Arnold Han

Linking T-cell receptor sequence to functional phenotype at the single-cell level

Although each T lymphocyte expresses a T-cell receptor (TCR) that recognizes cognate antigen and controls T-cell activation, different T cells bearing the same TCR can be functionally distinct. Each TCR is a heterodimer, and both α- and β-chains contribute to determining TCR antigen specificity. Here we present a methodology enabling integration of information about TCR specificity with information about T cell function. This method involves sequencing of TCRα and TCRβ genes, and amplifying functional genes characteristic of different T cell subsets, in single T cells. Because this approach retains information about individual TCRα-TCRβ pairs, TCRs of interest can be expressed and used in functional studies, for antigen discovery, or in therapeutic applications. We apply this approach to study the clonal ancestry and differentiation of T lymphocytes infiltrating a human colorectal carcinoma.

Dietary gluten triggers concomitant activation of CD4+ and CD8+ αβ T cells and γδ T cells in celiac disease

Celiac disease is an intestinal autoimmune disease driven by dietary gluten and gluten-specific CD4+ T-cell responses. In celiac patients on a gluten-free diet, exposure to gluten induces the appearance of gluten-specific CD4+ T cells with gut-homing potential in the peripheral blood. Here we show that gluten exposure also induces the appearance of activated, gut-homing CD8+ αβ and γδ T cells in the peripheral blood. Single-cell T-cell receptor sequence analysis indicates that both of these cell populations have highly focused T-cell receptor repertoires, indicating that their induction is antigen-driven. These results reveal a previously unappreciated role of antigen in the induction of CD8+ αβ and γδ T cells in celiac disease and demonstrate a coordinated response by all three of the major types of T cells. More broadly, these responses may parallel adaptive immune responses to viral pathogens and other systemic autoimmune diseases.

Identifying specificity groups in the T cell receptor repertoire

T cell receptor (TCR) sequences are very diverse, with many more possible sequence combinations than T cells in any one individual. Here we define the minimal requirements for TCR antigen specificity, through an analysis of TCR sequences using a panel of peptide and major histocompatibility complex (pMHC)-tetramer-sorted cells and structural data. From this analysis we developed an algorithm that we term GLIPH (grouping of lymphocyte interactions by paratope hotspots) to cluster TCRs with a high probability of sharing specificity owing to both conserved motifs and global similarity of complementarity-determining region 3 (CDR3) sequences. We show that GLIPH can reliably group TCRs of common specificity from different donors, and that conserved CDR3 motifs help to define the TCR clusters that are often contact points with the antigenic peptides. As an independent validation, we analysed 5,711 TCRβ chain sequences from reactive CD4 T cells from 22 individuals with latent Mycobacterium tuberculosis infection. We found 141 TCR specificity groups, including 16 distinct groups containing TCRs from multiple individuals. These TCR groups typically shared HLA alleles, allowing prediction of the likely HLA restriction, and a large number of M. tuberculosis T cell epitopes enabled us to identify pMHC ligands for all five of the groups tested. Mutagenesis and de novo TCR design confirmed that the GLIPH-identified motifs were critical and sufficient for shared-antigen recognition. Thus the GLIPH algorithm can analyse large numbers of TCR sequences and define TCR specificity groups shared by TCRs and individuals, which should greatly accelerate the analysis of T cell responses and expedite the identification of specific ligands.

Bam32 Links the B Cell Receptor to ERK and JNK and Mediates B Cell Proliferation but Not Survival

Bam32 is an adaptor protein recruited to the plasma membrane upon B cell receptor (BCR) crosslinking in a phosphoinositol 3-kinase (PI3K)-dependent manner; however, its physiologic function is unclear. To determine its physiologic function, we produced Bam32-deficient mice. Bam32(-/-) B cells develop normally but have impaired T-independent antibody responses in vivo and diminished responses to BCR crosslinking in vitro. Biochemical analysis revealed that Bam32 acts in a novel pathway leading from the BCR to MAPK/ERK Kinases (MEK1/2), MAPK/ERK Kinase Kinase-1 (MEKK1), extracellular signal-regulated kinase (ERK), and c-jun NH2-terminal kinase (JNK), but not p38 mitogen-activated protein kinase (p38). This pathway appears to be initiated by hematopoietic progenitor kinase-1 (HPK1), which interacts directly with Bam32, and differs from all previously characterized BCR signaling pathways in that it is required for normal BCR-mediated proliferation but not for B cell survival.

Non-equivalence of Wnt and R-spondin ligands during Lgr5 + intestinal stem-cell self-renewal

