Article

https://doi.org/10.1038/s41467-024-45573-9

TYK2 signaling promotes the developmentof autoreactive CD8+ cytotoxic T
lymphocytes and type 1 diabetesReceived: 16 August 2023

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Accepted: 25 January 2024

Keiichiro Mine 1,2 , Seiho Nagafuchi 1, Satoru Akazawa 3, Norio Abiru3,4,
Hitoe Mori1, Hironori Kurisaki5, Kazuya Shimoda 6, Yasunobu Yoshikai2,
Hirokazu Takahashi 1,7 & Keizo Anzai1

Tyrosine kinase 2 (TYK2), a member of the JAK family, has attracted attention
as a potential therapeutic target for autoimmune diseases. However, the roleof TYK2 in CD8+ T cells and autoimmune type 1 diabetes (T1D) is poorly
understood. In this study, we generate Tyk2 gene knockout non-obese diabetes (NOD) mice and demonstrate that the loss of Tyk2 inhibits the development of autoreactive CD8+ T-BET+ cytotoxic T lymphocytes (CTLs) byimpairing IL-12 signaling in CD8+ T cells and the CD8+ resident dendritic celldriven cross-priming of CTLs in the pancreatic lymph node (PLN). Tyk2-deﬁcient CTLs display reduced cytotoxicity. Increased inﬂammatory responses in
β-cells with aging are dampened by Tyk2 deﬁciency. Furthermore, treatment
with BMS-986165, a selective TYK2 inhibitor, inhibits the expansion of T-BET+
CTLs, inﬂammation in β-cells and the onset of autoimmune T1D in NOD mice.
Thus, our study reveals the diverse roles of TYK2 in driving the pathogenesis of T1D.

The immune system is strictly regulated to maintain homeostasis bybalancing immune tolerance and immunogenicity, and therefore, abreak in this balance leads to autoimmune disorders or increased
susceptibility to infectious agents1. It is thought that a combination ofgenetic and environmental factors contributes to the pathogenesis of
autoimmune diseases. Multifaceted approaches, although challenging,are needed to uncover the pathogenic mechanisms of autoimmune
disease and to develop effective preventive/therapeutic measuresagainst multifactorial diseases2.
Type 1 diabetes (T1D) is caused by the destruction of insulinproducing pancreatic β-cells and requires lifelong insulin therapy.
The number of patients with T1D worldwide in 2021 was approximately 8.4 million and is expected to increase rapidly because ofchanges in environment and lifestyle3. T1D is a multifactorial disease
related to genetic and environmental factors with a heterogeneousand complex pathogenesis4. Genetic studies of T1D revealed riskloci associated with immune responses5,6. A candidate susceptibility
gene of T1D is tyrosine kinase 2 (TYK2)5,6, a member of the Janus
kinase (JAK) family. TYK2 is expressed ubiquitously and involves inthe signal transduction of type 1 interferons (IFNs), interleukin (IL)−23, IL-12, IL-10, and IL-67. When these cytokines bind to cell surface
receptors, TYK2 is activated, phosphorylates the intracellular tail ofcytokine receptors, and recruits signal transducers and activators oftranscriptions (STATs) in concert with other JAKs. The recruited
STATs are phosphorylated, dimerized, and translocated to the
nucleus to regulate cytokine-speciﬁc gene expression and cytokine
secretion8. These TYK2 responses confer host defense against
microorganisms9–11. Of note, TYK2-deﬁcient patients have impaired
type 1 IFNs and IL-12 signal transduction, resulting in mycobacterial
and viral infections12.

1

Division of Metabolism and Endocrinology, Faculty of Medicine, Saga University, Saga, Japan. 2Division of Host Defense, Medical Institute of Bioregulation,
Kyushu University, Fukuoka, Japan. 3Department of Endocrinology and Metabolism, Unit of Translational Medicine, Nagasaki University Graduate School of
Biomedical Sciences, Nagasaki, Japan. 4Midori Clinic, Nagasaki, Japan. 5Department of Medical Science and Technology, Graduate School of Medical
Sciences, Kyushu University, Fukuoka, Japan. 6Division of Hematology, Diabetes, and Endocrinology, Department of Internal Medicine, Faculty of Medicine,
e-mail: sv7899@cc.saga-u.ac.jp
University of Miyazaki, Miyazaki, Japan. 7Liver Center, Saga University Hospital, Saga University, Saga, Japan.

Nature Communications | (2024)15:1337

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Article
Viruses are reported to be potential environmental factors related
to T1D13–15. Viral infection may contribute to the development of T1D by
several mechanisms including direct β-cell destruction, triggering
autoimmunity to β-cells, molecular mimicry, and induction of β-cell
dedifferentiation13,14,16. We previously reported that reduced Tyk2
expression in β-cells led to impaired antiviral defense, increasing therisk of β-cell-tropic virus-induced diabetes in mice11. In addition, we
found that a polymorphism in the promoter region of the TYK2 gene
(ClinVar, 440728), which decreased promoter activity, was a risk factor
for T1D, particularly in patients with T1D associated with ﬂu-like syndrome and who were autoantibody negative at the onset of disease17,18.
Although the role of TYK2 in virus-induced β-cell destruction iscontroversial11,19, these observations suggest that TYK2 is involved in
the pathogenesis of diabetes induced by virus.
However, TYK2 signaling is associated with the pathogenesis of
autoimmune diseases. Indeed, the loss-of-function mutations andinhibition of TYK2 suppressed autoimmune processes in mice andhumans20–23. The role of TYK2 in psoriasis, which predominantly
manifests as skin lesions24, is well characterized. In a mouse model of
imiquimod-induced psoriasis-like dermatitis, IL-17 and IL-22 secreted
from γδ T cells and Th17 upon IL-23 stimulation were involved in skininﬂammation and keratinocyte activation, which were improved by
TYK2 inhibitor treatment20,25. In patients with psoriasis, oral treatment
with a selective TYK2 inhibitor, BMS-986165 (deucravacitinib), wasefﬁcacious26. Furthermore, a TYK2 inhibitor ameliorated disease progression in murine models of spondyloarthritis, lupus nephritis, andinﬂammatory bowel disease21,22. These studies demonstrate the involvement of TYK2 in the development of autoimmune disease and its
therapeutic potential, although the precise role of TYK2 in autoimmune T1D remains unknown.
In this study, we generated Tyk2 knockout (KO) NOD mice andrevealed the role of TYK2 in driving the pathogenesis of autoimmune

Fig. 1 | Tyk2 deﬁciency suppresses autoimmune diabetes in NOD mice.
a Incidence of spontaneous diabetes in female Tyk2+/+ (n = 14), Tyk2+/− (n = 10), andTyk2−/− NOD mice (n = 13). Diabetes was deﬁned by a non-fasting blood glucose levelexceeding 250 mg/dL. b Insulitis scores of normoglycemic mice at 14w (Tyk2+/+,n = 8; Tyk2+/−, n = 8; Tyk2−/−, n = 8) and 24w (Tyk2+/+, n = 10; Tyk2+/−, n = 8; Tyk2−/−,n = 10). Score 0, no insulitis; Score 1, peri-insulitis; Score 2, inﬁltrative insulitis lessthan 50% of the islet area; and Score 3, inﬁltrative insulitis more than 50% of the islet
area (Supplementary Fig. 1d). c Insulin autoantibody levels in the serum of normoglycemic mice at 14w (Tyk2+/+, n = 17; Tyk2+/−, n = 22; Tyk2−/−, n = 21) and 24w(Tyk2+/+, n = 10; Tyk2+/−, n = 8; Tyk2−/−, n = 16). d Incidence of diabetes in female wild

Nature Communications | (2024)15:1337

https://doi.org/10.1038/s41467-024-45573-9

T1D. These ﬁndings may provide insight into the development of safeand effective prevention strategies for T1D.

Results

Tyk2 deﬁciency suppresses autoimmune T1D in NOD mice
To determine the role of TYK2 in autoimmune T1D, we generated Tyk2
gene KO NOD mice by backcrossing Tyk2KO.B6 mice onto the NOD
background for 10 generations. We analyzed 64 short tandem repeat(STR) loci in Tyk2KO.NOD mice and conﬁrmed that all tested lociexcept for D9Mit83, which is located in chromosome 9 that has Tyk2
gene, were of NOD origin (Supplementary Fig. 1a). In addition, we
conﬁrmed STRs at the insulin-dependent diabetes susceptibility (Idd)loci (Idd1 to Idd15) were of NOD origin (Supplementary Fig. 2b)27.
Because the incidence of diabetes is more prevalent in female NOD
mice28, this study used female mice. We analyzed the spontaneous
incidence of diabetes in mice (+/+, wild type; +/−, heterozygous Tyk2KO;−/−, homozygous Tyk2KO) in cohoused conditions after weaning tominimize the effect of different gut microbiota composition on diabetes development. As expected, the incidence of diabetes was
reduced in Tyk2−/− mice (15.3%) compared with their Tyk2+/+ (78.5%) andTyk2+/− (90.0%) littermates (Fig. 1a). In addition, the timing of diabetes
onset was delayed in Tyk2−/− mice (22 weeks of age (22w)) comparedwith their Tyk2+/+ (15w) and Tyk2+/− (16w) littermates (Fig. 1a). Comparable diabetes development was observed between Tyk2+/+ andTyk2+/− mice. Although TYK2 was reported to be associated withobesity29, our colony exhibited equivalent weight gain throughout the
study (Supplementary Fig. 2c). At 14w, a reduced number of islets withinsulitis were observed in Tyk2−/− mice compared with age-matched
Tyk2-sufﬁcient littermates, consistent with the reduction of diabetes
incidence in Tyk2−/− NOD mice (Fig. 1b and Supplementary Fig. 1d).
Serum anti-insulin autoantibodies (IAAs) were comparable among
14w mice irrespective of genotypes (Fig. 1c). We also analyzed

type NOD.SCID mice adoptively transferred with splenocytes from normoglycemic
female 14w Tyk2+/− NOD mice (n = 7), 14w Tyk2−/− NOD mice (n = 7), or 24w Tyk2−/−
NOD mice (n = 10). Splenocytes (1 ×107) were intravenously transferred into 6–8w
recipient mice. Diabetes was deﬁned by a non-fasting blood glucose level exceeding 250 mg/dL. e Incidence of diabetes in female Tyk2−/− NOD.SCID mice adoptivelytransferred with splenocytes (1 ×107) from normoglycemic female 14w Tyk2+/− NOD
mice (n = 8), 14w Tyk2−/− NOD mice (n = 6), or 24w Tyk2−/− NOD mice (n = 8). Data
represent the mean ± SEM. P-values were calculated using the log-rank test (a, d, e),and one-way ANOVA with Tukey’s posttest (b, c).

