Case Report Bone Marrow Transplantation As Treatment for X-Linked Immunodeficiency with Hyper-Igm

Bone Marrow Transplantation, (1998) 22, 1111–1114  1998 Stockton Press All rights reserved 0268–3369/98 $12.00 http://www.stockton-press.co.uk/bmt Case report Bone marrow transplantation as treatment for X-linked immunodeﬁciency with hyper-IgM

P Bordigoni1, B Auburtin1, A-S Carret1, A Schuhmacher1, J-C Humbert1, F Le Deist2 and D Sommelet1

1Bone Marrow Transplantation Unit, Children’s Hospital, Nancy; and 2INSERM U 429, Hoˆpital Necker, Paris, France

Summary: prone to autoimmune diseases and neutropenia, and are at increased risk of carcinomas of the liver, pancreas, or We report a 10-year-old boy with a severe form of biliary tree.1,6 immunodeficiency with hyper-IgM who underwent suc- For this reason, allogeneic bone marrow transplantation cessful bone marrow transplantation with his HLA- (BMT) from HLA-identical siblings, usually beneficial for matched sister as donor. Busulfan (20 mg/kg) and cyclo- patients with genetic disorders affecting marrow, could be phosphamide (200 mg/kg) were used as conditioning. performed in young patients with defects of CD40 L. We The post-transplant course was uneventful. He is alive report a successful allogeneic BMT in a child with a severe 25 months later with full hematological and immunolog- from of X-HIM. ical reconstitution. Keywords: X-linked immunodeficiency with hyper-IgM; bone marrow transplantation; CD40 ligand Materials and methods

Case report X-linked immunodeficiency with hyper-IgM (X-HIM) is A boy born in April 1986, without any family history of characterized by undetectable serum levels of IgG, IgA and immune deficiency, was seen for the first time at the age 1 IgE and normal or elevated levels of IgM. The underlying of 3 months for a localized bacille Calmette–Guerin (BCG) defect in these patients is the inability to switch from infection (regional adenitis) after vaccine. This episode IgM/IgD secretion to production of the other immunoglob- resolved after surgery. At the age of 5 months, he ulin isotypes, IgG, IgA and IgE. This rare primary disorder developed a PC pneumonia cured by trimethoprim-sulfame- of immunity is caused by mutations affecting the thoxazole. On the basis of his medical history and low 2,3 expression of CD40 ligand (CD40 L) on T lymphocytes. serum concentrations of IgG and IgA with hyper-IgM The gene encoding the CD40 L is located at Xq26 and (Table 1), he was considered to have X-HIM. Treatment 4 encompasses 13 kb of genomic DNA. CD40 L is a type with intravenous immune globulins was initiated. II integral transmembrane glycoprotein expressed primarly At the age of 6 years, he had recurrent pneumonia due by activated CD4ϩ lymphocytes.3 Interactions between ϩ ϩ to pyogenic bacteria and persistent diarrhea associated with CD40 L activated CD4 T cells and B lymphocytes (that oral and perianal ulcers, leading to severe failure of thrive constitutionally express CD40) serve as a fundamental (height: 10th percentile; weight: 5th percentile) and requir- 5 membrane signal for B cell growth and differentiation. ing total parenteral nutrition. He developed a seronegative This interaction normally mediates immunoglobulin class polyarthritis, successfully treated with nonsteroidal anti- switching by B cells. The consequence of mutations is that inflammatory agents. A cyclic, chronic neutropenia with CD40 L on T cells cannot interact with the CD40 glyco- defective myeloid differentiation in the bone marrow protein on the surface of B cells. In addition, interaction occurred in February 1991. Lack of sustained efficacy of between CD40 L activated T cells and antigen presenting intravenous immunoglobulin and granulocyte colony-sti- cells (which also express CD40) elicits an immune response mulating factor (5 ␮g/kg/day) was recorded. Different treat- to intracellular microorganisms. This may explain the sus- ments were begun including corticosteroids and colchicine, ceptibility of a subgroup of patients to severe opportunistic without effect and the subject had a poor quality of life. A infections particularly those due to Pneumocystis carinii diagnosis of X-HIM was confirmed by the finding of defec- 6 (PC), BCG and cryptosporidium infections. They are also tive expression of CD40 L on the patient’s activated T lymphocytes (F Le Deist, INSERM U429, Hopital Necker, Paris, France). A mutation in the TNF-homology domain Correspondence: Dr P Bordigoni, Unite´ de Transplantation Me´dullaire, Hoˆpital d’Enfants, Centre Hospitalier Universitaire de Nancy, rue du Mor- (L 155 P) was found. The mutation was also identified in van, 54511 Vandûuvre-les-Nancy, France the patient’s mother, but not in his HLA-identical sister. Received 24 February 1998; accepted 19 July 1998 Because of the occurrence of opportunistic infections, BMT as treatment for X-linked immunodeficiency P Bordigoni et al 1112 Table 1 Serum immunoglobulin concentrations and post immunization antibody responses before and after bone marrow transplantation

