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There are few reports of clinical presentation, genotype, and hematopoietic cell transplant (HCT) outcomes for T-B+NK+ SCID patients. Between 1981 and 2007, 8 of 84 SCID patients who received and/or were followed after HCT at UCSF had the T-B+NK+ phenotype. One additional T-B+NK+ SCID patient was identified as the sibling of a patient treated at UCSF. Chart reviews were performed. Molecular analyses of IL7R, IL2RG, JAK3 and the genes encoding the CD3 T-cell receptor components δ (CD3D), ε (CD3E), and ζ (CD3Z) were done. IL7R mutations were documented in 4 patients and CD3D mutations in 2 others. Three patients had no defects found. Only 2/9 patients had an HLA-matched related HCT donor. Both survived, and neither developed graft-versus-host disease. Five of 7 recipients of haploidentical grafts survived. Although the majority of reported cases of T-B+NK+ SCID are due to defects in IL7R, CD3 complex defects were also found in this series and should be considered when evaluating patients with T-B+NK+ SCID. Additional genes, mutations in which account for T-B+NK+ SCID, remain to be found. Better approaches to early diagnosis and HCT treatment are needed for patients lacking an HLA-matched related donor.
Classic SCID patients have a profound defect in T cell numbers and T and B cell function, while B cell and NK cell numbers are variably affected. Severe combined immunodeficiency (SCID) is lethal early in life unless the cellular immune system is reconstituted. Over 14 disease genes for SCID are known (1-29) and a classification of cellular phenotypes has been established that correlates lymphocyte subset profile with the effects of impairment of particular immune cell developmental pathways.
The T-B+NK+ SCID phenotype has been reported for patients with mutations of genes encoding IL-7Rα, CD3δ, CD3ε, and CD3ζ although IL2RG (encoding the common γ chain γc defective in X-linked SCID) and JAK3 mutations as causes of this phenotype have also been seen. (27, 30) Currently, the literature contains 49 cases of the IL-7Rα chain deficiency (12-15, 28, 31-38), 8 cases of CD3δ chain deficiency (39, 40), 3 cases of CD3ε chain deficiency (40), and 2 cases of CD3ζ chain deficiency. (41, 42)
Controversy exists regarding the need for conditioning prior to hematopoietic cell transplantation (HCT) in all forms of SCID. In particular, limited numbers of reports describe the clinical outcome of T-B+NK+ SCID patients after HCT, and it is not clear whether the presence of host NK cells has an impact on their outcome. (12, 13, 20, 32-34, 36, 43-50) Most published series of these patients were transplanted when the molecular cause of their SCID was still undiscovered. From 1981 to 2007, 8 T-B+NK+ SCID patients were transplanted (n=7) or followed shortly after transplant (n=1) at the University of California San Francisco (UCSF). An additional SCID patient was identified as a sibling of a patient who was treated at UCSF. The aim of our study was to study the incidence of the genetic defects leading to the T-B+NK+ SCID phenotype and evaluate the long-term engraftment and survival of these patients.
All studies were approved by the UCSF Institutional Review Board. From a database of 84 SCID patients transplanted at UCSF between 1981 and 2007, 8 T-B+NK+ patients were identified. An additional T-B+NK+ patient was identified as a sibling of a patient treated at UCSF. A retrospective chart review was performed to summarize their presentations and clinical courses.
Peripheral blood lymphocyte subsets were determined by flow cytometry. T-cell function was assayed using lymphocyte mitogen responses to phytohemagglutinin (PHA), concanavalin A (Con A), and pokeweed mitogen (PWM). A CD3+ T-lymphocyte count less than 300/μL and PHA response <10% of control were considered diagnostic of classic SCID when there was no evidence of maternal engraftment. (46) Serum immunoglobulin levels were analyzed via standard methods.
Seven of the 9 hematopoietic cell grafts were T-cell depleted using standard methods to prevent graft versus host disease (GVHD). (46) Patient 1a received donor stem cells depleted of mature T cells by an anti-CD2 monoclonal antibody and complement, while patient 1b received an undepleted graft from her post-BMT sibling. (48) Both of these patients received cyclosporine for GVHD prophylaxis.
Post-HCT chimerism was determined by in situ hybridization for sex-disparate recipients and donors, quantitative PCR using short tandem repeats (STR) of peripheral blood mononuclear cells (PBMC), or specific lineages separated by magnetic beads. (51, 52) For each leukocyte lineage, mixed chimerism was defined as the presence of 5-95% donor-derived cells.
DNA was prepared from pre-HCT blood or skin fibroblasts from patients and blood from parents if available. Mutational analysis of IL7R was performed for all patients by sequencing the flanking and coding regions of exons 1-8 of the gene via previously published primers. (28) All patients who tested negative for IL7R mutations were subsequently tested for mutations in the genes encoding CD3δ, CD3ε, and CD3ζ chain via previously published methods. (40, 42) All male patients who tested negative for mutations in the IL7R or CD3 subunit genes were tested for mutations in IL2RG via previously published methods. (11, 27) The remaining patients who tested negative for the above mutations were tested for JAK3 mutations.
