ABSTRACT:Chronic graft-versus-host-disease (cGVHD) is the primary nonrelapse limitation to a successful hematopoietic cell transplantation and is largely treated as a single biological entity. We hypothesized that there exist different biological subtypes of cGVHD. Using the Applied Biomarkers of Late Effects of Childhood Cancer (ABLE) network database, which is derived from the largest pediatric cGVHD cohort worldwide, we applied clustering analysis to subtype patients with cGVHD from the ABLE1.0 and 2.0 studies (51 patients with cGVHD and 158 with non-cGVHD). We found 3 distinct cGVHD subtypes: cGVHD-1 was characterized by an effector memory T-cell, cytotoxic natural killer cell, and early precursor B-cell predominant pattern; cGVHD-2 was phosphatidylcholine, cytokine, and plasma cell predominant; and cGVHD-3 had more naïve CD4+ T cells and naïve regulatory T cells, had later onset, and was the only subtype with measurable T-cell receptor excision circles. We partially replicated these subtypes using metabolomic data from a separate pediatric cohort of the Children's Oncology Group trial ASCT0031 (33 patients with cGVHD and 39 with non-cGVHD). Furthermore, cGVHD-1 was associated with serotherapy (predominantly antithymocyte globulin) exposure, and cGVHD-3 was associated with receiving peripheral blood stem cells from donors, total body irradiation, and no previous acute GVHD. cGVHD-2 was associated with liver involvement and cGVHD-2 and -3 with de novo cGVHD. Overall, none of the subtypes were closely associated with organ involvement. Contrasting each subtype against patients with non-cGVHD, the 3 subtypes shared common markers, all of which were used in our previous cGVHD diagnostic classifier. These findings suggest the presence of distinct biological subtypes of cGVHD that may help guide therapeutic strategies.

The large diversity of neuronal and glial cell types in the human brain is underpinned by foundational cell populations known as neural progenitor cells (NPCs). The dentate gyrus (DG) of the hippocampus, a key structure in learning and memory, maintains a tightly organized NPC population into adulthood across many mammalian species. However, the emergence, organization and persistence of NPCs in the human hippocampus remain poorly characterized. Reports of NPCs in the juvenile, adult, and aged periods have been variable, reflecting differences in identification criteria and highlighting the need for a unified framework across development. In this study, we provide a spatial and molecular map of the developmental trajectory of NPCs in the human DG, combining multimodal transcriptomic analysis within a neuroanatomical context. At mid-gestation, we observed changes in the structural and cellular arrangement of the hippocampus, coinciding with the emergence of a multicellular NPC layer within the DG, herein named the granular-hilar progenitor zone (GHPZ). Neurogenic transcriptomic signatures in the GHPZ were diminished by early infancy, coinciding with a reduction in NPC number as they progressed toward an astrocytic program. At childhood, the GHPZ dissolved with only sparse radial NPCs remaining in the DG. Lastly, we validated WNT signaling pathway-associated genes as NPC identity markers in the developing human DG, observing a decline in their expression after infancy. Our study defines the steep decline of NPCs from gestation to the postnatal period, identifies their progression to an astrocytic nature, and sets the molecular blueprint for NPC identification in the human DG. Highlights:Multimodal mapping of neural progenitor cells from gestational to postnatal stages in the human hippocampusFormation of the granular-hilar progenitor zone within the dentate gyrus at mid-gestationNeurogenic potential declines sharply from the prenatal period to childhood, with radial glia cells progressively acquiring astrocytic featuresDevelopmental modulation of the WNT signaling pathway accompanies radial glia cell transitions.

Neuronal subtype generation in the mammalian central nervous system is governed by competing genetic programs. The medial ganglionic eminence (MGE) produces two major cortical interneuron (IN) populations, somatostatin (Sst) and parvalbumin (Pvalb), which develop on different timelines. The extent to which external signals influence these identities remains unclear. Pvalb-positive INs are crucial for cortical circuit regulation but challenging to model in vitro. We grafted mouse MGE progenitors into diverse 2D and 3D co-culture systems, including mouse and human cortical, MGE, and thalamic models. Strikingly, only 3D human corticogenesis models promoted efficient, non-autonomous Pvalb differentiation, characterized by upregulation of Pvalb maturation markers, downregulation of Sst-specific markers, and the formation of perineuronal nets. Additionally, lineage-traced postmitotic Sst-positive INs upregulated Pvalb when grafted onto human cortical models. These findings reveal unexpected fate plasticity in MGE-derived INs, suggesting that their identities can be dynamically shaped by the environment.

