The frequency and avidity of committed cytotoxic T lymphocytes (cCTL) for donor HLA class I and class II antigens and their relation with graft vascular disease

Abstract

Cellular immune processes may trigger the development of graft vascular disease (GVD). CD4 and CD8 cytotoxic T lymphocytes that infiltrate the allograft could play a role in the development of GVD. We studied the presence of in vivo primed or committed CTL (cCTL) and their avidity for donor HLA class I and class II antigens in graft-infiltrating lymphocyte cultures propagated from endomyocardial biopsies derived from patients with and without signs of GVD. The fraction of cCTL with high avidity for HLA class I or class II antigens was estimated by the addition of anti-CD8 or anti-CD4 MoAbs to the cytotoxic phase of the limiting dilution analysis. In the first year after transplantation no difference in the frequency of donor-specific class I cCTL between patients with and without GVD was found. Addition of anti-CD8 MoAb revealed that most cultures predominantly consisted of cCTL with low avidity for donor HLA class I antigens, irrespective of the development of GVD at 1 year after transplantation. However, in patients who did not develop GVD, the frequency of cCTL with donor HLA class II specificity was significantly higher than in patients who did develop GVD. The avidity for donor HLA class II antigens was comparable in both groups. A high frequency of donor-specific cCTL for HLA class II antigens seems to be a protective factor against the development of GVD. These cCTL might be cytotoxic for cells involved in GVD development, e.g. activated endothelium and smooth muscle cells of donor origin.

INTRODUCTION

The long-term success of organ transplantation is limited by the development of chronic rejection. In the allograft, chronic rejection manifests itself as an occlusive disease of the donor vessels, which leads to ischaemia and thereby to organ dysfunction. In human cardiac allografts, it is characterized by a process of accelerated coronary artery disease, which can be visualized by angiography or intravascular ultrasound. The vascular lesions develop in the donor vessels, while the recipient vessels remain free from lesions. Therefore an allogeneic immune process is probably involved in the pathogenesis of graft vascular disease (GVD). Cells obtained from affected coronary vessels are a likely source to study cellular immune processes in relation to GVD. Unfortunately, such an analysis is limited by the material available. However, GVD involves vessels of different sizes in the graft [1], including the very small vessels that can be observed in endomyocardial biopsies (EMB) [2,3]. After heart transplantation (HTx), EMB are regularly taken to diagnose acute rejection. Recently, we have demonstrated that during the first post-operative year graft-infiltrating lymphocytes (GIL) propagated from EMB derived from patients who developed GVD at 1 year after HTx produced significantly more T-helper 1 (Th1) cytokines than the cultures from patients who remained free from GVD [4]. Not only the T-helper cells, but also CTL could play a role in the development of GVD. During acute rejection, donor-specific CTL propagated from EMB had mainly a high avidity for donor HLA class I and class II antigens, whereas CTL from non-rejection EMB had a low avidity for donor antigens [5–7]. The CTL with high avidity for donor HLA class I or class II antigens are resistant to in vitro inhibition with CD8 or CD4 MoAbs, respectively, indicating that these cells do not need the CD8 or CD4 molecule to stabilize their antigen binding [8,9]. On the other hand, low-avidity CTL can easily be inhibited.

In the present study, we analysed the cytotoxic capacity of GIL to donor HLA class I and class II antigens during the first post-operative year and their relation with GVD as diagnosed at 1 year after HTx. To study the relevance of CD8⁺ and CD4⁺ CTL during the development of GVD, we investigated the frequency of in vivo primed or committed CTL (cCTL) present within the graft and their avidity for donor HLA class I and class II antigens in the first year after HTx, thus before the diagnosis of GVD.

PATIENTS AND METHODS

Patients

We studied 89 cardiac allograft recipients transplanted consecutively between September 1987 and January 1991. Detection of acute rejection was performed by histological grading in EMB and defined as mononuclear cell infiltrates with myocyte damage. We refer to acute rejection when grade 3A or more is histologically diagnosed [10]. At 1 year after HTx, 18 patients had signs of GVD and 71 patients did not. GVD was visually assessed by coronary angiography taken at 1 year after HTx, and scored by one of us (A.H.M.M.B). GVD was defined as all vascular wall changes, including minimal wall irregularities.

