CAR-NK Cells: From Natural Basis to Design for Kill.

Chimeric antigen receptors (CARs) are fusion proteins with an extracellular antigen recognition domain and numerous intracellular signaling domains that have been genetically modified. CAR-engineered T lymphocyte-based therapies have shown great success against blood cancers; however, potential fatal toxicity, such as in cytokine release syndrome, and high costs are some shortcomings that limit the clinical application of CAR-engineered T lymphocytes and remain to overcome. Natural killer (NK) cells are the focal point of current immunological research owing to their receptors that prove to be promising immunotherapeutic candidates for treating cancer. However, to date, manipulation of NK cells to treat malignancies has been moderately successful. Recent progress in the biology of NK cell receptors has greatly transformed our understanding of how NK cells recognize and kill tumor and infected cells. CAR-NK cells may serve as an alternative candidate for retargeting cancer because of their unique recognition mechanisms, powerful cytotoxic effects especially on cancer cells in both CAR-dependent and CAR-independent manners and clinical safety. Moreover, NK cells can serve as an 'off-the-shelf product' because NK cells from allogeneic sources can also be used in immunotherapies owing to their reduced risk of alloreactivity. Although ongoing fundamental research is in the beginning stages, this review provides an overview of recent developments implemented to design CAR constructs to stimulate NK activation and manipulate NK receptors for improving the efficiency of immunotherapy against cancer, summarizes the preclinical and clinical advances of CAR-NK cells against both hematological malignancies and solid tumors and confronts current challenges and obstacles of their applications. In addition, this review provides insights into prospective novel approaches that further enhance the efficiency of CAR-NK therapies and highlights potential questions that require to be addressed in the future.

Myelin-specific CD8+ T cells exacerbate brain inflammation in CNS autoimmunity.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the CNS. Although CD4+ T cells are implicated in MS pathogenesis and have been the main focus of MS research using the animal model experimental autoimmune encephalomyelitis (EAE), substantial evidence from patients with MS points to a role for CD8+ T cells in disease pathogenesis. We previously showed that an MHC class I-restricted epitope of myelin basic protein (MBP) is presented in the CNS during CD4+ T cell-initiated EAE. Here, we investigated whether naive MBP-specific CD8+ T cells recruited to the CNS during CD4+ T cell-initiated EAE engaged in determinant spreading and influenced disease. We found that the MBP-specific CD8+ T cells exacerbated brain but not spinal cord inflammation. We show that a higher frequency of monocytes and monocyte-derived cells presented the MHC class I-restricted MBP ligand in the brain compared with the spinal cord. Infiltration of MBP-specific CD8+ T cells enhanced ROS production in the brain only in these cell types and only when the MBP-specific CD8+ T cells expressed Fas ligand (FasL). These results suggest that myelin-specific CD8+ T cells may contribute to disease pathogenesis via a FasL-dependent mechanism that preferentially promotes lesion formation in the brain.

FAS-mediated apoptosis impairment in patients with ALPS/ALPS-like phenotype carrying variants on CASP10 gene.

Autoimmune lymphoproliferative syndrome (ALPS) is a congenital disorder that results in an apoptosis impairment of lymphocytes, leading to chronic lymphoproliferation and autoimmunity, mainly autoimmune cytopenias. FAS gene defects are often responsible for the disease, the phenotype of which can vary from asymptomatic/mild forms to severe disease. More rarely, defects are associated to  other genes involved in apoptosis pathway, such as CASP10. Few data are available on CASP10-mutated patients. To date, two CASP10 mutations have been recognized as pathogenic (I406L and L258F) and others have been reported with controversial result on their pathogenicity (V410l, Y446C) or are known to be polymorphic variants (L522l). In this study, we evaluated apoptosis function in patients with an ALPS/ALPS-like phenotype carrying CASP10 variants. Molecular findings were obtained by next generation sequencing analysis of genes involved in immune dysregulation syndromes. Functional studies were performed after inducing apoptosis by FAS-ligand/TRIAL stimulation and analysing cell death and the function of CASP10, CASP8 and PARP proteins. We identified 6 patients with an ALPS (n = 2) or ALPS-like (n = 4) phenotype, carrying I406L (n = 1),V410l (n = 2),Y446C (n = 1) heterozygous CASP10 variants or the L522l polymorphisms (n = 2) associated with another polymorphic homozygote variant on CASP8 or a compound heterozygous mutation on TNFRSF13C. Apoptosis was impaired in all patients showing that such variants may play a role in the development of clinical phenotype.