Time (months) Diagnosis BMT stop Igg immunizationf Normal values before and after BMT −1 +3 +9 +12 ϩ15 ϩ23

IgGa 0.54 4.8 7.2 6.7 7.7 7.1 7.2 11.2 Ϯ 3 IgAa 0 0 0 0.51 0.56 0.7 0.7 1.3 Ϯ 0.7 IgMa 0.7 1.7 0.35 0.57 0.45 0.9 0.6 0.8 Ϯ 0.3 IgG1a NAb 1.1 4.2 4.5 NA 3.9 4.1 2.8–17.4 IgG2a NA 1.6 3.4 2.5 NA 2.1 2.4 0.8–5.3 IgG3a NA 0 0.2 0.2 NA 0.37 0.41 0.2–3.2 IgG4a NA 0 0.2 0.1 NA 0.02 0.05 0.1–1.7 Isohemagglutininsg 0 1 NA 4 16 32 32 16–64 (10−1) Poliovirus (10−1) 1 10 0 NA 40 120 80 128 Ͼ40 2 0 0 NA 50 50 80 128 Ͼ40 3 10 10 NA 80 120 80 512 Ͼ40 Diphteria toxoid 0 0 NA 0.50 NA 0.01 0.6 Ͼ0.1 (kUI/l) Tetanus toxoid 0 0.05 NA 4 3.5 0.4 0.9 Ͼ0.1 (UI/ml) Pneumoc NA 50 NA 600 NA 50 220 Ͼ100 Hibd (␮g/ml) NA 5.7 NA Ͻ0.5 NA 4.3 32.8 Ͼ0.5

aMg/dl. bNot available. cAntibody to Streptococcus pneumoniae (23 serotypes) expressed as the reciprocal of that serum dilution showing a positive signal compared with a standard serum (the pooled sera of 10 patients who had been successfully immunized) which was used for the calculation of the cutoff. dAnti-Haemophilus inﬂuenzae type b capsular polysaccharide (␮g antibody/ml). eImmunoglobulins iv. fImmunization with Hib conjugate vaccines, diphtheria, tetanus toxoids and poliovirus vaccines (1/97, 2/97, 3/97) and 23-valent pneumococcal polysaccharide vaccine (1/97). gAnti-A isohemagglutinins.

