Photodynamic Therapy Enhancement of Anti-Tumor Immunity

Abstract

Photodynamic therapy (PDT) is an FDA-approved modality for the treatment of early-stage disease and palliation of late-stage disease. Pre-clinical studies using mouse models and clinical studies in patients have demonstrated that PDT is capable of influencing the immune system. The effect of PDT on the generation of anti-tumor immunity is regimen-dependent and is tightly linked to the degree and nature of inflammation induced by PDT. However, the precise mechanism underlying PDT regulated adaptive anti-tumor immunity remains unclear. This review will focus on the current knowledge of immune regulation by PDT.

PDT Efficacy is Dependent upon Anti-Tumor Immunity

Photodynamic therapy (PDT) is an anti-tumor modality that rapidly destroys tumors by a multi-faceted mechanism. The efficacy of PDT has been shown to be dependent upon the induction of anti-tumor immunity. Initial studies in mice models demonstrated that long-term tumor control is diminished in immuno-compromised mice^1,2. PDT treatment of EMT6 tumors at a curative dose in BALB/c mice exhibited no long-term protection of tumors in either scid, which lack T and B cells, or nude mice, which lack T cells. The curative effect of PDT on EMT6 tumors was restored when scid mice were reconstituted with splenic T cells or bone marrow cells from BALB/c mice.

The dependence of PDT efficacy on the induction of anti-tumor immunity has also been demonstrated in clinical studies. Topical PDT of immuno-suppressed actinic keratoses and Bowen’s disease transplant patients had comparable initial response with those in immuno-competent patients ³. However, immuno-suppressed patients had an increased propensity to develop new lesions soon after treatment. Patient vulval intraepithelial neoplasia (VIN) biopsies expressing major histocompatibility class I molecules (MHC I) were more likely to respond to ALA-PDT than tumors that had down-regulated MHC I molecules ⁴. Recognition of MHC I is critical for CD8⁺ T cell activation and one mechanism used by tumors to evade immune recognition is down-regulation of MHC I molecules ⁵. The infiltration of CD8⁺ T cells was significantly increased in VIN responders compared with nonresponders following PDT.

PDT Augments Anti-Tumor Immunity

The induction of anti-tumor immunity by PDT was first established by Canti et al.⁶ who demonstrated that transfer of TDLN cells from PDT treated tumors to naïve hosts conferred resistance upon subsequent tumor challenge. In this study, PDT-treated mice that remained tumor free for 100 days were able to effectively control subsequent tumor challenge suggesting the presence of immune memory. Korbelik and Dougherty ⁷ later demonstrated that PDT treatment of murine tumors resulted in the generation of immune memory. Reconstitution of scid mice with PDT-cured splenocytes protected the mice against tumor rechallenge. Recent reports have demonstrated that clinical PDT also augments anti-tumor immunity. PDT treatment of basal cell carcinoma (BCC) increased systemic anti-tumor immunity to a BCC-associated tumor antigen ⁸. A recent study demonstrated that treatment of multifocal angiosarcoma of the head and neck with PDT resulted in increased immune cell reactivity against distant untreated tumors that correlated with tumor regression ⁹.

Although PDT can activate both humoral and cell-mediated adaptive anti-tumor immunity ¹⁰, the importance of the humoral arm of anti-tumor immunity remains unclear. The efficacy of PDT in mice and humans is dependent upon CD8⁺ T cells ^2,4,11. Therefore most mechanistic studies have focused on PDT modulation of cell-mediated adaptive immunity.

Mechanism of PDT Enhanced Anti-Tumor Immunity

A hallmark of PDT is the induction of acute inflammation that contributes considerably to the overall long-term control of tumor growth ¹². High-inflammatory PDT regimens induce significant acute inflammation characterized by increased expression of several pro-inflammatory cytokines including TNF-α, IL-1β, and IL-6 ^13–16, adhesion molecules E-selectin and ICAM-1 ¹⁶, and rapid accumulation of leukocytes into the treated tumor bed ^1,7,17 and TDLNs ¹⁸. The accumulation of leukocytes to the tumor is dominated by Gr-1 expressing leukocytes that morphologically resemble neutrophils. Depletion of Gr-1-expressing leukocytes resulted in diminished long-term tumor growth by PDT ^2,12,17,19.

Studies in pre-clinical and clinical settings have demonstrated that the degree of inflammation influences the anti-tumor adaptive immune response. PDT was initially considered a local treatment that generated a local inflammatory response. Subsequent studies have identified PDT as a systemic response characterized by increased systemic neutrophilia ²⁰, systemic release of pro-inflammatory cytokines ^16,21–25, induction of acute phase proteins ^16,20, and increased complement protein expression ^26–28. These acute inflammatory mediators augment the presence and activation of innate and adaptive immunity and the generation of anti-tumor immunity.

PDT enhancement of anti-tumor immunity appears to involve the stimulation of DCs by dying tumor cells ^10,29. Induction of acute inflammation by PDT results in the maturation and activation of dendritic cells ³⁰ and migration to the TDLNs where they are believed to stimulate T cell activation ^30,31. Studies have shown that incubation of PDT-treated tumor cells with immature DCs leads to enhanced DC maturation, activation, and ability to stimulate T cell activation ^29,32. A recent report demonstrated that PDT-generated tumor cell lysate induces IL-1α, IL-1β, and IL-6 secretion from DCs, suggesting PDT enhanced anti-tumor immunity is due in part to increased DC activation ³³.

