Efficacy and safety of donor lymphocyte infusion after allogeneic hematopoietic stem cell transplantation in pediatric patients

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We describe the long-term use of preemptive, prophylactic and therapeutic DLI with a gradual dose increase in half-log increments. Under close monitoring, we increased the DLI dose only in patients who had tolerated the previous dose without any signs of graft-versus-host disease (GVHD). In the preemptive cohort, we were able to prevent hematological relapse by using DLI in ten of the twelve patients (83%) showing minimal residual disease (MRD) positivity. We treated eleven patients with genetic disease and mixed chimerism who were at risk for graft rejection with preemptive DLI. In total, nine patients (82%) responded. Six patients (100%) of the prophylactic cohort with a very high risk of relapse had a successful outcome without relapse or GVHD. Three of the five patients (60%) of the therapeutic cohort were successfully treated with DLI. We observed acute GVHD (grade I and II) in only two patients (6%) who could be cured with immunosuppressive therapy. The results of our study indicate that DLI is a promising strategy and can effectively prevent relapse, graft rejection, and even cure relapse. The observed high response rates in our three cohorts may be attributed to the long-term use of DLI and the very low rate of GVHD to the gradual dose increase. Therefore, we consider DLI as a safe and highly effective therapeutic option when administered in a preemptive, prophylactic or therapeutic setting. donor lymphocyte infusion hematopoietic stem cell transplantation graft-versus-host disease graft-versus-leukemia effect donor chimerism Figures Figure 1 Figure 2 Figure 3 Introduction Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for pediatric patients with high-risk malignancies and genetic diseases [ 1 , 2 ]. In HSCT, hematopoietic stem cells from a healthy donor are transferred to the patient to reconstitute hematopoiesis and restore a functional immune system. However, relapse and graft rejection remain major reasons for treatment failure. In general, patients who experience relapse after HSCT have a dismal prognosis, with limited therapeutic options and poor long-term survival rates [ 3 , 4 ]. To improve outcomes, donor lymphocyte infusion (DLI) has emerged as a promising strategy to boost the graft-versus-leukemia (GVL) effect or graft-versus-tumor (GVT) effect and thereby reduce the risk of relapse in patients with malignant disease [ 5 ]. It is also an established immunotherapeutic option that is known to increase donor chimerism and prevent imminent graft failure after HSCT in patients with genetic disease. Data from various studies have demonstrated that DLI can effectively be used in patients who show early signs of relapse such as persisting minimal residual disease (MRD) or mixed chimerism (MC) [ 6 ]. Depending on the clinical setting, DLI can be administered as preemptive DLI to counter early signs of relapse or graft rejection, as prophylactic DLI in patients with a very high risk of relapse, or as therapeutic DLI in case of manifest hematological relapse. However, the efficacy of DLI treatment is limited by the induction of graft-versus-host disease (GVHD), the most common and adverse DLI effect. GVHD is a severe and potentially life-threatening donor-derived immune response against recipient tissues. It primarily affects the gastrointestinal tract, skin, and liver, and can manifest as an acute or chronic form potentially leading to severe long-term complications [ 7 ]. Given these risks, it is essential to identify the safest possible approach for DLI administration to minimize adverse effects such as GVHD. Although there is a growing body of practical recommendations and standardized protocols for the use of DLI in adults, comparable guidelines for pediatric patients are still lacking. Critical aspects such as the optimal timing, dosing, and frequency of DLI administration to maximize therapeutic benefit while minimizing the risk of GVHD remain subjects of ongoing debate. To contribute to this discussion, we present our single-center experience with the long-term use of DLI in pediatric patients after allogeneic HSCT, evaluating efficacy and safety across prophylactic, preemptive, and therapeutic cohorts. Patients and methods Study population The study was approved by the local institutional review board (2024-3396). We included 34 pediatric patients (20 male, 14 female) who received DLI after HSCT between 01.07.2006 and 30.06.2024 at the Department of Pediatrics, Jena University Hospital, Jena, Germany in a single-center retrospective cohort study. The median age at first DLI administration was 6.5 years and DLI administration was started at a median time of four months after HSCT. Thirty-one patients received allogeneic HSCT from a matched unrelated donor with a minimum of 9 out of 10 HLA molecular match. Three patients received haploidentical HSCT. The underlying diseases were genetic diseases (n=11), acute lymphoblastic leukemia (ALL) (n=10), acute myeloid leukemia (AML) (n=6), juvenile myelomonocytic leukemia (JMML) (n=2), chronic myeloid leukemia (CML) (n=1), myelodysplastic syndrome (n=1) and solid tumors (n=3). Based on the reasons for DLI administration we defined a preemptive cohort (n=23), a prophylactic cohort (n=6), and a therapeutic cohort (n=5). Patients of the preemptive cohort were divided into those receiving DLI because of MRD reappearance in patients with hematological malignancies (n=12) and those with mixed chimerism in patients with genetic disease (n=11). The prophylactic cohort included six patients with a very high risk of relapse. The therapeutic cohort consisted of four patients suffering from hematological relapse and one patient who was affected by recurrent autoimmune hemolytic crises. In case of multiple HSCT, we took only the first HSCT and following doses of DLI into consideration. Six Patients were excluded due to the fact, that complete remission (CR) was not achieved at the time of HSCT and one patient due to missing data. No patients were excluded based on other criteria like gender, disease type or conditioning regimen. Patient characteristics are shown in the Table 1. Table 1 Characteristics of patients Characteristics No. (%) Sex Male Female 20 (58.8) 14 (41.2) Median age in years 6.5 Stem cell source Unrelated donor Haploidentical donor 31 (91.2) 3 (8.8) Human leukocyte antigene match Unrelated donor 9/10 match 10/10 match Haploidentical donor 5/10 15 (44.1) 16 (47.1) 3 (8.8) Reason for donor lymphocyte infusion Preemptive cohort Detection of minimal residual disease Mixed chimerism Prophylactic cohort (high risk of relapse) Therapeutic cohort Hematological relapse Recurrent autoimmune hemolytic crises 23 (67.6) 12 11 6 (17.7) 5 (14.7) 4 1 Diagnosis Genetic disease Acute lymphoblastic leukemia Acute myeloid leukemia Juvenile myelomonocytic leukemia Chronic myeloid leukemia Myelodysplastic syndrome Solid tumor 11 (32.4) 10 (29.4) 6 (17.7) 2 (5.9) 1 (2.9) 1 (2.9) 3 (8.8) DLI All patients received at least two doses of DLI from the same donor who provided the hematopoietic stem cells. We collected DLI samples either at time of HSCT or after the decision for DLI was made. Therefore, DLI was mostly administered as a cryopreserved sample. In several cases patients received freshly isolated cells with the first DLI dose. We started at a median DLI dose of 1.0 x 10 5 CD3 cells/kg (range 1.0 x 10 4 – 1.0 x 10 6 CD3 cells/kg) in the preemptive cohort and 3.2 x 10 4 CD3 cells/kg (range 1.0 x 10 4 – 1.0 x 10 5 CD3 cells/kg) in the prophylactic cohort. In the therapeutic cohort, we started at a median DLI dose of 1.0 x 10 6 CD3 cells/kg (range 1.0 x 10 5 – 1.0 x 10 6 CD3 cells/kg) except for the patient who was affected by recurrent autoimmune hemolytic crises where we started at 3.2 x 10 4 CD3 cells/kg. In patients treated with DLI after haploidentical HSCT we administered at a median DLI dose of 3.2 x 10 4 CD3 cells/kg (range 1.0 x 10 4 – 1.0 x 10 5 CD3 cells/kg). We increased in half-log increments to a maximum of 3.2 x 10 7 CD3 cells/kg. All patients were closely monitored for symptoms of GVHD according to the classification by the Mount Sinai Acute GVHD International Consortium [8]. We increased the DLI dose only in patients who tolerated the previous dose without any symptoms of GVHD and administered DLI monthly with a median number of eight infusions (range: 2-48) during a median time of nine months. Disease monitoring Disease specific genetic markers were detected by standard sequencing methods and used to monitor the presence of minimal residual disease (MRD). MRD was closely monitored by polymerase chain reaction and flow cytometry. Donor chimerism was measured by short tandem repeat analyses from peripheral blood and bone marrow. In patients with malignant disease, the goal of treatment was complete donor chimerism. In non-malignant diseases, achieving a stable mixed donor chimerism is generally sufficient, as it typically ensures patient well-being and prevents the recurrence of disease-related symptoms [9]. Bone marrow aspirates from patients with hematological diseases were analyzed morphologically to monitor blast counts. Patients with solid tumors received regular imaging according to standard guidelines. We defined hematological relapse morphologically if a bone marrow smear contained more than 25% of blasts. Additional therapy Some of the patients received additional treatment with demethylating agents such as azacytidine. Others were treated with tyrosine kinase inhibitors such as ponatinib or sorafenib. By enhancing the former downregulated production of IL-15 in acute myeloid leukemia blasts, sorafenib increases the therapeutic potential of DLI [10]. Some patients also received antibody treatment. Four patients were administered the antibody blinatumomab, which immunologically couples the CD3 receptor of T cells to CD19-positive leukemia cells, inducing their apoptosis. Two patients were treated with inotuzumab ozogamicin, a monoclonal antibody directed against CD22. Four patients received zoledronic acid simultaneously to each DLI dose. Based on the current state of research, this bisphosphonate has been shown to enhance the graft-versus-malignancy effect by promoting γδ T-cell cytotoxicity [11, 12]. In the preemptive cohort, patients with ALL and a high MRD load received either zoledronic acid (n=4) or blinatumomab (n=3) or inotuzumab ozogamicin (n=1) in addition to DLI. In another patient with ALL we started with administering blinatumomab and switched to inotuzumab ozogamicin because of insufficient efficacy. We combined ponatinib with DLI in a patient with CML. In the prophylactic cohort, one patient received azacytidin and one zoledronic acid. In the therapeutic cohort, we administered DLI in combination with sorafenib (n=2) or azazytidine with zoledronic acid (n=1). Statistical analysis Outcome variables of interest were response to DLI, course of donor chimerism, acute and chronic GVHD (aGVHD and cGVHD), GVHD-free and relapse-free survival (GRFS) and overall survival (OS). We used electronic medical records and archived files to collect clinical, laboratory and demographic data. The patients’ 3-year GRFS and 5-year OS were estimated by Kaplan-Meier survival analyses and log-rank tests. The cumulative incidence of relapse was calculated using Gray´s test. GRFS was calculated between the date of first DLI and date of relapse, graft failure, death or first signs of aGVHD or cGVHD. Regardless of cause, OS was defined as interval from date of first DLI to date of death. Results Preemptive DLI We treated twelve patients with preemptive DLI because of MRD positivity. Ten patients responded and achieved a complete molecular remission (83%). In the other two patients of this cohort, hematological relapse could not be prevented. Because of high MRD load in ALL patients, DLI administration was combined with either zoledronic acid or inotuzumab ozogamicin or blinatumomab in one patient each. In another patient with ALL, two antibodies were administered one after the other. Yet only a combination of DLI and inotuzumab ozogamicin resulted in complete donor chimerism and molecular remission. We successfully combined DLI with ponatinib in one patient with CML. Nevertheless, two patients progressed to a hematological relapse that lead to death in one of them. We treated eleven patients with genetic disease with preemptive DLI. All of them showed mixed chimerism and were at risk for graft rejection. In total, nine patients (82%) were treated successfully. Six patients (55%) responded with increasing donor chimerism above 95%. Three patients developed a stable mixed chimerism at a median of 70%. The remaining two patients had a secondary graft failure and had to be retransplanted. Development of donor chimerism after HSCT is shown in Fig. 1 . The 3-year GRFS for all patients treated with preemptive DLI was 83% (Fig. 2 ) and the 5-year OS was 96% (Fig. 3 ). Prophylactic DLI We administered prophylactic DLI in six patients suffering from high-risk malignancies. One