C. J. Knoop

Donor-specific cellular immune response against human cardiac valve allografts

We studied the presence of donor-specific T lymphocytes in explanted human cardiac valve allografts in vivo. From five of seven explants we propagated lymphocyte cultures in an interleukin-2 conditioned medium. Phenotyping revealed the presence of T-cell receptors in more than 95% of the lymphocytes obtained in each culture. Donor-specific cytotoxicity was demonstrated in three patients with known HLA status of the donor. Cytotoxicity was directed against only HLA class I in one patient, and against class I and/or class II in the others. These results indicate that donor-specific cellular reactivity can be induced by transplantation of human cardiac valve allografts.

Anti-CD25 therapy reveals the redundancy of the intragraft cytokine network after clinical heart transplantation

BACKGROUND Despite blockade of the interleukin-2/interleukin 2 receptor (IL-2/IL-2R) pathway by the murine anti-CD25 (i.e., IL-2R alpha chain) monoclonal antibody BT563, cardiac rejection can still occur. In these cases, growth factors other than IL-2 may contribute to allograft rejection. We studied the expression of IL-15, a macrophage-derived cytokine associated with T-cell activation, which interacts with the beta and gamma chains of the IL-2R during rejection episodes under anti-CD25 therapy. METHODS We measured intragraft IL-15 mRNA expression and the number of IL-15- and CD68-positive cells in posttransplantation endomyocardial biopsies (EMBs; n=45) and in nontransplanted, donor-heart specimens (n=11) by competitive template reverse transcription-polymerase chain reaction and immunohistochemistry, respectively. RESULTS IL-15 mRNA expression was present in the majority of posttransplantation EMB specimens (91%, 41/45) and in nontransplanted donor-heart specimens (91%, 10/11). Relative IL-15 mRNA levels were neither associated with transplantation nor with rejection status. After transplantation, the number of IL-15- and CD68-positive cells significantly increased (P<0.001), but IL-15-positive cell counts did not reflect the histological rejection grade. Anti-CD25 treatment, in contrast to its effects on the IL-2/IL-2R complex, had no influence on intragraft IL-15 mRNA and protein production. In rejection EMB specimens, during (n=5) and after (n=8) anti-CD25 therapy, no differences in relative IL-15 mRNA levels, or in IL-15- and CD68-positive cell counts, were measured. CONCLUSIONS After heart transplantation, high numbers of IL-15- and CD68-positive cells infiltrate the graft. This phenomenon is independent of the rejection status. IL-15 remains present during blockade of the IL-2/IL-2R pathway by anti-CD25 monoclonal antibodies, and it may participate in T cell-dependent donor-directed immune responses, thereby explaining the occurrence of rejection in the absence of IL-2.

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.

Stimulation of immune-competent cells in vitro by human cardiac valve-derived endothelial cells

Both fresh and cryopreserved human cardiac valve allografts are transplanted without matching donor and recipient for blood group or human leukocyte antigens (HLA) and without the usual immunosuppressive therapy that follows organ transplantation. Calcification occurs in almost all transplanted valves, and in children acute valve failure is frequently seen. We hypothesized that failure of the human valve allografts could have an immunologic basis. This hypothesis was tested in a functional way by performing lymphocyte stimulation assays using fresh and cryopreserved valve pieces and endothelial cells derived from valve leaflets as stimulator. Human peripheral blood lymphocytes, both matched and mismatched for HLA antigens, were used as responder cells. The results were expressed as the stimulation index. Fresh valve pieces induced a significantly higher stimulation index (median, 9; range, 4 to 117) compared with the cryopreserved pieces (median, 2; range, 0 to 9; p = 0.002 by Wilcoxon test). The stimulation index was significantly reduced when lymphocytes matched for HLA-DR with the valve pieces were used (median, 1; range, 0 to 5) as compared with the HLA-DR-mismatched combination (median, 4; range, 2 to 117; p = 0.006, Wilcoxon test). Valve leaflet-derived endothelial cells were able to induce a median stimulation index of 8 (range, 3 to 15) when incubated with lymphocytes mismatched for HLA-A, -B, and -DR. In conclusion, stimulation of immune-competent cells in vitro is induced by both fresh and cryopreserved human valve pieces and by endothelial cells derived from fresh valve leaflets. The immune response can be reduced by using cryopreserved valves or by matching valve donor and responder lymphocytes for HLA-DR.(ABSTRACT TRUNCATED AT 250 WORDS)

FUNCTIONAL RESPONSES OF T CELLS BLOCKED BY ANTI-CD25 ANTIBODY THERAPY DURING CARDIAC REJECTION

BACKGROUND Despite anti-CD25 (interleukin [IL]-2 receptor alpha chain) monoclonal antibody (mAb) therapy, rejection can still occur. T-cell activation through the IL-2 receptor beta and gamma chains by IL-2 or other growth factors may contribute to this rejection. Recently, we have demonstrated that the T-cell growth factor IL-15 was abundantly present in rejecting cardiac grafts during anti-CD25 mAb treatment. METHODS To test whether IL-2- and IL-15-responsive T cells play an active role in rejection during anti-CD25 mAb therapy, we measured the frequency of IL-2- and IL-15-proliferative T cells in peripheral blood from treated patients during rejection (n=12). Measurements were made by limiting dilution analysis in the absence and presence of extra in vitro-added mouse anti-human CD25 mAb. RESULTS In the absence of anti-CD25 mAb, the frequencies of peripheral T cells responding to recombinant human (rh)IL-2 and rhIL-15 from patients were lower than those measured in samples of healthy controls (n=7): median of IL-2-responding T cells 78 per 10(6) (range 31-210 per 10(6)) vs. 154 per 10(6) (122-484 per 10(6), P=0.008) and median of IL-15-responding T cells 62 per 10(6) (range 19-207 per 10(6)) vs. 129 per 10(6) (range 79-192 per 10(6), P=0.02), respectively. In the presence of extra in vitro-added anti-CD25 mAb, frequencies of IL-2-responding T cells from patients significantly decreased, although a considerable number of T cells still proliferated on rhIL-2 (median 85%, range 46-100%). In contrast, the frequencies of IL-15 T cells still responding remained stable (median 2%, range 0-50%, P<0.001). CONCLUSIONS Treatment with anti-CD25 mAbs cannot provide complete suppression of T-cell function because significant numbers of IL-2- and IL-15-responsive T cells remain present in the peripheral blood of allograft recipients during anti-CD25 mAb treatment.

Human heart endothelial-cell-restricted allorecognition.

We studied the reactivity of cardiac graft-infiltrating cells, cultured from endomyocardial biopsy specimens, toward heart endothelial cells (Hec). In two cases, Hec derived from the specific donor heart or Hec compatible with the donor were lysed, but not the syngeneic B cell line. A vessel-derived endothelial cell line was not lysed. Panel studies suggest that the epitopes recognized are, in one case, a Hec-specific peptide presented in the context of HLA-Bw41 and, in the other case, a Hec-specific peptide presented by a subtype of HLA-B44. In conclusion, we showed that cardiac graft-infiltrating cells cultured from endomyocardial biopsy specimens can exhibit cytotoxic reactivity specifically directed against HLA-peptide complexes on Hec.

