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. 2003 Aug 18;198(4):569-80.
doi: 10.1084/jem.20030590.

Tumor regression and autoimmunity after reversal of a functionally tolerant state of self-reactive CD8+ T cells

Willem W Overwijk  1 , Marc R Theoret, Steven E Finkelstein, Deborah R Surman, Laurina A de Jong, Florry A Vyth-Dreese, Trees A Dellemijn, Paul A Antony, Paul J Spiess, Douglas C Palmer, David M Heimann, Christopher A Klebanoff, Zhiya Yu, Leroy N Hwang, Lionel Feigenbaum, Ada M Kruisbeek, Steven A Rosenberg, Nicholas P Restifo
Affiliations

Tumor regression and autoimmunity after reversal of a functionally tolerant state of self-reactive CD8+ T cells

Willem W Overwijk et al. J Exp Med. .

"V体育2025版" Abstract

Many tumor-associated antigens are derived from nonmutated "self" proteins VSports手机版. T cells infiltrating tumor deposits recognize self-antigens presented by tumor cells and can be expanded in vivo with vaccination. These T cells exist in a functionally tolerant state, as they rarely result in tumor eradication. We found that tumor growth and lethality were unchanged in mice even after adoptive transfer of large numbers of T cells specific for an MHC class I-restricted epitope of the self/tumor antigen gp100. We sought to develop new strategies that would reverse the functionally tolerant state of self/tumor antigen-reactive T cells and enable the destruction of large (with products of perpendicular diameters of >50 mm2), subcutaneous, unmanipulated, poorly immunogenic B16 tumors that were established for up to 14 d before the start of treatment. We have defined three elements that are all strictly necessary to induce tumor regression in this model: (a) adoptive transfer of tumor-specific T cells; (b) T cell stimulation through antigen-specific vaccination with an altered peptide ligand, rather than the native self-peptide; and (c) coadministration of a T cell growth and activation factor. Cells, vaccination, or cyto-kine given alone or any two in combination were insufficient to induce tumor destruction. Autoimmune vitiligo was observed in mice cured of their disease. These findings illustrate that adoptive transfer of T cells and IL-2 can augment the function of a cancer vaccine. Furthermore, these data represent the first demonstration of complete cures of large, established, poorly immunogenic, unmanipulated solid tumors using T cells specific for a true self/tumor antigen and form the basis for a new approach to the treatment of patients with cancer. .

