Background Impaired Fas-induced apoptosis of lymphocytes invitro is a principal feature of the autoimmune lymphoproliferativesyndrome (ALPS). We studied six children with ALPS whose lymphocyteshad normal sensitivity to Fas-induced apoptosis in vitro.
Methods Susceptibility to Fas-mediated apoptosis and the Fasgene were analyzed in purified subgroups of T cells and othermononuclear cells from six patients with ALPS type III.
Results Heterozygous dominant Fas mutations were detected inthe polyclonal double-negative T cells from all six patients.In two patients, these mutations were found in a fraction ofCD4+ and CD8+ T cells, monocytes, and CD34+ hematopoietic precursors,but not in hair or mucosal epithelial cells.
Conclusions Somatic heterozygous mutations of Fas can causea sporadic form of ALPS by allowing lymphoid precursors to resistthe normal process of cell death.
Fas (also called Apo-1 and CD95) is a cell-surface receptorbelonging to the tumor necrosis factor receptor (TNFR) superfamily1(Fas is the sixth member, TNFRSF6). Once triggered by its cognateligand (Fas ligand), Fas initiates a cascade of events withinthe cell that culminates in the death of the cell (apoptosis).This process involves the formation of the death-inducing signalingcomplex,2 consisting mainly of the Fas-associated death domainand the caspase 8 and caspase 10 proteins. The essential roleof Fas in lymphocyte homeostasis was initially recognized inMRL lpr/lpr mice, which have a germ-line autosomal recessivemutation of Fas.3 Subsequently, heterozygous dominant mutationsof Fas were found in children with the autoimmune lymphoproliferativesyndrome (ALPS),4,5,6 which is also known as the Canale–Smithsyndrome.7 The main features of this disease are splenomegaly,lymphadenopathy, hypergammaglobulinemia (IgG and IgA), and autoimmunity.8,9ALPS is characterized by the accumulation of a polyclonal populationof T cells called double-negative T cells. These lymphocytesdisplay the marker common to mature T cells, CD3, and / T-cell–antigenreceptors, but neither the CD4 nor the CD8 coreceptors (CD3+T-cell receptor /+ CD4–CD8–). They normally accountfor less than 2 percent of peripheral /+ T cells8 and are distinctfrom the double-negative thymocytes in the cortex of the thymus,which lack CD3 and T-cell receptors for antigen. The double-negativeT cells in patients with ALPS are poorly responsive to mitogensand antigens and fail to produce growth and survival factorssuch as interleukin-2.10 In Fas-deficient MRL lpr/lpr mice,the large population of double-negative T cells appears to originatefrom chronically activated CD8+ T cells that down-regulate theexpression of CD8 and fail to undergo apoptosis.3 In humans,double-negative T cells also seem to be antigen-exposed T cellsthat have escaped apoptosis.
ALPS is classified according to the underlying genetic defect.11In type 0 disease, homozygous Fas mutations usually cause acomplete deficiency of the Fas protein and a severe form ofthe disease.4,12,13 In ALPS type I, heterozygous Fas mutations(ALPS type Ia)14,15,16 or, more rarely, heterozygous mutationsin the gene for Fas ligand (ALPS type Ib)17 are usually associatedwith a partial defect in apoptosis mediated by Fas and its ligand.ALPS type II, which is characterized by resistance to Fas-mediatedapoptosis despite the presence of normal Fas ligand and Fas,has been found in two patients with caspase 10 mutations.18In ALPS type III, Fas-mediated apoptosis is also normal in vitro,19and the genetic defect is unclear. Patients with ALPS type IIImay not have all four classic features of the syndrome —lymphoproliferation, excessive numbers of double-negative Tcells, hypergammaglobulinemia, and autoimmune manifestations.Many cases of ALPS type III are sporadic, precluding the useof a genetic approach to identify the molecular defect. In thepresent study, we obtained lymphocytes from six children withALPS type III for an in-depth analysis.
Methods
Patients
Blood samples were obtained from the six patients, their parents,and five healthy controls. All subjects or their parents orguardians provided written informed consent, validated by theComité Consultatif pour la Protection des Personnes enRecherche Biomédicale. Table 1 summarizes the clinicalfeatures of the six patients.
