Treatment of recurrent pediatric myelodysplastic syndrome post hematopoietic stem cell transplantation.

Treatment of recurrent myelodysplastic syndrome (MDS) after hematopoietic cell transplantation (HCT) remains challenging. We present a 4-year-old girl experiencing early MDS relapse post-HCT treated with a multimodal strategy encompassing a second HCT and innovative targeted therapies. We underscore the potential of a comprehensive treatment approach in managing recurrent pediatric MDS.

Formate Supplementation Enhances Antitumor CD8+ T-cell Fitness and Efficacy of PD-1 Blockade.

The tumor microenvironment (TME) restricts antitumor CD8+ T-cell function and immunotherapy responses. Cancer cells compromise the metabolic fitness of CD8+ T cells within the TME, but the mechanisms are largely unknown. Here we demonstrate that one-carbon (1C) metabolism is enhanced in T cells in an antigen-specific manner. Therapeutic supplementation of 1C metabolism using formate enhances CD8+ T-cell fitness and antitumor efficacy of PD-1 blockade in B16-OVA tumors. Formate supplementation drives transcriptional alterations in CD8+ T-cell metabolism and increases gene signatures for cellular proliferation and activation. Combined formate and anti-PD-1 therapy increases tumor-infiltrating CD8+ T cells, which are essential for enhanced tumor control. Our data demonstrate that formate provides metabolic support to CD8+ T cells reinvigorated by anti-PD-1 to overcome a metabolic vulnerability in 1C metabolism in the TME to further improve T-cell function. SIGNIFICANCE: This study identifies that deficiencies in 1C metabolism limit the efficacy of PD-1 blockade in B16-OVA tumors. Supplementing 1C metabolism with formate during anti-PD-1 therapy enhances CD8+ T-cell fitness in the TME and CD8+ T-cell-mediated tumor clearance. These findings demonstrate that formate supplementation can enhance exhausted CD8+ T-cell function. See related commentary by Lin et al., p. 2507. This article is featured in Selected Articles from This Issue, p. 2489.

Severe congenital neutropenia due to jagunal homolog 1 (JAGN1) mutation: a case report and literature review.

Severe congenital neutropenia caused by jagunal homolog 1 (JAGN1) mutation is a rare condition resulting from maturation arrest secondary to endoplasmic reticulum stress response from impaired neutrophil protein glycosylation. Here, we report a case of a 4-year-old boy who presented with a history of recurrent infections and manifestations, including recurrent intracranial hemorrhage. A review of similar cases reported in the literature indicates that a bleeding diathesis has not been previously described in these patients. We hypothesize that this newly described association of bleeding complications in this patient with JAGN1 mutation is secondary to defective glycosylation in the normal functioning of platelets or clotting factors. Recurrent infections with intracranial hemorrhage, new focal neurologic defects, or altered mental status in a child should warrant a suspicion for this immunodeficiency for the prompt initiation of treatment and prophylaxis for life-threatening infections or trauma.

Mesenchymal and adrenergic cell lineage states in neuroblastoma possess distinct immunogenic phenotypes.

Apart from the anti-GD2 antibody, immunotherapy for neuroblastoma has had limited success due to immune evasion mechanisms, coupled with an incomplete understanding of predictors of response. Here, from bulk and single-cell transcriptomic analyses, we identify a subset of neuroblastomas enriched for transcripts associated with immune activation and inhibition and show that these are predominantly characterized by gene expression signatures of the mesenchymal lineage state. By contrast, tumors expressing adrenergic lineage signatures are less immunogenic. The inherent presence or induction of the mesenchymal state through transcriptional reprogramming or therapy resistance is accompanied by innate and adaptive immune gene activation through epigenetic remodeling. Mesenchymal lineage cells promote T cell infiltration by secreting inflammatory cytokines, are efficiently targeted by cytotoxic T and natural killer cells and respond to immune checkpoint blockade. Together, we demonstrate that distinct immunogenic phenotypes define the divergent lineage states of neuroblastoma and highlight the immunogenic potential of the mesenchymal lineage.

