T-Cell Depleted Haploidentical Transplantation in Children With Hematological Malignancies: A Comparison Between CD3+/CD19+ and TCRαβ+/CD19+ Depletion Platforms

Background T-cell depleted (TCD) haploidentical transplantation using CD3+/CD19+ and TCRαβ+/CD19+ depletion techniques has been increasingly used in children with hematological malignancies. We present a retrospective study aimed to compare transplant outcomes in children with leukemia receiving a TCD haploidentical transplant using either CD3+/CD19+ or TCRαβ+/CD19+ platforms. Methods A total of 159 children with leukemia (ALL=80) (AML=79) that received a TCD haploidentical transplantation using either CD3+/CD19+ (n=79) or TCRαβ+/CD19+ (n=80) platforms between 2005 and 2020 were included. Median age was 9 years in both groups. There were no differences in patient, donor, and transplant characteristics between groups except for donor KIR B genotype more frequent in the TCRαβ+/CD19+ group (91%) than in the CD3+/CD19+ group (76%) (p=0.009) and a high number of NK+ cells and lower CD19+ cells infused in the TCRαβ+/CD19+ group (35.32x106/kg and 0.06 x106/Kg) than in the CD3+/CD19 group (24.6x106/Kg and 0.25 x106/Kg) (p=0.04 and p=0.0001), respectively. Conditioning was based on TBF. Median follow-up for survivors was 11 years (range; 8-16 y) in CD3+/CD19+ group and 5 years (range; 2-9 y) in the TCRαβ+/CD19+ group. Results Engraftment kinetics were similar in both groups (13 days for neutrophils and 10 days for platelets). There was no difference in the incidence of acute GvHD II-IV (29 ± 5% in the CD3+/CD19+ group vs 38 ± 5% in the TCRαβ+/CD19+ group) and chronic GvHD (32 ± 5% vs 23 ± 4%, respectively). NRM was 23 ± 5% in the CD3+/CD19+group vs 21 ± 4% in the TCRαβ+/CD19+group. Relapse incidence was also similar, 32 ± 5% vs 34 ± 6%, respectively. DFS and OS were not different (45 ± 5% vs 45 ± 6% and 53 ± 6% vs 58 ± 6% respectively). As there were no differences on transplant outcomes between groups, we further analyzed all patients together for risk factors associated with transplant outcomes. On multivariate analysis, we identified that early disease status at transplant (HR: 0.16; 95%CI (0.07-0.35) (p=0.0001), presence of cGvHD (HR: 0.38; 95%CI (0.20-0.70) (p= 0.002), and donor KIR-B genotype (HR: 0.50; 95%CI (0.32-0.90) (p=0.04) were associated with better DFS. Conclusions Our data suggest that there are no advantages in transplant outcomes between TCD platforms. Risk factors for survival are dependent on disease characteristic, donor KIR genotype, and chronic GvHD rather than the TCD platform used.

Background: T-cell depleted (TCD) haploidentical transplantation using CD3+/CD19+ and TCRab+/CD19+ depletion techniques has been increasingly used in children with hematological malignancies. We present a retrospective study aimed to compare transplant outcomes in children with leukemia receiving a TCD haploidentical transplant using either CD3+/CD19+ or TCRab+/CD19+ platforms.

INTRODUCTION
Allogeneic hematopoietic stem cell transplantation is a wellstablished therapy for pediatric patients with high-risk hematological malignancies. A human leukocyte antigen (HLA)-identical sibling or an HLA well-matched unrelated donor has been considered the first option for allogeneic transplantation. However, such HLA identical donors are not always available for all patients, or even when having one, they do not have time to be waiting for donor availability (1)(2)(3)(4)(5). More than two decades ago, T-cell depleted (TCD) haploidentical transplantation (HaploSCT) was used as an alternative option for hematopoietic stem cell for allogeneic transplantation (6). Biological parents share at least one haplotype with their children and all of them can be considered as potential donors. Moreover, siblings and other relatives might be potential haploidentical donors, which contributes to most pediatric patients having at least one available donor in a timely manner. Although initially the use of haploidentical transplantation was hampered by many clinical complications, the advent of graft manipulation techniques and the use of post-transplant cyclophosphamide (post-Cy) have changed the haploidentical transplant landscape, rapidly increasing its use worldwide over the last few years. As such, HaploSCT is the allogeneic transplant modality and its use has the most growth in recent years not only in adults but also in children (7,8). Graft manipulation techniques have been used mainly in pediatric patients. They evolved from initial CD34+ selection to the recent CD45RA+ depletion, going by CD3+/CD19+ and TCRab+/CD19+ depletion platforms (6,(9)(10)(11). Nowadays, several ex vivo TCD HaploSCT platforms have gained clinical relevance (12). Considering that there are no prospective studies comparing transplant outcomes of different TCD HaploSCT platforms, data from observational and retrospective studies could be useful in guiding which TCD approach should be chosen. This retrospective, multivariable study was designed to compare transplant outcomes among pediatric patients with hematological malignancies receiving T-cell depletion haploidentical transplant using CD3+/CD19+ or TCRab+/ CD19+ platforms. We hypothesized that theoretical TCRab+/ CD19+ advantages in terms of graft composition would result on better transplant outcomes.

