MHC multimer

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MHC multimers are oligomeric forms of MHC molecules, designed to identify and isolate T-cells with high affinity to specific antigens amid a large group of unrelated T-cells.[1] Multimers generally range in size from dimers to octamers; however, some companies (such as Immudex) use even higher quantities of MHC per multimer. Multimers may be used to display class 1 MHC, class 2 MHC, or nonclassical molecules from species such as monkeys, mice, and humans.

Background

Since T-Cell Receptors have a low affinity for their MHC counterparts, it was historically problematic to label T cells effectively using single MHC-T-cell interactions.[2] However, in 1996 it was proposed by John Altman to use a complex of multiple MHC molecules to form a more stable bond between corresponding T-cells.[3]

Production

The most commonly used MHC Multimers are tetramers.[3] These are typically produced by biotinylating soluble MHC monomers, which are typically produced recombinantly in eukaryotic or bacterial cells. These monomers then bind to a backbone, such as streptavidin or avidin, creating a tetravalent structure. These backbones are conjugated with fluorochromes to subsequently isolate bound T-cells via flow cytometry.[4]

Potential clinical applications

MHC Multimers allow for a previously unattainable level of specificity in antigen-specific T-cell detection and isolation. This ability gives rise to several clinical applications. MHC Multimers allow for ex vivo selection and proliferation of T-cells specific to viral or tumor-related antigens, which can then be reintroduced to augment the immune system. MHC Multimers can also be used to eliminate graft-originating T-cells on transplant organs, ex vivo. MHC Multimers may also be used to eliminate harmful or unwanted T-cells in vivo, such as those that target self cells and lead to autoimmune disease.[4][5][6] Cancer immunotherapy and vaccine development can also be largely influenced by this technology.[7]

Sub-Types

MHC Tetramer

MHC Tetramers consist of four MHC molecules, a tetramerization agent and a fluorescently labeled protein (usually streptavidin). MHC tetramers are used to identify and label specific T-cells by epitope specific binding, allowing the antigen specific immune response to be analyzed in both animal model and in man.[8] MHC tetramers were originally developed using MHC class I molecules for the recognition of CD8 T cells,[9][10] but over the last decade they have allowed for the recognition of CD4 T cells by a wide variety of antigens. Tetramer assays are used for single-cell phenotyping and cell counting, and offer an important advantage over other methods, such as ELISPOT and single-cell PCR because they enable the recovery and further study of sorted cells. As a flow-cytometry-based application, tetramers are also easy to use and have a short assay time, similar to Antibody-based flow cytometry studies.[4]

MHC tetramers are used in studies of pathogen immunity and vaccine development, in evaluation of antitumor responses, in allergy monitoring and desensitization studies, and in autoimmunity.[4][11] They provide an ideal means to characterize the T cells that respond to a vaccine, and they have been used to test T cell responses in many vaccine systems, including influenza,[12] yellow fever,[13] tuberculosis,[14] HIV/SIV[15] and a large number of cancer vaccine trials,[16] including melanoma and chronic myeloid leukemia.[17] Class II tetramers have been used for analysis of a variety of human CD4 T cell responses to pathogens, including influenza A, Borrelia, Epstein-Barr virus, CMV, Mycobacterium tuberculosis, human T-lymphotropic virus 1, hepatitis C, anthrax, severe acute respiratory syndrome virus, human papillomavirus, and HIV.[4] Tetramer variants have been developed that, either radiolabelled or coupled to a toxin such as saporin, can be injected into live mice to modulate or even deplete specific T cell populations.[18][19] Peptide–MHC tetramers have also been used therapeutically.[20] For instance, cytomegalovirus-specific T cells have been enriched to high levels of purity using magnetic bead-based enrichment for use as a therapy for stem cell transplant patients.[11]

MHC Pentamer

Pentamers consist of five MHC-peptide headgroups, arranged in a planar configuration so that, unlike MHC tetramers, all of the headgroups can contact the CD8+ T cell. The headgroups are connected via flexible linkers to a coiled-coil multimerization domain, which in turn is connected to five fluorescent or biotin tags. Pentamers are available with APC, R-PE, or biotin labelling, and also unlabelled with separate tags for long-term storage. Pentamers offer enhanced brightness and avidity of staining compared with other multimer reagents.

