DNT Cells Research Articles

Loss of P2X7 receptor membrane expression in B220+ double-negative T lymphocytes of autoimmune MRL/lpr mice (143.32)

Abstract Lupus is an autoimmune disease influenced by multiple genetic loci. Fas-deficient MRL/lpr mice exhibit lupus and lymphoproliferative syndromes with massive accumulation of B220+CD4-CD8- (DN) T lymphocytes. Pathogenic B220+ DN T lymphocytes are derived from normal activated T lymphocytes which have not undergone activation-induced cell death due to inactivation of Fas. Normal activated peripheral T lymphocytes up-regulate the membrane phosphatase B220 before undergoing apoptosis. In Fas-deficient mice, activated T cells down-regulate CD4 or CD8 molecules but retain the expression of B220. P2X7R is an extracellular ATP-gated cell membrane receptor involved in the release of inflammatory cytokines and cell death. 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-mediated stimulation of P2X7R. This decrease in P2X7R activity parallels the increase in B220+ DN T-cell numbers in lymphoid organs. The rare B220+ T cells observed in normal mice are also resistant to ATP treatment. Moreover, B cells, which express B220 as a developmental marker, are also dramatically less sensitive to ATP treatment. Using rabbit anti-P2X7R sera, we found a strong reduction in P2X7R levels on B220+ cells. Our results prompt the conclusion that B220 expression strongly correlates with the negative regulation of ATP/P2X7R pathway on both T and B cells.

Somatic Fas Mutations Account for Nearly One Third of Autoimmune Lymphoproliferative Syndrome (ALPS) Cases with Previously Unknown Genetic Mutations.

Abstract 710 Background:Autoimmune lymphoproliferative syndrome (ALPS) is characterized by childhood onset of lymphadenopathy, hepatosplenomegaly, autoimmune cytopenias, elevated (>1%) double negative T (DNT; CD3+, TCRalpha-beta+, CD4−, CD8−, B220+) lymphocytes in peripheral blood and an increased risk of lymphoma; primarily due to impaired lymphocyte apoptosis. Most cases (65%; 173 individuals in our cohort), known as ALPS Type Ia, are associated with dominant heterozygous germline mutations in the gene TNFRSF6 encoding the protein for CD95 (Fas, Apo-1). Another 5% of patients have mutations in FasL (Type Ib), caspases (Type II) or NRAS (Type IV). However, approximately 30% of ALPS patients in our cohort have no mutation found upon genomic sequencing of DNA from peripheral blood. We designated these patients as ALPS Type III (16%) if they met all the criteria of ALPS, i.e., elevated DNT cell numbers, nonmalignant chronic lymphadenopathy/splenomegaly and defective lymphocyte apoptosis by in vitro assay; we called them ALPS phenotype (14%) if they had all the features as described above but did not demonstrate an in vitro apoptosis defect. Holzelova et. al. (NEJM 2004; 351:1409) previously identified somatic Fas mutations in DNT cell population in 6 patients with ALPS phenotype. However, there was no information as to the proportion of ALPS patients expressing a somatic Fas mutation or any differences in their clinical phenotype. Methods and Results:Over the last 5 years we sought to determine the proportion of ALPS Type III and ALPS phenotype patients with somatic Fas mutations in their DNT cell population and to further clinically characterize these patients. DNT cells were purified to >50% by magnetic bead separation and DNA was sequenced for Fas. We found somatic Fas mutations in the DNT cells of 11/31 (35.5%) patients; 5/15 with ALPS Type III (5 males; age range = 1-17 years; median = 12 years), and 6/16 with ALPS phenotype (3 males and 3 females; age range = 3 mo–48 years; median age = 1 year). All the mutations clustered to an approximately 150 base region of the intracellular domain of Fas (exons 7, 8 and 9), except for one mutation in exon 3. All mutations resulted in functional loss of normal Fas signaling based on the mutation type or having been previously observed in ALPS Typa Ia. The somatic ALPS patients showed a similar clinical phenotype to that of ALPS Type Ia with increased DNT cell numbers (Median 6%; Range 4-19%), and increased levels of biomarkers like serum vitamin B12, IL-10 and sFasL. All 4 patients with onset of symptoms in infancy have required long-term treatment of their refractory autoimmune cytopenias with mycophenolate mofetil for the last 2-8years, while one of them underwent splenectomy. Inability to demonstrate defective lymphocyte apoptosis by in vitro testing of unseparated lymphocytes in ALPS phenotype patients with somatic Fas mutations is likely due to the relatively small number of affected cells (<20%) in their peripheral blood. Additionally, we hypothesize that the late onset of clinical disease in ALPS Type III patients with somatic Fas mutations may reflect the time required to accumulate a threshold number of cells that are able to confer clinical manifestations. Conclusion:Thus, the majority of somatic ALPS mutations can be detected by sequencing exons 7-9 of isolated DNT cell DNA. Patients with somatic Fas mutations now comprise the second largest group of known genetic mutations in ALPS, followed by patients with caspase 10, NRAS, caspase 8 and FasL germline mutations, respectively. We recommend testing for somatic Fas mutations in ALPS Type III patients (particularly those with late onset) and all ALPS phenotype patients, using ungated DNT purities of >50%. These data also highlight the role of somatic mutations in the pathogenesis of nonmalignant hematological conditions in adults and children, as well as in clonal malignancies. Disclosures:No relevant conflicts of interest to declare.

Lymphocyte Homeostasis FAS Pathway Is Altered in Some Patients with Immune Thrombocytopenia.

