Background
Adoptive transfer of tumor-specific T cells can induce tumor regression in animal models and occasionally in patients with cancer [
1,
2]. However, the mechanisms for T cell mediated tumor regression are still under intensive investigation. Tumor-specific T cells process multiple effector molecules that can potentially participate in various pathways leading to tumor destruction in vivo. Previously, we have documented that tumor regression mediated by adoptive transfer of tumor-specific effector T cells could be independent of either perforin or IFN-γ pathways [
3,
4]. Recently, we also demonstrated that effector T cells lacking both perforin and IFN-γ could mediate regression of pulmonary metastases of melanoma and fibrosarcoma, albeit the efficacy was greatly reduced [
5], demonstrating that perforin/granzyme and IFN-γ-dependent mechanisms may have a compensatory role. However, the fact that tumor regression did occur in a system lacking both perforin and IFN-γ indicates that other mechanisms, such as TNF-mediated pathways, can orchestrate tumor regression [
5].
IFN-γ is known to play a central role in the immune surveillance against tumors [
6‐
8]. In several murine tumor models the therapeutic efficacy of adoptively transferred effector T cells strongly correlates with their tumor-specific IFN-γ release. Barth et al., and others observed a direct correlation between the therapeutic efficacy of tumor infiltrating lymphocytes (TIL) and their tumor-specific IFN-γ production in a murine sarcoma model [
9]. Similar correlations between therapeutic efficacy and the tumor specific IFN-γ production were found for effector T cells derived from lymph nodes (LN) draining the vaccine sites of MCA-205 sarcoma or B16BL6 melanoma tumor cell lines [
10‐
13]. We also recently showed that a T1 phenotype is crucial for their therapeutic efficacy [
14]. When therapeutic effector T cells from wt TVDLN are cultured in a T2 promoting cytokine milieu with IL-4 and anti-IL-12 antibody, they lost their therapeutic efficacy. So far, two major classes of effector molecules that have been identified. First, effector molecules are able to mediate the direct killing of tumor targets – perforin and granzymes in the granules of CTL and ligands for death receptors on the cell surface of T cells. Second, IFN-γ produced by tumor-specific T cells mediates tumor regression probably via the activation of host macrophages [
9,
15].
While these studies indicate that IFN-γ plays a critical role in the development of tumor immunity, we and others have recently shown, that IFN-γ is not essential for the priming of tumor specific effector cells in TVDLN or as an effector molecule of adoptively transferred T-cells [
4,
15,
16]. This observation led to the hypothesis that other T1 cytokines might play an essential role for the therapeutic efficacy of tumor-specific effector T cells and might compensate for the loss of IFN-γ in GKO mice.
Because no evidence for the generation of type 2 cytokine T cell immune responses was observed in GKO mice, we hypothesized that other type 1 cytokines produced by adoptively transferred T cells were critical for the therapeutic efficacy. LT-βR ligand, a membrane bound heterotrimer known as LT-α1β2, was found to be expressed abundantly on recently activated Th1 T cells [
17‐
19]. In addition, a recently described ligand for LT-βR (LIGHT) was found to be expressed on activated lymphocytes and shown to be able to induce secretion of chemokines and apoptosis of some tumor cell lines [
20‐
24]. Meanwhile, LT-βR was found to be expressed on non-lymphoid cells and the majority of tumor cell lines [
18,
25‐
27]. To investigate whether ligands for LT-βR, LT-α1β2 (and/or LIGHT), could be the effector molecules of effector T cells adoptive transfer experiments were designed. These studies examined how the presence or absence of IFN-γ or IFN-γ and perforin affected the contribution of LTα to T cell mediated-tumor regression. Effector T cells were generated from TVDLN of wt, GKO and adoptively transferred into wt or GKO mice with established 3 day pulmonary metastases of D5 tumor cells [
5]. Signaling through LT-βR was blocked by administration of LT-βR Fc after adoptive transfer of T cells. Effector T cells deficient of membrane bound lymphotoxin LT-α1β2 were also generated from TVDLN by vaccinating RAG1 mice reconstituted with naïve spleen cells from LKO mice. The therapeutic efficacy of LKO effector T cells in an adoptive immunotherapy model was compared in the presence or absence of IFN-γ neutralizing antibody. To delineate a potential role of LT-βR signaling in T cell mediated tumor regression, recombinant LT-α1β2 was used for the further investigation of the effect of LT-βR signaling on D5 tumor cells in vitro.
