Profiling of Natural Killer Interactions With Cancer Cells Using Mass Cytometry.

We developed a comprehensive method for functional assessment of the changes in immune populations and killing activity of peripheral blood mononuclear cells after cocultures with cancer cells using mass cytometry. In this study, a 43-marker mass cytometry panel was applied to a coculture of immune cells from healthy donors' peripheral blood mononuclear cells with diverse cancer cell lines. DNA content combined with classical CD45 surface staining was used as gating parameters for cocultures of immune cells (CD45high/DNAlow) with hematological (CD45low/DNAhigh) and solid cancer cell lines (CD45neg/DNAhigh). This strategy allows for universal discrimination of cancer cells from immune populations without the need for a specific cancer cell marker and simultaneous assessment of phenotypical changes in both populations. The use of mass cytometry allows for simultaneous detection of changes in natural killer, natural killer T cell, and T cell phenotypes and degranulation of immune populations upon target recognition, analysis of target cells for cytotoxic protein granzyme B content, and cancer cell death. These findings have broad applicability in research and clinical settings with the aim to phenotype and assess functional changes following not only NK-cancer cell interactions but also the effect of those interactions on other immune populations.

Simplified mass cytometry protocol for in-plate staining, barcoding, and cryopreservation of human PBMC samples in clinical trials.

With the increasing use of mass cytometry in clinical research, a simplified and standardized protocol for immunophenotyping human peripheral blood mononuclear cells (PBMCs) in clinical trials is needed. We present a simplified in-plate staining protocol for up to 80 samples, for laboratories of all mass cytometry expertise levels, aimed to generate reproducible datasets for large clinical cohorts. In this protocol, we provide details on the requirements to obtain meaningful results, spanning from sample quality, barcoding, and batch-freezing of stained samples.

Cytokine changes during immune-related adverse events and corticosteroid treatment in melanoma patients receiving immune checkpoint inhibitors.

BACKGROUND: 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. METHODS: 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. RESULTS: 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%). CONCLUSIONS: 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.

Standardized 11-color flow cytometry panel for the functional phenotyping of human T regulatory cells.

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 TGFß 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.