Baseline T cell dysfunction by single cell network profiling in metastatic breast cancer patients

Abstract

Background: We previously reported the results of a multicentric prospective randomized trial of chemo-refractory metastatic breast cancer patients testing the efficacy of two doses of TGFβ blockade during radiotherapy. Despite a lack of objective responses to the combination, patients who received a higher dose of TGFβ blocking antibody fresolimumab had a better overall survival when compared to those assigned to lower dose (hazard ratio of 2.73, p = 0.039). They also demonstrated an improved peripheral blood mononuclear cell (PBMC) counts and increase in the CD8 central memory pool. We performed additional analysis on residual PBMC using single cell network profiling (SCNP).

Methods: The original trial randomized metastatic breast cancer patients to either 1 or 10 mg/kg of fresolimumab, every 3 weeks for 5 cycles, combined with radiotherapy to a metastatic site at week 1 and 7 (22.5 Gy given in 3 doses of 7.5 Gy). Trial immune monitoring results were previously reported. In 15 patients with available residual blood samples, additional functional studies were performed, and compared with data obtained in parallel from seven healthy female donors (HD): SCNP was applied to analyze T cell receptor (TCR) modulated signaling via CD3 and CD28 crosslinking and measurement of evoked phosphorylation of AKT and ERK in CD4 and CD8 T cell subsets defined by PD-1 expression.

Results: At baseline, a significantly higher level of expression (p < 0.05) of PD-L1 was identified in patient monocytes compared to HD. TCR modulation revealed dysfunction of circulating T-cells in patient baseline samples as compared to HD, and this was more pronounced in PD-1+ cells. Treatment with radiotherapy and fresolimumab did not resolve this dyfunctional signaling. However, in vitro PD-1 blockade enhanced TCR signaling in patient PD-1+ T cells and not in PD-1- T cells or in PD-1+ T cells from HD.

Conclusions: Functional T cell analysis suggests that baseline T cell functionality is hampered in metastatic breast cancer patients, at least in part mediated by the PD-1 signaling pathway. These preliminary data support the rationale for investigating the possible beneficial effects of adding PD-1 blockade to improve responses to TGFβ blockade and radiotherapy.

Trial registration: NCT01401062 .

Keywords: Immunotherapy; Programmed Death-1 (PD-1); Radiotherapy; T cell receptor (TCR) signaling.

Conflict of interest statement

The authors declare no competing financial interests related to this manuscript, but SCF has received advisory/ speaker compensation from Sanofi, Regeneron, Elekta, EMD Serono, Merck, Astra Zeneca, Bayer and research support from Varian, Bristol-Myer Squibb, Regeneron, Eisai, Merck and Janssen, and SD has received compensation for consultant/advisory services from Lytix Biopharma, AstraZeneca, Mersana Therapeutics and EMD Serono, and research support form Lytix Biopharma and Nanobiotix.

Figures

Fig. 1

Increased PD-L1 expression and decreased TCR signaling in breast cancer patients PBMC compared to HD. a-b Samples were analyzed at baseline for expression of immunomodulatory receptors. a Significantly higher levels of PD-L1 on monocytes of breast cancer patients compared to healthy donors (HD). b Higher levels of PD-1 on CD4 T cells of breast cancer patients compared to HD. c, d TCR modulated signaling in CD8 T cells from HD and patients at baseline and during treatment, as measured by phosphorylation of AKT. c Analysis of all available samples from patients treated in arm 1 (1 mg/kg of fresolimumab) and arm 2 (10 mg/kg of fresolimumab) shows overall reduced TCR signaling in CD8 T cells of patients compared to HD. Each dot represents one individual. Unpaired t test, two-tailed, *p < 0.05, **P < 0.005. (D) pAKT values were plotted overtime for 7 patients with baseline and at least one post-treatment sample measurements

Fig. 2

PD-1+ CD4 and CD8 T cells show decreased TCR signaling compared to PD-1- T cells. a, b The response to TCR stimulation was measured using p-AKT and p-ERK as a readout. PD1+ T cells from both HD and patients collected at baseline show significantly reduced TCR modulated signaling compared to PD1− T cells, as measured by phosphorylation of AKT (a) and ERK (b). Significantly reduced TCR modulated AKT phosphorylation is also evident in PD1− T cells from patients compared to HD. Each dot represents one individual and data is represented as Log2Fold value. A log2Fold value of 1 implies a 2-fold change in Equivalent Number of Reference Fluorophores (ERFs) intensity whereas a value close to 2 implies a 4-fold change. *p < 0.05, **P < 0.005, ***P < 0005

Fig. 3

Anti-PD-1 antibody restores TCR signaling in PD-1+ CD4 T cells from breast cancer patients. a-d PBMC from HD (Left panels) and patients (Right panels) were stimulated with anti-CD3 and anti-CD28 in the presence of anti-PD-1 antibody pembrolizumab (anti-PD-1) or isotype control antibody (Isotype). Graphs show the results for PD1+ T cells, as measured by phosphorylation of AKT (a) and ERK (b). Stimulation in the presence of anti-PD-1 did not significantly increase the response of PD1+ CD4 T cells from HD, but increased the response of PD-1+ CD4 T cells from patients, achieving significance as measured by AKT phosphorylation and showing a trend to significance, as measured by ERK phosphorylation. Stimulation in the presence of anti-PD-1 did not significantly increase the response of PD1− CD4 T cells from HD and patients as measured by phosphorylation of AKT (c) or ERK (d). Each line corresponds to one individual. T test, two-tailed, *p < 0.05, **P < 0.005