Abstract 3514Poster Board III-451Patients with autoimmune lymphoproliferative syndrome (ALPS) are the only described patients in which a known genetic defect leads directly to autoimmunity. In these patients, mutations in FAS or FASL result in an inability of activated lymphocytes to die resulting in defective lymphocyte homeostasis. The disorder manifests itself in autoimmunity directed towards platelets, red cells and neutrophils in addition to lymphadenopathy, splenomegaly and an increased risk of lymphoma. Inheritance of the defect is dominant, however there is variable penetration and patients even within the same family and with the same mutation can have a different phenotype. A definitive diagnosis requires clinical symptoms, elevated (>1%) TCRab+CD3+CD4-CD8- (DNT) cells in the peripheral blood and the demonstration of defective lymphocyte apoptosis in vitro. Given the tendency for patients with ALPS to develop ITP, we hypothesised that patients with ITP may also have mutations or polymorphisms within this pathway and hence abnormal lymphocyte homeostasis as an etiology of their ITP. DNA from 88 children and adults with chronic ITP (48 post splenectomy, all more than 1 year from diagnosis) were screened for mutations in the FAS gene. None had the mutations typically associated with ALPS. However, in 6 patients single nucleotide polymorphisms (SNPs) were detected: 3 at 528(+6)C>T and 3 at 836C>T; one patient had both polymorphisms. Three of these patients also had haemolysis. In three patients, FAS induced apoptosis studies were normal (the remaining await analysis). DNT cells (the signature cells of ALPS) ranged from 0.5% to 2.9% in these 6 patients with all but one above the normal adult range (0.1-0.9%). DNT cells were also raised in 3 of 4 patients with no SNPs in FAS, (median 1.8 - range 1.1 to 6.1%). One individual with the 836C>T SNP had 2.9% DNT cells with an absolute number of 91 cells (DNT cells NR 2-17 cells) with virtually absent gamma-delta DNTs. In addition, this patient had evidence activation of T cells with raised CD4/HLA-DR. and CD8/HLA-DR and CD27+ B cells were <16% of B cells. This patient had severe ITP with a complete response to rituximab for the last 8 years. This data, although not quite conclusive, suggests that SNPs in the FAS pathway may contribute to the autoimmunity seen in patients with ITP. In addition, the presence of increased DNT cells, even in patients without SNPs in the FAS gene, suggests defect in the immune system not previously investigated and that the DNT cells may have a role in immune homeostasis. Disclosures:Cooper:GSK: Consultancy, Honoraria; Amgen: Consultancy. Bussel:Genzyme: Research Funding; GlaxoSmithKline: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cangene: Research Funding; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Immunomedics: Research Funding; Ligand: Membership on an entity's Board of Directors or advisory committees, Research Funding; Eisai, Inc: Research Funding; Sysmex: Research Funding; Scienta: Speakers Bureau; Shionogi: Membership on an entity's Board of Directors or advisory committees.

CT Scan Enumeration of Altered Spleen Status and Pulmonary Artery Size in Patients with Autoimmune Lymphoproliferative Syndrome (ALPS).

Abstract 4034Poster Board III-970 BackgroundALPS is a disorder of apoptosis resulting in elevated DNT (CD3+, TCRab+, CD4-, CD8-, B220+ double negative T lymphocyte) cells associated with childhood onset nonmalignant lymphadenopathy, splenomegaly and multilineage cytopenias due to splenic sequestration and autoimmune destruction. Patients with ALPS frequently have altered spleen status: splenomegaly or splenectomy, the latter often-related to refractory chronic AIHA and ITP. This altered spleen status has been associated with pulmonary arterial hypertension (PAH) in two patients with ALPS. Though traditionally diagnosed as elevated tricuspid regurgitant jet velocity > 2.6 m/sec, PAH can also be predicted using PA diameter, most commonly measured as the Main PA (MPA) diameter. Similar to Sickle Cell Disease patients who are evaluated for presence of PAH due to functional asplenia here we attempted to further elucidate the relationship between dilated pulmonary arteries (PA) and spleen status in ALPS patients. MethodsCT scans of 162 patients with ALPS were studied; their age range at the time of the study was 1-63yrs, median age 16yrs. The CT scans were obtained as part of the routine monitoring of their LPD. Patients' most recent complete chest, abdomen and pelvis CT scans were evaluated. In 97 patients spleen sizes in two scans done 1-12 years apart were also measured. Each scan was evaluated using Kodak Carestream PACS Version 10.1.sp1 software. Point of maximal distension was visually identified in the Main, Left, and Right PA. Artery diameters were measured perpendicular to the vessel wall. Measurement of spleen length consisted of summing the distance between the axial slice in which the spleen first appears to the slice where it appears last. Digital Image Processing (IP) measurements are also underway to compute spleen and liver volumes in these patients. Evaluation CriteriaSplenomegaly was defined as a CT estimated spleen length of > 9.76 cm. PA diameter was considered above the upper limits of normal if adults if MPA measured > 28.6 mm LPA > 22.1 mm; RPA> 19.8 mm. For children age specific sizes were adapted from published reports. Results and conclusionIn our cohort of 162 ALPS patients, 51% had splenomegaly, 42% had undergone splenectomy due to hypersplenism prior to CT scan evaluations and 7% had normal size spleens. 26% of them demonstrated dilated MPA. However, contingency table analysis yielded a P value = 0.436 (P < 0.05) evaluating an association between splenic abnormalities and dilated MPA. ANOVA analysis of an association between MPA diameter and broad patient classifications of normal spleen, splenectomy, and splenomegaly yielded P = .02 (α < 0.05) and non-significant Tukey HSD statistical tests, the odds ratio = 0.93 for an association between spleen length and MPA diameter. Correlation R2 = 0 between age, spleen length and MPA diameter, though this was only evaluated in 42 patients 17 years and older. Serial measurements of enlarged spleens in the 97 children and adults with at least 2 CT scans done at a median time of 4 yrs apart (range 1-12yrs) showed no further significant increase in spleen size with age. For most patients, their ALPS associated lymphoproliferative burden appears to be worse during early childhood and as they grow older it may become self-limited and stable. Further work is in progress to correlate changes in PA diameter with changes in spleen length, spleen volumes and age. There was no statistical correlation between MPA diameter and altered spleen status, though a quarter of these patients demonstrated dilated pulmonary arteries. Active surveillance for PAH by periodic echocardiograms is underway in these patients. Disclosures:No relevant conflicts of interest to declare.

