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Organ infiltration by donor T cells is critical to the development of acute graft-versus-host disease (aGVHD) in recipients after allogeneic hematopoietic stem cell transplant (allo-HCT). However, deconvoluting the transcriptional programs of newly recruited donor T cells from those of tissue-resident T cells in aGVHD target organs remains a challenge. Here, we combined the serial intravascular staining technique with single-cell RNA sequencing to dissect the tightly connected processes by which donor T cells initially infiltrate tissues and then establish a pathogenic tissue residency program in a rhesus macaque allo-HCT model that develops aGVHD. Our results enabled creation of a spatiotemporal map of the transcriptional programs controlling donor CD8+ T cell infiltration into the primary aGVHD target organ, the gastrointestinal (GI) tract. We identified the large and small intestines as the only two sites demonstrating allo-specific, rather than lymphodepletion-driven, T cell infiltration. GI-infiltrating donor CD8+ T cells demonstrated a highly activated, cytotoxic phenotype while simultaneously developing a canonical tissue-resident memory T cell (TRM) transcriptional signature driven by interleukin-15 (IL-15)/IL-21 signaling. We found expression of a cluster of genes directly associated with tissue invasiveness, including those encoding adhesion molecules (ITGB2), specific chemokines (CCL3 and CCL4L1) and chemokine receptors (CD74), as well as multiple cytoskeletal proteins. This tissue invasion transcriptional signature was validated by its ability to discriminate the CD8+ T cell transcriptome of patients with GI aGVHD from those of GVHD-free patients. These results provide insights into the mechanisms controlling tissue occupancy of target organs by pathogenic donor CD8+ TRM cells during aGVHD in primate transplant recipients.

Hematopoietic cell transplantation (HCT) is the only readily available cure for many life-threatening pediatric nonmalignant diseases (NMD), but most patients lack a matched related donor and are at higher risk for graft-versus-host disease (GVHD). Use of abatacept (Aba) to target donor T-cell activation has been safe and effective in preventing GVHD after unrelated donor (URD) HCT for malignant diseases (Aba2 trial). Our primary objective was to evaluate the tolerability of Aba added to standard GVHD prophylaxis (cyclosporine and mycophenolate mofetil) in pediatric patients with NMD undergoing URD HCT. In this single-arm, single-center phase 1 trial, 10 patients receiving reduced intensity or nonmyeloablative conditioning underwent URD HCT. Immune reconstitution was assessed longitudinally via flow cytometry and compared to pediatric patients on Aba2. Nine patients successfully engrafted, with 1 primary graft rejection in the setting of inadequate cell dose; secondary graft rejection occurred in 1 patient with concurrent cytomegalovirus viremia. Two deaths occurred, both unrelated to Aba. One patient developed probable posttransplant lymphoproliferative disease, responsive to rituximab and immune suppression withdrawal. No patients developed severe acute or chronic GVHD, and 8 patients were off systemic immunosuppression at 1 year. Immune reconstitution did not appear to be impacted by Aba, and preservation of naïve relative to effector memory CD4+ T cells was seen akin to Aba2. Thus, 4 doses of Aba were deemed tolerable in pediatric patients with NMD following URD HCT, with encouraging preliminary efficacy and supportive immune correlatives in this NMD cohort.

In the United States, Blacks and Hispanics are less likely than Whites to survive acute leukemias and other serious hematologic malignancies.1-3 Allogeneic hematopoietic cell transplantation (HCT) plays an important role in the treatment of high-risk hematologic malignancies, and 1 factor likely contributing to this disparity is the limited availability of 8/8 HLA-matched unrelated donors for persons of color (POC).4 Although most POC have access to 7/8 matched unrelated donors, mismatching is associated with increased risk for acute graft-versus-host disease (aGVHD), transplant-related mortality (TRM), and diminished overall survival (OS).

