Case report Split chimerism after allogeneic bone marrow ... - Nature

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Case report Split chimerism after allogeneic bone marrow transplantation in Chediak–Higashi syndrome S Yamazaki1, H Takahashi1, H Fujii1, T Miyamae1, M Mori1, K Fujioka1, T Funabiki1, S Yokota1, N Arai2 and K Ikuta2 1 Department of Pediatrics, Yokohama City University School of Medicine, Yokohama, Japan; and 2Department of Blood Transfusion, Yokohama City University School of Medicine, Yokohama, Japan

Summary: Chediak–Higashi Syndrome (CHS) is a hereditary multiorgan disease associated with a lymphoproliferative disorder termed ‘accelerated phase’ (AP). As AP is often life-threatening, hematopoietic stem cell transplantation has been proposed as the only curative treatment for CHS. Here, we report a 1-year-old Japanese boy with CHS who received an HLA-matched unrelated BMT at the AP stage, which resulted in split chimerism. We evaluated the chimerism status of isolated leukocytes and found that only a limited population of T and NK cells was of donor origin and the majority of these and other hematopoietic cells was of host origin. Clinical outcome was successful, and the patient is currently alive and well, free of AP and serious infections more than 18 months after BMT. Bone Marrow Transplantation (2003) 31, 137–140. doi:10.1038/sj.bmt.1703789 Keywords: Chediak–Higashi syndrome; split chimerism; T,NK and hematopoietic stem cell engraftment

Chediak–Higashi syndrome (CHS) is a rare autosomal recessive disease characterized by partial albinism, susceptibility to bacterial infections, giant lysosomes in granulocytes, defective phagocyte, lymphocyte and NK cell function, and cranial neuropathies.1 In total, 85% of patients with CHS experience a hemophagocytic-syndrome-like lymphoproliferative disorder termed ‘accelerated phase’ (AP), characterized by infiltration of multiple organs with activated T lymphocytes and macrophages. This leads to fever, hepatosplenomegaly, pancytopenia, coagulation disorders, and infiltration of the CNS. Recently, the lysosomal trafficking regulator gene LYST was found to be mutated in CHS, resulting in impaired lysosome-mediated cellular functions.2 Among these, defective cell surface expression of the immunoregulatory molecule, CTLA-4 (CD152), is considered to be responsible

Correspondence: Dr H Takahashi, Department of Pediatrics, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan Received 5 April 2002; accepted 17 September 2002

for the development of AP, because it is essential for the cessation of ‘overactivated’ immune responses.3 Since the development of AP is often life-threatening, allogeneic hematopoietic stem cell transplantation (HSCT) has been proposed as the sole curative therapy for CHS.4 In this report, we describe a patient with CHS who achieved split chimerism after HLA-matched unrelated BMT. It was characterized by limited numbers of donor-derived T cells, NK cells, and hematopoietic stem cells. We suggest that partial engraftment of T and NK cells is sufficient to prevent AP and improve clinical outcome.

Case Report A 4-month-year-old Japanese boy was referred to our hospital in November 1999 for investigation of splenomegaly, anemia, and thrombocytopenia. On physical examination, he presented with gray hair, fair skin, photophobia, and hepatosplenomegaly. Laboratory tests revealed pancytopenia and elevated transaminases. He was diagnosed as suffering from CHS on identification of disease-specific giant lysosomes (Chediak-Higashi granules) in erythroid and myeloid cells. After admission, he entered AP more than 15 times in 9 months, and was treated with G-CSF, corticosteroids, cyclosporin, and blood transfusions. Although he had no significant neurological symptoms except for the retardation of motor development, cerebrospinal fluid (CSF) examination showed increased cell counts of 38/mm3 and a neopterin level of 169 pmol/ml. MRI imaging of the brain revealed fluctuating inflammation even during periods of clinical stability. Since the patient had no sibling donor, allogeneic BMT from a phenotypically matched, genotypically one-locusmismatched unrelated donor was performed in August 2000. The conditioning regimen consisted of fractionated TBI 240 cGy daily for 5 days (total 12 Gy), followed by etoposide (VP-16) 60 mg/kg daily on day 4, and cyclophosphamide 60 mg/kg once daily on days 3 and 2 (total dose 120 mg/kg). He recieved 6.8 108/kg of bone marrow nucleated cells without any modifications such as T-cell depletion. A short course of methotrexate and FK506 was administered for graft-versus-host disease (GVHD) prophylaxis. It was noteworthy that two episodes of AP, induced by viral gastroenteritis and sepsis caused by central