Materials and methods
Mice
Female C57BL/6J (wt), GKO (C57BL/6-IFN-γ tm1Ts), and LKO (C57BL/6 -LTtm1Sdz) mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and maintained in a specific pathogen-free environment. Perforin and IFN-γ double deficient (PKO/GKO) mice were generated as described previously (5). Mice were generally 8 to 12 weeks old at the time of experimentation. Recognized principles of laboratory animal care were followed (Guide for the Care and Use of Laboratory Animals, National Research Council, 1996), and all animal protocols were approved by the Earle A. Chiles Research Institute Animal Care and Use Committee.
Tumor cell lines
D5 is a poorly immunogenic subclone of the spontaneously arising B16BL6 melanoma [
10] (provided by Dr. S. Shu, Cleveland Clinic Foundation, Cleveland, OH). An early passage of the original BL6 tumor was provided by Dr. E. Gorelick and was subcloned by limiting dilution culture in Dr. S. Shu's laboratory. D5 exhibits low to undetectable class I (H-2 D
b and K
b) expression and no class II expression. D5-G6 is a stable clone of D5 that was originally transduced with a murine GM-CSF retroviral MFG vector (provided by Dr. M. Arca, University of Michigan, Ann Arbor, MI) [
44]. D5-G6 cells secrete approximately 200 ng/ml/10
6 cells/24 h GM-CSF.
Culture conditions
Lymphocytes and tumor cells were cultured in complete medium (CM), which consisted of RPMI 1640 containing 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, and 50 μg/ml of gentamicin sulfate (Bio Whittaker, Walkersville, MD.). This was further supplemented with 50 mM 2-mercaptoethanol (Aldrich, Milwaukee, WI, USA.), and 10% fetal bovine serum (GIBCO BRL, Grand Island, NY). Tumor cells were harvested 2-to 3 times per week by brief trypsinization and maintained in T-75 or T-150 culture flasks.
Generation of effector T cells from TVDLN
D5-G6 tumor cells were harvested by trypsinization, washed twice with HBSS and resuspended at 2 × 107 cells per ml. One million D5-G6 tumor cells were injected s.c. into both hind and fore flanks of wt, or GKO mice. Eight days following vaccination, the draining superficial inguinal and auxiliary lymph nodes were harvested. TVDLN were resuspended at 2 × 106 cells per ml in CM and cultured in 24 well plates with 50 μl of a 1:40 dilution of 2c11 ascites (anti-CD3) as described previously [3]. After two days of activation the T cells were harvested and expanded in CM containing 60 IU rhIL-2/ml for three additional days. T cells were then harvested, washed twice in HBSS, counted and used in adoptive transfer and cytokine release assays.
Adoptive immunotherapy
Experimental pulmonary metastases were established by i.v. inoculation of 2 × 105 D5 tumor cells. Three days later effector T cells were adoptively transferred i.v. via tail vein. Starting on the day of T-cell infusion, mice received 90,000 IU recombinant human IL-2 (provided by Chiron, Emeryville, CA) i.p. once per day for four days. Animals were sacrificed 11 to 13 days following tumor inoculation by CO2 narcosis and their lungs were harvested and fixed in Fekete's solution. Where indicated, neutralizing LT-βR Fc or control human IgG were administered i.v. before the adoptive transfer of T cells and for the following three days. The number of pulmonary metastases was counted in a blinded fashion. Metastases that were too numerous to count accurately were known to be greater than 250 metastases and were assigned a value of 250.
Statistical analysis
The statistical significance of differences in the number of metastases between experimental groups was determined by the Wilcoxon rank sum test. Two-sided p values of < 0.05 were considered significant. Each treatment group consisted of at least 5 mice, and no animal was excluded from the statistical evaluations.
Apoptosis induction
D5 tumor cells were incubated with different concentration of recombinant mouse LT-α1β2 (Sigma, MO) with or without cycloheximide (CHX) (10 μg/ml) in 500 μl CM in 24 well plates. 24 hours later the cells were harvested, washed twice with ice cold HBSS and resuspended in 100 μl Annexin binding buffer. Apoptosis was determined by staining with Annexin-V-FITC (Pharmingen) and counterstaining with 10 μl propidium (50 g/ml in PBS). 15 minutes later, the cells were analyzed by FACS and the amount of apoptotic cells determined by calculating the percentage of cells staining positive with Annexin-V.