Tumor cells dictate anti-tumor immune responses by altering pyruvate utilization and succinate signaling in CD8+ T cells.

The tumor microenvironment (TME) is a unique metabolic niche that can inhibit T cell metabolism and cytotoxicity. To dissect the metabolic interplay between tumors and T cells, we establish an in vitro system that recapitulates the metabolic niche of the TME and allows us to define cell-specific metabolism. We identify tumor-derived lactate as an inhibitor of CD8+ T cell cytotoxicity, revealing an unexpected metabolic shunt in the TCA cycle. Metabolically fit cytotoxic T cells shunt succinate out of the TCA cycle to promote autocrine signaling via the succinate receptor (SUCNR1). Cytotoxic T cells are reliant on pyruvate carboxylase (PC) to replenish TCA cycle intermediates. By contrast, lactate reduces PC-mediated anaplerosis. The inhibition of pyruvate dehydrogenase (PDH) is sufficient to restore PC activity, succinate secretion, and the activation of SUCNR1. These studies identify PDH as a potential drug target to allow CD8+ T cells to retain cytotoxicity and overcome a lactate-enriched TME.

Low frequency of treatable pediatric disease alleles in gnomAD: An opportunity for future genomic screening of newborns.

Hematopoietic stem cell transplant (HSCT) can prevent progression of several genetic disorders. Although a subset of these disorders are identified on newborn screening panels, others are not identified until irreversible symptoms develop. Genetic testing is an efficient methodology to ascertain pre-symptomatic children, but the penetrance of risk-associated variants in the general population is not well understood. We developed a list of 127 genes associated with disorders treatable with HSCT. We identified likely pathogenic or pathogenic (LP/P) and loss-of-function (LoF) variants in these genes in the Genome Aggregation Database (gnomAD), a dataset containing exome and genome sequencing data from 141,456 healthy adults. Within gnomAD, we identified 59 individuals with a LP/P or LoF variant in 15 genes. Genes were associated with bone marrow failure syndromes, bleeding disorders, primary immunodeficiencies, osteopetrosis, metabolic disorders, and epidermolysis bullosa. In conclusion, few ostensibly healthy adults had genotypes associated with pediatric disorders treatable with HSCTs. Given that most of these disorders do not have biomarkers that could be cheaply and universally assessed on a standard newborn screen, our data suggest that genetic testing may be a complementary approach to traditional newborn screening methodology that has the potential to improve mortality and is not expected to lead to a high burden of false-positive results.

The aging lung: Physiology, disease, and immunity.

The population is aging at a rate never seen before in human history. As the number of elderly adults grows, it is imperative we expand our understanding of the underpinnings of aging biology. Human lungs are composed of a unique panoply of cell types that face ongoing chemical, mechanical, biological, immunological, and xenobiotic stress over a lifetime. Yet, we do not fully appreciate the mechanistic drivers of lung aging and why age increases the risk of parenchymal lung disease, fatal respiratory infection, and primary lung cancer. Here, we review the molecular and cellular aspects of lung aging, local stress response pathways, and how the aging process predisposes to the pathogenesis of pulmonary disease. We place these insights into context of the COVID-19 pandemic and discuss how innate and adaptive immunity within the lung is altered with age.

