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

Abstract

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.

Fig. 1

Graphic image of helper T lymphocyte (HTL) frequency estimate, with the 48-h assay (a), in the first week after heart transplantation (HTx) (•) (baseline frequency, f = 1/13 513 or 74 HTL/10⁶ peripheral blood mononuclear cells (PBMC)) and at day 44 after HTx during acute rejection (▴) (f = 1/2762 or 362 HTL/10⁶ PBMC) of patient HR. (b) Graphic image of the frequency estimate of the same sample during AR and in the first week after HTx with the 96-h assay. For each frequency determination the lower (dashed) line represents the upper 95% confidence limit, the middle (solid) line represents the estimate f, and the upper (dashed) line represents the lower 95% confidence limit. The zero term of the Poisson equation predicts that when a fraction of 0.37 of the test cultures are negative there is an average one HTL per well. The frequency of the HTL can be extrapolated from the graph by drawing a line from 0.37 to the point where it intersects with the fitted (solid) line.

Fig. 2

(a) Relationship between donor-specific helper T lymphocyte frequency (HTLf) and endomyocardial biopsy (EMB)-diagnosed rejection grade in patient RE who never experienced an episode with acute rejection. The error bars represent the standard error in the frequency estimate. (b) Kinetics of donor-specific HTLf in relation to EMB-diagnosed rejection grade in patient TH who experienced two episodes with acute rejection. The first rejection at day 29 was accompanied by an increase in donor-specific HTLf (□). HTLf with specificity for third-party HLA antigens (▪) did not increase. During the second rejection episode (day 63) donor-specific HTL did not increase.

Fig. 3

Relationship between donor-reactive helper T lymphocyte frequency (HTLf) in peripheral blood (PB) and histological rejection grade, and the first biopsy, during the first 3 months after heart transplantation; summary of the results in nine patients. There was no significant difference between HTLf found at the time of the first biopsy, at the time a grade 2 and at the time a grade 0 or 1 was diagnosed. When multifocal moderate acute rejection was found HTLf in PB were significantly higher than at the time of first biopsy (P = 0.0118), grade 0 or 1 biopsies (P = 0.0189) and at the time grade 2 was found (P = 0.0358).

Fig. 4

Relative helper T lymphocyte frequencies (HTLf) as percentage of baseline frequency during acute rejection, 1–2 weeks before an acute rejection was diagnosed histologically, and after anti-rejection therapy with steroids or rabbit ATG. HTLf were significantly increased during acute rejection (AR) compared with 1–2 weeks before rejection (P = 0.003, Wilcoxon signed rank test). After successful rejection treatment relative HTLf decreased significantly (P = 0.006, Wilcoxon signed rank test). Depicted are 12 rejection episodes in eight patients. Patients RO, JO, HR and TH experienced two rejection episodes. For explanation see Results.

Fig. 5

Summary of the relationship between donor-reactive helper T lymphocyte frequencies (HTLf) in peripheral blood (PB) and histological rejection grade in the endomyocardial biopsy (EMB). HTL frequencies from all nine patients are presented as percentage of baseline frequency. The baseline frequency is the frequency determined at the time the first biopsy was taken (6–8 days after heart transplantation). HTLf found in PB at the time grade 2 and grade 3 were diagnosed were significantly increased compared with grade 0–1 biopsies (P = 0.0320 and 0.0002, respectively). HTLf found at the time of a grade 3 biopsy was significantly (P = 0.0238) higher than at the time a grade 2 was diagnosed. The horizontal line is the 150% of baseline frequency; this line is used as cut-off point for the decision acute rejection (AR) or not (see Table 2).