Table 1. Clinical and Immunologic Characteristics of Six Patients with Autoimmune Lymphoproliferative Syndrome Type III.
Cell Culture, Apoptosis Assay, and Analysis of the T-Cell–Receptor Repertoire
Peripheral-blood mononuclear cells were isolated from freshlydrawn heparin-treated blood by means of Ficoll–Hypaquedensity gradient centrifugation. Apoptosis assays and repertoireanalysis were performed on activated T cells and whole blood,respectively, as previously described.16,20
Nucleic Acid Preparation, Amplification, and Detection of Fas Mutations
Total RNA was isolated from freshly isolated peripheral-bloodmononuclear cells and T cells that had been activated by ninedays of in vitro exposure to phytohemagglutinin. The reverse-transcriptase–polymerase-chain-reaction(RT-PCR) assay was performed as previously described.4,16 DNAextracted from phytohemagglutinin-activated lymphocytes or purifieddouble-negative T cells was amplified with oligonucleotidesspanning the nine Fas exons with the use of PCR conditions describedelsewhere,16 except that the annealing temperatures for exons4 and 7 were 58°C and 60°C, respectively. Leukocytesubgroups were purified by cell sorting (purification alwaysexceeded 95 percent) with a fluorescence-activated cell sorter(FACS) (FACStarPLUS, Becton Dickinson) from the peripheral-bloodmononuclear cells as described previously.21 DNA from thesesubgroups was amplified by nested PCR. Sequencing was performeddirectly on PCR products with the use of the Big Dye DNA SequencingKit (Perkin–Elmer) and an ABI PRISM 377 automated sequencer(Applied Biosystems). For quantification, PCR products correspondingto Fas exon 8 were ligated into the TOPO vector with the useof the TOPO-TA Cloning Instruction Manual (Invitrogen), andat least 60 clones were individually sequenced. Allele-specificPCR was performed with the successive use of two sets of primers.The first step amplified Fas exon 8 on both alleles; the secondstep amplified either the mutant or the wild-type allele. PCRproducts were separated on 1.5 percent agarose gel and transferredto a Hybridization Transfer Membrane (NEN Life Science Products).Oligonucleotide hybridization was performed as described previously8at 3°C below the melting temperature of the oligonucleotide.Descriptions of all oligonucleotide sequences and PCR conditionsare available on request.
Results
Identification of Fas Mutations
We studied six patients with phenotypic features of ALPS (Table 1)but with normal levels of Fas-mediated apoptosis of phytohemagglutinin-activatedT-cell blasts (Table 2) and no family history of ALPS. However,because of the strong association between an excess of double-negativeT cells and a defect in Fas-mediated apoptosis,11 we lookedfor Fas gene mutations in FACS-sorted double-negative T cellsfrom these patients and found heterozygous Fas mutations inall six (Table 2). Donor and acceptor splice-site mutationsof exon 8 were identified in Patient 1 and Patient 6, respectively,and a donor splice-site mutation of exon 7 was identified inPatient 5 (Table 2). These mutations are predicted to lead tothe splicing out of the corresponding exon on RNA. Patient 2had a nonsense mutation in exon 8, and Patient 3 had a missensemutation in exon 9 (D244V); Patient 4 had a deletion of 8 bpin exon 9 leading to a premature stop codon at position 227(Table 2). Identical Fas mutations or mutations leading to identicalchanges in the structure of Fas have been described in patientswith ALPS type Ia (Table 2) and have been shown to be dominant.14,15We also looked for Fas mutations in sorted double-negative Tcells from five healthy, age-matched controls and found none.
Table 2. Levels of Fas-Mediated Apoptosis and Heterozygous Fas Mutations in Six Patients with Autoimmune Lymphoproliferative Syndrome (ALPS) Type III and Three Patients with ALPS Type Ia.
In contrast to the results with purified double-negative T cells,mutant Fas products could not be detected by PCR performed onDNA from T-cell blasts that had been activated in vitro fornine days by exposure to phytohemagglutinin.
