SCID is a clinically defined group of monogenetic diseases characterized by life-threatening infections in early infancy. One variant of SCID is due to defects in the Zeta-chain-associated protein (ZAP-70; MIM+176947), which results in deficient signal transduction from the TCR to the nucleus and abnormal maturation and function of thymocytes. A characteristic finding is a complete lack of circulating CD8+ T cells, whereas CD4+ T cells are present, but without function.1 Although ZAP-70 is not expressed in B cells, humoral immunity is indirectly affected because of inadequate T-cell help. Currently, hematopoietic cell transplantation represents the only curative treatment.1
We diagnosed ZAP-70 deficiency in two brothers born to consanguineous parents of Turkish origin. The family history was positive for early infant deaths in two female cousins. The older brother (sibling 1) presented at the age of 5 months with severe life-threatening pneumonia caused by Pneumocystis jirovecii, requiring mechanical ventilation for 3 weeks. In addition he developed cholestatic liver disease. No infectious agent was isolated and a biopsy revealed evidence of toxic cholangitic hepatitis with portal fibrosis. Lymphocyte phenotyping and proliferation assays showed typical findings of ZAP-70 deficiency. A homozygous missense mutation (c.C1153T) was detected in Exon 10 of the ZAP70 gene leading to a change of the amino-acid sequence from arginine to cysteine at position 385 on protein level (p.Arg385Cys), a mutation in the kinase domain of ZAP-70 not yet described. The parents and the healthy sister were found to be heterozygous carriers of this mutation.
At the age of 8 months, the patient received an unmanipulated BM graft from a matched unrelated female donor following myeloablative conditioning with radioimmunotherapy (Yttrium-CD66-Ab, BM dose 17 Gy), Melphalan (140 mg/m2), Fludarabine (160 mg/m2) and ATG (Sangstat, rabbit 10 mg/kg). The transplant course was uneventful, resulting in complete hematological and immunological recovery and full-donor chimerism. Over the subsequent months, however, liver function further deteriorated leading ultimately to liver failure and the need for organ transplantation, which was performed successfully 2 years after BMT. Currently the patient is doing well with stable function of both grafts.
Shortly before the liver transplant, a younger brother (sibling 2) was born, also affected by ZAP-70 deficiency. The parents objected to any kind of conditioning before transplantation in this child because of potential toxicity. As both brothers turned out to be HLA-identical, SCT with a graft donated by sibling 1 was a consideration to obviate conditioning. This option, however, was rejected for medical and ethical reasons. Instead, we opted for an alternative approach, mimicking an HLA-identical HCT by combining two cell sources, namely highly purified CD34+ peripheral stem cells from the original donor for sibling 1, devoid of GvHD-inducing potential, and PBLs obtained from sibling 1, as a source of functioning, yet tolerant T cells derived from the matched unrelated female donor. The parents gave written informed consent to this procedure, in accordance with the recommendations of the local Ethics Committee. At the age of 4 weeks, sibling 2 received a transfusion of 40 mL of heparinized peripheral blood donated by sibling 1. The T-cell content was 1.2 × 107/kg (body weight of the recipient). No conditioning or GvHD prophylaxis was given. At 3 weeks later, CD8+ T cells (125 cells/μL) were detected in the peripheral blood of sibling 2, corresponding to a similar proportion of blood lymphocytes with female (donor) karyotype. No signs of GvHD were observed. At 4 weeks after this transfusion, purified peripheral stem cells from the common unrelated donor were transplanted, containing 1.0 × 107 CD34+cells/kg and 2.2 × 104 CD3+cells/kg. This was followed 2 weeks later by a second blood cell transfusion from sibling 1 (1.1 × 107/kg CD3+ cells). T-cell counts increased, T-cell functions became normal within a few weeks and Ab responses were positive after triple vaccination (hexavalent vaccine plus pneumococcal conjugate vaccine) (Table 1). The patient has remained free of serious infectious problems, for more than 2 years after treatment.
Repeat chimerism analysis (XX/XY-FISH) showed a mixed chimerism of CD3+ cells, with a stable proportion of 25–50% of donor type (selection by EasySep, Stemcell Technologies, Grenoble, France; purity >98%). In contrast, B cells, monocytes and granulocytes remained exclusively autologous, indicating lack of stem cell engraftment (Table 2). To differentiate whether the observed T-cell reconstitution was due to thymic maturation of precursor cells, delivered by the CD34+ cell graft, or by the peripheral expansion of mature T cells, delivered by blood cell transfusions from the brother, we analyzed the T-cell sub-populations in detail. Blood mononuclear cells were stained with antibodies to CD3, CD4, CD45RA and CCR7 (Becton Dickinson, Heidelberg, Germany); they were separated by FACS (FACSaria, Becton Dickinson) into naïve and memory cell compartments; and they were submitted to STR analysis to identify their origin. As indicated in Table 2, no naïve CD4+ T cells of donor type were identified, whereas memory CD4+ T cells were of mixed donor and recipient origin. CD8+ T cells were exclusively of memory phenotype and all originated from the donor. This finding indicates that T-cell reconstitution was exclusively the result of peripheral expansion of donor T cells and not of thymus-dependent T-cell maturation, indicating that the additional transplantation of purified CD34+ cells in our patient was most likely dispensable.
Successful serial marrow transplantation in sibling pairs with primary immunodeficiency has been described in single cases.2, 3, 4 In the setting described here, stem cell donation by the older brother was not possible because of his critical liver disease. The imitation of a matched sibling graft by the combination of two cell sources provided a chance to establish immune functions without conditioning and with a low risk of inducing GvHD. In our case, T-cell functions were restored exclusively by peripheral expansion of transfused mature T cells, as indicated by the lack of evidence of marrow engraftment and thymic maturation of donor cells. Most likely, thymic engraftment of lymphatic precursors was hampered by the occupation of thymic niches by autologous ZAP-70-deficient precursor cells. This approach to restore a T-cell system just by peripheral expansion of mature T cells has previously been described to be successful in single cases with complete thymic aplasia due to DiGeorge syndrome,5 and it may be particularly attractive in diseases with defects strictly confined to the T-cell system (ZAP-70 deficiency, CD3-deficiencies and DiGeorge syndrome),5 as autologous B cells will resume normal function once adequate T-cell help is established as demonstrated here. Although the long-term stability of immune functions in our patient provided by the expansion of transfused donor T cells cannot be predicted, our findings indicate that the strategy of transfusing peripheral T cells derived from an HLA-identical sibling donor as the sole therapeutical maneuver can be a potentially life-saving procedure.
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The authors declare no conflict of interest.
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Hönig, M., Schuetz, C., Schwarz, K. et al. Immunological reconstitution in a patient with ZAP-70 deficiency following transfusion of blood lymphocytes from a previously transplanted sibling without conditioning. Bone Marrow Transplant 47, 305–307 (2012) doi:10.1038/bmt.2011.71
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