Tumor necrosis factor-alpha (TNFalpha) and nitric oxide (NO), the product of inducible NO synthase (iNOS), mediate inflammatory and immune responses in the CNS under a variety of neuropathological situations. They are produced mainly by "activated" astrocytes and microglia, the two immune regulatory cells of the CNS. In this study we have examined the regulation of TNFalpha and iNOS gene expression in endotoxin-stimulated primary glial cultures, focusing on the role of mitogen-activated protein (MAP) kinase cascades. The bacterial lipopolysaccharide (LPS) was able to activate extracellular signal-regulated kinase (ERK) and p38 kinase subgroups of MAP kinases in microglia and astrocytes. ERK activation was sensitive to PD98059, the kinase inhibitor that is specific for ERK kinase. The activity of p38 kinase was inhibited by SB203580, a member of the novel class of cytokine suppressive anti-inflammatory drugs (CSAIDs), as revealed by blocked activation of the downstream kinase, MAP kinase-activated protein kinase-2. The treatment of glial cells with either LPS alone (microglia) or a combination of LPS and interferon-gamma (astrocytes) resulted in an induced production of NO and TNFalpha. The two kinase inhibitors, at micromolar concentrations, individually suppressed and, in combination, almost completely blocked glial production of NO and the expression of iNOS and TNFalpha, as determined by Western blot analysis. Reverse transcriptase-PCR analysis showed changes in iNOS mRNA levels that paralleled iNOS protein and NO while indicating a lack of effect of either of the kinase inhibitors on TNFalpha mRNA expression. The results demonstrate key roles for ERK and p38 MAP kinase cascades in the transcriptional and post-transcriptional regulation of iNOS and TNFalpha gene expression in endotoxin-activated glial cells.

Tissue-resident memory T cells (TRM cells) have been widely characterized in infectious disease settings; however, their role in mediating immunity to cancer remains unknown. Here 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, but lacked PD-1 or LAG-3, and were capable of making IFN-γ. CD103 expression on CD8 T cells was required for establishment of TRM cells in skin, but was dispensable for vitiligo development. Importantly, CD103+ CD8 TRM cells were critical for protection against melanoma re-challenge. This work establishes that CD103-dependent TRM cells play a key role in perpetuating anti-tumor immunity.

Abstract:Oxidative stress is known to induce cell death in a wide variety of cell types, apparently by modulating intracellular signaling pathways. In this study, we have examined the activation of mitogen-activated protein kinase (MAPK) cascades in relation to oxidant-induced cell death in an oligodendrocyte cell line, central glia-4 (CG4). Exposure of CG4 cells to hydrogen peroxide (H 2 O 2 ) resulted in an increased tyrosine phosphorylation of several protein species, including the abundantly expressed platelet-derived growth factor (PDGF) receptor and the activation of the three MAPK subgroups, i.e., extracellular signal-regulated kinase (ERK), p38 MAPK, and c-Jun Nterminal kinase (JNK). Dose-response studies showed differential sensitivities of PDGF receptor phosphorylation (Ͼ1 mM) and ERK/p38 MAPK (Ͼ0.5 mM) and JNK (Ͼ0.1 mM) activation by H 2 O 2 . The activation of ERK was inhibited by PD98059, a specific inhibitor of the upstream kinase, MAPK or ERK kinase (MEK). H 2 O 2 also activated MAPK-activated protein kinase-2, and this activation was blocked by SB203580, a specific inhibitor of p38 MAPK. The oxidant-induced cell death was indicated by morphological changes, decreased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction, and DNA fragmentation. These effects were suppressed dose-dependently by the MEK inhibitor PD98059. The results demonstrate that H 2 O 2 induces the activation of multiple MAPKs in oligodendrocyte progenitors and that the activation of ERK is associated with oxidant-mediated cytotoxicity.

Combinatorial use of iron oxide nanoparticles (IONPs) and an alternating magnetic filed (AMF) can induce local hyperthermia in tumors in a controlled and uniform manner. Heating B16 primary tumors at 43°C for 30 minutes activated dendritic cells (DCs) and subsequently CD8+ T cells in the draining lymph node (dLN) and conferred resistance against rechallenge with B16 (but not unrelated Lewis Lung carcinoma) given 7 days post hyperthermia on both the primary tumor side and the contralateral side in a CD8+ T cell-dependent manner. Mice with heated primary tumors also resisted rechallenge given 30 days post hyperthermia. Mice with larger heated primary tumors had greater resistance to secondary tumors. No rechallenge resistance occurred when tumors were heated at 45°C. Our results demonstrate the promising potential of local hyperthermia treatment applied to identified tumors in inducing anti-tumor immune responses that reduce the risk of recurrence and metastasis.

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.

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The generation of protective CD8

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