Early progression to active tuberculosis is a highly heritable trait driven by 3q23 in Peruvians

Abstract: Of the 1.8 billion people worldwide infected with Mycobacterium tuberculosis, 5–15% will develop active tuberculosis (TB). Approximately half will progress to active TB within the first 18 months after infection, presumably because they fail to mount an effective initial immune response. Here, in a genome-wide genetic study of early TB progression, we genotype 4002 active TB cases and their household contacts in Peru. We quantify genetic heritability (hg2) of early TB progression to be 21.2% (standard error 0.08). This suggests TB progression has a strong genetic basis, and is comparable to traits with well-established genetic bases. We identify a novel association between early TB progression and variants located in a putative enhancer region on chromosome 3q23 (rs73226617, OR = 1.18; P = 3.93 × 10−8). With in silico and in vitro analyses we identify rs73226617 or rs148722713 as the likely functional variant and ATP1B3 as a potential causal target gene with monocyte specific function

Multimodal memory T cell profiling identifies a reduction in a polyfunctional Th17 state associated with tuberculosis progression

Mycobacterium tuberculosis (M.tb) results in 10 million active tuberculosis (TB) cases and 1.5 million deaths each year, making it the world's leading infectious cause of death. Infection leads to either an asymptomatic latent state or TB disease. Memory T cells have been implicated in TB disease progression, but the specific cell states involved have not yet been delineated because of the limited scope of traditional profiling strategies. Furthermore, immune activation during infection confounds underlying differences in T cell state distributions that influence risk of progression. Here, we used a multimodal single-cell approach to integrate measurements of transcripts and 30 functionally relevant surface proteins to comprehensively define the memory T cell landscape at steady state (i.e., outside of active infection). We profiled 500,000 memory T cells from 259 Peruvians > 4.7 years after they had either latent M.tb infection or active disease and defined 31 distinct memory T cell states, including a CD4+CD26+CD161+CCR6+ effector memory state that was significantly reduced in patients who had developed active TB (OR = 0.80, 95% CI: 0.73-0.87, p = 1.21 x 10-6). This state was also polyfunctional; in ex vivo stimulation, it was enriched for IL-17 and IL-22 production, consistent with a Th17-skewed phenotype, but also had more capacity to produce IFNgamma than other CD161+CCR6+ Th17 cells. Additionally, in progressors, IL-17 and IL-22 production in this cell state was significantly lower than in non-progressors. Reduced abundance and function of this state may be an important factor in failure to control M.tb infection. ### Competing Interest Statement The authors have declared no competing interest

Dissecting the Genetics and Function of CD5+ B and invariant NKT cells using Congenic Lupus-Prone Mouse Strains

Systemic lupus erythematosus (SLE) is a complex multisystem autoimmune disorder characterized by the production of antibodies to nuclear antigens. Congenic strains derived from lupus-prone mice have been extremely useful in determining the pathogenesis of this condition. These mice, which harbor selected genetic elements containing susceptibility loci, can be used to identify key pathways and immune networks involved in disease initiation and progression. Alternatively, these same models can be used to identify the role of suppressive regulatory cells involved in preventing disease. In this thesis, the role and function of CD5+ and invariant Natural Killer T (iNKT) innate lymphocytes were investigated in the suppression of disease in New Zealand Black lupus-prone congenic mice. Through adoptive transfer and knockout experiments, CD5+ B cells, but not iNKT cells, were shown to suppress autoimmunity by inhibiting the frequencies of proinflammatory T cell subsets. As these CD5+ B cells secreted IL-10, which has been shown previously to suppress disease, the role of this cytokine was further investigated through knockout experiments that highlighted its critical role in maintaining CD5+ B, iNKT, and Natural Killer cell homeostasis. Finally, the immunogenetic basis of impaired iNKT cell function in the congenic mouse strains was investigated, revealing a critical role for Ly108 in iNKT cell development and function. Collectively, these findings highlight the role of CD5+ B cells and iNKT cells in SLE pathogenesis and demonstrate the power of congenic animals to dissect the genetic basis of disease.Ph.D

Genome editing to define the function of risk loci and variants in rheumatic disease

