CXCL9 and CXCL10 can be produced by antigen-presenting cells (dendritic cells or macrophages) and by tumor cells. Hoch et al. demonstrated that CXCL9 and CXCL10 co-localize with LAG3 ⁺ T cells expressing CCL4 or CXCL13 and contribute to the generation of a “hot” tumor microenvironment.

A T cell-inflamed tumor microenvironment is characterized by the accumulation and local activation of CD8⁺ T cells and Bat3-lineage dendritic cells, which together are associated with clinical response to anti-programmed cell death protein 1 (anti-PD-1)-based immunotherapy. Preclinical models have demonstrated a crucial role for the chemokine CXCL10 in the recruitment of effector CD8⁺ T cells into the tumor site, and a chemokine gene signature is also seen in T cell-inflamed tumors from patients. However, the cellular source of CXCL10 in human solid tumors is not known. To identify the cellular source of CXCL10 we analyzed 22 pretreatment biopsy samples of melanoma metastases from patients who subsequently underwent checkpoint blockade immunotherapy. We stained for CD45⁺ and Sox10⁺ cells with multiparameter immunofluorescence staining, and RNA in situ hybridization technology was used in concert to identify CXCL10 transcripts. The results were correlated with the expression levels of CXCL10 transcripts from bulk RNA sequencing and the best overall response to immune checkpoint inhibition (anti-PD-1 alone or with anti-CTLA-4) in the same patients. We identified CD45⁺ cells as the major cellular source for CXCL10 in human melanoma metastases, with additional CXCL10 production seen by Sox10⁺ cells. Up to 90% of CD45⁺ cells and up to 69% of Sox10⁺ cells produced CXCL10 transcripts. The CXCL10 staining result was consistent with the level of CXCL10 expression determined by bulk RNA sequencing. The percentages of CD45⁺ CXCL10⁺ cells and Sox10⁺ CXCL10⁺ cells independently predicted response (p<0.001). The average number of transcripts per cell correlated with the CD45⁺ cell infiltrate (R=0.37). Immune cells and melanoma cells produce CXCL10 in human melanoma metastases. Intratumoral CXCL10 is a positive prognostic factor for response to immunotherapy, and the RNAscope technique is achievable using paraffin tissue. Strategies that support effector T cell recruitment via induction of CXCL10 should be considered as a mechanism-based intervention to expand immunotherapy efficacy.

Immune checkpoint blockade is therapeutically successful for many patients across multiple cancer types. However, immune-related adverse events (irAE) frequently occur and can sometimes be life threatening. It is critical to understand the immunologic mechanisms of irAEs with the goal of finding novel treatment targets. Herein, we report our analysis of tissues from patients with irAE dermatitis using multiparameter immunofluorescence (IF), spatial transcriptomics, and RNA in situ hybridization (RISH). Skin psoriasis cases were studied as a comparison, as a known Th17-driven disease, and colitis was investigated as a comparison. IF analysis revealed that CD4+ and CD8+ tissue-resident memory T (TRM) cells were preferentially expanded in the inflamed portion of skin in cutaneous irAEs compared with healthy skin controls. Spatial transcriptomics allowed us to focus on areas containing TRM cells to discern functional phenotype and revealed expression of Th1-associated genes in irAEs, compared with Th17-asociated genes in psoriasis. Expression of PD-1, CTLA-4, LAG-3, and other inhibitory receptors was observed in irAE cases. RISH technology combined with IF confirmed expression of IFNγ, CXCL9, CXCL10, and TNFα in irAE dermatitis, as well as IFNγ within TRM cells specifically. The Th1-skewed phenotype was confirmed in irAE colitis cases compared with healthy colon.

To identify potential early biomarkers of treatment response and immune-related adverse events (irAE), a pilot immune monitoring study was performed in stage IV melanoma patients by flow cytometric analysis of peripheral blood mononuclear cells (PBMC). Overall, 17 patients were treated with either nivolumab or pembrolizumab alone, or with a combination of nivolumab and ipilimumab every three weeks. Of 15 patients for which complete response assessment was available, treatment responders (n = 10) as compared to non-responders (n = 5) were characterized by enhanced PD-1 expression on CD8⁺ T cells immediately before treatment (median ± median absolute deviation/MAD 26.7 ± 10.4% vs. 17.2 ± 5.3%). Responders showed a higher T cell responsiveness after T cell receptor ex vivo stimulation as determined by measurement of programmed cell death 1 (PD-1) expression on CD3⁺ T cells before the second cycle of treatment. The percentage of CD8⁺ effector memory (CD8⁺CD45RA⁻CD45RO⁺CCR7⁻) T cells was higher in responders compared to non-responders before and immediately after the first cycle of treatment (median ± MAD 39.2 ± 7.3% vs. 30.5 ± 4.1% and 37.7 ± 4.6 vs. 24.0 ± 6.4). Immune-related adverse events (irAE) were accompanied by a higher percentage of activated CD4⁺ (CD4⁺CD38⁺HLADR⁺) T cells before the second treatment cycle (median ± MAD 14.9 ± 3.9% vs. 5.3 ± 0.4%). In summary, PBMC immune monitoring of immune-checkpoint inhibition (ICI) treatment in melanoma appears to be a promising approach to identify early markers of treatment response and irAEs.

Chemokines and cytokines represent an emerging field of immunotherapy research. They are responsible for the crosstalk and chemoattraction of immune cells and tumor cells. For instance, CXCL9/10/11 chemoattract effector CD8⁺ T cells to the tumor microenvironment, making an argument for their promising role as biomarkers for a favorable outcome. The cytokine Interleukin-15 (IL-15) can promote the chemokine expression of CXCR3 ligands but also XCL1, contributing to an important DC-T cell interaction. Recruited cytotoxic T cells can be clonally expanded by IL-2. Delivering or inducing these chemokines and cytokines can result in tumor shrinkage and might synergize with immune checkpoint inhibition. In addition, blocking specific chemokine and cytokine receptors such as CCR2, CCR4 or Il-6R can reduce the recruitment of tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs) or regulatory T cells (Tregs). Efforts to target these chemokines and cytokines have the potential to personalize cancer immunotherapy further and address patients that are not yet responsive because of immune cell exclusion. Targeting cytokines such as IL-6 and IL-15 is currently being evaluated in clinical trials in combination with immune checkpoint-blocking antibodies for the treatment of metastatic melanoma. The improved overall survival of melanoma patients might outweigh potential risks such as autoimmunity. However, off-target toxicity needs to be elucidated.

Immune checkpoint inhibitors (ICIs) have transformed cancer treatment but are frequently associated with immune-related adverse events (irAEs). This article offers a novel synthesis of findings from both preclinical and clinical studies, focusing on the molecular mechanisms driving irAEs across diverse organ systems. It examines key immune cells, such as T cell subsets and myeloid cells, which are instrumental in irAE pathogenesis, alongside an in-depth analysis of cytokine signaling [interleukin (IL)-6, IL-17, IL-4), interferon γ (IFN-γ), IL-1β, tumor necrosis factor α (TNF-α)], integrin-mediated interactions [integrin subunits αITGA)4 and ITGB7], and microbiome-related factors that contribute to irAE pathology. This exploration of modifiable pathways uncovers new opportunities to mitigate irAEs by using available antibodies (Abs) that target key inflammatory molecules across tumor types, while ideally preserving the antitumor efficacy of ICIs.