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Fig. 2 | Increased inﬂammatory signature in β-cells with age is attenuated by
Tyk2 deﬁciency. a Principal component analysis (PCA) of transcriptome data of
puriﬁed β-cells pooled from 6w female Tyk2+/+ (n = 6), 6w female Tyk2−/− (n = 6), 11w
female Tyk2+/+ (n = 8), and 11w female Tyk2−/− NOD mice (n = 9). β-cells were puriﬁedusing FluoZin-3-AM and TMRE, and analyzed for gene expression proﬁles. b Gene
ontology (GO) biological process enrichment analysis of differentially expressed
genes between β-cells from 11w mice and 6w mice (11w Tyk2+/+ vs 6w Tyk2+/+, or 11w
Tyk2−/− vs 6w Tyk2−/−). Selected GO terms are shown. The two-sided adjusted pvalues were calculated using Benjamini-Yekutieli method (FDR p < 0.05) by GeneTrail. c GO biological process enrichment analysis of differentially expressed genes
in β-cells from 11w mice (11w Tyk2+/+ vs 11w Tyk2−/−). Selected GO terms are shown.
The two-sided adjusted p-values were calculated using Benjamini-Yekutieli method(FDR p < 0.05) by GeneTrail. d, e Gene expression proﬁles in puriﬁed β-cells. Data
are normalized by Z score. Selected genes are shown. Asterisks (*) indicate differentially expressed genes between 11w wild type vs 11w KO mice. Daggers (†) indicate
differentially expressed genes between 6w wild type vs 11w wild type mice. Double
daggers (‡) indicate differentially expressed genes between 6w KO vs 11w KO mice.
Criteria used for identifying differentially expressed genes were as follows: upregulated genes, Z score >2.0 and ratio <1.5; downregulated genes, Z score <−2.0 andratio >0.66. f Protein expression levels of MHC I (H-2Kd (Tyk2+/+, n = 9; Tyk2+/−, n = 12;
Tyk2−/−, n = 9) and H-2Db (Tyk2+/+, n = 7; Tyk2+/−, n = 9; Tyk2−/−, n = 9)) in β-cells from
11–12w female mice. Data represent the mean ± SEM. P-values were calculated using
Kruskal-Wallis test with Dunn’s posttest (f).

normoglycemic 24w mice to assess the long-term effects of Tyk2
deﬁciency. Increased numbers of inﬂamed islets and elevated levels of
serum IAAs were observed in 24w Tyk2−/− mice compared with 14w
Tyk2−/− mice (Fig. 1b, c). Because diabetic mice were excluded, levels of
insulitis and IAAs were understated in this analysis, especially in 24w
Tyk2+/+ and Tyk2+/− mice. These observations suggest that Tyk2 deﬁciency does not completely prevent islet autoimmunity but reduces
the progression rate of invasive insulitis leading to T1D onset.

Tyk2 in the host environment led to a reduced incidence of diabetes
induced by Tyk2+/− splenocytes compared with those in wild type host
NOD.SCID mice (P = 0.003) (Fig. 1e). Thus, these results suggest that
TYK2 expression in immune cells and in the host environment contributes to the development of autoimmune T1D.

TYK2 expression in immune cells and the host environment isassociated with the development of autoimmune T1D
We next asked whether TYK2 expression was critical for immune cells
and/or the host environment (other than immune cells) using an
adoptive transfer model. Because Tyk2+/+ and Tyk2+/− mice had
equivalent diabetes incidence (Fig. 1a) and Tyk2 KO allele had
neomycin-resistant cassette30, we used splenocytes derived fromTyk2+/− mice as a positive control. The phenotype of the transferredsplenocytes, deﬁned by CD44 and CD62L expressions (CD44loCD62L+,naïve; CD44hiCD62L-, effector memory; CD44hiCD62L+, central memory), were comparable (Supplementary Fig. 1e). NOD.SCID mice, whichlack T and B cells, adoptively transferred with Tyk2+/− splenocytes
developed diabetes within nine weeks after transfer, whereas micereceiving Tyk2−/− splenocytes had reduced diabetes incidence irrespective of the donor age (Fig. 1d). Next, to determine the role of TYK2
in the host environment, we generated Tyk2-deﬁcient NOD.SCID miceand conducted the transfer experiments. We revealed that a loss of
Nature Communications | (2024)15:1337

Reduced Fas expression and type I IFN signatures in β-cells fromTyk2-deﬁcient micePancreatic β-cells are a source of islet-associated autoantigens; therefore, we focused on β-cells as an important component of the hostenvironment. We performed transcriptome analysis of β-cells puriﬁedby FluoZin-3-AM and tetramethylrhodamine ethyl ester perchlorate(TMRE) from at least six female mice to minimize the effect of individual heterogeneity on T1D development31,32. β-cells from 6w Tyk2+/+
mice and 6w Tyk2−/− mice had similar gene expression proﬁles, whereas
β-cells from 11w mice had different gene expression proﬁles fromthose of 6w mice (Fig. 2a). This suggests that TYK2 signaling in β-cells
becomes active with age. These differences in gene expression proﬁlesin β-cells with age were conﬁrmed by hierarchical clustering that
divided the data into two clusters by age (Supplementary Fig. 1f). Gene
ontology (GO) analysis revealed that β-cells from 11w Tyk2+/+ andTyk2−/− mice were enriched in immune-related pathways comparedwith those from 6w mice (Fig. 2b). Comparison of β-cells from 11w
Tyk2+/+ and 11w Tyk2−/− mice showed that both genotypes were enriched in immune-related pathways (Fig. 2c). Indeed, inﬂammationrelated genes including Vcam1, B2m, and interferon stimulated genes
3Articlehttps://doi.org/10.1038/s41467-024-45573-9

Fig. 3 | Increased naïve CD8+ T cells and reduced IFN-γ-producing CD8+ T cells in
the PLN of Tyk2-deﬁcient NOD mice. a Cell number (#) or frequency among CD45+
cells (%) of CD8+ T cells in the PLN and pancreas measured by ﬂow cytometry.
Pancreas; 6w (Tyk2+/−, n = 6; Tyk2−/−, n = 6), 11w (Tyk2+/−, n = 5; Tyk2−/−, n = 9), 14w(Tyk2+/−, n = 11; Tyk2−/−, n = 15), and normoglycemic 24w (Tyk2−/−, n = 8) female NOD
mice. PLN; 6w (Tyk2+/−, n = 9; Tyk2−/−, n = 7), 11w (Tyk2+/−, n = 11; Tyk2−/−, n = 11), 14w(Tyk2+/−, n = 11; Tyk2−/−, n = 11), and normoglycemic 24w (Tyk2−/−, n = 3) female NOD
mice. b Frequency of naïve (CD44lo CD62L+), effector memory (CD44hi CD62L-), andcentral memory (CD44hi CD62L+) CD8+ T cells in the PLN of 6w female mice (Tyk2+/−,n = 6; Tyk2−/−, n = 6). c Frequency of Ki67+ cells among the indicated immune cells in
the PLN of 6w female mice (Tyk2+/−, n = 11; Tyk2−/−, n = 10). d (Left) Representative
ﬂow cytometry plots of IFN-γ and IL-17-producing CD8+ T cells upon PMA/iono
stimulation in the PLN of 6w female mice. (Right) The graphs indicate the frequency
of IFN-γ and IL-17-producing CD8+ T cells among CD8+ T cells in the PLN at theindicated ages (6w (Tyk2+/−, n = 9; Tyk2−/−, n = 7), 11w (Tyk2+/−, n = 9; Tyk2−/−, n = 10),and 14w (Tyk2+/−, n = 14; Tyk2−/−, n = 14)). NT not tested. Data represent the mean ±SEM. P-values were calculated using two-tailed Student’s t tests.

(ISGs), were highly expressed in β-cells from 11w mice compared with
6w mice irrespective of the Tyk2 genotypes (Fig. 2d).
However, Gbp2, an ISG, was expressed at lower levels in β-cells
from 11w Tyk2−/− mice compared with 11w Tyk2+/+ mice (Fig. 2e). Notably, the expression level of Fas (Cd95), a cell surface death receptor
involved in β-cell death33, was lower in β-cells from 11w Tyk2−/−
mice compared with 11w Tyk2+/+ mice (Fig. 2e). The expressions of
ISGs (Gbp3, Bst2, and Isg20), chemokines (Cxcl9 and Cxcl10), a type I
IFN receptor (Ifnar2), and type I IFNs (Ifnb, Ifna1) were slightly lower in
β-cells from 11w Tyk2−/− mice compared with 11w Tyk2+/+ mice, although
they did not reach statistical signiﬁcance (Fig. 2d, e). In contrast,
Cxcl13, a ligand for CXCR5 involved in B cell chemotaxis, was expressedat higher levels in β-cells from 11w Tyk2−/− mice compared with 11w
Tyk2+/+ mice (Fig. 2d). These results suggest that type I IFN signaling,
T cell migration into inﬂamed islets, and FAS-mediated β-celldeath are impaired in β-cells of Tyk2−/− mice compared with Tyk2sufﬁcient mice.
The increased expression of MHC I in β-cells was reported to be afeature of autoimmune T1D, and TYK2 inhibition prevented IFN-αinduced MHC I expression in human β-cells19,34. H-2k1, an MHC I
molecule, was expressed at lower levels, whereas B2m, a component of
MHC I, was expressed at higher levels, in β-cells from 11w Tyk2−/− micecompared with 11w Tyk2+/+ mice (Fig. 2d). We also analyzed the protein
expression levels of MHC I in β-cells and revealed that H-2Kd, but not
H-2Db, was slightly decreased in 11–12w Tyk2−/− mice compared withage-matched littermates (Fig. 2f). Together, these ﬁndings suggest thatthe increased inﬂammatory state in β-cells with age is attenuated by
Tyk2 deﬁciency, which may lead to the preservation of β-cells from
immune cell attack.
Type I IFN signaling in β-cells was associated with the presentationof autoantigens19,35; therefore, we compared the expression levels of
T1D-related autoantigens in β-cells. At 11w, gene expression levels of
T1D-related autoantigens including insulin (Ins1 and Ins2), GAD65(Gad2), IA-2 (Ptprn), ZnT-8 (Slc30a8), and IGRP (G6pc2) in β-cells werecomparable between genotypes (Supplementary Fig. 1g). However, at
6w, Ins1 and G6pc2 were expressed at lower levels in β-cells from Tyk2−/−
mice compared with Tyk2+/+ mice (Supplementary Fig. 1g).