All patients received preoperative blood transfusion, maintenance immunosuppression consisted of cyclosporin A (CsA) and low dose of steroids.

Culturing, phenotypic analysis and cell-mediated lympholysis of cultures

GIL were established from EMB in 96-well U-bottomed tissue culture plates (Costar, Cambridge, MA) in medium (RPMI 1640 Dutch Modification; Gibco, Paisley UK) supplemented with 10% pooled heat-inactivated human serum, 4 mml-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (= culture medium) in the presence of ≈ 30 U/ml exogenous IL-2 (lectin-free lymphocult T-LF; Biotest AG, Dreieich, Germany) and 1 × 10⁵ irradiated (30 Gy) autologous peripheral blood mononuclear cells (PBMC) per well [11]. GIL cultures propagated under these conditions contain in vivo cCTL [6,12]. The plates were incubated in a humidified atmosphere at 37°C in 5% CO₂.

When sufficient cell numbers was reached, the cultures were analysed by three-colour flow cytometry on a FACScan (Becton Dickinson, San Jose, CA) for the expression of cell surface markers. Screening was performed with the combination WT31 FITC (T cell receptor (TCR) αβ), CD4 PE and CD8 PerCP.

Subsequently, the cytotoxic capacity of GIL cultures was tested against donor cells or a panel of target cells sharing either HLA class I or class II antigens with the donor. Briefly, effector GIL were incubated with 2.5 × 10³⁵¹Cr-labelled target cells at different effector/target ratios in 200 μl culture medium. After 4 h of incubation supernatants were harvested and ⁵¹Cr-release was determined as described in the limiting dilution analysis (LDA). Cells not used for this test were stored at −140°C.

Allogeneic target cells

T cell blasts were obtained by culturing donor spleen cells for 7 days in culture medium supplemented with 1% phytohaemagglutinin (PHA)-M (Difco, Detroit, MI), and after 3 days half of the medium was replaced by culture medium supplemented with 10% v/v lymphocult-T (Biotest). These blasts served as target cells to determine donor class I-directed cytotoxicity. The T cell blasts can not be used as HLA class II targets [7].

Epstein–Barr virus (EBV)-transformed B cell lines (B-LCL) were obtained by infection of PBMC or spleen cells with the virus from the marmoset cell line B95-8 and addition of CsA as described by Moreau et al. [13]. These cells were maintained in culture medium supplemented with 10% heat-inactivated bovine calf serum (BCS; Hyclone, Logan, UT). The B-LCL served as target cells to measure HLA class I and II antigens. Because we determined especially the avidity for donor HLA class II antigens, we used B-LCL that did not share donor HLA class I, but shared only donor HLA class II antigens with the donor [7].

Limiting dilution analysis

We obtained outgrowth of GIL in ≈ 42% of the non-rejection EMB [14] and stored the cultures when enough cells were left after the standard cell-mediated lympholysis test. Only cultures showing donor-specific class I and/or class II cytotoxicity were analysed in the present study. We studied 19 GIL cultures propagated from EMB of eight patients without GVD and 10 patients with signs of GVD in their 1-year angiogram. The donors of these patients had several mismatches for HLA class I (HLA-A and -B) and/or HLA class II (HLA-DR) antigens (Tables 1 and 2). The selected cultures had to exhibit donor HLA class I-directed cytotoxicity in ⁵¹Cr-release assays in order to be able to determine the percentage of cells with high avidity for donor HLA class I antigens, and donor HLA class II-directed cytotoxicity to determine the percentage of cells with high avidity for donor HLA class II antigens. The days after HTx, days in cultures, phenotypic composition and number of HLA mismatches between donor and acceptor were comparable between patients with and without GVD (Tables 1 and 2).

Table 1.

Characteristics of the graft-infiltrating lymphocyte (GIL) cultures of which the fraction of cCTL and their avidity for donor HLA class I antigens were determined

Table 2.

