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Combination cytokine therapy inhibits tumor growth by generation of tumor-specific T-cell responses in a murine melanoma model
Cytokine 49 (2010) 287–293
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Cytokine journal homepage: www.elsevier.com/locate/issn/10434666
Combination cytokine therapy inhibits tumor growth by generation of tumor-specific T-cell responses in a murine melanoma model Amer N. Kalaaji a,*, Jun Lu a, Svetomir N. Markovic b, Esteban Celis c,1, Mark R. Pittelkow a,d a
Department of Dermatology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA Division of Hematology, Mayo Clinic, Rochester, MN, USA c Department of Immunology, Mayo Clinic, Rochester, MN, USA d Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA b
a r t i c l e
i n f o
Article history: Received 24 August 2009 Received in revised form 16 November 2009 Accepted 17 November 2009
Keywords: Cytokines Cytotoxic T lymphocytes Melanoma Therapy Tumor
a b s t r a c t Various cytokines, including interferon a (IFNa), tumor necrosis factor a (TNFa), and granulocyte–macrophage colony-stimulating factor (GM-CSF), have been used as adjuvant therapy for advanced-stage melanoma with some success but with marked toxicity, which appears to be related to higher doses. We investigated the efficacy of IFNa, GM-CSF, and TNFa in various combinations to induce antitumor and immune responses in a B16F10 murine melanoma model. These studies showed that GM-CSF, IFNa, and TNFa, when injected together intratumorally, mediated significant inhibition of tumor growth. Tumor regression correlated with local tumor necrosis and significant infiltration of T cells. In addition, this injected intralesional cytokine cocktail also induced lymphadenopathy, with an increase in both CD4+ and CD8+ T cells in the draining lymph nodes. Furthermore, tumor-specific CD8+ T cells were identified from draining lymph nodes. These investigations identify the combined effects of IFNa, GM-CSF, and TNFa in induction of the adaptive immune response and generation of antigen-specific T-cell reactivity. These results support potential clinical trials of the low-dose cytokine combination as adjuvant therapy for melanoma. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Malignant melanoma is occurring with increasing frequency. Over the past 40 years in the United States, the incidence of melanoma has increased 3-fold in middle-aged men and 5-fold in older men [1]. Various adjuvant therapies have been used, including immunotherapy with cytokines. Cytokines are small proteins that act in an autocrine or paracrine manner. They bind to specific receptors, with individual cytokines having multiple functions. Cytokine adjuvant therapy for advanced-stage melanoma has shown some success but also dose-dependent, marked toxicity [2]. Interferons are one of the earliest identified families of cytokines. In addition to its antiviral effects, interferon a (IFNa) has antiproliferative and immunomodulatory effects including increasing major histocompatibility complex class I antigen expression, which is crucial for cytotoxic T lymphocyte (CTL) antigen recogni-
Abbreviations: CTL, cytotoxic T lymphocyte; GM-CSF, granulocyte–macrophage colony-stimulating factor; IFNa, interferon a; TNFa, tumor necrosis factor a. * Corresponding author. E-mail addresses: [email protected], [email protected] (A.N. Kalaaji). 1 Present address: Department of Interdisciplinary Oncology, H. Lee Moffitt Cancer Center & Research Institute, University of South Florida, 12902 Magnolia Drive, Tampa, FL 33612, USA. 1043-4666/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2009.11.016
tion [3]. In addition, IFNa provides crucial signals to CD8+ T cells for strong expansion and development of effector functions and memory [4]. IFNa has also been shown to inhibit angiogenesis and to increase infiltration of CD4+ T cells into melanoma [5,6]. Although high-dose IFNa has been approved by the US Food and Drug Administration as adjuvant therapy for high-risk melanoma, three Eastern Cooperative Oncology Group trials have shown extensive toxicity, and overall survival benefits have been controversial [7,8]. Granulocyte–macrophage colony-stimulating factor (GM-CSF) is a cytokine produced by macrophages and T cells, which can mediate the proliferation, maturation, and migration of dendritic cells [9–11]. Regression of melanoma metastasis after intratumoral injection of GM-CSF has been demonstrated clinically [12,13]. This effect in melanoma appears to be mediated by GM-CSF, with enhancement of tumor-associated peptide processing by antigenpresenting cells. Tumor necrosis factor a (TNFa) is a cytokine with multiple biologic actions. TNFa enhances both neutrophil and lymphocyte migration in vivo [14,15]. TNFa also has been shown to enhance dendritic cell migration into lymph nodes [16] and to cause hemorrhagic necrosis of tumor nodules when injected intratumorally [17,18]. TNFa also has direct antitumor effects that are mediated by a specific destructive effect against the tumor vasculature.
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Some clinical responses have been observed when combining TNFa with the cytotoxic drug melphalan in isolated limb perfusion for patients with in-transit melanoma metastases [19,20]. The present study aimed to investigate the efficacy of various combinations of IFNa, GM-CSF, and TNFa as antitumor cytokines in a B16F10 murine melanoma model. We hypothesized that combined cytokine therapy may coordinate and synergize the distinct biologic actions of individual cytokines, thus enhancing the overall antitumor effects. In addition, the toxicity profile is proposed to improve with the lower dosages required for combination therapy. We show that GM-CSF, IFNa, and TNFa result in significant inhibition of tumor growth when injected in combination intratumorally. Tumor regression correlates with local tumor necrosis and marked infiltration of T cells. In addition, injection of the intralesional cytokine cocktail also induces lymphadenopathy, with an increase in both CD4+ and CD8+ T cells in the draining lymph nodes. Furthermore, tumor-specific CD8+ T cells were identified from draining lymph nodes, which suggests the generation of antigenspecific T-cell responses. 2. Materials and methods 2.1. Mice, cell lines, and cytokines Female C57/BL6 mice, 6–8 weeks of age, were obtained from The Jackson Laboratory (Bar Harbor, Maine). Melanoma cell line B16F10 was obtained from the American Type Culture Collection (Manassas, Virginia). The B16F10 cell line was maintained in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum, 4 mM L-glutamine, 1.5 g/L sodium bicarbonate, and 4.5 g/L glucose. Recombinant mouse GM-CSF (Cell Sciences, Inc., Canton, Massachusetts) was reconstituted in sterile 20 mM acetic acid, further diluted in 0.1% bovine serum albumin/PBS, and kept in aliquots at 20 °C. Recombinant mouse IFNaA (PBL Biomedical Laboratories, New Brunswick, New Jersey) was kept at 70 °C before being diluted to working concentration. Recombinant mouse TNFa was obtained from Sigma (St. Louis, Missouri), and reconstituted aliquots were stored frozen at 20 °C. 2.2. Assessment of antitumor effects Mice were injected on day 0 with 1 106 B16F10 cells into both hind footpads. Starting from day 3, mice received 100 lL cytokine solution injected into the right footpad and 100 lL normal saline into the left footpad three times per week (Monday, Wednesday, and Friday). Tumor growth was monitored three times per week and measured in two perpendicular dimensions; tumor size is reported as the product of the two dimensions. Different dosages of single cytokines and combinations of two or three cytokines were all tested. When studying cytokine combination therapy, 10 lg GM-CSF, 50 ng IFNaA, and 50 ng TNFa were mixed, in a final volume of 100 lL per injection. All experiments were routinely performed in groups of 10–20 mice. Tumor size was compared among treatment groups for statistical significance using log-rank Mantel–Haenzel tests at the 0.05 level with 95% confidence intervals. Three weeks after tumor challenge, three mice from each treatment group were sacrificed to quantitate tumor-specific immune responses. 2.3. Evaluation of T-cell expansion in vivo Three weeks after tumor challenge, the draining inguinal lymph nodes from both right and left sides were harvested. The total number of leukocytes was determined by counting the live cells with Trypan blue. The percentage of CD4+ and CD8+ T cells was
determined by flow cytometry. The absolute number of CD4+ and CD8+ T cells per lymph node was calculated by multiplying the percentage of T-cell subsets by total leukocyte number.