Discoveries in human genetic studies have revolutionized our understanding of complex rheumatic and autoimmune diseases, including the identification of hundreds of genetic loci and single nucleotide polymorphisms that potentially predispose individuals to disease. However, in most cases, the exact disease-causing variants and their mechanisms of action remain unresolved. Functional follow-up of these findings is most challenging for genomic variants that are in non-coding genomic regions, where the large majority of common disease-associated variants are located, and/or that probably affect disease progression via cell type-specific gene regulation. To deliver on the therapeutic promise of human genetic studies, defining the mechanisms of action of these alleles is essential. Genome editing technology, such as CRISPR-Cas, has created a vast toolbox for targeted genetic and epigenetic modifications that presents unprecedented opportunities to decipher disease-causing loci, genes and variants in autoimmunity. In this Review, we discuss the past 5-10 years of progress in resolving the mechanisms underlying rheumatic disease-associated alleles, with an emphasis on how genomic editing techniques can enable targeted dissection and mechanistic studies of causal autoimmune risk variants

Impaired B cell anergy is not sufficient to breach tolerance to nuclear antigen in Vκ8/3H9 lupus-prone mice.

BackgroundSystemic lupus erythematosus (SLE) is a severe autoimmune disease in which immune tolerance defects drive production of pathogenic anti-nuclear autoantibodies. Anergic B cells are considered a potential source of these autoantibodies due to their autoreactivity and overrepresentation in SLE patients. Studies of lupus-prone mice have shown that genetic defects mediating autoimmunity can breach B cell anergy, but how this breach occurs with regards to endogenous nuclear antigen remains unclear. We investigated whether B and T cell defects in congenic mice (c1) derived from the lupus-prone New Zealand Black strain can breach tolerance to nuclear self-antigen in the presence of knock-in genes (Vκ8/3H9; dKI) that generate a ssDNA-reactive, anergic B cell population.MethodsFlow cytometry was used to assess splenic B and T cells from 8-month-old c1 dKI mice and serum autoantibodies were measured by ELISA. dKI B cells stimulated in vitro with anti-IgM were assessed for proliferation and activation by examining CFSE decay and CD86. Cytokine-producing T cells were identified by flow cytometry following culture of dKI splenocytes with PMA and ionomycin. dKI B cells from 6-8-week-old mice were adoptively transferred into 4-month-old wild type recipients and assessed after 7 days via flow cytometry and immunofluorescence microscopy.Resultsc1 dKI mice exhibited B cell proliferation indicative of impaired anergy, but had attenuated autoantibodies and germinal centres compared to wild type littermates. This attenuation appeared to stem from a decrease in PD-1hi T helper cells in the dKI strains, as c1 dKI B cells were recruited to germinal centres when adoptively transferred into c1 wild type mice.ConclusionAnergic, DNA-specific autoreactive B cells only seem to drive profound autoimmunity in the presence of concomitant defects in the T cell subsets that support high-affinity plasma cell production

IL-10 Production Is Critical for Sustaining the Expansion of CD5⁺ B and NKT Cells and Restraining Autoantibody Production in Congenic Lupus-Prone Mice

<div><p>The development and progression of systemic lupus erythematosus is mediated by the complex interaction of genetic and environmental factors. To decipher the genetics that contribute to pathogenesis and the production of pathogenic autoantibodies, our lab has focused on the generation of congenic lupus-prone mice derived from the New Zealand Black (NZB) strain. Previous work has shown that an NZB-derived chromosome 4 interval spanning 32 to 151 Mb led to expansion of CD5⁺ B and Natural Killer T (NKT) cells, and could suppress autoimmunity when crossed with a lupus-prone mouse strain. Subsequently, it was shown that CD5⁺ B cells but not NKT cells derived from these mice could suppress the development of pro-inflammatory T cells. In this paper, we aimed to further resolve the genetics that leads to expansion of these two innate-like populations through the creation of additional sub-congenic mice and to characterize the role of IL-10 in the suppression of autoimmunity through the generation of IL-10 knockout mice. We show that expansion of CD5⁺ B cells and NKT cells localizes to a chromosome 4 interval spanning 91 to 123 Mb, which is distinct from the region that mediates the majority of the suppressive phenotype. We also demonstrate that IL-10 is critical to restraining autoantibody production and surprisingly plays a vital role in supporting the expansion of innate-like populations.</p></div

Multiple tolerance defects contribute to the breach of B cell tolerance in New Zealand Black chromosome 1 congenic mice

