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Tumor Regression and Autoimmunity after Reversal of a Functionally Tolerant State of Self-reactive CD8+ T Cells

Tumor Regression and Autoimmunity after Reversal of a Functionally Tolerant State of... Many tumor-associated antigens are derived from nonmutated “self” proteins. T cells infiltrat- ing 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 mm ), subcutaneous, unmanipulated, poorly immunogenic B16 tumors that were established for up to 14 d before the start of treat- ment. 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 an- tigen-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. Further- more, 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. Key words: adoptive cell transfer • immunotherapy • IL-2 • recombinant poxvirus • T cell epitope Introduction The observation that many tumor-associated antigens are attractive targets for immunotherapy, because they are nonmutated “self-antigens” has raised the question of shared among patients, obviating the need for personal- how to induce large numbers of highly active, self/tumor ized vaccine development (1–6). We and others have at- antigen-specific T cells capable of destroying large, estab- tempted numerous vaccination approaches targeting self/ lished tumors. Nonmutated self-antigens are particularly tumor antigens. However, despite the increased numbers of self/tumor antigen-specific T cells, the reproducible W.W. Overwijk, M.R. Theoret, and S.E. Finkelstein contributed equally induction of significant destruction of large, established to this work. Address correspondence to Nicholas P. Restifo, NCI, NIH, Building 10, Room 2B42, Bethesda, MD 20892-1502. Phone: (301) 496-4904; Abbreviations used in this paper: CM, culture media; rFPV, recombinant Fax: (301) 402-0922; email: [email protected] fowlpox virus; rVV, recombinant vaccinia virus. 569 The Journal of Experimental Medicine • Volume 198, Number 4, August 18, 2003 569–580 http://www.jem.org/cgi/doi/10.1084/jem.20030590 The Journal of Experimental Medicine cancers in mouse or in man using any self-antigen vac- The generation of autoreactive T cells in vivo may be cine-based approach remains an elusive goal (7–9). A state difficult because of the mechanisms of central and periph- of functional tolerance apparently exists in the tumor- eral tolerance and the impact of antigen expression by tu- bearing host. mor tissue on T cell function (14, 25–27). Altered peptide This state of functional tolerance, defined here as the co- ligands have been used to improve the reactivity of T existence of tumor-specific T cells and growing tumor cells specific for self/tumor antigens in mouse and man (9, cells, may be incomplete. There is evidence indicating that 28–30). For gp100, H-2D -restricted CD8 T cells ca- antigen expression by tumors leads to limited T cell activa- pable of recognizing B16 melanoma and normal melano- tion without tumor regression (10–12). Work in the spon- cytes could only be elicited when the altered peptide was taneous insulinoma model has indicated that effector “ex- used (31). We described previously the cloning of the un- haustion,” rather than tumor escape is the underlying mutated mouse homologue of gp100 (mgp100) from B16 mechanism of functional tolerance (13). Work using other melanoma (32). We have also described the identification antigenic systems indicates that antigens expressed by tu- of a peptide derived from human (h)gp100, KVPRN- mors actively tolerize tumor-specific T cells (14). Antigenic QDWL (hgp100 ), that represents an altered peptide 25–33 “ignorance” to tumors has also been proposed as a mecha- ligand form of mgp100 , EGSRNQDWL (31). Upon 25–33 nism for functional tolerance (15). Thus, the mechanisms establishing this model for vaccination against self/tumor underlying the observed functional tolerance of antitumor antigen, our next goal was to identify immunotherapeutic T cells remain unclear. strategies that would induce the regression of large estab- The modeling of self-reactivity is likely to be important lished tumors using CD8 T cells specific for a self-antigen. in the development of new immunotherapies that target self/tumor antigens, but many of the currently available tu- mor models that model the complete destruction of estab- Materials and Methods lished tumors target “foreign” antigens. The antigenic sys- Generation of Pmel-1 TCR Transgenic Mice. To study self/tu- tems most commonly used until now by us and others mor antigen-specific T cell responses to melanoma, we developed include chicken egg ovalbumin, -galactosidase from Esch- a transgenic mouse strain on a C57BL/6 background and named erichia coli, the L major histocompatibility antigen, and it pmel-1. RNA was isolated from clone 9, a gp100 -specific, 25–33 hemagglutinin or nucleoprotein derived from influenza A vi- H-2D –restricted CD8 T cell clone (31), and  and  TCR re- gions were amplified by 5-Rapid Amplification of cDNA Ends rus (14, 16–20). These models have shed valuable light on (5-RACE, Life Technologies) using constant region anti-sense basic immunologic principles (21), but it is unclear to what primers 1 (5-GGCTACTTTCAGCAGGAGGA-3) and 1 extent the results obtained from these models reflect im- (5-AGGCCTCTGCACTGATGTTC-3), respectively. 5- mune responses against true self/tumor antigens. For exam- RACE products were amplified with nested TCR  and  con- ple, the high levels of expression of foreign antigens under stant region primers 2 (5-GGGAGTCAAAGTCGGTGAAC- the control of constitutively activated viral promoters may 3) and 2 (5-CCACGTGGTCAGGGAAGAAG-3), and not mimic the expression of self-antigens that are the tar- cloned into pCR4TOPO TA sequencing vectors (Invitrogen). gets of tumor-specific T cells. Most importantly, each of TCR  and  transcripts were sequenced as V1/JTA19/C these model antigens has been selected for study because and V13S1/D1/J1S6/C1. Genomic cloning PCR primers they induce unusually strong T cell responses, perhaps not were designed based on the method described previously by Kouskoff et al. (33). The  and  genomic variable domains were reflecting T cell responses characteristic of those observed PCR amplified (Perkin-Elmer) with primers g1 (5-TCTC- against tumor antigens. CCGGGCTTCTCACTGCCTAGCCATGATGAAATCCT- We sought to study the activation and proliferation of TGAGTGTTTC-3) and g2 (5-GTAGCGGCCGCGTAAA- self-specific T cells that can infiltrate and destroy large, es- ATCTATCCTAGTGTTCCCCAGA-3) or g1 (5-GATCT- tablished tumors. Here, we investigate parameters that can CGAGAATCTGCCATGGGCACCAG-3) and g2 (5-GAT- be manipulated in vivo to transform a completely ineffec- ACCGCGGTTCCTTTCCAAGACCAT-3), respectively. The tive antitumor response into a productive one. To utilize a genomic variable domains were TA-cloned into pCR4TOPO tumor model that is more physiologically relevant, we used (Invitrogen), validated by sequencing, subcloned into TCR cas- the poorly immunogenic B16 melanoma, a highly aggres- sette vectors provided by Dr. D. Mathis (Harvard Medical School, sive tumor in C57BL/6 mice (22). A much-studied tumor, Boston, MA; reference 33), and co-injected into fertilized C57BL/6 B16 expresses no MHC class II and very low levels of embryos (Science Applications International Corporation) yield- ing three TCR transgenic founder lines. Unpublished transgenic MHC class I, although both are inducible upon treatment mice expressing the  / transgenic TCR with specificity for a with interferon- (23). The B16 melanoma expresses the K -restricted epitope from -galactosidase (34) were identically mouse homologue (pmel-17) of human gp100, an enzyme constructed in our laboratory and were used as controls in some involved in pigment synthesis that is expressed by the ma- experiments. jority of malignant melanoma cells, as well as by normal Mice and Tumor Cells. C57BL/6 and pmel-1 TCR trans- melanocytes. gp100 is a member of a family of “self” (i.e., genic mice were bred and housed at the National Institutes of unmutated), melanoma/melanocyte differentiation antigens Health (NIH) and Netherlands Cancer Institute animal facilities. that are widely expressed by melanoma cells. These anti- Rag-1 mice were obtained from Jackson ImmunoResearch gens are frequently the targets of T cells that infiltrate hu- Laboratories. B16 (H-2 ) is a gp100 spontaneous murine mela- man tumors (24). noma obtained from the National Cancer Institute tumor reposi- 570 Induction of Tumor Regression and Autoimmunity by Self-reactive CD8 T Cells The Journal of Experimental Medicine tory and maintained in culture media (CM) comprised of RPMI hgp100, or -galactosidase (31) or by subcutaneous injection 1640 with 10% heat-inactivated fetal bovine serum (Biofluids), with 100 l water/IFA emulsion containing 100 g of 0.03% l-glutamine, 100 g/ml streptomycin, 100 g/ml penicil- mgp100 , hgp100 , or -gal peptide followed by two 25–33 25–33 96–103 lin, and 50 g/ml gentamicin sulfate (NIH Media Center). daily intraperitoneal injections of 100 g anti-CD40 mAb puri- Peptides and Recombinant Poxviruses. All synthetic peptides fied from FGK45 hybridoma culture supernatant. rhIL-2 (a gift were synthesized using regular F-MOC chemistry. The syn- from Chiron Corp.) was administered intraperitoneally directly thetic, H-2D –restricted peptides hgp100 , KVPRNQDWL, after vaccination (100,000 Cetus Units or 600,000 IU rhIL-2 in 25–33 mgp100 , EGSRNQDWL, and NP , ASNENMETM, PBS, twice daily for 3–5 d). Tumors were measured with calipers 25–33 366–374 were synthesized by Peptide Technologies to a purity 99% by and the products of perpendicular diameters were recorded. Mice HPLC and amino acid analysis. All recombinant vaccinia viruses were killed once tumors