High dimensional flow cytometry for comprehensive leukocyte immunophenotyping (CLIP) in translational research

Abstract

New paradigms in translational research are focused on deep understanding of all aspects of the human immune system in response to diseases or perturbations such as vaccination or therapy. To obtain this knowledge, coordinated, comprehensive assessments by genomics, proteomics, and cytomics are necessary. One component of this assessment is comprehensive leukocyte immunophenotyping (CLIP) that not only provides a deep and broad description of the entire immune system at any given moment, but also encompasses all leukocyte lineages, including activation states, functional markers, and signaling molecules. As envisioned, a CLIP panel could study nearly 400 antigens utilizing 17-parameter flow cytometry. The CLIP panel is structured in a manner that tubes are grouped by lineage and, within lineage each of the tubes, while having some redundant markers, characterize distinct populations. To date, a preliminary 10 tube CLIP panel has been developed with the following 17 parameter tubes: T(reg), T(h₁₇), T(h₁/₂), B(general), B(naive/memory), B(intracellular), NK₁, NK₂, myeloid/monocyte, and dendritic cells (DC). Together these tubes have the potential to identify over 28,000 subsets of leukocytes. The feasibility of developing these tubes has been demonstrated, as well as their utility in describing complex alterations of the immune system in the context of disease and vaccination. The plethora of data accrued in the preliminary CLIP panel highlights the need for novel data analysis and reduction strategies, while at the same time illustrates the power of CLIP.

Figure 1. Schematic overview of tube development in CLIP

Figure 2. Gating strategy in T lineage tubes

Single-cell suspensions from healthy donors were stained with a combination of 14 antibodies and viable dye as described in table 1. Lymphocytes were identified based on their forward and side scatter properties. Subsequently, dead cells were excluded through the use of a viability dye. CD45 and CD3 were used to identify T cells (CD45⁺CD3⁺) among the previously selected living lymphocytes. CD4 T cells and CD8 T cells were identified with the expression of CD4 or CD8 antigens. A. Among CD4 and CD8 T cells, CD45-RA⁺ cells were separated by gating from CD45-RA⁻, and the expression of CCR7 and CD27 inside these 2 sub-populations was determined. Four sub-populations could be defined as follows; naïve (CD45-RA⁺) effector (CD45-RA⁻CD27⁻CCR7⁻), central memory (CD45-RA⁻CD27⁺CCR7⁺) and effector memory (CD45-RA⁻CD27⁺ CCR7⁻). B. Histograms of the expression of the 7 markers remaining in T1 tube after memory naïve separation. C. Histograms of the expression of the 7 markers remaining in T2 tube after memory naïve separation. D. Histograms of the expression of the 7 markers remaining in T3 tube after memory naïve separation. E. Representative dot plot of T cell specific population definition. Conventional T_reg were defined as CD4 T cells co-expressing CD25 and transcription factor FOxp3. T_h17 cells were defined as IL-17A expressing cells. T_h1 defined s IFNg expressing cells, T_h2 as IL-4 expressing cells. Also, IL-2 secreting cells can be identified.

Figure 2. Gating strategy in T lineage tubes

Single-cell suspensions from healthy donors were stained with a combination of 14 antibodies and viable dye as described in table 1. Lymphocytes were identified based on their forward and side scatter properties. Subsequently, dead cells were excluded through the use of a viability dye. CD45 and CD3 were used to identify T cells (CD45⁺CD3⁺) among the previously selected living lymphocytes. CD4 T cells and CD8 T cells were identified with the expression of CD4 or CD8 antigens. A. Among CD4 and CD8 T cells, CD45-RA⁺ cells were separated by gating from CD45-RA⁻, and the expression of CCR7 and CD27 inside these 2 sub-populations was determined. Four sub-populations could be defined as follows; naïve (CD45-RA⁺) effector (CD45-RA⁻CD27⁻CCR7⁻), central memory (CD45-RA⁻CD27⁺CCR7⁺) and effector memory (CD45-RA⁻CD27⁺ CCR7⁻). B. Histograms of the expression of the 7 markers remaining in T1 tube after memory naïve separation. C. Histograms of the expression of the 7 markers remaining in T2 tube after memory naïve separation. D. Histograms of the expression of the 7 markers remaining in T3 tube after memory naïve separation. E. Representative dot plot of T cell specific population definition. Conventional T_reg were defined as CD4 T cells co-expressing CD25 and transcription factor FOxp3. T_h17 cells were defined as IL-17A expressing cells. T_h1 defined s IFNg expressing cells, T_h2 as IL-4 expressing cells. Also, IL-2 secreting cells can be identified.

