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Racial/ethnic disparities in utilizing inpatient palliative care services are well documented in the adult literature. However, the impact of racial/ethnic disparities in the context of pediatric palliative care is less well understood even in high-acuity patient populations such as stem cell transplant patients. We investigated racial/ethnic differences in the utilization of inpatient palliative care consultations (IPCCs) for pediatric stem cell transplant patients.A retrospective cohort study was conducted using the Pediatric Health Information System database. A generalized linear mixed effects model was developed to assess demographic and clinical characteristics associated with the likelihood of receiving IPCC.Thirty-eight tertiary pediatric hospitals in the United States.Pediatric patients undergoing stem cell transplantation for any indication from January 2017 to December 2019.None.Of the 1,193 inpatient encounters studied, 12% (n = 143) included a palliative care consult. IPCC rates varied across hospitals with a median rate of 5.97% (interquartile range, 0.00-20.71). In multivariable analyses, Hispanic/Latinx patients were 59% less likely to receive IPCC compared with non-Hispanic White patients (odds ratio [OR], 0.41; 95% CI, 0.21-0.78). This difference persisted after adjusting for all other sociodemographic and clinical factors. In terms of the other clinical characteristics, having a malignant condition and mechanical ventilation were associated with significantly increased odds of receiving IPCC for the entire cohort (OR Malignancy: 1.93; 95% CI: 1.07-3.51; OR Mechanical Ventilation: 2.37; 95% CI: 1.36-4.13). The remainder of the variables were not found to be significantly associated with IPCC.Racial and ethnic differences exist in the likelihood of receiving palliative care consultations among hospitalized pediatric stem cell transplant recipients. Evaluating the impact of systemic racism and social determinants on palliative care medicine as well as standardizing early integration of IPCC may potentially mitigate disparities in this population.

High-risk neuroblastoma patients with end-induction residual disease commonly receive post-induction therapy in an effort to increase survival by improving the response before autologous stem cell transplantation (ASCT). The authors conducted a multicenter, retrospective study to investigate the efficacy of this approach.Patients diagnosed between 2008 and 2018 without progressive disease with a partial response or worse at end-induction were stratified according to the post-induction treatment: 1) no additional therapy before ASCT (cohort 1), 2) post-induction "bridge" therapy before ASCT (cohort 2), and 3) post-induction therapy without ASCT (cohort 3). χThe study cohort consisted of 201 patients: cohort 1 (n = 123), cohort 2 (n = 51), and cohort 3 (n = 27). Although the end-induction response was better for cohort 1 than cohorts 2 and 3, the outcomes for cohorts 1 and 2 were not significantly different (P = .77 for EFS and P = .85 for OS). Inferior outcomes were observed for cohort 3 (P.001 for EFS and P = .06 for OS). Among patients with end-induction stable metastatic disease, 3-year EFS was significantly improved for cohort 2 versus cohort 1 (P = .04). Cohort 3 patients with a complete response at metastatic sites after post-induction therapy had significantly better 3-year EFS than those with residual metastatic disease (P = .01).Prospective studies to confirm the benefits of bridge treatment and the prognostic significance of metastatic response observed in this study are warranted.

The COVID-19 pandemic underscores the need for rapid, safe, and effective vaccines. In contrast to some traditional vaccines, nanoparticle-based subunit vaccines are particularly efficient in trafficking antigens to lymph nodes, where they induce potent immune cell activation. Here, we developed a strategy to decorate the surface of oxidation-sensitive polymersomes with multiple copies of the SARS-CoV-2 spike protein receptor-binding domain (RBD) to mimic the physical form of a virus particle. We evaluated the vaccination efficacy of these surface-decorated polymersomes (RBDsurf) in mice compared to RBD-encapsulated polymersomes (RBDencap) and unformulated RBD (RBDfree), using monophosphoryl-lipid-A-encapsulated polymersomes (MPLA PS) as an adjuvant. While all three groups produced high titers of RBD-specific IgG, only RBDsurf elicited a neutralizing antibody response to SARS-CoV-2 comparable to that of human convalescent plasma. Moreover, RBDsurf was the only group to significantly increase the proportion of RBD-specific germinal center B cells in the vaccination-site draining lymph nodes. Both RBDsurf and RBDencap drove similarly robust CD4+ and CD8+ T cell responses that produced multiple Th1-type cytokines. We conclude that a multivalent surface display of spike RBD on polymersomes promotes a potent neutralizing antibody response to SARS-CoV-2, while both antigen formulations promote robust T cell immunity., We developed nanoscale polymer vesicles (polymersomes) decorated with multiple copies of SARS-CoV-2 antigen as a COVID-19 vaccine that elicits neutralizing antibodies and cellular immunity.

Chimeric antigen receptor (CAR) T-cell therapy has become a novel approach in the treatment of many hematologic malignancies. However, ocular adverse effects have not been well described. This report presents a case of a pediatric patient with relapsed B-cell acute lymphoblastic leukemia with ocular involvement treated with CAR T-cell therapy who developed an exudative retinal detachment likely secondary to an inflammatory response to CAR T-cell therapy. [ Ophthalmic Surg Lasers Imaging Retina . 2022;53:113–115.]

Biomacromolecules have long been at the leading edge of academic and pharmaceutical drug development and clinical translation. With the clinical advances of new therapeutics, such as monoclonal antibodies and nucleic acids, the array of medical applications of biomacromolecules has broadened considerably. A major on-going effort is to expand therapeutic targets within intracellular locations. Owing to their large sizes, abundant charges, and hydrogen-bond donors and acceptors, advanced delivery technologies are required to deliver biomacromolecules effectively inside cells. In this review, strategies used for the intracellular delivery of three major forms of biomacromolecules: nucleic acids, proteins, and peptides, are highlighted. An emphasis is placed on synthetic delivery approaches and the major hurdles needed to be overcome for their ultimate clinical translation.