MHC Pentamers have been used in the detection of antigen-speficic CD8+ T cells in flow cytometry,[11] and are cited in over 750 peer reviewed publications , including several in the journals Nature[21] and Science.[22][23] MHC Pentamers can also be used in tissue staining,[24] and in magnetic isolation of antigen-specific T cells.[25]

While Pentamers are licensed for research use only, in 2009 a special dispensation was granted for a team to use them for isolating EBV-specific T cells for mother-daughter transfer, for lifesaving treatment of EBV-associated lymphoma in the daughter.[26]

Pentamers are available for antigens from the following disease areas: Adenovirus, HCV, Malaria, SIV, Autoimmune disease, HIV, transplantation antigens, Trypanosoma, Cancer, HPV, Tuberculosis, Chlamydia, HTLV, Vaccinia, CMV, Influenza, VSV, EBV, LCMV, RSV, West Nile Virus, HBV, Listeria, Sendai Virus, Yellow Fever. Custom specificity Pentamers may also be commissioned.

Pentamers are currently used in research by academia, industry and clinicians, and research using Pentamers has appeared in the international media on several occasions.

MHC Dextramer

A form of MHC multimer developed and trademarked by Danish biotechnology company, Immudex in 2002. Dextramer reagents are fluorescently labeled with FITC, PE or APC, and contain MHC molecules attached to a dextran backbone, which are used to detect antigen-specific T-cells in fluid cells and solid tissue samples using flow cytometry. These T-cells contain T-cell receptors (TCR) that recognize a specific MHC-peptide complex displayed on the surface of antigen presenting cells allowing for detection, isolation, and quantification of these specific T-cell populations due to an improved signal-to-noise ratio not present in prior generations of multimers.[3][11][27]

Dextramers have been developed with a larger number of MHC-peptides for various human, mouse, and Rhesus Macaque genes involved in diseases including but not limited to: cancer, HIV, Epstein-Barr Virus (EBV), Cytomegalovirus (CMV), LCMV, Human Papillomavirus (HPV), BK polyomavirus, HTLV, Hepatitis, Mycobacterium, and graft-versus-host Disease.

Dextramer technology is currently used in academic and clinical research due to their increased specificity and binding affinity, which allows for increased avidity for specific T-cells and enhances staining intensity. This advantage is a result of the increased ability of Dextramers to bind multiple times to a single T-cell, improving the stability of this interaction as compared with other multimer technologies such as pentamers and tetramers. Further applications include the ability to isolate antigen specific T-cell populations as well as in situ detection using immunohistochemistry (IHC) for various disease states (e.g. solid tumors). These reagents are therefore important for future drug and vaccine development.[1][11][27][28][29]

Immudex has currently developed a CMV Dextramer assay for exploratory detection and quantification of CD8+ T-cells in blood samples, covering a broad range of epitopes to assist with screening and monitoring CMV progression in future clinical settings.[30] Currently Dextramers are only available with MHC class I molecules, and there is future development and research into the applications and production of MHC class II Dextramers.[31]

