B. Mochtar

Cytokine mRNA expression in endomyocardial biopsies during acute rejection from human heart transplants.

The immune response to an allograft is regulated by cytokines produced by cells infiltrating the allograft. However, the immunopathogenesis of allograft rejection is not completely understood. To investigate the role of cytokines after clinical heart transplantation, we analysed the expression of cytokine genes in sequentially taken endomyocardial biopsies (EMB) by using the reverse transcriptase‐polymerase chain reaction (RT‐PCR). We analysed 44 EMB from II recipients: 21 EMB before or during rejection, and 23 EMB without histological evidence of acute rejection. A strong correlation was found between IL‐2 gene expression and histologically proved rejection (16/21 versus 1/23 without rejection, P < 0.001; x2 test). Also, expression of IL‐4 and IL‐6 genes was more often found in EMB during rejection than in EMB without signs of rejection (IL‐4, 62% versus 35%; and IL‐6, 81% versus 39%. respectively). No relation with rejection or with immunological quiescence was observed for the presence of IL‐10 gene transcripts. IL‐10, but also IL‐6 mRNA were detectable in donor heart tissue before transplantation (9/10), In contrast, IL‐2 and IL‐4 gene transcripts were absent in these samples. These differences could not be explained by the presence or absence of T ceils, since the gene for the constant region of the β‐chain (CG) of the T cell receptor (TCR) not only was expressed in post‐transplant EMB but also in pre‐transplant donor heart tissue. Our results provide strong evidence that the immunoregulatory cytokines IL‐2, IL‐4 and IL‐6 are important local regulators in the graft during acute rejection. The role of IL‐10 in the immunologic response lo the transplanted organ needs further investigation.

Blockade of the interleukin (IL)-2/IL-2 receptor pathway with a monoclonal anti-IL-2 receptor antibody (BT563) does not prevent the development of acute heart allograft rejection in humans.

BACKGROUND Anti-interleukin (IL)-2 receptor (IL-2R) antibodies have been used as rejection prophylaxis after organ transplantation. Despite this induction treatment, acute rejections may occur. We wondered whether these rejections developed via the IL-2/IL-2R pathway. METHODS In a prospective trial using BT563, a murine IgG1 anti-IL-2R antibody, for rejection prophylaxis after heart transplantation, 20 patients were treated in combination with cyclosporine from the day of transplantation (group A). As a control group, 31 patients were also treated with BT563, but in these patients, cyclosporine treatment was initiated on day 3 (group B). RESULTS Three patients from group A and two patients from group B died in the first postoperative month (of causes not related to acute rejection) and were left out of the analysis of rejection incidence. Freedom from acute rejection at 1 week after transplantation in group A (14/17; 82%) was lower than in group B (16/29; 55%), although the difference did not reach statistical significance. There was no difference in either the number of acute rejection episodes at 12 weeks or the required rejection treatments between groups A and B. Infectious complications were evenly distributed in both groups. Immunohistochemistry showed that during acute rejection, in the presence of circulating BT563, IL-2R-bearing cells were present in only one of five rejection biopsies (20%), whereas these cells were often present (6/8, or 75%) in rejections occurring in the absence of BT563. The presence of BT563 was associated with a similar difference in the mRNA expression of IL-2 (2/5 vs. 6/8). CONCLUSIONS Apparently, despite adequate blockade of the IL-2/IL-2R pathway, patients may develop acute rejection, reflecting the redundancy of the cytokine network. The ever-present IL-15 may well be a candidate for overtaking the role of IL-2.

Differential avidity and cyclosporine sensitivity of committed donor-specific graft-infiltrating cytotoxic T cells and their precursors : relevance for clinical cardiac graft rejection

We have used limiting dilution analysis to study the qualitative and quantitative differences between graft-infiltrating cytotoxic T cell populations propagated from endomyocardial biopsies of heart transplant patients who experienced one or more acute rejection episodes and patients who never showed signs of rejection. Limiting dilution cultures were stimulated with autologous or donor cells both in the absence or in presence of cyclosporine and of CD8 in the cytotoxic phase. Almost all antigen-primed, committed cytotoxic T cells (cCTL) present in the graft of patients with rejections were CsA resistant. In contrast, in most patients of the nonrejector group, a substantial part of the cCTL could be inhibited by CsA. The CTL precursors (pCTL) in both groups were predominantly CsA sensitive. Addition of CD8 mAb during the cytotoxicity phase of the limiting dilution analysis was used to differentiate between CTL populations with high avidity for donor antigens and populations with low avidity. The predominant subpopulation in the graft of rejectors was a CsA-resistant cCTL with high avidity, while in the graft of most nonrejectors, cCTL with low avidity dominated. In most rejectors, CD8 mAb had only a minor influence on the pCTL frequency estimates, and thus on T cells with high avidity. CsA-sensitive pCTL with high avidity might represent an intermediate stage between the naive pCTL and mature, functional, CsA-insensitive cCTL with high avidity for donor antigens.

