All Publications

  • Transcript-indexed ATAC-seq for precision immune profiling. Nature medicine Satpathy, A. T., Saligrama, N., Buenrostro, J. D., Wei, Y., Wu, B., Rubin, A. J., Granja, J. M., Lareau, C. A., Li, R., Qi, Y., Parker, K. R., Mumbach, M. R., Serratelli, W. S., Gennert, D. G., Schep, A. N., Corces, M. R., Khodadoust, M. S., Kim, Y. H., Khavari, P. A., Greenleaf, W. J., Davis, M. M., Chang, H. Y. 2018


    T cells create vast amounts of diversity in the genes that encode their T cell receptors (TCRs), which enables individual clones to recognize specific peptide-major histocompatibility complex (MHC) ligands. Here we combined sequencing of the TCR-encoding genes with assay for transposase-accessible chromatin with sequencing (ATAC-seq) analysis at the single-cell level to provide information on the TCR specificity and epigenomic state of individual T cells. By using this approach, termed transcript-indexed ATAC-seq (T-ATAC-seq), we identified epigenomic signatures in immortalized leukemic T cells, primary human T cells from healthy volunteers and primary leukemic T cells from patient samples. In peripheral blood CD4+ T cells from healthy individuals, we identified cis and trans regulators of naive and memory T cell states and found substantial heterogeneity in surface-marker-defined T cell populations. In patients with a leukemic form of cutaneous T cell lymphoma, T-ATAC-seq enabled identification of leukemic and nonleukemic regulatory pathways in T cells from the same individual by allowing separation of the signals that arose from the malignant clone from the background T cell noise. Thus, T-ATAC-seq is a new tool that enables analysis of epigenomic landscapes in clonal T cells and should be valuable for studies of T cell malignancy, immunity and immunotherapy.

    View details for PubMedID 29686426

  • Copy number variation in Y chromosome multicopy genes is linked to a paternal parent-of-origin effect on CNS autoimmune disease in female offspring. Genome biology Case, L. K., Wall, E. H., Osmanski, E. E., Dragon, J. A., Saligrama, N., Zachary, J. F., Lemos, B., Blankenhorn, E. P., Teuscher, C. 2015; 16: 28-?


    The prevalence of some autoimmune diseases is greater in females compared with males, although disease severity is often greater in males. The reason for this sexual dimorphism is unknown, but it may reflect negative selection of Y chromosome-bearing sperm during spermatogenesis or male fetuses early in the course of conception/pregnancy. Previously, we showed that the sexual dimorphism in experimental autoimmune encephalomyelitis (EAE) is associated with copy number variation (CNV) in Y chromosome multicopy genes. Here, we test the hypothesis that CNV in Y chromosome multicopy genes influences the paternal parent-of-origin effect on EAE susceptibility in female mice.We show that C57BL/6 J consomic strains of mice possessing an identical X chromosome and CNV in Y chromosome multicopy genes exhibit sperm head abnormalities and female-biased sex ratio. This is consistent with X-Y intragenomic conflict arising from an imbalance in CNV between homologous X:Y chromosome multicopy genes. These males also display paternal transmission of EAE to female offspring and differential loading of microRNAs within the sperm nucleus. Furthermore, in humans, families of probands with multiple sclerosis similarly exhibit a female-biased sex ratio, whereas families of probands affected with non-sexually dimorphic autoimmune diseases exhibit unbiased sex ratios.These findings provide evidence for a mechanism at the level of the male gamete that contributes to the sexual dimorphism in EAE and paternal parent-of-origin effects in female mice, raising the possibility that a similar mechanism may contribute to the sexual dimorphism in multiple sclerosis.

