Validation of RNA-based molecular clonotype analysis for virus-specific CD8+ T-cells in formaldehyde-fixed specimens isolated from peripheral blood.
Recent advances in the field of molecular clonotype analysis have enabled detailed repertoire characterization of viably isolated antigen-specific T cell populations directly ex vivo. However, in the absence of a biologically contained FACS facility, peripheral blood mononuclear cell (PBMC) preparations derived from patients infected with agents such as HIV must be formaldehyde fixed to inactivate the pathogen; this procedure adversely affects nucleic acid template quality. Here, we developed and validated a method to amplify and sequence mRNA species derived from formaldehyde fixed PBMC specimens. Antigen-specific CD8+ cytotoxic T-lymphocyte populations were identified with standard fluorochrome-conjugated peptide-major histocompatibility complex class I tetramers refolded around synthetic peptides representing immunodominant epitopes from HIV p24 Gag (KRWII[M/L]GLNK/HLA B*2705) and CMV pp65 (NLVPMVATV/HLA A*0201 and TPRVTGGGAM/HLA B*0702), and acquired in separate laboratories with or without fixation. In the presence of proteinase K pre-treatment, the observed antigen-specific CD8+ T-cell repertoire determined by molecular clonotype analysis was statistically no different whether derived from fixed or unfixed PBMC. However, oligo-dT recovery methods were not suitable for use with fixed tissue as significant skewing of clonotypic representation was observed. Thus, we have developed a reliable RNA-based method for molecular clonotype analysis that is compatible with formaldehyde fixation and therefore suitable for use with primary human samples isolated by FACS outside the context of a biological safety level 3 containment facility.
van Bockel
David
D
Centre for Immunology, St Vincent's Hospital, Sydney, NSW, Australia. d.vanbockel@cfi.unsw.edu.au
Price
David A
DA
Asher
Tedi E
TE
Venturi
Vanessa
V
Suzuki
Kazuo
K
Warton
Kristina
K
Davenport
Miles P
MP
Cooper
David A
DA
Douek
Daniel C
DC
Kelleher
Anthony D
AD
D000818:Animals; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism* / Q000821:virology; D002469:Cell Separation; D002478:Cells, Cultured; D002999:Clone Cells; D022801:Complementarity Determining Regions / Q000235:genetics; D005404:Fixatives; D005434:Flow Cytometry; D005557:Formaldehyde; D019674:Genes, T-Cell Receptor beta; D015497:HIV-1 / Q000276:immunology / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D016130:Immunophenotyping; D051379:Mice; D012313:RNA / Q000032:analysis* / Q000097:blood / Q000378:metabolism; D038301:Virus Inactivation
D022801:Complementarity Determining Regions; D005404:Fixatives; D005557:Formaldehyde; D012313:RNA
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