The patients included 6 females and 3 males from 6 unrelated families. (Table 1) Parental consanguinity occurred in 1 family (P2). The mean age at diagnosis of SCID was 8.9 months (range 2 days to 16 months). Two children (P1b, P4b) were diagnosed soon after birth because of a family history of SCID. Two sisters (P5a and 5b) presented with autoimmune hemolytic anemia (AIHA), one of whom also had immune thrombocytopenic purpura (ITP). The remaining patients presented with typical clinical features of SCID including failure to thrive, diarrhea, and recurrent and/or opportunistic infections.
The primary immunologic characteristics of the patients at presentation are shown in Table 2. All patients presented with lymphopenia for their age. (53) The number of circulating CD3+ T lymphocytes was markedly depressed in all patients, ranging from undetectable to 238/μL. Maternal T-cell engraftment occurred in one patient (P5b). All patients had a decreased in vitro response to PHA of less than 10% of normal control except for one patient (P1b) where it was not measured. B cell numbers were normal to increased in all patients (79/μL to 1818/μL). As expected due to maternal transfer, Immunoglobulin levels showed variability in IgG levels. Circulating NK lymphocyte numbers were normal (range 63/μL to 728/μL).
Four patients from 2 families (1 and 5) were found to have IL7R mutations (Table 3). All had the previously published single nucleotide change in exon 5 cDNA 638C>T, causing the formation of a premature stop codon R206X. (13) The brother and sister pair P4a, P4b had a previously published nonsense mutation in exon 2 of CD3δ, cDNA 202C>T leading to the formation of a premature stop codon R68X. (39, 40) There were no mutations found in the coding or splice regions of the CD3E, CD3Z, IL2RG, or JAK3 genes. Adenosine deaminase and purine nucleoside phosphorylase levels were normal in 5 patients (P1a, P1b, P2, P4a, and P5a) in whom these were tested.
Despite significant infections or autoimmune disease in 7 of the 9 patients, all were treated with HCT (Table 3). The mean age at transplantation was 10.2 months (range 5 weeks to 16 months). From 1987 to 1994, the source of cells for transplant was bone marrow. After 1994, peripheral blood stem cells mobilized with G-CSF were used for transplantation. The only exceptions were one HCT with marrow from an HLA-identical sibling (P5b) and one marrow from an HLA-matched sibling affected with SCID who had previously been reconstituted by paternal haploidentical bone marrow (P1b). (48) No patients experienced graft-versus-host disease.Seven patients were treated with haploidentical related HCTs (Table 3). Two of the 7 patients did not receive conditioning prior to HCT.
Seven of the 9 patients (78%) remain alive a mean of 16 years post-HCT (range 6 to 23 years) (Table 3). All survivors have normal CD3 counts except for P1b and P4b. The in vitro response to PHA improved or normalized in all survivors. In addition to one patient who did not receive a conditioning regimen(P1a), the other 4 patients [who received conditioning regimens (P3, P4b, P5a, P6)] have full B-cell reconstitution. All survivors have been followed between 6-23 years post transplant. All are healthy except for P3, who has short stature, osteochondromas and bilateral cataracts, presumably from the TBI he received 22 years ago.
The majority of cases of T-B+NK+ SCID are reportedly caused by deficiency of the IL-7 receptor α chain, encoded by the IL7R gene. We found 4 of our 9 patients in 2 families had the IL-7 receptor alpha chain deficiency. Less commonly reported causes of T-B+NK+ SCID are defects in the genes encoding the CD3δ, CD3ε, and CD3ζ chains involved in the intracellular transmission of signals after TCR recognition. We discovered that 2 siblings in another family had the CD3δ chain deficiency. Sequencing for X-linked SCID, which is due to mutations in IL2RG, was also performed because it is the single most common genetic form of this disease and leaky phenotypes with substantial numbers of NK cells have been reported. Although no IL2RG defects were found in this series, sequencing for IL2RG along with IL7R and CD3D, CD3E, and CD3Z should be a considered when evaluating children with this immune phenotype. If these genes do not harbor defects, defects in JAK3 and CD45 should be considered. Although autosomal JAK3 defects are a less common cause of SCID than X-linked IL2RG, these genotypes are phenotypically similar in all other respects. Determining the genetic defect is important for genetic counseling and possibly in the approach to transplant.
