Metabolic reprogramming of tumor-associated macrophages using glutamine antagonist JHU083 drives tumor immunity in myeloid-rich prostate and bladder cancer tumors.

Glutamine metabolism in tumor microenvironments critically regulates anti-tumor immunity. Using glutamine-antagonist prodrug JHU083, we report potent tumor growth inhibition in urologic tumors by JHU083-reprogrammed tumor-associated macrophages (TAMs) and tumor-infiltrating monocytes (TIMs). We show JHU083-mediated glutamine antagonism in tumor microenvironments induces TNF, pro-inflammatory, and mTORC1 signaling in intratumoral TAM clusters. JHU083-reprogrammed TAMs also exhibit increased tumor cell phagocytosis and diminished pro-angiogenic capacities. In vivo inhibition of TAM glutamine consumption resulted in increased glycolysis, a broken TCA cycle, and purine metabolism disruption. Although the anti-tumor effect of glutamine antagonism on tumor-infiltrating T cells was moderate, JHU083 promoted a stem cell-like phenotype in CD8+ T cells and decreased Treg abundance. Finally, JHU083 caused a ubiquitous shutdown in glutamine utilizing metabolic pathways in tumor cells, leading to reduced HIF-1alpha, c-MYC phosphorylation, and induction of tumor cell apoptosis, all key anti-tumor features.

Adoptive transfer of CD49a+ Tissue resident memory cells reverses pulmonary fibrosis in mice.

Pulmonary fibrosis is a devastating disease with no effective treatments to cure, stop or reverse the unremitting, fatal fibrosis. A critical barrier to treating this disease is the lack of understanding of the pathways leading to fibrosis as well as those regulating the resolution of fibrosis. Fibrosis is the pathologic side of normal tissue repair that results when the normal wound healing programs go awry. Successful resolution of tissue injury requires several highly coordinated pathways, and this research focuses on the interplay between these overlapping pathways: immune effectors, inflammatory mediators and fibroproliferation in the resolution of fibrosis. Previously we have successfully prevented, mitigated, and even reversed established fibrosis using vaccinia vaccination immunotherapy in two models of murine lung fibrosis. The mechanism by which vaccinia reverses fibrosis is by vaccine induced lung specific Th1 skewed tissue resident memory (TRMs) in the lung. In this study, we isolated a population of vaccine induced TRMs - CD49a+ CD4+ T cells - that are both necessary and sufficient to reverse established pulmonary fibrosis. Using adoptive cellular therapy, we demonstrate that intratracheal administration of CD49a+ CD4+ TRMs into established fibrosis, reverses the fibrosis histologically, by promoting a decrease in collagen, and functionally, by improving lung function, without the need for vaccination. Furthermore, co-culture of in vitro derived CD49+ CD4+ human TRMs with human fibroblasts from individuals with idiopathic pulmonary fibrosis (IPF) results in the down regulation of IPF fibroblast collagen production. Lastly, we demonstrate in human IPF lung histologic samples that CD49a+ CD4+ TRMs, which can down regulate human IPF fibroblast function, fail to increase in the IPF lungs, thus potentially failing to promote resolution. Thus, we define a novel unappreciated role for tissue resident memory T cells in regulating established lung fibrosis to promote resolution of fibrosis and re-establish lung homeostasis. We demonstrate that immunotherapy, in the form of adoptive transfer of CD49a+ CD4+ TRMs into the lungs of mice with established fibrosis, not only stops progression of the fibrosis but more importantly reverses the fibrosis. These studies provide the insight and preclinical rationale for a novel paradigm shifting approach of using cellular immunotherapy to treat lung fibrosis.

Hematopoietic Bone Marrow Transplant to Treat Systemic EBV-positive T-cell Lymphoma of Childhood.