Background Melphalan is often used as the backbone agent for conditioning prior to A/B-T-cell depleted (A/B-TCD) hematopoietic cell transplant (HCT) due to lower rates of organ toxicity compared to busulfan or total-body irradiation, albeit with significant mucosal injury. Traditional dosing based on body-surface-area (BSA) may result in non-optimal melphalan exposure among certain patient subsets. Objectives As mucosal injury is linked to initiation of alloreactivity, we hypothesized that high exposure of melphalan predicted via a pharmacokinetic (PK) model would be associated with an increased risk of acute graft-versus-host disease (aGVHD). Study Design We performed an analysis of 85 patients who underwent A/B-TCD haploidentical HCT on 2 prospective trials using melphalan-based conditioning for treatment of malignancy at 3 centers from 2015 to 2024. Most patients (61.2%) received a total dose of melphalan at 140 mg/m2 using actual body weight; others received a dose adjusted for obesity or age <2 years. We analyzed outcomes based on whether melphalan exposure was above or below the median exposure for the group. Results The 100-day cumulative incidences of engraftment syndrome (ES), grade II-IV aGVHD, and grade III-IV aGVHD were 34.2%, 24.8%, and 17.1%, respectively. The 3-year cumulative incidence of chronic GVHD (cGVHD), non-relapse mortality (NRM), and relapse were 17.5%, 8.7%, 21.8%, respectively. The 3-year cumulative incidence of disease-free survival (DFS) and severe GVHD-relapse-free survival (GRFS) were 71.4% and 55.6%, respectively. ES was significantly associated with the subsequent development of aGVHD, both grade II-IV (41.4% vs. 17.3% in those with and without ES, P = .01) and grade III-IV (34.5% vs. 8.5% in those with and without ES; P = .003). Chronic GVHD occurred at significantly higher rates in patients with prior Grade II-IV (66.7% vs. 0% for Grade 0-I; P < .001) and Grade III-IV aGVHD (75% vs. 4.3% for Grade 0-II; P < .001). Compared to non-obese patients, the PK model predicted lower melphalan exposure (P = .02) in obese patients where adjusted ideal body weight was utilized, suggesting overcorrection of the dose. There was no impact of melphalan exposure on immunologic rejection. The median melphalan exposure was 6.81 mg*hr/L (range, 4.4-8.8). Compared to a melphalan exposure ≤6.8 mg*hr/L, a melphalan exposure >6.8 mg*hr/L was associated with a higher incidence of ES (48.8% vs. 19.1%; P = .005), grade II-IV aGVHD (39.3% vs. 10.1%; P = .002), and grade III-IV aGVHD (31.5% vs. 2.5%; P < .001). The 3-year incidence of cGVHD was 27.2% in those with high predicted melphalan exposure compared to 7.4% for low exposure (P = .03); with no difference in 3-year NRM incidence (9.2% vs. 7.7%; P = .82) or 3-year relapse incidence (16.8% vs. 27.6%; P = .31) for high compared to low exposure. However, GRFS was significantly worse in patients with high exposure (46.4%) vs. low exposure (64.6%; P = .02). Conclusions High melphalan exposure predicted by a validated population PK model is associated with an increased likelihood of developing ES and subsequently acute and chronic GVHD. Given that a substantial number of patients already require adjustment of standard BSA-based dosing for young age or obesity, a prospective trial of model-based dosing to individualize melphalan exposure is warranted to confirm these results.

