OMIP‐067: 28‐Color Flow Cytometry Panel to Evaluate Human T‐Cell Phenotype and Function (2020)

This 28-color flow cytometry panel was developed to detect frequencies and function of a broad spectrum of T-cell subsets from the blood of infants and adults following antigen-specific stimulation. This panel includes markers to characterize αβ and γδ T-cell subsets, T regulatory cells (Tregs), T follicular helper cells (TfH), and recent thymic emigrants (RTEs). Additionally, antibodies that measure cell surface activation molecules (CD154, CD69, HLA-DR, and CD38) and function through detection of cytokines (IL-8, IL-2, IL-4, IL-5, IL-13, IL-21, IFNγ, and TNF) are also included. This panel was optimized using PMA-stimulated, cryopreserved healthy adult and infant PBMCs as well as stimulation with CMV-specific peptides. Currently, this panel is being used to evaluate antigen-specific T-cell responses in adults and infants following immunization with an attenuated malaria vaccine (Table 1). T cells have an essential role in mediating protection against a variety of infectious diseases and tumors (1). Importantly, T cells are remarkably diverse, comprising multiple different subsets with distinct functions (2). Developing technologies that can efficiently and comprehensively assess the phenotypic and functional heterogeneity of different T-cell populations following vaccination or infection will be important for defining immune correlates of protection and enable development of more efficacious vaccines (3, 4). Thus, a single, comprehensive T-cell flow cytometry panel that can assess a broad array of phenotypic, activation markers, and cytokines would be highly efficient for high through-put human studies. This is especially true for clinical trials focused on infants or young children which may have a limited sampling of blood. Therefore, we have designed the first 28-color OMIP that provides a comprehensive phenotypic and functional analysis of αβ and γδ T-cell subsets within a single sample from donors of any age. Following antigen encounter, naïve CD4 and CD8 αβ T cells (Fig. 1A) rapidly expand and differentiate into memory and/or effector cells (5). Each of these discrete T-cell populations has distinct functional capabilities and can be identified with unique combinations of surface markers (Fig. 1B) (6). This panel includes the markers CCR7 and CD45RA to differentiate naïve, effector (Tef), effector memory (Tem), and central/transitional memory T cells (Tcm/Ttm). This panel also includes several markers to detect T-cell activation following antigen stimulation (Fig. 1C,F,G). CD38 and HLA-DR are upregulated on αβ and γδ T cells following antigen encounter and have been shown to correlate with protection following malaria vaccination (7). CD154, a CD4 T-cell activation marker, is essential for the development of high-affinity antibody responses and can license dendritic cell activation (8). Furthermore, CD154 expression can be used to determine the frequency of antigen specific CD4 T cells following in vitro stimulation as it is expressed on essentially all antigen specific cells (9). Finally, this panel also includes the activation marker CD69 to aid in the detection of low-frequency cytokine producing cells following antigen-specific stimulation in vitro. CD4 T cells have striking phenotypic and functional heterogeneity. Thus, this panel also includes markers to characterize two CD4 T-cell subsets that play important roles in regulating immune responses following vaccination. First, TfH cells are specialized CD4 T cells located in secondary lymphoid organs that are essential for helping B cells generate protective antibody responses (10). Non-naïve CXCR5+ CD4 T cells in the blood share a similar genetic, phenotypic, and functional profile as traditional TfH cells (11, 12) and are called blood TfH cells (Fig. 1D). Importantly, the frequency of blood TfH cells has been shown to directly correlate with protective antibody responses (11). Blood TfH cells can be further divided into distinct functional subsets using the markers PD-1 and CXCR3 (13), which we have also included in this panel (Fig. 1D). Second, Tregs play an essential role in negatively regulating immune cell-generated inflammation (14) and can impact vaccine responses (15). Thus, this panel uses the markers CD25 and CD127 to identify Tregs in the blood (Fig. 1E) (16). Activated T cells rapidly differentiate and acquire the capability of producing cytokines which can vary depending on multiple factors including the nature of the stimulating antigen, the innate milieu, and the age of the individual. This panel includes a wide array of markers to detect T-cell function (Fig. 1F–H). These include the canonical cytokines, IFNγ, TNF, and IL-2 which have been shown to be important in mediating protection against tumors and a number of infectious diseases, including malaria (7, 17, 