DNT Cells Research Articles

Erratum: Idd13 is involved in determining immunoregulatory DN T-cell number in NOD mice

Correction to: Genes and Immunity (2014) 15, 82–87; doi:10.1038/gene.2013.65; published online 16 January 2014 Since the publication of this paper, the authors have noted that reference 52 is a retracted paper. They wish to replace this with the following paper: Peterson DA, DiPaolo RJ, Kanagawa O, Unanue ER.

Chronic activation with a staphylococcal superantigen drives the expansion of CD4, CD8 double negative T cells and promotes multiorgan inflammation mimicking systemic lupus erythematosus in HLA class II transgenic mice. (HUM7P.306)

Abstract CD4 and CD8 coreceptors play an important role in the development and maturation of αβ TCR+ T cells. However, T cells lacking CD4 and CD8 (double negative T or DNT cells) are present in the periphery and are significantly expanded in systemic lupus erythematosus (SLE) and Sjogren’s syndrome. The antigens that cause expansion of these DNT cells are unknown. As T cell activation by superantigens does not require coreceptors, we hypothesized that superantigens could activate DNT cells and contribute to autoimmunity. HLA-DQ8 and HLA-DR3 transgenic mice lacking both CD4 and CD8 were generated by intercrossing CD4 and CD8 single knockout mice. The CD4.CD8 double knockout (DKO) mice harbored a diverse repertoire of DNT cells in the periphery, albeit at reduced numbers compared to wild type (WT) mice. DNT cells from DKO mice responded robustly to the superantigen, staphylococcal enterotoxin B, but not to HLA restricted conventional antigens. Implantation of mini-osmotic pumps delivering 10 or 50 μg of SEB continuously over 7 or 28 days induced a lupus-like disease in WT as well as DKO DR3 and DQ8 mice. However, the immunopathology was significantly more pronounced in DKO mice. Immunostaining revealed extensive infiltration of multiple organs including lungs, liver and kidneys with CD3+ T cells in DKO mice. Overall, our study unraveled the role of CD4 and CD8 coreceptors in T cell development/function and the role of superantigens and DNT cells in the pathogenesis of lupus.

Recurrent Oral Inflammation in Autoimmune Lymphoproliferative Syndrome

Abstract Background and aim: Autoimmune lymphoproliferative syndrome (ALPS) is a disorder of abnormal lymphocyte survival caused by dysregulation of the Fas apoptotic pathway. In ALPS defective lymphocyte apoptosis manifests as a chronic, nonmalignant lymphadenopathy and/or splenomegaly/hepatosplenomegaly, expansion of double negative T cell (DNTC) – CD4 - CD8 - TCRI±I²+ T cells, autoimmune cytopenias and other autoimmune diseases. Patients demonstrate oral lesions which have not yet been reported in the literature. The aim of the study was to present the oral status of children with ALPS, associated mainly with haematologic and immunologic disorders. Material and methods: Among almost 1500 patients with primary immune deficiency diagnosed between 1980 and 2012 at the Department of Immunology, Children's Memorial Hospital, Warsaw (Poland) 7 cases of ALPS were identified according to ALPS diagnostic criteria. Routine immunological parameters were evaluated (serum immunoglobulin levels, immunophenotyping of peripheral lymphocytes, DNT cells, proliferative response to mitogens), vitamin B12 level, haematological and biochemical laboratory tests. Clinical course was retrieved retrospectively from medical records and evaluated during control visits. All patients were offered dental evaluation and treatment. Clinical examinations included the status of marginal gingiva, oral mucosa (the presence and type of lesions) and presence of caries. Results and conclusions: Almost all patients presented with leukopenia and granulocytopenia during control visits. Unsatisfactory oral hygiene status, carious cavities and gingivitis were found in all patients. In six subjects oral ulcers were also noticed. In two of them gingivitis was assessed as severe (with redness, swelling and spontaneous gingival bleeding), in other - as mild. An association between hematological and immune disorders and the health status of the oral mucosa and gingivae was observed in all but one patient. Findings included pale oral mucosa, a smooth tongue, recurrent mucosal ulcers, and gingivitis with bleeding. Their onset was accompanied by deterioration in the patients' general health condition and the presence of local causative factors. R ecurrent oral inflammation/aphtae can be the first symptom of ALPS and should be taken into account in differential diagnosis by paediatrician or general practitioner. It indicates strong need for cooperation between the dentists and immunologists and haematologists.