Neurological disorders often originate from progressive brain network dysfunctions that start years before symptoms appear. How these changes emerge in the developing human brain remains elusive due to a lack of tractable model systems. Here, we show a cerebral organoid model of Tuberous Sclerosis Complex (TSC) that recapitulates hallmarks of epileptogenesis in vitro. We compare extracellular recordings of TSC organoids with intraoperative electrocorticography from TSC patients to reveal striking functional similarities, including high-frequency oscillations - an electrical biomarker for epileptogenic tissue. In TSC, a human-specific interneuron sub-type derived from the caudal ganglionic eminence drives network hyper-synchronization through increased spontaneous firing and altered excitability. Inhibiting overproliferation of its progenitors via long-term epidermal growth factor receptor inhibition prevented the onset of this pathological phenotype at functional and morphological levels. Our work shows that organoids allow mechanistic analysis of emerging neural network phenotypes, enabling anti-epileptogenic drug testing in a human brain development model. ### Competing Interest Statement J.A.K. is co-founder of A:Head bio AG and is an inventor on several patents relating to cerebral organoids. Austrian Academy of Sciences, https://ror.org/03anc3s24 FWF Austrian Science Fund, https://ror.org/013tf3c58, F7804-B FWF Austrian Science Fund, P35680 FWF Austrian Science Fund, P35369 Federal Ministry of Education, Science and Research, https://ror.org/03gng8t46 The Gilbert Family Foundation, 923017 Valencian Council for Education, Culture, University and Employment, CIPROM/2023/053 Instituto de Investigación Sanitaria La Fe Dutch Research Council, 09150172210057 Australian Research Council, FT230100138 Horizon 2023 Framework Programme (Marie Skłodowska-Curie), 101155338

Successful allogeneic hematopoietic cell transplantation (HCT) for genetic nonmalignant diseases (NMD) is dependent upon elimination of host hematopoietic stem cells (HSCs) and replacement with donor HSCs in stable numbers sufficient to correct the underlying disease. Donor myeloid chimerism (DMC) in peripheral blood (PB) is a surrogate marker for bone marrow HSC engraftment due to the rapid turnover of PB myeloid cells. Busulfan is commonly used during conditioning for patients with NMD, though its optimal exposure to avoid inadequate DMC is not fully known. Younger patients with NMD have more mixed chimerism compared to older patients when receiving melphalan-based conditioning, possibly due to increased marrow reserve; we hypothesized that a similar phenomenon would occur with busulfan-based conditioning. We performed a retrospective analysis of 105 consecutive patients with NMDs (median age of 3.9 years) undergoing first allogeneic HCT with myeloablative (cAUC ≥55 mg*hr/L) busulfan-based conditioning from 2007 to 2023 and evaluated risk factors for the development of mixed (<95%) and minimal (≤20%) DMC. Receiver operating curve analysis demonstrated that age <2.9 years was the cut-off associated with mixed DMC. The cohort was therefore divided into two age groups: <3 years (43.8% of patients) and ≥3 years; there was no difference in busulfan exposure between the groups. The 3-year cumulative incidence of developing mixed DMC was 20.3% (95% CI, 12.1% to 28.5%). Age was associated with mixed DMC by 3 years post-HCT: 45.2% (95% CI, 29.7% to 60.7%) in those <3 years versus 1.9% (95% CI, 0.1% to 5.4%) in those ≥3 years (P < .001). Busulfan exposure was also associated with mixed DMC: 24.8% (95% CI, 14.8% to 34.8%) in those with cAUC 55 to 74.9 mg*hr/L versus 5% (95% CI, 0.1% to 8.3%) in those with cAUC ≥75 mg*hr/L (P = .03). In patients ≥3 years of age, there was no impact of busulfan exposure on development of mixed DMC. The 3-year cumulative incidence of developing minimal DMC was 5.5% (95% CI, 0.8% to 10.2%). Only young age was significantly associated with minimal DMC by 3 years post-HCT: 12.8% (95% CI, 2.2% to 23.4%) in those <3 years versus 0% (95% CI, 0% to 6.1%) in those ≥3 years (P = .008). There was no impact of age or busulfan exposure on immunologic graft rejection, acute or chronic graft-versus-host-disease, or transplant-related mortality. There was no difference in sinusoidal obstruction syndrome (SOS) incidence based on busulfan exposure: 13.7% (95% CI, 6.3% to 21.1%) for a cAUC of 55 to 74.9 mg*hr/L compared to 20.8% (95% CI, 4.5% to 37.1%) for a cAUC of ≥75 mg*hr/L (P = .33). However, the incidence of SOS was impacted by patient age: 32.9% (95% CI, 19.2% to 46.6%) in patients <3 years compared to 1.7% (95% CI, 0.1% to 5%) in patients ≥3 years (P < .001). Age <3 years at time of HCT is the major risk factor for mixed and minimal DMC in patients with NMD who receive busulfan-based conditioning. Patients ≥3 years of age may not require busulfan exposures above 55 to 60 mg*hr/L to develop full DMC. Conversely, to avoid development of mixed DMC, patients <3 years may benefit from either HCT delay (when feasible) or higher (≥75 mg*hr/L) busulfan exposure, albeit carrying a high-risk for SOS development.

Fludarabine is commonly used during conditioning for allogeneic hematopoietic cell transplantation (HCT) to prevent rejection of donor cells, given its profound immunosuppressive effects on T cells, albeit with minimal activity against NK cells. In pediatric patients, traditional body-surface-area (BSA)-based dosing strategies may result in non-optimal exposure. Pharmacokinetic (PK) models have been developed that allow precise targeting of a goal exposure (area-under-the-curve [cAUC] of 20 mg·h/L), which may improve outcomes. We sought to determine whether the use of PK-guided model-based dosing (MBD) results in lower incidences of rejection in the context of other relevant predictors of rejection. We performed a retrospective analysis of 410 patients with a median age of 8 yr (range, 0.1 to 26.5) who underwent first allogeneic HCT for treatment of both malignant and non-malignant conditions between 2008 and 2024 with fludarabine-containing conditioning, including 239 who received BSA-based dosing and 171 who received MBD. The overall 6-mo cumulative incidence of immunologic rejection was 5.8% (95% CI, 3.4% to 8.2%). Factors associated with rejection included: (1) an absolute lymphocyte count obtained prior to start of lymphodepletion (absolute lymphocyte count [ALC]) ≥1.275 × 10^9/L (9.4% versus 1.1% for lower ALCs; P < .001); (2) using a female donor for a male recipient (10.9% versus 4% for other combinations; P = .009); (3) using an HLA-mismatched donor (9.7% versus 1.6% for 10/10 matched donors; P < .001); and (4) positive CMV serostatus of recipient and/or donor (7.3% versus 1.6% for CMV recipient negative/donor negative; P = .02). MBD of fludarabine was also associated with a lower risk of rejection (1.8% versus 8.5% for BSA-based dosing; P = .004) and this association was most apparent amongst those with two or three of the other identified risk factors. Multivariate analysis confirmed all five of these factors are independently associated with rejection; the hazard ratio of rejection for BSA-based dosing of fludarabine was 5.12 (1.51 to 17.28; P = .009). Use of MBD of fludarabine to target a cAUC of 20 mg·h/L is an important component of optimizing conditioning regimens to minimize risk of immunologic rejection. While the potential benefit of MBD with fludarabine was most significant in patients with other risk factors for rejection, the simplicity of this approach allows for its universal incorporation into conditioning regimens for all pediatric allogeneic HCT.