Systemic Epstein-Barr virus-positive T-cell lymphoma of childhood (S-EBV-TCL) is a rare disease for which there is no standard of care. S-EBV-TCL is often associated with hemophagocytic lymphohistiocytosis and is generally thought of on the spectrum of EBV-related disease. For the few reported cases of cure in the literature, hematopoietic stem cell transplant has been required because it is the only treatment that has induced complete remission in patients suffering from EBV-associated T-cell or natural killer cell lymphoproliferative diseases, except hemophagocytic lymphohistiocytosis. Here, we present the case of one patient who was successfully cured with a modified regimen of dose-adjusted EPOCH (etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin), followed by hematopoietic stem cell transplant using a reduced-intensity conditioning regimen.

Ensuring Intervention Fidelity of an Attention Control Arm in a Multisite Randomized Controlled Trial.

BACKGROUND: Intervention fidelity is a critical element of randomized controlled trials, yet reporting of intervention fidelity among attention control arms is limited. Lack of fidelity to attention control procedures can affect study outcomes by either overestimating or underestimating the efficacy of the intervention under examination. OBJECTIVES: This brief report describes the approach researchers took to promote fidelity to the attention control arm of a pediatric palliative care randomized controlled trial funded by the National Institutes of Health. METHODS: The Informational Meetings for Planning and Coordinating Treatment trial aims to determine the efficacy of a communication intervention that uses care team dyads (i.e., physicians partnered with nurses or advanced practice providers) to engage parents of children with cancer who have a poor prognosis in structured conversations about prognostic information, goals of care, and care planning. The intervention is compared with an attention control arm, which provides parents with structured conversations on common pediatric cancer education topics, such as talking to their child about their cancer, clinical trials, cancer treatment, side effects, and so forth. National Institutes of Health guidelines for assessing and implementing strategies to promote intervention fidelity were used to design (a) the attention control arm of a randomized controlled trial, (b) related attention control arm training, and (c) quality assurance monitoring. RESULTS: Attention control study procedures were designed to mirror that of the intervention arm (i.e., same number, frequency, and time spent in study visits). Cluster randomization was used to allocate care team dyads to one arm of the randomized controlled trial. Care team dyads assigned to the attention control arm participated in online training sessions to learn attention control procedures, the different roles of research team members, and quality assurance methods. Fidelity to attention control procedures is assessed by both the interveners themselves and a quality assurance team. DISCUSSION: Study design, training, and delivery are all critical to attention control fidelity. Baseline training often needs to be supplemented with booster training when time gaps occur between study start-up and implementation. Quality assurance procedures are essential to determine whether interveners consistently deliver attention control procedures correctly.

Successful Crizotinib-targeted Therapy of Pediatric Unresectable ERC1::ALK Fusion Sarcoma.

Anaplastic lymphoma kinase ( ALK )-fusion sarcomas are rare part of the emerging theoretically targetable tyrosine kinase RAS::MAPK pathway fusion myopericytic-ovoid sarcomas. We report our clinicopathologic and treatment experience with an ALK fusion sarcoma. A novel ELKS/RAB6-interacting/CAST family member 1 - unaligned ALK fusion infiltrative nonmetastatic low-grade sarcoma of the right hand of a 15-month-old male was treated with crizotinib, an ALK tyrosine kinase inhibitor as oral monotherapy, inducing complete radiographic and clinical resolution by 10 months and sustained response now over 12 months after elective discontinuation. Crizotinib can successfully be used to treat unresectable novel ALK fusion sarcomas.

TSC2 S1365A mutation potently regulates CD8+ T cell function and differentiation and improves adoptive cellular cancer therapy.

