The Ratios of CD8+ T Cells to CD4+CD25+ FOXP3+ and FOXP3- T Cells Correlate with Poor Clinical Outcome in Human Serous Ovarian Cancer

Ovarian cancer is an immune reactive malignancy with a complex immune suppressive network that blunts successful immune eradication. This suppressive microenvironment may be mediated by recruitment or induction of CD4⁺ regulatory T cells (Tregs). Our study sought to investigate the association of tumor-infiltrating CD4⁺CD25⁺FOXP3⁺ Tregs, and other immune factors, with clinical outcome in serous ovarian cancer patients. We performed immunofluorescence and quantification of intraepithelial tumor-infiltrating triple positive Tregs (CD4⁺CD25⁺FOXP3⁺), as well as CD4⁺CD25⁺FOXP3^-, CD3⁺ and CD8⁺ T cells in tumor specimens from 52 patients with high stage serous ovarian carcinoma. Thirty-one of the patients had good survival (i.e. > 60 months) and 21 had poor survival of < 18 months. Total cell counts as well as cell ratios were compared among these two outcome groups. The total numbers of CD4⁺CD25⁺FOXP3⁺ Tregs, CD4⁺CD25⁺FOXP3^-, CD3⁺ and CD8⁺ cells were not significantly different between the groups. However, higher ratios of CD8⁺/CD4⁺CD25⁺FOXP3⁺ Treg, CD8⁺/CD4⁺ and CD8/CD4⁺CD25⁺FOXP3^- cells were seen in the good outcome group when compared to the patients with poor outcome. These data show for the first time that the ratios of CD8⁺ to both CD4⁺CD25⁺FOXP3⁺ Tregs and CD4⁺CD25⁺FOXP3^- T cells are associated with disease outcome in ovarian cancer. The association being apparent in ratios rather than absolute count of T cells suggests that the effector/suppressor ratio may be a more important indicator of outcome than individual cell count. Thus, immunotherapy strategies that modify the ratio of CD4⁺CD25⁺FOXP3⁺ Tregs or CD4⁺CD25⁺FOXP3^- T cells to CD8⁺ effector cells may be useful in improving outcomes in ovarian cancer.     

Loss of the Inducible Hsp70 Delays the Inflammatory Response to Skeletal Muscle Injury and Severely Impairs Muscle Regeneration

Skeletal muscle regeneration following injury is a highly coordinated process that involves transient muscle inflammation, removal of necrotic cellular debris and subsequent replacement of damaged myofibers through secondary myogenesis. However, the molecular mechanisms which coordinate these events are only beginning to be defined. In the current study we demonstrate that Heat shock protein 70 (Hsp70) is increased following muscle injury, and is necessary for the normal sequence of events following severe injury induced by cardiotoxin, and physiological injury induced by modified muscle use. Indeed, Hsp70 ablated mice showed a significantly delayed inflammatory response to muscle injury induced by cardiotoxin, with nearly undetected levels of both neutrophil and macrophage markers 24 hours post-injury. At later time points, Hsp70 ablated mice showed sustained muscle inflammation and necrosis, calcium deposition and impaired fiber regeneration that persisted several weeks post-injury. Through rescue experiments reintroducing Hsp70 intracellular expression plasmids into muscles of Hsp70 ablated mice either prior to injury or post-injury, we confirm that Hsp70 optimally promotes muscle regeneration when expressed during both the inflammatory phase that predominates in the first four days following severe injury and the regenerative phase that predominates thereafter. Additional rescue experiments reintroducing Hsp70 protein into the extracellular microenvironment of injured muscles at the onset of injury provides further evidence that Hsp70 released from damaged muscle may drive the early inflammatory response to injury. Importantly, following induction of physiological injury through muscle reloading following a period of muscle disuse, reduced inflammation in 3-day reloaded muscles of Hsp70 ablated mice was associated with preservation of myofibers, and increased muscle force production at later time points compared to WT. Collectively our findings indicate that depending on the nature and severity of muscle injury, therapeutics which differentially target both intracellular and extracellular localized Hsp70 may optimally preserve muscle tissue and promote muscle functional recovery.     

