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Autoimmune lymphoproliferative syndrome due to somatic FAS mutation (ALPS-sFAS) combined with a germline caspase-10 (CASP10) variation
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Autoimmune lymphoproliferative syndrome due to somatic FAS mutation (ALPS-sFAS) combined with a germline caspase-10 (CASP10) variation Ana Martínez-Feito a , Josefa Melero b , Sergio Mora-Díaz a , Carmen Rodríguez-Vigil c , Ramón Elduayen d , Luis I. González-Granado e,f , Dolores Pérez-Méndez a , Elena Sánchez-Zapardiel a , Raquel Ruiz-García a , Miguela Menchén a , a ˜ ˜ h, Josefa Díaz-Madronero , Estela Paz-Artal a,f , Rafael del Orbe-Barreto g , Marta Rinón a,f,∗ Luis M. Allende a
Servicio de Inmunología, Hospital Universitario 12 de Octubre, Madrid, Spain Servicio de Inmunología, Hospital Universitario Infanta Cristina, Badajoz, Spain c Servicio de Hemato-Oncología Pediátrica, Hospital Universitario Miguel Servet, Zaragoza, Spain d Servicio de Hematología, Hospital Universitario Infanta Cristina, Badajoz, Spain e Unidad de Inmunodeficiencias e Infecciosas pediátricas, Unidad de Hemato-Oncología Pediátrica, Hospital Universitario 12 Octubre, Madrid, Spain f Instituto de Investigación i+12, Madrid, Spain g Servicio de Hematología, Hospital Universitario de Cruces, Bilbao, Spain h Servicio de Inmunología, Hospital Universitario de Cruces, Bilbao, Spain b
a r t i c l e
i n f o
Article history: Received 9 June 2015 Received in revised form 31 July 2015 Accepted 11 August 2015 Available online xxx Keywords: ALPS Apoptosis CASP10 FAS Immunodeficiency
a b s t r a c t Autoimmune lymphoproliferative syndrome (ALPS) is a primary immunodeficiency caused by impaired Fas/FasL-mediated apoptosis of lymphocytes and is characterized by chronic nonmalignant or benign lymphoproliferation, autoimmune manifestations and expansion of double negative (DN) T-cells (TCR˛ + CD4-CD8-). Most cases of ALPS are associated with germline (ALPS-FAS) or somatic (ALPS-sFAS) heterozygous FAS mutations or a combination of both. Here we report three unrelated patients with ALPS-sFAS. Only one of them showed impaired Fas function in PHA-activated T-cells. In this patient, the genetic analysis of the caspase-10 gene (CASP10) identified a heterozygous germline change in exon 9 (c.1337A>G) causing Y446C substitution in the caspase-10 protein. In addition, this patient had a dysregulated T- and B-cell phenotype; circulating lymphocytes showed expansion of T effector memory CD45RA+ (TEMRA) CD4 T-cells, effector memory CD8 T-cells, CD21low B-cells and reduced memory switched B-cells. Additionally, this patient showed altered expression in T-cells of several molecules that change during differentiation from naïve to effector cells (CD27, CD95, CD57 and perforin). Molecular alterations in genes of the Fas pathway are necessary for the development of ALPS and this syndrome could be influenced by the concurrent effect of other mutations hitting different genes involved in Fas or related pathways. © 2015 Published by Elsevier GmbH.
1. Introduction Autoimmune lymphoproliferative syndrome (ALPS) is a rare genetic disorder of lymphocyte homeostasis characterized by
∗ Corresponding author at. Servicio de Inmunología, Hospital Universitario 12 de Octubre, Avda. Córdoba s/n, Madrid-28041, Spain. E-mail address: [email protected] (L.M. Allende).
chronic benign lymphoproliferation, autoimmune manifestations and an increased risk of lymphoma (Fisher et al., 1995; Neven et al., 2011; Price et al., 2014; Rieux-Laucat et al., 1995). Proportions of CD3 + TcR˛+ CD4-CD8- double negative (DN) T-cells and some plasma biomarkers including interleukin-10 (IL-10), soluble FasL (sFasL), soluble CD25 (sCD25) and vitamin B12 are elevated in ALPS patients. Like other primary immunodeficiencies (PIDs), ALPS can be classified according to the underlying genetic defect. Most ALPS patients carry heterozygous germline (ALPS-FAS) or somatic FAS
http://dx.doi.org/10.1016/j.imbio.2015.08.004 0171-2985/© 2015 Published by Elsevier GmbH.
Please cite this article in press as: Martínez-Feito, A., et al., Autoimmune lymphoproliferative syndrome due to somatic FAS mutation (ALPS-sFAS) combined with a germline caspase-10 (CASP10) variation. Immunobiology (2015), http://dx.doi.org/10.1016/j.imbio.2015.08.004
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mutations (ALPS-sFAS) or both (Holzelova et al., 2004; Rieux-Laucat et al., 1995). In addition, germline mutations in FASL and CASP10 genes have been identified in a small number of ALPS patients (DelRey et al., 2006; Wang et al., 1999). ALPS patients with no known genetic defects are classified as ALPS-unknown (ALPS-U) (Oliveira et al., 2010). The partial penetrance observed in some ALPS patients suggests that, in addition to the germline FAS mutation, a second hit is required for the onset of the disease. Both alterations may cooperate in disrupting the switching off of the immune response affecting immune surveillance (Magerus-Chatinet et al., 2011). This work describes three unrelated ALPS-sFAS patients (P1, P2 and P3) born to non-consanguineous parents who had chronic nonmalignant noninfectious lymphadenopathy, splenomegaly, and autoimmune cytopenias and were positive for ALPS biomarkers. In addition, P3 is double heterozygous for a somatic FAS mutation and a germline CASP10 variation. We assessed the effects of ALPS genotypes on T- and B-cell phenotype. 2. Materials and methods 2.1. Immunophenotype analysis For direct immunofluorescence, peripheral whole blood was incubated using the corresponding monoclonal antibodies: anti-CCR7-FITC, anti-TCR␣-FITC, anti-TCRd-PE, anti-IgD-PE, anti-CD21-PE, anti-CD57-PE, anti-CD95-PE, anti-Perforin-PE, antiCD3-PerCP5.5, anti-CD45RA-PECy7 (all from BD Biosciences, Madrid, Spain); anti-CD27-PE, anti-CD8-PECy7, anti-CD19-PECy7, anti-CD8-APC, anti-CD4-APC, anti-CD4-APC-Alexa Fluor 750, and anti-CD45-APC-Alexa Fluor 750 (all from Beckman Coulter, Madrid, Spain). TcR V repertoire (Beckman Coulter) was determined in gated CD4, CD8 and DN T-cells. Proportions of T- and B-cells were analyzed by flow cytometry using a Navios Cytometer (Beckman Coulter). 