C1.7 monoclonal antibody designates high-avidity CD4+ cytotoxic T lymphocytes involved in clinical heart rejection

BACKGROUND It is assumed that not all donor-specific cytotoxic T lymphocytes (CTLs), but only those with a high avidity for donor antigens, can function as terminal effector cells in transplant rejection. METHODS In the present study, we searched for markers that would exclusively designate these high-avidity CTL. RESULTS FACS analysis of donor-specific CTL clones obtained from heart transplant patients revealed that high- and low-avidity CTL varied in their expression of p38, a surface molecule involved in signal transduction, which is stained by the antibody C1.7. High- and low-avidity CD8+ CTL and high-avidity CD4+ CTL expressed p38, whereas low-avidity CD4+ CTL did not. Noncytotoxic and naive CD4+ lymphocytes also lacked p38 surface expression. CONCLUSION Therefore, we conclude that p38 is a marker for CD4+ lymphocytes with the potency to damage the transplanted heart. Accordingly, p38 might be used to analyze the contribution of CD4+ CTL in immune responses, such as transplant rejection.

Kinetics of circulating cytotoxic T lymphocyte precursors that have a high avidity for donor antigens: Correlation with the rejection status of the human cardiac allograft

Studies on graft infiltrating cells demonstrated that accumulation of cytotoxic T lymphocytes (CTL) with high avidity for donor antigens (Ag) coincided with acute cardiac rejection. In the present study, we analyse whether such high-avidity CTL are present within the peripheral blood of cardiac transplant recipients and whether their kinetics correspond with the rejection status of the allograft. Using limiting dilution analysis (LDA), donor-specific CTL were enumerated in serial blood samples of seven patients. From each patient, 7-11 samples were obtained during the first year after transplantation and up to three samples were obtained at a later date. Enumerated donor-specific CTL were divided into CTL with high or low avidity for donor Ag, depending on their sensitivity to CD8-blocking. In contrast to the situation in the graft, the donor-specific CTL present within the peripheral blood were CTL precursors (pCTL) and not fully mature CTL (cCTL). The number of donor-specific pCTL among peripheral blood cells fluctuated irrespective of the rejection grade of the allograft, indicating that the frequency of circulating donor-specific CTL does not reflect the immunological status of the allograft. During acute cardiac rejection, 66% (median) of the circulating donor-specific pCTL had a high avidity for donor Ag. This percentage significantly exceeded pre- and postrejection values obtained during the first year post-transplantation (median, 39% and 37%, respectively). The disparity in avidity increased even further more than 1 year after transplantation, when stable engraftment was achieved. Among donor-specific pCTL in peripheral blood, those with a high avidity were absent (median, 0%). Hence the avidity of circulating donor-specific CTL might inform us about the immune status of the cardiac allograft.

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.

Pharmacodynamics of prophylactic antirej ection therapy with an anti interleukin-2 receptor monoclonal antibody (BT563) after heart and kidney transplantation

Abstract A mouse monoclonal antibody (BT563) directed to the α-chain of the IL-2 receptor was administrated immediately after transplantation in a dose of 10 mg/day prophylactically to 30 heart transplant recipients (HTx) and 40 renal transplant recipients (RTx) to induce immunosuppression. Plasma levels increased to a plateau level of 5300 ng/ml in HTx and 5900 ng/ml in RTx. BT563 plasma disappearance curves gives a mean T1/2, of respectively 39 h (range 14–112 h) and 42 h (range 8–122 h) for HTx and RTx respectively. The CD25 marker (IL-2R) on the peripheral blood lymphocytes disappeared within hours after the first gift and returned to normal within 0–20 days after the last gift. In HTx more often CD25+ cells were found in the presence of BT563 and more rejections occurred shortly after discontinuation of BT563 compared to the RTx group. Rejectors and non-rejectors within the HTX group did not differ with respect to the period of depletion of CD25 positive cells in the peripheral blood. In 56% of the patients a substantial IgM antibody response was detected. This response was similar for HTx and RTx and not related to rejection. The frequency of IgG responses was low in both HTx (13%) and RTx (21%) patients and the IgG response was not related with graft rejection or with antirejection treatment. Peripheral monitoring showed that mAb plasma levels, antimurine antibody responses and number of CD25 positive cells were not related with the clinical results. The mAb BT563 proved to be safe with respect to the generation of antimurine antibodies and, when given in combination with CsA, is a therapy with a potential for high efficacy.