Ruth Fritsch

Stepwise Differentiation of CD4 Memory T Cells Defined by Expression of CCR7 and CD27

To study the steps in the differentiation of human memory CD4 T cells, we characterized the functional and lineage relationships of three distinct memory CD4 subpopulations distinguished by their expression of the cysteine chemokine receptor CCR7 and the TNFR family member CD27. Using the combination of these phenotypic markers, three populations were defined: the CCR7+CD27+, the CCR7−CD27+, and the CCR7−CD27− population. In vitro stimulation led to a stepwise differentiation from naive to CCR7+CD27+ to CCR7−CD27+ to CCR7−CD27−. Telomere length in these subsets differed significantly (CCR7+CD27+ > CCR7−CD27+ > CCR7−CD27−), suggesting that these subsets constituted a differentiative pathway with progressive telomere shortening reflecting antecedent in vivo proliferation. The in vitro proliferative response of these populations declined, and their susceptibility to apoptosis increased progressively along this differentiation pathway. Cytokine secretion showed a differential functional capacity of these subsets. High production of IL-10 was only observed in CCR7+CD27+, whereas IFN-γ was produced by CCR7−CD27+ and to a slightly lesser extent by CCR7−CD27− T cells. IL-4 secretion was predominantly conducted by CCR7−CD27− memory CD4 T cells. Thus, by using both CCR7 and CD27, distinct maturational stages of CD4 memory T cells with different functional activities were defined.

Bet v 1, the major birch pollen allergen, and Mal d 1, the major apple allergen, cross-react at the level of allergen-specific T helper cells☆☆☆★★★

BACKGROUND Food allergy to apple is frequent in individuals allergic to tree pollen. The major allergens of birch, Bet v 1, and apple, Mal d 1, have been cloned and sequenced and display a high degree of sequence identity, leading to IgE cross-reactivity. OBJECTIVE We sought to investigate cross-reactivity between Bet v 1 and Mal d 1 at the level of allergen-specific T lymphocytes. METHODS PBMCs of 13 patients allergic to birch pollen with oral allergy syndrome to apple were stimulated with rBet v 1 and rMal d 1, respectively, thereby establishing allergen-specific T-cell lines and T-cell clones. rMal d 1-specific T-cell cultures were tested for reactivity with rBet v 1, and rBet v 1-specific T cells were analyzed for reactivity with apple allergen. Cytokine production patterns in response to specific stimulation were evaluated. A selection of cross-reacting T-cell clones was mapped for epitope specificity by the use of overlapping Bet v 1- derived peptides. RESULTS Nineteen Mal d 1-specific T-cell clones were produced, 79% of which cross-reacted with Bet v 1. Eight of 18 Bet v 1-specific T-cell clones cross-reacted with Mal d 1. Six peptides representing cross-reactive T-cell epitopes could be identified. The respective fragments from birch and apple displayed approximately 50% amino acid identity. Seventy percent of the cross-reactive T-cell clones revealed a T(H2)-like cytokine production pattern. CONCLUSION The results indicate that cross-reactivity between apple and birch pollen leading to the clinical oral allergy syndrome occurs not only at the serologic, but also at the cellular level.

Characterization of Autoreactive T Cells to the Autoantigens Heterogeneous Nuclear Ribonucleoprotein A2 (RA33) and Filaggrin in Patients with Rheumatoid Arthritis

The role of autoimmune reactions in the pathogenesis of rheumatoid arthritis (RA) is poorly understood. To address this issue we have investigated the spontaneous T cell response to two well-characterized humoral autoantigens in RA patients and controls: 1) the heterogeneous nuclear ribonucleoprotein A2, i.e., the RA33 Ag (A2/RA33), and 2) filaggrin in unmodified and citrullinated forms. In stimulation assays A2/RA33 induced proliferative responses in PBMC of almost 60% of the RA patients but in only 20% of the controls (patients with osteoarthritis or psoriatic arthritis and healthy individuals), with substantially stronger responses in RA patients (p < 0.00002). Furthermore, synovial T cells of seven RA patients investigated were also clearly responsive. In contrast, responses to filaggrin were rarely observed and did not differ between RA patients and controls. Analysis of A2/RA33-induced cytokine secretion revealed high IFN-γ and low IL-4 production in both RA and control PBMC, whereas IL-2 production was mainly observed in RA PBMC (p < 0.03). Moreover, A2/RA33-specific T cell clones from RA patients showed a strong Th1 phenotype and secreted higher amounts of IFN-γ than Th1 clones from controls (p < 0.04). Inhibition experiments performed with mAbs against MHC class II molecules showed A2/RA33-induced T cell responses to be largely HLA-DR restricted. Finally, immunohistochemical analyses revealed pronounced overexpression of A2/RA33 in synovial tissue of RA patients. Taken together, the presence of autoreactive Th1-like cells in RA patients in conjunction with synovial overexpression of A2/RA33 may indicate potential involvement of this autoantigen in the pathogenesis of RA.

Allergenic crossreactivities: Pollens and vegetable foods

ConclusionCross-sensitization to inhalant pollen allergens and ingested food allergens have been the focus of an increasing number of studies. During the last few years, the molecular characterization of the major allergens of most common pollens and foods has made it possible to identify some potential candidates of crossreactive IgE binding proteins. In this respect, the family of Bet v 1 homologs, the profilin family, and an until recently not completely characterized allergen cluster of approx 60 kDa have been described. These three allergenic structures constitute useful tools in diagnostic procedures, as the majority of vegetable allergy could be detected by this combination of allergens. Further information about crossreacting agents is needed in order to be able to advise pollen allergic patients fully about their risks of a possibly dangerous reaction to food.