2.4. Identifying tumor-specific CD8+ T Cells by CD107a mobilization assay Harvested lymph node cells were frozen and analyzed in batches. The cells were thawed the day before an experiment by overnight culture in CTL medium (Iscove’s modified Dulbecco’s medium with 10% fetal bovine serum, L-glutamine, penicillin/ streptomycin, and 100 units/mL interleukin-2). The following morning, cells were collected, washed, and resuspended to 107 cells/mL in CTL medium as effector cells. B16F10 melanoma cells were trypsinized using trypsin/EDTA solution (Gibco, Carlsbad, California), washed, and resuspended to 107 cells/mL in CTL medium as target cells. Mouse thymoma cell line EL4 was used as control target cells. The effector-to-target cell ratio used was generally 1 to 1. All assays were performed on 96-well V-bottom plates in triplicate for each condition. The following were added in order to each well: 1 lL of 2 mM monensin (Sigma) in 100% ethanol, 100 lL of target cells (106 B16F10 or EL4), 100 lL effector cells (106 lymph node cells), and 1 lL of fluorescein isothiocyanate-conjugated anti-CD107a (also known as lysosomal-associated membrane protein-1) antibody (BD PharMingen, San Jose, California). The wells were mixed with a multichannel pipette. The plate was centrifuged at 1200 rpm for 1 min and incubated at 37 °C for 6 h. After incubation, the plate was centrifuged at 1800 rpm for 1 min, and the supernatant was removed. Cell pellets were collected and washed with PBS plus 0.5 mM EDTA and 2% fetal bovine serum. Cells were stained with anti-CD8 phycoerythrin (BD PharMingen) and then analyzed by fluorescence-activated cell sorting.
3. Results 3.1. Antitumor effects of GM-CSF, IFNa, and TNFa combination therapy We first examined B16F10 tumor growth. After tumor challenge with 1 106 B16F10 melanoma cells in the right and left hind footpads, tumor size was closely monitored and compared in both footpads for 6 weeks. No differences were seen in tumor size or growth curve between the two sides during the time course (data not shown), which validates the feasibility of using the hind footpad on one side as a control. To evaluate the antitumor effects of each cytokine individually, GM-CSF alone, IFNaA alone, or TNFa alone was injected intratumorally into the right hind footpads, and PBS (control) was injected intratumorally into the left hind footpads, and tumor growth was compared. For each cytokine, three different doses were tested: 5, 10, and 20 lg of GM-CSF; 10, 50, and 100 ng of IFNaA; and 5, 10, and 50 ng of TNFa. All cytokines were injected three times per week for 6 weeks. As shown in Fig. 1, none of the individual cytokines—10 lg GM-CSF (Fig. 1A), 50 ng TNFa (Fig. 1B), and 50 ng IFNaA (Fig. 1C)—elicited significant antitumor effects; no differences in tumor size were seen between cytokine-injected and saline-injected footpads. Other test dosages yielded similar results (data not shown). To examine additive or synergistic effects of these cytokines, we tested the combinations of two cytokines in three combinations: 10 lg GM-CSF + 50 ng TNFa, 10 lg GM-CSF + 50 ng IFNaA, and 50 ng TNFa + 50 ng IFNaA. Although 2-cytokine combination therapy slightly inhibited tumor growth compared with saline injec-
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Fig. 1. Single-cytokine therapy fails to elicit antitumor effects. B16F10 tumor cells were injected into both hind footpads of mice on day 0. Starting on day 3, mice received intratumoral injection of 100 lL saline (circles) into the left hind footpad and 100 lL of a single cytokine (squares) into the right hind footpad three times per week. Tumor size was measured three times per week and calculated by multiplying two perpendicular dimensions. (A) Granulocyte–macrophage colony-stimulating factor (10 lg). (B) Tumor necrosis factor a (50 ng). (C) Interferon a (50 ng).
tion, the differences were not statistically significant (data not shown). To maximize the distinct biologic actions of the three cytokines, we evaluated the antitumor effects of all three cytokines combined (10 lg GM-CSF + 50 ng TNFa + 50 ng IFNaA). As shown in Fig. 2, tumors injected with the mixture of three cytokines were significantly smaller than control tumors injected with PBS (P < 0.05). After day 14, the growth rate of the cytokine cocktail-injected tumors decreased and tumor size plateaued. Compared with the unrestricted growth of PBS-injected tumors, the cytokine cocktail-treated tumors grew much slower from weeks 2 to 3 and failed to increase in size after 3 weeks. Experiments were terminated after 6 weeks, and mice were euthanized because large ulcerated tumors developed on their left hind footpads (saline controls). This
result demonstrates that the combination of GM-CSF, TNFa, and IFNa induces strong antitumor effects with marked suppression of tumor growth. Taken together, the above results strongly indicate that intratumoral injection of the mixture of GM-CSF, TNFa, and IFNa significantly potentiates and enhances the antitumor efficacy of local cytokine therapy. The marked difference between the 3-cytokine and 2-cytokine therapies demonstrates the synergistic actions that all three cytokines contribute to the overall antitumor effect, and possibly to inducing adaptive components of immune responses. 3.2. Tumor necrosis with infiltration of lymphocytes Histologic examination of tumor-bearing footpads receiving PBS injection showed intact melanoma tumor masses (Fig. 3). In contrast, tumors receiving the cytokine cocktail injection showed tumor necrosis with marked infiltration of neutrophils and lymphocytes (Fig. 3). This suggests that the antitumor effects of GMCSF, IFNa, and TNFa combination therapy may induce activation of an adaptive immune response. 3.3. T-cell expansion in draining lymph nodes
Fig. 2. Combination treatment with three cytokines (10 lg granulocyte–macrophage colony-stimulating factor, 50 ng tumor necrosis factor a, and 50 ng interferon a) significantly inhibits tumor growth. B16F10 tumor cells (1 106) were injected into both hind footpads of mice on day 0. Starting on day 3, mice received intratumoral injection of 100 lL phosphate-buffered saline (PBS; circles) into the left footpad and 100 lL of the cytokine cocktail (squares) into the right footpad three times per week. Tumor size was measured three times per week and calculated by multiplying two perpendicular dimensions. The difference in tumor size between PBS-treated footpads (n = 10) and cytokine-treated footpads (n = 10) was statistically significant (P < 0.05).
