Flow Cytometry.

Epstein–Barr virus (EBV)-transformed B cells were stained with monoclonal anti-CD25 conjugated with phycoerythrin or with isotype-matched control antibodies (Coulter) and were analyzed on an Epics Coulter V flow cytometer.

Western Blots.

Peripheral blood lymphocytes (2 × 10⁶) were lysed in SDS-sample buffer, electrophoresed on SDS/8% polyacrylamide gels, and transferred to nitrocellulose membranes (Hybond-C, Amersham). After nonspecific blocking with 5% milk solids in PBS, immunoblots with anti-IL-2Rα and anti-IL-2Rβ (Santa Cruz Biotechnology) were performed in PBS containing 0.05% Tween-20 and 1% milk solids. Bound antibody was detected with the secondary reagent horseradish peroxidase conjugated with donkey antibody to rabbit immunoglobulin (Amersham) and developed by enhanced chemiluminescence (ECL; Amersham).

Absence of CD1 Expression on Cortical Thymocytes.

Atypically for profound cellular immunodeficiency, the thymus was of normal size, although displaying minimal Hassall’s corpuscles and a lack of distinct cortical–medullary demarcation (Fig. ​(Fig.1,1, top row). Whereas immunohistochemical staining of thymus sections for CD3 (Fig. ​(Fig.11 bottom row), CD2, CD4, CD8, and class I and class II major histocompatibility complex proteins (not shown) revealed normal patterns of expression, staining for CD1 was completely negative (Fig. ​(Fig.11 second row).

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Figure 1

In immunohistological comparison of patient (Left) and normal age-matched (Right) thymus sections, hematoxylin and eosin (H&E) staining revealed fibrous septa separating lobules of lymphoid tissue as seen in normal thymus. However, although cortex and medulla are present in the patient thymus, there is a loss of the normally distinct demarcation between the two areas. Immunohistochemical analysis clearly showed lack of expression of CD1 (second row) but normal staining for CD3 (bottom row), while control samples stained strongly for all three markers. bcl-2 expression in the normal thymus is restricted to the medulla, with few positive cells present within the cortex, whereas the patient’s sample displays a uniformly high level of bcl-2 expression (third row). (Figure is printed at 74% of original magnification.)

The CD1 family consists of five members (a–e), all structurally related to class I major histocompatibility complex proteins (7). In normal thymus CD1a is highly expressed on cortical thymocytes and dramatically down-regulated upon progression into the medulla. These proteins may function as nonclassical antigen-presenting molecules, as CD1b has been shown to present mycobacterial lipoglycan antigens (8, 9). A mutation in CD1 was unlikely to be the primary cause of immunodeficiency, as patient monocytes up-regulated CD1 expression normally upon in vitro stimulation with granulocyte/macrophage colony-stimulating factor and IL-4 (10) (not shown). However, to conclusively exclude this possibility, sequencing of patient CD1a cDNA was performed; it was found to be normal. This suggested a failure to provide appropriate signals for CD1 up-regulation in thymocytes. Currently, the significance of CD1 deficiency during T cell differentiation is difficult to identify. It is possible that rather than serving simply as a nonclassical antigen-presenting molecule, CD1a may participate in mediating thymocyte–epithelium interactions and in its absence, be partially responsible for the loss of the distinct corticomedullary boundary.

Absence of IL-2R α Chain (CD25) Expression.

Since expression of CD25 occurs at an early stage in thymocyte differentiation, preceding CD1 up-regulation (11), we hypothesized that an IL-2 signal may be necessary for CD1 induction. Indeed, immunohistochemical analysis of patient thymus sections revealed an absence of CD25 expression (not shown). Similarly, an EBV-transformed cell line derived from patient peripheral B lymphocytes did not express detectable CD25 by flow cytometry, whereas EBV-transformed lines from normal individuals typically contained 20–40% CD25-positive cells (Fig. ​(Fig.22A). Western blot analysis of lysates prepared from patient peripheral blood lymphocytes confirmed the absence of CD25 protein (Fig. ​(Fig.22B). In contrast, expression of the IL-2R β chain was elevated in patient lymphocytes, presumably reflecting compensation for the lack of the α chain CD25.

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Figure 2

(A) EBV-transformed patient B lymphocytes (trace i) and normal control B lymphocytes (trace ii) were stained with anti-CD25 and analyzed by flow cytometry, demonstrating the absence of CD25 expression on patient cells (1.1% positive) compared with a normal control (44% positive). Isotype-matched antibody control staining of patient cells (1.8% positive) is shown in trace iii. LFL2, count. (B) Western blot analysis demonstrates the absence of IL-2Rα protein and elevated IL-2Rβ expression in patient peripheral blood lymphocytes compared with normal.

Two forms of CD25 (12–14) message were isolated from patient lymphocyte RNA by reverse transcriptase–polymerase chain reaction, representing the full-length sequence (coding the active receptor) and a secondary splicing product lacking exon 4 (coding an inactive receptor). All forms of the patient’s CD25 message were found to contain a 4-bp deletion at base pairs 60–64, resulting in a translational frameshift (Fig. ​(Fig.3).3). Translation proceeds for 20 amino acids before the deletion and resultant frameshift occurs, effectively ablating CD25 expression. A further 25 irrelevant amino acids are added before termination. The homozygous nature of the mutation was confirmed by analysis of sequences from the parents, who both demonstrated a normal and 4-bp-deleted allele.

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Figure 3

A 4-bp deletion in the patient’s CD25 gene results in a frameshift in protein translation. The sequence around the deletion (base pairs 60–64) is shown (MUT.) and compared with the sequence and reading frame of normal CD25 (WT.).

Dense Lymphocytic Infiltrates of Multiple Tissues Are Observed.

The phenotype of CD25 deficiency differs significantly from most other cellular immunodeficiencies, with a normal size thymus and most noticeably lymphocytic infiltration of multiple tissues (Fig. ​(Fig.4).4). Unlike CD25^−/− mice, which have infiltrates limited to the gastrointestinal tract (6), human CD25 deficiency manifests dense lymphocytic infiltration of lung (Fig. ​(Fig.44 Upper), liver (Fig. ​(Fig.44 Lower), gut, soft tissue, and bone, accompanied by tissue atrophy and chronic inflammation. The lymphocytic infiltration observed in CD25^−/− mice was suggested to occur as the result of inefficient antigen-mediated deletion of activated mature T cells (6). However, analysis of the human disease suggests that one cause of infiltration may lie in abnormal thymic differentiation.

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Figure 4

Light microscopy of hematoxylin and eosin-stained sections of patient lung biopsy (Upper; ×18,250) shows dilatation of airways, partially collapsed lung tissue, and dense infiltration of lymphocytes in the bronchial wall as well as in the lung parenchyma. No fungal elements were identified by specific staining and no viral particles were identified by electron microscopy (not shown). In addition, hepatitis B and C virus, EBV, and cytomegalovirus sequences were not detected by PCR. Light microscopy of liver sections (Lower; ×18,250) shows preserved lobular architecture, with marked infiltration of lymphocytes in the portal tracts. Consistent with the analysis of the lung biopsy, no fungal or viral elements were identified. Similar dense lymphocytic infiltrates were found in stomach and duodenal biopsies (not shown).

We thank Wilson Chan and Ilan Dalal for their technical assistance and A. Cohen and M. Julius for their critical review of the manuscript. This work was supported by the Medical Research Council of Canada and the Reichmann Immunodeficiency Fund.