Complete arrest from pro- to pre-B cells in a case of B cell-negative severe combined immunodeficiency (SCID) without recombinase activating gene (RAG) mutations

Abstract

The B-cell lineage in a patient with B-cell-negative severe combined immunodeficiency (SCID) was analysed by using antisurrogate light chain (SL) MoAbs. Peripheral CD3(+) T cells and CD19(+) B cells were absent in the patient. The common gamma (gamma c) chain was expressed normally on the patient's peripheral NK cells and his peripheral mononuclear cells did not possess any mutations in recombinase activating gene (RAG)-1, 2. Normal levels of expression of Ku70 and Ku80 protein were found by Western blot analysis. The patient did, however, display an increase in fibroblast sensitivity to irradiation. Furthermore, flow cytometric analyses of bone marrow cells showed that surface IgM and cytoplasmic mu positive cells were absent and that CD19(+) B cells were composed of only CD34(+) terminal deoxynucleotidyl transferase (TdT)(+) SL(+) pro-B cells. The complete arrest of pro- to pre-B cell development in the SCID patient's bone marrow suggests that some genes involved in V(D)J recombination, excepting the RAG gene, may play a causative role in the immunodeficiency.

Fig. 1

Pedigree and biological status. Peripheral blood counts of T, B and NK cells were determined by immunostaining with anti-CD2, anti-CD3, anti-CD4, anti-CD8, anti-CD19, anti-CD20 and anti-CD56 MoAbs. *NK cell origin; n.d., not done.

Fig. 2

Sensitivity of fibroblasts to irradiation. Primary skin fibroblasts obtained from the patient and normal control (ie lacking increased sensitivities to irradiation) were cultured. The fraction of fibroblasts surviving after increasing doses (0·5–6 Gy) of γ-radiation was determined. Results are expressed as survival relative to non-irradiated fibroblasts. ○, Normal fibroblast; •, patient's fibroblast.

Fig. 3

The expression levels of Ku as determined by Western blot. Western blot analysis with anti-Ku70, anti-Ku80 and positive control (anti-HO-2) MoAbs was performed. Lysates were obtained from EBV transformed B cells (normal controls; 1, 2 and 3, patient; 4).

Fig. 4

Flow cytometric analysis of bone marrow B cells from a normal age-matched control (left) and the patient (right). Bone marrow cells were stained with anti-SL (HSL96)-PE/anti-CD19-PerCP or anti-IgM-FITC/anti-SL (HSL96)-PE//anti-CD19-PerCP. The bone marrow cells after permeabilization were also stained with anti-TdT-FITC/anti-SL (HSL96)-PE//anti-CD19-PerCP. Lymphoid-gated cells were analysed by flow cytometry. Anti-SL/anti-IgM and anti-SL/anti-TdT are shown after being gated on CD19⁺ cells by means of three-colour immunofluorescence. The same results were obtained when anti-λ5 (HSL11) or anti-pre-B-receptor (HSL2) MoAbs were used.