Preferential expansion of CD8+ CD19-CAR T cells postinfusion and the role of disease burden on outcome in pediatric B-ALL.

T cells expressing CD19-specific chimeric antigen receptors (CD19-CARs) have potent antileukemia activity in pediatric and adult patients with relapsed and/or refractory B-cell acute lymphoblastic leukemia (B-ALL). However, not all patients achieve a complete response (CR), and a significant percentage relapse after CD19-CAR T-cell therapy due to T-cell intrinsic and/or extrinsic mechanisms. Thus, there is a need to evaluate new CD19-CAR T-cell products in patients to improve efficacy. We developed a phase 1/2 clinical study to evaluate an institutional autologous CD19-CAR T-cell product in pediatric patients with relapsed/refractory B-ALL. Here we report the outcome of the phase 1 study participants (n = 12). Treatment was well tolerated, with a low incidence of both cytokine release syndrome (any grade, n = 6) and neurotoxicity (any grade, n = 3). Nine out of 12 patients (75%) achieved a minimal residual disease-negative CR in the bone marrow (BM). High disease burden (≥40% morphologic blasts) before CAR T-cell infusion correlated with increased side effects and lower response rate, but not with CD19-CAR T-cell expansion. After infusion, CD8+ CAR T cells had a proliferative advantage over CD4+ CAR T cells and at peak expansion, had an effector memory phenotype with evidence of antigen-driven differentiation. Patients that proceeded to allogeneic hematopoietic cell transplantation (AlloHCT) had sustained, durable responses. In summary, the initial evaluation of our institutional CD19-CAR T-cell product demonstrates safety and efficacy while highlighting the impact of pre-infusion disease burden on outcomes. This trial was registered at www.clinicaltrials.gov as #NCT03573700.

Regnase-1 suppresses TCF-1+ precursor exhausted T-cell formation to limit CAR-T-cell responses against ALL.

Chimeric antigen receptor (CAR)-T-cell therapeutic efficacy is associated with long-term T-cell persistence and acquisition of memory. Memory-subset formation requires T-cell factor 1 (TCF-1), a master transcription factor for which few regulators have been identified. Here, we demonstrate using an immune-competent mouse model of B-cell acute lymphoblastic leukemia (ALL; B-ALL) that Regnase-1 deficiency promotes TCF-1 expression to enhance CAR-T-cell expansion and memory-like cell formation. This leads to improved CAR-T-mediated tumor clearance, sustained remissions, and protection against secondary tumor challenge. Phenotypic, transcriptional, and epigenetic profiling identified increased tumor-dependent programming of Regnase-1-deficient CAR-T cells into TCF-1+ precursor exhausted T cells (TPEX) characterized by upregulation of both memory and exhaustion markers. Regnase-1 directly targets Tcf7 messenger RNA (mRNA); its deficiency augments TCF-1 expression leading to the formation of TPEX that support long-term CAR-T-cell persistence and function. Regnase-1 deficiency also reduces exhaustion and enhances the activity of TCF-1- CAR-T cells. We further validate these findings in human CAR-T cells, where Regnase-1 deficiency mediates enhanced tumor clearance in a xenograft B-ALL model. This is associated with increased persistence and expansion of a TCF-1+ CAR-T-cell population. Our findings demonstrate the pivotal roles of TPEX, Regnase-1, and TCF-1 in mediating CAR-T-cell persistence and recall responses, and identify Regnase-1 as a modulator of human CAR-T-cell longevity and potency that may be manipulated for improved therapeutic efficacy.

T cell metabolism in homeostasis and cancer immunity.

T cells shape immune responses in cancer, autoimmunity and infection, in which CD4+ T helper (Th) and CD8+ T cells mediate effector responses that are suppressed by regulatory T (Treg) cells. The balance between effector T cell and Treg cell function orchestrates immune homeostasis and functional programming, with important contributions to the onset and progression of cancer. Cellular metabolism is dynamically rewired in T cells in response to environmental cues and dictates various aspects of T cell function. In this review, we summarize recent findings on how cellular metabolism modulates effector T cell and Treg cell functional fitness in homeostasis and cancer immunity, and highlight the therapeutic implications of targeting immunometabolic pathways for cancer and other diseases.

Contribution of donor- and recipient-associated factors to allergic transfusion reactions to platelets.