Study Eligibility Criteria, Patients, and Donor Characteristics
All consecutive pediatric patients who received a TCD HaploSCT with CD3+/CD19+ depletion (n=79) or TCRab+/CD19+ depletion (n=80) at our institution from January 2005 to January 2020 with hematological malignancies were included in this retrospective study. They were transplanted because of high-risk hematological malignancies and they lacked a suitable HLA-matched (related or unrelated) donor. Indications for allogeneic hematopoietic transplantation for children with acute lymphoblastic leukemia (ALL) included the following: poor cytogenetics, induction failure (defined as no remission at 1 month following induction treatment), persistent MRD with a cutoff point of 10 -3 and 2 nd complete remission (CR) or beyond. For acute myeloid leukemia (AML) patients, transplant criteria included intermediate or poor risk characteristics at diagnosis and also 2 nd CR or beyond. Details of transplantation criteria have been published elsewhere (13-15). MRD was performed using multiparametric flow cytometry, following the recommendations of the Euroflow consortium (16) using a FACS Canto II cytometer (BD Bioscience) and combinations of 8 markers per tube. A combination of markers capable of identifying the leukemia-associated immunophenotype (LAIP) was determined for each patient. This combination was used individually at all study times for levels of MRD. For quantitative estimation of MRD levels, a minimum of 500,000 events were acquired in each tube. In hypocellular samples in which the acquisition per tube was <2x10 5 events, the maximum theoretical sensitivity of the test was indicated (number of LAIP events)/(number of total events acquired) x100), since it did not reach 0.01. At least 20 LAIP events per tube were required for the test to be considered informative.
Patients, donors, parents, and/or their legal guardians gave written informed consent for donation and transplantation in accordance with the Helsinki Declaration. Patients, donors, and

Donor Hematopoietic Stem Cell Mobilization and Collection Procedures
Donor mobilization was performed as previously described (13-15). Briefly, donor peripheral blood stem cell progenitors were mobilized by administering subcutaneous granulocyte colonystimulating factor (10 mg/kg per day for 4 days) and were harvested by 1 or 2 large-volume leukapheresis procedures according to established protocols of the center using a continuous flow blood cell separator (Spectra OptiaMNC v.3.0. Terumo BCT, Lakewood, CO; USA or COBE Spectra TM, v.6.1, by Caridian BCT Europe, Garching, Germany) on the fifth day of mobilization and the day before infusion.

Graft Manipulation and Infusion Procedures
T-cell depletion was performed using CliniMACS Plus device or the fully automated Prodigy device after manipulations in a laminar-flow cabinet located in a clean room certified for sterile manipulations. All immuno-magnetic procedures were performed according to the manufacturer's standard protocol. Clinical grade reagents, disposable kits, and instrumentation were from Miltenyi Biotec (Bergisch Gladbach, Germany). Before and after depletion procedures, the apheresis, and final product cell composition were analyzed by flow cytometry (cell count and cell subpopulation CD34+, CD3+ ab+, CD3+ gd+, CD19+, and CD3-CD56+ cells, and their viability). Finally, the cell product was freshly infused via a central venous catheter on day 0.

KIR Ligand and KIR Genotyping Analysis
The KIR ligand HLA-C allotypes (C1 and C2) and the HLA-B allotypes (Bw4) were determined using high-resolution PCRsequence-based typing. Fifteen human KIR genes and two pseudogenes were analyzed by PCR with a KIR genotyping kit  (19). Donor characteristics are shown in Table 1.