Autoantibodies to hnRNP-A2 in SLE: identification of disease-specific linear epitopes and correlation with disease activity and clinical features

The autoantigen hnRNP-A2 (RA33) is targeted by autoantibodies (autoAbs) of patients with rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD). To define the humoral autoimmune response in more detail, a series of overlapping peptides covering the N-terminal part hnRNP-A2 known to harbour the major epitopes was studied by ELISA in sera from patients with SLE (n = 40), RA (n = 50), MCTD (n = 11) and other rheumatic diseases (n = 86) and from healthy subjects (n = 29). Anti-peptide reactivities were detected in 25% of SLE sera but only rarely in patients with RA or MCTD. Since most of the SLE patients investigated had inactive disease, we next studied sequential sera of 15 individual patients. Of those, only three patients were completely negative for hnRNP-A2 antibodies, while all the other patients showed at least one positive reaction during the observation period. These were directed to the full-length protein and/or to 4 of the 13 peptides used: p35–55, p50–70, p90–116 and p155–175. Interestingly, autoreactivities to the first three peptides were significantly associated with each other but not with reactivity to p155–175 or to the complete protein. This cluster of reactivity was also not linked to any clinical marker of disease. In contrast, p155–175 was strongly correlated with reactivity to the complete protein (P < 0.001) and both reactivities were associated with autoAb to dsDNA and correlated significantly with clinical disease activity, skin involvement and proteinuria (P < 0.01). Remarkably, immunohistochemical analysis revealed overexpression of hnRNP-A2 in affected skin of SLE patients. These data, together with previously published findings in murine SLE models, are suggestive of an involvement of hnRNP-A2 autoimmunity in the pathogenesis of SLE.

Characterization of autoreactive T cells to the autoantigens hnRNP-A2/RA33 and filaggrin in patients with rheumatoid arthritis and controls

In an attempt elucidate the role of autoimmune processes in the pathogenesis of rheumatoid arthritis (RA) we investigated the T cell responses to two autoantigens targeted by autoantibodies of patients with RA, (i) the heterogeneous nuclear ribonucleoprotein (hnRNP) A2/RA33 and (ii) filaggrin which is one of the target structures recognized by anti-citrulline antibodies. Stimulation assays were performed with peripheral blood mononuclear cells of 50 RA patients, 20 patients with osteoarthritis and 21 healthy control individuals using recombinant hnRNP-A2/RA33 as well as some fragments thereof and recombinant filaggrin both in unmodified and citrullinated form. Antigen-specific T cell clones (TCC) were obtained by cultivating T cell lines in the presence of antigen and IL-2 followed by limiting-dilution cloning. Proliferative responses to hnRNP-A2/RA33 were seen in 60% of the RA patients with a mean stimulation index (SI) of 3.5 ± 2.8 and were significantly higher than those observed in the control group (mean SI=1.7 ± 1, P < 0.00005). There was no correlation with the presence of anti-A2/RA33 autoantibodies nor with MHC genes, although more than 60% of the responsive patients carried the shared epitope. Results obtained with recombinant fragments indicated a major T cell epitope to be located in the N-terminal first RNA binding domain of the protein. Anti-A2/RA33 specific TCC (n = 16) derived from RA patients were almost exclusively CD4?, whereas only 7 of 12 TCC derived from controls showed this phenotype, and secreted high amounts of IFNg upon antigen stimulation as did all TCC derived from controls. Proliferative responses to filaggrin in either form were seen in only 25% of the RA patients tested and did not differ from those observed in the control group indicating that filaggrin-reactive T cells do presumably not drive the autoantibody response to citrullinated antigens. Taken together, a Th1 type autoimmune response to hnRNP-A2/RA33 was commonly observed in RA patients suggesting this nuclear protein to constitute a major T cell autoantigen which might be fuelling one of the pathological autoimmune reactions that drive the destructive processes effective in RA.

Characterization of RA33 (hnRNP-A2/B1)-autoreactive T cells in SLE-patients

SLE is a systemic autoimmune disease with distinct immunological characteristics including defective T cell functions, especially concerning IL2 production and proliferation. Furthermore, B-cell hyperactivity is observed leading to the formation of several characteristic autoantibodies (ab), among them ab to the heterogenous nuclear ribonucleoprotein A2/B1 (hnRNP/RA33). These antibodies are known to occur in over 20% of SLE patients. In order to elucidate the role of T cells and their influence in antibody production in SLE, we studied proliferation of PBMC to purified hnRNP-A2/B1 in 34 SLE patients and 21 healthy controls. While the stimulation indices (SI) in the healthy control group ranged from 0.5 to 3.5 (mean SI: 1.5 ± 0.9), the proliferative response of PBMC of the patient group ranged from 0.7 to 17 with a mean SI of 4.8 ± 4.0 (only 6 of 34 patients had an SI<2; P < 0.00004). We then proceeded to draw RA33-specific T cell clones (TCC) by cultivation and limiting-dilution cloning of T cell lines. The generated 30 TCC derived from SLE patients and 19 TCC from healthy controls did not reveal a significant difference in SI and produced either more IFNγ than IL4 or none of these cytokines at all, suggesting that these TCC were of T1 or T0, but not T2 phenotype. Interestingly though, while only 11% of healthy control patients showed a CD4-/CD8+ subtype and 16% displayed a CD4+/CD8+ phenotype, 37% of TCC derived from SLE patients were CD4-/CD8+ (and 20 % expressed CD4 as well as CD8). Our data reveal that more than 80 % of SLE-patients have a significant T cell reactivity (SI ≥ 2) to the nuclear protein hnRNP-A2/B1 indicating that the antibody response might be T cell driven. Furthermore, almost 60% of TCC derived from SLE patients were CD8+, which supports the importance of these T cells in SLE. Further studies will have to elucidate the pathogenetic implications of these findings.