Background: Optimal chemotherapy for treating mixed-phenotype acute leukemia (MPAL) and the role of hematopoietic stem cell transplantation (HSCT) remain uncertain. Major limitations in interpreting available data are MPAL's rarity and the use of definitions other than the currently widely accepted criteria: the World Health Organization 2016 (WHO2016) classification. Methods: To assess the relative efficacy of chemotherapy types for treating pediatric MPAL, the Children's Oncology Group (COG) Acute Leukemia of Ambiguous Lineage Task Force assembled a retrospective cohort of centrally reviewed WHO2016 MPAL cases selected from banking studies for acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Patients were not treated in COG trials; treatment and outcome data were captured separately. The findings were then integrated with the available, mixed literature to develop a prospective trial in pediatric MPAL. Results: The central review confirmed that 54 of 70 cases fulfilled WHO2016 criteria for MPAL. ALL induction regimens achieved remission in 72% of the cases (28 of 39), whereas AML regimens achieved remission in 69% (9 of 13). The 5-year event-free survival (EFS) and overall survival (OS) rates for the entire cohort were 72% ± 8% and 77% ± 7%, respectively. EFS and OS were 75% ± 13% and 84% ± 11%, respectively, for those receiving ALL chemotherapy alone without HSCT (n = 21). Conclusions: The results of the COG MPAL cohort and a literature review suggest that ALL chemotherapy without HSCT may be the preferred initial therapy. A prospective trial within the COG is proposed to investigate this approach; AML chemotherapy and/or HSCT will be reserved for those with treatment failure as assessed by minimal residual disease. Embedded biology studies will provide further insight into MPAL genomics.

We compared outcomes after hematopoietic cell transplantation in patients of African American (n=84) and Caucasian (n=215) descent with severe aplastic anemia. African Americans and Caucasians were matched for age, donor-recipient human leukocyte antigen match, graft type, and transplantation year. The median follow-up of surviving patients was 5 years. In multivariate analysis, overall mortality risks were higher for African Americans compared to Caucasians (relative risk 1.73, P=0.01). The 5-year probabilities of overall survival adjusted for interval from diagnosis to transplantation, and performance score was 58% for African Americans and 73% for Caucasians. The day-100 cumulative incidence of grade III-IV, but not grade II-IV acute graft-versus-host disease (GVHD), was higher in African Americans compared to Caucasians (29% vs. 13%, P=0.006). Although the 5-year cumulative incidence of chronic GVHD was not significantly different between the racial groups, African Americans were more likely to have extensive chronic GVHD compared to Caucasians (72% vs. 49%, P=0.06). Survival differences between Caucasians and African Americans can be attributed to multiple factors. Our data suggest that some of the observed survival differences between Caucasians and African Americans may be explained by higher rates of acute GVHD and severity of chronic GVHD.

Background: We sought to better define the role of hematopoietic cell transplantation (HCT) in first remission (CR1) for high-risk pediatric acute myeloid leukemia (AML). Procedures: Outcomes were compared among patients aged less than 21 years with cytogenetically defined poor-risk AML treated with chemotherapy, matched related (MRD), or unrelated donor (URD) transplantation in CR1. Poor-risk cytogenetics was defined as monosomy 7/del7q, monosomy 5/del 5q, abnormalities of 3q, t(6;9)(p23;q34), or complex karyotype. Included are patients treated on Children's Oncology Group trials or reported to the Center for International Blood and Marrow Transplant Research from 1989 to 2006. Results: Of the 233 patients, 123 received chemotherapy, 55 received MRD HCT, and 55 received URD HCT. The 5-year overall survival from the time of consolidation chemotherapy or transplant conditioning was similar: chemotherapy (43%±9%), MRD (46%±14%), or URD (50%±14%), P=0.99. Similarly, multivariate analysis demonstrated no significant differences in survival [(reference group=chemotherapy); MRD HR 1.08, P=0.76; URD HR 1.13, P=0.67] despite lower relapse risk with URD HCT (HR=0.43, P=0.01). Conclusions: Our findings do not provide support for the preferential use of HCT over chemotherapy alone for children with cytogenetically defined poor-risk AML in CR1.

