immunology.sciencemag.org/cgi/content/full/5/49/eaba7918/DC1

Supplementary Materials for

TOX is expressed by exhausted and polyfunctional human effector memory

CD8+ T cells

Takuya Sekine, André Perez-Potti, Son Nguyen, Jean-Baptiste Gorin, Vincent H. Wu, Emma Gostick, Sian Llewellyn-Lacey, Quirin Hammer, Sara Falck-Jones, Sindhu Vangeti, Meng Yu, Anna Smed-Sörensen, Ahmed Gaballa, Michael Uhlin,

Johan K. Sandberg, Christian Brander, Piotr Nowak, Paul A. Goepfert, David A. Price, Michael R. Betts, Marcus Buggert*

*Corresponding author. Email: marcus.buggert@ki.se

Published 3 July 2020, Sci. Immunol. 5, eaba7918 (2020)

DOI: 10.1126/sciimmunol.aba7918

This PDF file includes:

Fig. S1. Gating strategy and expression of TOX and TCF-1 in resting and activated CD8+ T cell populations. Fig. S2. Tox and Tcf7 expression in CD8+ T cells. Fig. S3. IR expression and distribution among different Seurat clusters. Fig. S4. UMAP analysis and Phenograph clustering of CD8+ T cell populations in relation to TOX and TCF-1. Fig. S5. UMAP analysis and Phenograph clustering of memory CD8+ T cells from HIV– and HIV+ viremic donors and expression of TOX and TCF-1 in virus-specific memory CD8+ T cells. Fig. S6. UMAP analysis and Phenograph clustering of memory CD8+ T cells from HIV– and HIV+ viremic donors and expression of TOX and TCF-1 in virus-specific memory CD8+ T cells. Fig. S7. Expression of TOX and TCF-1, functions, and memory markers in virus-specific memory CD8+ T cells. Fig. S8. Longitudinal assessment of TOX and TCF-1 in virus-specific memory CD8+ T cells. Fig. S9. Analysis of HIV-specific CD8+ T cell functionality in relation to TOX and TCF-1.

Fig. S1. Gating strategy and expression of TOX and TCF-1 in resting and activated CD8+ T cell populations. (A) Flow cytometric gating strategy for the identification of TN

(CCR7+CD45RA+), TCM (CCR7+CD45RA−), TEM (CCR7−CD45RA−), and TEMRA (CCR7−CD45RA+)

cells in the CD8+ lineage. (B) Bar-graph based on Western blot analysis of TOX/Actin ratio (left)

and TCF-1/Actin ratio (right) expression in resting naive and memory CD8+ T cells. (C) Left:

Western blot analysis of TOX and TCF-1 expression in resting naive and memory subsets of CD8+

T cells. GAPDH was used as a loading control. Right: Bar-graph of TOX/GAPDH ratio for the

different CD8+ T cell subsets.

Fig. S2. Tox and Tcf7 expression in CD8+ T cells. (A) Distribution of single-cells in the UMAP

space between n = 2 healthy donors. (B) The UMAP plots illustrate the distribution of different

genes in the UMAP space.

Fig. S3. IR expression and distribution among different Seurat clusters. (A) A bubble plot

showing expression of common inhibitory receptors for T cells in specific genes. Size represents

the percentage of cells within each cluster with non-zero expression of each gene, while color

represents the average normalized read counts for each cluster and gene.

Fig. S4. UMAP analysis and Phenograph clustering of CD8+ T cell populations in relation to TOX and TCF-1. (A) Flow cytometric gating strategy for the identification of CD8+ T cells. (B) UMAP dimensionality reduction plot generated from bulk CD8+ T cells after data concatenation (n

= 4 healthy donors). (C) Phenograph clusters and the ones (red) overlaid on the general UMAP

plot (grey). (D) Analysis of TOX and TCF-1 expression in functionally distinct populations.

GzmBlowperforinlow cells (red) were skewed toward a TOX−TCF-1+ phenotype, and

GzmBhighperforinhigh cells (orange) were skewed toward a TOX+TCF-1− phenotype.