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Figures

Figure 1.
Figure 1.
Despite large numbers of gp100-specific T cells, B16 melanoma grows normally in pmel-1 TCR transgenic mice. (A) Generation and characterization of pmel-1 TCR transgenic mice. Single cell suspensions of spleens from a 6-wk-old pmel-1 mouse and a nontransgenic C57BL/6 mouse littermate as well as pmel-1 splenocytes cultured with hgp10025–33 peptide were stained for CD8, Vβ13, and the activation markers CD25, CD44, CD62L, and CD69, and analyzed by FACS®. (B) Recognition of gp100 peptide by pmel-1 T cells. PBL from pmel-1 “D8” founder were cultured for 7 d with mgp10025–33 peptide, washed, and incubated with titrated doses of mgp10025–33 (native) or hgp10025–33 (altered) peptide. IFN-γ production was measured by ELISA on culture supernatants. (C) Pmel-1 T cells specifically recognize B16 melanoma. Cultured pmel-1 splenocytes (gray bars) or clone 9 (black bars) were coincubated with B16 melanoma, EL-4 thymoma, MCA207 sarcoma, MC38 colon carcinoma cells, or CM. Supernatants were assessed for IFN-γ production by ELISA. (D) B16 melanoma grows progressively in pmel-1 TCR transgenic mice. B16 cells were implanted in 6-wk-old pmel-1 T cell receptor transgenic mice and littermates not expressing the transgene. All experiments shown were performed independently at least two times with similar results.
Figure 2.
Figure 2.
Enhanced T cell and antitumor response by adoptively transferred T cells after vaccination with altered peptide ligand. (A) Enhanced antitumor efficacy upon vaccination with rVV encoding altered peptide ligand. Tumor growth was measured in C57BL/6 mice bearing 3-d B16 tumors that received pmel-1 splenocytes followed immediately by vaccination with rVVLacZ, rVVmgp100, rVVhgp100, or no treatment. (B) Increased numbers of pmel-1 T cells from mice immunized with rVV encoding altered peptide ligand. Lymphocytes from mice described in A were isolated from peripheral blood and stained for CD8 and Vβ13 before FACS® analysis. Numbers of CD8+Tm+ cells are depicted through time as percentage of total CD8+ cells. (C) Prolonged peptide/MHC dissociation time of altered peptide ligand. RMA-S cells were incubated with hgp10025–33 (closed symbols) or mgp10025–33 (open symbols, shown at corresponding concentrations) at 25°C overnight. The cells were washed three times, incubated at 37°C for the time designated, and added to 7-d cultured pmel-1 T cells for 24 h. IFN-γ in culture supernatants was quantified by ELISA. Experiments were performed independently at least twice with similar results.
Figure 3.
Figure 3.
Adoptive transfer of tumor-specific T cells combined with vaccination and IL-2 causes regression and cure of large, established tumors. B16 tumor was implanted subcutaneously into C57BL/6 mice treated by adoptive transfer of fresh pmel-1 splenocytes ± vaccination with rFPVhgp100 either 7 (A and B) or 14 d (C and D) after tumor inoculation. IL-2 was administered twice daily for six doses. Fresh or cultured splenocytes were effective in the treatment of large, established tumors. Splenocytes derived from an identically constructed TCR transgenic mouse with specificity for β-galactosidase were used as a control in some experiments (C and D) and were not therapeutic. Statistically significant tumor regression was seen in mice treated with pmel-1 cells given in combination with rFPVhgp100 and IL-2 in >20 independently performed experiments. There were at least five mice/group in all experiments. Mouse survival consistently correlated with tumor growth reduction.
Figure 4.
Figure 4.
Long-term (>1 yr) survival of mice bearing large, established B16 tumors after treatment with adoptive transfer of tumor-specific T cells combined with vaccination and IL-2 is associated with the development of vitiligo. C57BL/6 mice were treated with adoptive transfer of fresh or cultured pmel-1 transgenic splenocytes 14 d after inoculation with B16 melanoma and vaccinated with rFPVhgp100. IL-2 was administered twice daily for six doses. Mice treated with fresh naive or cultured transgenic T cells were cured of B16, and vitiligo was observed, which started at the former tumor site. At >1-yr after therapy, these mice remain tumor-free with progressive vitiligo. To illustrate the autoimmune vitiligo, a photograph of the cohort of 5/5 surviving mice treated with cultured pmel-1 cells from A is shown in B.
Figure 5.
Figure 5.
Endogenous host B or T lymphocytes are not required for the treatment of established B16 tumors. C57BL/6 Rag-1−/− knockout mice bearing subcutaneous B16 tumors established for 14 d were treated with 106 cultured pmel-1 cells given in combination with rFPVhgp100 and IL-2 as described previously. Similar results were obtained in five independently completed experiments.
Figure 6.
Figure 6.
Persistence and function of gp100 reactive T cells after adoptive transfer, vaccination, and administration of IL-2 in mice bearing subcutaneous B16 tumors. (A–C) Data derived from the same, representative experiment. (A) Representative treatment experiment using C57BL/6 mice bearing B16 tumors established for 3 d were treated with fresh pmel-1 T cells plus vaccination with rVVLacZ, rVVmgp100, or rVVhgp100 with or without IL-2. (B) Vaccine-induced specific T cells in peripheral blood. Numbers of CD8+ Tm+ cells isolated from PBL of groups from A are depicted as percentage of total CD8+ cells. (C) Effect of boost with vaccine and IL-2. Mice surviving to day 30 (continuation of B) were retreated by heterologous boosting with rFPVhgp100 plus IL-2. Numbers of CD8+Vβ13+ cells isolated from blood are depicted as percentage of total CD8+ cells. (D) Effect of IL-2 on the number and function of vaccine-induced gp100-specific T cells in tumor tissue. The absolute numbers used to calculate the ratios shown were obtained 7 d after treatment (14 d after tumor implantation) and are normalized for 300 mg of tumor tissue (the average weight of excised tumors on day 14). These numbers are as follows (IL-2/PBS): total lymphocyte gate (1,758:945), CD8+ T cells (1,271:619), CD8+Vβ13+ T cells (890:266), and CD8+Vβ13+ IFN-γ+ T cells (261:8). After a 4-h restimulation with 1 μM mgp10025–33 peptide, the number of CD8+Vβ13+ IFN-γ+ T cells measured from this group were (754:147) to give a ratio of 5:1 (not depicted).
Figure 7.
Figure 7.
Histological analysis reveals presence of Vβ13+ T cells in tumors and demonstrates the activating effects of IL-2. T cells and tissue architecture within tumors were visualized after vaccination with or without IL-2. C57BL/6 mice bearing 14-d B16 tumors received 107 pmel-1 splenocytes and vaccination with hgp10025–33 peptide in IFA and anti-CD40 with or without IL-2. As was the case for vaccination with rVVs or rFPVs encoding hgp100, vaccination with hgp10025–33 peptide in IFA and anti-CD40 after the adoptive transfer of pmel-1 cells was greatly enhanced with the addition of IL-2 in repeated experiments (not depicted). Tumors were excised 7 d after the vaccination and 4-μm frozen sections were stained with anti-Vβ13 mAb (top, arrows) or IgG control (bottom), counterstained with hematoxylin, and photographed at an original magnification of 200. (left) pmel-1 cells plus hgp100 vaccination. (right) pmel-1 cells plus hgp100 vaccination followed by IL-2 administration.
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