patient with relapsed JMML received azacytidine in addition and another patient with Ewing sarcoma received zoledronic acid additionally. As all patients of the prophylactic cohort are in complete remission, the safety of DLI administration is the highest priority. To prevent the occurrence of GVHD, we started at a lower median DLI dose than in the preemptive or therapeutic cohort and increased very carefully. Overall, all patients (100%) had a successful outcome without relapse or GVHD (Fig. 2 ) and the 5-year OS was 100% (Fig. 3 ). Therapeutic DLI Four patients were treated with therapeutic DLI due to hematological relapse. We administered DLI in addition to sorafenib in two patients with relapsed AML. Both patients showed a successful outcome and achieved complete molecular remission. The remaining two patients relapsed. The fifth patient of the therapeutic cohort, suffered from recurrent autoimmune hemolytic crises and was successfully treated with DLI. In the therapeutic cohort the GRFS was 60% (Fig. 2 ) and the 5-year OS 80% (Fig. 3 ). GVHD We observed acute GVHD only in two patients (6%) of the preemptive cohort. One patient who was treated with DLI because of mixed chimerism in genetic disease suffered from acute GVHD Grade I of the skin. We successfully treated him with methylprednisolone and extracorporeal photopheresis. Another patient with relapse in JMML developed GVHD grade II of the skin and intestine. He responded to our therapy with methylprednisolone and cyclosporine A. Both patients with GVHD could be cured in a short time period. However, both patients benefited from DLI therapy and responded with an increase of donor chimerism and complete remission, respectively. We observed no chronic GVHD in our patients. Discussion DLI remains the most widely used immunotherapeutic strategy to prevent or treat relapse following HSCT, particularly in hematological malignancies [ 13 ]. Numerous studies across both clinical and preclinical models have consistently demonstrated the efficacy of DLI in enhancing GVL and GVT effects. Although the sensitivity to DLI varies considerably between different malignant diseases, DLI has shown efficacy across a wide range of hematological malignancies [ 14 ]. Mixed donor chimerism in patients with malignant disease was found to be a significant risk factor for relapse. In contrast, however, complete donor chimerism in patients with non-malignant disease is not necessary, as there are no advantages in transplant outcomes in comparison to patients with mixed chimerism. However, a significant decline in donor chimerism in blood correlates with an increased risk of graft failure, and rejection may be prevented by timely DLI administration [ 15 ]. Overall, 82% of all patients included in this study responded to DLI administration. We report a GRFS of 83% in the preemptive cohort, 100% in the prophylactic cohort and 60% in the therapeutic cohort. The OS was 96% in the preemptive cohort, 100% in the prophylactic cohort and 80% in the therapeutic cohort. Compared to other studies about the efficacy of DLI after HSCT, we achieved similar to slightly better results in all categories. [ 1 , 2 , 16 ]. With a median of 8 doses of DLI per patient, only few centers perform infusions as frequently. Despite the limited number of patients in this study, the findings indicate a tendency for long-term therapy to be more effective in maintaining or achieving remission. Nevertheless, it should be noted that some of the patients received additional treatment as explained earlier. These co-interventions may have influenced outcomes and should be considered when interpreting the results. We started at a median DLI dose of 1.0 x 10 5 CD3 cells/kg in the preemptive cohort, 3.2 x 10 4 CD3 cells/kg in the prophylactic cohort and 1.0 x 10 6 CD3 cells/kg in the therapeutic cohort. For patients treated with DLI after haploidentical HSCT we started at a DLI dose of 1.0 x 10 4 CD3 cells/kg. Are starting doses were lower than in comparable studies, as a higher dose is associated with an increased risk of GVHD [ 14 , 16 ]. Moreover, there are currently no established guidelines for the administration of DLI in pediatric patients. According to the recently published practice recommendations for adults from the EBMT [ 14 ], a dose escalating regimen reduces the occurrence of GVHD in case of multiple DLI. Our practical experience aligned with these recommendations. In this study we increased DLI doses gradually in half-log increments and continued with the administration only in patients who tolerated the previous dose without any symptoms of GVHD. To maximize the safety of our treatment, we did not exceed a DLI dose of 3.2 x 10 7 CD3 cells/kg. Considering these aspects, we are able to report an extremely low rate of GVHD (5%) in all patients. Acute GVHD did not impact overall survival and there were no cases of chronic GVHD observed. Therefore, we consider DLI to be a safe, yet effective treatment option for patients in a preemptive, prophylactic and therapeutic setting. Declarations Author contribution D.W. and B.G. wrote the main manuscript text and D.W. prepared figures 1-3. D.W., J.E., C.W., S.W., M.H., G.B., T.M. and B.G reviewed the manuscript. Funding No financial support was taken. Data availability All data supporting the findings of this study are available upon request. Ethical approval All procedures were in accordance with the ethical standards. The study has been approved by the Jena University Hospital Ethics Committee (2024–3396). Patient consent statement Informed consent was obtained from the parents of the child included in this study. Permission to publish the data has been taken from the parents of the patient. Conflict of interest The authors declare that they have no conflict of interest. Author information Authors and Affiliations Department of Pediatric and Adolescent Medicine, Jena University Hospital, Jena, Germany Dinah Walther, Jana Ernst, Carola Wollenhaupt, Susan Wittig, Manuela Härtel, Grit Brodt, Till Milde & Bernd Gruhn Comprehensive Cancer Center Central Germany (CCCG), Jena, Germany Jana Ernst, Grit Brodt, Till Milde & Bernd Gruhn Hopp Children's Cancer Center Heidelberg (KiTZ), Heidelberg, Germany Till Milde Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Center Heidelberg (DKFZ), Heidelberg, Germany Till Milde References Hou MH, Lee CY, Ho CY, Yu TY, Hung GY, Huang FL, Chiou TJ, Liu CY, Yen HJ (2023) Donor lymphocyte infusion for prophylaxis and treatment of relapse in pediatric hematologic malignancies after allogeneic hematopoietic stem cell transplant. 