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Comparative Study
. 2004 Dec 15;173(12):7209-16.
doi: 10.4049/jimmunol.173.12.7209.

Vaccine-stimulated, adoptively transferred CD8+ T cells traffic indiscriminately and ubiquitously while mediating specific tumor destruction

Douglas C Palmer  1 , Sanjeeve Balasubramaniam, Ken-Ichi Hanada, Claudia Wrzesinski, Zhiya Yu, Shahram Farid, Marc R Theoret, Leroy N Hwang, Christopher A Klebanoff, Luca Gattinoni, Allan L Goldstein, James C Yang, Nicholas P Restifo
Affiliations
Comparative Study

Vaccine-stimulated, adoptively transferred CD8+ T cells traffic indiscriminately and ubiquitously while mediating specific tumor destruction

Douglas C Palmer et al. J Immunol. .

Abstract

It has been suggested that antitumor T cells specifically traffic to the tumor site, where they effect tumor destruction. To test whether tumor-reactive CD8(+) T cells specifically home to tumor, we assessed the trafficking of gp100-specific pmel-1 cells to large, vascularized tumors that express or do not express the target Ag. Activation of tumor-specific CD8(+) pmel-1 T cells with IL-2 and vaccination with an altered peptide ligand caused regression of gp100-positive tumors (B16), but not gp100-negative tumors (methylcholanthrene 205), implanted on opposing flanks of the same mouse. Surprisingly, we found approximately equal and very large numbers of pmel-1 T cells (>25% of all lymphocytes) infiltrating both Ag-positive and Ag-negative tumors. We also found evidence of massive infiltration and proliferation of activated antitumor pmel-1 cells in a variety of peripheral tissues, including lymph nodes, liver, spleen, and lungs, but not peripheral blood. Most importantly, evidence for T cell function, as measured by production of IFN-gamma, release of perforin, and activation of caspase-3 in target cells, was confined to Ag-expressing tumor. We thus conclude that CD8(+) T cell-mediated destruction of tumor is the result of specific T cell triggering at the tumor site. The ability to induce ubiquitous homing and specific tumor destruction may be important in the case of noninflammatory metastatic tumor foci.