Increased Presence of FOXP3+ Regulatory T Cells in Inflamed Muscle of Patients with Active Juvenile Dermatomyositis Compared to Peripheral Blood

Juvenile dermatomyositis (JDM) is an immune-mediated inflammatory disease affecting the microvasculature of skin and muscle. CD4⁺CD25⁺FOXP3⁺ regulatory T cells (Tregs) are key regulators of immune homeostasis. A role for Tregs in JDM pathogenesis has not yet been established. Here, we explored Treg presence and function in peripheral blood and muscle of JDM patients. We analyzed number, phenotype and function of Tregs in blood from JDM patients by flow cytometry and in vitro suppression assays, in comparison to healthy controls and disease controls (Duchenne’s Muscular Dystrophy). Presence of Tregs in muscle was analyzed by immunohistochemistry. Overall, Treg percentages in peripheral blood of JDM patients were similar compared to both control groups. Muscle biopsies of new onset JDM patients showed increased infiltration of numbers of T cells compared to Duchenne’s muscular dystrophy. Both in JDM and Duchenne’s muscular dystrophy the proportion of FOXP3⁺ T cells in muscles were increased compared to JDM peripheral blood. Interestingly, JDM is not a self-remitting disease, suggesting that the high proportion of Tregs in inflamed muscle do not suppress inflammation. In line with this, peripheral blood Tregs of active JDM patients were less capable of suppressing effector T cell activation in vitro, compared to Tregs of JDM in clinical remission. These data show a functional impairment of Tregs in a proportion of patients with active disease, and suggest a regulatory role for Tregs in JDM inflammation.     

Skeletal Muscle Regeneration by the Exosomes of Adipose Tissue-Derived Mesenchymal Stem Cells

Profound skeletal muscle loss can lead to severe disability and cosmetic deformities. Mesenchymal stem cell (MSC)-derived exosomes have shown potential as an effective therapeutic tool for tissue regeneration. This study aimed to determine the regenerative capacity of MSC-derived exosomes for skeletal muscle regeneration. Exosomes were isolated from human adipose tissue-derived MSCs (AD-MSCs). The effects of MSC-derived exosomes on satellite cells were investigated using cell viability, relevant genes, and protein analyses. Moreover, NOD-SCID mice were used and randomly assigned to the healthy control (n = 4), muscle defect (n = 6), and muscle defect + exosome (n = 6) groups. Muscle defects were created using a biopsy punch on the quadriceps of the hind limb. Four weeks after the surgery, the quadriceps muscles were harvested, weighed, and histologically analyzed. MSC-derived exosome treatment increased the proliferation and expression of myocyte-related genes, and immunofluorescence analysis for myogenin revealed a similar trend. Histologically, MSC-derived exosome-treated mice showed relatively preserved shapes and sizes of the muscle bundles. Immunohistochemical staining revealed greater expression of myogenin and myoblast determination protein 1 in the MSC-derived exosome-treated group. These results indicate that exosomes extracted from AD-MSCs have the therapeutic potential for skeletal muscle regeneration.     

骨骼肌卫星细胞的特性及其增殖与分化的调控

成体骨骼肌细胞的数量基本保持恒定,骨骼肌的再生主要依赖肌卫星细胞的增殖与分化。骨骼肌卫星细胞是能够被激活、进而分化为肌细胞的一类成肌细胞。现对肌卫星细胞的发生、体外培养以及增殖与分化的调控进行综述,并对能否通过激活肌卫星细胞的增殖来实现肌肉组织生长的调控进行探讨。     

骨骼肌卫星细胞生物学特性及临床应用前景

骨骼肌卫星细胞是位于肌细胞膜和基膜之间的具有增殖和分化潜力的生肌干细胞,对于出生后骨骼肌的生长、再生修复和维持具有重要的意义.主要对卫星细胞的含量与分布、细胞标志、可塑性和不均一性等生物学特性做了综述,最后展望了卫星细胞移植在临床上的应用前景.     