Expression of Mutant Alleles
The expression of the mutant alleles in T cells from Patient1 and Patient 2 was analyzed. An aberrant product of RT-PCRamplification of Fas DNA, as well as the expected normal-sizedproduct, was detected in cDNA prepared from resting peripheral-bloodmononuclear cells (Figure 1A). Sequencing of these RT-PCR productsrevealed a wild-type sequence and an abnormal product in whichexon 8 was missing (data not shown). The omission of exon 8in Fas messenger RNA could be the consequence of the nonsenseand splice-site mutations in DNA of double-negative T cellsfrom Patient 1 and Patient 2, respectively (Table 2). Indeed,such mutations were detected in genomic DNA from purified double-negativeT cells (Figure 1B) but not in that from phytohemagglutinin-stimulatedT cells.
Figure 1. Analysis of the Expression of Fas Mutations in Activated T Cells and Purified Double-Negative T Cells from Patient 1 and Patient 2.
Panel A shows the results of RT-PCR amplification of Fas in double-negative T cells in resting peripheral-blood mononuclear cells and phytohemagglutinin-activated T cells. Panel B shows the sequences of exon 8 of genomic Fas obtained from purified double-negative T cells and phytohemagglutinin-activated T cells. Panel C shows the FACS analysis of the percentages of double-negative T cells (CD3+ T-cell receptor [TCR] /+CD4–CD8–) in resting peripheral-blood mononuclear cells and phytohemagglutinin-activated T cells.
Moreover, the double-negative T cells of these patients, whichwere readily detectable among resting T cells (Figure 1C), wereundetectable after being incubated with phytohemagglutinin fornine days (Figure 1C) or after stimulation of T cells with antibodiesagainst T-cell receptors (data not shown). Thus, the absenceof mutant cells after in vitro stimulation by phytohemagglutininaccounts for the normal Fas-mediated apoptosis in unfractionatedlymphocytes from patients with ALPS type III.
Distribution of Fas Mutations
To determine the cellular distribution of the Fas mutationsin Patient 1 and Patient 2, we performed PCR analysis of genomicDNA from FACS-sorted peripheral-blood CD4+ or CD8+ T cells (hereaftercalled single-positive T cells), T-cell receptor /+ T cells,natural killer cells, B cells, monocytes, splenic CD34+ hematopoieticprogenitors, hair cells, and buccal epithelial cells. To ruleout cellular chimerism, the migration profiles of nine polymorphicmarkers on double-negative T cells and single-positive T cellswere determined (data not shown) and were found to be identical.
We first used a mutation-specific PCR method22,23 to determinewhich type of cell or tissue bears the Fas mutation, and wequantified the level of mutant cells in these populations. Bymeans of dilution experiments, we found that heterozygous Fasmutations could be detected by direct sequencing of PCR productsonly when more than 20 percent of cells carried the mutation(data not shown). With a more sensitive analysis, which entailedcloning and sequencing PCR products, we could detect cells withmutations when more than 1 percent of cells carried the mutation,a percentage compatible with the level of purity of FACS separationtechniques. The mutation in exon 8 was detected in all leukocytesubgroups from Patient 1, whichever method was used (Figure 2).Thus, in this patient, a mutant Fas was present in morethan 20 percent of lymphocytes and myeloid cells. The quantitativeanalysis showed that 100 percent of double-negative T cellsbut only 20 percent of single-positive T cells and 14 percentof monocytes carried the mutation, indicating that, with theexception of double-negative T cells, these different leukocytesubgroups contained a similar proportion of mutant cells. Themutation was undetectable in hair cells and buccal epithelialcells from Patient 1 on direct sequencing.
Figure 2. Hybridization of the PCR Products of Fas Exon 8 after Wild-Type–Specific and Mutation-Specific Amplifications.
Detection of the mutant allele after direct sequencing of PCR products is indicated by a plus sign, and the absence of the mutation by a minus sign. Numbers in parentheses are the percentages of mutant cells in a given cell population as determined by cloning and sequencing. SP denotes single-positive (CD4+ or CD8+) T cells, DN double-negative T cells, / T-cell receptor /+ T cells, NK natural killer cells, Mono monocytes, HP hematopoietic-progenitor–enriched cells, Buccal buccal epithelial cells, Cwt T cells from a wild-type control, C-1 a negative control included in the first amplification, and C-2 a negative control included in the second amplification (with the use of products of the first PCR).
