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

Cutaneous RANK–RANKL Signaling Upregulates CD8-Mediated Antiviral Immunity during Herpes simplex Virus Infection by Preventing Virus-Induced Langerhans Cell Apoptosis

Herpes simplex virus-type 1 (HSV-1) causes the majority of cutaneous viral infections. Viral infections are controlled by the immune system, and CD8+ cytotoxic T-lymphocytes (CTLs) have been shown to be crucial during the clearance of HSV-1 infections. Although epidermal Langerhans cells (LCs) are the first dendritic cells (DCs) to come into contact with the virus, it has been shown that the processing of viral antigens and the differentiation of antiviral CTLs are mediated by migratory CD103+ dermal DCs and CD8α+ lymph node–resident DCs. In vivo regulatory T-cells (Tregs) are implicated in the regulation of antiviral immunity and we have shown that signaling via the receptor activator of NF-κB (RANK) and its ligand RANKL mediates the peripheral expansion of Tregs. However, in addition to expanding Tregs, RANK–RANKL interactions are involved in the control of antimicrobial immunity by upregulating the priming of CD4+ effector T cells in LCMV infection or by the generation of parasite-specific CD8+ T cells in Trypanosoma cruzi infection. Here, we demonstrate that cutaneous RANK–RANKL signaling is critical for the induction of CD8-mediated antiviral immune responses during HSV-1 infection of the skin by preventing virus-induced LC apoptosis, improving antigen transport to regional lymph nodes, and increasing the CTL priming capacity of lymph node DCs

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 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

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

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

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

Alpha-MSH does not influence MHC I expression in CD8⁺ T cells or the capacity to recognize antigens.

<p>(<b>A</b>) Flow cytometric analysis of CD8⁺ T cells from wt or C57BL/6J^e/e mice (n = 5 mice each group) after stimulation with α-MSH (10⁻⁹ molar) for 48 h or 96 h. Cells were gated for CD8 and MHC class I expression is shown in a representative histogram overlay. (<b>B</b>) PBS and α-MSH stimulated CD8⁺ T cells from OT-1 mice equally recognize the relevant antigen presented by DC. BmDC were pulsed with SIINFEKL or HSV-1 gB peptides and co-cultured with PBS or α-MSH stimulated CD8⁺ T cells from OT-1 mice for 3 days (DC/T cell ratio  = 1/40). Subsequently, T cell proliferation was assessed by ³H-thymidine incorporation. One representative experiment out of three is shown.</p