FAS-L, IL-10, and double-negative CD4−CD8− TCR α/β+ T cells are reliable markers of autoimmune lymphoproliferative syndrome (ALPS) associated with FAS loss of function

AbstractAutoimmune lymphoproliferative syndrome (ALPS) is characterized by splenomegaly, lymphadenopathy, hypergammaglobulinemia, accumulation of double-negative TCRαβ+ CD4−CD8− T cells (DNT cells), and autoimmunity. Previously, DNT cell detection and a functional defect of T cells in a FAS-induced apoptosis test in vitro had been used for ALPS diagnosis. However, a functional defect can also be detected in mutation-positive relatives (MPRs) who remain free of any ALPS-related disease. In contrast, lymphocytes from patients carrying a somatic mutation of FAS exhibit normal sensitivity to FAS-induced apoptosis in vitro. We assessed the soluble FAS-L concentration in the plasma of ALPS patients carrying FAS mutations. Overall, we showed that determination of the FAS-L represents, together with the IL-10 concentration and the DNT cell percentage, a reliable tool for the diagnosis of ALPS.

Significantly Elevated Vitamin B12 Levels in Autoimmune Lymphoproliferative Syndrome (ALPS), a Rare Lymphoproliferative Disorder with Apoptosis Defect

Vitamin B12 is an essential micronutrient that plays a fundamental role in cell division and one-carbon metabolism. Vitamin B12 also plays an important role in hematopoiesis and maintaining the integrity of the nervous system. Vitamin B12 in serum is bound to two proteins, haptocorrin (HC) and transcobalamin (TC). HC is a glycosylated serum protein of ~68kDa that is produced mainly by myeloid cells and binds the majority of circulating vitamin B12 (70–90%). The exact function of this HC-bound vitamin B12 is unknown (holo-HC), but it is believed to be biologically unavailable to most cells. The remainder of vitamin B12 is bound to TC (holo-TC). TC is a 43 kDa non-glycosylated serum protein that is synthesized primarily in the enterocytes and carries vitamin B12 to the tissues via a specific receptor with a high affinity for holo-TC. Only vitamin B12 bound to TC is available for cellular uptake and use as coenzymes for L-methylmalonyl-CoA mutase and methionine synthase that synthesize succinyl-CoA and methionine, respectively.ALPS is a rare autoimmune disease in children resulting from defective lymphocyte apoptosis and is characterized by lymphoproliferation, peripheral accumulation of DNT cells (double-negative T cells; TCR alpha/beta +, CD3+ CD4− CD8−), often leading to autoimmune multilineage cytopenias. Type Ia ALPS with Fas mutations accounting for ~75% of all reported cases is the most prevalent,High serum vitamin B12 concentrations have been observed in patients with ALPS studied at NIH Clinical Center. Levels of Vitamin B12 detected in these patients range from 742 to 35365 pmol/L (median: 2489 pmol/L; mean: 4123 pmol/L; reference interval: 162–708 pmol/L) among 137 patients compiled for this study (Vitamin B12: 1pmol/L=1.355 pg/mL). To date, the mechanism of the elevated vitamin B12 concentrations in ALPS patients has not been determined and we sought to explore the source of elevated vitamin B12.Radio- and enzyme immunoassays, size-exclusion chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot, and flow cytometry techniques were used to identify and quantitate the source of the elevated vitamin B12 concentrations in peripheral blood specimens from ALPS patients and controls.Total and holo-HC levels were 36- and 32-fold higher than the upper limit of the reference interval (Total HC: 250–840 pmol/L; Holo-HC: 240–680 pmol/L), respectively, in ALPS patients. However, total and holo-TC levels (1210 pmol/L; reference interval: 500–1500 pmol/L and 143 pmol/L; reference interval: >40 pmol/L, respectively) were not elevated in ALPS patients. The elution profile from size-exclusion chromatography of vitamin B12-binding proteins did not reveal any abnormal vitamin B12-binding proteins in ALPS. SDS-PAGE and western blot analysis revealed the presence of HC in lymphocyte lysates of ALPS but not apparently healthy controls. Flow cytometry studies also showed elevated levels of HC in all white blood cell types from ALPS patients compared to controls.The results of this study show for the first time that elevated concentrations of vitamin B12 found in ALPS patients were due to increased leukocyte (including lymphocytes) expression of HC. Further studies are needed to elucidate the mechanism for the increased synthesis and secretion of HC from lymphocytes of ALPS patients, but altered cytokine profiles, including increased interleukin-10 levels in ALPS may contribute.

Hydroxychloroquine Diminishes Lymphoproliferation in the Fas Deficient MRL/lpr−/− Murine Model of Autoimmune Lymphoproliferative Syndrome (ALPS).

