Novel Role for Iron Transport in Immune Cell Function

An effective immune response relies on proliferation of lymphocytes and the generation of protective antibodies. Combined immunodeficiency (CID) is a genetic disorder characterized by abnormal development or function of T and B lymphocytes; however, early hematopoietic stem cell transplantation cures the disease. The underlying mutations are heterogeneous, but in several patients, no genetic defect has been identified.

Dr. Raif Geha and colleagues investigated two families of Middle Eastern origin in which affected CID individuals had severe, recurrent childhood infections, hypo- or agammaglobulinemia, intermittent thrombocytopenia, and mild anemia. Lymphocyte counts were normal, but the cells were dysfunctional. T cells failed to proliferate appropriately following several stimuli, and B-cell proliferation was also impaired. Additionally, the number of memory B cells was significantly reduced, and immunoglobulin class switching from IgM to IgG and IgE was defective. These abnormalities resulted in CID and rendered the patients susceptible to infection.

To identify the causative mutation, the research team performed whole-genome sequencing and demonstrated that two patients were homozygous for a missense mutation in the TFRC gene that codes for the transferrin receptor 1 (TfR1), and the obligate carrier father was heterozygous. The mutation segregated with disease phenotype in the extended family and was also present in the CID patient from the second family. Moreover, the mutation was not present in control individuals.

TfR1 is a transmembrane glycoprotein that consists of two monomers joined by disulfide bonds and facilitates the uptake of iron into cells. Iron (Fe³⁺) in the blood plasma is bound to circulating apotransferrin to form a holotransferrin molecule, which interacts with TfR1. This complex is then internalized by the cell through endocytosis. TfR1 maintains cellular iron homeostasis whereby low iron concentrations trigger the binding of regulatory proteins to the iron-responsive element in the 3' UTR of the TfR1 mRNA, which stabilizes the transcript and leads to increased levels of TfR1 protein and enhanced uptake of iron. The cytoplasmic tail of TfR1 contains an internalization motif of ²⁰YTRF²³, and the tyrosine residue is highly conserved in vertebrates. The T>C mutation in the TFRC gene of CID patients causes substitution of the tyrosine by a histidine residue, which disrupts the internalization signal leading to defective receptor endocytosis, which is consistent with the markedly increased levels of TfR1 on the surface of the cells. Lymphocytes are able to take up non–transferrin-bound iron, and in vitro supplementation with iron citrate supersaturates the transferrin molecules so that excess free iron is internalized independently of TfR1. Supplementation experiments with patient cells rescued the lymphocyte defects, demonstrating that insufficient iron uptake is the cause of CID. Further evidence for the pathogenic nature of the PfR1 mutation was provided by Tfrc^Y20H/Y20H knock-in mice that showed the same immunologic abnormalities as the CID patients.

Iron is essential for erythrocyte development and oxygen transport, and knockout mice lacking the TfR1 protein die in utero, so the mild anemia in the CID patients with dysfunctional TfR1 was an unexpected and intriguing finding. Supporting this observation, the expression of TfR1 on patient-derived erythroid precursor cells from bone marrow was only slightly elevated, and precursor cells from knock-in Tfrc^Y20H/Y20H mice showed normal internalization of TfR1. These findings implied that iron transport into red cells may use more than one mechanism. This prompted further research focusing on STEAP3, a conserved ferrireductase, which is highly expressed in red cell precursors but not in lymphocytes. STEAP3 co-localizes with TfR1and possesses a cytoplasmic YXXF internalization motif similar to the ²⁰YTRF²³ in TfR1. Elegant co-immunoprecipitation and transfection experiments with wild-type and an internalization-defective mutant STEAP3 demonstrated that the interaction of TfR1 and STEAP3 facilitates the entry of TfR1 into erythroid precursor cells. This compensatory mechanism is only present in red cells and prevents severe anemia in the CID patients. It is tempting to speculate that the alternative mechanism of iron uptake in erythrocytes, but not lymphocytes, evolved due to the absolute requirement of iron in red cells, which is essential to sustain life.