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• Surgery-induced DNT infiltration promotes cognitive dysfunction. • Hippocampal DNTs result in IL-17 upregulation postoperatively. • IL-17 directly induces activation of astrocytes and C3 upregulation via CEBPβ. • Inhibition of the IL-17/CEBPβ pathway attenuates postoperative neuroinflammation and cognitive dysfunction. CD3(+) CD4(−) CD8(−) double negative T cells (DNTs) manifest themselves in autoimmune diseases and associated inflammation. In the central nervous system, the increased presence of DNTs is associated with the progression of neurological conditions and brain injury. Active DNTs that produce IL-17 have been regarded as a pro-inflammatory phenotype. The IL-17 signaling pathway mediates neuroinflammatory responses by inducing glial activation and producing inflammatory factors. Neuroinflammation is considered integral to the pathogenesis of perioperative neurocognitive disorders (PNDs), commonly developed after surgery in susceptible patients. We and others have demonstrated a significant role for complement C3 in surgery-induced neuroinflammation and cognitive impairment but the regulatory mechanisms for this remain unexplored. We hypothesized that surgery induces DNT infiltration into the CNS that in turn upregulates complement C3 expression and this causes changes that contribute to cognitive impairment. Using an adult murine abdominal surgery model, we investigated perioperative changes in cognitive performance, quantifying the presence of T cell subsets and phenotype, IL-17 signaling pathway activation, glial cell activation and C3 expression in the brain. Postoperative IL-17 specific inhibitor GSK2981278 administration or preoperatively conditional CEBPβ knock-down by AAV9 viral vector were then applied to evaluate the effect of inhibiting IL-17 signaling pathway on postoperative C3 expression and cognitive performance. The results showed an increased hippocampus infiltration of DNTs with augmented IL-17 production, along with C3 upregulation and cognitive impairment. Both inhibition of IL-17 or knock-down of CEBPβ significantly suppressed C3 expression, synaptic engulfment by microglia and attenuated cognitive impairment. These findings indicate that DNTs promote postoperative neuroinflammation and cognitive impairment via the IL-17/CEBPβ/C3 pathway and targeting this IL-17 axis could be a potential therapeutic strategy to ameliorate postoperative neuroinflammation and cognitive impairment. [ABSTRACT FROM AUTHOR]

Abstract Background Psoriasis is a chronic immune-mediated skin condition. Although biologic treatments are effective in controlling psoriasis, some patients do not respond or lose response to these therapies. Thus, new strategies for psoriasis treatment are still urgently needed. Double-negative T cells (DNT) play a significant immunoregulatory role in autoimmune diseases. In this study, we aimed to evaluate the protective effect of DNT in psoriasis and explore the underlying mechanism. Methods We conducted a single adoptive transfer of DNT into an imiquimod (IMQ)-induced psoriasis mouse model through tail vein injection. The skin inflammation and IL-17A producing γδ T cells were evaluated. Results DNT administration significantly reduced the inflammatory response in mouse skin, characterized by decreased skin folds, scales, and red patches. After DNT treatment, the secretion of IL-17A by RORc+ γδlow T cells in the skin was selectively suppressed, resulting in an amelioration of skin inflammation. Transcriptomic data suggested heightened expression of NKG2D ligands in γδlow T cells within the mouse model of psoriasis induced by IMQ. When blocking the NKG2D ligand and NKG2D (expressed by DNT) interaction, the cytotoxic efficacy of DNT against RORc+IL17A+ γδlow T cells was attenuated. Using Ccr5 −/− DNT for treatment yielded evidence that DNT migrates into inflamed skin tissue and fails to protect IMQ-induced skin lesions. Conclusions DNT could migrate to inflamed skin tissue through CCR5, selectively inhibit IL-17-producing γδlow T cells and finally ameliorate mouse psoriasis. Our study provides feasibility for using immune cell therapy for the prevention and treatment of psoriasis in the clinic.

Background: Psoriasis is a chronic immune-mediated skin condition. Although biologic treatments are effective in controlling psoriasis, some patients do not respond or lose response to these therapies. Thus, new strategies for psoriasis treatment are still urgently needed. Double-negative T cells (DNT) play a significant immunoregulatory role in autoimmune diseases. In this study, we aimed to evaluate the protective effect of DNT in psoriasis and explore the underlying mechanism. Methods: We conducted a single adoptive transfer of DNT into an imiquimod (IMQ)-induced psoriasis mouse model through tail vein injection. The skin inflammation and IL-17A producing γδ T cells were evaluated. Results: DNT administration significantly reduced the inflammatory response in mouse skin, characterized by decreased skin folds, scales, and red patches. After DNT treatment, the secretion of IL-17A by RORc+ γδlow T cells in the skin was selectively suppressed, resulting in an amelioration of skin inflammation. Transcriptomic data suggested heightened expression of NKG2D ligands in γδlow T cells within the mouse model of psoriasis induced by IMQ. When blocking the NKG2D ligand and NKG2D (expressed by DNT) interaction, the cytotoxic efficacy of DNT against RORc+IL17A+ γδlow T cells was attenuated. Using Ccr5−/− DNT for treatment yielded evidence that DNT migrates into inflamed skin tissue and fails to protect IMQ-induced skin lesions. Conclusions: DNT could migrate to inflamed skin tissue through CCR5, selectively inhibit IL-17-producing γδlow T cells and finally ameliorate mouse psoriasis. Our study provides feasibility for using immune cell therapy for the prevention and treatment of psoriasis in the clinic. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the effects of early ART on the frequencies and phenotypes of double-negative(DN)T cells in acute HIV-infected individuals(AHIs). Methods: AHIs who received early ART were enrolled and the frequencies and phenotypes of DN T cells were detected by flow cytometry. Results: The frequencies of DN T cells were significantly lower in AHIs compared with healthy controls (HCs). Early ART failed to fully restore the frequencies of DN T cells. Moreover, the activation of DN T cell subsets, Vδ1 and Vδ2 T cells in AHIs were significantly increased and negatively associated with CD4+ T cell counts at baseline. Although early ART could normalize the activation of Vδ1 and Vδ2 T cells, Vδ1/2neg T cell activation was still higher in AHIs than that in HCs, which was negatively associated with CD4+ T cell counts after early ART. Conclusion: The changes in the frequencies and phenotypes of DN T cells are associated with HIV disease progression and CD4+ T cell recovery after early ART. [ABSTRACT FROM AUTHOR]