Figure 2. Gating strategy in T lineage tubes

Single-cell suspensions from healthy donors were stained with a combination of 14 antibodies and viable dye as described in table 1. Lymphocytes were identified based on their forward and side scatter properties. Subsequently, dead cells were excluded through the use of a viability dye. CD45 and CD3 were used to identify T cells (CD45⁺CD3⁺) among the previously selected living lymphocytes. CD4 T cells and CD8 T cells were identified with the expression of CD4 or CD8 antigens. A. Among CD4 and CD8 T cells, CD45-RA⁺ cells were separated by gating from CD45-RA⁻, and the expression of CCR7 and CD27 inside these 2 sub-populations was determined. Four sub-populations could be defined as follows; naïve (CD45-RA⁺) effector (CD45-RA⁻CD27⁻CCR7⁻), central memory (CD45-RA⁻CD27⁺CCR7⁺) and effector memory (CD45-RA⁻CD27⁺ CCR7⁻). B. Histograms of the expression of the 7 markers remaining in T1 tube after memory naïve separation. C. Histograms of the expression of the 7 markers remaining in T2 tube after memory naïve separation. D. Histograms of the expression of the 7 markers remaining in T3 tube after memory naïve separation. E. Representative dot plot of T cell specific population definition. Conventional T_reg were defined as CD4 T cells co-expressing CD25 and transcription factor FOxp3. T_h17 cells were defined as IL-17A expressing cells. T_h1 defined s IFNg expressing cells, T_h2 as IL-4 expressing cells. Also, IL-2 secreting cells can be identified.

Figure 2. Gating strategy in T lineage tubes

Single-cell suspensions from healthy donors were stained with a combination of 14 antibodies and viable dye as described in table 1. Lymphocytes were identified based on their forward and side scatter properties. Subsequently, dead cells were excluded through the use of a viability dye. CD45 and CD3 were used to identify T cells (CD45⁺CD3⁺) among the previously selected living lymphocytes. CD4 T cells and CD8 T cells were identified with the expression of CD4 or CD8 antigens. A. Among CD4 and CD8 T cells, CD45-RA⁺ cells were separated by gating from CD45-RA⁻, and the expression of CCR7 and CD27 inside these 2 sub-populations was determined. Four sub-populations could be defined as follows; naïve (CD45-RA⁺) effector (CD45-RA⁻CD27⁻CCR7⁻), central memory (CD45-RA⁻CD27⁺CCR7⁺) and effector memory (CD45-RA⁻CD27⁺ CCR7⁻). B. Histograms of the expression of the 7 markers remaining in T1 tube after memory naïve separation. C. Histograms of the expression of the 7 markers remaining in T2 tube after memory naïve separation. D. Histograms of the expression of the 7 markers remaining in T3 tube after memory naïve separation. E. Representative dot plot of T cell specific population definition. Conventional T_reg were defined as CD4 T cells co-expressing CD25 and transcription factor FOxp3. T_h17 cells were defined as IL-17A expressing cells. T_h1 defined s IFNg expressing cells, T_h2 as IL-4 expressing cells. Also, IL-2 secreting cells can be identified.

Figure 2. Gating strategy in T lineage tubes

Single-cell suspensions from healthy donors were stained with a combination of 14 antibodies and viable dye as described in table 1. Lymphocytes were identified based on their forward and side scatter properties. Subsequently, dead cells were excluded through the use of a viability dye. CD45 and CD3 were used to identify T cells (CD45⁺CD3⁺) among the previously selected living lymphocytes. CD4 T cells and CD8 T cells were identified with the expression of CD4 or CD8 antigens. A. Among CD4 and CD8 T cells, CD45-RA⁺ cells were separated by gating from CD45-RA⁻, and the expression of CCR7 and CD27 inside these 2 sub-populations was determined. Four sub-populations could be defined as follows; naïve (CD45-RA⁺) effector (CD45-RA⁻CD27⁻CCR7⁻), central memory (CD45-RA⁻CD27⁺CCR7⁺) and effector memory (CD45-RA⁻CD27⁺ CCR7⁻). B. Histograms of the expression of the 7 markers remaining in T1 tube after memory naïve separation. C. Histograms of the expression of the 7 markers remaining in T2 tube after memory naïve separation. D. Histograms of the expression of the 7 markers remaining in T3 tube after memory naïve separation. E. Representative dot plot of T cell specific population definition. Conventional T_reg were defined as CD4 T cells co-expressing CD25 and transcription factor FOxp3. T_h17 cells were defined as IL-17A expressing cells. T_h1 defined s IFNg expressing cells, T_h2 as IL-4 expressing cells. Also, IL-2 secreting cells can be identified.

Figure 3. Gating strategy in B lineage tubes

A. Single-cell suspensions from healthy donors were stained with a combination of 14 antibodies and viability dye as described in the material and methods and table 1 (B1, B2 and B3). Lymphocytes were identified based on their forward and side scatter properties. Subsequently, dead cells were excluded through the use of a viability dye. CD45 and CD19 were used to identify B cells (CD45⁺CD19⁺) among the previously selected living lymphocytes. B. Representative dot plot of the markers specific of Staining B1. C. Representative dot plot of the markers specific of Staining B2. D. Representative dot plot of the markers specific of Staining B3.

