, Volume 68, Issue 3, pp 191–204 | Cite as

Refinement of the canine CD1 locus topology and investigation of antibody binding to recombinant canine CD1 isoforms

  • Mette Schjaerff
  • Stefan M. Keller
  • Joseph Fass
  • Lutz Froenicke
  • Robert A. Grahn
  • Leslie Lyons
  • Verena K. Affolter
  • Annemarie T. Kristensen
  • Peter F. Moore
Original Article


CD1 molecules are antigen-presenting glycoproteins primarily found on dendritic cells (DCs) responsible for lipid antigen presentation to CD1-restricted T cells. Despite their pivotal role in immunity, little is known about CD1 protein expression in dogs, notably due to lack of isoform-specific antibodies. The canine (Canis familiaris) CD1 locus was previously found to contain three functional CD1A genes: canCD1A2, canCD1A6, and canCD1A8, where two variants of canCD1A8, canCD1A8.1 and canCD1A8.2, were assumed to be allelic variants. However, we hypothesized that these rather represented two separate genes. Sequencing of three overlapping bacterial artificial chromosomes (BACs) spanning the entire canine CD1 locus revealed canCD1A8.2 and canCD1A8.1 to be located in tandem between canCD1A7 and canCD1C, and canCD1A8.1 was consequently renamed canCD1A9. Green fluorescent protein (GFP)-fused canine CD1 transcripts were recombinantly expressed in 293T cells. All proteins showed a highly positive GFP expression except for canine CD1d and a splice variant of canine CD1a8 lacking exon 3. Probing with a panel of anti-CD1 monoclonal antibodies (mAbs) showed that Ca13.9H11 and Ca9.AG5 only recognized canine CD1a8 and CD1a9 isoforms, and Fe1.5F4 mAb solely recognized canine CD1a6. Anti-CD1b mAbs recognized the canine CD1b protein, but also bound CD1a2, CD1a8, and CD1a9. Interestingly, Ca9.AG5 showed allele specificity based on a single nucleotide polymorphism (SNP) located at position 321. Our findings have refined the structure of the canine CD1 locus and available antibody specificity against canine CD1 proteins. These are important fundamentals for future investigation of the role of canine CD1 in lipid immunity.


Canine CD1 genes Canine CD1 proteins Antibody recognition of canine CD1 Single nucleotide polymorphisms (SNPs) Antibody epitope identification Canine dendritic cells 



We kindly acknowledge Dr. Bruce Roe and his team at Advanced Center for Genome Technology, Oklahoma University for preliminary 454 Sanger sequencing of two of the three BAC sequences. We would like to thank Dr. Christopher Dascher (Havas Life, NY, USA) for input on guinea pig CD1 background, and we thank Dr. Steven Porcelli (Albert Einstein College of Medicine, NY, USA) for generously donating guinea pig hybridomas for use in this study. Furthermore, we thank Biolegend (San Diego, CA, USA) and AbD Serotec (Raleigh, NC, USA) for kindly donating anti-CD1 antibodies. We would like to thank Ken Jackson (UC Davis, CA, USA) for invaluable advice with cloning procedures and culture of BAC clones. This study was partly funded by a PhD scholarship from the Graduate School of Health and Medical Sciences, University of Copenhagen, Denmark. The PacBio analysis and bioinformatics analysis of this study was funded by two pilot grants received from the UC Davis DNA Technologies Core and the UC Davis Bioinformatics Core, respectively.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

251_2015_889_MOESM1_ESM.docx (95 kb)
ESM 1 (DOCX 95 kb)


