Solid tumors induce systemic immune suppression, including impaired B lymphopoiesis in the bone marrow (BM). This study elucidates a novel mechanism by which remote tumors disrupt early B cell development. We demonstrate that tumor-bearing hosts exhibit skewed BM hematopoiesis favoring myeloid lineages at the expense of lymphoid lineages, specifically B cells. This impairment originates at the Common Lymphoid Progenitors (CLPs) stage. Mechanistically, monocytes, particularly CXCR2⁺ monocytes within the BM of tumor bearing mice leads to significantly elevated secretion of inflammatory cytokines TNF-α and IL-1β. These cytokines suppress CLPs differentiation into B cells. Furthermore, the enhanced secretion of TNF-α and IL-1β is mainly amplified by elevated levels of myeloid-derived S100A8/A9 proteins in the tumor-bearing BM microenvironment. Collectively, these findings identify the S100A8/A9 / TNF-α/IL-1β axis as a critical pathway disrupting B lymphopoiesis in cancer, revealing potential therapeutic targets to restore immune competence and improve immunotherapy responses.

Obesity remains a worldwide health issue, with visceral adipose tissue as a leading driver of this pathology. As the executors of biological functions in living cells, proteins have their activity regulated by diverse post-translational modifications, including ubiquitination. However, obesity-related changes in ubiquitination of visceral adipose tissue (VAT) proteins are still poorly understood. Here, we obtained the global proteomic and ubiquitylomic data of epididymal VAT from lean and obese male mice by mass spectrometry. Our proteomic analyses revealed significant changes of metabolic pathways involved in fatty acid, acyl-CoA and branched chain amino acids metabolism in obese VAT. Intriguingly, a comparative analysis of proteomic and ubiquitylomic data highlighted discordance in the quantity changes of certain proteins and their ubiquitination levels. Notably, STEAP4 exhibited a markedly reduced protein level coupled with an enhanced K48-linked ubiquitination, suggesting a potential role for ubiquitination-mediated proteasome degradation in VAT dysfunction. Further in vitro experiments revealed that knockdown of STEAP4 in adipocytes impaired mitochondrial function of 3T3-L1 adipocytes. Collectively, this study introduces the first combined proteomic and ubiquitylomic examination of murine VAT, offering novel insights and potential therapeutic targets for obesity.

The OP9 culture system is an important in vitro model for B cell development. However, the complex nature of operations and the intrinsic variability of stromal cell functionality, which can be influenced by factors such as radiation exposure or contamination, pose considerable challenges to their wider application. Currently, there exists a paucity of studies documenting in vitro B cell differentiation culture systems that exclude stromal cells, and the experimental methodologies available for reference remain limited. This report elucidates a robust stromal cell-free culture system. Specifically, bovine serum albumin (BSA) or fetal bovine serum (FBS), in conjunction with interleukin-7 (IL-7), Flt3 ligand (Flt3L), and stem cell factor (SCF), were incorporated into X-VIVO15 medium. This system proficiently facilitates the directed differentiation of common lymphoid progenitor cells (CLP), defined as lineage-CD127 + CD117lowsca-1lowCD135+, into B lymphocytes in vitro, achieving an amplification factor of up to one hundredfold. We examined the roles of IL-7, Flt3L and SCF in differentiation of B cells from CLP in this culture system. Our findings indicate that IL-7 is a pivotal cytokine essential for B cell differentiation in vitro, demonstrating a notable synergistic impact when combined with SCF and FLT3L. Moreover, this system is capable of supporting the differentiation of hematopoietic stem cells (HSCs) and lymphoid-primed multipotent progenitor cells (LMPPs) into B cells in vitro. The findings substantiated the efficacy of the culture system in investigating the in vitro differentiation of bone marrow-derived progenitor cells into B cells and elucidated the specific roles of BSA, FBS and three cytokines (IL-7, FLT3L and SCF) in promoting efficient B lineage differentiation.