Hydroxychloroquine (HCQ) is an anti-malarial drug in clinical use for decades that is finding further use as a steroid sparing agent in the treatment of immune disorders such as chronic GVHD, lupus and rheumatoid arthritis. HCQ is a lysosomotropic agent with more recent evidence showing immunomodulatory anti-TNF activity. It is currently being explored as a cytotoxic antineoplastic/antimicrobial agent. Hence, studies were conducted to determine the efficacy of HCQ to control the lymphoproliferation associated with ALPS. ALPS is an inherited disorder of apoptosis leading to lymphoproliferation and autoimmunity. The majority of ALPS patients are classified as Type Ia (>70%), having germline mutations in Fas. Other ALPS patients are classified as Type Ib, II, IV or III, if they have mutations in FasL, casapases, NRAS, or no identified mutations, respectively. They often present with childhood onset autoimmune cytopenias with lymphadenopathy, splenomegaly, increased circulating double negative T cells (DNT; TCRa/b+CD3+CD4−CD8−), defective apoptosis in vitro, and have an increased risk of lymphoma. Similarly, MRL/lpr−/− mice homozygous for Fas mutations develop an ALPS-like disease with massive lymphadenopathy, splenomegaly, hypergammaglobulinemia, autoimmune glomerulonephritis, and expansion of DNT cells secondary to defective lymphocyte apoptosis leading to lymphomagenesis. Currently, there are no proven therapies for the lymphoproliferation underlying ALPS itself. PBMCs from normal controls and ALPS Type Ia patients were cultured in vitro with 0–120 ug/mL HCQ in the presence or absence of 50 uM of the pan-caspase inhibitor Z-VAD-FMK, the caspase 9 inhibitor Z-LEHD-FMK, or the caspase 8 inhibitor Z-IETD-FMK. A dose response was observed with a high degree of cell death noted at 120 ug/mL after 48 hours, with an LD50 of 40 ug/mL. HCQ induced cell death was through a caspase-independent mechanism based on no inhibition of cell death by Z-VAD-FMK, Z-LEHD-FMK, or Z-IETD-FMK. Further preclinical studies were conducted in the MRL/lpr−/− murine model of ALPS. Forty five, 8-week old female MRL/lpr−/− mice were treated with 30 or 60 mg/kg of HCQ by gavage in sterile PBS or PBS alone three days per week for up to 14 weeks. Reductions of the spleen weight (p = 0.045, 437±85 vs 240±27) and cellularity (p = 0.08, 217±32 vs 148±18) were observed in high dose HCQ treated mice compared to controls at 7 weeks. Reductions in cellularity were also noted in the lymph nodes (p = 0.032, 118±33 vs 38±6). There was a trend towards decreasing DNT percentages in the spleen (21±5 vs 14±2), LN (70±4 vs 67±2) and blood (57±6 vs 51±5). Additionally, the CBC showed only marginal reductions in the WBC count (4.2±0.5 vs 3.5±0.3), hematocrit (49±0.3 vs 46±1.0), polymorphonuclear cells (33±3.7 vs 29±3.5) and monocytes (23±3.2 vs 17±2.5). Future studies are planned to more clearly determine the effect of HCQ on autoimmune kidney disease. Based on our in vitro and in vivo data, HCQ is effective at reducing lymphoproliferative activity in Fas deficient MRL/lpr−/− mice. It is being further explored in early phase clinical trials as a lympholytic agent to shrink lymph nodes and abrogate hypersplenism in ALPS patients. HCQ may also prove beneficial for long term use, especially in children, as a steroid sparing agent for treating refractory autoimmune cytopenias in ALPS.

A primary study of the subgroups of T lymphocytes in MHV-3 induced chronic viral hepatitis

To study the contribution of T cell subsets in the pathogenesis of Murine hepatitis virus Type 3 (MHV-3) induced chronic viral hepatitis in C3H/Hej mice, ninety C3H/Hej mice were chosen to individually receive 10 plaque forming units (PFU) of MHV-3 intraperitoneally. The changes of virus titer and pathology in liver tissue were examined by standard plaque assay and by the hematoxylin/eosin (HE) staining method from 2 days post MHV-3 infection. The ratios of T cell subsets including CD3+CD4+CD8−, CD3+CD4−CD8+, CD3+CD4−CD8−, CD3+CD4+CD25+, CD3+CD4+CD25− and CD3+ CD4−CD25+ T lymphocyte of total T lymphocytes in blood, spleen and liver were examined at 0, 2, 4, 6, 8, 10, 12, 15, 20, 25, 30, 40 days post MHV-3 infection by flow cytosorting. We observed that the virus titer raised and showed persistent virus duplications and inflammatory changes in the livers of C3H/Hej mice from 2 days post MHV-3 infection. The double negative T cell (DN Treg cell) and CD4+CD25+ T cell ratios increased significantly from 2 days post MHV-3 infection in C3H/Hej mice, and CD3+CD4+CD8−, CD3+CD4−CD8+, CD3+CD4+CD25− and CD3+CD4−CD25+ T cell ratios decreased accordingly. In conclusion, the changes of virus titer and pathology in the livers of C3H/Hej mice post MHV-3 suggest their contribution to viral persistence. Further characterizations of DN Treg cells are that infection indicates that MHV-3 could induce the chronic inflammation in livers of C3H/Hej mice. The increase of the DN Treg cell and CD4+CD25+ T cell ratios in C3H/Hej mice post MHV-3 infection suggests that DN Treg cells and CD4+CD25+ T cells may both have important suppressive immunomodulation functions in the development of chronic viral hepatitis and have important roles in the virus persistent infection. Further characterizations of DNT cell and CD4+CD25+ T cell are under investigation.