MTORC1 integrates signaling from the immune microenvironment to regulate T cell activation, differentiation, and function. TSC2 in the tuberous sclerosis complex tightly regulates mTORC1 activation. CD8+ T cells lacking TSC2 have constitutively enhanced mTORC1 activity and generate robust effector T cells; however, sustained mTORC1 activation prevents generation of long-lived memory CD8+ T cells. Here we show that manipulating TSC2 at Ser1365 potently regulated activated but not basal mTORC1 signaling in CD8+ T cells. Unlike nonstimulated TSC2-KO cells, CD8+ T cells expressing a phosphosilencing mutant TSC2-S1365A (TSC2-SA) retained normal basal mTORC1 activity. PKC and T cell receptor (TCR) stimulation induced TSC2 S1365 phosphorylation, and preventing this with the SA mutation markedly increased mTORC1 activation and T cell effector function. Consequently, SA CD8+ T cells displayed greater effector responses while retaining their capacity to become long-lived memory T cells. SA CD8+ T cells also displayed enhanced effector function under hypoxic and acidic conditions. In murine and human solid-tumor models, SA CD8+ T cells used as adoptive cell therapy displayed greater antitumor immunity than WT CD8+ T cells. These findings reveal an upstream mechanism to regulate mTORC1 activity in T cells. The TSC2-SA mutation enhanced both T cell effector function and long-term persistence/memory formation, supporting an approach to engineer better CAR-T cells for treating cancer.

Distinct Myeloid Derived Suppressor Cell Populations Promote Tumor Aggression in Glioblastoma.

The diversity of genetic programs and cellular plasticity of glioma-associated myeloid cells, and thus their contribution to tumor growth and immune evasion, is poorly understood. We performed single cell RNA-sequencing of immune and tumor cells from 33 glioma patients of varying tumor grades. We identified two populations characteristic of myeloid derived suppressor cells (MDSC), unique to glioblastoma (GBM) and absent in grades II and III tumors: i) an early progenitor population (E-MDSC) characterized by strong upregulation of multiple catabolic, anabolic, oxidative stress, and hypoxia pathways typically observed within tumor cells themselves, and ii) a monocytic MDSC (M-MDSC) population. The E-MDSCs geographically co-localize with a subset of highly metabolic glioma stem-like tumor cells with a mesenchymal program in the pseudopalisading region, a pathognomonic feature of GBMs associated with poor prognosis. Ligand-receptor interaction analysis revealed symbiotic cross-talk between the stemlike tumor cells and E-MDSCs in GBM, whereby glioma stem cells produce chemokines attracting E-MDSCs, which in turn produce growth and survival factors for the tumor cells. Our large-scale single-cell analysis elucidated unique MDSC populations as key facilitators of GBM progression and mediators of tumor immunosuppression, suggesting that targeting these specific myeloid compartments, including their metabolic programs, may be a promising therapeutic intervention in this deadly cancer. One-Sentence Summary: Aggressive glioblastoma harbors two unique myeloid populations capable of promoting stem-like properties of tumor cells and suppressing T cell function in the tumor microenvironment.

A novel nano-iron supplement versus standard treatment for iron deficiency anaemia in children 6-35 months (IHAT-GUT trial): a double-blind, randomised, placebo-controlled non-inferiority phase II trial in The Gambia.

