Bone Marrow Transplantation, (1999) 23, 783–788 1999 Stockton Press All rights reserved 0268–3369/99 $12.00 http://www.stockton-press.co.uk/bmt Serum levels of IL-7 in bone marrow transplant recipients: relationship to clinical characteristics and lymphocyte count E Bolotin, G Annett, R Parkman and K Weinberg Divisions of Research Immunology and Bone Marrow Transplantation and Hematology/Oncology, Department of Pediatrics, Childrens Hospital Los Angeles, University of Southern California School of Medicine, Los Angeles, CA, USA Summary: interactions with the microenvironment of the bone marrow and lymphopoietic organs. The ability of the stromal cells IL-7 is produced by stromal cells and is the major lym- to support lymphopoiesis after BMT is adversely affected pho- and thymopoietic cytokine. IL-7 induces prolifer- by age, graft-versus-host disease (GVHD), irradiation, ation and differentiation of immature thymocytes, and immunosuppressive therapy and infections. However, the protects thymocytes from apoptosis by induction of bcl- recipients of allogeneic BMT who do not have GVHD and 2 expression. The regulation of IL-7 production is recipients of autologous BMT are also immune poorly characterized, although down-regulation by deficient.10–16 transforming growth factor-␤ (TGF-␤) has been There is growing evidence that production of T lympho- described. We measured the serum levels of IL-7 before cytes is dependent upon interleukin-7 (IL-7). IL-7 is a cyto- and after bone marrow transplant (BMT) in 32 children kine produced by bone marrow and thymic stroma. IL-7 undergoing BMT for genetic diseases (severe combined was originally identified by its ability to induce prolifer- immune deficiency (SCID) and thalassemia), aplastic ation of B cell progenitors in vitro and in vivo and was anemia, and acute lymphoblastic and non-lymphoblas- subsequently shown to stimulate T lymphopoiesis.17–26 tic leukemia (ALL and ANLL). Prior to BMT, the high- Recently, we have shown that administration of IL-7 sig- est IL-7 levels were observed in patients with SCID and nificantly enhances immune recovery in mice after T cell- ALL, ie those patients with genetic or acquired lympho- depleted BMT.27 Therefore, production of IL-7 may be one penia. Patients with thalassemia and ANLL had normal of the mechanisms regulating de novo production of T lym- levels of IL-7. Over the 8 weeks following BMT, the IL- phocytes after BMT. 7 levels of patients with SCID and ALL fell as the absol- In the present study we analyzed serum IL-7 levels by ute lymphocyte count (ALC) increased. No detectable ELISA in patients before and after BMT in relation to the change in IL-7 levels was observed in the patients with patients’ clinical characteristics and absolute lymphocyte thalassemia and ANLL. Levels of IL-7 were highest in count (ALC). We found that lymphopenic patients and the young infants with SCID compared to the age- patients less than 1 years old had higher levels of serum matched controls. Together, the data demonstrate that IL-7. Understanding of the mechanisms of endogenous IL- serum levels of IL-7 in lymphopenic patients are 7 response to lymphopenia may add to understanding of inversely related to patient age and the absolute lym- the mechanism of immune reconstitution after BMT and phocyte count (ALC). The inverse relationship to ALC chemotherapy. suggests that there is either direct regulation of stromal production or more likely, binding of secreted IL-7 to lymphocytes expressing IL-7 receptors. Materials and methods Keywords: interleukin-7; immune reconstitution; immune deficiency; bone marrow transplantation Patients Normal controls: Sixty-eight samples from controls were Immune deficiency after bone marrow transplantation tested for serum IL-7 levels. All serum samples were (BMT) contributes significantly to morbidity and mortality obtained from blood remaining after routine immunologic from a variety of infections, particularly fungal and viral, testing. Blood samples were obtained from infants undergo- which are normally controlled by functional T and B lym- ing open heart surgery for correction of congenital heart 1–3 phocytes. Lymphopenia is a common finding in patients disease (n ϭ 13), healthy siblings of patients undergoing 4–9 after BMT and its etiology is multifactorial. The growth BMT, or from BMT donors (n ϭ 55). Seventeen controls and differentiation of lymphocytes involve the complex less than 1 years old, 21 children older than 1 year, and thirty adults (Ͼ21 years of age) were included in the study. None of the patients with congenital heart disease had Correspondence: Dr E Bolotin, Divisions of Hematology/Oncology, and Research Immunology and Bone Marrow Transplantation, Childrens Hos- DiGeorge syndrome clinically and by fluorescence in situ pital Los Angeles, 4650 Sunset Boulevard, Los Angeles, CA 90027, USA hybridization for