title: Normalizing ELISPOT responses to T-cell counts: a novel approach for quantification of HCMV-specific CD4(+) and CD8(+) T-cell responses in kidney transplant recipients.
abstract: BACKGROUND
  Human cytomegalovirus (HCMV) is the most common opportunistic virus infection in solid organ transplant recipients. The analysis of HCMV-specific T-cell immunity after organ transplant is of relevant clinical interest.
  
  
  OBJECTIVE
  To analyze HCMV-specific CD4(+) and CD8(+) T-cell responses in healthy subjects and kidney transplant recipients (KTR).
  
  
  METHODS
  HCMV-specific T-cell responses were evaluated by interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) using overlapping 15-mer peptide pools of immediate early (IE)-1, IE-2, phosphoprotein 65 (pp65) (for stimulation of both CD4(+) and CD8(+) T-cell responses) and a pool of 34 short peptides (8-12 amino acids in length, for stimulation of CD8(+) T-cell responses). ELISPOT results were normalized to T-cell subset counts and their correlations with a reported dendritic cell (DC)-based assay, which simultaneously quantifies HCMV-specific CD4(+) and CD8(+) T-cell responses, were analyzed.
  
  
  RESULTS
  HCMV-seropositive KTR showed higher ELISPOT responses compared to HCMV-seropositive healthy subjects. IE-1 and pp65 ELISPOT responses were mediated mainly by CD8(+) T-cells and, to a lesser extent, CD4(+) T cells; IE-2 peptides appear to stimulate CD56(+) cells (natural killer cells). In HCMV-seropositive healthy subjects, ELISPOT results (expressed either as net spots/million cells or normalized to the corresponding T-cell count) significantly correlated with the DC assay. However, in HMCV-seropositive KTR, only normalized ELISPOT responses to overlapping 15-mer peptide pools significantly correlated with DC-assay responses.
  
  
  CONCLUSIONS
  The normalized ELISPOT represents a novel and simple approach for quantifying and monitoring HCMV-specific CD4(+) and CD8(+) T-cell responses in KTR.
authors: Molecular Virology Unit, Microbiology and Virology Department, Fondazione IRCCS Policlinico San Matteo, Via Taramelli 5, 27100 Pavia, Italy.
authors: Sandra A
authors: SA
authors: Calarota
authors: Molecular Virology Unit, Microbiology and Virology Department, Fondazione IRCCS Policlinico San Matteo, Via Taramelli 5, 27100 Pavia, Italy.
authors: Antonella
authors: A
authors: Chiesa
authors: Nephrology, Dialysis and Transplantation Unit, Fondazione IRCCS Policlinico San Matteo, Viale Golgi 19, 27100 Pavia, Italy.
authors: Lucia
authors: L
authors: Scaramuzzi
authors: Molecular Virology Unit, Microbiology and Virology Department, Fondazione IRCCS Policlinico San Matteo, Via Taramelli 5, 27100 Pavia, Italy.
authors: Kodjo M G
authors: KM
authors: Adzasehoun
authors: Molecular Virology Unit, Microbiology and Virology Department, Fondazione IRCCS Policlinico San Matteo, Via Taramelli 5, 27100 Pavia, Italy; Experimental Research Laboratories, Biotechnology Area, Fondazione IRCCS Policlinico San Matteo, Viale Golgi 19, 27100 Pavia, Italy.
authors: Giuditta
authors: G
authors: Comolli
authors: Nephrology, Dialysis and Transplantation Unit, Fondazione IRCCS Policlinico San Matteo, Viale Golgi 19, 27100 Pavia, Italy.
authors: Filippo
authors: F
authors: Mangione
authors: Nephrology, Dialysis and Transplantation Unit, Fondazione IRCCS Policlinico San Matteo, Viale Golgi 19, 27100 Pavia, Italy.
authors: Pasquale
authors: P
authors: Esposito
authors: Molecular Virology Unit, Microbiology and Virology Department, Fondazione IRCCS Policlinico San Matteo, Via Taramelli 5, 27100 Pavia, Italy. Electronic address: f.baldanti@smatteo.pv.it.
authors: Fausto
authors: F
authors: Baldanti
mesh_terms: D000328:Adult; D000368:Aged; D000956:Antigens, Viral / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D003587:Cytomegalovirus / Q000276:immunology*; D003586:Cytomegalovirus Infections / Q000276:immunology*; D058501:Enzyme-Linked Immunospot Assay / Q000592:standards*; D005260:Female; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D016030:Kidney Transplantation; D018655:Lymphocyte Count; D008297:Male; D008875:Middle Aged; D066027:Transplant Recipients
chemical_list: D000956:Antigens, Viral; D007371:Interferon-gamma
keywords: ELISPOT; Human cytomegalovirus; Interferon-γ; Kidney transplantation; T-cell counts