Human TSLP directly enhances expansion of CD8+ T cells

Abstract

Human thymic stromal lymphopoietin (TSLP) promotes CD4(+) T-cell proliferation both directly and indirectly through dendritic cell (DC) activation. Although human TSLP-activated DCs induce CD8(+) T-cell proliferation, it is not clear whether TSLP acts directly on CD8(+) T cells. In this study, we show that human CD8(+) T cells activated by T-cell receptor stimulation expressed TSLP receptor (TSLPR), and that TSLP directly enhanced proliferation of activated CD8(+) T cells. Although non-stimulated human CD8(+) T cells from peripheral blood did not express TSLPR, CD8(+) T cells activated by anti-CD3 plus anti-CD28 did express TSLPR. After T-cell receptor stimulation, TSLP directly enhanced the expansion of activated CD8(+) T cells. Interestingly, using monocyte-derived DCs pulsed with a cytomegalovirus (CMV)-specific pp65 peptide, we found that although interleukin-2 allowed expansion of both CMV-specific and non-specific CD8(+) T cells, TSLP induced expansion of only CMV-specific CD8(+) T cells. These results suggest that human TSLP directly enhances expansion of CD8(+) T cells and that the direct and indirect action of TSLP on expansion of target antigen-specific CD8(+) T cells may be beneficial to adoptive cell transfer immunotherapy.

Fig. 4

TSLPR and CD80 expression on blood myeloid DCs and monocyte-derived DCs. (a) Purified immature CD11c⁺ blood myeloid DCs and monocyte-derived DCs with or without maturation with IL-6, tumour necrosis factor-α, IL-1β and prostaglandin-E2, were stained with mAb to TSLPR or isotype control. Cell surface marker phenotypes were determined by flow cytometry. Filled histograms represent staining of DCs with TSLPR; open histograms represent isotype controls. Data represent one of three experiments. (b) Indicated DCs were cultured for 24 h with or without TSLP and stained with mAb to CD80 or isotype control. Filled histograms represent staining of DCs with CD80; open histograms represent isotype controls. Numbers in histograms indicate the mean fluorescence intensity. Data represent one of three experiments.

Fig. 1

TCR stimulation induces TSLPR expression on human CD8⁺ T cells. (a) PBMCs were stained with mAb to CD3, CD8 and TSLPR or isotype control. Cell surface marker phenotypes were determined by flow cytometry. Lower panels represent staining of CD3⁺ cells with TSLPR or isotype control. Numbers in quadrants indicate the percentages of cells for each given phenotype in CD3⁺ cells. Data represent one of five independent experiments. (b, c) Purified human CD8⁺ T cells were stimulated for 5 days (b) or indicated days (c) with immobilized anti-CD3 plus soluble anti-CD28, and numbers in quadrants indicate the percentages of cells for each given phenotype in CD3⁺CD8⁺ cells. Data shown from five individuals (b) and one of five independent experiments (c). (d) CD8⁺ T cells were stimulated with anti-CD28 and various concentrations of anti-CD3. Data shown are the percentages of TSLPR⁺ cells in CD3⁺CD8⁺ cells in three individual experiments (closed symbols). Horizontal bars indicate the mean of percentages of TSLPR⁺ cells and open symbols show isotype control from individuals. P-values as determined by the paired t-test. *P < 0·05.

Fig. 2

CD8⁺ T cells express TSLPR on their surface in mixed lymphocyte reaction. Purified human CD8⁺ T cells were cultured with allogeneic MoDCs for 7 days at a DC : T cell ratio of 1:2. Cell surface marker phenotypes were determined by flow cytometry as in Fig. 1b. Data represent one of five independent experiments.

Fig. 3

TSLP directly enhances expansion of functional CD8⁺ T cells induced by TCR stimulation. (a) Cell divisions of CFSE-labelled purified human CD8⁺ T cells stimulated with anti-CD3 plus anti-CD28 with or without TSLP for indicated days. Filled histograms represent CFSE dilution in CD8⁺ T cells. Data represent one of five individual experiments. (b) Closed circles indicate fold expansion of CD8⁺ T cells stimulated with anti-CD3 plus anti-CD28 with or without TSLP compared with the initial CD8⁺ T-cell number in five individual experiments. The lines indicate CD8⁺ T cells from the same individual. (**P < 0·05). (c) The percentages of IFN-γ producing cells in CD8⁺ T cells using intracellular cytokine staining. CD8⁺ T cells were stimulated with anti-CD3 plus anti-CD28 with or without TSLP or IL-2 and restimulated with PMA plus ionomycin. Data represent one of three independent experiments. (d) Expression levels of mRNA encoding Foxp3 were measured using the real-time quantitative RT-PCR. CD8⁺ T cells were stimulated with anti-CD3 plus anti-CD28 with or without TSLP or IL-2. Data represent one of three independent experiments.

Fig. 5

Human TSLP induces predominant expansion of CMV-specific CD8⁺ T cells. (a) Purified human CD8⁺ T cells were stimulated with autologous MoDCs pulsed with HLA-A24-restricted CMVpp65 peptide for 7 days with or without TSLP at a DC : T cell ratio of 1:2. Cells were stained with mAb to cell surface marker phenotypes and HLA-A2402-CMVpp65 tetramer. (b) Fold expansion of tetramer^-CD8⁺ T cells and tetramer⁺CD8⁺ T cells after 7 days of culture with (filled bars) and without (open bars) TSLP. Data represent one of 12 independent experiments. (c) Cell divisions of CFSE-labelled purified human CD8⁺ T cells stimulated for 7 days with autologous MoDCs pulsed with CMVpp65 peptide (upper panels) or with either TSLP (middle panels) or IL-2 (lower panels). Cells were stained with anti-CD8 and CMVpp65 tetramer. Numbers indicate the percentages of proliferated tetramer⁺CD8⁺ T cells (left panels) and proliferated tetramer^-CD8⁺ T cells (right panels) in CD8⁺ cells. Data represent one of five independent experiments.