DNT Cells Research Articles

Targeting chemotherapy-resistant leukemia by combining DNT cellular therapy with conventional chemotherapy

BackgroundWhile conventional chemotherapy is effective at eliminating the bulk of leukemic cells, chemotherapy resistance in acute myeloid leukemia (AML) is a prevalent problem that hinders conventional therapies and contributes to disease relapse, and ultimately patient death. We have recently shown that allogeneic double negative T cells (DNTs) are able to target the majority of primary AML blasts in vitro and in patient-derived xenograft models. However, some primary AML blast samples are resistant to DNT cell therapy. Given the differences in the modes of action of DNTs and chemotherapy, we hypothesize that DNT therapy can be used in combination with conventional chemotherapy to further improve their anti-leukemic effects and to target chemotherapy-resistant disease.MethodsDrug titration assays and flow-based cytotoxicity assays using ex vivo expanded allogeneic DNTs were performed on multiple AML cell lines to identify therapy-resistance. Primary AML samples were also tested to validate our in vitro findings. Further, a xenograft model was employed to demonstrate the feasibility of combining conventional chemotherapy and adoptive DNT therapy to target therapy-resistant AML. Lastly, blocking assays with neutralizing antibodies were employed to determine the mechanism by which chemotherapy increases the susceptibility of AML to DNT-mediated cytotoxicity.ResultsHere, we demonstrate that KG1a, a stem-like AML cell line that is resistant to DNTs and chemotherapy, and chemotherapy-resistant primary AML samples both became more susceptible to DNT-mediated cytotoxicity in vitro following pre-treatment with daunorubicin. Moreover, chemotherapy treatment followed by adoptive DNT cell therapy significantly decreased bone marrow engraftment of KG1a in a xenograft model. Mechanistically, daunorubicin increased the expression of NKG2D and DNAM-1 ligands on KG1a; blocking of these pathways attenuated DNT-mediated cytotoxicity.ConclusionsOur results demonstrate the feasibility and benefit of using DNTs as an immunotherapy after the administration of conventional chemotherapy.

Alpha beta double negative T cells in children with systemic lupus erythematosus: The relation to disease activity and characteristics

Objectives: We aimed to investigate alpha beta double negative (αβ DN) T-cell percentages in juvenile systemic lupus erythematosus (JSLE) and their relation to disease activity and organ involvement.Methods: This prospective study was carried out over a period of 12 months and included 21 JSLE patients and 20 healthy matched controls. SLE disease activity index 2000 (SLEDAI-2K) scores were recorded in addition to αβ DN T-cell percentages measurement at enrollment and after remission.Results: Enrolled patients (n = 21) were females with age range 10–17 years. Patients were followed up for a duration ranging 5–9 months. αβ DN T-cell percentages were higher in cases during activity [median (IQR): 3.7 (3.0–5.7)] versus remission [median (IQR): 1.4 (1.2–1.8)] and during activity and remission versus healthy controls [median (IQR): 1.0 (0.5–1.4)]. αβ DN T-cell percentages correlated positively with SLEDAI-2K score (p < .001). The mean αβ DN T-cell percentages varied significantly with different degrees of activity per SLEDAI-2K score (p = .002) and with the presence of neurological (p = .028) and hematological (p = .032) manifestations.Conclusion: αβ DNT cells percentages are elevated in patients with JSLE and their percentages correlate with disease activity. Further studies are needed in conjunction with the proinflammatory cytokine profile, apoptotic assays and histological findings.

Role of TRAIL pathway and NKG2D pathway in double negative T cell cell anti-tumor

Double negative T cell is a rare type of cells that regulates immune responses in a way that is unique. In the process of adoptive immunotherapy, it gradually becomes a hot spot, because of its obvious anti-tumor effect. However, the mechanism by which DNT cell kills tumor cell is not clear. Tumor necrosis factor-related apoptosis-inducing ligand(TRAIL) and natural-killer group 2, member D (NKG2D) pathway is a more important part of its killing mechanism, and NKG2D receptor can also regulate the production of soluble TRAIL, making the two channels to establish a certain link. In this paper, the effect of Double negative T cell and the mechanism of TRAIL and NKG2D pathway in DNT cell′s anticancer were described. Key words: Immunity; Pancreatic neoplasms; DNT cell; TRAIL; NKG2D

Effect of preventive treatment in EAE mouse model transplanted with DN T cells transformed from CD4 T cells