The pronounced T-cell infiltration at tumor sites suggests that the intratumoral cytokine cocktail injection may stimulate the activation and proliferation of immune effector cells in situ. These effects may contribute to the overall antitumor effect. To evaluate induction of antitumor immune responses, we examined the secondary lymphoid organs for T-cell proliferation. The draining inguinal lymph nodes from both experimental and control sides were harvested, and all cells were counted and stained for CD4 and CD8 T cells. The lymph nodes from the cytokine cocktail-injected side were significantly enlarged, and the total number of leukocytes harvested was more than 2-fold greater than from lymph nodes on the PBS-injected side (Fig. 4A). On the basis of percentage of CD4+ and CD8+ T-cell subsets determined by fluorescence-activated cell sorting, the absolute numbers of CD4+ and CD8+ T cells per lymph node increased more than 2-fold with the cytokine
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Fig. 3. Footpads (left panel) injected with phosphate-buffered saline (PBS) or cytokine cocktail (granulocyte–macrophage colony-stimulating factor, tumor necrosis factor a, and interferon a) were frozen and sectioned for pathologic analysis (right panel) (hematoxylin–eosin; original magnification, 10 [upper], 25 [lower]). Upper panel: no evidence of tumor regression is seen in PBS-injected footpads; there is no histologic infiltrate or tumor necrosis. Lower panel: the right footpad that received the cytokine cocktail injection shows tumor regression; corresponding histologic findings show tumor necrosis and lymphocytic infiltrate.
Fig. 4. Intratumoral injection of 3-cytokine cocktail—granulocyte–macrophage colony-stimulating factor, tumor necrosis factor a, and interferon a (black bars)— induces an increase in total leukocyte number (A) and both CD4+ and CD8+ T cell numbers (B) in draining lymph nodes (LN) from cytokine-injected sides versus saline-injected sides (white bars). Leukocytes were counted as total live cell numbers per LN. The number of CD4+ and CD8+ cells was calculated by multiplying the proportion of T-cell subsets (determined by flow cytometry) by the total leukocytes per LN. Results are representative of three separate experiments (P < 0.05).
cocktail therapy (P < 0.05) (Fig. 4B). The ratio of CD4 to CD8 cells did not change in either group. These results indicate that a locally administered cytokine cocktail of GM-CSF, TNFa, and IFNa induces an increase in total lymphocytes, with proportionate proliferation of CD4+ and CD8+ T cells at secondary lymphoid organs. The cell increase correlated closely with lymphadenopathy. 3.4. Generation of tumor-specific CD8 T-cell responses in vivo The above results demonstrated that an intratumorally administered cytokine cocktail resulted in local and systemic T-cell proliferation. To evaluate for generation of tumor-specific T-cell responses, we used flow cytometry to test the T cells from draining lymph nodes for surface mobilization of CD107a upon tumor stimulation. CD107a (lysosomal-associated membrane protein-1) is a vesicular membrane protein that becomes transiently mobilized to the cell surface during the degranulation process when CD8+ T
cells fuse with target cell membrane and release cytotoxic mediators such as perforin and granzymes [21]. Therefore, the CD107a mobilization assay can be used to rapidly identify tumor-cytolytic T cells using CD107a mobilization as a marker of degranulation upon interaction with tumor cells, without knowing the specific tumor antigen. For our experiments, cells harvested from inguinal lymph nodes of both sides were first incubated with B16F10 melanoma cells or the control tumor cell line EL4 in vitro for 5 h in the presence of fluorescently labeled anti-CD107a antibody and then stained with anti-CD8 antibody before flow analysis. The percentage of CD107a+/CD8+ T cells upon B16F10 stimulation versus control tumor stimulation was used to measure the tumor-lytic T-cell population. As shown in Fig. 5, only CD8+ T cells in the cytokine cocktail group had substantial tumor-specific cytotoxic function; 10.55% of CD8+ T cells degranulated upon recognizing B16F10 cells. After subtracting background killing of control tumor cells (2.38%), 8.17% of CTLs demonstrated recognition and direct killing of the melanoma tumor line within a short period of time. In contrast, T cells from lymph nodes of saline-administered sides had no specific CD107a+ staining toward B16F10. The small percentage of CD107a staining—1.14% toward B16F10 cells and 1.02% toward EL4 cells—showed no tumor specificity and represents nonspecific background. Both the cytokine-treated and control groups had a small percentage of background signal that could be due to activity of natural killer cells. The results of the CD107a assay strongly suggest that an intratumorally administered GM-CSF, TNFa, and IFNa cocktail induces tumor-specific T-cell responses that have the potential for important roles in the antitumor effects of this therapeutic strategy. The combined effects of the three cytokines on dendritic cells, tumor cells, and T cells would be expected to generate tumor-specific CTL responses through synergistic actions.
4. Discussion The antitumor effects of cytokines are generally considered to be multifactorial. Previous studies have suggested that systemically or locally delivered cytokines could inhibit proliferation of
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Fig. 5. Cytokine cocktail (granulocyte–macrophage colony-stimulating factor, tumor necrosis factor a, and interferon a) induces tumor-specific T cells identified by CD107a mobilization assay. Inguinal lymph nodes were harvested from the cytokine-treated sides (A) and the control sides (B) 14 days after tumor challenge. Lymphocytes were incubated with either B16F10 tumor cells or control tumor cell line EL4 for 6 h while co-staining with anti-CD107a–fluorescein isothiocyanate, followed by staining with antiCD8–phycoerythrin. Cell-surface expression of CD107a upon tumor stimulation was analyzed by flow cytometry. Plots are gated on CD8+ T cells. Results are representative of three separate experiments.