BCL-2 family proteins are central regulators of apoptosis and represent prime therapeutic targets for overcoming cell death resistance in malignancies. However, plasticity of anti-apoptotic members, such as MCL-1, often allows for a switch in cell death dependency patterns that lie outside the binding profile of targeted BH3-mimetics. Therefore discovery of therapeutics that effectively inactivate all anti-apoptotic members is a high priority. To address this we tested the potency of a hydrocarbon stapled BIM BH3 peptide (BIM SAHB A ) to overcome both BCL-2 and MCL-1 apoptotic resistance given BIM's naturally wide ranging affinity for all BCL-2 family multidomain members. BIM SAHB A effectively killed diffuse large B-cell lymphoma (DLBCL) cell lines regardless of their anti-apoptotic dependence. Despite BIM BH3's ability to bind all BCL-2 anti-apoptotic proteins, BIM SAHB A 's dominant intracellular target was MCL-1 and this specificity was exploited in sequenced combination BH3-mimetic treatments targeting BCL-2, BCL-XL, and BCL-W. Extending this MCL-1 functional dependence, mouse embryonic fibroblasts (MEFs) deficient in MCL-1 were resistant to mitochondrial changes induced by BIM SAHB A . This study demonstrates the importance of understanding BH3 mimetic functional intracellular affinities for optimized use and highlights the diagnostic and therapeutic promise of a BIM BH3 peptide mimetic as a potential MCL-1 inhibitor.

Chimeric antigen receptor (CAR) T-cell therapy has become the standard of care for children and young adults with relapsed and refractory B-cell acute lymphoblastic leukemia (B-ALL), and it is a highly promising therapy under investigation for adults with relapsed disease. Despite having potentially life-threatening toxicities, such as cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome, the benefits of CAR T-cell therapy far outweigh these risks, particularly as increased experience and improved supportive care measures are mitigating these toxicities. CAR T cells can result in complete remission for significant proportion of patients with relapsed and refractory B-ALL and permit them to proceed to potentially curative allogeneic hematopoietic stem cell transplantation (allo-HSCT). CAR T cells may also be curative by themselves. Herein lie the greatest challenges and questions for clinical investigators, specifically, how are CAR T cells best employed and how do we overcome mechanisms of resistance to them? The primary clinical question is the timing and even the necessity of allo-HSCT. Relative to resistance, we know that target antigen loss, specifically CD19, is a major contributor to resistance. However, current investigations of alternative targets, such CD22, and CAR T cells expressing dual targeting antigen receptors have demonstrated encouraging initial results and provide a high degree of optimism that the efficacy and the broader application of CAR T-cell therapy will gradually increase in B-ALL. That optimism is not as high and the challenges are increased for the application of CAR T cells in T-cell leukemias and acute myeloid leukemia due to the relative lack of suitable leukemia surface targets that are not also expressed on normal hematopoietic progenitors. Despite these significant challenges, considerable research is being conducted into the development of CAR T cells for these diseases utilizing unique technologies, which may be applicable to other diseases.

A diverse portfolio of SARS-CoV-2 vaccine candidates is needed to combat the evolving COVID-19 pandemic. Here, we developed a subunit nanovaccine by conjugating SARS-CoV-2 Spike protein receptor binding domain (RBD) to the surface of oxidation-sensitive polymersomes. We evaluated the humoral and cellular responses of mice immunized with these surface-decorated polymersomes (RBDsurf) compared to RBD-encapsulated polymersomes (RBDencap) and unformulated RBD (RBDfree), using monophosphoryl lipid A-encapsulated polymersomes (MPLA PS) as an adjuvant. While all three groups produced high titers of RBD-specific IgG, only RBDsurf elicited a neutralizing antibody response to SARS-CoV-2 comparable to that of human convalescent plasma. Moreover, RBDsurf was the only group to significantly increase the proportion of RBD-specific germinal center B cells in the vaccination-site draining lymph nodes. Both RBDsurf and RBDencap drove similarly robust CD4+ and CD8+ T cell responses that produced multiple Th1-type cytokines. We conclude that multivalent surface display of Spike RBD on polymersomes promotes a potent neutralizing antibody response to SARS-CoV-2, while both antigen formulations promote robust T cell immunity.

Chimeric antigen receptor (CAR) T-cell therapy has become the standard of care for children and young adults with relapsed and refractory B-cell acute lymphoblastic leukemia (B-ALL), and it is a highly promising therapy under investigation for adults with relapsed disease. Despite having potentially life-threatening toxicities, such as cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome, the benefits of CAR T-cell therapy far outweigh these risks, particularly as increased experience and improved supportive care measures are mitigating these toxicities. CAR T cells can result in complete remission for significant proportion of patients with relapsed and refractory B-ALL and permit them to proceed to potentially curative allogeneic hematopoietic stem cell transplantation (allo-HSCT). CAR T cells may also be curative by themselves. Herein lie the greatest challenges and questions for clinical investigators, specifically, how are CAR T cells best employed and how do we overcome mechanisms of resistance to them? The primary clinical question is the timing and even the necessity of allo-HSCT. Relative to resistance, we know that target antigen loss, specifically CD19, is a major contributor to resistance. However, current investigations of alternative targets, such CD22, and CAR T cells expressing dual targeting antigen receptors have demonstrated encouraging initial results and provide a high degree of optimism that the efficacy and the broader application of CAR T-cell therapy will gradually increase in B-ALL. That optimism is not as high and the challenges are increased for the application of CAR T cells in T-cell leukemias and acute myeloid leukemia due to the relative lack of suitable leukemia surface targets that are not also expressed on normal hematopoietic progenitors. Despite these significant challenges, considerable research is being conducted into the development of CAR T cells for these diseases utilizing unique technologies, which may be applicable to other diseases.

