C. R. Daane

Donor-specific cytokine production by graft-infiltrating lymphocytes induces and maintains graft vascular disease in human cardiac allografts.

BACKGROUND The development of graft vascular disease (GVD) in the allograft is a major impediment for long-term survival of heart transplant recipients. GVD may be mediated by cellular processes, in response to the transplanted heart, and regulated by cytokines. METHODS We studied donor-specific cytokine production patterns in graft-infiltrating lymphocyte cultures propagated from endomyocardial biopsies. The biopsies were derived from patients with and without signs of GVD, as diagnosed by angiography at 1 year after heart transplantation. RESULTS In the first year after transplantation, significantly more T-helper (Th) 1 cytokines (interleukin [IL]-2: P=0.04; interferon-gamma: P=0.01), but not Th2 (IL-4 and IL-6) cytokines, were produced by cultures of patients with GVD compared with patients without GVD. Thereafter, the Th1 cytokine levels in patients with GVD normalized to the levels of patients without GVD. Detectable levels of IL-6 were produced significantly more often (P=0.009) by cultures obtained more than 1 year after transplantation from patients with GVD. CONCLUSIONS The results suggest that high levels of Th1 cytokines produced by graft-infiltrating lymphocytes early after transplantation may be responsible for activation of vascular endothelium, leading to the migration and proliferation of smooth muscle cells that is characteristic of GVD. IL-6, produced later after transplantation, continues this process by promoting smooth muscle cell proliferation.

Peripheral monitoring of direct and indirect alloantigen presentation pathways in clinical heart transplant recipients

It has been reported that the response to alloantigens presented by the direct and indirect pathway may be of differential relevance after human kidney transplantation. Accordingly, we monitored these routes in peripheral blood mononuclear cells (PBMC) of heart transplant patients from before transplantation and up to 2 years thereafter in an attempt to find a correlation with the clinical status of the patients. Both before and after transplantation, comparable proportions of PBMC samples reacted in mixed lymphocyte culture to nondepleted donor spleen cells (direct route), but never to donor cells depleted for antigen-presenting cells (indirect route). In contrast, the latter route could easily be activated by a nominal antigen and persisted after transplantation, although the proportion of PBMC samples responding was significantly suppressed, irrespective of the occurrence of rejection. Consequently, complete removal of antigen-presenting cells from the stimulator population in a mixed lymphocyte culture with PBMC as responder is not a suitable tool for measuring indirect presentation of alloantigens, and therefore not relevant for monitoring the immunological status of heart transplant recipients.

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.

T helper frequencies in peripheral blood reflect donor-directed reactivity in the graft after clinical heart transplantation

We describe the usefulness of a fast (48‐h) limiting dilution assay (LDA) for the enumeration of human alloreactive helper T lymphocytes (HTL) in the peripheral blood, in relation to histologically defined rejection grades after heart transplantation. HTL frequencies (HTLf) in pretransplant samples varied from patient to patient, ranging from 106 to 625 HTL/106 peripheral blood mononuclear cells (PBMC). In the first week after heart transplantation (HTx), when immunosuppression was instituted, HTLf were significant lower (range 30–190 HTL/106). The level of HTL in the first week after HTx when rejection grade was 0 or 1A (ISHLT score) was considered to be the baseline frequency. This frequency did not correlate with the number of subsequent rejection episodes. During rejection (grade 3), donor‐specific HTLf were increased above their baseline frequencies (P = 0.01). Expressed as percentage of baseline frequencies, HTLf increased significantly during acute rejection (AR) compared with 1–2 weeks before rejection (P = 0.003). The increase was specific, since viral infections did not result in a rise of donor‐specific HTL, while also HTLf specific for third party HLA antigens were not elevated during rejection. Monitoring HTLf in peripheral blood with a shortened (48‐h) assay may serve as a non‐invasive method for detecting intragraft immunological reactivity. Demonstrating absence of donor‐specific reactivity may limit the number of invasive endomyocardial biopsy (EMB) procedures and allow tapering of immunosuppressive treatment.

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.

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.

Discrepancy between mRNA expression and production of IL-2 and IL-4 by cultured graft infiltrating cells propagated from endomyocardial biopsies

Abstract We studied whether acute rejection correlated with the cytokine production pattern and mRNA expression of interleukin‐2 (IL‐2) and interleukin‐4 (IL‐4) in lymphocyte cultures derived from endomyocardial biopsies (EMB) that were stimulated with B cell lines of donor origin. Unstimulated biopsy cultures neither expressed mRNA nor produced IL‐2 or IL‐4. All stimulated biopsy cultures contained mRNA transcripts for IL‐2 and IL‐4. In contrast, we found different IL‐2 and IL‐4 production patterns. Within the first 90 days after heart transplantation (HTx), higher levels of IL‐4 were measured in cultures derived from EMB with myocytolysis than in cultures from EMB without signs of myocytolysis. More than 90 days after HTx, this phenomenon was reversed and more IL‐4 was produced in cultures derived from EMB without myocytolysis. These differences were not detected for IL‐2 production.

Nonspecific immune reactivity of peripheral blood mononuclear cells is related to graft vascular disease

I N HEART transplant patients, high responses of donorspecific mixed lymphocyte culture (MLC) reactivity’ --’ or incrcascd numbers of IL-2 producing helper T-lymphocytcs (HTL)’ have been reported in relation to acute rcjcction. chronic rejection or in patients with stable graft. Thcsc tests also have been used to identify patients with donor-specific proliferative hypeor hypcr-reactivity.? 7 We I-eport hcrc on the relation of graft vascular disease (GVD) to donor-specific reactivity of peripheral blood mononuclear cells (PBMC) in the MLC and the HTL-assay as well as following ;I nonspecitic stimulation with tetanus toxoid (TET).

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.