References

  1. ↑ 1.0 1.1 Hadrup, Sine R.; Bakker, Arnold H.; et al. "Parallel Detection of Antigen-Specific T-Cell responses by multidimensional encoding of MHC multimers," Nature Methods, Vol. 6 (2009), pp. 520-526
  2. ↑ Nepom, Gerald T. "MHC Multimers: Expanding the Clinical Toolkit," Clinical Immunology, Vol 6 (2003), pp. 1-4.,Antigen. 106 (2003), pp. 1-4.
  3. ↑ 3.0 3.1 3.2 Bakker, Arnold; Schumacher, Tom. "MHC Multimer Technology: Current Status and Future Prospects," Current Opinion in Immunology, Vol. 17, No. 4 (August 2005), pp. 428-433.
  4. ↑ 4.0 4.1 4.2 4.3 4.4 Nepom, Gerald T. "MHC Class II Tetramers," Journal of Immunology, Vol. 188 (2012), pp. 2477-2482.
  5. ↑ Holman, Philmore O.; Walsh, Elizabeth R.; Jameson, Stephen C. "Characterizing the Impact of CD8 Antibodies on Class I MHC Multimer Binding," The Journal of Immunology, Vol. 174, No. 7, pp. 3986-3991.
  6. ↑ Hackett, Charles J.; Sharma, Opendra K. "Frontiers in peptide-MHC class II multimer technology," Nature Immunology, Vol. 3 (2002), pp. 887-889.
  7. ↑ Davis, Mark M.; Altman, John D.; Newell, Evan W. "Interrogating the Repertoire: Broadening the Scope of Peptide-MHC Multimer Analysis," Nature Reviews Immunology, Vol. 11, No. 8 (July 15, 2011), pp. 551-558.
  8. ↑ Erfle V, inventor; 2004 Jul. 15. MHC tetramers. United States patent US 20040137642.
  9. ↑ Altman, J. D, et al. 1996. Phenotypic analysis of antigen-specific T lymphocytes" Science 274: 94–96.
  10. ↑ Lebowitz, M. S,. 1999. Soluble, high-affinity dimers of T-cell receptors and class II major histocompatibility complexes: biochemical probes for analysis and modulation of immune responses" Cell. Immunol 192: 175–184.
  11. ↑ 11.0 11.1 11.2 11.3 11.4 Davis MM, Altman JD, Newell EW. "Interrogating the repertoire: broadening the scope of peptide-MHC multimer analysis". Nat Rev Immunol. 2011 Jul 15;11(8) 551-8. doi:10.1038/nri3020
  12. ↑ He, X. S. et al. Phenotypic changes in influenza-specific CD8+ T cells after immunization of children and adults with influenza vaccines" J. Infect. Dis 197, 803–811 (2008).
  13. ↑ Co, M. D., Kilpatrick, E. D. & Rothman, A. L. Dynamics of the CD8 T-cell response following yellow fever virus 17D immunization" Immunology 128, e718–e727 (2009).
  14. ↑ Wei, H. et al. DR*W201/P65 tetramer visualization of epitope-specific CD4 T-cell during M. tuberculosis infection and its resting memory pool after BCG vaccination" PLoS ONE 4, e6905 (2009).
  15. ↑ Betts, M. R. et al. Characterization of functional and phenotypic changes in anti-Gag vaccine-induced T cell responses and their role in protection after HIV-1 infection. Proc. Natl Acad. Sci. USA 102, 4512–4517 (2005).
  16. ↑ Pittet, M. J. et al. Ex vivo analysis of tumor antigen specific CD8+ T cell responses using MHC/peptide tetramers in cancer patients. Int. Immunopharmacol. 1, 1235–1247 (2001).
  17. ↑ Lee, P. P. et al. Characterization of circulating T cells specific for tumor-associated antigens in melanoma patients.
  18. ↑ Maile, R. et al. Antigen-specific modulation of an immune response by in vivo administration of soluble MHC class I tetramers" J. Immunol 167, 3708–3714 (2001).
  19. ↑ Yuan, R. R. et al. Targeted deletion of T-cell clones using alpha-emitting suicide MHC tetramers" Blood 104, 2397–2402 (2004).