RT-PCR
D5 cells were cultured in CM alone, with an indicated number of effector T cells generated as above, or with an indicated concentration of LT-α1β2. After 4–24 hours incubation, the total RNA was extracted from D5 cells, or after removal of T cells by washing three time with PBS, using the Qiagen Rneasy mini kit (Qiagen, CA). 2 μg of RNA was denatured and reversely transcribed to cDNA using the oligo dT (15) primer (Roche) and MMLV reverse transciptase (Invitrogen, CA). Thermocycling conditions were: denaturing at 94°C for 30', annealing at 55°C for 30', and extending at 72°C for 30'. A total of 25 cycles were performed. The DNA sequences of primers used are shown in Table
1.
Table 1
Primer sequences used for RT-PCR
MIP-1α | 5'-atg aag gtc tcc acc act gcc ctt g-3' | 5'-ggc att cag ttc cag gtc agt gat-3' |
MIP-1β | 5'-gtt ctc agc acc aat ggg ctc tga-3' | 5'-ctc tcc tga agt ggc tcc tcc tg-3' |
IP-10 | 5'-cct atc ctg ccc acg tgt tg-3' | 5'-cgc acc tcc aca tag ctt aca-3' |
RANTES | 5'-cat cct cac tgc agc cgc c-3' | 5'-cca agc tgg cta gga cta gag-3' |
MIG | 5'-atg aag tcc gct gtt ctt ttc-3' | 5'-tta tgt agt ctt cct tga acg ac-3' |
HPRT | 5'-gtt gga tac agg cca gac ttt gtt g-3' | 5'-gag ggt agg atg gcc tat agg ct-3' |
Measurement of cytokines
After activation and expansion TVDLN were washed, resuspended in CM, supplemented with IL-2 (60 IU/ml) and seeded at 4 × 106/2 ml/well in a 24 well plate. The cells were either cultured without further stimulation or stimulated with 2 × 105 D5, MCA-310 tumor cells, or immobilized anti-CD3 (positive control). Supernatants were harvested after 24 hours and assayed for the release of KC and RANTES by ELISA using commercially available reagents (Pharmingen). The concentration of cytokines in the supernatant was determined by regression analysis.
Chemotactic assay
D5 tumors cells (105 well) were plated in the bottom chamber of a 24 well transwell plate (Corning Costar, Cambridge, MA) in CM. Two hours later they were stimulated with or without LT-α1β2 (100 ng/ml). After 12 hours 3.5 × 105 DJ2PM macrophage cells were resuspended in 250 μl CM and plated into the upper chamber of a transwell plate (5 μm pore size). After 4 h the cells in the bottom chamber were trypsinized, harvested, and washed 2 × in PBS and stained with anti-CD11b antibody (Pharmingen). The number of macrophages that migrated into the lower well was determined by FACS analysis as the percentage of CD11b positively stained cells.
Discussion and conclusion
Previously, we have documented that granzyme, IFN-γ, and TNF are three primary effector mechanisms by which effector T cells could mediate tumor regression in adoptive transfer models [
3‐
5]. The contribution by TNF family members expressed by effector T cells is more difficult to measure and less well appreciated. Our previous publication indicated that TNF could mediate tumor regression if effector T cells were deficient of both perforin and IFN-γ [
15]. However, the blocking experiments with TNFR-Fc fusion could not completely abrogate the tumor regression mediated by the adoptive transfer of perforin and IFN-γ double deficient cells. Thus, other effector molecules expressed by effector T cells could play a role even if all three major effector molecules were absent or blocked. In our present study we identified that LT-βR signaling pathways also played a significant role if IFN-γ was absent in the system. One possible mechanism for LT-βR signaling is to stimulate chemokine secretion by D5 tumor cells and induce macrophage recruitment.
Cross linking of LT-βR on tumor cells by membrane bound ligands expressed on effector T cells after tumor stimulation contributed to tumor regression. In vitro experiments suggested a possible mechanism involving the recruitment of macrophages rather than a direct killing mechanism by LT-α1β2. According to this notion, Plautz et al. demonstrated that host macrophages are important for the cross-presentation of tumor antigens to adoptively transferred effector T cells during the phase of tumor eradication [
16]. A critical role of LT-βR has also been demonstrated in the infectious, autoimmune diseases and transplantation rejection models [
29‐
32]. Lucas et al. demonstrated that both TNFR and LT-βR pathways played important roles in protective immunity against
Mycobacterium bovis BCG infection and LT-βR signaling is critical for the development of Th1 immune response, iNOS activation of macrophage, and granuloma formation [
33]. LT-βR was used to reverse autoimmune diseases in various models [
29,
32] and to prevent transplant rejection [
31]. Our results added another important function of LT-βR as an important tumor regression mechanism independent of IFN-γ.