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.

Passive immunization against cytomegalovirus in allograft recipients. The Rotterdam heart transplant program experience

SummaryWe analyzed the results of passive immunization against CMV in 146 heart transplant recipients. The 65 seronegative recipients were prophylactically treated with anti-CMV immunoglobulins during and after the operation. Twenty-nine of these 65 patients received a seropositive donor heart. CMV infection occurred in 21/65 seronegative and in 40/81 seropositive recipients (difference not significant). The incidence of CMV infection in seronegative recipients of a CMV-matched donor heart (3/34) was significantly lower than in seronegative recipients of a positive donor heart and lower than in seropositive recipients, but no significant difference in infection rate was found between the two latter groups (18/29 vs. 40/81). Although primary infection more frequently resulted in CMV disease than secondary infection (11/21 vs. 10/40) no difference in incidence of disease was noted between seronegative and seropositive patients (11/65 vs. 10/81), nor was there a difference in the severity of symptoms following primary or secondary infection. There was a higher incidence of CMV disease in all patients who received a heart from a seropositive donor versus a seronegative donor. However, after transplantation of a heart from a seropositive donor the incidence (27%) of CMV disease observed in our passively immunized seronegative patients was the same as in the patients with naturally acquired seropositivity. There was no difference in the prevalence of coronary artery disease between patients with and without CMV infection or disease. We conclude that using the current passive immunization scheme the occurrence of CMV infection and disease is largely dependent on the serostatus of the donor.ZusammenfassungAn 146 Herztransplantationspatienten untersuchten wir den Effekt passiver Immunisierung gegen CMV. Die 65 seronegativen Herzempfänger wurden peri- und postoperativ prophylaktisch mit Anti-CMV-Immunglobulin behandelt. 29 dieser 65 Patienten erhielten ein seropositives Spenderherz. Bei 21 von 65 seronegativen und bei 40 von 81 seropositiven Empfängern kam es zur Infektion (Unterschied nicht signifikant). Die Inzidenz von CMV-Infektionen war bei seronegativen Empfängern eines entsprechenden Spenderherzens (3/34) signifikant niedriger als bei seronegativen Empfängern eines positiven Spenderherzens und niedriger als bei seropositiven Empfängern, jedoch ergab sich kein signifikanter Unterschied zwischen den beiden letztgenannten Gruppen (18/29 versus 40/81). Obwohl primäre Infektionen häufiger zur CMV-Erkrankung führten als sekundäre Infektionen (11/21 versus 10/40), ergab sich kein Unterschied in der Häufigkeit seronegativer und seropositiver Patienten (11/65 versus 10/81), oder im Schweregrad der Symptomatik nach primärer beziehungsweise sekundärer Infektion. Bei allen Patienten, die das Herz eines seropositiven Spenders erhielten, ergab sich eine höhere Inzidenz an CMV-Erkrankungen, als bei denen, die einen seronegativen Spender hatten. Nach der Transplantation eines Herzens von einem seropositiven Spender war jedoch bei unseren passiv immunisierten seronegativen Patienten die gleiche Inzidenz (27%) von CMV-Erkrankungen zu beobachten wie bei den Patienten mit natürlich erworbener Seropositivität. In der Prävalenz der koronaren Herzkrankheit ergab sich kein Unterschied zwischen Patienten mit und ohne CMV-Infektion oder Erkrankung. Demzufolge hängt das Auftreten einer CMV-Infektion und Erkrankung bei Anwendung des gegenwärtigen Schemas zur passiven Immunisierung weitgehend vom serologischen Status des Spenders ab.

Kinetics of IL-2 and IL-4 mRNA and protein production by graft-infiltrating lymphocytes responsible for rejection after clinical heart transplantation

During cardiac rejection we studied the kinetics of IL-2 and IL-4 mRNA and subsequent protein production by in vivo primed graft-infiltrating lymphocytes (GIL), using semiquantitative RT-PCR and ELISA. Following in vitro stimulation with either donor or third-party antigens, mRNA expression of IL-2 and IL-4 were already detectable 1-2 h after stimulation, while their protein production could be measured from 4 h onwards at least until 48 h. At both the mRNA and protein level, we measured a donor-specific signal for IL-2 and for IL-4 production (p = 0.02), while the relative donor-specific IL-2 mRNA level was significantly higher than the relative IL-4 mRNA level (p = 0.002). These observations suggest that after in vitro challenge with donor antigens, GIL obtained from rejecting cardiac allografts predominantly produce IL-2 mRNA and protein.