    View details for DOI 10.1186/s13059-015-0591-7

    View details for PubMedID 25886764

    View details for PubMedCentralID PMC4396973

  • Systemic lack of canonical histamine receptor signaling results in increased resistance to autoimmune encephalomyelitis. Journal of immunology (Baltimore, Md. : 1950) Saligrama, N., Case, L. K., Del Rio, R., Noubade, R., Teuscher, C. 2013; 191 (2): 614?22


    Histamine (HA) is a key regulator of experimental allergic encephalomyelitis (EAE), the autoimmune model of multiple sclerosis. HA exerts its effects through four known G-protein-coupled receptors: H1, H2, H3, and H4 (histamine receptors; H1-4R). Using HR-deficient mice, our laboratory has demonstrated that H1R, H2R, H3R, and H4R play important roles in EAE pathogenesis, by regulating encephalitogenic T cell responses, cytokine production by APCs, blood-brain barrier permeability, and T regulatory cell activity, respectively. Histidine decarboxylase-deficient mice (HDCKO), which lack systemic HA, exhibit more severe EAE and increased Th1 effector cytokine production by splenocytes in response to myelin oligodendrocyte gp35-55. In an inverse approach, we tested the effect of depleting systemic canonical HA signaling on susceptibility to EAE by generating mice lacking all four known G-protein-coupled-HRs (H1-4RKO mice). In this article, we report that in contrast to HDCKO mice, H1-4RKO mice develop less severe EAE compared with wild-type animals. Furthermore, splenocytes from immunized H1-4RKO mice, compared with wild-type mice, produce a lower amount of Th1/Th17 effector cytokines. The opposing results seen between HDCKO and H1-4RKO mice suggest that HA may signal independently of H1-4R and support the existence of an alternative HAergic pathway in regulating EAE resistance. Understanding and exploiting this pathway has the potential to lead to new disease-modifying therapies in multiple sclerosis and other autoimmune and allergic diseases.

    View details for DOI 10.4049/jimmunol.1203137

    View details for PubMedID 23772030

  • The Y chromosome as a regulatory element shaping immune cell transcriptomes and susceptibility to autoimmune disease. Genome research Case, L. K., Wall, E. H., Dragon, J. A., Saligrama, N., Krementsov, D. N., Moussawi, M., Zachary, J., Huber, S. A., Blankenhorn, E. P., Teuscher, C. 2013


    Understanding the DNA elements that constitute and control the regulatory genome is critical for the appropriate therapeutic management of complex diseases. Here, using chromosome Y (ChrY) consomic mouse strains on the C57BL/6J (B6) background, we show that susceptibility to two diverse animal models of autoimmune disease, experimental allergic encephalomyelitis (EAE) and experimental myocarditis, correlates with the natural variation in copy number of Sly and Rbmy multicopy ChrY genes. On the B6 background, ChrY possesses gene regulatory properties that impact genome-wide gene expression in pathogenic CD4+ T cells. Using a ChrY consomic strain on the SJL background, we discovered a preference for ChrY-mediated gene regulation in macrophages, the immune cell subset underlying the EAE sexual dimorphism in SJL mice, rather than CD4+ T cells. Importantly, in both genetic backgrounds, an inverse correlation exists between the number of Sly and Rbmy ChrY gene copies and the number of significantly upregulated genes in immune cells, thereby supporting a link between copy number variation of Sly and Rbmy with the ChrY genetic element exerting regulatory properties. Additionally, we show that ChrY polymorphism can determine the sexual dimorphism in EAE and myocarditis. In humans, an analysis of the CD4+ T cell transcriptome from male multiple sclerosis patients versus healthy controls provides further evidence for an evolutionarily conserved mechanism of gene regulation by ChrY. Thus, as in drosophila, these data establish the mammalian ChrY as a member of the regulatory genome due to its ability to epigenetically regulate genome-wide gene expression in immune cells.