reached 400 mm . All experiments were (rVVs) and recombinant fowlpox viruses (rFPVs) used in this pa- performed in a blinded, randomized fashion (measuring investiga- per have been described previously (31) and were either generated tor had no knowledge of the experimental group) and performed in our laboratory or provided by D. Panicali, L. Gritz, and A. Go- independently at least twice with similar results. mez-Yafal (Therion Biologics, Cambridge, MA). These viruses were constructed and purified as described by Earl et al. (35). Results In Vitro Activation of Pmel-1 T Cells and Cytokine Release As- say. Peripheral blood mononuclear cells or splenocytes from Despite Large Numbers of gp100-specific T Cells, B16 Mela- mice were depleted of erythrocytes by hypotonic lysis, cultured noma Grows Normally in Pmel-1 TCR Transgenic Mice. To in CM with 30 IU/ml rhIL-2 in the presence of 1 M hgp100 25–33 study self/tumor antigen-specific T cell responses to mela- peptide, and used on days 5–10 after start of the culture. For noma, we developed a transgenic mouse strain on a cytokine release assays, 10 T cells were cocultured in CM with C57BL/6 background and named it pmel-1. Pmel-1 trans- 10 target cells or 1 M of the indicated peptide. Supernatants genic mice expressed the V1V13 TCR from a cloned T were collected after 24 h and tested using an mIFN- ELISA kit cell (clone 9) described previously (31). Clone 9, like the (Endogen) according to the manufacturer’s protocol. pmel-1 TCR transgenic cells derived from it, recognized Histology. Skin and tumor samples were analyzed as de- an H-2D –restricted epitope corresponding to amino acids scribed previously (36). In brief, 4- m cryostat sections were 25–33 of gp100. Virtually all (95%) of the CD8 T cells air-dried overnight and fixed in acetone for 10 min at room temperature, preincubated in 5% (vol/vol) normal goat serum in pmel-1 TCR transgenic mice were V13 , amounting (Central Laboratory of the Netherlands Red Cross Blood Trans- to 20% of all splenocytes (Fig. 1 A). Pmel-1 T cells in fusion Service) and stained with 0.5 g/ml anti–mouse v13 blood and spleen generally expressed baseline levels of the mAb-FITC or mIgG1-FITC control Ab (BD Biosciences) in activation/effector markers CD25, CD44, and CD69, indi- PBS/1% BSA followed by rabbit anti-FITC (1:40,000; DakoCy- cating that most of the transgenic cells were in the naive tomation), biotinylated goat anti–rabbit antibody (1:400; Dako- state. The CD62L levels were as high if not higher than Cytomation) and streptavidin–biotin-conjugated alkaline phos- those found in normal splenocytes, which was also consis- phatase complex (1:100, ABC-protocol; DakoCytomation). tent with the finding that pmel-1 cells were largely naive. Color was developed using Permanent red chromogen substrate Upon in vitro stimulation with 1 M of the hgp100 25–33 (PRC kit; Cell Marque Corporation) and sections were counter- peptide, T cells from pmel-1 TCR transgenic mice prolif- stained with hematoxylin. erated extensively and developed an effector phenotype Flow Cytometry and Intracellular IFN- Assay. To obtain ap- propriate lymphocyte samples, mice were either tail-bled on indi- that included the up-regulation of CD25, CD44, and cated days after vaccination or killed and splenectomized for fresh CD69 and the partial down-regulation of CD62L (Fig. 1 splenocytes. Where designated, splenocytes were cultured as de- A). Functionally, pmel-1 T cells released IFN- after stim- scribed previously (31). Erythrocytes were removed by hypotonic ulation with mgp100, hgp100 peptide (Fig. 1 B), or B16 lysis or ficoll gradient separation, and cells were stained with the melanoma cells, which are naturally mgp100 (Fig. 1 C). indicated dilutions of mAbs against CD8a (1:20, clone 53–6.7), The transgenic T cells resembled the reactivity of the origi- CD4 (1:10, clone H129.19), V13 (1:10, clone MR12–3), CD25 nal clone 9, from which their / TCR was cloned (Fig. 1 (1:10, clone PC61), CD44 (1:10, clone IM7), CD62L (1:10, C and not depicted). Thus, the pmel-1 transgenic mouse clone MEL-14), and CD69 (1:10, clone H1.2F3). All antibodies b could be used as a source of naive T cells with specificity were purchased from BD Biosciences. H2-D –mgp100 tetra- 25–33 for the gp100 epitope. mers were a gift from M. Toebes and T.N.M. Schumacher 25–33 To determine the role of T cell precursor frequency in (Netherlands Cancer Institute, Amsterdam, Netherlands). Propid- ium iodide staining cells were excluded from analysis. Intracellu- the ability of the immune system to reject tumors, we in- lar IFN- assay was performed using the Cytofix/Cytoperm kit jected B16 tumor cells subcutaneously into pmel-1 TCR (BD Biosciences) according to the manufacturer’s recommenda- transgenic mice and nontransgenic littermates. Surprisingly, tions. Samples were analyzed using a FACScalibur™ flow cytom- tumors grew at the same rate in pmel-1 mice despite the eter and CELLquest™ software. presence of overwhelming numbers of CD8 gp100-spe- Adoptive Transfer and Tumor Treatment. Mice were injected cific T cells (Fig. 1 D). Likewise, the adoptive transfer of subcutaneously with 1–5 10 B16 melanoma cells and treated naive or in vitro–activated gp100-specific pmel-1 spleno- 6–7 with intravenous adoptive transfer of freshly isolated 10 fresh 6   cytes into tumor-bearing mice alone or in combination splenocytes (2 10 CD8 V13 T cells) or in vitro–acti- 6–7 with IL-2 had minimal effects on tumor growth or the sur- vated pmel-1 splenocytes (10 CD8 V13 T cells). Mice (n vival of tumor-bearing mice. Thus, the mere presence of 5 for all groups) were vaccinated by intravenous injection of 2 10 plaque-forming units of rVV or rFPV encoding mgp100, naive or activated tumor-specific T cells alone was insuffi- 571 Overwijk et al. The Journal of Experimental Medicine Figure 1. Despite large numbers of gp100-specific T cells, B16 melanoma grows normally in pmel-1 TCR transgenic mice. (A) Generation and char- acterization 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 lit- termate as well as pmel-1 splenocytes cultured with hgp100 peptide were stained for CD8, V13, and the activation markers CD25, CD44, CD62L, 25–33 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 mgp100 peptide, washed, and incubated with titrated doses of mgp100 (native) or hgp100 (altered) peptide. IFN- production was measured 25–33 25–33 25–33 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- 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. cient to cause the regression of the subcutaneous tumor, FACS analysis using mgp100-specific MHC class I tet- thereby indicating that the pmel-1 cells were functionally ramers showed that vaccination with rVVmgp100 induced tolerant to the tumor. a weak and sometimes undetectable T cell response, Immunization with Altered Peptide Ligand Results in Higher whereas use of rVVhgp100 markedly increased specific T Numbers of Self/Tumor Antigen-Specific T Cells That Have cell numbers (Fig. 2 B). Similar results were obtained when Limited Antitumor Efficacy. In an effort to break the func- we used a “minigene” rVV encoding the nine amino acid tionally tolerant state of pmel-1 cells, we immunized tu- minimal peptide determinant preceded by an ER-insertion mor-bearing wild-type C57BL/6 mice with rVVs or rFPVs, sequence (31; unpublished data), or when we used the syn- encoding either the self-antigen, mgp100, or the altered thetic 9-mer peptides emulsified in IFA and given with ligand, hgp100 (31). To track the activation and antitumor anti-CD40 antibody (37). Only immunization with the activity of naive pmel-1 T cells in vivo, we adoptively hgp100 effectively induced gp100-specific T cells, and 25–33 transferred pmel-1 splenocytes into otherwise normal, un- these T cell responses were cross-reactive with mgp100 . 25–33 manipulated mice bearing subcutaneous B16 tumors estab- These findings confirmed earlier observations that gp100 lished for 3 d. Vaccination with rVVmgp100 did not cause sequences outside the minimal determinant do not contrib- any significant antitumor effect. However, vaccination ute to the differential immunogenicity of rVVmgp100 and with rVVhgp100 induced a modest delay in subcutaneous rVVhgp100 (31). tumor growth in some experiments, one of which is shown Both hgp100 and mgp100 peptides contain the 25–33 25–33 (Fig. 2 A). optimal MHC anchor residues at the dominant anchor po- 572 Induction of Tumor Regression and Autoimmunity by Self-reactive CD8 T Cells The Journal of Experimental Medicine sitions, 5 (N) and 9 (L). However, the difference in the than the mouse peptide (31). Here, we extended these ob- three amino-terminal residues resulted in T cell recognition servations by evaluating the dissociation time of peptide at 100-fold lower concentrations of the hgp100 pep- from MHC I molecules on RMA-S cells, again observing 25–33 tide when compared with mgp100 (Fig. 1 B). We have significantly decreased binding at time zero of mgp100 , 25–33 25–33 assessed previously the binding efficiency of the peptides compared with hgp100 , followed by an accelerated dis- 25–33 for the H-2 D class I molecule, finding that the human sociation time for the mgp100 peptide (Fig. 2 C). 