Increased naïve CD8+ T cells in the PLN of Tyk2-deﬁcient mice

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TYK2 was reported to be involved in immune cell functions30,36, andtherefore we assessed immune cell proﬁles with age. T cell maturationin the thymus was comparable between Tyk2+/− and Tyk2−/− mice
(Supplementary Fig. 2a). There was no difference in the frequency andnumber of splenic immune cells (CD4+ T, CD8+ T, γδ T, and B cells) withsome exceptions (Supplementary Fig. 2b). Next, we assessed immune
cells in the pancreas (Supplementary Fig. 2c). Although the frequency
was equivalent between genotypes, the numbers of CD4+ T, CD8+ T, γδ
T, and B cells in the pancreas were decreased in 11–14w Tyk2−/− micecompared with Tyk2+/− mice (Fig. 3a, Supplementary Fig. 2d-f). The
phenotypes of CD4+ T and CD8+ T cells deﬁned by CD44 and CD62L
expression levels were also comparable between genotypes (Supplementary Fig. 3a). In addition, the frequency of IFN-γ or IL-17Aproducing T cells upon PMA/ionomycin stimulation, which areimportant effector cells in autoimmune T1D37, was similar between
genotypes (Supplementary Fig. 3b). Thus, there was reduced immune
cell migration into the pancreas of Tyk2−/− mice without cell-type speciﬁcity, and these migrated T cells had no defects in IFN-γ and IL-17A
production following T cell receptor stimulation.
Pancreatic lymph node (PLN) is a reservoir for islet-autoreactive T
cells38,39. Next, we analyzed the PLN and found that naïve CD8+ T cells
were increased in the PLN of 6w Tyk2−/− mice compared with agematched Tyk2+/− mice (Fig. 3b and Supplementary Fig. 3c). Ki67positive proliferating CD8+ T cells were slightly decreased in the PLN of
Tyk2−/− mice compared with age-matched Tyk2+/− mice (Fig. 3c). In
addition, IFN-γ-producing CD8+ T cells upon PMA/ionomycin stimulation were decreased in the PLN of 6w Tyk2−/− mice (Fig. 3d and Supplementary Fig. 3e). These defects were not observed in CD8+ T cells
from the inguinal LN (iLN) (Supplementary Fig. 3d, f). There wascomparable cytokine producing capacity of CD4+ T cells and similarfrequency of CD4+ CD25+ FOXP3+ Treg in the PLN between the genotypes (Supplementary Fig 3g, h). Although these observations in CD8+
T cells were restricted to the initiation phase of autoimmune T1D when
the PLN was crucial for the development of diabetes (~10w)39, theseresults suggest that the defective proliferation and/or reduced numberof CD8+ CTLs, which are the primary effector immune cells for β-cell4Articlehttps://doi.org/10.1038/s41467-024-45573-9

Fig. 4 | Defective APC function of CD8+ rDC in Tyk2-deﬁcient NOD mice.
a Frequency of proliferating CD44hi Tyk2+/− or Tyk2−/− CTV-labeled 8.3 CD8+ T cells in
the PLN of the indicated female recipient mice after 5d or 10d post transfer: 4 ×106
labeled naïve 8.3 CD8+ T cells were transferred into 6–8w female recipient mice.
“24w” indicates the age of the recipient mice. (Tyk2+/− → Tyk2+/−, n = 6; Tyk2−/− →Tyk2+/−, n = 8; Tyk2+/− → Tyk2−/−, n = 4; Tyk2−/− → Tyk2−/−, n = 4; Tyk2+/− → 24w Tyk2−/−,n = 3; Tyk2−/− → 24w Tyk2+/−, n = 5; Tyk2−/− → Tyk2+/− 10d, n = 6; Tyk2+/− → Tyk2−/− 10d,n = 6.) b Frequency of IGRP peptide-speciﬁc CD8+ T cells in the PLN of female mice
(6w (Tyk2+/−, n = 14; Tyk2−/−, n = 11), 11w (Tyk2+/−, n = 10; Tyk2−/−, n = 12). c (Left)
Representative ﬂow cytometry plots of resident DC (rDC) in the PLN of 6w mice.
rDCs are gated on MHC IImid CD11chi cells (Supplementary Fig. 5a, b). (Right) The
frequencies of CD8+ rDC and CD11b+ rDC among rDC are shown (Tyk2+/+, n = 4;

Tyk2+/−, n = 6; Tyk2−/−, n = 4). d Protein expression levels of H-2Kd and H-2Db in CD8+
rDC in the PLN of 6w female mice (Tyk2+/+, n = 4; Tyk2+/−, n = 6; Tyk2−/−, n = 4).
e Volcano plot of transcriptome data in CD8+ rDC in the PLN of 6w female mice
(Tyk2+/+, n = 4; Tyk2−/−, n = 4). The CD8+ rDC were sorted and RNA-sequencing analysis was performed. Pairwise comparisons of differentially expressed genes were
performed using DESeq2 and used to create the plot (FDR < 0.1, logFC > |0.58|).
ISGs and genes associated with APC function are highlighted. f (Left) Representative histogram and (right) proliferation of CTV labeled naïve 8.3 CD8+ T cells after
co-culture with CD8+ rDC and 1 ng/mL of IGRP peptide for 3d (Tyk2+/+, n = 4; Tyk2−/−,n = 5). Data represent the mean ± SEM. P-values were calculated using two-tailed
Student’s t test (a, b, and f) and one-way ANOVA with Dunnett’s posttest (c, d).

destruction38, in the PLN are responsible for the inhibition of autoimmune T1D development in Tyk2−/− NOD mice.

proliferation of islet-autoreactive CTLs39, the limited proliferation of
8.3 CD8+ T cells was observed in the iLN (Supplementary Fig. 4c).
Because IGRP epitopes were reported to appear and spread in the
middle-to-late stage of T1D42, defects in IGRP epitope spreading inTyk2−/− mice might correlate with the reduced proliferation of 8.3 CD8+
T cells in the PLN of Tyk2−/− mice. To exclude this possibility, we evaluated IGRP-speciﬁc CD8+ T cells (IGRP CD8+ T) using tetramers containing a mimotope of IGRP (NRP-V7). A comparable frequency of IGRP
CD8+ T cells was observed in the PLN of both Tyk2 genotypes (Fig. 4b).
Furthermore, the proliferation of 8.3 CD8+ T cells was also suppressedin the PLN of normoglycemic 24w Tyk2−/− recipient mice with advanced
insulitis compared with 14w Tyk2−/− mice, but not in the PLN of normoglycemic 24w Tyk2+/− recipient mice (Figs. 1b and 4a). These datasuggest that the defective proliferation of CD8+ T cells in the PLN of
Tyk2−/− mice is not due to reduced IGRP epitope spreading in Tyk2−/−
mice, but likely other factors.
NY8.3-NOD transgenic mice develop diabetes much earlier thanwild type NOD mice41. To assess the role of TYK2 under these highly
diabetogenic conditions, we analyzed the incidence of diabetes andfound that it was slightly reduced in Tyk2−/− NY8.3 NOD mice (60%)
compared with Tyk2-sufﬁcient NY8.3 NOD mice (Tyk2+/+, 87.5%; Tyk2+/−,
85.7%) (Supplementary Fig. 4d). The timing of diabetes onset wascomparable between the genotypes (Supplementary Fig. 4d). These
results suggest that Tyk2 deﬁciency is partially involved in the suppression of diabetes development even in the presence of abundantdiabetogenic CD8+ T cells.

Reduced proliferation of CD8+ T cells in the PLN of Tyk2deﬁcient mice
Antigen encountered naïve T cells become activated, proliferate, anddevelop into effector and memory T cells that exhibit the CD44hi
phenotype40. To investigate the role of TYK2 in the priming of naïve
CD8+ T cells, we used islet-speciﬁc glucose-6-phosphatase catalytic
subunit-related protein (IGRP)-speciﬁc CD8+ T cells (8.3 CD8+ T cells)
from NY8.3-NOD transgenic mice, which express a T cell receptor(TCR) speciﬁc for the islet autoantigen IGRP206-214 peptide41. The
transfer of Tyk2+/− or Tyk2−/− naïve 8.3 CD8+ T cells into 6–9w Tyk2+/−
recipient mice resulted in a similar upregulation of CD44 in thetransferred cells, and proliferation in the PLN 5d post transfer, whereas
their proliferation was suppressed in the PLN of age-matched Tyk2−/−
recipient mice (Fig. 4a and Supplementary Fig. 4a, b). These observations suggest that CD8+ T cell-extrinsic mechanisms have a role in the
proliferation of islet-autoreactive CTLs in the PLN. Indeed, Tyk2−/− 8.3
CD8+ T cells proliferated more in the PLN of Tyk2+/− recipient mice
10 days after transfer compared with those 5 days post transfer(Fig. 4a). Ten days after transfer, the number of proliferating 8.3 CD8+
T cells was increased in the PLN of Tyk2−/− mice compared with those
5 days after transfer (Fig. 4a), suggesting that the priming of CD8+
T cells was impaired but not abolished in the PLN of Tyk2−/− mice. In
agreement with a study showing the importance of the PLN for the

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MHC class I expression is reduced in Tyk2-deﬁcient CD8+
resident DCs
The results of the adoptive transfer (Fig. 4a) suggest that TYK2
expression in antigen presenting cells (APCs) was associated withthe efﬁcient proliferation of islet-autoreactive CTLs in the PLN. A
previous study reported the importance of TYK2 in antigen presentation by dendritic cells (DCs) for the expansion of IFN-γ+ CD8+
T cells following Listeria monocytogenes infection43. Next, we analyzed
DCs, which have a central role in the adaptive immune system via
antigen presentation to T cells (Supplementary Fig. 5a, b)40. In the PLN,
the frequency of CD11c+ MHC IImid resident DCs (rDCs), including CD8+
and CD11b+ subsets, and CD11c+ MHC IIhi migratory DCs (mDCs),
including the CD103+ CD11b-, CD103+ CD11b+, and CD103- CD11b+ subsets, were comparable irrespective of Tyk2 genotypes (Fig. 4c andSupplementary Fig. 5c). Comparable expression levels of MHC II (I-Ag7)
were observed in these DC subsets from all genotypes (Supplementary
Fig. 5d). However, we found that Tyk2 deﬁciency reduced the expression of MHC I (H-2Kd and H-2Db), a crucial molecule for presenting
antigens to CD8+ T cells40, in CD8+ rDC but not other subsets of DCs(Fig. 4d and Supplementary Fig. 5e, f). This MHC I reduction in CD8+
rDC was not speciﬁc for the PLN as comparable results were observedin the iLN and spleen (Supplementary Fig. 5g, h). The expression levelsof co-stimulatory molecules including CD40 and CD86 in CD8+ rDC
were comparable between genotypes (Supplementary Fig. 5i). Previous studies reported that CD8+ rDC have a role in the expansion of
CTLs by cross-priming and therefore we focused on CD8+ rDC40,44. We
analyzed the gene expression proﬁles of CD8+ rDC in the PLN andfound that Interferon regulatory factor 9 (Irf9) and ISGs including
Iﬁ27l2a, Iﬁ44, Iﬁ203, and Iﬁh1 were highly expressed in Tyk2+/+ CD8+ rDC
compared with Tyk2−/− CD8+ rDC (Fig. 4e), suggesting that type I IFN
signaling was upregulated in Tyk2+/+ CD8+ rDC (Supplementary Fig. 5j).
Tyk2−/− CD8+ rDC had higher expression levels of Androgen receptor(Ar), which might be associated with the suppression of APC functionsin DCs45, and Il4-induced 1 (Il4i1), which inhibits the proliferation of
T cells46, compared with Tyk2+/+ CD8+ rDC (Fig. 4e). The expression of
IL-12 and IL-23, which are involved in T cell differentiation via TYK2,
were comparable between genotypes.
To assess the APC function of CD8+ rDC, we performed an ex vivo
co-culture experiment. This revealed that Tyk2−/− CD8+ rDC (pulsedwith IGRP peptide or NIT-1 cells) induced lower levels of 8.3 CD8+
T cells expansion compared with Tyk2+/+ CD8+ rDC (Fig. 4f and Supplementary Fig. 5k). These results suggest that defective TYK2mediated type I IFN signaling in CD8+ rDC results in decreased MHC
I expression, leading to the impaired cross-priming of isletautoreactive CTLs in the PLN.

