Liver transplant-associated graft-versus-host disease : Transplantation

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Clinical Transplantation

Liver transplant-associated graft-versus-host disease

Smith, Douglas M.1; Agura, Edward1; Netto, George1; Collins, Robert2; Levy, Marlon1; Goldstein, Robert1; Christensen, Laura1; Baker, Judy1; Altrabulsi, Basel1; Osowski, Lori1 3; McCormack, Jeff1 4; Fichtel, Lisa1 5; Dawson, D. Brian2; Domiati-Saad, Rana2; Stone, Marvin1; Klintmalm, Goran1

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Abstract

Graft-versus-host disease (GVHD) is an uncommon but devastating complication of liver transplantation. GVHD results from the engraftment of T lymphocytes associated with the liver graft and is characterized by fever, skin rash, diarrhea, or pancytopenia usually occurring 2 to 6 weeks after the transplant. The syndrome may initially be difficult to differentiate from cytomegalovirus disease or drug-induced rash and pancytopenia. The diagnosis is confirmed by demonstrating large numbers of donor lymphocytes in the patient’s circulation. Treatment has consisted of either increasing or withdrawing immunosuppression. This is often ineffective, and the mortality rate in published reports is greater than 75% (1–30) (J Forsberg, M.D. Oklahoma University Health Sciences Center, Oklahoma City, OK, personal communication, 1999). Death is usually from infection, bleeding, or severe metabolic disorders resulting from desquamation and severe diarrhea.

Previous reports of liver transplant-associated GVHD (LTx-GVHD) have been case descriptions with little data on incidence or risk factors. We have reviewed our experience in a large liver transplant program to identify risk factors in the development of GVHD and to better characterize its presentation and course. We also examined the utility of human leukocyte antigen (HLA) testing, by serologic methods and by polymerase chain reaction (PCR)-based methods, in documenting the presence of donor lymphocytes. These data may aid in the early diagnosis and perhaps avoidance of some cases of LTx-GVHD.

MATERIALS AND METHODS

Study Design

This is a retrospective study of GVHD occurring after allogeneic liver transplantations performed at our center between January 1991 and December 1998. Cases were referred to the laboratory because of suspected GVHD and included if donor lymphoid chimerism was demonstrated. Clinical course and laboratory data were obtained from hospital records. Histologic sections from skin and large intestinal biopsies and postmortem examinations were reviewed. Potential risk factors were compared between patients with and without GVHD. Data were collected prospectively on all liver transplant patients and entered into a relational database. We did not search patient files to identify undiagnosed clinical cases. We identified one undiagnosed case that occurred before 1991. Data from that case are described but not used for statistical comparisons.

Immunosuppression

Immunosuppression in organ transplantation is an evolving field. At Baylor University Medical Center, liver transplant recipients are treated with different regimens especially designed to meet their specific needs. The immunosuppression regimen is customized according to age and the presence or absence of nephrotoxicity, neurotoxicity, hepatocellular carcinoma, and infectious hepatitis (B or C). These criteria are reflected in the multiple regimens that appear in the patients in this study. In this regard, the main component of our immunosuppressant regimen has been a calcineurin-inhibitor (cyclosporine and, more recently, tacrolimus) supplemented by a purine synthesis inhibitor (azathioprine) and steroids (prednisolone).

Cyclosporine therapeutic levels were between 250 and 350 ng/mL for the first 6 months, 200 to 250 ng/mL until the end of the first year, and 100 to 200 ng/mL afterward. Tacrolimus therapeutic levels were maintained between 10 and 15 ng/mL for the first month and then between 5 and 10 ng/mL. Azathioprine was used at a standard dose of 2 mg/kg per day for the first month, and then it was reduced to 1 mg/kg per day. Prednisolone dosing followed a rapid tapering regimen during the first week (200 mg/day 1, 160 mg/day 2, 120 mg/day 3, 80 mg/day 4, 40 mg/day 5, 20 mg/day 6, and 15 mg/day 7) and then was reduced to 10 mg per day by the end of year 1 and 5 mg per day by the end of year 2.

