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Long term Outcome of Non-Ablative Booster Bone Marrow Transplantation in Patients with Severe Combined Immunodeficiency

Severe combined immunodeficiency (SCID) is a fatal syndrome caused by mutations in at least 13 different genes. It is characterized by the absence of T-cells. Immune reconstitution can be achieved through non-ablative related donor bone marrow transplantation. However, the first transplant may not provide sufficient immunity. In these cases, booster transplants may be helpful. A prospective/retrospective study was conducted of 49 SCID patients (28.7 percent of 171 SCIDs transplanted over 30 years) who had received booster transplants to define the long term outcome, factors contributing to a need for a booster and factors that predicted success. Of the 49 patients, 31 (63 percent) are alive for up to 28 years. Age at initial transplantation was found to have a significant effect on outcome (mean of 194 days old for patients currently alive, versus a mean of 273 days old for those now deceased, p=0.0401). Persistent viral infection was present in most deceased booster patients. In several patients, the use of two parents as sequential donors resulted in striking T and B cell immune reconstitution. A majority of the patients alive today have normal or adequate T-cell function and are healthy. Non-ablative booster bone marrow transplantation can be life-saving for SCID.


Introduction
Severe combined immunodeficiency (SCID) is a fatal syndrome characterized by the absence of T cells and, in some molecular types, also of B or NK cells. 1,2 Without immune reconstitution by hematopoietic stem cell transplantation or gene therapy, infants with SCID will die in the first two years of life. The use of HLA identical or haploidentical allogeneic bone marrow stem cell transplantation without pre-transplant chemotherapy or post-Serum Immunoglobulin and Antibody Measurements-Serum IgG, IgA, IgM and IgE were quantified by single radial diffusion or nephelometry. 13 Anti-diphtheria and antitetanus antibodies were determined by tanned cell hemagglutination 14 or by an ELISA after standard vaccines had been administered, and isohemagglutinins were measured by a microtiter plate assay.
Flow Cytometry and T-Cell Function-Lymphocyte phenotypes were determined by immunofluorescent staining of PBMC or whole blood with labeled antibodies to CD3, CD4, CD8, CD14, CD16, CD20, CD45RA, CD45RO, CD132, CD56, TCRαβ, and TCRγδ from BD Biosciences, San Jose, CA and multi-color flow cytometry. Lymphocyte proliferation was assessed by measuring [ 3 H] thymidine incorporation into PBMCs following culture with the stimuli. 15 T-Cell Depletion-Donor bone marrow was rigorously depleted of T-cells by soybean lectin agglutination followed by two cycles of rosetting with sheep erythrocytes treated with aminoethylisothiuronium bromide, reducing the number of T-cells by a factor of 10,000. 3,16,17 Chimerism-This was detected using karyotyping, fluorescence in situ hybridization or short tandem repeats.
Statistical Methods-Statistical comparisons were made using the Mann-Whitney U test for non-parametric analyses and Student's t-test or Chi-square for parametric data. All analyses were performed using Stata 12 (College Park, Tx).

Results
Of the 49 patients receiving booster transplants, 31 (63 %) are alive today, a survival rate lower than the 80.3% survival rate in the 122 non-boosted SCIDs (Table 1) and the 75% survival rate for the entire group. The length of survival ranges from 0.33 to 27.6 years from their first transplant (Supplementary Figure 1).

Factors associated with need for booster transplantation
Infections and poor or no immune reconstitution-The average time for donor T cells to appear in SCID infants after a successful rigorously T cell depleted stem cell transplant is from 90 to 120 days post-transplantation. 15 If a SCID patient had no or poor T cell function at between 120 and 180 days post-transplantation, particularly if there was a chronic viral infection, he or she was considered for booster transplantation. If there was no T cell function or chimerism, the other parent was used as the donor for the booster transplant. If there was some but inadequate T cell function despite donor T cell chimerism, the donor used for the first transplant was used for the booster.
Age at initial transplantation-This was significantly correlated with need for a booster transplant. Patients who required booster transplantation were an average of 223 days old at initial transplantation (SD 131), whereas patients who did not require booster transplantation were an average of 165 days at initial transplantation (SD 152). This difference was significant (t=−2.3358, N=171, P=0.0207).