25–33 peptide bound to H-2 D with 100-fold greater efficiency Together, these results suggest that pmel-1 T cells can escape central and peripheral tolerance and are not com- pletely refractory to stimulation by self-antigen because the immunogen encoding the altered peptide ligand activated and expanded these self-reactive T cells and conferred lim- ited antitumor efficacy. Stimulation of Adoptively Transferred T Cells through Anti- gen-specific Vaccination and IL-2 Causes Regression and Long- term Cures of Large Established Tumors. Enhanced activation of adoptively transferred pmel-1 T cells by immunization with an altered peptide ligand resulted in inconsistent and limited antitumor effects, leading to growth retardation but not regression of small tumors. Therefore, we set out to determine other factors that might enhance self-specific T cell activation and antitumor effect. We have explored pre- viously the uses of a variety of T cell costimulatory, growth, and activation factors and found that in mouse models, IL-2 can augment the function of recombinant vi- rus–based vaccines (38). To determine if we could enhance the activation of antitumor T cells in a way that would en- able them to treat large, established, vascularized tumors, we adoptively transferred pmel-1 splenocytes into mice bearing subcutaneous B16 tumors established for 7 (Fig. 3, A and B) or 14 d (Fig. 3, C and D). This adoptive transfer was followed by vaccination with rFPV encoding the hgp100-altered peptide ligand and administration of IL-2. This combined treatment reproducibly induced tumor re- gression and even long-term cures of mice bearing estab- lished tumors larger than 50 mm (Fig. 3 C). Invariably, as observed in 20 independently performed experiments, the combination of adoptive transfer of self/ tumor-specific CD8 T cells, antigenic stimulation with an altered peptide ligand, and administration of IL-2 was re- quired to induce this strong antitumor effect. The regimen Figure 2. Enhanced T cell and antitumor response by adoptively trans- ferred 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 vacci- nation with rVVLacZ, rVVmgp100, rVVhgp100, or no treatment. (B) Increased numbers of pmel-1 T cells from mice immunized with rVV en- coding altered peptide ligand. Lymphocytes from mice described in A were isolated from peripheral blood and stained for CD8 and V13 be- fore FACS analysis. Numbers of CD8 Tm cells are depicted through time as percentage of total CD8 cells. (C) Prolonged peptide/MHC dis- sociation time of altered peptide ligand. RMA-S cells were incubated with hgp100 (closed symbols) or mgp100 (open symbols, shown 25–33 25–33 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- in culture supernatants was quantified by ELISA. Experiments were performed independently at least twice with similar results. 573 Overwijk et al. The Journal of Experimental Medicine 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 -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. is ineffective against tumors that are gp100-negative (un- able pattern throughout the mouse. Vitiligo is not observed published data). Administration of either pmel-1 cells when mice are treated with control -galactosidase–specific alone, IL-2 alone, vaccine alone, or any combination of T cells (unpublished data). A representative cohort of com- just two of the three of these immunotherapeutic compo- plete responder mice observed 1 yr after treatment corre- nents induced weak, if any, antitumor effects (Fig. 3, A and sponds to the group treated with cultured T cells, IL-2, and B). In an effort to use the model in a clinically relevant vaccination with rFPVhgp100, shown in Fig. 4 B. way, we have focused on the adoptive transfer of T cells. It is important to note that complete cures of tumors are However, in mice transgenic for the pmel-1 TCR, IL-2 not always observed when using the triple combination of given in combination with vaccination using the altered cells, vaccine, and cytokine, especially when doses of indi- form of gp100 was also therapeutically effective in the treat- vidual components are reduced (unpublished data). Thus, it ment of established B16 tumors (unpublished data). may be useful to elucidate the minimal optimal treatment T cells derived from fresh, naive splenocytes, or from cul- protocol required to reproducibly induce the regression of tured splenocytes activated ex vivo, could mediate the regres- large B16 tumors established for 14 d. 10 Cetus units (6 sion of large, established, subcutaneous tumors in vivo. All 10 IU) twice a day for 3 d was clearly sufficient when mice in Fig. 4 A that were treated with the combined regi- given in combination with 10 fresh or cultured pmel-1 men of adoptive transfer of pmel-1 cells (either with fresh or cells and 2 10 plaque-forming units of rFPVhgp100 to cultured T cells), IL-2, and vaccination with rFPVhgp100 reproducibly induce the regression of large B16 tumors es- were complete and long-term responders. These responses tablished for 14 d. We used adoptively transferred cells in have been followed for 1 yr, and are often accompanied by an effort to model a clinical possibility, but as mentioned the development of limited vitiligo, which generally begins at earlier, vaccination plus IL-2 is effective in pmel-1 TCR the site of tumor regression, and then spreads in an unpredict- transgenic mice bearing established tumors. 574 Induction of Tumor Regression and Autoimmunity by Self-reactive CD8 T Cells The Journal of Experimental Medicine 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 10 cultured pmel-1 cells given in combination with rFPVhgp100 and IL-2 as described previously. Similar results were obtained in five in- dependently completed experiments. the transferred cells after treatment. Data shown in Fig. 6 Figure 4. Long-term (1 yr) survival of mice bearing large, established (A–C) are obtained from the same, representative experi- B16 tumors after treatment with adoptive transfer of tumor-specific T ment. In this treatment model, using B16 tumors estab- cells combined with vaccination and IL-2 is associated with the develop- lished for 3 d, cell transfer combined with immunization ment of vitiligo. C57BL/6 mice were treated with adoptive transfer of fresh or cultured pmel-1 transgenic splenocytes 14 d after inoculation with the altered peptide ligand and IL-2 was required to with B16 melanoma and vaccinated with rFPVhgp100. IL-2 was adminis- observe the maximal therapeutic effect (Fig. 6 A). In other tered twice daily for six doses. Mice treated with fresh naive or cultured experiments, IL-2 could be replaced with either IL-7 or IL- transgenic T cells were cured of B16, and vitiligo was observed, which 15 (unpublished data). Cells plus rVV encoding the altered started at the former tumor site. At 1-yr after therapy, these mice re- main tumor-free with progressive vitiligo. To illustrate the autoimmune peptide ligand can induce limited destruction of small tu- vitiligo, a photograph of the cohort of 5/5 surviving mice treated with mor burden, but this treatment effect is not significant in cultured pmel-1 cells from A is shown in B. mice with large tumor burden (i.e., tumors established for 14 d). We found that the effect of IL-2 on the levels of pmel-1 The administration of a combination of cultured pmel-1 T cells, as a percentage of CD8 T cells or total nucleated T cells, vaccination, and IL-2 was required even in the ab- cells in blood, was modest (two- to fourfold, at most, com- sence of endogenous host B or T lymphocytes, as demon- pared with treatment groups receiving adoptively trans- strated by experiments in C57BL/6 Rag-1 knockout ferred pmel-1 cells and vaccination at any given time in mice bearing subcutaneous B16 tumors established for 14 d multiple experiments; Fig. 6 B). When measured as either (Fig. 5). The cultured cells that were transferred were percentage of all nucleated cells in blood or absolute num- 98% CD8 by FACS (unpublished data). Tumors bers in the spleen, administration of IL-2 increased the rel- shown in Fig. 5 were treated with a suboptimal number ative number of pmel-1 T cells maximally threefold. IL-2 (10 ) of pmel-1 cells, resulting in resurgent tumor growth. did not influence the induction of persistent memory T However, in experiments repeated more than five times, cells that could be reactivated by booster vaccination 32 d antitumor effects were at least as strong (and generally after priming (Fig. 6 C). stronger) in Rag-1 mice as those seen in wild-type We sought to understand the impact of IL-2 administra- C57BL/6 mice (unpublished data). tion on absolute numbers of pmel-1 T cells, and to measure IL-2 Increases the Number and Function of Self-specific, Tu- the effect it had on their localization and effector function. moricidal T Cells. To further understand the mechanisms We found that administration of IL-2 after adoptive trans- of how the antitumor effector function of pmel-1 cells was fer and vaccination increased the total number of lympho- enhanced by IL-2, we monitored the levels of pmel-1 T cytes infiltrating into 300 mg of solid tumor by 1.9-fold 7 d cells in the blood of mice using tetramer analysis to follow after vaccination (Fig. 6 D). The number of V13 CD8 575 Overwijk et al. The Journal of Experimental Medicine Figure 6. Persistence and function of gp100 reactive T cells after adoptive transfer, vaccination, and admin- istration of IL-2 in mice bearing subcutaneous B16 tu- mors. (A–C) Data derived from the same, repre- sentative 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 de- picted 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 V13 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 V13 T cells (890:266), and CD8 V13 IFN- T cells (261:8). After a 4-h restimulation with 1 M mgp100 peptide, the number of CD8 V13 25–33 IFN- T cells measured from this group were (754:147) to give a ratio of 5:1 (not depicted). Discussion T cells was increased by 3.3-fold, corresponding to the modest increase in V13 CD8 T cell levels in blood (Fig. Strengths and Weaknesses of the Pmel-1 TCR Transgenic 6 D). Strikingly, the number of V13 CD8 T cells that Model. Here, we have investigated parameters that can produced IFN- directly ex vivo and