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. 2006 Oct 15;66(20):10145-52.
doi: 10.1158/0008-5472.CAN-06-1822.

Attenuation of CD8(+) T-cell function by CD4(+)CD25(+) regulatory T cells in B-cell non-Hodgkin's lymphoma

Zhi-Zhang Yang  1 , Anne J Novak, Steven C Ziesmer, Thomas E Witzig, Stephen M Ansell
Affiliations

Attenuation of CD8(+) T-cell function by CD4(+)CD25(+) regulatory T cells in B-cell non-Hodgkin's lymphoma

Zhi-Zhang Yang et al. Cancer Res. .

Abstract

The underlying mechanisms by which tumor cells are resistant to CTL-mediated apoptosis are not clear. Using a human model of B-cell non-Hodgkin's lymphoma (B-cell NHL), we show that intratumoral T(reg) cells inhibit the proliferation and granule production of activated autologous infiltrating CD8(+) T cells. Our results also show that degranulation and subsequent cytotoxic activity of infiltrating CD8(+) T cells exposed to lymphoma B cells is completely attenuated by the presence of intratumoral T(reg) cells. Furthermore, we show that increased numbers of intratumoral T(reg) cells correlates with the number of CD8(+) T cells in biopsy specimens from patients with B-cell NHL, supporting the in vitro findings that intratumoral T(reg) cells inhibit proliferation of infiltrating CD8(+) T cells. Taken together, these data indicate that human lymphoma B cells are sensitive to autologous CTL-mediated cell death but are protected by the inhibitory function of intratumoral T(reg) cells.

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Figures

Figure 1
Figure 1
Identification of Foxp3-expressing cells in biopsy specimens of patients with B-cell NHL. Immunohistochemical analysis of Foxp3 expression in NHL tumor tissue was done as described in Materials and Methods. Expression of CD25, CD4, CD8, CD3, and CD20 was also performed. From the same region of a serial section of the tumor specimen. Original magnification,×40. Representative specimen (n = 11).
Figure 2
Figure 2
Intratumoral Treg cells inhibit the proliferation of infiltrating CD8+ T cells in B-cell NHL. Proliferation of infiltrating CD8+ T cells was determined by coculturing CFSE labeled CD8+ T cells with autologous CD4+CD25- or CD4+CD25+ T cells in the presence (activated) or absence of PHA (resting) for 3 days. The percentage of cells that lost CFSE expression were considered to have undergone proliferation. Representative of three independent experiments.
Figure 3
Figure 3
Intratumoral Treg cells dose-dependently inhibit the production of PFN and GzmB by infiltrating CD8+ T cells in B-cell NHL. A, intracellular staining of PFN or GzmB expression as well as coexpression of PFN and GzmB in CD8+T cells from fresh biopsy specimens of B-cell NHL. Isotype controls are shown for each sample (top). B, summary of the percentage of CD8+ T cells expressing PFN, GzmB, or both from 25 patient samples. C to D, intracellular expression of PFN (C) and GzmB (D) in infiltrating CD8+ T cells cocultured either alone or with infiltrating CD4+CD25- T cells or intratumoral Treg cells in the presence or absence of PHA for 3 days. Representative of three independent experiments.
Figure 4
Figure 4
Intratumoral Treg cells inhibit the degranulation of infiltrating CD8+ T cells in B-cell NHL. A, expression of CD107a on PHA-activated infiltrating CD8+ T cells in freshly isolated biopsy specimens of B-cell NHL at the indicated time points. B, CD107a expression on infiltrating CD8+ T cells exposed to Raji cells at the indicated E/T ratios. C, CD107a surface expression on activated CD8+ T cells exposed to Raji cells alone (i) or in the presence of CD4+CD25-T cells (ii)orTreg cells (iii). Resting CD8+T cells (iv) or activated CD8+ T cells in the absence of Raji (v) serve as negative controls.
Figure 5
Figure 5
Intratumoral Treg cells inhibit the cytotoxicity of infiltrating CD8+ T cells to lymphoma B cells. A, cytotoxic activity, measured by specific lysis of targets cells, was analyzed in activated (solid line) or resting (dashed line) CD8+ T cells at the indicated E/T ratio. Target cells include DoHH2, Karpas, and Raji cells (n = 3). Cytotoxic activity of resting or activated infiltrating CD8+ T cells cocultured with CD4+CD25- or CD4+CD25+ T cells toward Raji (n =3; B) or autologous lymphoma B-cells (n =6; C) at the indicated E/T ratios. The percentage of 7-AAD+ cells was used to calculate the % lysis of target cells by CD8+ T cells. Points, mean; bars, SD. *, P < 0.05.
Figure 6
Figure 6
Increased numbers of intratumoral Treg cells are associated with decreased numbers of infiltrating CD8+ T cells in B-cell NHL. A, relationship between the percentage of CD4+CD25+ Treg cells and the percentage of CD8+of CD3+ T cells in 22 fresh biopsy samples of B-cell NHL. Liner line was drawn to calculate R2. B, relationship between the percentage CD4+CD25+ T cells and the ratio of CD8+ to CD4+ T cells in 23 fresh biopsy samples of B-cell NHL. The liner lines were drawn based on percentage of CD4+CD25+ T cells. The value of 15% makes two populations.
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