Calm in the midst of cytokine storm: a collaborative approach to the diagnosis and treatment of hemophagocytic lymphohistiocytosis and macrophage activation syndrome.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS) were historically thought to be distinct entities, often managed in isolation. In fact, these conditions are closely related. A collaborative approach, which incorporates expertise from subspecialties that previously treated HLH/MAS independently, is needed. We leveraged quality improvement (QI) techniques in the form of an Evidence-Based Guideline (EBG) to build consensus across disciplines on the diagnosis and treatment of HLH/MAS. METHODS: A multidisciplinary work group was convened that met monthly to develop the HLH/MAS EBG. Literature review and expert opinion were used to develop a management strategy for HLH/MAS. The EBG was implemented, and quality metrics were selected to monitor outcomes. RESULTS: An HLH/MAS clinical team was formed with representatives from subspecialties involved in the care of patients with HLH/MAS. Broad entry criteria for the HLH/MAS EBG were established and included fever and ferritin ≥500 ng/mL. The rheumatology team was identified as the "gate-keeper," charged with overseeing the diagnostic evaluation recommended in the EBG. First-line medications were recommended based on the acuity of illness and risk of concurrent infection. Quality metrics to be tracked prospectively based on time to initiation of treatment and clinical response were selected. CONCLUSION: HLH/MAS are increasingly considered to be a spectrum of related conditions, and joint management across subspecialties could improve patient outcomes. Our experience in creating a multidisciplinary approach to HLH/MAS management can serve as a model for care at other institutions.

Patients with CD3G mutations reveal a role for human CD3γ in Treg diversity and suppressive function.

Integrity of the T-cell receptor/CD3 complex is crucial for positive and negative selection of T cells in the thymus and for effector and regulatory functions of peripheral T lymphocytes. In humans, CD3D, CD3E, and CD3Z gene defects are a cause of severe immune deficiency and present early in life with increased susceptibility to infections. By contrast, CD3G mutations lead to milder phenotypes, mainly characterized by autoimmunity. However, the role of CD3γ in establishing and maintaining immune tolerance has not been elucidated. In this manuscript, we aimed to investigate abnormalities of T-cell repertoire and function in patients with genetic defects in CD3G associated with autoimmunity. High throughput sequencing was used to study composition and diversity of the T-cell receptor ß (TRB) repertoire in regulatory T cells (Tregs), conventional CD4+ (Tconv), and CD8+ T cells from 6 patients with CD3G mutations and healthy controls. Treg function was assessed by studying its ability to suppress proliferation of Tconv cells. Treg cells of patients with CD3G defects had reduced diversity, increased clonality, and reduced suppressive function. The TRB repertoire of Tconv cells from patients with CD3G deficiency was enriched for hydrophobic amino acids at positions 6 and 7 of the CDR3, a biomarker of self-reactivity. These data demonstrate that the T-cell repertoire of patients with CD3G mutations is characterized by a molecular signature that may contribute to the increased rate of autoimmunity associated with this condition.

Modeling altered T-cell development with induced pluripotent stem cells from patients with RAG1-dependent immune deficiencies.

Primary immunodeficiency diseases comprise a group of heterogeneous genetic defects that affect immune system development and/or function. Here we use in vitro differentiation of human induced pluripotent stem cells (iPSCs) generated from patients with different recombination-activating gene 1 (RAG1) mutations to assess T-cell development and T-cell receptor (TCR) V(D)J recombination. RAG1-mutants from severe combined immunodeficient (SCID) patient cells showed a failure to sustain progression beyond the CD3(--)CD4(-)CD8(-)CD7(+)CD5(+)CD38(-)CD31(-/lo)CD45RA(+) stage of T-cell development to reach the CD3(-/+)CD4(+)CD8(+)CD7(+)CD5(+)CD38(+)CD31(+)CD45RA(-) stage. Despite residual mutant RAG1 recombination activity from an Omenn syndrome (OS) patient, similar impaired T-cell differentiation was observed, due to increased single-strand DNA breaks that likely occur due to heterodimers consisting of both an N-terminal truncated and a catalytically dead RAG1. Furthermore, deep-sequencing analysis of TCR-ß (TRB) and TCR-α (TRA) rearrangements of CD3(-)CD4(+)CD8(-) immature single-positive and CD3(+)CD4(+)CD8(+) double-positive cells showed severe restriction of repertoire diversity with preferential usage of few Variable, Diversity, and Joining genes, and skewed length distribution of the TRB and TRA complementary determining region 3 sequences from SCID and OS iPSC-derived cells, whereas control iPSCs yielded T-cell progenitors with a broadly diversified repertoire. Finally, no TRA/δ excision circles (TRECs), a marker of TRA/δ locus rearrangements, were detected in SCID and OS-derived T-lineage cells, consistent with a pre-TCR block in T-cell development. This study compares human T-cell development of SCID vs OS patients, and elucidates important differences that help to explain the wide range of immunologic phenotypes that result from different mutations within the same gene of various patients.

