Targeting abatacept-resistant T-helper-17 cells by aldehyde dehydrogenase inhibition

Summary IL-17-producing helper T (Th17) cells are long-lived and serve as central effector cells in chronic autoimmune diseases. The underlying mechanisms of Th17 persistence remain unclear. We demonstrated that abatacept, a CD28 antagonist, effectively prevented the development of skin disease in a Th17-dependent experimental autoimmune dermatitis model. Abatacept selectively inhibited the emergence of IL-7R-negative effector-phenotype T cells while allowing the survival and proliferation of IL-7R+ memory-phenotype cells. The surviving IL-7R+ Th17 cells expressed genes associated with alcohol/aldehyde detoxification and showed potential to transdifferentiate into IL-7R-negative effector cells. Inhibiting aldehyde dehydrogenase reduced IL-7R+ Th17 cells in vivo, independently of CD28, and exhibited additive effects when combined with abatacept. Our findings suggest that CD28 blockade prevents inflammation without eliminating persistent memory cells. These remaining memory cells can be targeted by other drugs, such as aldehyde dehydrogenase inhibitors, to limit their survival, thereby facilitating the treatment of chronic autoimmune diseases.


INTRODUCTION
Autoimmune diseases affect approximately 5% of the global population. 1 Biological drugs targeting proinflammatory cytokines are effective; however, disease recurrence during or after treatment is often problematic. 24][5] Th17 cells regulate inflammation by producing proinflammatory effector cytokines, such as IL-17, tumor necrosis factor alpha (TNF-a), IL-22, interferon gamma (IFNg), and granulocyte-macrophage colony-stimulating factor (GM-CSF).Furthermore, Th17 cells survive longer than other T cells.7][8] Understanding the mechanisms underlying long-term survival of autoreactive Th17 will be useful for deciding the optimal treatment of chronic autoimmune diseases.
CD28, the best-known costimulatory receptor, 9 mediates T cell activation through IL-2 production 10 and resistance to apoptosis. 11At the molecular level, CD28 ligation activates serine/threonine kinase Akt, nuclear factor kB (NF-kB), and the mechanistic Target of Rapamycin (mTOR) that stimulate uptake and metabolism of glucose for full activation and differentiation into effector cells. 12,13Abatacept, a fusion protein of CTLA-4 and immunoglobulin (Ig) antagonizes CD28 by competing with costimulatory ligands (CD80 and CD86), acts as a strong inhibitor of T cells, and has been used for the treatment of autoimmune diseases.CD28 serves as a primary checkpoint in T cell activation but also may be involved in the peripheral maintenance of T cell homeostasis such as Th17 cells.
Desmoglein 3 (DSG3) is an adhesion molecule that is primarily expressed on keratinocytes.It is also the target autoantigen in pemphigus vulgaris, an autoimmune blistering disease. 14T cells extracted from mice carrying a DSG3-specific T cell receptor (Dsg3H1 TCR Tg mouse; hereafter simply designated as Dsg3H1) are known to directly infiltrate the epidermis and induce cellular immunity in DSG3-bearing keratinocytes and cause interface dermatitis after adoptive transfer into Rag2À/À mice. 15,16sing the modified chronic experimental autoimmune dermatitis (EAD) model, we demonstrated that CD28 signal plays a key role in activation and effector function of Th17.Abatacept treatment completely blocked the development of skin inflammation by inhibiting activation and proliferation of effector T cells.In contrast, IL-7 receptor (IL-7R)-positive Th17 cells with memory-like phenotype were resistant to abatacept and remained in the body.To inhibit abatacept-resistant remaining Th17 cells in vivo, we extensively characterized this population and discovered that ALDH inhibitors can prevent the formation of this memory population.

