Abstract
BackgroundSIV and HIV predominantly replicate in lymphoid tissue, but the study of virus specific CD8+ T cells in intact lymphoid tissue is difficult, as traditional in situ tetramer staining requires fresh tissue.ResultsIn this report, we demonstrate a novel technique using Qdot 655-conjugated peptide-MHC multimers to directly visualize SIV specific cells in cryopreserved tissue biopsies from chronically SIVmac239 infected Rhesus macaques. Qdot 655 multimers showed similar sensitivity and specificity to APC-conjugated tetramers by flow cytometry analysis, but yielded ten-fold higher signal intensity when imaged by fluorescence microscopy. Using this technique, we detected CD8+ T cells which recognize an immunodominant epitope (Gag CM9) in the spleen, lymph nodes, ileum and colon. In all these tissues, the Gag CM9 positive cells were mainly located in the extra follicular T cell zone. In the ileum and colon, we found Gag CM9 positive cells concentrated in Peyer's patches and solitary lymphoid follicles, a pattern of localization not previously described.ConclusionsThe use of Qdot multimers provide an anatomic and quantitative evaluation of SIV specific CD8+ T cell responses in SIV pathogenesis, and may prove useful to studies of SIV specific CD8+ T cell responses elicited by vaccines and other immunotherapies in the non-human primate model.
Highlights
SIV and HIV predominantly replicate in lymphoid tissue, but the study of virus specific CD8+ T cells in intact lymphoid tissue is difficult, as traditional in situ tetramer staining requires fresh tissue
The T cells were stained with anti-CD3, anti-CD8 antibodies and Gag CM9 Qdot 655 multimers, or Gag CM9 APC tetramers or Qdot 655 conjugated with the MamuA*01 MHC Class I allele loaded with an irrelevant peptide FLP
Analysis by flow cytometry showed that all cells from the Gag CM9 T cell clone were CD3+CD8+ cells and more than 99% of the cells bound Gag CM9 Qdot 655 multimers or the Gag CM9 APC tetramer (Fig. 1A)
Summary
SIV and HIV predominantly replicate in lymphoid tissue, but the study of virus specific CD8+ T cells in intact lymphoid tissue is difficult, as traditional in situ tetramer staining requires fresh tissue. Previous studies have demonstrated in situ staining of tetramers in fresh, lightly fixed, or frozen tissue using a two step enhancement methodology to visualize tetramer positive cells [11,12,13]. This technique has proven suboptimal for frozen tissue, presenting such difficulties as low signal intensity and poor cell morphology. Tetramer staining requires fresh tissue that should be processed within 24 h for optimal staining results and does not permit the use of archived tissue samples
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