GzmBhighperforinlow cells (blue) were predominantly TOX+ and expressed variable levels of TCF-

1. The plot in the bottom right corner shows the relative abundance of these different phenotypes

for the three different functional profiles identified among memory CD8+ T cells (n = 4 healthy

donors). (E) Histograms showing expression of the indicated markers among TOX-TCF-1+ cells.

Fig. S5. UMAP analysis and Phenograph clustering of memory CD8+ T cells from HIV− and HIV+ viremic donors and expression of TOX and TCF-1 in virus-specific memory CD8+ T cells. (A) Stack bars showing the relative abundance of different memory subsets (TCM, TEM,

TEMRA) for three different TCF-1/TOX profiles (n = 30 HIV− donors and n = 17 HIV+ viremic

donors). (B) Significant correlations between memory subsets and TCF-1/TOX profiles.

Fig. S6. UMAP analysis and Phenograph clustering of memory CD8+ T cells from HIV− and HIV+ viremic donors and expression of TOX and TCF-1 in virus-specific memory CD8+ T cells. (A) Distribution of multiple inhibitory receptors in TOX+/−TCF-1+/− populations (n = 30 HIV−

donors in blue and n = 17 HIV+ viremic donors in red). (B) All phenograph clusters and (C) the

ones (red) overlaid on the UMAP plot (grey) generated from memory CD8+ T cells after data

concatenation (n = 2 HIV− donors and n = 2 HIV+ viremic donors). *P < 0.05, **P < 0.01, ***P <

0.001.

Fig. S7. Expression of TOX and TCF-1, functions and memory markers in virus-specific memory CD8+ T cells. (A) Flow cytometric identification of memory CD8+ T cells specific for Flu,

EBV, CMV, or HIV using MHC class I tetramers. (B) Scatter plots showing TOX+/−TCF-1+/−

population frequencies among virus-specific memory CD8+ T cells. (C) MFI expression of TOX in

virus-specific non-naive CD8+ T cells. (D) Correlation between the MFI of cytolytic molecules and

TOX. (E) Percent expression of TOX among memory subsets for virus-specific memory CD8+ T

cells. (F) Percent of different functions between TOX+/−TCF-1+/− populations for virus-specific

memory CD8+ T cells (Flu: n = 25 healthy donors; EBV: n = 27 healthy donors; CMV: n = 30

healthy donors; HIV: n = 25 healthy donors). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. S8. Longitudinal assessment of TOX and TCF-1 in virus-specific memory CD8+ T cells. (A) Longitudinal assessment of CMV-specific CD8+ T cells before, 14-60 days and 90-360 days

after bone marrow transplantation. (B) Top: Stack bars showing the relative abundance of

different TCF-1/TOX subsets for the three different time intervals after transplantation (n = 6

donors). Bottom: Scatter plots showing TOX+/−TCF-1+/− population frequencies among CMV-

specific memory CD8+ T cells at the different time intervals. (C) Plots showing the TOX+/−TCF-1+/−

distribution of CD38hiKi-67+ CD8+ T cells during symptomatic acute Flu infection.

Fig. S9. Analysis of HIV-specific CD8+ T cell functionality in relation to TOX and TCF-1. (A) Percent expression of TOX and TCF-1 in tetramer-defined HIV-specific CD8+ T cells from ECs

(n = 11; light red), HIV+ aviremic donors on ART (n = 15; red), and HIV+ viremic donors (n = 16;

dark red). (B) Functional responses in a representative EC (top) and a representative HIV+ viremic

donor (bottom) after stimulation of PBMCs with pools of peptides corresponding to known optimal

epitopes derived from HIV. (C) Colored graphs: percent frequency of TNF+ among all responsive

HIV-specific CD8+ T cells from the donor groups in A after stimulation of PBMCs with pools of

peptides corresponding to known optimal epitopes derived from HIV.

Donor-matched graphs: expression intensities of TOX and TCF-1 in TNF− versus TNF+ HIV-

specific CD8+ T cells. *P < 0.05, **P < 0.01, ***P < 0.001.