Testing for Lymphoid Chimerism

Whenever possible, liver transplant patients and donors were HLA typed by standard serologic methods for HLA A, B, DR, and DQ antigens on specimens obtained before transplantation. In 10 of 1,082 cases, HLA typing was not available on the donor or recipient. All suspected cases of GVHD were retyped by standard serologic methods for HLA A and B antigens on one or more samples obtained after the onset of symptoms, except for one patient whose donor showed no HLA A or B mismatches. Posttransplant HLA typing for HLA DR and DQ antigens was performed by serology on six patients by PCR using site-specific primers (PCR-ssp) on four patients and by PCR using site-specific oligonucleotide probes (PCR-ssop) on one patient. Two patients and their donors were tested using microsatellite markers (restriction fragment length polymorphisms), and one female patient was tested for cells with the donor’s Y chromosome using fluorescent in situ hybridization (FISH). Chimerism was established in tissue from postmortem examination in one case by single tandem repeat (STR) analysis using the AmpFLSTR Profiler Plus PCR Amplification Kit (PE Applied Biosystems, Foster City, CA) (31).

Statistical Analysis

Fisher’s exact test (two-tailed) was used to test for differences in categorical data, and the two-sample t test was used to test for differences in means of continuous data. Potential risk factors for GVHD were analyzed using logistic regression and the Wald chi-square test. A P value of less than 0.05 was considered significant for all tests.

RESULTS

Patient Population

The liver transplant program at Baylor University Medical Center was established in 1984; currently, approximately 150 transplantations are performed there annually. The first prospectively identified case of GVHD occurred in 1991. Between January 1, 1991, and December 31, 1998, 1,082 transplants were performed in 1,009 patients. We identified 12 patients with clinical signs of GVHD and laboratory evidence of engraftment with donor T lymphocytes. Sixty-two patients who died or underwent retransplantation within 21 days were not considered to be at risk for GVHD because no cases of GVHD were diagnosed within 21 days posttransplant.

Clinical Course

The clinical course was relatively consistent and characterized by fever, skin rash, diarrhea, or pancytopenia occurring 2 to 6 weeks posttransplantation (Table 1a). The patient’s surgical recovery was generally uneventful. Only two patients experienced an episode of acute cellular rejection. Nine of the 12 patients were discharged 7 to 17 days postsurgery (median=12 days). Three patients experienced early onset of GVHD and were never discharged from the hospital. The earliest sign of GVHD was fever in six patients and skin rash in two patients. In another eight patients, a skin rash appeared shortly after the onset of fever (median=4.5 days). One patient presented with both diarrhea and fever, and another presented with fever, diarrhea, and a skin rash. In total, six patients experienced diarrhea. Some patients presented with other gastrointestinal complaints such as nausea and vomiting or abdominal pain. Two patients presented with profound neutropenia, and all but one eventually became profoundly pancytopenic. Neutropenia and thrombocytopenia occurred after the onset of other signs of GVHD (median=9 days and 11 days later). This pattern of presentation is similar to that of other published cases of LTx-GVHD (Table 1b).

T1A-22
Table 1A:
Table 1a. Clinical course and treatment of 13 cases of LTx-GVHD at Baylor University Medical Center
T1B-22
Table 1B:
Table 1b. Clinical course and treatment of 32 reported cases of LTx-GVHD