Isolation and Biological Characteristics of Beijing Fatty Chicken Skeletal Muscle Satellite Cells

Abstract

Skeletal muscle satellite cells, a postulated multipotential stem cell population, play an essential role in the postnatal replenishment of skeletal muscles. In the present research, the skeletal muscle satellite cells were isolated from the pectorals of 15-day-old Beijing Fatty Chicken embryos using combined enzymatic digestion of 0.1% collagenase 1 and 0.25% trypsin. Myogenic markers such as MyoD, Pax7 and demin were detected, indicating their skeletal muscle satellite cell identity. Karyotype analysis showed that these in vitro cultured cells were genetically stable. Being exposed to bone morphogen and adipogenic factors, it was proved that they differentiated into osteocytes and adipocytes correspondingly.     

Activation of Skeletal Muscle Satellite Cells and the Role of Fibroblast Growth Factor Receptors

Specific, high-affinity binding of FGF2 was evaluated in cultured skeletal muscle satellite cells from young (3- to 4-week-old) and adult (9- to 12-month-old) rats prior to the first division in culture. Specific binding of FGF2 was detected on satellite cells from young rats at 18 h postplating, the earliest time examined, but specific binding was not detectable until 42 h on satellite cells from old rats. This correlates well with the delayed entry into the cell cycle exhibited by adult satellite cells and with the ability of satellite cells from rats of these ages to proliferate in response to FGF2. Patterns of tyrosine phosphorylation in whole cell extracts, following stimulation by FGF2, indicated specific FGF2 phosphorylation of proteins of 150/145, 90, 42, and 35 kDa in cells from both age groups. Several growth factors were evaluated for their ability to stimulate early entry of adult satellite cells into the cell cycle, and none of the following growth factors were able to activate proliferation of these cells: FGF2, IGF-1, IGF-2, PDGF-BB, TGF-β1, or TGF-β2. In addition, specific binding of FGF2 to 48-h cultures of adult satellite cells was not stimulated by FGF2, IGF-1, IGF-2, PDGF-BB, or TGF-β2, and specific binding was significantly decreased (P < 0.05) by FGF2 and TGF-β2. Specific binding was significantly lower in cells treated with PDGF-BB than in cells treated with either form of IGF but was greater than in cells treated with FGF2 or TGF-β2. The results of these experiments suggest that expression of functional FGF receptors on the surface of satellite cells may represent an important step in the activation of quiescent satellite cells.     

蛋肉鸡骨骼肌卫星细胞的特性比较及分析

目的:比较蛋、肉鸡骨骼肌卫星细胞在增殖、分化速度及在细胞因子作用下细胞周期等方面所存在的特性差异,为人类肌肉疾病的研究和千细胞治疗提供一定的理论依据。方法:采用两步酶消化法体外原代培养获得7日龄蛋肉鸡骨骼肌卫星细胞,利用血球计数板进行细胞计数绘制出二者的细胞生长曲线;通过流式细胞仪检测经细胞因子bFGF和Myostatin处理后,蛋肉鸡骨骼肌卫星细胞的细胞周期变化情况。结果:体外相同的培养条件下,肉鸡肌卫星细胞的增殖、分化速度大于蛋鸡肌卫星细胞;且经相同剂量的同种细胞因子处理后,蛋鸡卫星细胞对于Myostatin的抑制作用十分敏感,而肉鸡则对bFGF的促进增殖的作用反应强烈。结论:肉鸡肌卫星细胞的增殖、分化速度大于蛋鸡肌卫星细胞,且二者对于同种细胞因子的敏感程度不同。     