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Cytotherapy 10:842–856. https://doi.org/https://doi.org/10.1080/14653240802419328 Santoro N, Schmid C, de Witte M et al (2025) Current use of donor lymphocyte infusions after allogenic stem cell transplantation in Europe: a survey on behalf of the cellular therapy and immunobiology working party of the EBMT. https://doi.org/10.1038/s41409-025-02555-9 . Bone Marrow Transplantation Pagliuca S, Schmid C, Santoro N et al (2024) Donor lymphocyte infusion after allogeneic haematopoietic cell transplantation for haematological malignancies: basic considerations and best practice recommendations from the EBMT. Lancet Haematol 11:e448–e458. https://doi.org/10.1016/s2352-3026(24)00098-x Park M, Koh KN, Seo JJ, Im HJ (2011) Clinical implications of chimerism after allogeneic hematopoietic stem cell transplantation in children with non-malignant diseases. Korean J Hematol 46:258–264. https://doi.org/10.5045/kjh.2011.46.4.258 Harada K (2023) Pre-emptive and prophylactic donor lymphocyte infusion following allogeneic stem cell transplantation. Int J Hematol 118:158–168. https://doi.org/10.1007/s12185-023-03595-x Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7022685","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":479752742,"identity":"02144d8b-e316-4aee-a05b-f52483895e9d","order_by":0,"name":"Dinah Walther","email":"","orcid":"","institution":"Jena University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Dinah","middleName":"","lastName":"Walther","suffix":""},{"id":479752743,"identity":"a1f930d8-1e60-49b0-9bf2-2c2b08639bdf","order_by":1,"name":"Jana Ernst","email":"","orcid":"","institution":"Jena University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jana","middleName":"","lastName":"Ernst","suffix":""},{"id":479752744,"identity":"e71b232a-9b68-4cb4-abcc-8c1fb1900164","order_by":2,"name":"Carola Wollenhaupt","email":"","orcid":"","institution":"Jena University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Carola","middleName":"","lastName":"Wollenhaupt","suffix":""},{"id":479752745,"identity":"bfb5b2b3-096f-4bfd-9119-b3214f6dc564","order_by":3,"name":"Susan Wittig","email":"","orcid":"","institution":"Jena University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Susan","middleName":"","lastName":"Wittig","suffix":""},{"id":479752746,"identity":"b09b4d4b-6940-4e9c-a52d-a1c345ac3e4e","order_by":4,"name":"Manuela Härtel","email":"","orcid":"","institution":"Jena University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Manuela","middleName":"","lastName":"Härtel","suffix":""},{"id":479752747,"identity":"efccdc9b-2f24-4bf8-991c-e8360d2d1b4d","order_by":5,"name":"Grit Brodt","email":"","orcid":"","institution":"Jena University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Grit","middleName":"","lastName":"Brodt","suffix":""},{"id":479752748,"identity":"6b3dbc7f-ea34-48d3-83a6-75da8da35476","order_by":6,"name":"Till Milde","email":"","orcid":"","institution":"Jena University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Till","middleName":"","lastName":"Milde","suffix":""},{"id":479752749,"identity":"d627aba0-a6cd-4902-8015-a763a7668e12","order_by":7,"name":"Bernd Gruhn","email":"data:image/png;base64,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","orcid":"","institution":"Jena University Hospital","correspondingAuthor":true,"prefix":"","firstName":"Bernd","middleName":"","lastName":"Gruhn","suffix":""}],"badges":[],"createdAt":"2025-07-01 17:38:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7022685/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7022685/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86138008,"identity":"fc146efd-0512-43ac-8206-cb632b22b358","added_by":"auto","created_at":"2025-07-07 08:07:39","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":322202,"visible":true,"origin":"","legend":"\u003cp\u003eCourse of donor chimerism in patients with genetic disease. DLI, donor lymphocyte infusion\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7022685/v1/a785dd6443be21579b8bcd98.jpeg"},{"id":86138014,"identity":"9c8a630a-eeab-43ee-ae49-734476a0c240","added_by":"auto","created_at":"2025-07-07 08:07:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":205543,"visible":true,"origin":"","legend":"\u003cp\u003eGVHD-free and relapse-free survival in patients of the preemptive, prophylactic and therapeutic cohort. GVHD, graft-versus-host disease; DLI, donor lymphocyte infusion\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7022685/v1/57e153f0d8e8412d4547fc6f.png"},{"id":86139318,"identity":"ef6050d6-8af3-41f7-aa65-193dad9a75e8","added_by":"auto","created_at":"2025-07-07 08:15:39","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":234522,"visible":true,"origin":"","legend":"\u003cp\u003eOverall survival (OS) in patients of the preemptive, prophylactic and therapeutic cohort. DLI, donor lymphocyte infusion\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7022685/v1/75de6ce6f1f0c3d96ea5e6c9.jpeg"},{"id":87032166,"identity":"ec67cd0e-1de5-4242-b6fc-53faf56867cb","added_by":"auto","created_at":"2025-07-18 12:54:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1333619,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7022685/v1/1d210ca5-f4fa-4834-a2fa-3f92a97440bb.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Efficacy and safety of donor lymphocyte infusion after allogeneic hematopoietic stem cell transplantation in pediatric patients","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAllogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for pediatric patients with high-risk malignancies and genetic diseases [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In HSCT, hematopoietic stem cells from a healthy donor are transferred to the patient to reconstitute hematopoiesis and restore a functional immune system. However, relapse and graft rejection remain major reasons for treatment failure. In general, patients who experience relapse after HSCT have a dismal prognosis, with limited therapeutic options and poor long-term survival rates [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo improve outcomes, donor lymphocyte infusion (DLI) has emerged as a promising strategy to boost the graft-versus-leukemia (GVL) effect or graft-versus-tumor (GVT) effect and thereby reduce the risk of relapse in patients with malignant disease [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. It is also an established immunotherapeutic option that is known to increase donor chimerism and prevent imminent graft failure after HSCT in patients with