T cell acute lymphoblastic leukemia (T-ALL) and lymphoblastic lymphoma (T-LBL) in pediatric patients remain a high-risk hematologic malignancy with suboptimal outcomes in relapsed or refractory cases. Allogeneic hematopoietic stem cell transplantation (HSCT) is considered a potentially curative option, particularly for children with poor prognostic factors or refractory patients. In this retrospective study, we evaluated transplant outcomes in 135 pediatric patients (128 diagnosed with T-ALL and 7 with T-LBL). Isolated bone marrow involvement was documented in 100 cases, isolated extramedullary disease in 13 cases, and combined bone marrow and extramedullary involvement in another 22 patients. The cohort included recipients of related fully matched, unrelated, and haploidentical transplants, as well as different GVHD prophylaxis strategies (ex vivo T cell depletion and pharmacological prophylaxis). These factors were not considered exclusion criteria, as the primary focus of the present analysis was the assessment of relapse risk and its predictors. The 5-year cumulative incidence of non-relapse mortality was 7.6%, and of relapse or progression was 26%. The 5-year event-free survival (EFS) was 63%, and the 5-year overall survival (OS) was 65%. The principal factor influencing outcomes was active disease at the time of HSCT. In the cohort of patients transplanted in complete remission (CR), OS and EFS rates were 73.3% and 72.8%, respectively.

Hematopoietic stem cell transplantation (HSCT) is a curative option for children with high-risk acute lymphoblastic leukemia (ALL). This retrospective single-center study analyzed 236 pediatric ALL patients in complete remission who underwent allogeneic HSCT using ex-vivo T cell depletion between 2012 and 2021. The majority received haploidentical grafts (n = 202), while the remainder received matched unrelated donor (MUD) grafts (n = 34). At four years, event-free survival (EFS) and overall survival (OS) were 57

Background: Graft-versus-host disease (GVHD) refractory to immunosuppressive therapy (IST) remains a major complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT) in pediatric patients, often compounded by post-transplant viral infections. Adoptive regulatory T cell (Treg) therapy offers a strategy for GVHD control, while donor-derived memory T cells may aid in targeted immunological recovery. This study reports on the safety and outcomes of combined Treg and memory T cell therapy in this high-risk setting. Methods: Thirteen pediatric patients with steroid-refractory late acute or chronic GVHD received haploidentical Treg infusions. Treg cells were generated by ex-vivo polyclonal expansion in 7 cases or by direct flow-sorting (CD4+CD127low) using GMP-grade Tyto sorter (Miltenyi Biotec) after CD25⁺ magnetic enrichment. The median dose of Tregs was 0.5*106 Tregs/kg (0.25 - 1*106/kg), the median frequency of Tregs (FoxP3+) was 94% (58 - 98%). Eight patients with active viral infections also received donor memory T cells. Memory T cells were generated by direct flow cytometry-based sorting for CD45RO+ cells from donor leukapheresis products using GMP-grade Tyto sorter (Miltenyi Biotec). All patients had received multiple lines of prior IST, including glucocorticosteroids, cyclosporine, and biologics (tocilizumab, infliximab, abatacept, etarnecept, vedolizumab). IST was discontinued completely before Treg infusion. Nine patients recieved lymphodepletion with cyclophosphamide at 400 mg/m2. Patients were assessed for safety, immune reconstitution, responses to GVHD and viral infections, and survival, with a median follow-up of 582 days. Results: The Treg and Treg+Tmemory cell therapy was well tolerated with no severe infusion-related adverse events or new infectious complications. Eleven of 13 (84%) patients experienced either complete or partial response of GVHD manifestations. Seven of 8 (88%) patients with viral infections experienced control or resolution. Immune monitoring confirmed sustained peripheral presence of infused Treg and memory T cell populations, along with recovery of CD4 and CD8 effector/memory compartments and virus-specific T cells. In 6 patients reactivation of GVHD was recorded with good response to IST. At 1 year post-infusion, 10 patients were evaluable. Among them, 8 had no evidence of GVHD and were completely off IST. Conclusions: Combined haploidentical Treg and memory T cell therapy, with memory T cells generated solely by flow cytometry-based sorting, is feasible and safe in pediatric patients with refractory GVHD and viral infections. This approach resulted in complete IST withdrawal and GVHD control in the majority of evaluable patients at 1 year. The promising outcomes in immune reconstitution and viral control support further clinical investigation of this integrative cellular strategy for high-risk pediatric transplant recipients.