Bone marrow transplantation in Chediak–Higashi syndrome S Yamazaki et al

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venous catheter infection, were documented. After bone marrow recovery, nearly half of the granulocytes in peripheral blood contained Chediak–Higashi granules. Bone marrow smears also showed erythroid and myeloid cells with abnormal granules, suggesting failure of engraftment. To test the chimerism status of the patient, we performed microsatellite analysis using highly polymorphic variable number of tandem repeats (VNTRs) markers, and found that more than half of the peripheral mononuclear cells were of recipient origin (Figure 1). These findings indicate that the patient had achieved mixed chimerism. With the expectation of inducing a graft-versus-host-cell effect, we immediately began to taper off FK506. A week later, on day +45, acute GVHD (grade II, skin stage 3) was documented. The eruption decreased once on day +55, recurred on day +79, and persisted until it diminished spontaneously on day +117. Contrary to our expectations, examination of bone marrow smears disclosed that most of the erythroid and myeloid cells still contained Chediak– Higashi granules, indicating recipient derivation, despite two episodes of GVHD. Despite this recipient-dominant bone marrow recovery, clinical manifestations have improved markedly. No new signs of AP have occurred and neurological development is progressing satisfactorily. Some examinations also corroborate neurological improvement, with MRI showing diminishing infiltration of the CNS (Figure 2), and decreasing cell counts as well as neopterin level in the CSF. Regarding hematological and immunological parameters, neutropenia persists at approximately 500 106/l and cells still contain Chediak–Higashi granules. Peripheral blood T- and B-cell populations are nearly normal with normal antigen-specific responses (data not shown). NK cell activity is low but nearly normalized and stable at 12%, which is much better than pre-BMT (3%). We hypothesize that BMT resulted in split chimerism for several reasons. First, all granulocytes and erythroblasts are entirely recipient-derived. Second, CNS infiltration and NK cell activity have improved. Third, AP has never recurred after BMT when the patient developed an infection. Moreover, microsatellite analysis using VNTRs revealed mixed chimerism. To test this hypothesis further,

Figure 2 MRI image of the brain before and after BMT. Hyperintensity areas around the lateral ventricule and cerebellar peduncle observed before BMT (arrows in (a)) were diminished completely 6 months after BMT with no evidence of new foci (b).

Figure 3 Lineage-specific chimerism analysis after BMT. Myelocytes, monocytes, B cells and T cells (Dynabeads M 450-CD15, -CD14, -CD19, and -CD3, respectively, Dynal, Oslo, Norway), and NK cells and HSCs (MACS CD56 and AC133 MicroBeads, respectively, Miltenyi BioTec, Auburn, CA) were immunomagnetically collected either from peripheral blood (a) or bone marrow (b) and subjected to microsatellite analysis using HGH gene (a) or HUMACTBP2 gene (b) polymorphism, respectively. The arrow and arrowhead indicate donor-specific bands.

