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Comparative Study
, 202 (7), 907-12

Removal of Homeostatic Cytokine Sinks by Lymphodepletion Enhances the Efficacy of Adoptively Transferred Tumor-Specific CD8+ T Cells

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Comparative Study

Removal of Homeostatic Cytokine Sinks by Lymphodepletion Enhances the Efficacy of Adoptively Transferred Tumor-Specific CD8+ T Cells

Luca Gattinoni et al. J Exp Med.

Abstract

Depletion of immune elements before adoptive cell transfer (ACT) can dramatically improve the antitumor efficacy of transferred CD8+ T cells, but the specific mechanisms that contribute to this enhanced immunity remain poorly defined. Elimination of CD4+CD25+ regulatory T (T reg) cells has been proposed as a key mechanism by which lymphodepletion augments ACT-based immunotherapy. We found that even in the genetic absence of T reg cells, a nonmyeloablative regimen substantially augmented CD8+ T cell reactivity to self-tissue and tumor. Surprisingly, enhanced antitumor efficacy and autoimmunity was caused by increased function rather than increased numbers of tumor-reactive T cells, as would be expected by homeostatic mechanisms. The gammaC cytokines IL-7 and IL-15 were required for augmenting T cell functionality and antitumor activity. Removal of gammaC cytokine-responsive endogenous cells using antibody or genetic means resulted in the enhanced antitumor responses similar to those seen after nonmyeloablative conditioning. These data indicate that lymphodepletion removes endogenous cellular elements that act as sinks for cytokines that are capable of augmenting the activity of self/tumor-reactive CD8+ T cells. Thus, the restricted availability of homeostatic cytokines can be a contributing factor to peripheral tolerance, as well as a limiting resource for the effectiveness of tumor-specific T cells.

Figures

Figure 1.
Figure 1.
Lymphodepletion enhances antitumor efficacy of adoptively transferred CD8+ T cells. (a) 5 Gy TBI induces severe lymphopenia. Mice were treated with 5 Gy TBI or a nonmyeloablative chemotherapy regimen with 250 mg/kg Cytoxan + 50 mg/kg fludarabine. Absolute lymphocyte count was determined at the indicated time points. (b) Lymphodepletion augments antitumor responses. TBI or nonirradiated WT mice bearing 12-d-old established s.c. B16 tumors were left untreated or received adoptive transfer of 106 cultured pmel-1 T cells in conjunction with rFPhgp100 vaccination and rhIL-2. Data shown are representative of multiple independent experiments. Values represent the mean ± SEM. (c–e) Lymphodepletion does not result in increased numbers of adoptively transferred T cells. Absolute numbers of adoptively transferred pmel-1 cells (CD8+Thy1.1+) in the spleens (c) and in the blood (d) of tumor-bearing, TBI, and nonirradiated mice. Percentages of adoptively transferred pmel-1 cells (CD8+Thy1.1+) in the tumor (e) under conditions specified. (f–i). Lymphodepletion enhances effector functions of adoptively transferred T cells. 6 d after adoptive transfer, pmel-1 thy1.1+ cells were isolated from the spleens of irradiated and nonirradiated mice and co-cultured with irradiated splenocytes pulsed with the indicated doses of hgp10025–33. Unpulsed splenocytes were used as controls. Data shown are representative of two independent experiments.
Figure 2.
Figure 2.
Even in the absence of T reg cells, ablation enhances antitumor efficacy of adoptively transferred T cells. (a) Ablation augments tumor responses in Rag1−/− mice. TBI or nonirradiated Rag1−/− mice bearing 14-d-old established s.c. B16 tumors were left untreated or received adoptive transfer of 106 cultured pmel-1 T cells in conjunction with rFPhgp100 vaccination and rhIL-2. Values represent the mean ± SEM. (b) Ablation augments tumor responses in MHC class II−/− mice. TBI or nonirradiated MHC class II−/− mice bearing 14-d-old established s.c. B16 tumors were left untreated or received adoptive transfer of 106 cultured pmel-1 T cells in conjunction with rFPhgp100 vaccination and rhIL-2. Values represent the mean ± SEM. (c) Ablation of mice genetically devoid of T reg cells augments autoimmunity. 30 d after treatment, mice were evaluated in a blinded fashion for the development of vitiligo. The percentages of mice with vitiligo are shown.
Figure 3.
Figure 3.
Removal of NK cells enhances antitumor efficacy of adoptively transferred T cells. (a) Irradiation or administration of NK1.1 antibody efficiently depletes NK cells. Absolute numbers of NK1.1+ cells in the blood of tumor-bearing TBI Rag-1−/− mice and nonirradiated Rag-1−/− mice treated with NK1.1 antibody or Ig2aK antibody. Results are shown 3 d after the administration of 106 cultured pmel-1 T cells in conjunction with rFPhgp100 vaccination and rhIL-2. Values represent the mean ± SEM. (b) Removal of NK cells by antibody or by irradiation enhances antitumor efficacy of adoptively transferred T cells. Rag-1−/− mice bearing 9-d-old established s.c. B16 tumors received TBI or antibodies (anti-NK1.1 or Ig2aK antibody). Mice then received adoptive transfer of 106 cultured pmel-1 T cells in conjunction with rFPhgp100 vaccination and rhIL-2 or were left untreated. Values represent the mean ± SEM. (c) The genetic depletion of T cells, B cells, and NK cells recapitulates the effect of TBI. Rag-2−/−C −/− mice bearing 8-d-old established s.c. B16 tumors were either left untreated or received TBI. Where designated, mice received adoptive transfer of 106 cultured pmel-1 T cells in conjunction with rFPhgp100 vaccination and rhIL-2. Irradiation improved antitumor responses in control WT mice (not depicted). Values represent the mean ± SEM.
Figure 4.
Figure 4.
Increased access to supportive endogenous cytokines into irradiated hosts enhances antitumor efficacy of transferred T cells. (a and b) Treatment is impaired in irradiated mice genetically deficient in IL-15 or IL-7 and IL-15 versus WT mice. WT, IL-15−/−, or IL-7−/−IL-15−/− mice bearing 9-d-old established s.c. B16 tumors were left untreated or received adoptive transfer of 106 cultured pmel-1 T cells in conjunction with rFPhgp100 vaccination and rhIL-2 with or without TBI. Values represent the mean ± SEM. (c and d) Absence of both IL-7 and IL-15 impaired proliferative responses of adoptively transferred T cells. Absolute numbers of adoptively transferred pmel-1 cells (CD8+Thy1.1+) in the spleens of tumor-bearing, nonirradiated WT, IL-15−/−, or IL-7−/−IL-15−/− mice (c). Absolute numbers of adoptively transferred pmel-1 cells (CD8+Thy1.1+) in the spleens of tumor-bearing, irradiated WT, IL-15−/−, or IL-7−/−IL-15−/− mice (d). (e–h) Increased access to IL-7 and IL-15 enhances effector functions of adoptively transferred T cells. 6 d after adoptive transfer pmel-1 thy1.1+ cells were isolated from the spleens of TBI WT, IL-15−/−, or IL-7−/−IL-15−/− mice and co-cultured with irradiated splenocytes pulsed with the indicated doses of hgp10025–33. Unpulsed splenocytes were used as controls. Because of the limited numbers of cells isolated from IL-7−/−IL-15−/− mice, pmel-1 cells were tested only against 1 μg/ml of peptide and unpulsed control.

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