Treatment

Treatment usually consisted of increasing immunosuppression and supporting myelopoiesis by use of cytokines. Immunosuppression was increased with antilymphocyte antibody treatment in 10 patients (eight antithymocyte globulin, one Minnesota antilymphocytic globulin, one OKT3) and with an increased dosage of corticosteroid in 11 patients. When in use, azathioprine was discontinued. Ten patients were treated with granulocyte colony stimulating factor beginning a median of 7 days after the first signs of GVHD. One patient also received granulocyte monocyte colony stimulating factor. Seven patients showed substantial recovery of their neutrophil counts. The neutrophils of two patients were of donor origin. One of these was the only survivor. He demonstrated permanent engraftment of donor hematopoiesis and is off all immunosuppression more than 8 years posttransplant. In the remaining patients, the recovery of neutrophils was temporary or occurred after onset of overwhelming infection or organ failure. Eleven of the 12 patients died. Eight of these deaths were primarily the result of sepsis, one of severe gastrointestinal bleeding and erythroderma, and one of multiorgan failure resulting in respiratory arrest. Two patients experienced cerebrovascular accidents while septic and one patient an intracranial event of undetermined cause.

Diagnosis

When GVHD was clinically suspected, blood was sent to the laboratory to test for donor lymphocytes. HLA serologic typing showed the donor’s HLA class I antigens (HLA A and B) in all cases except case 12, in which the donor showed no mismatched HLA A or B antigens (Table 2). In five cases, engraftment was so complete that at least one specimen yielded only donor antigens. In six patients, both donor and recipient HLA A or B antigens were detected. Serologic typing for HLA DR or DQ antigens was performed on B lymphocytes from six patients. The HLA DR or DQ antigens of both donor and recipient were found in four, of the donor only in one, and of the patient only in one. Typing of HLA DR and DQ alleles by PCR methods was performed in six patients, and donor alleles were found in three patients. In each case, DNA was extracted from whole blood, which we found could decrease the sensitivity of donor lymphocyte detection. In cases 7 and 12, the blood leukocytes were only 3% and 6% lymphocytes, respectively. In case 7, the patient was believed to be recovering because HLA DR typing by PCR showed only recipient alleles; however, serologic testing of subsequent samples again showed the donor’s HLA antigens. In case 12, the diagnosis of GVHD was originally dismissed because the donor’s antigens were not detected. However, postmortem testing of samples of colonic tissue revealed evidence of chimerism by use of STR analysis. In two cases, restriction fragment length polymorphism testing also identified donor lymphocytes. Finally, in one case, the presence of cells with the male donor’s Y chromosome in a female recipient was documented with FISH analysis.

T2-22
Table 2:
Table 2. Results of testing for donor chimerism

Risk Factors Analysis

Because this is a retrospective analysis, the most statistically valid comparison is to compare the incidence of potential risk factors in the GVHD group with those in the group that was at risk but did not develop GVHD (Table 3). However, most clinical decisions are based on the risk that a patient with a particular risk factor will develop GVHD. We therefore used the potential risk factors identified in Table 3 to estimate the absolute risk and odds ratio associated with each factor (Table 5).

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Table 3:
Table 3. Characteristics of patients in this study
T5-22
Table 5:
Table 5. Risk factors for GVHD

Two age-related risk factors for GVHD were found: Patients with GVHD were significantly older, and the age difference between donor and recipient was significantly greater. The only diagnosis seen more frequently in GVHD patients was cryptogenic cirrhosis. A significantly higher proportion of donors was closely matched for HLA A and B antigens. No more than one mismatched HLA class I allele was present in the donor of four of our 12 cases (33%). This was consistent with published cases, in which six of 17 cases (35%) showed no more than one mismatched HLA class I allele in the donor. This occurred in only 35 patients (4%) who did not develop GVHD (P <0.001).

HLA Matching

Close matching of the HLA types of the donor and recipient was a significant risk factor for the development of GVHD. Histocompatibility test results for GVHD patients in our study are in Table 4. HLA DR matching alone showed no correlation with the incidence of GVHD. None of our cases were fully matched for HLA-DR alleles, and only two of the published cases were fully matched for HLA-DR alleles. There was a strong correlation of HLA A and B matching to the risk of development of GVHD. The risk did not increase significantly when the donor showed two HLA class I alleles mismatched. The risk increased to 10.3% when the donor showed no more than one HLA class I allele mismatched. The risk increased further to 22.2% when the donor also shared at least one HLA DR allele.

