Utility of Indocyanine Green for Sentinel Lymph Node Biopsy in Pediatric Sarcoma and Melanoma.

BACKGROUND: Indocyanine green (ICG) is a fluorescent dye with increasing use for adult sentinel lymph node biopsy (SLNB). The utility of ICG in pediatric oncology remains understudied. We aim to describe our experience using ICG for SLNB in pediatrics versus standard blue dye. METHODS: A retrospective review of pediatric patients with melanoma or sarcoma who underwent SLNB with technetium plus ICG or blue dye from 2014 to 2023 at a large academic children's hospital was conducted. RESULTS: Twenty-four patients were included; 58.3% were male with median age 13 years (range 4-21 years). The majority had a melanocytic tumor (91.7%) and 8.3% had sarcoma. All patients received technetium with concomitant blue dye (62.5%) or ICG (37.5%). ICG more reliably identified radioactive SLNs, compared to blue dye (mean 100% vs 78.3 ± 8.3%, p = 0.03). There was no significant difference in median operative time (ICG 82 min [68-203] vs blue dye 93 min [78-105], p = 0.84). Seven patients had positive SLNs (29.2%), with recurrence in 2 patients (8.3%) and 1 death (4.2%). There were no adverse events. CONCLUSION: ICG-directed SLNB in children is a safe and effective alternative to blue dye. Use of ICG did not add to operative time, and more often identified sentinel nodes versus blue dye. TYPE OF STUDY: Original Research Article, Retrospective Comparative Study. LEVEL OF EVIDENCE: III.

Neurotrophic factor Neuritin modulates T cell electrical and metabolic state for the balance of tolerance and immunity.

The adaptive T cell response is accompanied by continuous rewiring of the T cell's electric and metabolic state. Ion channels and nutrient transporters integrate bioelectric and biochemical signals from the environment, setting cellular electric and metabolic states. Divergent electric and metabolic states contribute to T cell immunity or tolerance. Here, we report that neuritin (Nrn1) contributes to tolerance development by modulating regulatory and effector T cell function. Nrn1 expression in regulatory T cells promotes its expansion and suppression function, while expression in the T effector cell dampens its inflammatory response. Nrn1 deficiency causes dysregulation of ion channel and nutrient transporter expression in Treg and effector T cells, resulting in divergent metabolic outcomes and impacting autoimmune disease progression and recovery. These findings identify a novel immune function of the neurotrophic factor Nrn1 in regulating the T cell metabolic state in a cell context-dependent manner and modulating the outcome of an immune response.

Unraveling the Therapeutic Benefit of Sequenced Chemo-immunotherapy.

Combination immune-checkpoint inhibition with chemotherapy is a clinical standard, yet concurrent administration may limit the full benefit of immunotherapy by blunting the proliferation and differentiation of CD8 T cells. Identifying patients in whom sequential chemo-immunotherapy or immunotherapy alone is feasible should be a priority to optimize long-term outcomes. See related article by Mariniello et al., p. 1833.

Hypoxia drives CD39-dependent suppressor function in exhausted T cells to limit antitumor immunity.

CD8+ T cells are critical for elimination of cancer cells. Factors within the tumor microenvironment (TME) can drive these cells to a hypofunctional state known as exhaustion. The most terminally exhausted T (tTex) cells are resistant to checkpoint blockade immunotherapy and might instead limit immunotherapeutic efficacy. Here we show that intratumoral CD8+ tTex cells possess transcriptional features of CD4+Foxp3+ regulatory T cells and are similarly capable of directly suppressing T cell proliferation ex vivo. tTex cell suppression requires CD39, which generates immunosuppressive adenosine. Restricted deletion of CD39 in endogenous CD8+ T cells resulted in slowed tumor progression, improved immunotherapy responsiveness and enhanced infiltration of transferred tumor-specific T cells. CD39 is induced on tTex cells by tumor hypoxia, thus mitigation of hypoxia limits tTex suppression. Together, these data suggest tTex cells are an important regulatory population in cancer and strategies to limit their generation, reprogram their immunosuppressive state or remove them from the TME might potentiate immunotherapy.

Tumor microenvironmental signals reshape chromatin landscapes to limit the functional potential of exhausted T cells.

Response rates to immunotherapy in solid tumors remain low due in part to the elevated prevalence of terminally exhausted T cells, a hypofunctional differentiation state induced through persistent antigen and stress signaling. However, the mechanisms promoting progression to terminal exhaustion in the tumor remain undefined. Using the low-input chromatin immunoprecipitation sequencing method CUT&RUN, we profiled the histone modification landscape of tumor-infiltrating CD8+ T cells throughout differentiation. We found that terminally exhausted T cells had unexpected chromatin features that limit their transcriptional potential. Terminally exhausted T cells had a substantial fraction of active chromatin, including active enhancers enriched for bZIP/AP-1 transcription factor motifs that lacked correlated gene expression, which was restored by immunotherapeutic costimulatory signaling. Reduced transcriptional potential was also driven by an increase in histone bivalency, which we linked directly to hypoxia exposure. Enforced expression of the hypoxia-insensitive histone demethylase Kdm6b was sufficient to overcome hypoxia, increase function, and promote antitumor immunity. Our study reveals the specific epigenetic changes mediated by histone modifications during T cell differentiation that support exhaustion in cancer, highlighting that their altered function is driven by improper costimulatory signals and environmental factors. These data suggest that even terminally exhausted T cells may remain competent for transcription in settings of increased costimulatory signaling and reduced hypoxia.

