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. 2016 May 1;22(9):2207-16.
doi: 10.1158/1078-0432.CCR-15-2273. Epub 2015 Dec 9.

The Antitumor Efficacy of IL2/IL21-Cultured Polyfunctional Neu-Specific T Cells Is TNFα/IL17 Dependent

Vy Phan-Lai  1 , Yushe Dang  1 , Ekram Gad  1 , Jennifer Childs  1 , Mary L Disis  2
Affiliations

The Antitumor Efficacy of IL2/IL21-Cultured Polyfunctional Neu-Specific T Cells Is TNFα/IL17 Dependent

Vy Phan-Lai et al. Clin Cancer Res. .

Abstract

Purpose: Infusion of HER2-specific T cells, derived from vaccine-primed patients and expanded with IL2/IL12, has induced tumor regression in a minority of patients with metastatic treatment-refractory HER2(+) breast cancer. We questioned whether alteration of cytokine growth factors used to culture vaccine-primed T cells could improve antitumor activity.

Experimental design: Using the TgMMTV-neu murine mammary tumor model, we cultured T cells derived from mice immunized with a previously defined neu class II peptide, p98-114 (neu p98), and evaluated different cytokine combinations for expansion.

Results: Infusion of neu p98-specific T-cell lines derived from all cytokine conditions evaluated resulted in significant antitumor activity compared with infused naïve splenocytes (P < 0.05). T cells cultured with IL2/IL21 could uniquely mediate complete regression of spontaneous mammary tumors. IL2/IL21 cultured neu-specific T cells demonstrated a different cytokine secretion pattern as compared with other cultured T cells; secreting high levels of TNFα and IL17 (P < 0.05). Moreover, tumor-infiltrating CD8(+) cells were significantly increased after the infusion of IL2/IL21 cultured T cells as compared with tumors treated with T cells expanded under other cytokine conditions (P < 0.001). The antitumor effect of the infusion of IL2/IL21 cultured cells was mediated by CD8 T cells. Depletion of TNFα or IL17, but not IFNγ, abrogated the tumor growth inhibition induced by the IL2/IL21 T cells and markedly decreased the influx of CD8 into tumors. Finally, IL2/IL21-cultured human antigen specific T cells also displayed a similar polyfunctional Th1/Th17 phenotype.

Conclusions: Expansion of HER2 vaccine-primed T cells with IL2/IL21 may have the potential to effectively mediate tumor regression when used in adoptive transfer. Clin Cancer Res; 22(9); 2207-16. ©2015 AACR.

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Conflict of interest statement

Conflict of Interest: MLD is a stockholder in Epithany and VentiRx and receives grant support from Celgene, EMD Serono, VentiRx and Seattle Genetics. The remaining authors have no COI.

Figures

Figure 1
Figure 1. Specific cytokines influence the number of antigen-specific T-cells and anti-tumor activity generated
(A) Cell growth under different cytokine conditions. Bars show mean fold expansions relative to IL-2 condition for all cytokine conditions. * indicates p<0.05. (B) neu p98 specific IFN-gamma secretion from T-cells cultured under different cytokine conditions. * indicates p<0.05; ** p<0.01 vs. IL2 only. Data is representative of two experiments. (C) Tumor growth (y-axis) over time (x-axis) in TgMMTV-neu mice implanted with MMC admixed with naïve splenocytes (-■-) or with T cell lines expanded in IL-2 (-▲-), or combined with IL-4 (-○□-), IL-7 (-□-), IL-12 (-△-), IL-15 (-▼□-), IL-18 (-◇□-), IL-21 (-●-), or IL-7/IL-15 (-◆-). Data points are the mean (± SE) of tumor size from five mice. (D) Tumor growth (y-axis) over time (x-axis) of spontaneous tumors developed in TgMMTV-neu mice treated with naïve splenocytes (■) or with T-cell lines expanded in IL-2 (▲), or combined with IL-21 (●), or IL-7/IL-15 (◆). Data points are the mean (± SE) of tumor size from 3 mice. * indicates p<0.05, *** p<0.001.
Figure 2
Figure 2. T cells expanded in IL-2/IL-21 condition demonstrate a polyfunctional Th1/Th17 phenotype
Bars are the mean (± SE) of duplicates for all culture conditions. (A) IFN-gamma, (B) TNF-alpha, (C) IL-17, (D) IL-5, (E) IL-10, (F) IL-13. * p<0.05, ** p<0.01, *** p<0.001 vs. IL-2. Results shown are representative of two experiments.
Figure 3
Figure 3. The anti-tumor efficacy of IL-2/IL-21 neu-specific T-cells is mediated by CD8+ T-cells
(A) Quantitation of CD8+ cells in tumors after infusion of IL-2, IL-2/IL-21, or IL-2/IL-7/IL-15 cultured p98 T-cells, or naïve T-cells. Columns and bars represent the mean (± SE) of 5 mice per group. ** indicates p<0.01; *** p<0.001. (B) Relative mRNA expression of Granzyme B to B-actin. Bars are the mean (± SE) of 2 mice per group, 3 replicates per mouse. *p<0.05. (C) Spontaneous tumors developed in TgMMTV-neu mice treated with naïve splenocytes (-○-), IL-2/IL-21 cultured T-cells only (-●-), or with anti-CD8 (-◆-), or with anti-CD4 (-▲-), or anti-CD22 antibody (-◇-). Data points are the mean of tumor size (± SE) from 4 mice. *** indicates p<0.001.
Figure 4
Figure 4. The anti-tumor efficacy of IL-2/IL-21 cultured neu-specific T-cells is IL-17 and TNF-alpha dependent
(A–C) Spontaneous tumors developed in TgMMTV-neu mice after infusion with naïve splenocytes (-○-), or with IL-2/IL-21 cultured T-cells alone (-●-), or with isotype control (-◇), or neutralizing anti-IL-17 (A), or TNF-alpha (B), or IFN-gamma (C) antibodies (-■-). Data points are the mean tumor size (± SE) of three mice. Data is representative of 2 separate experiments. *** indicates p<0.001. (D) Number of CD8+ cells % of total cells (y-axis) in isotype control, and TNF-alpha, IL-17 or IFN-gamma depleted mice after T-cell infusion (x-axis). Columns and bars represent the mean (± SE) of 5 mice per group. * p<0.05; ** p<0.01; *** p<0.001.
Figure 5
Figure 5. The expression of Th1/Th17 differentiation genes is increased in IL-2/IL-21 cultured CMV-specific T-cells
Relative mRNA expression to B-actin (y-axis) in IL-2 (white columns), IL-2/IL-12 (grey), and IL-2/IL-21 (black) cultured condition (x-axis). (A) IFN-gamma, (B) IL-17; (C) IL-21, (D) IRF-4, (E) RORc, and (F) TNF-alpha. Bars are the mean (± SE) of duplicates for all culture conditions. * p<0.05; **p<0.01. and *** p<0.001.
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