tumor cells, stimulate activity or proliferation of immune effector cells (including T cells, natural killer cells, macrophages, and dendritic cells), and inhibit angiogenesis in the tumor microenvironment [22,23]. Because different cytokines have unique biologic functions and act on different immune elements in combination, they may mediate novel activities by complementing and synergizing with each other. Our findings show that the combination of GM-CSF, IFNa, and TNFa injected intratumorally significantly inhibits B16F10 melanoma growth as compared with intratumoral injection of saline. Single-cytokine injections (Fig. 1) and combinations of two cytokines did not result in inhibition of growth of the melanomas. However, as shown in Fig. 2, coinjection of all three cytokines produced significant inhibition of melanoma growth; melanomas were smaller after cytokine therapy as compared with the contralateral saline-injected control tumors (Fig. 2). Histologically, the cytokine-injected melanomas have pronounced infiltration of leukocytes with observed tumor necrosis, which is not seen in the saline-injected melanomas (Fig. 3). The antitumor effect seen with the combination cytokine therapy is most likely a result of the synergistic effects of these cytokines. This combination takes advantage of the different actions of the three cytokines: the effect of GM-CSF on dendritic cells [9–11], the antiproliferative immunomodulatory effects of IFNa [5,6], and induction of tumor necrosis by TNFa. The mechanisms of TNFa-induced cell death might be mediated through TNFa-induced manganese superoxide dismutase, which would promote reactive oxygen species [24,25]. TNFa most likely also enhances both lymphocyte migration into the tumors and dendritic cell migration and activation [14–18]. A significant tumor inhibitory effect was observed only when all three cytokines were included as a combination therapy, which indicates that the different biologic
function of all three cytokines are indispensable and complementary in achieving effective therapeutic responses. Furthermore, the antimelanoma effect of these three cytokines extended beyond the injected tumor site. Lymph nodes removed from the same side as nearby cytokine-injected melanomas were enlarged compared with lymph nodes removed from the contralateral control side. In addition, both CD4+ and CD8+ T cells were increased in the draining lymph nodes of the cytokine-treated side as compared with the lymph nodes of the saline-injected side (Fig. 4). The combination of GM-CSF, IFNa, and TNFa appears not only to cause inhibition of growth of melanomas when injected intratumorally but also to enhance the immune response in nearby draining lymph nodes. These results suggest that, in addition to the local inflammatory responses induced by the cytokine cocktail, an adaptive immune response is activated by this therapeutic intervention. CD8+ CTLs are recognized as the most direct and effective elements of the immune system capable of generating antitumor immune responses. CD4+ helper T lymphocytes are considered to have an essential role in the generation and maintenance of CTLs through various mechanisms. Helper T cells also are critical for the activation and maturation of dendritic cells, the professional antigen-presenting cells for CTL priming. The proliferation of T cells both locally and in secondary lymphoid tissues in our study indicates that T-cell responses, possibly tumor-specific T-cell responses, are involved in the antitumor effect. We next determined whether this was a cytokine-induced, nonspecific inflammatory event, or whether the cytokine cocktail generated CTLs specific to B16F10 melanoma. We therefore examined CD8+ T cells from draining lymph nodes for surface mobilization of CD107a using flow cytometry. Recent studies have shown that CD107a mobilizes to the cell surface in response to tumor stimulation and is a rapid and reliable measure for identifying and
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Fig. 6. Effects of combination cytokine therapy take advantage of the synergistic interaction between the different effects of tumor necrosis factor a (TNFa), granulocyte– macrophage colony-stimulating factor (GM-CSF), and interferon a (IFNa), on tumor cells, dendritic cells, and T lymphocytes. TNFa induces tumor necrosis or apoptosis, releasing tumor antigen and inflammatory signals. The presentation of tumor antigen by dendritic cells is facilitated by GM-CSF and IFNa, and is a downstream effect of TNFa, which subsequently leads to tumor-specific T-cell activation and proliferation at tumor sites and in draining lymph nodes. In addition to increasing MHC (major histocompatibility complex) class I antigen presentation on antigen-presenting cells, IFNa also has direct effects on potentiating T-cell development and expansion.
isolating functional tumor-reactive T cells. As shown in Fig. 5, only CD8+ T cells obtained from the lymph nodes draining the cytokine cocktail-injected melanomas showed significant tumor-specific, functionally active CTLs. Therefore, the combination of IFNa, GMCSF, and TNFa was able to generate CD8+ T cells that were specific to and functionally active against the B16F10 melanoma. The identification of tumor-specific CTL responses induced by cytokine cocktail therapy is intriguing. Currently, most preclinical and clinical studies of melanoma immunotherapy aim to generate tumor-specific CTL responses by vaccines containing exogenous tumor antigen-like peptide/protein derived from tumor-associated antigens, irradiated tumor cells, or genetically modified tumor cells. How the tumor-specific T-cell responses are generated in this local 3-cytokine combination therapy is a critical question to answer; investigation is currently under way. We propose that the tumor necrosis induced by intralesional TNFa injection may provide devitalized tumor cells, which serve as a source of effective tumor antigens. With the combined effects of GM-CSF and TNFa, as well as release of a potential host of downstream inflammatory cytokines, dendritic cells migrate to the tumor site, become activated to effectively present melanoma antigen, and trigger T-cell activation. In addition, antigen-bound dendritic cells migrate to draining lymph nodes and further induce T cells to proliferate. The actions of IFNa potentially include enhanced infiltration of CD4+ T cells, as well as increased major histocompatibility complex class I expression [5,6]. The effects of our combination cytokine therapy are likely mediated by complex interactions of multiple downstream pathways that contribute to antigen processing and presentation, T-cell priming, trafficking, and proliferation (Fig. 6). In summary, we have shown that the combination of GM-CSF, IFNa, and TNFa inhibits melanoma growth in vivo and encompasses more than a localized intratumor effect by engaging a regional response, with enhanced lymphocyte numbers in lymph nodes
draining the cytokine-treated melanomas. Furthermore, the generation of CD8+ T cells specific for B16F10 melanoma was shown to develop with this cytokine combination. Many previous clinical trials have focused on therapy with single cytokines, such as highdose IFNa. These trials resulted in marked toxicity for patients with overall survival benefits that showed inconclusive outcomes in some studies [7,8,26]. On the basis of these findings, combinations of intratumorally injected cytokines should be examined in the clinical setting to take full advantage of their different biologic actions and robust, potentiating effects in treating melanoma. In addition to intratumoral administration, the cytokine combination therapy might also be investigated for possible systemic administration through subcutaneous injection or isolated limb perfusion. However, further research is needed to identify the effects of systemic versus intratumoral injection on the safety profile and treatment efficacy. The synergistic effects of this cytokine combination therapy (IFNa, GM-CSF, and TNFa) should allow for the use of lower doses than had previously been tested in clinical trials and may translate to more effective, less toxic therapy. References [1] SEER Cancer Statistics Review, 1975–2002 [database on the Internet]. Bethesda (MD): National Cancer Institute; 2002. Available from: http://seer.cancer.gov/ csr/1975_2002/ [cited 2007 Feb 19]. [2] Parkinson DR, Abrams JS, Wiernik PH, Rayner AA, Margolin KA, Van Echo DA, et al. Interleukin-2 therapy in patients with metastatic malignant melanoma: a phase II study. J Clin Oncol 1990;8:1650–6. [3] Isaacs A, Lindenmann J. Virus interference. I. The interferon. Proc R Soc Lond B Biol Sci 1957;147:258–67. [4] Mescher MF, Curtsinger JM, Agarwal P, Casey KA, Gerner M, Hammerbeck CD, et al. Signals required for programming effector and memory development by CD8+ T cells. Immunol Rev 2006;211:81–92. [5] Hakansson A, Gustafsson B, Krysander L, Hakansson L. Effect of IFN-a on tumor-infiltrating mononuclear cells and regressive changes in metastatic malignant melanoma. J Interferon Cytokine Res 1998;18:33–9.