TCRγδ+CD4−CD8− T Cells Suppress the CD8+ T-Cell Response to Hepatitis B Virus Peptides, and Are Associated with Viral Control in Chronic Hepatitis B

The immune mechanisms underlying failure to achieve hepatitis B e antigen (HBeAg) seroconversion associated with viral control in chronic hepatitis B (CHB) remain unclear. Here we investigated the role of CD4−CD8− T (double-negative T; DNT) cells including TCRαβ+ DNT (αβ DNT) and TCRγδ+ DNT (γδ DNT) cells. Frequencies of circulating DNT cell subsets were measured by flow cytometry in a retrospective cohort of 51 telbivudine-treated HBeAg-positive CHB patients, 25 immune tolerant carriers (IT), 33 inactive carriers (IC), and 37 healthy controls (HC). We found that γδ DNT cell frequencies did not significantly change during treatment, being lower at baseline (P = 0.019) in patients with HBeAg seroconversion after 52 weeks of antiviral therapy (n = 20) than in those without (n = 31), and higher in the total CHB and IT than IC and HC groups (P<0.001). αβ DNT cell frequencies were similar for all groups. In vitro, γδ DNT cells suppressed HBV core peptide-stimulated interferon-γ and tumor necrosis factor-α production in TCRαβ+CD8+ T cells, which may require cell–cell contact, and could be partially reversed by anti-NKG2A. These findings suggest that γδ DNT cells limit CD8+ T cell response to HBV, and may impede HBeAg seroconversion in CHB.

Idd13 is involved in determining immunoregulatory DN T-cell number in NOD mice

Immunoregulatory T cells have been identified as key modulators of peripheral tolerance and participate in preventing autoimmune diseases. CD4(-)CD8(-) (double negative, DN) T cells compose one of these immunoregulatory T-cell subsets, where the injection of DN T cells confers protection from autoimmune diabetes progression. Interestingly, genetic loci defining the function and number of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) coincide with at least some autoimmune disease susceptibility loci. Herein, we investigate the impact of major insulin-dependent diabetes (Idd) loci in defining the number of DN T cells. We demonstrate that although Idd3, Idd5 and Idd9 loci do not regulate DN T-cell number, NOD mice congenic for diabetes resistance alleles at the Idd13 locus show a partial restoration in DN T-cell number. Moreover, competitive and non-competitive bone marrow chimera experiments reveal that DN T-cell number is defined by a bone marrow-intrinsic, but DN T-cell-extrinsic, factor. This suggests that non-autonomous candidate genes define DN T-cell number in secondary lymphoid organs. Together, our results show that the regulation of DN T-cell number in NOD mice is at least partially conferred by alleles at the Idd13 locus.

The Functional Attitude and The Predictive Role Of An Unconventional Subset Of T Cells In Clinical Outcome Of Lymphoma Patients: αβ-Double Negative T Cells, Preliminary Data Of a Prospective Study

Background Many aspects of lymphoma pathophysiology indicate mutual interactions between the host immune system and lymphoma cells. These interactions may either promote or control lymphomagenesis. An unconventional subset of CD4-CD8- double-negative T cells (DNTs) has been recently described to contribute specifically to anti-tumor immunity. Indeed, DNTs are involved in immune regulation and tolerance as well as in host defense and inflammation, acting as regulatory T cells and/or cytotoxic T cells. DNTs are T lymphocytes which express either αβ or γδ T-cell receptors (TCR) and lack CD4, CD8 and CD56. In healthy human donors and murine models, they constitute about 1-5% of the lymphocytes in peripheral blood and in lymphoid organs. No data are available on the role of DNT cells in human anti-lymphoma immunity. Information from murine models suggests that expanded DNT cells would not impair host immunity against lymphoma and would perhaps stimulate it. DNT cells have also demonstrated to have a direct in vitro anti-tumor activity against lymphoma. Few data are available on the prognostic significance of DNTs in lymphomas, on their interaction with other immune cells, and on their functional attitude. Aims The aim of this study was to assess the frequency and the functional attitude of circulating DNTs in Lymphoma patients and healthy donors as controls, in order to assess the role of DNTs on clinical outcome and progression. Methods To test this population as prognostic factor on clinical outcome and progression of lymphoma disease, peripheral blood (PB) and bone marrow (BM) samples of 46 Lymphoma patients (pts), with non-Hodgkin's Lymphomas and classical Hodgkin Lymphoma were selected and prospectively collected at diagnosis and after one month till the end of chemo- or immuno-chemotherapy therapy. Blood samples were collected also at the time of relapse or progression. As control PB samples of 16 healthy donors were collected. Circulating DNT subsets (TCRαβ+ and TCRγδ+) were characterized for their ontogeny, tolerogenic or cytotoxic attitude and TCR clonality by staining with the following conjugated monoclonal antibodies (MoAbs) for surface and intracellular markers: CD3, CD4, CD8, CD56, CD45, TCRαβ, CD45Ra, CD45Ro, CCR7, CD27, CD28, CD30, CD69, GITR, CD95, CD178, CD152, IFN-γ, TNF-α, granzyme B, and perforin. Isotype-matched MoAbs were used as staining controls. For functional studies, DNTs were purified from PBMCs of patients through a negative selection by using specific MACS microbeads and then cultured for 2 weeks in complete medium supplemented with anti-CD3 (OKT3), rhIL-2 and rhIL-4. Data were acquired using an 8-colour flow cytometer and analyzed using Kaluza software. Data were compared among the groups using the Mann-Whitney non-parametric test or Kruskal–Wallis one-way analysis of variance. The study was approved by the local Ethics Committee and all patients provided their informed consent in accordance with the Declaration of Helsinki. Results The percentage (mean + SE) of DNTs in BM (2.367 ± 0.5891) of Lymphoma pts was lower than in PB samples (3.421 ± 0.981). Moreover we observed a significant decrease (p = 0.006) of circulating αβ-DNTs in pts with untreated lymphoma (23.7 ± 3.7) as compared with healthy controls (31.3 ± 3.4), and their number seemed to be modulated by disease relapse/progression or disease treatment. (fig.1). In Hodgkin's Lymphoma circulating αβ-DNTs were significantly increased as compared with other histotypes (p = 0.0001) (fig.2). Circulating αβ-DNTs were significantly decreased (p=0.006) in serial samples collected after treatment or at the time of disease relapse. Interestingly, after ex vivo expansion, DNTs acquired an immunomodulatory cytokine profile, characterized by the secretion of IFN-γ and granzyme B which are known as central components of anti-tumor immune responses (fig.3). Conclusions To date, no data have been reported on DNT phenotypic and functional characterization in Lymphoma patients. Our study has demonstrated for the first time that αβ-DNTs could play an important role in both the development and the progression of lymphomas. In addition, based on our preliminary results, it is likely that ex-vivo expanded DNTs exert an anti-tumor activity thus suggesting their possible use as a new strategy for adoptive immune-therapy. Disclosures: No relevant conflicts of interest to declare.