BACKGROUND: Pediatric hematology-oncology patients require frequent platelet transfusions to manage chemotherapy-induced thrombocytopenia, and allergic transfusion reactions (ATRs) are common. Risk for platelet-associated ATRs can result from recipient- or donor-specific factors. STUDY DESIGN AND METHODS: We report a rare case in which an individual platelet donor caused repeated ATRs in multiple recipients. This observation led us to conduct a retrospective study at a pediatric hematology-oncology center to identify donor- and recipient-associated risk factors for ATRs. RESULTS: Single-donor platelets from an individual donor precipitated ATRs in 78.6% (n = 11/14) of recipients and 66.7% (n = 12/18) of platelet transfusions. We found in a cohort of pediatric hematology-oncology patients that 12.6% of recipients and 1.0% of platelet transfusions were associated with ATRs. Recipients who were aged 4 to 18 years, male, and those with central nervous system or solid tumors and with a history of ATRs to platelets were more likely to experience ATRs. Donor-associated risk factors were not identified, and we did not implicate additional donors in our single-center cohort with a frequency of ATRs comparable to the index donor. Based on our findings, we developed a novel statistical model to identify recipients and donors prone to experiencing or mediating ATRs. CONCLUSIONS: Both donors and recipients contribute to ATRs. Identification of high-risk donors and recipients for further scrutiny and potential interventions can improve the safety of platelet transfusions.

Anti-TNF therapy in IBD exerts its therapeutic effect through macrophage IL-10 signalling.

OBJECTIVE: Macrophage interleukin (IL)-10 signalling plays a critical role in the maintenance of a regulatory phenotype that prevents the development of IBD. We have previously found that anti-tumour necrosis factor (TNF) monoclonal antibodies act through Fcγ-receptor (FcγR) signalling to promote repolarisation of proinflammatory intestinal macrophages to a CD206+ regulatory phenotype. The role of IL-10 in anti-TNF-induced macrophage repolarisation has not been examined. DESIGN: We used human peripheral blood monocytes and mouse bone marrow-derived macrophages to study IL-10 production and CD206+ regulatory macrophage differentiation. To determine whether the efficacy of anti-TNF was dependent on IL-10 signalling in vivo and in which cell type, we used the CD4+CD45Rbhigh T-cell transfer model in combination with several genetic mouse models. RESULTS: Anti-TNF therapy increased macrophage IL-10 production in an FcγR-dependent manner, which caused differentiation of macrophages to a more regulatory CD206+ phenotype in vitro. Pharmacological blockade of IL-10 signalling prevented the induction of these CD206+ regulatory macrophages and diminished the therapeutic efficacy of anti-TNF therapy in the CD4+CD45Rbhigh T-cell transfer model of IBD. Using cell type-specific IL-10 receptor mutant mice, we found that IL-10 signalling in macrophages but not T cells was critical for the induction of CD206+ regulatory macrophages and therapeutic response to anti-TNF. CONCLUSION: The therapeutic efficacy of anti-TNF in resolving intestinal inflammation is critically dependent on IL-10 signalling in macrophages.

Targeting REGNASE-1 programs long-lived effector T cells for cancer therapy.

Adoptive cell therapy represents a new paradigm in cancer immunotherapy, but it can be limited by the poor persistence and function of transferred T cells1. Here we use an in vivo pooled CRISPR-Cas9 mutagenesis screening approach to demonstrate that, by targeting REGNASE-1, CD8+ T cells are reprogrammed to long-lived effector cells with extensive accumulation, better persistence and robust effector function in tumours. REGNASE-1-deficient CD8+ T cells show markedly improved therapeutic efficacy against mouse models of melanoma and leukaemia. By using a secondary genome-scale CRISPR-Cas9 screening, we identify BATF as the key target of REGNASE-1 and as a rheostat that shapes antitumour responses. Loss of BATF suppresses the increased accumulation and mitochondrial fitness of REGNASE-1-deficient CD8+ T cells. By contrast, the targeting of additional signalling factors-including PTPN2 and SOCS1-improves the therapeutic efficacy of REGNASE-1-deficient CD8+ T cells. Our findings suggest that T cell persistence and effector function can be coordinated in tumour immunity and point to avenues for improving the efficacy of adoptive cell therapy for cancer.

Pediatric patients with acute lymphoblastic leukemia generate abundant and functional neoantigen-specific CD8+ T cell responses.