Reduced T-BET+ CD8+ T cells in the PLN of Tyk2-deﬁcient
NOD mice
Although we found that efﬁcient cross-priming and proliferationof CD8+ T cells was evident in the PLN of Tyk2-sufﬁcient mice compared with Tyk2−/− mice (Fig. 4a), the number of CD8+ T cells in the
PLN were comparable between the genotypes (Fig. 3a). Next, we characterized antigen-experienced CD8+ T cells that exhibited the CD44hi
phenotype. Because transcription factors (TFs) are key regulators of T
cell effector functions, we examined TFs in CD44hi CD8+ T cells in the
PLN. The frequency of EOMES, BLIMP-1, and TCF1-positive CD44hi
polyclonal or IGRP CD8+ T cells in the PLN were comparable between
genotypes (Supplementary Fig. 6a–c). We found that polyclonal and
IGRP-speciﬁc CD44hi CD8+ T cells positive for T-BET, an important TF
for the production of IFN-γ, were decreased in the PLN of Tyk2−/− micecompared with Tyk2-sufﬁcient mice (Fig. 5a, b), supporting the resultsof reduced IFN-γ-producing CD8+ T cells in the PLN of Tyk2−/− mice
(Fig. 3d). The expression levels of T-BET were also reduced in CD44hi
T-BET+ IGRP CD8+ T cells in the PLN of Tyk2−/− mice compared withthose of Tyk2+/− mice (Fig. 5c). Thus, these data suggest that the
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development of T-BET+ CTLs is impaired in the PLN of Tyk2−/−
NOD mice.
IL-12 is known to drive T-BET expression in T cells during theirpriming47. Next, to address the role of Tyk2 in CD8+ T cells during
priming, puriﬁed CD8+ T cells were stimulated ex vivo with anti-CD3/
CD28 beads in the presence or absence of IL-12. We found that the lossof Tyk2 in CD8+ T cells was not associated with the expansion of T-BET+
CD8+ T cells (Fig. 5d). However, T-BET expression level was impaired inTyk2−/− CD8+ T cells compared with Tyk2+/− CD8+ T cells when the cells
were stimulated with IL-12 (Fig. 5e). These data suggest that Tyk2−/−
CD8+ T cells develop CTLs that are expressed lower levels of T-BET
than those of Tyk2-sufﬁcient CD8+ T cells in the PLN due to defective
CD8+ T cell-intrinsic IL-12 signaling. In addition, the decreased numberof T-BET+ CTLs in the PLN of Tyk2−/− mice might be related to defective
APC functions of CD8+ rDC.
Chemotaxis is an important mechanism in the induction of insulitis. A previous study reported Cxcr3, a receptor for CXCL9 and
CXCL10, is a target gene of T-BET48. In addition, the expression of
Cxcl10 in β-cells has an important role in the chemotaxis of isletautoreactive T cells49. Next, we examined the expression of CXCR3 andfound that polyclonal and IGRP-speciﬁc CD44hi CXCR3+ CD8+ T cells
were decreased in the PLN of Tyk2−/− mice compared with age-matched
Tyk2-sufﬁcient littermates (Fig. 5f, g, and Supplementary Fig. 6d, e).
The decreased number of CD44hi CXCR3+ CD8+ T cells was speciﬁc for
the PLN of Tyk2−/− mice because there were no differences in the iLN
and spleen (Supplementary Fig. 6f). In contrast, the frequency of
CXCR3 expressing CD4+ T cells in the PLN was comparable between
genotypes (Supplementary Fig. 6e). Together, these observations
suggest that the comparable number of CD8+ T cells in the PLN
between Tyk2 genotypes (Fig. 3a) might be related to the developed
CTLs expressing CXCR3, exiting the PLN, and migrating to the islets,
resulting in similar numbers of CD8+ T cells in the PLN between the
genotypes but increased numbers of CD8+ T cells in the pancreas of
Tyk2-sufﬁcient mice compared with Tyk2−/− mice (Fig. 3a).

CTLs from Tyk2-deﬁcient mice display impaired cytotoxicity
Our data reveal that the CD8+ rDC-mediated cross-priming of CD8+
T cells and the development of islet-autoreactive CD8+ T-BET+ CTLs are
impaired but not abolished in the PLN of Tyk2−/− mice. It was demonstrated that stem-like CD8+ T cells in the PLN give rise to pathogenic
CD8+ T cells in the pancreas with upregulating effector functions38.
Next, to test whether the transition of gene expression proﬁles in isletautoreactive CTLs depends on Tyk2 genotypes, we performed transcriptome analysis of puriﬁed antigen-experienced CD44hi IGRP CD8+
T cells in the PLN and IGRP CD8+ T cells in the pancreas (Fig. 5h). The
expression status of CD44 and CD62L in the IGRP CD8+ T cells in the
PLN resembled those of polyclonal CD8+ T cells (Supplementary
Fig. 6g, h). In the pancreas, IGRP CD8+ T cells exhibited a CD62Lphenotype (Supplementary Fig. 6h). The gene expression proﬁles of
IGRP CD8+ T cells in the pancreas differed from those in the PLN38,
irrespective of the Tyk2 genotypes (Fig. 5h). Nevertheless, we foundthat 744 genes were differentially expressed and that Gzmb andFasl were upregulated in Tyk2+/+ IGRP CD8+ T cells in the pancreas
compared with Tyk2−/− cells (Fig. 5i; Group 3). In contrast, Tyk2−/− IGRP
CD8+ T cells puriﬁed from the pancreas upregulated apoptosis-related
genes, including Casp3 and Birc5, compared with Tyk2+/+ cells (Fig. 5i;
Group 4). However, binding of annexin V, a marker of early apoptosis,
in IGRP CD8+ T cells puriﬁed from the pancreas was comparable
between the genotypes (Supplementary Fig. 6i), suggesting thatTyk2−/− CD8+ T cells were not conspicuously prone to apoptosis in
the pancreas. These results suggest that the loss of Tyk2 impairs theupregulation of effector functions in islet-autoreactive CTLs in thepancreas.
A previous study reported that TYK2 had a role in CTL-mediatedtumor surveillance50. To assess the cytotoxic activity of CTLs against
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Fig. 5 | Defective development of CD8+ T-BET+ CTLs in the PLN and effector
function of CD8+ CTLs in the pancreas of Tyk2-deﬁcient NOD mice. a (Left)
Representative ﬂow cytometry plots and (right) graph indicate the frequency ofCD44hi T-BET+ polyclonal cells among CD8+ T cells in the PLN of 6w female mice
(Tyk2+/+, n = 6; Tyk2+/−, n = 12; Tyk2−/−, n = 7). b Frequency of CD44hi T-BET+ cells
among IGRP-speciﬁc CD8+ T cells in the PLN of 6w female mice (Tyk2+/−, n = 8; Tyk2−/−, n = 6). c Protein expression levels of T-BET in CD44hi T-BET+ IGRP-speciﬁc CD8+
T cells in the PLN of 6w female mice (Tyk2+/−, n = 9; Tyk2−/−, n = 6). Frequency ofCD44hi T-BET+ cells (d) and protein expression levels of T-BET (e) in unstimulated or
ex vivo-activated Tyk2+/− or Tyk2−/− CD8+ T cells with anti-CD3/CD28 beads and IL-2
in the presence or absence of IL-12 for 3d (Tyk2+/−, n = 4; Tyk2−/−, n = 4). f (Left)
Representative ﬂow cytometry plots and (right) graph indicate the frequency ofCD44hi CXCR3+ cells among polyclonal CD8+ T cells in the PLN of 6w female mice
(Tyk2+/+, n = 7; Tyk2+/−, n = 5; Tyk2−/−, n = 7). g Frequency of CD44hi CXCR3+ cells

among IGRP-speciﬁc CD8+ T cells in the PLN of 6w female mice (Tyk2+/−, n = 7; Tyk2−/−, n = 5). h PCA of transcriptome data of puriﬁed CD44hi IGRP-speciﬁc CD8+ T cells in
the PLN (Tyk2+/+, n = 3; Tyk2−/−, n = 3) and IGRP-speciﬁc CD8+ T cells in the pancreas(Tyk2+/+, n = 3; Tyk2−/−, n = 3). Gene expression proﬁles in the puriﬁed cells wereanalyzed by RNA-seq analysis. i Box plots show the pattern of differentiallyexpressed genes in IGRP-speciﬁc CD8+ T cells in the PLN (Tyk2+/+, n = 3; Tyk2−/−, n = 3)
and pancreas (Tyk2+/+, n = 3; Tyk2−/−, n = 3). Likelihood ratio tests were used todetermine differentially expressed genes between the genotypes (FDR < 0.1, foldchange >1.5). Selected genes are highlighted on the right side of each box plots.
Boxes and whiskers represent the median and the lower and upper hinges correspond to the 25th and 75th percentiles. The whiskers extend from the hinges to thevalues no further than 1.5 times the inter-quartile ranges from each box hinge. Data
represent the mean ± SEM. P-values were calculated using one-way ANOVA with
Dunnett’s posttest (a, f) and two-tailed Student’s t test (b–e, and g).

β-cells, ex vivo-activated 8.3 CD8+ T cells in the presence of IL-2 andIL-12 (8.3 CTLs) were cultured with NIT-1 cells, a pancreatic β-cell line
derived from female NOD mice51, and the expression levels of effector
molecules in the CTLs and the survival of NIT-1 cells were analyzed52.
After priming of the 8.3 CTLs, the expression levels of GZMB in the
CTLs were comparable between genotypes (Fig. 6a). However, the 8.3
CTLs cultured with NIT-1 cells had upregulated expression levels of
GZMB, which were attenuated by Tyk2 deﬁciency, blockade of interferon alpha and beta receptor subunit 1 (IFNAR1), and neutralization of
IFN-β, but not IFN-α (Fig. 6a, b). The expression levels of Ifnb1 in NIT-1
cells were upregulated after coculturing with 8.3 CTLs (Fig. 6c). These
results suggest that TYK2-mediated IFN-β signaling in CTLs is involvedin the enhancement of the expression levels of GZMB in the CTLs aftertarget cell encounter. Indeed, ISGs, including Irf7 and Isg20, wereupregulated in Tyk2+/+ IGRP CD8+ T cells in the pancreas compared with
Tyk2−/− IGRP CD8+ T cells (Fig. 5i; Group 3). The expression levels of
FASL and CASP3/7 in the 8.3 CTLs after NIT-1 cell encounter werecomparable between genotypes (Supplementary Fig. 6j). The cytotoxic function of Tyk2−/− 8.3 CTLs primed with IL-12 had a reduced
killing capacity compared with those of Tyk2+/− 8.3 CTLs (Fig. 6d).
Together, these results suggest that defective CTL-intrinsic IFN-β signaling related to Tyk2 deﬁciency reduces the cytotoxic activity of CTLs
after encountering β-cells.

