Tumor-associated macrophages trigger MAIT cell dysfunction at the HCC invasive margin.

Mucosal-associated invariant T (MAIT) cells represent an abundant innate-like T cell subtype in the human liver. MAIT cells are assigned crucial roles in regulating immunity and inflammation, yet their role in liver cancer remains elusive. Here, we present a MAIT cell-centered profiling of hepatocellular carcinoma (HCC) using scRNA-seq, flow cytometry, and co-detection by indexing (CODEX) imaging of paired patient samples. These analyses highlight the heterogeneity and dysfunctionality of MAIT cells in HCC and their defective capacity to infiltrate liver tumors. Machine-learning tools were used to dissect the spatial cellular interaction network within the MAIT cell neighborhood. Co-localization in the adjacent liver and interaction between niche-occupying CSF1R+PD-L1+ tumor-associated macrophages (TAMs) and MAIT cells was identified as a key regulatory element of MAIT cell dysfunction. Perturbation of this cell-cell interaction in ex vivo co-culture studies using patient samples and murine models reinvigorated MAIT cell cytotoxicity. These studies suggest that aPD-1/aPD-L1 therapies target MAIT cells in HCC patients.

Immunosuppressive CD29+ Treg accumulation in the liver in mice on checkpoint inhibitor therapy.

OBJECTIVE: Liver metastases are often resistant to immune checkpoint inhibitor therapy (ICI) and portend a worse prognosis compared with metastases to other locations. Regulatory T cells (Tregs) are one of several immunosuppressive cells implicated in ICI resistance of liver tumours, but the role played by Tregs residing within the liver surrounding a tumour is unknown. DESIGN: Flow cytometry and single-cell RNA sequencing were used to characterise hepatic Tregs before and after ICI therapy. RESULTS: We found that the murine liver houses a Treg population that, unlike those found in other organs, is both highly proliferative and apoptotic at baseline. On administration of αPD-1, αPD-L1 or αCTLA4, the liver Treg population doubled regardless of the presence of an intrahepatic tumour. Remarkably, this change was not due to the preferential expansion of the subpopulation of Tregs that express PD-1. Instead, a subpopulation of CD29+ (Itgb1, integrin ß1) Tregs, that were highly proliferative at baseline, doubled its size in response to αPD-1. Partial and full depletion of Tregs identified CD29+ Tregs as the prominent niche-filling subpopulation in the liver, and CD29+ Tregs demonstrated enhanced suppression in vitro when derived from the liver but not the spleen. We identified IL2 as a critical modulator of both CD29+ and CD29- hepatic Tregs, but expansion of the liver Treg population with αPD-1 driven by CD29+ Tregs was in part IL2-independent. CONCLUSION: We propose that CD29+ Tregs constitute a unique subpopulation of hepatic Tregs that are primed to respond to ICI agents and mediate resistance.

A Paradoxical Tumor Antigen Specific Response in the Liver.

Functional tumor-specific CD8+ T cells are essential for an effective anti-tumor immune response and the efficacy of immune checkpoint inhibitor therapy. In comparison to other organ sites, we found higher numbers of tumor-specific CD8+ T cells in primary, metastatic liver tumors in murine tumor models. Despite their abundance, CD8+ T cells in the liver displayed an exhausted phenotype. Depletion of CD8+ T cells showed that liver tumor-reactive CD8+ T failed to control liver tumors but was effective against subcutaneous tumors. Similarly, analysis of single-cell RNA sequencing data from patients showed a higher frequency of exhausted tumor-reactive CD8+ T cells in liver metastasis compared to paired primary colon cancer. High-dimensional, multi-omic analysis combining proteomic CODEX and scRNA-seq data revealed enriched interaction of SPP1+ macrophages and CD8+ tumor-reactive T cells in profibrotic, alpha-SMA rich regions in the liver. Liver tumors grew less in Spp1-/- mice and the tumor-specific CD8+ T cells were less exhausted. Differential pseudotime trajectory inference analysis revealed extrahepatic signaling promoting an intermediate cell (IC) population in the liver, characterized by co-expression of VISG4, CSF1R, CD163, TGF-ßR, IL-6R, SPP1. scRNA-seq of a third data set of premetastatic adenocarcinoma showed that enrichment of this population may predict liver metastasis. Our data suggests a mechanism by which extrahepatic tumors facilitate the formation of liver metastasis by promoting an IC population inhibiting tumor-reactive CD8+ T cell function.

The Gut Microbiome Controls Liver Tumors via the Vagus Nerve.