    View details for DOI 10.1101/gr.156703.113

    View details for PubMedID 23800453

  • Histamine H2 receptor signaling x environment interactions determine susceptibility to experimental allergic encephalomyelitis. FASEB journal : official publication of the Federation of American Societies for Experimental Biology Saligrama, N., Case, L. K., Krementsov, D. N., Teuscher, C. 2013


    Histamine and its receptors are important in both multiple sclerosis and experimental allergic encephalomyelitis (EAE). C57BL/6J (B6) mice deficient for the histamine H2 receptor (H2RKO) are less susceptible to EAE and exhibit blunted Th1 responses. However, whether decreased antigen-specific T-cell effector responses in H2RKO mice were due to a lack of H2R signaling in CD4(+) T cells or antigen-presenting cells has remained unclear. We generated transgenic mice expressing H2R specifically in T cells on the H2RKO background, and, using wild-type B6 and H2RKO mice as controls, induced EAE either in the presence or absence of the ancillary adjuvant pertussis toxin (PTX), which models the effects of infectious inflammatory stimuli on autoimmune disease. We monitored the mice for clinical signs of EAE and neuropathology, as well as effector T-cell responses using flow cytometry. EAE severity and neuropathology in H2RKO mice expressing H2R exclusively in T cells become equal to those in wild-type B6 mice only when PTX is used to elicit disease. EAE complementation was associated with frequencies of CD4(+)IFN-?(+) and CD4(+)IL-17(+) cells that are equal to or greater than those in wild-type B6, respectively. Thus, the regulation of encephalitogenic T-cell responses and EAE susceptibility by H2R signaling in CD4(+) T cells is dependent on gene × environment interactions.-Saligrama, N., Case, L. K., Krementsov, D. N., Teuscher, C. Histamine H2 receptor signaling × environment interactions determine susceptibility to experimental allergic encephalomyelitis.

    View details for DOI 10.1096/fj.13-239939

    View details for PubMedID 24371118

  • H1R expression by CD11B(+) cells is not required for susceptibility to experimental allergic encephalomyelitis CELLULAR IMMUNOLOGY Saligrama, N., Noubade, R., Case, L. K., Poynter, M. E., Teuscher, C. 2012; 278 (1-2): 27-34


    The histamine H(1) receptor (Hrh1/H(1)R) was identified as an autoimmune disease gene in experimental allergic encephalomyelitis (EAE), the principal autoimmune model of multiple sclerosis (MS). Previously, we showed that selective re-expression of H(1)R by endothelial cells or T cells in H(1)RKO mice significantly reduced or complemented EAE severity and cytokine responses, respectively. H(1)R regulates innate immune cells, which in turn influences peripheral and central nervous system CD4(+) T cell effector responses. Therefore, we selectively re-expressed H(1)R in CD11b(+) cells of H(1)RKO mice to test the hypothesis that H(1)R signaling in these cells contributes to EAE susceptibility. We demonstrate that transgenic re-expression of H(1)R by H(1)RKO-CD11b(+) cells neither complements EAE susceptibility nor T cell cytokine responses highlighting the cell-specific effects of Hrh1 in the pathogenesis of EAE and MS, and the need for cell-specific targeting in optimizing therapeutic interventions based on such genes.

    View details for DOI 10.1016/j.cellimm.2012.06.012

    View details for Web of Science ID 000311812000005

    View details for PubMedID 23121973

  • Combinatorial roles for histamine H1-H2 and H3-H4 receptors in autoimmune inflammatory disease of the central nervous system EUROPEAN JOURNAL OF IMMUNOLOGY Saligrama, N., Noubade, R., Case, L. K., del Rio, R., Teuscher, C. 2012; 42 (6): 1536-?


    Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system in which histamine (HA) and its receptors have been implicated in disease pathogenesis. HA exerts its effects through four different G protein-coupled receptors designated H(1)-H(4). We previously examined the effects of traditional single HA receptor (HR) knockouts (KOs) in experimental allergic encephalomyelitis (EAE), the autoimmune model of MS. Our results revealed that H(1) R and H(2) R are propathogenic, while H(3) R and H(4) R are antipathogenic. This suggests that combinatorial targeting of HRs may be an effective disease-modifying therapy (DMT) in MS. To test this hypothesis, we generated H(1) H(2) RKO and H(3) H(4) RKO mice and studied them for susceptibility to EAE. Compared with wild-type (WT) mice, H(1) H(2) RKO mice developed a less severe clinical disease course, whereas the disease course of H(3) H(4) RKO mice was more severe. H(1) H(2) RKO mice also developed less neuropathology and disrupted blood brain barrier permeability compared with WT and H(3) H(4) RKO mice. Additionally, splenocytes from immunized H(1) H(2) RKO mice produced less interferon(IFN)-? and interleukin(IL)-17. These findings support the concept that combined pharmacological targeting of HRs may be an appropriate ancillary DMT in MS and other immunopathologic diseases.

