Therapeutic depletion of CCR8+ tumor-infiltrating regulatory T cells elicits antitumor immunity and synergizes with anti-PD-1 therapy.

BACKGROUND: Modulation and depletion strategies of regulatory T cells (Tregs) constitute valid approaches in antitumor immunotherapy but suffer from severe adverse effects due to their lack of selectivity for the tumor-infiltrating (ti-)Treg population, indicating the need for a ti-Treg specific biomarker. METHODS: We employed single-cell RNA-sequencing in a mouse model of non-small cell lung carcinoma (NSCLC) to obtain a comprehensive overview of the tumor-infiltrating T-cell compartment, with a focus on ti-Treg subpopulations. These findings were validated by flow cytometric analysis of both mouse (LLC-OVA, MC38 and B16-OVA) and human (NSCLC and melanoma) tumor samples. We generated two CCR8-specific nanobodies (Nbs) that recognize distinct epitopes on the CCR8 extracellular domain. These Nbs were formulated as tetravalent Nb-Fc fusion proteins for optimal CCR8 binding and blocking, containing either an antibody-dependent cell-mediated cytotoxicity (ADCC)-deficient or an ADCC-prone Fc region. The therapeutic use of these Nb-Fc fusion proteins was evaluated, either as monotherapy or as combination therapy with anti-programmed cell death protein-1 (anti-PD-1), in both the LLC-OVA and MC38 mouse models. RESULTS: We were able to discern two ti-Treg populations, one of which is characterized by the unique expression of Ccr8 in conjunction with Treg activation markers. Ccr8 is also expressed by dysfunctional CD4+ and CD8+ T cells, but the CCR8 protein was only prominent on the highly activated and strongly T-cell suppressive ti-Treg subpopulation of mouse and human tumors, with no major CCR8-positivity found on peripheral Tregs. CCR8 expression resulted from TCR-mediated Treg triggering in an NF-κB-dependent fashion, but was not essential for the recruitment, activation nor suppressive capacity of these cells. While treatment of tumor-bearing mice with a blocking ADCC-deficient Nb-Fc did not influence tumor growth, ADCC-prone Nb-Fc elicited antitumor immunity and reduced tumor growth in synergy with anti-PD-1 therapy. Importantly, ADCC-prone Nb-Fc specifically depleted ti-Tregs in a natural killer (NK) cell-dependent fashion without affecting peripheral Tregs. CONCLUSIONS: Collectively, our findings highlight the efficacy and safety of targeting CCR8 for the depletion of tumor-promoting ti-Tregs in combination with anti-PD-1 therapy

Immune stimulation recruits a subset of pro-regenerative macrophages to the retina that promotes axonal regrowth of injured neurons

Abstract The multifaceted nature of neuroinflammation is highlighted by its ability to both aggravate and promote neuronal health. While in mammals retinal ganglion cells (RGCs) are unable to regenerate following injury, acute inflammation can induce axonal regrowth. However, the nature of the cells, cellular states and signalling pathways that drive this inflammation-induced regeneration have remained elusive. Here, we investigated the functional significance of macrophages during RGC de- and regeneration, by characterizing the inflammatory cascade evoked by optic nerve crush (ONC) injury, with or without local inflammatory stimulation in the vitreous. By combining single-cell RNA sequencing and fate mapping approaches, we elucidated the response of retinal microglia and recruited monocyte-derived macrophages (MDMs) to RGC injury. Importantly, inflammatory stimulation recruited large numbers of MDMs to the retina, which exhibited long-term engraftment and promoted axonal regrowth. Ligand-receptor analysis highlighted a subset of recruited macrophages that exhibited expression of pro-regenerative secreted factors, which were able to promote axon regrowth via paracrine signalling. Our work reveals how inflammation may promote CNS regeneration by modulating innate immune responses, providing a rationale for macrophage-centred strategies for driving neuronal repair following injury and disease

Single-cell RNA and protein profiling of immune cells from the mouse brain and its border tissues

This protocol enables single-cell multi-omic analysis of the mouse brain immune compartment to explore immune cell heterogeneity and activation in the healthy and diseased brain, using a workflow based on HD flow cytometry, scRNA-seq and CITE-seq. Brain-immune cross-talk and neuroinflammation critically shape brain physiology in health and disease. A detailed understanding of the brain immune landscape is essential for developing new treatments for neurological disorders. Single-cell technologies offer an unbiased assessment of the heterogeneity, dynamics and functions of immune cells. Here we provide a protocol that outlines all the steps involved in performing single-cell multi-omic analysis of the brain immune compartment. This includes a step-by-step description on how to microdissect the border regions of the mouse brain, together with dissociation protocols tailored to each of these tissues. These combine a high yield with minimal dissociation-induced gene expression changes. Next, we outline the steps involved for high-dimensional flow cytometry and droplet-based single-cell RNA sequencing via the 10x Genomics platform, which can be combined with cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and offers a higher throughput than plate-based methods. Importantly, we detail how to implement CITE-seq with large antibody panels to obtain unbiased protein-expression screening coupled to transcriptome analysis. Finally, we describe the main steps involved in the analysis and interpretation of the data. This optimized workflow allows for a detailed assessment of immune cell heterogeneity and activation in the whole brain or specific border regions, at RNA and protein level. The wet lab workflow can be completed by properly trained researchers (with basic proficiency in cell and molecular biology) and takes between 6 and 11 h, depending on the chosen procedures. The computational analysis requires a background in bioinformatics and programming in R

Junctional adhesion molecule-A is dispensable for myeloid cell recruitment and diversification in the tumor microenvironment.

Junctional adhesion molecule-A (JAM-A), expressed on the surface of myeloid cells, is required for extravasation at sites of inflammation and may also modulate myeloid cell activation. Infiltration of myeloid cells is a common feature of tumors that drives disease progression, but the function of JAM-A in this phenomenon and its impact on tumor-infiltrating myeloid cells is little understood. Here we show that systemic cancer-associated inflammation in mice enhanced JAM-A expression selectively on circulating monocytes in an IL1β-dependent manner. Using myeloid-specific JAM-A-deficient mice, we found that JAM-A was dispensable for recruitment of monocytes and other myeloid cells to tumors, in contrast to its reported role in inflammation. Single-cell RNA sequencing revealed that loss of JAM-A did not influence the transcriptional reprogramming of myeloid cells in the tumor microenvironment. Overall, our results support the notion that cancer-associated inflammation can modulate the phenotype of circulating immune cells, and we demonstrate that tumors can bypass the requirement of JAM-A for myeloid cell recruitment and reprogramming