BACKGROUND Studies comparing survival of adolescent and young adult (AYA) patients to that of younger patients with newly diagnosed acute myeloid leukemia (AML) have yielded conflicting results. In order to more accurately characterize relative survival and other outcomes of AYA patients, a cross-study analysis was conducted using data from recent trials conducted by the Children's Cancer Group (CCG) and Children's Oncology Group (COG). METHODS Data were combined from the CCG-2891, CCG-2941, CCG-2961, and AAML03P1 trials. The data set included 1840 patients, comprising 238 AYA and 1602 younger patients. RESULTS Overall survival was not significantly different in the 2 groups (AYA, 49% ± 7% versus younger, 54% ± 3% (± 2 standard errors), P =.058). Relapse was lower in AYA patients (30% ± 7% versus 41% ± 3%, P =.002), but treatment-related mortality (TRM) was higher (25% ± 6% versus 12% ± 2%, P <.001). After adjustment for other factors, older age remained strongly associated with TRM (hazard ratio = 2.30, 95% CI = 1.59-3.33, P <.001). Infection accounted for the excess TRM in AYA patients. CONCLUSIONS Survival in AYA and younger patients with newly diagnosed AML is similar; however, older patients are at higher risk for TRM. More effective strategies for preventing mortality from infection in AYA patients are needed.

Sickle cell disease (SCD) is increasingly appreciated as an inflammatory condition associated with alterations in immune phenotype and function. In this cross-sectional study we performed a multiparameter analysis of 18 immune markers in 114 paediatric SCD patients divided by treatment group [those receiving hydroxycrabamide (HC, previously termed hydroxyurea), chronic transfusion (CT), or no disease-modifying therapy] and 29 age-matched African American healthy controls. We found global elevation of most immune cell counts in SCD patients receiving no disease-modifying therapy at steady state. Despite the decrease in percentage of haemoglobin S associated with CT therapy, the abnormal cellular immune phenotype persisted in patients on CT. In contrast, in both univariate and multivariate analysis, treatment with HC was associated with normalization of the vast majority of leucocyte populations. This study provides additional support for HC treatment in SCD, as it appears that HC decreases the abnormally elevated immune cell counts in patients with SCD.

Transfusion-related alloimmunization is a potent barrier to the engraftment of allogeneic hematopoietic stem cells in patients with nonmalignant diseases (NMDs). Memory T cells, which drive alloimmunization, are relatively resistant to commonly used conditioning agents. Alefacept, a recombinant leukocyte function antigen-3/IgG1 fusion protein, targets CD2 and selectively depletes memory versus naive T cells. Three multiply transfused pediatric patients with NMD received a short course of high-dose i.v. alefacept (.25 mg/kg/dose on days −40 and −9 and .5 mg/kg/dose on days −33, −26, −19, and −12) before undergoing unrelated allogeneic transplant in the setting of reduced-intensity pretransplant conditioning and calcineurin inhibitor–based post-transplant graft-versus-host disease (GVHD) prophylaxis. Alefacept infusions were well tolerated in all patients. Peripheral blood flow cytometry was performed at baseline and during and after alefacept treatment. As expected, after the 5 weekly alefacept doses, each patient demonstrated selective loss of CD2hi/CCR7−/CD45RA− effector memory (Tem) and CD2hi/CCR7+/CD45RA− central memory (Tcm) CD4+ and CD8+ T cells with relative preservation of the CD2lo Tem and Tcm subpopulations. In addition, depletion of CD2+ natural killer (NK) cells also occurred. Neutrophil recovery was rapid, and all 3 patients had 100% sorted (CD3/CD33) peripheral blood donor chimerism by day +100. Immune reconstitution (by absolute neutrophil, monocyte, and lymphocyte counts) was comparable with a cohort of historical control patients. All 3 patients developed GVHD but are all now off immune suppression and >2 years post-transplant with stable full-donor engraftment. These results suggest that alefacept at higher dosing can deplete both memory T cells and NK cells and that incorporating CD2-targeted depletion into a reduced-intensity transplant regimen is feasible and safe in heavily transfused patients.