A diagnostic dilemma in AL(L)PS

Dear Editor, An 18-month-old female infant presented to the paediatric haematology service in 1985 with lymphadenopathy, hepatosplenomegaly, anaemia (Hb 6.3 g/dL, range 11.7– 14 g/dL) and thrombocytopenia (17×10, range 150– 400×10). Bone marrow and lymph node examinations were reported as ‘reactive’. Karyotyping was normal. Viral, autoimmune and metabolic screens were negative. Following a period of observation and external review, during which time her marrow blast count rose to 9 %, the decision was made to proceed with Medical Research Council protocol for acute lymphoblastic leukaemia, with complete normalisation of peripheral blood counts and regression of spleen size for the duration of treatment (1986–1988). Her subsequent disease course included recurrent sinus and respiratory tract infections, fluctuating cytopenias, chronic lymphadenopathy and an elective splenectomy in the setting of massive splenomegaly. She developed severe autoimmune haemolytic anaemia, requiring intense immunosuppression and ultimately plasma exchange followed by recurrent invasive pneumococcal septicaemia on ages 14, 16 and 18 years. She was referred for immunological assessment in 2009 when a biopsy of an epitrochlear lymph node revealed paracortical T cell zone expansion with evidence of clonal T cell rearrangements, subsequently confirmed on bone marrow biopsy. CT images of thorax, abdomen and pelvis showed no evidence of lymphoma. On review, it emerged that her father, paternal aunt and first cousin all had a previous diagnosis of lymphoma. Screening for autoimmune lymphoproliferative syndrome (ALPS) confirmed the presence of double negative (DN) αβT cells (18 %, normal<1.5%) andFasL 1.5 ng/mL (normal range<0.2 ng/mL). Genetic testing demonstrated heterozygous nonsense mutation in exon 9 of the Fas gene confirming the diagnosis. Her father was found to have the same mutation. She failed to respond to the meningococcal vaccine and to both conjugated and unconjugated pneumococcal vaccines. She had a low IgG (IgG 4.6, range 6–15 g/L) and absent IgG2 with an elevated IgA (IgA 5.65, range 0.7–4.0 g/L) and normal IgM (IgM 1.0, range 0.5–2.3 g/L). Hypogammaglobulinaemia, recurring infection and failure to respond to vaccination is consistent with common variable immune deficiency which has been reported in association with ALPS [1]. At age 28, she presented with left hand and forearm swelling following minor trauma. C4 was undetectable (<0.0167 g/L, range 0.14–0.54 g/L). Both C3 and C1 inhibitor levels were reduced at 0.55 g/L (range 0.75–1.65 g/L) and 0.38 mg/L (range 150–350 mg/L), respectively. C1q levels were 44 mg/L (normal range 50– 250 mg/L). Based on the history, reduced C4, C1 inhibitor and C1q levels, a diagnosis of acquired C1 inhibitor deficiency was made. The diagnostic criteria for ALPS are elevated CD3 TCRαβ CD4 CD8 DNT cells ≥1.5 % of the total lymphocytes along with chronic non-malignant, non-infectious C. Lee-Brennan :M. Keogan Department of Immunology, Beaumont Hospital, Beaumont, Dublin 9, Ireland

Phenotypic and functional analysis of double negative T cells in kidney. (P1002)

