Type 2 innate lymphoid cells (ILC2) contribute to immune homeostasis, protective immunity and tissue repair. Here we demonstrate that functional ILC2 can arise in the embryonic thymus, from shared T cell precursors, preceding the emergence of CD4 + CD8 + (double-positive) T cells. Thymic ILC2 migrated to mucosal tissues with colonization of the intestinal lamina propria. RORα expression repressed T cell development, while promoting ILC2 in the thymus. From RNA-seq, ATAC-seq and ChIP-seq data we propose a revised transcriptional circuit to explain the co-development of T cells and ILC2 from common progenitors in the thymus. When Notch signaling is present, Bcl11b dampens Nfil3/Id2 expression, permitting E protein-directed T cell commitment. However, concomitant expression of RORα overrides the Nfil3/Id2 repression, allowing Id2 to repress E proteins and promote ILC2 differentiation. Thus, we demonstrate that RORα expression represents a critical checkpoint at the bifurcation of the T cell and ILC2 lineages in the embryonic thymus.
Innate lymphoid cells (ILCs) are increasingly recognised as an innate immune counterpart of adaptive TH cells. In addition to their similar effector cytokine production, there is a strong parallel between the transcription factors that control the differentiation of TH1, TH2 and TH17 cells and ILC Groups 1, 2 and 3, respectively. Here, we review the transcriptional circuit that specifies the development of a common ILC progenitor and its subsequent programming into distinct ILC groups. Notch, GATA-3, Nfil3 and Id2 are identified as early factors that suppress B and T cell potentials and are turned on in favour of ILC commitment. Natural killer cells, which are the cytotoxic ILCs, develop along a pathway distinct from the rest of the helper-like ILCs that are derived from a common progenitor to all helper-like innate lymphoid cells (CHILPs). PLZF− CHILPs give rise to lymphoid tissue inducer cells while PLZF+ CHILPs have multi-lineage potential and could give rise to ILCs 1, 2 and 3. Such lineage specificity is dictated by the controlled expression of T-bet, RORα, RORγt and AHR. In addition to the type of transcription factors, the developmental stages at which these factors are expressed are crucial in specifying the fate of the ILCs.
Cells that constitutively diversify their immunoglobulin genes can be used for selection of novel antibodies and for refining existing affinities and specificities. Here, we report an adaptation of the chicken DT40 system wherein its capacity for somatic hypermutation is harnessed to evolve human antibodies expressed as single-chain variable fragments (scFvs). Expression of membrane-anchored scFvs from within the rearranged Igλ locus created self-diversifying scFv libraries from which we could both select scFvs of a desired specificity and evolve both the specificity and affinity of existing scFvs by iterative expansion and selection. From these scFvs, we were able to create fully human IgG antibodies with nanomolar affinities. We further enhanced the functionality of the system by creating a pool of DT40 scFv lines with high levels of mutation driven by the overexpression of a hyperactive variant of activation-induced deaminase. From this library, we successfully isolated scFvs that bound the spliceosome factor CWC15 and the cytokine human IFNγ. Our results demonstrate the flexibility and utility of DT40 for rapid generation of scFv repertoires and efficient selection, evolution and affinity maturation of scFv specificities.
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