without further stim- be manipulated in vivo to transform a functionally tolerant ulation was increased 34-fold by IL-2 administration. transgenic T cell into an activated one that mediates the re- These data illustrate that in the absence of IL-2, T cells are gression of large established tumors. As a tumor model, we present at the tumor site but not fully activated. Thus, IL-2 used the poorly immunogenic B16 melanoma. This tumor acted primarily as a costimulatory/activation factor rather failed to induce activation of adoptively transferred than purely as a growth factor. mgp100-specific pmel-1 T cells. Reports on the treatment Histological analysis of B16 tumors 7 d after treatment of of large, established tumors with systemic, rather than local 7-d tumors with pmel-1 cells plus vaccination showed spe- therapies, remain rare, but not unprecedented in the scien- cific T cell infiltration but no marked tumor cell death or tific literature (39). tissue destruction (Fig. 7, left). By contrast, when the adop- Most of the published mouse tumor models use preven- tive transfer of pmel-1 cells plus vaccination was followed tion of tumor implantation and growth as the measure of by administration of IL-2, we observed extensive tumor success. When the treatment of established tumors is re- cell death and loss of tissue integrity (Fig. 7, right). These ported, treated tumors are generally significantly smaller data show that strong vaccination by itself can induce pro- than those treated in the present set of experiments. Fur- liferation and tumor localization of antigen-specific T cells, thermore, many of the existing tumor systems target model but that these T cells do not effectively mediate tumor de- antigens that have been artificially inserted into the tumor struction unless provided with exogenous cytokine. genome, whereas the majority of human tumor–associated 576 Induction of Tumor Regression and Autoimmunity by Self-reactive CD8 T Cells The Journal of Experimental Medicine splenocytes, 95% of all CD8 T cells) in pmel-1 mice did not impact tumor growth. Apparently, large numbers of activated tumor-specific T cells are necessary but not suffi- cient to induce tumor destruction. This puzzling observa- tion, observed several years ago in mice by Ohashi and col- leagues (13), has been rediscovered in recent clinical trials (7–9, 42). Provision of nonspecific systemic inflammation or “dan- ger” signals by infection of mice with control vaccinia virus or injection of anti-CD40 mAb and IL-2 did not result in T cell activation or suppression of tumor growth. Further- more, in vitro–activated specific T cells neither proliferated in response to tumor nor slowed the growth of even tiny subcutaneous tumors. This suggests that direct presentation of mgp100 antigen by tumor cells or its cross-presentation by activated professional APCs is insufficient to overcome peripheral tolerance and induce T cell activation and prolif- eration. A possible explanation for this poor presentation may reside in the low affinity of the mgp100 peptide 25–33 for H-2 D and the resulting brief half-life of MHC– mgp100 complexes on APCs (Fig. 2 C; references 31, 25–33 43). Indeed, vaccination with the self-antigen, mgp100, in- duced only marginal T cell proliferation and no control of Figure 7. Histological analysis reveals presence of V13 T cells in tu- tumor growth (Fig. 2, A and B; and Fig. 6, A and B). mors and demonstrates the activating effects of IL-2. T cells and tissue ar- When tumor-bearing recipients were vaccinated with a chitecture within tumors were visualized after vaccination with or with- peptide epitope capable of binding H-2D with 100-fold out IL-2. C57BL/6 mice bearing 14-d B16 tumors received 10 pmel-1 greater affinity than its mouse counterpart (31), we saw a splenocytes and vaccination with hgp100 peptide in IFA and anti- 25–33 CD40 with or without IL-2. As was the case for vaccination with rVVs or greatly increased mgp100-specific T cell response (in which rFPVs encoding hgp100, vaccination with hgp100 peptide in IFA and 25–33 up to 80% of all CD8 T cells in the blood can be anti-CD40 after the adoptive transfer of pmel-1 cells was greatly en- mgp100-specific). It is possible that the use of an altered hanced with the addition of IL-2 in repeated experiments (not depicted). peptide ligand is not an absolute requirement in other can- Tumors were excised 7 d after the vaccination and 4- m frozen sections cer vaccines, particularly if the antigenic peptide of choice were stained with anti-V13 mAb (top, arrows) or IgG control (bottom), counterstained with hematoxylin, and photographed at an original mag- is, unlike the mgp100 peptide, highly capable of activating nification of 200. (left) pmel-1 cells plus hgp100 vaccination. (right) T cells. However, it is likely that T cells that have survived pmel-1 cells plus hgp100 vaccination followed by IL-2 administration. central and peripheral tolerance to self/tumor antigens gen- erally target peptides with poor avidity. Our observations in mice seem analogous to those observed in human mela- antigens used in current clinical efforts are nonmutated self- noma patients in which vaccination with the gp100 209–217 antigens. In studies focusing on adoptive immunotherapy, peptide is inefficient, unless that peptide is altered to in- investigators generally report on the treatment of small pul- crease its avidity for the HLA-A*0201 molecule (9). Thus, monary “metastases,” which are nodules created by the in- the optimization of self/tumor peptides for binding to travenous injection of tumor cells, which are treated with MHC molecules should be further explored in future can- lymphocytes injected by the same route. The present paper cer vaccine development. describes the use of large, vascularized subcutaneous tumors. One observation in mouse and in man is that therapeutic One aspect of the current model that does not model immunization designed to treat established tumor is gener- the human tumor–bearing situation is the use of a trans- ally ineffective. Where immunization can induce tumor plantable tumor rather than spontaneous melanomas aris- antigen-specific T cells, the destruction of an established ing and progressing in each mouse, which could be sub- tumor is not reproducibly observed. In the present model, jected to treatment. Spontaneous tumor models can be adoptive transfer of T cells was required in order for vac- more cumbersome and time-consuming to work with. cine and IL-2 to reproducibly induce the destruction of tu- However, the use of spontaneous tumors (40, 41) may mor. There apparently are quantitative or qualitative differ- more effectively model some aspects of tumor carcinogen- ences between cells expanded ex vivo and used for esis and treatment. Efforts to validate the findings of the adoptive transfer, and cells expanded in vivo as a result of present paper in a model of spontaneous tumor are cur- immunization. Cells induced by vaccination are likely to be rently ongoing in the laboratory. lower in number; furthermore, they may be quiescent or The Mere Presence of Tumor-reactive CD8 T Cells Is Insuf- refractory to activation by tumor cells, perhaps in a way ficient to Induce Tumor Regression. The presence of over- similar to the changes observed in T cells during chronic whelming numbers of mgp100-specific T cells (20% of all infection (44). Thus, we seek to use, in new clinical trials, 577 Overwijk et al. The Journal of Experimental Medicine the combined regimen of cells, IL-2, and vaccination with requirements defined the components of successful antitu- an altered peptide ligand described in this paper. mor treatment. Based on these data, clinical trials that use The use of lymphodepletion (nonmyeloablative condi- adoptive cell transfer used in combination with IL-2 and tioning) may enhance the function of adoptively transferred vaccination with an altered peptide ligand are being initi- T cells as shown previously (45, 46). Lymphodepletion ated in patients with cancer. may facilitate immune responses by deletion of endogenous We would like to thank D. Panicali, L. Gritz, and A. Gomez-Yafal regulatory T cells, or by increasing the availability of en- from the Therion Biologics for providing recombinant poxviruses dogenous immune activating cytokines such as IL-7 or IL- used in this work, T.N.M. Schumacher and M. Toebes for help 15. Results in the clinic are encouraging, but the consistent with tetramer production, P. van den Berk for producing FGK45 induction of complete responses in our patients with cancer anti-CD40 mAb, and J. Yang and P. Hwu for helpful discussions. remains the focus of our ongoing clinical efforts. We are This work was supported in part by a grant (KWF 2001-2562) now using the pmel-1 model to explore the immune- from the Dutch Cancer Society. enhancing mechanisms of nonmyeloablative conditioning. Submitted: 11 April 2003 IL-2 Enhances CD8 T Cell Function In Vivo. IL-2 can Revised: 1 July 2003 be clinically successful but the mechanism responsible for Accepted: 1 July 2003 the sometimes-dramatic clinical antitumor responses is un- clear (47). We had found previously that IL-2 could aug- ment the T cell response to a virus (38). These observa- References tions have been confirmed and expanded recently by 1. Houghton, A.N., J.S. Gold, and N.E. Blachere. 2001. Immu- others (48). 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Viral persistence alters CD8 T-cell cer immunotherapy. J. Immunother. Emphasis Tumor Immunol. immunodominance and tissue distribution and results in dis- 19:81–84. 580 Induction of Tumor Regression and Autoimmunity by Self-reactive CD8 T Cells The Journal of Experimental Medicine http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Experimental Medicine Pubmed Central