Skeletal Muscle Satellite Cells Are Committed to Myogenesis and Do Not Spontaneously Adopt Nonmyogenic Fates

The developmental potential of skeletal muscle stem cells (satellite cells) remains controversial. The authors investigated satellite cell developmental potential in single fiber and clonal cultures derived from MyoD^iCre/+;R26R^EYFP/+ muscle, in which essentially all satellite cells are permanently labeled. Approximately 60% of the clones derived from cells that co-purified with muscle fibers spontaneously underwent adipogenic differentiation. These adipocytes stained with Oil-Red-O and expressed the terminal differentiation markers, adipsin and fatty acid binding protein 4, but did not express EYFP and were therefore not of satellite cell origin. Satellite cells mutant for either MyoD or Myf-5 also maintained myogenic programming in culture and did not adopt an adipogenic fate. Incorporation of additional wash steps prior to muscle fiber plating virtually eliminated the non-myogenic cells but did not reduce the number of adherent Pax7+ satellite cells. More than half of the adipocytes observed in cultures from Tie2-Cre mice were recombined, further demonstrating a non-satellite cell origin. Under adipogenesis-inducing conditions, satellite cells accumulated cytoplasmic lipid but maintained myogenic protein expression and did not fully execute the adipogenic differentiation program, distinguishing them from adipocytes observed in muscle fiber cultures. The authors conclude that skeletal muscle satellite cells are committed to myogenesis and do not spontaneously adopt an adipogenic fate.     

Absence of CD34 on Murine Skeletal Muscle Satellite Cells Marks a Reversible State of Activation during Acute Injury

Background

Skeletal muscle satellite cells are myogenic progenitors that reside on myofiber surface beneath the basal lamina. In recent years satellite cells have been identified and isolated based on their expression of CD34, a sialomucin surface receptor traditionally used as a marker of hematopoietic stem cells. Interestingly, a minority of satellite cells lacking CD34 has been described.

Methodology/Principal Findings

In order to elucidate the relationship between CD34+ and CD34- satellite cells we utilized fluorescence-activated cell sorting (FACS) to isolate each population for molecular analysis, culture and transplantation studies. Here we show that unless used in combination with α7 integrin, CD34 alone is inadequate for purifying satellite cells. Furthermore, the absence of CD34 marks a reversible state of activation dependent on muscle injury.

Conclusions/Significance

Following acute injury CD34- cells become the major myogenic population whereas the percentage of CD34+ cells remains constant. In turn activated CD34- cells can reverse their activation to maintain the pool of CD34+ reserve cells. Such activation switching and maintenance of reserve pool suggests the satellite cell compartment is tightly regulated during muscle regeneration.     

Analysis of Suppressor and Non-Suppressor FOXP3+ T Cells in HIV-1-Infected Patients

Recently, it was shown that peripheral blood FOXP3+CD4+ T cells are composed of three phenotypic and functionally distinct subpopulations. Two of them having in vitro suppressive effects were characterized as resting Treg cells (rTregs) and activated Treg cells (aTregs). A third subset, identified as FOXP3+ non-Tregs, does not display any suppressor activity and produce high levels of Th1 and Th17 cytokines upon stimulation. In the present study we focus on the characteristics of these three subsets of FOXP3+CD4+ T cells in untreated HIV-1-infected patients. We found that the absolute counts of rTregs, aTregs and FOXP3+ non-Tregs were reduced in HIV-1 patients compared with healthy donors. The relative frequency of rTregs and aTregs was similar in HIV-1 patients and healthy donors, while the frequency of FOXP3+ non-Tregs was significantly higher in HIV-1 patients, reaching a maximum in those patients with the lower values of CD4 counts. Contrasting with the observations made in FOXP3- CD4+ T cells, we did not find a negative correlation between the number of rTregs, aTregs or FOXP3+ non-Tregs and virus load. Studies performed with either whole PBMCs or sorted aTregs and FOXP3+ non-Tregs cells showed that these two populations of FOXP3+ T cells were highly permissive to HIV-1 infection. Upon infection, FOXP3+ non-Tregs markedly down-regulates its capacity to produce Th1 and Th17 cytokines, however, they retain the ability to produce substantial amounts of Th2 cytokines. This suggests that FOXP3+ non-Tregs might contribute to the polarization of CD4+ T cells into a Th2 profile, predictive of a poor outcome of HIV-1-infected patients.     

