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. 2012 Apr;214(4):700-7; discussion 707-8.
doi: 10.1016/j.jamcollsurg.2011.12.034. Epub 2012 Feb 22.

Ex vivo interleukin-12-priming during CD8(+) T cell activation dramatically improves adoptive T cell transfer antitumor efficacy in a lymphodepleted host

Mark P Rubinstein  1 , Colleen A Cloud, Tracy E Garrett, Caitlin J Moore, Kristina M Schwartz, C Bryce Johnson, David H Craig, Mohamed L Salem, Chrystal M Paulos, David J Cole
Affiliations

Ex vivo interleukin-12-priming during CD8(+) T cell activation dramatically improves adoptive T cell transfer antitumor efficacy in a lymphodepleted host

Mark P Rubinstein et al. J Am Coll Surg. 2012 Apr.

Abstract

Background: Clinical application of adoptive T cell therapy has been hindered by an inability to generate adequate numbers of nontolerized, functionally active, tumor-specific T cells, which can persist in vivo. In order to address this, we evaluated the impact of interleukin (IL)-12 signaling during tumor-specific CD8(+) T cell priming in terms of persistence and antitumor efficacy using an established B16 melanoma tumor adoptive therapy model.

Study design: B6 mice were injected subcutaneously with B16 melanoma tumor cells. On day 12 of tumor growth, mice were preconditioned with cyclophosphamide (4mg dose, intraperitoneally), and 1 day later were treated by adoptive transfer of tumor-specific pmel-1 CD8(+) T cells primed ex vivo 3 days earlier with both IL-12 and antigen (hGP100(25-33) peptide) or antigen only. Tumors were measured biweekly, and infused donor T cells were analyzed for persistence, localization to the tumor, phenotype, and effector function.

Results: Adoptive transfer of tumor-specific CD8(+) T cells primed with IL-12 was significantly more effective in reducing tumor burden in mice preconditioned with cyclophosphamide compared with transfer of T cells primed without IL-12. This enhanced antitumor response was associated with increased frequencies of infused T cells in the periphery and tumor as well as elevated expression of effector molecules including granzyme B and interferon-γ (IFNγ).

Conclusions: Our findings demonstrate that ex vivo priming of tumor-specific CD8(+) T cells with IL-12 dramatically improves their in vivo persistence and therapeutic ability on transfer to tumor-bearing mice. These findings can be directly applied as novel clinical trial strategies.

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Figures

Figure 1
Figure 1
Phenotypic analysis of CD8+ T cells primed with or without IL-12. After priming for three days, pmelIL-12 or pmelsham T cells were analyzed by flow cytometry for the markers indicated on the far left: (A) shows cytokine receptor subunits and CD69, (B) shows costimulatory and inhibitory molecules, and (C) shows transcription factors. All results are representative of at least 2 independent experiments.
Figure 2
Figure 2
Priming pmel-1 CD8+ TCR transgenic T cells with IL-12 leads to enhanced granzyme B and IFNγ expression. (A) After priming for three days, pmelIL-12 or pmelsham T cells were directly analyzed for granzyme B expression. (B) PmelIL-12 or pmelsham T cells were cocultured with B6 splenocytes with or without relevant peptide in a flat 96-well plate for 6 hours. (C) as in ‘B’ except in a round 96-well plate. All results are representative of at least 3 independent experiments.
Figure 3
Figure 3
IL-12 primed CD8+ T cells exhibit improved localization to the tumor. PmelIL-12 or pmelsham T cells were adoptively transferred into B6 mice with day 7 established B16 tumors. Mice received either 5×106 (1×) or 2.5×107 T cells. One week later, spleens and tumors were harvested. Shown is the percentage of donor T cells (Thy1.1+) among total lymphocytes in the (A) spleen and (B) tumor. Each triangle represents one mouse. *, p < 0.05, indicate statistical analysis by Student’s t test.
Figure 4
Figure 4
IL-12-priming during CD8+ T cell activation leads to significantly enhanced anti-tumor activity. Mice with s.c. B16 tumors were treated on day 12 as indicated with CTX. On day 13, mice were adoptively transferred with pmelIL-12 (3×106) or pmelsham (3×106) T cells. Tumors were measured blindly twice weekly. Each line represents one mouse.
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