Tumor Regression and Autoimmunity after Reversal of a Functionally Tolerant State of Self-reactive CD8+ T Cells

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Abstract

Many tumor-associated antigens are derived from nonmutated “self” proteins. T cells infiltrat- ing 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 mm ), subcutaneous, unmanipulated, poorly immunogenic B16 tumors that were established for up to 14 d before the start of treat- ment. 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 an- tigen-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. Further- more, 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. Key words: adoptive cell transfer • immunotherapy • IL-2 • recombinant poxvirus • T cell epitope Introduction The observation that many tumor-associated antigens are attractive targets for immunotherapy, because they are nonmutated “self-antigens” has raised the question of shared among patients, obviating the need for personal- how to induce large numbers of highly active, self/tumor ized vaccine development (1–6). We and others have at- antigen-specific T cells capable of destroying large, estab- tempted numerous vaccination approaches targeting self/ lished tumors. Nonmutated self-antigens are particularly tumor antigens. However, despite the increased numbers of self/tumor antigen-specific T cells, the reproducible W.W. Overwijk, M.R. Theoret, and S.E. Finkelstein contributed equally induction of significant destruction of large, established to this work. Address correspondence to Nicholas P. Restifo, NCI, NIH, Building 10, Room 2B42, Bethesda, MD 20892-1502. Phone: (301) 496-4904; Abbreviations used in this paper: CM, culture media; rFPV, recombinant Fax: (301) 402-0922; email: [email protected] fowlpox virus; rVV, recombinant vaccinia virus. 569 The Journal of Experimental Medicine • Volume 198, Number 4, August 18, 2003 569–580 http://www.jem.org/cgi/doi/10.1084/jem.20030590 The Journal of Experimental Medicine cancers in mouse or in man using any self-antigen vac- The generation of autoreactive T cells in vivo may be cine-based approach remains an elusive goal (7–9). A state difficult because of the mechanisms of central and periph- of functional tolerance apparently exists in the tumor- eral tolerance and the impact of antigen expression by tu- bearing host. mor tissue on T cell function (14, 25–27). Altered peptide This state of functional tolerance, defined here as the co- ligands have been used to improve the reactivity of T existence of tumor-specific T cells and growing tumor cells specific for self/tumor antigens in mouse and man (9, cells, may be incomplete. There is evidence indicating that 28–30). For gp100, H-2D -restricted CD8 T cells ca- antigen expression by tumors leads to limited T cell activa- pable of recognizing B16 melanoma and normal melano- tion without tumor regression (10–12). Work in the spon- cytes could only be elicited when the altered peptide was taneous insulinoma model has indicated that effector “ex- used (31). We described previously the cloning of the un- haustion,” rather than tumor escape is the underlying mutated mouse homologue of gp100 (mgp100) from B16 mechanism of functional tolerance (13). Work using other melanoma (32). We have also described the identification antigenic systems indicates that antigens expressed by tu- of a peptide derived from human (h)gp100, KVPRN- mors actively tolerize tumor-specific T cells (14). Antigenic QDWL (hgp100 ), that represents an altered peptide 25–33 “ignorance” to tumors has also been proposed as a mecha- ligand form of mgp100 , EGSRNQDWL (31). Upon 25–33 nism for functional tolerance (15). Thus, the mechanisms establishing this model for vaccination against self/tumor underlying the observed functional tolerance of antitumor antigen, our next goal was to identify immunotherapeutic T cells remain unclear. strategies that would induce the regression of large estab- The modeling of self-reactivity is likely to be important lished tumors using CD8 T cells specific for a self-antigen. in the development of new immunotherapies that target self/tumor antigens, but many of the currently available tu- mor models that model the complete destruction of estab- Materials and Methods lished tumors target “foreign” antigens. The antigenic sys- Generation of Pmel-1 TCR Transgenic Mice. To study self/tu- tems most commonly used until now by us and others mor antigen-specific T cell responses to melanoma, we developed include chicken egg ovalbumin, -galactosidase from Esch- a transgenic mouse strain on a C57BL/6 background and named erichia coli, the L major histocompatibility antigen, and it pmel-1. RNA was isolated from clone 9, a gp100 -specific, 25–33 hemagglutinin or nucleoprotein derived from influenza A vi- H-2D –restricted CD8 T cell clone (31), and  and  TCR re- gions were amplified by 5-Rapid Amplification of cDNA Ends rus (14, 16–20). These models have shed valuable light on (5-RACE, Life Technologies) using constant region anti-sense basic immunologic principles (21), but it is unclear to what primers 1 (5-GGCTACTTTCAGCAGGAGGA-3) and 1 extent the results obtained from these models reflect im- (5-AGGCCTCTGCACTGATGTTC-3), respectively. 5- mune responses against true self/tumor antigens. For exam- RACE products were amplified with nested TCR  and  con- ple, the high levels of expression of foreign antigens under stant region primers 2 (5-GGGAGTCAAAGTCGGTGAAC- the control of constitutively activated viral promoters may 3) and 2 (5-CCACGTGGTCAGGGAAGAAG-3), and not mimic the expression of self-antigens that are the tar- cloned into pCR4TOPO TA sequencing vectors (Invitrogen). gets of tumor-specific T cells. Most importantly, each of TCR  and  transcripts were sequenced as V1/JTA19/C these model antigens has been selected for study because and V13S1/D1/J1S6/C1. Genomic cloning PCR primers they induce unusually strong T cell responses, perhaps not were designed based on the method described previously by Kouskoff et al. (33). The  and  genomic variable domains were reflecting T cell responses characteristic of those observed PCR amplified (Perkin-Elmer) with primers g1 (5-TCTC- against tumor antigens. CCGGGCTTCTCACTGCCTAGCCATGATGAAATCCT- We sought to study the activation and proliferation of TGAGTGTTTC-3) and g2 (5-GTAGCGGCCGCGTAAA- self-specific T cells that can infiltrate and destroy large, es- ATCTATCCTAGTGTTCCCCAGA-3) or g1 (5-GATCT- tablished tumors. Here, we investigate parameters that can CGAGAATCTGCCATGGGCACCAG-3) and g2 (5-GAT- be manipulated in vivo to transform a completely ineffec- ACCGCGGTTCCTTTCCAAGACCAT-3), respectively. The tive antitumor response into a productive one. To utilize a genomic variable domains were TA-cloned into pCR4TOPO tumor model that is more physiologically relevant, we used (Invitrogen), validated by sequencing, subcloned into TCR cas- the poorly immunogenic B16 melanoma, a highly aggres- sette vectors provided by Dr. D. Mathis (Harvard Medical School, sive tumor in C57BL/6 mice (22). A much-studied tumor, Boston, MA; reference 33), and co-injected into fertilized C57BL/6 B16 expresses no MHC class II and very low levels of embryos (Science Applications International Corporation) yield- ing three TCR transgenic founder lines. Unpublished transgenic MHC class I, although both are inducible upon treatment mice expressing the  / transgenic TCR with specificity for a with interferon- (23). The B16 melanoma expresses the K -restricted epitope from -galactosidase (34) were identically mouse homologue (pmel-17) of human gp100, an enzyme constructed in our laboratory and were used as controls in some involved in pigment synthesis that is expressed by the ma- experiments. jority of malignant melanoma cells, as well as by normal Mice and Tumor Cells. C57BL/6 and pmel-1 TCR trans- melanocytes. gp100 is a member of a family of “self” (i.e., genic mice were bred and housed at the National Institutes of unmutated), melanoma/melanocyte differentiation antigens Health (NIH) and Netherlands Cancer Institute animal facilities. that are widely expressed by melanoma cells. These anti- Rag-1 mice were obtained from Jackson ImmunoResearch gens are frequently the targets of T cells that infiltrate hu- Laboratories. B16 (H-2 ) is a gp100 spontaneous murine mela- man tumors (24). noma obtained from the National Cancer Institute tumor reposi- 570 Induction of Tumor Regression and Autoimmunity by Self-reactive CD8 T Cells The Journal of Experimental Medicine tory and maintained in culture media (CM) comprised of RPMI hgp100, or -galactosidase (31) or by subcutaneous injection 1640 with 10% heat-inactivated fetal bovine serum (Biofluids), with 100 l water/IFA emulsion containing 100 g of 0.03% l-glutamine, 100 g/ml streptomycin, 100 g/ml penicil- mgp100 , hgp100 , or -gal peptide followed by two 25–33 25–33 96–103 lin, and 50 g/ml gentamicin sulfate (NIH Media Center). daily intraperitoneal injections of 100 g anti-CD40 mAb puri- Peptides and Recombinant Poxviruses. All synthetic peptides fied from FGK45 hybridoma culture supernatant. rhIL-2 (a gift were synthesized using regular F-MOC chemistry. The syn- from Chiron Corp.) was administered intraperitoneally directly thetic, H-2D –restricted peptides hgp100 , KVPRNQDWL, after vaccination (100,000 Cetus Units or 600,000 IU rhIL-2 in 25–33 mgp100 , EGSRNQDWL, and NP , ASNENMETM, PBS, twice daily for 3–5 d). Tumors were measured with calipers 25–33 366–374 were synthesized by Peptide Technologies to a purity 99% by and the products of perpendicular diameters were recorded. Mice HPLC and amino acid analysis. All recombinant vaccinia viruses were killed once tumors reached 400 mm . All