2.2. ALPS biomarkers Plasma levels of IL-10 (Bender MedSystems, Labclinics, Madrid, Spain), sCD25, sFasL (R&D, Vitro, Madrid, Spain) and vitamin B12 (Beckman Coulter) were measured in duplicate by enzyme-linked immunosorbent assay. Serum immunoglobulin concentrations were determined by nephelometry (Beckman Coulter). 2.3. Isolation of DN T-cells DN T-cells were isolated from peripheral-blood mononuclear cells (PBMCs) by a magnetic Double negative T Cell Isolation Kit (Miltenyi Biotec, Madrid, Spain) (purities of DN T-cells exceeding 90%). 2.4. Cells and cell culture Human PBMCs were obtained from the blood of patients, relatives and healthy donors by Ficoll separation. T-cell blasts were generated from these cells by PHA stimulation (20 ng/L) (Sigma–Aldrich, Madrid, Spain) in the presence of IL-2 (100 IU/L) (Roche, Madrid, Spain), as described elsewhere (Del-Rey et al., 2006). 2.5. Fas function in T-cell blasts and in DN T-cells T-cell blasts (1 × 105 cells) from the patients and healthy controls were aliquoted in triplicate into 96-well plates and cultured with different concentrations of anti-Fas mAb (125, 250, or 500 ng/L) (Clon Apo1.3; Enzo Life Sciences, Madrid, Spain). After overnight incubation, apoptotic cells were detected by flow
cytometry with the use of annexin V-FITC (Immunostep, Salamanca, Spain). For Fas-induced apoptosis in DN T-cells, PBMCs (2 × 105 ) were aliquoted in triplicate into 96-well plates and cultures with different concentrations of anti-Fas mAb (250, 500 or 1000 ng/mL) (Clon Apo1.3) for 4 h in media without IL-2. After incubation, cells were washed and stained with annexin V-FITC or anti-CD95 FITC (BD Biosciences) and antibodies to anti-CD3APC-Cy7 (BD Biosciences), anti-CD4-APC, anti-CD8-APC (Beckman Coulter), anti-TcR˛-PECy7 (Immunostep), anti-B220-PE (Miltenyi Biotec) to identify apoptotic DN T-cells, as described elsewhere (Lo et al., 2013) 2.6. Molecular genetics DNA was extracted from whole blood or DN T-cells using a MagNa Pure Compact Nucleic Acid Isolation Kit (Roche, Madrid, Spain) and QIAamp DNA Blood Mini Kit (Qiagen, Madrid, Spain), respectively. Mutation analysis of the FAS gene was assessed as described elsewhere (Del-Rey et al., 2006). The CASP10 gene was amplified and sequenced according to standard methods (primers available upon request). 2.7. Statistical analysis Statistical comparisons were performed with unpaired Student t tests, with significance defined as P < 0.05(*), P < 0.01(**), or P < 0.001(***). 3. Results 3.1. Case report Patient 1 (P1) is a 56-year-old woman whose first manifestation was painless generalized lymphadenopathy (axillary, supraclavicular, cervical and retroauricular) and splenomegaly with no other clinical signs or symptoms. A primary hematologic disorder or bone marrow infiltration by malignant tumor cells was ruled out. Peripheral blood showed mild cytopenias (leucopenia 3400/mm3 and thrombocytopenia 125,000/mm3 ), and IgA hypergammaglobulinemia, but normal levels of IgG and IgM, and was negative for antinuclear antibodies (ANA). Extensive lymph node disease with multiple supradiaphragmatic and infradiaphragmatic enlarged nodes and splenomegaly were observed on PET scanning. No significant clinical or laboratory variations have been observed in 3 years of follow-up without treatment. Patient 2 (P2), a 13-year-old boy, was referred for evaluation of chronic splenomegaly. At age 10 years, seroreactivity to CMV was demonstrated, with splenomegaly that lasted for several months. Eight months before the consultation, he was hospitalized for a 2-week history of lymphadenopathy, splenomegaly, fever, thrombocytopenia (96,000/mm3 ) and neutropenia (700/mm3 ). Results of microbiological tests (blood cultures, CMV, EBV, HIV, Parvovirus B19, HSV, Leishmania) were negative. Bone marrow aspirate was normal. Fever, lymphadenopathy and cytopenias improved, but splenomegaly was persistent. On physical examination, he had cervical lymphadenopathy (less than 2 cm in diameter) and palpable spleen 6 cm below the left costal margin. Laboratory investigations revealed a hemoglobin level of 11.9 g/dL, platelet count of 118,000/mm3 , and WBC of 3800/mm3 (1200/mm3 neutrophils, 2500/mm3 lymphocytes). Liver and renal function tests were normal. A direct Coombs test and ANA test were both negative. At the present time, the patient is clinically stable, without cytopenias despite persistent splenomegaly. He started rapamycin treatment and responded well, the spleen returning to a normal size after 3 weeks of treatment with no reported side effects.
Please cite this article in press as: Martínez-Feito, A., et al., Autoimmune lymphoproliferative syndrome due to somatic FAS mutation (ALPS-sFAS) combined with a germline caspase-10 (CASP10) variation. Immunobiology (2015), http://dx.doi.org/10.1016/j.imbio.2015.08.004
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Demographic data Age Age at diagnosis Clinical data Splenomegaly Lymphadenopathy Autoimmunity Laboratory results Lymphocyte count (cells/l) T-cells CD3(%) CD4(%) CD8(%) DN (%) B-cells CD19(%) CD27 + (%) IgD-CD27+ (switching) (%) CD21Low (%) Serum immunoglobulins Ig G (mg/dl) Ig A (mg/dl) Ig M (mg/dl) Plasma biomarkers IL-10 (pg/ml) sFasL (pg/ml) sCD25 (U/ml) Vitamin B12 (pg/ml) Molecular genetics Somatic FAS mutation
P1
P2
P3
56 53
13 13
47 45
+ ++ +/−
++ +/− T, N
+++ ++ Aa , Tb , Nc
806
2410
5282
1200–3000
73 40 24 5.5
87 42 34 6.9
73 22 44 6.1
62–81 32–59 15–36 0–2.5
12 5 2.2 7.3
10 6.2 2.8 6.6
19 0.8 0.4 28a
8–20 8–45 5–25 1.5–9
1140 454 94
2480 654 58
1410 595 35
700–1600 70–400 40–230
128 1406 1462 >2000
208 1656 1623 >2000
66 682 1300 >2000
0–20 0–250 0–215 200–753
c.779A>G p.D260G WT
c.749G>A p.R250Q c.1337A>G p.Y446C
c.686T>A p.L229X Germline CASP10 substitution WTd a
b c d
Normal range
Anemia. Thrombopenia. Neutropenia. Wild type.
