Multiplex immunohistochemistry accurately defines the immune context of metastatic melanoma.

A prospective study explored the heterogeneous nature of metastatic melanoma using Multiplex immunohistochemistry (IHC) and flow cytometry (FACS). Multiplex IHC data quantitated immune subset number present intra-tumoral (IT) vs the tumor stroma, plus distance of immune subsets from the tumor margin (TM). In addition, mIHC showed a close association between the presence of IT CD8+ T cells and PDL1 expression in melanoma, which was more prevalent on macrophages than on melanoma cells. In contrast, FACS provided more detailed information regarding the T cell subset differentiation, their activation status and expression of immune checkpoint molecules. Interestingly, mIHC detected significantly higher Treg numbers than FACS and showed preferential CD4+ T cell distribution in the tumor stroma. Based on the mIHC and FACS data, we provide a model which defines metastatic melanoma immune context into four categories using the presence or absence of PDL1+ melanoma cells and/or macrophages, and their location within the tumor or on the periphery, combined with the presence or absence of IT CD8+ T cells. This model interprets melanoma immune context as a spectrum of tumor escape from immune control, and provides a snapshot upon which interpretation of checkpoint blockade inhibitor (CBI) therapy responses can be built.

The G(2) DNA damage checkpoint targets both Wee1 and Cdc25.

The onset of mitosis is controlled by the cyclin dependent kinase Cdc2p. Cdc2p activity is controlled through the balance of phosphorylation and dephosphorylation of tyrosine-15 (Y15) by the Wee1p kinase and Cdc25p phosphatase. In the fission yeast Schizosaccharomyces pombe, detection of DNA damage in G(2) activates a checkpoint that prevents entry into mitosis through the maintenance of Y15 phosphorylation of Cdc2p, thus ensuring DNA repair precedes chromosome segregation. The protein kinase Chk1p is the endpoint of this checkpoint pathway. We have previously reported that overexpression of Chk1p causes a wee1(+)-dependent G(2) arrest, and this or irradiation leads to hyperphosphorylation of Wee1p. Moreover, Chk1p directly phosphorylates Wee1p in vitro. These data suggested that Wee1p is a key target of Chk1p action in checkpoint control. However, cells lacking wee1(+) are checkpoint proficient and sustained Chk1p overexpression arrests cell cycle progression independently of Wee1p. Therefore, up-regulation of Wee1p alone cannot enforce a checkpoint arrest. Chk1p can also phosphorylate Cdc25p in vitro. These phosphorylation events are thought to promote the interaction with 14-3-3 proteins the cytoplasmic retention of the 14-3-3/Cdc25p complexes. However, we show here that the G(2) DNA damage checkpoint is intact in cells that regulate mitotic entry independently of Cdc25p. Further, these cells are still sensitive to Chk1p-mediated arrest, and so down-regulation of Cdc25p is also insufficient to regulate checkpoint arrest. Conversely, inactivation of both wee1(+) and cdc25(+ )abolishes checkpoint control. We also show that activation of the G(2) DNA damage checkpoint induces a transient increase in Wee1p levels. We conclude that the G(2) DNA damage checkpoint simultaneously signals via both up-regulation of Wee1p and down-regulation of Cdc25p, thus providing a double-lock mechanism to ensure cell cycle arrest and genomic stability.

Chk1 is a wee1 kinase in the G2 DNA damage checkpoint inhibiting cdc2 by Y15 phosphorylation.

The G2 DNA damage checkpoint ensures maintenance of cell viability by delaying progression into mitosis in cells which have suffered genomic damage. It is controlled by a number of proteins which are hypothesized to transduce signals through cell cycle regulators to delay activation of p34cdc2. Studies in mammalian cells have correlated induction of inhibitory tyrosine 15 (Y15) phosphorylation on p34cdc2 with the response to DNA damage. However, genetic studies in fission yeast have suggested that the major Y15 kinase, p107wee1, is not required for the cell cycle delay in response to DNA damage, although it is required for survival after irradiation. Thus, the target of the checkpoint, and hence the mechanism of cell cycle delay, remains unknown. We show here that Y15 phosphorylation is maintained in checkpoint-arrested fission yeast cells. Further, wee1 is required for cell cycle arrest induced by up-regulation of an essential component of this checkpoint, chk1. We observed that p107wee1 is hyperphosphorylated in cells delayed by chk1 overexpression or UV irradiation, and that p56chk1 can phosphorylate p107wee1 directly in vitro. These observations suggest that in response to DNA damage p107wee1 is phosphorylated by p56chk1 in vivo, and this results in maintenance of Y15 phosphorylation and hence G2 delay. In the absence of wee1, other Y15 kinases, such as p66mik1, may partially substitute for p107wee1 to induce cell cycle delay, but this wee1-independent delay is insufficient to maintain full viability. This study establishes a link between a G2 DNA damage checkpoint function and a core cell cycle regulator.

Developmentally regulated expression of a Haemonchus contortus surface antigen.

All life cycle stages of Haemonchus contortus were screened with antibody-secreting cell (ASC) probes generated from immune sheep 5 days after a challenge infection. An antigen with an approximate molecular weight of 70-90 kDa was found to be predominantly expressed in the 3rd-stage larvae, while a molecule with an approximate molecular weight of 28-31 kDa was recognised by the ASC-probes in the earlier developmental stages. Antisera generated against the purified 70-90 kDa molecule and antibodies eluted from the 70-90 kDa region in the 3rd-stage larvae were found to react with the 28-31 kDa region in the 2nd-stage larvae. These results suggest that the 70-90 kDa antigen, previously identified as a major 3rd-stage surface antigen, is synthesised as a lower molecular weight molecule in the earlier larval stages and assembled into a higher molecular weight complex through non-reducible covalent bonds.

Stage-specific expression of surface molecules by the larval stages of Haemonchus contortus.

Three monoclonal antibodies, Hc2, Hc22 and Hc6, were produced against a surface extract of L3 Haemonchus contortus and screened against both free living and parasitic stages of the parasite. Hc2 and Hc22 were of IgG2c isotype and their target epitopes were insensitive to periodate treatment. Hc6 was of IgM isotype and its reactivity was sensitive to periodate treatment of the antigen, suggesting that Hc6 is specific for a carbohydrate epitope. Western blotting of larval extracts and staining of live worms demonstrated that Hc2 was specific for the surface of second stage larvae and that the epitope was still present on the protective sheath of third stage larvae but absent from the L3 cuticle itself. Hc22 was found to be specific for a 70-90 kDa antigen on the surface of third stage infective larvae and cross reacted with a higher molecular weight species present on the surface of third stage Teladorsagia circumcincta. Hc6 showed strong binding to three high molecular weight proteins on immunoblots of third stage larvae only, but in contrast to Hc2 and Hc22, showed no binding to the surface of live larvae. These studies demonstrate unique expression patterns of stage specific antigens on the surface of free living and parasitic H. contortus larvae.