genetic disease. Data from various studies have demonstrated that DLI can effectively be used in patients who show early signs of relapse such as persisting minimal residual disease (MRD) or mixed chimerism (MC) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDepending on the clinical setting, DLI can be administered as preemptive DLI to counter early signs of relapse or graft rejection, as prophylactic DLI in patients with a very high risk of relapse, or as therapeutic DLI in case of manifest hematological relapse. However, the efficacy of DLI treatment is limited by the induction of graft-versus-host disease (GVHD), the most common and adverse DLI effect. GVHD is a severe and potentially life-threatening donor-derived immune response against recipient tissues. It primarily affects the gastrointestinal tract, skin, and liver, and can manifest as an acute or chronic form potentially leading to severe long-term complications [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven these risks, it is essential to identify the safest possible approach for DLI administration to minimize adverse effects such as GVHD. Although there is a growing body of practical recommendations and standardized protocols for the use of DLI in adults, comparable guidelines for pediatric patients are still lacking. Critical aspects such as the optimal timing, dosing, and frequency of DLI administration to maximize therapeutic benefit while minimizing the risk of GVHD remain subjects of ongoing debate. To contribute to this discussion, we present our single-center experience with the long-term use of DLI in pediatric patients after allogeneic HSCT, evaluating efficacy and safety across prophylactic, preemptive, and therapeutic cohorts.\u003c/p\u003e"},{"header":"Patients and methods","content":"\u003ch2\u003eStudy population\u003c/h2\u003e\n\u003cp\u003eThe study was approved by the local institutional review board (2024-3396). We included 34 pediatric patients (20 male, 14 female) who received DLI after HSCT between 01.07.2006 and 30.06.2024 at the Department of Pediatrics, Jena University Hospital, Jena, Germany in a single-center retrospective cohort study. The median age at first DLI administration was 6.5 years and DLI administration was started at a median time of four months after HSCT. Thirty-one patients received allogeneic HSCT from a matched unrelated donor with a minimum of 9 out of 10 HLA molecular match. Three patients received haploidentical HSCT. The underlying diseases were genetic diseases (n=11), acute lymphoblastic leukemia (ALL) (n=10), acute myeloid leukemia (AML) (n=6), juvenile myelomonocytic leukemia (JMML) (n=2), chronic myeloid leukemia (CML) (n=1), myelodysplastic syndrome (n=1) and solid tumors (n=3). Based on the reasons for DLI administration we defined a preemptive cohort (n=23), a prophylactic cohort (n=6), and a therapeutic cohort (n=5). Patients of the preemptive cohort were divided into those receiving DLI because of MRD reappearance in patients with hematological malignancies (n=12) and those with mixed chimerism in patients with genetic disease (n=11). The prophylactic cohort included six patients with a very high risk of relapse. The therapeutic cohort consisted of four patients suffering from hematological relapse and one patient who was affected by recurrent autoimmune hemolytic crises. In case of multiple HSCT, we took only the first HSCT and following doses of DLI into consideration. Six Patients were excluded due to the fact, that complete remission (CR) was not achieved at the time of HSCT and one patient due to missing data. No patients were excluded based on other criteria like gender, disease type or conditioning regimen. Patient characteristics are shown in the Table 1.\u003c/p\u003e\n\u003cp\u003eTable 1 Characteristics of patients\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"575\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 86.9565%;\"\u003e\n \u003cp\u003eCharacteristics\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.0435%;\"\u003e\n \u003cp\u003eNo. (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 86.9565%;\"\u003e\n \u003cp\u003eSex\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; Male\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; Female\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.0435%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e20 (58.8)\u003c/p\u003e\n \u003cp\u003e14 (41.2)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 86.9565%;\"\u003e\n \u003cp\u003e\u0026nbsp; Median age in years\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.0435%;\"\u003e\n \u003cp\u003e6.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 86.9565%;\"\u003e\n \u003cp\u003eStem cell source\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; Unrelated donor\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;Haploidentical donor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.0435%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e31 (91.2)\u003c/p\u003e\n \u003cp\u003e3 (8.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 86.9565%;\"\u003e\n \u003cp\u003eHuman leukocyte antigene match\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; Unrelated donor\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;9/10 match\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;10/10 match\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;Haploidentical donor\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;5/10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.0435%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e15 (44.1)\u003c/p\u003e\n \u003cp\u003e16 (47.1)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e3 (8.