Acute Graft versus Host Disease After Liver Transplantation: Patterns of Lymphocyte Chimerism

Authors


Corresponding author: Douglas M. Smith, dsmith@baylorhealth.edu

Abstract

The diagnosis of acute graft versus host disease (aGVHD) following liver transplantation can be difficult, since many of the clinical signs can be caused by drug reactions or viral infections. To establish criteria for the persistence of donor T-cells versus engraftment, we measured donor T-cells by short tandem repeat (STR) assays in 49 liver transplant patients for 8 or more weeks post-transplant.

Donor CD3+ T-cells were detected in 38 of 49 patients, on POD 2 with a mean level of 5%. The top of the 99% confidence interval for weeks 1, 2, 3, 4 and 8 were 11, 6, 3, 2 and 3%. Donor CD8+ T-cells were measured in eight patients. The level of CD8+ T-cells was much less than that for CD3+ T-cells, except in two cases of apparent aGVHD. One patient developed severe aGVHD with donor T-cells as high as 84%. The other had 10% donor T-cells for more than 16 weeks associated with fever and neutropenia.

We tested the sensitivity of PCR-ssp typing of HLA DR/DQ for donor T-cells. At least one donor type was detected in all samples with 1% or more donor DNA.

Thus, higher levels of donor T-cell chimerism, particularly with a high proportion of CD8+ T-cells, strongly supports a diagnosis of aGVHD.

Introduction

Acute graft versus host disease (aGVHD) is an under diagnosed, often fatal complication of liver transplantation that occurs in about 1% of cases, most often between 10 days to 6 weeks after transplantation (1–17). It most often presents with fever and either skin rash, diarrhea, or neutropenia. Once patients develop severe, neutropenia (<0.1 × 109/L) recovery is rare. Patients most often die from complications of bone marrow failure, either infections or bleeding. In its early stages, it is often difficult to differentiate from drug reactions or viral infections. Since T-lymphocytes are the effector cells of aGVHD, the demonstration of substantial donor T-lymphocyte chimerism is an essential tool for the diagnosis of post-transplant aGVHD (18–22). A quantitative assay for donor lymphocytes is also important to monitor the course of disease and response to treatment.

Chimerism analysis using short tandem repeat (STR) loci is a quantitative method that has been used in bone marrow transplant patients to monitor engraftment (23). It is sensitive to donor cells at levels of about 1%. If DNA is isolated from purified cell populations, this method can be used to quantitate the donor's contribution to any of the nucleated cell populations present in the blood. One of the principal aims of this study is to document the normal course of transient donor lymphocyte chimerism following liver transplantation and establish the levels that indicate the abnormal engraftment of donor lymphocytes and support the diagnosis of aGVHD. We report the results of donor lymphocyte chimerism studies by STR testing during the first two months after liver transplantation in 49 patients. We contrast the normal pattern of chimerism to one of these patients who clearly had fatal aGVHD and one patient who had abnormal persistence of donor T-cells associated with neutropenia, both of which resolved after removing immunosuppression.

Since some centers may not perform STR testing in their own laboratories, it may not be available with a short turnaround time, thus it would be helpful to have a more readily available screening test for lymphocyte chimerism. Since most HLA laboratories associated with transplant centers routinely perform HLA typing of class II alleles by PCR-ssp typing, we have also tested the sensitivity of this assay in detecting donor lymphocytes.

Methods

Study design and patient selection

This is a prospective study of 49 consecutive liver transplant patients who received orthotopic liver transplants at Baylor University Medical Center between December 2003 and June 2004 and who consented to participate. The study was approved by Baylor's Institutional Review Board. No lymphocyte depleting induction therapy was used. The patients received 1000 mg of hydrocortisone at reperfusion of the allograft, with a rapid taper of methylprednisolone from 100 to 20 mg per day for 5 days. The first three patients enrolled had blood drawn on days 1, 2, 3, 4 and 5 post-transplant. These samples were analyzed to determine the day of peak chimerism for subsequent first samples. A patient and donor sample was collected before transplantation. All patients had blood drawn on the second post-operative day (at least 24-h post-transplant) and at 1 week, 2 weeks, 3 weeks, 4 weeks and 8 weeks post-transplant. If any patients still had detectable donor cells at 8 weeks, they were tested monthly until the donor cells became undetectable.

Patients were monitored for the appearance of any clinical signs of aGVHD at each blood sample collection visit. If any signs or symptoms were present that might be suspicious for aGVHD, patients were referred to our bone marrow transplant program for further evaluation. Diagnosis of aGVHD was based on standard clinical and histopathologic methods and was graded based on previously published criteria (24). If the diagnosis of aGVHD was established, the patient was treated according to our currently established clinical protocols. Prospective data were collected on blood counts, fever, skin rash and diarrhea.

