Single-Cell Analysis of CD4 T Cells in Type 1 Diabetes: from Mouse to Man, How to Perform Mechanistic Studies

1886 Diabetes Volume 68, October 2019 Single-Cell Analysis of CD4 T Cells in Type 1 Diabetes: From Mouse to Man, How to Perform Mechanistic Studies Siddhartha Sharma,1 Jeremy Pettus,2 Michael Gottschalk,3 Brian Abe,1 Peter Gottlieb,4 and Luc Teyton1 Diabetes 2019;68:1886–1891 | https://doi.org/10.2337/dbi18-0064 Type 1 diabetes is the prototypical CD4 T cell–mediated acid at position b57, unlike any other HLA-DR, -DQ, or -DP autoimmune disease. Its genetic linkage to a single poly- molecules, HLA-DQ2 (HLA-DQb0201), and HLA-DQ8 (HLA- morphism at position 57 of the HLA class II DQb chain DQb0302) (Fig. 1). This genetic terrain allows multiple makes it unique to study the molecular link between HLA environmental factors to emerge as disease triggers (5). and disease. However, investigating this relationship has At first glance, an association of a CD4 T cell–mediated been limited by a series of anatomical barriers, the small disease with HLA class II gene products, whose function is size and dispersion of the insulin-producing organ, and to present peptides to CD4 T cells, appears easily explain- the scarcity of appropriate techniques and reagents to able. Over the years, the most obvious, nonexclusive fi interrogate antigen-speci c CD4 T cells both in man and theories have been tested: instability and poor peptide rodent models. Over the past few years, single-cell tech- binding of diabetogenic HLA class II molecules (6), unique nologies, paired with new biostatistical methods, have peptide repertoire of the same molecules (7), T cells fo- changed this landscape. Using these tools, we have cused on the recognition of HLA-DQb57 (8), failed thymic identified the first molecular link between MHC class II selection of autoreactive T cells (9), and abnormal T-cell and the onset of type 1 diabetes. The translation of these – observations to man is within reach using similar binding to autoimmune peptide MHC complexes (10). approaches and the lessons learned from rodent models. While all of those might bear truth and give some level of understanding of what the b57 residue might do, none could formally associate the mutation to a molecular Type 1 diabetes is a CD4 T cell–mediated autoimmune mechanism leading to diabetes. The closest one to explain- PERSPECTIVES IN DIABETES disease that results in the destruction of the pancreatic ing the association of the same mutation with a disease b-cells that produce insulin. Type 1 diabetes is also the was in the context of celiac disease, where the same poster child of autoimmune diseases linked to genetic HLA-DQ molecules are strongly predisposing to onset susceptibility. While more than 40 genes have been de- and also promote a frequent association with type 1 di- scribed in this inherited landscape (1), the association that abetes (11). In this instance, it was shown in transgenic stands out is with the HLA class II locus on chromosome HLA-DQ8 mice, and for some human CD4 T-cell clones, 2 6 with a P value of 10 123, suggesting not only influence that gliadin peptides were recognized by T-cell receptors but causality. This notion is reinforced by the fact that the (TCRs) bearing a negatively charged residue in the first fine mapping of this linkage with the HLA class II region segment of the CDR3b loop (12). Interestingly, most identifies a single polymorphism at position 57 of the native gliadin peptides are glutamine rich and neutral HLA-DQb chain as being responsible for most of the unless deamidated by tissue transglutaminase (13) but association (2,3), while non-HLA loci and genes likely play can still be presented by HLA-DQ2 and -DQ8 molecules, aroleininfluencing the progression to disease onset (4). which usually prefer peptides with negatively charged Only two common haplotypes of HLA-DQ carry a nonaspartic amino acids in their C-termini (7). The acidic residues 1Department of Immunology and Microbiology, The Scripps Research Institute, La Received 16 July 2019 and accepted 21 July 2019 Jolla, CA B.A. is currently affiliated with Division of Immunology & Rheumatology, Stanford 2 Division of Endocrinology and Metabolism, University of California, San Diego, San University School of Medicine, Stanford, CA. Diego, CA © 2019 by the American Diabetes Association. Readers may use this article as 3University of California, San Diego Medical Center, San Diego, CA long as the work is properly cited, the use is educational and not for profit, and the 4Department of Pediatrics and Department of Immunology & Microbiology, work is not altered. More information is available at http://www.diabetesjournals University of Colorado School of Medicine, and Barbara Davis Center for Diabetes, .org/content/license. Denver, CO Corresponding author: Luc Teyton, [email protected] diabetes.diabetesjournals.org Sharma and Associates 1887 also the making of T cell–detecting reagents such as MHC tetramers. The results from our structural studies were surprising; while we