Intratumoral CD4+CD25+ regulatory T-cell-mediated suppression of infiltrating CD4+ T cells in B-cell non-Hodgkin lymphoma - PubMed (original) (raw)

Intratumoral CD4+CD25+ regulatory T-cell-mediated suppression of infiltrating CD4+ T cells in B-cell non-Hodgkin lymphoma

Zhi-Zhang Yang et al. Blood. 2006.

Abstract

Most non-Hodgkin lymphomas (NHLs) are of B-cell origin, but the tumor tissue can be variably infiltrated with T cells. In the present study, we have identified a subset of CD4(+)CD25(+) T cells with high levels of CTLA-4 and Foxp3 (intratumoral T(reg) cells) that are overrepresented in biopsy specimens of B-cell NHL (median of 17% in lymphoma biopsies, 12% in inflammatory tonsil, and 6% in tumor-free lymph nodes; P = .001). We found that these CD4(+)CD25(+) T cells suppressed the proliferation and cytokine (IFN-gamma and IL-4) production of infiltrating CD4(+)CD25(-) T cells in response to PHA stimulation. PD-1 was found to be constitutively and exclusively expressed on a subset of infiltrating CD4(+)CD25(-) T cells, and B7-H1 could be induced on intratumoral CD4(+)CD25(+) T cells in B-cell NHL. Anti-B7-H1 antibody or PD-1 fusion protein partly restored the proliferation of infiltrating CD4(+)CD25(-) T cells when cocultured with intratumoral T(reg) cells. Finally, we found that CCL22 secreted by lymphoma B cells is involved in the chemotaxis and migration of intratumoral T(reg) cells that express CCR4, but not CCR8. Taken together, our results suggest that T(reg) cells are highly represented in the area of B-cell NHL and that malignant B cells are involved in the recruitment of these cells into the area of lymphoma.

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Figures

Figure 1.

Figure 1.

Antigen expression on CD4+CD25+ T cells from biopsy specimens of patients with B-cell NHL. (A) Dot plots showing CD25 expression on CD4+ T cells in freshly isolated cell suspensions from the tissue types indicated. (B) Frequency (mean ± SD) of CD4+CD25+ among normal peripheral blood mononuclear cells (PBMCs) (n = 6), cells from inflammatory tonsils (n = 6), and benign/reactive LNs (n = 6). *P < .001, compared with benign/reactive LNs. (C) The phenotypic analysis of CD4+CD25+ T cells in B-cell NHL. The line was drawn based on the isotype control. The set of histograms is a representative of 6 samples. (D) Dot plots showing intracellular staining of Foxp3 or CTLA-4 as well as their corresponding isotype control in CD3+ CD4+CD25+/- T cells. Patient sample nos. 1 to 4, nos. 6 to 11, and no. 24 were used in these experiments.

Figure 2.

Figure 2.

Intratumoral Treg cells suppressed autologous infiltrating CD4+CD25- T-cell proliferation. Isolated CD4+CD25- T cells (A) or CD25-depleted cell suspensions (B) from biopsy specimens of B-cell NHL were labeled with CFSE and cocultured with either CD4+CD25- T cells or CD4+CD25+ T cells in the presence or absence of PHA for 5 days. CFSEdim cells were measured as a percentage of the proliferated cells. The histogram shown is representative of 7 and 3 samples for panels B and C, respectively. Patient sample nos. 7 to 9 and nos. 20 to 23 were used in these experiments.

Figure 3.

Figure 3.

CD4+CD25+ T cells from B-cell NHL inhibit cytokine production of infiltrating CD4+CD25- T cells. (A) A representative ELISPOT image of IFN-γ and IL-4 secretion by an unsorted cell suspension and by CD4+ T cells isolated from biopsy specimens of B-cell NHL. (B) Bar graph showing the average number (± SE; n = 6) of IFN-γ and IL-4 spot-forming cells (SFCs) produced by 5 × 105 unsorted, CD4+ or non-CD4+ cells on PHA stimulation. (C) IFN-γ and IL-4 production by CD4+CD25- T cells determined by intracellular staining. IFN-γ- or IL-4-positive cells were determined by gating on the corresponding isotype control. (D) Bar graph showing the average number (± SE; n = 6) of IFN-γ and IL-4 SFCs produced by 5 × 105 total CD4+CD25- T cells incubated with or without CD4+CD25+ T cells on PHA stimulation. Significantly greater IFN-γ secretion (*P < .05) and IL-4 secretion (#P < .01) was seen when compared with cells incubated in the presence of Treg cells. Patient sample nos. 5 to 10 were used in these experiments.

Figure 4.

Figure 4.

The interaction between CCL22 and CCR4 is involved in the migration of CD4+CD25+ T cells to tumor sites of B-cell NHL. (A) Dot plots showing the expression of CCR4 and CCR8 on CD4+ T cells from biopsy specimens of B-cell NHL (n = 6). (B) Histograms showing intracellular expression of CCL17 and CCL22 in CD20+ lymphoma B cells from biopsy specimens of B-cell NHL (n = 6). The shaded histogram represents the isotype control staining, and the open histogram represents the CCL17 or CCL22 staining. (C) The bar graph showing CD4+CD25+ T-cell migration in response to supernatant of lymphoma B cells (SP). CD4+CD25+ T cells were labeled with calcein AM, and the fluorescence intensity of migrated cells was measured by fluorescent plate reader. Results are the mean ± SD, n = 6, *P < .01, compared with media alone; #P < .05, compared with supernatant. Patient sample nos. 3, 10, 11, 16, 17, and 20 to 23 were used in these experiments.

Figure 5.

Figure 5.

The interaction between PD-1 and B7-H1 is involved in intratumoral Treg cell-mediated inhibition of CD4+CD25+ T cells in B-cell NHL. (A) Dot plots showing the expression of B7-H1 and PD-1 on tumor-infiltrating CD4+CD25+/- T cells freshly isolated from B-cell NHL (n = 6). (B) Dot plots showing the expression of B7-H1 on resting (middle) or PHA-activated (right) CD4+CD25+ T cells (n = 3). (C) Histograms showing the effect of the interaction between B7-H1 and PD-1 on intratumoral Treg cell-mediated suppression of infiltrating CD4+CD25- T cells in B-cell NHL. CFSE-labeled CD4+CD25- T cells were cocultured either with CD4+CD25- or CD4+CD25+ T cells in the presence or absence of PHA stimulation for 5 days. Cells in the coculture system were treated with an anti-B7-H1 antibody or PD-1 fusion protein as well as their corresponding controls. The histogram is a representative of 7 samples using the B7-H1 antibody, and of 5 samples using PD-1 Fc. Patient sample no. 11, nos. 15 to 18, and nos. 20 to 23 were used in these experiments.

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