Patient 3 (P3) is a 47-year-old man. Since the age of 17, he had had slight enlargement of the spleen and of bilateral inguinal and axillary lymph nodes. At 21 years of age, he experienced acute recurrent episodes of autoimmune thrombocytopenia (2–3 every year) with remission under steroid therapy. He had neutropenia (<500/mm3 ) at the ages of 27, 37 and 41 years concomitantly with the idiopathic thrombocytopenic purpura flares. He had had one (transient) hemolytic crisis. At 35, he underwent splenectomy. Nonetheless, idiopathic thrombocytopenic purpura recurred, requiring multiple treatments (steroids, cyclosporine, methotrexate). Now he is alive and well off therapy. 3.2. Immunological characteristics, Fas function and molecular analysis The most prominent immunologic abnormalities in these ALPSsFAS patients included increased percentages of DN T-cells, high plasma levels of IL-10, sFasL, vitamin B12, and sCD25, and serum IgA hypergammaglobulinemia (Table 1). The patients fulfilled clinical and phenotypic criteria for ALPS, but the demonstration of an apoptosis defect is critical for the diagnosis of the syndrome. T-cell blasts from P1 and P2 showed conserved Fas-induced apoptosis using an agonistic anti-Fas mAb (Apo1.3); however, Fas-induced cell death was decreased in T-cell blasts from P3 (Fig. 1A). ALPSsFAS patients have the same clinical phenotype and biomarker profile as ALPS-FAS patients, but they do not display defects in Fasinduced apoptosis in T-cell blasts. Germline mutations in the FAS gene were ruled out in all three patients, and then somatic FAS mutations were suspected.
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Somatic FAS mutations are mainly restricted to DN T-cells in ALPS-sFAS patients. All three patients (P1, P2 and P3) showed impaired Fas-induced apoptosis in DN T-cells using a recently reported modified FasT Kill assay for patients with suspicion of ALPS-sFAS (Fig. 1B) (Lo et al., 2013). We confirmed heterozygous somatic FAS mutations in isolated DN T-cells of these patients (Table 1). P1 and P2 presented new somatic mutations in the FAS gene (c.686T>A, p.L229X, Genbank KJ958564 and c.779A>G, p.D260G, KM114217, respectively), while the somatic FAS mutation found in P3 (c.749G>A, p.R250Q) was described previously (Neven et al., 2011). The three mutations found are located in exon 9 (intracellular domain) of the FAS gene and none of them were found as single-nucleotide polymorphisms. The mutation in P1 was outside the death domain of FAS and led to a premature stop codon. Missense mutations in P2 and P3 were located in the death domain of FAS, presumably exerting a dominant negative effect as the majority of mutations affecting the intracytoplasmic tail of Fas result in defective trimer formation (Siegel et al., 2000). Interestingly, impaired Fas function was observed in PHAactivated T-cell blasts from P3 (Fig. 1A), a result that was not consistent with the somatic FAS mutation identified. Then, DNA sequencing of the caspase-10 (CASP10) gene showed a heterozygous germline change 1337A>G in exon 9 causing the Y446C substitution in the predicted protease domain of the caspase-10 protein. This variant was previously associated with ALPS and it has also been reported with an allele frequency ranging from 1.6 to 2% in a healthy Caucasian population and < 0.5% in African Americans (Campagnoli et al., 2006; Cerutti et al., 2007; Zhu et al., 2006). Pedigree analysis in the family of P3 confirmed that the Y446C variant was inherited from his healthy father. Other authors showed that Y446C had decreasing caspase-10 activity without exerting a dominant negative effect on the WT protein (Cerutti et al., 2007). Since PRF1 and UNC13D (involved in perforin secretion) genes have been suggested to play a role in some ALPS patients, we ruled out mutations in P3 in both genes (Aricò et al., 2013; 2006). 3.3. T- and B-cell phenotypes To explore the effects of the ALPS genotypes on T- and B-cells, we analyzed peripheral blood lymphocytes from three ALPSsFAS patients. P3 showed lymphocytosis and an inversion in the CD4+/CD8+ ratio (0.5) (Table 1). To compare the cell phenotype of CD4 and CD8 T-cells in the patients, we stained CCR7 and CD45RA to distinguish between naïve (CCR7 + CD45RA+), central memory (CM) (CCR7 + CD45RA-), effector memory (EM) (CCR7-CD45RA-) and TEMRA (CCR7-CD45RA+) subsets, similar to a widely used classification for CD4 and CD8 T-cells (Rensing-Ehl et al., 2014). We observed significantly higher counts of TEMRA CD4 and EM CD8 T-cells in P3 than healthy controls (Figs. 2 and 3). In contrast, P1 and P2 did not present alterations in CD4+ and CD8 + T-cell subset frequencies. Moreover, CD4 and CD8 + T-cell subsets were further assessed for the expression of several molecules that change during differentiation from naïve to effector cells (Ruiz-García et al., 2014). The CD4 + T-cells from P3 showed dysregulated expression of CD27, CD95 and CD57 molecules. Interestingly, CD27 expression in CD4 T-cells was significantly lower in P3 than in the controls (mean ± SD, 69.9 ± 5.2 vs 91.9 ± 4.5 in controls, P < 0.0001). The differences in CD27 expression affected EM CD4 T-cells (mean ± SD, 26.9 ± 4.2 vs 66.9 ± 13.1 in controls, P < 0.0005). P3 showed higher levels of CD95 only when we compared naïve CD4 + CD95+ Tcells (mean ± SD, 33.8 ± 3.6 vs 7.2 ± 4.7 in controls, P < 0.0001). CD57 expression in CD4 T-cells was significantly higher in P3 than in the controls (mean ± SD, 41.0 ± 4.2 vs 9.1 ± 11.9 in controls, P < 0.0018). The differences in CD57 expression affected naïve (mean ± SD, 33.3 ± 3.3 vs 6.2 ± 14.8 in controls, P < 0.022), CM
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Fig. 1. Induction of Fas-induced apoptosis by different doses of agonistic anti-Fas mAb. A, Fas function in PHA-activated T-cell blasts. B, Fas function in DN T-cells. Columns show the percentage of apoptotic cells (Annexin V+) (mean ± SD of two independent experiments).
(mean ± SD, 21.6 ± 2.9 vs 2.9 ± 2.1 in controls, P < 0.0001) and EM (mean ± SD, 58.8 ± 4.1 vs 15.1± 17.2 in controls, P < 0.0028) CD4 T-cells (Fig. 2). In P3, CD8 + T-cells showed dysregulated expression of perforin, CD57 and CD27 molecules. Perforin expression in CD8 T-cells was significantly higher in P3 than in the controls (mean ± SD, 49.5 ± 3.0 vs 29.0 ± 12.7 in controls, P < 0.0381), CD57 expression was significantly higher in P3 (mean ± SD, 64.1 ± 5.0 vs 26.1 ± 11.2 in controls, P < 0.0008) and CD27 expression was significantly reduced in P3 (mean ± SD, 42.1 ± 4.8 vs 73.9 ± 13.5 in controls, P < 0.0043) (Fig. 3). Furthermore, P3 showed a restricted CD8 and DN T-cell repertoire with increased expression of families TCRV5.3 and 16, respectively (Fig. 4). Regarding B-cells, P3 had reduced proportions of memory B-cells (CD19 + CD27+) (mean ± SD, 1.06 ± 0.25 vs 18.24 ± 8.92 in controls, P < 0.005), memory unswitched B-cells (CD19 + IgD + CD27+) (mean ± SD, 0.56 ± 0.2 vs 7.26 ± 3.8 in controls, P < 0.0088), memory switched B-cells (CD19 + IgD-CD27+) (mean ± SD, 0.53 ± 0.15 vs 10.9 ± 5.4 in controls, P < 0.0049) and increased proportions of CD21low B-cells (mean ± SD, 20.0 ± 11.3 vs 6.28 ± 2.6 in controls, P < 0.0003). Serum IgM levels were also reduced (mean ± SD, 32 ± 3.5; normal range 40–230 mg/dl) (Table 1).
