GFAP and desmin expression in lymphatic tissues leads to difficulties in distinguishing between glial and stromal cells

Recently, we found many immune cells including antigen presenting cells neurally hard wired in the T-cell zone of most lymphoid organs like amongst others, lymph nodes in rats, mice and humans. Single immune cells were reached by single neurites and enclosed with a dense neural meshwork. As it is well known that axons are always accompanied by glial cells, we were able to identify Schwann cells in the hilum, medullary and capsule region, like expected. Unexpected was the result, that we found oligodendrocyte-like cells in these regions, myelinating more than one axon. Likewise important was the finding, that one of the standard glial markers used, a polyclonal GFAP antibody equally bound to desmin and therefore marked nearly all stromal cells in cortical, paracortical and medullary cord regions. More detailed analysis showed that these results also appeared in many other non-lymphoid organs. Therefore, polyclonal GFAP antibodies are only conditionally usable for immunohistochemical analysis in peripheral tissues outside the central nervous system. It remains to be elucidated, if the binding of the GFAP antibody to desmin has its reason in a special desmin variant that can give stromal cells glial character.

Numerical Simulation of Thrombolysis in Robot-Assisted Retinal Vein Cannulation

Robot-assisted retinal cannulation is an eye surgical procedure which can dissolve the obstruction by using robot to inject anticoagulant into occluded vessel. The current research on the critical parameters of cannulation for human is scarce because of the immature technology. Considering the influence of microneedle, this work investigated the effects of drug concentration, injection velocity, injection position, and size of clot on cannulation by theoretical analysis and finite element analysis. For finite element analysis, the multiphysics continuum model was established to demonstrate species transport and reaction which simulates the entire lytic process of the occlusive clot, and four cell zones were established to describe the generation of plasmin (PLS) with the addition of tissue-type plasminogen activator (tPA) and fibrinolysis of clot by importing subroutines into each cell zone under the conditions of constant clot size and variable size, respectively. The results imply that the most efficient value of tPA concentration is 50 nM, injection velocity is 60 mm/s for clot length of 0.1 mm, and the best position to insert the cannula is 0.5 mm in front of the thrombus. For different clot lengths of 0.1 mm to 0.6 mm, the optimal range of tPA concentration and injection velocity is from 20 nM to 70 nM and from 40 mm/s to 60 mm/s, respectively, and explores the reasonable injection position of 0.3 mm to 0.5 mm in front of clot in a vein of 100 μm. This conclusion can be used to perform robot-assisted cannulation surgery to improve fibrinolytic efficiency.

CXCR5-negative natural killer cells ameliorate experimental autoimmune myasthenia gravis by suppressing follicular helper T cells

Background Recent studies have demonstrated that natural killer (NK) cells can modulate other immune components and are involved in the development or progression of several autoimmune diseases. However, the roles and mechanisms of NK cells in regulating experimental autoimmune myasthenia gravis (EAMG) remained to be illustrated. Methods To address the function of NK cells in experimental autoimmune myasthenia gravis in vivo, EAMG rats were adoptively transferred with splenic NK cells. The serum antibodies, and splenic follicular helper T (Tfh) cells and germinal center B cells were determined by ELISA and flow cytometry. The roles of NK cells in regulating Tfh cells were further verified in vitro by co-culturing splenocytes or isolated T cells with NK cells. Moreover, the phenotype, localization, and function differences between different NK cell subtypes were determined by flow cytometry, immunofluorescence, and ex vivo co-culturation. Results In this study, we found that adoptive transfer of NK cells ameliorated EAMG symptoms by suppressing Tfh cells and germinal center B cells. Ex vivo studies indicated NK cells inhibited CD4+ T cells and Tfh cells by inducing the apoptosis of T cells. More importantly, NK cells could be divided into CXCR5- and CXCR5+ NK subtypes according to the expression of CXCR5 molecular. Compared with CXCR5- NK cells, which were mainly localized outside B cell zone, CXCR5+ NK were concentrated in the B cell zone and exhibited higher expression levels of IL-17 and ICOS, and lower expression level of CD27. Ex vivo studies indicated it was CXCR5- NK cells not CXCR5+ NK cells that suppressed CD4+ T cells and Tfh cells. Further analysis revealed that, compared with CXCR5- NK cells, CXCR5+ NK cells enhanced the ICOS expression of Tfh cells. Conclusions These findings highlight the different roles of CXCR5- NK cells and CXCR5+ NK cells. It was CXCR5- NK cells but not CXCR5+ NK cells that suppressed Tfh cells and inhibited the autoimmune response in EAMG models.

A CD146 FACS Protocol Enriches for Luminal Keratin 14/19 Double Positive Human Breast Progenitors

Human breast cancer is believed to arise in luminal progenitors within the normal breast. A subset of these are double positive (DP) for basal and luminal keratins and localizes to a putative stem cell zone within ducts. We here present a new protocol based on a combination of CD146 with CD117 and CD326 which provides an up to thirty fold enrichment of the DP cells. We show by expression profiling, colony formation, and morphogenesis that CD146high/CD117high/CD326high DP cells belong to a luminal progenitor compartment. While these DP cells are located quite uniformly in ducts, with age a variant type of DP (vDP) cells, which is mainly CD146-negative, accumulates in lobules. Intriguingly, in specimens with BRCA1 mutations known to predispose for cancer, higher frequencies of lobular vDP cells are observed. We propose that vDP cells are strong candidates for tracing the cellular origin of breast cancer.

High-resolution 3D imaging and topological mapping of the lymph node conduit system

The conduit network is a hallmark of lymph node microanatomy, but lack of suitable imaging technology has prevented comprehensive investigation of its topology. We employed an extended-volume imaging system to capture the conduit network of an entire murine lymph node (≈280,000 segments). The extensive 3D images provide a comprehensive overview of the regions supplied by conduits including perivascular sleeves, and distinctive “follicular reservoirs” within B cell follicles, surrounding follicular dendritic cells. A 3D topology map of conduits within the T cell zone showed homogeneous branching, but conduit density was significantly higher in the superficial T cell zone compared to the deep zone, where distances between segments are sufficient for T cells to lose contact with fibroblastic reticular cells. This topological mapping of the conduit anatomy can now aid modeling of its roles in lymph node function, as we demonstrate by simulating T cell motility in the different T cell zones.

Spatial distribution and function of T follicular regulatory cells in human lymph nodes

T follicular regulatory (Tfr) cells are a population of CD4+ T cells that express regulatory T cell markers and have been shown to suppress humoral immunity. However, the precise mechanisms and location of Tfr-mediated suppression in the lymph node (LN) microenvironment are unknown. Using highly multiplexed quantitative imaging and functional assays, we examined the spatial distribution, suppressive function, and preferred interacting partners of Tfr cells in human mesenteric LNs. We find that the majority of Tfr cells express low levels of PD-1 and reside at the border between the T cell zone and B cell follicle, with very few found in the germinal centers (GCs). Although PD-1+ Tfr cells expressed higher levels of CD38, CTLA-4, and GARP than PD-1Neg Tfr cells, both potently suppressed antibody production in vitro. These findings highlight the phenotypic diversity of human Tfr cells and suggest that Tfr-mediated suppression is most efficient at the T-B border and within the follicle, not in the GC.