title: Raman spectroscopic modeling of early versus late T-lymphocyte activation via differential spectral detection of receptor expression.
abstract: The proven efficacy of renal transplantation has made it the definitive treatment for end-stage renal disease. Despite its wide acceptance, transplantation has been limited by organ shortages. In the face of this, preservation of allograft longevity is essential. The predominately T cell-driven process of acute rejection (AR) can lead to graft dysfunction and even graft loss. As a marker for AR screening, serum creatinine has a low sensitivity and specificity. This has warranted the development of more accurate screening/diagnostic tools such as Raman Spectroscopy (RS) which has been demonstrated in previous studies to accurately quantify T cell activation. In this study we further explore its application by modeling the dynamic process of cell surface receptor expression during T cell activation. 50 mitogen (Concanavalin A and pokeweed) activated T cells were stained with CD69, CD25, and CD71 monoclonal antibodies (mAbs) at 48 and 72 hour time points. In parallel, 50 activated T cells were analyzed using RS at these same time periods. At 4 8h there was high expression of the CD69 cell surface receptor detected via mAb staining with no appreciable binding of CD25/CD71 fluorescent tag. In conjunction, 48 hour RS-analyzed T cells demonstrated a significant peak difference at the 1585 cm(-1) position which represented a 63% (p=0.01) increase in peak magnitude when compared with the 72 hour samples. By contrast, the 72 hour data demonstrated an attenuation of the CD69 expression and increased CD25/CD71 expression. The corresponding RS analysis showed two significant peak differences at the 903 cm(-1) and 1449 cm(-1) positions that were not present at 48 h. These differences in Raman shifts resulted in a 40% (p=0.04) and a 59% (p=0.001) increase in peak magnitudes at these positions, respectively. This study serves to further validate RS as a screening modality capable of not only detecting T cells early in the activation process through the spectral signatures associated with CD69, but also quantifying the persistent expression of CD25 and CD71. This provides a foundation for the development of a system capable of the accurate assessment of acute and maintenance immunosuppression efficacy at the molecular level.
authors.affiliation: Department of Surgery, Section of Transplant Surgery, Suite 6-C, Wayne State University School of Medicine, 4201 St. Antoine BLVD, Detroit, MI 48201, United States; Department of Biomedical Engineering, Smart Sensor and Integrated Microsystems, 3100 Eng. Bldg, 5050 Anthony Wayne Drive, Detroit, MI 48202, United States. Electronic address: kristilb@usc.edu.
authors.forename: Kristian L
authors.initials: KL
authors.lastname: Brown
authors.affiliation: Department of Biomedical Engineering, Smart Sensor and Integrated Microsystems, 3100 Eng. Bldg, 5050 Anthony Wayne Drive, Detroit, MI 48202, United States.
authors.forename: Olena Y
authors.initials: OY
authors.lastname: Palyvoda
authors.affiliation: Department of Biomedical Engineering, Smart Sensor and Integrated Microsystems, 3100 Eng. Bldg, 5050 Anthony Wayne Drive, Detroit, MI 48202, United States.
authors.forename: Gregory W
authors.initials: GW
authors.lastname: Auner
authors.affiliation: Department of Biomedical Engineering, Smart Sensor and Integrated Microsystems, 3100 Eng. Bldg, 5050 Anthony Wayne Drive, Detroit, MI 48202, United States; Department of Biomedical Engineering, Smart Sensor and Integrated Microsystems, 3100 Eng. Bldg, 5050 Anthony Wayne Drive, Detroit, MI 48202, United States.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Gruber
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry; D015703:Antigens, CD / Q000235:genetics / Q000276:immunology; D000945:Antigens, Differentiation, T-Lymphocyte / Q000235:genetics / Q000276:immunology; D003208:Concanavalin A / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D016130:Immunophenotyping; D053645:Interleukin-2 Receptor alpha Subunit / Q000235:genetics / Q000276:immunology; D037181:Lectins, C-Type / Q000235:genetics / Q000276:immunology; D008213:Lymphocyte Activation / Q000187:drug effects; D008934:Mitogens / Q000494:pharmacology*; D011043:Pokeweed Mitogens / Q000494:pharmacology*; D061251:Primary Cell Culture; D011990:Receptors, Transferrin / Q000235:genetics / Q000276:immunology; D013059:Spectrum Analysis, Raman; D013194:Staining and Labeling; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D000099192:CD69 Antigens
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D003208:Concanavalin A; D053645:Interleukin-2 Receptor alpha Subunit; D037181:Lectins, C-Type; D008934:Mitogens; D011043:Pokeweed Mitogens; D011990:Receptors, Transferrin; D000099192:CD69 Antigens; C068126:CD71 antigen; C508017:IL2RA protein, human
keywords: Acute rejection; CD25 receptor expression; CD69 receptor expression; CD71 receptor expression; Raman Spectroscopy; Renal transplantation