Study Design, Endpoints, Definitions, and Statistical Analysis
This study was designed to retrospectively compare transplant outcomes between two TCD haploidentical transplant cohorts. Primary endpoints were disease-free survival (DFS), relapse incidence (RI), and non-relapse mortality (NRM). Secondary endpoints were engraftment kinetics and early and late immune reconstitution. Graft-versus-Host disease (GvHD) and infections incidence were also analyzed. GvHD was diagnosed and graded according to previously described definitions (22)(23)(24).
Phenotyping of NK cells, T lymphocytes, T lymphocyte subsets, and B lymphocytes was performed on fresh samples of whole blood by multi-parametric flow cytometry as previously described (25). Immune reconstitution was analyzed at day +15, +30, +60, +90, +180, +270, +1 year, and 2 years following transplantation. DFS was defined as time from transplantation to either relapse or death of any cause. Relapse was defined as morphological or clinical evidence of recurrence in peripheral blood, bone marrow, or extramedullary sites. NRM was defined as any cause of death other than relapse. Categorical variables were summarized as frequencies and percentages. Continuous variables were presented as medians and ranges. Comparison of two means has been made with the non-parametric Mann-Whitney U test. Cumulative incidence was used to estimate the relapse and NRM probabilities (26). Death in remission was treated as a competing event to calculate the cumulative relapse incidence. Relapse was considered to be the competing event for calculating cumulative incidence of NRM. Gray's test was used to assess differences between relapse incidence and NRM. Probabilities of OS, LFS, and EFS were calculated according to the Kaplan and Meier method. The significance of differences between survival probabilities was estimated by the log-rank test (Mantel-Cox). The multivariate analysis of survival was evaluated using a proportional hazard regression model (27
Nausea and vomiting, diarrhea, and hemorrhagic cystitis were the most common toxicities in both groups. Twelve out of 80 patients developed VOD in the TCRab+/CD19+ group, whereas only 2 out of 79 did in a CD3+/CD19+ group (p=0.006). Data regarding toxicity profile are provided in Supplementary Table S1.

Infections
In all, 149 infectious episodes were diagnosed using microbiological and/or clinical criteria in the CD3+/CD19+ group and 170 in the TCRab+/CD19+ group. All patients suffered some kind of infection during transplant admission. Taking together all infection episodes, there were no significant differences in the incidence of infection complications between groups. Viral infections were the most frequent ones in both groups (52% in the CD3+/CD19+ group vs 63% in the TCRab+/CD19+ group). However, there was a trend to higher incidence of viral infections in the TCRab+/CD19+ group with a higher incidence of HHV-6 in the TCRab+/CD19+ group, whereas VZV infection was more frequent in CD3+/CD19+ group.
HHV-6 reactivation occurred in 23 patients and eleven developed encephalitis diagnosed by neurological symptoms, positive PCR for HHV-6 in cerebrospinal fluid, and the absence of other identified etiologies of CNS dysfunction. Main neurological symptoms were alteration of consciousness, amnesia, seizures, hypothermia, hyponatremia, and pruritus. Treatment was mainly based in foscarnet. One patient died by HHV6 disseminated disease.
Bacterial infections were the second group of infections comprising 39% of all in the CD3+/CD19+ group and 29% in the TCRab+/CD19+ group. However, there was a higher incidence of Clostridium species in the TCRab+/CD19+ group (30% of bacterial episodes) compared with 12% in the CD3 +/CD19+ group. Detailed data regarding bacterial, viral, and fungal infections are provided in Supplementary Table S2.
T cells and their subsets progressively increase since day +60 in both groups become the predominant lymphocyte population on day +90 and afterward. There was a trend for a better B-cell reconstitution in the TCRab+/CD19+ group from day +270. Detailed data on absolute lymphocyte count (ALC), NK cells, T lymphocytes, T lymphocyte subsets, and B lymphocytes following transplant are provided in Supplementary Table S3.

Acute and Chronic Graft Versus Host Disease
Thirty-one patients developed aGvHD in the CD3+/CD19+ group (grade I: 13, grade II: 8, grade III: 7 and grade IV: 3) and 40 (grade I: 13, grade II: 12, grade III: 8 and grade IV: 7) in TCRab+/CD19+ group. The median time to aGvHD was 30 days (range, 10-90 days) in the CD3+/CD19+ group and 29 days (range, 10-95 days) in the TCRab+/CD19+ group. The cumulative incidence of aGvHD was 29±5% in the CD3+/CD19+ group and 38±5% in the TCRab+/ CD19+ group. These differences were not statistically significant. Acute GvHD treatment in the first line was based on steroids and in the second line ruxolitinib and immunomodulatory therapy with photopheresis and mesenchymal cells. GvHD was the main cause of death in 7 patients.