Background: Iron deficiency anaemia (IDA) is the leading cause of years lost to disability in most sub-Saharan African countries and is especially common in young children. The IHAT-GUT trial assessed the efficacy and safety of a novel nano iron supplement, which is a dietary ferritin analogue termed iron hydroxide adipate tartrate (IHAT), for the treatment of IDA in children under 3 years of age. Methods: In this single-country, randomised, double-blind, parallel, placebo-controlled, non-inferiority Phase II study in The Gambia, children 6-35 months with IDA (7≤Hb < 11 g/dL and ferritin<30 µg/L) were randomly assigned (1:1:1) to receive either IHAT, ferrous sulphate (FeSO4) or placebo daily for 3 months (85 days). The daily iron dose was 12.5 mg Fe equivalent for FeSO4 and the estimated dose with comparable iron-bioavailability for IHAT (20 mg Fe). The primary efficacy endpoint was the composite of haemoglobin response at day 85 and correction of iron deficiency. The non-inferiority margin was 0.1 absolute difference in response probability. The primary safety endpoint was moderate-severe diarrhoea analysed as incidence density and prevalence over the 3 months intervention. Secondary endpoints reported herein include hospitalisation, acute respiratory infection, malaria, treatment failures, iron handling markers, inflammatory markers, longitudinal prevalence of diarrhoea and incidence density of bloody diarrhoea. Main analyses were per-protocol (PP) and intention-to-treat (ITT) analyses. This trial is registered with clinicaltrials.gov (NCT02941081). Findings: Between Nov 2017 and Nov 2018, 642 children were randomised into the study (214 per group) and included in the ITT analysis, the PP population included 582 children. A total of 50/177 (28.2%) children in the IHAT group achieved the primary efficacy endpoint, as compared with 42/190 (22.1%) in the FeSO4 group (OR 1.39, 80% CI 1.01-1.91, PP population) and with 2/186 (1.1%) in the placebo group. Diarrhoea prevalence was similar between groups, with 40/189 (21.2%) children in the IHAT group developing at least one episode of moderate-severe diarrhoea over the 85 days intervention, compared with 47/198 (23.7%) in the FeSO4 group (OR 1.18, 80% CI 0.86-1.62) and 40/195 (20.5%) in the placebo group (OR 0.96, 80% CI 0.7-1.33, PP population). Incidence density of moderate-severe diarrhoea was 2.66 in the IHAT group and 3.42 in the FeSO4 group (RR 0.76, 80% CI 0.59-0.99, CC-ITT population).There were 143/211 (67.8%) children with adverse events (AEs) in the IHAT group, 146/212 (68.9%) in the FeSO4 group and 143/214 (66.8%) in the placebo group. There were overall 213 diarrhoea-related AEs; 35 (28.5%) cases reported in the IHAT group compared with 51 (41.5%) cases in the FeSO4 group and 37 (30.1%) cases in the placebo group. Interpretation: In this first Phase II study conducted in young children with IDA, IHAT showed sufficient non-inferiority compared to standard-of-care FeSO4, in terms of ID correction and haemoglobin response, to warrant a definitive Phase III trial. In addition, IHAT had lower incidence of moderate-severe diarrhoea than FeSO4, with no increased adverse events in comparison with placebo. Funding: The Bill & Melinda Gates Foundation (OPP1140952).

Differential Effects of Iron Chelates vs. Iron Salts on Induction of Pro-Oncogenic Amphiregulin and Pro-Inflammatory COX-2 in Human Intestinal Adenocarcinoma Cell Lines.

We previously showed that two iron compounds that are orally ingested by humans, namely ferric EDTA and ferric citrate, can induce an oncogenic growth factor (amphiregulin) in human intestinal epithelial adenocarcinoma cell lines. Here, we further screened these iron compounds, plus four other iron chelates and six iron salts (i.e., 12 oral iron compounds in total), for their effects on biomarkers of cancer and inflammation. Ferric pyrophosphate and ferric EDTA were the main inducers of amphiregulin and its receptor monomer, IGFr1. Moreover, at the maximum iron concentrations investigated (500 µM), the highest levels of amphiregulin were induced by the six iron chelates, while four of these also increased IGfr1. In addition, we observed that ferric pyrophosphate promoted signaling via the JAK/STAT pathway by up-regulating the cytokine receptor subunit IFN-γr1 and IL-6. For pro-inflammatory cyclooxygenase-2 (COX-2), ferric pyrophosphate but not ferric EDTA elevated intracellular levels. This, however, did not drive the other biomarkers based on COX-2 inhibition studies and was probably downstream of IL-6. We conclude that of all oral iron compounds, iron chelates may particularly elevate intracellular amphiregulin. Ferric pyrophosphate additionally induced COX-2, probably because of the high IL-6 induction that was observed with this compound.

Can people living with and beyond colorectal cancer make lifestyle changes with the support of health technology: A feasibility study.

