Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official. Federal government websites often end in . gov or . mil. Before sharing sensitive information, make sure you’re on a federal government site. VSports app下载.

Https

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely. V体育官网.

. 2005 Jun;115(6):1616-26.
doi: 10.1172/JCI24480.

Acquisition of full effector function in vitro paradoxically impairs the in vivo antitumor efficacy of adoptively transferred CD8+ T cells

Luca Gattinoni  1 , Christopher A Klebanoff, Douglas C Palmer, Claudia Wrzesinski, Keith Kerstann, Zhiya Yu, Steven E Finkelstein, Marc R Theoret, Steven A Rosenberg, Nicholas P Restifo
Affiliations

"VSports注册入口" Acquisition of full effector function in vitro paradoxically impairs the in vivo antitumor efficacy of adoptively transferred CD8+ T cells

Luca Gattinoni et al. J Clin Invest. 2005 Jun.

Abstract

T cell differentiation is a progressive process characterized by phenotypic and functional changes. By transferring tumor-specific CD8+ T cells into tumor-bearing mice at various stages of differentiation, we evaluated their efficacy for adoptive immunotherapy VSports手机版. We found that administration of naive and early effector T cells, in combination with active immunization and IL-2, resulted in the eradication of large, established tumors. Despite enhanced in vitro antitumor properties, more-differentiated effector T cells were less effective for in vivo tumor treatment. Several events may underlie this paradoxical phenomenon: (a) downregulation of lymphoid-homing and costimulatory molecules; (b) inability to produce IL-2 and access homeostatic cytokines; and (c) entry into a proapoptotic and replicative senescent state. While the progressive acquisition of terminal effector properties is characterized by pronounced in vitro tumor killing, in vivo T cell activation, proliferation, and survival are progressively impaired. These findings suggest that the current methodology for selecting T cells for transfer is inadequate and provide new criteria for the generation and the screening of optimal lymphocyte populations for adoptive immunotherapy. .

