L. M. B. Vaessen

The detection of cytotoxic T cells with high-affinity receptors for donor antigens in the transplanted heart as a prognostic factor for graft rejection

Alloreactive T lymphocytes are the initiators and effectors of acute rejection of organ transplants, and T cells with high-affinity receptors for antigen might be especially implicated in this process. It has been shown that the cytotoxic capacity of CTL with low affinity for alloantigens can be inhibited with CD8 mAb, while high-affinity CTL are not affected. To investigate whether the presence of such high-affinity cells in human heart transplants may be predictive for acute rejection, we analyzed their frequency in cultures derived from endomyocardial biopsies in 19 patients, 9 of whom had never experienced acute rejection and 10 who had had one or more rejection episodes. IN the rejectors, already before histological signs of rejection (myocyte damage) had developed, significantly higher donor-reactive CTL frequencies were found compared with the nonrejectors (medians of 10,586 vs 1,169 reactive cells per 10(6) tested cells, P = 0.002). After CD8 inhibition, the difference between rejectors and nonrejectors was even more pronounced (P < 0.001). In patients with rejection, the number of CD8-resistant, high-affinity CTL was higher than 1000 per million cells in all cases, while in patients who had never experienced rejection this number was less than 1000. As these CTL characteristics are already present before the first histological signs of rejection have developed, this might be used as a prognostic factors.

A double-blind, placebo-controlled study of monoclonal anti-interleukin-2 receptor antibody (BT563) administration to prevent acute rejection after kidney transplantation.

In a double-blind, randomized, placebo-controlled trial, BT563, a murine IgGt anti-IL-2R antibody, was given as a rejection prophylaxis after kidney transplantation. Drug-related side effects were not observed. During the 10-day course of BT563, no rejections (0/27) were found, whereas a rejection episode occurred in 7 patients (7/29) (P=0.01) during placebo treatment. Within the first 4 postoperative weeks, freedom from rejection in the BT563 group and in the placebo group was 96% vs. 76% (P=0.05). Due to rejection in the placebo group, 2 grafts were lost. At 3 months, an overall rejection incidence in the BT563 and placebo group was found of 3/27 (11%) vs. 8/29 (28%) patients (P=0.18). Infectious complications were distributed equally between the 2 groups. CMV disease, found in 3 placebo-treated patients, occurred after rejection treatment (2/3). Within the BT563 group, 1 patient lost his graft due to renal artery thrombosis, 2 grafts were lost as a result of technical failure, and 2 patients had a squamous cell carcinoma that could be treated curatively. We conclude that the use of the anti-IL-2R mAb BT563 effectively prevents rejection after kidney transplantation without increasing infectious complications.

Alloreactive lymphoid infiltrates in human heart transplants: Loss of class II-directed cytotoxicity more than 3 months after transplantation

From 535 endomyocardial biopsies (87 heart transplant recipients) 283 cell cultures could be generated. All cultures tested contained T lymphocytes and in most cases CD4 was the predominant phenotype at any time posttransplant. A significantly higher proportion of CD8-dominated cultures was found among cultures from biopsies without myocytolysis. In the first 3 months post transplant 57% of cultures showed cytotoxicity against both class I and class II mismatched donor major histocompatibility complex (MHC) antigens, changing to an incidence of 33% at greater than 90 days. This proved to be due to a significant decrease in the number of cultures with human leukocyte antigen class II-directed cytotoxicity. This study shows that early after transplantation a heart transplant is infiltrated with activated donor-specific cytotoxic T cells which recognize a broad spectrum of mismatched donor MHC antigens, and that in time this spectrum becomes more restricted.