Effect of adopting a new histological grading system of acute rejection after heart transplantation

Background Treatment policy of acute rejection after heart transplantation has been changed after adopting the ISHLT endomyocardial biopsy grading system in 1991. Objective To determine the effect of this policy change on clinical outcome after transplantation. Methods The outcome of 147 patients who had a transplant before (early group, median follow up 96 months) and 114 patients who had a transplant after (late group, median follow up 41 months) the introduction of the ISHLT biopsy grading system was studied retrospectively. Initially “moderate rejection” according to Billingham’s conventional criteria was treated. From January 1991 grade 3A and higher was considered to require intensification of immunosuppression. Results There were some differences between the two groups: recipients (50 v 44 years) as well as donors (28 v 24 years) were older in the “late group” and more patients of this group received early anti-T cell prophylaxis (92% v 56%). Despite more extensive use of early prophylaxis more rejection episodes were diagnosed (2.4 v1.4) and considerably more courses of rejection treatment were instituted in the late compared with the early group (3.2v 1.5). There were no deaths because of rejection in the late group, however, more infections occurred within the first year (mean 1.8 v 1.4) and more non-skin malignancies within the first 41 months were diagnosed (8 of 57 v 6 of 147, 95% CIs of difference includes 0). The incidence of graft vascular disease in the late group has been comparable to the early group until now. Conclusion The interpretation of the ISHLT grading system resulted in lowering of the threshold for the diagnosis of rejection thereby increasing the number of rejections and subsequently the immunosuppressive load and its complications.

In vitro and in vivo effects of BT563, an anti‐interleukin‐2 receptor monoclonal antibody

Abstract BT563, a murine anti‐IL‐2R MoAb, was found to be more potent than anti‐Tac in inhibiting proliferation in the mixed lymphocyte reaction. Results obtained with 33 B3.1 in these experiments were similar to those with BT563. The anti‐IL‐2R MoAb 2A3 was shown to be a suitable agent for monitoring the effect of BT563 on peripheral blood. IL‐2R‐positive cells were not detected in peripheral blood samples from 1 h after the first dose until 8 days after the last dose. Plasma trough levels were measured in patients receiving 5 or 10 mg daily. The administration of BT563 to allograft recipients did not lead to clinically significant side effects.

The avidity of allospecific cytotoxic T lymphocytes (CTL) determines their cytokine production profile

Donor‐specific CTL present within the cardiac allograft during a rejection episode are distinct from those that populate the cardiac allograft in the absence of rejection. Whereas the former generally have a high avidity for donor cells, the latter mainly have a low avidity for donor cells. This observation made us reason that high‐avidity CTL are implicated in transplant rejection, whereas low‐avidity CTL are not. In the present study, we analyse whether both CTL subsets were distinct with respect to their IL‐2, IL‐4, IL‐6 and interferon‐gamma (IFN‐γ) secretion pattern. CTL clones with either a high or a low avidity for donor antigens were stimulated with donor cells, third party cells, or immobilized anti‐CD3 MoAb and the amount of cytokine released was measured. High‐ and low‐avidity CTL clones were found to differ with respect to their IFN‐γ production profile. Stimulation with donor cells resulted in IFN‐γ secretion by high‐avidity CTL clones, but not by low‐avidity CTL clones. CD3 stimulation, in contrast, led to secretion of equivalent amounts of IFN‐γ by both CTL subsets. These observations indicate that low‐avidity CTL are fully capable of producing IFN‐γ, but, in contrast to high avidity CTL, fail to do so when they encounter donor cells. As IFN‐γ favours the occurrence of transplant rejection, this observation emphasizes the relevance of high‐avidity CTL in the rejection process. Additionally, the data show that the cytokine production profile of CTL depends on the nature of the stimulus.

Polyclonal versus monoclonal rejection prophylaxis after heart transplantation: a randomised study.

Recent studies comparing the effects of induction therapy with polyclonal antilymphocyte globulins (ALG) or with monoclonal T-cell-specific antibodies are not unanimous. Therefore, 55 heart recipients were allocated to either 7-day courses of polyclonal ALG (n = 28) or of monoclonal OKT3 (n = 27). Additionally, azathioprine and low dose steroids were given. There were no severe side effects after OKT3; the course of ALG, however, had to be discontinued in 20 patients because of extensive flares. No differences between the two groups were found in freedom from rejection or in the incidence of infection. The 1- and 2-year survival was 96% in both groups. Although monoclonal and polyclonal induction therapies are equally effective for rejection prophylaxis, OKT3 may be preferred because of a lack of important side effects. However, the fact that a shorter course of ALG is equally effective may be in favour of ALG.