New differentiation pathway for double-negative regulatory T cells that regulates the magnitude of immune responses

AbstractRecent studies have demonstrated that in peripheral lymphoid tissues of normal mice and healthy humans, 1% to 5% of αβ T-cell receptor–positive (TCR+) T cells are CD4−CD8− (double-negative [DN]) T cells, capable of down-regulating immune responses. However, the origin and developmental pathway of DN T cells is still not clear. In this study, by monitoring CD4 expression during T-cell proliferation and differentiation, we identified a new differentiation pathway for the conversion of CD4+ T cells to DN regulatory T cells. We showed that the converted DN T cells retained a stable phenotype after restimulation and that furthermore, the disappearance of cell-surface CD4 molecules on converted DN T cells was a result of CD4 gene silencing. The converted DN T cells were resistant to activation-induced cell death (AICD) and expressed a unique set of cell-surface markers and gene profiles. These cells were highly potent in suppressing alloimmune responses both in vitro and in vivo in an antigen-specific manner. Perforin was highly expressed by the converted DN regulatory T cells and played a role in DN T-cell–mediated suppression. Our findings thus identify a new differentiation pathway for DN regulatory T cells and uncover a new intrinsic homeostatic mechanism that regulates the magnitude of immune responses. This pathway provides a novel, cell-based, therapeutic approach for preventing allograft rejection.

New differentiation pathway for double-negative regulatory T-cells that regulates the magnitude of immune responses (87.9)

Abstract CD4−CD8− (double-negative, DN) T-cells are capable of down-regulating the immune response. However, the origin and developmental pathway of DN T-cells are still unclear. In this study, we showed that proliferated CD4+ T-cells could convert to DN T-cells after 4–5 rounds of either allogeneic or syngeneic DC triggered proliferation, IL-2 and IL-15 enhanced the conversion. The disappearance of CD4 molecular on converted DN T-cells was a result of CD4 gene silence. Converted DN T-cells were resistant to AICD and expressed unique cell markers and gene profile. Moreover, DN T-cells retained a stable phenotype after re-stimulation. DN T-cells converted from CD4+ T-cells exerted powerful inhibition on same, but not third party, alloantigen triggered proliferation of naïve T cells. Perforin, a cytotoxic lymphocyte related cytokine, was highly expressed by DN T-cells, played a role in DN T-cell mediated suppression. Transferring of DN T-cells alone could prevent allograft rejection and the onset of autoimmune disease in vivo. This previously unknown pathway of differentiating DN regulatory T-cells represents a negative feedback mechanism of immune response. The results of using ex vivo CD4+ T-cells converted DN T-cells in transplantation and autoimmune disease models support the concept and the feasibility of potentially utilizing this novel cell-based therapy.

Role of Somatic Fas Mutations in the Pathogenesis of Autoimmune Lymphoproliferative Syndrome (ALPS).

ALPS is a disorder of lymphocyte apoptosis associated with expansion of double negative T cells (DNT; TCRα/β+CD3+CD4−CD8−), an in vitro apoptosis defect and affected individuals typically present in early childhood with nonmalignant lymphadenopathy, splenomegaly, and multilineage cytopenias due to splenic sequestration and/or peripheral autoimmune destruction. ALPS classifications include Type Ia, Ib, and II associated with mutations in Fas, FasL, and Caspases, respectively, and Type III when the apoptosis pathway mutation is unidentified. Among our cohort of more than 200 patients, ALPS Type Ia with germline Fas mutations constitute the most common subtype (over 70%). These patients show an in vitro Fas mediated lymphocyte apoptosis defect and have a known increased risk of lymphoma. However, there are a number of patients that have an “ALPS-phenotype” manifesting typical historical and clinical features of ALPS and present with elevated circulating DNT cells, but have no demonstrable in vitro apoptosis defect. Based on the work of Holzelova et. al (NEJM , 2004, 351:1409) in which six such patients were reported with somatic Fas mutations in their DNT cell subsets, we sought to characterize our patient set without defined mutations. DNT cells from 20 patients, including 8 with ALPS-phenotype and 12 with ALPS Type III, were isolated using magnetic bead separation of frozen PBMCs. The sensitivity for PCR followed by sequencing required >20% abnormal cells using a mixture of normal and ALPS Type Ia PBMCs. However, the sensitivity proved to be lower (40%) using purified DNT cells from patients with potential somatic Fas mutations. Therefore, we chose an ungated DNT purity of 50% as a minimum requirement. Ungated DNT purities for the 20 evaluated patients ranged from 51% to 92%, with a mean of 66%. All nine exons of the Fas gene were PCR amplified prior to sequencing.We found that 3/8 (37.5%) patients with ALPS-phenotype had somatic mutations in the Fas gene: 825G−>T, 846(−9)del11, and 846(−1)G−>T, with the first two mutations being new. Only 1/12 (8.3%) ALPS Type III patients had a somatic Fas muation: 851delAG, which also represents a new mutation. These four somatic mutations were considered significant as two were in a splice site and two were in coding regions. The ALPS Type III patient is atypical given a late age of presentation at age 17 and had milder cytopenias. The three ALPS-phenotype patients with somatic Fas mutations presented similar to cases of “classic” ALPS, with an early age of presentation (median, 1 yr; range, 4 months to 4 years), cytopenias and features of autoimmunity; two require chronic immunosuppression with Cellcept (mycophenolate mofitil) for chronic persistent cytopenias, one having previously undergone surgical splenectomy. From these data we conclude that patients with ALPS-phenotype or atypical ALPS Type III should be screened for somatic mutations in their peripheral blood DNT cell subset using ungated DNT purities of >50%, with priority given to evaluation by sequencing of exons 7–9 of the Fas gene. It is not yet evident if there is an increased risk of lymphoma in patients with somatic Fas mutations as is the case with intracellular germline Fas mutations. Somatic Fas mutations explain the molecular basis of a small subset of patients and are more readily observed in ALPS-phenotype patients.