SIRT1 negatively regulates invasive and angiogenic activities of the extravillous trophoblast.

PROBLEM: Dysregulation of extravillous trophoblast (EVT) invasion leads to pregnancy complications, such as pre-eclampsia, fetal growth restriction, and placenta accreta. The aim of this study was to explore the role of SIRT1 in EVT invasion and its underlying mechanism. METHOD OF STUDY: SIRT1-specific siRNA was transfected into Swan 71 cells, an immortalized first trimester trophoblast cell line. The Boyden chamber invasion assay, the scratch wound healing assay, and cell proliferation assay were performed. The expression levels of epithelial-to-mesenchymal transition (EMT) markers, matrix metalloproteinase-2 (MMP-2), MMP-9, p-Akt, Akt, p-p38MAPK, p38MAPK, p-ERK, ERK, p-JNK, JNK, Fas, and Fas ligand (FasL) were examined by western blot. Tube formation assay was conducted by using Matrigel. RESULTS: SIRT1 knockdown by siRNA significantly enhanced invasion and migration as well as the expression of MMP-2, MMP-9, and EMT markers in Swan 71 cells, but reduced proliferation. The effects of SIRT1 knockdown on invasion, migration, proliferation, and endothelial-like tube formation in Swan 71 cells were reversely regulated by blockade of Akt and p38MAPK signaling. In addition, SIRT1 knockdown markedly promoted colocalization of Swan 71 cells to human umbilical vein endothelial cell (HUVEC) networks and induced reduction in Fas and enhancement of FasL. Conditioned media of SIRT1 knockdown-Swan 71 cells caused reduction in cell proliferation and augmentation of cytotoxicity along with increased Fas expression in HUVECs. CONCLUSION: Our results suggest that SIRT1 may be associated with placental development by controlling EVT invasion and spiral artery remodeling via modulation of EMT, MMP-2, MMP-9, Akt/p38MAPK signaling, and Fas/FasL.

The anti-cancer drug gefitinib accelerates Fas-mediated apoptosis by enhancing caspase-8 activation in cancer cells.

Fas/CD95 plays a pivotal role in T cell-mediated cytotoxicity. Accumulating evidence has suggested that resistance to Fas-mediated apoptosis contributes to the escape of cancer cells from immune destruction, and allows to undergo proliferation and outgrowth of cancer cells. In this study, we found that the anti-cancer drug gefitinib, a tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR), has an ability to enhance Fas-mediated cytotoxicity. In the presence of nontoxic concentrations of gefitinib, Fas-induced activation of caspase-8 and subsequent apoptosis was dramatically promoted, suggesting that gefitinib increases the sensitivity to Fas-mediated apoptosis. Interestingly, the effects of gefitinib were observed in EGFR or p53 knockout (KO) cells. These observations indicate that both EGFR and p53 are dispensable for the enhancement. On the other hand, gefitinib clearly downregulated heat shock protein 70 (HSP70) as previously reported. Considering that HSP70 contributes to protection of cells against Fas-mediated apoptosis, gefitinib may increase the sensitivity to Fas-mediated apoptosis by downregulating HSP70. Thus, our findings reveal novel properties of gefitinib, which may provide insight into the alternative therapeutic approaches of gefitinib for Fas-resistant tumors.

Growth-dependent phenotype in FasL-deficient mandibular/alveolar bone.

