The Neuropeptide Alpha-Melanocyte-Stimulating Hormone Is Critically Involved in the Development of Cytotoxic CD8+ T Cells in Mice and Humans

BACKGROUND: The neuropeptide alpha-melanocyte-stimulating hormone is well known as a mediator of skin pigmentation. More recently, it has been shown that alpha-melanocyte-stimulating hormone also plays pivotal roles in energy homeostasis, sexual function, and inflammation or immunomodulation. Alpha-melanocyte-stimulating hormone exerts its antiinflammatory and immunomodulatory effects by binding to the melanocortin-1 receptor, and since T cells are important effectors during immune responses, we investigated the effects of alpha-melanocyte-stimulating hormone on T cell function. METHODOLOGY/PRINCIPAL FINDINGS: T cells were treated with alpha-melanocyte-stimulating hormone, and subsequently, their phenotype and function was analyzed in a contact allergy as well as a melanoma model. Furthermore, the relevance of alpha-melanocyte-stimulating hormone-mediated signaling for the induction of cytotoxicity was assessed in CD8(+) T cells from melanoma patients with functional and nonfunctional melanocortin-1 receptors. Here we demonstrate that the melanocortin-1 receptor is expressed by murine as well as human CD8(+) T cells, and we furthermore show that alpha-melanocyte-stimulating hormone/melanocortin-1 receptor-mediated signaling is critical for the induction of cytotoxicity in human and murine CD8(+) T cells. Upon adoptive transfer, alpha-melanocyte-stimulating hormone-treated murine CD8(+) T cells significantly reduced contact allergy responses in recipient mice. Additionally, the presented data indicate that alpha-melanocyte-stimulating hormone via signaling through a functional melanocortin-1 receptor augmented antitumoral immunity by up-regulating the expression of cytotoxic genes and enhancing the cytolytic activity in tumor-specific CD8(+) T cells. CONCLUSIONS/SIGNIFICANCE: Together, these results point to an important role of alpha-melanocyte-stimulating hormone in MHC class I-restricted cytotoxicity. Therefore, treatment of contact allergies or skin cancer with alpha-melanocyte-stimulating hormone or other more stable agonists of melanocortin-1 receptor might ameliorate disease or improve antitumoral immune responses

Alpha-MSH/MC-1R signaling induces cytotoxicity in CD8⁺ T cells.

<p>(<b>A</b>) CD8⁺ T cells were isolated from wt or C57BL/6J^e/e mice and stimulated with PBS or α-MSH. Subsequently, real-time PCR analyses were performed and the relative mRNA expression levels of granzyme A, granzyme B, perforin, CTLA-4, and Fas in α-MSH stimulated CD8⁺ T cells vs. PBS treated controls are shown (gene expression in PBS treated CD8⁺ T cells  = 1). * <i>P</i><0.05 vs. PBS stimulated CD8⁺ T cells. (<b>B</b>) Increased cytolytic activity in α-MSH stimulated CD8⁺ T cells. CD8⁺ T cells were purified from wt or C57BL/6J^e/e mice and stimulated with PBS or α-MSH. Subsequently, the cytotoxic activity of CD8⁺ T cells was analyzed 6 h after co-culture with chromium-loaded L1210/3 target cells. One representative out of three independent experiments is shown. * <i>P</i><0.05 vs. PBS stimulated CD8⁺ T cells.</p

Alpha-MSH/MC-1R signaling is involved in the induction of cytotoxic activity in human CD8⁺ T cells.

<p>(<b>A</b>) MC-1R is expressed in human CD8⁺ T cells. RT- PCR of purified CD8⁺ T cells from two healthy donors after stimulation with α-MSH or PBS. (<b>B</b>) CD8⁺ T cells from healthy donors with skin type 1 or skin type 2/3 as well as melanoma patients with skin type 1 or skin type 2/3 were stimulated with PBS or α-MSH and subjected to real-time PCR analyses. Relative mRNA expression levels of granzyme B, perforin, CTLA-4, and Fas in α-MSH stimulated CD8⁺ T cells and PBS treated controls from 6–12 patients in each group are shown.</p

Alpha-MSH is not secreted by B16-OVA melanoma cells or up-regulated in mice bearing B16-OVA tumors.