Introduction Autologous chimeric antigen receptor (CAR) T cells induce high rate of deep remissions among children with relapsed/refractory B-precursor acute lymphoblastic leukemia (R/R B-ALL). In a significant proportion of cases cure is achieved only with HSCT as post-CAR-T consolidation. Seeking to combine the cytoreductive and curative power of HSCT with the antigen-specific activity of CAR-T we devised an approach with simultaneous infusion of hematopoietic stem cell graft and CAR-T cells, derived from the same donor. Two graft-versus-host disease prevention platforms were used: ex-vivo T cell depletion and post-transplant cyclophosphamide. We report here pooled analysis of prospective trial patients and compassionate use program. Patients and methods A total of 39 pts with R/R BCP-ALL (n=35), B-NHL/leukemia (n=3) and MPAL (n=1) (16 female, 23 male, median age 9,6 y) were enrolled. All patients had disease relapse after multiple lines of treatment including previous HSCT(n=13), blinatumomab (n=22), inotuzumab (n=2), blinatumomab+inotuzumab (n=6) and CAR-T cell infusion(n=12). The disease burden was above 5% for bone marrow blasts among 16 patients, with a median blast count of 50% (6 – 90%). MRD was detectable by flow cytometry among 13 patients with a median leukemia population of 0.15% (0.002 - 6.9%), and five patients had MRD-negative complete remission. The extramedullary disease was seen among 5 patients. The median proportion of CD19-positive cells in the leukemic population was 100% (0–100%), while the median proportion of CD22-positive cells was 100% (36–100%). Fourteen (36%) pts received treosulfan-based myeloablative preparative regimen and TBI-based regimen was used in 25 (64%) pts. Twenty-six patients received T-cell depleted graft, 25 from haploidentical donors and one from match related. In this subgroup CAR-T products included CD19 CAR- T (n=3), CD19-22 tandem CAR-T (n=20), and cocktail CD19 and CD22 CAR-T (n=2). Thirteen pts were transplanted with T-replete bone marrow with PtCy-based GVHD prophylaxis, 3 from matched sibling and 10 from haploidentical donors. In this subgroup CAR-T cells were infused on day +5 after HSCT and were represented by either by CD19-22 tandem CAR- T (n=1) or by CD19 and CD22 cocktail product (n=12). In the PtCy group GVHD prophylaxis included abatacept, vedolizumab and CsA or baricitinib Results There was no suspected conflict between the graft and CAR-T cells. Primary engraftment was observed in 37 of 39 pts (one pt died before engraftment and one relapsed early), the median time to neutrophil and platelet recovery was 13 and 17 days, respectively, all engrafted patients achieved MRD-negative CR at day +28 after HSCT. The median time to CAR-T cell peak expansion was 14 days. Cytokine release syndrome (CRS) occurred in 22 (56%) patients and only two pts had grade >2 CRS. Also 22 patients had neurologic events (ICANS grade 1, n=14, grade 2, n=6 and grade 3, n=2). Eleven patients had aGvHD, grade 2 (n=9), grade 3(n=1) and grade 4 (n=1), 5 patients had mild chronic GvHD. There were no significant differences in CAR-T toxicity between depletion and PtCy groups. Leukemia relapse was observed in nine children at a median time of 174 days (range, 95-1641 days) after allograft, 2 pts had CD 19 negative relapse. Twenty-four (62%) patients are alive (22 in CR, 2 with disease progression) with a median follow-up of 2,6 years (0,34-5,4), 15 patients died. Eight deaths were due to progressive disease. Six were in CR and one before engraftment. Causes of non-relapse mortality were VOD+pneumonia (n=1), CMV (n=1), Mucormycosis (n=1), septic event (n=3) and COVID19 (n=1). EFS and OS at 2,6 years are 59% (95%CI:42- 76) and 56% (95%CI:39- 72), respectively, without differences between T-cell depletion and ptCy groups. Conclusion Our early experience suggests that allogenic CAR-T cells can be safely infused simultaneously with the hematopoietic stem cell graft both on the platform of T cell depletion and PtCy-based GVHD prophylaxis. The infusions did not compromise engraftment and GvHD control, while specific CAR-T toxicity was mild and manageable. We have documented CAR-T expansion and persistence. Prospective testing of the approach is warranted.

