Mechanosensing is critical for axon growth in the developing brain.

During nervous system development, neurons extend axons along well-defined pathways. The current understanding of axon pathfinding is based mainly on chemical signaling. However, growing neurons interact not only chemically but also mechanically with their environment. Here we identify mechanical signals as important regulators of axon pathfinding. In vitro, substrate stiffness determined growth patterns of Xenopus retinal ganglion cell axons. In vivo atomic force microscopy revealed a noticeable pattern of stiffness gradients in the embryonic brain. Retinal ganglion cell axons grew toward softer tissue, which was reproduced in vitro in the absence of chemical gradients. To test the importance of mechanical signals for axon growth in vivo, we altered brain stiffness, blocked mechanotransduction pharmacologically and knocked down the mechanosensitive ion channel piezo1. All treatments resulted in aberrant axonal growth and pathfinding errors, suggesting that local tissue stiffness, read out by mechanosensitive ion channels, is critically involved in instructing neuronal growth in vivo.This work was supported by the German National Academic Foundation (scholarship to D.E.K.), Wellcome Trust and Cambridge Trusts (scholarships to A.J.T.), Winston Churchill Foundation of the United States (scholarship to S.K.F.), Herchel Smith Foundation (Research Studentship to S.K.F.), CNPq 307333/2013-2 (L.d.F.C.), NAP-PRP-USP and FAPESP 11/50761-2 (L.d.F.C.), UK EPSRC BT grant (J.G.), Wellcome Trust WT085314 and the European Research Council 322817 grants (C.E.H.); an Alexander von Humboldt Foundation Feodor Lynen Fellowship (K.F.), UK BBSRC grant BB/M021394/1 (K.F.), the Human Frontier Science Program Young Investigator Grant RGY0074/2013 (K.F.), the UK Medical Research Council Career Development Award G1100312/1 (K.F.) and the Eunice Kennedy Shriver National Institute Of Child Health & Human Development of the National Institutes of Health under Award Number R21HD080585 (K.F.).This is the author accepted manuscript. The final version is available from Nature Publishing Group via https://doi.org/10.1038/nn.439

Cis interactions make immune checkpoint blockade more trans -parent

PD-L1:CD80 cis-heterodimer formation preferentially blocks CTLA-4 trans-signaling while allowing CD28-mediated effector T cell activation

A sweet alternative: maintaining M2 macrophage polarization

Glycolytic metabolism functions as a backup mechanism for M2 macrophage polarization when oxidative phosphorylation is disrupted

TB or not TB: Type 3 ILCs may decide

Mouse models are used to show that type 3 innate lymphoid cells provide early protection against infection

Ex Vivo Expansion of Th2-Polarizing Immunotherapeutic iNKT Cells from Human Peripheral Blood

iNKT cells, classified as innate lymphocytes with invariant TCRs, have been highlighted as a putative, “off-the-shelf” cellular immunotherapeutic strategy for the treatment of malignant and nonmalignant diseases. However, their paucity in human blood limits their immunotherapeutic applications. Herein we describe a rigorously optimized 21-day ex vivo expansion method to achieve log-fold increases in immunotherapeutic human iNKT cells

CyTOF ® for the Masses

Mass cytometry has revolutionized immunophenotyping, particularly in exploratory settings where simultaneous breadth and depth of characterization of immune populations is needed with limited samples such as in preclinical and clinical tumor immunotherapy. Mass cytometry is also a powerful tool for single-cell immunological assays, especially for complex and simultaneous characterization of diverse intratumoral immune subsets or immunotherapeutic cell populations. Through the elimination of spectral overlap seen in optical flow cytometry by replacement of fluorescent labels with metal isotopes, mass cytometry allows, on average, robust analysis of 60 individual parameters simultaneously. This is, however, associated with significantly increased complexity in the design, execution, and interpretation of mass cytometry experiments. To address the key pitfalls associated with the fragmentation, complexity, and analysis of data in mass cytometry for immunologists who are novices to these techniques, we have developed a comprehensive resource guide. Included in this review are experiment and panel design, antibody conjugations, sample staining, sample acquisition, and data pre-processing and analysis. Where feasible multiple resources for the same process are compared, allowing researchers experienced in flow cytometry but with minimal mass cytometry expertise to develop a data-driven and streamlined project workflow. It is our hope that this manuscript will prove a useful resource for both beginning and advanced users of mass cytometry

Donor CD8+ T Cells Facilitate Graft-Versus-Tumor Effect Via Alloantigen Rather Than Tumor-Specific Antigen Recognition Following Murine Myeloablative BMT

Abstract Previous data from our group and others has shown that donor CD8+ Tcells mediate graft-versus-tumor (GVT) function in murine myeloablative bone marrow transplantation (BMT) via Perforin/Fas ligand-dependent mechanisms. However, there has to date been no analysis of the mechanism of tumor recognition (i.e allo- versus tumor-specific antigen recognition) by donor CD8+ T cells following myeloablative MHC-mismatched BMT. In order to test the hypothesis that donor CD8 T-cells require allo-antigen recognition to maintain graft-versus-tumor effect, we developed stable full chimeras by transplanting T-cell depleted bone marrow (TCD BM) from C57BL/6 (H-2b) donor mice into myeloablated BALB/c (H-2d) hosts given 800 cGy total body irradiation (TBI) and evaluated the in vivo ability of CD8+ TCR+ splenocytes from these donors to kill the BCL1 tumor (a BALB/c-derived B-cell lymphoma carrying a detectable tumor-specific idiotype which can be monitored via peripheral blood flow cytometric analysis). These chimeras showed complete donor chimerism in myeloid, B- and T-lymphocytic lineages by day +100 following transplantation, and splenocytes from these chimeras exhibited tolerance to host-type but not third-party alloantigens. BALB/c hosts were given 800 cGy TBI on day -1, followed on day 0 by intravenous administration of 500 BCL1 tumor cells and infusion of 0.3x 106 CD8+ T cells of C57 origin (H-2b+) sorted by flow cytometric analysis from the either spleens of the chimeric mice or from the spleens of untreated wild-type C57BL/6 mice. All hosts were given 5 x 106 T cell-depleted wild-type C57 bone marrow cells. All mice were observed for clinical signs of graft-versus-host disease (GVHD) and mortality through day +100. Autopsy was performed at death to assess for sub-clinical target organ involvement with GVHD or tumor. Donor chimerism and BCL1 status was assessed at day +28 and day +100 by 3-color flow cytometric analysis for donor-specific MHC versus T-, B- and myeloid lineage markers as well as tumor-specific idiotype in the peripheral blood (or in the spleen at time of death for animals dying prior to day +100). All animals receiving BCL1 tumor cells and sorted CD8+T cells from wild-type untreated C57 donors cleared tumor idiotype but succumbed to GVHD. All animals receiving tumor cells and sorted chimeric C57 CD8+ T cells remained free of clinical or pathologic evidence of GVHD, but died with tumor progression. Control myeloablated animals given C57 TCD BM alone with BCL1 tumor cells all succumbed to tumor, whereas those receiving C57 whole bone marrow with tumor demonstrated tumor survival without GVHD. The data indicate that chimeric donor peripheral CD8+ T cells, which lose their capacity to induce lethal GVHD in BALB/c hosts, also lose the capacity to eradicate BALB/c-type lymphoma cells. We conclude that CD8+ T cell-induced GVT effect in this model is dependent upon alloantigen rather than tumor-specific antigen recognition

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn’s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods