P. de Kuiper

Down-regulated donor-specific T-cell reactivity during successful tapering of immunosuppression after kidney transplantation

Stable cadaveric renal transplant patients were routinely converted from cyclosporin A (CsA) to either azathioprine (AZA) or mycophenolate mofetil (MMF) 1 year after transplantation to reduce the side effects of long‐term immunosuppressive therapy. Thereafter, the AZA and MMF dose was gradually tapered to 50% at 2 years after transplantation. We questioned whether a reduction of immunosuppressive treatment results in a rise of donor‐specific T‐cell reactivity. Before transplantation (no immunosuppression), 1 year (high dose immunosuppression) and 2 years (low dose immunosuppression) after transplantation, the T‐cell reactivity of peripheral blood mononuclear cells (PBMC) against donor and third‐party spleen cells was tested in mixed lymphocyte cultures (MLC) and against tetanus toxoid (TET) to test the general immune response. We also measured the frequency of donor and third‐party reactive helper (HTLpf) and cytotoxic (CTLpf) T‐lymphocyte precursors in a limiting dilution assay. Donor‐specific responses, calculated by relative responses (RR = donor/third‐party reactivity), were determined. Comparing responses after transplantation during high dose immunosuppression with responses before transplantation (no immmunosuppression), the donor‐specific MLC‐RR (P = 0·04), HTLp‐RR (P = 0·04) and CTLp‐RR (P = 0·09) decreased, while the TET‐reactivity did not change. Comparing the responses during low dose with high dose immunosuppression, no donor‐ specific differences were found in the MLC‐RR, HTLp‐RR and CTLp‐RR, although TET‐reactivity increased considerably (P = 0·0005). We observed a reduction in donor‐specific T‐cell reactivity in stable patients after renal transplantation during in vivo high dose immunosuppression. Tapering of the immunosuppressive load had no rebound effect on the donor‐specific reactivity, while it allowed recovery of the response to nominal antigens.

Cytolytic activity in T cell clones derived from human synovial rheumatoid membrane: inhibition by synovial fluid

A panel of T cell clones was derived from the synovial membrane of a patient with rheumatoid arthritis (RA). We investigated whether T cell clones with cytolytic properties were present and whether T cell cytotoxicity was influenced by the presence of synovial fluid. These issues were studied using anti‐CD3 and lectin‐induced cytotoxicity assays. The majority of the T cell clones derived from the synovial membrane showed cytotoxic properties although non‐cytotoxic clones were also found. Three clones (N11, N6 and N15) showed strong cytotoxicity (more than 40% lysis at an effector‐to‐target cell ratio of 10:1) whereas three clones (N16, N4 and N14) were non‐cytotoxic (less than 20% lysis at an effector‐to‐target cell ratio of 10:1). The induction of cytotoxicity in the anti‐CD3‐driven system was shown to be dependent on the dose of anti‐CD3 present. When synovial fluid was added to these assays a strong inhibition of cytotoxicity was found. This inhibition of cytotoxicity was found with synovial fluid samples of RA patients, as well as with non‐RA synovial fluids. Both anti‐CD3 and lectin‐dependent cytotoxicity assays were strongly inhibited. In conclusion, T cell clones with cytotoxic activity can be isolated from rheumatoid synovial membrane. In the presence of synovial fluid these cytotoxic cells are inhibited to exert their cytotoxic function.

Donor-specific CTL frequencies in peripheral blood in relation to graft vascular disease after clinical heart transplantation

Abstract Cellular mechanisms may play a role in the development of graft vascular disease (GVD). We previously demonstrated that GVD correlated with an increase of donor‐specific T‐helper 1 cytokine production by graft‐infiltrating lymphocytes but not by peripheral blood mononuclear cells (PBMC). These T‐helper 1 cytokines aid the generation of cytotoxic T‐lymphocytes (CTL). In the present report, we investigated whether there is a relationship between the frequency of donor‐specific CTL precursors (pCTL) in PBMC and the development of GVD. We tested PBMC samples of five patients with GVD and five patients without GVD in the periods 3–6 months, 1 year, and 3 years after heart transplantation. At all time points, GVD was not related to the number of pCTL. In conclusion, donor‐specific cellular tests in peripheral blood could not be related to GVD. Apparently, donor‐specific reactions associated with the induction of GVD can only be monitored in the graft.

Anti-CD25 therapy affects the death signals of activated T-cells after clinical heart transplantation.

IL-2 was initially defined as the T-cell growth factor mediating allograft rejection. However, recent experimental studies have shown that IL-2 also regulates the mechanism by which the immune system eliminates activated T-cells. IL-2 is required for the apoptosis of antigen specific T-cells by upregulating FAS receptors. To define whether inhibition of the IL-2 pathway by immunosuppressive agents prevents activation induced cell death (AICD) of activated T-cells, we measured intragraft mRNA expression of IL-2, IL-15, CD25, FAS, FASL, and characterized the phenotype of graft infiltrating T-cells (CD3, CD25) by real time RT-PCR and immunohistochemistry, respectively. Analysis was performed in EMB (n536) from cardiac allograft recipients who were treated with anti-CD25 mAb induction therapy (Daclizumab, n58) or with placebo (n56), in combination with CsA, steroids, and mycophenolate mofetil. Treatment with anti-CD25 mAb significantly affected the IL-2 pathway. In the presence of circulating anti-CD25 mAb, the first 4 months post-transplant, extremely low intragraft IL-2, and CD25 mRNA transcription levels and low numbers of activated CD25 positive T-cells were found compared to the levels measured in EMB from placebo treated patients (5-10 fold, p, 0.0001). The inhibition of the IL-2 pathway during anti-CD25 mAb therapy was associated with reduced FASL mRNA expression and low numbers of CD3 positive T-cells in the graft. Significantly lower FASL mRNA expression levels and CD3 positive T-cells were measured in EMB of anti-CD25 mAb treated patients than in EMB from patients who received placebo (p50.01 and p50.02, respectively). No effect of anti-CD25 therapy was found on intragraft IL-15 and FAS mRNA expression levels. In conclusion, our data suggest that immune activation in the absence of IL-2 is associated with impaired apoptosis of activated T-cells in the graft. Manipulation of the immune system by immunosuppressive agents such as anti-CD25 mAb might prevent the induction of activation induced cell death (AICD) of alloreactive T-cells by hindering the IL-2 dependent FASL expression.