In Situ Staining and Laser Capture Microdissection of Lymph Node Residing SIV Gag-Specific CD8+ T cells--A Tool to Interrogate a Functional Immune Response Ex Vivo.

Annelie Tjernlund,Jessica M Lindvall,Louis J Picker,Tao Peng,Adam Burgener,Lars Öhrmalm,Lawrence Corey,Jia Zhu,Kristina Broliden,M Juliana Mcelrath,Zandrea Ambrose

doi:10.1371/journal.pone.0149907

Abstract

While a plethora of data describes the essential role of systemic CD8+ T cells in the control of SIV replication little is known about the local in situ CD8+ T cell immune responses against SIV at the intact tissue level, due to technical limitations. In situ staining, using GagCM9 Qdot 655 multimers, were here combined with laser capture microdissection to detect and collect SIV Gag CM9 specific CD8+ T cells in lymph node tissue from SIV infected rhesus macaques. CD8+ T cells from SIV infected and uninfected rhesus macaques were also collected and compared to the SIV GagCM9 specific CD8+ T cells. Illumina bead array and transcriptional analyses were used to assess the transcriptional profiles and the three different CD8+ T cell populations displayed unique transcriptional patterns. This pilot study demonstrates that rapid and specific immunostaining combined with laser capture microdissection in concert with transcriptional profiling may be used to elucidate phenotypic differences between CD8+ T cells in SIV infection. Such technologies may be useful to determine differences in functional activities of HIV/SIV specific T cells.

Highlights

Replication of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), the simian equivalent of HIV, is highly variable, as is the host immune response to the infection
Humans that have an overrepresentation of HLA-BÃ27, HLA-BÃ57 or HLA-BÃ28 alleles and rhesus macaques (RMs) that have an overrepresentation of Mamu-AÃ01, Mamu-BÃ08, or Mamu-BÃ17 alleles are associated with a slow HIV/SIV disease progression [12]
A rapid immunofluorescent staining combined with laser capture microdissection (LCM) was used to detect and collect GagCM9+cellsSIV+RMs, CD8+ cellsSIV+RMs and CD8+cellsSIV-RMs in cryopreserved tissue sections of lymph nodes obtained from chronically SIV infected and uninfected RMs (Fig 1)

Summary

Introduction

Replication of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), the simian equivalent of HIV, is highly variable, as is the host immune response to the infection. While a plethora of data describes the essential role of systemic CD8+ T cells in the control of HIV/SIV replication, little is known about the local in situ CD8+ T cell immune responses against HIV/SIV at the intact tissue level [17,18,19]. Since HIV/SIV predominantly replicate in lymphoid tissue it is of major importance to study the in situ immune response, including the CD8+ T cell response, against HIV/SIV directly in these tissues [20,21,22,23,24,25,26]. We have previously shown that it is possible to detect GagCM9 specific CD8+ T cells in cryopreserved lymphoid tissue from chronically SIV infected RMs with the use of GagCM9 Qdot 655 multimers (Qdot 655 conjugated with the Mamu-AÃ01 MHC Class I allele loaded with the SIVmac239 peptide Gag181-189CM9; Gag CM9) [27]. This report describes a pilot study to evaluate the use of these Gag CM9 Qdot 655 multimers for in situ staining followed by laser capture microdissection (LCM) and the subsequent gene transcriptional profiles of these cell populations

Methods

Results

Conclusion