Abstract Double negative T cells (DNT cells) are the main subset in kidney lymphocytes. In peripheral blood represent less than a 5%, but they gradually accumulate in lymph nodes and spleens of deficient FasL mice (gld). The role of these cells in kidney and how they mediate suppression/regulation are unknown. We performed a comprehensive phenotypic and functional analysis of this population in kidney. Hypothesis: DNT cells modulate immune responses in kidney. They have a suppressive function on cytokine production, playing a role in protection cells from injury. Methods: Purified T lymphocytes from mice were analyzed for expressing surface markers and cytokines secretion. B6 wild type, gld mice and ischemic reperfusion injury mice model were used. Surface and intracellular staining were done and analyzed with flow cytometry. Results: There are more DNT cells in the kidney (B6 and gld) than renal draining lymph nodes or inguinal lymph nodes. The percentage decreases in kidney after reperfusion ischemic injury. DNT cells showed an increased expression of activation markers such as CD44/CD69/CD28 compared to CD4+ and CD8+ in kidney. DNT cells exhibit a low secretion of cytokine like IL-17, IL4 or IFN gamma. Conclusion: Our data shows that double negative T cells have the potential to modulate immune responses in kidney. This is promising for better control of kidney injury or autoimmunodiseases.

Unusual selection and peripheral homeostasis for immunoregulatory CD4− CD8−T cells

Immunoregulatory CD4(-) CD8(-) (double-negative; DN) T cells exhibit a unique antigen-specific mode of suppression, yet the ontogeny of DN T cells remains enigmatic. We have recently shown that 3A9 T-cell receptor (TCR) transgenic mice bear a high proportion of immunoregulatory 3A9 DN T cells, facilitating their study. The 3A9 TCR is positively selected on the H2(k) MHC haplotype, is negatively selected in mice bearing the cognate antigen, namely hen egg lysozyme, and there is absence of positive selection on the H2(b) MHC haplotype. Herein, we take advantage of this well-defined 3A9 TCR transgenic model to assess the thymic differentiation of DN T cells and its impact on determining the proportion of these cells in secondary lymphoid organs. We find that the proportion of DN T cells in the thymus is not dictated by the nature of the MHC-selecting haplotype. By defining DN T-cell differentiation in 3A9 TCR transgenic CD47-deficient mice as well as in mice bearing the NOD.H2(k) genetic background, we further demonstrate that the proportion of 3A9 DN T cells in the spleen is independent of the MHC selecting haplotype. Together, our findings suggest that immunoregulatory DN T cells are subject to rules distinct from those imposed upon CD4 T cells.

The predictive role of a�-double negative T cells in clinical outcome of patients with lymphoma: preliminary data of prospective study.

The predictive role of a�-double negative T cells in clinical outcome of patients with lymphoma: preliminary data of prospective study.

Loss of P2X7 Receptor Plasma Membrane Expression and Function in Pathogenic B220+ Double-Negative T Lymphocytes of Autoimmune MRL/lpr Mice

Lupus is a chronic inflammatory autoimmune disease influenced by multiple genetic loci including Fas Ligand (FasL) and P2X7 receptor (P2X7R). The Fas/Fas Ligand apoptotic pathway is critical for immune homeostasis and peripheral tolerance. Normal effector T lymphocytes up-regulate the transmembrane tyrosine phosphatase B220 before undergoing apoptosis. Fas-deficient MRL/lpr mice (lpr mutation) exhibit lupus and lymphoproliferative syndromes due to the massive accumulation of B220+ CD4–CD8– (DN) T lymphocytes. The precise ontogeny of B220+ DN T cells is unknown. B220+ DN T lymphocytes could be derived from effector CD4+ and CD8+ T lymphocytes, which have not undergone activation-induced cell death due to inactivation of Fas, or from a special cell lineage. P2X7R is an extracellular ATP-gated cell membrane receptor involved in the release of proinflammatory cytokines and TNFR1/Fas-independent cell death. P2X7R also regulate early signaling events involved in T-cell activation. We show herein that MRL/lpr mice carry a P2X7R allele, which confers a high sensitivity to ATP. However, during aging, the MRL/lpr T-cell population exhibits a drastically reduced sensitivity to ATP- or NAD-mediated stimulation of P2X7R, which parallels the increase in B220+ DN T-cell numbers in lymphoid organs. Importantly, we found that this B220+ DN T-cell subpopulation has a defect in P2X7R-mediated responses. The few B220+ T cells observed in normal MRL+/+ and C57BL/6 mice are also resistant to ATP or NAD treatment. Unexpectedly, while P2X7R mRNA and proteins are present inside of B220+ T cells, P2X7R are undetectable on the plasma membrane of these T cells. Our results prompt the conclusion that cell surface expression of B220 strongly correlates with the negative regulation of the P2X7R pathway in T cells.

Immunogenetic Characterization of Primary Immune Thrombocytopenia (ITP) in Adults: Preliminary Results of the Unit Study.

