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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 use even higher quantities of MHC per multimer. Multimers may be used to display class 1 MHC, class 2 MHC, or nonclassical molecules (e.g. CD1d) from species such as monkeys, mice, and humans.

Background

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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

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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

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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

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MHC tetramer

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MHC tetramers consist of four MHC molecules, a tetramerization agent and a fluorescently labeled protein (usually streptavidin). Streptavidins have also been generated with 6 or 12 binding sites for MHC.[8] 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 human.[9] MHC tetramers were originally developed using MHC class I molecules for the recognition of cytotoxic T cells,[10][11] 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][12] 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,[13] yellow fever,[14] tuberculosis,[15] HIV/SIV[16] and a large number of cancer vaccine trials,[17] including melanoma and chronic myeloid leukemia.[18] 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.[19][20] Peptide–MHC tetramers have also been used therapeutically.[21] 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.[12]

MHC pentamer

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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-specific CD8+ T cells in flow cytometry,[12] and are cited in over 750 peer reviewed publications [1], including several in the journals Nature[22] and Science.[23][24] MHC pentamers can also be used in tissue staining,[25] and in magnetic isolation of antigen-specific T cells.[26]

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.[27]

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 [2][3] on several occasions.

MHC Dextramer®

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A form of MHC multimer developed and trademarked by the 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][12][28]

Dextramer® reagents 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][12][28][29][30]

Immudex 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.[31] MHC Dextramer® reagents are available with MHC class I and MHC class II molecules.[32]