Purpose: Tumor-infiltrating lymphocytes (TILs) have shown remarkable clinical responses in some patients with advanced solid tumors. As a rare subset of TILs, CD4−/CD8− double-negative T cells (DNTs) were poorly known. This study aims to investigate the characteristics and function of CD3+CD4−CD8− TILs (double-negative TIL, DN-TILs) derived from solid tumor. Methods: DN-TILs were derived and expanded ex vivo from resected gastric carcinoma tissue and phenotyped by flow cytometry. The cytotoxicity of DN-TILs was determined against established tumor cell lines in vitro or through in vivo adoptive transfer into xenograft models. K562 cells were transferred with the HLA gene to verify whether the cytotoxicity of DN-TILs was MHC-independent. Results: Flow cytometric analysis revealed a high-purity population of DN-TILs (> 97%) within CD3+ TILs, which expanded more than 800-folds in 2 weeks, consisting of a mixture of alpha–beta (αβ) and gamma–delta (γδ) T-cell receptor (TCR)—expressing cells (with the majority being αβ-TCR, > 95%). Using single-cell RNA sequencing, the expanded DN-TILs were categorized into four main subsets, Natural Killer T cells (approximately 80%, 5563 in 7028), Progenitor cells, Germ cells and T helper2 cells. DN-TILs exhibited a broad anticancer cytotoxicity in a donor-unrestricted manner against various cancer cell lines derived from pancreatic cancer (Panc-1), gastric cancer (HGC-27), ovarian cancer (SKOV-3), malignant melanoma (A375). The cytotoxicity was MHC-independent, which was not altered in K562 transferring with HLA gene or not. DN-TILs significantly reduced tumor volume in xenograft models with superior tumor-homing ability and low off-target toxicity. Conclusion: Gastric carcinoma derived DN-TIL can target tumor cells in vitro and in vivo. DN-TILs have the potential to be used as a adoptive cell therapy for solid cancers with both the advantages of DNT and TIL. [ABSTRACT FROM AUTHOR]

Background: Immune function in the genital mucosa balances reproduction with protection against pathogens. As women age, genital infections, and gynecological cancer risk increase, however, the mechanisms that regulate cell-mediated immune protection in the female genital tract and how they change with aging remain poorly understood. Unconventional double negative (DN) T cells (TCRαβ + CD4-CD8-) are thought to play important roles in reproduction in mice but have yet to be characterized in the human female genital tract. Using genital tissues from women (27–77 years old), here we investigated the impact of aging on the induction, distribution, and function of DN T cells throughout the female genital tract. Results: We discovered a novel site-specific regulation of dendritic cells (DCs) and unconventional DN T cells in the genital tract that changes with age. Human genital DCs, particularly CD1a + DCs, induced proliferation of DN T cells in a TFGβ dependent manner. Importantly, induction of DN T cell proliferation, as well as specific changes in cytokine production, was enhanced in DCs from older women, indicating subset-specific regulation of DC function with increasing age. In human genital tissues, DN T cells represented a discrete T cell subset with distinct phenotypical and transcriptional profiles compared to CD4 + and CD8 + T cells. Single-cell RNA and oligo-tag antibody sequencing studies revealed that DN T cells represented a heterogeneous population with unique homeostatic, regulatory, cytotoxic, and antiviral functions. DN T cells showed relative to CD4 + and CD8 + T cells, enhanced expression of inhibitory checkpoint molecules and genes related to immune regulatory as well as innate-like anti-viral pathways. Flow cytometry analysis demonstrated that DN T cells express tissue residency markers and intracellular content of cytotoxic molecules. Interestingly, we demonstrate age-dependent and site-dependent redistribution and functional changes of genital DN T cells, with increased cytotoxic potential of endometrial DN T cells, but decreased cytotoxicity in the ectocervix as women age, with implications for reproductive failure and enhanced susceptibility to infections respectively. Conclusions: Our deep characterization of DN T cell induction and function in the female genital tract provides novel mechanistic avenues to improve reproductive outcomes, protection against infections and gynecological cancers as women age. [ABSTRACT FROM AUTHOR]

Simple Summary: Consensus Molecular Subtypes have recently been proposed based on molecular and immune landscape of colorectal carcinoma (CRC). Only mismatch repair deficient and hyper-mutated CRC (CRCdMMR) can obtain clinical benefits from immune checkpoint blockades; on the other hand, mismatch repair proficient CRCs (CRCpMMR) have very limited therapeutic options. This study establishes that CRCpMMR displays an immunosuppressive microenvironment containing abundant tumor-associated macrophages (TAMs) and neutrophils, with a reduction in double negative T lymphocytes and B cells and increased exhausted tumor-infiltrating lymphocytes. Poor immunogenicity in CRCpMMR is further supported by interferon gamma (IFN-γ) unresponsiveness of both tumor cells and TAMs. Colorectal carcinoma (CRC) represents a lethal disease with heterogeneous outcomes. Only patients with mismatch repair (MMR) deficient CRC showing microsatellite instability and hyper-mutated tumors can obtain clinical benefits from current immune checkpoint blockades; on the other hand, immune- or target-based therapeutic strategies are very limited for subjects with mismatch repair proficient CRC (CRCpMMR). Here, we report a comprehensive typing of immune infiltrating cells in CRCpMMR. We also tested the expression and interferon-γ-modulation of PD-L1/CD274. Relevant findings were subsequently validated by immunohistochemistry on fixed materials. CRCpMMR contain a significantly increased fraction of CD163+ macrophages (TAMs) expressing TREM2 and CD66+ neutrophils (TANs) together with decrease in CD4−CD8−CD3+ double negative T lymphocytes (DNTs); no differences were revealed by the analysis of conventional and plasmacytoid dendritic cell populations. A fraction of tumor-infiltrating T-cells displays an exhausted phenotype, co-expressing PD-1 and TIM-3. Remarkably, expression of PD-L1 on fresh tumor cells and TAMs was undetectable even after in vitro stimulation with interferon-γ. These findings confirm the immune suppressive microenvironment of CRCpMMR characterized by dense infiltration of TAMs, occurrence of TANs, lack of DNTs, T-cell exhaustion, and interferon-γ unresponsiveness by host and tumor cells. Appropriate bypass strategies should consider these combinations of immune escape mechanisms in CRCpMMR. [ABSTRACT FROM AUTHOR]

Autoimmune lymphoproliferative syndrome (ALPS) is a rare genetic disorder of lymphocyte homeostasis, resulting from mutations in the Fas apoptotic pathway. It is characterized by noninfectious and non-malignant chronic lymphoproliferation and an increased risk of lymphoid malignancy. The diagnosis of this condition usually combines chronic lymphadenopathy and/or splenomegaly exceeding 6 months, autoimmune cytopenias, with an elevated level of CD3+CD4-CD8-Tαβ lymphocytes, known as "double-negative" T cells. Differential diagnosis includes infections, autoimmune diseases or malignancies. Although clinical examination and laboratory tests are highly suggestive, this disease goes widely unrecognized. We here report, for the first time, the case of ALPS, a Moroccan patient, and aged 8 years, with recurrent fever, splenomegaly and adenopathies. Paraclinical examinations revealed chronic pancytopenia, higher than normal Tαβ double negative lymphocytes, hypergammaglobulinemia, and elevated serum levels of soluble FAS ligand. The diagnosis of ALPS was made. First-line treatment included corticosteroids and immunoglobulins. Then the patient received mycophenolate followed by Sirolimus. This treatment resulted in better clinical and laboratory tests results. Our aim is to raise awareness of this rare condition, which may be under-diagnosed, among physicians. [ABSTRACT FROM AUTHOR]

