A variant of SCID with specific immune responses and predominance of gamma delta T cells

Abstract

We describe here a patient with a clinical and molecular diagnosis of recombinase activating gene 1-deficient (RAG1-deficient) SCID, who produced specific antibodies despite minimal B cell numbers. Memory B cells were detected and antibodies were produced not only against some vaccines and infections, but also against autoantigens. The patient had severely reduced levels of oligoclonal T cells expressing the alphabeta TCR but surprisingly normal numbers of T cells expressing the gammadelta TCR. Analysis at a clonal level and TCR complementarity-determining region-3 spectratyping for gammadelta T cells revealed a diversified oligoclonal repertoire with predominance of cells expressing a gamma4-delta3 TCR. Several gammadelta T cell clones displayed reactivity against CMV-infected cells. These observations are compatible with 2 non-mutually exclusive explanations for the gammadelta T cell predominance: a developmental advantage and infection-triggered, antigen-driven peripheral expansion. The patient carried the homozygous hypomorphic R561H RAG1 mutation leading to reduced V(D)J recombination but lacked all clinical features characteristic of Omenn syndrome. This report describes a new phenotype of RAG deficiency and shows that the ability to form specific antibodies does not exclude the diagnosis of SCID.

Figure 1

Multifocal monoclonal EBV-induced lymphoproliferation. (A) CT scan of the lung demonstrating large ovaloid lesions. (B and C) Polymorphic lymphoproliferation of the lung consisting of CD20⁺ B cells that coexpressed EBV LMP-1. (D) Clonality analysis of lymphoproliferative lesions. IgH gene scan profiles from lung and mastoid biopsy DNA are shown.

Figure 2

B cell phenotype. (A) Proportion of CD19⁺ B cells in the patient (Pt) and an age-matched control (Co) patient without immunological abnormalities. (B) Expression of IgD and the memory marker CD27 on CD19⁺ B cells.

Figure 3

Predominance of activated γδ T cells. (A) TCR expression among CD3⁺ lymphocytes in the patient and a healthy control. (B) Expression of CD4 and CD8 among CD3⁺TCRαβ⁺ and CD3⁺TCRγδ⁺ T cells. (C) Expression of CD4 and CD45RA among CD3⁺TCRαβ⁺ and CD3⁺TCRγδ⁺ T cells. (D) Expression of CD4 and HLA-DR among CD3⁺TCRαβ⁺ and CD3⁺TCRγδ⁺ T cells.

Figure 4

T cell proliferative responses. PBMCs from the patient and a healthy control were labeled with the fluorescent dye CFSE and incubated in medium or stimulated with PHA or with anti-CD3/anti-CD28 beads. Five days later, cells were stained with anti-CD4 and anti-CD8 and analyzed by flow cytometry. CFSE dilution is plotted as an overlay of cells incubated in medium (dotted line) versus cells incubated with the indicated stimulus (bold line).

Figure 5

T cell repertoire. (A) Vβ chain expression among CD8⁺ T cells as determined by flow cytometry. (B) CDR3 length profile of various Vβ populations as determined by immunoscope analysis. The relative intensity of the bands is plotted as a function of CDR3 size. Since the nomenclature of the used antibodies is based on Wei et al. (37), the corresponding IGMT nomenclature is also provided in order to correlate the cytometry data with spectratyping results.

Figure 6

Oligoclonal T cell expansions. Heteroduplex analysis of PCR products was performed by nondenaturing PAGE and visualized by ethidium bromide staining.

Figure 7

γδ T cell repertoire analysis. (A) Vδ chain usage among total γδ TCR⁺CD3⁺ T cells. (B) CDR3 length profile of Vδ1–Vδ3 populations using Jδ1–Jδ3–specific primers as determined by immunoscope analysis. (C) Vγ chain usage among total γδ TCR⁺CD3⁺ T cells. (D) CDR3 length profile of Vγ populations using primers specific for Vγ4, VγIF (specific for subgroup I), and Vγ9 (specific for subgroup II).

Figure 8

V(D)J junction sequences of γδ T cells. The V(D)J segments identified are indicated in bold; N sequences are underlined. The number of times a sequence was found among PCR products and among clone sequences is given in parenthesis.

Figure 9

CMV reactivity of Vδ2^– γδ T cell clones. Two Vγ9–Vδ2 and 3 Vδ2^–Vγ9^– clones from healthy controls as well as 6 γδ T cell clones using different combination of T cell receptors from the patient were tested for CMV reactivity. γδ TCR usage of the clones was analyzed with various antibodies. The clones were incubated with CMV-infected and uninfected fibroblasts, and TNF release was quantified in a bioassay on WEHI cells (43). *The antibody used for characterization of these clones reacts with all 3 γ chain proteins.