T4-22
Table 4:
Table 4. HLA typing and histocompatibility testing results

This risk may be underestimated because some cases of GVHD may not have been diagnosed. There were only three patients in the total liver transplant group that showed no HLA A and B antigens mismatched and at least one HLA DR matched. One of the patients was diagnosed clinically with GVHD (case 12). Both of the other patients died from sepsis within 40 days after transplant, and one of these patients showed evidence of donor cell chimerism based on STR testing of postmortem tissues.

A positive T-lymphocyte crossmatch did not prevent GVHD. One patient who developed GVHD showed a positive T-lymphocyte crossmatch, which is consistent with the overall incidence of positive T-lymphocyte crossmatches.

Age-Related Risk Factors

Both the recipient’s age and the difference between the ages of the donor and recipient were strongly associated with an increased risk of GVHD. Because these variables are related to one another, the relative contribution of each is uncertain. When the age of the recipient is assumed to have a linear effect on the risk of GVHD, each additional year increases the patient’s risk by a factor of 1.2 (P <0.001). This assumes that risk increases at a constant rate with age, which may not be true. Recipients 65 years and older were found to have a ninefold increase in the risk of GVHD (P <0.001). When the difference between the age of the donor and recipient is assumed to have a linear effect on the risk of GVHD, each additional year increases the patient’s risk by a factor of 1.06 (P <0.005). When we grouped the differences in age between the recipient and donor, recipients 30 to 39 years older than their donor showed a fourfold increase in the incidence of GVHD, but this increase was not statistically significant. Recipients more than 40 years older than their donor showed a 10-fold increase in the incidence of GVHD (P <0.001). All but one of our patients with GVHD showed at least one of the age-related or HLA matching risk factors, whereas only 15% of the overall liver transplant patients showed one of these risk factors.

Congenital Immunodeficiency

An additional undiagnosed case of GVHD occurred before the period of this study. We include a description to illustrate what may be an additional risk factor, namely, a preexisting cellular immunodeficiency. This patient was a 15-year-old boy with dwarfism who also demonstrated an unclassified combined immunodeficiency, which resulted in decreased delayed hypersensitivity skin tests and required periodic intravenous immunoglobulin. He experienced chronic sinusitis and numerous warts. He developed jaundice and was found to have micronodular cirrhosis of unknown origin. He received a liver transplant from a 9-year-old male donor. His postoperative course was uneventful until the 10th postoperative day when he became febrile. He developed nausea, vomiting, diarrhea, and a diffuse maculopapular erythematous rash on the 11th day. By the 13th day, he appeared septic and became obtunded. He experienced a grand mal seizure and respiratory distress requiring intubation and ventilation. His neutrophil count dropped from 8,400/μL to 370/μL on the 13th day, and a bone marrow biopsy showed severe aplasia. His counts continued to drop to less than 100 WBC/μL and 7,000 platelets/μL. He developed pulmonary infiltrates, and blood cultures grew gram-negative rods and Candida. Multiorgan failure developed, and he died on the 20th day. Major autopsy findings included Candida infection causing diffuse bilateral pulmonary consolidation, pseudomembranous colitis, massive gastrointestinal bleeding, diffuse lymphadenopathy, and bone marrow aplasia.

DISCUSSION

GVHD is associated with various medical interventions such as bone marrow transplantation, blood transfusion, and certain types of solid organ transplantation. GVHD after transfusion or solid organ transplantation, in which the donor is usually HLA mismatched, has a high mortality rate (>90%) (32–36). The United Network for Organ Sharing registry has listed GVHD as a reportable cause of death for liver transplant patients since 1994. The registry data shows only 19 deaths from GVHD of 23,452 liver transplants. The incidence of GVHD in our series was 1%; therefore, GVHD is probably underdiagnosed and underreported.