Figure 1 Microsatellite analysis after BMT. Genomic DNA either pre- or post-BMT, as well as from donor cells, was extracted and subjected to microsatellite analysis using PCR amplification of the polymorphic region of HUMACTBP2 gene. The arrow indicates a donor-specific band. Bone Marrow Transplantation

we sorted T cells (CD3+), NK cells (CD56+), B cells (CD19+), monocytes (CD14+), and myeloid cells (CD15+) using monoclonal antibodies labeled with immunomagnetic beads, and subjected these subpopulations to microsatellite analysis. As shown in Figure 3a, only limited numbers of T and NK cells were derived from the donor, the majority being recipient-derived. Next, we analyzed whether donor-derived hematopoietic stem cells


(HSCs) were even present, because mature T and NK cells might only be transferred concurrently with BMT. HSCs were sorted by AC133 immunomagnetic beads from bone marrow cells and chimerism assessed. As shown in Figure 3b, most HSCs indeed appeared to be of recipient origin; however, there was a small donor-derived population as well. Additionally, expression of CTLA-4 molecules on CD8-positive T cells which was not detected before BMT, was confirmed by fluorescence-activated cell sorter analysis (data not shown).

Discussion Since the development of AP is often a life-threatening event in patients with CHS, HSCT is proposed as the only curative treatment, especially for cases developing AP early in childhood. In our case, the first AP occurred 3 months after birth and recurred frequently even after he was admitted to hospital, so HSCT had to be carried out as soon as possible. There are some encouraging reports for HSCT in CHS.4–6 Despite myeloablative conditioning our patient did not achieve full chimerism, and engraftment of donor cells was restricted to a limited population of only T and NK cells. For patients with CHS, engraftment of T cells is the most important factor in preventing AP. Haddad et al4 reported four of seven long-term survivors, who retain a stable mixed chimerism without recurrence of AP.4 If only a small population of T cells that expresses normal CTLA-4 is enough for this purpose, aggressive HSCT conditioning may not be necessary.7 Unlike in malignant diseases where fully ablative conditioning is required to eliminate the tumor, most nonmalignant diseases could probably be cured with low levels of donor chimerism.8 Moreover, using nonmyeloablative conditioning regimens as a means of reducing short- and long-term toxicity, rapid engraftment might offer significant advantages.7–9 Split chimerism after HSCT is commonly reported, especially in cases of congenital immunodeficiency syndrome such as severe combined immunodeficiency (SCID).10,11 However, the pattern of split chimerism in our case diverges somewhat from that found in SCID. In the cases of SCID, T cells were predominantly of donor origin, whereas B, NK, and myeloid cells were either of recipient, donor, or mixed origin.10,11 In our case, B, myeloid, and erythroid cells are solely of recipient origin whereas T and NK cells are mixed. Among them, the most characteristic difference in our case is the coexistence of both recipient- and donor-derived T cells, which indicates immune tolerance. Induction of a graft-versus-host-cell effect by immediate reduction of the dose of immunosuppresant seemed to be ineffective against host blood cells. On the contrary, immune tolerance was easily obtained after two episodes of GVHD, possibly because the underlying disease was CHS. It is also reported that the number of CD3-positive T cells transplanted is the most important factor for engraftment,12 and it is now understood that Tcell depletion increases the risk of failure of engraftment.13 However, the precise mechanisms of the development of split chimerism as well as the induction of immune

tolerance remain to be elucidated. In our case, AP occurred just before transplantation, so such upregulated immunological activity may itself have contributed to incomplete engraftment. From the results of chimerism analysis of AC133-positive cells, we anticipate that T and NK cells of donor origin may persist indefinitely, although longer follow-up is necessary. Since the immunosuppressive function of CTLA-4 depends entirely on the antigen specificity of the corresponding T-cell receptor, the fact that AP has never developed even at the time of various infections (ie antigenic stimuli) after BMT indicates that T-cell neogenesis and thymic education are indeed occurring. Alhough BMT appeared to have failed at the cytological level, it was clinically successful, and the patient is currently alive and well with no recurrence of AP on viral infection for 18 months. Additionally, bacterial infection has not recurred although myelocytes and monocytes are all of recipient origin, and mild neutropenia persists. We have no explanation as to why the differentiation of donor-derived HSCs is restricted to only T and NK cells. Therefore, close clinical monitoring is necessary to determine the status of immune function, to assess the consequences of stable mixed chimerism, and to evaluate the long-term benefits of BMT.

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