The effects of renal transplantation on circulating dendritic cells

The effects of immunosuppressive agents on T cell function have been well characterized but virtually nothing is known about the effects of renal transplantation on human dendritic cells (DCs). With the use of flow cytometry, we studied the kinetics of myeloid and plasmacytoid DCs in peripheral blood of 24 kidney allograft recipients before and after transplantation, and in 23 donors before and after kidney donation. All patients were treated with tacrolimus, mycophenolate mofetil and prednisone. Surgery resulted in a strong decline in the number of myeloid and plasmacytoid DCs, both in kidney donors and in their recipients. However, in donors this effect was transient, as the numbers of both DC subsets had normalized completely by the third postoperative month. In contrast, the recovery of myeloid DC counts in kidney transplant recipients was only incomplete at the end of the 3‐month follow‐up, despite tapering of immunosuppression. The seven patients who required additional immunosuppressive treatment because of acute rejection experienced an even more marked decrease in DC counts in the early postoperative period compared with patients who remained rejection‐free. Surgical procedures markedly affect the numbers of circulating myeloid and plasmacytoid DCs. Immunosuppressive drugs have important additional in vivo effects on this cell type and impair the reconstitution of the myeloid DC subset in peripheral blood after renal transplantation.

Acute rejection in heart transplant patients is associated with the presence of committed donor-specific cytotoxic lymphocytes in the graft but not in the blood.

In vivo‐activated, commuted donor‐specific cytotoxic lymphocytes (cCTL) can be propagated and expanded from endomyocardial biopsies (EMB) in IL‐2‐enriched medium especially during an acute rejection episode. We report here our efforts to detect these cCTL by the same technique in peripheral blood at the moment of rejection and when no rejection was diagnosed. During or just before rejection, significantly less frequent (P < 0·01) donor reactive cCTL were found in PBL samples (two out of 20) than in the simultaneously taken EMB samples (13 out of 19). Donor B‐LCL and/or third‐party B‐LCL were lysed by 15 PBL samples. Inhibition studies revealed that this lysis was due to LAK‐like cytotoxicity. The results show that peripheral blood does not reflect intra‐graft events, which is probably the reason for the irrcproduciblc results of diagnosis of rejection by monitoring immunological parameters in the peripheral blood.

Preferential depletion of blood myeloid dendritic cells during acute cardiac allograft rejection under controlled immunosuppression.

Allo‐Ag presentation to Ag‐specific T‐lymphocytes by donor or recipient dendritic cells (DCs) induces acute rejection (AR) after solid organ transplantation. It is postulated that myeloid (mDC) and plasmacytoid (pDC) subsets circulate differentially between bone marrow, heart and lymphoid tissues after cardiac transplantation (HTx). We investigated peripheral blood DC subset distribution, maturation and lymphoid homing properties in relation to endomyocardial biopsy (EMB) rejection grade after clinical HTx. Twenty‐one HTx recipients under standard immunosuppression were studied in a 9‐month follow‐up. mDC and pDC numbers were analyzed by flow cytometry in fresh venous whole blood samples collected during the EMB procedures and before histological diagnosis of AR. Subsets were further characterized for maturation marker CD83 and lymphoid homing chemokine receptor CCR7. Although numbers of both DC subsets remained low for the whole post‐HTx period, we observed a negative association of mDCs with rejection grade. Repeated measurements analysis revealed that only mDCs decreased during AR episodes. Rejectors had lower mDC numbers after a 3‐month follow‐up compared to nonrejectors. Furthermore, patients during AR exhibited low proportions of mDCs positive for CD83 or CCR7. These findings suggest peripheral blood mDC depletion in association with selective lymphoid homing of this subset during AR after clinical HTx.

The direct and indirect allogeneic presentation pathway during acute rejection after human cardiac transplantation

Alloreactive T cells may be activated via a direct or an indirect antigen presentation pathway. We questioned whether the frequency of interferon (IFN)‐γ producing cells determined by enzyme‐linked immunospot (ELISPOT) assay is an effective tool to monitor the direct and/or indirect presentation pathway. Secondly, we wondered whether early and late acute rejection (AR) are associated with both pathways. Before (n = 15), during (n = 18) and after (n = 16) a period of AR, peripheral blood mononuclear cell (PBMC) samples were tested from 13 heart transplant recipients. The direct presentation pathway was always present. The number of IFN‐γ producing cells reactive to this pathway increased significantly (P = 0·04) during AR and the number decreased (P = 0·005) after AR therapy. In contrast, the indirect allogeneic presentation pathway was present in only eight of 18 AR samples. When the indirect presentation pathway was detectable, it increased significantly during AR. Five of eight of these AR occurred more than 6 months after transplantation. The ELISPOT assay, enumerating alloreactive IFN‐γ producing cells, is a valuable tool to determine the reactivity via both the direct and the indirect presentation pathway. The direct presentation pathway always plays a role in AR, while the indirect pathway contributes especially to late AR.

