ELISPOT in Autoimmune Diseases

ELISPOT and diabetesELISPOT assays are among the most-sensitive and -specific methods available for the detection of both T cell and B cell responses. Major advantages are its relatively fast and easy performance, high sensitivity (detection level of one cell out of one million), its potential for high throughput screening and no requirement for expensive instruments.

The high sensitivity of the ELISPOT assay is particularly useful in autoimmune studies, as autoreactive T and B cell responses are typically of much lower frequency than those found for viral and vaccine-associated immune responses.

For example in human diabetes Type 1, a chronic autoimmune disease leading to selective destruction of insulin producing ß-cells, T cells play a key role, but the detection of these lymphocytes is difficult. Fortunately, cytokine secretion by autoantigen reactive T cells can be demonstrated in individual cells with the use of the ELISPOT assay, offering preclinical diagnoses and immune surrogate end points for clinical trials. Many studies have investigated cytokine production (e.g. IFN-γ, IL-5, IL-10, IL-13) by antigen-reactive peripheral blood mononuclear cells with the use of the ELISPOT assay and related T cell responses with β-cell function. Read more about the use of T cell ELISPOT in type 1 diabetes research.

Autoreactive B cell responses are evident in systemic autoimmune diseases such as Systemic lupus erythematous (SLE) and Rheumatoid arthritis (RA). In these diseases, B cell tolerance is broken triggering the formation of autoreactive B cells. Preimmune B cells in bone marrow are polyreactive expressing a wide repertoire of antigen receptors that recognize both non-self and self-antigens. These B cells will pass a series of self-tolerance checkpoints and only B cells that recognize non-self antigens will enter the mature B cell pool. The potentially harmful autoreactive B cells are eliminated from the repertoire by an unknown mechanism. In systemic autoimmunity this mechanism does not function properly and impaired early B cell tolerance occurs in patients with various autoimmune diseases. The formation of autoreactive B cells is influenced by a composite of factors including genetic susceptibility and environmental factors. For example, clinical observations and experimental animal models have demonstrated that autoimmune diseases can be initiated or worsened by infections. When an infection is involved, autoimmunity is thought to be initiated by molecular mimicry (structural cross-reactivity between the microbial antigens and self-antigens) leading to the activation of autoreactive B cells. The detection and enumeration of autoreactive B cells can be best studied by the ELISPOT assay as exemplified in the manuscript from Enghard et al. 2011.

 

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Examples of studies using our ELISPOT assays:

Click on the authors for the abstract of the below mentioned acticles or find them in our Reference Database.

Arif S, Tree TI, Astill TP, Tremble JM, Bishop AJ, Dayan CM, Roep BO, and Peakman M.
Autoreactive T cell responses show proinflammatory polarization in diabetes but a regulatory phenotype in health.
J Clin Invest 113:451-63 (2004).
U-CyTech products used in this study:
Human IFN-γ ELISPOT kit
Human IL-4 ELISPOT kit
Human IL-10 ELISPOT kit
 
Enee E, Martinuzzi E, Blancou P, Bach JM, Mallone R, and van Endert P.
Equivalent specificity of peripheral blood and islet-infiltrating CD8+ T lymphocytes in spontaneously diabetic HLA-A2 transgenic NOD mice.
J Immunol 180:5430-8 (2008).
U-CyTech products used in this study:
Mouse IFN-γ ELISPOT antibody pair

Han G, Wang R, Chen G, Wang J, Xu R, Feng J, Yu M, Wu X, Qian J, Shen B, and Li Y.
Gene delivery GAD 500 autoantigen by AAV serotype 1 prevented diabetes in NOD mice: transduction efficiency do not play important roles.
Immunol Lett 115:110-6 (2008). Abstract
U-CyTech products used in this study:
Mouse IFN-γ ELISPOT kit
Mouse IL-4 ELISPOT kit
Mouse IL-10 ELISPOT kit

Haanstra KG, Endell J, Estevao D, Kondova I, and Jonker M.
Blocking T cell co-stimulation using a CD80 blocking small molecule reduces delayed type hypersensitivity responses in rhesus monkeys.
Clin Exp Immunol 158:91-8 (2009).
U-CyTech products used in this study:
Monkey IFN-γ ELISPOT kit
Monkey species: Macaca mulatta

Martin S, Wolf-Eichbaum D, Duinkerken G, Scherbaum WA, Kolb H, Noordzij JG, and Roep BO.
Development of type 1 diabetes despite severe hereditary B-lymphocyte deficiency.
N Engl J Med 345:1036-40 (2001).
U-CyTech products used in this study:
Human IFN-γ ELISPOT kit
Human IL-4 ELISPOT kit
Human IL-5 ELISPOT kit
Human IL-10 ELISPOT kit
Human IL-13 ELISPOT kit

Pinkse GG, Tysma OH, Bergen CA, Kester MG, Ossendorp F, van Veelen PA. Keymeulen B. Pipeleers D. Drijfhout JW, and Roep BO.
Autoreactive CD8 T cells associated with beta cell destruction in type 1 diabetes.
Proc Natl Acad Sci U S A 102:18425-30 (2005).
U-CyTech products used in this study:
Human IFN-γ ELISPOT kit
Human Granzyme B ELISPOT kit
Human IL-10 ELISPOT kit
 
Raz I, Elias D, Avron A, Tamir M, Metzger M, and Cohen IR.
Beta-cell function in new-onset type 1 diabetes and immunomodulation with a heat-shock protein peptide (DiaPep277): a randomised, double-blind, phase II trial.
Lancet 358:1749-53 (2001).
U-CyTech products used in this study:
Human IFN-γ ELISPOT kit
Human IL-4 ELISPOT kit
Human IL-10 ELISPOT kit
Human IL-13 ELISPOT kit
 
van der Meide PH, de Labie MC, Ruuls SR, Groenestein RJ, Botman CA, Olsson T, and Dijkstra CD.
Discontinuation of treatment with IFN-beta leads to exacerbation of experimental autoimmune encephalomyelitis in
Lewis rats. Rapid reversal of the antiproliferative activity of IFN-beta and excessive expansion of autoreactive T cells as disease promoting mechanisms.
J Neuroimmunol 84:14-23 (1998).
U-CyTech products used in this study:
Rat IFN-γ antibody used in ELISPOT
 
van Halteren AG, van Etten E, de Jong EC, Bouillon R, Roep BO, and Mathieu C.
Redirection of human autoreactive T-cells Upon interaction with dendritic cells modulated by TX527, an analog of 1,25 dihydroxyvitamin D(3).
Diabetes 51:2119-25 (2002).
U-CyTech products used in this study:
Human IFN-γ ELISPOT kit
Human IL-2 ELISPOT kit
Human IL-10 ELISPOT kit
Human IL-13 ELISPOT kit
 

Islet-specific CTL cloned from a type 1 diabetes patient cause beta-cell destruction after engraftment into HLA-A2 transgenic NOD/scid/IL2RG null mice.
PLoS ONE 7:e49213 (2012).
U-CyTech products used in this study:
Human IFN-γ ELISPOT kit
Human Granzyme B ELISPOT kit
Human IL-10 ELISPOT kit