Pathogenic Th17-dependent mouse model of chronic skin inflammation
We previously reported a Th17-dependent EAD model in mice. 16Briefly, naive CD4 + T cells extracted from Dsg3H1 mice 15 were purified and activated in vitro under Th17 polarizing condition.Subsequently, when these Th17 cells were transferred into lymphocyte-deficient Rag2 knockout mice, they induced IL-17-dependent subacute skin inflammation, both histologically and immunologically resembling psoriasis. 16nfortunately, severe weight loss and rapid death of recipient mice following cell transfer prevented us from analyzing persistent autoimmune disease in this model ( 16 and unpublished).
To investigate long-term Th17 cell survival in the persistent EAD model, we used sublethally irradiated wild-type mice as recipients.Additionally, during T cell differentiation culture, we added IL-1b, known to promote the development of long-lived pathogenic Th17 cells (pTh17; Figure 1A and STAR methods section).The resulting pTh17 cells exhibited stronger production of IL-17A compared with normally skewed Th17 cells (nTh17).Furthermore, pTh17 cells produced IL-17F (Figure S1A), which is suggested to be produced by more epigenetically committed Th17 cells. 17Genes encoding IL-23 receptor and GM-CSF (Il23r and Csf2, respectively) are higher in pTh17 than nTh17 (Figure S1B).Consequently, we termed the induced cells ''Dsg3H1-pTh17,'' representing pathogenic Th17-skewed Dsg3H1 cells.
When Dsg3H1-pTh17 cells were transferred into irradiated syngeneic wild-type C57BL/6 mice, they induced dermatitis at a slower rate than in Cd3e KO recipients (which lack endogenous T cells and showed EAD with kinetics similar to Rag2 KO recipients) (Figure 1B).The skin inflammation, typically affecting the ears, back, neck, and/or tail, persisted for at least a month without the death of recipient mice (Figure 1C and data not shown).Thickened skin with a massive infiltration of mononuclear cells in epidermal and dermal tissues was evident (Figure 1C).In the affected skin, strong expression of cytokines, such as IL-17A, IFNg, IL-6, and TNF-a, were detected, indicating severe inflammation caused by Dsg3H1-pTh17 (Figure 1D).
Using congenically labeled donor T cells (pTh17 prepared from Dsg3H1 transgenic, CD45.1 congenic mice) allowed us to discriminate transferred cells via fluorescence-activated cell sorting (FACS) analyses in the recipients (CD45.2).In support of Th17-dependent inflammation, the transferred donor Dsg3H1-pTh17 cells (CD45.1 + ) were detected in epidermal and dermal tissues after 2 weeks, as well as in skindraining lymph nodes and spleen (Figure 1E, upper panels).In contrast, Dsg3H1 T cells skewed into Th1 (Dsg3H1 Th1; Figure 1E lower panels) did not persist in the skin nor induce skin inflammation in vivo.Moreover, Dsg3H1-pTh17 cells neither caused any inflammation nor exhibited survival beyond 2 weeks in vivo (data not shown) in non-irradiated wild-type mice.Taken together, we have successfully developed a chronic EAD model induced by the injection of pathogenic Th17 cells reactive to a defined autoantigen in the skin.

CD28 blockade by abatacept prevents Th17-mediated skin inflammation
Abatacept, a human CTLA-4 Ig that inhibits CD28 signaling, was previously shown to ameliorate human psoriasis. 18,19However, its impact on skin-reactive helper T cells remains unknown.Therefore, we conducted tests using abatacept in our model.Remarkably, mice that received Dsg3H1-pTh17 cells and were treated with abatacept showed nearly complete prevention of skin lesions, indicating the critical role of CD28 signaling in skin inflammation (Figures 2A and 2B).
To facilitate tracking of transferred cells and assessment of proliferative responses, we utilized congenic marker (CD45.1)and a proliferation reporter dye (CTV) (Figures 2C and 2D).Abatacept-treated recipients showed fewer donor cells compared with control mice (Figure 2C).While a significant increase in the proliferation of donor CD4 + cells was observed in mice treated with control Ig, abatacept-treated mice exhibited inhibition of proliferative responses in transferred pTh17 cells (Figure 2D).Glucose transporter 1 (GLUT1), a target gene of CD28 and a hallmark indicator of glucose metabolism and extensive T cell proliferation, 12,13 showed significantly lower expression in donor cells derived from abatacept-treated recipients (Figure 2E).These findings suggest that abatacept exerts an inhibitory effect on pTh17 cells.