BACKGROUND: Rates of cancer survival are increasing, with more people living with and beyond cancer. Lifestyle recommendations for cancer survivors are based largely on extrapolation from cancer prevention recommendations. This feasibility study was designed to investigate diet and physical activity variables linked to primary prevention and digital behaviour change interventions in cancer survivors and delivered by an oncology dietitian to plan for future research. METHODS: In this 2-month feasibility study, participants who had completed treatment for colorectal cancer were invited to complete online food diaries, underwent physical activity assessment, attended fortnightly telephone consultations with an oncology dietitian and completed an evaluation form. The baseline food diaries were used to help participants pick two lifestyle changes to focus on throughout the intervention. Demographic and clinical data were analysed using descriptive statistics. RESULTS: In total, 996 patients were screened for eligibility; of these, 78 were eligible to approach and 69 were approached, resulting in 20 participants consenting to take part. Overall, the intervention was acceptable with 65% of participants completing an online food diary and 70% engaging with the dietitian over the telephone. The intervention received good feedback, with 100% of those completing the evaluation form reporting they felt supported and found it helpful. CONCLUSIONS: The present study offers preliminary evidence that a lifestyle intervention delivered by an oncology dietitian using digital behaviour change interventions (DBCIs) to cancer survivors is feasible and accepted by participants and providers.

Cutting Edge: mTORC2 Regulates CD8+ Effector and Memory T Cell Differentiation through Serum and Glucocorticoid Kinase 1.

The mechanistic target of rapamycin is an essential regulator of T cell metabolism and differentiation. In this study, we demonstrate that serum- and glucocorticoid-regulated kinase 1 (SGK1), a downstream node of mechanistic target of rapamycin complex 2 signaling, represses memory CD8+ T cell differentiation. During acute infections, murine SGK1-deficient CD8+ T cells adopt an early memory precursor phenotype leading to more long-lived memory T cells. Thus, SGK1-deficient CD8+ T cells demonstrate an enhanced recall capacity in response to reinfection and can readily reject tumors. Mechanistically, activation of SGK1-deficient CD8+ T cells results in decreased Foxo1 phosphorylation and increased nuclear translocation of Foxo1 to promote early memory development. Overall, SGK1 might prove to be a powerful target for enhancing the efficacy of vaccines and tumor immunotherapy.

Discovery of DRP-104, a tumor-targeted metabolic inhibitor prodrug.

6-Diazo-5-oxo-l-norleucine (DON) is a glutamine antagonist that suppresses cancer cell metabolism but concurrently enhances the metabolic fitness of tumor CD8+ T cells. DON showed promising efficacy in clinical trials; however, its development was halted by dose-limiting gastrointestinal (GI) toxicities. Given its clinical potential, we designed DON peptide prodrugs and found DRP-104 [isopropyl(S)-2-((S)-2-acetamido-3-(1H-indol-3-yl)-propanamido)-6-diazo-5-oxo-hexanoate] that was preferentially bioactivated to DON in tumor while bioinactivated to an inert metabolite in GI tissues. In drug distribution studies, DRP-104 delivered a prodigious 11-fold greater exposure of DON to tumor versus GI tissues. DRP-104 affected multiple metabolic pathways in tumor, including decreased glutamine flux into the TCA cycle. In efficacy studies, both DRP-104 and DON caused complete tumor regression; however, DRP-104 had a markedly improved tolerability profile. DRP-104's effect was CD8+ T cell dependent and resulted in robust immunologic memory. DRP-104 represents a first-in-class prodrug with differential metabolism in target versus toxicity tissue. DRP-104 is now in clinical trials under the FDA Fast Track designation.

Interrogation of T Cell-Enriched Tumors Reveals Prognostic and Immunotherapeutic Implications of Polyamine Metabolism.