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Cytokine journal homepage: www.elsevier.com/locate/issn/10434666
Combination cytokine therapy inhibits tumor growth by generation of tumor-specific T-cell responses in a murine melanoma model Amer N. Kalaaji a,*, Jun Lu a, Svetomir N. Markovic b, Esteban Celis c,1, Mark R. Pittelkow a,d a
Department of Dermatology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA Division of Hematology, Mayo Clinic, Rochester, MN, USA c Department of Immunology, Mayo Clinic, Rochester, MN, USA d Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA b
a r t i c l e
i n f o
Article history: Received 24 August 2009 Received in revised form 16 November 2009 Accepted 17 November 2009
Keywords: Cytokines Cytotoxic T lymphocytes Melanoma Therapy Tumor
a b s t r a c t Various cytokines, including interferon a (IFNa), tumor necrosis factor a (TNFa), and granulocyte–macrophage colony-stimulating factor (GM-CSF), have been used as adjuvant therapy for advanced-stage melanoma with some success but with marked toxicity, which appears to be related to higher doses. We investigated the efficacy of IFNa, GM-CSF, and TNFa in various combinations to induce antitumor and immune responses in a B16F10 murine melanoma model. These studies showed that GM-CSF, IFNa, and TNFa, when injected together intratumorally, mediated significant inhibition of tumor growth. Tumor regression correlated with local tumor necrosis and significant infiltration of T cells. In addition, this injected intralesional cytokine cocktail also induced lymphadenopathy, with an increase in both CD4+ and CD8+ T cells in the draining lymph nodes. Furthermore, tumor-specific CD8+ T cells were identified from draining lymph nodes. These investigations identify the combined effects of IFNa, GM-CSF, and TNFa in induction of the adaptive immune response and generation of antigen-specific T-cell reactivity. These results support potential clinical trials of the low-dose cytokine combination as adjuvant therapy for melanoma. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Malignant melanoma is occurring with increasing frequency. Over the past 40 years in the United States, the incidence of melanoma has increased 3-fold in middle-aged men and 5-fold in older men [1]. Various adjuvant therapies have been used, including immunotherapy with cytokines. Cytokines are small proteins that act in an autocrine or paracrine manner. They bind to specific receptors, with individual cytokines having multiple functions. Cytokine adjuvant therapy for advanced-stage melanoma has shown some success but also dose-dependent, marked toxicity [2]. Interferons are one of the earliest identified families of cytokines. In addition to its antiviral effects, interferon a (IFNa) has antiproliferative and immunomodulatory effects including increasing major histocompatibility complex class I antigen expression, which is crucial for cytotoxic T lymphocyte (CTL) antigen recogni-
Abbreviations: CTL, cytotoxic T lymphocyte; GM-CSF, granulocyte–macrophage colony-stimulating factor; IFNa, interferon a; TNFa, tumor necrosis factor a. * Corresponding author. E-mail addresses: [email protected], [email protected] (A.N. Kalaaji). 1 Present address: Department of Interdisciplinary Oncology, H. Lee Moffitt Cancer Center & Research Institute, University of South Florida, 12902 Magnolia Drive, Tampa, FL 33612, USA. 1043-4666/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2009.11.016
tion [3]. In addition, IFNa provides crucial signals to CD8+ T cells for strong expansion and development of effector functions and memory [4]. IFNa has also been shown to inhibit angiogenesis and to increase infiltration of CD4+ T cells into melanoma [5,6]. Although high-dose IFNa has been approved by the US Food and Drug Administration as adjuvant therapy for high-risk melanoma, three Eastern Cooperative Oncology Group trials have shown extensive toxicity, and overall survival benefits have been controversial [7,8]. Granulocyte–macrophage colony-stimulating factor (GM-CSF) is a cytokine produced by macrophages and T cells, which can mediate the proliferation, maturation, and migration of dendritic cells [9–11]. Regression of melanoma metastasis after intratumoral injection of GM-CSF has been demonstrated clinically [12,13]. This effect in melanoma appears to be mediated by GM-CSF, with enhancement of tumor-associated peptide processing by antigenpresenting cells. Tumor necrosis factor a (TNFa) is a cytokine with multiple biologic actions. TNFa enhances both neutrophil and lymphocyte migration in vivo [14,15]. TNFa also has been shown to enhance dendritic cell migration into lymph nodes [16] and to cause hemorrhagic necrosis of tumor nodules when injected intratumorally [17,18]. TNFa also has direct antitumor effects that are mediated by a specific destructive effect against the tumor vasculature.
