Dynamic niches in the origination and differentiation of haematopoietic stem cells.

Haematopoietic stem cells (HSCs) are multipotent, self-renewing progenitors that generate all mature blood cells. HSC function is tightly controlled to maintain haematopoietic homeostasis, and this regulation relies on specialized cells and factors that constitute the haematopoietic 'niche', or microenvironment. Recent discoveries, aided in part by technological advances in in vivo imaging, have engendered a new appreciation for the dynamic nature of the niche, identifying novel cellular and acellular niche components and uncovering fluctuations in the relative importance of these components over time. These new insights significantly improve our understanding of haematopoiesis and raise fundamental questions about what truly constitutes a stem cell niche.

Cellular Therapy for Children with Central Nervous System Tumors: Mining and Mapping the Correlative Data.

PURPOSE OF REVIEW: Correlative studies should leverage clinical trial frameworks to conduct biospecimen analyses that provide insight into the bioactivity of the intervention and facilitate iteration toward future trials that further improve patient outcomes. In pediatric cellular immunotherapy trials, correlative studies enable deeper understanding of T cell mobilization, durability of immune activation, patterns of toxicity, and early detection of treatment response. Here, we review the correlative science in adoptive cell therapy (ACT) for childhood central nervous system (CNS) tumors, with a focus on existing chimeric antigen receptor (CAR) and T cell receptor (TCR)-expressing T cell therapies. RECENT FINDINGS: We highlight long-standing and more recently understood challenges for effective alignment of correlative data and offer practical considerations for current and future approaches to multi-omic analysis of serial tumor, serum, and cerebrospinal fluid (CSF) biospecimens. We highlight the preliminary success in collecting serial cytokine and proteomics from patients with CNS tumors on ACT clinical trials.

Attenuation of PKCδ enhances metabolic activity and promotes expansion of blood progenitors.

A finely tuned balance of self-renewal, differentiation, proliferation, and survival governs the pool size and regenerative capacity of blood-forming hematopoietic stem and progenitor cells (HSPCs). Here, we report that protein kinase C delta (PKCδ) is a critical regulator of adult HSPC number and function that couples the proliferative and metabolic activities of HSPCs. PKCδ-deficient mice showed a pronounced increase in HSPC numbers, increased competence in reconstituting lethally irradiated recipients, enhanced long-term competitive advantage in serial transplantation studies, and an augmented HSPC recovery during stress. PKCδ-deficient HSPCs also showed accelerated proliferation and reduced apoptosis, but did not exhaust in serial transplant assays or induce leukemia. Using inducible knockout and transplantation models, we further found that PKCδ acts in a hematopoietic cell-intrinsic manner to restrict HSPC number and bone marrow regenerative function. Mechanistically, PKCδ regulates HSPC energy metabolism and coordinately governs multiple regulators within signaling pathways implicated in HSPC homeostasis. Together, these data identify PKCδ as a critical regulator of HSPC signaling and metabolism that acts to limit HSPC expansion in response to physiological and regenerative demands.

HLA Haploidentical Stem Cell Transplant with Pretransplant Immunosuppression for Patients with Sickle Cell Disease.

Allogeneic stem cell transplantation (HCT) is curative in patients with severe sickle cell disease (SCD), but a significant number of patients lack an HLA-identical sibling or matched unrelated donor. Mismatched related (haploidentical) HCT with post-transplant cyclophosphamide (PTCY) allows expansion of the donor pool but is complicated by high rates of graft failure. In this report we describe a favorable haploidentical HCT approach in a limited cohort of SCD patients with significant comorbidities. To reduce the risk of graft failure we administered the conditioning regimen of rabbit antithymocyte globulin, busulfan, and fludarabine preceded with 2 courses of pretransplant immunosuppressive therapy (PTIS) with fludarabine and dexamethasone. Graft-versus-host disease (GVHD) prophylaxis consisted of PTCY on days +3 and +4 followed by tacrolimus and mycophenolate mofetil starting on day +5. Four patients (ages 13, 19, 19, and 23 years) received T cell-replete haploidentical stem cell infusion. All patients engrafted with 99.9% to 100% donor chimerism, and all patients continued with stable engraftment at the last follow-up (5 to 11 months post-transplant). Time to neutrophil engraftment was 14 to 26 days. Two patients had high levels of donor-specific anti-HLA antibodies, which required the implementation of an antibody management protocol. This facilitated neutrophil engraftment on day +16 and day +26, respectively. One patient developed grade I acute GVHD, which resolved. Three patients developed mild, limited skin GVHD that responded to conventional immunosuppressive therapy. Human herpesvirus-6 viremia was detected in 3 patients but resolved without treatment. One patient developed asymptomatic cytomegalovirus viremia that responded appropriately to standard therapy with ganciclovir. The prompt, stable engraftment and low toxicity in the post-transplant period makes PTIS with haploidentical transplant a promising option for patients with SCD.