FASL (CD178) is known for its role in triggering apoptosis, mostly in relation with immune cells but additional functions have been reported more recently, including those in bone development. Examination of postnatal FasL-deficient mice (gld) showed an increased bone deposition in adult mice when compared with wild types. However, a different phenotype was observed prenatally, when the gld bone was underdeveloped. The aim of the following investigation was to evaluate this indication for an growth-dependent bone phenotype of gld mice and to search for the 'switch point'. This study focused on the mandibular/alveolar bone as an important structure for tooth anchorage. In vivo micro-computed tomography (CT) analysis was performed at different stages during the first month (6, 12 and 24 days) of postnatal bone development. In 6-day-old gld mice, a decrease in bone volume/tissue volume (BV/TV), trabecular thickness and trabecular number was revealed. In contrast, the 12-day-old gld mice showed an increased BV/TV and trabecular thickness in the alveolar bone. The same observation applied for bone status in 24-day-old gld mice. Therefore, changes in the bone phenotype occurred between day 6 and 12 of the postnatal development. The switch point is likely related to the changing proportion of bone cells at these stages of development, when the number of osteocytes increases. Indeed, the immunohistochemical analysis of FASL localized this protein in osteoblasts, whereas osteocytes were mostly negative at examined stages. The impact of FASL particularly on osteoblasts would agree with an earlier in vivo observed effect of FASL deficiency on expression of Mmp2, typical for osteoblasts, in the gld mandibular/alveolar bone. Notably, an age-dependent bone phenotype was reported in Mmp2-deficient mice.

Soluble Fas ligand blocks destructive corneal inflammation in mouse models of corneal epithelial debridement and LPS induced keratitis.

Neutrophil-mediated inflammation plays a critical role in corneal damage following injury or infection. Previous studies demonstrated that membrane-bound FasL (mFasL) induces neutrophil chemokine production. However, the extracellular domain of mFasL is normally cleaved by matrix metalloproteinases to release a soluble form of FasL (sFasL) and sFasL antagonizes mFasL-mediated chemokine production. Therefore, we hypothesized that sFasL could be used to prevent neutrophil-mediated corneal inflammation associated with injury and bacterial keratitis. To test this hypothesis, GFP-only, sFasL-GFP, or mFasL-GFP were expressed in the corneal stroma of C57BL/6 mice, using intra-stromal injections of plasmid DNA or adenoviral vectors (AV) and the role of mFasL and sFasL in corneal inflammation was examined in models of corneal injury and LPS-induced keratitis. Our work addresses an important area of disagreement in the field of FasL, with regard to the mechanism by which sFasL regulates ocular inflammation. Herein, we demonstrate that an intrastromal injection of GFP-only, sFasL-GFP, or mFasL-GFP plasmid DNA resulted in GFP expression throughout the corneal stroma for up to two weeks with little to no evidence of inflammation in the GFP-only and sFasL-GFP groups and mild corneal inflammation in the mFasL-GFP group. Similarly, following epithelial debridement, corneas expressing GFP-only or sFasL-GFP showed no significant signs of corneal inflammation, with clear corneas at 15 days post debridement. By contrast, epithelial debridement of corneas expressing mFasL-GFP triggered persistent corneal inflammation and the development of central corneal opacities that was blocked by sFasL. Similar to the mFasL-GFP plasmid DNA, intrastromal injection of mFasL-GFP AV triggered mild corneal inflammation, but it was transient and resolved by day 10 with corneas remaining clear out to 30 days post injection. Nevertheless, intrastromal expression of mFasL-GFP AV exacerbated LPS-induced keratitis, corneal opacity, and neovascularization, while sFasL-GFP AV expression prevented LPS-induced keratitis, resulting in a clear cornea. Histological analysis of corneas with LPS-induced keratitis revealed a robust infiltration of macrophages and neutrophils and sFasL expression specifically blocked the neutrophil influx. Overall, our data demonstrate that stromal expression of mFasL is inflammatory, while sFasL is non-inflammatory, and opposes the effects of mFasL in mouse models of epithelial debridement and LPS-induced keratitis. These data demonstrate that a delicate balance between sFasL and mFasL regulates ocular inflammation. This study further identifies sFasL as a potent inhibitor of neutrophil-mediated corneal damage, and supports the potential use of sFasL in the treatment of neutrophil-mediated keratitis. These results strongly support the hypothesis that, in the immune privileged environment of the eye, the isoform of FasL regulates immune privilege and determines the extent of inflammation: mFasL promotes inflammation and sFasL blocks inflammation.

Nonapoptotic functions of Fas/CD95 in the immune response.

