Modulation of cell signaling networks after CTLA4 blockade in patients with metastatic melanoma

Abstract

Background: The effects on cell signalling networks upon blockade of cytotoxic T lymphocyte-associated antigen-4 (CTLA4) using the monoclonal antibody tremelimumab were studied in peripheral blood mononuclear cell (PBMC) samples from patients with metastatic melanoma.

Methodology/principal: Findings Intracellular flow cytometry was used to detect phosphorylated (p) signaling molecules downstream of the T cell receptor (TCR) and cytokine receptors. PBMC from tremelimumab-treated patients were characterized by increase in pp38, pSTAT1 and pSTAT3, and decrease in pLck, pERK1/2 and pSTAT5 levels. These changes were noted in CD4 and CD8 T lymphocytes but also in CD14 monocytes. A divergent pattern of phosphorylation of Zap70, LAT, Akt and STAT6 was noted in patients with or without an objective tumor response.

Conclusions/significance: The administration of the CTLA4-blocking antibody tremelimumab to patients with metastatic melanoma influences signaling networks downstream of the TCR and cytokine receptors both in T cells and monocytes. The strong modulation of signaling networks in monocytes suggests that this cell subset may be involved in clinical responses to CTLA4 blockade.

Clinical trial registration: clinicaltrials.gov; Registration numbers NCT00090896 and NCT00471887.

Conflict of interest statement

Competing Interests: Robert Balderas is an employee of BD Biosciences, the company from which the authors purchased most of the antibodies and reagents for this work. Jesus Gomez-Navarro was an employee of Pfizer Inc, the maker of tremelimumab, at the time that this research was conducted. He now works at Millennium Pharmaceuticals. Antoni Ribas has received research funding and honoraria from Pfizer Inc, the maker of tremelimumab. However, these confilcts did not influence the conduct of the research described in this manuscript, nor the adherence to the PLoS ONE policies on sharing data and materials.

Figures

Figure 1. Outline of flow cytometry staining, cellular barcoding and phosphoflow analysis.

a) Fixation, permeabilization, fluorescent barcoding and surface and intracellular staining sequence for pre- and post-tremelimumab treatment samples. b) Gating pyramid. After gating by morphology, pre- and post-dosing samples were resolved based on the Pacific orange barcoding cell labeling. c) The cells from pre- (negative for Pacific orange) and post-treatment (positive for Pacific orange) time points were then separated as CD3+ (T lymphocytes) and CD14+ (monocytes). The CD3+ cells were gated on CD4+ to analyze T helper cells, and CD4− cells to analyze for CD8+ cytotoxic T lymphocytes (CTLs). d) Intracellular phosphoproteins were analyzed in each cell subset by mean fluorescence intensity (MFI).

Figure 2. Basal levels of TCR and cytokine signaling pathways in samples taken from patients in the NRA study of tremelimumab administered together with MART-126–35 peptide pulsed dendritic cells (DC).

The MFI of phosphoproteins was measured in CD3+CD4+ (T helper), CD3+CD4− (mainly representing CD8+ CTLs) and CD3−CD14+ (monocyte) cell subsets. PBMC were obtained from patients before (Pre) and after (Post) receiving the combined therapy. a) pLck (pY505), b) pZAP70 (pY292) and c) pLAT (pY171) representative of proximal T cell receptor (TCR) activation, which was only analyzed in CD3 gates including CD4 and CD8 cells. d) pAKT (pT308), e) p38 (pT180/pY182), f) pERK1/2 (T202/204), correspond to the main intracellular signaling pathways downstream of surface receptors, including the TCR. g) Cyclin D1 and h) bcl2 were measured as whole protein content for surrogate evidence for modulation of the cell cycle and apoptosis, respectively. i) Phospho-STAT1 (pY701), j) pSTAT3 (pY705), k) pSTAT5 (Y694) and l) pSTAT6 (Y641) were studied as a measure of STAT signaling downstream of cytokine receptors. In all cases, p values were calculated using a two-sided paired t-test, and significant results are denoted with a line comparing pre- and post-dosing samples with an asterisk to represent the significance level as follows: *p < 0.05; **p < 0.01; ***p < 0.001. Y-axis =  MFI - mean fluorescent intensity. Each dot represents the average of six replicates in most instances, and twenty-four replicates for pSTAT 1 and pSTAT5. The horizontal bar in each scatter column indicates medians. The vertical bar in each scatter column indicates standard error.

Figure 3. Modulation of CTLA4 and phosphoprotein expression in monocytes exposed ex vivo to tremelimumab.

Monocytes were cultured in increasing concentrations of tremelimumab for 48 hours, after which were analyzed for intracellular flow cytometry. Data is presented as fold change from the baseline mean fluorescence intensity (MFI) at increasing concentrations of tremelimumab (0, 0.1, 1. 10. 15 and 100 µg/ml) presented in a semilogarithmic plot. Red triangles represent monocytes samples obtained from three patients with metastatic melanoma (MD: melanoma donors); black circles are results from monocytes analyzed from five healthy subjects (HD: healthy donors). a) Intracellular CTLA4 expression; b) pp38(pT180/pY182); c) pErk1/2 (T202/204); d) pAkt (pT308); e) pSTAT1(pY701); f) pSTAT3 (pY705); g) pSTAT5 (Y694); h) pSTAT6 (Y641). **p

Figure 4. Basal levels of TCR and cytokine signaling pathway activation in samples taken from patients in the GA study administering single agent tremelimumab.

PBMC obtained from patients before (Pre) and after (Post) single agent tremelimumab were analyzed by phosphoflow as described in Figure 2 within each of the three cell subsets: CD4+, CD8+ (gated as CD3+CD4−) and CD14+ cells. a) pLck (pY505), b) pZAP70 (pY292), and c) pLAT (pY171) representative of proximal T Cell receptor (TCR) activation, only analyzed in CD3 subsets. d) pAKT (pT308), e) pp38 (pT180/pY182) and f) pERK1/2 (T202/204) as intracellular signaling pathways downstream of surface receptors. g) Phospho-STAT1 (pY701), h) pSTA3 (pY705), i) pSTAT5 (Y694), and j) pSTAT6 (Y641) as a measure of cytokine receptor signaling. Statistically significant results are denoted with asterisks as described in Figure 2.

Figure 5. Analysis of signaling patterns in samples from clinically responding and non-responding patients in the GA study administering single agent tremelimumab.

Phosphoflow results from patients before (Pre) and after (Post) single agent tremelimumab separated among patients with a clinical response (R) or no response (NR). Samples were analyzed by phosphoflow as described in Figure 2. a and b) pZAP70 (pY292) in CD4+ and CD8++ cell subsets. c, d and e) pAkt (pT308) in CD4++, CD8+ and CD14+ cell subsets. f, g, and h) pSTAT6 (Y641) in all three cell subsets. Statistically significant results are denoted with asterisks as described in Figure 2.