Glucocorticoid-induced tumor necrosis factor receptor stimulation enhances the multifunctionality of adoptively transferred tumor antigen-specific CD8+ T cells with tumor regression

Abstract

We have reported for the first time the significance of effector T-cell multifunctionality in antitumor immunity, suggesting that the appearance of multifunctional/polyfunctional tumor-specific CD8(+) T cells in vivo is a critical determinant of the success of antitumor immunotherapy, and a strategy to induce multifunctionality in effector cells is required for the successful immunotherapy of hosts with progressing tumor. Glucocorticoid-induced tumor necrosis factor receptor (GITR) stimulation has been shown to enhance antitumor immune response. However, its functional impact on adoptively transferred T cells remains unclear. Here, we analyzed the impact of GITR stimulation in vivo on the functional profiles of adoptively transferred CD8(+) T cells specific for murine fibrosarcoma CMS5. GITR stimulation was found to enhance multifunctionality (interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha production and CD107a mobilization as a degranulation marker) in transferred cells at the single-cell level. These cells exhibited upregulated expression of CD25 in draining lymph nodes and increased infiltration in tumor. Mice that received T-cell therapy with GITR stimulation showed reduced Foxp3(+)CD4(+) T cells among tumor infiltrating lymphocytes and increased in vivo cytotoxic T lymphocytes (CTL) activity even with progressing tumor, resulting in enhanced tumor regression. These data strengthen the idea that effector T-cell multifunctionality is a sensitive immune correlate for successful immunotherapy against malignancy and provide an immunological rationale for effective T-cell therapy combined with GITR stimulation.

Figure 1

Glucocorticoid‐induced tumor necrosis factor receptor (GITR) stimulation combined with adoptive T‐cell therapy induces tumor regression in hosts with progressing tumor. BALB/c mice (n = 5 per group) were inoculated subcutaneously with 1 × 10⁶ CMS5 cells on day 0. On day 7, mice were administrated with the anti‐GITR mAb DTA‐1 or PBS, and a total of 1 × 10⁶ CD8⁺ cells purified from splenocytes isolated from DUC18 transgenic mice were adoptively transferred intravenously in the groups indicated as adoptive cell transfer; tumor growth was monitored over time. The mean tumor diameter of each group is represented as the average + SD of five mice. Results are representative of three independent experiments.

Figure 2

Glucocorticoid‐induced tumor necrosis factor receptor (GITR) stimulation enhances the multifunctionality of adoptively transferred tumor‐specific CD8+ T cells. BALB/c mice (n = 5 per group) were injected subcutaneously with 1 × 10⁶ CMS5 cells. Adoptive transfer of 1 × 10⁶ CFSE‐labeled DUC18 CD8⁺ cells was performed with administration of DTA‐1 or PBS on day 7 of tumor challenge. Three days after adoptive cell transfer (ACT), we used polychromatic flow cytometry to determine antigenic peptide 9 m‐specific cytokine production and CD107a mobilization by transferred CD8⁺ T cells harvested from draining lymph nodes. (a) Representative histograms for CFSE and dot plots for CFSE versus IFN‐γ, TNF‐α, or CD107a are shown. (b) MFI of IFN‐γ, TNF‐α, and CD107a staining at each cell division for cells derived from mice that received ACT with DTA‐1 or PBS are represented as the means + SD of five mice. (c) Responses are grouped and color‐coded according to the number of acquired functions (IFN‐γ, TNF‐α, CD107a). Data are summarized in the pie chart, where each wedge represents the frequency of CFSE⁺CD8⁺ cells expressing all three functions as IFN‐γ⁺TNF‐α⁺CD107a⁺ (3), any two functions including IFN‐γ⁺TNF‐α⁺CD107a⁻, IFN‐γ⁺TNF‐α^–CD107a⁺, and IFN‐γ⁻TNF‐α⁺CD107a⁺ (2), a single function including IFN‐γ⁺TNF‐α^–CD107a⁻, IFN‐γ⁻TNF‐α⁺CD107a⁻, and IFN‐γ⁻TNF‐α^–CD107a⁺ (1), or no function (0). Results are representative of three independent experiments. (d) Functional composition of the CD8⁺ T‐cell response. Every possible combination of responses is shown in the x‐axis. Boxes represent interquartile ranges; the line in the box represents the median, and minimum/maximum lines are shown. (e) Analysis of the frequency of each population with different numbers of acquired functions in cells at each cell division, as assessed by CFSE intensity. Results are representative of three independent experiments.