Increasing evidence demonstrates a close relationship between daily diet and homeostasis of the body’s internal environment, particularly hematopoietic system homeostasis. Hematopoietic stem cells (HSCs) are located at the top of the hematopoietic system and have the ability to self-renew and differentiate into various types of immune cells. They play an important role in maintaining body stability and health. Studies have shown that different diets can lead to changes in HSC homeostasis, thereby affecting immune function and overall health status of the body. However, there is a scarcity of comprehensive reviews on how different diets affects HSC function. Therefore, this review summarizes the current progression in research on the effects of a high-fat diet (HFD) and energy-restricted diet on HSC function. HFD has a predominantly negative effect on HSCs, as does severe energy-restricted diet (SERD). Conversely, moderate energy-restricted diet (dietary restriction, DR) promotes the repopulation of HSCs but seriously impairs the differentiation of HSCs into lymphoid lineage. Further study of the influence of different diets on HSCs may aid in designing rational dietary guidelines to optimize the hematopoietic and immune functions of the body, which has significant implications for clinical medical practices.

Chronic inflammation in adipose tissue is widely recognized as a pivotal link connecting obesity to a spectrum of related chronic diseases, including type 2 diabetes, non-alcoholic fatty liver disease, and cardiovascular disorders. In this pathogenic process, the dysregulated interaction between adipocytes and adipose-resident immune cells plays a critical regulatory role; however, the underlying mechanisms governing this abnormal interaction remain largely unknown. In this study, we showed that upregulated β2-microglobulin expression in hypertrophic adipocytes during obesity not only mediated the activation of adipose-resident CD8+ T cells in a cell contact-dependent manner but also facilitated iron overload and the ferroptosis of adipocytes, thereby promoting the M1 polarization of adipose tissue macrophages. Conversely, specific ablation of β2-microglobulin in adipocytes effectively suppressed the activation and accumulation of adipose-resident CD8+ T cells, as well as adipocyte ferroptosis and M1 polarization, ultimately preventing high-fat diet-induced obesity and its related inflammation and metabolic disorders. Additionally, adeno-associated virus-mediated adipose-targeted knockdown of β2-microglobulin has been demonstrated to therapeutically alleviate high-fat diet-induced obesity, as well as its related chronic inflammation and metabolic disorders. Furthermore, our bioinformatic analysis of human adipose transcriptome data revealed a strong correlation between adipose β2-microglobulin and obesity. More importantly, β2-microglobulin is significantly upregulated in adipocytes isolated from patients with obesity. Thus, our findings highlight the pivotal role of adipocytes in obesity-associated chronic inflammation and metabolic disorders via β2-microglobulin-dependent mechanisms.

Autoreactive CD8+ T cells play a key role in type 1 diabetes (T1D), but the antigen spectrum that activates autoreactive CD8+ T cells remains unclear. Endoplasmic reticulum stress (ERS) has been implicated in β-cell autoantigen generation. Here, we analyzed the major histocompatibility complex class I (MHC-I)-associated immunopeptidome (MIP) of islet β-cells under steady and ERS conditions and found that ERS reshaped the MIP of β-cells and promoted the MHC-I presentation of a panel of conventional self-peptides. Among them, OTUB258-66 showed immunodominance, and the corresponding autoreactive CD8+ T cells were diabetogenic in nonobese diabetic (NOD) mice. High glucose intake upregulated pancreatic OTUB2 expression and amplified the OTUB258-66-specific CD8+ T-cell response in NOD mice. Repeated OTUB258-66 administration significantly reduced the incidence of T1D in NOD mice. Interestingly, peripheral blood mononuclear cells (PBMCs) from patients with T1D, but not from healthy controls, showed a positive IFN-γ response to human OTUB2 peptides. This study provides not only a new explanation for the role of ERS in promoting β-cell-targeted autoimmunity but also a potential target for the prevention and treatment of T1D. The data are available via ProteomeXchange with the identifier PXD041227.