A heterozygous mutation in the PTPN2 gene has recently been described in several patients exhibiting symptoms of immune dysregulation. The gene encodes a ubiquitous non-receptor T-cell protein tyrosine phosphatase that exerts a negative feedback on the JAK-STAT pathway. Limited clinical data are available advocating the use of JAK inhibitors as an effective treatment for autoimmune complications of PTPN2 deficiency. However, the mechanism of pathogenesis for these complications suggests this possibility. We report on a 32-year-old male patient with interstitial lung disease, cytopenia, and lymphadenopathy accompanied by de-novo deletion in PTPN2. The patient has been receiving systemic steroid treatment for decades, which has resulted in hormone dependence as well as therapy-related adverse side effects. After the diagnosis of PTPN2 deficiency, treatment with the JAK inhibitor ruxolitinib was initiated at a dose of 15 mg per day, which was escalated to 30 mg daily after 1 month. The steroid treatment was discontinued within 3 months. At the 9- and 16-month checkpoint, after 6 and 13 months correspondingly of monotherapy with ruxolitinib at a dosage of 30 mg per day, the patient had stable blood counts, lymphadenopathy decreased, and the lung interstitial disease improved. Thus, according to our experience, JAK inhibitors are able to alleviate the PTPN2 deficiency symptoms, including hematological changes and interstitial lung damage.

BACKGROUND:Functional immune reconstitution (IR) is a key factor in determining the success of hematopoietic stem cell transplantation (HSCT). IR depends on a number of factors and is typically delayed by ex vivo T cell depletion of the graft. αβ T cell depletion (αβ TCD) platform was reported to be associated with improved IR. OBJECTIVE:In this retrospective study, we've focused on a homogeneous cohort of 262 children with acute leukemia first transplanted in complete remission, and investigate whether very early recovery of NK and T cells, as well as αβ and γδ T subsets, is associated with clinical outcomes. STUDY DESIGN:The grafts were obtained from apheresis products and processed by αβ TCD method. IR of lymphocyte subpopulations was measured in peripheral blood (PB) on day +30 after HSCT by flow cytometry. RESULTS:The study suggests that in the early post-HSCT period, higher absolute number of T cells, despite being far below the normal range and having a rather limited T cell receptors repertoire, is associated with radically improved non-relapse mortality (NRM). Multivariate analysis confirmed the independent effect of T-cell IR on NRM. Our results show that each 10-fold increase in T cell PB count is associated with a 2.1-fold reduction in NRM (cause-specific Hazard Ratio (csHR) 0.47), independent from other important factors such as aGVHD or serotherapy use. CONCLUSION:Based on these results we suggest that T cells recovery on day +30 after HSCT can be used in predicting NRM risk in the setting of αβ TCD HSCT. Early intervention for IR improvement can be planned.