TYK2 inhibitor has the potential to prevent the development of
autoimmune T1D

Nature Communications | (2024)15:1337

Recently, BMS-986165, a selective TYK2 inhibitor53, was reported toinhibit autoimmune diseases including psoriasis in mice andhumans21,54, although its effects on autoimmune T1D are unknown.
Our results suggest that TYK2 is a potential target for preventing
autoimmune T1D. To test whether BMS-986165 inhibited thedevelopment of CTLs, puriﬁed Tyk2+/+ CD8+ T cells were stimulated
ex vivo with anti-CD3/CD28 beads, IL-2, and IL-12, in the presence orabsence of BMS-986165. The expression levels of T-BET in stimulated CD8+ T cells were reduced BMS-986165 dose-dependently(Fig. 7a, and Supplementary Fig. 7a). Consistent with the T-BET
expression levels, the β-cell killing capacity of stimulated 8.3 CD8+
T cells was impaired BMS-986165 dose-dependently (Fig. 7b), supporting the results of reduced killing capacity in Tyk2−/− 8.3 CD8+
T cells (Fig. 6d). Next, we assessed CXCR3 expression levels in thestimulated CD8+ T cells. Re-culture of stimulated CD8+ T cells for 2d
induced CXCR3 expression in the cells (Fig. 7c)55. However, recultured CD8+ T cells primed with BMS-986165 had reduced CXCR3
expression levels dose-dependently (Fig. 7c), suggesting that the
reduction of CXCR3 in CTLs was due to a CTL-intrinsic mechanism.
Next, to test the effect of BMS-986165 on developed CTLs, we
treated ex vivo stimulated CD8+ T cells with BMS-986165 for 2d and7Articlehttps://doi.org/10.1038/s41467-024-45573-9

Fig. 6 | Loss of Tyk2 impairs the cytotoxic activity of CD8+ CTLs. a Protein
expression levels of GZMB in the unstimulated 8.3 CD8+ T cells, ex vivo-activated
8.3 CD8+ T cells, or ex vivo-activated 8.3 CD8+ T cells cocultured with NIT-1 cells(Tyk2+/−, n = 6, Tyk2−/−, n = 6). Puriﬁed 8.3 CD8+ T cells were activated with IL-2, IL-12,
and anti-CD3/28 beads for 3d. For coculture, ex vivo-activated 8.3 CD8+ T cells were
cultured with NIT-1 cells for 24 h (CTLs:NIT-1 = 1:1). b Protein expression levels of
GZMB in Tyk2+/− 8.3 CTLs after 24 h of coculture with NIT-1 cells in the presence of
IFNAR1 blocking antibody (n = 7), IFN-β neutralizing antibody (n = 7), or IFN-α
neutralizing antibody (n = 7). Puriﬁed Tyk2+/− 8.3 CD8+ T cells were activated with IL-

2, IL-12, and anti-CD3/28 beads for 3d, and cultured with NIT-1 cells. c Gene
expression levels of Ifnb1 in NIT-1 cells after coculture with ex vivo-activated 8.3
CD8+ T cells (0 h, n = 4; 1 h, n = 5; 2 h, n = 5; 6 h, n = 4; 12 h, n = 3). d The killing
capacity of ex vivo-activated 8.3 CTLs against NIT-1 cells at the indicated ratios.
Puriﬁed 8.3 CD8+ T cells were activated with IL-2 and CD3/28 beads in the presenceor absence of IL-12 for 3d. NIT-1 cells were cultured with activated 8.3 CD8+ T cells
for 24 h, and then the survival of NIT-1 cells was analyzed (1:0.1, n = 4; 1:1, n = 3; 1:2,
n = 3). Data represent the mean ± SEM. P-values were calculated using one-way
ANOVA with Dunnett’s posttest (c) and two-tailed Student’s t test (a, b, and d).

Fig. 7 | BMS-986165 prevents the development of autoimmune T1D. a (Left)
Representative histogram and (right) protein expression levels of T-BET in stimulated Tyk2+/+ CD8+ T cells. Puriﬁed Tyk2+/+ CD8+ T cells were activated with IL-2, IL-12,
and anti-CD3/28 beads for 3d in the presence or absence of BMS-986165 (unstimulated control, n = 3; stimulated cells, n = 4). b The β-cell killing capacity of exvivo-activated Tyk2+/+ 8.3 CD8+ T cells. Puriﬁed 8.3 CD8+ T cells were primed with IL2, IL-12, and anti-CD3/28 beads for 3d in the presence or absence of BMS-986165.
NIT-1 cells were cultured with activated 8.3 CD8+ T cells for 24 h, and then thesurvival of NIT-1 cells was analyzed (n = 5). c Protein expression levels of CXCR3 in
stimulated Tyk2+/+ CD8+ T cells. Ex vivo activated CD8+ T cells with IL-2, IL-12, andanti-CD3/28 beads for 3d in the presence or absence of BMS-986165 were recultured without BMS-986165 for 48 h and the induction of CXCR3 was analyzed(unstimulated control and stimulated cells without BMS-986165, n = 3; stimulated
cells with BMS-986165, n = 4). Day 0 data indicate the CXCR3 expression levels justafter ex vivo stimulation. Gene expression levels of Cxcl10 (d) and Isg15 (e) in NIT-1
cells stimulated with 100 U/mL of IFN-β or 0.1 ng/mL of IFN-γ in the presence orabsence of BMS-986165 for 3 h (n = 3). f Incidence of diabetes in vehicle (n = 6) or
BMS-986165-treated (n = 6) wild type female NOD mice for 4w from 6w of age. Mice
were dosed by oral gavage once daily with BMS-986165 30 mg/kg in vehicle(EtOH:TPGS:PEG300, 5:5:90) or vehicle alone. Diabetes was deﬁned by a nonfasting blood glucose level exceeding 250 mg/dL. g Incidence of diabetes in vehicle(n = 6) or BMS-986165-treated (n = 4) wild type female NOD mice for 4w from 12w ofage. Data represent the mean ± SEM. P-values were calculated using one-way
ANOVA with Dunnett’s posttest (a–e) and log-rank test (f, g).

found that the expression levels of T-BET and CXCR3 were
unchanged (Supplementary Fig. 7b). However, BMS-986165 inhibited IL-12 and IL-18-induced bystander IFN-γ production in CD44hi
CD8+ T cells dose-dependently (Supplementary Fig. 7c). These
results suggest that TYK2 has a role in the development of CD8+
T-BET+ CXCR3+ CTLs and bystander activation of CD8+ CTLs uponcytokines stimulation, and is dispensable for the maintenance of
T-BET and CXCR3 expression in CD8+ CTLs.

Next, to test whether BMS-986165 inhibited inﬂammatory
responses in β-cells, NIT-1 cells with or without BMS-986165 were stimulated with type I or type II IFNs. BMS-986165 inhibited IFN-βinduced Cxcl10, Isg15, and Oas2 expressions dose-dependently(Fig. 7d, e, and Supplementary Fig. 7e). IFN-β-induced Cxcl9, Cxcl13,
and Fas expression levels were reduced but not signiﬁcantly suppressed by BMS-986165 treatment (Supplementary Fig. 7e). BMS986165 had no effect on IFN-γ-induced these genes expression levels in

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8

Article
NIT-1 cells, because TYK2 is not associated with type II IFN signaling,
conﬁrming the high selectively of the TYK2 inhibitor. Together, theseresults demonstrate that BMS-986165 inhibits the development andbystander activation of CD8+ CTLs, and type I IFN-induced gene
expressions in β-cells.
To demonstrate the effects of BMS-986165 on the development of
autoimmune T1D in vivo, we administrated the inhibitor to wild type
NOD mice by oral gavage once-daily at 30 mg/kg for 4 weeks from 6w
of age. NOD mice treated with BMS-986165 had reduced diabetes
incidence (1/6) compared with vehicle control mice (5/6) (p = 0.025,
Fig. 7f). The timing of diabetes onset was delayed in the inhibitor
treated mice (32w) compared with vehicle treated mice (19w) (Fig. 7f).
In contrast, treatment with BMS-986165 at the same dose from 12w ofage resulted in comparable diabetes incidence (3/4) with vehicle
control mice (4/6) (p = 0.99, Fig. 7g). However, the timing of diabetes
onset was delayed in the inhibitor treated mice (30w) compared withvehicle treated mice (20w) (Fig. 7g). These results suggest that treatment with a TYK2 inhibitor started at an early stage of T1D may be apotential strategy to prevent the development of autoimmune T1D.