Heparan Sulfate 6-O-endosulfatases (Sulfs) Coordinate the Wnt Signaling Pathways to Regulate Myoblast Fusion during Skeletal Muscle Regeneration

Skeletal muscle regeneration is mediated by satellite cells (SCs). Upon injury, SCs undergo self-renewal, proliferation, and differentiation into myoblasts followed by myoblast fusion to form new myofibers. We previously showed that the heparan sulfate (HS) 6-O-endosulfatases (Sulf1 and -2) repress FGF signaling to induce SC differentiation during muscle regeneration. Here, we identify a novel role of Sulfs in myoblast fusion using a skeletal muscle-specific Sulf double null (Sulf^SK-DN) mouse. Regenerating Sulf^SK-DN muscles exhibit reduced canonical Wnt signaling and elevated non-canonical Wnt signaling. In addition, we show that Sulfs are required to repress non-canonical Wnt signaling to promote myoblast fusion. Notably, skeletal muscle-relevant non-canonical Wnt ligands lack HS binding capacity, suggesting that Sulfs indirectly repress this pathway. Mechanistically, we show that Sulfs reduce the canonical Wnt-HS binding and regulate colocalization of the co-receptor LRP5 with caveolin3. Therefore, Sulfs may increase the bioavailability of canonical Wnts for Frizzled receptor and LRP5/6 interaction in lipid raft, which may in turn antagonize non-canonical Wnt signaling. Furthermore, changes in subcellular distribution of active focal adhesion kinase (FAK) are associated with the fusion defect of Sulf-deficient myoblasts and upon non-canonical Wnt treatment. Together, our findings uncover a critical role of Sulfs in myoblast fusion by promoting antagonizing canonical Wnt signaling activities against the noncanonical Wnt pathway during skeletal muscle regeneration.     

鸡骨骼肌卫星细胞的分离培养、鉴定及生物学特性研究

采用I型胶原酶和胰蛋白酶二步消化,经体外培养获得鸡骨骼肌卫星细胞,并通过检测肌卫星细胞特异基因的表达进行鉴定。结果表明：二步消化法适用于鸡骨骼肌卫星细胞的分离和获取,此方法分离得到的鸡骨骼肌卫星细胞表达卫星细胞特异的标志基因desmin和Pax7,并具有良好的增殖和分化能力,为鸡骨骼肌细胞增殖、分化和再生机制的研究提供技术平台。      

CD4+FOXP3+ Regulatory T Cells Exhibit Impaired Ability to Suppress Effector T Cell Proliferation in Patients with Turner Syndrome

Objective

We investigated whether the frequency, phenotype, and suppressive function of CD4⁺FOXP3⁺ regulatory T cells (Tregs) are altered in young TS patients with the 45,X karyotype compared to age-matched controls.

Design and Methods

Peripheral blood mononuclear cells from young TS patients (n = 24, 17.4–35.9 years) and healthy controls (n = 16) were stained with various Treg markers to characterize their phenotypes. Based on the presence of thyroid autoimmunity, patients were categorized into TS (–) (n = 7) and TS (+) (n = 17). Tregs sorted for CD4⁺CD25^bright were co-cultured with autologous CD4⁺CD25⁻ target cells in the presence of anti-CD3 and -CD28 antibodies to assess their suppressive function.