4. Discussion We have described the clinical, immunologic and molecular findings of three ALPS-sFAS patients carrying a somatic FAS mutation that affects the intracellular domain of the FAS gene. Notably, P3 has an additional alteration in the CASP10 gene that affects caspase-10 function (Zhu et al., 2006). Variations in the CASP10 gene should be taken into account, since ALPS does not behave as a classic monogenic disease and could result from the concurrence of more than one genetic alteration. The Y446C variation in the CASP10 gene has previously been reported as a single substitution in two patients (Dianzani Autoimmune Lymphoproliferative Disease and atypical ALPS patients) (Campagnoli et al., 2006; Zhu et al., 2006) and in combination with another germline mutation in the FAS gene in two ALPS-FAS patients (Campagnoli et al. 2006; Cerutti et al. 2007). The somatic mutation of FAS and the germline CASP10 substitution in P3 might cooperate in the alteration of homeostasis; the latter can be a subtle alteration, insufficient to cause ALPS alone, but in combination with another mutation may act as a disease modifier for ALPS, resulting in a more severe ALPS phenotype. The
Please cite this article in press as: Martínez-Feito, A., et al., Autoimmune lymphoproliferative syndrome due to somatic FAS mutation (ALPS-sFAS) combined with a germline caspase-10 (CASP10) variation. Immunobiology (2015), http://dx.doi.org/10.1016/j.imbio.2015.08.004
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Fig. 2. CD4 T-cell phenotype. A, Representative dot plots showing CD45RA and CCR7 expression in P3 vs control CD4 T-cell subsets (naïve, central memory [CM], effector memory [EM] and TEMRA). B, Representative dot plots of CD57 expression on CD4 T-cell subsets. Columns show the distribution of CD4 T-cell subsets in ALPS-sFAS patients vs controls (n = 19) (mean ± SD of three and two independent experiments, respectively).
Please cite this article in press as: Martínez-Feito, A., et al., Autoimmune lymphoproliferative syndrome due to somatic FAS mutation (ALPS-sFAS) combined with a germline caspase-10 (CASP10) variation. Immunobiology (2015), http://dx.doi.org/10.1016/j.imbio.2015.08.004
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Fig. 3. CD8 T-cell phenotype. A, Representative dot plots showing CD45RA and CCR7 expression of P3 vs control CD8 T-cell subsets (naïve, central memory [CM], effector memory [EM] and TEMRA). B, Representative dot plots of perforin, CD57 and CD27 expression on CD8 T-cells. Columns show the distribution of CD8 T-cell subsets in ALPS-sFAS patients vs controls (n = 19) (mean ± SD of three and two independent experiments, respectively).
Fig. 4. TCRV repertoire was determined by flow cytometry in gated CD4+, CD8+ and DN T-cells from PBMCs of P3 (Beckman Coulter, Madrid, Spain).
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decreased Fas induced apoptosis in T-cell blasts from P3 could be due to an additional defect in CASP10 (Y446C) inherited from his healthy father, but we cannot rule out defects in other genes involved in the Fas pathway. By contrast, Fas-induced cell death was conserved in T-cell blasts from the parents (data not shown). Indeed, all biomarkers of ALPS analyzed in P3’s parents were in the normal range, the only relevant finding being high levels of vitamin B12 in P3’s father. Some reports have been published showing a molecular explanation for the variable penetrance seen in ALPS. The two-hit hypothesis argues that, as well as a heterozygous germline FAS mutation, a somatic FAS second hit affecting the wild type allele is necessary to produce the disease (Magerus-Chatinet et al., 2011; Rensing-Ehl et al., 2014). Additionally, modifying alterations have been described in genes other than FAS (PRF1, UNC13D, XIAP, CASP10, and OPN) (Aricò et al., 2013; Boggio et al., 2013; Campagnoli et al., 2006; Cerutti et al., 2007; Chiocchetti et al., 2004; Clementi et al., 2004, 2006; Zhu et al., 2006). P3 presented a more dysregulated T- and B-cell phenotype than either P1 or P2 and this could be related to an exhausted phenotype. He showed a predominantly CD4 and CD8 T-cell effector phenotype, these cells express a combination of markers (CD57, CD95 and perforin) usually observed on effector and TEMRA T-cells, a similar abnormal differentiation pattern was also seen in DN T-cells (Rensing-Ehl et al., 2014). It is necessary to clarify the contribution of the different T-cell subsets, DN-T cells and B-cells to the immunological derangements in ALPS. Clinically, P3 was splenectomized due to refractory cytopenias, but he has not suffered invasive bacterial infections by encapsulated bacteria, despite the elevated risk of severe postsplenectomy invasive bacterial infections (Neven et al., 2014). In summary, ALPS-sFAS and ALPS-FAS patients are clinically indistinguishable, but those with ALPS-sFAS have a later age of disease onset, and lower rates of splenectomy, and further there is no evidence of a neoplastic lymphoproliferative disorder in ALPSsFAS, unlike in ALPS-FAS (Dowdell et al., 2010). Here, we report the first ALPS patient with a combination of a somatic FAS mutation and a germline CASP10 substitution. The combined effect of these two molecular alterations may have contributed to the development of severe ALPS with more autoimmune complications, a dysregulated phenotype and no history of bacterial infections despite splenectomy. Further work in this area should explore the ability to switch off the immune response and the link to lymphocyte lifespan regulation and maintenance of peripheral tolerance (Nagata 1997). Conflict of interest The authors declare that they have no conflicts of interest. Acknowledgments AM performed the laboratory work for this study and drafted the manuscript; JM, CRV, RE, LIGG, ROB, MR recruited the patients; SMD, DPM, ESZ, RRG, MM, JDM collaborated in the laboratory work for this study; EPA reviewed the manuscript; and LMA designed the research and drafted the manuscript. The authors would like to thank the patients and their families for their participation. This study was supported by a grant from the Spanish Health Research Fund (PI11/1591) to LMA. The project has been co-financed with FEDER funds. References Aricò, M., Boggio, E., Cetica, V., Melensi, M., Orilieri, E., Clemente, N., Cappellano, G., Buttini, S., Soluri, M.F., Comi, C., Dufour, C., Pende, D., Dianzani, I., Ellis, S.R., Pagliano, S., Marcenaro, S., Ramenghi, U., Chiocchetti, A., Dianzani, U., 2013. Variations of the UNC13D gene in patients with autoimmune lymphoproliferative syndrome. PLoS One 8 (July (7)), e68045.
