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Differential Hox gene expression is central for specification of axial neuronal diversity in the spinal cord. Here, we uncover an additional function of Hox proteins in the developing spinal cord, restricted to B cluster Hox genes. We found that members of the HoxB cluster are expressed in the trunk neural tube of chicken embryo earlier than Hox from the other clusters, with poor antero-posterior axial specificity and with overlapping expression in the intermediate zone (IZ). Gain-of-function experiments of HoxB4, HoxB8 and HoxB9, respectively, representative of anterior, central and posterior HoxB genes, resulted in ectopic progenitor cells in the mantle zone. The search for HoxB8 downstream targets in the early neural tube identified the leucine zipper tumor suppressor 1 gene (Lzts1), the expression of which is also activated by HoxB4 and HoxB9. Gain- and loss-of-function experiments showed that Lzts1, which is expressed endogenously in the IZ, controls neuronal delamination. These data collectively indicate that HoxB genes have a generic function in the developing spinal cord, controlling the expression of Lzts1 and neuronal delamination.

Differential Hox gene expression is central for specification of axial neuronal diversity in the spinal cord. Here, we uncover an additional function of Hox proteins in the developing spinal cord, restricted to B cluster Hox genes. We found that members of the HoxB cluster are expressed in the trunk neural tube of chicken embryo earlier than Hox from the other clusters, with poor antero-posterior axial specificity and with overlapping expression in the intermediate zone (IZ). Gain-of-function experiments of HoxB4, HoxB8 and HoxB9, respectively, representative of anterior, central and posterior HoxB genes, resulted in ectopic progenitor cells in the mantle zone. The search for HoxB8 downstream targets in the early neural tube identified the leucine zipper tumor suppressor 1 gene (

Cancer immunotherapy by anti-PD-1 or anti-PD-L1 antibodies is now established as an efficient approach to restore the cytotoxic activity of exhausted T-cells. However, the strong and durable response observed in non-small cell lung cancer (NSCLC) patients is limited to a fraction of the treated population. Biomarkers, such as PD-L1 expression on tumor cells or tumor mutational burden, are approved as predictive markers of the response to anti-PD-1 or anti-PD-L1 antibodies. However, they are far from perfect to select patients eligible to immunotherapy. Since exhausted T-cells express more than one checkpoint inhibitor, it is hypothesized that an anti-PD1 antibody may not be sufficient to restore the activity of exhausted cells. Multiplex detection of the major immune checkpoints, i.e., PD-1, LAG-3 and TIM-3, on T-cells within the tumor microenvironment could, on one hand, predict the poor response to a single agent and could, on the other hand, predict the best combination of antibodies targeting more than one immune checkpoint. Here we present an automated sequential chromogenic multiplex assay allowing the assessment of the expression of PD-1, LAG-3 and TIM-3 on CD3+/CD8+ cells on a single FFPE tumor tissue section. Briefly, a tissue section is sequentially stained, digitized, unstained and restained with antibodies targeting the five markers. Images of the whole slide are then analyzed by digital pathology. First, a newly developed software is used to co-register the 5 virtual slides and perform colors deconvolution. Detection of positive cells is performed for each marker independently, using Indica Lab’s HALO software. Then, individual cells can be analyzed to identify complex phenotypes and assess their density on the entire tissue section. In addition, tissue segmentation tools are used to assess densities of exhausted T-cells expressing the major immune checkpoints in parenchyma, tumor stroma and invasive margin regions. The quantitative evaluation of T-cells’ exhaustion based on the expression of more than one immune checkpoint could improve patient stratification and lead to individualized combinations of immunotherapy agents. Citation Format: Aurélie Collignon, Assil Benchaaben, Anna Martirosyan, Matthieu Duval, Emilie Bonzom, Emmanuel Prestat, Christophe Haond, Jacques Fieschi. T-cell exhaustion assessment using a fully automated sequential chromogenic multiplex assay [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A182.