Peripheral Lymphocyte Subsets in the Management of PTLD G.L. Gupte, S.V. Beath, P.L. Amlot, C. James, D.A. Kelly, P.J. McKiernan, and J. DeVille De Goyet P OSTTRANSPLANT LYMPHOPROLIFERATIVE disorder (PTLD) secondary to Epstein-Barr virus (EBV) infection occurs in 10% to 30% of recipients follow- ing intestinal transplantation. 1 Although it is agreed that immunosuppression should be reduced when PTLD is identified, there is no consensus about the surveillance and management of PTLD. EBV viral load measured by poly- merase chain reaction (PCR) has been demonstrated to be useful in detecting host’s immune response to the virus but does not reliably predict the onset of the onset of PTLD. 2 Primary infection by EBV normally provokes a profound immune response in which there is an increase in CD8+- activated cell markers such as HLA-DR or CD45RO. Monitoring of this response should provide dynamic infor- mation on the impact of EBV in immune-suppressed patients and possibly predict the onset of the PTLD and help in the management of PTLD. The aim of this study was to evaluate the immune responsiveness of intestinal transplant recipients using pe- ripheral blood lymphocyte subset analysis and to relate this to the onset and subsequent management of PTLD. MATERIALS AND METHODS Patients Thirteen children underwent intestinal transplantation from Feb- ruary 1997 to May 2000. Two died within 6 weeks, leaving 11 included in this study who were followed up for at least 12 months. The diagnoses in the 11 children were short bowel syndrome (eight), Hirchsprung disease (two), and microvillous inclusion disease (one). The children received either combined liver and bowel transplant (nine), or isolated small bowel (one), or multivis- ceral (one). The mean age of the children at the time of transplant was 29 months (range 9 to 95 months). Methods Blood samples for examination of peripheral lymphocyte subsets and EBV PCR were collected before and after the diagnosis and subsequent management of PTLD. EBV viraemic children not developing PTLD and non-EBV viraemic children had blood samples during their routine follow-up attendances (once every 3 to 6 months). Analysis of Peripheral Lymphocyte Subsets (PLS). Two flow co- lour analysis of the PLS was done by using a FACScan (Beckton Dickinson, Oxford, UK) flow cytometer, and Consort 30 software by method previously described. 3 Blood counts for CD4 count, CD8 count, CD8+CD69, and CD8+HLA-DR were expressed as the total percentage of lymphocyte count. Detection of EBV viraemia and PTLD. EBV genome copies of 200 copies per million peripheral blood lymphocytes detected by a quantitative polymerase chain reaction assay were suggestive of EBV viraemia. 4 PTLD was diagnosed in patients who were system- ically unwell with evidence of lymphoma cells in tissue (gastric, duodenal, and bone marrow aspirate) or in a lymph node biopsy demonstrated by histology and immunohistochemistry. Statistical Analysis SPSS program was used for analysis. RESULTS Eleven children were followed up over 4 years with a median follow-up of 26 months. Three children remained EBV PCR-negative (group I) and eight children became EBV PCR-positive, 2 to 20 months (median time 8 months) after transplantation (group II). Of the eight children with EBV viraemia, five developed PTLD 5 to 21 months (median time 15 months) after transplantation with a median time of detection of PTLD being 15 months. All children with PTLD had immunosuppression reduced by 50% immediately. Of the five children diagnosed of PTLD three children survived (group III) and two children died due to PTLD (group IV). Of the three survivors, one recovered after reducing immunosuppression and two did not respond to reduced immunosuppression alone and received autologous HLA-matched cytotoxic T lymphocytes (CTL) from EBV-positive healthy blood donors. The three remaining patients with primary EBV infection have not yet developed PTLD and are being closely monitored. None of the nonviraemic patients developed PTLD. Risk Factors for Death From PTLD (Group IV) There was a trend for decreased CD4:CD8 ratio in group IV as compared to groups III, II, and I (see Table 1). Within 4 to 12 weeks of diagnosis the HLA-DR response reduced drastically in group IV as compared to group III (P = .0368), as see in Fig 1. From the Liver Unit, Birmingham Children’s Hospital (G.L.G., S.V.B., C.J., D.A.X., P.J.M., J.D.G.), Birmingham, UK, and De- partment of Clinical Immunology, Royal Free Hospital, (P.L.A.), London, UK. Address reprint requests to Dr Sue Beath, Consultant Hepatologist, Birmingham Children’s Hospital, Steelhouse Lane, Birmingham B4 6NH, UK. E-mail: sue.beath@bhamchildrens.wmids.nhs.uk 0041-1345/02/$–see front matter © 2002 by Elsevier Science Inc. PII S0041-1345(02)03064-6 655 Avenue of the Americas, New York, NY 10010 1782 Transplantation Proceedings, 34, 1782–1783 (2002)