BACKGROUND:Atopic dermatitis (AD) is a chronic and recurrent inflammatory skin disease characterized by severe pruritus and eczematous lesions. Heterogeneity of AD has been reported among different racial groups according to clinical, molecular and genetic differences.OBJECTIVE:This study aimed to conduct an in-depth transcriptome analysis of AD in Chinese population.METHODS:We performed single-cell RNA sequencing (scRNA-seq) analysis of skin biopsies from five Chinese adult patients with chronic AD and from four healthy controls, combined with multiplexed immunohistochemical analysis in whole-tissue skin biopsies. We explored the functions of IL19 in vitro.RESULTS:ScRNA-seq analysis was able to profile a total of 87,853 cells, with keratinocytes (KCs) in AD manifesting highly expressed keratinocyte activation and pro-inflammatory genes. KCs demonstrated a novel IL19+ IGFL1+ subpopulation that increased in AD lesions. Inflammatory cytokines IFNG, IL13, IL26 and IL22 were highly expressed in AD lesions. In vitro, IL19 directly downregulated KRT10 and LOR in HaCaT cells and activated HaCaT cells to produce TSLP.CONCLUSION:Abnormal proliferation and differentiation of keratinocytes contribute immensely to the pathogenesis of AD, whereas AD chronic lesions have witnessed significant presence of IL19+ IGFL1+ KCs, which may be involved in the disruption of the skin barrier, the connection and magnification of Th2 and Th17 inflammatory responses, and mediation of skin pruritus. Furthermore, progressive activation of multiple immune axes dominated by Type 2 inflammatory reaction occur in AD chronic lesions.

玫瑰痤疮是一种慢性炎症性皮肤病,免疫反应在其发病中发挥重要作用.除了固有免疫反应异常,如TLR-2、LL-37与肥大细胞形成的促炎驱动环,适应性免疫也参与玫瑰痤疮的发生发展,两种类型的免疫反应形成交错互相影响的复杂网络,共同介导玫瑰痤疮的发病.此外,近年来还有研究发现皮肤神经系统失调、维生素D3、内质网应激等也在玫瑰痤疮的免疫发病机制中发挥作用.本综述对玫瑰痤疮的免疫发病机制研究进展进行归纳和总结.

The peptides repertoire presented to CD8+ T cells by major histocompatibility complex (MHC) class I molecules is referred to as the MHC I-associated peptidome (MIP), which regulates thymus development, peripheral survival and function during lifetime of CD8+ T cells. Type 1 diabetes (T1D) is an organ-specific autoimmune disease caused by pancreatic β cells destruction mediated primarily by autoreactive CD8+ T cells. Non-obese diabetic (NOD) mouse is an important animal model of T1D. Here, we deeply analyzed the MIP derived from NOD mice thymus and pancreas, and demonstrated that the thymus MIP source proteins partially shared with the MIP source proteins derived from NOD mice pancreas and β cell line. One H-2Kd restricted peptide SLC35B126-34 which was shared by MIP derived from both NOD mice pancreatic tissues and islet β-cell line, but absent in MIP from NOD thymus tissues, showed ability to stimulate IFN-γ secretion and proliferation of NOD mice splenic CD8+ T cells. The global view of the MHC I-associated self-peptides repertoire in the thymus and pancreas of NOD mice may serve as a biological reference to identify potential autoantigens targeted by autoreactive CD8+ T cells in T1D. Data are available via ProteomeXchange with identifier PXD031966. SIGNIFICANCE: The peptides repertoire presented to CD8+ T cells by major histocompatibility complex (MHC) class I molecules is referred to as the MHC I-associated peptidome (MIP). The MIP presented by thymic antigen presenting cells (APCs) is crucial for shaping CD8+ T cell repertoire and self-tolerance, while the MIP presented by peripheral tissues and organs is not only involved in maintaining periphery CD8+ T cell survival and homeostasis, but also mediates immune surveillance and autoimmune responses of CD8+ T cells under pathological conditions. Type 1 diabetes (T1D) is an organ-specific autoimmune disease caused by the destruction of pancreatic β cells, mediated primarily by autoreactive CD8+ T cells. Non-obese diabetic (NOD) mouse is one of important animal models of spontaneous autoimmune diabetes that shares several key features with human T1D. The global view of the MHC I-associated self-peptides repertoire in the thymus and pancreas of NOD mice may serve as a good biological reference to identify potential autoantigens targeted by autoreactive CD8+ T cells in T1D. It has great significance for further clarifying the immune recognition and effect mechanism of autoreactive CD8+ T cells in the pathogenesis of T1D, and then developing antigen-specific immune intervention strategies.