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 86.9565%;\"\u003e\n \u003cp\u003eReason for donor lymphocyte infusion\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; Preemptive cohort\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;Detection of minimal residual disease\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;Mixed chimerism\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; Prophylactic cohort (high risk of relapse)\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; Therapeutic cohort\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;Hematological relapse\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;Recurrent autoimmune hemolytic crises\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.0435%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e23 (67.6)\u003c/p\u003e\n \u003cp\u003e12 \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e11 \u0026nbsp;\u003c/p\u003e\n \u003cp\u003e6 (17.7)\u003c/p\u003e\n \u003cp\u003e5 (14.7)\u003c/p\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 86.9565%;\"\u003e\n \u003cp\u003eDiagnosis\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; Genetic disease\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; Acute lymphoblastic leukemia\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; Acute myeloid leukemia\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; Juvenile myelomonocytic leukemia\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; Chronic myeloid leukemia\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;Myelodysplastic syndrome\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp; Solid tumor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 13.0435%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e11 (32.4)\u003c/p\u003e\n \u003cp\u003e10 (29.4)\u003c/p\u003e\n \u003cp\u003e6 (17.7)\u003c/p\u003e\n \u003cp\u003e2 (5.9)\u003c/p\u003e\n \u003cp\u003e1 (2.9)\u003c/p\u003e\n \u003cp\u003e1 (2.9)\u003c/p\u003e\n \u003cp\u003e3 (8.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eDLI\u003c/h2\u003e\n\u003cp\u003eAll patients received at least two doses of DLI from the same donor who provided the hematopoietic stem cells. We collected DLI samples either at time of HSCT or after the decision for DLI was made. Therefore, DLI was mostly administered as a cryopreserved sample. In several cases patients received freshly isolated cells with the first DLI dose. We started at a median DLI dose of 1.0 x 10\u003csup\u003e5\u003c/sup\u003e CD3 cells/kg (range 1.0 x 10\u003csup\u003e4\u003c/sup\u003e \u0026ndash; 1.0 x 10\u003csup\u003e6\u0026nbsp;\u003c/sup\u003eCD3 cells/kg) in the preemptive cohort and 3.2 x 10\u003csup\u003e4\u003c/sup\u003e CD3 cells/kg (range 1.0 x 10\u003csup\u003e4\u003c/sup\u003e \u0026ndash; 1.0 x 10\u003csup\u003e5\u0026nbsp;\u003c/sup\u003eCD3 cells/kg) in the prophylactic cohort. In the therapeutic cohort, we started at a median DLI dose of 1.0 x 10\u003csup\u003e6\u003c/sup\u003e CD3 cells/kg (range 1.0 x 10\u003csup\u003e5\u003c/sup\u003e \u0026ndash; 1.0 x 10\u003csup\u003e6\u0026nbsp;\u003c/sup\u003eCD3 cells/kg) except for the patient who was affected by recurrent autoimmune hemolytic crises where we started at 3.2 x 10\u003csup\u003e4\u003c/sup\u003e CD3 cells/kg. In patients treated with DLI after haploidentical HSCT we administered at a median DLI dose of 3.2 x 10\u003csup\u003e4\u003c/sup\u003e CD3 cells/kg (range 1.0 x 10\u003csup\u003e4\u003c/sup\u003e \u0026ndash; 1.0 x 10\u003csup\u003e5\u0026nbsp;\u003c/sup\u003eCD3 cells/kg). We increased in half-log increments to a maximum of 3.2 x 10\u003csup\u003e7\u003c/sup\u003e CD3 cells/kg. All patients were closely monitored for symptoms of GVHD according to the classification by the Mount Sinai Acute GVHD International Consortium [8]. We increased the DLI dose only in patients who tolerated the previous dose without any symptoms of GVHD and administered DLI monthly with a median number of eight infusions (range: 2-48) during a median time of nine months.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eDisease monitoring\u003c/h2\u003e\n\u003cp\u003eDisease specific genetic markers were detected by standard sequencing methods and used to monitor the presence of minimal residual disease (MRD). MRD was closely monitored by polymerase chain reaction and flow cytometry.\u003c/p\u003e\n\u003cp\u003eDonor chimerism was measured by short tandem repeat analyses from peripheral blood and bone marrow. In patients with malignant disease, the goal of treatment was complete donor chimerism. In non-malignant diseases, achieving a stable mixed donor chimerism is generally sufficient, as it typically ensures patient well-being and prevents the recurrence of disease-related symptoms [9].\u003c/p\u003e\n\u003cp\u003eBone marrow aspirates from patients with hematological diseases were analyzed morphologically to monitor blast counts. Patients with solid tumors received regular imaging according to standard guidelines. We defined hematological relapse morphologically if a bone marrow smear contained more than 25% of blasts.\u003c/p\u003e\n\u003ch2\u003eAdditional therapy\u003c/h2\u003e\n\u003cp\u003eSome of the patients received additional treatment with demethylating agents such as azacytidine. Others were treated with tyrosine kinase inhibitors such as ponatinib or sorafenib. By enhancing the former downregulated production of IL-15 in acute myeloid leukemia blasts, sorafenib increases the therapeutic potential of DLI [10]. Some patients also received antibody treatment. Four patients were administered the antibody blinatumomab, which immunologically couples the CD3 receptor of T cells to CD19-positive leukemia cells, inducing their apoptosis. Two patients were treated with inotuzumab ozogamicin, a monoclonal antibody directed against CD22.\u003c/p\u003e\n\u003cp\u003eFour patients received zoledronic acid simultaneously to each DLI dose. Based on the current state of research, this bisphosphonate has been shown to enhance the graft-versus-malignancy effect by promoting \u0026gamma;\u0026delta; T-cell cytotoxicity [11, 12].\u003c/p\u003e\n\u003cp\u003eIn the preemptive cohort, patients with ALL and a high MRD load received either zoledronic acid (n=4) or blinatumomab (n=3) or inotuzumab ozogamicin (n=1) in addition to DLI. In another patient with ALL we started with administering blinatumomab and switched to inotuzumab ozogamicin because of insufficient efficacy. We combined ponatinib with DLI in a patient with CML. In the prophylactic cohort, one patient received azacytidin and one zoledronic acid. In the therapeutic cohort, we administered DLI in combination with sorafenib (n=2) or azazytidine with zoledronic acid (n=1).\u003c/p\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eOutcome variables of interest were response to DLI, course of donor chimerism, acute and chronic GVHD (aGVHD and cGVHD), GVHD-free and relapse-free survival (GRFS) and overall survival (OS). We used electronic medical records and archived files to collect clinical, laboratory and demographic data. The patients\u0026rsquo; 3-year GRFS and 5-year OS were estimated by Kaplan-Meier survival analyses and log-rank tests. The cumulative incidence of relapse was calculated using Gray\u0026acute;s test. GRFS was calculated between the date of first DLI and date of relapse, graft failure, death or first signs of aGVHD or cGVHD. Regardless of cause, OS was defined as interval from date of first DLI to date of death.