Katelyn T. Byrne

Sunlight triggers cutaneous lupus through a CSF-1-dependent mechanism in MRL-Faslpr mice

Sunlight (UVB) triggers cutaneous lupus erythematosus (CLE) and systemic lupus through an unknown mechanism. We tested the hypothesis that UVB triggers CLE through a CSF-1-dependent, macrophage (Mø)-mediated mechanism in MRL-Faslpr mice. By constructing mutant MRL-Faslpr strains expressing varying levels of CSF-1 (high, intermediate, none), and use of an ex vivo gene transfer to deliver CSF-1 intradermally, we determined that CSF-1 induces CLE in lupus-susceptible MRL-Faslpr mice, but not in lupus-resistant BALB/c mice. UVB incites an increase in Møs, apoptosis in the skin, and CLE in MRL-Faslpr, but not in CSF-1-deficient MRL-Faslpr mice. Furthermore, UVB did not induce CLE in BALB/c mice. Probing further, UVB stimulates CSF-1 expression by keratinocytes leading to recruitment and activation of Møs that, in turn, release mediators, which induce apoptosis in keratinocytes. Thus, sunlight triggers a CSF-1-dependent, Mø-mediated destructive inflammation in the skin leading to CLE in lupus-susceptible MRL-Faslpr but not lupus-resistant BALB/c mice. Taken together, CSF-1 is envisioned as the match and lupus susceptibility as the tinder leading to CLE.

Immune-Mediated Regression of Established B16F10 Melanoma by Intratumoral Injection of Attenuated Toxoplasma gondii Protects against Rechallenge

Immune recognition of tumors can limit cancer development, but antitumor immune responses are often blocked by tumor-mediated immunosuppression. Because microbes or microbial constituents are powerful adjuvants to stimulate immune responses, we evaluated whether intratumoral administration of a highly immunogenic but attenuated parasite could induce rejection of an established poorly immunogenic tumor. We treated intradermal B16F10 murine melanoma by intratumoral injection of an attenuated strain of Toxoplasma gondii (cps) that cannot replicate in vivo and therefore is not infective. The cps treatment stimulated a strong CD8+ T cell–mediated antitumor immune response in vivo that regressed established primary melanoma. The cps monotherapy rapidly modified the tumor microenvironment, halting tumor growth, and subsequently, as tumor-reactive T cells expanded, the tumors disappeared and rarely returned. The treatment required live cps that could invade cells and also required CD8+ T cells and NK cells, but did not require CD4+ T cells. Furthermore, we demonstrate that IL-12, IFN-γ, and the CXCR3-stimulating cytokines are required for full treatment efficacy. The treatment developed systemic antitumor immune activity as well as antitumor immune memory and therefore might have an impact against human metastatic disease. The approach is not specific for either B16F10 or melanoma. Direct intratumoral injection of cps has efficacy against an inducible genetic melanoma model and transplantable lung and ovarian tumors, demonstrating potential for broad clinical use. The combination of efficacy, systemic antitumor immune response, and complete attenuation with no observed host toxicity demonstrates the potential value of this novel cancer therapy.

Autoimmune melanocyte destruction is required for robust CD8+ memory T cell responses to mouse melanoma

A link between autoimmunity and improved antitumor immunity has long been recognized, although the exact mechanistic relationship between these two phenomena remains unclear. In the present study we have found that vitiligo, the autoimmune destruction of melanocytes, generates self antigen required for mounting persistent and protective memory CD8+ T cell responses to melanoma. Vitiligo developed in approximately 60% of mice that were depleted of regulatory CD4+ T cells and then subjected to surgical excision of large established B16 melanomas. Mice with vitiligo generated 10-fold larger populations of CD8+ memory T cells specific for shared melanoma/melanocyte antigens. CD8+ T cells in mice with vitiligo acquired phenotypic and functional characteristics of effector memory, suggesting that they were supported by ongoing antigen stimulation. Such responses were not generated in melanocyte-deficient mice, indicating a requirement for melanocyte destruction in maintaining CD8+ T cell immunity to melanoma. Vitiligo-associated memory CD8+ T cells provided durable tumor protection, were capable of mounting a rapid recall response to melanoma, and did not demonstrate phenotypic or functional signs of exhaustion even after many months of exposure to antigen. This work establishes melanocyte destruction as a key determinant of lasting melanoma-reactive immune responses, thus illustrating that immune-mediated destruction of normal tissues can perpetuate adaptive immune responses to cancer.