Chimerism measurement using short tandem repeats

A single 8.5 mL tube of ACD anticoagulated blood was divided and used to isolated CD3+ and CD8+ cells using monoclonal antibody coated immunomagnetic beads (Dynal Biotech, Oslo, Norway). Genomic DNA was isolated from these cells. The donor and patient's cells were identified by testing for short tandem repeat (STR) genetic markers using a commercial kit (AmpFLSTR profiler plus, Applied Biosystems, Foster City, CA). Pre-transplant samples from the donor and the patient were tested and STR markers that differed between the donor and recipient were selected. The informative STR markers were then monitored in post-transplant blood samples. STR results are expressed as a percentage (relative value) of donor-derived T-lymphocytes in the patient's blood. All samples were tested for donor CD3+ T-cells. Eight cases were selected for further testing of CD8+ T-cell chimerism. In these cases, all samples that were positive for CD3+ T-cells and one subsequent negative sample were tested for CD8+ donor T-cells.

HLA typing by PCR-ssp of chimeric DNA samples

A commercial HLA typing kit was used to perform low-resolution HLA-DRB1, DRB3, 4, 5 and DQB1 typing by PCR-ssp (One Lambda, Canoga Park, CA, catalog # LSSP2L) according to the manufacturer's instructions. DNA samples were selected to represent a range of donor chimerism from 0 to 10%. The 0% samples were the first sample after transplant that did not have detectable donor DNA by STR testing. The patient and donor HLA types had previously been determined at the time of transplant. The PCR-ssp reactions that represented the donor type were examined for detectable PCR products, in order to determine the sensitivity of this test for the detection of donor DNA.

Statistical methods

Comparisons between study population and overall population were analyzed using nonparametric statistical methods. The χ2 test was used to compare differences in percentages. Continuous data were analyzed using Wilcoxon rank sum test. Spearman's rank correlation test was used to test the significance of the correlation between donor age and POD 2. A p-value of <0.05 was considered statistically significant.

Results

Patient characteristics

The subjects of this study were representative of the population of patients who have been transplanted within the last three years at our institution (Table 1). The patients were all adults, with a similar age and gender distribution. The donors also had a similar age and gender distribution. There was considerable variability in immunosuppressive drug regimens per protocol but the study population was similar to our overall population (25).

Table 1.  Patient characteristics
ParameterTest populationAll patients within 3 years
Number49381
Patient age (range)52 (24–72)50 (18–73)
Patient gender (M/F)32/17257/123
Donor age (range)36 (8–72)40 (7–82)
Donor gender (M/F)31/17231/141
Initial immunosuppression
 Cyclo-steroids-sirolomus10%11%
 Cyclo-other0%7%
 Tacro-steroids-MMF47%47%
 Tacro-other40%29%
 Other2%6%

Donor lymphocyte chimerism

In the three patients that were followed from the first until the fifth post-operative day, donor lymphocytes were already present on the first post-operative day and remained fairly constant during the first week (data not shown). Thus, the passenger lymphocytes from the donor equilibrate quickly in the patient and POD 2 (24–48-h post-transplant) is representative of the peak.

Our study shows that 38 of 49 liver transplant patients had donor T-cell chimerism on POD 2 and 23 remained positive by the end of the first week. At two, three four and eight weeks post-transplant 13, 7, 5 and 3 patients had detectable donor lymphocytes. In one case, the patient had aGVHD (severe diarrhea, fever, skin rash and profound pancytopenia, which was eventually fatal) and was not used in our statistical calculations. One patient, who did not demonstrate many of the typical signs of aGVHD, had a persistence of 8–11% donor T-cells for 16 weeks associated with fever and a progressive neutropenia, which resolved after his removal from immunosuppression.

The mean level of donor CD3-T-cells fell consistently over the period studied. The mean levels were 4.8, 2.4, 1.1, 0.5, 0.3 and 0.2% at 2 days, 1, 2, 3, 4 and 8 weeks post-transplant (Figure 1). Although some means are less than 1%, it should be noted that the STR assay has a limit of detection of about 1% on individual tests. The upper limit of the 99% confidence interval for weeks 1, 2, 3, 4 and 5 were 11, 6, 3, 2 and 3%, respectively. There was no significant difference in the level of chimerism in those patients who had episodes of acute rejection (data not shown).

Figure 1.

The level of CD3+ T-cell chimerism in 49 patients during the first 2 months after liver transplantation. Error bars represent the 95% confidence interval for 48 patients, excluding patient 36. Patient 36 developed severe aGVHD, which became symptomatic 12 days post-transplant and he died 63 days post-transplant. Patient 7 developed progressive neutropenia, which resolved after removal of immunosuppression.

