Niroshana Anandasabapathy

Sec6/8 Complex Is Recruited to Cell–Cell Contacts and Specifies Transport Vesicle Delivery to the Basal-Lateral Membrane in Epithelial Cells

In budding yeast, the Sec6/8p complex is essential for generating cell polarity by specifying vesicle delivery to the bud tip. We show that Sec6/8 homologs are components of a cytosolic, approximately 17S complex in nonpolarized MDCK epithelial cells. Upon initiation of calcium-dependent cell-cell adhesion, approximately 70% of Sec6/8 is rapidly (t(1/2) approximately 3-6 hr) recruited to sites of cell-cell contact. In streptolysin-O-permeabilized MDCK cells, Sec8 antibodies inhibit delivery of LDL receptor to the basal-lateral membrane, but not p75NTR to the apical membrane. These results indicate that lateral membrane recruitment of the Sec6/8 complex is a consequence of cell-cell adhesion and is essential for the biogenesis of epithelial cell surface polarity.

GRAIL: An E3 Ubiquitin Ligase that Inhibits Cytokine Gene Transcription Is Expressed in Anergic CD4+ T Cells

T cell anergy may serve to limit autoreactive T cell responses. We examined early changes in gene expression after antigen-TCR signaling in the presence (activation) or absence (anergy) of B7 costimulation. Induced expression of GRAIL (gene related to anergy in lymphocytes) was observed in anergic CD4(+) T cells. GRAIL is a type I transmembrane protein that localizes to the endocytic pathway and bears homology to RING zinc-finger proteins. Ubiquitination studies in vitro support GRAIL function as an E3 ubiquitin ligase. Expression of GRAIL in retrovirally transduced T cell hybridomas dramatically limits activation-induced IL-2 and IL-4 production. Additional studies suggest that GRAIL E3 ubiquitin ligase activity and intact endocytic trafficking are critical for cytokine transcriptional regulation. Expression of GRAIL after an anergizing stimulus may result in ubiquitin-mediated regulation of proteins essential for mitogenic cytokine expression, thus positioning GRAIL as a key player in the induction of the anergic phenotype.

Type I interferons and microbial metabolites of tryptophan modulate astrocyte activity and central nervous system inflammation via the aryl hydrocarbon receptor

Astrocytes have important roles in the central nervous system (CNS) during health and disease. Through genome-wide analyses we detected a transcriptional response to type I interferons (IFN-Is) in astrocytes during experimental CNS autoimmunity and also in CNS lesions from patients with multiple sclerosis (MS). IFN-I signaling in astrocytes reduces inflammation and experimental autoimmune encephalomyelitis (EAE) disease scores via the ligand-activated transcription factor aryl hydrocarbon receptor (AHR) and the suppressor of cytokine signaling 2 (SOCS2). The anti-inflammatory effects of nasally administered interferon (IFN)-β are partly mediated by AHR. Dietary tryptophan is metabolized by the gut microbiota into AHR agonists that have an effect on astrocytes to limit CNS inflammation. EAE scores were increased following ampicillin treatment during the recovery phase, and CNS inflammation was reduced in antibiotic-treated mice by supplementation with the tryptophan metabolites indole, indoxyl-3-sulfate, indole-3-propionic acid and indole-3-aldehyde, or the bacterial enzyme tryptophanase. In individuals with MS, the circulating levels of AHR agonists were decreased. These findings suggest that IFN-Is produced in the CNS function in combination with metabolites derived from dietary tryptophan by the gut flora to activate AHR signaling in astrocytes and suppress CNS inflammation.

Two isoforms of otubain 1 regulate T cell anergy via GRAIL.

The active ubiquitin E3 ligase GRAIL is crucial in the induction of CD4 T cell anergy. Here we show that GRAIL is associated with and regulated by two isoforms of the ubiquitin-specific protease otubain 1. In lethally irradiated mice reconstituted with bone marrow cells from T cell receptor–transgenic mice retrovirally transduced to express the genes encoding these proteases, otubain 1–expressing cells contained negligible amounts of endogenous GRAIL, proliferated well and produced large amounts of interleukin 2 after antigenic stimulation. In contrast, cells expressing the alternatively spliced isoform, otubain 1 alternative reading frame 1, contained large amounts of endogenous GRAIL and were functionally anergic, and they proliferated poorly and produced undetectable interleukin 2 when stimulated in a similar way. Thus, these two proteins have opposing epistatic functions in controlling the stability of GRAIL expression and the resultant anergy phenotype in T cells.

Flt3L controls the development of radiosensitive dendritic cells in the meninges and choroid plexus of the steady-state mouse brain

As shown by analyses of morphology, gene expression, antigen-presenting function, and Flt3 dependence, the steady-state mouse brain contains a population of DCs that exhibits similarities to splenic DCs and differences from microglia.