In transfusion- or transplant-associated GVHD, one of the main tissues damaged is the bone marrow. This results in severe pancytopenia and immunodeficiency, and most patients die from infection. The clinical course of GVHD in our patients was similar to that of transfusion-associated GVHD except for the lack of liver dysfunction (32–36). GVHD symptoms usually began 2 to 6 weeks after transplantation. Fever was the earliest sign in most patients; however, skin rash and diarrhea are more specific and usually occurred soon after the onset of symptoms. Profound neutropenia and thrombocytopenia followed a few days later. Because patients presenting with fever and skin rash are often suspected of having a drug reaction and those presenting with fever and diarrhea or other gastrointestinal symptoms are often suspected of having cytomegalovirus infection, the diagnosis of GVHD is often delayed.

When a patient presents with clinical signs of GVHD, demonstration of large numbers of circulating donor T lymphocytes strongly supports this diagnosis. Serologic typing of HLA A and B antigens is performed on separated T lymphocytes, and in most cases it will distinguish between donor and recipient T lymphocytes with a sensitivity of approximately 10% donor cells. Serologic typing of HLA DR antigens is performed on separated B lymphocytes, so this testing cannot rule out engraftment with donor T lymphocytes. Although T lymphocytes do not normally express HLA DR antigens, they do carry the HLA DR gene in their DNA. Therefore, HLA DR typing by PCR-ssp or PCR-ssop using DNA isolated from separated T lymphocytes can be used to detect donor T lymphocytes. This may be useful when the only differences between the patient and donor HLA types are in the HLA-DR alleles. Other genetic tests, such as microsatellite markers, can also be used to detect donor cells as long as DNA is isolated from separated T lymphocytes. In two of our cases, we were misled because DNA testing was performed on DNA from whole blood leukocytes, which were predominantly neutrophils. In two cases of LTx-GVHD reported by Hahn and Baliga (24), donors’ cells were successfully detected using PCR-ssp typing on DNA isolated from CD8+ cells separated on magnetic beads. When there is a difference in the gender of the donor and recipient, FISH analysis of X and Y chromosomes may be used to detect donor cells. FISH analysis is quantitative; however, it does not distinguish between T or B lymphocytes. In many cases, we also demonstrated engraftment of donor B lymphocytes, and in some cases donor myeloid cells, but not as consistently as the engraftment of donor T lymphocytes.

Donor lymphocytes may normally persist some time after liver transplantation. This should be kept in mind when interpreting the results of testing for donor T lymphocytes. To quantify the persistence of donor lymphocytes, Schlitt et al. (37) studied posttransplant lymphocyte populations from 16 liver transplant patients using flow cytometry. All recipients demonstrated detectable donor lymphocytes 1 week after transplantation, which averaged 7.5% and ranged from 1% to 24%. The second week, donor lymphocytes were found in eight of the 16 patients and averaged 2.7%. After the second week, only two patients demonstrated detectable donor lymphocytes. The serologic methods for HLA typing require at least 10% cytotoxicity to be called a positive reaction; thus, donor antigens would not have been detected after the first week in any of these patients. The sensitivity of PCR-based HLA typing varies depending on the method, but in our experience it was no more sensitive than serologic typing. Some methods for microsatellite testing may be more sensitive and may detect donor cells in the first few weeks after transplant. Thus, patients presenting with signs of GVHD should be re-HLA typed by HLA class I serology if there are informative HLA class I differences. If there are no informative HLA class I differences, then other tests for polymorphic genetic markers should be performed on DNA isolated from T lymphocytes.

Basic requirements for the development of GHVD were described by Billingham (38) and Simonsen (39). They concluded that (a) the graft must contain immunologically competent cells, (b) the host must be sufficiently different from the graft to be seen as antigenically foreign, and (c) the host must be incapable of mounting an effective rejection of the graft. The liver graft contains lymphoid cells in hilar lymph nodes and in the liver parenchyma. Norris et al. (32) were able to isolate 1 to 2.5×106 lymphocytes/200 mg of liver biopsy tissue, which represents a dose of approximately 1 to 3×108 lymphocytes/kg to the recipient. In comparison, an allogeneic bone marrow graft contains approximately 3 to 6×107 lymphocytes/kg. The liver recipient’s HLA antigens are almost always foreign to the donor. The development of LTx-GVHD then depends primarily on whether the host can mount an effective rejection of the donor lymphocytes.

