Supplementary Materials for

Trastuzumab emtansine (T-DM1) renders HER2+ breast cancer highly

susceptible to CTLA-4/PD-1 blockade

Philipp Müller,* Matthias Kreuzaler, Tarik Khan, Daniela S. Thommen, Kea Martin,

Katharina Glatz, Spasenija Savic, Nadia Harbeck, Ulrike Nitz, Oleg Gluz, Michael von

Bergwelt-Baildon, Hans Kreipe, Sai Reddy, Matthias Christgen, Alfred Zippelius*

*Corresponding author. E-mail: ph.mueller@unibas.ch (P.M.); alfred.zippelius@usb.ch (A.Z.)

Published 25 November 2015, Sci. Transl. Med. 7, 315ra188 (2015)

DOI: 10.1126/scitranslmed.aac4925

The PDF file includes:

Fig. S1. Effect of ansamitocin P3 and DM1 on the maturation of DCs.

Fig. S2. CD8/CD4 ratios in human breast tumors before and after T-DM1

monotherapy.

Fig. S3. H&E staining of treated Fo5 tumors.

Fig. S4. Patterns of intratumoral immune cell subsets after T-DM1 therapy.

Fig. S5. Tumor growth and tumor-infiltrating immune cell subsets in mice

receiving trastuzumab instead of T-DM1.

Fig. S6. Luminex-based cytokine and growth factor measurement in T-DM1– and

α-CTLA-4/PD-1–treated tumors.

Fig. S7. T cell function in mice receiving trastuzumab instead of T-DM1.

Fig. S8. Helios expression, CD8/CD4 and CD8/Treg ratios, and splenic Tregs.

Fig. S9. Helios expression and Tregs in mice receiving trastuzumab instead of T-

DM1.

www.sciencetranslationalmedicine.org/cgi/content/full/7/315/315ra188/DC1

Fig. S1. Effect of ansamitocin P3 and DM1 on the maturation of DCs.

(A) DCs were treated as indicated (24 h) and analyzed for their viability and fold upregulation

of the maturation markers CD40, CD80, MHC-II, and CD86. LPS was used as positive

control (Ansa = AnsamitocinP3). (B) Representative histograms of DCs treated as in A. (C)

Cytokine secretion of DCs treated as in A. Experiments were performed in duplicates and

were reproduced at least twice. Graphs show mean ± SD.

Fig. S2. CD8/CD4 ratios in human breast tumors before and after T-DM1 monotherapy.

(A) Ratio of CD8/CD4 positive cells from 24 matched breast cancer biopsies, before (pre-

therapeutic) and after T-DM1 monotherapy (T-DM1-mono). There were no significant

differences between the two groups as analyzed by unpaired student t-test (p = 0.1524); pre-

therapeutic: mean ± SEM, 5.017 ± 0.7860; T-DM1-mono: mean ± SEM, 3.479 ± 0.7062.

(B/C) Representative matched tumor sections from two independent patients, before and after

T-DM1 monotherapy, were stained as indicated (scale bar = 100 μm).

Fig. S3. H&E staining of treated Fo5 tumors.

Representative tumor sections from Fo5 tumors treated as indicated [COMBO is T-DM1 (one

dose) and α-CTLA-4/-PD-1]; scale bar = 50 μm.

Fig. S4. Patterns of intratumoral immune cell subsets after T-DM1 therapy.

(A) TCRγ/δ T cells as % of all CD45+ cells. (B) B cells as % of all CD45+ cells. (C) NKT

cells as % of all CD45+ cells. (D) NK cells as % of all CD45+ cells (left), and EOMES

expression on tumor-infiltrating NK cells (middle and right); two independent, pooled

experiments with n ≥ 11. Data in A-C are depicted as mean ± SEM. In D, the left graph

depicts data as mean ± SE, whereas the right graph contains box and whiskers plots, with

whiskers showing min to max.

Fig. S5. Tumor growth and tumor-infiltrating immune cell subsets in mice receiving

trastuzumab instead of T-DM1.

(A) Survival of mock-treated (PBS only) Fo5 tumor-bearing mice and mice receiving

trastuzumab (pooled data from two independent experiments n=12). (B) Tumor size of mice

treated as indicated (pooled data from three independent experiments, n=17), showing mean ±

SEM. (C) Summary of immune cell subsets and CD45- cells as determined by FACS (mice

from B). Subsets are depicted as % of all acquired live events.

Fig. S6. Luminex-based cytokine and growth factor measurement in T-DM1– and α-

CTLA-4/-PD-1–treated tumors.

M-CSF and FGF-basic production in tumors from Fig. 4D. Data are depicted as box and

whiskers plots with whiskers showing min to max.

Fig. S7. T cell function in mice receiving trastuzumab instead of T-DM1.

(A) Granzyme B production in CD8 T cells (pooled data from three independent experiments;

n=16). (B) IFNγ production in CD8 T cells (pooled data from two independent experiments;

n=12). Data on the right are depicted as box and whiskers plots with whiskers showing min to

max.

Fig. S8. Helios expression, CD8/CD4 and CD8/Treg ratios, and splenic Tregs.

(A) CD4 T cells from Fo5 tumors treated as indicated were stained for FoxP3 and Helios. (B)

CD8/CD4 and CD8/Treg ratios from mice treated as indicated (pooled data from at least six

independent experiments (n ≥32). (C) Peripheral regulatory T cells from spleen and draining

(dLN) as well as non-draining (ndLN) lymph nodes were analyzed by FACS (n ≥17). Mean ±

SEM is indicated for data in B and C. P values in C were calculated using the Mann-Whitney

test.

Fig. S9. Helios expression and Tregs in mice receiving trastuzumab instead of T-DM1.

(A) CD4 T cells from Fo5 tumors treated as indicated were stained for FoxP3 and Helios. (B)

Treg frequencies in mice treated as indicated [pooled data from three independent

experiments (n ≥16)]. Data in B are depicted as box and whiskers plots with whiskers

showing min to max.