Discussion
In the context of autoimmune disease, the role of TYK2 in CD4+ T cells,
but not CD8+ T cells, has been well described36,56. Our study reveals that
IL-12 signaling via TYK2 upregulated T-BET expression in CD8+ CTLs
during their priming, leading to the upregulation of CXCR3. In addition, the efﬁcient cross-priming of CTLs by CD8+ rDC may be associated with MHC I expression levels in CD8+ rDC, probably induced bytype I IFN signaling via TYK2. These data provide evidence that TYK2
has a critical role in the generation of autoreactive CD8+ CTLs. However, increased naïve CD8+ T cells and decreased IFN-γ-producing CD8+
T cells in the PLN of Tyk2−/− mice were limited in the initiation phase of
T1D (Fig. 3b, d) when the PLN is dispensable for T1D development39.
This is likely because loss or inhibition of TYK2 did not completely
inhibit the development of T-BET+ CTLs (Figs. 5a and 7a), the numberof CTLs in the PLN of Tyk2−/− mice became comparable to that in the
PLN of Tyk2-sufﬁcient mice in accordance with aging. In addition to theimpaired development of CD8+ T-BET+ CTLs in the PLN of Tyk2−/− mice,the reduced killing capacity of Tyk2−/− CD8+ CTLs against β-cells
probably contributes to the suppression of the development of autoimmune T1D. Importantly, TYK2-mediated IFN-β signaling was associated with the upregulation of effector molecule GZMB in CD8+ CTLs
after β-cell encounter (Fig. 6a, b). Thus, TYK2 is involved in thedevelopment of CD8+ T-BET+ CTLs via IL-12 signaling and the inductionof effector functions via type I IFN signaling after encounteringtarget cells.
Consistent with an earlier study57, a similar frequency of Treg wasobserved regardless of the Tyk2 genotypes (Supplementary Fig. 3h).
The non-involvement of TYK2 in Treg differentiation and maintenance
may have led to the efﬁcient suppression of autoreactive CTLs in Tyk2−/−
mice57. Although the differentiation of Th1 and Th17 cells in the PLN
was comparable between Tyk2 genotypes, bystander IFN-γ production
by CD44hi CD4+ T cells was impaired by TYK2 inhibition (Supplementary Fig. 7d), suggesting the pathogenic role of autoreactive CD4+
T cells may be also defective in Tyk2−/− mice.
Our data suggest that TYK2 signaling in β-cells becomes activewith age. Because the expression levels of chemokines, ISGs, and MHC
I molecules were lower in the β-cells from 6w mice compared withthose from 11w mice (Fig. 2d, e), the increased inﬂammatory signatureof β-cells with age may be caused by inﬁltrating immune cells or the
local islet environment induced by immune cells. We found that Cxcl9
and Cxcl10 expressions in β-cells were upregulated with age, whichwere slightly attenuated by Tyk2 deﬁciency, at least in part, due toimpaired type I IFN signaling in β-cells (Fig. 7d, e, and Supplementary
Fig. 7e). In addition to the low Cxcl9 and Cxcl10 expressions, reduced
MHC I and Fas expressions in the β-cells of Tyk2−/− mice may contribute
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to the preservation of β-cells from CTLs attack. In the early phase of
T1D development, TYK2 inhibition might reduce the expression levelsof islet-associated autoantigens in β-cells (Supplementary Fig. 1g). In
contrast to these preservation effects of TYK2 inhibition in β-cells, a
recent study suggested that TYK2 inhibition lead to reduced β-cellmass related to the defective formation of pancreatic endocrine
precursors35. Consequently, TYK2 may has a different role in β-cells
depending on the stage of T1D and/or developmental stage of β-cells.
Together, these ﬁndings suggested that the mechanisms described above act in concert to inhibit the development of autoimmune
T1D in Tyk2−/− NOD mice. In support of this, a recent study reported
that the loss-of-function variant rs34536443 in TYK2 was associatedwith a lower risk of autoimmune disease including T1D23. Thus, TYK2 isinvolved in the pathogenesis of autoimmune T1D in mice and humans.
Treatment with the TYK2 inhibitor BMS-986165 impaired thedevelopment of CD8+ T-BET+ CXCR3+ CTLs, bystander activation of
CD8+ CTLs, and chemokine and ISG expressions in β-cells dosedependently. Of note, a reduced T1D progression rate was evident
when the treatment was started at 6w but not at 12w of age, suggestingthat the initiation or early phase of T1D is the therapeutic window for
the TYK2 inhibitor. The abundant presence of IFN-γ-producing CD8+
T cells in the PLN during the initiation phase of T1D (Fig. 3d) may
explain why intervention in the initiation phase of T1D is optimal forthe efﬁcient suppression of T1D development. Even though the overall
incidence of diabetes was not reduced, mice treated with the TYK2
inhibitor from 12w had a delayed onset of T1D compared with vehicle
treated mice (Fig. 7g). These observations suggest that early intervention with a TYK2 inhibitor may be a potent immunotherapeuticstrategy for autoimmune T1D, and treatment with the inhibitor in thelate stage of T1D may also be effective at suppressing the autoimmune
process. The effect of the TYK2 inhibitor on autoimmune T1D will
be better deﬁned by studies using large group sizes. In addition,
because our conclusions were based on experiments using female
mice, further studies with male mice will be needed.
TYK2 is involved in anti-microorganism responses via cytokine
signaling11,12,43. Clinical studies reported potential adverse effects of
BMS-986165, including nasopharyngitis, diarrhea, nausea, and upper
respiratory tract infection23,26. In the clinical trial, BMS-986165 wasconsidered safe on the basis of observations of a slight increase but
low rate of serious viral infection26. However, it was reported that somepatients with T1D, especially with acute onset and fulminant T1D,
exhibited severe or mild ﬂu-like syndromes at disease onset17,58–62. In
contrast to the role of TYK2 in autoimmune T1D described in thispaper, we reported that reduced TYK2 expression was a risk factor for
β-cell tropic virus-induced diabetes and impaired insulin
secretion11,17,18,63. Of note, the importance of anti-viral defense in β-cells
to prevent β-cell destruction caused by viruses was reported based onin vivo ﬁndings11,32,64,65. Our studies suggest that the role of TYK2 in T1D
depends on causal factors. In contrast, TYK2 inhibition reducesinﬂammation in β-cells in response to cytokines and a mimic of viralRNA, leading to the preservation of β-cells19,35. Although the role ofviruses in T1D is controversial, the advantages and risks of immunomodulation should be considered when developing treatments forT1D. An anti-diabetogenic virus vaccine, which is currently underdevelopment66, might be a potent option to eliminate the potential
viral contribution to β-cell destruction in patients taking immunosuppressant drugs such as TYK2 inhibitors and anti-CD3 antibodies,regardless of the mechanisms of T1D development induced by viruses.
In addition to the increased infection risk, there is a potential risk oflung cancer, non-Hodgkin lymphoma, and reduced β-cell mass withTYK2 inhibition35,67, indicating that further safety assessments will beneeded for long-term treatment with TYK2 inhibitors.
In conclusion, our study demonstrated that the blockade of
TYK2 signaling in autoimmune T1D model NOD mice impaired
the development and effector functions of CD8+ CTLs, the efﬁcient9Articlecross-priming of CD8+ CTLs by CD8+ rDC, and the expression of
inﬂammatory molecules in β-cells, leading to the inhibition of theonset of overt autoimmune T1D. The evaluation of candidate genes bymultifaceted approaches will aid our understanding of the complex
role of genes involved in multifactorial diseases and help the development of the safe and effective preventive/therapeutic strategies.

Methods
Animals and cells
Wild type NOD/shiJcl mice were obtained from Clea Japan, Inc. (Tokyo,
Japan) and housed in our facility. Female mice were used for all
experiments. Tyk2 KO mice on a C57BL/6J genetic background weregenerated by backcrossing 129/Sv-derived Tyk2 KO mice to C57BL/6J
mice for 12 times11,30. Tyk2KO mice on a NOD/shiJcl genetic background(Tyk2KO.NOD) were generated by backcrossing with NOD/shiJcl micemore than ten times. The STR loci were analyzed by ICLAS Monitoring
Center (Kanagawa, Japan). The microsatellite repeat polymorphisms at
the Idd loci were tested by PCR27. Tyk2 heterozygous and homozygote
mice were healthy and fertile. NOD.SCID mice were obtained from theJackson Laboratory Japan, Inc. (RRID:IMSR_JAX:001303) (Yokohama,Japan) and bred in our facility. Tyk2KO mice on a NOD.SCID genetic
background (Tyk2KO.NOD.SCID) were generated by crossing with
Tyk2KO.NOD mice. The Prkdcscid gene mutation was determined by
PCR68. NY8.3 NOD mice were provided by S. Akazawa and N. Abiru(Nagasaki University, Nagasaki, Japan) (RRID:IMSR_JAX:005868)41.
Tyk2KO mice on a NY8.3 NOD genetic background (Tyk2KO 8.3 NOD)
were generated by crossing with Tyk2KO.NOD mice. Blood glucose
levels were measured in tail vein blood using Glutest Ai (Sanwa,
Nagoya, Japan). Diabetes was deﬁned by a non-fasting blood glucose
level exceeding 250 mg/dL. All mice were maintained on a 12-hour
light/dark cycle in a temperature-controlled facility under speciﬁc
pathogen-free conditions at 23 ± 2 °C with 50 ± 10% humidity, andprovided with sterile food and water ad libitum. SCID mice were fed
with sterile water. Experimental and control animals were housed inco-housed condition. All animals were euthanized by cervical dislocation under isoﬂurane anesthesia. This study was approved by the
Saga University Animal Care and Use Committee and conducted inaccordance with the regulations on animal experimentation at Saga
University.
NIT-1 cells, a pancreatic β-cell line derived from female NOD
mice51, were obtained from ATCC (Manassas, VA, USA). The cells were
cultured and maintained in F-12K medium (Gibco) containing 10% fetal
bovine serum and 1% penicillin and streptomycin (Wako) in 5%
CO2 at 37 °C.

Histology
Pancreas specimens were immersed in 10% (w/v) formaldehyde overnight at 4 °C and then embedded in parafﬁn. Cut sections (3 µm) were
subjected to hematoxylin and eosin (H&E) staining. Insulitis scores
were deﬁned as follows: 0, no insulitis; 1, peri-insulitis; 2, inﬁltrative
insulitis less than 50% of the islet area; and 3, inﬁltrative insulitis morethan 50% of the islet area (Supplementary Fig. 1d).

ELISA
IAA levels in serum were measured using an ELISA kit (Cusabio, Huston, TX, USA) according to the manufacturer’s instructions. The opticaldensity was determined at 450 nm.

Adoptive transfer experiments
Donor splenocytes were puriﬁed from the spleens of non-diabetic 14w
or 24w female mice. Splenocytes (1×107) were intravenously transferred into 6–8w wild type or Tyk2KO.NOD.SCID female recipient mice.
Blood glucose levels were measured in tail vein blood once weekly
using Glutest Ai (Sanwa). To analyze the proliferation of CD8+ T cells
in LNs, naïve 8.3 CD8+ T cells from the LNs of non-diabetic female
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NY8.3 NOD mice were puriﬁed using two rounds of the naïve CD8+ T
cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) with LScolumns (Miltenyi Biotec). The average purity of the sorted cells was
96%. Puriﬁed naïve 8.3 CD8+ T cells were labeled using a CellTraceviolet cell proliferation kit (Invitrogen, Waltham, MA, USA) according
to the manufacturer’s protocol. Then, 4 ×106 labeled naïve 8.3 CD8+
T cells were transferred into 6–9w or 24w recipient mice. Next, 5 or 10d
after the transfer, the PLN and iLN were harvested and the proliferationof CTV labeled cells were analyzed using a FACSVerse ﬂow cytometer(BD Biosciences).

Islets isolation and β-cell sorting
Islets were isolated using 1 mg/ml collagenase-P (Roche) andHistopaque-1077 (Sigma-Aldrich, Tokyo, Japan) as previously described with minor modiﬁcations69. Puriﬁcation of β-cells was performed
as previously described31,32. Brieﬂy, isolated islets were dissociatedusing TrypLE express (Gibco BRL, Grand Island, NY, USA) for 9 min at
37 °C with vortexing every 3 min. Dissociated islet cells were washedwith RPMI-1640 containing 10% FBS and 1% PcSM, and resuspended in
the medium. Dissociated islet cells were stained with FluoZin-3-AM(Invitrogen), TMRE (Life Technologies, Carlsbad, CA, USA), anti-CD45
antibody (BioLegend, San Diego, CA, USA), and anti- MHC class I
antibody (H-2Kd or H-2Db) (BioLegend) for 30 min at 37 °C. Then, 1 mg/
mL propidium iodide (Sigma-Aldrich) was added to the cell suspension
just before sorting to exclude dead cells. FluoZin-3-AM-positive, TMREpositive, and CD45-negative cells were sorted using MA900 (Sony,Tokyo, Japan), and data were analyzed using FlowJo software (TreeStar, Ashland, OR, USA).