Circulating precursors of human CD1c+ and CD141+ dendritic cells

The Liu and Nussenzweig groups identify the immediate precursor of CD1c+ and CD141+ dendritic cells in the circulation of healthy donors. These precursor cells (hpre-cDC) were detectable in cord blood, bone marrow, blood, and peripheral lymphoid organs.

Brain dendritic cells: biology and pathology

Dendritic cells (DC) are the professional antigen-presenting cells of the immune system. In their quiescent and mature form, the presentation of self‐antigens by DC leads to tolerance; whereas, antigen presentation by mature DC, after stimulation by pathogen‐associated molecular patterns, leads to the onset of antigen-specific immunity. DC have been found in many of the major organs in mammals (e.g. skin, heart, lungs, intestines and spleen); while the brain has long been considered devoid of DC in the absence of neuroinflammation. Consequently, microglia, the resident immune cell of the brain, have been charged with many functional attributes commonly ascribed to DC. Recent evidence has challenged the notion that DC are either absent or minimal players in brain immune surveillance. This review will discuss the recent literature examining DC involvement within both the young and aged steady-state brain. We will also examine DC contributions during various forms of neuroinflammation resulting from neurodegenerative autoimmune disease, injury, and CNS infections. This review also touches upon DC trafficking between the central nervous system and peripheral immune compartments during viral infections, the new molecular technologies that could be employed to enhance our current understanding of brain DC ontogeny, and some potential therapeutic uses of DC within the CNS.

Classical Flt3L-dependent dendritic cells control immunity to protein vaccine

Protective immunity to protein vaccines is controlled by Flt3L-dependent classical LN-resident dendritic cells, and dampened by migratory dendritic cells.

Flt3L Dependence Helps Define an Uncharacterized Subset of Murine Cutaneous Dendritic Cells

Skin-derived dendritic cells (DC) are potent antigen presenting cells with critical roles in both adaptive immunity and tolerance to self. Skin DC carry antigens and constitutively migrate to the skin draining lymph nodes (LN). In mice, Langerin-CD11b− dermal DC are a low-frequency, heterogeneous, migratory DC subset that traffic to LN (Langerin-CD11b-migDC). Here, we build on the observation that Langerin-CD11b− migDC are Fms-like tyrosine kinase 3 ligand (Flt3L) dependent and strongly Flt3L responsive, which may relate them to classical DCs. Examination of DC capture of FITC from painted skin, DC isolation from skin explant culture, and from the skin of CCR7 knockout mice which accumulate migDC, demonstrate these cells are cutaneous residents. Langerin-CD11b-Flt3L responsive DC are largely CD24(+) and CX3CR1low and can be depleted from Zbtb46-DTR mice, suggesting classical DC lineage. Langerin-CD11bmigDC present antigen with equal efficiency to other DC subsets ex vivo including classical CD8α cDC and Langerin+CD103+ dermal DC. Finally, transcriptome analysis suggests a close relationship to other skin DC, and a lineage relationship to other classical DC. This work demonstrates that Langerin- CD11b− dermal DC, a previously overlooked cell subset, may be an important player in the cutaneous immune environment.

Efficacy and safety of CDX-301, recombinant human Flt3L, at expanding dendritic cells and hematopoietic stem cells in healthy human volunteers.

Fms-like tyrosine kinase-3 ligand (Flt3L) uniquely binds the Flt3 (CD135) receptor expressed on hematopoietic stem cells (HSCs), early progenitor cells, immature thymocytes and steady-state dendritic cells (DCs) and induces their proliferation, differentiation, development and mobilization in the bone marrow, peripheral blood and lymphoid organs. CDX-301 has an identical amino-acid sequence and comparable biological activity to the previously tested rhuFlt3L, which ceased clinical development over a decade ago. This Phase 1 trial assessed the safety, pharmacokinetic, pharmacodynamic and immunologic profile of CDX-301, explored alternate dosing regimens and examined the impact of rhuFlt3L on key immune cell subsets. Thirty healthy volunteers received CDX-301 (1–75 μg/kg/day) over 5–10 days. One event of Grade 3 community-acquired pneumonia occurred. There were no other infections, dose-limiting toxicities or serious adverse events. CDX-301 resulted in effective peripheral expansion of monocytes, hematopoietic stem and progenitor cells and key subsets of myeloid DCs and plasmacytoid DCs, with no clear effect on regulatory T cells. These data from healthy volunteers support the potential for CDX-301, as monotherapy or in combination with other agents, in various indications including allogeneic HSC transplantation and immunotherapy, but the effects of CDX-301 will need to be investigated in each of these patient populations.