Human autologous mixed lymphocyte reaction as an in vitro model for autoreactivity to apoptotic antigens

Abstract

Recent studies have indicated that cells undergoing apoptosis are the source of autoantigens which drive autoimmune responses in systemic lupus erythematosus (SLE). It has been recognized for many years that in vitro stimulation of T cells with irradiated major histocompatibility complex (MHC) class II-bearing autologous cells results in T-cell proliferation with immunological specificity and memory, namely the autologous mixed lymphocyte reaction (AMLR). The nature of the major stimulants in the AMLR is still unclear. We investigated whether apoptotic fragments from irradiated cells act as antigenic stimulators for AMLR or nucleohistone-primed T cells. T-cell proliferation in the primary AMLR was significantly suppressed by the presence of a caspase inhibitor Z-Val-Ala-Asp-CH2F (Z-VAD.fmk), indicating that apoptotic antigens released from irradiated autologous feeder cells act as stimulators of AMLR T cells. This inhibitory effect of Z-VAD was not caused by toxic effects, because the T-cell response to the mitogen phytohaemagglutinin (PHA) was not inhibited by Z-VAD. A nucleohistone preparation was shown to contain antigens that are important in the AMLR, as culture with nucleohistone (but not with thyroglobulin or hen-egg lysozyme) primed T cells to respond with secondary kinetics in a subsequent AMLR that was also suppressed by Z-VAD. Our data provide evidence that the AMLR constitutes a model for the evaluation of cellular and molecular mechanisms that may be relevant to the pathogenesis of SLE and similar autoimmune diseases.

Figure 1

(a) Gel electrophoresis of nucleohistone (NH). A 7·5-µg sample of NH preparation was loaded onto gels, as described in the text. Arrows indicate molecular-weight markers, and histone positions are presented on the right of the gel. (b) Ethidium bromide-stained agarose-gel electrophoresis of NH. Lane 1, DNA base pair (bp) marker; lane 2, NH sample.

Figure 2

Primary and secondary immune responses in a human autologous mixed lymphocyte reaction (AMLR). (a) Primary AMLR. Responder cells (1 × 10⁵) were cultured in the absence of feeder cells (closed triangles) or in the presence of 1 × 10⁵ (open circles), 4 × 10⁵ (closed circles) or 6 × 10⁵ (open triangles) autologous-irradiated feeder cells, in triplicate wells for 9 days. (b) Secondary AMLR. Viable cells (1 × 10⁵) from the day-10 primary AMLR (panel a) were cultured with 0·5 × 10⁵ (data not shown), 1 × 10⁵ (open circles), 4 × 10⁵ (closed circles), or 6 × 10⁵ (open triangles) autologous-irradiated feeder cells, for 7 days. T-cell proliferation assays were carried out with these cultured cells. Each point represents the mean value of triplicate wells, and error bars indicate the standard deviation (SD) (representative of five experiments). c.p.m., counts per minute.

Figure 3

Detection of apoptotic cells with fractional DNA content based on cellular DNA content analysis. Non-irradiated (a) and gamma-irradiated (b) feeder cells were cultured for 48 hr. Some of the latter cells were cultured with 5 µm (c), 10 µm (d), 20 µm (e), or 30 µm (f) caspase inhibitor Z-Val-Ala-Asp-CH₂F (Z-VAD.fmk) from the beginning of culture. The cells were then fixed in 70% ethanol, permeabilized and stained with propidium iodide (PI), and their fluorescence was measured by flow cytometry. The population of early and late apoptotic cells is represented by the M1 fraction.

Figure 4

The effect of the caspase inhibitor Z-Val-Ala-Asp-CH₂F (Z-VAD.fmk) on the autologous mixed lymphocyte reaction (AMLR) of normal individuals (representative of four experiments). The day of the AMLR culture is designated on the x-axis, and proliferative responses, in counts per minute (c.p.m.), designated by vertical bars. The results represent the mean of triplicate wells (1 × 10⁵ responding cells and 4 × 10⁵ stimulating cells/well), and error bars indicate the standard deviation (SD). The AMLR proliferative response in the presence of 10 µm Z-VAD (black bar) is shown compared to that in the presence of 15 µm dimethylsulphoxide (DMSO) (grey bar) (control). The white bar corresponds to the c.p.m. measured in the absence of stimulating cells. The P-value is shown above the bars.

Figure 5

Lack of inhibitory effect of the caspase inhibitor Z-Val-Ala-Asp-CH₂F (Z-VAD.fmk) on T-cell proliferation of phytohaemagglutinin (PHA)-stimulated cells. T-cell proliferation assays were carried out for 3 days with peripheral blood mononuclear cells (PBMC) from healthy normal donors stimulated with PHA (0·5, 1, 2 or 4 µg/ml) in the absence of Z-VAD, or in the presence of 10 or 30 µm Z-VAD. Each bar represents the mean value of triplicate wells (2 × 10⁵ cells per well), and error bars indicate the standard deviation (SD). Data are representative of five experiments. c.p.m., counts per minute.

Figure 6

(a) Peripheral blood mononuclear cells (PBMC) from normal individuals were initially stimulated with 50 µg/ml bovine nucleohistone (NH) (closed circles), 10 µg/ml hen-egg lysozyme (HEL) (open circles), 10 µg/ml bovine thyroglobulin (Tg) (open triangles) or medium alone (open diamonds), for 11 days. T-cell proliferation assays were carried out with these cells stimulated with an equivalent number of irradiated autologous feeder cells. (b) Day-11 NH-primed cells were challenged with feeder cells (closed circles) and day-11 Tg-primed cells were stimulated with 10 µg/ml bovine Tg alone (open circles), feeder cells alone (open triangles), or Tg plus feeder cells (closed triangles). Each point represents the mean value of triplicate wells (1 × 10⁵ primed cells and 1 × 10⁵ feeder cells per well), and error bars indicate the standard deviation (SD). The data are representative of five experiments. *P < 0·05 comparing Tg-primed cells stimulated with feeder cells alone or with Tg plus feeder cells. c.p.m., counts per minute.

Figure 7

The effects of caspase inhibitor Z-Val-Ala-Asp-CH₂F (Z-VAD.fmk) on the secondary autologous mixed lymphocyte reaction (AMLR) response following nucleohistone (NH) priming. Peripheral blood mononuclear cells (PBMC) were initially stimulated with 50 µg/ml bovine NH for 11 days. T-cell proliferation assays were carried out with these cells stimulated with equivalent numbers of irradiated autologous feeder cells in the presence of 10 µm (closed squares) or 30 µm (closed triangles) Z-VAD, or medium alone (closed circles). Cultures with 15 µm (open squares) or 45 µm (open triangles) dimethylsulphoxide (DMSO) were used as controls for 10 µm and 30 µm Z-VAD, respectively. Each point represents the mean value of triplicate wells (1 × 10⁵ primed-cells and 1 × 10⁵ feeder cells per well), and error bars indicate the standard deviation (SD). *P < 0·001 comparing NH-primed cells cultured in the presence of 30 µm Z-VAD with DMSO controls.