Double-Negative T Cells, Activated by Xenoantigen, Lyse Autologous B and T Cells Using a Perforin/Granzyme-Dependent, Fas-Fas Ligand-Independent Pathway

The ability to control the response of B cells is of particular interest in xenotransplantation as Ab-mediated hyperacute and acute xenograft rejection are major obstacles in achieving long-term graft survival. Regulatory T cells have been proven to play a very important role in the regulation of immune responses to self or non-self Ags. Previous studies have shown that TCRalphabeta+CD3+CD4-CD8- (double-negative (DN)) T cells possess an immune regulatory function, capable of controlling antidonor T cell responses in allo- and xenotransplantation through Fas-Fas ligand interaction. In this study, we investigated the possibility that xenoreactive DNT cells suppress B cells. We found that DNT cells generated from wild-type C57BL/6 mice expressed B220 and CD25 after rat Ag stimulation. These xenoreactive B220+CD25+ DNT cells lysed activated, but not naive, B and T cells. This killing, which took place through cell-cell contact, required participation of adhesion molecules. Our results indicate that Fas ligand, TGF-beta, TNF-alpha, and TCR-MHC recognition was not involved in DNT cell-mediated syngenic cell killing, but instead this killing was mediated by perforin and granzymes. The xenoreactive DNT cells expressed high levels of granzymes in comparison to allo- or xenoreactive CD8+ T cells. Adoptive transfer of DNT cells in combination with early immune suppression by immunosuppressive analog of 15-deoxyspergualin, LF15-0195, significantly prolonged rat heart graft survival to 62.1 +/- 13.9 days in mice recipients. In conclusion, this study suggests that xenoreactive DNT cells can control B and T cell responses in perforin/granzyme-dependent mechanisms. DNT cells may be valuable in controlling B and T cell responses in xenotransplantation.

Valproic Acid (VPA), a Histone Deacetylase (HDAC) Inhibitor, Diminishes Lymphoproliferation in the Fas Deficient MRL/lpr−/− Murine Model of Autoimmune Lymphoproliferative Syndrome (ALPS).

ALPS is an inherited disorder of apoptosis leading to lymphoproliferation and autoimmunity. ALPS Type Ia, Ib and II are associated with germline mutations in Fas, FasL and Casapase 8 or 10, respectively; patients in whom no mutations have been identified are classified as Type III. The vast majority of patients are ALPS Type Ia (greater than 70%). They often present with childhood onset autoimmune cytopenias associated with lymphadenopathy, splenomegaly, increased double negative T cells (DNT; TCRα/β+CD3+CD4−CD8−), defective apoptosis by in vitro assay, and have an increased risk of lymphoma. Similarly, MRL/lpr−/− mice homozygous for Fas mutations develop an ALPS-like disease with massive lymphadenopathy, splenomegaly, hypergammaglobulinemia, autoimmune glomerulonephritis, and expansion of DNT cells secondary to defective lymphocyte apoptosis leading to lymphomagenesis. Currently, there are no proven therapies for the lymphoproliferation underlying ALPS itself, although complications like autoimmune cytopenias and post-splenectomy sepsis are manageable. Hence, studies were conducted to determine the efficacy of valproic acid (VPA) to control the lymphoproliferation associated with ALPS. VPA is a histone deacetylase (HDAC) inhibitor in clinical use for the last four decades as an anticonvulsant in children and adults, and recently being explored as an anti-neoplastic agent.PBMCs from normal controls and ALPS Type Ia patients were cultured in vitro with 0–4 mM VPA in the presence or absence of 50 uM of the pan-caspase inhibitor Z-VAD-FMK. A dose response was observed with a high degree of cell death noted at 4 mM after 48 hours, with an LD50 of 2 mM. VPA appeared to induce cell death by both caspase-dependent and -independent mechanisms based on partial inhibition of VPA-induced cell death by Z-VAD-FMK. Further preclinical studies were conducted in the MRL/lpr−/− murine model of ALPS. Twenty, 8-week old female MRL/lpr−/− mice were treated intraperitoneally (i.p.) with 500 mg/kg of VPA in sterile PBS or PBS alone five days per week for 8 weeks. Significant reduction of the spleen weight (p=0.034) and cellularity (p=0.0001) was observed in VPA treated (n=10) mice compared to controls (n=10). Reductions in size and cellularity were also observed in the lymph nodes (p=0.09 and 0.0002, respectively). A concomitant decrease (p<0.05) in DNT cells was observed in the spleen (11.2±0.6 vs 8.1±0.4) and blood (9.3±0.96 vs 5.5±0.6). Serum drug levels peaked (462±10 ug/mL) by 2 hours post-i.p. injection of VPA, where-as a 2.5 fold increase in histone acetylation was observed in the spleen at 4 hours, following a single injection. Analysis of the effects of VPA on autoimmune renal disease in these animals is underway. Based on our in vitro and in vivo data, VPA is effective at reducing lymphoproliferative activity in Fas deficient MRL/lpr−/− mice. It is being further explored in early phase clinical trials as a lympholytic agent to shrink lymph nodes and abrogate hypersplenism in ALPS patients.