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system (CNS). Experimental autoimmune encephalomyelitis (EAE) has long served as an animal model for human MS. Both diseases are characterized by inflammation and demyelination of the CNS. Lots of study confirmed that Th1 and Th17 cells could aggravate the illness, while Foxp3+ regulatory T cells (Treg cells) could inhibit the autoimmune reaction and limit disease progression. However, it has been studied that both the number and immune suppression function of Treg cells were reduced in EAE mice, and although myelin specific Treg cells accumulated in the CNS, it failed to control autoimmune inflammation. Based on that, promoting immune regulation by other regulatory cells may be a new strategy to restore immune homeostasis and control EAE. Many studies have demonstrated that in peripheral lymphoid tissues of normal mice and healthy humans, 1% to 3% of T cell receptor positive T cells are double negative T cells (DN T cells), which capable of down regulating immune responses. We have identified a new differentiation pathway for the conversion of CD4 T cells to DN T cells, which could large amount DN T cells in vitro in a short time. We have showed that the converted DN T cells retained a stable phenotype and expressed a unique set of cell surface markers and gene profiles. These cells were highly potent in suppressing allo- and auto-immune responses both in vitro and in vivo in an antigen specific manner. Adoptive cell therapy using these antigen specific DN T cells could prevent and cure autoimmune diabetes and promote islet allograft survival in NOD mice. In a ConA mediated liver injury model, CD4 T cell converted DN T cells exerted significant protection against immune mediated liver injury and maintained the homeostasis of the immune system. Up to now, there is no report on the role of these converted DNT cells in mouse EAE model. In this study, we further explored the protection and its mechanism of DN T cells on EAE. CD4 T cells were converted to antigen specific DN T cells in vitro with MOG35–55 stimulation. Then we adoptive transferred these antigen specific DN T cells to mice before EAE immunization. Time of disease onset, EAE score, mouse weight, lymphocyte infiltration were compared between EAE control group and DNT pre-treatment group, the inhibitory function of DN T cells on pathogenic CD4 T cells from EAE model were further explored. Our data showed that a single adoptive transfer of MOG35–55 primed DN T cells significantly lowered the EAE score, prevented the weight loss and reduced lymphocytes infiltration in brain tissue. DN T cells pre-treatment group contained lower plasma IL-2 and IL-4 levels compared to EAE control group. In vitro suppression assays showed DN T cells significantly suppressed the proliferation of inflammatory CD4 T cells from EAE mice. Our data suggests that DN T cells can significantly reduce the EAE severity. This preventive effect is partly due to DN T cells’ suppression on the proliferation and cytokine production of inflammatory CD4 T cells. DN T cells may have a potential clinical application in the prevention and treatment of MS.

Differential Effect of Cytomegalovirus Infection with Age on the Expression of CD57, CD300a, and CD161 on T-Cell Subpopulations.

Immunosenescence is a progressive deterioration of the immune system with aging. It affects both innate and adaptive immunity limiting the response to pathogens and to vaccines. As chronic cytomegalovirus (CMV) infection is probably one of the major driving forces of immunosenescence, and its persistent infection results in functional and phenotypic changes to the T-cell repertoire, the aim of this study was to analyze the effect of CMV-seropositivity and aging on the expression of CD300a and CD161 inhibitory receptors, along with the expression of CD57 marker on CD4+, CD8+, CD8+CD56+ (NKT-Like) and CD4−CD8− (DN) T-cell subsets. Our results showed that, regardless of the T-cell subset, CD57−CD161−CD300a+ T-cells expand with age in CMV-seropositive individuals, whereas CD57−CD161+CD300a+ T-cells decrease. Similarly, CD57+CD161−CD300a+ T-cells expand with age in CMV-seropositive individuals in all subsets except in DN cells and CD57−CD161+CD300a− T-cells decrease in all T-cell subsets except in CD4+ T-cells. Besides, in young individuals, CMV latent infection associates with the expansion of CD57+CD161−CD300a+CD4+, CD57−CD161−CD300a+CD4+, CD57+CD161−CD300a+CD8+, CD57−CD161−CD300a+CD8+, CD57+CD161−CD300a+NKT-like, and CD57+CD161−CD300a+DN T-cells. Moreover, in young individuals, CD161 expression on T-cells is not affected by CMV infection. Changes of CD161 expression were only associated with age in the context of CMV latent infection. Besides, CD300a+CD57+CD161+ and CD300a−CD57+CD161+ phenotypes were not found in any of the T-cell subsets studied except in the DN subpopulation, indicating that in the majority of T-cells, CD161 and CD57 do not co-express. Thus, our results show that CMV latent infection impact on the immune system depends on the age of the individual, highlighting the importance of including CMV serology in any study regarding immunosenescence.

