Donor-specific immune regulation by CD8 lymphocytes expanded from rejecting human cardiac allografts.

To assess whether regulatory T cells are present in rejecting human cardiac allografts, we performed functional analyses of graft lymphocytes (GLs) expanded from endomyocardial biopsies (EMB; n = 5) with histological signs of acute cellular rejection. The GL cultures were tested for their proliferative capacity and regulatory activity on allogeneic-stimulated peripheral blood mononuclear cells (PBMC) of the patient (ratio PBMC:GLs = 5:1). Three of these GL cultures were hyporesponsive to donor antigens and suppressed the antidonor proliferative T-cell response of PBMC, but not the anti-third-party response. Interestingly, it was the CD8(+) GL subset of these cultures that inhibited the antidonor response (65-91% inhibition of the proportion of proliferating cells); the CD4(+) GLs of the expanded GL cultures were not suppressive. In conclusion, CD8(+) GLs expanded from rejecting human cardiac allografts can exhibit donor-specific immune regulatory activities in vitro. We suggest that during acute cellular rejection, GLs may not only consist of graft-destructing effector T cells, but also of cells of the CD8(+) type with the potential to specifically inhibit antidonor immune reactivity.

FoxP3+ T cells can be expanded from rejecting cardiac allografts.

A specific subset of T cells, the FoxP3+ regulatory T cells, control effector T-cell responses to self and foreign antigens. In transplant patients, we and others have shown that high intragraft FOXP3 mRNA levels are associated with acute rejection, suggesting that immune regulation is dependent on immune activation. To study whether transplanted grafts harbor FoxP3+ T cells and to functionally analyze them, graft infiltrating lymphocytes (GILs) must be propagated from the transplanted tissue. In the present study, we analyzed whether FoxP3+ T cells can be grown from endomyocardial biopsies (EMBs; n = 5) from patients after heart transplantation during acute cellular rejection. After 18 to 21 days of culture, 0.5 to 1.0 x 10(6) GILs were cultured from the EMBs. Of these GILs, 10.6% (median; range, 1.6%-17.1%) stained positive for FoxP3. Thus Foxp3+ T cells can be grown from EMBs, providing the tools to functionally characterize these cells in depth in forthcoming studies.

Poor expression of T cell-derived cytokines and activation and proliferation markers in early rheumatoid synovial tissue.

We compared the state of activation and proliferation of T cells in synovial tissue (ST) from rheumatoid arthritis (RA) patients in early and late stages of the disease to find out whether T-cell-driven immune responses vary during the course of the disease. ST was obtained from 12 patients with early RA (< 1 year) and 12 patients with longstanding RA (> 5 years). T cells and interferon-gamma (IFN-gamma)-positive cells were detected in ST using immunohistologic methods. To determine the percentage of T cells expressing the interleukin-2 receptor, IFN-gamma, or the proliferation associated antigen Ki-67, immunofluorescence double-staining techniques were used. The scores for the number of T cells and for the expression of IFN-gamma as well as the percentages of T cells expressing CD25, IFN-gamma, or Ki-67 in rheumatoid synovium were not dependent on disease duration. These results do not support the assumption that the responsiveness of T cells in ST of RA patients differs between early and late stages of the disease. The data indicate that at present no arguments exist that the effect of T-cell-directed interventions on synovial inflammation might vary in different stages of the disease.

Distribution of T cells and signs of T-cell activation in the rheumatoid joint: implications for semiquantitative comparative histology.

A prerequisite for comparative histology of synovial tissue by means of biopsies is insight into the distribution of a marker under study. This investigation focuses on the variation in the presence of T cells and signs of T-cell activation within the rheumatoid joint. For this purpose, multiple slides from several pieces of synovial tissue from different parts of a joint were stained and scored for the expression of CD3, CD25, HLA-DR, Ki67 and interferon-gamma. The variation in scores for the presence of T cells and markers of activation was more pronounced in slides prepared from different pieces of tissue than in slides from one piece of tissue. Based on multiple analysis of variance, methods are suggested to establish a reliable overall score for the expression of a certain marker within a joint. Following validation, such methods may prove to be useful by allowing semiquantitative histology of synovial tissue for studies on arthritis.

T cells cloned from human rheumatoid synovial membrane functionally represent the Th1 subset.

The presence of activated T cells in the synovial membrane of patients with rheumatoid arthritis (RA) suggests a role for these cells in the pathogenesis of the disease. Recent evidence indicates that human T cells may fall into functional categories dependent on their cytokine profile and cytotoxic capacity. The human Th1 subset is cytolytic and produces high levels of IFN-gamma whereas the Th2 type of T cell produces IL-4. In order to investigate whether Th1 or Th2 type cells are present in the inflammatory synovial membrane in RA, a panel of synovial membrane derived T-cell clones (n = 19) was generated and studied functionally. Anti-CD3-induced cytotoxicity assays were performed to demonstrate the cytotoxic potential of clones. Except for two, all clones were cytolytic in this test. Clone cells were activated to initiate cytokine production and assessment of the cytokine levels showed that all clones produced large amounts of IFN-gamma (18 out of 19 clones: over 50,000 pg/ml) whereas IL-4 was absent or present in minimal amounts (17 out of 19 clones: less than 1000 pg/ml). The production of IL-1, IL-2 and IL-6 was variable. The functional characteristics of the clones studied indicate that they may resemble the Th1 subtype of T cells. Our data suggest a relation between Th1-type functions the chronic inflammation characteristic of RA.

Interleukin-6 activity in paired samples of synovial fluid. Correlation of synovial fluid interleukin-6 levels with clinical and laboratory parameters of inflammation.

Paired synovial fluid (SF) samples obtained from the knees of 12 arthritis patients were studied to establish a relation between parameters of local inflammatory activity and SF interleukin-6 (IL-6) levels. Local disease activity was scored using joint temperature, swelling and pain as clinical parameters of inflammation. SF samples were assayed for laboratory parameters of inflammation such as leucocyte content, the percentage polymorphonuclear cells, the pH, and for immunoglobulin levels (IgG, IgM). SF IL-6 concentrations were determined using the B9-bioassay. Within individual patients the local activity of inflammation as measured using clinical parameters was found to be related to the local SF IL-6 level. When considering the total group of patients, a correlation (P less than 0.001) was found between the clinical parameters of local inflammation and the SF IL-6 levels. Furthermore, IL-6 levels were found to correlate with leucocyte counts (P less than 0.02), the percentage of polymorphonuclear cells (P less than 0.10), the pH value (P less than 0.01), but not with SF IgM and IgG concentrations.