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Autoimmune lymphoproliferative syndrome due to somatic FAS mutation (ALPS-sFAS) combined with a germline caspase-10 (CASP10) variation Ana Martínez-Feito a , Josefa Melero b , Sergio Mora-Díaz a , Carmen Rodríguez-Vigil c , Ramón Elduayen d , Luis I. González-Granado e,f , Dolores Pérez-Méndez a , Elena Sánchez-Zapardiel a , Raquel Ruiz-García a , Miguela Menchén a , a ˜ ˜ h, Josefa Díaz-Madronero , Estela Paz-Artal a,f , Rafael del Orbe-Barreto g , Marta Rinón a,f,∗ Luis M. Allende a
Servicio de Inmunología, Hospital Universitario 12 de Octubre, Madrid, Spain Servicio de Inmunología, Hospital Universitario Infanta Cristina, Badajoz, Spain c Servicio de Hemato-Oncología Pediátrica, Hospital Universitario Miguel Servet, Zaragoza, Spain d Servicio de Hematología, Hospital Universitario Infanta Cristina, Badajoz, Spain e Unidad de Inmunodeficiencias e Infecciosas pediátricas, Unidad de Hemato-Oncología Pediátrica, Hospital Universitario 12 Octubre, Madrid, Spain f Instituto de Investigación i+12, Madrid, Spain g Servicio de Hematología, Hospital Universitario de Cruces, Bilbao, Spain h Servicio de Inmunología, Hospital Universitario de Cruces, Bilbao, Spain b
a r t i c l e
i n f o
Article history: Received 9 June 2015 Received in revised form 31 July 2015 Accepted 11 August 2015 Available online xxx Keywords: ALPS Apoptosis CASP10 FAS Immunodeficiency
a b s t r a c t Autoimmune lymphoproliferative syndrome (ALPS) is a primary immunodeficiency caused by impaired Fas/FasL-mediated apoptosis of lymphocytes and is characterized by chronic nonmalignant or benign lymphoproliferation, autoimmune manifestations and expansion of double negative (DN) T-cells (TCR˛ + CD4-CD8-). Most cases of ALPS are associated with germline (ALPS-FAS) or somatic (ALPS-sFAS) heterozygous FAS mutations or a combination of both. Here we report three unrelated patients with ALPS-sFAS. Only one of them showed impaired Fas function in PHA-activated T-cells. In this patient, the genetic analysis of the caspase-10 gene (CASP10) identified a heterozygous germline change in exon 9 (c.1337A>G) causing Y446C substitution in the caspase-10 protein. In addition, this patient had a dysregulated T- and B-cell phenotype; circulating lymphocytes showed expansion of T effector memory CD45RA+ (TEMRA) CD4 T-cells, effector memory CD8 T-cells, CD21low B-cells and reduced memory switched B-cells. Additionally, this patient showed altered expression in T-cells of several molecules that change during differentiation from naïve to effector cells (CD27, CD95, CD57 and perforin). Molecular alterations in genes of the Fas pathway are necessary for the development of ALPS and this syndrome could be influenced by the concurrent effect of other mutations hitting different genes involved in Fas or related pathways. © 2015 Published by Elsevier GmbH.
1. Introduction Autoimmune lymphoproliferative syndrome (ALPS) is a rare genetic disorder of lymphocyte homeostasis characterized by
∗ Corresponding author at. Servicio de Inmunología, Hospital Universitario 12 de Octubre, Avda. Córdoba s/n, Madrid-28041, Spain. E-mail address: [email protected] (L.M. Allende).
chronic benign lymphoproliferation, autoimmune manifestations and an increased risk of lymphoma (Fisher et al., 1995; Neven et al., 2011; Price et al., 2014; Rieux-Laucat et al., 1995). Proportions of CD3 + TcR˛+ CD4-CD8- double negative (DN) T-cells and some plasma biomarkers including interleukin-10 (IL-10), soluble FasL (sFasL), soluble CD25 (sCD25) and vitamin B12 are elevated in ALPS patients. Like other primary immunodeficiencies (PIDs), ALPS can be classified according to the underlying genetic defect. Most ALPS patients carry heterozygous germline (ALPS-FAS) or somatic FAS
http://dx.doi.org/10.1016/j.imbio.2015.08.004 0171-2985/© 2015 Published by Elsevier GmbH.
Please cite this article in press as: Martínez-Feito, A., et al., Autoimmune lymphoproliferative syndrome due to somatic FAS mutation (ALPS-sFAS) combined with a germline caspase-10 (CASP10) variation. Immunobiology (2015), http://dx.doi.org/10.1016/j.imbio.2015.08.004
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mutations (ALPS-sFAS) or both (Holzelova et al., 2004; Rieux-Laucat et al., 1995). In addition, germline mutations in FASL and CASP10 genes have been identified in a small number of ALPS patients (DelRey et al., 2006; Wang et al., 1999). ALPS patients with no known genetic defects are classified as ALPS-unknown (ALPS-U) (Oliveira et al., 2010). The partial penetrance observed in some ALPS patients suggests that, in addition to the germline FAS mutation, a second hit is required for the onset of the disease. Both alterations may cooperate in disrupting the switching off of the immune response affecting immune surveillance (Magerus-Chatinet et al., 2011). This work describes three unrelated ALPS-sFAS patients (P1, P2 and P3) born to non-consanguineous parents who had chronic nonmalignant noninfectious lymphadenopathy, splenomegaly, and autoimmune cytopenias and were positive for ALPS biomarkers. In addition, P3 is double heterozygous for a somatic FAS mutation and a germline CASP10 variation. We assessed the effects of ALPS genotypes on T- and B-cell phenotype. 2. Materials and methods 2.1. Immunophenotype analysis For direct immunofluorescence, peripheral whole blood was incubated using the corresponding monoclonal antibodies: anti-CCR7-FITC, anti-TCR␣-FITC, anti-TCRd-PE, anti-IgD-PE, anti-CD21-PE, anti-CD57-PE, anti-CD95-PE, anti-Perforin-PE, antiCD3-PerCP5.5, anti-CD45RA-PECy7 (all from BD Biosciences, Madrid, Spain); anti-CD27-PE, anti-CD8-PECy7, anti-CD19-PECy7, anti-CD8-APC, anti-CD4-APC, anti-CD4-APC-Alexa Fluor 750, and anti-CD45-APC-Alexa Fluor 750 (all from Beckman Coulter, Madrid, Spain). TcR V repertoire (Beckman Coulter) was determined in gated CD4, CD8 and DN T-cells. Proportions of T- and B-cells were analyzed by flow cytometry using a Navios Cytometer (Beckman Coulter). 