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

Autoimmune lymphoproliferative syndrome (ALPS), first described in 1967 by Canale and Smith,¹  was a human disease characterized by a benign lymphoproliferative disease and autoimmune cytopenia. The molecular basis of ALPS was identified in 1995 via the demonstration of mutations in the FAS gene in patients with lymphocyte apoptosis defects^2,3 ; the subsequent discovery of mutations in other genes in several ALPS patients has led to the classification of the condition^4,5  into subgroups: ALPS-0 and -Ia with homozygous and heterozygous inherited mutations of the FAS gene, respectively; ALPS-Ib with mutations in the gene encoding FAS-ligand (FAS-L)⁶ ; ALPS-IIa and -IIb with mutations in CASPASE-10⁷  and CASPASE-8⁸  genes, respectively; ALPS-III with unknown genetic defects but with a similar clinical and immunologic phenotype to mild ALPS; and finally ALPS-IV with NRAS mutations.⁹  A new subgroup called ALPS-Im¹⁰  includes individuals carrying somatic mosaic FAS mutation. The pathognomonic ALPS immunologic features are as follows: nonmalignant lymphoproliferation (splenomegaly and/or adenopathy) along with increase of double-negative CD4⁻CD8⁻ TCRαβ⁺ T-lymphocyte counts (DNT cells), hypergammaglobulinemia, and autoimmune abnormalities

Elevated levels of IL-10 protein in the plasma and in lymphoid tissues have been reported in ALPS patients.^11,12  In this study, we have measured the FAS-L concentrations in the plasma of ALPS to establish whether these proteins constitute useful circulating markers in supporting ALPS diagnosis.

The study protocol was approved by the local independent ethics committee at Hôpital Necker-Enfants Malades and informed consent was obtained from the patients or their families prior to study entry in accordance with the Declaration of Helsinki.

The percentage of DNT cells was determined by flow cytometry as previously described.¹³ 

Apoptosis assays were performed as previously described.¹³ 

Plasma IL-10 and FAS-ligand were determined in EDTA plasma samples using commercially available enzyme-linked immunosorbent assay (ELISA; R&D Systems, Lille, France, and MBL; Beckman Coulter, Paris, France) according to the manufacturers' instructions.

All analyses were performed using Prism (GraphPad Software, San Diego, CA). Populations were compared using the Mann-Whitney test.

The plasma FAS-L concentration (which is known to be elevated in Fas- or Fas-L–defective mice¹⁴ ) was measured in 54 patients carrying either a homozygous FAS mutation (ALPS-0; n = 3), a germinal heterozygous FAS mutation (ALPS-Ia; n = 41), or a somatic heterozygous FAS mutation (ALPS-Im; n = 10). We also tested samples from 12 ALPS-like patients meeting clinical and laboratory criteria for ALPS (including lymphoproliferative syndrome or pancytopenia) but in whom no causative mutations have been identified, from 24 MPRs as well as samples from 41 healthy relatives carrying a wild-type FAS gene (HRs), and from 21 age-matched healthy controls (HCs). All FAS-mutated patients had significantly higher plasma FAS-L concentrations than controls (Figure 1A). We also observed a moderate increase of the FAS-L concentration in the plasma of some of the ALPS-like patients and some of the MPRs (Figure 1A).

Given that ALPS patients with FAS mutation treated with immunosuppressive drugs (open triangles in Figure 1A; n = 18: 3 ALPS-0, 8 ALPS-Ia, and 7 ALPS-Im) exhibit lower levels of FAS-L than untreated patients (dark circles), we compared the FAS-L concentration in the plasma of 8 patients before they received any treatment and then during immunosuppressive treatment (methotrexate [n = 1], pyrimethamine plus sulfadoxine [n = 1], azathioprine [n = 1], 6-mercaptopurine [n = 2], or azathioprine plus 6-mercaptopurine [n = 3]; Figure 1B). A significant drop in the plasma FAS-L level was observed in all patients following introduction of the immunosuppressive regimen. Nevertheless, these values remained above normal range. Of note, all 8 patients showed clinical improvement (substantiated by shrinking of the tumoral syndrome and remission of autoimmunity) following introduction of the immunosuppressive regimen.

We then assessed the plasma IL-10 concentrations in the present cohort. We confirmed in this ALPS cohort that the IL-10 concentration was variably but significantly increased in the plasma of FAS-mutated patients as previously described.^11,15,16  It was only moderately elevated in some ALPS-like patients (Figure 1C). We occasionally noticed slightly elevated plasma IL-10 values in a few healthy relatives. The plasma IL-10 concentration was also measured in 6 patients before or during immunosuppressive treatment (methotrexate [n = 1], pyrimethamine plus sulfadoxine [n = 1], azathioprine [n = 1], or azathioprine plus 6-mercaptopurine [n = 3]) (Figure 1D). The IL-10 concentration dropped in 5 of 6 treated ALPS-I patients showing clinical improvement.