Characteristics of the graft-infiltrating lymphocyte (GIL) cultures of which the fraction of cCTL and their avidity for donor HLA class II antigens were determined

The cultures were thawed in culture medium and irradiated (30 Gy) third-party B-LCL (5 × 10³/well) were added as feeder. These B-LCL did not share HLA antigens with the donor and acceptor, to avoid de novo activation in vitro of precursor CTL that can mature to cCTL by restimulating with donor antigens. Thus, only the activity of in vivo activated cCTL were quantified in the LDA. When sufficient cell numbers were reached, limiting dilution cultures were set up in 96-well U-bottomed tissue culture plates (Costar). Graded numbers of responder GIL were titrated in eight double dilutions starting from 5000 to 39 cells per well in 24 replicates with 5 × 10⁴ irradiated (30 Gy) autologous PBMC as feeder in 200 μl culture medium supplemented with 20 U recombinant IL-2 (Biotest). After 7 days of culture, the microcultures were split in two. Half of the split wells were tested in the absence of MoAb and the other half were tested in the presence of CD8 MoAb in case of determining the avidity for donor HLA class I antigens and CD4 MoAb for determining the avidity for donor HLA class II antigens. Each well was individually tested for cytolytic activity against 2.5 × 10³⁵¹Cr-labelled target cells. T cell blasts of donor origin were used as targets when the avidity for donor HLA class I antigens was tested. B-LCL were used as targets when the avidity for donor HLA class II antigens was tested [7]. These B-LCL lacked donor HLA class I molecules. After 4 h of incubation at 37°C in 5% CO₂ the supernatants were harvested (Skatron harvesting system: Skatron-AS, Lier, Norway) and the release of ⁵¹Cr was determined in a γ-counter (Packard Instruments, Downers Grove, IL). Maximum and spontaneous release were determined in four-fold and defined by incubation of target cells with culture medium in the presence and absence of Triton X-100 (5% v/v in Tris buffer), respectively.

The percentage of specific lysis was calculated according to the formula:

The fraction of cCTL with high avidity for donor class I (CD8) or class II (CD4) HLA antigens was calculated using the formula:

The microcultures were considered cytolytic when the percentage lysis exceeded 10%.

As a control for specificity, 5000 cells per well were cultured in quadruple and the capacity to lyse third party PHA blasts or B-LCL (these targets cells did not share HLA antigens of the donor) and the K562 cell line was tested.

CD8 inhibition

FK18 (a kind gift of Dr F. Koning, Department of Immunohematology and Bloodbank, University Hospital Leiden, The Netherlands), a mouse antibody of the IgG3 subclass, recognizes the gp32 chain of the human CD8 molecule [15]. A 1:500 dilution of ascitic fluid totally inhibited the cytotoxic capacity of CD8-dependent CTL clones, but did not affect target cell lysis by CD8-independent CTL clones. CD8 clones were not affected by the CD4 MoAb RIV6 [7]. Before addition of the target cells to the microcultures, FK18 was added to half of the split well cultures and was incubated for 30 min at 37°C in a humidified atmosphere containing 5% CO₂.

CD4 inhibition

RIV6 (a kind gift of Dr M. F. Leerling, RIVM, Bilthoven, The Netherlands) is a mouse antibody of the IgG2a subclass directed against the human CD4 molecule. At a concentration of 1 μg/ml RIV6 totally inhibited the cytotoxic capacity of CD4-dependent CTL clones, but did not affect target cell lysis by CD4-independent CTL clones. CD4 clones were not affected by FK18 [7]. Before addition of the target cells to the microcultures, RIV6 was added to half of the split well cultures and was incubated for 30 min at 37°C in a humidified atmosphere containing 5% CO₂.

Statistical analysis

Data concerning the cytotoxicity against donor HLA class I and class II antigens were analysed with Fisher's Exact Test (Tables 3 and 4). The CTL frequency (CTLf; expressed as the number of CTL per 10⁶ cells) and the 95% confidence interval (CI) were calculated by the Jackknife procedure for maximum likelihood [16]. The calculated frequencies were accepted when the goodness-of-fit did not exceeded 12. The significance of differences between the patient groups was analysed with the Mann–Whitney U-test.