Background Regulatory T cells (Tregs) have been a therapeutic target of interest since early pre-clinical work revealed that their depletion led to enhanced tumor control. Despite continuing advances in the development of novel cellular-, antibody- and chemotherapeutic-based strategies to increase anti-tumor immunity, Treg presence and activity within the tumor microenvironment remains a complicating factor to their clinical efficacy. To overcome dosing limitations and off-target effects from antibody-based Treg deletional strategies, we investigated the ability to target FOXP3, the master regulator of Treg development, maintenance, and suppressive function using hydrocarbon stapled alpha-helical peptides (SAHs). We developed SAHs in the likeness of a portion of the native FOXP3 antiparallel coiled-coil homodimerization domain, in an effort to impede FOXP3 transcriptional function. SAHs overcome three major protein-protein interaction (PPI) therapeutic hurdles, namely: cellular penetrance, target specificity, and secondary structure stability. Our overall goal is to use these SAHs as investigatory drugs to demonstrate proof-of concept of the druggability of FOXP3. We aim to show their utility to further understand FOXP3 transcriptional dynamics and explore their potential use in altering the immune landscape in combination with other immune-focused therapies. Methods Utilizing the FOXP3 crystal structure as a guide, we developed a number of single and double SAH peptides corresponding to the leucine zipper homodimerization domain (DD) of FOXP3 (SAH-FOXP3DDs). We tested the ability of SAH-FOXP3DDs to bind FOXP3, to access the intracellular compartment, to alter Treg transcriptional and phenotypic profiles, and to inhibit Treg-mediated immune suppression. Results Select SAH-FOXP3DDs bound recombinant FOXP3ΔN and the FOXP3 leucine zipper (LZCC) domain with high affinity and dose-dependently inhibited binding of FOXP3 to cognate DNA in vitro. Lead SAH-FOXP3DDs were cell permeable and showed no non-specific toxicity to T cells at high concentrations. Flow cytometric and qRT-PCR analysis of treated Tregs revealed dose-dependent changes in protein and gene expression of several FOXP3 targets suggestive of FOXP3-specific transcriptional alteration. Treatment of Tregs with lead SAHs effectively inhibited in vitro Treg-mediated T cell suppression and induced global gene expression changes corresponding to loss of function Foxp3 in vivo. Conclusions This work supports the ability of SAHs to target transcription factors, particularly as a method of interrogating specific PPI functions, and provides strong proof-of-principle evidence that FOXP3 is druggable. SAH-FOXP3DDs will not only further our understanding of FOXP3 transcriptional control but will serve as prototype therapeutics whereby we can explore their ability to amplify anti-tumor immunity in pre-clinical tumor models.

BIM is the master BH3-only BCL-2 family regulator of lymphocyte survival. To understand how long-term loss of BIM affects apoptotic resistance in T cells we studied animals with T cell-specific deletion of Bim. Unlike CD19(CRE) Bim(fl/fl) animals, LCK(CRE) Bim(fl/fl) mice have pronounced early lymphocytosis followed by normalization of lymphocyte counts over time. This normalization occurred in mature T cells, as thymocyte development and apoptotic sensitivity remained abnormal in LCK(CRE) Bim(fl/fl) mice. T cells from aged mice experienced normalization of their absolute cell numbers and responses against various apoptotic stimuli. mRNA expression levels of BCL-2 family proteins in CD4(+) and CD8(+) T cells from young and old mice revealed upregulation of several BH3-only proteins, including Puma, Noxa, and Bmf. Despite upregulation of various BH3 proteins, there were no differences in anti-apoptotic BCL-2 protein dependency in these cells. However, T cells had continued resistance to direct BIM BH3-induced mitochondrial depolarization. This study further highlights the importance of BIM in cell death maintenance in T cells and provides new insight into the dynamism underlying BH3-only regulation of T cell homeostasis versus induced cell death and suggests that CD4(+) and CD8(+) T cells compensate differently in response to loss of Bim.

Despite the therapeutic promise of phospholipid-based nanocarriers, a major obstacle to their widespread clinical translation is a susceptibility to fatty acid ester hydrolysis, leading to lack of quality control and inconsistencies in self-assembly formulations. Using electrospray ionization mass spectrometry fragmentation in combination with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we have demonstrated a method to detect hydrolysis of one or both of the fatty acid esters in a PEGylated phospholipid, DSPE-PEG, in conditions commonly applied during nanocarrier production. Because such carriers are increasingly being used to deliver peptide-based therapeutics, we further investigated the hydrolysis of phospholipid esters in conditions used for solid-phase peptide synthesis and high-performance liquid chromatography of peptides. We ultimately detail a synthetic strategy to reliably produce pure phospholipid-peptide bioconjugates (peptide amphiphiles), while avoiding unintended or unnoticed hydrolyzed byproducts that could lead to polymorphic nanotherapeutics with dampened therapeutic efficacy. We believe that such an approach could help standardize phospholipid-peptide-based therapeutic development, testing, and clinical translation.

Outcomes for patients with high-risk neuroblastoma (HR-NBL) are significantly improved with the addition of immunotherapy (dinutuximab + cytokines) following autologous hematopoietic stem cell transplantation (auto-HSCT). We hypothesized that the immune system is not fully reconstituted at the initiation of immunotherapy. To test this hypothesis, we evaluated hematologic and immune subsets in 34 patients with HR-NBL before and after auto-HSCT. We found that absolute T, B, and NK cell counts at the time of immunotherapy were below normal in 80% of patients. Patients with residual disease at the time of transplantation had significantly lower absolute lymphocyte counts (ALC; P = .008), lower CD16+ cell counts (P = .009), and an abnormal ratio of cytokine-releasing to cytotoxic NK cells at the time of dinutuximab treatment. In addition, the preparative regimen used for auto-HSCT predicted immune recovery. Finally, higher total white blood cell count (P = .013) and ALC (P = .013) at 3 months after completion of therapy were measured in patients who remained in remission compared with those who relapsed. Our results indicate that most patients with HR-NBL do not have full immune reconstitution at the time of dinutuximab treatment after auto-HSCT, and that immune recovery may correlate with disease-related outcomes in patients with high-risk disease.

We report the development of a new technology for monitoring multidimensional protein-protein interactions (PPIs) inside live mammalian cells using split RNA polymerase (RNAP) tags. In this new system, a protein-of-interest is tagged with an N-terminal split RNAP (RNAPN) and multiple potential binding partners are each fused to orthogonal C-terminal RNAPs (RNAPC). Assembly of RNAPN with each RNAPC is highly dependent on interactions between the tagged proteins. Each PPI-mediated RNAPN-RNAPC assembly transcribes from a separate promoter on a supplied DNA substrate, thereby generating a unique RNA output signal for each PPI. We develop and validate this new approach in the context of the Bcl-2 family of proteins. These key regulators of apoptosis are important cancer mediators, but are challenging to therapeutically target due to imperfect selectivity that leads to either off-target toxicity or tumor resistance. We demonstrated binary (1×1) and ternary (1×2) Bcl-2 PPI analyses by imaging fluorescent protein translation from mRNA outputs. Next, we performed a 1×4 PPI network analysis by direct measurement of four unique RNA signals via RT-qPCR. Finally, we used these new tools to monitor pharmacological engagement of Bcl-2 protein inhibitors, and uncover inhibitor-dependent competitive PPIs. The split RNAP tags improve upon other protein fragment complementation (PFC) approaches by offering both multidimensionality and sensitive detection using nucleic acid amplification and analysis techniques. Furthermore, this technology opens up new opportunities for synthetic biology applications due to the versatility of RNA outputs for cellular engineering applications.