experiments were (rVVs) and recombinant fowlpox viruses (rFPVs) used in this pa- performed in a blinded, randomized fashion (measuring investiga- per have been described previously (31) and were either generated tor had no knowledge of the experimental group) and performed in our laboratory or provided by D. Panicali, L. Gritz, and A. Go- independently at least twice with similar results. mez-Yafal (Therion Biologics, Cambridge, MA). These viruses were constructed and purified as described by Earl et al. (35). Results In Vitro Activation of Pmel-1 T Cells and Cytokine Release As- say. Peripheral blood mononuclear cells or splenocytes from Despite Large Numbers of gp100-specific T Cells, B16 Mela- mice were depleted of erythrocytes by hypotonic lysis, cultured noma Grows Normally in Pmel-1 TCR Transgenic Mice. To in CM with 30 IU/ml rhIL-2 in the presence of 1 M hgp100 25–33 study self/tumor antigen-specific T cell responses to mela- peptide, and used on days 5–10 after start of the culture. For noma, we developed a transgenic mouse strain on a cytokine release assays, 10 T cells were cocultured in CM with C57BL/6 background and named it pmel-1. Pmel-1 trans- 10 target cells or 1 M of the indicated peptide. Supernatants genic mice expressed the V1V13 TCR from a cloned T were collected after 24 h and tested using an mIFN- ELISA kit cell (clone 9) described previously (31). Clone 9, like the (Endogen) according to the manufacturer’s protocol. pmel-1 TCR transgenic cells derived from it, recognized Histology. Skin and tumor samples were analyzed as de- an H-2D –restricted epitope corresponding to amino acids scribed previously (36). In brief, 4- m cryostat sections were 25–33 of gp100. Virtually all (95%) of the CD8 T cells air-dried overnight and fixed in acetone for 10 min at room temperature, preincubated in 5% (vol/vol) normal goat serum in pmel-1 TCR transgenic mice were V13 , amounting (Central Laboratory of the Netherlands Red Cross Blood Trans- to 20% of all splenocytes (Fig. 1 A). Pmel-1 T cells in fusion Service) and stained with 0.5 g/ml anti–mouse v13 blood and spleen generally expressed baseline levels of the mAb-FITC or mIgG1-FITC control Ab (BD Biosciences) in activation/effector markers CD25, CD44, and CD69, indi- PBS/1% BSA followed by rabbit anti-FITC (1:40,000; DakoCy- cating that most of the transgenic cells were in the naive tomation), biotinylated goat anti–rabbit antibody (1:400; Dako- state. The CD62L levels were as high if not higher than Cytomation) and streptavidin–biotin-conjugated alkaline phos- those found in normal splenocytes, which was also consis- phatase complex (1:100, ABC-protocol; DakoCytomation). tent with the finding that pmel-1 cells were largely naive. Color was developed using Permanent red chromogen substrate Upon in vitro stimulation with 1 M of the hgp100 25–33 (PRC kit; Cell Marque Corporation) and sections were counter- peptide, T cells from pmel-1 TCR transgenic mice prolif- stained with hematoxylin. erated extensively and developed an effector phenotype Flow Cytometry and Intracellular IFN- Assay. To obtain ap- propriate lymphocyte samples, mice were either tail-bled on indi- that included the up-regulation of CD25, CD44, and cated days after vaccination or killed and splenectomized for fresh CD69 and the partial down-regulation of CD62L (Fig. 1 splenocytes. Where designated, splenocytes were cultured as de- A). Functionally, pmel-1 T cells released IFN- after stim- scribed previously (31). Erythrocytes were removed by hypotonic ulation with mgp100, hgp100 peptide (Fig. 1 B), or B16 lysis or ficoll gradient separation, and cells were stained with the melanoma cells, which are naturally mgp100 (Fig. 1 C). indicated dilutions of mAbs against CD8a (1:20, clone 53–6.7), The transgenic T cells resembled the reactivity of the origi- CD4 (1:10, clone H129.19), V13 (1:10, clone MR12–3), CD25 nal clone 9, from which their / TCR was cloned (Fig. 1 (1:10, clone PC61), CD44 (1:10, clone IM7), CD62L (1:10, C and not depicted). Thus, the pmel-1 transgenic mouse clone MEL-14), and CD69 (1:10, clone H1.2F3). All antibodies b could be used as a source of naive T cells with specificity were purchased from BD Biosciences. H2-D –mgp100 tetra- 25–33 for the gp100 epitope. mers were a gift from M. Toebes and T.N.M. Schumacher 25–33 To determine the role of T cell precursor frequency in (Netherlands Cancer Institute, Amsterdam, Netherlands). Propid- ium iodide staining cells were excluded from analysis. Intracellu- the ability of the immune system to reject tumors, we in- lar IFN- assay was performed using the Cytofix/Cytoperm kit jected B16 tumor cells subcutaneously into pmel-1 TCR (BD Biosciences) according to the manufacturer’s recommenda- transgenic mice and nontransgenic littermates. Surprisingly, tions. Samples were analyzed using a FACScalibur™ flow cytom- tumors grew at the same rate in pmel-1 mice despite the eter and CELLquest™ software. presence of overwhelming numbers of CD8 gp100-spe- Adoptive Transfer and Tumor Treatment. Mice were injected cific T cells (Fig. 1 D). Likewise, the adoptive transfer of subcutaneously with 1–5 10 B16 melanoma cells and treated naive or in vitro–activated gp100-specific pmel-1 spleno- 6–7 with intravenous adoptive transfer of freshly isolated 10 fresh 6   cytes into tumor-bearing mice alone or in combination splenocytes (2 10 CD8 V13 T cells) or in vitro–acti- 6–7 with IL-2 had minimal effects on tumor growth or the sur- vated pmel-1 splenocytes (10 CD8 V13 T cells). Mice (n vival of tumor-bearing mice. Thus, the mere presence of 5 for all groups) were vaccinated by intravenous injection of 2 10 plaque-forming units of rVV or rFPV encoding mgp100, naive or activated tumor-specific T cells alone was insuffi- 571 Overwijk et al. The Journal of Experimental Medicine Figure 1. Despite large numbers of gp100-specific T cells, B16 melanoma grows normally in pmel-1 TCR transgenic mice. (A) Generation and char- acterization 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 lit- termate as well as pmel-1 splenocytes cultured with hgp100 peptide were stained for CD8, V13, and the activation markers CD25, CD44, CD62L, 25–33 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 mgp100 peptide, washed, and incubated with titrated doses of mgp100 (native) or hgp100 (altered) peptide. IFN- production was measured 25–33 25–33 25–33 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- 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. cient to cause the regression of the subcutaneous tumor, FACS analysis using mgp100-specific MHC class I tet- thereby indicating that the pmel-1 cells were functionally ramers showed that vaccination with rVVmgp100 induced tolerant to the tumor. a weak and sometimes undetectable T cell response, Immunization with Altered Peptide Ligand Results in Higher whereas use of rVVhgp100 markedly increased specific T Numbers of Self/Tumor Antigen-Specific T Cells That Have cell numbers (Fig. 2 B). Similar results were obtained when Limited Antitumor Efficacy. In an effort to break the func- we used a “minigene” rVV encoding the nine amino acid tionally tolerant state of pmel-1 cells, we immunized tu- minimal peptide determinant preceded by an ER-insertion mor-bearing wild-type C57BL/6 mice with rVVs or rFPVs, sequence (31; unpublished data), or when we used the syn- encoding either the self-antigen, mgp100, or the altered thetic 9-mer peptides emulsified in IFA and given with ligand, hgp100 (31). To track the activation and antitumor anti-CD40 antibody (37). Only immunization with the activity of naive pmel-1 T cells in vivo, we adoptively hgp100 effectively induced gp100-specific T cells, and 25–33 transferred pmel-1 splenocytes into otherwise normal, un- these T cell responses were cross-reactive with mgp100 . 25–33 manipulated mice bearing subcutaneous B16 tumors estab- These findings confirmed earlier observations that gp100 lished for 3 d. Vaccination with rVVmgp100 did not cause sequences outside the minimal determinant do not contrib- any significant antitumor effect. However, vaccination ute to the differential immunogenicity of rVVmgp100 and with rVVhgp100 induced a modest delay in subcutaneous rVVhgp100 (31). tumor growth in some experiments, one of which is shown Both hgp100 and mgp100 peptides contain the 25–33 25–33 (Fig. 2 A). optimal MHC anchor residues at the dominant anchor po- 572 Induction of Tumor Regression and Autoimmunity by Self-reactive CD8 T Cells The Journal of Experimental Medicine sitions, 5 (N) and 9 (L). However, the difference in the than the mouse peptide (31). Here, we extended these ob- three amino-terminal residues resulted in T cell recognition servations by evaluating the dissociation time of peptide at 100-fold lower concentrations of the hgp100 pep- from MHC I molecules on RMA-S cells, again observing 25–33 tide when compared with mgp100 (Fig. 1 B). We have significantly decreased binding at time zero of mgp100 , 25–33 25–33 assessed previously the binding efficiency of the peptides compared with hgp100 , followed by an accelerated dis- 25–33 for the H-2 D class I molecule, finding that the human sociation time for the mgp100 peptide (Fig. 2 C). 