Introduction: The characteristics of the tumor immune microenvironment (TIME) are closely related to immunotherapy. Breast cancer can benefit from immunotherapy, and its TIME is still unclear. Methods: We utilized mass cytometry to explore the immune cell heterogeneity in breast cancer. Double‐negative T cells (DNTs) from healthy volunteers (HBs) were enriched in vitro. Flow cytometry was used to detect the cell surface receptors of cancer cells and DNT cells. The correlation between immune checkpoints and the abundance of immune cells or prognosis of breast cancer was analyzed by the TCGA database. The messenger RNA (mRNA) expression of functional genes was performed by quantitative real‐time PCR. Results: We found that the frequencies of Granzyme B (GZMB)+CD8+T and GZMB+DNT cells in cancer tissues (CA) of breast cancer were lower than those in blood samples of patients (PB), and the frequencies of programmed cell death protein 1 (PD1)+CD8+T and PD1+DNT cells in CA were higher than those in PB. DNTs from HBs had a cytotoxic effect on MDA‐MB‐231. LAG3Ab could upregulate the mRNA expression of interferon gamma and perforin by increasing T‐BET transcription to enhance the cytotoxicity of DNT cells in vitro. Conclusion: Our study revealed the suppressive status of TIME in breast cancer and supported DNT cells had the potential to be applied as a novel adoptive cell therapy for TNBC either alone or combined with LAG3Ab. [ABSTRACT FROM AUTHOR]

OBJECTIVES: The 2015 Workshop of the Society for Hematopathology/European Association for Haematopathology aimed to review primary immunodeficiency and related lymphoproliferations. METHODS: Primary immunodeficiencies were divided into immune dysregulation, DNA repair defects, low immunoglobulins, and combined immunodeficiencies. RESULTS: Autoimmune lymphoproliferative syndrome (ALPS) is a prototypical immune dysregulation-type immunodeficiency, with defects in T-cell signaling or apoptosis, expansion of T-cell subsets, and predisposition to hemophagocytic lymphohistiocytosis. DNA repair defects directly predispose to malignancy. Low immunoglobulin immunodeficiencies such as common variable immunodeficiency (CVID) have underlying T-cell repertoire abnormalities predisposing to autoimmunity and B-cell lymphoproliferations. The full spectrum of B-cell lymphoproliferative disorders occurs in primary immunodeficiency. CONCLUSIONS: Lymphoproliferations in primary immunodeficiency mirror those in other immunodeficiency settings, with monomorphic B- and sometimes T lymphoproliferative disorders enriched in DNA repair defects. Distinctive T-cell subset expansions in ALPS, CVID, and related entities can mimic lymphoma, and recognition of double-negative T-cell or cytotoxic T-cell expansions is key to avoid overdiagnosis.

Abstract Introduction The characteristics of the tumor immune microenvironment (TIME) are closely related to immunotherapy. Breast cancer can benefit from immunotherapy, and its TIME is still unclear. Methods We utilized mass cytometry to explore the immune cell heterogeneity in breast cancer. Double‐negative T cells (DNTs) from healthy volunteers (HBs) were enriched in vitro. Flow cytometry was used to detect the cell surface receptors of cancer cells and DNT cells. The correlation between immune checkpoints and the abundance of immune cells or prognosis of breast cancer was analyzed by the TCGA database. The messenger RNA (mRNA) expression of functional genes was performed by quantitative real‐time PCR. Results We found that the frequencies of Granzyme B (GZMB)+CD8+T and GZMB+DNT cells in cancer tissues (CA) of breast cancer were lower than those in blood samples of patients (PB), and the frequencies of programmed cell death protein 1 (PD1)+CD8+T and PD1+DNT cells in CA were higher than those in PB. DNTs from HBs had a cytotoxic effect on MDA‐MB‐231. LAG3Ab could upregulate the mRNA expression of interferon gamma and perforin by increasing T‐BET transcription to enhance the cytotoxicity of DNT cells in vitro. Conclusion Our study revealed the suppressive status of TIME in breast cancer and supported DNT cells had the potential to be applied as a novel adoptive cell therapy for TNBC either alone or combined with LAG3Ab.

Immune regulation of female reproductive function plays a crucial role in fertility, as alterations in the relationship between immune and reproductive processes result in autoimmune subfertility or infertility. The breakdown of immune tolerance leads to ovulation dysfunction, implantation failure, and pregnancy loss. In this regard, immune cells with regulatory activities are essential to restore self-tolerance. Apart from regulatory T cells, double negative T regulatory cells (DNTregs) characterized by TCRaβ+/ γδ+CD3+CD4-CD8- (and negative for natural killer cell markers) are emerging as effector cells capable of mediating immune tolerance in the female reproductive system. DNTregs are present in the female reproductive tract of humans and murine models. However, their full potential as immune regulators is evolving, and studies so far indicate that DNTregs exhibit features that can also maintain tolerance in the female reproductive microenvironment. This review describes recent progress on the presence, role and mechanisms of DNTregs in the female reproductive system immune regulation and tolerance. In addition, we address how DNTregs can potentially provide a paradigm shift from the known roles of conventional regulatory T cells and immune tolerance by maintaining and restoring balance in the reproductive microenvironment of female fertility. [ABSTRACT FROM AUTHOR]

Liver transplantation (LTx) is currently the only effective therapy for patients with end-stage liver diseases, but post-transplant infection is a key issue for morbidity and mortality. In this study, we found that pre-transplant patients with an expansion of double-negative T (DNT) cells (CD3+CD4−CD8− T cells) had an increased incidence of infections within the first 6 months after LTx. These DNT cells also negatively correlated with their CD4/CD8 ratio. Compared to patients who had no infections after LTx, these DNT cells expressed more CD25, especially in the memory compartment. The receiver operating characteristic (ROC) analysis showed that the threshold area under the ROC curve of DNT cells which could be used to distinguish LTx patients with post-transplant infections from patients without infections after LTx was 0.8353 (95% CI: 0.6591–1.000). The cut-off for the pre-LTx DNT cell level was 11.35%. Although patients with post-transplant infections had decreased levels of CD4/CD8 T cells, CD8+ T cells in these patients were more exhausted, with higher PD-1 expression and lower IFNγ secretion. The increased levels of DNT cells in patients with post-transplant infections were still observed 2 weeks after LTx, with higher proportions of memory DNT cells. In conclusion, increased levels of DNT cells in pre-LTx patients may be valuable for the prognosis of post-transplant infections, especially within the first 6 months after LTx. [ABSTRACT FROM AUTHOR]

Immune regulation of female reproductive function plays a crucial role in fertility, as alterations in the relationship between immune and reproductive processes result in autoimmune subfertility or infertility. The breakdown of immune tolerance leads to ovulation dysfunction, implantation failure, and pregnancy loss. In this regard, immune cells with regulatory activities are essential to restore self-tolerance. Apart from regulatory T cells, double negative T regulatory cells (DNTregs) characterized by TCRαβ+/γδ+CD3+CD4–CD8– (and negative for natural killer cell markers) are emerging as effector cells capable of mediating immune tolerance in the female reproductive system. DNTregs are present in the female reproductive tract of humans and murine models. However, their full potential as immune regulators is evolving, and studies so far indicate that DNTregs exhibit features that can also maintain tolerance in the female reproductive microenvironment. This review describes recent progress on the presence, role and mechanisms of DNTregs in the female reproductive system immune regulation and tolerance. In addition, we address how DNTregs can potentially provide a paradigm shift from the known roles of conventional regulatory T cells and immune tolerance by maintaining and restoring balance in the reproductive microenvironment of female fertility.