Background: Treatment options for relapse after allo-SCT remain limited with poor outcomes. Second Allo-SCT provides benefit to a small subset of patients (pts). However, relapse rates after second Allo-SCT are high and novel approaches to improve outcomes after second Allo-SCT are needed. Conventional total body irradiation (TBI) can reduce relapse rates, but greater radiation doses cannot be delivered safely without increasing the toxicity to surrounding normal tissues and organs. Total marrow irradiation (TMI) techniques permit dose escalation while substantially reducing doses delivered to normal tissues relative to conventional TBI. We conducted a phase I study to assess the feasibility of combining TMI with a fludarabine-melphalan conditioning regimen for second or beyond Allo-SCT to decrease relapse. Methods: Inclusion criteria required a matched/mismatched donor and relapse after previous allo-SCT. GVHD prophylaxis was tacrolimus and mycophenolate mofetil, plus ATG for mismatched donors (NCT02333162). From December 2015 to May 2020, twenty-two pts with were consented to this 3 + 3 phase I trial design; one pt failed to proceed and was replaced. 21 pts (18 AML, 2 MDS/MPN, one ALL) were treated with linac-based volumetric modulated arc therapy (VMAT) technique using 6 MV photons. TMI was provided twice daily at 1.5 Gy per fraction starting with a total dose of 6 Gy, with dose escalation to 9 Gy and 12 Gy. Mucositis was an adverse event of special interest and assessed by the WHO Mucositis Score on days 7, 14, 21, and 28 post-transplant to investigate the combined mucosal injury from alkylating chemotherapy and radiation. Results: Baseline characteristics are summarized in Table 1. The median age was 56 years. All had matched donors except for one mismatched unrelated donor. 6 pts were in CR2, 4 pts in CR3, and 11 pts had refractory disease prior to 2nd SCT. 3 pts treated on dose level 6 Gy had no DLTs. At dose level 2 (9 Gy), the first pt of the cohort of six pts had grade 3 mucositis as a DLT on day 7; it resolved by day 21. The first 2 pts at dose level 3 (12 Gy) had no DLT, but the 3rd pt had DLT of grade 3 mucositis on day 7, resolving by day 14. Two more pts were treated and none had DLT. A sixth pt with refractory disease was treated but had graft failure with concurrent bacterial, fungal, and viral infections. This pt also developed grade 3 mucositis on day 14 that progressed to grade 4 on day 21 but resolved by day 28. Thus, we determined the MTD for TMI was 12 Gy; 9 Gy was an alternative dose for less fit or older pts. SCT outcomes were listed in Table 1. As mentioned above, 1 pt had primary graft failure, and one pt failed to have platelet engraftment due to early relapse. 11 pts developed aGVHD with cumulative aGVHD at 58%, and 4 pts developed cGVHD with cumulative cGVHD at 33%. With median follow up of 11 months (1 - 30 months), 6 pts had disease relapse after a median of 205 days (46-464 days); 10 pts have died (5 from PD; 2 from infection, 1 from GVHD, 1 from PTLD, 1 from second malignancy). Two year PFS and OS were 48% and 50% respectively, which compare favorably to the 2 years PFS and OS of 17.5% and 22.6% from our historical data with 65 pts who had 2nd Allo-SCT prior to this TMI trial at our center (Fan et al. Exp Hematol Oncol 2019). Conclusion: We recommend 12 Gy of VMAT-TMI as the MTD combined with fludarabine-melphalan (140mg/m2) as a conditioning regimen for second allo-SCT. Grade 3-4 mucositis was transient, peaking at days 7-14, and resolving by day 21-28. 9 Gy may be appropriate for less fit or older pts. The 2 year PFS and OS of 48% and 50% after second Allo-SCT for this novel TMI-based regimen are sufficiently encouraging to warrant further study. Table 1 Disclosures Liu: Agios: Honoraria, Other: Regional Advisory board meeting; BMS: Research Funding; Karyopharm: Research Funding. Stock:Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Research to Practice: Honoraria; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kite: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Morphosys: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Research Funding; Novartis: Research Funding; Leukemia and Lymphoma Society: Research Funding; American Society of Hematology: Honoraria; Adaptive Biotechnologies: Consultancy, Membership on an entity's Board of Directors or advisory committees; UpToDate: Honoraria; Servier: Consultancy, Membership on an entity's Board of Directors or advisory committees. Larson:Novartis, Takeda, CVS/Caremark, Celgene, Amgen, Epizyme: Consultancy; Astellas, Celgene, Daiichi Sankyo, Novartis, Rafael Pharmaceuticals, Cellectis, Forty Seven: Research Funding. Kline:Verastem: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite/Gilead: Speakers Bureau; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Merck: Research Funding. Riedell:Novartis: Consultancy, Honoraria, Research Funding; Kite Pharmaceuticals/Gilead: Honoraria, Research Funding; Bayer: Honoraria; Celgene/Bristol-Myers Squibb Company: Honoraria, Research Funding; Verastem Oncology: Honoraria; Morphosys: Research Funding; Karyopharm Therapeutics: Honoraria. Bishop:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Autolus: Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Speakers Bureau; CRSPPR Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Incyte: Honoraria, Speakers Bureau.

Therapeutic manipulation of the BCL-2 family using BH3 mimetics is an emerging paradigm in cancer treatment and immune modulation. For example, peptides mimicking the BIM BH3 helix can directly target the full complement of anti- and pro-apoptotic BCL-2 proteins to trigger apoptosis. This study has incorporated the potent BH3 &alpha, helical death domain of BIM into peptide amphiphile (PA) nanostructures designed to facilitate cellular uptake and induce cell death. This study shows that these PA nanostructures are quickly incorporated into cells, are able to specifically bind BCL-2 proteins, are stable at physiologic temperatures and pH, and induce dose-dependent apoptosis in cells. The incorporation of a cathepsin B cleavable linker between the BIM BH3 peptide and the hydrophobic tail resulted in increased intracellular accumulation and mitochondrial co-localization of the BIM BH3 peptide while also improving BCL-2 family member binding and apoptotic reactivation. This PA platform represents a promising new strategy for intracellular therapeutic peptide delivery for the disruption of intracellular protein:protein interactions.