PubMed Disclaimer

VSports app下载 - Figures

Figure 1
Figure 1
Acquisition of full effector function in vitro impairs antitumor efficacy. Pmel-1 CD8+ T cells at progressive stages of differentiation were generated using multiple in vitro stimulations with 1 μM hgp10025–33 and 30 IU/ml of rhIL-2. Seven days following the last stimulation, phenotypic and functional assays were performed. (A) Phenotypic differences in naive and effector subgroups. Flow cytometry analysis for the expression of lymphoid-homing molecules CD62L, CCR7, and β7 integrin; costimulatory molecule CD27; activation/effector markers CD69, CD44, CD25, and granzyme-B (GZM-B) on CD8-enriched pmel-1 naive, early effector, intermediate effector, and effector T cells. Mean fluorescence intensity (MFI) values after gating on CD8+ cells are shown. Data shown are representative of 3 independent experiments. (B) Functional differences in naive and effector subgroups. IFN-γ and IL-2 release and cytotoxic assay against hgp10025–33–pulsed MCA-205 targets. MCA-205 cells plus the irrelevant influenza nucleoprotein peptide were used as control. In the cytotoxic assay, values for control target cells (0–14%) were subtracted. Data shown are representative of 3 independent experiments. (C and D) Differentiation of antitumor T cells is inversely associated with in vivo effectiveness. WT mice bearing 10-day-old established s.c. B16 tumors were sublethally irradiated and left untreated as control or received adoptive transfer of 1 × 106 (when not otherwise indicated) CD8-enriched pmel-1 naive, early effector, intermediate effector, and effector T cells in conjunction with rFPhgp100 vaccination and exogenous rhIL-2 (36 μg per dose). Results for tumor area are the mean of measurements from 5 mice per group (± SEM). Data shown are representative of 7 independent experiments.
Figure 2
Figure 2
Cytofluorometry and Western blot analysis of effective and impaired CD8+ T cells. Pmel-1 CD8+ T cells at progressive stages of differentiation were generated using multiple in vitro stimulations with 1 μM hgp10025–33 and 30 IU/ml of rhIL-2. (A) Flow cytometry for the surface expression of IL-7Rα and KLRG-1. Results after gating for CD8+ cells are shown. (B) Detection of BID and its activated, truncated C-terminal cleavage products (tBID) p15 and p13 and BAD by Western blot analysis.
Figure 3
Figure 3
The differentiation state of CD8+ T cells is inversely related to their proliferative capacity. Pmel-1 CD8+ T cells at progressive stages of differentiation were generated using multiple in vitro stimulations with 1 μM hgp10025–33 and 30 IU/ml of rhIL-2. (A) In vitro proliferation of effector subgroups. Early effector, intermediate effector, and effector T cells were labeled with CFSE and were cultured with 1 μM hgp10025–33 peptide–pulsed on irradiated splenocytes in the presence of 30 IU/ml rhIL-2. Cells were harvested after 4 days, and their CFSE content was determined. Results after gating on Vβ13+ population are shown. This experiment was performed twice with similar results. (B) In vivo proliferation of effector subgroups. Absolute numbers of adoptively transferred pmel-1 cells (CD8+Vβ13+) in the spleens of tumor-bearing, sublethally irradiated WT mice. Mice were treated with rFPhgp100, rhIL-2 (36 μg per dose), and 1 × 106 pmel-1 early effector, intermediate effector, and effector T cells. Data shown are the mean of 2 mice per group. This experiment was performed twice, with similar results.
Figure 4
Figure 4
Subfractionated CD62Lhigh and CD62Llow early effector cells possess similar properties in vitro. (A and B) Phenotypic characterization of CD62Lhigh and CD62Llow subsets. Five days after the initial stimulation with antigen and IL-2, CD62Lhigh and CD62Llow populations were sorted by MACS CD62L-positive selection column and analyzed by flow cytometry on day 6 for the expression of IL-7Rα; lymphoid-homing molecules β7 integrin and CCR7; the costimulatory molecule CD27; activation/effector markers CD44, CD69, CD25, and granzyme-B; and the senescence marker KLRG-1. MFI values after gating on CD8+Vβ13+ cells are shown. The MFI was determined for all groups at the same time under the same cytoflourometric settings using the same reagents and isotype-matched controls. Data shown are representative of 2 independent experiments. (C) CD62Lhigh and CD62Llow subsets have similar functional qualities. 51Cr, IFN-γ, and IL-2 release against hgp10025–33–pulsed MCA-205 targets. MCA-205 cells plus influenza nucleoprotein peptide (np) were used as control. Where error bars are not visible, it is because they are obscured by the symbols used. Data shown are representative of 2 independent experiments. (D) CD62Lhigh and CD62Llow subsets have similar proliferative capacity. CD62Lhigh and CD62Llow cells were labeled with CFSE and were cultured with hgp10025–33 peptide–pulsed on irradiated splenocytes in the presence of 30 IU/ml of rhIL-2. Cells were harvested from day 1 to day 4, and their CFSE content was determined. Results after gating on CD8+Vβ13+ population are shown. This experiment was performed twice, with similar results.
Figure 5
Figure 5