Figure 4. Gating strategy in NK lineage tubes

A. Single-cell suspensions from healthy donors were stained with a combination of 14 antibodies and viability dye as described in the material and methods and table 1. Natural killer cells were identified based on their forward and side scatter properties. Subsequently, dead cells were excluded through the use of a viability dye. CD45 and CD3 were used to identify NK cells (CD45⁺CD3⁻) among the previously selected living lymphocytes. CD16 and CD56 were used to identify NK among the prior populations CD16⁺ and/or CD56⁺. B. Representative dot plot of the markers specific of Staining NK1. C. Representative dot plot of the markers specific of Staining NK2

Figure 5. Gating strategy in myeloid/monocytic lineage tubes

Single-cell suspensions from healthy donors were stained with a combination of 14 antibodies and viability dye as described in the material and methods and table 1. Monocytes were identified based on their forward and side scatter properties. Subsequently, dead cells were excluded through the use of a viability dye. CD45 and CD14 were used to identify monocytes (CD45⁺CD14⁺) among the previously selected living monocytic gate.

Figure 6. Gating strategy in dendritic cell lineage tubes

A. Single-cell suspensions from healthy donors were stained with a combination of 14 antibodies and viability dye as described in the material and methods and Table 1. Dendritic cells were identified based on their forward and side scatter properties (same as lymphocyte). Subsequently, dead cells were excluded through the use of a viability dye. CD45 and lineage were used to identify DC cells (CD45⁻Lineage⁻). Expression of HLA-DR was required, and we identified myeloid dendritic cells (CD11c⁺) from plasmacytoid dendritic cells (CD123⁺) with the expression of CD11c and CD123. B. Myeloid dendritic cells (CD11c⁺) were analyzed for the expression of cytokines such as IFNa, TNFa or activation markers CD83, CD25, CD40, CD33 C. Plasmacytoid dendritic cells (CD123⁺) were analyzed for the expression of cytokines such as IFNa, TNFa or activation markers CD83, CD25, CD40. Due to low number of cells, all figures are smoothed histograms.

Figure 7. Cell loss during staining procedure

Single-cell suspensions from healthy donors were stained with Treg (A) and Th17 (B), and we counted cell numbers at each steps of the staining prior to the centrifugation. The graphs represent the total cell number at each step of the staining procedure. A. 1: wash after ACK lysis, 2: wash after ACK lysis, 3:wash after viability staining, 4:wash after extracellular staining, 5: wash after fix and permeabilization, 6: wash after intracellular staining. B. 1: wash after ACK lysis, 2: wash after ACK lysis, 3:wash after viability staining, 4:wash after extracellular staining, 5: wash after fix and permeabilization, 6: wash after intracellular staining, 7:wash after streptavidin binding.

Figure 8. Fresh vs Frozen

Freshly isolated single-cell suspensions from healthy donors or frozen cells from the same donor were stained with T2 staining. A comparison was made between the percentages of cells expressing the cell surface markers. Fresh lymphocytes (A) are compared to frozen lymphocytes (B). Presented are some of the dot plot combinations for the T2 staining. The arrows point out the main differences observed.

Figure 9. Antibodies mixture stability

Single-cell suspensions from healthy donors were stained with T1 staining every day with a fresh mix along with a mix that was prepared at day 0. The graphs represent dot plots for CD4 and CD8 T cells stained with the premixed ‘cocktail’ of reagents from day 0 to day 4.

Figure 10. Staining variability

Single-cell suspensions from healthy donors were stained with T1 staining (n = 4). CD8 T cells were identified as described previously. The graphs represent the percentage of each marker of positive CD8 T cells at 4 different time points. (A) Each time point is represented and (B) is the mean and standard error of the mean of 6 time points.

Figure 11. Probability State Modeling (PSM)

A probability state model was constructed with Gemstone Verity Software House and was applied to CD4 T cells and CD8 T cells. For these displays, the data were gated on light scatter, viability, and expression of CD45, CD3, and either CD4 or CD8 as described earlier. The y-axis is fluorescence intensity while the x-axis displays transitions from naïve to memory and effector populations. Here it is possible to visualize relative intensity of marker expression along with putative transitions among these populations. A. The graph reveals the proportions of memory and naïve subsets as in the previous figure 2A, for CD4⁺ T cells. B. The graph reveals the proportions of memory and naïve subsets as in the previous figure 2A, for CD8⁺ T cells. C. For this model, the data were gated on light scatter, viability, and expression of CD45, CD3, CD4, CD25^high, and FoxP3 as described earlier. The y-axis is fluorescence intensity while the x-axis displays transitions from naïve to memory and effector populations. This graphs reveals the coordinate expression patterns of memory and naïve markers as well as activation markers through the transitions.