Prophylactic donor lymphocyte infusion (pDLI) is a potential intervention to prolong remission for patients receiving allogeneic hematopoietic stem cell transplantation (allo-SCT), however, the optimal timing and dose are unknown. We conducted a prospective trial exploring the feasibility of early withdrawal of immunosuppression (WOI) at day 60 followed by dose escalation of pDLI after alemtuzumab-based, T-cell depleted conditioning for patients with high-risk hematologic malignancies. pDLI were administered at day 75 to day 90 and again in 4-8 week intervals with receipt of up to 5 pDLI infusions. Fourty-six patients with matched-related donors (MRD) and 29 patients with matched-unrelated donors (MUD) were considered. Twenty-eight MRD patients were able to undergo WOI, 26 patients (93%) received at least 1 DLI, 16 patients (57%) received 3+, and 7 patients (25%) received 5 pDLI. Only 7 MUD patients were able to undergo WOI, 4 (57%) received at least 1 pDLI, 1 patient (14%) received 3 DLI, and no patients received all 5. Median PFS for patients on the study was 366 days. The estimated 2-year PFS and OS rates for all patients were 41% (95% CI, 32-54%) and 51% (95% CI, 41-63%) compared with 57% (95% CI, 41-77%) and 67% (95% CI, 52-86%) for patients who received at least one pDLI. In addition, MRD patients receiving pDLI had faster immune re-constitution and improved donor chimerism. Our trial proposes a novel dosage and treatment schedule for pDLI that is tolerable for patients who have received MRD allo-SCT and leads to improved outcomes.

The BCL-2 family of proteins orchestrates a complex signaling network that governs the balance between cellular survival and death. A comprehensive understanding of the mechanistic interactions between these proteins continues to evolve in normal and malignant cells. The functional variation by individual BCL-2 proteins in different cell types has driven clinical therapeutic development in targeting individual BCL-2 members with the goal of fine-tuning cell death in diseased cells. Given the importance of understanding and validating the effect of activating or inhibiting BCL-2 protein interactions in individual cells, the methods used to measure apoptotic cell death have undergone increased scrutiny. Here, we describe two in vitro flow cytometry-based methods that are useful in measuring BCL-2 proteins and mitochondrial-based cell death in complex cell populations.

Background Second allogeneic hematopoietic stem cell transplant (HCT) remains as an option for disease relapse after initial HCT. Methods We analyzed retrospectively the outcomes of 65 consecutive patients who underwent a second HCT for disease relapse at the University of Chicago. Univariate and multivariate analysis were conducted, and a scoring system was generated to select the patients who would benefit second HCT. Results All except four patients received T cell depleted (TCD) first HCT. The majority of patients had AML (n = 47) and high risk MDS (n = 5). The median age at second HCT was 45 years (11–73). 13 patients (20%) achieved CR before second HCT. 98% (n = 64) and 72% (n = 47) patients achieved neutrophil and platelet engraftment at a median interval of 10 and 18 days, respectively, following the second HCT. With a median follow up of 23 (5.5–140) months for survivors after second HCT, the estimated 2 years PFS was 17.5% and the 2 years OS was 22.6%. The day 100 cumulative incidence of non-relapse mortality rate was 23.6%, and the cumulative incidence of aGVHD and cGVHD were 16.9% and 7.7% respectively at 1 year after second HCT. In univariate analysis, patients with remission duration after first HCT of > 12 months and those in CR before second HCT had significantly better PFS and OS. A scoring system using disease status before second HCT (CR = 0 vs. non-CR = 1), and remission duration after first HCT ( 12 months = 0) was generated as an approach to classify patients into different risk categories in the purpose to provide guidance to the transplant physician to inform the outcomes to potential patients undergoing 2nd HCT. A score of 12 months after first HCT with acceptable rates of GVHD and donor engraftment.

Proteins and their interactions in and out of cells must be well-orchestrated for the healthy functioning and regulation of the body. Even the slightest disharmony can cause diseases. Therapeutic peptides are short amino acid sequences (generally considered

Peptides synthesized in the likeness of their native interaction domain(s) are natural choices to target protein–protein interactions (PPIs) due to their fidelity of orthostatic contact points between binding partners. Despite therapeutic promise, intracellular delivery of biofunctional peptides at concentrations necessary for efficacy remains a formidable challenge. Peptide amphiphiles (PAs) provide a facile method of intracellular delivery and stabilization of bioactive peptides. PAs consisting of biofunctional peptide headgroups linked to hydrophobic alkyl lipid-like tails prevent peptide hydrolysis and proteolysis in circulation, and PA monomers are internalized via endocytosis. However, endocytotic sequestration and steric hindrance from the lipid tail are two major mechanisms that limit PA efficacy to target intracellular PPIs. To address these problems, we have constructed a PA platform consisting of cathepsin-B cleavable PAs in which a selective p53-based inhibitory peptide is cleaved from its lipid tail within endosomes, allowing for intracellular peptide accumulation and extracellular recycling of the lipid moiety. We monitor for cleavage and follow individual PA components in real time using a Förster resonance energy transfer (FRET)-based tracking system. Using this platform, we provide a better understanding and quantification of cellular internalization, trafficking, and endosomal cleavage of PAs and of the ultimate fates of each component.