Background: To date, fewer than 20 patients have been identified as having germline biallelic mutations in the coronin-1A gene (CORO1A) and its protein with clinical features of combined immunodeficiency characterized by T-cell lymphopenia ranging from the severe phenotype to the mild phenotype, recurrent infections, and lymphoproliferative disorders. However, the effects of CORO1A protein disruption on actin-dependent functions in primary cells have not been fully delineated. Objective: We sought to characterize the underlying defects of actin-dependent cellular functions in a female patient with combined immunodeficiency caused by a novel missense variant in the CORO1A gene in combination with a de novo heterozygous microdeletion of chromosome 16p11.2 and also to provide evidence of the pathogenicity of this gene mutation. Methods: To identify the genetic defect, next-generation sequencing followed by Sanger confirmation and array comparative genomic hybridization were performed. Western blot and quantitative PCR tests were used to assess the effects on the protein. Flow cytometry and live microscopy were performed to investigate cellular motility and immune cell counts and function. Results: We demonstrated that the CORO1A hemizygous variant c.19C>T, p. A7C induces significant decreases in cellular levels of the CORO1A protein while leaving mRNA concentrations unaffected. The observed mutation resulted in impaired natural killer cell cytotoxicity and platelet calcium signaling. In addition, primary granulocytes and mesenchymal cells showed significant defects in motility. Conclusion: Collectively, we added new data about the CORO1A gene as a key player in actin cytoskeleton dynamics and cell signaling. Our findings expand the clinical spectrum regarding CORO1A protein deficiency and confirm the importance of a personalized therapeutic approach for each patient.

The expanded newborn screening (NBS) program in the Russian Federation was initiated in 2023, among which severe combined immunodeficiency (SCID) is screened using TREC/KREC assays. Here, we report a rare case of a TP63-associated disease identified through this NBS program. Dried blood spots from newborns were initially screened for TREC/KREC levels, and those with values below the cut-off underwent confirmatory testing and further genetic analysis, including whole-exome sequencing (WES). A male newborn was identified with significantly reduced TREC values, indicative of T cell lymphopenia. Genetic analysis revealed a heterozygous NM_003722.5:c.1027C>T variant in TP63, leading to the p.(Arg343Trp) substitution within the DNA binding domain. This mutation has been previously associated with Ectrodactyly–Ectodermal Dysplasia–Cleft lip/palate syndrome (EEC) syndrome and shown to reduce the transactivation activity of TP63 in a dominant-negative manner. This case represents one of the few instances of immune system involvement in a patient with a TP63 mutation, highlighting the need for further investigation into the immunological aspects of TP63-associated disorders. Our findings suggest that comprehensive immunological evaluation should be considered for patients with TP63 mutations to better understand and manage potential immune dysfunctions.

Разработка дополнительного метода диагностики Х-сцепленного лимфопролиферативного синдрома второго типа на основании исследования мурамилдипептид-стимулированной экспрессии фактора некроза опухоли моноцитамиФедеральное государственное бюджетное учреждение «Национальный медицинский исследовательский центр детской гематологии, онкологии и иммунологии имени Дмитрия

Newborn screening (NBS) for severe inborn errors of immunity (IEI), affecting T lymphocytes, and implementing measurements of T cell receptor excision circles (TREC) has been shown to be effective in early diagnosis and improved prognosis of patients with these genetic disorders. Few studies conducted on smaller groups of newborns report results of NBS that also include measurement of kappa-deleting recombination excision circles (KREC) for IEI affecting B lymphocytes. A pilot NBS study utilizing TREC/KREC detection was conducted on 202,908 infants born in 8 regions of Russia over a 14-month period. One hundred thirty-four newborns (0.66‰) were NBS positive after the first test and subsequent retest, 41