Introduction:Epidemiological studies have suggested that dietary factors, especially high consumption of high glycaemic index carbohydrates and sugars, may trigger or exacerbate the progression of type 1 diabetes. We aimed to provide experimental evidence to confirm this relevance and to explore the underlying mechanisms.Methods:NOD mice were given sustained high-glucose drinking or glucose-free water and observed for the incidence of type 1 diabetes and islet inflammation. RNAseq was performed to detect the transcriptome changes of the NOD islet beta cell line NIT-1 after high glucose treatment, and mass spectrometry was performed to detect the proteome changes of NIT-1-cells-derived sEVs.Results:Sustained high glucose drinking significantly aggravates islet inflammation and accelerates the onset of type 1 diabetes in NOD mice. Mechanistically, high glucose treatment induces aberrant ER stress and up-regulates the expression of autoantigens in islet beta cell. Moreover, high glucose treatment alters the proteome of beta-cells-derived sEVs, and significantly enhances the ability of sEVs to promote DC maturation and stimulate immune inflammatory response.Discussion:This study provides evidence for negative effect of high glucose intake as a dietary factor on the pathogenesis of type 1 diabetes in genetically predisposed individuals. Therefore, avoiding high sugar intake may be an effective disease prevention strategy for children or adults susceptible to type 1 diabetes.

Background Studies have shown that autoimmune response contributes to chronic hepatitis B (CHB) development. Aim This study aimed to identify autoantibodies in the sera of patients with CHB and to investigate the association of autoimmune response with disease severity in CHB. Methods Proteins from human liver carcinoma cell line HepG2 were separated by two-dimensional electrophoresis. The candidate autoantigens were recognized by serum autoantibodies from Chinese CHB patients. Immunohistochemical staining was performed to determine the hepatic expression of the autoantigen in CHB patients with different inflammatory grades. Enzyme-linked immunosorbent assay (ELISA) was conducted to measure the prevalence and the levels of serum autoantibody in CHB patients with different disease severity. Flow cytometry analysis was carried out to assess the autoreactive T cell response in the peripheral circulation of CHB patients. Results ErbB-3-binding protein-1 (EBP-1) was identified as an autoantigen of serum autoantibodies in CBP patients. EBP-1 protein expression was upregulated in the liver of CHB patients with high-grade hepatic inflammation. The prevalence and levels of serum anti-EBP1 IgG were significantly increased in CHB patients with severe diseases compared with those with mild or moderate diseases, but none was detectable in the healthy controls. EBP-1 peptides induced proinflammatory cytokine expression in CD4 + T cells from CHB patients. Conclusion Our results demonstrate the presence of an autoantibody against EBP-1 in the sera as well as EBP-1-reactive T cells in the peripheral blood of CHB patient. EBP-1-induced autoimmune response is positively associated with the disease severity, suggesting that EBP-1-induced autoimmune response possibly contributes to progressive liver failure.

