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The tryptic FAD-peptide carrying the flavin in 8alpha-(N3)histidyl linkage as natural hapten was isolated by HPLC from the bacterial enzyme 6-hydroxy-d-nicotine oxidase. The same flavin protein linkage is found in the mitochondrial succinate dehydrogenase flavoprotein subunit, the predominant flavoprotein with covalently bound FAD in mitochondria of cardiomyocytes. Peripheral blood mononuclear cells (PBMC) were isolated from four patients with acute myocarditis, seven patients with dilated cardiomyopathy (DCM) and from four healthy control individuals. The response of PBMC to the FAD-peptide was evaluated by measuring proliferation ([3H]-dThd incorporation) and cytokine secretion [interferon (IFN)-gamma]. PBMC from all patients with acute myocarditis showed positive responses to the FAD-peptide, in contrast to PBMC from patients with DCM or control individuals. Following the recovery of the patients from the acute inflammation of the heart, PBMC no longer exhibited a proliferation response to the FAD-peptide. A chemically synthesized FAD-free peptide with identical amino acid sequence induced no response of PBMC. The results are consistent with a recall response by activated T cells, specific for the normally cryptic mitochondrial flavin-hapten, which may be liberated following cardiomyocyte destruction during the inflammation of the heart.

The expression of CR2 (CD21) by synovial B and T lymphocytes of patients suffering from various forms of arthritis was analysed with cytofluorometry and with reverse transcriptase-polymerase chain reaction. CR2 (CD21) cell surface protein was detected in normal quantities on peripheral B cells, but was almost absent on synovial B lymphocytes of the same patients. This reduction was most severe in patients with rheumatoid arthritis, but also observed in all other cases. CR2 (CD21) did not reappear after in vitro culture. CR2 (CD21) mRNA was also strongly reduced in synovial B and T lymphocytes. Synovial fluid B lymphocytes were larger than peripheral blood B lymphocytes, while T cells from the same patients showed no size differences. We conclude that synovial fluid B lymphocytes have undergone an irreversible step towards terminal differentiation. The presence or absence of CR2 (CD21) mRNA in peripheral versus synovial T cells indicates that CR2 (CD21) is also differentially expressed by T lymphocytes.

CD21, the complement receptor type 2 (CR2), binds the complement fragments iC3b, C3dg and C3d, interacts with CD23 (the low-affinity receptor for IgE), and binds IFN-alpha. This 145 kDa glycoprotein merits particular interest because it plays a pivotal role in the activation and proliferation of B cells by lowering the signal threshold. In human disease CD21 is important as a receptor for Epstein-Barr virus and HIV. CD21 is primarily expressed on B lymphocytes and follicular dendritic cells, but has also been reported on T cells. We established a semi-quantitative PCR and compared the CD21 mRNA levels of B and T lymphocytes with the expression of the CD21 glycoprotein on the surface of the respective cells by flow cytometry. The B cell lines Raji and Ramos and the T cell lines Jurkat and Molt4 expressed equal amounts of CD21 mRNA, but differed in surface staining. To address the question to which extent primary human B and T lymphocytes express CD21 mRNA and membrane-bound CD21 glycoprotein, we separated B cells, CD4+ and CD8+ T cells from peripheral blood mononuclear cells of healthy donors. B lymphocytes and CD4+ or CD8+ T cells expressed similar amounts of CD21 mRNA. Nevertheless only B cells, but not CD4+ or CD8+ T cells, expressed detectable amounts of CD21 on their cell surface. Expression of the CD21 exon 11 has been reported being restricted to follicular dendritic cells only. To the contrary, we found that both purified B and T cell subpopulations expressed CD21 mRNA with and without exon 11.

