A Simple Method for the Detection of CD154 (CD40L) on Peripheral Blood Lymphocytes
Authors:
V. Thon; M. Vlková; J. Litzman; J. Lokaj
Authors‘ workplace:
Ústav klinické imunologie a alergologie LF MU, Univerzitní centrum pro primární imunodeficience, Masarykova univerzita, Fakultní nemocnice u sv. Anny v Brně
Published in:
Epidemiol. Mikrobiol. Imunol. 59, 2010, č. 3, s. 147-154
Overview
Objective:
CD154 (also called CD40L) is a transmembrane glycoprotein predominantly expressed on the surface membrane of activated CD4+ T cells. Its receptor CD40 is present on all B cells, but also on other cells. The interaction CD154-CD40 is necessary for the optimal development of the adaptive immune response and also has consequences for the modulation of the inflammatory response. A defect in the expression of CD154 is pathognomonic of congenital immunodeficiency called X-linked Hyper-IgM syndrome (XHIGM). To detect the abnormality of CD154 is essential for making the diagnosis of XHIGM.
Material and methods:
We worked out a microtest for the detection of CD154 expression on in vitro activated CD4+ T cells in whole blood and compared it with that on isolated cells from peripheral blood. Heparinized peripheral blood was activated with phorbol 12-myristate 13-acetate and ionomycin for 4 hours, labeled with monoclonal antibodies and analyzed by flow cytometry. Considering that the CD4 marker on the plasma membrane surface decreases during the activation, CD4+ T cells are mostly recognized as CD5+/CD8- cells. Their activation is monitored based on the expression of CD69. Three--colour immunofluorescence staining was used for simultaneous detection of CD154.
Results:
Ten blood donors were tested. As little as 0.5 ml of heparinized whole blood is needed to complete the test. Optimal time for activation and detection of CD154 on T lymphocytes is 4 hours. We found that the number of CD4 molecules on the surface of T cells decreases during the activation. The expression of CD154 in our whole blood microtest is fully comparable with that in the test on isolated leukocytes.
Conclusion:
The presented microtest for the detection of CD154 on activated lymphocytes in whole blood is fast and blood saving, since as little as 0.5 ml of blood is needed to complete it. It can be recommended as the initial test for suspected hyper-IgM syndrome in children. We demonstrate that this screening method can help to detect also carriers of XHIGM.
Key words:
CD154, CD40 ligand (CD40L), CD40, hyper-IgM syndrome, flow cytometry.
Sources
1. Aghamohammadi, A., Parvaneh, N., Rezaei, N., Moazzami, K. et al. Clinical and Laboratory Findings in Hyper-IgM Syndrome with Novel CD40L and AICDA Mutations. J. Clin. Immunol., 2009, 29, 6, p. 769–776.
2. Allen, R. C., Armitage, R. J., Conley, M. E., Rosenblatt, H. et al. CD40 ligand gene defects responsible for X-linked hyper-IgM syndrome. Science, 1993, 259, 5097, p. 990–993.
3. Bartlett, A., McCall, J., Ameratunga, R., Munn, S. The kinetics of CD154 (CD40L) expression in peripheral blood mononuclear cells of healthy subjects in liver allograft recipients and X-linked hyper-IgM syndrome. Clin. Transplant., 2000, 14, 6, p. 520–528.
4. Callard, R. E., Smith, S. H., Herbert, J., Morgan, G. et al. CD40 ligand (CD40L) expression and B cell function in agammaglobulinemia with normal or elevated levels of IgM (HIM). Comparison of X-linked, autosomal recessive, and non-X-linked forms of the disease, and obligate carriers. J. Immunol., 1994, 1, 153, p. 3295–3306.
5. Durandy, A., Taubenheim, N., Peron, S., Fischer, A. Pathophysiology of B-cell intrinsic immunoglobulin class switch recombination deficiencies. Adv. Immunol., 2007, 94, p. 275–306.
6. Etzioni, A., Ochs, H. D. The hyper IgM syndrome – an evolving story. Pediatr. Res., 2004, 56, 4, p. 519–525.
7. Farrington, M., Grosmaire, L. S., Nonoyama, S., Fischer, S. H. et al. CD40 ligand expression is defective in a subset of patients with common variable immunodeficiency. Proc. Natl. Acad. Sci. USA, 1994, 1, 91, p. 1099–1103.