CD95 (also known as Fas) is a member of the tumor necrosis factor receptor (TNFR) superfamily. Its cognate ligand, CD95L, is implicated in immune homeostasis and immune surveillance. Mutations in this receptor are associated with a loss of apoptotic signaling and have been detected in an autoimmune disorder called autoimmune lymphoproliferative syndrome (ALPS) type Ia, which shares some clinical features with systemic lupus erythematosus (SLE). In addition, deletions and mutations of CD95 have been described in many cancers, which led researchers to initially classify this receptor as a tumor suppressor. More recent data demonstrate that CD95 engagement evokes nonapoptotic signals that promote inflammation and carcinogenesis. Transmembrane CD95L (m-CD95L) can be cleaved by metalloproteases, releasing a soluble ligand (s-CD95L). Soluble and membrane-bound CD95L show different stoichiometry (homotrimer versus multimer of homotrimers, respectively), which differentially affects CD95-mediated signaling through molecular mechanisms that remain to be elucidated. This review discusses the biological roles of CD95 in light of recent experiments addressing how a death receptor can trigger both apoptotic and nonapoptotic signaling pathways.

Activation of Fas/FasL pathway and the role of c-FLIP in primary culture of human cholangiocarcinoma cells.

Intrahepatic cholangiocarcinoma (iCCA) represents a heterogeneous group of malignancies emerging from the biliary tree, often in the context of chronic bile ducts inflammation. The immunological features of iCCA cells and their capability to control the lymphocytes response have not yet been investigated. The aims of the present study were to evaluate the interaction between iCCA cells and human peripheral blood mononuclear cells (PBMCs) and the role of Fas/FasL in modulating T-cells and NK-cells response after direct co-culture. iCCA cells express high levels of Fas and FasL that increase after co-culture with PBMCs inducing apoptosis in CD4+, CD8+ T-cells and in CD56+ NK-cells. In vitro, c-FLIP is expressed in iCCA cells and the co-culture with PBMCs induces an increase of c-FLIP in both iCCA cells and biliary tree stem cells. This c-FLIP increase does not trigger the caspase cascade, thus hindering apoptotis of iCCA cells which, instead, underwent proliferation. The increased expression of Fas, FasL and c-FLIP is confirmed in situ, in human CCA and in primary sclerosing cholangitis. In conclusion our data indicated that iCCA cells have immune-modulatory properties by which they induce apoptosis of T and NK cells, via Fas/FasL pathway, and escape inflammatory response by up-regulating c-FLIP system.

Death of adrenocortical cells during murine acute T. cruzi infection is not associated with TNF-R1 signaling but mostly with the type II pathway of Fas-mediated apoptosis.

Earlier studies from our laboratory demonstrated that acute experimental Trypanosoma cruzi infection promotes an intense inflammation along with a sepsis-like dysregulated adrenal response characterized by normal levels of ACTH with raised glucocorticoid secretion. Inflammation was also known to result in adrenal cell apoptosis, which in turn may influence HPA axis uncoupling. To explore factors and pathways which may be involved in the apoptosis of adrenal cells, together with its impact on the functionality of the gland, we carried out a series of studies in mice lacking death receptors, such as TNF-R1 (C57BL/6-Tnfrsf1a tm1Imx or TNF-R1-/-) or Fas ligand (C57BL/6 Fas-deficient lpr mice), undergoing acute T. cruzi infection. Here we demonstrate that the late hypercorticosterolism seen in C57BL/6 mice during acute T. cruzi infection coexists with and hyperplasia and hypertrophy of zona fasciculata, paralleled by increased number of apoptotic cells. Apoptosis seems to be mediated mainly by the type II pathway of Fas-mediated apoptosis, which engages the mitochondrial pathway of apoptosis triggering the cytochrome c release to increase caspase-3 activation. Fas-induced apoptosis of adrenocortical cells is also related with an exacerbated production of intra-adrenal cytokines that probably maintain the late supply of adrenal hormones during host response. Present results shed light on the molecular mechanisms dealing with these phenomena which are crucial not only for the development of interventions attempting to avoid adrenal dysfunction, but also for its wide occurrence in other infectious-based critical illnesses.

Interferon-γ affects leukemia cell apoptosis through regulating Fas/FasL signaling pathway.