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003ePreemptive DLI\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe treated twelve patients with preemptive DLI because of MRD positivity. Ten patients responded and achieved a complete molecular remission (83%). In the other two patients of this cohort, hematological relapse could not be prevented. Because of high MRD load in ALL patients, DLI administration was combined with either zoledronic acid or inotuzumab ozogamicin or blinatumomab in one patient each. In another patient with ALL, two antibodies were administered one after the other. Yet only a combination of DLI and inotuzumab ozogamicin resulted in complete donor chimerism and molecular remission. We successfully combined DLI with ponatinib in one patient with CML. Nevertheless, two patients progressed to a hematological relapse that lead to death in one of them.\u003c/p\u003e \u003cp\u003eWe treated eleven patients with genetic disease with preemptive DLI. All of them showed mixed chimerism and were at risk for graft rejection. In total, nine patients (82%) were treated successfully. Six patients (55%) responded with increasing donor chimerism above 95%. Three patients developed a stable mixed chimerism at a median of 70%. The remaining two patients had a secondary graft failure and had to be retransplanted. Development of donor chimerism after HSCT is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The 3-year GRFS for all patients treated with preemptive DLI was 83% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and the 5-year OS was 96% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eProphylactic DLI\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe administered prophylactic DLI in six patients suffering from high-risk malignancies. One patient with relapsed JMML received azacytidine in addition and another patient with Ewing sarcoma received zoledronic acid additionally. As all patients of the prophylactic cohort are in complete remission, the safety of DLI administration is the highest priority. To prevent the occurrence of GVHD, we started at a lower median DLI dose than in the preemptive or therapeutic cohort and increased very carefully. Overall, all patients (100%) had a successful outcome without relapse or GVHD (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and the 5-year OS was 100% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTherapeutic DLI\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFour patients were treated with therapeutic DLI due to hematological relapse. We administered DLI in addition to sorafenib in two patients with relapsed AML. Both patients showed a successful outcome and achieved complete molecular remission. The remaining two patients relapsed. The fifth patient of the therapeutic cohort, suffered from recurrent autoimmune hemolytic crises and was successfully treated with DLI. In the therapeutic cohort the GRFS was 60% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and the 5-year OS 80% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGVHD\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe observed acute GVHD only in two patients (6%) of the preemptive cohort. One patient who was treated with DLI because of mixed chimerism in genetic disease suffered from acute GVHD Grade I of the skin. We successfully treated him with methylprednisolone and extracorporeal photopheresis. Another patient with relapse in JMML developed GVHD grade II of the skin and intestine. He responded to our therapy with methylprednisolone and cyclosporine A. Both patients with GVHD could be cured in a short time period. However, both patients benefited from DLI therapy and responded with an increase of donor chimerism and complete remission, respectively. We observed no chronic GVHD in our patients.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDLI remains the most widely used immunotherapeutic strategy to prevent or treat relapse following HSCT, particularly in hematological malignancies [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Numerous studies across both clinical and preclinical models have consistently demonstrated the efficacy of DLI in enhancing GVL and GVT effects. Although the sensitivity to DLI varies considerably between different malignant diseases, DLI has shown efficacy across a wide range of hematological malignancies [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Mixed donor chimerism in patients with malignant disease was found to be a significant risk factor for relapse. In contrast, however, complete donor chimerism in patients with non-malignant disease is not necessary, as there are no advantages in transplant outcomes in comparison to patients with mixed chimerism. However, a significant decline in donor chimerism in blood correlates with an increased risk of graft failure, and rejection may be prevented by timely DLI administration [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOverall, 82% of all patients included in this study responded to DLI administration. We report a GRFS of 83% in the preemptive cohort, 100% in the prophylactic cohort and 60% in the therapeutic cohort. The OS was 96% in the preemptive cohort, 100% in the prophylactic cohort and 80% in the therapeutic cohort. Compared to other studies about the efficacy of DLI after HSCT, we achieved similar to slightly better results in all categories. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. With a median of 8 doses of DLI per patient, only few centers perform infusions as frequently. Despite the limited number of patients in this study, the findings indicate a tendency for long-term therapy to be more effective in maintaining or achieving remission.\u003c/p\u003e \u003cp\u003eNevertheless, it should be noted that some of the patients received additional treatment as explained earlier. These co-interventions may have influenced outcomes and should be considered when interpreting the results.