sclerosing cholangitis, severe chronic inflammatory bowel T and B lymphocyte phenotyping was performed by disease and because of the known poor outcome of patients indirect immunofluorescence using the following specific who develop such complications, the parents gave informed monoclonal antibodies: anti-CD2, CD3, CD4, CD8, CD16, consent for BMT with the sister acting as donor. Condition- HLA DR, CD19, CD56 and CD14 (all from Becton ing consisted of busulfan (5 mg/kg body weight per day Dickinson). Proliferation assays in response to PHA and for 4 days) followed by cyclophosphamide (50 mg/kg body tetanus toxoid (Pasteur Diagnostic, Marne-la-Coquette, weight per day for 4 days). Prophylaxis against graft-ver- France, final dilution: 1/250) were performed as previously sus-host disease consisted of cyclosporine (3 mg/kg/day as described.7 Normal response was determined as an index a continuous i.v. infusion on days Ϫ1 to 30 and 6.25 mg/kg above 100 for PHA, and as above 15 for tetanus toxoid. twice daily from days 31 to 180 post BMT) and methotrex- Serum immunoglobulin isotypes and IgG subclass concen- ate (15 mg/m2 on day 1 and 10 mg/m2 on days 3, 6 and 11 trations were measured using nephelometry. Isohemaggluti- after BMT). Prophylaxis against infections included hospi- nins were determined by an agglutination assay. talization in a laminar air-flow isolation unit, oral adminis- The patient was immunized at 12, 13 and 14 months after tration of non-absorbable antibiotics and treatment with i.v. BMT with a series of three tetanus and diphtheria toxoids, immune globulin for 6 months (200 mg/kg per week for 3 poliovirus and Haemophilus influenzae type B conjugate months and 400 mg/kg per month for the 3 following vaccines (Hib), and a series of two (12 and 13 months post months). Prophylaxis against PC consisted of trimethoprim- BMT) 23-valent pneumococcal polysaccharide vaccines sulfamethoxazole administered from day Ϫ7 to day Ϫ2 and (Pvax). Antibodies to poliovirus, tetanus and diphtheria from day ϩ30 to day ϩ360. On 3 October 1995, the patient toxoids, Hib capsular polysaccharide and to 23-pneumococ- received 2.7 ϫ 108 nucleated marrow cells/kg from his cal serotypes were detected before and after immunization sister. by enzyme-linked immunosorbent assay tests. Expression of CD40 L was evaluated with a CD40 immunoglobulin fusion protein (CD40-Fc) as described Methods previously.3,9 Mutations in the gene encoding the CD40 L Polymorphonuclear and mononuclear cells were isolated were assessed by LD Notarangelo (Department of Pedi- from heparin-treated blood by exposure to dextran followed atrics, University of Brescia, Brescia, Italy) in the setting of by Ficoll–Hypaque centrifugation. Flow cytometry was the CD40 L mutation database organized by the European performed according to standard protocols with a FACScan Society for Immune Deficiency. We used polymerase chain flow cytometer (Becton Dickinson, San Diego, CA, USA). reaction amplification of minisatellite sequences and of a BMT as treatment for X-linked immunodeficiency P Bordigoni et al 1113 Y-chromosome-specific sequence to document chimerism Table 2 Expression of the CD40 ligand by activated T cells from the in lymphocytes and polymorphonuclear cells. patient before and after BMT