Non-Relapse Mortality
The cumulative incidence of NRM was 23±5% in the CD3+/CD19 + group. Seventeen patients died with a median time to death of 103 days (range: 30-1391 days). In the TCRab+/CD19+ group, the cumulative incidence of NRM was 21±4%. Fifteen patients died with a median time to death of 86 days (range: 27-543 days). The leading cause of death was relapse of disease in both (21% and 19%, respectively) followed by infection (9% and 14%, respectively). These differences were not statistically significant. The primary causes of death are given in Table 2.

Relapse
Twenty-three patients relapsed in the CD3+/CD19+ group and twenty-four in the TCRab+/CD19+ group. Median time to relapse was 164 days (range: 23-1286 days) in CD3+/CD19+ group and 206 days (range: 37-1876) in the TCRab+/CD19+ group. The cumulative incidence of relapse was 32±5% in CD3 +/CD19+ group and 34±6% in the TCRab+/CD19+ group. This difference was not statistically significant. Nine patients underwent a second hematopoietic transplant after relapse treatment and four are alive and in complete remission.
We analyzed the patients transplanted after a previous allograft and whose OS was also similar: 60± % after the 1 st transplant and 47 ±7% beyond (p=ns).

Secondary Risk Factors Analysis
Considering that there were no differences in transplant outcomes between both groups, we analyzed risk factors associated to transplant outcomes for the whole group. Univariate analysis (UVA) showed that NRM was associated with the following variables: disease status at transplant (9±4% in 1 st CR, 28±6% in 2 nd CR, and 27±6% in >2 nd CR; p=0.005); amount of NK cells on day +30 (11±4% in patients with ≥ 300/µL NK cells and 30±5% in patients with < 300/µL NK cells; p=0.005), and donor kinship (6 ±4% with sibling donors and 24±3% with parents as donors; , and cGvHD (20±6% in patients with cGvHD and 37±4% in patients without cGvHD; p=0.03). However, relapse was associated with disease status and cGvHD in MVA ( Table 3).