OBJECTIVE: Imbalance of hematopoietic cell proliferation and apoptosis is one of the major causes of leukemia. Enhanced cell proliferation and reduced apoptosis lead to hemocytes accumulation. Fas/FasL signaling pathway promotes cell apoptosis. This study investigated the impact of interferon γ (IFN-γ) on chronic myelogenous leukemia cell proliferation and apoptosis to elucidate its interaction with Fas/FasL signaling pathway. PATIENTS AND METHODS: Leukemia K562 cells were routinely cultivated and treated with 10 U/ml, 100 U/ml, and 1000 U/ml interferon for 12 h, 24 h, and 48 h, respectively. MTT assay was applied to test cell proliferation. TUNEL assay was adopted to determine cell apoptosis. Western blot was selected to detect Fas/FasL expression. RESULTS: Different concentrations of IFN-γ inhibited cell proliferation at various time points. IFN-γ at 1000 U/ml treatment for 48 h exhibited the strongest suppressive effect on cell proliferation (p < 0.05). IFN-γ intervention enhanced K562 cell apoptosis with concentration and time dependence (p < 0.05). Fas and FasL proteins expressions upregulated after treated by IFN-γ following dose elevation and time extension (p < 0.05). CONCLUSIONS: IFN-γ inhibits leukemia K562 cell proliferation and promotes cell apoptosis via facilitating Fas and FasL proteins expressions.

SIRT2-mediated FOXO3a deacetylation drives its nuclear translocation triggering FasL-induced cell apoptosis during renal ischemia reperfusion.

We have found that Fas/FasL-mediated "extrinsic" pathway promoted cell apoptosis induced by renal ischemic injury. This study is to elucidate the upstream mechanism regulating FasL-induced extrinsic pathway during renal ischemia/reperfusion. Results demonstrated that when SIRT2 was activated by renal ischemia/reperfusion, activated SIRT2 could bind to and deacetylate FOXO3a, promoting FOXO3a nuclear translocation which resulted in an increase of nuclear FOXO3a along with FasL expression and activation of caspase8 and caspase3, triggering cell apoptosis during renal ischemia/reperfusion. The administration of SIRT2 inhibitor AGK2 prior to renal ischemia decreased significantly the number of apoptotic renal tubular cells and alleviated ultrastructure injury. These results indicate that inhibition of FOXO3a deacetylation might be a promising therapeutic approach for renal ischemia /reperfusion injury.

A novel mechanism of rs763110 polymorphism contributing to cervical cancer risk by affecting the binding affinity of C/EBPß and OCT1 complex to chromatin.

Recently, several studies have showed that FAS (rs2234767, rs1800682) and FASL (rs763110) functional single nucleotide polymorphisms (SNPs) were associated with the risk of various cancers. However, the association between cervical cancer risk and the three SNPs above remained inconclusive. In this work, we performed a two-stage case-control study on 1155 cervical cancer patients and 1252 matched healthy controls to determine the roles of the mentioned SNPs in cervical cancer susceptibility. We genotyped the FAS rs2234767, rs1800682, and FASL rs763110 polymorphisms using PCR-TaqMan assays. Results revealed that the rs763110 TT genotype significantly increased the risk of cervical cancer compared with the CC/CT genotype (adjusted OR = 1.70, 95% CI = 1.19-2.42). However, we did not observe any association between the cervical cancer risk and the rs2234767 and rs1800682 polymorphisms. The immunohistochemistry assay showed that patients carrying the rs763110 TT genotype presented a lower cancerous FASL expression than that of the CC/CT genotypes. Chromatin immunoprecipitation (ChIP) and Sequential Chromatin immunoprecipitation assays also demonstrated that OCT1 was recruited to the FASL promoter region and regulated the FASL gene transcription by interacting with C/EBPß. In conclusion, this study provided evidence indicating that the rs763110 variant in the FASL promoter was associated with the risk of cervical cancer by affecting the binding affinity of the C/EBPß/OCT1 complex to chromatin.

Fluoride reduced the immune privileged function of mouse Sertoli cells via the regulation of Fas/FasL system.