Metabolic features of the tumor microenvironment (TME) antagonize anti-tumor immunity. We hypothesized that T cell infiltrated tumors with a known antigen should exhibit superior clinical outcomes, though some fare worse given unfavorable metabolic features leveraging T cell-infiltrated (Thi), human papillomavirus-related (HPV+) head and neck squamous cell carcinomas (HNSC) to test this hypothesis. Expression of 2,520 metabolic genes were analyzed among Thi HPV+ HNSCs stratified by high-risk molecular subtype. RNAseq data from The Cancer Genome Atlas (TCGA; 10 cancer types), single cell RNAseq data, and an immunotherapy-treated melanoma cohort were used to test the association between metabolic gene expression and clinical outcomes and contribution of tumor versus stromal cells to metabolic gene expression. Polyamine (PA) metabolism genes were overexpressed in high-risk, Thi HPV+ HNSCs. Genes involved in PA biosynthesis and transport were associated with T cell infiltration, recurrent or persistent cancer, overall survival status, primary site, molecular subtype, and MYC genomic alterations. PA biogenesis gene sets were associated with tumor intrinsic features while myeloid cells in HPV+ HNSCs were enriched in PA catabolism, regulatory, transport, putrescine, and spermidine gene set expression. PA gene set expression also correlated with IFNγ or cytotoxic T cell ssGSEA scores across TCGA tumor types. PA transport ssGSEA scores were associated with poor survival whereas putrescine ssGSEA scores portended better survival for several tumor types. Thi melanomas enriched in PA synthesis or combined gene set expression exhibited worse anti-PD-1 responses. These data address hurdles to anti-tumor immunity warranting further investigation of divergent polyamine metabolism in the TME.

Fusion-negative rhabdomyosarcoma with diffuse bony metastases and remarkable chemosensitivity.

In this report, we describe the case of an adolescent male with an unusual case of fusion-negative, paratesticular alveolar rhabdomyosarcoma who presented with spontaneous tumour lysis syndrome and diffuse bony metastases throughout the axial and appendicular skeleton with additional significant bone marrow involvement. Both spontaneous tumour lysis syndrome and diffuse bony metastases are extremely unusual for rhabdomyosarcoma. On the backbone of standard vincristine, dactinomycin and cyclophosphamide (VAC) chemotherapy, the only local control was orchiectomy at 15 weeks, with no radiation administered due to the initially diffuse nature of the disease and rapid response to chemotherapy. Following 43 weeks of VAC, a year-long maintenance phase with pazopanib was given which was well tolerated. The patient remains in remission now 4 years after completion of therapy.

Small Extracellular Vesicles Secreted by Nigrostriatal Astrocytes Rescue Cell Death and Preserve Mitochondrial Function in Parkinson's Disease.

Extracellular vesicles (EVs) are emerging as powerful players in cell-to-cell communication both in healthy and diseased brain. In Parkinson's disease (PD)-characterized by selective dopaminergic neuron death in ventral midbrain (VMB) and degeneration of their terminals in striatum (STR)-astrocytes exert dual harmful/protective functions, with mechanisms not fully elucidated. Here, this study shows that astrocytes from the VMB-, STR-, and VMB/STR-depleted brains release a population of small EVs  in a region-specific manner. Interestingly, VMB-astrocytes secreted the highest rate of EVs, which is further exclusively increased in response to CCL3, a chemokine that promotes robust dopaminergic neuroprotection in different PD models. The neuroprotective potential of nigrostriatal astrocyte-EVs is investigated in differentiated versus undifferentiated SH-SY5Y cells exposed to oxidative stress and mitochondrial toxicity. EVs from both VMB- and STR-astrocytes counteract H2 O2 -induced caspase-3 activation specifically in differentiated cells, with EVs from CCL3-treated astrocytes showing a higher protective effect. High resolution respirometry further reveals that nigrostriatal astrocyte-EVs rescue neuronal mitochondrial complex I function impaired by the neurotoxin MPP+ . Notably, only EVs from VMB-astrocyte fully restore ATP production, again specifically in differentiated SH-SY5Y. These results highlight a regional diversity in the nigrostriatal system for the secretion and activities of astrocyte-EVs, with neuroprotective implications for PD.