8. Haskard, D. O. Cell adhesion molecules in rheumatoid arthritis. Curr. Opin. Rheumatol., 1995, 7, 3, p. 229–234.
9. Hassan, G. S., Merhi, Y., Mourad, W. M. CD154 and its receptors in inflammatory vascular pathologies. Trends Immunol., 2009, 30, 4, p. 165–172.
10. Chatzigeorgiou, A., Lyberi, M., Chatzilymperis, G., Nezos, A., Kamper, E. CD40/CD40L signaling and its implication in health and disease. Biofactors, 2009, 35, 6, p. 474–483.
11. Korthäuer, U., Graf, D., Mages, H. W., BriŹre, F. et al. Defective expression of T-cell CD40 ligand causes X-linked immunodeficiency with hyper-IgM. Nature, 1993, 361, 6412, p. 539–541.
12. Kral, V., Jilek, D., Pohorska, J., Mikulova, S. et al. A ten-month-old boy with serious lung finding and poor weight gain. Contribution of laboratory search for early diagnostics of hyper-IgM syndrome. A case report. Allergy, 2007, 92, suppl. 83, p. 520.
13. Laman, J. D., de Smet, B. J., Schoneveld, A., van Meurs, M. CD40-CD40L interactions in atherosclerosis. Immunol. Today, 1997, 18, 6, p. 272–277.
14. Litzman, J., Lokaj, J., Thon, V. Syndrom hyperimunoglobulinémie M – klinický a laboratorní obraz. Klinická Imunológia a alergológia, 1998, 8, 4, p. 6–9.
15. Moschese, V., Litzman, J., Callea, F., Chini, L. et al. A novel form of non-X-linked hyperIgM associated with growth and pubertal disturbances and with lymphoma development. J. Pediatr., 2006, 148, 3, p. 404–406.
16. Ochs, H. D. Patients with abnormal IgM levels: assessment, clinical interpretation, and treatment. Ann. Allergy Asthma Immunol., 2008, 100, p. 509–511.
17. O‘Gorman, M. R., Zaas, D., Paniagua, M., Corrochano, V. et al. Development of a rapid whole blood flow cytometry procedure for the diagnosis of X-linked hyper-IgM syndrome patients and carriers. Clin. Immunol. Immunopathol., 1997, 85, 2, p. 172–181.
18. Pelchen-Matthews, A., Parsons, I. J., Marsh, M. Phorbol ester-induced downregulation of CD4 is a multistep process involving dissociation from p56lck, increased association with clathrin-coated pits, and altered endosomal sorting. J. Exp. Med., 1993, 178, 4, p. 1209–1222.
19. Rondina, M. T., Lappé, J. M., Carlquist, J. F., Muhlestein, J. B. et al. Soluble CD40 ligand as a predictor of coronary artery disease and long-term clinical outcomes in stable patients undergoing coronary angiography. Cardiology, 2008, 109, 3, p. 196–201.
20. Rosen, F. S., Kevy, S. V., Merler, E., Janeway, C. A. et al. Recurent bacterial infections and dysgammaglobulinemia: deficiency of 7S gammaglobulins in the presence of elevated 19S gamma-globulines. Report of two cases. Pediatrics, 1961, 28, p. 182–195.
21. Ruegg, C. L., Rajasekar, S., Stein, B. S., Engleman, E. G. Degradation of CD4 following phorbol-induced internalization in human T lymphocytes. Evidence for distinct endocytic routing of CD4 and CD3. J. Biol. Chem., 1992, 267, 26, p. 18837–18843.
22. Thon, V., Wolf, H. M., Sasgary, M., Litzman, J. et al. Defective integration of activating signals derived from the T cell receptor (TCR) and costimulatory molecules in both CD4+ and CD8+ T lymphocytes of common variable immunodeficiency (CVID) patients. Clin. Exp. Immunol., 1997, 110, p. 174–181.
23. Unek, I. T., Bayraktar, F., Solmaz, D., Ellidokuz, H. et al. Enhanced levels of soluble CD40 ligand and C-reactive protein in a total of 312 patients with metabolic syndrome. Metabolism, 2009, (Epub ahead of print).
24. van Kooten, C., Banchereau, J. CD40-CD40 ligand. J. Leukoc. Biol., 2000, 67, 1, p. 2–17.
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Epidemiology, Microbiology, Immunology
2010 Issue 3
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