deletion of the DiGeorge syndrome criti- Received 10 August 1998; accepted 16 November 1998 cal region on chromosome 22q11. IL-7 levels in BMT patients E Bolotin et al 784 BMT recipients: All BMT recipients were patients treated (R&D Systems, Minneapolis, MN, USA), according to the on the BMT unit at Childrens Hospital Los Angeles from manufacturer’s instructions. A standard curve was prepared 1992 to 1994. Thirty-two BMT patients were included in for each plate, plotting OD vs different concentrations of the study. Five pre-BMT diagnoses were selected for study recombinant human IL-7. All standards and samples were because they included children of various ages, had either tested in duplicate. normal lymphocyte counts or had congenital or acquired lymphopenias, and had either received or not received Statistical analyses chemotherapy pre-BMT. The diagnostic groups were sev- ere combined immune deficiency (SCID), acute lympho- Mean values and standard deviation were calculated using blastic leukemia (ALL), acute non-lymphoblastic leukemia routine methods. The probability of the differences was (ANLL), ␤-thalassemia major (THAL) and aplastic anemia determined with Student’s t-test, when two groups were (AA). The diagnosis of SCID was based on history and analyzed, and with the use of ANOVA and Student–New- physical examination, lymphocyte count, immunopheno- man–Keuls multiple-range tests to define the unique subsets type, and lack of proliferative responses to mitogens and within the study, when multiple groups were analyzed. All antigens. The patients with ALL had received multi-agent analyses were carried out at the alpha ϭ 0.05 level of sig- chemotherapy and were in either first or second remission. nificance. Correlation statistics were based on the calcu- ANLL patients were transplanted in first remission after lation of the Pearson correlation coefficient. four courses of chemotherapy with dexamethasone, cyto- sine arabinoside, 6-thioguanine, etoposide and adriamycin (DCTER).28 The THAL and AA patients had received sup- Results portive care with transfusions. One patient with AA had received 10 doses of anti-thymocyte globulin (ATG) before Patient characteristics his matched unrelated donor (MUD) BMT. The patient characteristics are shown in Table 1. There BMT preparation, source, and post-BMT care: All patients were seven patients with SCID, 12 with ALL, six with except five received pre-BMT conditioning with busulfan ANLL, three with thalassemia, and four with AA. There (1 mg/kg p.o. every 6 h for 16 doses total) followed by were no statistically significant differences between patients cyclophosphamide (50 mg/kg i.v. daily for four doses). One with regard to age, sex and race (not shown), except for patient with SCID received four doses of anti-thymocyte the SCID patients who were younger than the rest of the globulin (ATG) before his histocompatible BMT, and did patients and were predominantly boys. The mean age of Ϯ not receive marrow ablative therapy. The preparative regi- the patients with SCID was 0.53 0.32 years, compared Ϯ men for the four patients with AA consisted of total lymph- to the mean of 7.0 2.8 years for the rest of the patients. oid irradiation (TLI), cyclophosphamide 50 mg/kg i.v. daily The high proportion of boys in the SCID group was due for two doses, and ATG. to the presence of four patients with X-linked SCID. The GVHD prophylaxis was administered to all patients, type of donor cells used was related to the diagnosis: hap- except those who received autologous marrow. Recipients loidentical, T cell-depleted BMT was only used for the of allogeneic marrow, who were less than 10 years of age SCID patients, autologous BMT only for the ALL and received methotrexate (MTX) 10 mg/m2 on days 1, 3, 6 ANLL patients, and MUD BMT only for the ALL and and 11 after BMT, while those greater than 10 years of age AA patients. received MTX plus cyclosporin A (CsA). Recipients of T cell-depleted marrow were treated with methylprednisolone Normal control 2 mg/kg/day from days 5–18 and ATG every other day for 5–7 days from days 5–18. Recipients of matched unrelated Normal absolute lymphocyte count (ALC): The ALC in the ϫ 3 donor (MUD) marrow received MTX, CSA, and ATG. control group ranged from 1176 to 3986 10 /ml. Higher The research protocols for BMT and investigation of ALC were observed in children less than 1 year old, com- Ϯ ϫ 3 immune function in BMT, normal donors, and cardiac sur- pared to older children and adults (2696 698 10 /ml; Ϯ ϫ 3 Ϯ ϫ 3 gery patients were approved by the Committee on Clinical 2143 1012 10 /ml, 1610 837 10 /ml, Investigations (Institutional Review Board) of the Childrens respectively). Hospital Los Angeles. Normal IL-7 levels: The serum IL-7 levels were low in all Ϯ ϭ Blood samples normal adults (2.82 1.3 pg/ml, n 30) and children over 1 year of age (4.32 Ϯ 3.9 pg/ml, n ϭ 21).