Treatment resistance in T-cell acute lymphoblastic leukemia (T-ALL) is associated with phosphatase and tensin homolog (PTEN) deletions and resultant phosphatidylinositol 3'-kinase (PI3K)-AKT pathway activation, as well as MYC overexpression, and these pathways repress mitochondrial apoptosis in established T-lymphoblasts through poorly defined mechanisms. Normal T-cell progenitors are hypersensitive to mitochondrial apoptosis, a phenotype that is dependent on the expression of proapoptotic BIM. In a conditional zebrafish model, MYC downregulation induced BIM expression in T-lymphoblasts, an effect that was blunted by expression of constitutively active AKT. In human T-ALL cell lines and treatment-resistant patient samples, treatment with MYC or PI3K-AKT pathway inhibitors each induced BIM upregulation and apoptosis, indicating that BIM is repressed downstream of MYC and PI3K-AKT in high-risk T-ALL. Restoring BIM function in human T-ALL cells using a stapled peptide mimetic of the BIM BH3 domain had therapeutic activity, indicating that BIM repression is required for T-ALL viability. In the zebrafish model, where MYC downregulation induces T-ALL regression via mitochondrial apoptosis, T-ALL persisted despite MYC downregulation in 10% of bim wild-type zebrafish, 18% of bim heterozygotes and in 33% of bim homozygous mutants (P=0.017). We conclude that downregulation of BIM represents a key survival signal downstream of oncogenic MYC and PI3K-AKT signaling in treatment-resistant T-ALL.

Mantle cell lymphoma (MCL) is a highly aggressive B-cell lymphoma resistant to conventional chemotherapy. Although defined by the characteristic t(11;14) translocation, MCL has not been recapitulated in transgenic mouse models of cyclin D1 overexpression alone. Indeed, several genetic aberrations have been identified in MCL that may contribute to its pathogenesis and chemoresistance. Of particular interest is the frequent biallelic deletion of the proapoptotic BCL-2 family protein BIM. BIM exerts its pro-death function via its α-helical BH3 death domain that has the dual capacity to inhibit antiapoptotic proteins such as BCL-2 and MCL-1 and directly trigger proapoptotic proteins such as the mitochondrial executioner protein BAX. To evaluate a functional role for Bim deletion in the pathogenesis of MCL, we generated cyclin D1-transgenic mice harboring Bim-deficient B cells. In response to immunization, Eμ(CycD1)CD19(CRE)Bim(fl/fl) mice manifested selective expansion of their splenic mantle zone compartment. Three distinct immune stimulation regimens induced lymphomas with histopathologic and molecular features of human MCL in a subset of mice. Thus, deletion of Bim in B cells, in the context of cyclin D1 overexpression, disrupts a critical control point in lymphoid maturation and predisposes to the development of MCL. This genetic proof of concept for MCL pathogenesis suggests an opportunity to reactivate the death pathway by pharmacologic mimicry of proapoptotic BIM.

We present a clinicopathologic study of the youngest reported child with lethal cyclophosphamide-induced cardiotoxicity after hematopoietic stem cell transplantation for β-thalassemia major and the 1st pediatric report of the use of extracorporeal membrane oxygenation as a therapeutic modality to bridge the patient to myocardial recovery. Despite improvement in myocardial function while on extracorporeal membrane oxygenation, at autopsy 11 days after the onset of cardiac dysfunction, epicardial hemorrhage and extensive myocardial hemorrhagic infarction were revealed. Histopathologic and ultrastructural examination of the myocardium revealed extensive coagulative necrosis of cardiomyocytes, endothelial damage, fibrin thrombi, and subendothelial and interstitial fibrin. We review the literature on cyclophosphamide-induced cardiotoxicity and describe its clinicopathologic characteristics. Our findings point to endothelial damage leading to thrombotic microangiopathy and ischemic tissue injury as the most likely pathogenesis.

Children undergoing intense cancer treatment frequently require total parenteral nutrition (TPN). Rarely, vitamins are removed due to hypersensitivity to the carrier vehicle in the formulation. We present the case of a 5-year-old patient with stage 4, high-risk neuroblastoma who developed altered mental status, ataxia, and tachycardia during consolidative autologous stem cell transplantation. Skin findings and brain MRI were consistent with thiamine (vitamin B1) deficiency and Wernicke encephalopathy. Vitamin B1 administration rapidly reversed all skin and neurologic symptoms. This case highlights the importance of close monitoring of micronutrients in pediatric patients receiving prolonged courses of chemotherapy and stem cell transplantation. Pediatr Blood Cancer © 2015 Wiley Periodicals, Inc.

A crucial component of regulating organismal homeostasis is maintaining proper cell number and eliminating damaged or potentially malignant cells. Apoptosis, or programed cell death, is the mechanism responsible for this equilibrium. The intrinsic apoptotic pathway is also especially important in the development and maintenance of the immune system. Apoptosis is essential for proper positive and negative selection during B- and T-cell development and for efficient contraction of expanded lymphocytes following an immune response. Tight regulation of the apoptotic pathway is critical, as excessive cell death can lead to immunodeficiency while apoptotic resistance can lead to aberrant lymphoproliferation and autoimmune disease. Dysregulation of cell death is implicated in a wide range of hematological malignancies, and targeting various components of the apoptotic machinery in these cases is an attractive chemotherapeutic strategy. A wide array of compounds has been developed with the purpose of reactivating the intrinsic apoptotic pathway. These compounds, termed BH3 mimetics are garnering considerable attention as they gain greater clinical oncologic significance. As their use expands, it will be imperative to understand the effects these compounds have on immune homeostasis. Uncovering their potential immunomodulatory activity may allow for administration of BH3 mimetics for direct tumor cell killing as well as novel therapies for a wide range of immune-based directives. This review will summarize the major proteins involved in the intrinsic apoptotic pathway and define their roles in normal immune development and disease. Clinical and preclinical BH3 mimetics are described within the context of what is currently known about their ability to affect immune function. Prospects for future antitumor immune amplification and immune modulation are then proposed.

Peptides and peptide-conjugates, comprising natural and synthetic building blocks, are an increasingly popular class of biomaterials. Self-assembled nanostructures based on peptides and peptide-conjugates offer advantages such as precise selectivity and multifunctionality that can address challenges and limitations in the clinic. In this review article, we discuss recent developments in the design and self-assembly of various nanomaterials based on peptides and peptide-conjugates for medical applications, and categorize them into two themes based on the driving forces of molecular self-assembly. First, we present the self-assembled nanostructures driven by the supramolecular interactions between the peptides, with or without the presence of conjugates. The studies where nanoassembly is driven by the interactions between the conjugates of peptide-conjugates are then presented. Particular emphasis is given to in vivo studies focusing on therapeutics, diagnostics, immune modulation and regenerative medicine. Finally, challenges and future perspectives are presented.