Because LT-βR-Fc can block LT-α1β2 and LIGHT, another ligand of the TNF superfamily expressed on activated T-cells and immature DC [
21,
34], both LT-α1β2 and LIGHT on effector T cells could contribute to the tumor regression observed in our experiments. In addition to LT-βR, LIGHT can bind to other two receptors, herpes virus entry mediator (HVEM) and decoy receptor 3/TR6 [
21,
35]. Several studies indicate that LIGHT can trigger apoptosis as well as cell activation depending on the expression of different receptors on the targeted cells [
20,
22]. Shaikh et al. showed that the constitutive expression of LIGHT on T cells led to inflammation and tissue destruction [
36]. Tamada et al. showed that expression of LIGHT by transplanted tumors led to increased lymphocytic infiltrates, tumor necrosis and enhanced T cell cyotoxicity [
37]. Similarly, Schrama et al. demonstrated that targeting LT-α3 to tumor resulted in tumor destruction via the formation of lymphoid-like structure in tumor sites [
38]. It has been well documented that LT-βR signaling, and to a lessor extent, TNFR signaling is critical for the development and maturation of secondary lymphoid tissues [
18,
39]. One critical function of LT-βR is the activation of a chemokine-driven positive feedback loop required for the organization of lymphoid follicles [
40]. Interestingly, a recent reported LT-βR signaling by LIGHT at tumor sites could lead to eradiation of well-established tumors via the recruitment of immune cells, including naïve T cells, and the formation of lymphoid-like structure inside tumors [
41]. We hypothesized that one important function for LT-βR in our model is the activation of a similar chemokine-driven positive feedback loop by the adoptively transferred effector T cells that results in the recruitment of host innate cells, such as macrophages and dendritic cells, indirectly contributing to the tumor destruction process. Although it is conceivable that blocking with LT-βR might prevent the initial infiltration of adoptive effector T cells into the lungs, we did not observe a difference in the trafficking of CFSE-labeled effector T cells with or without LT-βR Fc treatment (data not shown). Thus, at least for our pulmonary metastases model, the effect of LT-βR blockage was unlikely due to the prevention of T cell trafficking. The fact that the therapeutic efficacy of wt effector T cells was not affected by LT-βR blockage is an additional argument against this possibility. The chemokines RANTES, MCP-1 and KC are induced in most inflammatory conditions and their expression correlates with the influx of macrophages into inflammatory sites [
42‐
44]. In our in vitro experiments we detected the expression of RANTES, IP-10, KC and MCP-1 by D5 tumor cells after incubation with recombinant LT-α1β2. While growing tumors can likely counteract the immune system to insure their progression, it is of interest to note that the effector T cells may co-opt tumor cells themselves to contribute to their own demise. Further investigations into this paradox are warranted.
Together with other published data, our current study suggests that tumor-reactive T cells are capable of mediating tumor regression via a number of compensatory effector mechanisms. Recently, the clinical significance of tumor infiltrating lymphocytes was highlighted in multiple studies of human tumors, including colon cancer, ovarian cancer, and lymphoma [
46‐
48]. Because not all possible effector molecules were examined, it will be of great interest to examine which particular effector mechanisms can be directly correlated to the patient's survival. Subsequently, strategies that induce these properties in T cells ex vivo could be applied to the adoptive immunotherapy of cancer, while alternatives that can induce these properties in vivo may serve as a useful adjunct for cancer vaccine strategies.
Acknowledgements
We would like to thank Drs. S. Santé, M. Croft, and Carl F. Ware (La Jolla Institute for Allergy and Immunology, La Jolla, California, USA) for advice and reagents. This work was supported by the American Cancer Society (LIB-106810) (H.-M. H.), and National Cancer Institute, National Institute of Health, Department of Health and Human Services CA80964 (B.A.F.), CA92254 (B.A.F.), CA107243 (H-M. H.), the M.J. Murdock Charitable Trust and the Chiles Foundation. H.W. and CHP were Chiles Foundation visiting fellows.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
BAF and H-MH conceived initial experiments to assess the role of LT-α as an anti-tumor effector mechanism. HW, CHP, BAF and H-MH designed and performed adoptive transfer experiments. Chemotaxis and molecular studies were designed and performed experiments by HW, NKE and H-MH. FAH and BAF was directly involved in drafting and revising the manuscript. HW, BAF, and H-MH were involved in data interpretation and the preparation and critical review of the manuscript.