Metabolic support of tumour-infiltrating regulatory T cells by lactic acid.

Regulatory T (Treg) cells, although vital for immune homeostasis, also represent a major barrier to anti-cancer immunity, as the tumour microenvironment (TME) promotes the recruitment, differentiation and activity of these cells1,2. Tumour cells show deregulated metabolism, leading to a metabolite-depleted, hypoxic and acidic TME3, which places infiltrating effector T cells in competition with the tumour for metabolites and impairs their function4-6. At the same time, Treg cells maintain a strong suppression of effector T cells within the TME7,8. As previous studies suggested that Treg cells possess a distinct metabolic profile from effector T cells9-11, we hypothesized that the altered metabolic landscape of the TME and increased activity of intratumoral Treg cells are linked. Here we show that Treg cells display broad heterogeneity in their metabolism of glucose within normal and transformed tissues, and can engage an alternative metabolic pathway to maintain suppressive function and proliferation. Glucose uptake correlates with poorer suppressive function and long-term instability, and high-glucose conditions impair the function and stability of Treg cells in vitro. Treg cells instead upregulate pathways involved in the metabolism of the glycolytic by-product lactic acid. Treg cells withstand high-lactate conditions, and treatment with lactate prevents the destabilizing effects of high-glucose conditions, generating intermediates necessary for proliferation. Deletion of MCT1-a lactate transporter-in Treg cells reveals that lactate uptake is dispensable for the function of peripheral Treg cells but required intratumorally, resulting in slowed tumour growth and an increased response to immunotherapy. Thus, Treg cells are metabolically flexible: they can use 'alternative' metabolites in the TME to maintain their suppressive identity. Further, our results suggest that tumours avoid destruction by not only depriving effector T cells of nutrients, but also metabolically supporting regulatory populations.

Mitochondrial stress induced by continuous stimulation under hypoxia rapidly drives T cell exhaustion.

Cancer and chronic infections induce T cell exhaustion, a hypofunctional fate carrying distinct epigenetic, transcriptomic and metabolic characteristics. However, drivers of exhaustion remain poorly understood. As intratumoral exhausted T cells experience severe hypoxia, we hypothesized that metabolic stress alters their responses to other signals, specifically, persistent antigenic stimulation. In vitro, although CD8+ T cells experiencing continuous stimulation or hypoxia alone differentiated into functional effectors, the combination rapidly drove T cell dysfunction consistent with exhaustion. Continuous stimulation promoted Blimp-1-mediated repression of PGC-1α-dependent mitochondrial reprogramming, rendering cells poorly responsive to hypoxia. Loss of mitochondrial function generated intolerable levels of reactive oxygen species (ROS), sufficient to promote exhausted-like states, in part through phosphatase inhibition and the consequent activity of nuclear factor of activated T cells. Reducing T cell-intrinsic ROS and lowering tumor hypoxia limited T cell exhaustion, synergizing with immunotherapy. Thus, immunologic and metabolic signaling are intrinsically linked: through mitigation of metabolic stress, T cell differentiation can be altered to promote more functional cellular fates.

Metabolic Regulation of T Cell Immunity.

It is becoming increasingly clear that cellular metabolism plays a critical role in the propagation of appropriate, effective, and pathologic immune responses. In this chapter, we detail the metabolic pathways involved in T cell activation and differentiation, highlighting specific factors responsible for directing the processes that lead to metabolic programming at important stages in the dynamic life cycle of this immune cell lineage. Additionally, this chapter will discuss how key metabolites are acquired, touching on the factors and conditions regulating the expression of crucial transporter molecules in response to activation and pathological states.

MicroRNA-17 Modulates Regulatory T Cell Function by Targeting Co-regulators of the Foxp3 Transcription Factor.

Regulatory T (Treg) cells are important in maintaining self-tolerance and immune homeostasis. The Treg cell transcription factor Foxp3 works in concert with other co-regulatory molecules, including Eos, to determine the transcriptional signature and characteristic suppressive phenotype of Treg cells. Here, we report that the inflammatory cytokine interleukin-6 (IL-6) actively repressed Eos expression through microRNA-17 (miR-17). miR-17 expression increased in Treg cells in the presence of IL-6, and its expression negatively correlated with that of Eos. Treg cell suppressive activity was diminished upon overexpression of miR-17 in vitro and in vivo, which was mitigated upon co-expression of an Eos mutant lacking miR-17 target sites. Also, RNAi of miR-17 resulted in enhanced suppressive activity. Ectopic expression of miR-17 imparted effector-T-cell-like characteristics to Treg cells via the de-repression of genes encoding effector cytokines. Thus, miR-17 provides a potent layer of Treg cell control through targeting Eos and additional Foxp3 co-regulators.