Increased numbers of circulating donor-specific T helper lymphocytes after human heart valve transplantation

Implantation of cryopreserved human donor heart valves for either congenital or acquired cardiac disease has been performed since the last three decades. Although the clinical outcome is good, long‐term valve degeneration resulting in dysfunction has been observed. A specific immunological response of the recipient against the allograft has been proposed as one of the factors involved in this process. Helper T lymphocytes play an important intermediate role in cellular and humoral immune response. Increasing numbers of circulating donor‐specific helper T lymphocytes precursors (HTLp) correlate with graft rejection after organ transplantation. To investigate whether cryopreserved human donor heart valves are able to induce a donor‐specific T helper response, we monitored the HTLp frequencies (HTLpf) in peripheral blood samples of 13 patients after valve allograft transplantation by use of a limiting dilution assay followed by an interleukin‐2 bioassay. Prior to transplantation, HTLpf specific for donor and third‐party antigens showed individual baseline levels. After allografting, the antidonor frequencies significantly increased in 11 of the 13 patients (P = 0·02). This was not found for stimulation with third‐party spleen cells (P = 0·68), which indicates a donor‐specific response. Maximal donor‐specific HTLpf were already found at 1–2 months after operation. Valve allograft transplantation induces an increase in the numbers of donor‐specific HTLp in peripheral blood of the patients. Analogous to organ transplantation, these HTLp may play a crucial role in events that lead to valve damage. Therefore, monitoring of HTLp in peripheral blood samples might be informative for donor valve degeneration (rejection) and subsequently valve allograft failure.

Cytokine gene expression profiles in human endomyocardial biopsy (EMB) derived lymphocyte cultures and in EMB tissue

Abstract The reverse transcriptase polymerase chain reaction (RT‐PCR) technique was used to analyse cytokine gene expression in relation to acute cardiac rejection. Expression of interleukins, IL‐2, IL‐4, IL‐6 and IL‐10 mRNA was studied in sequential endomyocardial biopsies (EMB) and in graft‐infiltrating lymphocyte (GIL) cultures propagated from EMB taken after heart transplantation. The cytokine gene expression of GIL propagated from EMB taken during an episode of rejection and of immunological quiescence was comparable. In contrast, posttransplant EMB showed selective IL‐2 gene expression when rejection was diagnosed. IL‐4 mRNA was absent in pretransplant EMB but present in posttransplant EMB taken during periods of rejection and of immunological quiescence. Both IL‐6 and IL‐10 transcripts were found in pre‐ and posttransplant EMB. These findings confirmed that IL‐2 is specifically involved in cardiac rejection, while IL‐4 may play a role in immune responses leading to graft rejection or graft tolerance.

Donor heart endothelial cells as targets for graft infiltrating lymphocytes after clinical cardiac transplantation

The in vitro cytotoxic reactivity of allograft infiltrating cells cultured from endomyocardial biopsies was tested against endothelial cells (EC) isolated from the own donor heart. EC derived from pieces of atrium were found to be proper targets for graft infiltrating cytotoxic T cells from four patients. The specificity of this cytotoxicity was further analysed by cold target inhibition studies and blocking with anti-CD3, anti-CD4 or anti-CD8 monoclonal antibodies. The experiments revealed that, besides a clearly HLA directed recognition, a more heterogeneous, multispecific response can be found, which might be partially explained by the activity of EC specific T cell clones. We conclude that this system provides a valuable approach to investigate the reactivity of graft infiltrating cells against EC in relation to the clinical course of the transplantation.