Les télomères sont composés d'une séquence répétée de nucléotides double brin TTAGGG et sont associés à de nombreuses protéines dont le complexe « Shelterin ». Leur rôle principal est de maintenir la stabilité du génome lors de la réplication cellulaire grâce à un mécanisme de copie de la séquence répétée par le complexe télomérase. L'ensemble des pathologies impliquant une dérégulation de la maintenance des télomères sont maintenant réunies sous le terme de téloméropathies. L'objectif de notre étude était de décrire les caractéristiques clinico-biologiques et thérapeutiques dans une cohorte de patients atteints de téloméropathie de recrutement hématologique suivis en médecine interne au centre hospitalier universitaire de Lille. Il s'agit d'une étude rétrospective, unicentrique réalisée au sein du service de médecine interne-immunologie clinique, centre de référence des maladies auto-immunes et systémique rares au CHU de Lille entre 2005 et 2020 incluant l'ensemble des patients suivis pour téloméropathie. Des cas index et des apparentés ont été inclus. Les 15 patients étudiés étaient issus de 10 familles indépendantes. Soixante pour cent étaient porteurs d'une mutation du gène TERC à l'état hétérozygote. Soixante-sept pour cent avaient une atteinte hématologique comprenant notamment une macrocytose avec anémie et/ou thrombopénie, 20 % une atteinte hépatique et 27 % une atteinte pulmonaire de type fibrosante. L'immunophénotypage lymphocytaire montrait la présence d'une population lymphocytaire T double négative avec une expression de TCR γδ pour 5 (33 %) d'entre eux. Quarante-sept pour cent des patients n'avaient reçu aucun traitement. Vingt-sept pour cent étaient sous androgène. Vingt pour cent avaient reçu de la ciclosporine et 13 % du sérum anti-lymphocytaire dans un contexte de diagnostic erroné d'une aplasie acquise. Il est important pour le médecin interniste d'être sensibilisé à la complexité des téloméropathies, diagnostic différentiel des aplasies médullaires acquises idiopathiques, pour pouvoir optimiser la prise en charge et éviter les thérapeutiques inappropriées. L'allogreffe de cellules souches hématopoïétiques est le seul traitement potentiellement curatif. Notre analyse a rapporté des particularités à l'immunophénotypage lymphocytaire non décrites à notre connaissance dont l'imputabilité physiopathologique reste à démontrer. Telomeres are composed of a repeated sequence of double-stranded nucleotides TTAGGG and numerous proteins including the Shelterin complex. Their main role is to maintain the stability of the genome during cell replication through a mechanism of copying the repeted sequence by the telomerase complexe. All the diseases involving a deregulation of this complex are now grouped together under the term telomeropathies. They are difficult to diagnose and manage. Our objective was to describe the clinico-biological characteristics and treatments used, in patients affected by telomeropathies previously seen by an hematologist followed at the Lille University Hospital Center. This is a retrospective, single-center study carried out within the department of internal medicine-clinical immunology, Reference center for rare autoimmune and systemic diseases at Lille University Hospital Center between 2005 and 2020 including all patients followed for telomeropathy. Probands and relatives were included. Fifteen patients were studied from 10 independant families. Sixty percent had an heterozygous TERC gene mutation. Sixty seven percent had haematological diseases including macrocytosis, anemia and/or thrombocytopenia, 20 % had a fibrotic hepatic disease, 27 % had a fibrotic pulmonary disease. Lymphocyte immunophenotyping showed a double negative T lymphocyte population with γδ TCR expression in 5 (33 %) patients. Forty-seven percent of the patients had not received any treatment. Twenty-seven percent were on androgen therapy. Twenty percent had received cyclosporine and 13 % anti-lymphocyte serum in the context of initial misdiagnosis. It is important to be aware of the complexity of telomeropathies, a differential diagnosis of immune aplastic anemia, in order to optimize management and avoid inappropriate treatments. Allografting of hematopoietic stem cells is the only potentially curative treatment. Our analysis found particularities in immunophenotyping lymphocyte not previously described to our knowledge, whose physiopathological imputability remains to be demonstrated. [ABSTRACT FROM AUTHOR]

Susceptibility to autoimmune diabetes is a complex genetic trait. Linkage analyses exploiting the NOD mouse, which spontaneously develops autoimmune diabetes, have proved to be a useful tool for the characterization of some of these traits. In a linkage analysis using 3A9 TCR transgenic mice on both B10.BR and NOD.H2k backgrounds, we previously determined that both the Idd2 and Idd13 loci were linked to the proportion of immunoregulatory CD4-CD8- double negative (DN) T cells. In addition to Idd2 and Idd13, five other loci showed weak linkage to the proportion of DN T cells. Of interest, in an interim analysis, a locus on chromosome 12 is linked to DN T cell proportion in both the spleen and the lymph nodes. To determine the impact of this locus on DN T cells, we generated two congenic sublines, which we named Chr12P and Chr12D for proximal and distal, respectively. While 3A9 TCR:insHEL NOD.H2k-Chr12D mice were protected from diabetes, 3A9 TCR:insHEL NOD.H2k-Chr12P showed an increase in diabetes incidence. Yet, the proportion of DN T cells was similar to the parental 3A9 TCR NOD.H2k strain for both of these congenic sublines. A genome-wide two dimensional LOD score analysis reveals genetic epistasis between chromosome 12 and the Idd13 locus. Altogether, this study identified further complex genetic interactions in defining the proportion of DN T cells, along with evidence of genetic epistasis within a locus on chromosome 12 influencing autoimmune susceptibility. [ABSTRACT FROM AUTHOR]

Abstract Background Though immune checkpoint blockade (ICB) against PD-1 has shown success in the treatment of lung cancer, not all patients respond. We have previously shown that adoptive transfer of double negative T (DNT) cells expanded from healthy donors can target leukemia but their role in treating established lung cancer is not clear. Here we explore the role of human DNT cells in targeting late-stage established lung cancer either alone or in combination with Nivolumab (anti-PD-1 antibody) and describe underlying mechanisms. Methods DNT cells from resected lung cancer tissue of patients were analyzed by flow cytometry to determine their infiltration and PD-1 expression. Expansion capacity and anti-tumor function of lung cancer patient and healthy donor DNT cells were compared. Late-stage lung cancer xenograft models were developed to determine the anti-tumor effect of DNT cells alone or in combination with anti-PD-1 antibody, and the level of tumor-infiltrating DNT cells was quantified by histology and characterized by flow cytometry. Results Patient-derived tumor infiltrating lymphocytes contained a lower frequency of DNT cells with a higher expression of PD-1 relative to normal lung tissue. Ex vivo expanded patient- and healthy donor-derived DNT cells showed similar levels of cytotoxicity against lung cancer cells in vitro. Healthy donor-derived DNT cells significantly inhibited the growth of late-stage lung cancer xenografts, which was further augmented by anti-PD-1 through increased DNT cell tumor infiltration. Conclusion This study supports the use of DNT cells for adoptive cellular therapy against lung cancer either alone or in combination with anti-PD-1.

