Here we report a young adult, Pt 86, with ALPS due to a point mutation in the TNFSF6 gene encoding FasL. He meets the defining criteria for ALPS by exhibiting chronic lymphadenopathy, splenomegaly, autoimmune hepatitis, elevated numbers of CD4-CD8-TCRαβ+ T cells and an apoptosis defect. However, unlike most patients with ALPS, he has no abnormality of the Fas receptor gene TNFRSF6, or of downstream intracellular signaling molecules that transmit the death signal. Rather, his FasL is defective. Thus, he can be classified as having a previously undocumented form of heterozygous ALPS (Type Ib) reserved by analogy with gld mice for cases arising because of FasL mutations.
Pt 86 has notable features that are not typical of ALPS patients with Fas mutations. These include hypogammaglobulinemia and granulomatous infiltration of the spleen, lymph nodes and liver. His hypogammaglobulinemia in this setting may be due to an abnormality in T cell help rather than a primary B cell defect. Paradoxically both the patient and his father, who carries the same FasL mutation on one of his alleles, have elevated circulating B cell numbers. In addition, Pt 86, but not his father, has a decreased percentage of CD3+
T cells, known to represent a regulatory subset, while the percentage of his CD3+
T cells is elevated, consistent with ongoing activation in vivo. Clinical evidence for Pt 86 in support of a cellular immune defect is his history of difficulty in controlling multiple viral infections: he has suffered from cutaneous warts and chronic lesions of molluscum contagiosum; and he had three discrete episodes of herpes zoster in the absence of immunosuppressive medications. Recent studies suggest that FasL ligation to Fas on T cells can act as a positive costimulatory signal in antigen-specific T cell activation [36
]. Moreover, direct Fas engagement has also been observed to provide a costimulatory effect on T cell proliferation [38
]. Thus, ineffectual Fas-FasL interactions might produce a T cell activation defect in a fashion similar to that seen with mutations in CD40-ligand (CD154) in X-linked hyper-IgM syndrome. Interestingly, the adult immunoglobulin levels for Pt 86 suggest a partial isotype switch defect with low IgA and IgG and IgM in the upper range of normal. However, if this were of substantial magnitude, one should observe T cell activation defects in patients with heterozygous and homozygous Fas mutations, nevertheless, they are substantially milder than those in CD154 deficiency. We have seen instances of herpes zoster infection and poor antibody responses following immunizations in ALPS Type Ia patients. However, cellular immune defects are not characteristic of gld
mice indicating that there is a partial signaling effect or some other reason for these clinical findings [7
Pt 86 originally presented with prolonged granulomatous interstitial pneumonitis that developed after a respiratory infection, and granulomas were evident on histological evaluation of his splenic, lymph node and liver tissues. Interestingly, after some viral infections the FasL-deficient gld
mice also develop inflammatory responses in the pulmonary interstitium that persist long after the infectious agent has disappeared [39
]. Nonetheless, the cause of the granulomas in Pt 86 remains unclear. No infectious etiology was found and the condition remained stable without antimicrobial intervention. Although his histology resembled sarcoidosis, Pt 86 lacked the cutaneous, neurological, and ocular features of that disease. Further patients with ALPS Type Ib must be studied in order to elucidate the relationship between FasL defects and granulomas. Lymph node biopsy of the FasL defective patient described by Del-Rey et al
] revealed sinus histiocytosis but no granulomas. Since their patient had a complete loss of function while Pt 86 has only partial loss of FasL function, it is possible that the difference in histology reflects this qualitatitive difference in function.
Another interesting difference between the patient reported by Del-Rey et al
] and patient 86 lies in the impressive history of recurrent infections in the former despite chronically elevated immunoglobulins. In contrast, Pt 86 has suffered from relatively few infections despite his pan-hypogammaglobulinemia. This difference may reflect virtually absent FasL function in the Del-Rey patient and partial function in Patient 86. However, like the Del-Rey patient, Pt 86 has experienced recurrent episodes of herpes zoster suggesting that this patient has altered cellular and humoral immune function as suggested by his depressed immunoglobulin levels.
The identification of Pt 86 and the present studies of the basis and mechanisms by which his mutation impairs apoptosis afford new insights into FasL biology. While the lpr
murine mutations in Fas or FasL, respectively manifest their phenotype in the homozygous state [7
], most ALPS patients including Pt 86 carry heterozygous mutations [3
]. Because human populations are usually not inbred, it was important to understand how heterozygous mutations impair cell death effector functions. Since Fas transduces the death signal as a homotrimeric complex, we showed that individual mutant Fas proteins, as seen in nearly all patients with ALPS Type Ia, dominantly inhibit apoptosis mediated by wild type Fas [4
]. Now corresponding analyses of mutations in FasL have been made possible by the discovery of Pt 86.
Molecular modeling predicts that this A530G mutation is at a surface-exposed residue near the Fas/FasL interface, suggesting that the mutation directly impairs Fas/FasL interactions. We showed that the mutant FasL chain associates readily with the wild-type chain and thereby dominantly interferes with apoptosis induction by wild-type FasL. By contrast, the gld mutation likely impairs Fas binding indirectly through disrupting the structure of the FasL trimer. The mutation in Pt 55C is a highly conservative one in which the methionine normally found at position 86 within the transmembrane domain of FasL in humans is replaced with another hydrophobic residue, valine, which is in fact found at this site in mice. Thus, it was not surprising that the FasL of Pt 55C can associate successfully with wild type FasL and does not interfere with the apoptotic killing mediated by wild type FasL.
Assuming that the wild type and Pt 86 mutant forms of FasL protein are synthesized equally, have equal stability (which would be difficult to confirm experimentally), and participate in trimer formation at random, the trimeric complexes generated by combinations of these molecules would include one-eighth of them comprised entirely of mutant FasL peptides, three quarters with one or two mutant FasL peptides, and one-eighth with only wild type FasL. Thus, some residual amount of normal FasL trimers would be expected and this might explain the residual apoptosis in PBLs of Pt 86 and his father (Figure ).
The father of patient 86 carries the same mutation as his son, and while he is reported to have had significant adenopathy during childhood, he does not currently manifest sufficient clinical features for the diagnosis of ALPS to be made. Variable expressivity of heterozygous mutations was confirmed early in our studies of ALPS Type Ia families with Fas mutations [4
]. The present data reveal parallel findings in ALPS Type Ib families with FasL mutations. Further investigations into other gene contributions may reveal the molecular basis for variable penetrance in ALPS.