Patient 36 developed severe diarrhea, fever and a skin rash on POD 12 at which time he had 25% donor CD3+ T-cells. His chimeric population peaked at 84% and he developed profound bone marrow aplasia. He died on POD 63 of probable sepsis.

Patient 12 had a prolonged persistence of donor T-cells but initially had no signs of aGVHD. However, he developed progressive neutropenia, which prompted the discontinuation of his immunosuppression, at which point both the donor cell chimerism and the neutropenia resolved.

Donor CD8+ T-cell chimerism was always less than the CD3+ T-cell chimerism measured on the same sample, except in the two patients that had suspected aGVHD. In both of those cases, the level of CD8+ T-cell chimerism was generally equal to or greater than the CD3+ T-cell chimerism (Figure 2).

Figure 2.

A comparison of CD3+ and CD8+ T-cell chimerism in eight patients.

Since we have previously found that the use of donors more than 40 years younger than the recipient was associated with a higher risk of aGVHD (17), we looked to see whether there was any correlation between donor age and the level of chimerism on POD 2. There was no such correlation (Figure 3).

Figure 3.

Correlation of donor age with the POD 2 level of CD3+ T-cell chimerism.

Sensitivity of HLA typing by PCR-ssp in detecting donor T-cells

Eight DNA samples were selected from those used in the STR testing, which represented donor DNA levels of 0–10%. The 0% samples were selected from the first samples where a chimeric patient had dropped to undetectable levels by STR testing. All of the samples that had donor DNA detectable by STR testing had at least one donor HLA type detected by PCR-ssp typing (Table 2). In one of the samples that had no donor DNA detectable by STR testing, one donor HLA type was detected. In all cases where a donor HLA type was detected, the HLA-DQ type was found.

Table 2.  Detection of donor HLA class II alleles by PCR-ssp in DNA samples with varying amounts of donor DNA
% Donor DNADonor HLA types detectedDonor HLA types not detected
0.0NoneDR4,7 DR53 DQ2,8
0.0NoneDR12 DR52
0.0DQ5DR1
1.0DR52 DQ5DR1,17
1.5DQ2DR4,7 DR53 DQ8
4.9DQ7All others shared with recipient
5.7DQ6DR15 DR51
9.8DR4 DR53 DQ8None

Discussion

In a patient who has had a recent liver transplant and develops fever, skin rash, diarrhea or profound leukopenia, the presence of large numbers of donor-derived T-lymphocytes in the patient's blood is strong supportive evidence for aGVHD (18–22). However, we have had little information regarding the normal pattern of T-lymphocyte chimerism in patients who have received a liver transplant. One study has been published using flow cytometry to detect donor lymphocytes (26) in 16 liver transplant recipients. The authors selected patient/donor pairs where the donor differed for an HLA type to which there was an available monoclonal antibody. According to the authors, a sensitivity of 1% donor cells was obtainable by their assay. The authors found only two patients who had detectable donor-derived T-lymphocytes by the third week post-transplant. This is similar to our 7 out of 49 patients with detectable donor lymphocytes at three weeks. Since monoclonal antibodies are not available for all HLA types, it is not possible to perform this test in all patients that are suspected of having aGVHD. However, all individuals have distinct STR patterns that can be used to test any patient/donor pair and it is a readily available clinical test that is often used for engraftment studies in bone marrow transplant patients.

In another study, Taylor et al. (27) used HLA typing by PCR-ssp to screen 33 patients with a clinical suspicion of aGVHD for evidence of donor cell chimerism. Seven patients had detectable donor DNA and were further characterized by flow cytometry. The level of donor CD3+ cells at the time of onset of symptoms in five of the patients range from 20 to 50%. Two patients had levels of 4 and 8% four weeks after transplant. Five of these patients had died and one was alive at 4 months post-transplant but still had signs of GVHD. The one long-term survivor had 8% donor cells at four weeks post-transplant. All of the patients who did not have detectable donor DNA were later diagnosed with other causes of their symptoms or recovered without treatment.

The initial level of donor T-cell chimerism, 24–48-h post-transplant, averaged 5% with a range of 0–24%. Lymphocytes in the blood equilibrate quickly with the circulating pool of T-cells in the patient which totals approximately 3.5 × 1011. This suggests that the liver contains about 1.8 × 1010 passenger T lymphocytes. This is very similar to the number that can be calculated from the results of lymphocyte isolations from liver biopsies (28) and is about 5-fold higher than is received in a bone marrow transplant.