CD28 drives expression of proinflammatory genes in pathogenic Th17
The robust effects of abatacept prompted further investigation into the fundamental roles of CD28 signaling in pTh17 cells.Therefore, we performed an RNA-sequencing analysis on fully differentiated Dsg3H1-pTh17 cells that were restimulated with plate-immobilized antibodies (Figure 3A).Importantly, the ''CD3-stimulated'' and ''CD3+CD28-stimulated'' samples showed distinct clustering in principal-component analysis of RNA-sequencing data, as early as 2 h after restimulation (Figure 3B).
Analysis of differentially expressed genes (DEGs) revealed that the rapid induction (at 2 h) of effector cytokines and transcriptional factors critically depend on CD28 signaling (Figure 3C).Specifically, we found that most CD28-dependent genes were cytokines (including those encoding IL-21, IL-2, TNF-a, GM-CSF, CCL4, XCL1, CCL20, IL-2, IL-31, and Tnfrsf4) and transcriptional regulators (Nfkbid, Fos, Nfkbia, Maff, and Atf3) that play pivotal roles in inflammation.These genes coexisted with an antiapoptotic protein, Bcl-XL (encoded by Bcl2l1) (Figure 3C).A gene set enrichment analysis (GSEA) demonstrated that ''CD3+CD28-stimulated cells'' exhibited a strong bias toward the ''INFLAMMATORY RESPONSE'' signature compared with cells stimulated by CD3 alone (Figure 3D).We confirmed these findings by reactivating Dsg3H1-pTh17 cells using the physiologic cognate peptide recognized by Dsg3H1 T cells 15 (Figure S2A).We observed that secondary proliferation (Figure S2B) and the production of effector cytokines (Figure S2C) were significantly augmented by CD28 signaling.These findings indicate that signals mediated by CD28 are critically involved in the secondary response of pTh17 cells.

Abatacept inhibits IL-7R neg inflammatory T cells but not IL-7R pos memory T cells
Our model allowed us to examine the phenotypes of transferred pTh17 cells following CD28 blockade by abatacept.One week after transfer, we conducted an extensive analysis of donor Dsg3H1-pTh17 cells isolated from lymph nodes using a combination of congenic markers (CD45.1), a proliferation reporter dye (CTV), and other markers associated with effector/memory responses.We observed that the majority of proliferated CTV-diluted (CTV dil ) cells exhibited a low/negative ( neg ) phenotype for the IL-7 receptor a (hereafter referred to as IL-7R), and those from abatacept-treated mice were much fewer in number compared with control mice (Figures 4A and 44B left panels).In contrast, donor-derived cells that survived in abatacept-treated mice were mostly IL-7R positive ( pos ).Moreover, the number of CTV dil IL-7R pos cells was not affected by abatacept, indicating that these cells proliferated independently of CD28 signaling (Figure 4B right).
We compared transcriptome data between the CTV dil IL-7R pos and CTV dil IL-7R neg populations in donor cells isolated from control mice (Figure 4C).Genes highly expressed by CTV dil IL-7R neg cells included effector molecules (such as Gzmb and Fasl), cytokines (Il21, Ifng, Spp1; osteopontin), chemokine receptors (Cxcr5 and Cx3cr1), transcription factors (TF) (Nr4a1 and Nr4a2), and cell surface molecules (Havcr2 and Tigit), all of which suggested strong T cell activation (Figure 4D).In contrast, IL-7R pos cells expressed genes related to Th17 cells (Satb1, Mafb, Ccr4, Ccr6, and Rara; Figure 4D).These findings suggested that inflammatory cells in the IL-7R neg population were inhibited by abatacept.To directly examine cytokine expression, we sorted CTV dil IL-7R pos and CTV dil IL-7R neg cells from recipients treated with control Ig or abatacept.An equal number of sorted cells were then restimulated with the cognate antigenic Dsg3H1 peptide.Regardless of the treatment, CTV dil  IL-7R neg cells produced higher levels of IFNg, whereas CTV dil IL-7R pos cells expressed IL-2 (Figures 4E and 4F).Both populations produced a comparable amount of IL-17A, indicating that they are subpopulations of Th17 cells (Figure 4E).Importantly, the sorted cells proliferated similarly (Figure 4F), indicating that unlike naive T cells, Th17 cells did not become anergic following CD28 blockade.
We conducted experiments to investigate whether IL-7R-positive and -negative pTh17 cells can transdifferentiate into each other and set up a transfer experiment into secondary recipients (Figure 4G).We observed that most CTV dil IL-7R pos cells, when transferred into secondary recipients, became IL-7R neg , whereas most CTV dil IL-7R neg cells remained IL-7R neg (Figure 4H).Taken together, these results suggested that pTh17 cells comprise two distinct populations.Abatacept appeared to block the IL-7R neg effector-like Th17 cells but allowed the survival of IL-7R pos memory-like cells that have the potential to transdifferentiate into IL-7R neg effector cells.