DISCUSSION
Nowadays, allogeneic hematopoietic stem cell transplantation from haploidentical donors is a suitable option for those pediatric patients lacking a HLA-identical donor. The advent of T-cell depletion techniques has made possible a wider use of haploidentical transplant in pediatric patients. Over the last two decades, we have been witnesses of a rapid evolution in T-cell depletion techniques. These techniques have evolved from the initial almost total T and B cells depletion by means of CD34+ isolation and CD3+/CD19+ depletion to more recently graft manipulation techniques such as TCRab+/CD19+ or CD45RA+ depletion that resulted to partial TCD holding back NK and some T-cell subset (gd and memory T cells, respectively) in the graft (12,(28)(29)(30). TCRab+/CD19+ depletion method that retains gd T cells in the inoculum has theoretical advantages over CD3+/CD19+ depletion because gd T-cells in the graft could improve antiinfectious and anti-leukemic effect. However, there are neither prospective nor retrospective reported studies comparing both options and it is not clear which one would be the better option. Therefore, the primary objective of this analysis was to compare DFS and other transplantation outcomes after haploidentical donor transplantation using CD3+/CD19+ or TCRab+/CD19+ depletion procedures. To our knowledge, this is the first study comparing both TCD procedures in pediatric haploidentical transplant setting. Our results clearly suggest that there are no relevant differences in terms of transplant outcomes between the two groups of patients. Patients' characteristics are similar between groups for the most potential variables affecting transplant outcomes. It is noteworthy that in both groups there is a high proportion of patients transplanted in the advance phase of disease (more than 20%) and with MRD positive (more than 30%) at the time of transplant. Disease status at transplant is associated not only with relapse incidence but also with NRM and obviously importantly impacts on DFS. Considering that disease status and MRD status at transplant are the most important wellknown variables influencing the transplant outcome, this might explain the absence of differences between groups.
Engraftment kinetics are also similar in both groups, although a trend to faster platelet engraftment is observed in TCRab+/CD19+ likely mirroring the higher number of CD34+ cells infused in that group of patients. Graft failure incidence is also similar in both groups with a very low impact on the cause of death in both groups. Beyond HLA disparity, there are other potential risk factors for graft failure in the TCD haploidentical transplant. Recently, patient age and the pre-transplant number of CD3 +CD8+ cells have been reported as risk factors in the TCD haploidentical transplant setting (21). The use of serotherapy, as part of conditioning regimen, might increase the probability of primary engraftment but increase the risk of delayed immune reconstitution (31).
The toxicity profile is very similar in both owing to the fact that there were no differences in the conditioning regimen used. However, the higher incidence of SOS/VOD in TCRab+/CD19+ group is remarkable. There is no clear explanation for this finding as there are no differences in race/ethnicity or intensity of conditioning between groups, but it may be because the new broader diagnostic criteria of VOD in children proposed by EBMT caused a real increase in VOD incidence. It is important to the use of Busulfan TDM to decrease the incidence of VOD after transplant. Despite this, SOS/VOD has no important impact as the cause of death in both groups maybe due to defibrotide treatment (32).
Early phase of immune reconstitution is dominated by a rapid rise of NK cell population in both groups with no differences (13, 15,25). T-cell reconstitution kinetics are also similar but a better CD4+ cells and B cells reconstitution is observed in the TCRab+/CD19+ group by 9 months after transplant. However, it does not seem to impact on infection complication likely due to the fact that most of viral and bacterial complication occurred very early following transplantation when a deep lymphopenia happens in both groups of patients. Nevertheless, the high incidence of HHV-6 infections in both groups but especially in the TCRab+/CD19+ group is remarkable (33,34). HHV-6, a T-lymphotropic herpesvirus, can productively infect gd T lymphocytes, inducing important phenotypic and cytopathic changes. Thus, by directly attacking gd T cells, HHV-6 may seek to escape the immune control of the host and thereby establish persistent infection (35). This may be the reason of increased incidence for HHV6 with ab depletion.
NK cells reconstitution in the early phase after transplant has been considered to have an impact on transplant outcomes (13, 15,36,37). A rapid NK cell reconstitution may prevent infections complications. However, a high incidence of CMV reactivation and other viral complications in haploidentical transplant setting especially when using post-Cy as GvHD prophylaxis has been published (38,39). Infections are the main cause of death apart from relapse in the whole group and still continue being an unsolved clinical problem. As expected, NRM is clearly associated with disease status at transplant. A rapid NK cell recovery is associated with a reduction on NRM because of a trend of lower incidence of severe GvHD.
Relapse after transplant still represents the main cause of transplant failure. Several mechanisms of immune evasion have been described such as genomic loss of HLA, a relapse modality first described in haploidentical transplant, the transcriptional downregulation of HLA class II molecules, and the enforcement of inhibitory checkpoints between T cells and leukemia (40). As expected, disease status at transplant and cGvHD are both associated with relapse incidence in MVA as previously described (13,15).
NK cells can also exert a notable GvL effect with no GvHD. However, it is not clear whether this GvL effect is mediated by NK cell alloreactivity based on donor/recipient KIR mismatching or by the presence of KIR activating receptors in donors with KIR-B genotype (15,41). Likely, the use of T-cell depletion or post-Cy as GvHD prophylaxis plays a key role on NK cell reconstitution and the NK cell associated GvL (42,43).
Finally, the MVA of transplant outcomes shows that patients transplanted in advanced phase of disease and those transplanted from a donor with KIR A genotype, had worse DFS due to a very high risk of relapse and mortality. Conversely, those patients that developed chronic GvHD did better in terms of DFS due to lower relapse incidence, no matter the transplant group. We acknowledge several limitations to the present study. The study limitations are mainly those derived from its retrospective nature. It covers a broad time range during which there have been several major changes in this field. In addition, being TCRab+/CD19+ depletion a more recent procedure, the follow-up in these patients was shorter than those transplanted with CD3+/CD19+ depletion procedure. Notwithstanding limitations inherent in all retrospective studies, we believe that the present study provides a valuable information to the hematopoietic transplant community regarding risk factors for transplant outcomes among pediatric patients receiving TCD haploidentical transplant.
Maybe in the next future, combination of myeloablative conditioning with T regulatory and T conventional cells as immunotherapy might reduce relapse incidence with no increase of risk of severe GvHD (44,45).
In conclusion, the study suggests that there are no advantages in transplant outcomes between both TCD platforms. Risk factors for survival are clearly dependent on disease characteristic, donor KIR genotype, and chronic GvHD rather than the TCD platform used.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Ethics committee of Hospital Niño Jesus. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.

AUTHOR CONTRIBUTIONS
MG-V is responsible for caring patients and statistical analysis and wrote the manuscript. BM cared for the patients. IL cared for the patients and collected data. JZ, ES, JI, and JS performed the graft manipulation. JR collected the data. JV performed the HLA study. AC, LA, and MR performed the cytometry analysis. MD wrote the manuscript. All authors contributed to the article and approved the submitted version.

ACKNOWLEDGMENTS
Acknowledgments are deserved by all members of the clinical laboratory, clinicians, and nurses of Hematology/Oncology department at Hospital Infantil Universitario "Niño Jesús" (Madrid, Spain) involved in patients care as well as the contributions made by the patients themselves and their families.