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Some clinical responses have been observed when combining TNFa with the cytotoxic drug melphalan in isolated limb perfusion for patients with in-transit melanoma metastases [19,20]. The present study aimed to investigate the efficacy of various combinations of IFNa, GM-CSF, and TNFa as antitumor cytokines in a B16F10 murine melanoma model. We hypothesized that combined cytokine therapy may coordinate and synergize the distinct biologic actions of individual cytokines, thus enhancing the overall antitumor effects. In addition, the toxicity profile is proposed to improve with the lower dosages required for combination therapy. We show that GM-CSF, IFNa, and TNFa result in significant inhibition of tumor growth when injected in combination intratumorally. Tumor regression correlates with local tumor necrosis and marked infiltration of T cells. In addition, injection of the intralesional cytokine cocktail also induces lymphadenopathy, with an increase in both CD4+ and CD8+ T cells in the draining lymph nodes. Furthermore, tumor-specific CD8+ T cells were identified from draining lymph nodes, which suggests the generation of antigenspecific T-cell responses. 2. Materials and methods 2.1. Mice, cell lines, and cytokines Female C57/BL6 mice, 6–8 weeks of age, were obtained from The Jackson Laboratory (Bar Harbor, Maine). Melanoma cell line B16F10 was obtained from the American Type Culture Collection (Manassas, Virginia). The B16F10 cell line was maintained in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum, 4 mM L-glutamine, 1.5 g/L sodium bicarbonate, and 4.5 g/L glucose. Recombinant mouse GM-CSF (Cell Sciences, Inc., Canton, Massachusetts) was reconstituted in sterile 20 mM acetic acid, further diluted in 0.1% bovine serum albumin/PBS, and kept in aliquots at 20 °C. Recombinant mouse IFNaA (PBL Biomedical Laboratories, New Brunswick, New Jersey) was kept at 70 °C before being diluted to working concentration. Recombinant mouse TNFa was obtained from Sigma (St. Louis, Missouri), and reconstituted aliquots were stored frozen at 20 °C. 2.2. Assessment of antitumor effects Mice were injected on day 0 with 1 106 B16F10 cells into both hind footpads. Starting from day 3, mice received 100 lL cytokine solution injected into the right footpad and 100 lL normal saline into the left footpad three times per week (Monday, Wednesday, and Friday). Tumor growth was monitored three times per week and measured in two perpendicular dimensions; tumor size is reported as the product of the two dimensions. Different dosages of single cytokines and combinations of two or three cytokines were all tested. When studying cytokine combination therapy, 10 lg GM-CSF, 50 ng IFNaA, and 50 ng TNFa were mixed, in a final volume of 100 lL per injection. All experiments were routinely performed in groups of 10–20 mice. Tumor size was compared among treatment groups for statistical significance using log-rank Mantel–Haenzel tests at the 0.05 level with 95% confidence intervals. Three weeks after tumor challenge, three mice from each treatment group were sacrificed to quantitate tumor-specific immune responses. 2.3. Evaluation of T-cell expansion in vivo Three weeks after tumor challenge, the draining inguinal lymph nodes from both right and left sides were harvested. The total number of leukocytes was determined by counting the live cells with Trypan blue. The percentage of CD4+ and CD8+ T cells was
determined by flow cytometry. The absolute number of CD4+ and CD8+ T cells per lymph node was calculated by multiplying the percentage of T-cell subsets by total leukocyte number.
2.4. Identifying tumor-specific CD8+ T Cells by CD107a mobilization assay Harvested lymph node cells were frozen and analyzed in batches. The cells were thawed the day before an experiment by overnight culture in CTL medium (Iscove’s modified Dulbecco’s medium with 10% fetal bovine serum, L-glutamine, penicillin/ streptomycin, and 100 units/mL interleukin-2). The following morning, cells were collected, washed, and resuspended to 107 cells/mL in CTL medium as effector cells. B16F10 melanoma cells were trypsinized using trypsin/EDTA solution (Gibco, Carlsbad, California), washed, and resuspended to 107 cells/mL in CTL medium as target cells. Mouse thymoma cell line EL4 was used as control target cells. The effector-to-target cell ratio used was generally 1 to 1. All assays were performed on 96-well V-bottom plates in triplicate for each condition. The following were added in order to each well: 1 lL of 2 mM monensin (Sigma) in 100% ethanol, 100 lL of target cells (106 B16F10 or EL4), 100 lL effector cells (106 lymph node cells), and 1 lL of fluorescein isothiocyanate-conjugated anti-CD107a (also known as lysosomal-associated membrane protein-1) antibody (BD PharMingen, San Jose, California). The wells were mixed with a multichannel pipette. The plate was centrifuged at 1200 rpm for 1 min and incubated at 37 °C for 6 h. After incubation, the plate was centrifuged at 1800 rpm for 1 min, and the supernatant was removed. Cell pellets were collected and washed with PBS plus 0.5 mM EDTA and 2% fetal bovine serum. Cells were stained with anti-CD8 phycoerythrin (BD PharMingen) and then analyzed by fluorescence-activated cell sorting.
3. Results 3.1. Antitumor effects of GM-CSF, IFNa, and TNFa combination therapy We first examined B16F10 tumor growth. After tumor challenge with 1 106 B16F10 melanoma cells in the right and left hind footpads, tumor size was closely monitored and compared in both footpads for 6 weeks. No differences were seen in tumor size or growth curve between the two sides during the time course (data not shown), which validates the feasibility of using the hind footpad on one side as a control. To evaluate the antitumor effects of each cytokine individually, GM-CSF alone, IFNaA alone, or TNFa alone was injected intratumorally into the right hind footpads, and PBS (control) was injected intratumorally into the left hind footpads, and tumor growth was compared. For each cytokine, three different doses were tested: 5, 10, and 20 lg of GM-CSF; 10, 50, and 100 ng of IFNaA; and 5, 10, and 50 ng of TNFa. All cytokines were injected three times per week for 6 weeks. As shown in Fig. 1, none of the individual cytokines—10 lg GM-CSF (Fig. 1A), 50 ng TNFa (Fig. 1B), and 50 ng IFNaA (Fig. 1C)—elicited significant antitumor effects; no differences in tumor size were seen between cytokine-injected and saline-injected footpads. Other test dosages yielded similar results (data not shown). To examine additive or synergistic effects of these cytokines, we tested the combinations of two cytokines in three combinations: 10 lg GM-CSF + 50 ng TNFa, 10 lg GM-CSF + 50 ng IFNaA, and 50 ng TNFa + 50 ng IFNaA. Although 2-cytokine combination therapy slightly inhibited tumor growth compared with saline injec-
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Fig. 1. Single-cytokine therapy fails to elicit antitumor effects. B16F10 tumor cells were injected into both hind footpads of mice on day 0. Starting on day 3, mice received intratumoral injection of 100 lL saline (circles) into the left hind footpad and 100 lL of a single cytokine (squares) into the right hind footpad three times per week. Tumor size was measured three times per week and calculated by multiplying two perpendicular dimensions. (A) Granulocyte–macrophage colony-stimulating factor (10 lg). (B) Tumor necrosis factor a (50 ng). (C) Interferon a (50 ng).