  1. Adams EJ (2014) Lipid presentation by human CD1 molecules and the diverse T cell populations that respond to them. Curr Opin Immunol 26:1–6. doi: 10.1016/j.coi.2013.09.005 CrossRefPubMedGoogle Scholar
  2. Aureli A et al (2007) CD1a and CD1e allele frequencies in an Italian population from the Abruzzo region. Int J Immunopathol Pharmacol 20:415–419PubMedGoogle Scholar
  3. Barral DC, Brenner MB (2007) CD1 antigen presentation: how it works. Nat Rev Immunol 7:929–941. doi: 10.1038/nri2191 CrossRefPubMedGoogle Scholar
  4. Beckman EM, Porcelli SA, Morita CT, Behar SM, Furlong ST, Brenner MB (1994) Recognition of a lipid antigen by CD1-restricted alpha beta + T cells. Nature 372:691–694. doi: 10.1038/372691a0 CrossRefPubMedGoogle Scholar
  5. Benam KH, Kok WL, McMichael AJ, Ho LP (2011) Alternative spliced CD1d transcripts in human bronchial epithelial cells. PLoS One 6, e22726. doi: 10.1371/journal.pone.0022726 PubMedCentralCrossRefPubMedGoogle Scholar
  6. Bonish B et al (2000) Overexpression of CD1d by keratinocytes in psoriasis and CD1d-dependent IFN-gamma production by NK-T cells. J Immunol 165:4076–4085CrossRefPubMedGoogle Scholar
  7. Brigl M, Brenner MB (2004) CD1: antigen presentation and T cell function. Annu Rev Immunol 22:817–890. doi: 10.1146/annurev.immunol.22.012703.104608 CrossRefPubMedGoogle Scholar
  8. Calabi F, Jarvis JM, Martin L, Milstein C (1989) Two classes of CD1 genes. Eur J Immunol 19:285–292. doi: 10.1002/eji.1830190211 CrossRefPubMedGoogle Scholar
  9. Cao X, Sugita M, Van Der Wel N, Lai J, Rogers RA, Peters PJ, Brenner MB (2002) CD1 molecules efficiently present antigen in immature dendritic cells and traffic independently of MHC class II during dendritic cell maturation. J Immunol 169:4770–4777CrossRefPubMedGoogle Scholar
  10. Chu CC, Di Meglio P, Nestle FO (2011) Harnessing dendritic cells in inflammatory skin diseases. Semin Immunol 23:28–41. doi: 10.1016/j.smim.2011.01.006 PubMedCentralCrossRefPubMedGoogle Scholar
  11. Colonna M (2010) Skin function for human CD1a-reactive T cells. Nat Immunol 11:1079–1080. doi: 10.1038/ni1210-1079 CrossRefPubMedGoogle Scholar
  12. Dascher CC (2007) Evolutionary biology of CD1 Curr Top Microbiol Immuno l 314:3–26Google Scholar
  13. de Jong A et al (2014) CD1a-autoreactive T cells recognize natural skin oils that function as headless antigens. Nat Immunol 15:177–185. doi: 10.1038/ni.2790 PubMedCentralCrossRefPubMedGoogle Scholar
  14. de Jong A, Pena-Cruz V, Cheng TY, Clark RA, Van Rhijn I, Moody DB (2010) CD1a-autoreactive T cells are a normal component of the human alphabeta T cell repertoire. Nat Immunol 11:1102–1109. doi: 10.1038/ni.1956 PubMedCentralCrossRefPubMedGoogle Scholar
  15. Dossa RG, Alperin DC, Hines MT, Hines SA (2014) The equine CD1 gene family is the largest and most diverse yet identified. Immunogenetics 66:33–42. doi: 10.1007/s00251-013-0741-6 CrossRefPubMedGoogle Scholar
  16. Dougan SK, Kaser A, Blumberg RS (2007) CD1 expression on antigen-presenting cells. Curr Top Microbiol Immunol 314:113–141PubMedGoogle Scholar
  17. Gan LH, Pan YQ, Xu DP, Li M, Lin A, Yan WH (2010) Polymorphism of human CD1a, CD1d, and CD1e in exon 2 in Chinese Han and She ethnic populations. Tissue Antigens 75:691–695. doi: 10.1111/j.1399-0039.2010.01443.x CrossRefPubMedGoogle Scholar
  18. Hickson RE, Cann RL (1997) Mhc allelic diversity and modern human origins. J Mol Evol 45:589–598CrossRefPubMedGoogle Scholar
  19. Hiromatsu K et al (2002) Characterization of guinea-pig group 1 CD1 proteins. Immunology 106:159–172PubMedCentralCrossRefPubMedGoogle Scholar
