[Show abstract][Hide abstract] ABSTRACT: Previously, we showed that nine intradermal injections of a plasmid in which the HSVtk suicide gene is expressed from a melanocyte-specific promoter (Tyr-HSVtk), combined with a plasmid expressing heat shock protein 70 (CMV-hsp70), along with systemic ganciclovir, kills normal melanocytes and raises a CD8+ T cell response that is potent enough to eradicate small, 3-day established B16 tumors. We show in this study that, in that regimen, hsp70 acts as a potent immune adjuvant through TLR-4 signaling and local induction of TNF-alpha. hsp70 is required for migration of APC resident in the skin to the draining lymph nodes to present Ags, derived from the killing of normal melanocytes, to naive T cells. The addition of a plasmid expressing CD40L increased therapeutic efficacy, such that only six plasmid injections were now required to cure large, 9-day established tumors. Generation of potent immunological memory against rechallenge in cured mice accompanied these therapeutic gains, as did induction of aggressive autoimmune symptoms. Expression of CD40L, along with hsp70, increased both the frequency and activity of T cells activated against melanocyte-derived Ags. In this way, addition of CD40L to the hsp70-induced inflammatory killing of melanocytes can be used to cure large established tumors and to confer immunological memory against tumor cells, although a concomitant increase in autoimmune sequelae also is produced.
The Journal of Immunology 10/2006; 177(6):4168-77. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have shown that inflammatory killing of normal melanocytes by intradermal injection of plasmids expressing HSVtk from a melanocyte specific promoter, along with hsp70 from a CMV promoter, cures small established B16 melanomas. However, destruction of normal cells is also accompanied by induction of regulatory T cells which, through a TGF-β-dependent mechanism, restrain the activity of CD8+ T cells specific for melanocyte/ melanoma associated antigens, restrict induction of autoimmunity and inhibit memory against melanoma re-challenge. Hsp70 is critical for this effect in vivo. We have now shown that hsp70 expression locally at the site of melanocyte killing induces TNF-α which is associated with migration of ClassII+ve/CD11c+ cells to the draining lymph nodes. These CD11c+ve cells can directly activate naive T cells against melanocyte expressed antigens. The ability of these cells to prime T cell responses against melanocyte expressed antigens is lost in TNF-α-/- mice. Moreover, therapy is lost in TLR4 knockout mice indicating that hsp70 acts through stimulation of Toll Like Receptors on cells locally resident in the skin. When a plasmid expressing CD40L was co-expressed with Ty r- HSVtk+hsp70, this regimen was able to cure large, 9 day established tumors with the same number of plasmid injections. CD40L plasmid, along with melanocyte killing, increased the frequency (by tetramer staining) and potency (IFN-γ ELISPOT) of antigen specific T cells compared to the use of Tyr-HSVtk and hsp70 alone and induces significant splenomegaly in treated mice. Moreover, animals treated with inflammatory melanocyte killing+pCD40L developed aggressive autoimmune vitiligo and alopecia, and cured mice developed immunological memory against B16 rechallenge. CD40L plasmid caused depletion of CD4+CD25+ Treg cells from the spleens and LN of mice 8 days following the first of three id injections, indicating a novel mechanistic link between the magnitude of anti melanoma T cell responses, CD40 ligation and Treg activity/survival. Finally, we have now extended these studies to show that the inflammatory killing of normal prostate tissue in vivo by a single dose of adenoviral- mediated gene delivery can also cure established TrampC2 tumors seeded subcutaneously in immunocompetent mice. Therapy was associated with weight reductions of about 50% of prostates from cured mice, indicating induction of a potent anti-prostate immune reaction. Taken together, our data show that by manipulating in vivo mechanisms which normally restrain autoimmune responses, increased anti-tumor responses can be induced following the killing of normal cells. However, such strategies also run the risk of increasing autoimmune related side effects.
[Show abstract][Hide abstract] ABSTRACT: We have reported that i.d. injection of plasmids encoding hsp70 and a suicide gene transcriptionally targeted to melanocytes generates specific proinflammatory killing of melanocytes. The resulting CD8+ T cell response eradicates systemically established B16 tumors. Here, we studied the consequences of that CD8+ T cell response on the phenotype of preexisting tumor. In suboptimal protocols, the T cell response selected B16 variants, which grow extremely aggressively, are amelanotic and have lost expression of the tyrosinase and tyrosinase-related protein 2 (TRP-2) antigens. However, expression of other melanoma-associated antigens, such as gp100, was not affected. Antigen loss could be reversed by long-term growth in culture away from immune-selective pressures or within 96 hours by treatment with the demethylating agent 5-azacytidine (5-Aza). When transplanted back into syngeneic animals, variants were very poorly controlled by further vaccination. However, a combination of vaccination with 5-Aza to reactivate antigen expression in tumors in situ generated highly significant improvements in therapy over treatment with vaccine or 5-Aza alone. These data show that inflammatory killing of normal cells activates a potent T cell response targeted against a specific subset of self-antigens but can also lead to the immunoselection of tumor variants. Moreover, our data indicate that emergence of antigen loss variants may often be due to reversible epigenetic mechanisms within the tumor cells. Therefore, combination therapy using vaccination and systemic treatment with 5-Aza or other demethylating agents may have significant therapeutic benefits for antitumor immunotherapy.