Sulforaphane exhibits antiviral activity against pandemic SARS-CoV-2 and seasonal HCoV-OC43 coronaviruses in vitro and in mice.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), has incited a global health crisis. Currently, there are limited therapeutic options for the prevention and treatment of SARS-CoV-2 infections. We evaluated the antiviral activity of sulforaphane (SFN), the principal biologically active phytochemical derived from glucoraphanin, the naturally occurring precursor present in high concentrations in cruciferous vegetables. SFN inhibited in vitro replication of six strains of SARS-CoV-2, including Delta and Omicron, as well as that of the seasonal coronavirus HCoV-OC43. Further, SFN and remdesivir interacted synergistically to inhibit coronavirus infection in vitro. Prophylactic administration of SFN to K18-hACE2 mice prior to intranasal SARS-CoV-2 infection significantly decreased the viral load in the lungs and upper respiratory tract and reduced lung injury and pulmonary pathology compared to untreated infected mice. SFN treatment diminished immune cell activation in the lungs, including significantly lower recruitment of myeloid cells and a reduction in T cell activation and cytokine production. Our results suggest that SFN should be explored as a potential agent for the prevention or treatment of coronavirus infections.

Re-engineered BCG overexpressing cyclic di-AMP augments trained immunity and exhibits improved efficacy against bladder cancer.

In addition to its role as a TB vaccine, BCG has been shown to elicit heterologous protection against many other pathogens including viruses through a process termed trained immunity. Despite its potential as a broadly protective vaccine, little has been done to determine if BCG-mediated trained immunity levels can be optimized. Here we re-engineer BCG to express high levels of c-di-AMP, a PAMP recognized by stimulator of interferon genes (STING). We find that BCG overexpressing c-di-AMP elicits more potent signatures of trained immunity including higher pro-inflammatory cytokine responses, greater myeloid cell reprogramming toward inflammatory and activated states, and enhances epigenetic and metabolomic changes. In a model of bladder cancer, we also show that re-engineered BCG induces trained immunity and improved functionality. These results indicate that trained immunity levels and antitumor efficacy may be increased by modifying BCG to express higher levels of key PAMP molecules.

Signatures of GVHD and relapse after posttransplant cyclophosphamide revealed by immune profiling and machine learning.

The key immunologic signatures associated with clinical outcomes after posttransplant cyclophosphamide (PTCy)-based HLA-haploidentical (haplo) and HLA-matched bone marrow transplantation (BMT) are largely unknown. To address this gap in knowledge, we used machine learning to decipher clinically relevant signatures from immunophenotypic, proteomic, and clinical data and then examined transcriptome changes in the lymphocyte subsets that predicted major posttransplant outcomes. Kinetics of immune subset reconstitution after day 28 were similar for 70 patients undergoing haplo and 75 patients undergoing HLA-matched BMT. Machine learning based on 35 candidate factors (10 clinical, 18 cellular, and 7 proteomic) revealed that combined elevations in effector CD4+ conventional T cells (Tconv) and CXCL9 at day 28 predicted acute graft-versus-host disease (aGVHD). Furthermore, higher NK cell counts predicted improved overall survival (OS) due to a reduction in both nonrelapse mortality and relapse. Transcriptional and flow-cytometric analyses of recovering lymphocytes in patients with aGVHD identified preserved hallmarks of functional CD4+ regulatory T cells (Tregs) while highlighting a Tconv-driven inflammatory and metabolic axis distinct from that seen with conventional GVHD prophylaxis. Patients developing early relapse displayed a loss of inflammatory gene signatures in NK cells and a transcriptional exhaustion phenotype in CD8+ T cells. Using a multimodality approach, we highlight the utility of systems biology in BMT biomarker discovery and offer a novel understanding of how PTCy influences alloimmune responses. Our work charts future directions for novel therapeutic interventions after these increasingly used GVHD prophylaxis platforms. Specimens collected on NCT0079656226 and NCT0080927627 https://clinicaltrials.gov/.