Proteasome inhibition suppresses essential immune functions of human CD4+ T cells

Abstract

The proteasome constitutes the central proteolytic component of the highly conserved ubiquitin-proteasome system, which is required for the maintenance and regulation of basic cellular processes, including differentiation, proliferation, cell cycling, gene transcription and apoptosis. Here we show that inhibition of proteasomal proteolytic activity by the proteasome inhibitors bortezomib and lactacystin suppresses essential immune functions of human CD4(+) T cells activated by allogeneic dendritic cells (DCs). In activated CD4(+) T cells, proteasome inhibition induces apoptosis accompanied by rapid accumulation and stabilization of the tumour suppressor protein p53. Activated CD4(+) T cells surviving proteasome inhibition undergo inhibition of proliferation by induction of G(1) phase cell-cycle arrest. Induction of G(1) arrest is accompanied by the accumulation of cyclin-dependent kinase inhibitors p21(WAF1/CIP1) and p27(KIP1) and the disappearance of cyclin A, cyclin D2 and proliferating cell nuclear antigen, proteins known to regulate G(1) to S phase cell-cycle transitions. Expression of the activation-associated cell surface receptors CD25, CD28, CD120b and CD134 as well as production of interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), interleukin-4 (IL-4) and IL-5 is suppressed in response to proteasome inhibition in CD4(+) T cells activated by DCs. Expression of CD25, IFN-gamma, TNF-alpha, IL-4 and IL-5 is known to be mediated by the transcriptional activity of nuclear factor of activated T cells (NFAT), and we show here that proteasome inhibition suppresses activation and nuclear translocation of NFATc2 in activated CD4(+) T cells. Thus, the proteasome is required for essential immune functions of activated CD4(+) T cells and can be defined as a molecular target for the suppression of deregulated and unwanted T-cell-mediated immune responses.

Figure 1

Inhibition of proteasomal proteolytic subunits and proteasomal protein degradation by bortezomib and lactacystin in activated CD4⁺ T cells. (a) Immunoblot analysis of expression of the proteasomal proteolytic subunits β1, β1i, β2, β2i, β5 and β5i in CD4⁺ T cells activated for 5 days with allogeneic DCs. (b) Immunoblot analysis of affinity labelling of the proteasomal proteolytic subunits β1, β1i, β2, β2i, β5 and β5i in activated CD4⁺ T cells using the specific affinity probe BioALVS. Cells were exposed for 1 hr to the indicated concentrations of bortezomib (Bor) or lactacystin (LC). (c) Immunoblot analysis of accumulation of polyubiquitinated proteins in activated CD4⁺ T cells using the FK2 monoclonal antibody which detects mono- and polyubiquitinated proteins. Immunoblots were performed in triplicate with similar results.

Figure 2

Apoptosis induced by proteasome inhibition in activated CD4⁺ T cells. (a) Flow cytometric analysis of cytoplasmic uptake of propidium iodide (PI) and binding of annexin V to the plasma membrane externalization of phosphatidylserine of resting CD4⁺ T cells and CD4⁺ T cells activated for 4 days with allogeneic dendritic cells (DCs) at a DC : T-cell ratio of 1 : 10. Before flow cytometric analysis, cells were exposed for 24 hr to dimethylsulphoxide (DMSO), bortezomib (Bor) or lactacystin (LC). (b) Specific apoptosis of resting and activated CD4⁺ T cells exposed for 24 hr to bortezomib or lactacystin. Specific apoptosis was determined by flow cytometry and calculated as described in the Materials and methods. Data are given as mean values ± SE of the mean of six independent experiments carried out in triplicate. (c) Immunoblot analyses of accumulation of p53 and polyubiquitinated p53 (p53-Ub_n) in activated CD4⁺ T cells exposed for the indicated times to bortezomib or DMSO. Amounts of β-actin are demonstrated as a control of equal protein loading. Immunoblots were performed in triplicate with similar results. (d) Demonstration of the purity of the CD4⁺ T-cell population and the ratio of CD4⁺ T cells and DCs in the coculture experiments for CD4⁺ T-cell activation. Cell surface expression of CD4 and CD209 was determined by flow cytometry after 5 days of coculture. A dot plot of one representative experiment out of 10 is shown.

