N. H. P. M. Jutte

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.

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.

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.

Vitrification of human islets of Langerhans

Cryopreservation of human islets of Langerhans by vitrification was studied. Isolated islets were divided into four groups: (1) control islets which were cultured for 6 days, (2) islets which were vitrified after 2 days of culture, (3) control islets which were cultured for 10-13 days, and (4) islets which were vitrified after 6-9 days of culture. After warming, islets from groups 2 and 4 were cultured for 4 days. The thus treated islets were investigated with respect to insulin secretion in the presence of 2.5 or 25 mM glucose, capacity to survive during postwarming culture, and morphology. The insulin secretion in islets from all groups could be stimulated by an increase of the concentration of glucose from 2.5 to 25 mM. No significant differences were observed between the insulin secretions of the vitrified and control islets or between the islets vitrified after 2 and 6-9 days of culture. It is concluded that human islets of Langerhans cryopreserved by vitrification are functional in vitro.

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.

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.

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)

Vitrification of mouse islets of Langerhans: Comparison with a more conventional freezing method

The possibility of cryopreservation of islets of Langerhans by vitrification using a mixture of cryoprotectants was investigated and the results were compared with a more conventional freezing method using Me2SO as cryoprotectant. Isolated mouse islets were divided into three groups: (1) control islets cultured for 6 days, (2) islets which were cryopreserved by vitrification after 2 days of culture, and (3) islets frozen in 1.5 M Me2SO after 2 days of culture. After warming, islets from groups 2 and 3 were cultured for 4 days. The thus treated islets were investigated with respect to insulin secretion in the presence of 2.5 or 25 mM glucose, survival during postwarming culture, morphology, and capability to reverse streptozotocin-induced diabetes. The insulin secretion in islets from all groups could be stimulated by a factor 5 or more by an increase in the concentration of glucose from 2.5 to 25 mM. The secretion of insulin in the presence of 2.5 mM glucose was similar in all groups of islets. The secretion of insulin in the presence of 25 mM glucose was slightly but not significantly lower in the cryopreserved islets than in the control noncryopreserved islets. The survival of islets during postwarming culture was comparable after cryopreservation with both methods, and islets from both groups could lower serum glucose in streptozotocin diabetic mice. We conclude that islets cryopreserved by the vitrification method are functional in vitro and in vivo. This method is quick, simple, and cheap because the use of complicated freezing equipment is avoided.

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.

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.