  20. ↑ Cobbold, M. et al. Adoptive transfer of cytomegalovirus-specific CTL to stem cell transplant patients after selection by HLA–peptide tetramers" J. Exp. Med 202, 379–386 (2005).
  21. ↑ Jiang; Clark, Liu, Wagers, Fuhlbrigge, Kupper (2012). "Skin infection generates non-migratory memory CD8+ T(RM) cells providing global skin immunity". Nature 483 (7388): 227–31. doi:10.1038/nature10851. PMC 3437663. PMID 22388819. 
  22. ↑ Saveanu; Carroll O, Weimershaus M, Guermonprez P, Firat E, Lindo V, Greer F, Davoust J, Kratzer R, Keller SR, Niedermann G, van Endert P. (2009). "IRAP identifies an endosomal compartment required for MHC class I cross-presentation.". Science 325 (5937): 213–7. doi:10.1126/science.1172845. PMID 19498108. 
  23. ↑ Bannard; Kraman, Fearon (2009). "Secondary replicative function of CD8+ T cells that had developed an effector phenotype". Science 323 (5913): 505–9. doi:10.1126/science.1166831. PMC 2653633. PMID 19164749. 
  24. ↑ Panoskaltsis-Mortari; Taylor PA, Riddle MJ, Shlomchik MA, Blazar BR. (2008). "In situ identification of allospecific B cells using pentamers.". Blood 111 (7): 3904–5. doi:10.1182/blood-2007-12-127415. PMC 2275041. PMID 18362221. 
  25. ↑ Griffioen, Genetic engineering of virus-specific T cells with T-cell receptors recognizing minor histocompatibility antigens for clinical application.; van Egmond HM, Barnby-Porritt H, van der Hoorn MA, Hagedoorn RS, Kester MG, Schwabe N, Willemze R, Falkenburg JH, Heemskerk MH. (2008). "Genetic engineering of virus-specific T cells with T-cell receptors recognizing minor histocompatibility antigens for clinical application". Haematologica 93 (10): 1535–43. doi:10.3324/haematol.13067. PMID 18768532. 
  26. ↑ Uhlin; Okas M, Gertow J, Uzunel M, Brismar TB, Mattsson J. (2009). "A novel haplo-identical adoptive CTL therapy as a treatment for EBV-associated lymphoma after stem cell transplantation". Cancer Immunol Immunother 59 (3): 473–7. doi:10.1007/s00262-009-0789-1. PMID 19908041. 
  27. ↑ 27.0 27.1 Rosaely Casalegno-Garduno, Anita Schmitt, Junxia Yao, Xinchao Wang, Xun Xu, Mathias Freund, Michael Schmitt. "Multimer technologies for detection and adoptive transfer of antigen-specific T cells". Cancer Immunol Immunother. 2010, 59:195–202.
  28. ↑ Jorgen Schøller, Mahavir Singh, Lesley Bergmeier, Katja Brunstedt, Yufei Wang, Trevor Whittall, Durdana Rahman, J. Pido-Lopez, T. Lehner. "A recombinant human HLA-class I antigen linked to dextran elicits innate and adaptive immune responses". Journal of Immunological Methods, 2010, 360, 1–9
  29. ↑ Pascal Batard, Daniel A. Peterson, Estelle Devêvre, Philippe Guillaume, Jean-Charles Cerottini, Donata Rimoldi, Daniel E. Speiser, Lars Winther, Pedro Romero. "Dextramers: New generation of fluorescent MHC class I/peptide multimers for visualization of antigen-specific CD8+ T cells". Journal of Immunological Methods, 2006, 310, 136–148.
  30. ↑ Hadrup SR ; Strindhall J ; Kollgaard T ; et al. "Longitudinal studies of clonally expanded CD8 T cells reveal a repertoire shrinkage predicting mortality and an increased number of dysfunctional cytomegalovirus-specific T cells in the very elderly". Journal of Immunology, 2006, 176 (4), 2645-2653.
  31. ↑ Massilamany et al. "Detection of autoreactive CD4 T cells using major histocompatibility complex class II dextramers". BMC Immunology, 2011, 12:40.
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