Complete Multilineage CD4 Expression Defect Associated With Warts Due to an Inherited Homozygous CD4 Gene Mutation

Abstract

Idiopathic T-CD4 lymphocytopenia (ICL) is a rare and heterogeneous syndrome characterized by opportunistic infections due to reduced CD4 T-lymphocytes (<300 cells/μl or <20% T-cells) in the absence of HIV infection and other primary causes of lymphopenia. Molecular testing of ICL has revealed defects in genes not specific to CD4 T-cells, with pleiotropic effects on other cell types. Here we report for the first time an absolute CD4 lymphocytopenia (<0.01 CD4⁺ T-cells/μl) due to an autosomal recessive CD4 gene mutation that completely abrogates CD4 protein expression on the surface membrane of T-cells, monocytes, and dendritic cells. A 45-year-old female born to consanguineous parents consulted because of exuberant, relapsing, and treatment-refractory warts on her hands and feet since the age of 10 years, in the absence of other recurrent infections or symptoms. Serological studies were negative for severe infections, including HIV 1/2, HTLV-1, and syphilis, but positive for CMV and EBV. Blood analysis showed the absence of CD4⁺ T-cells (<0.01%) with repeatedly increased counts of B-cells, naïve CD8⁺ T-lymphocytes, and particularly, CD4/CD8 double-negative (DN) TCRαβ⁺ TCRγδ^- T-cells (30% of T-cells; 400 cells/μl). Flow cytometric staining of CD4 using monoclonal antibodies directed against five different epitopes, located in two different domains of the protein, confirmed no cell surface membrane or intracytoplasmic expression of CD4 on T-cells, monocytes, and dendritic cells but normal soluble CD4 plasma levels. DN T-cells showed a phenotypic and functional profile similar to normal CD4⁺ T-cells as regards expression of maturation markers, T-helper and T-regulatory chemokine receptors, TCRvβ repertoire, and in vitro cytokine production against polyclonal and antigen-specific stimuli. Sequencing of the CD4 gene revealed a homozygous (splicing) mutation affecting the last bp on intron 7-8, leading to deletion of the juxtamembrane and intracellular domains of the protein and complete abrogation of CD4 expression on the cell membrane. These findings support previous studies in CD4 KO mice suggesting that surrogate DN helper and regulatory T-cells capable of supporting antigen-specific immune responses are produced in the absence of CD4 signaling and point out the need for better understanding the role of CD4 on thymic selection and the immune response.

Keywords: CD4; CD4 lymphopenia; double-negative T-cells (DNTs); idiopathic CD4 lymphocytopenia; warts.

Figure 1

Photographs of warts present in the patient's feet.

Figure 2

Schematic representation of the CD4 protein molecule (A) and CD4 surface membrane expression in the patient's (vs. healthy donor) blood CD8⁻ TCRγδ⁻ T-cells, monocytes, and dendritic cells (B). Panel (A) shows a schematic representation of the CD4 protein molecule including the localization of epitopes identified by the distinct antibody clones used in this study (–31). Amino acid positions that conform each domain are indicated between brackets. Black arrows depict the domain that contains those epitopes that the antibody clones are directed to. Panel (B) shows CD4 surface membrane expression levels for CD8⁻ TCRγδ⁻ T-cells, monocytes, and dendritic cells for the different anti-CD4 antibody clones tested in the patient (black histogram) compared to a representative healthy donor (gray histogram) and an isotype control (red dash line), and the staining for a negative population (CD8⁺ T-cells) in the patient (green line) and the healthy control (blue line). DCs, dendritic cells.

Figure 3

Nucleotide sequences of CD4 PCR products for the regions of interest and their localization in the CD4 gene. Panel (A) shows a representative sequence of CD4 focused on the region of interest. Gray underlining shows the nucleotide sequence affected by the major deletion (NM_000616: r.1157_1278del), and orange top line shows the nucleotide sequence affected by the minor deletion (NM_000616: r.1157_1185del). Panels (B,C) show mRNA electropherograms obtained from analysis of the minor (NM_000616: r.1157_1185del) and major (NM_000616: r.1157_1278del) deletions detected in the patient's CD4 cDNA, respectively, and the patient's relatives (C). Panels (D–F) show genomic DNA electropherograms from: (D), wild type CD4 gene; (E), patient's homozygous mutated CD4 gene; and (F), heterozygous mutated CD4 gene from the patient's parents and children. Panels (G,H) display a schematic representation of the localization in wild type CD4 protein of the mutated (green) nucleotide bases and the deleted (red) amino acid sequences, including the minor (G) and major (H) deletions, observed in the truncated CD4 cDNA molecules detected in the CD4^null patient here reported. Panel (I) shows the family pedigree (men are represented as squares and women as circles), including the key clinical manifestations and, between brackets, the results of CD4 DNA analysis in a coded/graphic format: half-shaded circles and squares, heterozygous DNA; fully shaded circles and squares, homozygous mutated DNA; and unshaded circles and squares, wild type DNA.