Resident memory T cells in the skin mediate durable immunity to melanoma

Resident memory CD8 T cells maintained in vitiligo-affected skin mediate long-lived protection against melanoma. Resident memory to cancer Melanoma patients with vitiligo are more likely to have a positive outcome, but the mechanism behind this association has remained unclear. Now, Malik et al. report that skin-resident memory T (TRM) cells specific to melanoma antigens are maintained in vitiligo-affected skin. These cells persist and function independently of the lymphoid compartment, suggesting that the vitiligo lesions provide a niche for the TRM cells. The TRM cells provide durable memory to the tumor, even in pigmented skin. These data suggest that skin TRM cells are critical for maintaining antitumor immunity. Tissue-resident memory T (TRM) cells have been widely characterized in infectious disease settings; however, their role in mediating immunity to cancer remains unknown. We report that skin-resident memory T cell responses to melanoma are generated naturally as a result of autoimmune vitiligo. Melanoma antigen–specific TRM cells resided predominantly in melanocyte-depleted hair follicles and were maintained without recirculation or replenishment from the lymphoid compartment. These cells expressed CD103, CD69, and CLA (cutaneous lymphocyte antigen), but lacked PD-1 (programmed cell death protein–1) or LAG-3 (lymphocyte activation gene–3), and were capable of making IFN-γ (interferon-γ). CD103 expression on CD8 T cells was required for the establishment of TRM cells in the skin but was dispensable for vitiligo development. CD103+ CD8 TRM cells were critical for protection against melanoma rechallenge. This work establishes that CD103-dependent TRM cells play a key role in perpetuating antitumor immunity.

BRAF Inhibition Alleviates Immune Suppression in Murine Autochthonous Melanoma

Steinberg and colleagues show that the BRAF-inhibitor PLX4720 enhanced intratumoral Treg apoptosis and decreased both the proportion and the per-cell immunosuppressive function of MDSCs, thus informing the design of combinatorial therapies for melanoma. A growing body of evidence suggests that BRAF inhibitors, in addition to their acute tumor growth–inhibitory effects, can also promote immune responses to melanoma. The present study aimed to define the immunologic basis of BRAF-inhibitor therapy using the Braf/Pten model of inducible, autochthonous melanoma on a pure C57BL/6 background. In the tumor microenvironment, BRAF inhibitor PLX4720 functioned by on-target mechanisms to selectively decrease both the proportions and absolute numbers of CD4+Foxp3+ regulatory T cells (Treg) and CD11b+Gr1+ myeloid-derived suppressor cells (MDSC), while preserving numbers of CD8+ effector T cells. In PLX4720-treated mice, the intratumoral Treg populations decreased significantly, demonstrating enhanced apopotosis. CD11b+ myeloid cells from PLX4720-treated tumors also exhibited decreased immunosuppressive function on a per-cell basis. In accordance with a reversion of tumor immune suppression, tumors that had been treated with PLX4720 grew with reduced kinetics after treatment was discontinued, and this growth delay was dependent on CD8 T cells. These findings demonstrate that BRAF inhibition selectively reverses two major mechanisms of immunosuppression in melanoma and liberates host-adaptive antitumor immunity. Cancer Immunol Res; 2(11); 1044–50. ©2014 AACR.

IL-10 Immunomodulation of Myeloid Cells Regulates a Murine Model of Ovarian Cancer

Elevated levels of IL-10 in the microenvironment of human ovarian cancer and murine models of ovarian cancer are well established and correlate with poor clinical prognosis. However, amongst a myriad of immunosuppressive factors, the actual contribution of IL-10 to the ovarian tumor microenvironment, the mechanisms by which it acts, and its possible functional redundancy are unknown. We previously demonstrated that elimination of the myeloid-derived suppressor cell (MDSC) compartment within the ovarian tumor ascites inhibited tumor progression and, intriguingly, significantly decreased local IL-10 levels. Here we identify a novel pathway in which the tumor-infiltrating MDSC are the predominant producers of IL-10 and, importantly, require it to develop their immunosuppressive function in vivo. Importantly, we demonstrate that the role of IL-10 is critical, and not redundant with other immunosuppressive molecules, to in vivo tumor progression: blockade of the IL-10 signaling network results in alleviation of MDSC-mediated immunosuppression, altered T cell phenotype and activity, and improved survival. These studies define IL-10 as a fundamental modulator of both MDSC and T cells within the ovarian tumor microenvironment. Importantly, IL-10 signaling is shown to be necessary to the development and maintenance of a permissive tumor microenvironment and represents a viable target for anti-tumor strategies.