Cancer cells subvert the natural balance between cellular life and death, achieving immortality through pathologic enforcement of survival pathways and blockade of cell death mechanisms. Pro-apoptotic BCL-2 family proteins are frequently disarmed in relapsed and refractory cancer through genetic deletion or interaction-based neutralization by overexpressed antiapoptotic proteins, resulting in resistance to chemotherapy and radiation treatments. New pharmacologic strategies are urgently needed to overcome these formidable apoptotic blockades. We harnessed the natural killing activity of BCL-2–interacting mediator of cell death (BIM), which contains one of the most potent BH3 death domains of the BCL-2 protein family, to restore BH3-dependent cell death in resistant hematologic cancers. A hydrocarbon-stapled peptide modeled after the BIM BH3 helix broadly targeted BCL-2 family proteins with high affinity, blocked inhibitory antiapoptotic interactions, directly triggered proapoptotic activity, and induced dose-responsive and BH3 sequence–specific cell death of hematologic cancer cells. The therapeutic potential of stapled BIM BH3 was highlighted by the selective activation of cell death in the aberrant lymphoid infiltrates of mice reconstituted with BIM-deficient bone marrow and in a human AML xenograft model. Thus, we found that broad and multimodal targeting of the BCL-2 family pathway can overcome pathologic barriers to cell death.

Regulatory T cells (Tregs) are critical for the maintenance of immune tolerance and control of immune responses to foreign agents. Tregs depend upon the transcriptional protein FOXP3 for their ontogeny and genetic orchestration of their suppressive mechanisms. While T cells have the ability to target tumor-associated antigens from many cancer subtypes, these T cells are often suppressed in patients with immunologically “silenced” tumors. The tolerizing function of Tregs can limit effective anti-tumor T cell-mediated killing, even in the presence of immunotherapies, including checkpoint inhibition, and their presence predicts reduced survival in patients with a myriad of cancers. Currently, there exists no means of depleting Tregs in patients without also targeting activated effector T cells. In an effort to overcome this, we have specifically inhibited Tregs by targeting FOXP3 through molecular mimicry using peptides designed to inhibit FOXP3 homodimerization. Homodimerization of FOXP3 is mediated by an antiparallel coiled-coil interaction between the leucine-zipper domains of two FOXP3 proteins. A series of single and double hydrocarbon-stapled alpha-helical (SAH) peptides were synthesized using the leucine zipper dimerization domain (DD) interface of FOXP3 (SAH-FOXP3DDs) as a molecular template. SAH-FOXP3DDs, but not native peptides, specifically bind FOXP3's leucine zipper domain with nanomolar affinity. Unlike stapled peptide point mutant controls, lead SAH-FOXP3DDs also showed dose-dependent inhibition of FOXP3 binding to cognate DNA. Lead SAH-FOXP3DDs are cell permeable and nontoxic to T cells as measured by absence of non-specific LDH-release and apoptosis induction following treatment. Flow cytometric analysis of treated Tregs measured changes in expression of a number of Treg/T cell - associated proteins including increases in FOXP3 and CD127 and decreases in CD25, while no changes were observed in treated conventional CD4+ T cells (Tcons) or with cells treated with point-mutant control peptides. In addition, treated Tregs demonstrated mRNA expression changes of FOXP3-regulated genes including Foxp3, CD127, IL2 and Ctla. Treatment of freshly isolated and in vitro expanded Tregs with lead SAH-FOXP3DD compounds inhibited Treg-mediated suppression of Tcon proliferation in response to CD3/CD28 activation, while no effects were measured with a point-mutant control. We believe that SAH-FOXP3DDs will be powerful molecular probes to further our understanding of FOXP3 transcriptional control. Additionally, they may be a foundation for prototype therapeutics to target Tregs in cancer patients in an effort to amplify the endogenous anti-tumor immune response alone or in combination with other cancer immunotherapies. Citation Format: Rachel Eclov, Marie Fefferman, Ravand Samaeekia, James L. LaBelle. FOXP3 dimerization by stapled alpha-helical peptides alters regulatory T cell function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3850.

BIM is the master BH3-only BCL-2 family regulator of lymphocyte survival. To understand how long-term loss of BIM affects apoptotic resistance in T and B cells we examined peripheral blood counts in mice with T or B cell-specific deletion of Bim. In contrast to CD19CRE Bimfl/fl animals, LCKCRE Bimfl/fl mice had pronounced early lymphocytosis followed by lymphocyte count normalization as they aged. To explore BCL-2 family specific mechanism(s) leading to this normalization we compared cell death sensitivity and BCL-2 expression patterns of young versus old LCKCRE Bimfl/fl T cells. Young (6-12 weeks) and old (>40 weeks) LCKCRE Bimfl/fl mice had consistently abnormal thymocyte development hallmarked by a decrease in CD4+8+ double positive (DP) cells and accumulation of double negative (DN) and single positive (SP) cells as previously described in global Bim knockout mice. Thymocytes maintained cell death resistance to a number of apoptotic stimuli throughout all ages, reflecting a consistent loss of BIM. In contrast to thymocytes, mature splenic-derived LCKCRE Bimfl/fl T cells from young mice were equally sensitive to apoptotic stimuli as WT controls, indicating BCL-2 family compensation. Expression profiling in these cells revealed upregulation of other BH3-only proteins, including Noxa = Puma << Bmf, suggesting that these proteins functionally compensated for the loss of BIM. To determine if this was a T cell-intrinsic process and if the presence of other BH3-only proteins with pan-BCL-2 family binding affinities were required for lymphocyte number and apoptotic normalization, we analyzed peripheral blood from mice globally deleted in Bim, Bid, and Puma (TKO). TKO, like LCKCRE Bimfl/fl mice, developed profound early lymphocytosis followed by normalization as they aged. A subset of TKO mice developed T-ALL hallmarked by upregulation of Bmf and Bcl-w, an under characterized anti-apoptotic BCL-2 family protein, suggesting that BCL-W allowed for malignant T cell survival despite the presence of BMF. To determine if such a relationship exists in human disease, expression profiling of pediatric T-ALL patient samples revealed a significant negative correlation between BIM and BCL-W expression in developmentally immature and mature T-ALL. Our findings suggest a model in which the loss of BIM in T cells can be compensated for through upregulation of BMF, a BH3-only protein mechanistically similar to BIM. Loss of other ‘direct activating' BH3-only proteins increases the chances of malignant cell escape despite BMF upregulation, particularly when BCL-W is overexpressed. This work provides a developmental framework between BIM and BMF in T cells and for a BIM:BCL-W axis in T-ALL. Overexpression of BCL-W in BIMlow-expressing T-ALL may be an additional target responsible for early immature T-ALL therapeutic resistance. Citation Format: Lindsey M. Ludwig, Lauren E. Roach, Samuel G. Katz, Jill K. Fisher, Melissa Burns, Matthew R. Schnorenberg, Riyue Bao, Makda Zewde, Yusuke Nakamura, Alejandro Gutierrez, Loren D. Walensky, James L. LaBelle. BCL-2 family compensation regulates T cell homeostasis and reveals a BIM:BCL-W axis in T-ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 294.