Today, the use of anti-CD19 biomedical cell products (BMCPs) for the treatment of B-cell malignancies yields impressive results and is becoming ever more popular. Several bioreactors have been developed that allow the manufacturing of high-quality cell products for clinical use. Choosing an appropriate bioreactor is an important step in this process. The aim of this study was to characterize and compare immunophenotypic and functional properties of anti-CD19 BMCPs manufactured using the automated CliniMACS Prodigy system (Miltenyi Biotec, Germany) and the manual G-Rex 10M-CS platform (Wilson Wolf, USA). The manufacturing of BMCPs and subsequent CAR T-cell therapy were carried out at the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology of Ministry of Healthcare of Russia. We used T cells from patients who had undergone HSCT as well as from autologous and allogeneic donors. In this study, we employed 26 anti-CD19 BMCPs manufactured using the automated CliniMACS Prodigy system in accordance with the GMP requirements as well as 25 cell products produced with the G-Rex platform. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. Quality control was ensured throughout the entire manufacturing cycle and included assessment of cell composition and survival, transduction efficiency, cell expansion, expression of exhaustion markers, and CD19-specific antitumor activity. Our research showed that both manufacturing platforms generate stable high-quality products with sufficient cell expansion, viability and T cell transduction for subsequent CAR-T therapy. However, the median transduction efficiency of the BMCPs produced using the CliniMACS Prodigy platform was statistically significantly higher than that of the BMCPs manufactured using the G-Rex bioreactor (41% vs 26%). The study showed that in the anti-CD19 BMCPs, there was a predominance of Tcm subpopulation over Tem subpopulation, a low expression of exhaustion markers and a pronounced CD19-specific activity. Nevertheless, the percentage of Tcm cells in the BMCPs manufactured using the CliniMACS Prodigy platform was statistically significantly higher than in the BMCPs produced using the G-Rex bioreactor (86.7% CD8 + Tcm cells and 82.3% CD4 + Tcm cells for CliniMACS Prodigy vs 69.0% CD8 + Tcm cells and 72.0% CD4 + Tcm cells for G-Rex). Despite the lower number of anti-CD19 CAR-T cells in the final cell products obtained with the G-Rex bioreactor, in all processes this amount was sufficient for subsequent CAR-T therapy. Thus, the CliniMACS Prodigy and G-Rex platforms can be used to produce high-quality anti-CD19 BMCPs.

Objectives: Patients with precursor B-cell ALL/lymphoma who have relapsed after allogeneic hematopoietic stem cell transplantation (allo-HSCT) have a dismal prognosis. We evaluated the efficacy and safety of donor-derived CAR-T cell therapy in children with post-HSCT ALL/lymphoma relapse. Patients and methods: Twenty children with r/r BCP-ALL/lymphoma were enrolled in a compassionate-use program (m/f ratio=15:5, median age 9,0 years). Eighteen patients had BCPALL, 2 had B cell lymphoblastic Lymphoma with BM involvement, all relapsing after multiple lines of treatment, including previous HSCT(n=20), blinatumomab (n=17), inotozumab (n=4) and CAR-T cell infusion(n=5). Allo-CAR-T were derived from previous HSCT donors (haplo n=16, MRD n=4). The CAR-T cells were targeting CD19 and CD22 (n=14), CD19 (n=5) or CD22 (n=1). At the time of allogeneic CAR-T application, the disease burden was high MRD (n=9), overt leukemia (n=7), and extramedullary lesions(n=4), five patients had extramedullary disease in addition to BM involvement The first CAR-T cell doses were 100х103/kg (n=18), 160х103/kg (n=1), 350х103/kg (n=1), 7 patients received 2nd dose 100x103/kg (n=2), 500x103/kg (n=1) and 900x103/kg(n=4) within median 10 days after first infusion. Lymphodepletion included fludarabine, cyclophosphamide +/- cytarabine, 8 pts received prophylactic tocilizumab at day-1, 12 pts received abatacept at days -1, +7, +14, +28 Results: Cytokine release syndrome (CRS) occurred in 15 (75%) patients and was grade ≤3; all pts were treated with tocilizumab with good effect. Four patients had neurologic events (ICANS grade 1 n=3, ICANS grade 5 n=1). 5 patients had the signs of aGVHD (skin grade 1, n=4, liver=gut grade 4, n=1). The median time to CAR-T cell peak expansion was 14 days. ORR was 85%, twelve patients achieved complete MRD-negative remission, five had MRD negative remission in BM with CT detectable extramedullary lesion, 3 patients had disease progression, one of them with CD19 loss. Nine patients received consolidative HSCT after CAR-T cells, all from alternative donors, four patients relapsed, one with CD19 loss. Fifteen (75%) patients are alive, 11 (73%) of them in CR (two patients without 2-d HSCT), one with stable disease and 3 with disease progression, 5 patients died (2 due to ICAN and sepsis, 3 in PD). Median time of follow-up for survivors was 1 years (range, 0,2 - 4,6). Conclusions: Our early experience suggests that donor-derived lymphoid antigen-directed CAR-T cells expand in vivo and provide CR with manageable safety profile. Prospective testing and further research of the approach is warranted.