In Patient 2, the Fas mutation was found in all leukocyte subgroupsby means of the mutation-specific PCR method. In contrast, themutation was detected in double-negative T cells by direct sequencing(Figure 2) but not in other leukocyte subgroups, suggestingthat it was carried by less than 20 percent of cells in theother leukocyte populations. The mutation-specific quantitativemethod indicated that 100 percent of double-negative T cellsfrom the blood and spleen of Patient 2 were mutant cells, whereasonly 10 percent of single-positive T cells carried the mutation.
A purified CD34+CD19–CD7– population, which wasknown to be enriched for hematopoietic progenitors, was obtainedfrom a spleen sample from Patient 2. Mutation-specific PCR revealedmutant cells in this population (Figure 2). We estimate, takinginto account the purification efficiency of FACS, that lessthan 2 percent of these cells carried the mutation (Figure 2).The Fas mutation was undetectable in nonhematopoietic cells,even when the mutation-specific PCR was used (Figure 2). Allthese results suggest that in both Patient 1 and Patient 2,Fas mutations originated in hematopoietic stem cells or possiblyearlier, in mesenchymal precursors. The specific accumulationof Fas mutants in the double-negative T-cell compartment underlinesthe essential role of Fas in the control of lymphocyte homeostasisin the periphery.
T-Cell–Receptor Repertoire and the Origin of Double-Negative T Cells
Somatic Fas mutations have been described in rare cases of asyndrome involving monoclonal or oligoclonal double-negativeT cells.24 We therefore analyzed the population of T-cell–receptor chains in double-negative T cells from both patients usingantibodies against the chains and found polyclonal T-cell populationsof double-negative and single-positive T cells in both patients(Figure 3).
Figure 3. Analysis of the T-Cell–Receptor (TCR) Repertoire on Single-Positive (CD4+ or CD8+) T Cells and Double-Negative (CD4–CD8– TCR /+) T Cells from Patient 1, Patient 2, and 80 Controls.
Values in controls are expressed as the mean (horizontal line in each box), with the standard deviation (top and bottom of the box) and 95 percent confidence interval (I bar).
Discussion
We found that patients with mosaicism carrying heterozygousFas mutations in hematopoietic cells have an ALPS phenotype.In the light of our findings, the classification of ALPS requiresrevision, with patients such as ours possibly denoted as belongingto a subgroup with mosaic ALPS type I, or ALPS type Im. In supportof this reclassification is the fact that the clinical phenotypeassociated with these somatic mutations is indistinguishablefrom that of ALPS type I.
Our study demonstrates that peripheral lymphocytes with a dominantsomatic Fas mutation exhibit a selective advantage (Figure 4).Germ-line mutations of Fas have been reported to impair Fas-inducedapoptosis of lymphocytes in patients with ALPS type Ia.14,15By resisting apoptosis, the mutant cells accumulate and becomedouble-negative T cells. This interpretation is consistent withdata from Fas-deficient chimeric mice25 and can account forthe lymphadenopathy and splenomegaly in all six of our patients.Indeed, a lymph node from Patient 4 showed paracortical expansionconsisting of double-negative T cells, a histologic pictureindistinguishable from that seen in patients with ALPS typeI (data not shown). Similarly, the relatively large proportionof mutant cells in peripheral lymphocytes from Patient 2, ascompared with the smaller proportion of mutant cells among hematopoieticprogenitors, suggests that Fas mutations provide a selectiveadvantage (by protecting against apoptosis) during hematopoiesis,a finding consistent with observations in MRL lpr/lpr mice.26
Figure 4. Hematopoietic Development and Peripheral Proliferation of Fas-Sensitive and Fas-Deficient Cells.