Factors influencing survival of the boosted patients
Age at Initial Transplantation-The effect of age at the time of the first transplantation on survival of the boosted patients is displayed in Supplementary Figure 2. The average age at initial transplantation for those who are currently alive was 194 days (S.D. 111) and for those who are deceased, the average age at initial transplantation was 273 days (S.D. 148). This difference was found to be significant (t=−2.1117, N=49, P=0.0401).
Sex, Race and Ethnicity-No significant differences in survival were found. Seventyone percent of non-Hispanic white patients survive, whereas only 50 percent of the 8 Hispanic and 4 black patients survive (X 2 =5.3566, N=49 P=0.253).
Type of Molecular Defect-The sample sizes were too small to evaluate statistically whether the molecular defect had an effect on mortality (Table 1).  Table 1). However, the average number of cells per kilogram for the "booster" transplants was significantly lower (z=7.517, N=200, P<0.0001), as the patients were older and weighed more (z=−10408, N=200, P<0.0001).

Donor Source of Transplanted
Transplantation Interval-The mean interval between the first and second transplants in living patients was 1262 days (S.D. 1737, N=31) vs. 326 days for deceased patients (S.D. 323, N=18). The difference between means was significant (t=−2.2534, N=49, P=0.0289).

Graft-versus-Host
Disease-Of all 171 SCID patients transplanted at this institution since 1982, 54 (32%) developed GVHD. Among the entire group, those who had GVHD were not more likely to require a booster transplant. Only 14 (28.6%) of the 49 boosted patients experienced GVHD following their first transplant and only one developed it after a booster transplant The latter patient developed fatal grade IV GVHD after a chemoablated matched unrelated cord blood transplant elsewhere. The boosted patients who had GVHD following their original transplant were no more likely to require more than one booster transplant than those who did not (X 2 =4.8782, N=49 P=0.181).
Infections-Eleven of the 18 deceased patients died of one or more clinically apparent viral infections: three of cytomegalovirus, one of EBV lymphoproliferative disease, two of rotavirus, two of adenovirus, two of varicella, two of parainfluenza 3, and one of a herpes simplex infection. One patient died of a fungal infection, one of gram negative sepsis, one of an undefined neurologic disease, two of pulmonary complications, one of hemorrhage following surgery and one of graft-vs-host disease.
Other than viral infections, Pneumocystis jiroveci pneumonia and oral moniliasis were found to be most common at presentation and resolved with appropriate therapy. All but 4 deceased patients had a clinically documented chronic viral infection, whereas 93.33% of living patients have never had a clinically documented chronic viral infection. This difference was highly significant (X 2 =24.85, N=47 P<0.0000).
Current Clinical Statuses-The 31 living patients' current clinical statuses were evaluated in 6 categories and a score was calculated with 6 being the most unhealthy. Patients were included in this evaluation if they had been seen within the last 2 years or had responded to a recent questionnaire (N=28). 18 The categories were regular antibiotic use, ADHD, neurological issues, gastrointestinal issues, receiving Cs or lower in school, and being in the 5 th percentile or below in height or weight. The average clinical total score was 1.8 (S.D. 1.6).