Patient Control Results Expression of CD40 La (%b) The patient’s clinical course after BMT was uneventful. An Before After (1 year) absolute neutrophil count greater than 500 per cubic milli- c meter occurred by day ϩ26, and platelet counts were self- CD69 92 96 91 Ϯ 5 CD40-Fcd 05054 sustaining to levels above 50 000/␮l after day ϩ34. Neither GVHD nor infectious complications were observed. Of aCD40 L expression by activated T cells.3 note, clinical manifestations of X-HIM syndrome, parti- bPercentage of positive cells. cularly chronic inflammatory bowel disease and oral and cControl for cell activation (cells stained with anti-CD69 Moab). perianal ulcers, disappeared in our patient within days of dCD40 immunoglobulin fusion protein.3 BMT, probably as a result of chemotherapy or/and cyclosporine treatment. The patient is currently very well 25 published observation was recently reported by the Necker- months post BMT, has had no significant infections, is Enfants Malades team in Paris.9 The indication was some- attending school, and he is in the 25th percentile for height what different in that BMT was performed in an infant and weight. DNA analysis revealed that more than 95% of because of a family history of two fatal cases of X-HIM lymphocytes and polymorphonuclear cells were of donor syndrome and the occurrence in the patient of PC pneumo- origin. He had full recovery of immune functions by 18 nitis at the age of 7 months. Nevertheless, characteristics months after transplantation. (preparative regimen and GVHD prophylaxis) and outcome of BMT were similar. Moreover, Levy et al,10 in the context B cell immunity (Table 1) of a European survey, reported three other transplants in ϩ children with X-HIM, of whom one died of CMV infection The percentage of B cells (CD19 cells) and absolute B cell and one is alive and well 3 years after transplantation. Fol- counts reached normal levels by 10 months after BMT. low-up is limited for the third boy. Our patient has required After cessation of i.v. immune globulin, normal serum IgM, no antibiotic or antifungal agents since transplantation. IgA, IgG and subclass IgG (IgG1, IgG2, IgG3) values were Moreover, BMT has markedly improved his quality of life. achieved respectively 7, 15, 11 and 15 months after trans- Durable and full T and B cell engraftment was achieved plantation. However, the patient still has subnormal levels within the first 2 months after transplantation. Expression of IgG4. Normal isohemagglutinin titers were achieved 11 of CD40 L by the activated recipient T cells was equivalent months after BMT. In response to immunization, he pro- to the expression of the ligand by an age-matched control duced normal or borderline levels of specific antibodies to subject. Most patients with X-HIM syndrome have symp- poliovirus, tetanus and diphteria toxoids during the first toms consistent with immunoglobulin deficiency which trimester of the second year post BMT. Immunization with include recurrent respiratory infections, otitis media, dia- Hib conjugate vaccine and Pvax unconjugated vaccine rrhoea, stomatitis and oral ulcers.1,6 Nevertheless, an resulted in protective antibody concentrations 18 months increased incidence of PC pneumonia, particularly as the after transplant. presenting feature (43% in a recently published cohort10) in the first year of life, and of chronic, watery diarrhoea T cell immunity with or without cryptosporidium infection has been CD4ϩ, CD8ϩ, CD4ϩ/CD45RAϩ and CD4ϩ/CD45ROϩ reported. These features, consistent with cell-mediated cells reached normal values within the first 8–9 months immunodeficiency, may be explained by disrupted after BMT. Percentage, but not absolute numbers of CD3ϩ CD40/CD40L interactions which lead to reduced IL12 pro- cells took 8 months to normalize. T cell functions became duction by antigen-presenting cells and a defective immune 11 normal within 18 months post BMT. The time to a normal response to intracellular microorganisms. response to PHA was 9 months. Before BMT, in vitro pro- In addition, certain other clinically poor prognostic fea- liferative responses to T cell mitogens were normal, tures deserve emphasis, including sclerosing cholangitis, whereas impairment of antigen-specific, T cell proliferation severe neurological disease and increased incidence of responses (ie tetanus toxoid) was observed.8 After immuni- tumors. In the same, recently published cohort of patients 10 zation, the patient had a normal proliferative response to with CD40 L deficiency, 20% of the boys had sclerosing this antigen 17 months post-BMT. cholangitis, 7% developed hepatic failure necessitating Moreover, expression of the CD40 L by activated T cells liver transplantation and 10.7% progressed to cirrhosis. from the recipient was equivalent to the expression of the Finally, 23% died, mostly from infections early in life ligand by age-matched control subject T cells (Table 2). and/or from liver disease after the first decade. The current survival rate at 25 years is no more than 20%.10 In conclusion, a subgroup of patients with X-HIM syn- Discussion drome presents with clinical and/or laboratory signs of T cell deficiency with a clinical picture of combined immuno- We report a successful BMT in a 10-year-old boy with a deficiency. It has not been possible to correlate such a sev- severe form of X-HIM syndrome. The first and only similar ere phenotype with molecular defects of the CD40 L gene. BMT as treatment for X-linked immunodeficiency P Bordigoni et al 1114 Nevertheless, establishing a database (CD40 L base) of 6 Banatvala N, Davies J, Kanariou M et al. Hypogammaglobuli- CD40 L mutations4,12 could perhaps lead to the recognition nemia associated with normal or increased IgM: a case series of a molecular basis for the clinical heterogeneity of X- review. Arch Dis Child 1994; 71: 150–152. HIM syndrome and may help to better define the indi- 7 Bordigoni P, Bene MC, Bach JF et al. Improvement of cellular cations for allogeneic BMT. immunity and IgA production in immunodeficient children after treatment with synthetic serum thymic factor. Lancet 1982; 1: 293–297. References 8 Ameratunga R, Lederman HM, Sullivan KE et al. Defective 1 Notarangelo LD, Duse M, Ugazio AG. Immunodeficiency antigen-induced lymphocyte proliferation in the X-linked with Hyper-IgM (HIM). 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