In the bone marrow, a mutant hematopoietic stem cell (in red), carrying a heterozygous Fas mutation, emerges during the course of the self-renewal or generation of these cells. It has been estimated that less than 2 percent of hematopoietic stem cells are mutated. The progeny of putative common lymphoid precursors, such as T cells, B cells, and natural killer cells, and the progeny of putative common myeloid precursors, such as monocytes and granulocytes, all contain a higher percentage of mutant cells (estimated at 10 to 20 percent). Therefore, the resistance of mutant hematopoietic stem cells to Fas-mediated apoptosis is probably the selective advantage that accounts for the increased proportion of mutant cells observed in peripheral leukocytes. In the bloodstream or lymphoid organs of patients with ALPS, some T-cell proliferations (driven by bacterial or viral antigens) are well controlled, probably by Fas-independent pathways. In contrast, other T-cell responses (perhaps driven by self-antigens), normally regulated by Fas, lead to uncontrolled proliferation and the accumulation of mutant double-negative T cells. The resistance to Fas-mediated cell death allows the proliferation or the persistence of cells that should otherwise disappear. TCR denotes T-cell receptor.
We identified Fas mutations in freshly purified double-negativeT cells but not in phytohemagglutinin-activated T cells. Thenormal in vitro response to Fas-induced apoptosis by activatedT cells is consistent with the absence of Fas mutations in suchcells. This result might be due to the high death rate of double-negativeT cells in vitro. Double-negative T cells are believed to originatefrom activated peripheral single-positive T cells that havereceived a death-inducing signal but cannot die, owing to adefect in Fas signaling.3,27,28 A similar abnormality of double-negativeT cells has been described in patients with ALPS type I, suggestingthat a signal required for the survival of double-negative Tcells is lacking in tissue-culture medium.10,29,30 In vivo,this signal could be provided by self-antigens, and chronicstimulation of apoptosis-resistant cells by autoantigens couldaccount for the autoimmune manifestations and lymphoproliferationof ALPS. However, autoimmune manifestations were observed inonly four of our six patients. In addition, there is no clearcorrelation between the numbers of double-negative T cells andautoimmunity, and whether these cells recognize and respondto self-antigens is also unknown.
A proportion of all hematopoietic cells from our patients carriedFas mutations, whereas they were not found in hair cells orbuccal epithelial cells. Several mechanisms could account forthis finding. One is chimerism, which could be the consequenceof transplacental passage of maternal blood or cell fusion froman aborted dizygote twin.31,32 This possibility was excluded,since the population of both the double-negative T cells (containingmutant cells alone) and the single-positive T cells (containingan excess of wild-type cells) had similar patterns of DNA polymorphicmarkers (data not shown). Therefore, these mutations must haveresulted from a somatic mutation that occurred during embryonicor fetal development, or after birth. Although Fas mutationswere not detected in cells from the mouth or hair (originatingfrom ectoderm), their presence in germ cells (of endodermicorigin), which would indicate a mutation early in embryogenesis,was not formally ruled out. Analyses of additional tissues wouldbe required to narrow the timing of the mutational events.
The clinical features of our patients with ALPS type III resemblethose of patients with ALPS type I who have identical or similargerm-line Fas mutations. In some other conditions, however,mosaicism is usually associated with a mild phenotype, becauseof somatic reversions to the wild type.33 In such cases, wild-typerevertant cells can have a selective advantage, enabling theirexpansion and the partial restoration of the wild-type phenotype.34,35,36Our findings in patients with acquired ALPS represent an exampleof dominant somatic mutations' conferring a selective advantageof mutant cells over normal cells. Nevertheless, a 10-year follow-upshowed that the proportion of mutant lymphocytes was steadyover time and that the cells did not outgrow normal cells.
Healthy relatives of patients with ALPS type I can carry aninherited dominant Fas mutation,14,15,16 thereby illustratingthe partial clinical penetrance of some Fas mutations. Mutationsaffecting the intracellular domain of Fas are associated withgreater clinical penetrance than mutations affecting the extracellulardomain.15,16 Notably, all the mutations we found affected theintracellular domain. They are predicted to generate abnormalFas molecules and have been associated with full penetranceof the disease in patients with ALPS type I.14,15,16
In conclusion, we found that some patients with ALPS type IIIhave somatic Fas mutations in cells of hematopoietic lineagesin the absence of any malignant condition. This situation isan example of a nonmalignant, genetically acquired disease inwhich a selective advantage (resistance to death) is conferredby Fas mutations.