    View details for DOI 10.1002/eji.201141859

    View details for Web of Science ID 000304997800018

    View details for PubMedID 22678907

  • Histamine H-4 Receptor Optimizes T Regulatory Cell Frequency and Facilitates Anti-Inflammatory Responses within the Central Nervous System JOURNAL OF IMMUNOLOGY del Rio, R., Noubade, R., Saligrama, N., Wall, E. H., Krementsov, D. N., Poynter, M. E., Zachary, J. F., Thurmond, R. L., Teuscher, C. 2012; 188 (2): 541-547


    Histamine is a biogenic amine that mediates multiple physiological processes, including immunomodulatory effects in allergic and inflammatory reactions, and also plays a key regulatory role in experimental allergic encephalomyelitis, the autoimmune model of multiple sclerosis. The pleiotropic effects of histamine are mediated by four G protein-coupled receptors, as follows: Hrh1/H(1)R, Hrh2/H(2)R, Hrh3/H(3)R, and Hrh4/H(4)R. H(4)R expression is primarily restricted to hematopoietic cells, and its role in autoimmune inflammatory demyelinating disease of the CNS has not been studied. In this study, we show that, compared with wild-type mice, animals with a disrupted Hrh4 (H(4)RKO) develop more severe myelin oligodendrocyte glycoprotein (MOG)(35\x{2013}55)-induced experimental allergic encephalomyelitis. Mechanistically, we also show that H(4)R plays a role in determining the frequency of T regulatory (T(R)) cells in secondary lymphoid tissues, and regulates T(R) cell chemotaxis and suppressor activity. Moreover, the lack of H(4)R leads to an impairment of an anti-inflammatory response due to fewer T(R) cells in the CNS during the acute phase of the disease and an increase in the proportion of Th17 cells.

    View details for DOI 10.4049/jimmunol.1101498

    View details for Web of Science ID 000299323700006

    View details for PubMedID 22147765

  • Chromosome Y Regulates Survival Following Murine Coxsackievirus B3 Infection G3-GENES GENOMES GENETICS Case, L. K., Toussaint, L., Moussawi, M., Roberts, B., Saligrama, N., Brossay, L., Huber, S. A., Teuscher, C. 2012; 2 (1): 115-121


    Coxsackievirus B3 (CVB3) contributes to the development of myocarditis, an inflammatory heart disease that predominates in males, and infection is a cause of unexpected death in young individuals. Although gonadal hormones contribute significantly to sex differences, sex chromosomes may also influence disease. Increasing evidence indicates that Chromosome Y (ChrY) genetic variants can impact biological functions unrelated to sexual differentiation. Using C57BL/6J (B6)-ChrY consomic mice, we show that genetic variation in ChrY has a direct effect on the survival of CVB3-infected animals. This effect is not due to potential Sry-mediated differences in prenatal testosterone exposure or to differences in adult testosterone levels. Furthermore, we show that ChrY polymorphism influences the percentage of natural killer T cells in B6-ChrY consomic strains but does not underlie CVB3-induced mortality. These data underscore the importance of investigating not only the hormonal regulation but also ChrY genetic regulation of cardiovascular disease and other male-dominant, sexually dimorphic diseases and phenotypes.