Commensal microbes drive intestinal inflammation by IL-17-producing CD4+ T cells through ICOSL and OX40L costimulation in the absence of B7-1 and B7-2.

The costimulatory B7-1 (CD80)/B7-2 (CD86) molecules, along with T-cell receptor stimulation, together facilitate T-cell activation. This explains why in vivo B7 costimulation neutralization efficiently silences a variety of human autoimmune disorders. Paradoxically, however, B7 blockade also potently moderates accumulation of immune-suppressive regulatory T cells (Tregs) essential for protection against multiorgan systemic autoimmunity. Here we show that B7 deprivation in mice overrides the necessity for Tregs in averting systemic autoimmunity and inflammation in extraintestinal tissues, whereas peripherally induced Tregs retained in the absence of B7 selectively mitigate intestinal inflammation caused by Th17 effector CD4(+) T cells. The need for additional immune suppression in the intestine reflects commensal microbe-driven T-cell activation through the accessory costimulation molecules ICOSL and OX40L. Eradication of commensal enteric bacteria mitigates intestinal inflammation and IL-17 production triggered by Treg depletion in B7-deficient mice, whereas re-establishing intestinal colonization with Candida albicans primes expansion of Th17 cells with commensal specificity. Thus, neutralizing B7 costimulation uncovers an essential role for Tregs in selectively averting intestinal inflammation by Th17 CD4(+) T cells with commensal microbe specificity.

Cutting edge: committed Th1 CD4+ T cell differentiation blocks pregnancy-induced Foxp3 expression with antigen-specific fetal loss.

Pregnancy stimulates induced Foxp3 expression among maternal CD4(+) T cells with fetal specificity. Although sustained maternal regulatory CD4(+) T cell (Treg) expansion is essential for maintaining fetal tolerance during pregnancy, the necessity for Foxp3(+) cells with fetal specificity remains undefined. In this study, we demonstrate that mitigating Treg differentiation among maternal CD4(+) T cells with a single surrogate fetal specificity elicits Ag-specific fetal loss. Using recombinant Listeria monocytogenes to prime stably differentiated Th1 CD4(+) T cells with fetal I-A(b):2W1S55-68 specificity refractory to pregnancy-induced Foxp3 expression, we show that Ag delivery by cytoplasmic L. monocytogenes causes selective loss of 2W1S(+) offspring through CD4 cell- and IFN-γ-dependent pathways. In contrast, CD4(+) T cells primed by L. monocytogenes restricted from the cell cytoplasm are markedly more plastic for induced Foxp3 expression, with normal pregnancy outcomes. Thus, committed Th1 polarization blocks pregnancy induced Treg differentiation among maternal CD4(+) T cells with fetal specificity and triggers Ag-specific fetal loss.

Accelerating regulatory progress in multi-institutional research.

