T Lymphocyte Immunophenotyping With Super Bright Antibody Conjugates

14-color flow cytometry panel

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Here we present a 14-color immunophenotyping panel used to characterize human T lymphocytes. This analysis takes advantage of the diverse collection of fluorescent antibodies available, including those labeled with Invitrogen eBioscience Super Bright polymer dyes, a suite of exceptionally bright fluorophores excited by the violet laser. Optimized for use in flow cytometry, the Super Bright dyes allow for expanded use of violet laser excitation, promote streamlined multicolor panel design, and enable detection of low-abundance cell targets.

Recently expanded to include 6 fluorescence detectors for the violet laser (405 nm), the Invitrogen Attune NxT Flow Cytometer is capable of detecting multiple Super Bright dyes in a single immunophenotyping panel, greatly expanding the number of fluorescent parameters that can be simultaneously measured. The unique acoustic-assisted hydrodynamic focusing technology of the Attune NxT cytometer enables rapid, accurate, and sensitive detection at high flow rates without compromising data quality, as demonstrated with this 14-color data set (Figure 1).

Summary of methods and panel development

Human peripheral blood mononuclear cells (PBMCs) were prepared from whole blood using density gradient separation, and then stimulated by treating with 5 μg/mL concanavalin A for 3 days. Unstimulated cells were also tested, and fluorescence-minus-one (FMO) controls were prepared for marker boundary placement [1]. Cell samples were labeled with the optimal titer for each antibody conjugate, and single-color compensation controls were prepared using the Invitrogen AbC Total Antibody Compensation Kit for antibody conjugate compensation and Invitrogen ArC Amine-Reactive Compensation Bead Kit for compensation of the Invitrogen LIVE/DEAD Fixable Dead Cell Stain. Table 1 shows the specifications for the 14-color T cell immunophenotyping panel, including the Attune NxT V6 instrument configuration and the panel reagents. The T cell phenotypic markers used in this panel identify differentiation profiles, activation, exhaustion status, and co-stimulatory activity. Instead of using conventional intracellular markers for cytokines (e.g., IFN-γ) or transcription master regulators (e.g., T-bet), only surface markers were selected for this panel so that fixation and permeabilization steps were not required. This streamlined and shortened the sample preparation process, while also reducing cell loss due to multiple wash steps. For example, to detect T helper 1 (Th1), Th2, and Th17 subpopulations, we used the surface markers CD45RA, CD196 (CCR6), CD183 (CXCR3), and CD194 (CCR4) [2]. Likewise, T regulatory (Treg) cells, often detected with the surface marker CD25 and the intracellular marker FoxP3, were instead identified using only surface markers CD25 and CD127 [3,4]. Upon activation or in diseases such as hematologic malignancies, T cells up-regulate expression of activation markers such as CD25, CD134 (OX40), and CD278 (ICOS, inducible co-stimulator). Expression of CD279 (PD-1), an immune checkpoint receptor, is used to identify T cells as exhausted or quiescent. Another immune checkpoint receptor, CD223 (LAG-3), negatively regulates cellular proliferation, activation, and homeostasis of T cells, and plays a role in Treg suppressive activity [5]. Table 2 lists the T cell subpopulations and corresponding immunophenotypes identified by this panel.

Table 1. Specifications for the 14-color T cell immunophenotyping panel, including the Attune NxT V6 instrument configuration and the panel reagents (antibody target, clone, and fluorophore).

Table 2. Summary of results from the 14-color T cell immunophenotyping panel.

Data acquisition and analysis

Samples were acquired on the recently introduced 4-laser Attune NxT V6 Flow Cytometer, which has the ability to detect 6 violet laser–excited fluorophores, with a flow rate of 200 μL/minute. Data were analyzed using Attune NxT v2.6 software. Corrections for spectral overlap were performed using standard instrument autocompensation procedures. The results show well-characterized cell populations, with the expected differences between unstimulated and stimulated PBMCs ex vivo (Table 2).

References

1. Maecker HT, Trotter J (2006) Cytometry A 69:1037–1042.
2. Gosselin A, Monteiro P, Chomont N et al. (2010) J Immunol 184:1604–1616.
3. Sakaguchi S, Miyara M, Costantino CM et al. (2010) Nat Rev Immunol 10:490-500.
4. Yu N, Li X, Song W et al. (2012) Inflammation 35:1773–1780.
5. Huang CT, Workman CJ, Flies D et al. (2004) Immunity 21:503–513.