Amran Nur

Functional tumor-specific CD8+ T cells are essential for effective anti-tumor immune response and immune checkpoint inhibitor therapy. Here we show that, compared to other organ sites, primary, metastatic liver tumors in murine models contain a higher number of tumor-specific CD8+ T cells which are also dysfunctional. High-dimensional, multi-omic analysis of patient samples reveals a higher frequency of exhausted tumor-reactive CD8+ T cells and enriched interactions between these cells and SPP1+ macrophages in profibrotic, alpha-SMA rich regions specifically in the liver. Differential pseudotime trajectory inference analysis reveals that extrahepatic signaling promotes an intermediate cell (IC) population in the liver, characterized by co-expression of VISG4, CSF1R, CD163, TGF-βR, IL-6R, and SPP1. Analysis of premetastatic adenocarcinoma patient samples reveals enrichment of this population may predict liver metastasis. These findings suggest a mechanism by which extrahepatic tumors drive liver metastasis by promoting an IC population that inhibits tumor-reactive CD8+ T cell function.

Abstract 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.

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 β1) 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.

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 nut¬meg 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.