References

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  1. ^ a b Hadrup, Sine R.; Bakker, Arnold H.; et al. (2009). "Parallel Detection of Antigen-Specific T-Cell responses by multidimensional encoding of MHC multimers". Nature Methods. 6 (7): 520–526. doi:10.1038/nmeth.1345. PMID 19543285. S2CID 1285613.
  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. ^ a b c 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. ^ a b c d e Nepom, Gerald T (2012). "MHC Class II Tetramers". Journal of Immunology. 188 (6): 2477–2482. doi:10.4049/jimmunol.1102398. PMC 3297979. PMID 22389204.
  5. ^ Holman, Philmore O.; Walsh, Elizabeth R.; Jameson, Stephen C. (2005). "Characterizing the Impact of CD8 Antibodies on Class I MHC Multimer Binding". The Journal of Immunology. 174 (7): 3986–3991. doi:10.4049/jimmunol.174.7.3986. PMID 15778355.
  6. ^ Hackett, Charles J.; Sharma, Opendra K. (2002). "Frontiers in peptide-MHC class II multimer technology". Nature Immunology. 3 (10): 887–889. doi:10.1038/ni1002-887. PMID 12352960. S2CID 43206855.
  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. ^ Fairhead, M; Veggiani, G; et al. (2014). "SpyAvidin Hubs Enable Precise and Ultrastable Orthogonal Nanoassembly". Journal of the American Chemical Society. 136 (35): 1528–35. doi:10.1021/ja505584f. PMC 4183622. PMID 25111182.
  9. ^ Erfle V, inventor; 2004 Jul. 15. MHC tetramers. United States patent US 20040137642.
  10. ^ Altman, J. D; et al. (1996). "Phenotypic analysis of antigen-specific T lymphocytes". Science. 274 (5284): 94–96. Bibcode:1996Sci...274...94A. doi:10.1126/science.274.5284.94. PMID 8810254. S2CID 12667633.
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  12. ^ a b c d e Davis, MM; Altman, JD; Newell, EW (Jul 2011). "Interrogating the repertoire: broadening the scope of peptide-MHC multimer analysis". Nat Rev Immunol. 11 (8): 551–8. doi:10.1038/nri3020. PMC 3699324. PMID 21760610.
  13. ^ He, X. S.; et al. (2008). "Phenotypic changes in influenza-specific CD8+ T cells after immunization of children and adults with influenza vaccines". J. Infect. Dis. 197 (6): 803–811. doi:10.1086/528804. PMID 18279048.
  14. ^ Co, M. D., Kilpatrick, E. D. & Rothman, A. L. Dynamics of the CD8 T-cell response following yellow fever virus 17D immunization" Immunology 2009; 128, e718–e727
  15. ^ 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).
  16. ^ Betts, M. R.; et al. (2005). "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 (12): 4512–4517. Bibcode:2005PNAS..102.4512B. doi:10.1073/pnas.0408773102. PMC 552973. PMID 15753288.
  17. ^ Pittet, M. J.; et al. (2001). "Ex vivo analysis of tumor antigen specific CD8+ T cell responses using MHC/peptide tetramers in cancer patients". Int. Immunopharmacol. 1 (7): 1235–1247. doi:10.1016/s1567-5769(01)00048-0. PMID 11460305.
  18. ^ Lee, P. P. et al. Characterization of circulating T cells specific for tumor-associated antigens in melanoma patients.
  19. ^ Maile, R.; et al. (2001). "Antigen-specific modulation of an immune response by in vivo administration of soluble MHC class I tetramers". J. Immunol. 167 (7): 3708–3714. doi:10.4049/jimmunol.167.7.3708. PMID 11564786.
  20. ^ Yuan, R. R.; et al. (2004). "Targeted deletion of T-cell clones using alpha-emitting suicide MHC tetramers". Blood. 104 (8): 2397–2402. doi:10.1182/blood-2004-01-0324. PMID 15217835.
  21. ^ Cobbold, M.; et al. (2005). "Adoptive transfer of cytomegalovirus-specific CTL to stem cell transplant patients after selection by HLA–peptide tetramers". J. Exp. Med. 202 (3): 379–386. doi:10.1084/jem.20040613. PMC 2213070. PMID 16061727.
  22. ^ 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. Bibcode:2012Natur.483..227J. doi:10.1038/nature10851. PMC 3437663. PMID 22388819.
  23. ^ Saveanu L, 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. Bibcode:2009Sci...325..213S. doi:10.1126/science.1172845. PMID 19498108. S2CID 35719774.
  24. ^ Bannard; Kraman, Fearon (2009). "Secondary replicative function of CD8+ T cells that had developed an effector phenotype". Science. 323 (5913): 505–9. Bibcode:2009Sci...323..505B. doi:10.1126/science.1166831. PMC 2653633. PMID 19164749.
  25. ^ 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.
  26. ^ Griffioen M, 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.
  27. ^ 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. PMC 11030917. PMID 19908041. S2CID 21605915.
  28. ^ a b Casalegno-Garduno, Rosaely; Schmitt, Anita; Yao, Junxia; Wang, Xinchao; Xu, Xun; Freund, Mathias; Schmitt, Michael (2010). "Multimer technologies for detection and adoptive transfer of antigen-specific T cells". Cancer Immunol Immunother. 2010 (59): 195–202. doi:10.1007/s00262-009-0778-4. PMC 11030699. PMID 19847424. S2CID 27882640.
  29. ^ Schøller, Jorgen; Singh, Mahavir; Bergmeier, Lesley; Brunstedt, Katja; Wang, Yufei; Whittall, Trevor; Rahman, Durdana; Pido-Lopez, J.; Lehner, T. (2010). "A recombinant human HLA-class I antigen linked to dextran elicits innate and adaptive immune responses". Journal of Immunological Methods. 2010 (360): 1–9. doi:10.1016/j.jim.2010.05.008. PMID 20542039.
  30. ^ Batard, Pascal; Peterson, Daniel A.; Devêvre, Estelle; Guillaume, Philippe; Cerottini, Jean-Charles; Rimoldi, Donata; Speiser, Daniel E.; Winther, Lars; Romero, Pedro (2006). "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. doi:10.1016/j.jim.2006.01.006. PMID 16516226.
  31. ^ 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
  32. ^ 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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