2.2. ALPS biomarkers Plasma levels of IL-10 (Bender MedSystems, Labclinics, Madrid, Spain), sCD25, sFasL (R&D, Vitro, Madrid, Spain) and vitamin B12 (Beckman Coulter) were measured in duplicate by enzyme-linked immunosorbent assay. Serum immunoglobulin concentrations were determined by nephelometry (Beckman Coulter). 2.3. Isolation of DN T-cells DN T-cells were isolated from peripheral-blood mononuclear cells (PBMCs) by a magnetic Double negative T Cell Isolation Kit (Miltenyi Biotec, Madrid, Spain) (purities of DN T-cells exceeding 90%). 2.4. Cells and cell culture Human PBMCs were obtained from the blood of patients, relatives and healthy donors by Ficoll separation. T-cell blasts were generated from these cells by PHA stimulation (20 ng/L) (Sigma–Aldrich, Madrid, Spain) in the presence of IL-2 (100 IU/L) (Roche, Madrid, Spain), as described elsewhere (Del-Rey et al., 2006). 2.5. Fas function in T-cell blasts and in DN T-cells T-cell blasts (1 × 105 cells) from the patients and healthy controls were aliquoted in triplicate into 96-well plates and cultured with different concentrations of anti-Fas mAb (125, 250, or 500 ng/L) (Clon Apo1.3; Enzo Life Sciences, Madrid, Spain). After overnight incubation, apoptotic cells were detected by flow
cytometry with the use of annexin V-FITC (Immunostep, Salamanca, Spain). For Fas-induced apoptosis in DN T-cells, PBMCs (2 × 105 ) were aliquoted in triplicate into 96-well plates and cultures with different concentrations of anti-Fas mAb (250, 500 or 1000 ng/mL) (Clon Apo1.3) for 4 h in media without IL-2. After incubation, cells were washed and stained with annexin V-FITC or anti-CD95 FITC (BD Biosciences) and antibodies to anti-CD3APC-Cy7 (BD Biosciences), anti-CD4-APC, anti-CD8-APC (Beckman Coulter), anti-TcR˛-PECy7 (Immunostep), anti-B220-PE (Miltenyi Biotec) to identify apoptotic DN T-cells, as described elsewhere (Lo et al., 2013) 2.6. Molecular genetics DNA was extracted from whole blood or DN T-cells using a MagNa Pure Compact Nucleic Acid Isolation Kit (Roche, Madrid, Spain) and QIAamp DNA Blood Mini Kit (Qiagen, Madrid, Spain), respectively. Mutation analysis of the FAS gene was assessed as described elsewhere (Del-Rey et al., 2006). The CASP10 gene was amplified and sequenced according to standard methods (primers available upon request). 2.7. Statistical analysis Statistical comparisons were performed with unpaired Student t tests, with significance defined as P < 0.05(*), P < 0.01(**), or P < 0.001(***). 3. Results 3.1. Case report Patient 1 (P1) is a 56-year-old woman whose first manifestation was painless generalized lymphadenopathy (axillary, supraclavicular, cervical and retroauricular) and splenomegaly with no other clinical signs or symptoms. A primary hematologic disorder or bone marrow infiltration by malignant tumor cells was ruled out. Peripheral blood showed mild cytopenias (leucopenia 3400/mm3 and thrombocytopenia 125,000/mm3 ), and IgA hypergammaglobulinemia, but normal levels of IgG and IgM, and was negative for antinuclear antibodies (ANA). Extensive lymph node disease with multiple supradiaphragmatic and infradiaphragmatic enlarged nodes and splenomegaly were observed on PET scanning. No significant clinical or laboratory variations have been observed in 3 years of follow-up without treatment. Patient 2 (P2), a 13-year-old boy, was referred for evaluation of chronic splenomegaly. At age 10 years, seroreactivity to CMV was demonstrated, with splenomegaly that lasted for several months. Eight months before the consultation, he was hospitalized for a 2-week history of lymphadenopathy, splenomegaly, fever, thrombocytopenia (96,000/mm3 ) and neutropenia (700/mm3 ). Results of microbiological tests (blood cultures, CMV, EBV, HIV, Parvovirus B19, HSV, Leishmania) were negative. Bone marrow aspirate was normal. Fever, lymphadenopathy and cytopenias improved, but splenomegaly was persistent. On physical examination, he had cervical lymphadenopathy (less than 2 cm in diameter) and palpable spleen 6 cm below the left costal margin. Laboratory investigations revealed a hemoglobin level of 11.9 g/dL, platelet count of 118,000/mm3 , and WBC of 3800/mm3 (1200/mm3 neutrophils, 2500/mm3 lymphocytes). Liver and renal function tests were normal. A direct Coombs test and ANA test were both negative. At the present time, the patient is clinically stable, without cytopenias despite persistent splenomegaly. He started rapamycin treatment and responded well, the spleen returning to a normal size after 3 weeks of treatment with no reported side effects.
Please cite this article in press as: Martínez-Feito, A., et al., Autoimmune lymphoproliferative syndrome due to somatic FAS mutation (ALPS-sFAS) combined with a germline caspase-10 (CASP10) variation. Immunobiology (2015), http://dx.doi.org/10.1016/j.imbio.2015.08.004
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Demographic data Age Age at diagnosis Clinical data Splenomegaly Lymphadenopathy Autoimmunity Laboratory results Lymphocyte count (cells/l) T-cells CD3(%) CD4(%) CD8(%) DN (%) B-cells CD19(%) CD27 + (%) IgD-CD27+ (switching) (%) CD21Low (%) Serum immunoglobulins Ig G (mg/dl) Ig A (mg/dl) Ig M (mg/dl) Plasma biomarkers IL-10 (pg/ml) sFasL (pg/ml) sCD25 (U/ml) Vitamin B12 (pg/ml) Molecular genetics Somatic FAS mutation
P1
P2
P3
56 53
13 13
47 45
+ ++ +/−
++ +/− T, N
+++ ++ Aa , Tb , Nc
806
2410
5282
1200–3000
73 40 24 5.5
87 42 34 6.9
73 22 44 6.1
62–81 32–59 15–36 0–2.5
12 5 2.2 7.3
10 6.2 2.8 6.6
19 0.8 0.4 28a
8–20 8–45 5–25 1.5–9
1140 454 94
2480 654 58
1410 595 35
700–1600 70–400 40–230
128 1406 1462 >2000
208 1656 1623 >2000
66 682 1300 >2000
0–20 0–250 0–215 200–753
c.779A>G p.D260G WT
c.749G>A p.R250Q c.1337A>G p.Y446C
c.686T>A p.L229X Germline CASP10 substitution WTd a
b c d
Normal range
Anemia. Thrombopenia. Neutropenia. Wild type.