The lack of immune tolerance after hematopoietic cell transplantation (HCT) can result in chronic graft-versus-host disease (cGvHD), which is the primary nonrelapse limitation of successful HCT. To date, immune tolerance has been considered a single biological entity, but we hypothesized that post-HCT immune tolerance could develop through multiple pathways. Using the ABLE network database, which comprises measurements of 75 cell populations, 10 cytokines and chemokines, lymphocyte population telomere length, KRECs and TRECs, and 132 metabolites from the largest pediatric cGvHD cohort (n = 241), we applied clustering analysis to patients with primary immune tolerance (PIT; no acute GvHD [aGvHD] or cGvHD) and patients with secondary immune tolerance (SIT; previous aGvHD and no cGvHD) to test whether subtypes could be identified. The evaluation of PIT identified 3 subtypes. PIT-1, associated with postpubertal age, lower thymic output, and increased ST2 compared to PIT-2 and PIT-3, is effector memory T cell-predominant. PIT-2, associated with prepubertal age, normal thymic output, increased B cell development, and longer lymphocyte telomeres, has a naïve T cell-predominant pattern. PIT-3, associated with postpuberty, higher thymic output, and malignancy, is dominated by increased PD1+ regulatory T cells and helper T cells and decreased long-chain acylcarnitine. We partially replicated these PIT subtypes using metabolomic data from a separate pediatric cohort of the Children’s Oncology Group trial ASCT0031 (n = 24 PIT patients). Previously resolved aGvHD had a minimal impact on the overall patterns of SIT-1 and SIT-2 compared to PIT-1 and PIT-2, except for time delays in the expansion of some immune cells. PIT-3 and SIT-3 were dominated by late increases in phosphatidylcholines (lysophosphatidylcholine precursors) and long-chain lysophosphatidylcholines (LYSOC20:4 and LYSOC16:2), respectively. This is the first time that distinct biological patterns of immune reconstitution after HCT are identified, which on validation and potentially could aid the development of future strategies for tolerance induction.

ABSTRACT Background Thrombopoietin receptor agonists (TPO‐RAs) have demonstrated efficacy in treating clinically significant thrombocytopenia, including chemotherapy‐induced thrombocytopenia in adults. However, data regarding their safety and efficacy in pediatric, adolescents, and young adult (AYA) patients with hematologic malignancies are limited. Methods We retrospectively identified 15 pediatric and AYA patients aged 25 years or younger with hematologic malignancies treated with a TPO‐RA at UCSF Benioff Children's Hospitals between 2015 and 2023. Platelet counts and transfusion requirements were compared before and after TPO‐RA therapy. Results The median age at TPO‐RA initiation was 16 years (range: 7–25 years). Nine patients (60%) had a history of bleeding or comorbidity that predisposed to severe bleeding risk. Eleven patients received romiplostim and four patients received eltrombopag. The median platelet count significantly increased from 24 × 10 9 /L at baseline to 54 × 10 9 /L after 3 weeks of any TPO‐RA therapy ( p = 0.029). Monthly platelet transfusion requirements significantly decreased from a median of 15 to two units after TPO‐RA therapy ( p = 0.007). Fourteen of the 15 patients (93%) achieved a sustained platelet count >50,000/µL within 8 weeks, with a median time to response of 3 weeks. No TPO‐RA‐related adverse events were observed. Conclusion TPO‐RAs were effective in managing refractory thrombocytopenia in pediatric and young adult patients being treated for hematologic malignancies, with a favorable safety profile, even among patients with multiple comorbidities. These findings warrant further investigation through prospective clinical trials to confirm efficacy and establish clinical guidelines for this population.