Autoreactive CD8 + T cells play an indispensable key role in the destruction of pancreatic islet β-cells and the initiation of type 1 diabetes (T1D). Insulin is an essential β-cell autoantigen in T1D. An HLA-A*0201-restricted epitope of insulin A chain (mInsA 2-10 ) is an immunodominant ligand for autoreactive CD8 + T cells in NOD. β2m null . HHD mice. Altered peptide ligands (APLs) carrying amino acid substitutions at T cell receptor (TCR) contact positions within an epitope are potential to modulate autoimmune responses via triggering altered TCR signaling. Here, we used a molecular simulation strategy to guide the generation of APL candidates by substitution of L-amino acids with D-amino acids at potential TCR contact residues (positions 4 and 6) of mInsA 2-10 , named mInsA 2-10 DQ4 and mInsA 2-10 DC6, respectively. We found that administration of mInsA 2-10 DQ4, but not DC6, significantly suppressed the development of T1D in NOD. β2m null . HHD mice. Mechanistically, treatment with mInsA 2-10 DQ4 not only notably eliminated mInsA 2-10 autoreactive CD8 + T cell responses but also prevented the infiltration of CD4 + T and CD8 + T cells, as well as the inflammatory responses in the pancreas of NOD. β2m null .HHD mice. This study provides a new strategy for the development of APL vaccines for T1D prevention.

Objective To study the effect of high glucose concentration on the transcriptome expression profile of islet β cells derived from type 1 diabetes (T1D) mice and to explore the possible mechanism of high glucose environment in the pathogenesis of T1D. Methods The non-obese diabetic (NOD) mice-derived islet β cell line NIT-1 in logarithmic growth phase were divided into high glucose-treated (HG) and control (NIT1) group. The cells in the NIT1 group were routinely cultured in F-12K medium, while HG group were treated with 20 mmol/L glucose. Whole transcriptome profile was analyzed using RNA-sequencing technology to obtain the differentially expressed genes (DEGs). And the DEGs were further analyzed by GO annotation and KEGG pathway enrichment analysis. Results A total of 5 548 DEGs were detected between the NIT1 group and HG group. Among them, 2 701 DEGs were up-regulated in the HG group, which were significantly enriched in endoplasmic reticulum stress, endoplasmic reticulum protein processing, ubiquitin-mediated protein degradation, autophagy and apoptosis pathways. And the 2 847 down-regulated genes were mainly concentrated in ribosome, metabolism, biosynthesis, cell cycle and DNA replication. Moreover, the mRNA levels of some important T1D auto-antigens were significantly increased in the HG group. Conclusion High glucose environment significantly changes the whole-transcriptome profile of islet β cells, and promotes the abilities of antigen processing and presentation, suggesting that high glucose might be associated with the induction of autoimmune response of islet cells and the incidence and development of T1D.

Adaptive CD8+ T cells were observed to contribute to the initiation and progression of obesity-induced visceral adipose tissue (VAT) chronic inflammation that is critically linked to metabolic disorders. Numerous peptides presented by the major histocompatibility complex (MHC) class I molecules at the cell surface are collectively termed as MHC I-associated immunopeptidome (MIP) for the interaction with CD8+ T cells. We conducted the in-depth mapping of MIP of VAT from lean and obese mice using large-scale high-resolution mass spectrometry and observed that obesity significantly alters the landscape of VAT MIPs. Additionally, the obese VAT-exclusive MIP source proteome reflected a distinct obesity-associated signature. A peptide derived from lactate dehydrogenase A (LDHA) or B chain, named LDHA237-244, was identified as an obese VAT-exclusive immunogenic peptide that was capable of eliciting pro-inflammatory CD8+ T cells responses. Our findings suggest that certain immunogenic peptides generated by obesity may trigger CD8+ T cell-mediated VAT inflammation.