25–33 peptide bound to H-2 D with 100-fold greater efficiency Together, these results suggest that pmel-1 T cells can escape central and peripheral tolerance and are not com- pletely refractory to stimulation by self-antigen because the immunogen encoding the altered peptide ligand activated and expanded these self-reactive T cells and conferred lim- ited antitumor efficacy. Stimulation of Adoptively Transferred T Cells through Anti- gen-specific Vaccination and IL-2 Causes Regression and Long- term Cures of Large Established Tumors. Enhanced activation of adoptively transferred pmel-1 T cells by immunization with an altered peptide ligand resulted in inconsistent and limited antitumor effects, leading to growth retardation but not regression of small tumors. Therefore, we set out to determine other factors that might enhance self-specific T cell activation and antitumor effect. We have explored pre- viously the uses of a variety of T cell costimulatory, growth, and activation factors and found that in mouse models, IL-2 can augment the function of recombinant vi- rus–based vaccines (38). To determine if we could enhance the activation of antitumor T cells in a way that would en- able them to treat large, established, vascularized tumors, we adoptively transferred pmel-1 splenocytes into mice bearing subcutaneous B16 tumors established for 7 (Fig. 3, A and B) or 14 d (Fig. 3, C and D). This adoptive transfer was followed by vaccination with rFPV encoding the hgp100-altered peptide ligand and administration of IL-2. This combined treatment reproducibly induced tumor re- gression and even long-term cures of mice bearing estab- lished tumors larger than 50 mm (Fig. 3 C). Invariably, as observed in 20 independently performed experiments, the combination of adoptive transfer of self/ tumor-specific CD8 T cells, antigenic stimulation with an altered peptide ligand, and administration of IL-2 was re- quired to induce this strong antitumor effect. The regimen Figure 2. Enhanced T cell and antitumor response by adoptively trans- ferred 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 vacci- nation with rVVLacZ, rVVmgp100, rVVhgp100, or no treatment. (B) Increased numbers of pmel-1 T cells from mice immunized with rVV en- coding altered peptide ligand. Lymphocytes from mice described in A were isolated from peripheral blood and stained for CD8 and V13 be- fore FACS analysis. Numbers of CD8 Tm cells are depicted through time as percentage of total CD8 cells. (C) Prolonged peptide/MHC dis- sociation time of altered peptide ligand. RMA-S cells were incubated with hgp100 (closed symbols) or mgp100 (open symbols, shown 25–33 25–33 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- in culture supernatants was quantified by ELISA. Experiments were performed independently at least twice with similar results. 573 Overwijk et al. The Journal of Experimental Medicine 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 -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. is ineffective against tumors that are gp100-negative (un- able pattern throughout the mouse. Vitiligo is not observed published data). Administration of either pmel-1 cells when mice are treated with control -galactosidase–specific alone, IL-2 alone, vaccine alone, or any combination of T cells (unpublished data). A representative cohort of com- just two of the three of these immunotherapeutic compo- plete responder mice observed 1 yr after treatment corre- nents induced weak, if any, antitumor effects (Fig. 3, A and sponds to the group treated with cultured T cells, IL-2, and B). In an effort to use the model in a clinically relevant vaccination with rFPVhgp100, shown in Fig. 4 B. way, we have focused on the adoptive transfer of T cells. It is important to note that complete cures of tumors are However, in mice transgenic for the pmel-1 TCR, IL-2 not always observed when using the triple combination of given in combination with vaccination using the altered cells, vaccine, and cytokine, especially when doses of indi- form of gp100 was also therapeutically effective in the treat- vidual components are reduced (unpublished data). Thus, it ment of established B16 tumors (unpublished data). may be useful to elucidate the minimal optimal treatment T cells derived from fresh, naive splenocytes, or from cul- protocol required to reproducibly induce the regression of tured splenocytes activated ex vivo, could mediate the regres- large B16 tumors established for 14 d. 10 Cetus units (6 sion of large, established, subcutaneous tumors in vivo. All 10 IU) twice a day for 3 d was clearly sufficient when mice in Fig. 4 A that were treated with the combined regi- given in combination with 10 fresh or cultured pmel-1 men of adoptive transfer of pmel-1 cells (either with fresh or cells and 2 10 plaque-forming units of rFPVhgp100 to cultured T cells), IL-2, and vaccination with rFPVhgp100 reproducibly induce the regression of large B16 tumors es- were complete and long-term responders. These responses tablished for 14 d. We used adoptively transferred cells in have been followed for 1 yr, and are often accompanied by an effort to model a clinical possibility, but as mentioned the development of limited vitiligo, which generally begins at earlier, vaccination plus IL-2 is effective in pmel-1 TCR the site of tumor regression, and then spreads in an unpredict- transgenic mice bearing established tumors. 574 Induction of Tumor Regression and Autoimmunity by Self-reactive CD8 T Cells The Journal of Experimental Medicine 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 10 cultured pmel-1 cells given in combination with rFPVhgp100 and IL-2 as described previously. Similar results were obtained in five in- dependently completed experiments. the transferred cells after treatment. Data shown in Fig. 6 Figure 4. Long-term (1 yr) survival of mice bearing large, established (A–C) are obtained from the same, representative experi- B16 tumors after treatment with adoptive transfer of tumor-specific T ment. In this treatment model, using B16 tumors estab- cells combined with vaccination and IL-2 is associated with the develop- lished for 3 d, cell transfer combined with immunization ment of vitiligo. C57BL/6 mice were treated with adoptive transfer of fresh or cultured pmel-1 transgenic splenocytes 14 d after inoculation with the altered peptide ligand and IL-2 was required to with B16 melanoma and vaccinated with rFPVhgp100. IL-2 was adminis- observe the maximal therapeutic effect (Fig. 6 A). In other tered twice daily for six doses. Mice treated with fresh naive or cultured experiments, IL-2 could be replaced with either IL-7 or IL- transgenic T cells were cured of B16, and vitiligo was observed, which 15 (unpublished data). Cells plus rVV encoding the altered started at the former tumor site. At 1-yr after therapy, these mice re- main tumor-free with progressive vitiligo. To illustrate the autoimmune peptide ligand can induce limited destruction of small tu- vitiligo, a photograph of the cohort of 5/5 surviving mice treated with mor burden, but this treatment effect is not significant in cultured pmel-1 cells from A is shown in B. mice with large tumor burden (i.e., tumors established for 14 d). We found that the effect of IL-2 on the levels of pmel-1 The administration of a combination of cultured pmel-1 T cells, as a percentage of CD8 T cells or total nucleated T cells, vaccination, and IL-2 was required even in the ab- cells in blood, was modest (two- to fourfold, at most, com- sence of endogenous host B or T lymphocytes, as demon- pared with treatment groups receiving adoptively trans- strated by experiments in C57BL/6 Rag-1 knockout ferred pmel-1 cells and vaccination at any given time in mice bearing subcutaneous B16 tumors established for 14 d multiple experiments; Fig. 6 B). When measured as either (Fig. 5). The cultured cells that were transferred were percentage of all nucleated cells in blood or absolute num- 98% CD8 by FACS (unpublished data). Tumors bers in the spleen, administration of IL-2 increased the rel- shown in Fig. 5 were treated with a suboptimal number ative number of pmel-1 T cells maximally threefold. IL-2 (10 ) of pmel-1 cells, resulting in resurgent tumor growth. did not influence the induction of persistent memory T However, in experiments repeated more than five times, cells that could be reactivated by booster vaccination 32 d antitumor effects were at least as strong (and generally after priming (Fig. 6 C). stronger) in Rag-1 mice as those seen in wild-type We sought to understand the impact of IL-2 administra- C57BL/6 mice (unpublished data). tion on absolute numbers of pmel-1 T cells, and to measure IL-2 Increases the Number and Function of Self-specific, Tu- the effect it had on their localization and effector function. moricidal T Cells. To further understand the mechanisms We found that administration of IL-2 after adoptive trans- of how the antitumor effector function of pmel-1 cells was fer and vaccination increased the total number of lympho- enhanced by IL-2, we monitored the levels of pmel-1 T cytes infiltrating into 300 mg of solid tumor by 1.9-fold 7 d cells in the blood of mice using tetramer analysis to follow after vaccination (Fig. 6 D). The number of V13 CD8 575 Overwijk et al. The Journal of Experimental Medicine Figure 6. Persistence and function of gp100 reactive T cells after adoptive transfer, vaccination, and admin- istration of IL-2 in mice bearing subcutaneous B16 tu- mors. (A–C) Data derived from the same, repre- sentative 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 de- picted 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 V13 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 V13 T cells (890:266), and CD8 V13 IFN- T cells (261:8). After a 4-h restimulation with 1 M mgp100 peptide, the number of CD8 V13 25–33 IFN- T cells measured from this group were (754:147) to give a ratio of 5:1 (not depicted). Discussion T cells was increased by 3.3-fold, corresponding to the modest increase in V13 CD8 T cell levels in blood (Fig. Strengths and Weaknesses of the Pmel-1 TCR Transgenic 6 D). Strikingly, the number of V13 CD8 T cells that Model. Here, we have investigated parameters that can produced IFN- directly ex vivo and without further stim- be