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The absence of tumor necrosis factor (TNF) causes lethal infection by Leishmania major in normally resistant C57BL/6J (B6.WT) mice. The underlying pathogenic mechanism of this fatal disease has so far remained elusive. We found that B6.WT mice deficient for the tnf gene (B6.TNF−/−) displayed not only a non-healing cutaneous lesion but also a serious infection of the liver upon L. major inoculation. Infected B6.TNF−/− mice developed an enlarged liver that showed increased inflammation. Furthermore, we detected an accumulating monocyte-derived macrophage population (CD45+F4/80+CD11bhiLy6Clow) that displayed a M2 macrophage phenotype with high expression of CD206, arginase-1, and IL-6, supporting the notion that IL-6 could be involved in M2 differentiation. In in vitro experiments, we demonstrated that IL-6 upregulated M-CSF receptor expression and skewed monocyte differentiation from dendritic cells to macrophages. This was countered by the addition of TNF. Furthermore, TNF interfered with the activation of IL-6-induced gp130-signal transducer and activator of transcription (STAT) 3 and IL-4-STAT6 signaling, thereby abrogating IL-6-facilitated M2 macrophage polarization. Therefore, our results support the notion of a general role of TNF in the inflammatory activation of macrophages and define a new role of IL-6 signaling in macrophage polarization downstream of TNF.

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SummaryThe mature, resting immunoglobulin (Ig) M, IgD + B lymphocyte can be induced by T cells to proliferate, switch isotype, and differentiate into Ig-secreting or memory cells. Furthermore, B cell activation results in the de novo expression or loss of a number of cell surface molecules that function in cell recirculation or further interaction with T cells. Here, a novel fluorescent technique reveals that T-dependent B cell activation induces cell surface changes that correlate with division cycle number. Furthermore, striking stepwise changes are often centered on a single round of cell division. Particularly marked was the consistent increase in IgG1 + B cells after the second division cycle, from an initial level of <3% IgG1 + to a plateau of~40% after six cell divisions. The relationship between the percentage oflgG1 + B cells and division number was independent of time after stimulation, indicating a requirement for cell division in isotype switching. IgD expression became negative after four divisions, and a number of changes centered on the sixth division, including the loss of IgM, CD23, and B220. The techniques used here should prove useful for tracking other differentiation pathways and for future analysis of the molecular events associated with stepwise differentiation at the single cell level.activation by T cells, mature B lymphocytes prorate and develop into Ig-secreting or memory cells (1). T cell-activated B cells also alter their functional characteristics such as Ig isotype, and expression of cell surface markers in response to cell contact and cytokine-mediated signals. The best studied example is the response to the cytokine IL-4, which induces the activated B cell to switch isotype from IgM to IgG1 and IgE (2, 3). As B cell differentiation is usually associated with mitosis, it has been difficult to directly assess whether cell division is required for differentiation, although a number of previous reports have linked the isotype switching mechanism with cell division (4-8). A technique for simultaneously tracking the division cycle history of stimulated cells and examining the cell surface phenotype has been developed by Lyons and Parish (9). Here, we use this method to track the relationship between division cycle number and B cell Ig isotype expression induced by the combination of plasma membranes from activated T cells and T cell-derived lymphokines (10). The results indicate that expression of surface IgG1 and other markers of B cell activation correlate with cell division number, making possible further molecular analysis of the process. Materials and MethodsPreparation and Stimulation of B Cells. Small, dense resting B cells were prepared by Percoll density gradient ~om anti-Thyl, -CD4, -CD8, and complement-treated CBA/H mouse spleens as described (10). B cells to be labeled with carboxyfluorescein, diacerate succinimidyl ester (CFSE; Molecular Probes, Inc., Eugene, OR) cells were washed twice in PBS containing 0.1% BSA and resuspended in this solution at 107 cells/ml. CFSE was t...

Clonally transmissible cancers are somatic cell lineages that are spread between individuals via the transfer of living cancer cells. There are only three known naturally occurring transmissible cancers, and these affect dogs, soft-shell clams, and Tasmanian devils, respectively. The Tasmanian devil transmissible facial cancer was first observed in 1996, and is threatening its host species with extinction. Until now, this disease has been consistently associated with a single aneuploid cancer cell lineage that we refer to as DFT1. Here we describe a second transmissible cancer, DFT2, in five devils located in southern Tasmania in 2014 and 2015. DFT2 causes facial tumors that are grossly indistinguishable but histologically distinct from those caused by DFT1. DFT2 bears no detectable cytogenetic similarity to DFT1 and carries a Y chromosome, which contrasts with the female origin of DFT1. DFT2 shows different alleles to both its hosts and DFT1 at microsatellite, structural variant, and major histocompatibility complex (MHC) loci, confirming that it is a second cancer that can be transmitted between devils as an allogeneic, MHC-discordant graft. These findings indicate that Tasmanian devils have spawned at least two distinct transmissible cancer lineages and suggest that transmissible cancers may arise more frequently in nature than previously considered. The discovery of DFT2 presents important challenges for the conservation of Tasmanian devils and raises the possibility that this species is particularly prone to the emergence of transmissible cancers. More generally, our findings highlight the potential for cancer cells to depart from their hosts and become dangerous transmissible pathogens.