Table 3.

Donor-specific cytotoxic characteristics analysed per patient of graft-infiltrating lymphocyte (GIL) cultures from patients with and without graft vascular disease (GVD) during the first year after transplantation

Table 4.

Donor-specific cytotoxicity analysed per graft-infiltrating lymphocyte (GIL) culture from patients with and without graft vascular disease (GVD) during the first year after transplantation

RESULTS

GIL with donor-specific HLA class I and class II CTL

We studied from 89 patients 531 GIL cultures propagated from acute rejection and non-rejection EMB, and analysed per patient (Table 3) and per culture (Table 4) the cytotoxic reactivity for donor HLA class I and class II antigens. Moreover, we investigated whether this cytotoxicity was discriminating for GVD. There was no difference in non-donor-specific cytotoxicity, donor HLA class I or class II, or both donor HLA class I and II cytotoxicity of the GIL cultures between the patients with and without GVD, neither when the cytotoxic reactivity per patient was analysed (Table 3a), nor when all cultures together were analysed (Table 4a). The cytotoxic capacity during acute rejection episodes was not discriminating for GVD development (Tables 3b and 4b).

The frequency of cCTL for donor HLA class I and their avidity for class I antigens

We determined the frequency of cCTL and the fraction of high-avidity cCTL specific for donor class I antigens in five cultures from patients with GVD and five cultures from patients without GVD. Since we found previously that acute rejection correlated with an increased number of high-avidity CTL for donor HLA class I antigens [5,6], we only analysed cultures derived from non-rejection EMB.

The cCTLf reactive to target cells expressing donor HLA class I antigens was not different between the two patient groups (Table 5). When anti-CD8 MoAbs were added during the CML phase also no difference in cCTLf was found (Table 5). Looking at the avidity of the cCTL, we demonstrated that most cultures during the first year after HTx predominantly consisted of cCTL with low avidity for donor HLA class I antigens, irrespective of GVD development at 1 year after transplantation (Fig. 1a).

Table 5.

Frequencies of donor-specific cCTL with their percentage of CD8 inhibition in graft-infiltrating lymphocyte (GIL) cultures from patients with or without graft vascular disease (GVD)

Fig. 1.

The fraction of graft-infiltrating lymphocyte (GIL) cultures propagated from endomyocardial biopsies (EMB) taken during the first preoperative year from patients with and without graft vascular disease (GVD) at 1 year after transplantation having a high avidity for donor HLA class I (a) and class II (b) antigens.

The cCTL did not lyse third party PHA blasts or B-LCL and the K562 cell line. Therefore we conclude that the cytotoxic capacity was donor-specific.

The cCTL frequency of donor HLA class II and their avidity for class II antigens

The cCTLf and the fraction of high-avidity cCTL for donor class II antigens were measured in nine patients with GVD and five patients without GVD. Acute rejection was already found to be associated with high-avidity CTL for donor HLA class II antigens in a previous study [7]. Therefore we only determined GIL cultures derived from non-rejection EMB.

In patients who did not develop GVD at 1 year after transplantation the cCTLf with donor class II specificity was significantly higher than the frequency in the patients who had GVD (Table 6). Also, in the presence of anti-CD4 MoAb significantly higher frequencies were found in patients without GVD (Table 6). The percentage cells with high avidity in the cultures for donor HLA class II antigens was comparable between the patient groups (P = 0.19) (Fig. 1b).

Table 6.

Frequencies of donor-specific cCTL with their percentage of CD4 inhibition in graft-infiltrating lymphocyte (GIL) cultures from patients with or without graft vascular disease (GVD)

Remarkable was the higher frequency of cCTL directed against donor HLA class II antigens compared with those cCTL directed against donor HLA class I antigens in patients who remained free from GVD (P = 0.10) (Tables 5 and 6).

