvement was typical for overlap syndromes (67.6%). A family history of rheumatic disease was reported in 17.2% of patients and was associated with early disease onset (P < 0.005). CONCLUSION: In this nationwide register, a descriptive classification of patients with disease manifestations characteristic of SSc in five groups allows to include a broader spectrum of patients with features of SSc.

Analysis of the human CD21 transcription unit reveals differential splicing of exon 11 in mature transcripts and excludes alternative splicing as the mechanism causing solubilization of CD21.

CD21 is found in a soluble form at low levels in normal human sera and at elevated levels in sera from patients with EBV-associated diseases and B-CLL. Ablation of complement, injection of recombinant soluble CD21 and knock-out of CD21 in mice by gene targeting interfere with T-cell-dependent immune responses, suggesting that in vivo-generated soluble CD21 may exert immunoregulatory functions. Soluble CD21 has a molecular weight of 130,000/135,000, which is equivalent to the entire extracellular domain. Soluble forms of membrane-anchored molecules may be generated by proteolytic cleavage of the extracellular portion or by the exclusion of the hydrophobic transmembrane region via alternative splicing. To delineate whether alternative splicing of CD21 mRNA creates transcripts encoding for the soluble form of CD21 we analyzed by PCR CD21 expression in PBLs, spleen, tonsils, bone marrow and in various cell lines. We found that all CD21 mRNA species contained the transmembrane exons, thus excluding alternative splicing as a factor contributing to the serum pool of soluble CD21. Differential splicing of the CD21 transcription unit has also been suggested for exon 11. Within the CD21 gene exons 3, 7 and 11 have a high degree of homology. However, we found in malignant human cell lines and primary lymphocytes from blood, bone marrow, tonsils and spleen that exon 11, but not exon 3 or 7, is subject to alternative splicing. We cloned and sequenced the intron preceding exon 11 and found that the surrounding splice sites match consensus splice sites. In conclusion, we show that human CD21 exon 11 is alternatively spliced in malignant cell lines of lymphoid origin and in purified lymphocytes from blood, tonsils, bone marrow and spleen. We found that both exon 11 lacking and exon 11 containing transcripts are always coexpressed and the ratio of the two forms is > 1. Moreover, we exclude the possibility that alternative splicing of the transmembrane region is the mechanism leading to soluble CD21.

Frequencies and interactions of regulatory T cells. I. The balance between help and suppression regulates the primary immune response to keyhole limpet hemocyanin in vitro.

Frequencies and efficiencies of regulatory T cells from non-immunized mice were estimated in several assay systems differing from each other in cellular composition and antigen dose (NIP-KLH). The NIP-specific and total IgM responses were quantified. Using 10(4) syngeneic B cells and 50 micrograms/ml NIP-KLH, helper (Th) cells from 5 day immune donors were detected in frequencies of 1:3000-1:4000 in lymphnode and spleen T cells, with an efficiency of 70-90 ng IgM/Th cell in C57B1/6 mice. In non-immune spleen T cells, Th cells were observed in frequencies of 1:16,000-1:38,000, with comparable efficiency, but these Th cells appeared suppressed at increased T cell doses. Polyclonal activation led to the appearance of multiple independently regulated populations of Th cells with similar efficiencies. In the presence of 10(5) syngeneic spleen cells, treated once with anti-Thy-1 antibody and complement and 50 micrograms/ml NIP-KLH, suppressor activity was observed in the same T cell population. Similar to help, suppression fluctuated with increasing T cell numbers. Using 1 x 10(6) spleen cells and 50 micrograms/ml NIP-KLH as assay system, T cells enhanced the responses. Again, several independently regulated populations were observed, with efficiencies slightly higher than those of the above-described Th cells. By maintaining the cellular components of the assay system constant (10(4) B cells) and titrating the antigen, Th cell frequencies showed little variation up to 100 micrograms/ml NIP-KLH and were always suppressed at higher T cell numbers. At 200 micrograms/ml NIP-KLH, the frequency was increased to approximately 1:2000, and not suppressed, i.e., was identical to the frequency observed in mice immunized 5 days previously. Efficiencies increased with increasing doses of antigen. The results strongly indicate that regulatory T cell function shows "plasticity", in the sense that the appearance and the frequencies of helping and suppressing T cell populations highly depend on the micro-environment present in culture.

Monoclonal autoantibodies from patients with autoimmune diseases: synovial fluid B lymphocytes of a patient with rheumatoid arthritis produced an IgG lambda antibody recognizing J-sequences of Ig kappa chains in a conformation-dependent way.

Synovial fluid B cells from a patient with seronegative rheumatoid arthritis were immortalized by electrofusion. The specificity of clone FKN-E12 (IgG1 lambda) was analysed by screening a phage display random peptide library. One heptamer sequence was identified (RASFp1 = HLTFGPG). Three human IgG kappa antibodies contained a highly homologous sequence (xLTFGPG) at the junction of V- and J-regions. Homologies were also found in distinct humans (J kappa 3, J kappa 4) and murine (J kappa 5) J kappa-sequences (TFGPG, LTFGxG), and to a lower degree in all remaining J kappa-sequences (TFGxG). Binding and binding inhibition assays showed that FKN-E12 bound to kappa light chains tested in a conformation-dependent way: it reacted only with IgG kappa or IgA kappa chains adhered to a plastic surface, but not in soluble form. In conclusion, FKN-E12 detects a conformational epitope on probably all kappa light chains, which could be definded by screening a phage library displaying linear epitopes.