The canonical Wnt/β-catenin signalling pathway governs diverse developmental, homeostatic and pathological processes. Palmitoylated Wnt ligands engage cell-surface frizzled (FZD) receptors and LRP5 and LRP6 co-receptors, enabling β-catenin nuclear translocation and TCF/LEF-dependent gene transactivation. Mutations in Wnt downstream signalling components have revealed diverse functions thought to be carried out by Wnt ligands themselves. However, redundancy between the 19 mammalian Wnt proteins and 10 FZD receptors and Wnt hydrophobicity have made it difficult to attribute these functions directly to Wnt ligands. For example, individual mutations in Wnt ligands have not revealed homeostatic phenotypes in the intestinal epithelium—an archetypal canonical, Wnt pathway-dependent, rapidly self-renewing tissue, the regeneration of which is fueled by proliferative crypt Lgr5+ intestinal stem cells (ISCs). R-spondin ligands (RSPO1–RSPO4) engage distinct LGR4–LGR6, RNF43 and ZNRF3 receptor classes, markedly potentiate canonical Wnt/β-catenin signalling, and induce intestinal organoid growth in vitro and Lgr5+ ISCs in vivo. However, the interchangeability, functional cooperation and relative contributions of Wnt versus RSPO ligands to in vivo canonical Wnt signalling and ISC biology remain unknown. Here we identify the functional roles of Wnt and RSPO ligands in the intestinal crypt stem-cell niche. We show that the default fate of Lgr5+ ISCs is to differentiate, unless both RSPO and Wnt ligands are present. However, gain-of-function studies using RSPO ligands and a new non-lipidated Wnt analogue reveal that these ligands have qualitatively distinct, non-interchangeable roles in ISCs. Wnt proteins are unable to induce Lgr5+ ISC self-renewal, but instead confer a basal competency by maintaining RSPO receptor expression that enables RSPO ligands to actively drive and specify the extent of stem-cell expansion. This functionally non-equivalent yet cooperative interaction between Wnt and RSPO ligands establishes a molecular precedent for regulation of mammalian stem cells by distinct priming and self-renewal factors, with broad implications for precise control of tissue regeneration.

A Highly Focused Antigen Receptor Repertoire Characterizes γδ T Cells That are Poised to Make IL-17 Rapidly in Naive Animals.

Interleukin (IL)-17 plays a key role in immunity. In acute infections, a rapid IL-17 response must be induced without prior antigen exposure, and γδ T cells are the major initial IL-17 producers. In fact, some γδ T cells make IL-17 within hours after an immune challenge. These cells appear to acquire the ability to respond to IL-1 and IL-23 and to make IL-17 naturally in naïve animals. They are known as the natural Tγδ17 (nTγδ17) cells. The rapidity of the nTγδ17 response, and the apparent lack of explicit T cell receptor (TCR) engagement for its induction have led to the view that this is a cytokine (IL-1, IL-23)-mediated response. However, pharmacological inhibition or genetic defects in TCR signaling drastically reduce the nTγδ17 response and/or their presence. To better understand antigen recognition in this rapid IL-17 response, we analyzed the antigen receptor repertoire of IL-1R+/IL-23R+ γδ T cells, a proxy for nTγδ17 cells in naïve animals directly ex vivo, using a barcode-enabled high throughput single-cell TCR sequence analysis. We found that regardless of their anatomical origin, these cells have a highly focused TCR repertoire. In particular, the TCR sequences have limited V gene combinations, little or no junctional diversity and much reduced or no N region diversity. In contrast, IL-23R− cells at mucosal sites similar to most of the splenic γδ T cells and small intestine epithelial γδ lymphocytes expressed diverse TCRs. This remarkable commonality and restricted repertoire of IL-1R+/IL-23R+ γδ T cells underscores the importance of antigen recognition in their establishment/function.

Erratum: Structure and conserved RNA binding of the PAZ domain

This corrects the article DOI: nature02129

Clonal expansion and interrelatedness of distinct B-lineage compartments in multiple myeloma bone marrow

Only some multiple myeloma patients benefit from cellular immunotherapies. A single-cell, high-throughput methodology was developed that determines the phenotypic range of a given Bor plasma cell clone, which could aid identification of those most likely to respond. Multiple myeloma is characterized by the clonal expansion of malignant plasma cells in the bone marrow. But the phenotypic diversity and the contribution of less predominant B-lineage clones to the biology of this disease have been controversial. Here, we asked whether cells bearing the dominant multiple myeloma immunoglobulin rearrangement occupy phenotypic compartments other than that of plasma cells. To accomplish this, we combined 13-parameter FACS index sorting and t-Stochastic Neighbor Embedding (t-SNE) visualization with high-throughput single-cell immunoglobulin sequencing to track selected B-lineage clones across different stages of human B-cell development. As expected, the predominant clones preferentially mapped to aberrant plasma cell compartments, albeit phenotypically altered from wild type. Interestingly, up to 1.2% of cells of the predominant clones colocalized with B-lineage cells of a normal phenotype. In addition, minor clones with distinct immunoglobulin sequences were detected in up to 9% of sequenced cells, but only 2 out of 12 of these clones showed aberrant immune phenotypes. The majority of these minor clones showed intraclonal silent nucleotide differences within the CDR3s and varying frequencies of somatic mutations in the immunoglobulin genes. Therefore, the phenotypic range of multiple myeloma cells in the bone marrow is not confined to aberrant-phenotype plasma cells but extends to low frequencies of normal-phenotype B cells, in line with the recently reported success of B cell–targeting cellular therapies in some patients. The majority of minor clones result from parallel nonmalignant expansion. Cancer Immunol Res; 5(9); 744–54. ©2017 AACR.

Corrigendum: Linking T-cell receptor sequence to functional phenotype at the single-cell level

210 VOLUME 33 NUMBER 2 FEBRUARY 2015 NATURE BIOTECHNOLOGY Erratum: Reinventing tech transfer Brady Huggett Nat. Biotechnol. 32, 1184–1191 (2014); published online 5 December 2014; corrected after print 9 December 2014 In the version of this article initially published online, the name of the University of Pennsylvania president Amy Gutmann was misspelled. The error has been corrected in the HTML and PDF versions of the article.