Abatacept inhibits effector signature but allows survival of persistent memory cells
We conducted RNA sequencing, to directly compare the entire donor T cell population isolated from recipients treated with either control Ig or abatacept (Figures 5A and 5B).As anticipated from the in vitro data, cells from abatacept-treated mice showed decreased expression of cytokines and transcription factors (TFs) associated with effector function (Figures 5C and 5D).The cytokines downregulated by abatacept treatment included those typically expressed by activated helper T cells, such as Ifng (Th1), Il4 (Th2), Il21 (T follicular helper; Tfh), and Spp1.The TFs downregulated by abatacept also encompassed lineage-specific TFs such as Tbx21 (T-bet; Th1), Foxp3 (Treg), Bcl6 (Tfh), and those involved in activation and function (Eomes, Tox, Tox2, Nfatc1, Nr4a2, Ezh2, and Batf), indicating a clear inhibition of effector function.
Conversely, T cells recovered from abatacept-treated recipients did not show significant upregulation of cytokines or chemokines, except for Ccl1.However, they exhibited upregulation of unique transcriptional factors (Nr1d1, Satb1, Bhlhe41, Myb, and Foxq1).SATB1 20 and NR1D1 (REV-ERBa) [21][22][23] have been reported to be involved in the development and function of Th17 cells.Myb is known to be essential in CD62L pos stem cell memory development. 24,25These findings collectively support the notion that CD28 blockade by abatacept inhibited the effector function of Th17 cells while preserving a unique IL-7R pos memory population.

Abatacept-resistant memory Th17 cells exhibit genes for aldehyde dehydrogenases
We further investigated the genes that may function on T cells extracted from abatacept-treated mice.Cells from control mice showed genes associated with ''glycoprotein metabolic process,'' ''response to virus,'' and ''carbohydrate derivative catabolic process'' signatures, suggesting the reliance of the cells depends on glycolysis for proliferation and effector function (Figure 6A).In contrast, cells from abatacept-treated mice showed genes linked to ''cholesterol metabolism pathways,'' ''carbohydrate biosynthesis process,'' and ''amino acid metabolism process,'' including Dhcr24, Acsl3, Them4, and Acss2 (Figures 6A and 6B).These findings align with previous reports that highlight the importance of cholesterol and lipid metabolism in the survival and pathogenicity of Th17 cells. 26,27n addition to genes involved in energy acquisition, we observed a significant upregulation of genes related to alcohol metabolism in donor cells treated with abatacept (categorized in ''ethanol oxidation''; Aldh2, Acss2, Aldh1b1, Acat1, Fpgs, Uros, Mthfd1, Acsl3, Aldoc, Aldh6a1, etc.; Figure 6C).We were particularly intrigued by the upregulation of aldehyde dehydrogenase (ALDH) genes (Aldh2, Aldh1b1, and Aldh6a1) for several reasons.First, ALDH plays a role in regulating stemness in hematopoiesis 28 and malignancy. 29Second, ALDH is involved in mitochondrial function. 30Third, ALDH may contribute to T cell survival. 31Fourth, ALDH expression has been reported in supporting Treg survival in humans. 32Importantly, we also found that Aldh2 was upregulated in IL-7R pos donor T cells extracted from both control and abatacept-treated mice (compared with IL-7R neg donor T cells.)(Figure 6D) Consequently, Aldh2 was upregulated in both IL-7R pos cells and T cells isolated from abatacept-treated mice (that are enriched in IL-7R pos memory cells in independent cohorts (Figure 6E).Therefore, our model reveals that memory-phenotype pTh17 cells exhibit a unique metabolic pathway that may involve ALDH for both survival and function.