The role of conditioning in HCT for SCID remains controversial. Engraftment without pretransplant chemotherapy may be possible because SCID patients lack T-cell immune function and cannot easily reject their grafts. (32) This is certainly true for children with HLA matched sibling donors and both patients (P1b and P5b) in this study with HLA matched HCT did not receive pretransplant chemotherapy and durably engrafted. However, engraftment may be more problematic for mismatched alternative donor transplants. A European analysis of 178 SCID patients treated with HLA non-identical T-cell depleted BMT showed that a conditioning regimen of busulfan and cyclophosphamide resulted in higher engraftment rates. (49) However, a large European experience with 475 SCID patients did not show significant differences in survival between regimens that included conditioning and those that did not. (47) A U.S. center with 48 SCID patients saw a trend towards increased survival in the group that received conditioning, although statistical significance was not achieved, possibly because of the small patient numbers. (35) It is believed that NK cells mediate engraftment resistance. (54-56) Recently, a prospective pilot study of 15 consecutive patients undergoing haplocompatible transplants for SCID without conditioning showed that megadoses of CD34+ cells with a fixed dose of CD3+ cells resulted in 87% survival at a median of 39 months post transplant. (57) All of the NK- SCID patients engrafted but only 43% of the NK+ SCID patients who did not have detectable maternal cells pre-transplant engrafted without conditioning, further suggesting that host NK cells are capable of mediating donor hematopoietic stem cell rejection. In our experience (M. Cowan, unpublished data), the presence of NK cells in patients with SCID is associated with normal NK function in vitro.
Our patient numbers were too small to yield statistical conclusions regarding conditioning in haploidentical transplants. However, analysis of the individual patients receiving T-cell depleted haploidentical transplants showed that those treated with conditioning had a higher survival rate than those treated without conditioning. This is despite the fact that the patients with conditioning presented on average with more pre-transplant morbidity, were transplanted later that those patients without conditioning, and received similar doses of CD34+ stem cells. Furthermore, the one NK+ SCID patient who died (P4a with CD3δ deficiency) was resistant to engraftment and required 2 additional HCTs with successively more myeloablative conditioning regimens. This suggests that transplant without conditioning would have been unsuccessful as well. However, our sample size was small and larger numbers of patients are needed to address the issues of whether host NK cells mediate graft resistance and whether conditioning improves survival and long-term engraftment in haploidentical transplants for NK+ SCID patients.
Analysis of the surviving patients who successfully engrafted showed evidence of mixed T chimerism with variable amounts of B and NK mixed chimerism. Our sample size was too small to determine whether the conditioning regimen affected donor chimerism.
It is well known that hematopoietic cell transplantation can cure SCID, with the highest success rates occurring with HLA identical related donors. Our outcome data for T-B+NK+ SCID patients at UCSF is similar to that from other centers; our patients have had 100% survival following HLA identical related transplants and 71% survival following haploidentical transplants. (32, 35, 47) However, HLA identical related donors are frequently not available and active infection often prevents a prolonged search for unrelated donors.
Determining the genetic defect is important for genetic counseling and possibly in the approach to transplant. For example, in patients with IL-7Ra deficient SCID, B cells can function once T cell immunity is restored so that myeloablative therapy may not be needed in order to restore both T and B cell immunity. (58) Consistent with this, patients with IL-7Ra deficient SCID in this case series (P1a, P1b, P5a, P5b) appeared to require less conditioning for engraftment compared to patients with other genetic defects and yet all had restoration of B cell function post transplant.
In order to improve the survival of SCID patients, it is important to determine optimal transplant regimens. Randomized, controlled, multi-center studies of treatment protocols are needed to find the optimal approach for children with SCID. However, such studies have not yet been done, in part due to the rarity of SCID and the multiplicity of different infectious exposures and underlying mutations and phenotypes that characterize SCID. Nonetheless, collaboration in this effort is imperative in order to give these children the best possible outcomes.
Those patients who were diagnosed at a young age because of family history of SCID (P1b, P4b) had no infectious complications, improved survival, and far fewer complications post HCT compared to patients who were diagnosed later and suffered from infectious complications prior to their diagnosis and treatment of SCID. The two patients who died in this case series after haploidentical HCT succumbed to infectious complications. P2 received a haploidentical transplant at 12.5 months without conditioning but, despite evidence of engraftment, succumbed to RSV infection. P4a presented with cytomegalovirus (CMV) pneumonia and hepatitis at 14 months of age. As a result his conditioning regimen was attenuated to prevent disseminated CMV infection. Unfortunately he did not engraft despite 3 haploidentical transplants, each with successively more myeloablative conditioning regimens. He ultimately succumbed to disseminated CMV infection. Our experience reinforces the published data that survival is improved if SCID is diagnosed at an early age, prior to infectious complications, and transplant occurs before 3.5 months of age. (20, 47, 49) As a result, it is critical to diagnose infants with SCID via newborn screening programs (59).
We are grateful to Gabriel Tsao M.D. and Irene H. Jun, M.D. and for their invaluable work in reviewing patient charts and Thierry Giffon Ph.D. for valuable assistance in the genetic analyses of our patients. This work was made possible by the American Academy of Pediatrics Residency Research Grant and the Stanford University School of Medicine Medical Scholars Grant. MJC and JP are supported by NIH U54 AI082973-01.