Thalidomide and its analogues have distinct and opposing effects on TNF-alpha and TNFR2 during co-stimulation of both CD4(+) and CD8(+) T cells

Abstract

Thalidomide (Thd) is clinically useful in a number of conditions where its efficacy is probably related to its anti-TNF-alpha activity. More recently, Thd has also been shown to co-stimulate T cells and second generation co-stimulatory (IMiD trade mark ) analogues are currently being assessed in the treatment of cancer patients. However, in contrast to their known suppressive effects during inflammatory stimuli, the effects of Thd/IMiDs on TNF-alpha and TNF receptors (TNFRs) during T cell co-stimulation are not known. We sought to determine the effect of Thd, two clinically relevant IMiDs (CC-4047, ACTIMID trade mark and CC-5013, REVIMID trade mark ) and a non-stimulatory SelCID analogue (CC-3052) on TNF-alpha production and on the expression and shedding of TNFRs during co-stimulation. We found that co-stimulation of PBMC with Thd/IMiDs, but not CC-3052, prevented alphaCD3-induced T cell surface expression of TNFR2 and thereby reduced soluble TNFR2 (sTNFR2) levels. However, there was no effect on total (surface/intracellular) TNFR2 protein expression, suggesting inhibition of trafficking to the cell membrane. The extent of co-stimulation by Thd/IMiDs (assessed by CD69/CD25 expression and IL-2/sIL-2Ralpha production) was similar for CD4+ and CD8+ T lymphocytes and correlated with TNFR2 inhibition. Co-stimulation, but not the early inhibitory effect on TNFR2, was IL-2-dependent and led to increased TNF-alpha production by both CD4+ and CD8+ T lymphocytes. The clinical relevance of this observation was confirmed by the elevation of serum TNF-alpha during REVIMID trade mark treatment of patients with advanced cancer. Together, these results suggest a possible role for TNF-mediated events during co-stimulation and contrast with the TNF inhibitory effects of Thd and its analogues during inflammatory stimuli.

Fig. 1

Co-stimulatory CC-4047 strongly inhibits the secretion of sTNFR2 by αCD3-stimulated PBMC cultures (but not LPS-stimulated cultures) whereas non-co-stimulatory SelCID CC-3052 augments sTNFR2. Divergent effects of CC-4047 (Ο), CC-3052 (□), and Thd (Δ) on the production of sTNFR2, sTNFR1 & TNF-α by PBMC-stimulated with αCD3 (a–c) and whole blood cultures stimulated with LPS (d–f). Cultures were incubated ± Thd/analogues (10 µg/ml) and supernatants collected at the indicated times as per Materials and methods. Results are expressed as percentage change compared to control cultures with DMSO alone. Data presented are from four normal donors used in two separate experiments.

Fig. 1

Co-stimulatory CC-4047 strongly inhibits the secretion of sTNFR2 by αCD3-stimulated PBMC cultures (but not LPS-stimulated cultures) whereas non-co-stimulatory SelCID CC-3052 augments sTNFR2. Divergent effects of CC-4047 (Ο), CC-3052 (□), and Thd (Δ) on the production of sTNFR2, sTNFR1 & TNF-α by PBMC-stimulated with αCD3 (a–c) and whole blood cultures stimulated with LPS (d–f). Cultures were incubated ± Thd/analogues (10 µg/ml) and supernatants collected at the indicated times as per Materials and methods. Results are expressed as percentage change compared to control cultures with DMSO alone. Data presented are from four normal donors used in two separate experiments.

Fig. 2

CC-4047 and CC-5013 strongly reduced the surface expression of TNFR2 on αCD3 stimulated CD4⁺ and CD8⁺ T cells during PBMC co-stimulation. PBMC were incubated ± Thd/analogues (10 µg/ml) for 48 h. Three-colour flow cytometric analysis was performed by surface staining stimulated PBMC with anti-CD25 FITC, anti-TNFR2 PE and anti-CD4/CD8 PerCP plus appropriate isotype matched and compensation controls as described in Materials and methods. (a) Dot plots represent TNFR2 versus CD25 (IL-2 receptor) gated on (a) CD4 and (b) CD8 T cell subsets. Histograms show mean fluorescent intensity of TNFR2 expression on CD25⁺ T cells. This effect was highly consistent when performed in 10 separate experiments each using PBMC from two normal donors although data from a single representative experiment are shown.