Previous investigations have demonstrated the adverse impacts of fluoride on Sertoli cells (SCs), such as oxidative stress and apoptosis. SCs are the crucial cellular components that can create the immune privileged environment in testis. However, the effect of fluoride on SCs immune privilege is unknown. In this study, mouse SCs were exposed to sodium fluoride with varying concentrations of 10-5, 10-4, and 10-3 mol/L to establish the model of fluoride-treated SCs (F-SCs) in vitro. After 48 h of incubation, F-SCs were transplanted underneath the kidney capsule of mice for 21 days, or cocultured with spleen lymphocytes for another 48 h. Immunohistochemical analysis of GATA4 in SCs grafts underneath kidney capsule presented less SCs distribution and obvious immune cell infiltration in F-SCs groups. In addition, the levels of FasL protein and mRNA in non-cocultured F-SCs decreased with the increase of fluoride concentration. When cocultured with F-SCs, lymphocytes presented significantly high cell viability and low apoptosis in F-SCs groups. Protein and mRNA expressions of FasL in cocultured F-SCs and Fas in lymphocytes were reduced, and the caspase 8 and caspase 3 mRNA levels were also decreased in fluoride groups in a dose-dependent manner. These findings indicated that fluoride influenced the testicular immune privilege through disturbing the Fas/FasL system.

[Recent progress in research of pathogeneses of Stevens-Johnson syndrome and toxic epidermal necrolysis].

Stevens-Johnson syndrome(SJS)and toxic epidermal necrolysis(TEN)are life-threatening cutaneous adverse drug reactions that induce widespread epidermal necrosis. Ocular and cutaneous diseases are common chronic sequelae of SJS and TEN. Several concepts have been proposed to explain the pathogenesis of severe cutaneous adverse drug reactions. Recent advances in genetic, pharmacogenomics and immunologic studies have provided evidences of genetic predispositions, drug metabolism and cytokines related to SJS and TEN. With regard to keratinocyte death, several cell death mediators, such as Fas/FasL, granulysin and TNF, have been proposed to play an important role in the pathogeneses of SJS and TEN. A subset of T lymphocytes, including regulatory T cells, may also play a role. This review summarizes the pathogeneses of SJS and TEN mainly from the aspects of genetic susceptibilities, drug metabolism, and immune cells and cytokines. (Chin J Ophthalmol, 2016, 52: 708-713).

TRPC6 channel activation promotes neonatal glomerular mesangial cell apoptosis via calcineurin/NFAT and FasL/Fas signaling pathways.

Glomerular mesangial cell (GMC) proliferation and death are involved in the pathogenesis of glomerular disorders. The mechanisms that control GMC survival are poorly understood, but may include signal transduction pathways that are modulated by changes in intracellular Ca(2+) ([Ca(2+)]i) concentration. In this study, we investigated whether activation of the canonical transient receptor potential (TRPC) 6 channels and successive [Ca(2+)]i elevation alter neonatal GMC survival. Hyperforin (HF)-induced TRPC6 channel activation increased [Ca(2+)]i concentration, inhibited proliferation, and triggered apoptotic cell death in primary neonatal pig GMCs. HF-induced neonatal GMC apoptosis was not associated with oxidative stress. However, HF-induced TRPC6 channel activation stimulated nuclear translocation of the nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1). HF also increased cell death surface receptor Fas ligand (FasL) level and caspase-8 activity in the cells; effects mitigated by [Ca(2+)]i chelator BAPTA, calcineurin/NFAT inhibitor VIVIT, and TRPC6 channel knockdown. Accordingly, HF-induced neonatal GMC apoptosis was attenuated by BAPTA, VIVIT, Fas blocking antibody, and a caspase-3/7 inhibitor. These findings suggest that TRPC6 channel-dependent [Ca(2+)]i elevation and the ensuing induction of the calcineurin/NFAT, FasL/Fas, and caspase signaling cascades promote neonatal pig GMC apoptosis.

[A role of the Fas system in the pathogenesis of ischemic stroke]. / Rol' sistemy Fas v patogeneze ishemicheskogo insul'ta.

The Fas system can promote several biological effects due to their activation after ischemic stroke: apoptosis, inflammation, proliferation, differentiation. Fas interacts with adapter proteins activating a number of signaling pathways, including MAPK, NFKB, JNK, ERK, phosphorylation of cytoskeletal proteins, and caspase-dependent apoptosis. Fas expressed by neuronal progenitor cells from the subventricular zone does not induce apoptosis in healthy adult humans. During motion and differentiation of these cells, Fas regulates their morphological structure by the phosphorylation/dephosphorylation of cytoskeletal elements. An increase in the Fas and Fas ligand expression is observed in response to stroke injury. Fas responsible not only for cell death and inflammation but also for neuronal plasticity which occupies a central place in the processes of sanogenesis.