tion, the differences were not statistically significant (data not shown). To maximize the distinct biologic actions of the three cytokines, we evaluated the antitumor effects of all three cytokines combined (10 lg GM-CSF + 50 ng TNFa + 50 ng IFNaA). As shown in Fig. 2, tumors injected with the mixture of three cytokines were significantly smaller than control tumors injected with PBS (P < 0.05). After day 14, the growth rate of the cytokine cocktail-injected tumors decreased and tumor size plateaued. Compared with the unrestricted growth of PBS-injected tumors, the cytokine cocktail-treated tumors grew much slower from weeks 2 to 3 and failed to increase in size after 3 weeks. Experiments were terminated after 6 weeks, and mice were euthanized because large ulcerated tumors developed on their left hind footpads (saline controls). This
result demonstrates that the combination of GM-CSF, TNFa, and IFNa induces strong antitumor effects with marked suppression of tumor growth. Taken together, the above results strongly indicate that intratumoral injection of the mixture of GM-CSF, TNFa, and IFNa significantly potentiates and enhances the antitumor efficacy of local cytokine therapy. The marked difference between the 3-cytokine and 2-cytokine therapies demonstrates the synergistic actions that all three cytokines contribute to the overall antitumor effect, and possibly to inducing adaptive components of immune responses. 3.2. Tumor necrosis with infiltration of lymphocytes Histologic examination of tumor-bearing footpads receiving PBS injection showed intact melanoma tumor masses (Fig. 3). In contrast, tumors receiving the cytokine cocktail injection showed tumor necrosis with marked infiltration of neutrophils and lymphocytes (Fig. 3). This suggests that the antitumor effects of GMCSF, IFNa, and TNFa combination therapy may induce activation of an adaptive immune response. 3.3. T-cell expansion in draining lymph nodes
Fig. 2. Combination treatment with three cytokines (10 lg granulocyte–macrophage colony-stimulating factor, 50 ng tumor necrosis factor a, and 50 ng interferon a) significantly inhibits tumor growth. B16F10 tumor cells (1 106) were injected into both hind footpads of mice on day 0. Starting on day 3, mice received intratumoral injection of 100 lL phosphate-buffered saline (PBS; circles) into the left footpad and 100 lL of the cytokine cocktail (squares) into the right footpad three times per week. Tumor size was measured three times per week and calculated by multiplying two perpendicular dimensions. The difference in tumor size between PBS-treated footpads (n = 10) and cytokine-treated footpads (n = 10) was statistically significant (P < 0.05).
The pronounced T-cell infiltration at tumor sites suggests that the intratumoral cytokine cocktail injection may stimulate the activation and proliferation of immune effector cells in situ. These effects may contribute to the overall antitumor effect. To evaluate induction of antitumor immune responses, we examined the secondary lymphoid organs for T-cell proliferation. The draining inguinal lymph nodes from both experimental and control sides were harvested, and all cells were counted and stained for CD4 and CD8 T cells. The lymph nodes from the cytokine cocktail-injected side were significantly enlarged, and the total number of leukocytes harvested was more than 2-fold greater than from lymph nodes on the PBS-injected side (Fig. 4A). On the basis of percentage of CD4+ and CD8+ T-cell subsets determined by fluorescence-activated cell sorting, the absolute numbers of CD4+ and CD8+ T cells per lymph node increased more than 2-fold with the cytokine
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Fig. 3. Footpads (left panel) injected with phosphate-buffered saline (PBS) or cytokine cocktail (granulocyte–macrophage colony-stimulating factor, tumor necrosis factor a, and interferon a) were frozen and sectioned for pathologic analysis (right panel) (hematoxylin–eosin; original magnification, 10 [upper], 25 [lower]). Upper panel: no evidence of tumor regression is seen in PBS-injected footpads; there is no histologic infiltrate or tumor necrosis. Lower panel: the right footpad that received the cytokine cocktail injection shows tumor regression; corresponding histologic findings show tumor necrosis and lymphocytic infiltrate.
Fig. 4. Intratumoral injection of 3-cytokine cocktail—granulocyte–macrophage colony-stimulating factor, tumor necrosis factor a, and interferon a (black bars)— induces an increase in total leukocyte number (A) and both CD4+ and CD8+ T cell numbers (B) in draining lymph nodes (LN) from cytokine-injected sides versus saline-injected sides (white bars). Leukocytes were counted as total live cell numbers per LN. The number of CD4+ and CD8+ cells was calculated by multiplying the proportion of T-cell subsets (determined by flow cytometry) by the total leukocytes per LN. Results are representative of three separate experiments (P < 0.05).
cocktail therapy (P < 0.05) (Fig. 4B). The ratio of CD4 to CD8 cells did not change in either group. These results indicate that a locally administered cytokine cocktail of GM-CSF, TNFa, and IFNa induces an increase in total lymphocytes, with proportionate proliferation of CD4+ and CD8+ T cells at secondary lymphoid organs. The cell increase correlated closely with lymphadenopathy. 3.4. Generation of tumor-specific CD8 T-cell responses in vivo The above results demonstrated that an intratumorally administered cytokine cocktail resulted in local and systemic T-cell proliferation. To evaluate for generation of tumor-specific T-cell responses, we used flow cytometry to test the T cells from draining lymph nodes for surface mobilization of CD107a upon tumor stimulation. CD107a (lysosomal-associated membrane protein-1) is a vesicular membrane protein that becomes transiently mobilized to the cell surface during the degranulation process when CD8+ T
cells fuse with target cell membrane and release cytotoxic mediators such as perforin and granzymes [21]. Therefore, the CD107a mobilization assay can be used to rapidly identify tumor-cytolytic T cells using CD107a mobilization as a marker of degranulation upon interaction with tumor cells, without knowing the specific tumor antigen. For our experiments, cells harvested from inguinal lymph nodes of both sides were first incubated with B16F10 melanoma cells or the control tumor cell line EL4 in vitro for 5 h in the presence of fluorescently labeled anti-CD107a antibody and then stained with anti-CD8 antibody before flow analysis. The percentage of CD107a+/CD8+ T cells upon B16F10 stimulation versus control tumor stimulation was used to measure the tumor-lytic T-cell population. As shown in Fig. 5, only CD8+ T cells in the cytokine cocktail group had substantial tumor-specific cytotoxic function; 10.55% of CD8+ T cells degranulated upon recognizing B16F10 cells. After subtracting background killing of control tumor cells (2.38%), 8.17% of CTLs demonstrated recognition and direct killing of the melanoma tumor line within a short period of time. In contrast, T cells from lymph nodes of saline-administered sides had no specific CD107a+ staining toward B16F10. The small percentage of CD107a staining—1.14% toward B16F10 cells and 1.02% toward EL4 cells—showed no tumor specificity and represents nonspecific background. Both the cytokine-treated and control groups had a small percentage of background signal that could be due to activity of natural killer cells. The results of the CD107a assay strongly suggest that an intratumorally administered GM-CSF, TNFa, and IFNa cocktail induces tumor-specific T-cell responses that have the potential for important roles in the antitumor effects of this therapeutic strategy. The combined effects of the three cytokines on dendritic cells, tumor cells, and T cells would be expected to generate tumor-specific CTL responses through synergistic actions.