Patient 3 (P3) is a 47-year-old man. Since the age of 17, he had had slight enlargement of the spleen and of bilateral inguinal and axillary lymph nodes. At 21 years of age, he experienced acute recurrent episodes of autoimmune thrombocytopenia (2–3 every year) with remission under steroid therapy. He had neutropenia (<500/mm3 ) at the ages of 27, 37 and 41 years concomitantly with the idiopathic thrombocytopenic purpura flares. He had had one (transient) hemolytic crisis. At 35, he underwent splenectomy. Nonetheless, idiopathic thrombocytopenic purpura recurred, requiring multiple treatments (steroids, cyclosporine, methotrexate). Now he is alive and well off therapy. 3.2. Immunological characteristics, Fas function and molecular analysis The most prominent immunologic abnormalities in these ALPSsFAS patients included increased percentages of DN T-cells, high plasma levels of IL-10, sFasL, vitamin B12, and sCD25, and serum IgA hypergammaglobulinemia (Table 1). The patients fulfilled clinical and phenotypic criteria for ALPS, but the demonstration of an apoptosis defect is critical for the diagnosis of the syndrome. T-cell blasts from P1 and P2 showed conserved Fas-induced apoptosis using an agonistic anti-Fas mAb (Apo1.3); however, Fas-induced cell death was decreased in T-cell blasts from P3 (Fig. 1A). ALPSsFAS patients have the same clinical phenotype and biomarker profile as ALPS-FAS patients, but they do not display defects in Fasinduced apoptosis in T-cell blasts. Germline mutations in the FAS gene were ruled out in all three patients, and then somatic FAS mutations were suspected.
3
Somatic FAS mutations are mainly restricted to DN T-cells in ALPS-sFAS patients. All three patients (P1, P2 and P3) showed impaired Fas-induced apoptosis in DN T-cells using a recently reported modified FasT Kill assay for patients with suspicion of ALPS-sFAS (Fig. 1B) (Lo et al., 2013). We confirmed heterozygous somatic FAS mutations in isolated DN T-cells of these patients (Table 1). P1 and P2 presented new somatic mutations in the FAS gene (c.686T>A, p.L229X, Genbank KJ958564 and c.779A>G, p.D260G, KM114217, respectively), while the somatic FAS mutation found in P3 (c.749G>A, p.R250Q) was described previously (Neven et al., 2011). The three mutations found are located in exon 9 (intracellular domain) of the FAS gene and none of them were found as single-nucleotide polymorphisms. The mutation in P1 was outside the death domain of FAS and led to a premature stop codon. Missense mutations in P2 and P3 were located in the death domain of FAS, presumably exerting a dominant negative effect as the majority of mutations affecting the intracytoplasmic tail of Fas result in defective trimer formation (Siegel et al., 2000). Interestingly, impaired Fas function was observed in PHAactivated T-cell blasts from P3 (Fig. 1A), a result that was not consistent with the somatic FAS mutation identified. Then, DNA sequencing of the caspase-10 (CASP10) gene showed a heterozygous germline change 1337A>G in exon 9 causing the Y446C substitution in the predicted protease domain of the caspase-10 protein. This variant was previously associated with ALPS and it has also been reported with an allele frequency ranging from 1.6 to 2% in a healthy Caucasian population and < 0.5% in African Americans (Campagnoli et al., 2006; Cerutti et al., 2007; Zhu et al., 2006). Pedigree analysis in the family of P3 confirmed that the Y446C variant was inherited from his healthy father. Other authors showed that Y446C had decreasing caspase-10 activity without exerting a dominant negative effect on the WT protein (Cerutti et al., 2007). Since PRF1 and UNC13D (involved in perforin secretion) genes have been suggested to play a role in some ALPS patients, we ruled out mutations in P3 in both genes (Aricò et al., 2013; 2006). 3.3. T- and B-cell phenotypes To explore the effects of the ALPS genotypes on T- and B-cells, we analyzed peripheral blood lymphocytes from three ALPSsFAS patients. P3 showed lymphocytosis and an inversion in the CD4+/CD8+ ratio (0.5) (Table 1). To compare the cell phenotype of CD4 and CD8 T-cells in the patients, we stained CCR7 and CD45RA to distinguish between naïve (CCR7 + CD45RA+), central memory (CM) (CCR7 + CD45RA-), effector memory (EM) (CCR7-CD45RA-) and TEMRA (CCR7-CD45RA+) subsets, similar to a widely used classification for CD4 and CD8 T-cells (Rensing-Ehl et al., 2014). We observed significantly higher counts of TEMRA CD4 and EM CD8 T-cells in P3 than healthy controls (Figs. 2 and 3). In contrast, P1 and P2 did not present alterations in CD4+ and CD8 + T-cell subset frequencies. Moreover, CD4 and CD8 + T-cell subsets were further assessed for the expression of several molecules that change during differentiation from naïve to effector cells (Ruiz-García et al., 2014). The CD4 + T-cells from P3 showed dysregulated expression of CD27, CD95 and CD57 molecules. Interestingly, CD27 expression in CD4 T-cells was significantly lower in P3 than in the controls (mean ± SD, 69.9 ± 5.2 vs 91.9 ± 4.5 in controls, P < 0.0001). The differences in CD27 expression affected EM CD4 T-cells (mean ± SD, 26.9 ± 4.2 vs 66.9 ± 13.1 in controls, P < 0.0005). P3 showed higher levels of CD95 only when we compared naïve CD4 + CD95+ Tcells (mean ± SD, 33.8 ± 3.6 vs 7.2 ± 4.7 in controls, P < 0.0001). CD57 expression in CD4 T-cells was significantly higher in P3 than in the controls (mean ± SD, 41.0 ± 4.2 vs 9.1 ± 11.9 in controls, P < 0.0018). The differences in CD57 expression affected naïve (mean ± SD, 33.3 ± 3.3 vs 6.2 ± 14.8 in controls, P < 0.022), CM
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Fig. 1. Induction of Fas-induced apoptosis by different doses of agonistic anti-Fas mAb. A, Fas function in PHA-activated T-cell blasts. B, Fas function in DN T-cells. Columns show the percentage of apoptotic cells (Annexin V+) (mean ± SD of two independent experiments).
(mean ± SD, 21.6 ± 2.9 vs 2.9 ± 2.1 in controls, P < 0.0001) and EM (mean ± SD, 58.8 ± 4.1 vs 15.1± 17.2 in controls, P < 0.0028) CD4 T-cells (Fig. 2). In P3, CD8 + T-cells showed dysregulated expression of perforin, CD57 and CD27 molecules. Perforin expression in CD8 T-cells was significantly higher in P3 than in the controls (mean ± SD, 49.5 ± 3.0 vs 29.0 ± 12.7 in controls, P < 0.0381), CD57 expression was significantly higher in P3 (mean ± SD, 64.1 ± 5.0 vs 26.1 ± 11.2 in controls, P < 0.0008) and CD27 expression was significantly reduced in P3 (mean ± SD, 42.1 ± 4.8 vs 73.9 ± 13.5 in controls, P < 0.0043) (Fig. 3). Furthermore, P3 showed a restricted CD8 and DN T-cell repertoire with increased expression of families TCRV5.3 and 16, respectively (Fig. 4). Regarding B-cells, P3 had reduced proportions of memory B-cells (CD19 + CD27+) (mean ± SD, 1.06 ± 0.25 vs 18.24 ± 8.92 in controls, P < 0.005), memory unswitched B-cells (CD19 + IgD + CD27+) (mean ± SD, 0.56 ± 0.2 vs 7.26 ± 3.8 in controls, P < 0.0088), memory switched B-cells (CD19 + IgD-CD27+) (mean ± SD, 0.53 ± 0.15 vs 10.9 ± 5.4 in controls, P < 0.0049) and increased proportions of CD21low B-cells (mean ± SD, 20.0 ± 11.3 vs 6.28 ± 2.6 in controls, P < 0.0003). Serum IgM levels were also reduced (mean ± SD, 32 ± 3.5; normal range 40–230 mg/dl) (Table 1).