Gastrin-mediated activation of cyclin D1 transcription involves beta-catenin and CREB pathways in gastric cancer cells.

Gastrin and its precursors promote proliferation in different gastrointestinal cells. Since mature, amidated gastrin (G-17) can induce cyclin D1, we determined whether G-17-mediated induction of cyclin D1 transcription involved Wnt signaling and CRE-binding protein (CREB) pathways. Our studies indicate that G-17 induces protein, mRNA expression and transcription of the G(1)-specific marker cyclin D1, in the gastric adenocarcinoma cell line AGSE (expressing the gastrin/cholecystokinin B receptor). This was associated with an increase in steady-state levels of total and nonphospho beta-catenin and its nuclear translocation, indicating the activation of the Wnt-signaling pathway. In addition, G-17-mediated increase in cyclin D1 transcription was significantly attenuated by axin or dominant-negative (dn) T-cell factor 4(TCF4), suggesting crosstalk of G-17 with the Wnt-signaling pathway. Mutational analysis indicated that this effect was mediated through the cyclic AMP response element (CRE) (predominantly) and the TCF sites in the cyclin D1 promoter, which was also inhibited by dnCREB. Furthermore, G-17 stimulation resulted in increased CRE-responsive reporter activity and CREB phosphorylation, indicating an activation of CREB. Chromatin immunoprecipitation studies revealed a G-17-mediated increase in the interaction of beta-catenin with cyclin D1 CRE, which was attenuated by dnTCF4 and dnCREB. These results indicate that G-17 induces cyclin D1 transcription, via the activation of beta-catenin and CREB pathways.

The nature and mechanisms of DN regulatory T-Cell mediated suppression

Regulatory T cells have been reported to enhance survival of transplanted allografts. We have recently identified and cloned a novel CD3 +CD4 −CD8 − (double negative, DN) regulatory T cell from mice that were given a single class I mismatched donor lymphocyte infusion and permanently accepted donor-specific skin allografts. When infused into naïve syngeneic mice, these DN T cells prolonged the survival of class I mismatched donor skin allografts. Here we further characterize the nature and mechanism of DN T-cell mediated suppression. This present study reveals that DN T cells are able to specifically eliminate activated syngeneic CD8 + T cells that share the same T cell receptor (TCR) specificity as DN T cells in vitro. Similarly, we found that, along with an increase of recipient DN T cells in the peripheral blood, anti-donor CD8 + T cells were also eliminated in vivo following a donor lymphocyte infusion. We further demonstrate that DN T regulatory cells do not mediate suppression by competition for growth factors or antigen presenting cells (APC) nor by modulation of APC, but require cell contact with the activated target CD8 + T cells. This contact can be mediated either by the TCR on CD8 + T cells that recognize constitutively expressed or acquired MHC molecules on DN T cells, or by the TCR on DN T cells that recognize constitutively expressed MHC molecules on CD8 + T cells. Together, these data extend our previous findings, and expand the conditions in which DN T cells can potentially be used to specifically suppress allogeneic immune responses.

Immunophenotypic profiles in families with autoimmune lymphoproliferative syndrome

AbstractAutoimmune lymphoproliferative syndrome (ALPS) type Ia is caused by inherited defects in apoptosis and is characterized by nonmalignant lymphoaccumulation, autoimmunity, and increased α/β+ double-negative T cells (α/β+-DNT cells). This study reports immunophenotypic findings in 166 members of 31 families with ALPS type Ia, associated with genetic mutations in theTNFRSF6 gene encoding Fas. The ALPS type Ia probands (n = 31) and relatives having both a Fas mutation and clinically proven ALPS (n = 28) showed significant expansion of CD8+T cells, α/β+-DNT cells, γ/δ+-DNT cells, CD3+/ HLA-DR+ T cells, CD8+/CD57+ T cells, and CD5+ B cells. Relatives with Fas mutations, but without all the required criteria for ALPS (n = 42), had expansions of CD8+ T cells, α/β+-DNT cells, and γ/δ+-DNT cells. Interestingly, relatives without a Fas mutation and with no features of ALPS (n = 65) demonstrated a small but significant expansion of CD8+ T cells, both DNT cell subsets, and CD5+ B cells. As compared to unrelated healthy controls, lymphocyte subset alterations were greatest in the probands, followed by the relatives with mutations and ALPS. Probands and relatives with mutations and ALPS also showed a lower number of CD4+/CD25+ T cells that, in combination with an independent increase in HLA-DR+ T cells, provided a profile predictive of the presence of clinical ALPS. Because quantitative defects in apoptosis were similar in mutation-positive relatives regardless of the presence of clinical ALPS, factors, other than modifiers of the Fas apoptosis pathway, leading to these distinctive immunophenotypic profiles most likely contribute to disease penetrance in ALPS.

TcR-alpha/beta(+) CD4(-)CD8(-) T cells in humans with the autoimmune lymphoproliferative syndrome express a novel CD45 isoform that is analogous to murine B220 and represents a marker of altered O-glycan biosynthesis.