AbstractAbstract 2192 Background.In adults, ITP displays variable clinical presentation and different response to steroid, Rituximab, and other immune suppressive agents. The pathophysiological differences underlying these different behaviours are mostly unknown and a better knowledge of this biological heterogeneity might help identifying more targeted and rationale treatments for this disease. Purpose.To identify immunogenetic features distinguishing ITP patients from control and different subsets of ITP patients based on clinical presentation and responsiveness to steroid or Rituximab. Several biological analyses were based on the model of autoimmune lymphoprolipherative syndrome (ALPS), a pediatric disease due to genetic defects decreasing function of the Fas death receptor involved in shutting off the immune response and cytotoxic cell function. ALPS displays polyclonal lymphoproliferation, peripheral blood (PB) expansion of TCRαβ+ T-cells double negative for CD4 and CD8 (DN T-cells) and autoimmune manifestations frequently including thrombocytopenia. ALPS is mostly due to deleterious mutations of the Fas gene, but the +1239A>C single nucleotide polymorphism (SNP) of the osteopontin gene (OPN) and several variations of the perforin gene (PRF1) can act as disease modifier. Moreover, defective Fas function and these OPN and PRF1 variations are also displayed by subsets of patients with multiple sclerosis, type-I diabetes mellitus, systemic lupus erythematosus, progressive sclerosis, and chronic inflammatory demyelinating polyneuropathy. Patients and Methods.Adult patients with ITP and matched controls were selected for the analyses and stratified in different clinical subsets according to the disease severity and responsiveness to therapy (asymptomatic, steroid or Rituximab sensitive vs. steroid or Rituximab refractory). Analyses included evaluation of Fas-mediated apoptosis in T cell cultures; proportions of DN-T cells in PB; typing of the +1239A>C SNP of OPN and sequencing of PRF1. All patients were also investigated for TCR monoclonality, whereas BCR monoclonality was analysed in Rituximab-untreated patients only. Results.Analysis of Fas function and DN T-cell expansion was assessed in 100 ITP patients and showed that they displayed higher frequency of defective Fas function than the controls (17/100 vs. 5/100; P<0.05). Expansion of DN T-cells was detected in 2/17 (12%) patients displaying defective Fas function and 3/83 (4%) of those with normal Fas function. Analysis of PRF1 and OPN was performed in 64 patients. Sequencing of PRF1 detected three patients carrying two rare variations; two carried the N252S amino acid substitution (previously described in ALPS) and one the novel R385W amino acid substitution. The overall frequency of these rare variations was higher in the patients than in the controls (4.7% vs. 0.8%, P<0.05). By contrast, the OPN +1239A>C SNP displayed a similar distribution in the patients and the controls. TCR monoclonality was assessed in 76 patients and was detected in 4 of them (5%). BCR monoclonality was assessed in 17 patients in PB and bone marrow and it was always absent. No statistical differences of these parameters were detected comparing patients refractory vs. sensitive to either Rituximab or steroid treatments. However, a trend was found for DN T-cell expansion that tended to be more frequent in Rituximab resistant vs. sensitive patients (0/13 vs. 4/20, P=0.13). Conclusions.These preliminary analyses detected some differences between ITP patients and controls suggesting that defects involved in ALPS development may play a role in adult ITP too. Increasing the patient number is needed to confirm these data and, possibly, to detect differences between clinical subgroups. Disclosures:Off Label Use: Rituximab in ITP.

Expanding Spectrum of Malignancies in ALPS: A Cancer Predisposing Syndrome?.