The technique of ab T cell depletion (abTCD) is a well-established method of hematopoietic stem cell transplantation (HSCT) for children with acute leukemia owing to the low rates of graft-versus-host disease and nonrelapse mortality (NRM). The graft-versus-leukemia effect is generally ascribed to natural killer (NK) cells conserved within the graft. It is not known whether NK-related factors affect the outcome of abTCD HSCT, however. The aim of this retrospective study was to explore the impact of NK alloreactivity (based on donor-recipient killer immunoglobulin-like receptor [KIR] mismatch), graft NK cell dose, and blood NK cell recovery on day +30 post-HSCT on the incidences of leukemia relapse and NRM. The pediatric acute leukemia cohort comprised 295 patients who underwent their first HSCT from a haploidentical donor in complete remission. During post hoc analysis, the total cohort was divided into subcohorts by diagnosis (acute lymphoblastic leukemia [ALL]/acute myeloid leukemia [AML]), NK alloreactivity prediction (KIR match/KIR mismatch), graft NK cell dose (less than versus greater than the median value), and blood NK cell recovery on day +30 post-HSCT (less than versus greater than the median value). We also investigated the influence of serotherapy (antithymocyte globulin [ATG] group) versus abatacept + tocilizumab combination [aba+toci] group) on relapse risk in the

Graft-versus-host disease (GVHD) remains the main life-threatening immunologic complication of hematopoietic stem cell transplantation. Despite modern pharmacological approaches for preventing and treating GVHD, there remains a need for new approaches to cure GVHD. Currently, more and more clinical experience is emerging globally in the field of using regulatory T-cell (T reg ) therapies for the treatment of refractory GVHD. Manufacturing cell products for T reg therapies has a wide range of protocol variations. We have developed an approach of T reg manufacturing for cell therapy and present data from our experience in manufacturing a haploidentical T reg cell product by combining CD25 + immunomagnetic selection with closed system flowbased cell sorting methods. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. The study describes the processes of producing 9 T reg cell products. According to the manufacturing protocol described here, it is possible to obtain a cell product that meets the quality control requirements necessary for approval for clinical use. Quality control includes the assessment of the cell composition, viability, and microbiological safety of the product, and is performed at all major stages of production. The final cell product is characterized by consistently high levels of FoxP3-expressing T reg (median: 98%), with a median cell viability of 99.1%, and has a high potential for functional efficacy. Thus, the protocol for producing T reg cell products by combining CD25 + immunomagnetic selection with flow cytometry-based cell sorting methods can be used for the clinical treatment of GVHD.

A patient presented with overlapping clinical and laboratory features of 2 rare autoinflammatory diseases, NLRP1-associated autoinflammation with arthritis and dyskeratosis and familial multiple self-healing palmoplantar carcinoma. Her severe inflammatory attack was treated with the IL-1 receptor-α inhibitor anakinra along with the Janus kinase inhibitor ruxolitinib. Three years into the treatment, the patient’s inflammatory symptoms are completely in remission.