Bone Marrow Transplantation (2001) 27, 1095–1100  2001 Nature Publishing Group All rights reserved 0268–3369/01 $15.00 www.nature.com/bmt Immunobiology Oligoclonal expansion of CD4 + CD28 - T lymphocytes in recipients of allogeneic hematopoietic cell grafts and identification of the same T cell clones within both CD4 + CD28 + and CD4 + CD28 - T cell subsets M Hirokawa 1 , T Horiuchi 1 , Y Kawabata 1 , A Kitabayashi 1 , H Saitoh 1 , Y Ichikawa 1 , T Matsutani 2 , T Yoshioka 2 , Y Tsuruta 2 , R Suzuki 2 and AB Miura 1 1 Department of Internal Medicine III, Akita University School of Medicine, Akita; and 2 Department of Immunology, Shionogi Institute for Medical Science, Osaka, Japan Summary: Recipients of allogeneic bone marrow grafts have clon- ally expanded CD8 + CD28 - T lymphocytes during the early period after transplantation, which leads to skew- ing of T cell receptor (TCR) repertoires. Here, we have addressed the question of whether clonal expansion of CD28 - T cells is also observed in CD4 + T lymphocytes after human allogeneic hematopoietic cell transplan- tation. We found that the fraction of T cells lacking CD28 expression in the CD4 + subset was increased after transplantation, and expanded CD4 + CD28 - T lympho- cytes carrying certain TCRBV subfamilies showed lim- ited TCR diversity. In order to further study the ontogeny of CD4 + CD28 - T cells, we analyzed the complementarity-determining region 3 (CDR3) of the TCR-b chain of CD4 + CD28 + and CD4 + CD28 - cells. We identified the same T cell clones within both CD4 + CD28 - and CD4 + CD28 + T cell subsets. These results suggest that both subsets are phenotypic variants of the same T cell lineage. Bone Marrow Transplantation (2001) 27, 1095–1100. Keywords: TCR-b; CDR3; clone; CD4; CD28 The CD28 molecule is a disulfide-linked homodimer expressed on the surface of T cells and it binds to the natu- ral ligand B7 family members, CD80/CD86, expressed on antigen-presenting cells, resulting in costimulation of T cell activation. 1 In humans, all thymocytes and the vast majority of peripheral blood T cells express CD28 at birth. An increase of the proportion of CD8 + T cells lacking CD28 expression has been demonstrated in the aged population, 2 HIV-infected individuals 3,4 and bone marrow transplant recipients. 5,6 In contrast, in healthy individuals, only a few percent of CD4 + T cells lack CD28, 3 and expansion of CD4 + CD28 - T cells is scarcely reported in rheumatoid arthritis patients. 7,8 Correspondence: Dr M Hirokawa, Akita University School of Medicine, Department of Internal Medicine III, 1-1-1 Hondo, Akita 010–8543, Japan Received 19 January 2001; accepted 13 March 2001 Loss of CD28 expression has recently been implicated in lymphocyte senescence. 9,10 We have previously reported the oligoclonal expansions of CD8 + CD28 - T lymphocytes in recipients of allogeneic bone marrow grafts and the dem- onstration of identical T cell clones within both CD8 + CD28 - and CD8 + CD28 + T cell subsets. 11 These results sug- gest that clonally expanded CD8 + CD28 - T cells after allo- geneic hematopoietic cell transplantation (HCT) are derived from the CD8 + CD28 + T cell subset, presumably by an antigen-driven mechanism. In the present study, we have addressed the question of whether clonal expansion of CD4 + CD28 - T cells is also observed in recipients of allo- geneic hematopoietic cell grafts and whether these cells are the descendents of CD4 + T cells expressing CD28. Materials and methods Patients Informed consent was obtained from the patients before blood samples were collected. All the patients were con- ditioned with myeloablative chemoradiotherapy, mostly consisting of fractionated total body irradiation (12 Gy in six fractions) and cyclophosphamide (60 mg/kg/day for 2 days), followed by infusion of allogeneic marrow or blood stem cell grafts from HLA-matched donors. All the patients received cyclosporin A and short-term methotrexate for prophylaxis of acute graft-versus-host disease (GVHD). 12 Engraftment was achieved in all patients, and confirmed by recovery of hematopoiesis and the presence of donor- derived sex chromosomes or mismatched antigens on red cells. All patients were seropositive for cytomegalovirus (CMV). Patients were monitored for CMV infection by weekly CMV antigenemia assays from when the granulo- cyte count reached 500/ml until day 100 after transplan- tation. Patients who were positive for CMV antigenemia assay received prophylactic ganciclovir (5 mg/kg/day, 3 days a week) from the time the granulocyte count became greater than 1000/ml.