AbstractAbstract 2149 Background:Autoimmune lymphoproliferative syndrome (ALPS) is a disorder of apoptosis characterized by lymphadenopathy, splenomegaly and peripheral accumulation of TCR αβ+ CD4-/CD8- double-negative T lymphocytes (DNT cells), often leading to multilineage cytopenias. ALPS-FAS with germline FAS mutations is the most common form of ALPS and has been associated with an increased risk for lymphoma (Straus, Blood 2001). We have now identified additional malignancies in our cohort, further underscoring the importance of cancer surveillance in ALPS patients. Objective:This is an update of the ALPS-associated cancers within the NIH cohort consisting of 240 individuals with ALPS-FAS from 116 families and 106 patients with ALPS-U (undetermined genetic defect). Patients and demographics:We previously reported 11 patients in our ALPS cohort with Hodgkin lymphoma (HL) and 9 patients with B cell Non-Hodgkin lymphoma (NHL)(Rao and Oliveira, Blood 2011). Here we report 6 additional types of malignancies associated with ALPS in 5 patients. Four of these patients have an intracellular FAS mutation. The frequency of malignancies within our ALPS cohort is 7.2% (25/346 patients) with a male:female gender distribution of 20:5. The age at the time of cancer diagnosis ranged from 5 to 60 years (median 22years). Results:As outlined in the Table below, ALPS-FAS patient #45.2 developed squamous cell carcinoma of the tongue that eventually led to his death from complications. His son also had ALPS-FAS with lymphoma. ALPS-FAS patient # 197.1 developed testicular cancer with malignant mixed germ cell tumor with seminoma and embryonal carcinoma elements. Marked leukocytosis during the first cycle of BEP chemotherapy led to a diagnosis of chronic myelogenous leukemia (CML) based on detecting a BCR-ABL translocation by PCR in peripheral blood and bone marrow.A patient with ALPS-FAS who previously had lymphoma developed histiocytic sarcoma and died following failed alloBMT. ALPS-FAS patient #121.13 developed acinic tumor of the parotid gland. A patient with ALPS-U (i.e. undetermined genetic defect) developed Ph+ acute lymphoblastic leukemia treated with an allogenic matched unrelated donor stem cell transplant and he is currently doing well. [Display omitted] Conclusion:Although lymphoma continues to be the most common type of malignancy seen in ALPS patients, we have observed additional types of cancers in this population suggesting a broader cancer predisposition than previously thought. While none of these additional cancers, except for squamous cell carcinoma, have been previously associated with somatic FAS mutation in the Catalogue of Somatic Mutations in Cancer (http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/), germline mutations affecting the intracellular component of the Fas protein confer a greater risk for developing malignancy as 22 malignancies were noted among 200 ALPS-FAS patients with such intracellular mutations. While this limited sample size does not allow us to definitively associate these cancers with ALPS, our findings highlight the need for active cancer surveillance in ALPS patients. Moreover, ALPS should be suspected in patients with sporadic lymphomas and other cancers that have a pertinent clinical and family history of cytopenias, hypersplenism and lymphoproliferation. Disclosures:No relevant conflicts of interest to declare.

The BH3 Mimetic, Small Molecule, ABT-737, Was Effective As Lympholytic Therapy in the MRL/lpr−/− Mouse Model of Autoimmune Lymphoproliferative Syndrome (ALPS)

Abstract 695▪▪This icon denotes a clinically relevant abstractAutoimmune Lymphoproliferative Syndrome (ALPS) is an inherited disease associated with defects of lymphocyte apoptosis leading to lymphoproliferation and autoimmunity. The majority of ALPS patients have mutations in the FAS gene. MRL/lpr−/− (lpr) mice are used as a model for ALPS and are homozygous for FAS mutations, presenting with massive lymphadenopathy and splenomegaly associated with hypergammaglobulinemia, glomerulonephritis, and expansion of a rare population of TCR ab+CD4−CD8− (Double-negative, DN) T cells that are characteristic of ALPS. Currently, there are no proven therapies for the lymphoproliferation underlying ALPS besides immunosuppressive agents like high dose steroids and rapamycin. ABT-737 is a BH3 mimetic agent that binds and inactivates anti-apoptotic proteins of the BCL-2 family such as BCL-XL and BCL-2. An oral equivalent to this drug, Navitoclax, is currently being tested in Phase 1 studies for the treatment of diverse tumors. ABT-737 is potentially of interest for the treatment of ALPS, given that it directly activates the intrinsic pathway of apoptosis, independent of the FAS pathway. We tested the BH3 mimetic ABT-737, with and without dexamethasone, in the lpr mouse model as a potential therapeutic agent for the lymphoproliferative disease. Lpr mice (N=30) were treated daily via IP injections with dexamethasone (4mg/kg) plus ABT-737 (50mg/kg) (Dex+ABT-737), dexamethasone alone (Dex), ABT-737 alone (ABT-737) or vehicle only (VEH) for 8 weeks. Vehicle treated mice developed very severe lymphoproliferation, with mean lymph node (LN) and spleen (SPL) weights of 750 mg and 465 mg, respectively. Cellularity reached LN= 85×106 cells; SPL=290×106 cells. Dex+ABT-737 treated mice had the greatest reduction in lymph node and spleen weights (final weight of 101 mg, p<0.0001; and 213 mg, p<0.005, respectively) and cellularity (LN=4×106, SPL=63×106, p<0.001), followed by ABT-737 alone (LN 453 mg, p=0.02; SPL 118 mg, p=ns; LN=20×106, p=0.001; SPL= 118×106, p=0.01), then Dex (LN 542 mg, p=ns; SPL 450mg, p=ns; LN 45×106, p=ns; SPL 241×106, p=ns). Although lymphoid cellularity was significantly lower with treatment, there was no bone marrow toxicity based on cell numbers. Additionally, there were significant reductions in DNT cells in the spleen, lymph nodes and blood with Dex+ABT-737 (p<0.005) and ABT-737 only (p<0.01) compared to vehicle. The DNT cells also appeared to be more sensitive to treatment as they were reduced to a greater extent than CD4, CD8 or B cells. ABT-737 was also able to kill patient derived EBV-transformed B cell lines in vitro, through induction of uncoupling of BAX and BAK from BCL-XL and consequent activation of the intrinsic pathway of apoptosis with release of cytochrome c and AIF. These studies provide the first evidence for the utility of this class of drugs in the treatment of a non-malignant lymphoproliferative disorder. Disclosures:No relevant conflicts of interest to declare.