<p>(<b>A</b>) Alpha-MSH was quantified in culture supernatants from B16-OVA melanoma cells at indicated time points by radioimmunoassays. (<b>B</b>) Alpha-MSH was quantified in the serum as well as in tumor lysates from tumor-bearing wt and C57BL/6J^e/e mice 21 days after subcutaneous injection of 100,000 B16-OVA melanoma cells (n = 5 mice each group).</p

Reduced tumor growth in mice injected with α-MSH treated CD8⁺ T cells.

<p>(<b>A</b>) Wt mice were inoculated with 1×10⁵ B16-OVA cells and injected with 5×10⁶ PBS or α-MSH stimulated CD8⁺ T cells from OT-1 mice at d8 and d10 after inoculation of tumor cells. Tumor growth was monitored over time in n = 18 mice in three independent experiments. * <i>P</i><0.05 vs. transfer of PBS stimulated CD8⁺ T cells. (<b>B</b>) Immunofluorescence staining of tumor tissue at d22 after inoculation of tumor cells using antibodies directed against CD8 and granzyme B. Original magnification: 400×, scale bar  = 25 µm. Cells expressing CD8 and granzyme B are marked by arrows. (<b>C</b>) MC-1R signaling is important for MHC class I-restricted anti-tumoral immunity. Wt and C57BL/6J^e/e mice were inoculated with indicated concentrations of B16 cells and tumor growth was monitored over time. One representative out of three independent experiments is shown. * <i>P</i><0.05 vs. wt. (<b>D</b>) Immunofluorescence staining of contralateral and tumor-draining lymph nodes from wt mice (i and ii) as well as C57BL/6J^e/e mice (iii and iv) at d20 after inoculation of 25,000 B16 cells using antibodies directed against CD8 and granzyme B. Original magnification: 300×, scale bar  = 25 µm. Cells expressing CD8 and granzyme B are marked by arrows.</p

Alpha-MSH stimulated CD8⁺ T cells from wt mice suppress contact allergy responses.

<p>(<b>A</b>) Twenty-four h before challenge, DNFB-sensitized recipients were injected intravenously with 5×10⁶ CD8⁺ T cells from wt donors that had been stimulated with PBS or α-MSH and co-cultured with DNBS-pulsed DC prior to adoptive cell transfer. Data are shown as mean ear swelling ± SD and are representative of 15 mice in three independent experiments. * <i>P</i><0.05 vs. transfer of PBS stimulated CD8⁺ T cells. (<b>B</b>) Lymphocyte infiltrations and apoptotic cell death were determined by H&E (original magnification: 200×, scale bar  = 25 µm) or immunofluorescence staining of ear skin (original magnification: 300×, scale bar  = 25 µm). (<b>C</b>) MC-1R mediated signaling in CD8⁺ T cells is required for the suppression of contact allergy. Twenty-four h before challenge, recipients were injected intravenously with 5×10⁶ PBS or α-MSH stimulated CD8⁺ T cells from C57BL/6J^e/e mice. Data are shown as mean ear swelling ± SD and are representative of 10 mice.</p

MC-1R is expressed on murine CD8⁺ T cells and up-regulated after stimulation with α-MSH.

<p>(<b>A</b>) RT- PCR analysis of CD4⁺ and CD8⁺ T cells after stimulation with α-MSH or PBS. (<b>B</b>) FACS analyses of CD8⁺ and CD4⁺ T cells from wt mice (total number of animals analyzed n = 15) after stimulation of cells with PBS or α-MSH. Representative dot blots are shown for each experimental group and cells are gated for CD8 and CD4, respectively.</p