DISCUSSION

Chronic rejection is a frequently encountered complication after organ transplantation. The pathogenesis of GVD is still unknown. However, as the vascular lesions develop in donor vessels and remain absent in recipient vessels, allogeneic immune processes may be involved in the pathogenesis of GVD. In the search for immunological parameters, we found no relation between GVD and the number of acute rejection periods, preoperative reactive antibodies against a panel of lymphocytes, number of HLA mismatches, in vitro growth or phenotypic composition of GIL derived from EMB [14,17]. We found, however, that certain characteristics of donor-specific Th cells are associated with the occurrence of GVD. Both IL-2 mRNA expression in EMB [18] and production of high levels of Th1 cytokines (IL-2 and interferon-gamma (IFN-γ)) by GIL derived from EMB [4] were especially found in patients with GVD. In the present study we investigated the possible link between GVD and the frequency or nature of CTL propagated from GIL.

First, we studied whether the presence of donor-directed CTL within the transplanted heart is associated with GVD. We found that the frequency of the GIL cultures lytic to donor HLA class I and class II antigens does not discriminate patients with GVD from patients who remain free from GVD in the first year after transplantation.

Second, we analysed 19 cultures from 10 patients with GVD and eight patients without signs of GVD in more detail. We found no differences in phenotype of the GIL cultures between patients with and without GVD. This confirms our previous study, which also showed that the phenotypic composition of the cultures does not correlate with GVD [14]. The EMB-derived cultures consisted of a mixture of CD8 and/or CD4-resistant and -susceptible donor-reactive CTL. During the first year after HTx, the frequency of donor-specific HLA class I cCTL did not differentiate the patients with GVD from those without GVD. The same was true when the cytotoxicity was studied in the presence of CD8 MoAb. The cultures contained predominantly cCTL with low avidity for donor HLA class I antigens, irrespective of the development of GVD at 1 year after HTx. During the first post-operative year, the frequency of cCTL to HLA class II donor antigens was found to be significantly higher in patients who remained free from GVD than in patients with angiographic signs of GVD at 1 year after transplantation. The fraction cCTL with high avidity for donor HLA class II antigens in the GIL cultures was comparable between these patient groups.

These data suggest that a high frequency of donor HLA class II-directed cCTL protects for GVD. As a possible explanation for this phenomenon, we suggest the following model. Shortly after transplantation, MHC class II antigens are predominantly expressed on the vascular wall of donor origin [19] due to tissue damage as the result of ischaemia and reperfusion [20,21]. Macrophages infiltrate the graft (GIL) and release cytokines to their microenvironment. These cytokines cause an up-regulation on donor cells of donor HLA class I and class II antigens and expression of lymphocyte binding ligands (intercellular adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), etc.) in the graft. The permeability of the microvessels increases and donor-specific activated mononuclear cells (macrophages and lymphocytes) enter the graft. Subsequently, in patients who remain free from GVD, allo-activated cCTL specific for donor HLA class II antigens accumulate at sites with activated donor cells. Thereafter donor cells with HLA class II antigens on their surface, which are responsible for the development of GVD (endothelial cells and smooth muscle cells), are killed, thereby preventing the generation of GVD. At the same time the frequency of cCTL specific for donor HLA class I antigens is still too low to be cytotoxic. Previously, our research group demonstrated that cCTL from GIL are able to lyse donor endothelial cells [22].

We have also reported a high production of Th1 cytokines by GIL derived from patients with GVD in the first year after HTx compared with those from patients without GVD [4]. This consequently results in an up-regulation of HLA class I and class II antigens on vascular endothelial cells of patients with GVD [23]. In case of GVD, the frequency of both the donor-directed HLA class I and class II cCTL is apparently too low to lyse the activated vascular wall cells and the characteristic process of GVD development continues: the activated endothelial cells and mononuclear cells secrete growth factors, which promote proliferation of smooth muscle cells, ultimately leading to intima thickening.

Dong et al. [24] demonstrated that indeed endothelial cell damage was less evident in the vessels with greater intimal disease severity, probably by a decreased number of HLA class II-specific CTL. In future, our proposed model for GVD has to be confirmed by an investigation of growth factor production by vascular cells in the presence of CTL from patients with and without GVD.

In conclusion, cCTL that infiltrate the human cardiac allograft could play a role in the development of GVD. Especially the high frequency of donor-specific cCTL for HLA class II antigens seems to be a protective factor in the development of GVD.