Cancer arises from the accumulation of genetic alterations, which can lead to the production of mutant proteins not expressed by normal cells. These mutant proteins can be processed and presented on the cell surface by major histocompatibility complex molecules as neoepitopes, allowing CD8+ T cells to mount responses against them. For solid tumors, only an average 2% of neoepitopes predicted by algorithms have detectable endogenous antitumor T cell responses. This suggests that low mutation burden tumors, which include many pediatric tumors, are poorly immunogenic. Here, we report that pediatric patients with acute lymphoblastic leukemia (ALL) have tumor-associated neoepitope-specific CD8+ T cells, responding to 86% of tested neoantigens and recognizing 68% of the tested neoepitopes. These responses include a public neoantigen from the ETV6-RUNX1 fusion that is targeted in seven of nine tested patients. We characterized phenotypic and transcriptional profiles of CD8+ tumor-infiltrating lymphocytes (TILs) at the single-cell level and found a heterogeneous population that included highly functional effectors. Moreover, we observed immunodominance hierarchies among the CD8+ TILs restricted to one or two putative neoepitopes. Our results indicate that robust antitumor immune responses are induced in pediatric ALL despite their low mutation burdens and emphasize the importance of immunodominance in shaping cellular immune responses. Furthermore, these data suggest that pediatric cancers may be amenable to immunotherapies aimed at enhancing immune recognition of tumor-specific neoantigens.

IL-6 Promotes T Cell Proliferation and Expansion under Inflammatory Conditions in Association with Low-Level RORγt Expression.

IL-6 is a critical driver of acute and chronic inflammation and has been reported to act as a T cell survival factor. The influence of IL-6 on T cell homeostasis is not well resolved. We demonstrate that IL-6 signaling drives T cell expansion under inflammatory conditions but not during normal homeostasis. During inflammation, IL-6Rα-deficient T cells are unable to effectively compete with wild type T cells. IL-6 promotes T cell proliferation, and this is associated with low-level expression of the RORγt transcription factor. T cells upregulate Rorc mRNA at levels substantially diminished from that seen in Th17 cells. Blockade of RORγt through genetic knockout or a small molecule inhibitor leads to T cell expansion defects comparable to those in IL-6Rα-deficient T cells. Our results indicate that IL-6 plays a key role in T cell expansion during inflammation and implicates a role for the transient induction of low-level RORγt.

Haploidentical Donor Transplantation Using a Novel Clofarabine-containing Conditioning Regimen for Very High-risk Hematologic Malignant Neoplasms.

Recurrent/refractory hematologic malignancies have a poor prognosis, and there is a need for novel treatment regimens that can be tolerated by this heavily pretreated patient group. Clofarabine has antileukemic activity with an acceptable toxicity profile. In a phase I clinical trial (NCT00824135), we substituted clofarabine for fludarabine in a well-established reduced-intensity conditioning regimen for a T cell-depleted, mismatched-related (haploidentical) donor transplant backbone and explored the maximum tolerated dose of clofarabine in this combination in 15 patients undergoing hematopoietic cell transplantation for recurrent/refractory or secondary leukemia. Clofarabine was well tolerated at a dose of 50 mg/m/d for 5 days in this regimen, with minimal treatment-related mortality in a heavily pretreated group of high-risk patients. All patients exhibited quick hematopoietic recovery, with median times to neutrophil and platelet engraftment being 11 and 16 days, respectively. Transient elevation of transaminases was the most common toxicity-observed in 13 patients (86.7%), with 6 (40%) grade III or above. Three patients (20%) developed hepatic veno-occlusive disease. Eleven patients (73.3%) died, with the most common cause of death being disease relapse (in 9 patients [60%]), followed by treatment-related mortality (in 2 patients [13.3%]). Four (26.6%) of the patients are long-term survivors.

PI3K orchestration of the in vivo persistence of chimeric antigen receptor-modified T cells.

In vivo persistence of chimeric antigen receptor (CAR)-modified T cells correlates with therapeutic efficacy, yet CAR-specific factors that support persistence are not well resolved. Using a CD33-specific CAR in an acute myeloid leukemia (AML) model, we show how CAR expression alters T cell differentiation in a ligand independent manner. Ex vivo expanded CAR-T cells demonstrated decreased naïve and stem memory populations and increased effector subsets relative to vector-transduced control cells. This was associated with reduced in vivo persistence. Decreased persistence was not due to specificity or tumor presence, but to pre-transfer tonic signaling through the CAR CD3ζ ITAMs. We identified activation of the PI3K pathway in CD33 CAR-T cells as responsible. Treatment with a PI3K inhibitor modulated the differentiation program of CAR-T cells, preserved a less differentiated state without affecting T cell expansion, and improved in vivo persistence and reduced tumor burden. These results resolve mechanisms by which tonic signaling of CAR-T cells modulates their fate, and identifies a novel pharmacologic approach to enhance the durability of CAR-T cells for immunotherapy.

