The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

Summary

The ability to monitor T cell responses in detail in vivo is important for the development of our understanding of the immune response. Here we describe the use of fluorescent target arrays (FTAs) in an in vivo T cell assay that assesses >250 parameters simultaneously by flow cytometry.

Abstract

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into >250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8⁺ and CD4⁺ T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8⁺ T cell-mediated killing of FTA target cells and CD4⁺ T cell-meditated help of FTA B cell target cells in real time in vivo by flow cytometry. Since >250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g. the cumulative magnitude of the response) and a qualitative (e.g. functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.

Introduction

T cells play a central role in the adaptive immune response and are often targeted for manipulation in immunotherapy. CD4⁺ effector T cells respond to foreign antigen by secreting cytokines that regulate many aspects of immunity and can also directly help B cells to manufacture antibodies. CD8⁺ cytotoxic T cells (CTLs) can also respond to foreign antigen by secreting cytokines as well as playing a central role in directly killing cells expressing a foreign antigen. The fundamental interaction that initiates these T cell effector functions involves the interaction of the T cell receptor (TCR) with foreign peptides displayed on MHC molecules on the....

Protocol

Note: Mice used under this protocol were handled according to the guidelines of the Australian National University Animal Experimentation Ethics Committee and mice were euthanized by cervical dislocation.

1. Dye and Peptide Preparation

1. Dye preparation
   Note: Dyes are prediluted at different concentrations to allow labeling of cells at discrete fluorescence intensities. CFSE is used at seven concentrations made through 3.5-fold serial dilutions, and CTV and CPD are used at six different concentrations made through 3.7-fold serial dilutions (see Tables 2-4). The stock concentrations of dyes listed in T....

Results

As an example of the use of the FTA assay, a BALB/c mouse was immunized with recombinant vaccinia virus (VV) expressing HIV-I epitopes (VV-HIV) and responses to the HIV-I CTL epitopes, Gag, Gag mut, Env and Pol, the VV CTL epitopes F2L and F2L mut, and the HIV-I T_H cell epitope, Gag Th (as described in²) were assessed using a 252 parameter FTA assay (Figure 1B). Epitope variants of Gag (Gag mut) and F2L (F2L mut) are not expressed in the VV-HIV vector and therefore responses against.......

Discussion

The advantage of FTA-based assays is that they allow the discrimination of >250 viable and fully functional target cell populations from a single host animal by flow cytometry. This provides a level of complexity to in vivo flow cytometry based assays that has not been possible before. This is highlighted in the 2 animal experiments shown above, where responses to 7 distinct viral epitopes at 6 concentrations could be monitored in replicates of 6 simultaneously in a single animal allowing parameters such as .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
RPMI	Sigma	R8758
Fetal calf serum	Serana	FCS-500
CTV	Invitrogen	C34557
CFDA, SE	Invitrogen	C1157
CPD	eBioscience	65-0840-90
anti-B220 PerCp-Cy5.5	eBioscience	110730
anti-CD69 brilliant violet 605	Biolegend	104529
PKH-26	Sigma	PKH26GL-1KT
Vortex	Scientific Industries Inc
Centrifuge	Eppendorf
Flow cytometer (Fortessa or equivalent with blue, red and violet laser source)	BD Bioscience