Figure 3

Multifunctional T cells exhibit more cytokine production and degranulation than cells with less functionality. MFI of IFN‐γ, TNF‐α, and CD107a staining of antigen specific tri‐, bi‐, or mono‐functional cells from mice subjected to adoptive cell transfer combined with DTA‐1 administration 7 days after tumor challenge are shown. Data represent the mean + SD of five mice. Differences between groups were examined for statistical significance using Student's t‐test. *P < 0.01, **P < 0.05.

Figure 4

Glucocorticoid‐induced tumor necrosis factor receptor (GITR)‐induced effector T‐cell multifunctionality is associated with their enhanced proliferation, increased expression of CD25, and augmented infiltration into tumor. (a,b) CFSE‐labeled DUC18 CD8⁺ T cells were adoptively transferred and administered DTA‐1 or PBS on day 7 after tumor challenge (five mice per group). (a) Lymphocytes were harvested from draining lymph nodes (DLN), non‐draining lymph nodes (NDLN), or tumor, and analyzed for their proliferation and cell surface expression of CD25 or CD69 by flow cytometry. (b) We analyzed 9m peptide‐specific cytokine production, CD107a mobilization, and CD25 surface expression by transferred CD8⁺ T cells harvested from DLN in mice treated with DTA‐1 or PBS. MFI of CD25 staining for tri‐functional, bi‐functional, mono‐functional, or non‐functional cells are indicated as the mean + SD of five mice. Data show the CD8⁺Vβ8.3⁺ population. Results are representative of three independent experiments.

Figure 5

Glucocorticoid‐induced tumor necrosis factor receptor (GITR) stimulation combined with adoptive cell transfer (ACT) reduces the ratio of Foxp3+ cells among tumor infiltrating CD4⁺ T cells. (a,b) BALB/c mice (n = 5 per group) were inoculated subcutaneously with 1 × 10⁶ CMS5 cells on day 0, and received 1 × 10⁶ DUC18 mouse‐derived CD8⁺ T cells combined with administration of DTA‐1 or PBS on day 7. Three days after ACT, lymphocytes in draining lymph nodes (DLN), non‐draining lymph nodes (NDLN), spleen, or tumor were harvested and stained with anti‐Foxp3 and anti‐CD4 mAb. (a) Representative histogram for Foxp3 staining among CD4⁺ cells in each organ is indicated. Similar results were obtained in three independent experiments. (b) The ratio of Foxp3⁺ cells among CD4⁺ cells in each organ is indicated. Data are the mean + SE of three independent experiments. Differences between groups were examined for statistical significance using Student's t‐test. *P < 0.01.

Figure 6

Glucocorticoid‐induced tumor necrosis factor receptor (GITR) expression is prominent on effector cells in draining lymph nodes (DLN) and on Treg in tumors. BALB/c mice were inoculated subcutaneously with 1 × 10⁶ CMS5 cells on day 0, and CFSE‐labeled DUC18 CD8⁺ T cells were adoptively transferred on day 2 or day 7. Three days after adoptive cell transfer, lymphocytes in DLN, non‐draining lymph nodes (NDLN), spleen, or tumor were harvested and stained with anti‐CD8, anti‐CD4, anti‐Foxp3, and anti‐GITR mAb. For CD8⁺ T cells, the solid line indicates the staining of CFSE‐positive effector T cells, and the dotted line represents CFSE‐negative non‐specific CD8⁺ T cells. For CD4⁺ T cells, the solid line indicates Foxp3⁺CD4⁺ T cells, and the dotted line represents Foxp3⁻CD4⁺ T cells. The filled histogram represents control staining.

Figure 7

Glucocorticoid‐induced tumor necrosis factor receptor (GITR) stimulation enhances the in vivo CTL activity in mice that received adoptive cell transfer on day 7 of tumor challenge. 3 × 10⁵ DUC18 CD8⁺ cells were transferred into day 2‐CMS5 tumor‐bearing mice or day 7‐CMS5 tumor‐bearing mice in the absence or presence of DTA‐1 treatment. CFSE‐labeled target cells were prepared and transferred on day 10. Twenty hours later, target cells were harvested from spleens and analyzed by flow cytometry. The percentage of specific lysis of target cells is indicated with representative histograms, in which 9m‐pulsed and ‐unpulsed target cells were labeled with high and low concentrations of CFSE respectively. The control indicates the result of naive mice in the absence of therapy. Data are the mean + SD of three mice. *P < 0.01, **P < 0.05. Results are representative of three independent experiments.