The neoepitope landscape in pediatric cancers.

BACKGROUND: Neoepitopes derived from tumor-specific somatic mutations are promising targets for immunotherapy in childhood cancers. However, the potential for such therapies in targeting these epitopes remains uncertain due to a lack of knowledge of the neoepitope landscape in childhood cancer. Studies to date have focused primarily on missense mutations without exploring gene fusions, which are a major class of oncogenic drivers in pediatric cancer. METHODS: We developed an analytical workflow for identification of putative neoepitopes based on somatic missense mutations and gene fusions using whole-genome sequencing data. Transcriptome sequencing data were incorporated to interrogate the expression status of the neoepitopes. RESULTS: We present the neoepitope landscape of somatic alterations including missense mutations and oncogenic gene fusions identified in 540 childhood cancer genomes and transcriptomes representing 23 cancer subtypes. We found that 88% of leukemias, 78% of central nervous system tumors, and 90% of solid tumors had at least one predicted neoepitope. Mutation hotspots in KRAS and histone H3 genes encode potential epitopes in multiple patients. Additionally, the ETV6-RUNX1 fusion was found to encode putative neoepitopes in a high proportion (69.6%) of the pediatric leukemia harboring this fusion. CONCLUSIONS: Our study presents a comprehensive repertoire of potential neoepitopes in childhood cancers, and will facilitate the development of immunotherapeutic approaches designed to exploit them. The source code of the workflow is available at GitHub ( https://github.com/zhanglabstjude/neoepitope ).

Autoimmune susceptibility imposed by public TCRß chains.

Although the TCR repertoire is highly diverse, a small fraction of TCR chains, referred to as public, preferentially form and are shared by most individuals. Prior studies indicated that public TCRß may be preferentially deployed in autoimmunity. We hypothesized that if these TCRß modulate the likelihood of a TCRαß heterodimer productively engaging autoantigen, because they are widely present in the population and often high frequency within individual repertoires, they could also broadly influence repertoire responsiveness to specific autoantigens. We assess this here using a series of public and private TCRß derived from autoimmune encephalomyelitis-associated TCR. Transgenic expression of public, but not private, disease-associated TCRß paired with endogenously rearranged TCRα endowed unprimed T cells with autoantigen reactivity. Further, two of six public, but none of five private TCRß provoked spontaneous early-onset autoimmunity in mice. Our findings indicate that single TCRß are sufficient to confer on TCRαß chains reactivity toward disease-associated autoantigens in the context of diverse TCRα. They further suggest that public TCR can skew autoimmune susceptibility, and that subsets of public TCR sequences may serve as disease- specific biomarkers or therapeutic targets.

Preferential Use of Public TCR during Autoimmune Encephalomyelitis.

How the TCR repertoire, in concert with risk-associated MHC, imposes susceptibility for autoimmune diseases is incompletely resolved. Due largely to recombinatorial biases, a small fraction of TCRα or ß-chains are shared by most individuals, or public. If public TCR chains modulate a TCRαß heterodimer's likelihood of productively engaging autoantigen, because they are pervasive and often high frequency, they could also broadly influence disease risk and progression. Prior data, using low-resolution techniques, have identified the heavy use of select public TCR in some autoimmune models. In this study, we assess public repertoire representation in mice with experimental autoimmune encephalomyelitis at high resolution. Saturation sequencing was used to identify >18 × 10(6) TCRß sequences from the CNSs, periphery, and thymi of mice at different stages of autoimmune encephalomyelitis and healthy controls. Analyses indicated the prominent representation of a highly diverse public TCRß repertoire in the disease response. Preferential formation of public TCR implicated in autoimmunity was identified in preselection thymocytes, and, consistently, public, disease-associated TCRß were observed to be commonly oligoclonal. Increased TCR sharing and a focusing of the public TCR response was seen with disease progression. Critically, comparisons of peripheral and CNS repertoires and repertoires from preimmune and diseased mice demonstrated that public TCR were preferentially deployed relative to nonshared, or private, sequences. Our findings implicate public TCR in skewing repertoire response during autoimmunity and suggest that subsets of public TCR sequences may serve as disease-specific biomarkers or influence disease susceptibility or progression.

Differential T Cell Cytokine Receptivity and Not Signal Quality Distinguishes IL-6 and IL-10 Signaling during Th17 Differentiation.

