Science 28Feb2020

Stadtmaueret al.,Science 367 , eaba7365 (2020) 28 February 2020 9of12

IP 10 113

Time (Days)

0.0

0.2

0.4

0.6

0.8

1.0

Cell Proportion

NY-ESO-1 TCR

TRAC Mutations

TRBC Mutations

PDCD1 Mutations

0.0

0.2

0.4

0.6

0.8

1.0

Cell Proportion

IP 113 IP 113

NY-ESO-1 TCR- NY-ESO-1 TCR+

Time (Days)

Wild-type

1 Mutation

2 Mutations

3 Mutations

Infusion Product

(IP)

Post-Infusion

(Day 10)

Post-Infusion

(Day 113)

RIPOR2TECR

GIMAP4UBE2V2

ICAM2SARS

C12orf65RAP1A

CCND3PTPRC

AC133644.2TAGAP

MAT2BTRAT1

FCMRCCR7

ATP5F1ECRIP1

CHCHD2H3F3A

HLACD52−A

S100A4FOS

AL138963.3TCF7

PNRC1HLA−E

SNHG8SYTL3

PABPC1PIK3R1

LEPROTL1PDCD4

SH3BGRL3IL7R

TMSB10CAPG

ENO1B2M

ACTBCFL1

SERF2TXN

DDIT4MIF

PTPRCAPJUNB

PIK3IP1BTG1

CD69TPT1

TSC22D3STK17A

ZFP36L1JUN

PTMAFTH1

CIRBPZFP36

HLAEIF1−B

EEF1A1HLA−C

ABRACLMYL6

CST7FTL

AHNRNPFTP6V0E1

SELPLGIL32

PRDX1TPI1

GAPDHACTR3

RAC2PPIA

S100A11STMN1

TMSB4XARPC5

NDFIP2DYNLL1

(IP) (Day 10) (Day 113)

0

max

UMAP1

(^2) PA
MU
NY-ESO-1 TCR
TCF7
A
B
C
D
E
NYCE T cell Infusion Product (IP)
NY-ESO-1 TCR+
11
6
6
2
0
1
1
PDCD1
TRAC
TRBC
− 2 −10 1 2
Expression
(43 Total Cells; 16 Unedited Cells)
84 104 43 8
Fig. 6. Single-cell RNA sequencing of patient UPN39 CRISPR-Cas9–
engineered NYCE T cells pre- and postinfusion.(A) Venn diagram showing
relative numbers of NY-ESO-1 TCR–positive cells withTRAC,TRBC,and/orPDCD1
mutations in the NYCE T cell infusion product (IP) (day 0). (B) Proportions of
preinfusion (IP, day 0) and postinfusion (days 10 and 113) wild-type T cells with
TRAC,TRBC,orPDCD1mutations or expressing the NY-ESO-1 TCR transgene.
Numbers of cells belonging to each of these categories are listed below the graph.
(C)AnalysisofNY-ESO-1TCR–positive (right) and NY-ESO-1 TCR–negative (left)
cells without mutations (wild type) or with single, double, or triple mutations at day 0
(NYCE T cell infusion product) and day 113 post–NYCE T cell infusion. Numbers of
analyzed cells for each time point are listed above the bars. (D) Uniform manifold
approximation and projection (UMAP) plots of gene expression data. Analysis
was performed on all T cells integrated across time points, but only NY-ESO-1 TCR–
expressing cells, split by time point, are shown (top). The increase inTCF7
expression is indicative of an acquired central memory phenotype (bottom, same
cells). (E) Heatmap showing scaled expression of differentially expressed genes
in NY-ESO-1 TCR–positive T cells across time points. Color scheme is based on
scaled gene expression from–2 (purple) to 2 (yellow).
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