BackgroundAlloHSCT serves as a long-term curative intervention for CAYA with varied disorders but poses risks like refractory ADV infections due to impaired T-cell immunity (George/Cairo, BJH, 2012). Here, we report data from the Viral Cytotoxic T-Lymphocyte Consortium (VIRCTLC), showing the safety and efficacy of employing familial ADV-specific CTLs in AlloHSCT recipients.ObjectiveAssess safety and effectiveness of familial ADV-specific CTLs for refractory ADV post-AlloHSCT.Design/MethodsPatients post-AlloHSCT showing refractory ADV were eligible. Refractory ADV infection was defined by increasing serum RT-PCR DNA (by 1 log) after 7 days or persistent quantitative RT-PCR DNA copies after 14 days of appropriate anti-viral therapy, and/or known resistance/intolerance to anti-viral agents. Related donors were matched at ≥3 HLA (A, B, or DRB1) loci and had an adequate T-cell IFN-γ response to ADV specific MACS PepTivators®. Direct selection was used to expedite cell product generation, in a point-of-care setting. Donor peripheral blood mononuclear cells were collected using non-mobilized apheresis. ADVCTLs were isolated using the CliniMACS ® Prodigy following stimulation with specific MACS® GMP PepTivator® AdV5 Hexon, provided by Miltenyi Biotec®. ADV-specific CTLs were enriched using a Cytokine Capture System (CCS). Target cell dose was 0.5 × 104 CD3+ cells/kg (recipient weight) for haploidentical related donors and 2.5 × 104 CD3+ cells/kg for matched related donors. Repeated doses were permitted every 2 weeks in the absence of a complete response (CR) and adverse events. The following were used to define response after initial 5 doses: CR - undetected ADV PCR, partial response (PR) - at least one log decrease from baseline, progressive disease (PD) – at least one log increase from baseline, and patients with stable disease.ResultsOf 15 enrolled patients (10F, 5M; ages 1-19), all post-AlloHSCT, 8 vsCTLs were derived from the original familial HSCT donors, and 7 from third-party donors (5 maternal, 2 paternal). The mean number of ADV CTL infusions was 3.7 (range: 1-16). Thirteen patients achieved CR as defined and 2 achieved PR. The overall response (OR) was 100% and the CR was 88%. The average time to OR was 33 days (range 6-112 days). Day 100 and 365 overall survival post-HSCT was 86.1% (CI95: 49.4-95.7) and 70.5% (CI95: 38.9-87.8), respectively (Figure 1). Among CR patients, Day 100 and 365 survival was 83.9% (CI95: 55.0-94.3) and 74.6% (CI95: 39.8-91.1), respectively. ADV-related mortality at both time points was 0% (Figure 2). One patient developed acute grade 2 skin GVHD possibly related to infusion, which resolved.ConclusionPreliminary data affirm that donor ADV-specific CTLs are safe and effective in treating refractory ADV cases post-AlloHSCT. Manufacturing is rapid and reliable. Supported by FDA RO1006301A1. Enrollment is ongoing.

Sinusoidal obstructive syndrome (SOS), or veno-occlusive disease, of the liver has been recognized as a complex, life-threatening complication in the posthematopoietic stem cell transplant (HSCT) setting. The diagnostic criteria for SOS have evolved over the last several decades with a greater understanding of the underlying pathophysiology, with 2 recent diagnostic criteria introduced in 2018 (European Society of Bone Marrow Transplant [EBMT] criteria) and 2020 (Cairo criteria). We sought out to evaluate the performance characteristics in diagnosing and grading SOS in pediatric patients of the 4 different diagnostic criteria (Baltimore, Modified Seattle, EBMT, and Cairo) and severity grading systems (defined by the EBMT and Cairo criteria). Retrospective chart review of children, adolescent, and young adults who underwent conditioned autologous and allogeneic HSCT between 2017 and 2021 at a single pediatric institution. A total of 250 consecutive patients underwent at least 1 HSCT at UCSF Benioff Children's Hospital San Francisco for a total of 307 HSCT. The day 100 cumulative incidence of SOS was 12.1%, 21.1%, 28.4%, and 28.4% per the Baltimore, Modified Seattle, EBMT, and Cairo criteria, respectively (P < .001). We found that patients diagnosed with grade >4 SOS per the Cairo criteria were more likely to be admitted to the Pediatric Intensive Care Unit (92% versus 58%, P = .035) and intubated (85% versus 32%, P = .002) than those diagnosed with grade >4 per EBMT criteria. Age <3 years-old (HR 1.76, 95% [1.04 to 2.98], P = .036), an abnormal body mass index (HR 1.69, 95% [1.06 to 2.68], P = .027), and high-risk patients cantly associated with SOS per the Cairo criteria. We demonstrate that age <3 years, abnormal body mass index, and other high-risk criteria associate strongly with subsequent SOS development. Patients with moderate to severe SOS based on Cairo severity grading system may correlate better with clinical course based on ICU admissions and intubations when compared to the EBMT severity grading system. (c) 2024 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.

