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Immune checkpoint inhibitors (ICIs) often cause immune-related adverse events (irAEs), most of which are treated with corticosteroids despite evidence suggesting that corticosteroids may blunt antitumor efficacy. We sought to identify cytokine changes that correlate with irAEs and study the impact of corticosteroid treatment on cytokine levels. We analyzed expression of 34 cytokines in 52 melanoma patients who developed irAEs during therapy with ICIs. Luminex serum assay was performed at baseline, 1, 2, and 3 months after starting ICI. Baseline cytokine levels and longitudinal log2 fold-change was compared with incidence and grade of irAEs. Cytokine patterns were compared between patients based on development of irAEs and steroid treatment. There were no differences in baseline cytokine levels between patients who developed grade 1–2 irAEs (N = 28) vs. grade 3–4 irAEs (N = 24). Dermatitis patients (N = 8) had significantly higher baseline Ang-1 (p = 0.006) and CD40L (p = 0.005). Pneumonitis patients (N = 4) had significantly higher baseline IL-17 (p = 0.009). Colitis patients (N = 8) had a trend toward decreased GCSF (p = 0.08). Through Spearman’s correlation analysis, patients who developed irAEs without receiving corticosteroids (N = 23) exhibited harmonization of cytokine fold-change, with 0/276 pairwise comparisons demonstrating significant divergence. In contrast, corticosteroid treatment in patients with irAEs (N = 15) altered fold-change to a discordant pattern (42/276 diverged, 15.2%). This discordant cytokine pattern in patients receiving corticosteroids is similar to the cytokine pattern in patients who did not develop irAEs (N = 8) during the longitudinal profiling period (41/276, 14.9%). Baseline levels of certain cytokines correlate with specific irAEs in melanoma patients receiving ICIs. irAEs drive a concordant pattern of cytokine fold-change, which is disrupted by corticosteroid treatment.

Phenotypic and functional assessment of Natural Killer (NK) cell activity against cancer cells in a clinical setting requires the integration of labor-intensive multicolor flow cytometry, chromium (Cr) release assays, and CD107a degranulation assays. To merge these into a single high-throughput assay, we developed a method for profiling NK-cancer cell interactions and simultaneous target killing using mass cytometry. In this study, a multiparameter 43-marker mass cytometry panel was applied to a co-culture of immune cells from healthy donors with diverse target cancer cell lines. DNA content, combined with classical CD45 surface staining, were used as gating parameters for co-cultures of immune cells (CD45high/DNAlow) with hematological (CD45low/DNAhigh) and solid cancer cell lines (CD45neg /DNAhigh), recapitulating flow cytometry forward and side scatter. This allows for discrimination of cancer cells from immune populations based on relative “size” and simultaneous assessment of phenotypical changes in both populations. In addition, the sensitivity of mass cytometry allows for the simultaneous detection of changes in NK cell phenotype and their degranulation upon target recognition, and for the analysis of target cells for cytotoxic protein granzyme B content, and cell death. These findings have broad applicability in clinical settings with the aim to phenotype and assess functional changes following NK-cancer cell interactions.

Background Angiogenesis is mediated by both the vascular endothelial growth factor (VEGF) family and the angiopoietin (Ang1-2)/Tie-2 pathway.1-3 We demonstrated that increased soluble VEGF and ang-2 is associated with decreased benefit to immune checkpoint inhibitors (ICIs).4 We then initiated a clinical trial combining pembrolizumab and ang-1/2 inhibitor (trebananib) (NCT03239145) with expansion cohorts in microsatellite stable (MSS) colorectal cancer (CRC), ovarian and renal cell cancer.5 We present the correlative analysis using high-dimensional single-cell mass cytometry (CyTOF) to characterize the effects of the combination therapy and examine differences between patients according to clinical benefit. Methods We used two separate CyTOF panels to monitor 48 markers of innate and adaptive immune populations in 26 evaluable patients who received the PR2D of trebananib (30mg/kg). Mass cytometry assay was performed on peripheral blood mononuclear cells of 26 patients at baseline (C1D1), 16 patients at cycle 3 day 1 (C3D1), and 4 patients at cycle 9 day 1 (C9D1). We compared immune cell markers between patients with clinical benefit (CB) and patients with no clinical benefit (NCB). Results Of 26 patients (16 CRC, 8 ovarian, 2 RCC), 11 patients had confirmed PR (3) or SD (8) resulting in CB of 42.3% while 15 patients had NCB. Independent of CB, there were statistically significant decreases from C1D1 to C3D1 in naive CD8+ T cells (p=0.03), CD4+ T central memory cells (p=0.05), and PD-1+/CD4+ and CD8+ T cells (p Conclusions Our findings suggest that the activity of anti-PD-1 and ang-2 peptibody (trebananib) combination is hindered by an increase in immunosuppressive myeloid cells leading to decrease in memory and effector T cell populations. The association between baseline activated NK cell and the expansion of cytolytic NK cells with favorable outcomes should be further explored. Trial Registration NCT03239145 Ethics Approval The study was approved by the Institutional Review Board (IRB) at Dana-Farber Cancer Institute for NCT03239145. Consent Written informed consent was obtained from the patient for participation in this study and publication of data. References Gerald D, Chintharlapalli S, Augustin HG, Benjamin LE. Angiopoietin-2: an attractive target for improved antiangiogenic tumor therapy. Cancer Research 2013;73:1649-57. Augustin HG, Koh GY, Thurston G, Alitalo K. Control of vascular morphogenesis and homeostasis through the angiopoietin-Tie system. Nature Reviews Molecular Cell Biology 2009;10:165-77. Rahma OE, Hodi FS. The Intersection between Tumor Angiogenesis and Immune Suppression. Clin Cancer Res 2019;15;25(18):5449-5457. Yuan J, Zhou J, Hodi FS, et al. Pretreatment serum VEGF is associated with clinical response and overall survival in advanced melanoma patients treated with ipilimumab. Cancer Immunol Res 2014 Feb;2(2):127-32. Rahma OE, Cleary J, Hodi FS, et al. Phase Ib study of pembrolizumab and trebananib (angiopoietin-2 inhibitor [Ang-2]): Preliminary analysis of the colorectal cancer (CRC) cohort. GI ASCO, San Francisco, 2019.

T regulatory cells (Tregs) are a cell subset that can suppress immune responses to maintain homeostasis and self-tolerance. In some scenarios, the immunosuppressive nature could be associated to other pathological developments such as autoimmune diseases and cancers. Due to the importance of Tregs in disease pathogenesis, we developed and validated an 11-color flow cytometry panel for phenotypic and functional detection of Treg markers using healthy human donor peripheral blood mononuclear cells (PBMCs). Our panel contains 4 Treg surface proteins and 2 functional cytokines as well as T-lymphocyte lineage markers CD3, CD4, and CD8. Our data shows an increase in expression of markers CD25, FoxP3, CTLA4, GITR and intracellular cytokines IL4 and TGFβ when comparing unstimulated samples to CD3/CD28 bead stimulated samples. This 11-color panel can be used to functionally evaluate immunosuppressive Tregs in human PBMC samples.