Evaluation of the Fas/FasL signaling pathway in diabetic rat testis.

We investigated the role of the Fas/Fas ligand (FasL) signaling pathway in diabetic male infertility. Male rats were divided into two groups: a control group and a streptozotocin induced diabetic group. Thirty days after induction of diabetes, samples of testes were harvested and fixed in 10% formalin for light microscopy. Germ cell apoptosis was determined using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate in situ nick end-labeling (TUNEL) and immunostaining of caspase 8 and active caspase 3. We also investigated the expressions of Fas and FasL using immunohistochemistry. Streptozotocin-induced diabetes caused severe histopathological damage and increased apoptotic tubule and apoptotic cell indices, caspase 8 and caspase 3 expressions, and Fas and FasL-immunopositive cells in the rat testes. We suggest that the Fas/FasL signaling pathway may play a role in male infertility caused by diabetes.

CD8+CD122+CD49dlow regulatory T cells maintain T-cell homeostasis by killing activated T cells via Fas/FasL-mediated cytotoxicity.

The Fas/FasL (CD95/CD178) system is required for immune regulation; however, it is unclear in which cells, when, and where Fas/FasL molecules act in the immune system. We found that CD8(+)CD122(+) cells, which are mostly composed of memory T cells in comparison with naïve cells in the CD8(+)CD122(-) population, were previously shown to include cells with regulatory activity and could be separated into CD49d(low) cells and CD49d(high) cells. We established in vitro and in vivo experimental systems to evaluate the regulatory activity of CD122(+) cells. Regulatory activity was observed in CD8(+)CD122(+)CD49d(low) but not in CD8(+)CD122(+)CD49d(high) cells, indicating that the regulatory cells in the CD8(+)CD122(+) population could be narrowed down to CD49d(low) cells. CD8(+)CD122(-) cells taken from lymphoproliferation (lpr) mice were resistant to regulation by normal CD122(+) Tregs. CD122(+) Tregs taken from generalized lymphoproliferative disease (gld) mice did not regulate wild-type CD8(+)CD122(-) cells, indicating that the regulation by CD122(+) Tregs is Fas/FasL-dependent. CD122(+) Tregs taken from IL-10-deficient mice could regulate CD8(+)CD122(-) cells as equally as wild-type CD122(+) Tregs both in vitro and in vivo. MHC class I-missing T cells were not regulated by CD122(+) Tregs in vitro. CD122(+) Tregs also regulated CD4(+) cells in a Fas/FasL-dependent manner in vitro. These results suggest an essential role of Fas/FasL as a terminal effector of the CD122(+) Tregs that kill activated T cells to maintain immune homeostasis.

Memory T cell-driven differentiation of naive cells impairs adoptive immunotherapy.

Adoptive cell transfer (ACT) of purified naive, stem cell memory, and central memory T cell subsets results in superior persistence and antitumor immunity compared with ACT of populations containing more-differentiated effector memory and effector T cells. Despite a clear advantage of the less-differentiated populations, the majority of ACT trials utilize unfractionated T cell subsets. Here, we have challenged the notion that the mere presence of less-differentiated T cells in starting populations used to generate therapeutic T cells is sufficient to convey their desirable attributes. Using both mouse and human cells, we identified a T cell-T cell interaction whereby antigen-experienced subsets directly promote the phenotypic, functional, and metabolic differentiation of naive T cells. This process led to the loss of less-differentiated T cell subsets and resulted in impaired cellular persistence and tumor regression in mouse models following ACT. The T memory-induced conversion of naive T cells was mediated by a nonapoptotic Fas signal, resulting in Akt-driven cellular differentiation. Thus, induction of Fas signaling enhanced T cell differentiation and impaired antitumor immunity, while Fas signaling blockade preserved the antitumor efficacy of naive cells within mixed populations. These findings reveal that T cell subsets can synchronize their differentiation state in a process similar to quorum sensing in unicellular organisms and suggest that disruption of this quorum-like behavior among T cells has potential to enhance T cell-based immunotherapies.