Immune Reconstitution
Lymphocyte Enumeration and T-Cell Function-All infants lacked T-cells prior to initial transplantation. As expected, transplants that resulted in improved immune function were more often found in patients who are now alive. Shown in Table 2 are the latest results of immune evaluations in all patients. The absolute numbers of CD3 (z=3.609, p=0.0003) and CD4 (z=4.096, P<0.0001) positive T cells were significantly higher in the surviving patients, but the percentage of CD45RA positive T cells was not significantly different when compared to that of the deceased (z=1.535, p=0.1247). Improved T-cell function, measured by lymphocyte proliferation assays, was used to assess immune reconstitution. If a patient had one response greater than 50,000 CPM to any of the mitogens tested, the transplant was considered to have "improved T-cell function." Seventy-five of 130 transplants given to these patients resulted in improved T-cell function; 62 (78%) of these transplants were in patients who are currently living, while only 13 were in patients who are deceased. This difference was found to be highly significant (p<0.0000 X 2 =32.4369, N=129). Mean responses to PHA (cpm) at the latest evaluations were also significantly higher in the living patients (z= 4.210, p<0.0001) ( Table 2).
B-cell Function-B-cell function has proven difficult to reconstitute in SCID patients. 3,[19][20][21] Two patients reported here (#'s 9 and 18, Table 2) were given boosters solely to gain B-cell function, and both failed. Currently, only 9 (29%) of the boosted patients have normal B cell function and do not require IG (Table 2). By contrast, 57 of the 98 (58%) non-boosted SCID patients who survive have B cell function and no longer require IG replacement. 21

Patients with Two Parental Donors
In 7 of the 10 surviving patients who were given a booster transplant only with marrow from the other parent, T-cell function improved remarkably and became normal in 6 of these cases. One CD3 epsilon-deficient SCID patient received her first transplant from her father, but because she showed no immune function at 183 days post-transplantation, she was given a rigorously T-cell-depleted maternal marrow transplant (Figure 1). One month following the administration of the second transplant (maternal marrow), the patient's T-cell proliferation improved to 53,237 CPM to Con A. However, T-cell chimerism studies at the time demonstrated that the proliferating T-cells were 100% of paternal origin. She subsequently demonstrated T-cell chimerism from both parents, but the paternal T-cells dominated. Because her T-cell function was still not normal, a third transplant was given, this time again from her father, and she subsequently developed and maintained normal Tcell function and remains healthy at age 24. She also has normal B-cell function and does not require IVIG. One boy with IL7Rα-Def SCID who received his first two transplants from his mother had no immune reconstitution at 408 days post-transplantation ( Figure 3). A rigorously T-celldepleted haploidentical transplant was then given from his father. He subsequently developed normal T-cell function and normal immunoglobulin levels. His T-cell chimerism is paternal. He is now 3.5 years old and healthy.
A Jak3-Def SCID also had a remarkable improvement in her T-cell function following a transplant from the other parent, as previously reported. 22 She had received two paternal transplants without achieving adequate T-cell function or engraftment. Finally, following a third transplant of maternal origin, normal T-cell function was achieved. Chimerism studies have shown that she has 2% paternal cells and 98% maternal T-cells. She also has normal Bcell function and does not require IVIG. She is now 8.5 years old and healthy.