Cell Therapy With Autologous Tolerogenic Dendritic Cells Induces Allograft Tolerance Through Interferon-Gamma and Epstein-Barr Virus-Induced Gene 3

Innovative therapeutic strategies are needed to diminish the impact of harmful immunosuppression in transplantation. Dendritic cell (DC)-based therapy is a promising approach for induction of antigen-specific tolerance. Using a heart allograft model in rats, we analyzed the immunoregulatory mechanisms by which injection of autologous tolerogenic DCs (ATDCs) plus suboptimal immunosuppression promotes indefinite graft survival. Surprisingly, we determined that Interferon-gamma (IFNG), a cytokine expected to be propathogenic, was threefold increased in the spleen of tolerant rats. Importantly, its blockade led to allograft rejection [Mean Survival Time (MST) = 25.6 ± 4 days], showing that IFNG plays a critical role in immunoregulatory mechanisms triggered by ATDCs. IFNG was expressed by TCRαβ(+) CD3(+) CD4(-) CD8(-) NKRP1(-) cells (double negative T cells, DNT), which accumulated in the spleen of tolerant rats. Interestingly, ATDCs specifically induced IFNG production by DNT cells. ATDCs expressed the cytokinic chain Epstein-Barr virus-induced gene 3 (EBI3), an IL-12 family member. EBI3 blockade or knock-down through siRNA completely abolished IFNG expression in DNT cells. Finally, EBI3 blockade in vivo led to allograft rejection (MST = 36.8 ± 19.7 days), demonstrating for the first time a role for EBI3 in transplantation tolerance. Taken together our results have important implications in the rationalization of DC-based therapy in transplantation as well as in the patient immunomonitoring follow-up.

Histone Deacetylase 9 Deficiency Protects against Effector T Cell-mediated Systemic Autoimmunity

Co-repressor histone deacetylase 9 (HDAC9) plays a key role in the development and differentiation of many types of cells, including regulatory T cells. However, the biological function of HDAC9 in T effector cells is unknown. Systemic autoimmune diseases like lupus, diabetes, and rheumatoid arthritis have dysfunctional effector T cells. To determine the role of HDAC9 in systemic autoimmunity, we created MRL/lpr mice with HDAC9 deficiency that have aberrant effector T cell function. HDAC9 deficiency led to decreased lympho-proliferation, inflammation, autoantibody production, and increased survival in MRL/lpr mice. HDAC9-deficient mice manifested Th2 polarization, decreased T effector follicular cells positive for inducible co-stimulator, and activated T cells in vivo compared with HDAC9-intact MRL/lpr mice. HDAC9 deficiency also resulted in increased GATA3 and roquin and decreased BCL6 gene expression. HDAC9 deficiency was associated with increased site-specific lysine histone acetylation at H3 (H3K9, H3K14, and H3K18) globally that was localized to IL-4, roquin, and peroxisome proliferator-activated receptor-γ promoters with increased gene expression, respectively. In kidney and spleen, HDAC9 deficiency decreased inflammation and cytokine and chemokine production due to peroxisome proliferator-activated receptor γ overexpression. These findings suggest that HDAC9 acts as an epigenetic switch in effector T cell-mediated systemic autoimmunity.

The Transcription Factor PU.1 Regulates γδ T Cell Homeostasis

BackgroundT cell development results in the generation of both mature αβ and γδ T cells. While αβ T cells predominate in secondary lymphoid organs, γδ T cells are more abundant in mucosal tissues. PU.1, an Ets family transcription factor, also identified as the spleen focus forming virus proviral integration site-1 (Sfpi1) is essential for early stages of T cell development, but is down regulated during the DN T-cell stage.Methodology/Principal FindingsIn this study, we show that in mice specifically lacking PU.1 in T cells using an lck-Cre transgene with a conditional Sfpi1 allele (Sfpi1 lck−/−) there are increased numbers of γδ T cells in spleen, thymus and in the intestine when compared to wild-type mice. The increase in γδ T cell numbers in PU.1-deficient mice is consistent in γδ T cell subsets identified by TCR variable regions. PU.1-deficient γδ T cells demonstrate greater proliferation in vivo and in vitro.Conclusions/SignificanceThe increase of γδ T cell numbers in Lck-Cre deleter strains, where deletion occurs after PU.1 expression is diminished, as well as the observation that PU.1-deficient γδ T cells have greater proliferative responses than wild type cells, suggests that PU.1 effects are not developmental but rather at the level of homeostasis. Thus, our data shows that PU.1 has a negative influence on γδ T cell expansion.