Protective CD8 Memory T Cell Responses to Mouse Melanoma Are Generated in the Absence of CD4 T Cell Help

Background We have previously demonstrated that temporary depletion of CD4 T cells in mice with progressive B16 melanoma, followed by surgical tumor excision, induces protective memory CD8 T cell responses to melanoma/melanocyte antigens. We also showed that persistence of these CD8 T cells is supported, in an antigen-dependent fashion, by concurrent autoimmune melanocyte destruction. Herein we explore the requirement of CD4 T cell help in priming and maintaining this protective CD8 T cell response to melanoma. Methodology and Principal Findings To induce melanoma/melanocyte antigen-specific CD8 T cells, B16 tumor bearing mice were depleted of regulatory T cells (Treg) by either temporary, or long-term continuous treatment with anti-CD4 (mAb clone GK1.5). Total depletion of CD4 T cells led to significant priming of IFN-γ-producing CD8 T cell responses to TRP-2 and gp100. Surprisingly, treatment with anti-CD25 (mAb clone PC61), to specifically deplete Treg cells while leaving help intact, was ineffective at priming CD8 T cells. Thirty to sixty days after primary tumors were surgically excised, mice completely lacking CD4 T cell help developed autoimmune vitiligo, and maintained antigen-specific memory CD8 T cell responses that were highly effective at producing cytokines (IFN-γ, TNF-α, and IL-2). Mice lacking total CD4 T cell help also mounted protection against re-challenge with B16 melanoma sixty days after primary tumor excision. Conclusions and Significance This work establishes that CD4 T cell help is dispensable for the generation of protective memory T cell responses to melanoma. Our findings support further use of CD4 T cell depletion therapy for inducing long-lived immunity to cancer.

Autoimmune Vitiligo Does Not Require the Ongoing Priming of Naive CD8 T Cells for Disease Progression or Associated Protection against Melanoma

Vitiligo is a CD8 T cell–mediated autoimmune disease that has been shown to promote the longevity of memory T cell responses to melanoma. However, mechanisms whereby melanocyte/melanoma Ag-specific T cell responses are perpetuated in the context of vitiligo are not well understood. These studies investigate the possible phenomenon of naive T cell priming in hosts with melanoma-initiated, self-perpetuating, autoimmune vitiligo. Using naive pmel (gp10025–33–specific) transgenic CD8 T cells, we demonstrate that autoimmune melanocyte destruction induces naive T cell proliferation in skin-draining lymph nodes, in an Ag-dependent fashion. These pmel T cells upregulate expression of CD44, P-selectin ligand, and granzyme B. However, they do not downregulate CD62L, nor do they acquire the ability to produce IFN-γ, indicating a lack of functional priming. Accordingly, adult thymectomized mice exhibit no reduction in the severity or kinetics of depigmentation or long-lived protection against melanoma, indicating that the continual priming of naive T cells is not required for vitiligo or its associated antitumor immunity. Despite this, depletion of CD4 T cells during the course of vitiligo rescues the priming of naive pmel T cells that are capable of producing IFN-γ and persisting as memory, suggesting an ongoing and dominant mechanism of suppression by regulatory T cells. This work reveals the complex regulation of self-reactive CD8 T cells in vitiligo and demonstrates the overall poorly immunogenic nature of this autoimmune disease setting.

Abstract A36: Treatment of established dermal murine B16F10 melanoma with an attenuated Toxoplasma gondii eliminates the treated tumor and stimulates systemic antitumor immunity.

While the surgical removal of dermal melanoma cures that lesion, it does nothing to stimulate antitumor immunity that could potentially identify and eliminate occult metastatic disease. An immune based treatment that eliminates the primary dermal melanoma could also potentially generate systemic immunity that will protect against metastatic disease. We have utilized an attenuated strain of Toxoplasma gondii (cps) to break tumor-mediated immunosuppression, stimulate an antitumor immune response that eliminates the dermal tumor, and generate systemic antitumor immunity that leads to rejection of subsequent dermal or intravenous challenges with B16F10. T. gondii is an obligate intracellular eukaryotic parasite that infects virtually any mammalian species. The cps strain is a uracil auxotroph that can be grown in vitro but is unable to replicate in vivo. Despite its lack of infectivity, it enters cells and stimulates a strong T-cell mediated immune response characterized by long lasted CD8 effector cells. The presence of cps in the tumor microenvironment modifies the phenotype of tumor infiltrating leukocytes, and along with the expected anti-Toxoplasma immune response there is antigen-spreading so that tumor antigens are responded to and the immune system recognizes subsequent tumor challenges when there is no associated cps. The treatment requires CD8 and NK cells for efficacy but does not require CD4 cells. The treatment also requires that the host express IL-12 and Ifn-g. The treatment requires live cps for efficacy and is effective in mice that are latently infected with another strain of T. gondii, so it could function in the high percentage of humans with latent T. gondii infection and an established immune response against T. gondii. Citation Format: Steven Fiering, Jason R. Baird, Katelyn Byrne, Patrick Lizotte, David Mullins, Mary Jo turk. Treatment of established dermal murine B16F10 melanoma with an attenuated Toxoplasma gondii eliminates the treated tumor and stimulates systemic antitumor immunity. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A36.