Abstract Background The advents of novel immunotherapies have revolutionized the treatment of cancer. Adoptive cellular therapies using chimeric antigen receptor T (CAR-T) cells have achieved remarkable clinical responses in B cell leukemia and lymphoma but the effect on solid tumors including lung cancer is limited. Here we present data on the therapeutic potential of allogeneic CD3+CD4−CD8− double negative T (DNT) cells as a new cellular therapy for the treatment of lung cancer and underlying mechanisms. Methods DNTs were enriched and expanded ex vivo from healthy donors and phenotyped by flow cytometry. Functionally, their cytotoxicity was determined against primary and established non-small-cell lung cancer (NSCLC) cell lines in vitro or through in vivo adoptive transfer into xenograft models. Mechanistic analysis was performed using blocking antibodies against various cell surface and soluble markers. Furthermore, the role of IL-15 on DNT function was determined. Results We demonstrated that ex vivo expanded DNTs can effectively lyse various human NSCLC cells in vitro and inhibit tumor growth in xenograft models. Expanded DNTs have a cytotoxic phenotype, as they express NKp30, NKG2D, DNAM-1, membrane TRAIL (mTRAIL), perforin and granzyme B, and secrete IFNγ and soluble TRAIL (sTRAIL). DNT-mediated cytotoxicity was dependent on a combination of tumor-expressed ligands for NKG2D, DNAM-1, NKp30 and/or receptors for TRAIL, which differ among different NSCLC cell lines. Furthermore, stimulation of DNTs with IL-15 increased expression of effector molecules on DNTs, their TRAIL production and cytotoxicity against NSCLC in vitro and in vivo. Conclusion Healthy donor-derived DNTs can target NSCLC in vitro and in vivo. DNTs recognize tumors via innate receptors which can be up-regulated by IL-15. DNTs have the potential to be used as a novel adoptive cell therapy for lung cancer either alone or in combination with IL-15.

Acute kidney injury (AKI) due to cisplatin is a significant problem that limits its use as an effective chemotherapeutic agent. T cell receptor+CD4-CD8- double negative (DN) T cells constitute the major T cell population in the human and mouse kidney, express programmed cell death protein (PD)-1, and protect from ischemic AKI. However, the pathophysiological roles of DN T cells in cisplatin-induced AKI is unknown. In this study, wild-type mice were treated with cisplatin (30 mg/kg) or vehicle, and the effects on kidney DN T cell numbers and function were measured. In vitro experiments evaluated effects of kidney DN T cells on cisplatin-induced apoptosis and PD ligand 1 (PD-L1) in renal epithelial cells. Adoptive transfer experiments assessed the therapeutic potential of DN T cells during cisplatin-induced AKI. Our results show that kidney DN T cell population increased at 24 h and declined by 72 h after cisplatin treatment. Cisplatin treatment increased kidney DN T cell proliferation, apoptosis, CD69, and IL-10 expression, whereas CD62L, CD44, IL-17A, interferon-γ, and TNF-α were downregulated. Cisplatin treatment decreased both PD-1 and natural killer 1.1 subsets of kidney DN T cells with a pronounced effect on the PD-1 subset. In vitro kidney DN T cell coculture decreased cisplatin-induced apoptosis in kidney proximal tubular epithelial cells, increased Bcl-2, and decreased cleaved caspase 3 expression. Cisplatin-induced expression of PD ligand 1 was reduced in proximal tubular epithelial cells cocultured with DN T cells. Adoptive transfer of DN T cells attenuated kidney dysfunction and structural damage from cisplatin-induced AKI. These results demonstrate that kidney DN T cells respond rapidly and play a protective role during cisplatin-induced AKI. [ABSTRACT FROM AUTHOR]

Background: The role of circulating CD4−/CD8− double-negative T cells (DNTs) in the immune response to melanoma is poorly understood, as are the effects of checkpoint inhibitors on T cell subpopulations. Methods: We performed a basal and longitudinal assessment of circulating immune cells, including DNTs, in metastatic melanoma patients treated with checkpoint blockade in a single-center cohort, and examined the correlations levels of immune cells with clinical features and therapy outcomes. Results: Sixty-eight patients (48 ipilimumab, 20 PD1 inhibitors) were enrolled in the study. Our analysis indicated that better outcomes were associated with normal LDH, fewer than three metastatic sites, an ECOG performance status of 0, M1a stage, lower WBC and a higher lymphocyte count. The increase in lymphocyte count and decrease of DNTs were significantly associated with the achievement of an overall response. The median value of DNT decreased while the CD4+ and NK cells increased in patients that responded to treatment compare to those who did not respond to treatment. Conclusions: DNT cells change during treatment with checkpoint inhibitors and may be adept at sensing the immune response to melanoma. The complementary variation of DNT cells with respect to CD4+ and other immune actors may improve the reliability of lymphocyte assessment. Further investigation of DNT as a potential target in checkpoint inhibitor resistant melanoma is warranted.

Autoimmune Lymphoproliferative Syndrome (ALPS) is an autoimmune disease that has been reported in over 2200 patients. It is a rare, genetic disease where pathogenic variants occur in the extrinsic pathway of apoptosis. Various mutations in different genes, such as FAS, FASL , and CASP10 , can result in ALPS. Most commonly, pathogenic variants occur in the FAS receptor. This malfunctioning pathway allows for the abnormal accumulation of lymphocytes, namely CD3 + TCRαβ+CD4 − CD8− (double negative (DN) T) cells, which are a hallmark of the disease. This disease usually presents in childhood with lymphadenopathy and splenomegaly as a result of lymphoproliferation. Over time, these patients may develop cytopenias or lymphomas because of irregularities in the immune system. Current treatments include glucocorticoids, mycophenolate mofetil, sirolimus, immunoglobulin G, and rituximab. These medications serve to manage the symptoms and there are no standardized recommendations for the management of ALPS. The only curative therapy is a bone marrow transplant, but this is rarely done because of the complications. This review serves to broaden the understanding of ALPS by discussing the mechanism of immune dysregulation, how the symptoms manifest, and the mechanisms of treatment. Additionally, we discuss the epidemiology, comorbidities, and medications relating to ALPS patients across the United States using data from Cosmos. • Autoimmune lymphoproliferative syndrome affects over 2200 patients in the U.S. • Over 55 mutations in FAS and FASL result in autoimmune lymphoproliferative syndrome. • Of the medications used for ALPS, glucocorticoids were most prescribed. [ABSTRACT FROM AUTHOR]

Autoimmune lymphoproliferative syndrome (ALPS) is an inherited syndrome characterized by abnormal lymphocyte survival caused by failure of apoptotic mechanisms to maintain lymphocyte homeostasis. This failure leads to the clinical manifestations of non-infectious and non-malignant lymphadenopathy, splenomegaly, and autoimmune pathology, most commonly, autoimmune cytopenias. Since ALPS was first characterized in the early 1990s, insights in disease biology have improved both diagnosis and management of this syndrome. Sirolimus is the best-studied and most effective corticosteroid-sparing therapy for ALPS and should be considered first-line for patients in need of chronic treatment. This review highlights practical clinical considerations for the diagnosis and management of ALPS. Further studies could reveal new proteins and regulatory pathways that are critical for lymphocyte activation and apoptosis.