DNT cell inhibits the growth of pancreatic carcinoma via NKG2D and MICA pathway in vivo

Objective: To investigate the effects of natural killer group 2 member D (NKG2D) and its ligands major histocompatibility complex class Ⅰ chain-related molecules A(MICA) on DNT cell killing pancreatic carcinoma. Methods: Antibodies adsorption was used to separate DNT cell from human peripheral blood. Human pancreatic tumor models were established via implanting BXPC-3 cells into nude mice, and then mice were randomly divided mice into 3 groups, blank group, gemcitabine group and DNT group. Mice weights and mice tumor volumes were measured every 5 days. After 40 days mice were euthanized at cervical dislocation method. Tumor weights were measured. Relative tumor volume and tumor inhibition rate were calculated. Western blot and qPCR were used to detect the expressions of NKG2D and MICA in the transplanted tumors of the three groups. Results: The blank group tumor volume and weight of blank group were significantly larger than those of gemcitabine group and DNT group (858.7±35.7 mg, 251.1±19.7 mg, 278.5±17.3 mg, P<0.001), but there were no significantly difference between DNT group and gemcitabine group. The tumor inhibition rate of gemcitabine and DNT cell were 40.4% and 35.5%. Western blot and qPCR showed that MICA mRNA and protein levels in blank group were significantly higher than those in DNT group (P=0.001, P=0.003). NKG2D mRNA and protein levels in blank group were significantly lower than those in DNT cells group (P<0.001, P=0.001). Conclusion: DNT cell can significantly inhibit the growth of pancreatic carcinoma in vivo, and the mechanism may be involved in abnormal expressions of MICA and NKG2D.

CTLA-4 haploinsufficiency in a patient with an autoimmune lymphoproliferative disorder

CTLA-4 haploinsufficiency in a patient with an autoimmune lymphoproliferative disorder

Autoimmune lymphoproliferative syndrome caused by homozygous FAS mutations with normal or residual protein expression

Autoimmune lymphoproliferative syndrome caused by homozygous FAS mutations with normal or residual protein expression

Human Double-Negative Regulatory T Cells Selectively Suppress mTOR Signaling and Metabolic Reprogramming of Conventional T Cells

Regulatory T (Treg) cells have been shown to be involved in downregulating immune responses in autoimmunity, transplant rejection, and graft-versus-host disease (GvHD). The subpopulation of TCRαβ+ CD4- CD8- (double-negative, DN) T cells has been described to suppress immune responses in both mice and humans. Of particular interest, infusion and/or activation of murine DN T cells specifically suppressed alloreactive T cells and prevented development of GvHD after allogeneic hematopoietic stem cell transplantation (SCT). Moreover, clinical studies in patients after SCT revealed an inverse correlation between the frequency of circulating DN T cells and the severity of GvHD, suggesting a therapeutic potential of human DN T cells. However, the molecular mechanism of suppression still remains unclear. To gain a better understanding of DN T-cell functionality, we investigated whether human DN T cells modulate distinct TCR signaling processes in conventional T cells. We found that DN T cells selectively inhibit the mechanistic target of rapamycin (mTOR) signaling pathway but not activation of mitogen-activated protein kinases (MAPK). The crucial function of mTOR signaling was confirmed by treating effector T cells with a chemical activator of protein kinase Akt, which induces mTOR phosphorylation. Indeed, enforced activation of the mTOR pathway rendered conventional T cells unsusceptible to DN T cell-mediated suppression. Given that mTOR is a critical regulator of cellular metabolism, we further determined the impact of DN T cells on the metabolic framework of conventional T cells. Intriguingly, DN T cells significantly diminished upregulation of the glycolytic machinery, expression of glucose transporters and glucose uptake in conventional T cells. These findings indicate that DN T cells inhibit metabolic reprogramming of conventional T cells by abrogating mTOR signaling, thereby inducing a quiescent phenotype. Further understanding of the mechanisms involved in human DN T-cell suppression may have important implications for using them as a cellular-based therapy to limit alloreactive immune responses. DisclosuresNo relevant conflicts of interest to declare.