Liver cancer ranks amongst the deadliest cancers. Nerves have emerged as an understudied regulator of tumor progression. The parasympathetic vagus nerve influences systemic immunity via acetylcholine (ACh). Whether cholinergic neuroimmune interactions influence hepatocellular carcinoma (HCC) remains uncertain. Liver denervation via hepatic vagotomy (HV) significantly reduced liver tumor burden, while pharmacological enhancement of parasympathetic tone promoted tumor growth. Cholinergic disruption in Rag1KO mice revealed that cholinergic regulation requires adaptive immunity. Further scRNA-seq and in vitro studies indicated that vagal ACh dampens CD8+ T cell activity via muscarinic ACh receptor (AChR) CHRM3. Depletion of CD8+ T cells abrogated HV outcomes and selective deletion of Chrm3 on CD8 + T cells inhibited liver tumor growth. Beyond tumor-specific outcomes, vagotomy improved cancer-associated fatigue and anxiety-like behavior. As microbiota transplantation from HCC donors was sufficient to impair behavior, we investigated putative microbiota-neuroimmune crosstalk. Tumor, rather than vagotomy, robustly altered fecal bacterial composition, increasing Desulfovibrionales and Clostridial taxa. Strikingly, in tumor-free mice, vagotomy permitted HCC-associated microbiota to activate hepatic CD8+ T cells. These findings reveal that gut bacteria influence behavior and liver anti-tumor immunity via a dynamic and pharmaceutically targetable, vagus-liver axis.

Survivability of Lactobacillus plantarum in nutmeg (Myristica fragrans Houtt) flesh extract and its effect on the performance of broiler chicken.

Objective: This study aimed to see if increasing the concentration of nutmeg flesh extract in vitro could increase the growth of Lactobacillus plantarum bacteria and if it had any effect on broiler chicken performance. Materials and Methods: Different concentrations of nutmeg flesh extract (5, 10, 15, and 20/100 ml distilled water) were combined with 10 ml L. plantarum (bacterial concentration 1 × 109 cfu/ml) to produce synbiotics. A total of 250 unsexed Lohmann broiler chickens were reared together from 0 to 7 days of age in the in vivo study. Beginning on day 8, synbiotics nutmeg flesh extract and L. plantarum were added to the ration in amounts of 0.5, 1, 1.5, and 2 ml/kg for T1, T2, T3, and T4, respectively, while no synbiotics were added to the control diet (T0). Results: The levels of nutmeg flesh extract had a significant (p < 0.05) effect on L. plantarum growth. In the survival test against gastric acid, bile salts, and temperature, the addition of nutmeg flesh extract (20/100 ml distilled water) significantly (p < 0.05) maintained the population of L. plantarum. In vivo studies showed that the T1,T2,T3, and T4 groups gained more body weight (p < 0.05) than the T0 group during the rearing period but had no effect (p > 0.05) on the internal organ weight and carcass of broiler chickens. Conclusions: Nutmeg flesh extract could stimulate the growth of L. plantarum bacteria, and using it as a synbiotic could improve broiler chicken performance.

Centering Community Voices: Establishment of a Community Advisory Board at a Student-Run Free Clinic.

Student-run free clinics (SRFCs) are common throughout the U.S. and have potential to meet the needs of both health professions trainees and patients in underserved communities. Here, we describe our SRFC's initial process for recruiting, implementing, and evaluating a Community Advisory Board to better align clinic offerings with community needs.

Combined influence of inspiratory loading and locomotor subsystolic cuff inflation on cardiovascular responses during submaximal exercise.

It is unknown if simultaneous stimulation of the respiratory and locomotor muscle afferents via inspiratory loading (IL) and locomotor subsystolic cuff inflation (CUFF) influences the cardiovascular responses during exercise. We hypothesized that combined IL and CUFF (IL + CUFF) will result in greater increases in blood pressure (MAP) and systemic vascular resistance (SVR) than IL and CUFF alone during exercise. Eight adults (6 males/2 females) were enrolled and performed four 10-min bouts of constant-load cycling eliciting 40% maximal oxygen uptake on a single day. For each exercise bout, the first 5 min consisted of spontaneous breathing. The second 5 min consisted of voluntary hyperventilation (i.e., breathing frequency of 40 breaths/min) with IL (30% maximum inspiratory pressure), CUFF (80 mmHg), IL + CUFF, or no intervention (CTL) in randomized order. During exercise, cardiac output and MAP were determined via open-circuit acetylene wash-in and manual sphygmomanometry, respectively, and SVR was calculated. Across CTL, IL, CUFF, and IL + CUFF, MAP was greater with each condition (CTL: 97 ± 14; IL: 106 ± 13; CUFF: 114 ± 14; IL + CUFF: 119 ± 15 mmHg, all P < 0.02). Furthermore, SVR was greater with IL + CUFF compared with IL, CUFF, and CTL (CTL: 6.6 ± 1.1; IL: 7.5 ± 1.4; CUFF: 7.5 ± 1.3; IL + CUFF: 8.2 ± 1.4 mmHg·L-1·min-1, all P < 0.02). Cardiac output was not different across conditions (CTL: 15.2 ± 3.8; IL: 14.8 ± 3.7; CUFF: 15.6 ± 3.5; IL + CUFF: 14.7 ± 4.3 L/min, all P > 0.05). These data demonstrate that simultaneous stimulation of respiratory and locomotor muscle afferent feedback results in additive MAP and SVR responses than IL and CUFF alone during submaximal exercise. These findings have important clinical implications for populations with exaggerated locomotor and respiratory muscle reflex feedbacks.NEW & NOTEWORTHY Reflexes arising from the respiratory and locomotor muscles influence cardiovascular regulation during exercise. However, it is unclear how the respiratory and locomotor muscle reflexes interact when simultaneously stimulated. Herein, we demonstrate that stimulation of the respiratory and locomotor muscle reflexes yielded additive cardiovascular responses during submaximal exercise.