Results

Despite a lower frequency of CD4⁺ T cells in the TS (-) and TS (+) patients (mean 30.8% and 31.7%, vs. 41.2%; P = 0.003 and P < 0.001, respectively), both groups exhibited a higher frequency of FOXP3⁺ Tregs among CD4⁺ T cells compared with controls (means 1.99% and 2.05%, vs. 1.33%; P = 0.029 and P = 0.004, respectively). There were no differences in the expression of CTLA-4 and the frequency of Tregs expressing CXCR3⁺, and CCR4⁺CCR6⁺ among the three groups. However, the ability of Tregs to suppress the in vitro proliferation of autologous CD4⁺CD25⁻ T cells was significantly impaired in the TS (–) and TS (+) patients compared to controls (P = 0.003 and P = 0.041). Meanwhile, both the TS (–) and TS (+) groups had lower frequencies of naïve cells (P = 0.001 for both) but higher frequencies of effector memory cells (P = 0.004 and P = 0.002) than did the healthy control group.

Conclusions

The Tregs of the TS patients could not efficiently suppress the proliferation of autologous effector T cells, despite their increased frequency in peripheral CD4⁺ T cells.     

CD8+CD45RA+CCR7+FOXP3+ T Cells with Immunosuppressive Properties: A Novel Subset of Inducible Human Regulatory T Cells

CD8 T cells stimulated with a suboptimal dose of anti-CD3 Abs (100 pg/ml) in the presence of IL-15 retain a naive phenotype with expression of CD45RA, CD28, CD27, and CCR7 but acquire new functions and differentiate into immunosuppressive T cells. CD8(+)CCR7(+) regulatory T cells (Tregs) express FOXP3 and prevent CD4 T cells from responding to TCR stimulation and entering the cell cycle. Naive CD4 T cells are more susceptible to inhibition than memory cells. The suppressive activity of CD8(+)CCR7(+) Tregs is not mediated by IL-10, TGF-β, CTLA-4, CCL4, or adenosine and relies on interference with very early steps of the TCR signaling cascade. Specifically, CD8(+)CCR7(+) Tregs prevent TCR-induced phosphorylation of ZAP70 and dampen the rise of intracellular calcium in CD4 T cells. The inducibility of CD8(+)CCR7(+) Tregs is correlated with the age of the individual with PBLs of donors older than 60 y yielding low numbers of FOXP3(low) CD8 Tregs. Loss of CD8(+)CCR7(+) Tregs in the elderly host may be of relevance in the aging immune system as immunosenescence is associated with a state of chronic smoldering inflammation.     

Time-Series Clustering of lncRNA-mRNA Expression during the Adipogenic Transdifferentiation of Porcine Skeletal Muscle Satellite Cells

Skeletal muscle satellite cells (SMSCs), which are multifunctional muscle-derived stem cells, can differentiate into adipocytes. Long-chain non-coding RNA (lncRNA) has diverse biological functions, including the regulation of gene expression, chromosome silencing, and nuclear transport. However, the regulatory roles and mechanism of lncRNA during adipogenic transdifferentiation in muscle cells have not been thoroughly investigated. Here, porcine SMSCs were isolated, cultured, and induced for adipogenic differentiation. The expressions of lncRNA and mRNA at different time points during transdifferentiation were analysed using RNA-seq analysis. In total, 1005 lncRNAs and 7671 mRNAs showed significant changes in expression at differential differentiation stages. Time-series expression analysis showed that the differentially expressed (DE) lncRNAs and mRNAs were clustered into 5 and 11 different profiles with different changes, respectively. GO, KEGG, and REACTOME enrichment analyses revealed that DE mRNAs with increased expressions during the trans-differentiation were mainly enriched in the pathways for lipid metabolism and fat cell differentiation. The genes with decreased expressions were mainly enriched in the regulation of cell cycle and genetic information processing. In addition, 1883 DE mRNAs were regulated by 193 DE lncRNAs, and these genes were related to the controlling in cell cycle mainly. Notably, three genes in the fatty acid binding protein (FABP) family significantly and continuously increased during trans-differentiation, and 15, 13, and 11 lncRNAs may target FABP3, FABP4, and FABP5 genes by cis- or trans-regulation, respectively. In conclusion, these studies identify a set of new potential regulator for adipogenesis and cell fate and help us in better understanding the molecular mechanisms of trans-differentiation.     