PI3K/mTOR-dependent IL-22 production modulates polyfunctional T cell responses in viral hepatitis.

Abstract IL-22 is an important cytokine in modulating the consequences of inflammation in lesions, including the liver. However, the role of IL-22 in Lymphocytic Choriomeningitis Virus (LCMV)-induced viral hepatitis remains obscure. Here, we report that LCMV-induced IL-23 release promoted IL-22 production. And gdT cells and double negative T cells (DNT, CD3+ CD4− CD8− γδTCR− NK1.1−) were the main sources of IL-22 and IL-17 productions following LCMV infection in the liver. Interestingly, we also found that IL-23-stimulated IL-17 production in γδ T cells, and DNT cells were totally dependent on RORγt and PI3K/mTOR, but independent in AhR activities. Although IL-22 production in these two lymphocyte subsets depended on PI3K/mTOR, unlike IL-17 production, IL-22 production required additional signals from RORγt and AhR. Furthermore, IL-22 appeared to act as an important mediator in T cell responses in LCMV-induced viral hepatitis, despite the fact that the IL-22 receptor is restricted to being expressed on non-hematopoitic cells. IL-22−/− mice increased the recruitment of activated T cells to the liver and enhanced polyfunctional cytokine productions in effector CD4+ and CD8+ T cells in LCMV-induced acute or persistent viral hepatitis. Thus, our data revealed a novel connection between IL-23-PI3K/mTOR signaling pathway and the regulation of IL-22/IL-17 productions in the liver, and showed a novel role of IL-22 in modulating the plurifunctionality of anti-viral T cell responses during acute and persistent LCMV infection.

A novel differentiation pathway from CD4⁺ T cells to CD4⁻ T cells for maintaining immune system homeostasis.

CD4+ T lymphocytes are key players in the adaptive immune system and can differentiate into a variety of effector and regulatory T cells. Here, we provide evidence that a novel differentiation pathway of CD4+ T cells shifts the balance from a destructive T-cell response to one that favors regulation in an immune-mediated liver injury model. Peripheral CD4−CD8−NK1.1− double-negative T cells (DNT) was increased following Concanavalin A administration in mice. Adoptive transfer of DNT led to significant protection from hepatocyte necrosis by direct inhibition on the activation of lymphocytes, a process that occurred primarily through the perforin-granzyme B route. These DNT converted from CD4+ rather than CD8+ T cells, a process primarily regulated by OX40. DNT migrated to the liver through the CXCR3-CXCL9/CXCL10 interaction. In conclusion, we elucidated a novel differentiation pathway from activated CD4+ T cells to regulatory DNT cells for maintaining homeostasis of the immune system in vivo, and provided key evidence that utilizing this novel differentiation pathway has potential application in the prevention and treatment of autoimmune diseases.

Immune cell subsets at birth may help to predict risk of late-onset sepsis and necrotizing enterocolitis in preterm infants

BackgroundParameters predicting late-onset sepsis (LOS) and necrotizing enterocolitis (NEC) in preterm infants would be valuable. Ten-color flow-cytometry enables the estimation of cellular immune status requiring only small sample volumes. AimsIdentifying predictive parameters for LOS and NEC in the cellular immune status of preterm infants. Study design and subjectsIn this prospective study in 40 preterm infants (week 26+0 to 30+6) and 10 healthy full-term newborn infants (control group, week 37+0 to 40+6), flow cytometric analyses of lymphocyte subpopulations were performed between the 2nd and the 6th day of life, with a follow-up until the preterm infant reached the calculated gestational age of week 40. Patients' episodes of infections and NEC were analyzed according to the NEO-KISS criteria of the German National Reference Center. ResultsTen preterm infants showed events within the first week of life and were excluded from the analysis. Of the other 30, five developed NEC, twelve LOS. In patients with LOS, the proportion of double-negative (DN) T cells was significantly elevated compared to patients without LOS, while immune-regulatory CD56bright and CD56negCD16+ NK cells were significantly decreased (p<0.05). Patients with NEC showed a reduction in the NK cell proportion (<3.7%) and significantly decreased naïve cytotoxic CD45RA+CD62L+ T cells (p<0.05). ConclusionNK cells and DN-T cell counts within the first week of life may be predictors for NEC and LOS in preterm infants. In order to identify patients at risk early, further analysis of these populations might be of interest.

