Enhanced apoptosis of T cells in common variable immunodeficiency (CVID): role of defective CD28 co-stimulation

Abstract

CVID is a primary immune disorder in which hypogammaglobulinaemia may be associated with a number of T cell defects including lymphopenia, anergy, impaired lymphocyte proliferation and deficient cytokine secretion. In this study we show that T cells of CVID subjects, in comparison with control T cells, undergo spontaneous apoptosis in culture and markedly accelerated apoptosis after gamma-irradiation. Although costimulation of the CD28 receptor following engagement of the TCR/CD3 receptor normally provides a second signal necessary for IL-2 secretion, CD28 costimulation in CVID does not significantly increase IL-2 production, nor does this combination of activators enhance the survival of irradiated CVID T cells, as it does for cultured normal T cells. Addition of IL-2 enhances CVID T cell survival, suggesting that the IL-2 signalling pathways are normal. CVID T cells have similar expression of Bcl-2 to control T cells. CD3 stimulation up-regulates T cell expression of bcl-xL mRNA for normal T cells, but anti-CD28 does not augment bcl-xL expression for CVID subjects with accelerated apoptosis. Defects of the CD28 receptor pathway, leading to cytokine deprivation and dysregulation of bcl-xL, could lead to poor T cell viability and some of the cellular defects observed in CVID.

Fig. 1

Cell cycle analysis of T cells of one CVID subject after permeabilization and staining with propidium iodide at 0, 24, 48, and 72 h of culture. The percentage of cells in the apoptotic fraction is indicated in each histogram. (Results similar to three other CVID subjects studied; two normal subjects showed 2·3% of apoptotic cells after 72 h of culture).

Fig. 2

Flow cytometric detection of APO2.7–PECy5 in CD4⁺ and CD8⁺ T cells after 24 h of culture. The numbers show the percentages of CD4⁺ and CD8⁺ T cells that are positively stained for APO2.7 for one CVID subject (ratio CD4/CD8 = 3·0) in comparison with one representative control (ratio CD4/CD8 = 1·5) (five CVID and four normal controls studied).

Fig. 3

T cells of 10 normal subjects (upper panel) or 16 CVID subjects (lower panel) were tested for survival before and following γ-irradiation. T cells were cultured in medium alone (□), or stimulated with plate-immobilized anti-CD3 alone (shaded bars) or with anti-CD3 plus anti-CD28 (hatched bars) for 16 h and then exposed to 15 Gy of γ-irradiation. Percent viability of each group was assessed by CD3 staining and propidium iodide exclusion. CVID T cells had significantly poorer T cell viability on days 1 and 2 after irradiation (**P < 0·01; *P < 0·05, respectively). CD3 stimulation enhanced CVID T cell viability on day 2 (†P < 0·05) and normal T cell viability on days 2 and 5 after irradiation (††P < 0·05). CD28 costimulation further increased normal T cell viability on day 5 (‡P < 0·05). The boxes represent the range, the line inside the box represents the median value.

Fig. 4

Pretreatment with IL-2 enhances the survival of normal (upper panel) and CVID (lower panel) T cells after γ-irradiation. Normal or CVID T cells were cultured overnight and then kept either in the original medium, or in medium with recombinant 100 U/ml IL-2 added prior to irradiation. The data refer to five normal controls and eight CVID subjects. The boxes represent the range, the line inside the box represents the median value. (*P < 0·05; **P < 0·01 difference for IL-2-treated versus no IL-2-treated, significant for both groups).

Fig. 5

T cells of CVID subjects were examined for up-regulation of bcl-xL gene expression, testing cells incubated with no stimulators, or with anti-CD3, or anti-CD3 plus anti-CD28 in a polymerase chain reaction (PCR) using primers specific for bcl-xL or actin, as a sample control. The positive control was Jurkat cells transfected with bcl-x. The upper panel shows PCR results for two normals and four CVID subjects; a, no stimulation; b, CD3 stimulation; c, CD3 + CD28 stimulation. CVID subjects had enhanced bcl-xL expression after CD3 stimulation which was not augmented by addition of anti-CD28. The actin sample controls are shown (lower panels) for each. M, Molecular weight markers; +, positive control.