Supported by grants from INSERM, the Ministère de laRecherche (ACI-jeune chercheur), the Ligue Nationale contrele Cancer, la Ligue Parisienne contre le Cancer, the Associationpour la Recherche contre le Cancer, the Programme Hospitalierde Recherche Clinique (AOR01070, and the European Commission(QLRT-2000-01395). Dr. Holzelova is the recipient of a fellowshipfrom the Fondation pour la Recherche Médicale and Ministèredes Affaires Etrangères. Mr. Vonarbourg is the recipientof a fellowship from the Ligue Nationale contre le Cancer.
Source Information
From INSERM Unité 429 (E.H., C.V., M.-C.S., F.S., A.F., F.L.D., F.R.-L.) and Unité d'Immunologie–Hématologie Pédiatrique (A.-M.P., S.B., A.F.), Hôpital Necker–Enfants Malades, Paris; the Academic Unit of Child Health, Booth Hall Children's Hospital, Manchester, United Kingdom (P.D.A.); the Institute of Immunology, 2nd Medical School, Charles University, Prague, Czech Republic (E.H., J.B.); and the Unité d'Immuno-Hématologie, Hôpital Robert Debré, Paris (E.V.).
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Reamon-Buettner, S M, Borlak, J
(2006). Somatic mutations in cardiac malformations.. J. Med. Genet.
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Roesler, J., Izquierdo, J.-M., Ryser, M., Rosen-Wolff, A., Gahr, M., Valcarcel, J., Lenardo, M. J., Zheng, L.
(2005). Haploinsufficiency, rather than the effect of an excessive production of soluble CD95 (CD95{Delta}TM), is the basis for ALPS Ia in a family with duplicated 3' splice site AG in CD95 intron 5 on one allele. Blood
106: 1652-1659
[Abstract][Full Text]
Beier, C. P., Wischhusen, J., Gleichmann, M., Gerhardt, E., Pekanovic, A., Krueger, A., Taylor, V., Suter, U., Krammer, P. H., Endres, M., Weller, M., Schulz, J. B.
(2005). FasL (CD95L/APO-1L) Resistance of Neurons Mediated by Phosphatidylinositol 3-Kinase-Akt/Protein Kinase B-Dependent Expression of Lifeguard/Neuronal Membrane Protein 35. J. Neurosci.
25: 6765-6774
[Abstract][Full Text]
Yan, M.-D., Hong, C.-C., Lai, G.-M., Cheng, A.-L., Lin, Y.-W., Chuang, S.-E.
(2005). Identification and characterization of a novel gene Saf transcribed from the opposite strand of Fas. Hum Mol Genet
14: 1465-1474
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Clementi, R., Locatelli, F., Dupre, L., Garaventa, A., Emmi, L., Bregni, M., Cefalo, G., Moretta, A., Danesino, C., Comis, M., Pession, A., Ramenghi, U., Maccario, R., Arico, M., Roncarolo, M. G.
(2005). A proportion of patients with lymphoma may harbor mutations of the perforin gene. Blood
105: 4424-4428
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Fischer, K., Voelkl, S., Heymann, J., Przybylski, G. K., Mondal, K., Laumer, M., Kunz-Schughart, L., Schmidt, C. A., Andreesen, R., Mackensen, A.
(2005). Isolation and characterization of human antigen-specific TCR{alpha}{beta}+ CD4-CD8- double-negative regulatory T cells. Blood
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[Abstract][Full Text]
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T-cell–antigen receptors, but neither the CD4 nor the CD8 coreceptors (CD3+ T-cell receptor 
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+ T cells, natural killer cells, B cells, monocytes, splenic CD34+ hematopoietic progenitors, hair cells, and buccal epithelial cells. To rule out cellular chimerism, the migration profiles of nine polymorphic markers on double-negative T cells and single-positive T cells were determined (data not shown) and were found to be identical. 
A 20210 prothrombin gene and C
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