Abatacept together with ALDH inhibitor targets memory Th17 cells
We aimed to explore the role of ALDH in Th17 activity both in vitro and in vivo.We used cyanamide and disulfiram, which are traditionally used as anti-alcoholic drugs.Treating DsgH1-Th17 cells with cyanamide or disulfiram inhibited cytokine production at lower doses and induced cell death at higher doses (Figure 7A).Subsequently, we treated EAD mice with cyanamide, either alone or in combination with abatacept (Figure 7A).We observed that mice treated with cyanamide showed a marked reduction in both IL-7R pos CTV dil and IL-7R neg CTV dil donor cells (Figure 7B).In contrast, abatacept alone selectively reduced the IL-7R neg CTV dil population (but not IL-7R pos CTV dil ) (Figure 7C).Notably, recipient mice treated with a combination of abatacept and cyanamide showed fewer CTV dil cells than those receiving single treatments, suggesting an additive effect.
Finally, we confirmed the effects of the two drugs by examining actual cytokine expression in CTV dil cells.As presented in Figure 7D, mice treated with abatacept or cyanamide alone showed a significant reduction in IL-17-producing cells, and those treated with the combination showed an additive effect.In contrast to IL-17, IFNg production was almost completely inhibited by abatacept alone, whereas cyanamide alone or in combination with abatacept had minimal effects on IFNg (Figure 7E).
These findings suggested that the inhibition of ALDH and CD28 affects self-reactive pathogenic Th17 cells through distinct mechanisms.ALDH inhibition had inhibitory effects on the survival and IL-17 expression of IL-7R pos memory Th17 cells, whereas CD28 inhibition primarily affected the differentiation into effector Th17 cells.Importantly, the combination of both treatments had the most pronounced effect in reducing both memory and effector Th17 cell populations.

ALDH2 correlates with IL-17 production in human cancer
Lastly, we investigated whether ALDH2 expression is functionally correlated with Th17 activity in humans, particularly in a cancer context.Upon re-examining of The Cancer Genome Atlas data, we observed a weak but significant correlation between ALDH2 and the IL-17A gene in certain types of cancers (Figure 8).Notably, IL-17A expression was evident in a limited fraction of patients, and it showed a significant correlation with high ALDH2 expression.Although these findings are preliminary, they suggest that ALDH2 may play a role in Th17 activity in humans.

