A reversion of an IL2RG mutation in combined immunodeficiency providing competitive advantage to the majority of CD8+ T cells

Abstract

Mutations in the common gamma chain (γc, CD132, encoded by the IL2RG gene) can lead to B(+)T(-)NK(-) X-linked severe combined immunodeficiency, as a consequence of unresponsiveness to γc-cytokines such as interleukins-2, -7 and -15. Hypomorphic mutations in CD132 may cause combined immunodeficiencies with a variety of clinical presentations. We analyzed peripheral blood mononuclear cells of a 6-year-old boy with normal lymphocyte counts, who suffered from recurrent pneumonia and disseminated mollusca contagiosa. Since proliferative responses of T cells and NK cells to γc -cytokines were severely impaired, we performed IL2RG gene analysis, showing a heterozygous mutation in the presence of a single X-chromosome. Interestingly, an IL2RG reversion to normal predominated in both naïve and antigen-primed CD8(+) T cells and increased over time. Only the revertant CD8(+) T cells showed normal expression of CD132 and the various CD8(+) T cell populations had a different T-cell receptor repertoire. Finally, a fraction of γδ(+) T cells and differentiated CD4(+)CD27(-) effector-memory T cells carried the reversion, whereas NK or B cells were repeatedly negative. In conclusion, in a patient with a novel IL2RG mutation, gene-reverted CD8(+) T cells accumulated over time. Our data indicate that selective outgrowth of particular T-cell subsets may occur following reversion at the level of committed T progenitor cells.

Figure 1.

Increased numbers and disturbed differentiation of CD8⁺ T cells. (A) Absolute numbers of total and CD4⁺ and CD8⁺ T cells, CD4/CD8 ratio, CD3⁻CD56⁺ NK cells and CD19⁺ B cells. Dotted lines indicate upper and lower limits of normal for corresponding age (changing at age 10 years), for CD4/CD8 ratio only the lower limit of normal is shown. (B) Distribution of naïve, memory and effector-memory T cells in both CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells (i.e. CD45RA⁺CD27⁺, CD45RA⁻CD27⁺, CD45RA^+or−CD27⁻, respectively), at 11 years of age compared to values in a 10-year old healthy control. Lower panels: B-cell differentiation (naïve, non-switched or class-switched memory, i.e. sIgD⁺CD27⁻sIgD⁺CD27⁺ or sIgD⁻CD27⁺).

Figure 2.

T-cell proliferation in response to recall antigens and cytokines is diminished. Analysis of proliferation of healthy control and patient CFSE-labeled cells after 6 days of culture with the indicated stimuli. (A) Proliferation of CD4⁺ and CD8⁺ T cells activated with αCD3/αCD28 monoclonal antibodies. (B) Proliferation of antigen-specific CD4⁺ T cells to recall antigens. The number indicate percentages of divided cells minus the background in unstimulated samples. (C) Proliferation of T cells in response to CD132 cytokines IL-2, IL-15 and IL-7. The number indicate percentages of undivided cells. No stim = unstimulated condition.

Figure 3.

X-linked inheritance of the IL2RG gene locus and mutation analysis. (A) Pedigree of the family indicates the X-linked inheritance by the index (III-2) who is alive and well, and the affected brother (II:2) who died from X-CID. The patient’s mother (II:1), one of her sisters (II:4), and the grandmother (I:2) are carriers of the mutated IL2RG gene (circles with dots). (B) Gene sequence analysis is shown for the patient in leukocytes, and sorted total T, B and NK cell subsets. Fifty percent of T cells had a reversion mutation (back mutation, indicated in red). (C) Percentage reversion in T-cell subsets at t=2 years; (naïve CD45RA⁺CD27⁺, memory CD45RA⁻CD27⁺, effector CD45RA^+/−CD27⁻). See also Online Supplementary Table S1.

Figure 4.

Decreased expression of CD132 on lymphocytes of the X-CID patient. CD132 expression on lymphocyte subsets upon 3-day stimulation using antiCD3/antiCD28 monoclonal antibodies. Filled histograms: unstimulated cells, black line: activated cells.

Figure 5.

TCR-β analysis in reverted cells shows a diverse repertoire. (A) CD8⁺CD45RA⁻ (memory) subset and the CD8⁺CD45RA⁺CD27⁺ (naïve) were sorted using FACS (>95% purity). Both samples were prepared for high throughput sequencing using a primer set that covers the complete TCR-β repertoire. From both samples approximately 10,000 TCR-β sequences were sequenced using high throughput sequencing. Each clone is depicted as a dot showing its relative frequency to the complete subset (percentage of total reads). As a comparison we used control samples. (B) Vβ-usage of the same samples used in (A) was determined. Use of V-genes (and J-genes, data not shown) was comparable to that of healthy controls (data not shown).