目的 建立高脂饮食(high fat diet,HFD)诱导的小鼠非酒精性脂肪肝(non-alcoholic fatty liver disease,NAFLD)模型,动态监测其血清生化指标、肝脏病理形态学及肝脏浸润T淋巴细胞变化,从而探讨NAFLD早期肝病变动态进展特征.方法 4～6周龄C57BL/6雄性小鼠随机分成正常饮食(normal-chow diet,NCD)组和HFD组,喂养2～16周后,禁食不禁饮12h后获取血清和肝组织样本,检测血清肝脏相关生化指标水平,肝脏H&E和油红O染色观察组织病理学改变,提取肝浸润淋巴细胞,并检测CD3+CD4+、CD3+CD8+、CD8+CD44+T淋巴细胞的频率及CD8+T淋巴细胞分泌IFN-γ的能力.结果 与NCD组相比,HFD组小鼠体质量和附睾脂肪质量在第4周明显增加,而肝脏指数在2～8周内降低,第10周开始持续上升;HFD喂养小鼠血清肝损伤指标于第8周开始上升,之后持续增加;H&E和油红O染色显示第10周小鼠肝细胞发生明显脂肪样变,并在第16周时出现片状气球样变,可见点状坏死和炎细胞浸润等病理改变;流式染色结果显示,HFD喂养2～8周内肝浸润CD3+CD8+,CD8+CD44+T细胞的频率及CD8+T细胞分泌IFN-γ的能力下降;在HFD持续喂养10周后,肝CD3+CD8+,CD8+CD44+T细胞的频率及CD8+T细胞分泌IFN-γ的能力增加.结论 在本实验条件下,HFD诱导的小鼠NAFLD肝损伤模型,早期病变始于HFD喂养8到10周,并动态观察到HFD喂养初期肝脏浸润CD8+T细胞的功能受到抑制,而在肝脏出现明显病变的同时CD8+T细胞也明显激活,频率上升及促炎反应增强.提示CD8+T细胞激活可能与肝脏病理进展密切相关,并明确了该模型中肝浸润CD8+T细胞激活扩增的阶段,为进一步研究其活化机制奠定实验基础.

目的 1型糖尿病(type l diabetes,T1D)是由于胰岛β细胞受自身免疫系统的破坏,而导致胰岛素缺乏和血糖升高的一种自身免疫性疾病,胰岛β细胞内质网应激(endoplasmic reticulum stress,ERS)是其发病的重要因素.甘露糖是一种葡萄糖的差向异构体,新近发现其具有减缓甚至阻断T1D进展的作用,但具体机制仍待进一步阐明.方法 通过ERS诱导剂(TG/TM)处理NIT-1细胞,荧光定量PCR检测内质网应激标志物mRNA表达水平,CCK-8法检测细胞生存活力,台盼蓝法鉴定细胞生死状态.结果 给予适宜浓度的甘露糖预处理,可改善ERS诱导剂引起的胰岛β细胞系(NIT-1)细胞活力下降并逆转ERS诱导剂引发的BiP、ATF4及CHOP mRNA水平升高.结论 甘露糖可抑制NIT-1细胞的过度ERS,进而减少凋亡,促进存活,为甘露糖用于治疗T1D提供了新的理论依据.

Autoreactive CD8 + T cells, which play an indispensable role in β cell destruction, represent an emerging target for the prevention of type 1 diabetes (T1D). Altered peptide ligands (APLs) can efficiently induce antigen-specific T cells anergy, apoptosis or shifts in the immune response. Here, we found that HLA-A*0201-restricted CD8 + T cell responses against a primary β-cell autoantigen insulin epitope InsB1 5–14 were present in both NOD. β2m null .HHD NOD mice and T1D patients. We generated several APL candidates for InsB1 5 –14 by residue substitution at the p6 position. Only H6F exhibited an inhibitory effect on mInsB1 5– 14 -specific CD8 + T cell responses in vitro. H6F treatment significantly reduced the T1D incidence, which was accompanied by diminished autoreactive CD8 + T cell responses to mInsB1 5-14 , inhibited infiltration of CD8 + and CD4 + T cells in the pancreas and reduced pro-inflammatory cytokine production in pancreatic and splenic T cells in NOD. β2m null .HHD mice. Mechanistically, H6F treatment significantly augmented a tiny portion of CD8 + CD25 + Foxp3 + T cells in the spleen and especially in the pancreas. This subset exhibited typical Treg phenotypes and required peptide-specific restimulation to exert immunosuppressive activity. Therefore, this APL H6F may be a promising candidate with potential clinical application value for antigen-specific prevention of T1D.