DISCUSSION
We demonstrated that CD28 blockade selectively inhibits effector Th17 cells that are highly differentiated, leading to the complete inhibition of dermatitis.Traditionally, it has been thought that naive T cells that receive TCR signals without CD28 activation become anergic or unresponsive, thereby contributing to tolerance. 9Given that the CD28/PI-3K/AKT axis is a hallmark of glycolysis, 12 it is plausible that abatacept inhibits aerobic glycolysis, which is essential for extensive proliferation and the expression of effector cytokines.However, we observed that abatacept did not inhibit the proliferation of memory-like T cells.This abatacept-resistant memory-phenotype Th17 population may explain the persistence of the disease, leading to recurrence during or after treatment.Recently, certain immunosuppressants, such as rapamycin, 33 MEK inhibitors, 34 and tyrosine kinase inhibitors, 35 have been found to induce long-term memory populations and sustain chronic immune responses.Therefore, in the context of autoimmunity, a potential drawback of CTLA-4 Ig is that although it may ameliorate inflammation by blocking CD28, it could also generate persistent, long-term memory Th17 cells by preventing exhaustion.Indeed, our data, along with previous research, indicate that memory-like Th17 cells can give rise to pathogenic effector cells (as shown in our data and by others 6 ).
Regarding the mechanisms underlying the persistence of Th17 cells, Muranski et al. 8 have suggested the possibility of stemness, whereas Karmaus et al. 7 have proposed the existence of two metabolically distinct populations.Our data support the existence of two distinct Th17 populations that show different responses to CD28 blockade.
Interestingly, in our study, the remaining memory-like Th17 cells expressed ALDH genes and can be targeted through systemic inhibition of ALDH.Therefore, ALDH not only controls stem cells but is also involved in the detoxification of endogenously produced aldehydes.7][38] Aldehydes are known to inhibit T cells, as exemplified by excessive alcohol consumption negatively impacting follicular helper T cells and attenuating immune responses. 39,40Aldh2-deficient mice show impaired T cell responses, which are associated with altered metabolism. 31Our data suggested that the inhibition of ALDH may lead to increased intracellular aldehyde concentrations in memory T cells, potentially affecting immune function.
An important question arises: does the genetic diversity of ALDH genes influence T cell immune responses in humans?The most wellknown single-nucleotide polymorphism (SNP) in the ALDH2 gene causes loss of function and is predominantly found in the East Asian population. 41Despite the population with this SNP having low ethanol consumption, it is associated with cancer susceptibility and progression.This suggests that the detoxification of endogenous aldehydes by ALDH is not negligible in tumorigenesis and progression.Furthermore, our preliminary data demonstrated a positive correlation between ALDH2 and IL-17A expression in certain cancers, such as head and neck carcinoma, colon adenocarcinoma, and esophageal cancer.This finding suggested a potential contribution of ALDH activity to memory or effector Th17 responses, which may be beneficial in the context of cancer.
In conclusion, we demonstrated a unique role of ALDH regulating Th17 cell responses.This systemic control of ALDH may hold promise for designing future treatment strategies for diseases involving T cell responses.

Limitations of the study
Firstly, we used irradiated wild-type mice as recipients rather than Rag2 knockout mice.Consequently, we did not address the potential contributions of recipient-derived lymphocytes in the establishment of EAD, including epitope spreading, autoantibody production, and T regulatory cell activity.
Secondly, although we demonstrated the persistence of CTV dil IL-7R pos cells after abatacept treatment and their ability to produce IL-7R neg effector cells, the limited cell numbers prevented us from directly confirming whether this phenomenon contributed to disease recurrence after discontinuing treatment.
Thirdly, the effects of abatacept and ALDH inhibitors on tissue resident memory T cells remain unclear and warrants further investigation.Fourthly, in terms of clinical relevance, comprehensive analyses examining whether ALDH expression is functionally correlated with Th17 cells in human autoimmune dermatitis or other autoimmune diseases are currently lacking.Future studies should address these points to provide a more comprehensive understanding of the topic.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

METHODS DETAILS
Th17-mediated EAD model The in vitro activation procedure of Dsg3H1 T cells was modified from Nishimoto et al. 16 Lymph node cells from 4-6-week-old Dsg3H1 mice were subjected to magnetic sorting using naive CD4 microbeads (Miltenyi) and LS columns (Miltenyi), following the manufacturer's protocol.The sorted T cells were cultured in a 24-well tissue culture plate (Corning) coated with anti-CD3 and anti-CD28 mAbs (2 mg/mL each; Biolegend) at a density of 2 3 10 5 /mL in 1 mL of T cell culture media (RPMI medium supplemented with 10% fetal calf serum (FCS), penicillin/ streptomycin, non-essential amino acid solution, HEPES solution, sodium pyruvate solution, and 55 mM 2-mercaptoethanol [RPMI and supplements were all procured from Nacalai Tesque, Kyoto, Japan, except 2-ME: Gibco]).To induce pathogenic Th17, mouse IL-6 (20 ng/mL), mouse IL-23 (20 ng/mL), human TGF-b (2 ng), mouse IL-1b (10 ng/mL), anti-mouse IFN-g (5 mg/mL), and anti-mouse IL-4 (5 mg/mL) were added to the culture.Three days later, cells were harvested from the plate and further expanded in the presence of mouse IL-23 (20 ng/mL) and mouse IL-2 (20 ng/mL) for another 3 days.To induce control Th1, IL-12 (20 ng/mL) and anti-mouse IL-4 (5 mg/mL) were added to the culture.Three days later, cells were harvested from the plate and further expanded in the presence of mouse IL-2 (20 ng/mL) for another 3 days.On the day of transfer, 4-5 million expanded T cells were resuspended in phosphate-buffered saline (PBS) and intravenously injected into 5Gy-irradiated wild-type C57BL/6NCrSlc or non-irradiated B6 CD3e KO mice.In some experiments, donor Dsg3H1 TCR Tg mice were further bred to (RNKAEFHQSVISQYR) in 0.2 mL of T cell culture media.In Figures 2 and 2 mg/ml anti-CD28 was additionally added to the indicated wells.After three days, cytokine production and cell proliferation were measured using Cytometric Bead Array (BD) and Cell Count Reagent SF, respectively, following the manufacturers' instructions.