Phosphoproteomic profiling of mouse primary HSPCs reveals new regulators of HSPC mobilization.

Protein phosphorylation is a central mechanism of signal transduction that both positively and negatively regulates protein function. Large-scale studies of the dynamic phosphorylation states of cell signaling systems have been applied extensively in cell lines and whole tissues to reveal critical regulatory networks, and candidate-based evaluations of phosphorylation in rare cell populations have also been informative. However, application of comprehensive profiling technologies to adult stem cell and progenitor populations has been challenging, due in large part to the scarcity of such cells in adult tissues. Here, we combine multicolor flow cytometry with highly efficient 3-dimensional high performance liquid chromatography/mass spectrometry to enable quantitative phosphoproteomic analysis from 200 000 highly purified primary mouse hematopoietic stem and progenitor cells (HSPCs). Using this platform, we identify ARHGAP25 as a novel regulator of HSPC mobilization and demonstrate that ARHGAP25 phosphorylation at serine 363 is an important modulator of its function. Our approach provides a robust platform for large-scale phosphoproteomic analyses performed with limited numbers of rare progenitor cells. Data from our study comprises a new resource for understanding the molecular signaling networks that underlie hematopoietic stem cell mobilization.

Locoregional delivery of IL-13Rα2-targeting CAR-T cells in recurrent high-grade glioma: a phase 1 trial.

Chimeric antigen receptor T cell (CAR-T) therapy is an emerging strategy to improve treatment outcomes for recurrent high-grade glioma, a cancer that responds poorly to current therapies. Here we report a completed phase I trial evaluating IL-13Rα2-targeted CAR-T cells in 65 patients with recurrent high-grade glioma, the majority being recurrent glioblastoma (rGBM). Primary objectives were safety and feasibility, maximum tolerated dose/maximum feasible dose and a recommended phase 2 dose plan. Secondary objectives included overall survival, disease response, cytokine dynamics and tumor immune contexture biomarkers. This trial evolved to evaluate three routes of locoregional T cell administration (intratumoral (ICT), intraventricular (ICV) and dual ICT/ICV) and two manufacturing platforms, culminating in arm 5, which utilized dual ICT/ICV delivery and an optimized manufacturing process. Locoregional CAR-T cell administration was feasible and well tolerated, and as there were no dose-limiting toxicities across all arms, a maximum tolerated dose was not determined. Probable treatment-related grade 3+ toxicities were one grade 3 encephalopathy and one grade 3 ataxia. A clinical maximum feasible dose of 200 × 106 CAR-T cells per infusion cycle was achieved for arm 5; however, other arms either did not test or achieve this dose due to manufacturing feasibility. A recommended phase 2 dose will be refined in future studies based on data from this trial. Stable disease or better was achieved in 50% (29/58) of patients, with two partial responses, one complete response and a second complete response after additional CAR-T cycles off protocol. For rGBM, median overall survival for all patients was 7.7 months and for arm 5 was 10.2 months. Central nervous system increases in inflammatory cytokines, including IFNγ, CXCL9 and CXCL10, were associated with CAR-T cell administration and bioactivity. Pretreatment intratumoral CD3 T cell levels were positively associated with survival. These findings demonstrate that locoregional IL-13Rα2-targeted CAR-T therapy is safe with promising clinical activity in a subset of patients. ClinicalTrials.gov Identifier: NCT02208362 .

Inclusion of 4-1BB Costimulation Enhances Selectivity and Functionality of IL13Rα2-Targeted Chimeric Antigen Receptor T Cells.