Autoimmune lymphoproliferative syndrome (ALPS), caused by inherited defects in apoptosis secondary to mutations in genes encoding Fas/CD95/APO-1 and Fas ligand (Fasl)/CD95L, is characterized by nonmalignant lymphadenopathy and splenomegaly, increased T cell receptor alpha/beta(+) CD4(-)CD8(-) T cells (alpha/beta(+) double-negative T cells [alpha/beta(+)-DNT cells]), autoimmunity, hypergammaglobulinemia, and cytokine abnormalities. The alpha/beta(+)-DNT cells are immunophenotypically and functionally similar to alpha/beta(+)-DNT cells that accumulate in lpr and gld mice, which bear genetic mutations in Fas and FasL. In these mice, alpha/beta(+)-DNT cells express the B-cell-specific CD45R isoform B220. We show that alpha/beta(+)-DNT cells of ALPS patients, with either Fas or FasL mutations, also express B220. In addition, also similar to LPR/gLD mice, they have an unusual population of B220-positive CD4(+) T cells. B220 expression, together with our finding of characteristic lectin binding profiles, demonstrates that cell surface O-linked glycoproteins have undergone specific modifications, which may have consequences for lymphocyte trafficking, cell-cell interactions, and access to alternative apoptosis pathways.

IL-12 Enhances Lymphoaccumulation by Suppressing Cell Death of T Cells in MRL- lpr/lpr Mice

Lymphoaccumulation occurs in MRL- lpr/lpr mice, because double-negative T cells (DNT cells) cannot be deleted due to their Fas mutation, i.e., lpr. We show here that IL-12 enhances in lymphoaccumulation by suppressing the negative T cells in MRL- lpr/lpr mice. It has been reported that viable DNT cells from MRL-lpr/lpr mice undergo rapid apoptosis in ordinary cell culture without additional stimulation, suggesting that unknownin vivo factors other than lpr suppress the apoptosis. In the present study, we found that plasma IL-12p40 monomer and/or homodimer level increased with age in MRL- lpr/lpr but not in MRL-+/+ mice, and the increase in IL-12 correlated well with lymphoaccumulation. Requirement of IL-12 in lymphoaccumulation and in suppressed cell death of DNT cells of MRL- lpr/lpr mice was assessed. When an antibody neutralizing IL-12 was injected into old MRL- lpr/lpr mice with high plasma IL-12 level, lymphoaccumulation was diminished. When IL-12p40- or IL-12p70-encoding plasmid was administered to young MRL- lpr/lpr mice before the plasma IL-12 level increases, lymphoaccumulation was enhanced. The ordinary cell culture-induced cell death of DNT cells from MRL- lpr/lpr mice was suppressed in the presence of IL-12. Since DNT cells produce IFN-γ, a potent inducer of IL-12, the INF-γ induced-IL-12 may enhance lymphoaccumulation in MRL-lpr/lpr mice.

Increase in CD3 + CD4 - T lymphocytes in patients with AIDS and disseminated Mycobacterium avium-intracellulare complex infection: a prospective study

In a retrospective study, an increase in double-negative (CD3 + CD4 - CD8 -) (DN) T lymphocytes has been shown to be an independent predictor of disseminated Mycobacterium avium complex (D.MAC) infection in patients with less than 100 CD4 + T cells per mm 3. To better characterize this cell expansion, a prospective study was designed. From July 1995 to April 1997, 206 HIV-infected patients with less than 100 CD4 + T cells per mm 3 were prospectively followed up and immunophenotyped. The median followup was 1.1 year (+/-0.5 year), and 14 new D.MAC infections were diagnosed among 84 first AIDS-defining events. In univariate and multivariate analyses, D.MAC infections were the only opportunistic infection with a significant increase in DN T-cell percentage (median = 6.6; range = 1.7 to 24.5, P = 0.004) compared with patients without any opportunistic infection. This alteration in T-lymphocyte count could constitute a predictor for D.MAC infection in clinical practice.

Distribution of double-negative (CD4- CD8-, DN) T subsets in blood and synovial fluid from patients with rheumatoid arthritis.

Double-negative (CD4- CD8-) T (DNT) cells have been postulated to be potentially autoreactive. However, the role of DNT cells in rheumatoid arthritis (RA) has received limited attention. We investigated the distribution of DNT subsets in peripheral blood (PB) and synovial fluid (SF) from patients with active RA to determine whether these cells have relevance to RA. Two-colour flow cytometric analysis was performed to detect DNT cells in PB from 35 RA patients, 26 healthy controls and in SF aspirated from 19 inflamed rheumatoid joints. The subsets of DNT cells, i.e those expressing T cell receptor alphabeta (alphabeta DNT) or gammadelta (gammadelta DNT) were simultaneously examined. Our results showed that DNT cells constituted a very minor subset of PB lymphocytes. When expressed as a percentage of total lymphocytes, alphabeta DNT levels in normal individuals ranged from 0.27 to 2.08% (average 0.76%), while those of gammadelta DNT ranged from 1.02 to 11.42% (average 3.23%). Compared with normal individuals, RA patients had a similar distribution of alphabeta DNT cells in both PB and SF. However, RA patients had significantly lower levels of gammadelta DNT cells in PB than control subjects (1.38 +/- 1.08% vs 3.23 +/- 2.12%, p<0.05), while the levels of gammadelta DNT cells in SF of RA patients were higher than those in PB from RA patients and normal controls. The difference between PB and SF in RA was statistically significant (3.90 +/- 1.88% vs 1.38 +/- 1.08%, p<0.05). A higher level of gammadelta DNT in SF than their paired PB was consistently noted from nine available paired samples. Our findings suggest that gammadelta NT cells, but not alphabeta DNT cells, are probably relevant to RA. The lower percentage of circulating gammadelta DNT cells might have resulted from migration from the circulation into the synovium, suggesting a role for gammadelta DNT cells in the pathogenesis of rheumatoid synovitis.