One factor in the rejection of donor lymphocytes is the immunocompetence of the recipient. We have described one patient with an inherited immunodeficiency who developed GVHD. This situation is rare, but it illustrates the potential risk of performing a liver transplant in a patient with defective cellular immunity. Reyes et al. (40) reported a similar case of GVHD after a liver and small bowel transplant in an 8-month-old child with a combined immunodeficiency.

Another factor in the rejection of donor lymphocytes is the differences in HLA type of the donor and recipient. Close matching of HLA types has been associated with transfusion-associated GVHD in immunocompetent recipients. In a review of 15 cases by Petz et al. (33), 12 patients showed no HLA mismatches and one patient showed no HLA A and B mismatches and one HLA-DR mismatch. In another report by Ohto and Anderson (36), six of eight cases of transfusion-associated GVHD in infants occurred after transfusion from a donor homozygous for a single HLA haplotype that was shared with the recipient. The effects of HLA matching on hematopoietic graft rejection can also be seen in the experience with bone marrow transplants using unrelated donors. The University of Washington (41) reported that the incidence of bone marrow graft rejection after a transplant from an HLA-matched donor was 2%. The incidence of rejection was not affected if the donor demonstrated multiple HLA class II (DR or DQ) mismatches or if the donor demonstrated a single HLA class I (A, B, or C) mismatch. However, if the donor was mismatched for multiple HLA class I antigens, the incidence of graft rejection was 29%. If the donor showed both a single HLA class I antigen and an HLA class II antigen mismatch, the incidence of graft rejection was 12%. We would therefore expect that multiple HLA class I antigen mismatches between the liver donor and the recipient would tend to protect the recipient from GVHD, which our data support. The highest risk of GVHD (22%) was found in patients who showed no more than one HLA A and B mismatch and shared at least one HLA-DR antigen. This conclusion is also supported by published reports, in which nine of 21 patients who developed GVHD showed no more than one HLA A and B mismatch. A limitation of our study is that HLA C locus typing was not available. It would be interesting to know whether the patients that were closely HLA A and B matched but did not develop GVHD showed additional HLA C mismatches.

Only three patients were matched for HLA A and B antigens and at least one HLA-DR antigen. All of these patients died of sepsis within 40 days of their transplant. One patient had GVHD (case 12). Another patient, who was not suspected of having GVHD on the basis of clinical signs, showed evidence of chimerism when STR analysis was performed on autopsy tissue. It is likely that this group is at even greater risk of GVHD, but the small numbers of HLA A- and B-matched transplants and the possibility that we may not have diagnosed all cases of GVHD make this difficult to determine. Kiuchi et al. (26) reported a case of GVHD in a patient who received a liver transplant from a living donor who was homozygous for an HLA haplotype shared with the recipient. A review of their 280 cases of living donor liver transplants showed only four cases of one-way matching of HLA haplotypes. Whitington et al. (27) also reported a case of GVHD in an infant after a liver transplant from a one-way HLA-matched parent.

Two age-related factors affected the risk of GVHD in our study: the age of the patient and the use of a donor more than 40 years younger than the patient. GVHD risk was considerably higher in the patients older than 65 years. The older recipients may be less able to reject the donor’s lymphocytes, or GVHD may be more severe in older patients and more likely to be diagnosed. The severity of GVHD in patients receiving allogeneic bone marrow transplants has been reported to increase with age. The relationship of GVHD to the donor’s age may be related to the number of lymphocytes present in a young donor’s liver, or it may be related to a qualitative difference in the graft’s lymphocyte populations. These risk factors were statistically significant in our study; however, the median age of patients in published reports is only 50 years, and four cases occurred in pediatric patients. It will be important to determine whether these age-related risk factors are seen in other liver transplant centers.