<div><p>Lupus is characterized by a loss of B cell tolerance leading to autoantibody production. In this study, we explored the mechanisms underlying this loss of tolerance using B6 congenic mice with an interval from New Zealand Black chromosome 1 (denoted c1(96–100)) sufficient for anti-nuclear antibody production. Transgenes for soluble hen egg white lysozyme (sHEL) and anti-HEL immunoglobulin were crossed onto this background and various tolerance mechanisms examined. We found that c1(96–100) mice produced increased levels of IgM and IgG anti-HEL antibodies compared to B6 mice and had higher proportions of germinal center B cells and long-lived plasma cells, suggesting a germinal center-dependent breach of B cell anergy. Consistent with impaired anergy induction, c1(96–100) double transgenic B cells showed enhanced survival and CD86 upregulation. Hematopoietic chimeric sHEL mice with a mixture of B6 and c1(96–100) HEL transgenic B cells recapitulated these results, suggesting the presence of a B cell autonomous defect. Surprisingly, however, there was equivalent recruitment of B6 and c1(96–100) B cells into germinal centers and differentiation to splenic plasmablasts in these mice. In contrast, there were increased proportions of c1(96–100) T follicular helper cells and long-lived plasma cells as compared to their B6 counterparts, suggesting that both B and T cell defects are required to breach germinal center tolerance in this model. This possibility was further supported by experiments showing an enhanced breach of anergy in double transgenic mice with a longer chromosome 1 interval with additional T cell defects.</p></div

c1(70–100) DTg mice have an enhanced breach of B cell tolerance.

<p>(A) Scatter plots showing serum levels of IgM^a anti-HEL, IgG anti-HEL and IgG anti-ssDNA Abs from 8 week-old to 4 month-old B6 (open circles), c1 (96–100) (filled circles) and c1(70–100) (filled triangles) mice, as measured by ELISA. (B) Shown on the left are flow plots gated upon B220⁺ cells indicating the regions used to gate GC B cells. Scatter plots show the proportion of GC cells within the B220⁺ subset of B6 DTg (open circles), c1(96–100) DTg (filled circles) or c1(70–100) DTg (filled triangles) mice. (C) Flow plots gated on CD4⁺ T cells showing the regions used to gated CXCR5^hi cells (regions were established as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179506#pone.0179506.g004" target="_blank">Fig 4G</a>). Scatter plots show the proportion of these cells within the CD4+ T cell subset in B6 DTg (open circles), c1(96–100) DTg (filled circles), or c1(70–100) DTg (filled triangles) mice. (D) Representative flow cytometry contour plots showing the gating used to identify the proportion of IFN-γ-, IL-17-, and IL-21-producing CD3⁺CD4⁺ T cells in c1(70–100) DTg mice. The quadrants used to define positively and negatively stained cells are indicated. For each condition, the upper plots show unstimulated cells and the lower plots show cells stimulated with PMA and ionomycin. Scatter plots showing the proportion of cytokine-producing T cells in B6 (open circles), c1(96–100) (filled circles) and c1(70–100) (filled triangles) mice. Each symbol represents an individual mouse. Horizontal lines represent the mean. The asterisks indicate p values <0.05 (*) or <0.001 (**). Statistical analyses were performed using a Mann-Whitney <i>U</i> test.</p

The expansion of peritoneal B1a B cells, splenic CD5⁺ B cells, and NKT cells localizes to an NZB-derived interval spanning 91 to 123 Mb on chromosome 4.

<p>(A) Figure illustrating the NZB chromosome 4 congenic strains used in these studies. D4Mit markers demarcate the known boundaries of introgression. Splenic and peritoneal immune cell frequencies were measured by flow cytometry from 4 month old mice. (B) Splenic CD5⁺ B cells were measured based on FSC, SSC, CD19, and CD5 staining. (C) Frequencies of peritoneal cells were identified by granularity and size and gated as CD19⁺ and CD5⁺. (D) Splenic NKT cells were gated based on size and granularity using FSC, SSC, and gated as PBS57 Tetramer⁺ NK1.1^-. (E) Splenic NK cells were measured based on FSC, SSC, and gated as NK1.1⁺ and PBS57 Tetramer^-. Each point represents a single mouse, with the lines indicating the median of each group. Statistical analyses were carried out using a Kruskal-Wallis test with select Dunn multiple comparison posttests to control B6 mice. * P < 0.05, ** P < 0.01, ***P <0.001.</p