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. 2009 Aug 15;183(4):2365-72.
doi: 10.4049/jimmunol.0900721. Epub 2009 Jul 22.

Chimeric NKG2D T cells require both T cell- and host-derived cytokine secretion and perforin expression to increase tumor antigen presentation and systemic immunity

Amorette Barber  1 , Charles L Sentman
Affiliations

Chimeric NKG2D T cells require both T cell- and host-derived cytokine secretion and perforin expression to increase tumor antigen presentation and systemic immunity

Amorette Barber et al. J Immunol. .

Erratum in

  • J Immunol. 2014 Aug 1;193(3):1513

Abstract

Treatment of mice bearing established ovarian tumors with T cells expressing chimeric NKG2D receptors (chNKG2D) develop protective host immune responses to tumor Ags. In this study, the mechanisms that chNKG2D T cells require to induce host immunity against ovarian tumors and which of the host immune cells are involved in tumor elimination were determined. Treatment with chNKG2D T cells led to a sustained, increased IFN-gamma production by host NK, CD4(+), and CD8(+) T cells in the spleen and at the tumor site and this continued for many weeks after T cell injection. Tumor Ag presentation was enhanced in chNKG2D T cell-treated mice, and there were greater numbers of tumor-specific T cells at the tumor site and in draining lymph nodes after treatment with chNKG2D T cells. The increase in host cell cytokine secretion and Ag presentation was dependent on chNKG2D T cell-derived perforin, IFN-gamma, and GM-CSF. Host immune mechanisms were involved in tumor elimination because inhibition of tumor growth was limited in mice that lacked perforin, IFN-gamma, NK cells, or T and B cells (Rag1(-/-)). There was no role for host-derived GM-CSF or CD1-dependent NKT cells, because mice deficient in these were able to clear tumors as well as treated wild-type B6 mice. In summary, chNKG2D T cells required both cytotoxicity and cytokine secretion as well as the participation of host immune cells for development of a host antitumor immune response and complete efficacy.