4. Discussion We have described the clinical, immunologic and molecular findings of three ALPS-sFAS patients carrying a somatic FAS mutation that affects the intracellular domain of the FAS gene. Notably, P3 has an additional alteration in the CASP10 gene that affects caspase-10 function (Zhu et al., 2006). Variations in the CASP10 gene should be taken into account, since ALPS does not behave as a classic monogenic disease and could result from the concurrence of more than one genetic alteration. The Y446C variation in the CASP10 gene has previously been reported as a single substitution in two patients (Dianzani Autoimmune Lymphoproliferative Disease and atypical ALPS patients) (Campagnoli et al., 2006; Zhu et al., 2006) and in combination with another germline mutation in the FAS gene in two ALPS-FAS patients (Campagnoli et al. 2006; Cerutti et al. 2007). The somatic mutation of FAS and the germline CASP10 substitution in P3 might cooperate in the alteration of homeostasis; the latter can be a subtle alteration, insufficient to cause ALPS alone, but in combination with another mutation may act as a disease modifier for ALPS, resulting in a more severe ALPS phenotype. The
Please cite this article in press as: Martínez-Feito, A., et al., Autoimmune lymphoproliferative syndrome due to somatic FAS mutation (ALPS-sFAS) combined with a germline caspase-10 (CASP10) variation. Immunobiology (2015), http://dx.doi.org/10.1016/j.imbio.2015.08.004
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Fig. 2. CD4 T-cell phenotype. A, Representative dot plots showing CD45RA and CCR7 expression in P3 vs control CD4 T-cell subsets (naïve, central memory [CM], effector memory [EM] and TEMRA). B, Representative dot plots of CD57 expression on CD4 T-cell subsets. Columns show the distribution of CD4 T-cell subsets in ALPS-sFAS patients vs controls (n = 19) (mean ± SD of three and two independent experiments, respectively).
Please cite this article in press as: Martínez-Feito, A., et al., Autoimmune lymphoproliferative syndrome due to somatic FAS mutation (ALPS-sFAS) combined with a germline caspase-10 (CASP10) variation. Immunobiology (2015), http://dx.doi.org/10.1016/j.imbio.2015.08.004
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Fig. 3. CD8 T-cell phenotype. A, Representative dot plots showing CD45RA and CCR7 expression of P3 vs control CD8 T-cell subsets (naïve, central memory [CM], effector memory [EM] and TEMRA). B, Representative dot plots of perforin, CD57 and CD27 expression on CD8 T-cells. Columns show the distribution of CD8 T-cell subsets in ALPS-sFAS patients vs controls (n = 19) (mean ± SD of three and two independent experiments, respectively).
Fig. 4. TCRV repertoire was determined by flow cytometry in gated CD4+, CD8+ and DN T-cells from PBMCs of P3 (Beckman Coulter, Madrid, Spain).
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decreased Fas induced apoptosis in T-cell blasts from P3 could be due to an additional defect in CASP10 (Y446C) inherited from his healthy father, but we cannot rule out defects in other genes involved in the Fas pathway. By contrast, Fas-induced cell death was conserved in T-cell blasts from the parents (data not shown). Indeed, all biomarkers of ALPS analyzed in P3’s parents were in the normal range, the only relevant finding being high levels of vitamin B12 in P3’s father. Some reports have been published showing a molecular explanation for the variable penetrance seen in ALPS. The two-hit hypothesis argues that, as well as a heterozygous germline FAS mutation, a somatic FAS second hit affecting the wild type allele is necessary to produce the disease (Magerus-Chatinet et al., 2011; Rensing-Ehl et al., 2014). Additionally, modifying alterations have been described in genes other than FAS (PRF1, UNC13D, XIAP, CASP10, and OPN) (Aricò et al., 2013; Boggio et al., 2013; Campagnoli et al., 2006; Cerutti et al., 2007; Chiocchetti et al., 2004; Clementi et al., 2004, 2006; Zhu et al., 2006). P3 presented a more dysregulated T- and B-cell phenotype than either P1 or P2 and this could be related to an exhausted phenotype. He showed a predominantly CD4 and CD8 T-cell effector phenotype, these cells express a combination of markers (CD57, CD95 and perforin) usually observed on effector and TEMRA T-cells, a similar abnormal differentiation pattern was also seen in DN T-cells (Rensing-Ehl et al., 2014). It is necessary to clarify the contribution of the different T-cell subsets, DN-T cells and B-cells to the immunological derangements in ALPS. Clinically, P3 was splenectomized due to refractory cytopenias, but he has not suffered invasive bacterial infections by encapsulated bacteria, despite the elevated risk of severe postsplenectomy invasive bacterial infections (Neven et al., 2014). In summary, ALPS-sFAS and ALPS-FAS patients are clinically indistinguishable, but those with ALPS-sFAS have a later age of disease onset, and lower rates of splenectomy, and further there is no evidence of a neoplastic lymphoproliferative disorder in ALPSsFAS, unlike in ALPS-FAS (Dowdell et al., 2010). Here, we report the first ALPS patient with a combination of a somatic FAS mutation and a germline CASP10 substitution. The combined effect of these two molecular alterations may have contributed to the development of severe ALPS with more autoimmune complications, a dysregulated phenotype and no history of bacterial infections despite splenectomy. Further work in this area should explore the ability to switch off the immune response and the link to lymphocyte lifespan regulation and maintenance of peripheral tolerance (Nagata 1997). Conflict of interest The authors declare that they have no conflicts of interest. Acknowledgments AM performed the laboratory work for this study and drafted the manuscript; JM, CRV, RE, LIGG, ROB, MR recruited the patients; SMD, DPM, ESZ, RRG, MM, JDM collaborated in the laboratory work for this study; EPA reviewed the manuscript; and LMA designed the research and drafted the manuscript. The authors would like to thank the patients and their families for their participation. This study was supported by a grant from the Spanish Health Research Fund (PI11/1591) to LMA. The project has been co-financed with FEDER funds. References Aricò, M., Boggio, E., Cetica, V., Melensi, M., Orilieri, E., Clemente, N., Cappellano, G., Buttini, S., Soluri, M.F., Comi, C., Dufour, C., Pende, D., Dianzani, I., Ellis, S.R., Pagliano, S., Marcenaro, S., Ramenghi, U., Chiocchetti, A., Dianzani, U., 2013. Variations of the UNC13D gene in patients with autoimmune lymphoproliferative syndrome. PLoS One 8 (July (7)), e68045.
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