Serial transfer model
For the experiment presented in Figures 4G and 4H, spleen and lymph node cells were pooled from 3 to 5 recipients 1 week after transfer.Donor cells were sorted as described above, and then 3 3 10 5 cells were intravenously transferred to 5Gy-irradiated C57BL/6N mice.The mice were analyzed 1 week later.

RNA sequencing
Total RNA was isolated using the RNeasy Plus Micro Kit (Qiagen).Libraries were prepared using the TruSeq stranded mRNA Library kit and sequenced on a NovaSeq 6000 (Illumina) to obtain 150-bp paired-end reads.HISAT2 version 2.1.0was used to map the RNA-seq data to the mouse genomic DNA sequences (mm10).Read counts, fragments per kilobase of exon per million mapped fragments, and transcripts per million were calculated using featureCounts version 1.6.3.The samples were clustered using the Wald method based on Euclidean distances of the normalized counts, utilizing the stats (Version 3.6.1)and ggplots (Version 3.0.1.1)R packages.Then, DEGs were identified using DESeq2 version 1.30.1.

Quantitative reverse transcription-PCR
Total RNA was reverse-transcribed by High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher.)Resulting cDNA was amplified with SsoFast EvaGreen Supermix (Bio-Rad) and CFX Connect Real-time PCR system by according to manufacturers' protocols.Primers used for PCR are listed in the key resources table.

Bioinformatics
Enrichment of GO biological processes was performed using Metascape. 42GSEA 43 was conducted using the GSEA desktop application (ver.4.2.3).Pathway analysis and molecular characterization information were obtained using Ingenuity Pathway Analysis (IPA; Qiagen).

Analysis of human cancer database
Analysis of The Cancer Genome Atlas (TCGA) database for gene correlation was conducted with the assistance of the Timer 2.0 resource. 44,45ta deposition All RNA sequence data have been publicly deposited on NCBI under accession #DRA016062 (run numbers DRR457517-DRR457541).

QUANTIFICATION AND STATISTICAL ANALYSIS
All statistical analyses, except for RNA-seq data, were performed using GraphPad PRISM software (ver.8.4.3).Student's T-test was used for comparing two groups, while multiple comparisons of one-way ANOVA were used for databases involving more than three groups.Cumulative incidence of dermatitis in experiments was analyzed using the Kaplan-Meier method with log rank tests.For gene correlation analysis, partial Spearman's correlation was determined through TIMER 2.0 analysis.Detailed statistical information for each experiment and the number of replicates can be found in the corresponding figure or figure legends.

Figure 1 .
Figure 1.Highly polarized skin-reactive Th17 cells induce chronic dermatitis upon transfer (A) Schematic representation of the induction of pathogenic Th17 cells from desmoglein-3-specific Dsg3H1-TCR transgenic mice.Refer to the STAR methods section for details.(B) Development and kinetics of dermatitis in recipient mice.Cumulative results from three experiments.Kaplan-Meier method with log rank test.(C) Macroscopic (tail and ear) and microscopic (ear skin) views of dermatitis.Scale bars: 200 mM.(D) mRNA expression of inflammatory cytokines in epidermal tissue two weeks after transfer.Two-tailed t test (N of 4 and 7).(E) Survival of transferred T cells two weeks after transfer.Donor T cells were detected by the CD45.1 congenic marker ex vivo.The data represent one representative datapoint from more than 10 mice.