Microarray
We used an Affymetrix Clariom D Assay Mouse Array for the β-celltranscriptome analysis. To identify differentially-expressed genes, we
calculated the Z scores and ratios (non-log-scale fold-change) from thenormalized signal intensities for each probe to compare control andexperimental samples. Criteria used for identifying differentiallyexpressed genes were as follows: upregulated genes, Z score >2.0 andratio <1.5; downregulated genes, Z score <−2.0 and ratio >0.66. The
accession number for the data is GEO: GSE235670. Microarray analysis
was supported by Cell Innovator (Fukuoka, Japan).

Flow cytometry analysis
To isolate immune cells from the LNs, thymus, or spleen, tissues wereminced and enzymatically dissociated with 1 mg/mL collagenase D(Roche Applied Science) and 25 U/mL DNase I (Fujiﬁlm Wako, Osaka,
Japan) in DMEM (Fujiﬁlm Wako) for 30 min at 37 °C. The digested tissues were ﬁltered through a 70-μm cell strainer with a syringe plunger.
Red blood cell lysis was performed by incubation with ammoniumchloride-potassium lysis buffer for 4 min on ice. Isolated cells werepreincubated with CD16/32 blocker (clone 93; BioLegend) for 5 minutes at 4 °C to prevent nonspeciﬁc staining, and stained for 20 min at
4 °C with antibodies. We added 1 mg/mL propidium iodide to the cellsuspension to exclude dead cells.
To isolate immune cells from the pancreas, pancreas was perfusedwith 1 mg/mL collagenase D, 0.5 mg/mL trypsin inhibitor (FujiﬁlmWako), and 25 U/mL DNase I (Fujiﬁlm Wako) through the bile duct. The
perfused pancreas was removed carefully from the intestine, stomach,spleen, and LNs, and dissociated for 30 min at 37 °C. Then, 5 mL of
DMEM containing 10% FBS and 1% PcSM was added to the dissociatedpancreas and shaken for 10 s to completely dissociate the tissue. The
digested pancreas was ﬁltered through a 70-μm cell strainer with asyringe plunger. Red blood cell lysis was performed by incubation withammonium-chloride-potassium lysis buffer for 4 min on ice. The
digested pancreas was centrifuged and resuspended in 40% Percoll(GE Healthcare Life Science, Buckinghamshire, UK) layered on 70%
Percoll, and centrifuged at 70 g for 20 min. Cells were collected from10

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the Percoll interface. Isolated cells were preincubated with CD16/32
blocker (BioLegend) for 5 min at 4 °C, and stained for 20 min at 4 °C
with antibodies. We added 1 mg/mL propidium iodide to the cell suspension to exclude dead cells (Supplementary Fig. 2c).
To stain IFN-γ and IL-17A, cells were stimulated with 25 ng/mL PMA(Fujiﬁlm Wako) and 1 mg/mL ionomycin (Fujiﬁlm Wako) for 6 h at
37 °C. Then, 10 mg/mL Brefeldin A (Fujiﬁlm Wako) was added for thelast 5 h of incubation. Intracellular staining was performed using a
Cytoﬁx/Cytoperm Fixation/Permeabilization Solution kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. The intracellular staining of FOXP3, Ki67, T-BET, EOMES, TCF-1,
GZMB, and BLIMP-1, was performed using the Foxp3/Transcription
factor staining buffer set (Invitrogen) according to the manufacturer’s
instructions. Dead cells were excluded using a Zombie Aqua ﬁxableviability kit (BioLegend). AnnexinV staining was performed with
Annexin V binding buffer (BioLegend) according to the manufacturer’s
instructions. Stained cells were analyzed by FACSVerse ﬂow cytometer(BD Biosciences) and data were analyzed using FlowJo software(Tree Star).
The following antibodies were used in this study. Details of theantibody information are shown in Supplementary Table 1. FITC antiCD8a (53-6.7), FITC anti-CD62L (MEL-14), FITC anti-CD4 (RM4-5), FITC
anti- RT1B (I-A g7) (OX-6), PE anti-CD4 (GK1.5), PE anti-CD3 (17A2), PE
anti-FOXP3 (MF-14), PE anti-IFN-γ (XMG1.2), PE anti-KI67 (16A8), PE
anti-T-bet (4B10), PE anti-BLIMP-1 (5E7), PE anti-CD40 (3/23), PE
Annexin V, PerCP Cy5.5 anti-CD3 (17A2), PerCP Cy5.5 anti-GZMB(QA16A02), PerCP Cy5.5 anti-CD44 (1M7), APC anti-CD19 (1D3), APC
anti-CD44 (1M7), APC anti-CD25 (PC61), APC anti-Cxcr3 (CXCR3-173),
APC anti-CD86 (GL-1), APC anti-CD103 (2E7), APC anti-H-2Db (KH95),
APC-Cy7 anti-CD3 (17A2), APC-Cy7 anti-TCR γ/δ (GL3), APC-Cy7 antiCD45 (30-F11), APC-Cy7 anti-CD8a (53-6.7), PE-Cy7 anti-TCR γ/δ (GL3),
PE-Cy7 anti-CD8a (53-6.7), PE-Cy7 anti-CD4 (RM4-5), PE-Cy7 anti-PD1(RMP1-30), BV421 anti-H2-Kd (SF-1.1), BV421 anti-Cxcr3 (CXCR3-173),
BV421 anti-CD103 (2E7), and BV510 anti-CD45 (30-F11) were purchasedfrom BioLegend. PE anti-CD3 (500A2), PE anti-CD11b (M1/70), PerCP
Cy5.5 anti-CD19 (1D3), PE anti-CD44 (1M7), PE-Cy7 anti-EOMES(Dan11mag), and PE-Cy7 anti-CD11c (N418) were purchased fromeBioscience. BV450 anti-CD4 (RM4-5), APC anti-IL-17A (TC11-18H10),
and BV450 anti-CD11b (M1/70) were purchased from BD Biosciences.
PE anti-TCF1 (C63D9) was purchased from Cell Signaling Technology.

Quantitative PCRmRNA was extracted using Isogen II (Nippon gene, Tokyo, Japan) andcDNA was synthesized using a High-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) according to the
manufacturer’s instructions. PCR was performed on a PCR thermal
cycler (Takara, Tokyo, Japan) and real-time PCR was performed using
QuantStudio 3 (Thermo Fisher Scientiﬁc, Waltham, MA, USA). The
relative quantiﬁcation value is expressed as 2−ΔΔCt, where ΔΔCt is thedifference between the mean Ct value of duplicate measurements ofthe sample and the endogenous Actb control. Actb: primer set ID,
MA050368 (Takara Bio); Cxcl10: primer set ID, MA118556 (Takara Bio);
Cxcl9: forward, 5′-CCTAGTGATAAGGAATGCACGATG-3′, reverse, 5′CTAGGCAGGTTTGATCTCCGTTC-3′; Cxcl13: forward, 5′-CATAGATC
GGATTCAAGTTACGCC-3′, reverse, 5′-GTAACCATTTGGCACGAGGAT
TC-3′; Fas: forward, 5′-CTGCGATTCTCCTGGCTGTGAA-3′, reverse, 5′CAACAACCATAGGCGATTTCTGG-3′; Isg15: forward, 5′-CATCCTGGTGAGGAACGAAAGG-3′, reverse, 5′-CTCAGCCAGAACTGGTCTTCGT-3′;
Oas2: forward, 5′-CCTTGGAAAGTGCCAGTACCTA-3′, reverse, 5′CCTTGGTCCTGCCACAAGAT-3′, and Ifnb1: forward, 5′-GCCTTTGCC
ATCCAAGAGATGC-3′, reverse, 5′-ACACTGTCTGCTGGTGGAGTTC-3′.

RNA-sequencing analysis
To analyze CD44hi IGRP CD8+ T cells and CD8+ rDC in the PLN, NOD
mice aged 6w were used. Approximately 300–600 cells were sorted
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and used for the analysis. To analyze IGRP CD8+ T cells in the pancreas,
NOD mice aged 10–12w were used. Approximately 600 cells were
sorted and used for the analysis. Complementary DNA was preparedusing a SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing(Clontech Laboratories, CA, USA). Libraries were prepared using a
Nextera XT DNA Library Prep Kit (Illumine, San Diego, CA, USA).
Samples were run on a NovaSeq 6000 (Illumina). An average of 64
million paired reads were generated per sample. The accession number for the data is SRA: PRJNA992677. RNA-seq analysis was supported
by Takara Bio Inc. (Japan).

Ex vivo co-culture assay
To obtain FACS-sorted CD8+ rDCs, spleens, iLN, and PLN from nondiabetic 6w–8w female mice were minced and enzymatically dissociated with 1 mg/mL collagenase D (Roche Applied Science) and25 U/mL DNase I in DMEM for 30 min at 37 °C, as described above. The
digested tissues were ﬁltered through a 70-μm cell strainer with asyringe plunger. Red blood cell lysis was performed by incubation withammonium-chloride-potassium lysis buffer for 5 min on ice. The
digested tissues were centrifuged and resuspended in 40% Percoll (GEHealthcare Life Science) layered on 70% Percoll, and centrifuged at
70 g for 20 min. Cells were collected from the Percoll interface. Then,isolated cells were preincubated with CD16/32 blocker (BioLegend) for
5 minutes at 4 °C, and stained for 5 min at 4 °C with biotinylated antiCD3 antibody (17A2; BioLegend) to exclude CD3-positive T cells in thecell suspension using anti-biotin microbeads (Miltenyi Biotec) and LS
columns (Miltenyi Biotec). Next, the cells were stained for 5 min at 4 °C
with biotinylated anti-CD19 antibody (1D3; BioLegend) to exclude
CD19-positive B cells in the cell suspension using anti-biotin
microbeads and LS columns. The cells were stained with antibodies(FITC anti-RT1B (OX-6), PE anti-CD11b (M1/70), APC anti-CD103 (2E7),
APC-Cy7 anti-CD8α (53-6.7), PE-Cy7 anti-CD11c (N418), BV510 antiCD45 (30-F11)) and sorted using MA900 (Sony). To obtain naïve 8.3
CD8+ T cells, naïve CD8+ T cells were isolated from the LNs of NY 8.3
NOD mice using two rounds of the naïve 8.3 CD8+ T cell isolation kit(Miltenyi Biotec) with LS columns (Miltenyi Biotec), following the
manufacturer’s instructions. Puriﬁed naïve 8.3 CD8+ T cells werelabeled using a CellTrace violet cell proliferation kit (Invitrogen)according to the manufacturer’s protocol.
For the co-culture assay (naïve CD8+ T, CD8+ rDC, and IGRP peptide), 2 ×104 sorted CD8+ rDC and 1 ×105 CTV labeled naïve 8.3 CD8+
T cells (DC:CD8+ T ratio = 1:5) were mixed and cultured in U-bottom 96well plates in RPMI-1640 with 10% FBS, 1% PcSM, 50 μM 2-mercaptoethanol, and 1 ng/mL IGRP206-214 (VYLKTNVFL). Cultures of CTVlabeled naïve 8.3 CD8+ T cells and 1 ng/mL IGRP206-214 peptide without
CD8+ rDC for 3d were used as a control.
For co-culture assays with NIT-1 cells (naïve CD8+ T, CD8+ rDC, andNIT-1 cells without IGRP peptide), 2 ×104 sorted CD8+ rDC and 1 ×105
CTV labeled naïve 8.3 CD8+ T cells (DC:CD8+ T ratio = 1:5) were mixed
and cultured in U-bottom 96-well plates in RPMI-1640 with 10% FBS, 1%
PcSM, 50 μM 2-mercaptoethanol, and 1 × 104 NIT-1 cells. Cultures ofCTV-labeled naïve 8.3 CD8+ T cells and NIT-1 cells without CD8+ rDC for
3d were used as controls. The cells were cultured at 37 °C and 5% CO2
for 72 h and then the dye dilution was analyzed using a FACSVerse ﬂow
cytometer (BD Biosciences), and data were analyzed using FlowJo
software (Tree Star).