Chimeric antigen receptor (CAR) T cell immunotherapy is emerging as a powerful strategy for cancer therapy; however, an important safety consideration is the potential for off-tumor recognition of normal tissue. This is particularly important as ligand-based CARs are optimized for clinical translation. Our group has developed and clinically translated an IL13(E12Y) ligand-based CAR targeting the cancer antigen IL13Rα2 for treatment of glioblastoma (GBM). There remains limited understanding of how IL13-ligand CAR design impacts the activity and selectivity for the intended tumor-associated target IL13Rα2 versus the more ubiquitous unintended target IL13Rα1. In this study, we functionally compared IL13(E12Y)-CARs incorporating different intracellular signaling domains, including first-generation CD3ζ-containing CARs (IL13ζ), second-generation 4-1BB (CD137)-containing or CD28-containing CARs (IL13-BBζ or IL13-28ζ), and third-generation CARs containing both 4-1BB and CD28 (IL13-28BBζ). In vitro coculture assays at high tumor burden establish that second-generation IL13-BBζ or IL13-28ζ outperform first-generation IL13ζ and third-generation IL13-28BBζ CAR designs, with IL13-BBζ providing superior CAR proliferation and in vivo antitumor potency in human xenograft mouse models. IL13-28ζ displayed a lower threshold for antigen recognition, resulting in higher off-target IL13Rα1 reactivity both in vitro and in vivo. Syngeneic mouse models of GBM also demonstrate safety and antitumor potency of murine IL13-BBζ CAR T cells delivered systemically after lymphodepletion. These findings support the use of IL13-BBζ CARs for greater selective recognition of IL13Rα2 over IL13Rα1, higher proliferative potential, and superior antitumor responsiveness. This study exemplifies the potential of modulating factors outside the antigen targeting domain of a CAR to improve selective tumor recognition. Significance: This study reveals how modulating CAR design outside the antigen targeting domain improves selective tumor recognition. Specifically, this work shows improved specificity, persistence, and efficacy of 4-1BB-based IL13-ligand CARs. Human clinical trials evaluating IL13-41BB-CAR T cells are ongoing, supporting the clinical significance of these findings.

Antigen-dependent IL-12 signaling in CAR T cells promotes regional to systemic disease targeting.

Chimeric antigen receptor (CAR) T cell therapeutic responses are hampered by limited T cell trafficking, persistence, and durable anti-tumor activity in solid tumors. However, these challenges can be largely overcome by relatively unconstrained synthetic engineering strategies. Here, we describe CAR T cells targeting tumor-associated glycoprotein-72 (TAG72), utilizing the CD28 transmembrane domain upstream of the 4-1BB co-stimulatory domain as a driver of potent anti-tumor activity and IFNγ secretion. CAR T cell-mediated IFNγ production facilitated by IL-12 signaling is required for tumor cell killing, which is recapitulated by engineering an optimized membrane-bound IL-12 (mbIL12) molecule in CAR T cells. These T cells show improved antigen-dependent T cell proliferation and recursive tumor cell killing in vitro, with robust in vivo efficacy in human ovarian cancer xenograft models. Locoregional administration of mbIL12-engineered CAR T cells promotes durable anti-tumor responses against both regional and systemic disease in mice. Safety and efficacy of mbIL12-engineered CAR T cells is demonstrated using an immunocompetent mouse model, with beneficial effects on the immunosuppressive tumor microenvironment. Collectively, our study features a clinically-applicable strategy to improve the efficacy of locoregionally-delivered CAR T cells engineered with antigen-dependent immune-modulating cytokines in targeting regional and systemic disease.

Tumor inflammation-associated neurotoxicity.

Cancer immunotherapies have unique toxicities. Establishment of grading scales and standardized grade-based treatment algorithms for toxicity syndromes can improve the safety of these treatments, as observed for cytokine release syndrome (CRS) and immune effector cell associated neurotoxicity syndrome (ICANS) in patients with B cell malignancies treated with chimeric antigen receptor (CAR) T cell therapy. We have observed a toxicity syndrome, distinct from CRS and ICANS, in patients treated with cell therapies for tumors in the central nervous system (CNS), which we term tumor inflammation-associated neurotoxicity (TIAN). Encompassing the concept of 'pseudoprogression,' but broader than inflammation-induced edema alone, TIAN is relevant not only to cellular therapies, but also to other immunotherapies for CNS tumors. To facilitate the safe administration of cell therapies for patients with CNS tumors, we define TIAN, propose a toxicity grading scale for TIAN syndrome and discuss the potential management of this entity, with the goal of standardizing both reporting and management.