Aberrant T-Cell Antigen Receptor-Mediated Responses in Autoimmune Lymphoproliferative Syndrome Frederick D. Goldman,* ,1 Rajeev Vibhakar,* Jennifer M. Puck,† Stephen E. Straus,‡ Zuhair K. Ballas,§ Clay Hollenback,* Thomas Loew,* Anthony Thompson,* Kejing Song, and Robert T. Cook Departments of *Pediatrics, §Medicine, and Pathology at the University of Iowa Hospitals and Clinics and Veterans Affairs Medical Center, 200 Hawkins Drive, Iowa City, Iowa 52242; and the †Genetics and Molecular Biology Branch, National Human Genome Research Institute, and ‡National Center for Complementary and Alternative Medicine and Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892 Autoimmune Lymphoproliferative Syndrome (ALPS) is a disorder of defective lymphocyte apoptosis due to mutations of the Fas receptor and other molecules in the Fas signaling pathway. In addition to accumulation of CD4  CD8  double-negative (DN) T cells, many patients display a dysregulated cytokine pattern with dysfunc- tional T cells, suggesting Fas defects may impact path- ways of T-cell activation/differentiation. Here, we report two novel mutations in the Fas receptor resulting in an ALPS phenotype. Utilizing flow cytometry, we found an- ti-CD3 activated CD4  T cells from these patients were incapable of fully upregulating activation markers (CD25, CD69, and CD40L) or producing interferon- and IL-2. Additionally, DN T cells were unable to transduce proximal T-cell antigen receptor signals or produce cy- tokines. Furthermore, DN T cells overexpressed CD57 and phenotypically resembled end-stage effector cells. As DN T cells were essentially anergic, the clinical man- ifestations of autoimmunity are more likely to be a con- sequence of aberrant cytokine secretion within the CD4  T-cell subpopulation. © 2002 Elsevier Science (USA) Key Words: autoimmune lymphoproliferative syn- drome; T-cell antigen receptor; CD4; signal transduc- tion; Fas receptor; cytokines. INTRODUCTION Autoimmune Lymphoproliferative Syndrome (ALPS) is a congenital disorder characterized by defective T-lym- phocyte apoptosis (1, 2). Originally described by Smith and Canale, the clinical features of this syndrome include childhood onset of lymphadenopathy; splenomegaly; and varying degrees of autoimmune hemolytic anemia, thrombocytopenia, and neutropenia (3). In addition, many patients with ALPS have a propensity to develop other autoimmune disorders and lymphomas later in life (4). Immunologically, there is extensive follicular hyper- plasia and paracortical expansion of lymphatic tissue; hypergammaglobulinemia; circulating autoantibodies; peripheral blood lymphocytosis; and, perhaps most nota- bly, the accumulation of CD4 - CD8 - double-negative (DN) T cells (5, 6). The molecular basis of the disease has been shown to be due to several genes involved in apoptosis, including the Fas receptor (TNFRSF6), Fas ligand (TNFSF6), and caspase 10 (7–9). The signaling pathway mediated through the Fas receptor plays a key role in lympho- cyte homeostasis. As a consequence of defective apoptosis, DN T cells, which are normally short-lived, as well as other types of lymphocytes, fail to be deleted and remain in circulation. Similarly, mice with ho- mozygous mutations in Fas (lpr) or FasL (gld) gene exhibit many of the immune abnormalities and clinical characteristics seen in ALPS patients, including the accumulation of DN T cells (6, 10). Fas deficiency has been shown to affect T-cell func- tion, and in particular signals generated through the T-cell antigen receptor (TCR) (4). The connection be- tween the TCR and Fas signaling pathways is not fully understood, nor is it known why Fas mutations lead to autoimmune disease. Several studies have demon- strated that ALPS patients have normal basal circu- lating levels of various cytokines, including IL-2, IFN-, TNF, and IL-6, but elevated levels of IL-10 (11, 12). Other studies have found decreased IL-2 and IFN- and increased IL-4 and IL-5 secretion from ALPS patient T cells (12). This type of cytokine profile, an exaggerated Th2 and diminished Th1 response, is typical of other “autoimmune” disorders, including sys- temic lupus erythematosus and chronic GVHD (13, 14). Here we describe two novel Fas mutations leading to ALPS phenotypes. Using freshly isolated lymphocytes from these patients, we studied T-cell antigen receptor- (TCR) generated events in CD4 + T cells, as well as other T-cell subpopulations. Importantly, we deter- mined that CD4 + T cells failed to upregulate surface 1 To whom correspondence and reprint requests should be ad- dressed. Fax: (319) 356-7659. E-mail: Frederick-Goldman@ uiowa.edu. Clinical Immunology Vol. 104, No. 1, July, pp. 31–39, 2002 doi:10.1006/clim.2002.5249 1521-6616/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved. 31