Efficacy and Safety of Allogeneic Double Negative T Cell As a Cellular Therapy for AML and Its Underlying Mechanism

Acute myeloid leukemia (AML) is the most common form of adult acute leukemia that is associated with a low long-term survival rate. While chemotherapy achieves remission in more than 70% of the treated AML patients, most relapse due to residual chemotherapy-resistant AML populations. Allogeneic hematopoietic stem cell transplantation reduces the relapse rate and demonstrates the efficacy of a cell-mediated treatment for the chemotherapy-resistant disease. However, its wide application is limited by donor availability and associated toxicity such as graft-versus-host disease (GvHD). Hence, there is a need for a new treatment approach that targets chemotherapy-resistant AML blasts with minimal side effects. The goals of this study were to characterize allogeneic CD3+ CD4-CD8- double negative T cells (DNTs) as a potential new therapy for AML patients, and to dissect its underlying mechanisms.Using a flow cytometry-based in vitro killing assay, we demonstrated that the allogeneic DNTs expanded from healthy volunteers were cytotoxic against 23/29 primary AML patient blasts in a dose-dependent manner. Thirteen blast samples were obtained from chemotherapy refractory or relapsing AML patients and, of those, 9 were susceptible to DNTs. Further, the average level of specific killing mediated by DNTs against chemotherapy-susceptible and -resistant blasts were comparable (19.30% ± 3.34% and 15.91% ± 3.63%, respectively). The anti-leukemia activity of DNTs was further validated in AML-NSG xenograft models. A single infusion of DNTs into mice pre-engrafted with primary AML blasts from chemoresponsive, chemorefractory, and relapsed patients significantly reduced the leukemia burden. While a single injection of DNTs resulted in a significant reduction of leukemia burden from 30% to 12.8%, it was further reduced to 2.6% after three injections of DNTs. Furthermore, the survival of NSG mice administered with a lethal dose of AML cell line, MV4-11, in DNT-treated group was significantly superior to the untreated group (57% vs. 0% survival on day 34, respectively). Although residual blasts were observed from the DNT -treated group, their susceptibility to DNT cell-mediated cytotoxicity remained comparable to blasts obtained from the PBS-treated group and primary AML blasts initially used for engraftment, suggesting that AMLs do not develop resistance to DNT-mediated cytotoxicity. In contrast to its potent cytolytic activity against leukemic cells, DNTs did not target allogeneic peripheral blood mononuclear cells (PBMC) and hematopoietic stem/progenitor cells (HSPC) in vitro. Administration of allogeneic DNTs into NSG mice engrafted with human HSPC had no effect on the engraftment level of human hematopoietic cells and their differentiation into different lineages. Further, in contrast to human PBMC, infusion of human DNTs did not cause xenogeneic GvHD in mice, collectively demonstrating the selective cytotoxic activity of allogeneic DNTs against leukemic cells. Using blocking assays, we showed that HLA-class I, NKG2D, and DNAM-1 were involved in DNT cell-mediated cytotoxicity against AML, whereas HLA-class II and T cell receptor did not play a significant role. We detected high levels of IFNγ release by DNTs upon encounter of susceptible AML targets. While IFNγ treatment alone did not induce AML cell death, neutralizing IFNγ reduced and pretreating AML cells with recombinant IFNγ increased their susceptibility to DNT cell killing. IFNγ treatment induced higher expressions of NKG2D and DNAM-1 ligands and blocking of NKG2D and DNAM-1 partially abrogated the effect of IFNγ on the AML-DNT interaction. Collectively, these studies demonstrated the safety and efficacy of allogeneic DNT therapy as a potential treatment for AML patients, including those with chemotherapy-resistant leukemia, and revealed the important molecules for the anti-leukemic activity of DNTs. DisclosuresNo relevant conflicts of interest to declare.