CD62L-selectin identifies CD8+ T cells with superior in vivo antitumor properties. (A) CD62Lhigh subset possesses superior in vivo antitumor properties. WT mice bearing 10-day-old established subcutaneous B16 tumors were sublethally irradiated and left untreated as control or received adoptive transfer of 1 × 106 of pmel-1 CD62Lhigh or CD62Llow cells with exogenous rhIL-2 (36 μg per dose) with or without rFPhgp100 vaccination. Results for tumor area are the mean of measurements from 5 mice per group (± SEM). Data shown are representative of 3 independent experiments. (B and C) CD62Lhigh subset expands vigorously in vivo. (B) Absolute numbers of adoptively transferred CD8+ pmel-1cells in the spleen of tumor-bearing, sublethally irradiated WT mice. Mice were treated with FPhgp100, rhIL-2 (36 μg per dose), and 1 × 106 of pmel-1 CD62Lhigh or CD62Llow cells. Data shown are the mean of 2 mice per group. This experiment was performed twice, with similar results. (C) Percentage of CD8+ pmel-1cells in the tumor. Mice were treated with FPhgp100, rhIL-2 (36 μg per dose), and 1 × 106 of pmel-1 CD62LhighLy5.1 or CD62LlowThy1.1 cells. Data shown are the mean of 2 mice per group. This experiment was performed twice with similar results. (D) CD62L expression is required to maximize the antitumor efficacy of adoptively transferred T cells. WT mice bearing 10-day-old established s.c. B16 tumors were sublethally irradiated and left untreated as control or received adoptive transfer of 1 × 106 one-week-cultured pmel-1 CD62L+/+ cells or pmel-1 CD62L–/– cells in conjunction with rFPhgp100 vaccination and exogenous rhIL-2 (36 μg per dose). Tumor area results are the mean of measurements from 5 mice per group (± SEM). Data shown are representative of 3 independent experiments.
Figure 6
Figure 6
Antigen presentation by BM-derived APCs is critical for tumor treatment. (A) Generation of BM chimeras. We generated chimeras by transferring 5 × 106 BM cells from β2m–/– and WT mice into lethally irradiated WT or β2m–/– mice. (B–D) Results from BM chimeras indicate that BM-derived APCs expressing MHC class I are required for tumor treatment. Chimeras bearing 10-day-old established s.c. B16 tumors were sublethally irradiated and left untreated as controls or received adoptive transfer of 1 × 106 pmel-1 cells cultured for 1 week in conjunction with rFPhgp100 vaccination and exogenous rhIL-2 (36 μg per dose). Results for tumor area are the mean of measurements from 5 mice per group (± SEM). Data shown are representative of 2 independent experiments.
Figure 7
Figure 7
IL-15 uncouples T cell proliferation from differentiation and preserves antitumor efficacy in vivo. Pmel-1 splenocytes were cultured and restimulated weekly in the presence of 1 μM hgp10025-33 and CM containing 30 UI/ml of rhIL-2 (Pmel-1IL-2) or 10 ng/mL rhIL-15 (Pmel-1IL-15). (A) T cells expand similarly in media containing IL-2 and IL-15. Pmel-1IL-2 and pmel-1IL-15 cells were enumerated using trypan blue exclusion at indicated time points. Data shown are representative of 2 independent experiments. (B) IL-15 preserves the expression of lymphoid-homing molecules after multiple in vitro stimulations. Flow cytometry analysis for the expression of the lymphoid-homing molecule CD62L on pmel-1IL-2 and pmel-1IL-15 after 14 and 21 days of culture. Results after gating for CD8+ cells are shown. Data are representative of 2 independent experiments. (C) IL-15 preserves in vivo antitumor efficacy of T cells after multiple in vitro stimulations. WT mice bearing 10-day-old established subcutaneous B16 tumors were sublethally irradiated and left untreated as control or received adoptive transfer of 1 × 106 pmel-1IL-2 or pmel-1IL-15 cells cultured for 14 and 21 days in conjunction with rFPhgp100 vaccination and exogenous rhIL-2 (36 μg per dose). Tumor area results are the mean of measurements from 5 mice per group (± SEM). Data shown are representative of 2 independent experiments. (D) Correlation between CD62L expression on adoptively transferred T cells and slope of tumor growth curve. Results were pooled from 2 independent experiments.
Figure 8
Figure 8
A schematic illustration summarizing results indicating the inverse relationship of in vitro and in vivo effector functions of adoptively transferred naive and effector T cell subsets. After primary antigen stimulation, naive CD8+ T cells proliferate and progressively differentiate into terminally differentiated effector T cells. Phenotypic and functional changes characterize the differentiation process. The gradual acquisition of complete effector functions (dashed red line) is associated with the progressive inability of T cells to cause tumor regression upon adoptive transfer (black line). Such mechanisms initially involve the downregulation of lymphoid-homing and costimulatory molecules, which results in a poor in vivo activation of T cells. Other mechanisms occur later and include the inability to produce IL-2 and access homeostatic cytokines, the imbalance of proapoptotic and anti-apoptotic signals, and the acquisition of a state of replicative senescence. +, low expression; ++, intermediate expression; +++, high expression.
See this image and copyright information in PMC

Comment in

References

    1. Rosenberg SA, Yang JC, Restifo NP. Cancer immunotherapy: moving beyond current vaccines. Nat. Med. 2004;10:909–915. - VSports注册入口 - PMC - PubMed
    1. Dudley ME, Rosenberg SA. Adoptive-cell-transfer therapy for the treatment of patients with cancer. Nat. Rev. Cancer. 2003;3:666–675. - PMC - PubMed
    1. Childs RW, Barrett J. Nonmyeloablative allogeneic immunotherapy for solid tumors. Annu. Rev. Med. 2004;55:459–475. - PubMed
    1. Dudley ME, et al. Cancer regression and autoimmunity in patients after clonal repopulation with antitumor lymphocytes. Science. 2002;298:850–854. - PMC (VSports) - PubMed
    1. Rosenberg SA, Dudley ME. Cancer regression in patients with metastatic melanoma after the transfer of autologous antitumor lymphocytes. Proc. Natl. Acad. Sci. U. S. A. 2004;101(Suppl. 2):14639–14645. - PMC - PubMed

MeSH terms