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Figures

Figure 1
Figure 1. Host NK cells and T cells have increased IFNγ production after treatment with chNKG2D T cells
One, three, and seven days after T cell injection, spleen cells from wtNKG2D (white bars) or chNKG2D T cell (black bars) treated tumor bearing mice were cultured in media for 24 hours. (A) Cell-free supernatants were assayed for IFNγ or (B) cells were assayed for IFNγ production by intracellular staining. Cells were gated on either CD3+Ly5.1+, CD8+CD3+, CD4+CD3+, or NK1.1+CD3− as indicated. The average of each group (n=4) is shown as percent IFNγ+ cells + SD. Treatment with chNKG2D T cells significantly increased IFNγ secretion compared to control treated mice (*-p<0.05). Data are representative of at least 5 separate experiments.
Figure 2
Figure 2. Tumor bearing mice treated with chNKG2D T cells have a sustained IFNγ response from host cells
(A) ID8-GFP cells were injected i.p. and mice were treated with wtNKG2D T cells (white bars) or chNKG2D T cells (black bars) one week later. Three, five, eight, or eleven weeks after tumor cell injection, peritoneal wash cells and spleen cells from T cell treated or naive mice (grey bars) were cultured in media for 24 hours. Cell-free supernatants were collected and assayed for IFNγ or for nitric oxide. (B) Intracellular staining for IFNγ was performed on peritoneal wash cells and spleen cells cultured as described in (A). Cells were gated on either CD8+CD3+, CD4+CD3+, or NK1.1+CD3− as indicated. The average of each group (n=4) is shown + SD. Treatment with chNKG2D T cells significantly increased IFNγ and nitric oxide secretion compared to control treated mice (*-p<0.05). Data are representative of 2 separate experiments.
Figure 3
Figure 3. Induction of host IFNγ production requires chNKG2D T cell- derived cytokines and perforin
(A) Spleen cells from tumor bearing mice treated with B6-derived wtNKG2D (white bars) or chNKG2D T cell (black bars), or chNKG2D T cells derived from GM-CSF- (hashed bars), IFNγ-(striped bars), or perforin-deficient mice (grey bars) or (B) spleen cells from mice deficient in IFNγR1 treated with wtNKG2D (hashed bars) or chNKG2D (striped bars) T cells were cultured in media for 24 hours. Cell-free supernatants were collected and assayed for IFNγ by (A, B) ELISA or (C) intracellular staining. Cells were gated on either NK1.1+ CD3−, CD8+CD3+, or CD4+CD3+ as indicated. The average of each group (n=4) is shown + SEM. Treatment with chNKG2D T cells significantly increased IFNγ secretion compared to wtNKG2D T cell treated mice (*-p<0.05). Treatment with chNKG2D T cells deficient in effector molecules secreted significantly less IFNγ compared to chNKG2D T cell treated mice (§-p<0.05). Data are representative of at least 2 separate experiments.
Figure 4
Figure 4. Treatment with chNKG2D T cells increases tumor antigen presentation and requires chNKG2D T cell expression of GM-CSF, IFNγ, and perforin for antigen presentation
(A) ID8-GFP-tOva or (B) ID8-GFP cells were injected i.p. and mice were treated with wtNKG2D T cells (white bars), chNKG2D T cells (black bars), or PBS (grey bars) one week later. Seven days after T cell injection, cells isolated from the mediastinal lymph node and spleen were cultured with CFSE-labeled OT-I T cells. OT-I T cell proliferation was measured after 72 hours of culture. (C) Lymph node cells isolated from ID8-GFP bearing mice treated as described in figure 4B were pulsed with different concentrations of Ova peptide as indicated, and were cultured with CFSE-labeled OT-I T cells. OT-I T cell proliferation was measured after 72 hours of culture. (D) ID8-GFP-tOva cells were injected i.p. and mice were treated with wtNKG2D T cells or chNKG2D T cells from B6 mice or chNKG2D T cells from GM-CSF- (hashed bars), IFNγ-(striped bars), or perforin-deficient mice (grey bars) one week later. Seven days after T cell injection, cells from the mediastinal lymph node and spleen were cultured as described in (A). The average of each group + SD is shown (n=4). Treatment with chNKG2D T cells significantly increased tumor antigen specific T cell proliferation compared to wtNKG2D T cell treated mice (*-p<0.05). Treatment with chNKG2D T cells deficient in effector molecules had significantly less T cell proliferation compared to chNKG2D T cell treated mice (§-p<0.05). Data are representative of 2 separate experiments.
Figure 5
Figure 5. ChNKG2D T cell treatment increases T cell trafficking to the tumor site
(A) ID8-GFP-tOva cells or (B) ID8-GFP cells were injected i.p. One week later, mice were treated i.p. with wtNKG2D T cells (white bars), chNKG2D T cells (black bars), or PBS (grey bars), and CFSE-labeled OT-I T cells were transferred i.v. Absolute number of OT-I T cells in the mediastinal lymph node and peritoneal cavity was determined three days after T cell injection. The average of each group (n=4) is shown + SD. Treatment with chNKG2D T cells significantly increased OT-I T cell trafficking in vivo compared to control treated mice (*-p<0.05). Data are representative of 2 independent experiments.
Figure 6
Figure 6. ChNKG2D T cells require host cell-derived perforin and IFN γ for in vivo efficacy
ID8-GFP cells were injected i.p. into (A) B6 or B6.IFNγ−/− mice or (B) B6, B6.Perforin−/−, or B6.GM-CSF−/− mice. After seven days, mice received wtNKG2D (WT) or chNKG2D (CH) T cells i.p. Tumor burden was determined after 8 weeks by measuring the absolute number of GFP+ cells in the peritoneal wash or number of solid tumors on the peritoneum. Each data point represents an individual and the average of each group is shown. Data are from at least two separate experiments. * p<0.05- compared with WT T cell treatment. § p<0.05, compared with CH T cell treatment.
Figure 7
Figure 7. ChNKG2D T cells require host immune cells for in vivo efficacy
ID8-GFP cells were injected i.p. into (A) B6 or B6.Rag1−/− mice or (B) B6 mice injected with anti-NK1.1 mAbs (PK136) or control mouse gamma globulin (msgg) or (C) B6 or B6.CD1−/− mice. After seven days, mice received wtNKG2D (WT) or chNKG2D (CH) T cells i.p. Tumor burden was determined after 8 weeks by measuring the absolute number of GFP+ cells in the peritoneal wash or number of solid tumors on the peritoneum. Each data point represents an individual and the average of each group is shown. Data are from two separate experiments. * p<0.05, compared with WT T cell treatment. § p<0.05, compared with CH T cell treatment.
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