expected the CDR3b negative charge to sit in proximity to the positive patch of the b57 residue and establish a salt bridge, shifting the TCR over the COOH- terminal part of the p-MHC complex, the TCR was found in a normal diagonal position putting the CDR3b far away from b57 (8). However, biophysical studies demonstrated that the complementation of charges between TCR and p-MHC were operating through Coulombic interactions, a phenomenon that allows surfaces of opposite charges to enhance dehydration and increase on-rates of binding interaction. In any case, this deep knowledge of I-Ag7 and HLA-DQ molecules could not establish a direct link between MHC class II, position b57, and type 1 diabetes. In the absence of — Figure 1 Depiction of the three-dimensional structure of HLA-DQ8, a rodent model and MHC tetramers, the celiac disease the prototypical diabetogenic molecule. In this top view of the molecule, the peptide binding groove is horizontal and limited at observation could not be tested further, while the absence the top by the a helix of the a chain (gray) and at the bottom by the a of antigen-specific reagents was impeding our studies in helix of the b chain (purple); the peptide is in yellow with its 9th mice. In addition, studying type 1 diabetes offers addi- residue represented in spheres. Position b57, also represented in spheres, and colored in green, limits the outside of the P9 pocket tional challenges that were insurmountable for decades where the P9 residue of the peptide is sitting. The nature of the both in mouse and man. The two most challenging were relationship b57-P9 residue and its interpretation by TCRs will drive thesizeoftheorganthatproducesinsulin(,1.5 g of tissue anti–b-cell autoimmunity. Image was generated using pdb 1JK6 in a human) and the asynchrony of the lesions across the from the Protein Data Bank (39). ;1 million islets. These numbers and the low efficiency of the autoimmune process, which takes on average 15% of a life span to reach completion (5 years in humans, occupy the P9 pocket of MHC and compensate for the loss 15 weeks in mice), likely translate to a very small number of the aspartic acid at position b57, which is an integral of anti–b-cell–specific CD4 T cells locally and in circulation. part of the outside wall of this MHC pocket (14) (Fig. 1). This situation is very consequential, making the diagnosis The need for side chains anchoring into pockets for of the preclinical phase of disease extremely difficult and peptide binding as we see for MHC class I and HLA-DR mechanistic studies very challenging. molecules has been lost for HLA-DQ molecules (14,15), As often in science, advancements in technology open allowing more diverse and promiscuous binding. As a con- access to the next level of understanding. About a decade sequence of this mode of binding, peptide repertoire is ago, microfluidic systems allowed the isolation of single much broader for HLA-DQ than for HLA-DR molecules, cells in microchambers that can be used as reaction ves- but the affinities of peptide binding are much lower, often sels. Concomitant with the rise of next-generation in the mid to high micromolar range (12). This biophys- sequencing, this new engineering gave birth to the rapidly ical detail is often overlooked, although it informs us of expanding world of single-cell technologies. We can now two important convergent features of autoimmunity: probe with single-cell resolution genetic differences, dif- first, because there is a threshold to activate T cells, ferential gene expression, genome-wide epigenetic mod- low affinity peptides must be abundant to compensate ifications, and large sets of unique proteins. Most for short binding half-lives; second, homozygosity of the importantly, these approaches have allowed us to inter- susceptibility HLA genes, as often observed in autoim- rogate very small numbers of cells as we expect in biopsies munity, is essential to increase cell surface expression of or circulating blood. As it stands today, single-cell tech- the diabetogenic peptide–MHC complexes. In type 1 di- nologies have allowed us to refine our translational studies abetes, this latter issue is further compounded by the fact from mouse to man and approach mechanistic under- that HLA-DQ2/DQ8 heterozygotes are also at a higher standing of disease, and, most importantly, might open risk of disease due to the expression of transdimers in the possibility of an early preclinical diagnosis and the which the b57 position always lacks the normal aspartic monitoring of treatment in man. acid (16). In an effort to understand how neutral peptides bound to HLA-DQ8 could select TCRs with a negatively A Nonexhaustive Review of Single-Cell Technologies charged residue in their CDR3b loop, we extended the Bulk analysis techniques have been the primary method observation to the mouse model and a neutral peptide utilized thus far to understand autoimmunity in type from hen egg lysozyme in order to gain structural in- 1 diabetes.

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