18). Also included are cytokines IL-4, IL-5, and IL-13 which are produced by CD4 T helper (Th) 2 cells. Such cytokines can play an important role in protection against certain parasitic infections but also are associated with allergy and asthma and can counter regulate Th1 responses. Of note, newborns and infants are more pre-disposed to producing Th2 cytokines and so they are important to assess in vaccine studies of infants (19, 20). Finally, IL-21 was included in the panel for its essential role in TfH-mediated generation of protective antibody responses (10) as well as its function in maintaining T-cell memory (21) which could both serve as a predictors of vaccine efficacy. Over the course of aging, thymic output decreases and the T-cell compartment shifts to a more antigen-experienced phenotype. Because this panel was in part designed to study infant PBMCs from a clinical vaccine trial, it was necessary to include phenotypic and functional markers that are more prominent at this stage of life. This panel uses CD31 in combination with CD45RA to identify RTEs (Fig. 1I), which are enriched in infants (22). Furthermore, infant T cells preferentially secrete IL-8 upon stimulation more so than Th1, Th2, or Th17 cytokines (23). Therefore, IL-8 was included as it would optimize the assessment of infant T-cell responses (Fig. 1I). γδ T cells are a group of unconventional T cells that have been implicated in the response to a number of different types of infection (24). The two major subsets in human blood can be identified by expression of either the Vδ1 or Vδ2 chain of the T-cell receptor (Fig. 1J). Vδ1+ and other Vδ2− γδ T cells are similar to conventional αβ T cells in that they have a diverse, private repertoire which becomes focused upon antigen encounter (25). Naïve Vδ1+ or Vδ1/2− γδ T cells can be identified using the markers CD27 and CD127, while those that have undergone clonal expansion can be identified with CD27 and CX3CR1 (Fig. 1J). Vδ2+ T cells on the other hand typically have somewhat invariant TCRs. Most Vδ2+ T cells are paired with the Vγ9 chain (Fig. 1J) and play an essential role in generating immunity to malaria following vaccination (18, 26). However, a small subset of Vδ2+ T cells are Vγ9− and are more similar to Vδ1+ and Vδ1/2− γδ T cells in their response to pathogens (27). Previously published T cell focused OMIPs are solely devoted to either phenotyping or function and are intended for use on adult subjects. This panel will be highly useful for a “one-shot” comprehensive analysis of T-cell phenotype and function in subjects of any age. OMIP-056 focuses on T-cell responses (4) and is similar to our panel. However, our panel additionally includes markers for extensive γδ T cell, TfH, and RTE characterization as well as more functional markers including IL-8 and IL-21. OMIP-058 is also somewhat similar to our panel in that it covers some αβ and γδ T-cell phenotyping (28), but some major differences are that there are no markers to identify TfH cells, Tregs, or RTEs and there are no functional markers. Additionally, our panel contains markers to identify γδ T-cell differentiation state, which are lacking in OMIP-058. OMIP-060 (3) contains similar T-cell functional markers as this panel, but there are no markers for extensive γδ T cell, TfH, and RTE characterization as in ours. Furthermore, OMIP-060 has a marker for only one Th2 cytokine (IL-13), while our panel contains markers for all three (IL-4,5,13). We would like to thank BD Biosciences for providing conjugated test antibodies and advice on high parameter fluorescence flow cytometry through a Cooperative Research and Development Agreement with the Vaccine Research Center, NIAID, NIH. In addition, we would like to thank the VRC flow core facility for instrument characterization and maintenance. This work was supported by funding from the NIH. MIFlowCyt MIFlowCyt-Compliant Items Appendix S1. Supporting Information. Online Figure 1 Surface marker titrations. Each of the indicated surface markers underwent six 2-fold dilutions as indicated on the x-axis of each dot plot. Cells were gated on live total lymphocytes. The very left cluster in each plot is a no stain control, and the optimal dilution for the final panel is indicated by a red box. Online Figure 2 CD38 and HLA-DR titrations. Surface titrations for CD38 and HLA-DR antibodies as in Online Figure 1. Cells were gated on either live total lymphocytes (top row) or live CD3+ T cells (bottom row). The very left cluster in each plot is a no stain control, and the optimal dilution for the final panel is indicated by a red box. Online Figure 3 Intracellular marker titrations. Each of the indicated intracellular markers underwent six 2-fold dilutions as indicated on the x-axis of each dot plot. Cells for each marker titration were gated on live CD3+ T cells with the exception of CD3 which was gated on live total lymphocytes. The