    View details for DOI 10.1534/g3.111.001610

    View details for Web of Science ID 000312410700013

    View details for PubMedID 22384388

  • Genetics of experimental allergic encephalomyelitis supports the role of T helper cells in multiple sclerosis pathogenesis ANNALS OF NEUROLOGY Blankenhorn, E. P., Butterfield, R., Case, L. K., Wall, E. H., del Rio, R., Diehl, S. A., Krementsov, D. N., Saligrama, N., Teuscher, C. 2011; 70 (6): 887-896


    The major histocompatibility complex (MHC) is the primary genetic contributor to multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE), but multiple additional interacting loci are required for genetic susceptibility. The identity of most of these non-MHC genes is unknown. In this report, we identify genes within evolutionarily conserved genetic pathways leading to MS and EAE.To identify non-MHC binary and quantitative trait loci (BTL/QTL) important in the pathogenesis of EAE, we generated phenotype-selected congenic mice using EAE-resistant B10.S and EAE-susceptible SJL mice. We hypothesized that genes linked to EAE BTL/QTL and MS-GWAS can be identified if they belong to common evolutionarily conserved pathways, which can be identified with a bioinformatic approach using Ingenuity software.Many known BTL/QTL were retained and linked to susceptibility during phenotype selection, the most significant being a region on chromosome 17 distal to H2 (Eae5). We show in pathway analysis that T helper (T(H))-cell differentiation genes are critical for both diseases. Bioinformatic analyses predicted that Eae5 is important in CD4 T-effector and/or Foxp3(+) T-regulatory cells (Tregs), and we found that B10.S-Eae5(SJL) congenic mice have significantly greater numbers of lymph node CD4 and Tregs than B10.S mice.These results support the polygenic model of MS/EAE, whereby MHC and multiple minor loci are required for full susceptibility, and confirm a critical genetic dependence on CD4 T(H)-cell differentiation and function in the pathogenesis of both diseases.

    View details for DOI 10.1002/ana.22642

    View details for Web of Science ID 000298332300007

    View details for PubMedID 22190363

  • Activation of p38 MAPK in CD4 T cells controls IL-17 production and autoimmune encephalomyelitis BLOOD Noubade, R., Krementsov, D. N., del Rio, R., Thornton, T., Nagaleekar, V., Saligrama, N., Spitzack, A., Spach, K., Sabio, G., Davis, R. J., Rincon, M., Teuscher, C. 2011; 118 (12): 3290-3300


    Although several transcription factors have been shown to be critical for the induction and maintenance of IL-17 expression by CD4 Th cells, less is known about the role of nontranscriptional mechanisms. Here we show that the p38 MAPK signaling pathway is essential for in vitro and in vivo IL-17 production by regulating IL-17 synthesis in CD4 T cells through the activation of the eukaryotic translation initiation factor 4E/MAPK-interacting kinase (eIF-4E/MNK) pathway. We also show that p38 MAPK activation is required for the development and progression of both chronic and relapsing-remitting forms of experimental allergic encephalomyelitis (EAE), the principal autoimmune model of multiple sclerosis. Furthermore, we show that regulation of p38 MAPK activity specifically in T cells is sufficient to modulate EAE severity. Thus, mechanisms other than the regulation of gene expression also contribute to Th17 cell effector functions and, potentially, to the pathogenesis of other Th17 cell-mediated diseases.

    View details for DOI 10.1182/blood-2011-02-336552

    View details for Web of Science ID 000295120900016

    View details for PubMedID 21791428

  • Mapping the complement C1q binding site in Haemonchus contortus calreticulin MOLECULAR AND BIOCHEMICAL PARASITOLOGY Naresha, S., Suryawanshi, A., Agarwal, M., Singh, B. P., Joshi, P. 2009; 166 (1): 42-46


    Haemonchus contortus is an economically important gastrointestinal parasite of domestic animals. The parasite secretes calreticulin (CalR), a Ca(++) binding protein which modulates the host immune response. One way by which this protein acts is by inhibiting the classical complement pathway by binding to complement C1q protein. Understanding CalR-C1q interaction is important to develop methods to enhance host immune response. In this study, we have mapped the regions in the N-domain of CalR that facilitates C1q binding by generating small recombinant fragments of the domain and using synthetic peptides. In addition to already identified C1q binding motifs in human CalR, two additional sites in the N-domain of H. contortus were revealed with the following sequences-GKYYDDAKRD and the AKFPKKFT. The significance of multiple C1q binding motifs in CalR is discussed in relation to host-parasite interactions.