The functional T-cell repertoire in the inflamed joint of a patient with rheumatoid arthritis was analysed at the clonal level. Using limiting dilution techniques and selecting for growth of in vivo activated and/or autoreactive T cells, 149 T-cell lines were established. They were tested in a proliferation assay for reactivity against an autologous Epstein-Barr virus (EBV)-transformed B-cell line and a panel of auto-antigens and foreign antigens. Seventy-five lines (approximately 50%) could be stimulated. Thirty-six lines (approximately 24%) were antigen-reactive. They were stimulated by human collagens type I (15), II (10), IV (7) or V (4), cartilage proteoglycans (4), Mycobacterium tuberculosis (15), the 60 kDa heat-shock protein of M. bovis (13) or tetanus toxoid (10). T-cell lines were either monoreactive (19), bireactive (6), or multireactive (11), i.e. they were stimulated by either one, by two, or by more antigens in the panel. About half of the antigen-reactive lines were at the same time autoreactive towards the autologous B-cell line. These data suggest the existence of multispecific autoreactive T-cell receptors comparable to multireactive or natural autoantibodies and prove the presence of autoantigen-reactive T cells in the inflamed joints of patients with rheumatoid arthritis.

The T cell receptor (TCR) V beta gene segment repertoire of T lymphocytes derived from peripheral blood of two healthy individuals and synovial tissue, synovial fluid and peripheral blood of three rheumatoid arthritis (RA) patients was analyzed. A sensitive assay based on the amplification of cDNA by the polymerase chain reaction (PCR) was used to analyze the levels of expression of 20 TCR V beta gene segment families. The relative expression of V beta gene segments in lymphocytes derived from peripheral blood, synovial tissue and synovial fluid was conserved over 155 days in one patient. V beta 9 transcripts were undetectable in the cells of this individual. In the two other patients the frequency of V beta 2 transcripts in synovial T cells of affected joints was significantly higher than in their peripheral blood lymphocytes. Dominance of distinct rearrangements among the V beta 2 transcripts from the synovial cells of these patients support the idea that the synovial T cell response is driven by antigen.

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.

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.

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.

A limiting dilution system was established in order to estimate frequencies of interleukin-2 (IL-2)-responsive, autoreactive and alloreactive T cells in samples of peripheral blood (PBL) and synovial fluid lymphocytes (SFL), from patients with rheumatoid arthritis (RA) and lyme disease, as well as from healthy donors and a patient with osteoarthrosis. The frequencies of IL-2-dependent T-cell colony formation were significantly higher in patients with RA and lyme disease (median: 1/287) as compared to controls (median: 1/1,313) indicating a preactivation of T cells in these patients in vivo. Autoreactivity was measured by the proliferative response of T-cell lines to autologous irradiated PBL as stimulating cells. The frequencies of autoreactive T cells in blood were significantly higher in patients (median: 1/2,615) as compared to controls (median: 1/19,607). There was no significant difference in autoreactive T-cell frequencies between the patients' SFL (median: 1/3,185) and PBL (median: 1/2,615). In every case the frequency of alloreactive T cells exceeded the frequency of autoreactive T cells. Most autoreactive T-cell lines were also alloreactive and were shown to be MHC Class II-restricted. There is evidence of a down regulation of autoreactive T cells by suppressor cells in peripheral blood in two cases with elevated autoreactive T-cell frequencies (one RA patient and one control patient suffering from a viral infection). In contrast, no suppression of autoreactive T cells was observed in the RA patients' SFL or in PBL and SFL from patients with lyme disease. These results suggest that the chronic inflammation observed in RA and lyme disease may be supported by an elevated number of autoreactive T cells in the absence of suppressive mechanisms.

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.

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.

EL-4 lymphoma cells are able to suppress the primary humoral immune response of spleen cells to sheep erythrocytes or 4-hydroxy-3-iodo-5-nitrophenylacetyl-keyhole limpet haemocyanin (NIP-KLH) in vitro. Typically, the cells suppress very efficiently in small numbers, but lose this capacity as the numbers increase. Three clones were analysed and this fluctuation in function was paralleled by a fluctuation in the expression of CD8 (Ly-2). Clones were incubated for 2 days, starting from different seeding concentrations, and analysed with cytofluorography. Neither Thy-1 nor CD5 (Ly-1), H-2 K, H-2 D, LFA-1 (all positive) nor CD4 (L3T4) or MEL-14 (both negative) were influenced by this treatment. In contrast, cells were CD8- at high seeding concentrations, and CD8+ at low seeding concentrations. When cell cultures grew to higher densities, the cells again lost the capacity to express CD8. Experiments testing the suppressive capacity of individual EL-4 clones after preculture at different densities or in the presence of antibodies against CD8 suggest that the efficiency of suppression may well be correlated to the amount of CD8 expressed.