Discussion
Our studies demonstrate that non-ablative booster transplantation is an effective means of enhancing immune system reconstitution following an unsatisfactory initial non-ablative Tcell-depleted HLA-haploidentical bone marrow transplant. The explanation for the higher rate of failure of haploidentical transplants (as opposed to HLA identical transplants) is unknown but appears to be related to the necessity to rigorously T cell deplete. In most cases, booster transplants were effective in improving immune function. No pre-transplant conditioning was used for any of the first or booster haploidentical transplants. Conditioning was used only prior to matched unrelated donor umbilical cord blood transplants. Omitting toxic chemoablative agents prior to bone marrow transplantation in SCID allows the patients to avoid later infertility, veno-occlusive disease and damage to the lungs, endocrine organs, or brain. [23][24][25] No increased incidence of GVHD following the first transplant was found when boosted patients were compared to non-boosted patients. In both groups, most of the donors were mismatched haploidentical parents whose marrow was rigorously T-cell depleted. Therefore, there was no need in either group for immunosuppressive drugs to be given for GVHD prophylaxis post-transplantation.
As with non-booster transplanted SCID patients, opportunistic viral infections and malnutrition were the main factors associated with mortality in booster-transplanted SCID patients. 3 Chronic viral infections were the most lethal complication among booster SCID patients, and in most patients these viral infections were present prior to their original transplant. All but four of 18 deceased patients in this study had clinically documented chronic viral infections and these were the direct cause of death in eleven. Early diagnosis and isolation are key to preventing infection in all SCIDS. 4,26 The underlying molecular defect had little effect on the need for a booster transplant, with the exception of RAG1 and RAG2-deficiency, where 6 of 7 transplanted SCIDs required booster transplants. In the case of ADA-deficient SCID, often considered problematic for achieving engraftment, 27 this Center has transplanted 26 such patients over the past 30 years. Twenty (77%) survive and only 6 required booster transplants. Three of the six deceased had been given booster transplants. Two received successful gene therapy, 28 one received an ablated transplant elsewhere, and two are receiving PEG-ADA. The other fifteen are alive and well and are chimeric with related donor T cells after rigorously T celldepleted non-ablative haploidentical parental (n=10, 2 boosted) or HLA identical (n=5) bone marrow transplants.
The use of bone marrow from both parental donors sequentially can improve immune reconstitution in some patients. In some cases, we found that the recipients became double parental chimeras, although usually chimerism with one parent's cells dominated. This is somewhat similar to the situation seen when multiple cord blood units are given to one recipient, in which case chimerism from one particular unit becomes dominant. 29 Although the factors that determine dominance of one donor over another have not been clarified, immune-mediated mechanisms are suspected. This was clearly the case in one such patient in our group who failed two paternal T-cell-depleted haploidentical bone marrow transplants but rapidly became immune reconstituted after her mother's T-cell-depleted haploidentical bone marrow transplant. 22 In that case, we suspected the later-identified transplacentallytransferred maternal T-cells rejected the paternal marrow transplants. Closer HLA matching of one haploidentical parent to the patient as opposed to the other parent's matching was examined, but review of the HLA typing data (not shown) found that in only one of the examples given was there such a possibility.
Booster transplantation has proven to be an effective means of prolonging life in SCID patients. Though the survival rate among booster patients (63%) is lower than the overall SCID survival rate at this institution (75%), 2 this is most likely due to the fact that the patients who received boosters were older at the time of initial transplantation and were sicker, primarily with chronic viral infections. Without receiving a booster transplant, these patients would not have survived. Clinically, most surviving booster transplanted patients are doing well. The majority have adequate T-cell function. As in our previous studies of the entire group, age at transplantation was a key factor in survival, 4 most likely because the older patients were already infected with viral agents. Recognition of the beneficial effect of very young age on treatment outcome was an important factor in securing approval for newborn screening for SCID. 26 (3000 words)

Supplementary Material
Refer to Web version on PubMed Central for supplementary material. The development of T cell function following sequential bone marrow transplants in a girl with CD3 epsilon deficient SCID. Her father was the first donor, but due to the lack of T cell function at 183 days post-transplantation, she received a rigorously T cell-depleted booster transplant from her mother. Following that, she developed some T cell function but chimerism studies revealed that most of the dividing cells were from her father. A third rigorously T cell-depleted transplant was then given from her father and she has subsequently gone on to have excellent long term T cell reconstitution. Subsequent T cell chimerism studies have revealed some chimerism from both parents, with the dominant chimerism being from the father. She does not require IVIG therapy although her B cells are all host.   Development of T cell function in another IL7Rα-Def SCID boy following three rigorously T cell depleted haploidentical parental bone marrow transplants. The mother was the donor for the first two, but T cell function failed to develop after either transplant, so a third transplant was given from the father and was subsequently followed by the development of sustained normal T cell function and paternal T cell chimerism.  Table 1 Comparisons of Age at Initial Transplant and Survival in Boosted and Non-Boosted SCIDs According to Molecular Type