Characterization of the Immunoregulatory Function of Human TCRαβ+ CD4-CD8- Double-Negative T Cells

AbstractAbstract 2776Regulatory T (Treg) cells have been shown to be involved in downregulating immune responses in autoimmunity, transplant rejection, and graft-versus-host disease. Moreover, the important role of Treg cells in tumor progression has also been extensively investigated and Treg-mediated immune suppression has emerged as a crucial mechanism of tumor evasion. Recently, a novel subset of TCRαβ+ CD4−CD8− (double-negative, DN) T cells has been characterized to specifically suppress immune responses in mice. Here we demonstrate that human DN T cells belong to the family of inducible Treg cells with potent suppressor activity towards CD4+ and CD8+ T-cell responses. Resting DN T cells failed to suppress responder cells, whereas antigen presenting cell (APC)-stimulated DN T-cells and freshly isolated CD4+CD25+ Treg cells revealed a strong suppressive activity. Of importance, when more potent stimulators such as allogeneic dendritic cells were used for activation of responder T cells, CD4+CD25+ Treg cells were unable to mediate any suppressor function, while APC-primed DN T cells were still able to suppress. The suppressive activity of DN T cells is neither mediated indirectly by modulation of APCs nor by competition for T-cell growth factors. Furthermore, DN T-cell mediated suppression towards responder T cells requires cell-cell contact and is TCR dependent. Based on the importance of Treg cells in tumor immunity, we determined the frequency of circulating DN T cells in patients with malignant diseases. Of interest, the DN T-cell pool revealed an increased frequency in cancer patients compared to healthy controls, indicating that these suppressor cells might limit tumor-specific T-cell responses and thereby impair tumor immunosurveillance. Taken together, our results demonstrate that human DN T cells are a new subset of inducible Treg cells exerting a very potent suppressive activity towards cellular immune responses. Further understanding of the mechanisms involved in human DN T-cell suppression may have important implications for novel immunotherapies. Disclosures:No relevant conflicts of interest to declare.

Adoptive transfer of DNT cells induces long-term cardiac allograft survival and augments recipient CD4+Foxp3+ Treg cell accumulation

Regulatory T (Treg) cells play an important role in the regulation of immune responses but whether Treg will induce tolerance in transplant recipients in the clinic remains unknown. Our previous studies have shown that TCRαβ+CD3+CD4−CD8−NK1.1− (double negative, DN) T cells suppress T cell responses and prolong allograft survival in a single locus MHC-mismatched mouse model. In this study, we investigated the role of DNT cells in a more robust, fully MHC-mismatched BALB/c to C57BL/6 transplantation model, which may be more clinically relevant. Adoptive transfer of DNT cells in combination with short-term rapamycin treatment (days 1–9) induced long-term heart allograft survival (101±31 vs. 39±13days rapamycin alone, p<0.01). Furthermore adoptive transfer DNT cells augmented CD4+Foxp3+ Treg cells accumulation in transplant recipients while depletion of CD4+ Treg cells by anti-CD25 inhibited the effect of DNT cells on long-term graft survival (48±12days vs. 101±31days, p<0.001). In conclusion, DNT cells combined with short-term immunosuppression can prolong allograft survival, which may be through the accumulation of CD4+Foxp3+ Treg cells in the recipient. Our result suggests that allograft tolerance may require the co-existence of different type Treg cell phenotypes which are affected by current immunosuppression.

Antigen dose‐dependent suppression of murine IgE responses is mediated by CD4−CD8− double‐negative T cells

The IgE response against protein antigens is profoundly influenced by the dose used for sensitization. The aim of the study was to identify immune cells that are involved in antigen dose-dependent regulation of IgE formation. Wild-type mice as well as T helper (Th)1-deficient IL-12p40(-/-) and IFN-gamma(-/-) mice were immunized by repeated intraperitoneal injection of either low doses (K01 mice) or high doses (K100 mice) of keyhole limpet haemocyanin adsorbed to aluminium hydroxide. Splenocytes of immunized mice were restimulated in vitro and antigen-dependent T cell proliferation and cytokine production were measured. The frequency of regulatory T cell subsets among splenocytes from K01 and K100 mice was compared using fluorocytometry and RT-PCR analysis. Splenocytes or T cell subpopulations were transferred into naïve mice and the effect of lymphocyte transfer on IgE production after priming of recipients with low antigen doses was determined. Specific IgE production was considerably impaired in K100 mice. Antigenic restimulation revealed hypoproliferation of K100 splenocytes and reduced production of Th2 cytokines IL-4, IL-5 and IL-13, but no induction of IFN-gamma production. Moreover, lymphocytes from K01 and K100 mice did not show significant differences in the expression of molecules associated with the phenotype or activity of conventional regulatory T cells. Transfer of splenocytes or purified T cells from K100 mice substantially suppressed the induction of IgE production in the recipients in an antigen- and isotype-specific manner. Neither CD4(+) nor CD8(+) T cells from K100 mice were able to inhibit IgE formation; instead, we identified CD4(-)CD8(-) double-negative T cells (dnT cells) as the principal T cell population, which potently suppressed IgE production. Our data demonstrate that CD4(-)CD8(-) dnT cells play a major role in the regulation of IgE responses induced by high antigen doses.