Ex vivo cytotoxicity assay
To assess the effector functions of CD8+ T cells against β-cells, ex vivo
activated 8.3 CD8+ T cells and NIT-1 cells were co-cultured as previously
described52. Brieﬂy, 8.3 CD8+ T cells were isolated from the LNs of nondiabetic NY 8.3 NOD female mice using two rounds of the CD8+ T cellisolation kit (Miltenyi Biotec) with LS columns (Miltenyi Biotec), following the manufacturer’s instructions. The isolated cells were cultured in U-bottom 96-well plates in RPMI-1640 with 10% FBS, 1% PcSM,
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50 μM 2-mercaptoethanol, 30 U/mL IL-2, 5 ng/mL IL-12, and Dynabeads
Mouse T-activator CD3/CD28 (Thermo Fisher) (8.3 CD8+ T:Beads = 1:1)
in the presence or absence of BMS-986165 at 37 °C and 5% CO2 for 3d.
Then, 1 ×105 NIT-1 cells were cultured in ﬂat-bottom 96-well plates in
F-12K with 10% FBS, 1% PcSM, and incubated at 37 °C and 5% CO2 for
24 h. For the co-culture cytotoxicity assay, ex vivo activated 8.3 CD8+
T cells were added to a plate with NIT-1 cells, and co-cultured in F-12K
with 10% FBS, 1% PcSM, and 1 μg/mL IGRP206-214 (VYLKTNVFL) at
37 °C and 5% CO2 for 24 h (NIT-1:CD8+ T ratio = 1:0.1, 1:1, 1:2). To
assess the viability of NIT-1 cells, the supernatants were removed
and washed three times with PBS(-) to remove 8.3 CD8+ T cells.
After washing, 100 μL of F-12K with 10% FBS, 1% PcSM, and 20 μL of
CellTiter 96 (Promega, Tokyo, Japan) were added and incubated at
37 °C and 5% CO2 for 2 h. The absorbance was determined at 450 nm.
The % killing was calculated as follows: % killing = 100 − ((Abs of NIT-1
cells cultured with 8.3 CD8+ T cells)/(Abs of untreated NIT-1 cells) ×100). To analyze the gene expression in NIT-1 cells, supernatants were
removed and washed three times with PBS(-) to remove 8.3 CD8+
T cells. After washing, RNA was extracted using Isogen II (Nippon gene)and cDNA was synthesized using a High-capacity cDNA reverse transcription kit (Applied Biosystems) according to the manufacturer’s
instructions.
To assess the cytokine production in 8.3 CD8+ T cells, ex vivo
activated 8.3 CD8+ T cells and NIT-1 cells were co-cultured in F-12K with
10% FBS, 1% PcSM, and 1 μg/mL IGRP206-214 (VYLKTNVFL) at 37 °C and
5% CO2 for 24 h (NIT-1:CD8+ T ratio = 1:1) with or without 10 μg/mL ofantibodies (anti-mouse IFNAR1 antibody (MAR1-5A3; Leinco Technologies, Fenton, MO, USA), mouse IgG1 isotype control (MOPC-21;
BioXCell, Lebanon, NH, USA), anti-mouse IFN-β antibody (HDB-4A7;
ichorbio, Wantage, UK), mouse IgG2a isotype control (BioXCell), antimouse IFN-α antibody (TIF-3C5; ichorbio), and Armenian hamster IgG
isotype control (RIP; BioXCell)), and 40 μg/mL Brefeldin A (SigmaAldrich) was added for the last 4 h of incubation. To test the Caspase 3
and 7 activation in 8.3 CD8+ T cells, the cells were incubated with
CellEvent Caspase-3/7 green ﬂow cytometry assay kit (Invitrogen)according to the manufacture’s protocol.

BMS-986165
BMS-986165 (deucravacitinib) (6-cyclopropaneamido-4-{[2-methoxy3-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl]amino}-N-(2H3)methylpyridazine-3-carboxamide) was purchased from Selleck Biotech (Tokyo,
Japan). To test the inhibitory effects of BMS-986165 on the development of autoimmune T1D, wild type NOD mice were dosed by oral
gavage once daily with BMS-986165 30 mg/kg in vehicle (EtOH:TPGS:PEG300, 5:5:90) or vehicle alone as previously described53.
To assess the inhibitory effects of BMS-986165 in the priming of
CTLs ex vivo, CD8+ T cells were isolated using two rounds of the CD8+ T
cell isolation kit (Miltenyi Biotec) with LS columns (Miltenyi Biotec)
and cultured with 30 U/mL IL-2, 5 ng/mL IL-12, and Dynabeads Mouse
T-activator CD3/CD28 (Thermo Fisher) (CD8+ T:beads = 1:1) in thepresence or absence of BMS-986165 at 37 °C and 5% CO2 for 3d. To test
the induction of CXCR3 in CTLs, primed CTLs were washed and recultured in U-bottom 96-well plates in RPMI-1640 with 10% FBS, 1%
PcSM, 50 μM 2-mercaptoethanol at 37 °C and 5% CO2 for 2d. To assess
the inhibitory effects of BMS-986165 in bystander activation of T cells,
5 ×105 splenocytes were cultured in 12-well plates in RPMI-1640 with
10% FBS, 1% PcSM, 50 μM 2-mercaptoethanol, and stimulatedwith 20 ng/mL IL-12 (BioLegend) and 20 ng/mL IL-18 (BioLegend)
at 37 °C and 5% CO2 for 24 h with or without BMS-986165; 40 μg/mL
Brefeldin A (Sigma-Aldrich) was added for the last 5 h of the incubation. To assess the inhibitory effects of BMS-986165 in cytokineinduced gene expressions in NIT-1 cells, 5 ×105 NIT-1 cells were cultured
in 12-well plates in F-12K with 10% FBS and 1% PcSM, and stimulatedwith 100 U/mL IFN-β (Pbl Assay Science, Piscataway, NJ, USA) and
0.1 ng/mL IFN-γ (BioLegend) at 37 °C and 5% CO2 for 3 h (for Cxcl9,
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Cxcl10, Cxcl13, Isg15, and Oas2) or 24 h (for Fas) in the presence orabsence of BMS-986165.

Data analysis
Heatmaps were generated with the R (3.6.0) package pheatmap, andclustering was performed using the word.D2 method. Enrichment
analysis was performed by GeneTrail70. PCA analysis was performed
using ClustVis71 and RNAseqChef72. Volcano plots were generatedusing ggVolcanoR73. Likelihood ratio tests were performed to determine differentially expressed genes between genotypes using
RNAseqChef72.

Statistical analysis
Data are expressed as the means ± SEM. Statistical signiﬁcancebetween two groups was determined using two-tailed Student’s t test.
For the analysis of diabetes incidence, Kaplan–Meier survival curves
were estimated by the log rank test. Statistical signiﬁcance of insulitis
levels were analyzed using the Mann–Whitney U-test. To analyze differences among three or more groups, statistical signiﬁcance wasdetermined using one-way ANOVA followed by post hoc Dunnett’s or
Tukey’s multiple-comparisons tests, and two-way ANOVA with Tukey’s
post hoc test. P < 0.05 was considered statistically signiﬁcant. Statistical analysis was performed using Prism 9 (9.5.1) (GraphPad Software).
Differentially expressed genes between pairwise comparisons were
performed using DESeq2 (FDR < 0.1, fold change >1.5) in iDEP.9674. For
the cluster analysis of IGRP CD8+ T cells in the PLN and pancreas,
likelihood ratio tests were used to determine differentially expressed
genes between the genotypes (FDR < 0.1, fold change >1.5) using
RNAseqChef72. No a priori statistical methods were used to determine
the sample size. Sample size sufﬁciency was based on previous
experiments from our laboratory and others. The investigators were
not blinded to the allocation of mice, samples, and outcome data
analyses.

Reporting summary
Further information on research design is available in the Nature
Portfolio Reporting Summary linked to this article.

Data availability
All data analyzed in this study are included in this article and supplementary information, or have been made available in public repositories. The microarray data generated in this study have been
deposited in the Gene Expression Omnibus under accession code
GSE235670. The RNA-sequencing data generated in this study havebeen deposited in the Sequence Read Archive under accession code
PRJNA992677. The representative ﬂow cytometry data generated inthis study have been deposited in the Mendeley Data under https://doi.
org/10.17632/b5bybfddnn.1[https://data.mendeley.com/datasets/
b5bybfddnn/1]. Source data are provided with this paper.

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Acknowledgements
This work was supported by a Japan IDDM Network Research Grant(S.N.), Japan Diabetes Foundation and Costco Wholesale Japan Ltd.
(K.M.), and the NOVARTIS Foundation (Japan) for the Promotion of Science (K.M.). We sincerely thank Shinichiro Sawa, Kota Yanagitani, Shinichi Koizumi, Naoto Noguchi, Shinya Hatano, and Yumiko Tsugitomi for
their counsel on experiments, and Yumiko Kitada, Akiko Takano, Chieko
Ogawa, Saori Fuchigami, Rasheda Perveen, Yoshifumi Morita, and Hiroaki Wakimoto for helping to prepare the manuscript. We appreciatetechnical support from the Analytical Research Center for Experimental
Sciences, Saga University. We thank Edanz (https://jp.edanz.com/ac) for
editing a draft of this manuscript.

Author contributions
Conceptualization, K.M., K.A., and S.N.; Investigation, K.M.; writing original draft preparation, K.M.; writing - review and editing, H.T., H.M.,
S.A., N.A., H.K., K.S., K.A., Y.Y., and S.N.; supervision, S.N.; project
administration, K.M.; resources, S.A., N.A., K.S., Y,Y., and H.K.; funding
acquisition, K.M., and S.N. All authors have read and agreed to the
published version of the manuscript.

Competing interests
The authors declare no competing interests.

Additional information
Supplementary information The online version contains
supplementary material available athttps://doi.org/10.1038/s41467-024-45573-9.
Correspondence and requests for materials should be addressed to
Keiichiro Mine.
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