Figure 2 .
Figure 2. Prevention of dermatitis by abatacept, a CD28 antagonist (A and B) Development and kinetics of dermatitis.A group of mice received human CTLA-4 Ig (abatacept; 200 mg/body, every 3 days) after the transfer of CellTrace Violet (CTV)-labeled pTh17 cells prepared from Dsg3H1-TCR Tg CD45.1 congenic mice.Control mice received the same dose of human Ig.Data from three independent experiments.(C and D) In vivo detection of donor cell proliferation one week after transfer.Gated CD4 + CD45.1 + donor cells (C) were evaluated for CTV dilution (D, left histogram), and the proportion of CTV-diluted cells is shown in (D).(N = 6 each) (E).Gated donor cells (C) were stained with Glut1 antibody (N = 3 each).Two-tailed t test.

Figure 4 .
Figure 4. CD28 blockade inhibits IL-7R neg inflammatory T cells but not IL-7R pos memory-phenotype Th17 cells (A and B) Transferred donor T cells were examined for the expression of IL-7R.Abatacept significantly prevented the emergence of CTV-diluted (dil) IL-7Rnegative (neg) population (B, left) but showed no effects on the IL-7R-positive (pos) counterpart.(Two-tailed t test, N of 6 and 7, three experiments).(C) Cell sorting for RNA sequence.(D) A heatmap showing cytokines, chemokines, receptors, and transcriptional factors differentially expressed between CTV dil IL-7R neg and CTV dil IL-7R pos donor cell populations.(N = 3 each, one experiment).(E and F) Cytokine production and proliferation of donor T cells recovered from treated recipient mice.(G and H) A scheme (G) and the result (H) for a serial transfer experiment.Two experiments.

Figure 5 .Figure 6 .
Figure 5. CD28 blockade inhibits effector function but leaves unique memory-phenotype T cells (A) Sorting strategy for RNA-sequencing analysis.CD4 + T cells from recipients were first enriched magnetically, and CD4 + CD45.1 + donor T cells were purified by FACS.(B) Principal-component analysis of RNA-sequencing data.(N = 5 each).(C) A volcano plot of RNA-sequencing data representing genes expressed in CD4 + donor T cells.Cytokine genes ( ) and transcriptional regulators ( ) are highlighted.represents significant DEGs (log2Fc > 2, P adj < 0.05).(D) Heatmaps demonstrating the expression of cytokine genes and transcriptional regulators.The experiment was performed once.

Figure 7 .
Figure 7. Cyanamide, an ALDH inhibitor, inhibits memory-like Th17 cells independently of CD28 blockade (A) Effects of an ALDH inhibitor on pTh17 cells in vitro.Dsg3-H1 pTh17 cells were restimulated in vitro with or without inhibitors.Three days later, cytokine production and cell viability were evaluated.Multiple comparisons by one-way ANOVA using no inhibitor as control (****; p < 0.0001).(B) Treatment of EAD mice with an ALDH inhibitor in vivo.Groups of mice were treated with daily cyanamide (cya) with or without abatacept (aba).(C and D) FACS analysis.For (D), lymph node cells from the treated mice were isolated and stimulated ex vivo to induce cytokines (see STAR methods).IL-17A pos (C) and IFNg pos (E) cells were analyzed using a separate staining.One-way ANOVA (*p < 0.05, **p < 0.001, ****p < 0.0001).The number of mice is indicated in the graphs.Each experiment was performed two times.

TABLE
d RESOURCE AVAILABILITY B Lead contact B Materials availability B Data and code availability d EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS B Animals d METHODS DETAILS B Th17-mediated EAD model , with food and water provided ad libitum.All animal experiments were conducted according to the approved protocol (#80006) of the Animal Ethics Committee of Keio University Medical School.