Transplant associated thrombotic microangiopathy (TA-TMA) is a complication of hematopoietic cell transplant (HCT) associated with endothelial injury resulting in severe end organ damage, acute and long-term morbidity, and mortality. Myeloablative conditioning is a known risk factor, though specific causative agents have not been identified. We hypothesized that the combination of cyclophosphamide and thiotepa (CY + TT) is particularly toxic to the endothelium, placing patients at elevated risk for TA-TMA. We conducted a retrospective review of pediatric and young adult patients who received conditioned autologous and allogeneic HCT between 2012 and August 2023 at UCSF Benioff Children's Hospital, San Francisco. We excluded patients undergoing gene therapy or triple tandem transplants for brain tumors. Neuroblastoma tandem transplants were classified a single transplant occurrence. High dose N-acetylcysteine (NAC) prophylaxis was incorporated into the institutional standard of care from December 2016-May 2019 and May 2022-August 2023. Defibrotide was given prophylactically to patients deemed high-risk for sinusoidal obstruction syndrome (SOS) per institutional guidelines or on clinical trial NCT#02851407 for SOS prophylaxis or NCT#03384693 for TA-TMA prophylaxis. Kaplan-Meier analysis was used to estimate the 1-year cumulative incidence of TA-TMA. Univariate analysis was performed for each of the potential risk factors of interest using log-rank tests and bivariate analysis with Cox regression models using backward selection and hazard ratios were built using all covariates with a univariate P-value < .2 for allogeneic HCT. SPSS (v29) was used to estimate all summary statistics, cumulative incidences, and uni- and bi-variate analyses. A total of 558 transplants were performed with 43 patients developing TA-TMA, for a 1-year cumulative incidence of 8.6% (95% CI, 5.9-11.3) and 7.2% (95% CI, 2.9-11.5) in allogeneic and autologous HCTs, respectively (P = .62). In allogeneic recipients (n = 417), the 1-year cumulative incidence of TA-TMA with CY + TT as part of conditioning was 35.7% (95% CI, 15.7-55.7) compared to 11.7% (95% CI, 7.2-16.2) with either CY or TT alone, and 1.2% (95% CI, 0-2.8) if neither agent was included in the conditioning regimen (P < .001). Use of either CY or TT (HR = 10.14; P = .002) or CY + TT (HR = 35.93; P < .001), viral infections (HR = 4.3; P = .017) and fungal infections (HR = 2.98; P = 0.027) were significant factors resulting in increased risk for developing TA-TMA. In subjects undergoing autologous HCT (n = 141), the 1-year cumulative incidence of TA-TMA with CY + TT was 19.6% (95% CI, 8.8-30.6) while TA-TMA did not occur in patients receiving either CY or TT alone or when neither were included (P < .001). TA-TMA occurred only in patients with neuroblastoma receiving CY + TT as part of their conditioning. For autologous patients who received CY + TT, those who were CMV seronegative at the time of HCT had an incidence of TA-TMA of 6.7% (95% CI, 0.1-15.7) compared to 38.1% (95% CI, 35-41.2) for those CMV seropositive (P = .007). These data show that CY or TT alone or in combination as part of pre-transplant conditioning prior to HCT increase the incidence of TA-TMA. Alternative conditioning excluding the combination of CY + TT should be considered whenever possible to limit the development of TA-TMA.

Viral infections remain a major risk in immunocompromised pediatric patients, and virus-specific T cell (VST) therapy has been successful for treatment of refractory viral infections in prior studies. We performed a phase II multicenter study (NCT03475212) for the treatment of pediatric patients with inborn errors of immunity and/or post allogeneic hematopoietic stem cell transplant with refractory viral infections using partially-HLA matched VSTs targeting cytomegalovirus, Epstein-Barr virus, or adenovirus. Primary endpoints were feasibility, safety, and clinical responses (>1 log reduction in viremia at 28 days). Secondary endpoints were reconstitution of antiviral immunity and persistence of the infused VSTs. Suitable VST products were identified for 75 of 77 clinical queries. Clinical responses were achieved in 29 of 47 (62%) of patients post-HSCT including 73% of patients evaluable at 1-month post-infusion, meeting the primary efficacy endpoint (>52%). Secondary graft rejection occurred in one child following VST infusion as described in a companion article. Corticosteroids, graft-versus-host disease, transplant-associated thrombotic microangiopathy, and eculizumab treatment correlated with poor response, while uptrending absolute lymphocyte and CD8 T cell counts correlated with good response. This study highlights key clinical factors that impact response to VSTs and demonstrates the feasibility and efficacy of this therapy in pediatric HSCT.