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Figures

FIGURE 1
FIGURE 1
Activated CD8+ T cells infiltrate to tumor and mediate its regression. A, Tumor alone does not induce pmel-1 activation. Pmel-1-transgenic mice were implanted with s.c. B16 for 11 days, then received vaccine encoding hgp100 and IL-2 stimulation, or were left untreated. Spleens and tumors were harvested and analyzed cytofluorometrically for pmel-1 T cells (Vβ13+/CD8+) 5 days after vaccination. The FACS plots shown are gated on CD8+ lymphocytes and analyzed for pmel-1 T cells, Vβ13, and expression of CD44 or CD62L. B, Tumors bearing transgenic T cells do not infiltrate into tumor in the naive state. In an experiment similar to that in A, B16 tumors were harvested from untouched or vaccinated, tumor-bearing, pmel-1-transgenic mice. Samples were then analyzed for the presence of pmel-1 T cells in the tumor. C, B16 regression after vaccination of pmel-1-transgenic T cells. Mice were randomized, and tumors were measured in a blinded fashion using digital calipers. The products of the perpendicular diameters are presented as the mean ± SEM. Experiments were performed independently at least twice with similar results.
FIGURE 2
FIGURE 2
Pmel-1 T cells destroy tumor in an Ag-dependent fashion. A, B16, but not MCA-205, expresses gp100. mRNA was extracted from B16 and MCA-205 lysates. cDNA was synthesized and subjected to PCR using gp100 and β-actin intron-spanning primers for 25, 30, and 35 cycles. B, Activated pmel-1 T cells recognize B16, but not MCA-205, in vitro. T cells (105) were cocultured in CM with 105 target cells that were or were not pulsed with 1 μM human gp10025–33. Supernatants were collected after 24 h and assayed using a mouse IFN-γ ELISA kit. C, Pmel-1 T cells mediate the destruction of established B16, but not MCA-205. C57BL/6n mice were implanted with both B16 and MCA-205 tumors on opposing flanks and treated after 7 days with cultured pmel-1 T cells, with or without rFPVhgp100 and IL-2. Mice were randomized, and tumors were measured in a blinded fashion using digital calipers. The products of the perpendicular diameters are presented as the mean ± SEM. Experiments were performed independently at least three times with similar results.
FIGURE 3
FIGURE 3
Tumor-specific T cell trafficking and growth kinetics were similar in gp100+ and gp100− tissues. A, Pmel-1 T cells traffic to Ag- and non-Ag-containing tissues. Seven-day established B16 and MCA-205 dual tumor-bearing C57BL/6n recipients were treated with cultured pmel-1 GFP+ T cells, with or without rFPVhgp100 and IL-2. On the days indicated, B16 and MCA tumors, eye, spleen (SPL), liver, lung, blood (PBL), B16 inguinal draining lymph node (B16 LN), and MCA-205 inguinal draining lymph node (MCA LN) were harvested and cytofluorometrically analyzed for pmel-1 (GFP+/CD8+). Values represent the percentage of GFP+CD8+ events compared with total CD8+ cells. B, Equal numbers of pmel-1 T cells extravasated into both B16 and MCA-205. Tumors were harvested and imbedded in OCT (Miles, Elkhart, IN) from perfused mice 5 days after treatment with rFPVhgp100, IL-2, and pmel-1 T cells where indicated. Frozen sections were stained for Vβ13 (green) and examined using confocal microscopy. Results are representative of multiple fields. C, The total numbers of pmel-1 T cells were similar in both B16 and MCA-205. Five days after adoptive transfer of pmel-1 Thy1.1+ T cells and in vivo stimulation, tumors of comparable size (∼20 mm2) were harvested from at least three mice and pooled. The product of the total percentage of Thy1.1+/CD8+ and the total cell count was used to calculate the total pmel-1. The results shown are presented as the mean ± SEM of three independently performed experiments.
FIGURE 4
FIGURE 4
Pmel-1 T cells proliferate in multiple tissues, regardless of Ag. A, After in vivo stimulation, pmel-1 T cells proliferate in many tissues. Four days after the indicated treatments, tumor bearing C57BL/6n mice were injected i.p. with BrdU; 2 h after injection, mice were bled and perfused, and organs were homogenized and extracellularly stained for Vβ13 and CD8 expression. To free anti-BrdU binding sites, samples were treated with DNase I, then intracellularly stained with anti-BrdU PE. In FACS plots, no treatment (NT) is gated on lymphocytes; pmel-1 and pmel-1 rFPV IL2 are gated on CD8+ lymphocytes. B, Pmel-1 T cells proliferate in both gp100+ and gp100− tissues after activation. In an experiment similar to that described above, B16 and MCA-205 tumors were harvested from dual tumor-bearing mice that received pmel-1 T cells, rFPVhgp100, and IL-2. All Vβ13+CD8+ events were analyzed for BrdU staining. Experiments were independently performed twice with similar results.
FIGURE 5
FIGURE 5
Differential function of pmel-1 T cells in B16 and MCA-205. A, Pmel-1 T cells up-regulate IFN-γ in B16, but not in MCA-205. Pmel-1 transgenics were implanted with B16 or MCA-205 for 11 days, then administered rFPVhgp100 and IL-2. After 12 days, tumors were harvested, homogenated, extracellularly stained for Vβ13 and CD8, and intracellularly stained for IFN-γ. Each dot plot represents an individual mouse and is gated on the entire CD8+ population. B, Pmel-1 T cells release perforin in B16, but not MCA-205. Pmel-1 T cells were adoptively transferred and in vivo stimulated in C57BL/6n mice bearing both B16 and MCA-205 tumors for 7 days. Five days after treatment with pmel-1 T cells, rFPVhgp100, and IL-2, tumors were harvested and imbedded in freezing medium. Frozen sections were stained for Vβ13 (green) and perforin (red) and imaged using confocal microscopy. Results are representative of multiple fields. C, Activation of caspase 3 in B16 infiltrated with pmel-1 T cells. In an experiment similar to that described above, tumors were harvested from tumor-bearing mice that received rFPVhgp100, IL-2, and pmel-1 T cells where indicated. Sections were stained for Vβ13 (green) and active caspase 3 (red) and were imaged using confocal microscopy. Results are representative of multiple fields.
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