CD4+ T-cell help in the tumor milieu is required for recruitment and cytolytic function of CD8+ T lymphocytes - PubMed (original) (raw)

CD4+ T-cell help in the tumor milieu is required for recruitment and cytolytic function of CD8+ T lymphocytes

Rinke Bos et al. Cancer Res. 2010.

Abstract

CD4 help for CD8(+) T lymphocytes prevents tolerance and promotes the survival of effector and memory CD8(+) T cells. Here, we describe additional helper functions that require CD4(+) T cells within the tumor environment. CD8(+) T-cell recruitment, proliferation, and effector function within the tumor were greatly enhanced by tumor-specific CD4(+) T cells. Recruitment of CD8(+) T cells was accelerated by IFN-γ-dependent production of chemokines. Production of interleukin-2 by tumor resident CD4(+) T cells enhanced CD8(+) T-cell proliferation and upregulated expression of granzyme B. These results highlight a novel role for tumor-specific CD4(+) T cells in promoting CD8(+) T-cell recruitment and cytolytic function, two previously unappreciated aspects of tumor-specific CD4 help.

©2010 AACR.

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Figures

Figure 1

Figure 1. Anti-tumor efficacy of Clone-1 CD8+ T cells

RIP-Tag2-HA mice were immunized and Clone-1 cells (0.3×106) with or without 0.3×106 SFE cells were injected i.v. A. On the indicated days PBL were analyzed by FACS for Thy1.1+ cells. B. Effector function of Clone-1 cells was measured in the spleen and draining lymph node of the vaccination site at day 6 by intra-cellular staining of Granzyme B and IFN-γ. C. Glucose levels in the blood were measured at the indicated time points and each line represents one mouse. Data are representative of 2 independent experiments with 4 mice per group.

Figure 2

Figure 2. CD4+ T cells are required in the tumor environment, for accumulation of Clone-1 cells and anti-tumor immunity

A. RIP-Tag2-HA mice were immunized and Clone-1 cells (0.3×106) with or without 0.3×106 SFE or DO11.10 cells were injected i.v. One group of mice received HA-Kd peptide and 200 μg poly(I:C) in IFA s.c. in the left flank and SFE peptide and 200 μg poly(I:C) in IFA s.c. in the right flank. Pancreata were analyzed by FACS at day 6. Data are cumulative over 4 experiments. B. Glucose levels in the blood were measured at the indicated time points and each line represents one mouse. Data are representative of 2 independent experiments with 4 mice per group.

Figure 3

Figure 3. Tumor-specific CD4+ T cells induce an inflammatory environment that promotes recruitment of Clone-1 cells

A. RIP-Tag2-HA mice were immunized with or without injection of 0.3×106 SFE cells. At day 6, 1×106 in vitro activated Clone-1 cells were injected and recruitment of Clone-1 cells to the pancreas was analyzed 40 hours later. The indicated neutralizing antibodies were injected at day 6 and 7. Data are cumulative over 3 experiments. **, P<0.005. B. RIP-Tag2-HA mice were immunized with or without injection of SFE cells (0.3×106) 6 days prior to isolation of pancreata and one group was injected with IFN-γ neutralizing antibodies at day 4 and 5. 200 μg of cell lysate was used for a cytokine array. Array images are from 30 minute exposures to X-ray film and quantitated data show relative changes between conditions. Data are representative of 2 independent experiments.

Figure 4

Figure 4. CD4+ T cells in the tumor environment stimulate proliferation and survival

RIP-Tag2-HA mice were immunized and Clone-1 cells (0.3×106) with or without 0.3×106 SFE or DO11.10 cells were injected i.v. Tumor infiltrating Clone-1 cells were analyzed at day 6 by FACS for proliferation (A: Ki-67) and Bim expression (B). The gate of the Ki-67 staining was based on the Thy1.1− Ki-67− cells in the pancreas. Control stainings in the histograms show staining with the Bim antibody of Bim−/− splenocytes. Dot plots and histograms are representative examples of each condition and bar graphs depict cumulative data of 3 experiments with 2–4 mice per group.

Figure 5

Figure 5. The role of IL-2 production by CD4+ T cells

A. RIP-Tag2-HA mice were immunized and Clone-1 cells (0.3×106) with or without 0.3×106 SFE, 2×106 IL-2−/− SFE or 2×106 SFE cells were injected i.v. The latter group was injected with IFN-γ neutralizing antibodies at day 4 and 5. Pancreata were analyzed by FACS at day 6. Data are cumulative over 2 experiments with 7 mice per group. B. Mice were injected as described in A. Expression of markers for proliferation (B: Ki-67) and survival (C: Bim) on Clone-1 cells were analyzed by FACS at day 6. Data are cumulative over 3 experiments with 9 mice per group. D. RIP-Tag2-HA mice were immunized with or without i.v. injection of 0.3×106 SFE or 2×106 SFE IL-2−/−. At day 6, 1×106 in vitro activated Clone-1 cells were injected and recruitment of Clone-1 cells to the pancreas was analyzed 4 days later. Data are cumulative over 3 experiments with 9 mice per group.

Figure 6

Figure 6. IL-2 production by CD4+ T cells is critical for increased effector function and anti-tumor efficacy of Clone-1 cells

A. RIP-Tag2-HA mice were immunized and Clone-1 cells (0.3×106) with or without 0.3×106 SFE, 0.3×106 DO11.10, 2×106 IL-2−/− SFE or 2×106 SFE cells were injected i.v. The latter group was injected with IFN-γ neutralizing antibodies at day 4 and 5. Pancreata were isolated at day 6. Data are cumulative over 3 experiments with 9 mice per group. B. RIP-Tag2-HA mice were immunized and Clone-1 cells (1×105) with or without 1×105 SFE, 7×105 IL-2−/− SFE or 7×105 SFE cells were injected i.v. The latter group was injected with IFN-γ neutralizing antibodies daily starting at day 4 through 13 followed by injections every 2 days. Glucose levels in the blood were measured at the indicated time points. Data are representative of 2 independent experiments with 3–5 mice per group. Each line represents one mouse.

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