  20. Kearse M et al (2012) Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28:1647–1649. doi: 10.1093/bioinformatics/bts199 PubMedCentralCrossRefPubMedGoogle Scholar
  21. Kielbasa SM, Wan R, Sato K, Horton P, Frith MC (2011) Adaptive seeds tame genomic sequence comparison Genome Res 21:487–493 doi: 10.1101/gr.113985.110
  22. Kojo S, Adachi Y, Tsutsumi A, Sumida T (2000) Alternative splicing forms of the human CD1D gene in mononuclear cells. Biochem Biophys Res Commun 276:107–111. doi: 10.1006/bbrc.2000.3450 CrossRefPubMedGoogle Scholar
  23. Krumsiek J, Arnold R, Rattei T (2007) Gepard: a rapid and sensitive tool for creating dotplots on genome scale Bioinformatics 23:1026–1028 doi:10.1093/bioinformatics/btm039Google Scholar
  24. Layre E et al. (2009) Mycolic acids constitute a scaffold for mycobacterial lipid antigens stimulating CD1-restricted T cells Chemistry & biology 16:82–92 doi: 10.1016/j.chembiol.2008.11.008
  25. Lindblad-Toh K et al (2005) Genome sequence, comparative analysis and haplotype structure of the domestic dog. Nature 438:803–819. doi: 10.1038/nature04338 CrossRefPubMedGoogle Scholar
  26. Looringh van Beeck FA, Leegwater PA, Herrmann T, Broere F, Rutten VP, Willemse T, Van Rhijn I (2013) Tandem repeats modify the structure of the canine CD1D gene. Anim Genet 44:352–355. doi: 10.1111/age.12002
  27. Looringh van Beeck FA et al (2008) Two canine CD1a proteins are differentially expressed in skin. Immunogenetics 60:315–324. doi: 10.1007/s00251-008-0297-z-- PubMedCentralCrossRefPubMedGoogle Scholar
  28. Malik R, Smits B, Reppas G, Laprie C, O'Brien C, Fyfe J (2013) Ulcerated and nonulcerated nontuberculous cutaneous mycobacterial granulomas in cats and dogs. Vet Dermatol 24(146–153):e132–143. doi: 10.1111/j.1365-3164.2012.01104.x Google Scholar
  29. Marsella R, Girolomoni G (2009) Canine models of atopic dermatitis: a useful tool with untapped potential. J Invest Dermatol 129:2351–2357. doi: 10.1038/jid.2009.98 CrossRefPubMedGoogle Scholar
  30. Martin LH, Calabi F, Milstein C (1986) Isolation of CD1 genes: a family of major histocompatibility complex-related differentiation antigens. Proc Natl Acad Sci U S A 83:9154–9158PubMedCentralCrossRefPubMedGoogle Scholar
  31. Miller MM et al (2005) Characterization of two avian MHC-like genes reveals an ancient origin of the CD1 family. Proc Natl Acad Sci U S A 102:8674–8679. doi: 10.1073/pnas.0500105102 PubMedCentralCrossRefPubMedGoogle Scholar
  32. Moody DB, Zajonc DM, Wilson IA (2005) Anatomy of CD1-lipid antigen complexes. Nat Rev Immunol 5:387–399. doi: 10.1038/nri1605 CrossRefPubMedGoogle Scholar
  33. Moore PF, Schrenzel MD, Affolter VK, Olivry T, Naydan D (1996) Canine cutaneous histiocytoma is an epidermotropic Langerhans cell histiocytosis that expresses CD1 and specific beta 2-integrin molecules. Am J Pathol 148:1699–1708PubMedCentralPubMedGoogle Scholar
  34. Olivry T (2011) Is the skin barrier abnormal in dogs with atopic dermatitis? Vet Immunol Immunopathol 144:11–16. doi: 10.1016/j.vetimm.2011.07.014 CrossRefPubMedGoogle Scholar
  35. Olivry T (2012) What can dogs bring to atopic dermatitis research? Chem Immunol Allergy 96:61–72. doi: 10.1159/000331884 CrossRefPubMedGoogle Scholar
  36. Olivry T, Naydan DK, Moore PF (1997) Characterization of the cutaneous inflammatory infiltrate in canine atopic dermatitis. Am J Dermatopathol 19:477–486CrossRefPubMedGoogle Scholar
  37. Popa I, Remoue N, Hoang LT, Pin D, Gatto H, Haftek M (2011) Portoukalian JAtopic dermatitis in dogs is associated with a high heterogeneity in the distribution of protein-bound lipids within the stratum corneum. Arch Dermatol Res 303:433–440. doi: 10.1007/s00403-011-1120-5 CrossRefPubMedGoogle Scholar