4. Discussion The antitumor effects of cytokines are generally considered to be multifactorial. Previous studies have suggested that systemically or locally delivered cytokines could inhibit proliferation of
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Fig. 5. Cytokine cocktail (granulocyte–macrophage colony-stimulating factor, tumor necrosis factor a, and interferon a) induces tumor-specific T cells identified by CD107a mobilization assay. Inguinal lymph nodes were harvested from the cytokine-treated sides (A) and the control sides (B) 14 days after tumor challenge. Lymphocytes were incubated with either B16F10 tumor cells or control tumor cell line EL4 for 6 h while co-staining with anti-CD107a–fluorescein isothiocyanate, followed by staining with antiCD8–phycoerythrin. Cell-surface expression of CD107a upon tumor stimulation was analyzed by flow cytometry. Plots are gated on CD8+ T cells. Results are representative of three separate experiments.
tumor cells, stimulate activity or proliferation of immune effector cells (including T cells, natural killer cells, macrophages, and dendritic cells), and inhibit angiogenesis in the tumor microenvironment [22,23]. Because different cytokines have unique biologic functions and act on different immune elements in combination, they may mediate novel activities by complementing and synergizing with each other. Our findings show that the combination of GM-CSF, IFNa, and TNFa injected intratumorally significantly inhibits B16F10 melanoma growth as compared with intratumoral injection of saline. Single-cytokine injections (Fig. 1) and combinations of two cytokines did not result in inhibition of growth of the melanomas. However, as shown in Fig. 2, coinjection of all three cytokines produced significant inhibition of melanoma growth; melanomas were smaller after cytokine therapy as compared with the contralateral saline-injected control tumors (Fig. 2). Histologically, the cytokine-injected melanomas have pronounced infiltration of leukocytes with observed tumor necrosis, which is not seen in the saline-injected melanomas (Fig. 3). The antitumor effect seen with the combination cytokine therapy is most likely a result of the synergistic effects of these cytokines. This combination takes advantage of the different actions of the three cytokines: the effect of GM-CSF on dendritic cells [9–11], the antiproliferative immunomodulatory effects of IFNa [5,6], and induction of tumor necrosis by TNFa. The mechanisms of TNFa-induced cell death might be mediated through TNFa-induced manganese superoxide dismutase, which would promote reactive oxygen species [24,25]. TNFa most likely also enhances both lymphocyte migration into the tumors and dendritic cell migration and activation [14–18]. A significant tumor inhibitory effect was observed only when all three cytokines were included as a combination therapy, which indicates that the different biologic
function of all three cytokines are indispensable and complementary in achieving effective therapeutic responses. Furthermore, the antimelanoma effect of these three cytokines extended beyond the injected tumor site. Lymph nodes removed from the same side as nearby cytokine-injected melanomas were enlarged compared with lymph nodes removed from the contralateral control side. In addition, both CD4+ and CD8+ T cells were increased in the draining lymph nodes of the cytokine-treated side as compared with the lymph nodes of the saline-injected side (Fig. 4). The combination of GM-CSF, IFNa, and TNFa appears not only to cause inhibition of growth of melanomas when injected intratumorally but also to enhance the immune response in nearby draining lymph nodes. These results suggest that, in addition to the local inflammatory responses induced by the cytokine cocktail, an adaptive immune response is activated by this therapeutic intervention. CD8+ CTLs are recognized as the most direct and effective elements of the immune system capable of generating antitumor immune responses. CD4+ helper T lymphocytes are considered to have an essential role in the generation and maintenance of CTLs through various mechanisms. Helper T cells also are critical for the activation and maturation of dendritic cells, the professional antigen-presenting cells for CTL priming. The proliferation of T cells both locally and in secondary lymphoid tissues in our study indicates that T-cell responses, possibly tumor-specific T-cell responses, are involved in the antitumor effect. We next determined whether this was a cytokine-induced, nonspecific inflammatory event, or whether the cytokine cocktail generated CTLs specific to B16F10 melanoma. We therefore examined CD8+ T cells from draining lymph nodes for surface mobilization of CD107a using flow cytometry. Recent studies have shown that CD107a mobilizes to the cell surface in response to tumor stimulation and is a rapid and reliable measure for identifying and
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Fig. 6. Effects of combination cytokine therapy take advantage of the synergistic interaction between the different effects of tumor necrosis factor a (TNFa), granulocyte– macrophage colony-stimulating factor (GM-CSF), and interferon a (IFNa), on tumor cells, dendritic cells, and T lymphocytes. TNFa induces tumor necrosis or apoptosis, releasing tumor antigen and inflammatory signals. The presentation of tumor antigen by dendritic cells is facilitated by GM-CSF and IFNa, and is a downstream effect of TNFa, which subsequently leads to tumor-specific T-cell activation and proliferation at tumor sites and in draining lymph nodes. In addition to increasing MHC (major histocompatibility complex) class I antigen presentation on antigen-presenting cells, IFNa also has direct effects on potentiating T-cell development and expansion.
isolating functional tumor-reactive T cells. As shown in Fig. 5, only CD8+ T cells obtained from the lymph nodes draining the cytokine cocktail-injected melanomas showed significant tumor-specific, functionally active CTLs. Therefore, the combination of IFNa, GMCSF, and TNFa was able to generate CD8+ T cells that were specific to and functionally active against the B16F10 melanoma. The identification of tumor-specific CTL responses induced by cytokine cocktail therapy is intriguing. Currently, most preclinical and clinical studies of melanoma immunotherapy aim to generate tumor-specific CTL responses by vaccines containing exogenous tumor antigen-like peptide/protein derived from tumor-associated antigens, irradiated tumor cells, or genetically modified tumor cells. How the tumor-specific T-cell responses are generated in this local 3-cytokine combination therapy is a critical question to answer; investigation is currently under way. We propose that the tumor necrosis induced by intralesional TNFa injection may provide devitalized tumor cells, which serve as a source of effective tumor antigens. With the combined effects of GM-CSF and TNFa, as well as release of a potential host of downstream inflammatory cytokines, dendritic cells migrate to the tumor site, become activated to effectively present melanoma antigen, and trigger T-cell activation. In addition, antigen-bound dendritic cells migrate to draining lymph nodes and further induce T cells to proliferate. The actions of IFNa potentially include enhanced infiltration of CD4+ T cells, as well as increased major histocompatibility complex class I expression [5,6]. The effects of our combination cytokine therapy are likely mediated by complex interactions of multiple downstream pathways that contribute to antigen processing and presentation, T-cell priming, trafficking, and proliferation (Fig. 6). In summary, we have shown that the combination of GM-CSF, IFNa, and TNFa inhibits melanoma growth in vivo and encompasses more than a localized intratumor effect by engaging a regional response, with enhanced lymphocyte numbers in lymph nodes
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