manipulated in vivo to transform a functionally tolerant ulation was increased 34-fold by IL-2 administration. transgenic T cell into an activated one that mediates the re- These data illustrate that in the absence of IL-2, T cells are gression of large established tumors. As a tumor model, we present at the tumor site but not fully activated. Thus, IL-2 used the poorly immunogenic B16 melanoma. This tumor acted primarily as a costimulatory/activation factor rather failed to induce activation of adoptively transferred than purely as a growth factor. mgp100-specific pmel-1 T cells. Reports on the treatment Histological analysis of B16 tumors 7 d after treatment of of large, established tumors with systemic, rather than local 7-d tumors with pmel-1 cells plus vaccination showed spe- therapies, remain rare, but not unprecedented in the scien- cific T cell infiltration but no marked tumor cell death or tific literature (39). tissue destruction (Fig. 7, left). By contrast, when the adop- Most of the published mouse tumor models use preven- tive transfer of pmel-1 cells plus vaccination was followed tion of tumor implantation and growth as the measure of by administration of IL-2, we observed extensive tumor success. When the treatment of established tumors is re- cell death and loss of tissue integrity (Fig. 7, right). These ported, treated tumors are generally significantly smaller data show that strong vaccination by itself can induce pro- than those treated in the present set of experiments. Fur- liferation and tumor localization of antigen-specific T cells, thermore, many of the existing tumor systems target model but that these T cells do not effectively mediate tumor de- antigens that have been artificially inserted into the tumor struction unless provided with exogenous cytokine. genome, whereas the majority of human tumor–associated 576 Induction of Tumor Regression and Autoimmunity by Self-reactive CD8 T Cells The Journal of Experimental Medicine splenocytes, 95% of all CD8 T cells) in pmel-1 mice did not impact tumor growth. Apparently, large numbers of activated tumor-specific T cells are necessary but not suffi- cient to induce tumor destruction. This puzzling observa- tion, observed several years ago in mice by Ohashi and col- leagues (13), has been rediscovered in recent clinical trials (7–9, 42). Provision of nonspecific systemic inflammation or “dan- ger” signals by infection of mice with control vaccinia virus or injection of anti-CD40 mAb and IL-2 did not result in T cell activation or suppression of tumor growth. Further- more, in vitro–activated specific T cells neither proliferated in response to tumor nor slowed the growth of even tiny subcutaneous tumors. This suggests that direct presentation of mgp100 antigen by tumor cells or its cross-presentation by activated professional APCs is insufficient to overcome peripheral tolerance and induce T cell activation and prolif- eration. A possible explanation for this poor presentation may reside in the low affinity of the mgp100 peptide 25–33 for H-2 D and the resulting brief half-life of MHC– mgp100 complexes on APCs (Fig. 2 C; references 31, 25–33 43). Indeed, vaccination with the self-antigen, mgp100, in- duced only marginal T cell proliferation and no control of Figure 7. Histological analysis reveals presence of V13 T cells in tu- tumor growth (Fig. 2, A and B; and Fig. 6, A and B). mors and demonstrates the activating effects of IL-2. T cells and tissue ar- When tumor-bearing recipients were vaccinated with a chitecture within tumors were visualized after vaccination with or with- peptide epitope capable of binding H-2D with 100-fold out IL-2. C57BL/6 mice bearing 14-d B16 tumors received 10 pmel-1 greater affinity than its mouse counterpart (31), we saw a splenocytes and vaccination with hgp100 peptide in IFA and anti- 25–33 CD40 with or without IL-2. As was the case for vaccination with rVVs or greatly increased mgp100-specific T cell response (in which rFPVs encoding hgp100, vaccination with hgp100 peptide in IFA and 25–33 up to 80% of all CD8 T cells in the blood can be anti-CD40 after the adoptive transfer of pmel-1 cells was greatly en- mgp100-specific). It is possible that the use of an altered hanced with the addition of IL-2 in repeated experiments (not depicted). peptide ligand is not an absolute requirement in other can- Tumors were excised 7 d after the vaccination and 4- m frozen sections cer vaccines, particularly if the antigenic peptide of choice were stained with anti-V13 mAb (top, arrows) or IgG control (bottom), counterstained with hematoxylin, and photographed at an original mag- is, unlike the mgp100 peptide, highly capable of activating nification of 200. (left) pmel-1 cells plus hgp100 vaccination. (right) T cells. However, it is likely that T cells that have survived pmel-1 cells plus hgp100 vaccination followed by IL-2 administration. central and peripheral tolerance to self/tumor antigens gen- erally target peptides with poor avidity. Our observations in mice seem analogous to those observed in human mela- antigens used in current clinical efforts are nonmutated self- noma patients in which vaccination with the gp100 209–217 antigens. In studies focusing on adoptive immunotherapy, peptide is inefficient, unless that peptide is altered to in- investigators generally report on the treatment of small pul- crease its avidity for the HLA-A*0201 molecule (9). Thus, monary “metastases,” which are nodules created by the in- the optimization of self/tumor peptides for binding to travenous injection of tumor cells, which are treated with MHC molecules should be further explored in future can- lymphocytes injected by the same route. The present paper cer vaccine development. describes the use of large, vascularized subcutaneous tumors. One observation in mouse and in man is that therapeutic One aspect of the current model that does not model immunization designed to treat established tumor is gener- the human tumor–bearing situation is the use of a trans- ally ineffective. Where immunization can induce tumor plantable tumor rather than spontaneous melanomas aris- antigen-specific T cells, the destruction of an established ing and progressing in each mouse, which could be sub- tumor is not reproducibly observed. In the present model, jected to treatment. Spontaneous tumor models can be adoptive transfer of T cells was required in order for vac- more cumbersome and time-consuming to work with. cine and IL-2 to reproducibly induce the destruction of tu- However, the use of spontaneous tumors (40, 41) may mor. There apparently are quantitative or qualitative differ- more effectively model some aspects of tumor carcinogen- ences between cells expanded ex vivo and used for esis and treatment. Efforts to validate the findings of the adoptive transfer, and cells expanded in vivo as a result of present paper in a model of spontaneous tumor are cur- immunization. Cells induced by vaccination are likely to be rently ongoing in the laboratory. lower in number; furthermore, they may be quiescent or The Mere Presence of Tumor-reactive CD8 T Cells Is Insuf- refractory to activation by tumor cells, perhaps in a way ficient to Induce Tumor Regression. The presence of over- similar to the changes observed in T cells during chronic whelming numbers of mgp100-specific T cells (20% of all infection (44). Thus, we seek to use, in new clinical trials, 577 Overwijk et al. The Journal of Experimental Medicine the combined regimen of cells, IL-2, and vaccination with requirements defined the components of successful antitu- an altered peptide ligand described in this paper. mor treatment. Based on these data, clinical trials that use The use of lymphodepletion (nonmyeloablative condi- adoptive cell transfer used in combination with IL-2 and tioning) may enhance the function of adoptively transferred vaccination with an altered peptide ligand are being initi- T cells as shown previously (45, 46). Lymphodepletion ated in patients with cancer. may facilitate immune responses by deletion of endogenous We would like to thank D. Panicali, L. Gritz, and A. Gomez-Yafal regulatory T cells, or by increasing the availability of en- from the Therion Biologics for providing recombinant poxviruses dogenous immune activating cytokines such as IL-7 or IL- used in this work, T.N.M. Schumacher and M. Toebes for help 15. Results in the clinic are encouraging, but the consistent with tetramer production, P. van den Berk for producing FGK45 induction of complete responses in our patients with cancer anti-CD40 mAb, and J. Yang and P. Hwu for helpful discussions. remains the focus of our ongoing clinical efforts. We are This work was supported in part by a grant (KWF 2001-2562) now using the pmel-1 model to explore the immune- from the Dutch Cancer Society. enhancing mechanisms of nonmyeloablative conditioning. Submitted: 11 April 2003 IL-2 Enhances CD8 T Cell Function In Vivo. IL-2 can Revised: 1 July 2003 be clinically successful but the mechanism responsible for Accepted: 1 July 2003 the sometimes-dramatic clinical antitumor responses is un- clear (47). We had found previously that IL-2 could aug- ment the T cell response to a virus (38). These observa- References tions have been confirmed and expanded recently by 1. Houghton, A.N., J.S. Gold, and N.E. Blachere. 2001. Immu- others (48). 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Viral persistence alters CD8 T-cell cer immunotherapy. J. Immunother. Emphasis Tumor Immunol. immunodominance and tissue distribution and results in dis- 19:81–84. 580 Induction of Tumor Regression and Autoimmunity by Self-reactive CD8 T Cells The Journal of Experimental Medicine

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