    View details for DOI 10.1016/j.molbiopara.2009.02.007

    View details for Web of Science ID 000266088900007

    View details for PubMedID 19428671

  • Autoimmune disease-associated histamine receptor H-1 alleles exhibit differential protein trafficking and cell surface expression JOURNAL OF IMMUNOLOGY Noubade, R., Saligrama, N., Spach, K., del Rio, R., Blankenhorn, E. P., Kantidakis, T., Milligan, G., Rincon, M., Teuscher, C. 2008; 180 (11): 7471-7479


    Structural polymorphisms (L263P, M313V, and S331P) in the third intracellular loop of the murine histamine receptor H(1) (H(1)R) are candidates for Bphs, a shared autoimmune disease locus in experimental allergic encephalomyelitis and experimental allergic orchitis. The P-V-P haplotype is associated with increased disease susceptibility (H(1)R(S)) whereas the L-M-S haplotype is associated with less severe disease (H(1)R(R)). In this study, we show that selective re-expression of the H(1)R(S) allele in T cells fully complements experimental allergic encephalomyelitis susceptibility and the production of disease-associated cytokines while selective re-expression of the H(1)R(R) allele does not. Mechanistically, we show that the two H(1)R alleles exhibit differential cell surface expression and altered intracellular trafficking, with the H(1)R(R) allele being retained within the endoplasmic reticulum. Moreover, we show that all three residues (L-M-S) comprising the H(1)R(R) haplotype are required for altered expression. These data are the first to demonstrate that structural polymorphisms influencing cell surface expression of a G protein-coupled receptor in T cells regulates immune functions and autoimmune disease susceptibility.

    View details for Web of Science ID 000257507300044

    View details for PubMedID 18490747

  • SNPs upstream of the minimal promoter control IL-2 expression and are candidates for the autoimmune disease-susceptibility locus Aod2/Idd3/Eae3 GENES AND IMMUNITY del Rio, R., Noubade, R., Subramanian, M., Saligrama, N., Diehl, S., Rincon, M., Teuscher, C. 2008; 9 (2): 115-121


    IL-2, a T-cell growth and differentiation factor, plays an important role in immune homeostasis. Previously, we identified IL2 as a candidate for Aod2, a quantitative trait locus (QTL) controlling susceptibility to autoimmune ovarian dysgenesis (AOD) induced by day 3 neonatal thymectomy. Here, we report the identification of single-nucleotide polymorphisms (SNPs) in a region upstream of the minimal IL2 promoter (-2.8 kb to -300 bp), which distinguish AOD-susceptible A/J and AOD-resistant C57BL/6J (B6/J) mice. Six of the SNPs (-1010 C --> T, -962 C --> T, -926/-925 Delta Delta --> AC, -921 T --> C, -914 T --> C and -674 G --> A) contribute to the enhanced transcriptional activity of the extended B6/J promoter relative to A/J. Importantly, the -1010 SNP resides within a canonical AP-1-binding motif with the C --> T transition at this site abrogating AP-1 binding. Moreover, these SNPs segregate with differential production of IL-2 by CD4(+) T cells as well as susceptibility alleles at Idd3 and Eae3, QTL controlling insulin-dependent diabetes mellitus and experimental allergic encephalomyelitis. These are the first SNPs identified within the extended murine IL2 promoter that control differential IL-2 transcription in CD4(+) T cells, and, as such, they are not only candidates for Aod2, but are also candidates for a shared autoimmune disease-susceptibility locus underlying Idd3 and Eae3.

    View details for DOI 10.1038/sj.gene.6364455

    View details for Web of Science ID 000253815600004

    View details for PubMedID 18200031

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