DNT cell inhibits the growth of pancreatic carcinoma via abnormal expressions of NKG2D and MICA in vivo

This research aimed to investigate the effects of natural killer group 2 member D (NKG2D) and its ligands major histocompatibility complex class I chain-related molecules A(MICA) in DNT cell killing pancreatic carcinoma. Antibodies adsorption was used to separate DNT cell from human peripheral blood. Human pancreatic tumor models were established via implanting BXPC-3 cells into nude mice. Then randomly divided mice into blank group, gemcitabine group and DNT group. Mice weights and mice tumor volumes were measured every 5 days. 50 days later mice were euthanized at cervical dislocation method. Tumor weights were measured. Relative tumor volume and tumor inhibition rate were calculated. Western blot and qPCR were used to detect the expressions of NKG2D and MICA in the transplanted tumors of the three groups. DNT cell significantly increased over time. The blank group tumor volume and weight were significantly larger than the other groups (p < 0.001, p < 0.001), but there were no significantly difference between DNT group and gemcitabine group (p > 0.05). Gemcitabine and DNT cell tumor inhibition rate were 40.4% and 35.5%. Western blot and qPCR showed that MICA mRNA and protein levels in blank group were significantly higher than DNT group (p = 0.001, p = 0.003). NKG2D mRNA and protein levels in blank group were significantly lower than DNT cells group (p < 0.001, p = 0.001). In conclusion DNT cell can significantly inhibit the growth of pancreatic carcinoma in vivo, and the mechanism may be involved in abnormal expressions of MICA and NKG2D.

Intraoperative exercises during surgery: surgical trainees rating of performance and disruption

RESULTS: DNT cells can be detected in pancreatic cancer patients in peripheral T-cells. In addition, we observed that the proportion of DNT cells was higher than in high-grade tumor (p 0.05); the staining intensity of DR5 appeared to have a lower strong staining in tumor tissue samples compared to adjacent tissue; 17% (8/48) vs 35% (17/48); (p<0.05). Clinicopathologic analysis demonstrated that DR4 expression was associated with tumor differentiation (p<0.05) and tumor pathologic stage (p<0.05). Similarly, DR5 expression did not show any significant association except tumor differentiation (p<0.05).

Expression of DNT cells in pancreatic cancer and anti-tumor efficacy via TRAIL-receptors DR4 and DR5

Expression of DNT cells in pancreatic cancer and anti-tumor efficacy via TRAIL-receptors DR4 and DR5

Double-negative T resident memory cells of the lung react to influenza virus infection via CD11c(hi) dendritic cells.

Immunity to Influenza A virus (IAV) is controlled by conventional TCRαβ(+) CD4(+) and CD8(+) T lymphocytes, which mediate protection or cause immunopathology. Here, we addressed the kinetics, differentiation, and antigen specificity of CD4(-)CD8(-) double-negative (DN) T cells. DNT cells expressed intermediate levels of TCR/CD3 and could be further divided in γδ T cells, CD1d-reactive type I NKT cells, NK1.1(+) NKT-like cells, and NK1.1(-) DNT cells. NK1.1(-) DNT cells had a separate antigen-specific repertoire in the steady-state lung, and expanded rapidly in response to IAV infection, irrespectively of the severity of infection. Up to 10% of DNT cells reacted to viral nucleoprotein. Reinfection experiments with heterosubtypic IAV revealed that viral replication was a major trigger for recruitment. Unlike conventional T cells, the NK1.1(-) DNT cells were in a preactivated state, expressing memory markers CD44, CD11a, CD103, and the cytotoxic effector molecule FasL. DNT cells resided in the lung parenchyma, protected from intravascular labeling with CD45 antibody. The recruitment and maintenance of CCR2(+) CCR5(+) CXCR3(+) NK1.1(-) DNT cells depended on CD11c(hi) dendritic cells (DCs). Functionally, DNT cells controlled the lung DC subset balance, suggesting they might act as immunoregulatory cells. In conclusion, we identify activation of resident memory NK1.1(-) DNT cells as an integral component of the mucosal immune response to IAV infection.

Long-term remission after allogeneic hematopoietic stem cell transplantation in LPS-responsive beige-like anchor (LRBA) deficiency

Long-term remission after allogeneic hematopoietic stem cell transplantation in LPS-responsive beige-like anchor (LRBA) deficiency

Dysregulation of IL12 Signaling As a Novel Cause of an Autoimmune Lymphoproliferative like Syndrome