Abstract Naïve CD4 T cells can be converted to double-negative regulatory T cells (DNT) by mature dendritic cells (mDCs) and IL-2 stimulation, with IL-2 enhancing the proliferation and Perforin expression of DNT. However, the molecules that affect the conversion of DNT are still not clear. Here, we investigated the effects of Ox40 on the conversion and function of DNT in vitro and in vivo without IL-2. We found that OX86 (an Ox40 agonist) increased the conversion rate of DNT but failed to enhance the suppressive function of DNT. Ox40 deficiency profoundly decreased the conversion rate and suppressive function of DNT. This suppression decline was caused by effects of Ox40 on proliferation and apoptosis independent of Perforin, Granzyme B and Fas ligand. Ox40 deficiency influenced the regulatory function of DNT through multiple signals, such as Cxcr3 , Cd160 and Cd30 , independently of Prf , Gzmb and Fasl. In conclusion, we elucidated that Ox40 promotes the conversion and maintenance of DNT. Ox40 deficiency reduced the regulatory function of DNT both in vitro and in vivo by regulating proliferation, apoptosis, and suppression-related genes. Highlights • Ox40 but not CD28 or CD154 deficiency profoundly decreased conversion rate of DN Tregs from CD4 T cells. • Ox40 deficiency markedly decreased the suppressive function of DN Tregs. • Ox40 affected DN Tregs' suppressive function by survival and gene expression independently of Prf , Gzmb and Fasl. [ABSTRACT FROM AUTHOR]

Several immune regulatory cell types participate in the protection against autoimmune diseases such as autoimmune diabetes. Of these immunoregulatory cells, we and others have shown that peripheral CD4−CD8− double negative (DN) T cells can induce antigen-specific immune tolerance. Particularly, we have described that diabetes-prone mice exhibit a lower number of peripheral DN T cells compared to diabetes-resistant mice. Identifying the molecular pathways that influence the size of the DN T cell pool in peripheral lymphoid organs may thus be of interest for maintaining antigen-specific immune tolerance. Hence, through immunogenetic approaches, we found that two genetic loci linked to autoimmune diabetes susceptibility, namely Idd2 and Idd13, independently contribute to the partial restoration of DN T cell proportion in secondary lymphoid organs. We now extend these findings to show an interaction between the Idd2 and Idd13 loci in determining the number of DN T cells in secondary lymphoid organs. Using bioinformatics tools, we link potential biological pathways arising from interactions of genes encoded within the two loci. By focusing on cell cycle, we validate that both the Idd2 and Idd13 loci influence RAD51 expression as well as DN T cell progression through the cell cycle. Altogether, we find that genetic interactions between Idd2 and Idd13 loci modulate cell cycle progression, which contributes, at least in part, to defining the proportion of DN T cells in secondary lymphoid organs. [ABSTRACT FROM AUTHOR]

TCRαβ + CD3 + CD4 − CD8 − “double negative” (DN) T cells comprise a small subset of mature peripheral T cells. The origin and function of DN T cells are somewhat unclear and discussed controversially. While DN T cells resemble a rare and heterogeneous T cell subpopulation in healthy individuals, numbers of TCRαβ + DN T cells are expanded in several inflammatory conditions, where they also exhibit distinct effector phenotypes and infiltrate inflamed tissues. Thus, DN T cells may be involved in systemic inflammation and tissue damage in autoimmune/inflammatory conditions, including SLE, Sjögren's syndrome, and psoriasis. Here, the current understanding of the origin and phenotype of DN T cells, and their role in the instruction of immune responses, autoimmunity and inflammation will be discussed in health and disease. [ABSTRACT FROM AUTHOR]

Despite increasing evidence that epididymal immune disorders can lead to infertility, the cells and mechanisms underlying epididymal immunity remain poorly understood. In this study, we propose a rather exhaustive overview of innate and adaptive immune cells present in the murine caput and cauda epididymis. Using flow cytometry and a wide set of markers, we screened the broadest panel of immune cells ever, in this organ. For the first time, we unequivocally quantified the innate populations of monocytes, macrophages, and dendritic cells subtypes. We also revealed the presence of B cells, gamma delta T cells, and double negative T cells in the murine epididymis. They were localized by immunofluorescence stainings, and appeared to be all present in the interstitium and epithelium along the organ, but with respective preferential regional distribution. Altogether, these findings provide new insights on the actors and potential mechanisms involved in the immune responses against genital tract ascending pathogens and in the setting and maintenance of tolerance toward the sperm cells. [ABSTRACT FROM AUTHOR]

Drinking water exposure to arsenic is known to cause immunotoxicity. Our previous studies demonstrated that monomethylarsonous acid (MMA +3 ) was the major arsenical species presented in mouse thymus cells after a 30 d drinking water exposure to arsenite (As +3 ). MMA +3 was also showed to be ten times more toxic than As +3 on the suppression of IL-7/STAT5 signaling in the double negative (DN) thymic T cells. In order to examine the genotoxicity induced by low to moderate doses of MMA +3 , isolated mouse thymus cells were treated with 5, 50 and 500 nM MMA +3 for 18 h in vitro . MMA +3 suppressed the proliferation of thymus cells in a dose dependent manner. MMA +3 at 5 nM induced DNA damage in DN not double positive (DP) cells. Differential sensitivity to double strand breaks and reactive oxygen species generation was noticed between DN and DP cells at 50 nM, but the effects were not seen at the high dose (500 nM). A stronger apoptotic effect induced by MMA +3 was noticed in DN cells than DP cells at low doses (5 and 50 nM), which was negated by the strong apoptosis induction at the high dose (500 nM). Analysis of intracellular MMA +3 concentrations in DN and DP cells, revealed that more MMA +3 accumulated in the DN cells after the in vitro treatment. Collectively, these results suggested that MMA +3 could directly induce strong genotoxicity in the early developing T cells in the thymus. The DN cells were much more sensitive to MMA +3 induced genotoxicity and apoptosis than DP cells, probably due to the higher intracellular levels of MMA +3 . [ABSTRACT FROM AUTHOR]

The autoimmune/inflammatory disorder psoriasis is characterized by keratinocyte proliferation and immune cell infiltration of the skin. TCR + CD3 + CD4 − CD8 − “double negative” (DN) T cells can derive from CD8 + T cells through the down-regulation of CD8. The inhibitory molecule programmed death (PD-)1 is expressed on activated T cells and plays a role in the maintenance of peripheral tolerance. A subset of DN T cells, characterized by the expression of PD-1, has recently been demonstrated to be self-reactive. We demonstrate that a majority of DN T cells exhibits effector memory phenotypes, express IFN-γ, and fail to proliferate. DN T cells from psoriasis patients are characterized by reduced DNA methylation of the IFNG gene and increased PD-1 expression. Furthermore, PD-1 positive DN T cells infiltrate the epidermis in psoriatic skin lesions. Our observations offer additional insight into the molecular pathophysiology of plaque psoriasis and show promise as potential disease biomarkers and/or therapeutic targets for future interventions. [ABSTRACT FROM AUTHOR]

Arsenite (As+3) exposure is known to cause immunotoxicity in human and animal models. Our previous studies demonstrated that As+3 at 50-500nM concentrations induced both genotoxicity and nongenotoxicity in mouse thymus cells. Developing T cells at CD4-CD8- double negative (DN) stage, the first stage after early T cells are transported from bone marrow to thymus, were found to be more sensitive to As+3 toxicity than the T cells at CD4+CD8+ double positive (DP) stage in vitro. Induction of Mdr1 (Abcb1) and Mrp1 (Abcc1), 2 multidrug resistance transporters and exporters of As+3, was associated with the reversal of As+3-induced double strand breaks and DNA damage. In order to confirm that the thymus cell populations have different sensitivity to As+3 in vivo, male C57BL/6J mice were exposed to 0, 100, and 500 ppb As+3 in drinking water for 30 d. A significant decrease in DN cell percentage was observed with exposure to 500 ppb As+3. Low to moderate concentrations of As+3 were shown to induce higher genotoxicity in sorted DN than DP cells in vitro. Calcein AM uptake and Mdr1/Mrp1 mRNA quantification results revealed that DN cells not only had limited As+3 exporter activity, but also lacked the ability to activate these exporters with As+3 treatments, resulting in a higher accumulation of intracellular As+3. Knockdown study of As+3 exporters in the DN thymic cell line, D1 using siRNA, demonstrated that Mdr1 and Mrp1 regulate intracellular As+3 accumulation and genotoxicity. Taken together, the results indicate that transporter regulation is an important mechanism for differential genotoxicity induced by As+3 in thymocytes at different developmental stages. [ABSTRACT FROM AUTHOR]

Objectives: The 2015 Workshop of the Society for Hematopathology/European Association for Haematopathology aimed to review primary immunodeficiency and related lymphoproliferations. Methods: Primary immunodeficiencies were divided into immune dysregulation, DNA repair defects, low immunoglobulins, and combined immunodeficiencies. Results: Autoimmune lymphoproliferative syndrome (ALPS) is a prototypical immune dysregulation-type immunodeficiency, with defects in T-cell signaling or apoptosis, expansion of T-cell subsets, and predisposition to hemophagocytic lymphohistiocytosis. DNA repair defects directly predispose to malignancy. Low immunoglobulin immunodeficiencies such as common variable immunodeficiency (CVID) have underlying T-cell repertoire abnormalities predisposing to autoimmunity and B-cell lymphoproliferations. The full spectrum of B-cell lymphoproliferative disorders occurs in primary immunodeficiency. Conclusions: Lymphoproliferations in primary immunodeficiency mirror those in other immunodeficiency settings, with monomorphic B- and sometimes T lymphoproliferative disorders enriched in DNA repair defects. Distinctive T-cell subset expansions in ALPS, CVID, and related entities can mimic lymphoma, and recognition of double-negative T-cell or cytotoxic T-cell expansions is key to avoid overdiagnosis. [ABSTRACT FROM AUTHOR]

Environmental arsenic exposure is a public health issue. Immunotoxicity induced by arsenic has been reported in humans and animal models. The purpose of this study was to evaluate mechanisms of As +3 and MMA +3 toxicity in mouse thymus cells. Because we know that MMA +3 inhibits IL-7 signaling in mouse bone marrow pre-B cells, we studied the influence of As +3 and MMA +3 on T cell development in the thymus at the earliest stage of T cell development (CD4-CD8-, double negative, DN) which requires IL-7 dependent signaling. We found in a DN thymus cell line (D1) that a low concentration of MMA +3 (50 nM) suppressed IL-7 dependent JAK1, 3 and STAT5 signaling. As +3 suppressed STAT5 and JAK3 at higher concentrations (500 nM). Cell surface expression of the IL-7 receptor (CD127) was also suppressed by 50 nM MMA + 3, but was increased by 500 nM As +3 , indicating possible differences in the mechanisms of action of these agents. A decrease in cyclin D1 protein expression was observed in D1 cells exposed to As +3 at 500 nM and MMA +3 starting at 50 nM, suggesting that arsenic at these environmentally-relevant doses suppresses early T cell development through the inhibition of IL-7 signaling pathway. [ABSTRACT FROM AUTHOR]

Lupus nephritis (LN) is a challenging problem that affects 50% of patients with systemic lupus erythematosus (SLE) without effective therapy. Here, we report that A77 1726, the active metabolite of leflunomide, effectively inhibits development of LN and attenuates the generalized autoimmune features. A77 1726 suppresses the expansion of double negative (DN) T cells, and inhibits T and B cell activation. Intriguingly, A77 1726 treatment significantly increases CD4 + Foxp3 + regulatory T cells but suppresses potential “pathogenic” IL-17-producing DN T cells in lymph nodes. In vitro experiment shows that A77 1726 potentiates the conversion of naive CD4 + CD25 − T cells into CD4 + CD25 + Foxp3 + inducible regulatory T cells (iTregs) by inhibiting Akt. Taken together, our data indicate that the therapeutic effects of A77 1726 in murine LN are mediated, at least in part, by augmenting iTregs which suppress pathogenic IL-17-producing DN T cells through an Akt-dependent mechanism. [ABSTRACT FROM AUTHOR]

Background Autoimmune lymphoproliferative syndrome (ALPS) is a human disorder of T cell homeostasis caused by mutations that impair FAS-mediated apoptosis. A defining characteristic of ALPS is the expansion of double negative T cells (DNTC). Relatively little is known about how defective FAS-driven cell death and the Bcl-2 apoptotic pathway intersect in ALPS patients. Objective We studied changes in Bcl-2 family member expression in ALPS to determine whether the Bcl-2 pathway might provide a therapeutic target. Methods We used flow cytometry to analyze the expression of pro- and anti-apoptotic Bcl-2 family members in T cells from 12 ALPS patients and determined the in vitro sensitivity of ALPS DNTC to the pro-apoptotic BH3 mimetic, ABT-737. Results The pro-apoptotic molecule, Bim, was significantly elevated in DNTC. Although no general pattern of individual anti-apoptotic Bcl-2 family members emerged, increased expression of Bim was always accompanied by increased expression of at least 1 anti-apoptotic Bcl-2 family member. Strikingly, Bim levels in DNTC correlated significantly with serum IL-10 in ALPS patients, and IL-10 was sufficient to mildly induce Bim in normal and ALPS T cells via a Janus kinase/signal transducer and activator of transcription 3–dependent mechanism. Finally, ABT-737 preferentially killed ALPS DNTC in vitro . Conclusion Combined, these data show that an IL-10/Janus kinase/signal transducer and activator of transcription 3 pathway drives Bim expression in ALPS DNTC, which renders them sensitive to BH3 mimetics, uncovering a potentially novel therapeutic approach to ALPS. [ABSTRACT FROM AUTHOR]

