Studying MHC Class II Peptide Loading and Editing In Vitro.

HLA-DM is now known to have a major contribution to the selection of immunodominant epitopes. A better understanding of the mechanisms controlling epitope selection can be achieved by examination of the biophysical behavior of MHC class II molecules upon binding of antigenic peptides and of the effect of DM on the interactions. Using purified soluble molecules, in this chapter we describe several in vitro methods for measuring peptide binding to HLA-DR molecules and the effects of HLA-DM on this interaction.

Publisher Correction: A shared Runx1-bound Zbtb16 enhancer directs innate and innate-like lymphoid lineage development.

In the original PDF version of this Article, which was published on 16 October 2017, the publication date was incorrectly given as 11 October 2017. This has now been corrected in the PDF; the HTML version of the paper was correct from the time of publication.

A shared Runx1-bound Zbtb16 enhancer directs innate and innate-like lymphoid lineage development.

Zbtb16-encoded PLZF is a signature transcription factor (TF) that directs the acquisition of T-helper effector programs during the development of multiple innate lymphocyte lineages, including natural killer T cell, innate lymphoid cell, mucosal-associated invariant T cell and γδ lineages. PLZF is also essential in osteoblast and spermatogonial development. How Zbtb16 itself is regulated in different lineages is incompletely understood.

The Innate Lymphoid Cell Precursor.

The discovery of tissue-resident innate lymphoid cell populations effecting different forms of type 1, 2, and 3 immunity; tissue repair; and immune regulation has transformed our understanding of mucosal immunity and allergy. The emerging complexity of these populations along with compounding issues of redundancy and plasticity raise intriguing questions about their precise lineage relationship. Here we review advances in mapping the emergence of these lineages from early lymphoid precursors.

Single-cell analysis defines the divergence between the innate lymphoid cell lineage and lymphoid tissue-inducer cell lineage.

The precise lineage relationship between innate lymphoid cells (ILCs) and lymphoid tissue-inducer (LTi) cells is poorly understood. Using single-cell multiplex transcriptional analysis of 100 lymphoid genes and single-cell cultures of fetal liver precursor cells, we identified the common proximal precursor to these lineages and found that its bifurcation was marked by differential induction of the transcription factors PLZF and TCF1. Acquisition of individual effector programs specific to the ILC subsets ILC1, ILC2 and ILC3 was initiated later, at the common ILC precursor stage, by transient expression of mixed ILC1, ILC2 and ILC3 transcriptional patterns, whereas, in contrast, the development of LTi cells did not go through multilineage priming.

Innate and Adaptive Humoral Responses Coat Distinct Commensal Bacteria with Immunoglobulin A.

Immunoglobulin A (IgA) is prominently secreted at mucosal surfaces and coats a fraction of the intestinal microbiota. However, the commensal bacteria bound by IgA are poorly characterized and the type of humoral immunity they elicit remains elusive. We used bacterial flow cytometry coupled with 16S rRNA gene sequencing (IgA-Seq) in murine models of immunodeficiency to identify IgA-bound bacteria and elucidate mechanisms of commensal IgA targeting.

PLZF expression maps the early stages of ILC1 lineage development.

Among the variety of tissue-resident NK-like populations recently distinguished from recirculating classical NK (cNK) cells, liver innate lymphoid cells (ILC) type 1 (ILC1s) have been shown to represent a distinct lineage that originates from a novel promyelocytic leukaemia zinc finger (PLZF)-expressing ILC precursor (ILCP) strictly committed to the ILC1, ILC2, and ILC3 lineages. Here, using PLZF-reporter mice and cell transfer assays, we studied the developmental progression of ILC1s and demonstrated substantial overlap with stages previously ascribed to the cNK lineage, including pre-pro-NK, pre-NK precursor (pre-NKP), refined NKP (rNKP), and immature NK (iNK). Although they originated from different precursors, the ILC1 and cNK lineages followed a parallel progression at early stages and diverged later at the iNK stage, with a striking predominance of ILC1s over cNKs early in ontogeny.

Elevated T cell receptor signaling identifies a thymic precursor to the TCRαβ(+)CD4(-)CD8β(-) intraepithelial lymphocyte lineage.

The origin and developmental pathway of intestinal T cell receptor αβ(+) CD4(-)CD8β(-) intraepithelial lymphocytes (unconventional iIELs), a major population of innate-like resident cytolytic T cells, have remained elusive. By cloning and expressing several TCRs isolated from unconventional iIELs, we identified immature CD4(lo)CD8(lo)(DP(lo))CD69(hi)PD-1(hi) thymocytes as the earliest postsignaling precursors for these cells. Although these precursors displayed multiple signs of elevated TCR signaling, a sizeable fraction of them escaped deletion to selectively engage in unconventional iIEL differentiation.

Total synthesis of a functional designer eukaryotic chromosome.

Rapid advances in DNA synthesis techniques have made it possible to engineer viruses, biochemical pathways and assemble bacterial genomes. Here, we report the synthesis of a functional 272,871-base pair designer eukaryotic chromosome, synIII, which is based on the 316,617-base pair native Saccharomyces cerevisiae chromosome III. Changes to synIII include TAG/TAA stop-codon replacements, deletion of subtelomeric regions, introns, transfer RNAs, transposons, and silent mating loci as well as insertion of loxPsym sites to enable genome scrambling.

Studying MHC class II peptide loading and editing in vitro.

HLA-DM is now known to have a major contribution to the selection of immunodominant epitopes. A better understanding of the mechanisms controlling epitope selection can be achieved by examination of the biophysical behavior of major histocompatibility complex (MHC) class II molecules upon binding of antigenic peptides and the effect of DM on the interactions. Using purified soluble molecules, in this chapter, we describe several in vitro methods for measuring peptide binding to HLA-DR molecules and the effects of HLA-DM on the interactions.