IntroductionAutoimmune lymphoproliferative syndrome (ALPS) is characterized by abnormal lymphocyte homeostasis caused by defective apoptosis. Mutations in genes involved in the Fas death receptor pathway (FAS, FASLG or CASP10 genes) are the cause for the pathogenesis of ALPS. However, in 20-30% of all ALPS cases, collectively classified as ALPS-U (undetermined), the genetic defect is still unknown. The objective of this study was to employ whole-exome sequencing to search for novel gene candidates underlying ALPS-U or ALPS-like disease. Resulting candidates should be validated and their impact on the Fas death receptor pathway studied.MethodsPeripheral blood samples were collected from 26 patients diagnosed with ALPS (based on clinical phenotype and accumulation of DNT cells), relatives and healthy controls. PCR and Sanger sequencing were performed to check for germline and somatically acquired FAS, FASLG and CASP10 mutations. Whole-exome sequencing was carried out for all patients with unknown mutation and their parents. Candidate genes were identified by a KEGG-based protein interaction analysis interface of our in-house developed proprietary MySQL database driven workbench, termed SNuPy (Single Nucleotide Polymorphism Database). STRING 9.1 was used to identify high confidence (≥0.900) interaction partners of the Fas pathway. Single nucleotide variations (SNVs) were verified by PCR and Sanger sequencing. The impact of detected mutations on the candidate genes´ expression and functionality was analyzed by immunoblot. The candidate genes´ impact on Fas death receptor pathway and Fas/FasL expression was examined by flow cytometry, ELISA and qRT-PCR.ResultsOut of 26 analyzed ALPS cases 4 unrelated patients harbored heterozygous germline mutations in the death domain of the Fas receptor (p.Q282K, p.R249G, p.NVQ265-267KQT) or the extracellular domain (p.R191C), respectively. Two siblings with homozygous FASLGtruncating mutation (A69fs*138) and complete loss of FasL surface expression as a consequence were identified. Whole-exome sequencing also identified a homozygous R212* mutation in the IL12/IL23 receptor-component IL12RB1 (Interleukin 12 receptor, beta 1 subunit) in a patient who presented with classical ALPS symptoms (chronic non-malignant, noninfectious lymphadenopathy, splenomegaly, hepatomegaly, elevation of DNT cells, autoimmune cytopenias with polyclonal hypergammaglobulinemia, persistently increased vitamin B12 and IL10 levels). The p.R212* mutation in IL12RB1 leads to premature protein truncation by a stop codon gain, resulting in a complete loss of cell surface expression of IL12RB1 in the patient. IL12 is a factor known to regulate expression of both Fas and FasL. IL12 signaling was abrogated as demonstrated by deficient downstream STAT4 phosphorylation and IFNγ production. Low FasL expression on T-cells and low soluble FasL plasma levels were probably due to lack of IFNγ mediated transcriptional activation of FasL. In contrast to healthy controls, prolonged IL12 stimulation did not trigger upregulation of FasL nor apoptosis in the IL12RB1 deficient and FasL deficient (A69fs*138) patients, indicating that IL12 mediated apoptosis is FasL dependent. Heterozygous carriers of the IL12RB1 or the FASLG mutation showed an intermediate response but were asymptomatic and sub-clinically affected (showing e.g. moderate elevation of DNT cells).ConclusionOur data show that IL12 employs Fas signaling to achieve T-cell apoptosis and reveal IL12 signaling deficiency as a new cause of ALPS like disease via its impact on FasL expression. DisclosuresNo relevant conflicts of interest to declare.

CD4+ T-Cell Converted Double Negative T-Cells Suppress B-Cells Proliferation and IGG Production in Mice.

We have reported that CD4+ T-cells could be converted to CD3+CD4-CD8-(DN) T-cells, the converted DN T-cells were very potent in suppressing antigen-specific T cells responses in vitro and prolonged islet allograft survival in vivo. In this study, we further tested the functional potential of CD4+ T-cells converted DN T-cells on B-cells. We found that the proliferation of B-cells were significantly suppressed by DN T-cells and IgG secretion of stimulated B cells was also inhibited by DN T-cells in vitro and in vivo. The suppression was cell dose dependent and was relied on cell-cell contact. Perforin, a cytotoxic lymphocyte related cytokine, was highly expressed by DN T-cells, played an important role in DN T-cell mediated suppression, the potency of DN T-cells derived from perforin KO mice to suppress the proliferation of B-cells was significantly lower than that from wild type mice. Our study suggested that ex vivo CD4+ T-cells converted DN T-cells have the potential to down-regulate immune responses by suppressing B cells proliferation and IgG production. It supports the concept and the feasibility of potentially utilizing this novel cell-based therapeutic approach clinically for the prevention and treatment of B-cell mediated rejection.