Antigen-specific human T-cell colony formation in a liquid culture system.

We have developed a limiting dilution microculture T-cell cloning technique to generate antigen-specific T-cell clones. Cultures contained peripheral blood T-cells, irradiated autologous mononuclear cells (feeders), human AB serum and recombinant IL-2. Colonies were enumerated at 14 days. Under these conditions the cloning efficiency expressed as the frequency of proliferating cells in response to PHA, ranged from 1/1.07 to 1/3.35. In response to tetanus toxoid, the frequency of proliferating cells ranged from 1/415 to 1/5655. Average colony size ranged from 0.7 x 10(5) to 6.2 x 10(5) cells with a mean T-cell recovery of 2.5 x 10(5) cells per microculture. Restimulation of 14-day tetanus toxoid T-cell colonies revealed marked proliferation to PHA and tetanus toxoid but only background responses to PPD. These studies document the development of a rapid, antigen-specific liquid culture T-cell cloning system which is likely to detect T-cell clones that are representative of the original T-cell population. The clones are of sufficient size to be useful in studies of antigen cross-reactivity.

Impaired generation of high-affinity interleukin-2 receptors in the autologous mixed lymphocyte reaction in rheumatoid arthritis.

We examined the expression of high-affinity interleukin (IL)-2 receptors (IL-2R) as well as Tac and HLA-DR antigens on peripheral blood (PB) T cells from 11 rheumatoid arthritis (RA) patients and 8 healthy controls induced in the autologous mixed lymphocyte reaction (AMLR). The proportion of HLA-DR- and Tac-bearing T cells and expression of these activation antigens were higher in patients relative to controls (P less than 0.01) in freshly isolated unstimulated PB mononuclear cells. AMLR stimulation of RA T cells failed to induce an increase in the proportion of HLA-DR and Tac-bearing T cells which was observed in health controls. After AMLR stimulation the number of high-affinity IL-2R were significantly lower in RA patients compared with controls (P less than 0.01). The number of high-affinity IL-2R on patient T cells correlated strongly with AMLR reactivity as measured by [3H]thymidine incorporation (r = 0.821, P = 0.002). The results suggest that the AMLR defect in RA may result from impaired generation of high-affinity IL-2R.

Elevation of a gamma delta T cell subset in peripheral blood and synovial fluid of patients with rheumatoid arthritis.

We examined the levels of TcR delta 1+ T cells (total gamma delta T cell) and delta TCS1+ (gamma delta T cell subset) T cells in the peripheral blood (PB) and synovial fluid (SF) of 16 patients with rheumatoid arthritis (RA) and compared them to the levels in PB of patients with Felty's syndrome (FS) and 21 healthy control subjects (NML). Synovial fluid from eight patients with seronegative spondyloarthropathies (SSA) was also examined. The results demonstrated elevated levels of the delta TCS1+ subset in the PB of RA and FS patients relative to NML (P less than 0.05). No such differences were observed in the levels of PB TcR delta 1+ T cells. The results did not appear to reflect a non-specific inflammatory response since delta TCS1 T cells were elevated in the SF of RA patients relative to SSA SF and NML PB. delta TCS1 T cells in SSA PB and SSA SF were comparable to NML PB. TcR delta 1+ T cells levels in RA SF were higher than SSA SF levels but were comparable to those of NML PB. Taken together, the results support a pathogenic role for delta TCS1+ T cells in RA.

Reconstitution of impaired autologous mixed lymphocyte reactivity in rheumatoid arthritis.

We examined the ability of recombinant IL-2 to reconstitute the autologous mixed lymphocyte reaction (AMLR) defect in peripheral blood mononuclear cells (PBM) from patients with rheumatoid arthritis (RA). Our results revealed an ability to fully reconstitute RA AMLRs with pharmacologic concentrations (100 units/ml), but not physiologic concentrations (10 units/ml) of IL-2. Full reconstitution of RA AMLRs was achieved whether IL-2 was added as the initiation of culture or at 48 or 72 hours prior to termination of the cultures. Impaired IL-2 production was noted throughout the time course of the RA AMLRs. Neither an inhibitor of IL-1 nor IL-2 was detected in AMLR culture supernatants. Moreover, IL-1 in pharmacologic concentrations up to 50 units/ml failed to reconstitute impaired AMLR reactivity. In 2 patients whose AMLRs failed to reconstitute fully with 100 units/ml IL-2, addition of 10 units/ml IL-1 in combination with IL-2 fully reconstituted the AMLR defect. The results may suggest that defective IL-2 generation alone cannot fully account for impaired AMLR reactivity in RA patients.

Serial levels of soluble interleukin 2 receptor in the peripheral blood of patients with rheumatoid arthritis: correlations with disease activity.

We serially assayed soluble interleukin 2 receptor (sIL-2R) levels in the peripheral blood (PB) of 22 patients with rheumatoid arthritis (RA) followed for a period of 12 months, and correlated these levels with disease activity. We examined the relationship between the direction in which each of the disease measures changed between assessments and the direction of change in sequential sIL-2R levels. In 22 of the 25 (88%) instances where there was a 30% change in the active joint count between sequential assessments, the direction of change of the PB sIL-2R level was found to be in parallel (chi 2 = 11.7, p less than 0.008). Our results suggest that serial sIL-2R levels are a useful means of confirming clinically significant changes in disease activity in patients with RA, irrespective of therapy.

Impaired autologous mixed lymphocyte reaction (AMLR) reactivity of peripheral blood T cell subsets in rheumatoid arthritis.