Figure 3

Inhibition of proliferation of activated and restimulated CD4⁺ T cells by proteasome inhibition. (a) Cells were activated for 4 days with allogeneic dendritic cells (DCs) at a DC : T-cell ratio of 1 : 10. Cells were subsequently exposed for 24 hr to dimethyl sulphoxide (DMSO), bortezomib (Bor) or lactacystin (LC), and incorporation of [³H]thymidine was determined as described in the Materials and methods. Data are given as mean values ± SE of the means of six independent experiments carried out in triplicate. (b) Cells were activated for 4 days with allogeneic DCs at a DC : T-cell ratio of 1 : 10, subsequently exposed for 24 hr to DMSO, Bor or LC and thereafter restimulated for 24 hr with the same allogeneic DCs used for initial activation, third-party allogeneic DCs or lectin. Data are given as mean values ± SE of the mean of six independent experiments carried out in triplicate. Incorporation of [³H]thymidine during restimulation was determined as described in the Materials and methods.

Figure 4

Induction of G₁ cell-cycle arrest and regulation of cell cycle- and proliferation-related proteins by proteasome inhibition in activated CD4⁺ T cells. (a) Flow cytometric analysis of DNA content of CD4⁺ T cells activated for 4 days with allogeneic dendritic cells (DCs) at a DC : T-cell ratio of 1 : 10 and subsequently exposed for 24 hr to dimethyl sulphoxide (DMSO), bortezomib (Bor) or lactacystin (LC). (b) Percentages of activated CD4⁺ T cells in G₁ cell-cycle phase after exposure of the cells for 24 hr to DMSO, Bor or LC. Data are given as mean values ± SE of the mean of six independent experiments carried out in triplicate. (c) Immunoblot analysis of intracellular amounts of p21^WAF1/CIP1, p27^KIP1, proliferating cell nuclear antigen (PCNA), cyclin A and cyclin D2 in activated CD4⁺ T cells exposed for the indicated times to Bor or for 24 hr to DMSO. Amounts of β-actin are demonstrated as a control of equal protein loading. Immunoblots were performed in triplicate with similar results.

Figure 5

Suppression of cell surface expression of activation-associated receptors by proteasome inhibition in activated and restimulated CD4⁺ T cells. (a) Cell surface expression of CD25, CD28, CD27, CD120b, CD95 and CD134 in CD4⁺ T cells activated for 4 days with allogeneic dendritic cells (DCs) at a DC : T-cell ratio of 1 : 10 and subsequently exposed for 24 hr to dimethyl sulphoxide (DMSO), bortezomib (Bor) or lactacystin (LC). Data are given as mean values ± SE of the mean of five independent experiments carried out in triplicate. (b) Cell surface expression of CD25, CD28 and CD134 in restimulated CD4⁺ T cells. Cells were activated for 4 days with allogeneic DCs at a DC : T-cell ratio of 1 : 10, subsequently exposed for 24 hr to DMSO or Bor and thereafter restimulated for 24 hr with the same allogeneic DCs used for initial activation, third-party allogeneic DCs or lectin. Data are given as mean values ± SE of the mean of four independent experiments carried out in triplicate. Cell surface expression of the activation-associated receptors was determined in viable cells by flow cytometry as described in the Materials and methods.

Figure 6

Inhibition of production of T-cell cytokines by proteasome inhibition in activated and restimulated CD4⁺ T cells. Inhibition of cytokine production in activated CD4⁺ T cells restimulated with (a) the same allogeneic dendritic cells (DCs) used for initial activation, (b) third-party allogeneic DCs, and (c) lectin. Cells were activated for 4 days with allogeneic DCs at a DC : T-cell ratio of 1 : 10, subsequently exposed for 24 hr to dimethyl sulphoxide (DMSO) or bortezomib (Bor) and thereafter restimulated for 24 hr. After restimulation for 24 hr, amounts of interleukin-2 (IL-2), soluble IL-2 receptor α (IL-2sRα), tumour necrosis factor-α (TNF-α), interferon-γ (IFN-γ), IL-4 and IL-5 were determined in the culture supernatant by enzyme-linked immunosorbent assay as described in the Materials and methods. Data are given as mean values ± SE of the mean of five independent experiments carried out in triplicate.

Figure 7

Suppression of nuclear translocation and abundance of nuclear factor of activated T cells (NFAT) c2 by proteasome inhibition in activated CD4⁺ T cells. Cells were activated for 4 days with allogeneic dendritic cells (DCs) at a DC : T-cell ratio of 1 : 10 and subsequently exposed for the indicated times to dimethyl sulphoxide (DMSO) or bortezomib (Bor). Amounts of NFATc2 were determined in the nuclear and cytoplasmic fractions of the cells by immunoblotting as described in the Materials and methods. Amounts of lamin A/C and β-actin are demonstrated as controls of equal protein loading of the nuclear and cytoplasmic fractions, respectively. Immunoblots were performed in triplicate with similar results.