Severe combined immunodeficiency disorders (SCID) are a group of primary immunodeficiencies resulting from any one of a diverse group of mutations impacting T-cell development. SCID is diagnosed and classified through assessment of the lymphocyte subset(s) affected and by the mechanisms responsible for the primary immune defect. Regardless of the genetics involved, patients invariably succumb to an early death without medical intervention. In the past, patients were primarily identified either by previous family history, physical manifestations, or after the onset of symptoms. However, the introduction of newborn screening for SCID has allowed the pediatrician to identify these patients at a much earlier age, greatly improving their survival. Currently, 23 states include SCID testing for T-cell deficiencies in their newborn screening platform. Protocols for confirmatory testing and medical intervention after a positive screen vary slightly from state-to-state. However, the standard curative treatment remains stem cell transplantation, although depending on the genetic cause of the disease, enzyme replacement and gene therapy may also be considered. [ Pediatr Ann . 2015;44(7):e181–e187.]

Children undergoing intense cancer treatment frequently require total parenteral nutrition (TPN). Rarely, vitamins are removed due to hypersensitivity to the carrier vehicle in the formulation. We present the case of a 5-year-old patient with stage 4, high-risk neuroblastoma who developed altered mental status, ataxia, and tachycardia during consolidative autologous stem cell transplantation. Skin findings and brain MRI were consistent with thiamine (vitamin B1) deficiency and Wernicke encephalopathy. Vitamin B1 administration rapidly reversed all skin and neurologic symptoms. This case highlights the importance of close monitoring of micronutrients in pediatric patients receiving prolonged courses of chemotherapy and stem cell transplantation.

B7 molecules provide important costimulatory signals to T cells, and B7 genes have been introduced into B7-negative tumor cells to enhance their immunogenicity. However, the role of B7 molecules in inducing tumor immunity is controversial because of conflicting results and reports of differential signaling through the B7 molecules and their ligands CD28 and CTLA-4. In this study, we compared the effect of B7-1 (CD80) and B7-2 (CD86) on the induction of T-cell immunity to C1498, a murine myelogenous leukemia. When cultured with exogenous cytokines in vitro, C1498/B7-1 and C1498/B7-2 induced syngeneic CD8+ T cells to kill parental C1498. In vivo, C1498/B7-1 grew progressively after subcutaneous injection, whereas C1498/B7-2 completely regressed after transient growth in naive mice. Spontaneous rejection of C1498/B7-2 resulted in immunity to challenge doses of C1498 and C1498/B7-1. Antibody-depletion studies in vivo showed that CD8+ T cells rejected C1498/B7-2, whereas only natural killer cells affected the growth of C1498/B7-1. Two approaches were used to determine whether preferential interaction of B7-1 with CTLA-4 contributed to the failure of C1498/B7-1 to activate CD8+ T cells in vivo. First, CTLA-4 specific monoclonal antibody was used to block B7-1–CTLA-4 interaction. Second, CTLA-4 deletional mutant (−/−) bone marrow chimeras were used as tumor hosts. In both systems, there was a significant increase in the rate of rejection of C1498/B7-1 tumors. Resistance to C1498/B7-1 in CTLA-4−/− hosts was mediated by CD8+ T cells. Blocking or deletion of CTLA-4 did not affect the growth of parental C1498, indicating that B7-1 was important for the induction of CD8+ T-cell immunity in the absence of CTLA-4.

Hydrocarbon stapling is a chemical approach to restoring and fortifying the natural α-helical structure of peptides that otherwise unfold when taken out of context from the host protein. By iterating the peptide sequence, staple type, and sites of insertion, discrete compositions can be generated to suit a diversity of biochemical, structural, proteomic, cellular, and drug development applications. Here, we reinforce key design considerations to avoid pitfalls and maximize progress when applying stapled peptides in chemistry and biology research.

In murine models of allogeneic bone marrow transplantation (BMT), MHC-mismatched recipients given a delayed infusion of donor leukocytes (DLI) at 21 days posttransplant develop significant GVHD whereas MHC-matched recipients do not. The current study was initially designed to test the hypothesis that small numbers of T cells in the MHC-mismatched donor bone marrow (BM) graft exacerbated graft-vs-host disease (GVHD) when DLI was administered at 21 days after BMT. Ex vivo depletion of Thy1+ cells from the donor BM had no impact on the severity of GVHD after DLI. However, depletion of donor T cells in vivo with a Thy1 allele-specific mAb given after BMT resulted in significantly more severe GVHD after DLI. Similar results were obtained in a MHC-matched model of allogeneic BMT, indicating that this was a general phenomenon and not model dependent. These results indicated that a population of donor-derived Thy1+ cells suppressed graft-vs-host reactivity after DLI. Results of experiments with thymectomized recipients demonstrated that an intact thymus was required for generation of the immunoregulatory donor cells. Experiments using TCR β-chain knockout mice as BM donors indicated that the immunosuppressive Thy1+ cells coexpressed αβTCR heterodimers. Similar experiments with CD4 and CD8 knockout donor BM suggested that the immunoregulatory Thy1+αβTCR+ cells consisted of two subpopulations: a CD4+CD8− subpopulation and a CD4−CD8− subpopulation. Together, these results show that thymus-derived, Thy1+αβTCR+ donor cells generated early after allogeneic BMT suppress the graft-vs-host reactivity of T cells given as DLI. These cells may mediate dominant peripheral tolerance after allogeneic BMT.