Science - USA (2022-04-15)

and isopropanol precipitation and then RNAeasy
Plus Mini kit (Qiagen) for clean-up. After anal-
ysis on the 2100 Bioanalyzer (Agilent), the se-
quencing libraries were prepared using the
SMARTer Stranded RNA-seq Kit (Clontech).
The resulting library was sequenced on the
HiSeq 4000 platform (Illumina) in Stanford
Functional Genomics Facility. For each sample
in the bulk RNA-seq library, 75–base pair (bp)

paired-end reads were acquired from the se-

quencer. We aligned the reads to the human

reference genome (NCBI GRCh38) using STAR

v2.7.0e ( 47 ). Gene counts were quantified and

normalized (TPM) with Salmon ( 48 ). DEGs were

determined using the DESeq function (ad-

justedP< 0.05, fold change > 2) in the DESeq2

R package ( 49 ). Heatmaps were generated with

seaborn.clustermap in python. Gene Ontology

analysis plots were generated with the R pack-

age“clusterProfiler.”To generate gene sets

for GSEA, we selected the top 200 genes up-

regulated in Ly49+CD8+T cells compared with

Ly49–CD8+T cells in EAE mice ( 7 ) and the

previously reported CD4+Tregsignature genes

identified in mice ( 17 ). These mouse genes

were converted to homolog genes in humans

and constituted as gene sets for the subsequent

Liet al.,Science 376 , eabi9591 (2022) 15 April 2022 10 of 13

Fig. 6. Exacerbated autoimmunity in
Klra6creDTA mice after viral infection.
(A) Frequency of Ly49+CD8+T cells
in the blood of DTA mice (N= 8) and
Klra6creDTA mice (N= 5) 0, 5, 8, and
12 days after LCMV-Armstrong infection.
P< 0.01; **P< 0.001; repeated
measures (RM) two-way analysis of
variance (ANOVA) followed by multiple
comparisons test. p.i., postinfection.
(B) Representative contour plots and
summarized scatter plots displaying
frequency and absolute number of
PD1+CXCR5+CD4+T cells (TFH) and
CD38–CD95+GC B cells in the spleen
of DTA mice (N= 8) andKlra6creDTA mice
(N= 5) 30 days after LCMV-Armstrong
infection. P< 0.05; P< 0.01; Mann-
Whitney test. Tfh, T follicular helper cell.
(C) Representative kidney pathology
of day-30 LCMV-infected DTA mice and
Klra6creDTA mice assessed by PAS staining
(630X; scale bars, 20mm). (D) IgG
deposition in glomeruli of the kidneys
of DTA mice (N= 8) andKlra6creDTA mice
(N= 5) 30 days after LCMV infection
accessed by immunofluorescence staining
(400X; scale bars, 20mm) and quantified
by ImageJ. P< 0.05; Mann-Whitney
test. DAPI, 4′,6-diamidino-2-phenylindole.
(E) Frequency of Ly49+CD8+T cells in the
blood of DTA mice (N= 6) andKlra6creDTA
mice (N= 5) after influenza A-PR8
infection. P< 0.05; P< 0.01; **P<
0.0001; RM two-way ANOVA followed by
multiple comparisons test. (F) Microscopy
of representative H&E staining of lung
sections from DTA mice andKlra6creDTA
mice 60 days after influenza infection
(top, 20X; scale bars, 2 mm) (bottom,
630X; scale bars, 50mm). Representative
data from two independent experiments are
shown. The means ± SEMs are indicated.

AB

PD1

CXCR5

DTA

CD38

CD95

Klra6creDTA

C

D

IgGDAPI 400x

630x

DTA Klra6creDTA

DTA Klra6creDTA

E

F

DTA Klra6creDTA

02468101214

0

2

4

6

8

10

Days p.i.

Ly49

+/CD8

+ T(%)

Ly49+ CD8

**** ****

****

**

*

DTA

Klra6creDTA

024681012

0

2

4

6

8

Days p.i.

Ly49

+/CD8

+ T(%)

Ly49+ CD8

Klra6creDTA

DTA

**

***

*** ***

DTA

Klra6

creDTA

0

20

40

60

80

100

Cell

number

(

10

4 )

GC B number

DTA

Klra6

creDTA

0

10

20

30

40

50

Cell number (

10

4 )

Tfh number

DTA

Klra6

creDT

A

0

2

4

6

8

PD1

+CXCR

+ 5

/CD

+ 4

T (%

)

Tfh %

DTA

Klra6

creDTA

0

1000

2000

3000

4000

5000

Integrated Intensity (

10

3 )

DTA

Klra

6 cr

eDTA

0

1

2

3

4

5

CD3

8

CD95

+/B cells (%

)

GC B%

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