Cancer Research 04/2005; 65(5):2009-17. · 8.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We hypothesized that it might be possible to use intentional pathological-like killing of normal cells (melanocytes) to generate immune responses against tumor cells (melanoma). Recently, we demonstrated that i.d. injections of two plasmids, in which the HSVtk suicide gene is transcriptionally targeted to melanocytes (Tyr-HSVtk) and the hsp70 protein is expressed from the CMV promoter, generated localized killing of normal melanocytes within a highly inflammatory environment. This primed a CD8 T cell response which cleared 6 day established tumors. However, it was also rapidly suppressed in vivo by a population of CD25 regulatory T cells which prevented autoimmune vitiligo in tumor cured mice. We have now investigated additional interventions by which the potency of the inflammatory killing of melanocytes may be enhanced. Co-injection of a plasmid expressing CD40L led to dramatically enhanced immunotherapeutic efficacy against B16 tumors (cure of >80% of mice bearing 9 day established B16 tumors) where suboptimal treatments with Tyr-HSVtk and CMV-hsp70 alone were ineffective. pCD40L increased the number of tetramer positive, antigen specific T cells directed against a melanocyte specific antigen in the LN. CD40L also led to the generation of potent immune memory in tumor cured mice (90% of rechallenged animals protected long term), and autoimmunity, effects that were not observed in mice cured of smaller tumors by Tyr-HSVtk+CMX//Q-hsp70. Interestingly, local expression of CD40L caused the disappearance of putative regulatory T cells from both the LN and the spleen within 8 days of the first plasmid injection, compatible with the ability to clear large established tumors, induction of memory and autoimmunity. Thus, inflammatory killing of normal cell types in vivo represents a novel way to induce autoimmune T cell reactivity which is cross reactive to tumors bearing shared SELF antigens. By manipulating the normal in vivo mechanisms which restrain these responses, increased anti-tumor responses can be induced but at the risk of also increasing autoimmune related side effects. Therefore, we also studied other ways to augment the T cell response without increasing autoimmune sequelae. In the non curative vaccination protocol, the CD8 T cell response leads to the immunoselection of tumor cell variants which have lost expression of the TRP-2 and tyrosinase melanocyte/melanoma antigens. Antigen expression could be re-induced in tumors by treatment with 5-Azacytidine and vaccination plus systemic 5-Azacytidine generated therapy against antigen loss variants which were otherwise poorly treated by further rounds of vaccination alone. These data show that protocols aimed at re-inducing/maintaining expression of tumor associated antigens may enhance the value of successful vaccination strategies which fail in part because of the emergence of antigen loss variants.
[Show abstract][Hide abstract] ABSTRACT: We describe a simple technology used to cure an established metastatic disease. Intradermal injection of plasmid DNA encoding a transcriptionally targeted cytotoxic gene, along with hsp70, not only promoted tissue-specific, inflammatory killing of normal melanocytes, but also induced a CD8(+) T-cell-dependent, antigen-specific response in mice that eradicated systemically established B16 tumors. This CD8(+) T cell response was subsequently suppressed in vivo within a few days. The data demonstrate that deliberate destruction of normal tissue can be exploited to generate immunity against a malignant disease originating from that tissue. This approach obviates the need to identify tumor antigens and does not require complex isolation of tumor cells or their derivatives. In addition, it provides a model system for studying the mechanisms underlying the etiology and control of autoimmune diseases. Finally, despite targeting normal tissue, therapy could be separated from development of overt autoimmune symptoms, suggesting that the strategy may be valuable against tumors derived from both non-essential and essential tissue types.
[Show abstract][Hide abstract] ABSTRACT: Most tumor vaccination strategies depend upon the molecular identification of specific tumor rejection antigens and/or the use of patient derived tumor materials. In addition, there is a strong correlation between the use of cancer vaccines and the induction of autoimmune reactivity. We hypothesised that in situ, inflammatory killing of a normal tissue from which a tumor derives may generate autoimmune reactivity to self antigens expressed in that tissue as well as in the tumor cells. Here we report that simple intradermal injection of 2 plasmids promoted tissue specific, inflammatory killing of melanocytes and induced an immune response that eradicated systemically established B16 tumors. The therapeutic response was CD8+T cell dependent but, significantly, was rapidly suppressed in vivo and did not induce autoimmune disease in animals in which tumors had been cured. The T cell response was characterized by selection of B16 tumor variants which grow extremely aggressively in vivo, are amelanotic and which have lost expression of the tyrosinase and TRP-2 antigens. The mechanism of antigen loss was, at least in part, through epigenetic mechanisms. These could be reversed by long term growth in culture or, more rapidly, by treatment with the de-methylating agent 5-azacytidine. Depletion of putative regulatory T cells within the CD25+ T cell population improved therapeutic efficacy. Finally, vaccination could be further improved using adoptive transfer of activated T cells with specificity for an antigen that was not a target of the initial T cell response raised by melanocyte killing. These data demonstrate that inflammatory killing of a normal tissue can lead to the immunoselection of tumor cell growth variants. They also raise the prospect that such vaccinations could be effectively used in combination with other conventional anti tumor treatment modalities.This is the first time that deliberate destruction of normal tissue has been used to generate an effective immune response against malignant disease. The technique circumvents the need to identify specific rejection antigens associated with a tumor and does not necessitate the recovery and preparation of tumor cells or its derivatives. It also offers a generalized approach to other malignancies involving non-essential tissues. Finally, despite targeting normal tissue, therapy could be separated from autoimmunity. This offers the possibility of similar strategies against tumors deriving from essential tissues.