\u003c/p\u003e \u003cp\u003eWe started at a median DLI dose of 1.0 x 10\u003csup\u003e5\u003c/sup\u003e CD3 cells/kg in the preemptive cohort, 3.2 x 10\u003csup\u003e4\u003c/sup\u003e CD3 cells/kg in the prophylactic cohort and 1.0 x 10\u003csup\u003e6\u003c/sup\u003e CD3 cells/kg in the therapeutic cohort. For patients treated with DLI after haploidentical HSCT we started at a DLI dose of 1.0 x 10\u003csup\u003e4\u003c/sup\u003e CD3 cells/kg. Are starting doses were lower than in comparable studies, as a higher dose is associated with an increased risk of GVHD [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Moreover, there are currently no established guidelines for the administration of DLI in pediatric patients. According to the recently published practice recommendations for adults from the EBMT [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], a dose escalating regimen reduces the occurrence of GVHD in case of multiple DLI. Our practical experience aligned with these recommendations. In this study we increased DLI doses gradually in half-log increments and continued with the administration only in patients who tolerated the previous dose without any symptoms of GVHD. To maximize the safety of our treatment, we did not exceed a DLI dose of 3.2 x 10\u003csup\u003e7\u003c/sup\u003e CD3 cells/kg.\u003c/p\u003e \u003cp\u003eConsidering these aspects, we are able to report an extremely low rate of GVHD (5%) in all patients. Acute GVHD did not impact overall survival and there were no cases of chronic GVHD observed. Therefore, we consider DLI to be a safe, yet effective treatment option for patients in a preemptive, prophylactic and therapeutic setting.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contribution\u0026nbsp;\u003c/strong\u003eD.W. and B.G. wrote the main manuscript text and D.W. prepared figures 1-3. D.W., J.E., C.W., S.W., M.H., G.B., T.M. and B.G reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eNo financial support was taken.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e All data supporting the findings of this study are available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e All procedures were in accordance with the ethical standards. The study has been approved by the Jena University Hospital Ethics Committee (2024\u0026ndash;3396).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient consent statement\u003c/strong\u003e Informed consent was obtained from the parents of the child included in this study. Permission to publish the data has been taken from the parents of the patient.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e The authors declare that they have no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cu\u003eAuthor information\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Pediatric and Adolescent Medicine, Jena University Hospital, Jena, Germany\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDinah Walther, Jana Ernst, Carola Wollenhaupt, Susan Wittig, Manuela H\u0026auml;rtel, Grit Brodt, Till Milde \u0026amp; Bernd Gruhn\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComprehensive Cancer Center Central Germany (CCCG), Jena, Germany\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJana Ernst, Grit Brodt, Till Milde \u0026amp; Bernd Gruhn\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHopp Children\u0026apos;s Cancer Center Heidelberg (KiTZ), Heidelberg, Germany\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTill Milde\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Cooperation Unit Pediatric Oncology, German Cancer Research Center Heidelberg (DKFZ), Heidelberg, Germany\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTill Milde\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHou MH, Lee CY, Ho CY, Yu TY, Hung GY, Huang FL, Chiou TJ, Liu CY, Yen HJ (2023) Donor lymphocyte infusion for prophylaxis and treatment of relapse in pediatric hematologic malignancies after allogeneic hematopoietic stem cell transplant. 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Korean J Hematol 46:258\u0026ndash;264. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.5045/kjh.2011.46.4.258\u003c/span\u003e\u003cspan address=\"10.5045/kjh.2011.46.4.258\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarada K (2023) Pre-emptive and prophylactic donor lymphocyte infusion following allogeneic stem cell transplantation. Int J Hematol 118:158\u0026ndash;168. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12185-023-03595-x\u003c/span\u003e\u003cspan address=\"10.1007/s12185-023-03595-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"donor lymphocyte infusion, hematopoietic stem cell transplantation, graft-versus-host disease, graft-versus-leukemia effect, donor chimerism","lastPublishedDoi":"10.21203/rs.3.rs-7022685/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7022685/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study evaluates the efficacy and safety of donor lymphocyte infusion (DLI) after allogeneic hematopoietic stem cell transplantation (HSCT) in pediatric patients. We describe the long-term use of preemptive, prophylactic and therapeutic DLI with a gradual dose increase in half-log increments. Under close monitoring, we increased the DLI dose only in patients who had tolerated the previous dose without any signs of graft-versus-host disease (GVHD). In the preemptive cohort, we were able to prevent hematological relapse by using DLI in ten of the twelve patients (83%) showing minimal residual disease (MRD) positivity. We treated eleven patients with genetic disease and mixed chimerism who were at risk for graft rejection with preemptive DLI. In total, nine patients (82%) responded. Six patients (100%) of the prophylactic cohort with a very high risk of relapse had a successful outcome without relapse or GVHD. Three of the five patients (60%) of the therapeutic cohort were successfully treated with DLI. We observed acute GVHD (grade I and II) in only two patients (6%) who could be cured with immunosuppressive therapy. The results of our study indicate that DLI is a promising strategy and can effectively prevent relapse, graft rejection, and even cure relapse. The observed high response rates in our three cohorts may be attributed to the long-term use of DLI and the very low rate of GVHD to the gradual dose increase. Therefore, we consider DLI as a safe and highly effective therapeutic option when administered in a preemptive, prophylactic or therapeutic setting.\u003c/p\u003e","manuscriptTitle":"Efficacy and safety of donor lymphocyte infusion after allogeneic hematopoietic stem cell transplantation in pediatric patients","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-07 08:07:34","doi":"10.21203/rs.3.rs-7022685/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ab2bbde2-b913-4f37-96b1-7eb98f6f473e","owner":[],"postedDate":"July 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-18T12:53:48+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-07 08:07:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7022685","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7022685","identity":"rs-7022685","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}