How a large number of cytokines differentially signal through a small number of signal transduction pathways is not well resolved. This is particularly true for IL-6 and IL-10, which act primarily through STAT3 yet induce dissimilar transcriptional programs leading alternatively to pro- and anti-inflammatory effects. Kinetic differences in signaling, sustained to IL-10 and transient to IL-6, are critical to this in macrophages. T cells are also key targets of IL-6 and IL-10, yet how differential signaling in these cells leads to divergent cellular fates is unclear. We show that, unlike for macrophages, signal duration cannot explain the distinct effects of these cytokines in T cells. Rather, naive, activated, activated-rested, and memory CD4(+) T cells differentially express IL-6 and IL-10 receptors in an activation state-dependent manner, and this impacts downstream cytokine effects. We show a dominant role for STAT3 in IL-6-mediated Th17 subset maturation. IL-10 cannot support Th17 differentiation because of insufficient cytokine receptivity rather than signal quality. Enforced expression of IL-10Rα on naive T cells permits an IL-10-generated STAT3 signal equivalent to that of IL-6 and equally capable of promoting Th17 formation. Similarly, naive T cell IL-10Rα expression also allows IL-10 to mimic the effects of IL-6 on both Th1/Th2 skewing and Tfh cell differentiation. Our results demonstrate a key role for the regulation of receptor expression rather than signal quality or duration in differentiating the functional outcomes of IL-6 and IL-10 signaling, and identify distinct signaling properties of these cytokines in T cells compared with myeloid cells.

Gut Microbial Dysbiosis Due to Helicobacter Drives an Increase in Marginal Zone B Cells in the Absence of IL-10 Signaling in Macrophages.

It is clear that IL-10 plays an essential role in maintaining homeostasis in the gut in response to the microbiome. However, it is unknown whether IL-10 also facilitates immune homeostasis at distal sites. To address this question, we asked whether splenic immune populations were altered in IL-10-deficient (Il10(-/-)) mice in which differences in animal husbandry history were associated with susceptibility to spontaneous enterocolitis that is microbiome dependent. The susceptible mice exhibited a significant increase in splenic macrophages, neutrophils, and marginal zone (MZ) B cells that was inhibited by IL-10 signaling in myeloid, but not B cells. The increase in macrophages was due to increased proliferation that correlated with a subsequent enhancement in MZ B cell differentiation. Cohousing and antibiotic treatment studies suggested that the alteration in immune homeostasis in the spleen was microbiome dependent. The 16S rRNA sequencing revealed that susceptible mice harbored a different microbiome with a significant increase in the abundance of the bacterial genus Helicobacter. The introduction of Helicobacter hepaticus to the gut of nonsusceptible mice was sufficient to drive macrophage expansion and MZ B cell development. Given that myeloid cells and MZ B cells are part of the first line of defense against blood-borne pathogens, their increase following a breach in the gut epithelial barrier would be protective. Thus, IL-10 is an essential gatekeeper that maintains immune homeostasis at distal sites that can become functionally imbalanced upon the introduction of specific pathogenic bacteria to the intestinal track.

Chronic TLR Stimulation Controls NLRP3 Inflammasome Activation through IL-10 Mediated Regulation of NLRP3 Expression and Caspase-8 Activation.

While the molecular mechanisms promoting activation of the Nod-like Receptor (NLR) family member NLRP3 inflammasome are beginning to be defined, little is known about the mechanisms that regulate the NLRP3 inflammasome. Acute (up to 4 hours) LPS stimulation, followed by ATP is frequently used to activate the NLRP3 inflammasome in macrophages. Interestingly, we observed that the ability of LPS to license NLRP3 is transient, as prolonged (12 to 24 hours) LPS exposure was a relatively ineffective priming stimulus. This suggests that relative to acute LPS, chronic LPS exposure triggers regulatory mechanisms to dampen NLRP3 activation. Transfer of culture supernatants from macrophages stimulated with LPS for 24 hours dramatically reduced ATP- and nigericin-induced NLRP3 inflammasome activation in naïve macrophages. We further identified IL-10 as the secreted inflammasome-tolerizing factor that acts in an autocrine manner to control activation of the NLRP3 inflammasome. Finally, we demonstrated that IL-10 dampens NLRP3 expression to control NLRP3 inflammasome activation and subsequent caspase-8 activation. In conclusion, we have uncovered a mechanism by which chronic, but not acute, LPS exposure induces IL-10 to dampen NLRP3 inflammasome activation to avoid overt inflammation.