• pSS patients showed lower serum and saliva melatonin levels, which were negatively associated with the disease activity. • Melatonin administration decreased the expression of melatonin effector/receptor system in peripheral blood of pSS patients. • Melatonin might inhibit responses of peripheral Th17 and DN T cells in pSS through nuclear receptors-dependent manner. • Overexpression of the melatonin effector/receptor system in pSS patients appeared to be involved in the disease. Primary Sjögren's syndrome (pSS) is a complex systemic autoimmune disease whose clinical spectrum extends from sicca syndrome to systemic involvement. T helper 17 cells (Th17) and CD4−CD8− (double negative, DN) T cells are actively involved in the pathogenesis of pSS. Melatonin shows important immunoregulatory functions in multiple T cell-mediated autoimmune diseases. However, the effects of melatonin on the immune cells of pSS patients are unclear. Hence, this study was aimed to evaluate the effects of melatonin on the immune responses of peripheral pathogenic Th17 and DN T cells from pSS patients, and explore the underlying receptor-related mechanism. The concentration of serum and saliva melatonin of pSS patients and healthy controls (HCs) were detected using Enzyme-linked immunosorbent assays (ELISA). Expression of arylalkylamine N-acetyltransferase (AANAT) and hydroxyindole O-methyltransferase (HIOMT) were conducted in labial glands samples by immunohistochemistry. The mechanism underlying the effects of melatonin on Th17 and DN T cells responses in peripheral blood from pSS was investigated by quantitative real-time polymerase chain reaction (RT-PCR), flow cytometry, ELISA, cell viability, and proliferation assays. Serum and saliva melatonin levels were lower in pSS patients than in HCs, which were negatively correlated with disease activity. The expression levels of melatonin's biosynthetic enzymes (AANAT, HIOMT) and nuclear receptors (RORα, RORγ) were significantly increased in peripheral blood mononuclear cells (PBMCs) from pSS patients. Furthermore, in vitro melatonin administration decreased the expression of melatonin effector/receptor system in peripheral blood of pSS patients. More importantly, Melatonin inhibited pathogenic responses of peripheral Th17 and DN T cells in PBMCs from pSS, which was independent of melatonin membrane receptors. However, melatonin nuclear receptor antagonist SR1001 enhanced the inhibitory ability of melatonin on Th17 and DN T cells production, and agonist SR1078 weakened the effects of melatonin. Additionally, overexpression of the melatonin effector/receptor system in pSS patients appeared to be involved in the disease, due to that melatonin effector/receptor system expression was correlated with the frequency of Th17 or DN T cells. Melatonin relieved the inflammatory responses of Th17 and DN T cells in PBMCs from pSS patients in a nuclear receptors-dependent manner, suggesting that melatonin might be beneficial to pSS. [ABSTRACT FROM AUTHOR]

A central objective in organ transplantation and the treatment or prevention of autoimmune disease is the achievement of antigen-specific immune tolerance. An additional challenge in bone marrow transplantation for the treatment of hematological malignancies is the prevention of graft-vs-host disease (GVHD) while maintaining graft-vs-tumor activity. Interestingly, CD4-CD8- (double negative, DN) T cells, which exhibit a unique antigen-specific immunoregulatory potential, appear to exhibit all of the properties to respond to these challenges. Herein, we review the therapeutic potential of immunoregulatory DN T cells in various immunopathological settings, including graft tolerance, GVHD, cancer and autoimmunity.

Purpose: Autoimmune diseases are thought to be caused by a loss of self-tolerance of the immune system. One candidate marker of immune dysregulation in autoimmune disease is the presence of increased double negative T cells (DNTs) in the periphery. DNTs are characteristically elevated in autoimmune lymphoproliferative syndrome, a systemic autoimmune disease caused by defective lymphocyte apoptosis due to Fas pathway defects. DNTs have also been found in the peripheral blood of adult patients with systemic lupus erythematosus (SLE), where they may be pathogenic. DNTs in children with autoimmune disease have not been investigated. Methods: We evaluated DNTs in pediatric patients with SLE, mixed connective tissue disease (MCTD), juvenile idiopathic arthritis (JIA), or elevated antinuclear antibody (ANA) but no systemic disease. DNTs (CD3CD56TCRαβCD4CD8) from peripheral blood mononuclear cells were analyzed by flow cytometry from 54 pediatric patients including: 23 SLE, 15 JIA, 11 ANA and 5 MCTD compared to 28 healthy controls. Results: Sixteen cases (29.6 %) had elevated DNTs (≥2.5 % of CD3CD56TCRαβ cells) compared to 1 (3.6 %) control. Medication usage including cytotoxic drugs and absolute lymphocyte count were not associated with DNT levels, and percentages of DNTs were stable over time. Analysis of multiple phenotypic and activation markers showed increased CD45RA expression on DNTs from patients with autoimmune disease compared to controls. Conclusion: DNTs are elevated in a subset of pediatric patients with autoimmune disease and additional investigations are required to determine their precise role in autoimmunity. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: Growing evidence suggests that IL-17-producing T cells, lacking both CD4 and CD8 molecules and defined as double negative (DN) cells, play a pivotal role in the pathogenesis of a number of systemic autoimmune disorders. We recently demonstrated that this T-cell subset is expanded in the peripheral blood (PB) of patients with primary Sjögren's syndrome (pSS), produces IL-17 and accumulates in minor salivary glands (MSGs). We aimed to investigate glandular and PB DN T cells in early pSS in order to verify a possible correlation with MSGs histological patterns and clinical parameters. Methods: Paired samples of PB mononuclear cells and MSGs from pSS patients were evaluated at the diagnosis by flow cytometry and immunofluorescence staining respectively. Histological analysis to identify histological scores, B/T cell segregation and the presence of germinal center (GC)-like structures was also performed. Results: In early stages of pSS, circulating DN T cells appear to be not yet expanded and inversely correlated with circulating CD4+Th17 cells. The number of infiltrating DN T cells were associated with extent of glandular involvement, presence of GC-like structures and dryness symptoms and were inversely correlated with circulating DN T cells. Conclusions: Our findings suggest that DN T cells are actively involved in the pathogenic mechanisms leading to glandular dysfunction and damage in pSS and may play a role in ectopic lymphoneogenesis development occurring during the disease. [Copyright &y& Elsevier]

A central objective in organ transplantation and the treatment or prevention of autoimmune disease is the achievement of antigen-specific immune tolerance. An additional challenge in bone marrow transplantation for the treatment of hematological malignancies is the prevention of graft-vs-host disease (GVHD) while maintaining graft-vs-tumor activity. Interestingly, CD4-CD8- (double negative, DN) T cells, which exhibit a unique antigen-specific immunoregulatory potential, appear to exhibit all of the properties to respond to these challenges. Herein, we review the therapeutic potential of immunoregulatory DN T cells in various immunopathological settings, including graft tolerance, GVHD, cancer, and autoimmunity. [ABSTRACT FROM AUTHOR]