PURPOSE: Multi-institutional collaborations are necessary in order to create large and robust data sets that are needed to answer important comparative effectiveness research (CER) questions. Before scientific work can begin, a complex maze of administrative and regulatory requirements must be efficiently navigated to avoid project delays. INNOVATION: Staff from research, regulatory, and administrative teams involved in three HMO Research Network (HMORN) multi-institutional collaborations developed and employed novel approaches: to secure and maintain Institutional Review Board (IRB) approvals; to enable data sharing, and to expedite subawards for two data-only minimal risk studies. These novel approaches accelerated required processes and approvals while maintaining regulatory, human subjects, and institutional protections. CREDIBILITY: Outcomes from the processes described here are compared with processes outlined in the research and regulatory literature and with processes that have been used in previous multisite research collaborations. CONCLUSION AND DISCUSSION: Research, regulatory, and administrative staff are essential contributors to the success of multi-institutional collaborations. Their flexibility, creativity, and effective communication skills can lead to the development of efficient approaches to achieving the necessary oversight for these complex projects. Elements of these specific strategies can be adapted and used by other research networks. Other efforts in these areas should be evaluated and shared. The processes that help develop a "learning research system" play an important and complementary role in sustaining multi-institutional research collaborations.

Regulatory T cells and the immune pathogenesis of prenatal infection.

Pregnancy in placental mammals offers exceptional comprehensive benefits of in utero protection, nutrition, and metabolic waste elimination for the developing fetus. However, these benefits also require durable strategies to mitigate maternal rejection of fetal tissues expressing foreign paternal antigens. Since the initial postulate of expanded maternal immune tolerance by Sir Peter Medawar 60 years ago, an amazingly elaborate assortment of molecular and cellular modifications acting both locally at the maternal-placental interface and systemically have been shown to silence potentially detrimental maternal immune responses. In turn, simultaneously maintaining host defense against the infinite array of potential pathogens during pregnancy is equally important. Fortunately, resistance against most infections is preserved seamlessly throughout gestation. On the other hand, recent studies on pathogens with unique predisposition for prenatal infections have uncovered distinctive holes in host defense associated with the reproductive process. Using these infections to probe the response during pregnancy, the immune suppressive regulatory subset of maternal CD4 T cells has been increasingly shown to dictate the inter-workings between prenatal infection susceptibility and pathogenesis of ensuing pregnancy complications. Herein, the recent literature suggesting a necessity for maternal regulatory T cells (Tregs) in pregnancy-induced immunological shifts that sustain fetal tolerance is reviewed. Additional discussion is focused on how expansion of maternal Treg suppression may become exploited by pathogens that cause prenatal infections and the perilous potential of infection-induced immune activation that may mitigate fetal tolerance and inadvertently inject hostility into the protective in utero environment.

B7-1/B7-2 blockade overrides the activation of protective CD8 T cells stimulated in the absence of Foxp3+ regulatory T cells.

Although T cell activation has been classically described to require distinct, positive stimulation signals that include B7-1 (CD80) and B7-2 (CD86) costimulation, overriding suppression signals that avert immune-mediated host injury are equally important. How these opposing stimulation and suppression signals work together remains incompletely defined. Our recent studies demonstrate that CD8 Teff activation in response to cognate peptide stimulation is actively suppressed by the Foxp3(+) subset of CD4 cells, called Tregs. Here, we show that the elimination of Treg suppression does not bypass the requirement for positive B7-1/B7-2 costimulation. The expansion, IFN-γ cytokine production, cytolytic, and protective features of antigen-specific CD8 T cells stimulated with purified cognate peptide in Treg-ablated mice were each neutralized effectively by CTLA-4-Ig that blocks B7-1/B7-2. In turn, given the efficiency whereby CTLA-4-Ig overrides the effects of Treg ablation, the role of Foxp3(+) cell-intrinsic CTLA-4 in mitigating CD8 Teff activation was also investigated. With the use of mixed chimera mice that contain CTLA-4-deficient Tregs exclusively after the ablation of WT Foxp3(+) cells, a critical role for Treg CTLA-4 in suppressing the expansion, cytokine production, cytotoxicity, and protective features of peptide-stimulated CD8 T cells is revealed. Thus, the activation of protective CD8 T cells requires positive B7-1/B7-2 costimulation even when suppression by Tregs and in particular, Treg-intrinsic CTLA-4 is circumvented.