It is thought that DC activation by PDT is the result of sensing endogenous danger signals released by dying tumor cells ^30,34–37. The danger model was initially proposed by Matzinger ³⁸ and suggests that cells express endogenous danger signals in response to damage or physical/chemical insult/stress. These danger signals are referred to as damage-associated molecular patterns (DAMPs) that are immunostimulatory by interacting with pattern-recognition receptors (PRRs) expressed on innate immune cells. These specialized receptors are non-clonal, germline-encoded molecules classified as RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), and Toll-like receptors (TLRs) ³⁹. It appears that PDT treatment of tumor cells induces expression of multiple danger signals capable of activating antigen presenting cells and generating anti-tumor immunity. Heat shock protein 70 (HSP70) is a danger signal that interacts with TLRs (Toll-like receptors) 2 and 4 ⁴⁰. PDT induces expression of HSP70 ⁴¹ and the level of expression correlates with DC maturation ⁴² and the extent of inflammation ³⁶. Other HSPs and danger signals released by PDT treated tumor cells have been reported to stimulate DC activation ^29,43. Additionally, complement opsonization of PDT-treated tumor cells augments immune recognition of PDT-generated vaccines ⁴⁴.

PDT regimens that lead to acute inflammation induce a rapid influx of Gr-1^hiCD11b⁺F4/80⁻CD11c⁻ neutrophils into treated tumors leading to enhanced anti-tumor immunity ^12,18 and strong primary and memory T cell responses ¹⁸. Furthermore, the efficacy of PDT is critically dependent on Gr-1 expressing leukocytes ^12,19. Mice defective in neutrophil homing (Cxcr2^−/−) to secondary lymphoid tissue lack the ability to mount strong CD8⁺ T cell responses following PDT ¹⁸. We therefore postulate that neutrophils are instrumental in modulating anti-tumor immunity following PDT. The mechanism by which neutrophils instruct adaptive anti-tumor immunity is an area of extensive investigation. These leukocytes have been shown to regulate anti-pathogen immunity through (1) secretion of chemokines and granule proteins that recruit monocytes and dendritic cells ⁴⁵ (2) activation of DCs via cell-to-cell contact and secretion of TNF-α ^46,47 (3) secretion of IFN-γ that stimulates monocytes and T cell differentiation ^45,48. We have previously demonstrated that neutrophils express cell-surface TNF-α following PDT ¹⁸. Neutrophils rapidly accumulate within the treated tumor bed and peak between 4 and 8 hours post treatment. These leukocytes acquire cell-surface expression of TNF-α and accumulate in the TDLN by 4h post treatment. We predict that these TNF-α expressing leukocytes directly interact with DCs in the TDLN, licensing them and promoting T cell activation and increased adaptive anti-tumor immunity. Generation of CD8⁺ effector and memory T cell formation is generally dependent on CD4⁺ T cells ^49–51. However, PDT-induced anti-tumor immunity may be CD4⁺ T cell independent and augmented by NK cells ⁵². Furthermore, Korbelik et al. ¹ demonstrated that transfer of CD8⁺ T cells alone could significantly restore PDT efficacy in SCID mice.

In addition to directly augmenting anti-tumor immunity by enhanced DC and T cell activation, PDT can also affect tumor-derived immuno-suppression. Immunosuppressive T_regulatory cells (T_regs) dampen the generation of anti-tumor immunity ^53,54. A recent study demonstrated that PDT in combination with low-dose cyclophosphamide depletes T_regs, potentiating the immune response leading to increased cures and the generation of immune memory ⁵⁵. Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells recruited by the tumor and suppress adaptive anti-tumor immunity ⁵⁶. Although the role of MDSCs in PDT has not been reported, it is tempting to speculate that PDT causes the destruction of MDSCs through a similar mechanism of tumor destruction initiated by PDT. Myeloid cells are extremely phagocytic, capable of phagocytosing photosensitizer and upon tumor illumination become apoptotic and necrotic. One could predict that PDT-initiated MDSC destruction may potentiate anti-tumor immunity.

The effects of PDT on the immune system appear not only to augment immune cell reactivity against tumors but also to suppress immune cell activation. Although the precise mechanism of immuno-suppression is unclear, the effect of PDT on the immune system appears dependent on the treatment regimen, the photosensitizer type, and the treatment area ^18,57. PDT induced immune suppression of cutaneous and transdermal PDT involves a large surface area ^57–59. Immune suppression generated by PDT can be adoptively transferred to naïve recipients and is mediated primarily by T cells ⁶⁰.

Conclusion

PDT is an effective therapy for a growing number of malignancies in the US and Europe. Pre-clinical and clinical studies have demonstrated that PDT eliminates tumors by direct tumor cell death and indirectly by augmenting anti-tumor immunity. The mechanism underlying PDT enhancement of anti-tumor immunity remains unclear and is under current investigation. Defining the mechanism by which PDT augments anti-tumor immunity will allow the development of protocols to eliminate primary tumor growth while augmenting systemic anti-tumor immunity to control disseminating disease. We hope to develop protocols to use PDT as an adjuvant in combination with other treatment modalities. PDT was shown to be an effective adjuvant in combination with surgery in patients with non-small-cell lung caner with plural spread ⁶¹. A recent report using a pre-clinical mouse model of reticulum cell sarcoma demonstrated that PDT can be effectively combined with low-dose cyclophosphamide to generate durable anti-tumor immunity while augmenting long-term cures ⁵⁵. PDT is a promising anti-tumor modality that is receiving considerable attention as a therapeutic tool alone and in combination with other treatment regimens.

Figure 1.