We examined AMLR reactivity of unseparated T cells and CD4+ and CD8+ T cell subsets in peripheral blood from 11 rheumatoid arthritis (RA) patients and 10 healthy controls. T cell subsets were isolated by negative selection using complement mediated cytotoxicity. AMLR reactivity of six patients (designated RA-L was reduced below the range of the controls' responses. Five patients (designated RA-N) exhibited normal AMLR reactivity. We observed impaired AMLR reactivity of CD4+ T cells from RA-L relative to RA-N and healthy controls (P < 0.05). CD4+ T cell reactivity of RA-L was reconstituted to normal with pharmacological doses of recombinant interleukin-2 (IL-2) (100 U/ml). In contrast, CD8+ T cells from RA-L in the presence of 100 U/ml IL-2 exhibited markedly impaired AMLR reactivity relative to RA-N and healthy controls (P < 0.05). Dose-response studies revealed partial reconstitution of CD4 T cells with physiological concentrations of IL-2 (10 U/ml). To examine the possibility that in vivo pre-activation of T cells in RA accounted for the findings, T cells or subsets were cultured alone for 7 days in the presence of 100 U/ml IL-2. A trend toward enhanced reactivity of CD4+ and CD8+ T cells in L-RA relative to N-RA and healthy controls was observed, but the differences were not statistically significant. There was no correlation between reactivity of T cells alone in the presence of IL-2 and AMLR reactivity. The results suggest the possibility that abnormal AMLR reactivity of CD4+ and CD8+ T cell subsets in RA may arise as a consequence of different pathophysiological mechanisms.

Impaired late suppression of Epstein-Barr virus (EBV)-induced immunoglobulin synthesis: a common feature of autoimmune disease.

We examined regulation of Epstein-Barr virus-induced plaque-forming cell generation in peripheral blood mononuclear cells from several autoimmune and seronegative diseases and correlated these results with Epstein-Barr virus-induced proliferation. We confirmed the defective regulation of Epstein-Barr virus-induced plaque-forming cells in peripheral blood mononuclear cells of patients with rheumatoid arthritis and scleroderma. Peripheral blood mononuclear cells from patients with seronegative arthropathies and chronic infective inflammation (cystic fibrosis) had normal regulation of Epstein-Barr virus-induced plaque-forming cells. Peripheral blood mononuclear cells from rheumatoid arthritis had excessive plaque-forming cell generation in the face of a normally regulated decrease in Epstein-Barr virus-induced proliferation. In contrast, peripheral blood mononuclear cells from scleroderma had defective suppression of both Epstein-Barr virus-induced proliferation and plaque-forming cell generation. Thus, impaired regulation of Epstein-Barr virus-induced plaque-forming cell generation is a common feature of autoimmune disease and demonstrates some specificity for these disorders.

Serum phospholipase A2 correlates with disease activity in rheumatoid arthritis.

We previously demonstrated a marked elevation of the proinflammatory enzyme phospholipase A2 (PLA2) in all synovial fluids and some sera of patients with rheumatoid arthritis (RA). Since PLA2 was found to induce inflammatory changes in the skin and joints of experimental animals, we tested whether the serum level of PLA2 correlates with the clinical activity of RA. In the group of 51 patients with classical or definite RA, 13 (25%) had high serum levels of PLA2 (over 2 standard deviations above the normal mean). Comparison of clinical disease activity in patients with high levels of PLA2 with those with normal PLA2 levels showed that patients with high PLA2 levels had a significantly higher joint count, more swollen joints, much higher Landsbury index, lower functional class, lower hemoglobin, lymphopenia and higher erythrocyte sedimentation rate (ESR). To more accurately assess the relationship between the PLA2 level and disease activity in RA, we formulated 2 indices. Clinical index consisted of the Landsbury index, number of swollen joints and duration of morning stiffness. Laboratory index consisted of hemoglobin, absolute number of peripheral blood lymphocytes, platelet count and ESR. Our results showed that both indices correlated strongly with PLA2 activity (p less than 0.0001). The results support the hypothesis that PLA2 plays a pathogenetic role in RA and suggest that serum PLA2 levels may serve as an additional measure of disease activity.

Elevated soluble interleukin-2 receptor levels in the sera and synovial fluids of patients with rheumatoid arthritis.

In a previous study, we used an enzyme-linked immunosorbent assay to measure soluble human interleukin-2 receptors (IL-2R), and found that when activated lymphocytes produce cell-associated IL-2R, they also release a soluble form of IL-2R into culture supernatants in vitro. Soluble IL-2R have also been detected circulating in vivo at low levels in the serum of healthy individuals, and at abnormal levels in a variety of diseases, particularly those where immune dysfunction is thought to play an important role. We therefore evaluated serum IL-2R levels in 77 patients with rheumatoid arthritis (RA), and compared them with levels in 46 age-matched healthy controls. Nineteen additional RA patients with concurrently obtained sera and synovial fluid (SF) samples were compared with 14 patients with osteoarthritis of the knee or hip. The serum IL-2R levels were significantly elevated in RA patients, compared with the control groups (P less than 0.0001). Serum IL-2R levels in the RA patients did not correlate with disease activity as determined by a variety of clinical and laboratory parameters. RA SF IL-2R levels were significantly higher than corresponding RA serum IL-2R levels (P = 0.0001). No such difference was noted in the osteoarthritis group, where serum and SF IL-2R levels were comparable with serum levels in healthy controls. These findings support the hypothesis that in vivo lymphocyte activation plays an important role in RA; moreover, soluble IL-2R measurement in serum and SF may be a very useful way to identify patients at risk for, or manifesting, a chronic immune-mediated inflammatory arthropathy.