  38. Porcelli S, Brenner MB, Greenstein JL, Balk SP, Terhorst C, Bleicher PA (1989) Recognition of cluster of differentiation 1 antigens by human CD4-CD8-cytolytic T lymphocytes. Nature 341:447–450. doi: 10.1038/341447a0 CrossRefPubMedGoogle Scholar
  39. Porcelli SA (1995) The CD1 family: a third lineage of antigen-presenting molecules. Adv Immunol 59:1–98CrossRefPubMedGoogle Scholar
  40. Porcelli SA, Modlin RL (1999) The CD1 system: antigen-presenting molecules for T cell recognition of lipids and glycolipids. Annu Rev Immunol 17:297–329. doi: 10.1146/annurev.immunol.17.1.297 CrossRefPubMedGoogle Scholar
  41. Porcelli SA, Segelke BW, Sugita M, Wilson IA, Brenner MB (1998) The CD1 family of lipid antigen-presenting molecules. Immunol Today 19:362–368CrossRefPubMedGoogle Scholar
  42. Ribeiro MG et al (2008) Nocardiosis: an overview and additional report of 28 cases in cattle and dogs. Rev Inst Med Trop Sao Paulo 50:177–185CrossRefPubMedGoogle Scholar
  43. Ryser S et al (2014) UVB-induced skin inflammation and cutaneous tissue injury is dependent on the MHC class I-like protein. CD1d. J Invest Dermatol 134:192–202. doi: 10.1038/jid.2013.300 PubMedCentralCrossRefPubMedGoogle Scholar
  44. Seshadri C et al (2013) Human CD1a deficiency is common and genetically regulated. J Immunol 191:1586–1593. doi: 10.4049/jimmunol.1300575 PubMedCentralCrossRefPubMedGoogle Scholar
  45. Spada FM et al (2000) Self-recognition of CD1 by gamma/delta T cells: implications for innate immunity. J Experimental medicine 191:937–948CrossRefGoogle Scholar
  46. Van Rhijn I, Ly D, Moody DB (2013) CD1a, CD1b, and CD1c in immunity against mycobacteria. Adv Exp Med Biol 783:181–197. doi: 10.1007/978-1-4614-6111-1_10 CrossRefPubMedGoogle Scholar
  47. Van Rhijn I, Moody DB (2015) CD1 and mycobacterial lipids activate human T cells. Immunol Rev 264:138–153. doi: 10.1111/imr.12253 CrossRefPubMedGoogle Scholar
  48. Vincent MS, Gumperz JE, Brenner MB (2003) Understanding the function of CD1-restricted T cells. Nat Immunol 4:517–523. doi: 10.1038/ni0603-517 CrossRefPubMedGoogle Scholar
  49. Vincent MS, Leslie DS, Gumperz JE, Xiong X, Grant EP, Brenner MB (2002) CD1-dependent dendritic cell instruction. Nat Immuno l 3:1163–1168. doi: 10.1038/ni851 CrossRefGoogle Scholar
  50. Woolfson A, Milstein C (1994) Alternative splicing generates secretory isoforms of human CD1 Proc. Natl Acad Sci USA 91:6683–6687CrossRefGoogle Scholar
  51. Zaba LC, Krueger JG, Lowes MA (2009) Resident and “inflammatory” dendritic cells in human skin. J Invest Dermatol 129:302–308. doi: 10.1038/jid.2008.225 PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Mette Schjaerff
    • 1
    • 2
  • Stefan M. Keller
    • 2
  • Joseph Fass
    • 3
  • Lutz Froenicke
    • 4
  • Robert A. Grahn
    • 5
  • Leslie Lyons
    • 6
  • Verena K. Affolter
    • 2
  • Annemarie T. Kristensen
    • 1
  • Peter F. Moore
    • 2
  1. 1.Department of Veterinary Clinical and Animal Sciences, Faculty of Health and Medical SciencesUniversity of CopenhagenFrederiksberg CDenmark
  2. 2.Department of Veterinary Pathology, Microbiology and ImmunologyUniversity of CaliforniaDavisUSA
  3. 3.UC Davis Genome Center – Bioinformatics CoreUniversity of CaliforniaDavisUSA
  4. 4.UC Davis Genome Center - DNA Technologies and Expression Analysis CoresUniversity of CaliforniaDavisUSA
  5. 5.Veterinary Genetics LaboratoryUniversity of CaliforniaDavisUSA
  6. 6.Lyons Feline and Comparative Genetics, Department of Veterinary Medicine and SurgeryUniversity of MissouriColombiaUSA

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