We have previously found that liver transplantation from young donors into older recipients was associated with an increased risk of aGVHD (17). Therefore, in the current study, we looked for any correlation between donor age and the POD 2 donor T-cell level. We did not find such a correlation. Thus, the dose of T-cells cannot explain our previous finding, however, there may be qualitative differences in the T-cell populations present in younger donors (29).

We found two cases of suspected aGVHD in our study. Patient 36 had an undisputable case of aGVHD with the entire spectrum of signs and symptoms. The diagnosis of aGVHD in patient 7 was not clear-cut, since he did not develop a skin rash or diarrhea but the persistence of donor cells >10% for 18 weeks after transplant was so unusual that it supports the diagnosis. In our previous series of 11 cases of aGVHD following liver transplantation, two cases presented with neutropenia and fever and did not have a skin rash or diarrhea. In those cases the patients' pancytopenia became profound and both patients died of sepsis. Therefore, we felt that it was important to treat this patient early. There was no other evidence of a viral illness and he was not on any drugs that are known to have a significant risk of neutropenia. Thus we strongly suspect that this was a case of aGVHD but the diagnosis is not certain.

In case 36 where the patient clearly developed aGVHD, the POD 2 and POD 7 donor T-cell levels were very close to the mean level for the whole group. Thus, these levels did not predict his likelihood to develop aGVHD. However, his level of CD8+ donor T-cells was higher than his level of CD3+ donor T-cells. The only other patient who had similar higher ratios of CD8 to CD3 donor T-cell levels in nearly all samples was patient #7 who also had suspected aGVHD. Thus a preponderance of CD8+ donor T-cells may be an additional indication of aGVHD; however, this finding needs confirmation.

aGVHD most often occurs between 10 days and 6 weeks post-transplant; thus this is the most likely time that the laboratory will be asked to test for donor T-cell chimerism. The patient in this study who developed clear-cut aGVHD had 25% donor CD3+ T-cells at the time he became symptomatic. Since the upper limit of the 99% confidence interval was 11% and 6% at week 1 and week 2 respectively, this level was clearly abnormal. In addition, this patients' level continued to climb to 84%. Any level above the 99% confidence interval for that time point may be considered clearly abnormal; however it is less clear how high a level is characteristic of aGVHD. All of the patients that we have seen that have had CD3+ T-cells levels greater than 20% any time after 1 week post-transplant, have had characteristic signs of aGVHD (additional cases of aGVHD are discussed in a companion paper). We have had one case of aGVHD, which had donor CD3+ T-cell levels of 7% at the time he presented with a characteristic skin rash. The rash was confirmed as being caused by aGVHD by showing that the infiltrating lymphocytes were mostly of donor origin by STR markers. Thus, any patient with symptoms of aGVHD and more than 20% donor CD3 T-cells can be diagnosed with aGVHD with considerable confidence. However, lower levels that are still above the 99% confidence limits may still be consistent with aGVHD, but one should be cautious in making the diagnosis at these levels. Additional, evidence such as a high proportion of CD8+ donor T-cells, a rising level of donor T-cells or the demonstration of donor T-cells in a skin biopsy of a rash may help in the diagnosis.

Based on all of this experience, we would make the following recommendations for the laboratory testing of transplant patients with suspected aGVHD;

  • • Repeat the patients HLA class I typing by serologic methods using purified T-cells and/or class II typing by PCR-ssp.
  • • PCR-ssp is more sensitive at detecting donor cells; however, it may be too sensitive to normal levels of chimerism. Levels above 10% should be detected by serologic typing, while PCR-ssp may detect levels around 1%.
  • • If donor HLA antigens are detected, then use STR testing to quantify the contribution of donor T-cells. CD3 donor fractions above 20% are highly suggestive of aGVHD. Lower levels should be interpreted in light of the patient's clinical signs and should be followed to see if they are rising.
  • • Follow-up STR testing of CD3 and/or CD8 populations about twice a week to monitor the patient's response to treatment.
  • • If neutrophil counts recover, then testing of additional populations may be helpful to determine whether hematopoiesis is of patient or donor origin (in particular, CD15).

It is essential that STR testing be performed on purified cell populations since a large number of recipient myeloid cells can dilute the contribution of donor T-cells. This would give a false impression that there were few donor cells. In addition, rapid turnaround time for STR testing is very important if it is to be used to guide treatment.

In summary, we have found that donor T-cell chimerism is very common following liver transplantation but usually disappears within 1–3 weeks. Even a single value greater than 20% more than one week post-transplant is highly supportive of the diagnosis of aGVHD. Levels between below 20% and above the 99% confidence interval may be consistent with aGVHD but should be interpreted with caution.

Acknowledgment

This study was funded by the Baylor Health Care System Foundation.

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