A higher than normal percentage of the DNT cells is a hallmark of FAS deficiency in both humans and mice.¹⁷  We therefore analyzed the proportion of DNT cells in blood samples of patients of our cohort. In agreement with previous studies, the DNT cell counts were significantly higher in ALPS patients with FAS mutation, compared with controls (Figure 1E). However, the proportion of the DNT cells was within the normal range in 3 ALPS-Ia patients. This is probably related to the immunosuppressive treatment, since half of the treated ALPS-I patients exhibited a normal DN T-cell percentage (Figure 1F). Indeed, when the proportion of the DNT cells was measured in the same group of patients before treatment, all values were above normal values (Figure 1F). We observed significant increase in the proportion of DNT cells in 9 of 12 ALPS-like patients. We also noted a rare, moderate increase in the percentage of DNT cells in 2 HRs and 3 MPRs.

As set out in Table 1, analysis of the percentage of donors of each category exhibiting the increase in 0, 1, 2, or 3 of the studied markers (ie, the percentage of DNT cells, and the plasma IL-10 and FAS-L concentrations) showed that 100% of ALPS-0 and ALPS-Im patients exhibited the concomitant increase in value for the 3 markers. Only 6 (14%) ALPS-Ia patients (4 untreated and 2 treated) did not exhibit an increase in IL-10 concentration. We did not observe a concomitant increase in the 3 markers in ALPS patients without FAS mutations.

These observations indicated that these markers are therefore more informative than the Fas-induced apoptosis assay. Indeed, the latter was unable to detect ALPS-Im, as a consequence of the death of the mutant cells in vitro,¹⁰  and apoptosis defect is observed in MPRs (Figure S1, available on the Blood website; see the Supplemental Materials link at the top of the online article).

Another important observation was the consequence of the immunosuppressive treatment on these parameters. All 3 parameter values correlated with extent of lymphadenopathy as previously described for IL-10.^11,15  Therefore, one has to consider whether the patients were treated to interpret the results. Indeed, values close to the normal range could result from immunosuppressive treatment. Moreover, these parameters could be useful for monitoring the treatment response.

When measured on samples from ALPS-like patients, rare and moderate increases in these markers could be observed. This finding indicates that these ALPS-like cases are probably different entities and it demonstrates the specificity of increases in these parameters in FAS-deficient patients and thus their helpful contribution to the diagnosis of ALPS.

Overall, we show here that the percentage of DNT cells and the plasma concentrations of FAS-L and IL-10 provide useful tools for the diagnosis of ALPS, a diagnosis that becomes definitive when a FAS mutation is identified. In addition, they allow (1) discrimination between ALPS-Ia patients and MPRs and (2) detection of ALPS-Im patients, and may also be useful markers for monitoring treatment efficacy.

The authors acknowledge Chantal Harre, Corinne Jacques, Nathalie Lambert, and Stephanie Ndaga for technical support.

This work was supported by grants from Inserm, the Agence Nationale de la Recherche (ANR, Paris, France; grant no. 05-MRAR-017, E-rare 2007 [epinostics] and grant no. 08-GENOPATH-015), the Association pour la Recherche contre le Cancer (ARC, Paris, France), and the European STReP (Specific Targeted Re-search Projects; autorome). A.M.-C. was supported by postdoctoral fellowship from ARC and the European STReP.

Contribution: A.M.-C. designed and performed research, collected, analyzed, and interpreted data, and wrote the paper; M.-C.S. performed research; M.S.L. performed research and wrote the paper; B.N. collected and analyzed data and provided clinical information and samples from patients; C.S. and N.D. performed research and analyzed data; P.D.A., B.B.-M., C.G., J.B., S.B., J.-L.C., M.D., A.F., C.F., B.F., O.H., C.G., O.L., E.S., C.T., and C.P. provided clinical information and blood sample from ALPS patients; F.L.D. designed research; A.F. provided essential clinical information from patients, designed research, interpreted data, and wrote the paper; and F.R.-L. designed research, interpreted data, and wrote the paper.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Frédéric Rieux-Laucat, Inserm U768, Hôpital Necker-Enfants Malades, 149 Rue de Sèvres, 75015 Paris, France; e-mail: frederic.rieux-laucat@inserm.fr.