very left cluster in each plot is a no stain control, and the optimal dilution for the final panel is indicated by a red box. Online Figure 4 BUV563 and BV570 SSE into PE. (A) Dot plots representing cells from two different donors that were either PMA/Ionomycin-stimulated or unstimulated and stained with panel iteration #1. CD45RA BUV563 spreads heavily into the PE channel in all plots. (B) Dot plots representing cells from two different donors that were either PMA/Ionomycin-stimulated or unstimulated and stained with panel iteration #2. CD45RA BV570 spreads heavily into the PE channel in all plots. (C) Dot plots representing cells from two different donors that were either PMA/Ionomycin-stimulated or unstimulated and stained with the final panel (iteration #13). Clear resolution of cell populations is observed in the BUV563, BV570, and PE channels. Online Figure 5 gd T cell antibody clone comparisons. Comparison of total gd T cell staining (A) and Vd2 gd T cell staining (B) using different combinations of pan-gd TCR and Vd2 TCR antibody clones on PBMCs from two different donors. Antibody clones are in parentheses on the axes. Black numbers within each plot indicate frequency of total gd T cells (A) or Vd2 gd T cells (B), and red numbers indicate difference in frequency between staining with B6 clone vs 123R3 clone. Online Figure 6 Vg9 gd T cell antibody clone comparisons. Comparison of total gd T cell staining (A) and Vg9 staining of Vd2+ gd T cells (B) using different combinations of Vg9 antibody clones with the REA591 pan-gd TCR antibody and 123R3 Vd2 TCR antibody on PBMCs from two different donors. Vg9 antibody clones are in parentheses on the axes. Black numbers within each plot indicate frequency of total gd T cells (A) or Vg9+/− of Vd2+ gd T cells (B), and red numbers indicate difference in frequency between staining with 7A5 clone vs the average of the others. Online Figure 7 Late-stage panel modifications. (A) SSE from CD25 PE/Cy5 into B660. (B) Comparison of Treg (CD25+CD127− CD4 T cells) resolution using CD25 on two different fluorochromes. (C) Comparison of RTE (CD31+CD45RA+ CD4 T cells) resolution using CD31 on two different fluorochromes. Online Figure 8 FMO controls for markers in final panel iteration with large SSE. (A) Unstimulated PBMCs were used to compare CXCR3 PE/Dazzle 594 staining of blood TfH cells with the full panel or IL-8 FMO. (B) Unstimulated PBMCs were used to compare CD25 BB700 staining of Tregs with full panel or HLA-DR FMO control. (C) PMA-stimulated (top row) or unstimulated (bottom row) PBMCs were used to compare CD69 BB790 staining in non-naïve CD4 T cells (top row) or CD27 BV785 staining in CD4 T cells (bottom row) with the full panel or CD127 FMO. (D) PMA-stimulated PBMCs were used to compare CD127 PE/Cy7 staining (top row) or CD27 BV785 staining (bottom row) in CD4 T cells with the full panel or CD69 FMO. Online Figure 9 Infant sample gating tree. Infant donor PBMCs stained with the full 28-color panel (Online Table 2). (A) Lineage gating of T cells. After gating on single cells, lymphocytes, and live CD3+ T cells, a pan-TCRgd antibody was used to separate gd and ab T cells, which were further subdivided into CD4+ and CD8+ ab T cells. (B) CCR7 and CD45RA antibodies were used to gate CD4+ and CD8+ ab T cell naïve and memory subsets. Naive T cells (naïve), Central and transitional memory T cells (Tcm/Ttm), Effector memory T cells (Tem), and Effector T cells (Tef). (C) CD38 and HLA-DR were used to identify activated non-naïve CD4 and CD8 ab T cells and gd T cells. (D) CXCR5+ was used to identify blood T follicular helper (TfH) cells of non-naïve CD4 T cells, and blood TfH subsets were further characterized using PD-1 and CXCR3. (E) CD25 and CD127 were used to identify T regulatory cells (Tregs). (F-G) Gating to measure T cell function following no stimulation or stimulation with cell activation cocktail. Cytokine staining is shown following gating on either non-naïve CD4 T cells (F) or non-naïve CD8 T cells (G). (H) CD31 and CD45RA were used to identify recent thymic emigrants (RTEs). RTE function was measured by IL-8 following no stimulation or stimulation with cell activation cocktail. (I) TCR Vd1 and Vd2 antibodies were used to identify gd T cell subsets. Vd1 and Vd1/Vd2− (not shown) T cell subsets were further analyzed for naïve (CD27+CD127+) vs clonal expansion (CX3CR1+CD127−). Vg9 was also used to identify Vg9+ or Vg9− Vd2+ T cells. Online Figure 10 Panel staining following antigen-specific stimulation. Donor PBMCs were either unstimulated or stimulated with a CMV peptide cocktail and then stained with the full 28-color panel. Cytokine staining is shown following gating on either non-naïve CD4 T cells (top) or non-naïve CD8 T cells (bottom). Numbers within plots indicate frequencies of indicated cytokines of respective non-naïve T cells. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.