Accumulation of CCR4+ CTLA-4hi FOXP3+CD25hi Regulatory T Cells in Colon Adenocarcinomas Correlate to Reduced Activation of Conventional T Cells

Background

Colorectal cancer usually gives rise to a specific anti-tumor immune response, but for unknown reasons the resulting immunity is not able to clear the tumor. Recruitment of activated effector lymphocytes to the tumor is important for efficient anti-tumor responses, while the presence of regulatory T cells (Treg) down-modulate tumor-specific immunity. We therefore aimed to determine homing mechanisms and activation stage of Treg and effector T cell infiltrating colon tumors compared to cells from the unaffected mucosa in patients suffering from colon adenocarcinoma.

Methodology/Principal Findings

Lymphocytes were isolated from unaffected and tumor mucosa from patients with colon adenocarcinoma, and flow cytometry, immunohistochemistry, and quantitative PCR was used to investigate the homing mechanisms and activation stage of infiltrating Treg and conventional lymphocytes. We detected significantly higher frequencies of CD25^highFOXP3⁺CD127^low putative Treg in tumors than unaffected mucosa, which had a complete demethylation in the FOXP3 promotor. Tumor-associated Treg had a high expression of CTLA-4, and some appeared to be antigen experienced effector/memory cells based on their expression of αEβ7 (CD103). There were also significantly fewer activated T cells and more CTLA-4⁺ conventional T cells susceptible to immune regulation in the tumor-associated mucosa. In contrast, CD8⁺granzyme B⁺ putative cytotoxic cells were efficiently recruited to the tumors. The frequencies of cells expressing α4β7 and the Th1 associated chemokine receptor CXCR3 were significantly decreased among CD4⁺ T cells in the tumor, while frequencies of CD4⁺CCR4⁺ lymphocytes were significantly increased.

Conclusions/Significance

This study shows that CCR4⁺CTLA4^hi Treg accumulate in colon tumors, while the frequencies of activated conventional Th1 type T cells are decreased. The altered lymphocyte composition in colon tumors will probably diminish the ability of the immune system to effectively attack tumor cells, and reducing the Treg activity is an important challenge for future immunotherapy protocols.     

PD-1 and Tim-3 Pathways Regulate CD8+ T Cells Function in Atherosclerosis

T cell-mediated immunity plays a significant role in the development of atherosclerosis (AS). There is increasing evidence that CD8⁺ T cells are also involved in AS but their exact roles remain unclear. The inhibitory receptors programmed cell death-1 (PD-1) and T cell immunoglobulin and mucin domain 3 (Tim-3) are well known inhibitory molecules that play a crucial role in regulating CD8⁺ T cell activation or tolerance. Here, we demonstrate that the co-expression of PD-1 and Tim-3 on CD8⁺ T cells is up-regulated in AS patients. PD-1⁺ Tim-3⁺ CD8⁺ T cells are enriched for within the central T (T_CM) cell subset, with high proliferative activity and CD127 expression. Co-expression of PD-1 and Tim-3 on CD8⁺ T cells is associated with increased anti-atherogenic cytokine production as well as decreased pro-atherogenic cytokine production. Blockade of PD-1 and Tim-3 results in a decrease of anti-atherogenic cytokine production by PD-1⁺ Tim-3⁺ CD8⁺ T cells and in an augmentation of TNF-α and IFN-γ production. These findings highlight the important role of the PD-1 and Tim-3 pathways in regulating CD8⁺ T cells function in human AS.