Preventing GVHD is of particular importance because effective treatment is lacking. Potential preventative strategies may include avoiding some high-risk donor–recipient pairs, treating donors to reduce the number of transplanted lymphocytes, or reducing immunosuppression in high-risk recipients. One third of cases occur when the donor’s HLA type is closely matched with that of the recipient. Avoiding such closely matched donors may prevent some cases; however, logistics may make it difficult to avoid all such close matches. HLA matching should be considered when using living related donors who are more likely to have close HLA matches.

It is common to treat small bowel donors with antilymphocyte antibodies to reduce the risk of GVHD (42). Treatment of the donor with corticosteroids is also common. None of the donors associated with our cases of GVHD had received systemic steroids. It may be beneficial to treat donors with antilymphocyte antibodies and systemic steroids when a high-risk donor–recipient pair is identified. Relatively few donors would be affected. Donors likely to present a high risk of GVHD on the basis of HLA-matching criteria would occur in 4% of donor–recipient pairs, whereas 15% of donors would have one of the HLA matching or age-related risk factors.

Reducing immunosuppression after transplantation from a high-risk HLA-matched donor may help the recipient’s immune system reject the donor lymphocytes. This would increase the risk of graft rejection, so it would need to be performed with great caution. Recently, we identified one patient with a donor who showed no HLA A and B mismatches and another patient with a donor who showed one HLA A and B mismatch, one shared HLA DR antigen, and who was more than 40 years younger than the patient. Consequently, in both cases immunosuppression was decreased by administration of azathioprine and steroids, instead of tacrolimus, and monitoring twice weekly by biopsy for histologic signs of acute cellular rejection or signs of bile duct injury. Treatment with tacrolimus was resumed only when the patients showed both clinical and histologic signs of early rejection. Both patients and grafts have survived more than 1 year. Neither patient developed any signs of GVHD. We believe that the risk associated with acute cellular rejection and its treatment are far outweighed by the risk of mortality if clinical GVHD develops. We expect that by treating patients with “old fashioned” azathioprine and corticosteroids alone, clinical rejection will develop in more than three fourths, but it will not be the uncontrolled rejection that would be seen with no immunosuppression and that would increase the risk to patients. Rejection that develops under these circumstances can easily be manipulated. With the immune reaction against the liver, we postulate that the passenger lymphocytes will be rejected, thus eliminating the risk of GVHD.

Reducing immunosuppression after the onset of symptoms has also been tried in several cases with varying success. In a recent case reported by Lehner et al. (30), all immunosuppression was withheld from postoperative day 83 until the donor’s lymphocytes could no longer be detected on postoperative day 91. This patient recovered; however, this patient never developed severe pancytopenia. The available cases do not clearly support either increasing or decreasing immunosuppression after the onset of symptoms, but reducing immunosuppression may allow the patient to reject the donor lymphocytes and reduce the chances of opportunistic infections, particularly if the patient has not yet developed severe pancytopenia.

We have described the clinical presentation and identified risk factors associated with the development of GVHD after liver transplantation. We conclude that GVHD is probably underdiagnosed; thus, it is important for those who care for liver transplant patients to be familiar with its signs and symptoms. The diagnosis should be considered when a patient presents 2 to 6 weeks posttransplant with fever, skin rash, diarrhea, or pancytopenia. Serologic typing of the patient for the donor’s HLA antigens is usually sufficient to document engraftment of donor T lymphocytes. When other tests using DNA are necessary, it is important to use DNA isolated from purified T lymphocytes. The incidence of GVHD in our patients was 1%, but the risk was higher in closely matched HLA recipients, recipients older than 65 years, and recipients whose donor was more than 40 years younger. Additional studies are needed to confirm these risk factors and to further evaluate methods of prevention.

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