Fig. 2

CC-4047 and CC-5013 strongly reduced the surface expression of TNFR2 on αCD3 stimulated CD4⁺ and CD8⁺ T cells during PBMC co-stimulation. PBMC were incubated ± Thd/analogues (10 µg/ml) for 48 h. Three-colour flow cytometric analysis was performed by surface staining stimulated PBMC with anti-CD25 FITC, anti-TNFR2 PE and anti-CD4/CD8 PerCP plus appropriate isotype matched and compensation controls as described in Materials and methods. (a) Dot plots represent TNFR2 versus CD25 (IL-2 receptor) gated on (a) CD4 and (b) CD8 T cell subsets. Histograms show mean fluorescent intensity of TNFR2 expression on CD25⁺ T cells. This effect was highly consistent when performed in 10 separate experiments each using PBMC from two normal donors although data from a single representative experiment are shown.

Fig. 3

CC-4047 did not alter the level of total cellular TNFR2 (intracellular plus surface) in T cells during PBMC co-stimulation. Unstimulated PBMC and cells stimulated with αCD3 ± CC-4047 (10 µg/ml) for 24 h (TNFR2) (the last 6 h in the presence of the protein transport inhibitor, Brefeldin A) were harvested and processed as described in Materials and methods. Lymphocytes were gated on forward scatter (FSC) versus side scatter (SSC) properties and displayed as two-colour dot-plots, with quadrants set according to isotype-matched controls. No difference was seen in the expression of intracellular TNFR2 during the co-stimulation of CD4⁺ or CD8⁺ T cells. The data presented are representative of three separate experiments in which very similar results were obtained.

Fig. 4

The relative extent of TNFR2 inhibition by CC-4047, CC-5013 and Thd corresponds to the degree of co-stimulation by these compounds. PBMC cultures were co-incubated for 24 h (IL-2), 72 h (sIL-2R) and 120 h (proliferation) ± Thd/analogues (10 µg/ml) as described in Materials and methods. Cell culture supernatants were assayed for IL-2 and sIL-2R by ELISA. Proliferation was assessed by [³H]-thymidine incorporation. Results are expressed as percentage change in (a) IL-2, (b) sIL-2R and (c) proliferation compared to control cultures. Data are from four normal donors used in two separate experiments.

Fig. 5

Co-stimulation by CC-4047 and subsequent TNF-α production, but not early inhibition of TNFR2, is dependent on IL-2-mediated signalling. PBMC cultures were stimulated with αCD3 in the presence of CC-4047 (10 µg/ml) and anti-IL-2 mAb (0·1–0·005 µg/ml) as described in Materials and methods. Results are expressed as percentage values relative to cultures co-stimulated with αCD3 + CC-4047 (0% represents αCD3 alone). Cultures with increasing concentrations of anti-IL-2 mAb show a reduction in (a) supernatant IL-2 levels and (b) supernatant TNF-α levels. Data shown are from four normal donors in two separate experiments.

Fig. 6

Co-stimulation-induced IL-2 and TNF-α are derived from both CD4⁺ and CD8⁺ T cells. Unstimulated PBMC and cells stimulated with αCD3 ± CC-4047 (10 µg/ml) for 96 h (IL-2/TNF-α) (the last 6 h in the presence of the protein transport inhibitor, Brefeldin A) were harvested and processed as described in Materials and methods. Lymphocytes were gated on forward scatter (FSC) versus side scatter (SSC) properties and displayed as two-colour dot-plots, with quadrants set according to isotype-matched controls. The induction of intracellular IL-2 and TNF-α was detected in both CD4⁺ and CD8⁺ T cells. Data shown are from one normal donor and are representative of very similar data obtained from three donors.

Fig. 6

Co-stimulation-induced IL-2 and TNF-α are derived from both CD4⁺ and CD8⁺ T cells. Unstimulated PBMC and cells stimulated with αCD3 ± CC-4047 (10 µg/ml) for 96 h (IL-2/TNF-α) (the last 6 h in the presence of the protein transport inhibitor, Brefeldin A) were harvested and processed as described in Materials and methods. Lymphocytes were gated on forward scatter (FSC) versus side scatter (SSC) properties and displayed as two-colour dot-plots, with quadrants set according to isotype-matched controls. The induction of intracellular IL-2 and TNF-α was detected in both CD4⁺ and CD8⁺ T cells. Data shown are from one normal donor and are representative of very similar data obtained from three donors.

Fig. 7

Treatment of advanced cancer patients with CC-5013/REVIMID™ results in increased TNF-α and sIL-2 receptor levels. Patient serum samples were obtained from a small phase I study as detailed in Materials and methods. Serum was collected at baseline and twice after commencing CC-5013 treatment (weeks 1–3 and weeks 4–5) and assayed for (a) sIL-2R and (b) TNF-α by ELISA. Each symbol represents a single patient.