Obesity and type 2 diabetes are linked with chronic, low-grade inflammation in visceral adipose tissue (VAT). A unique population of VAT-resident CD4+Foxp3+ Tregs plays a crucial role in regulating VAT inflammation and metabolic homeostasis. VAT-resident Tregs display a highly restricted TCR repertoire, suggesting they recognize certain autoantigen(s) in VAT. A dramatic reduction of VAT-resident Tregs has been shown to closely correlate with obesity-related VAT chronic inflammation and metabolic disorders. Oral tolerance strategy may modulate inflammatory response to autoantigens by several mechanisms including induction of autoantigen-specific Tregs. Here, we explored the effects and cellular mechanism of oral administration of VAT pooled antigens on high-fat diet (HFD)-induced metabolic disorders in mice. Indeed, we found that oral treatment of VAT mixture antigens effectively inhibited gain in body weight and fat mass, ameliorated serum lipid parameters, and improved insulin sensitivity in HFD mice. This strategy was shown to significantly restore HFD-induced decrease of VAT-resident Tregs, accompanied by a hampered M2-type to M1-type macrophages phenotypic switch as well as decreased CD8+ T cells infiltration in VAT. Thus, oral administration of VAT antigens may be a novel and safe strategy against obesity and its related metabolic disorders.

Immunogenicity is a key factor that influences whether a peptide presented by major histocompatibility complex (MHC) can be a T cell epitope. However, peptide immunization experiments have shown that approximately half of MHC class I-binding peptides cannot elicit a T cell response, indicating the importance of analyzing the variables affecting the immunogenicity of MHC-binding peptides. In this study, we hierarchically investigated the contribution of the binding stability and affinity of peptide–MHC complexes to immunogenicity based on the available quantitative data. We found that the immunogenicity of peptides presented by human leukocyte antigen (HLA) class I molecules was still predictable using the experimental binding affinity, although approximately one-third of the peptides with a binding affinity stronger than 500 nM were non-immunogenic, whereas the immunogenicity of HLA-II-presented peptides was predicted well using the experimental affinity and even the predicted affinity. The positive correlation between the binding affinity and stability was only observed in peptide–HLA-I complexes with a binding affinity stronger than 500 nM, which suggested that the stability alone could not be used for the prediction of immunogenicity. A characterization and comparison of the ‘holes’ in the CD8+ and CD4+ T cell repertoire provided an explanation for the observed differences between the immunogenicity of peptides presented by HLA class I and II molecules. We also provided the optimal affinity threshold for the potential CD4+ and CD8+ T cell epitopes. Our results provide important insights into the cellular immune response and the accurate prediction of T cell epitopes.

ChgA has recently been identified as the autoantigen for diabetogenic CD4(+) T cells in NOD mice and T1D patients. However, autoreactive CD8(+) T-cell responses targeting ChgA haven't been studied yet. Here several HLA-A*0201-restricted peptides derived from mChgA and hChgA were selected by an integrated computational prediction approach, followed by an HLA-A*0201 binding assay. MChgA10-19 and mChgA(43-52) peptides, which bound well with HLA-A*0201 molecule, induced significant proliferation and IFN-γ-releasing of splenocytes from diabetic NOD.β2m(null).HHD mice. Notably, flow cytometry analysis found that mChgA(10-19) and mChgA(43-52) stimulated the production of IFN-γ, perforin, and IL-17 by splenic CD8(+) T cells of diabetic NOD.β2m(null).HHD mice. Furthermore, hChgA(10-19) and hChgA(43-52)-induced IFN-γ releasing by specific CD8(+) T cells were frequently detected in recent-onset HLA-A*0201-positive T1D patients. Thus, this study demonstrated that autoreactive CD8(+) T cells targeting ChgA were present in NOD.β2m(null).HHD mice and T1D patients, and might contribute to pathogenesis of T1D through secreting proinflammatory cytokines and cytotoxic molecules.