Corrigendum: To Ki or Not to Ki: Re-Evaluating the Use and Potentials of Ki-67 for T Cell Analysis

A Corrigendum on
To Ki or Not to Ki: Re-Evaluating the Use and Potentials of Ki-67 for T Cell Analysis

By Di Rosa F, Cossarizza A and Hayday AC (2021). Front. Immunol. 12:653974. doi: . 10.3389/fimmu.2021.653974

In the original article, there was a mistake in the legend for Figure 1 as published. On the Viable cells, no “dump” gate, “CD16” was written instead of “CD19”. The correct legend appears below.

FIGURE 1

Figure 1 Example of CD8 T cell naïve/memory subset analysis by T_DS assay. HD PBMCs were stained with the viability dye eFluor 780 (eF780), the DNA dye Hoechst-33342, and fluorochrome conjugated mAbs against surface markers and Ki-67, as described (16). An example of flow cytometry analysis is shown. (A) Gating of viable single CD8 T cells in 6 steps: 1) DNA-A/-W singlets. Single cells having 2n≤ DNA content ≤4n were selected on the DNA-area (A) versus (vs) DNA-width (W) plot; 2) Time exclusion. Stable acquisition over time (seconds) was monitored on the time vs DNA-A plot and any events collected in case of pressure fluctuations were excluded; 3) Viable cells, no “dump”. Cells expressing CD4, CD14 and CD19, and dead cells were excluded; 4) FSC A/SSC-A “relaxed” gate. A “relaxed gate was used on the FSC-A vs SSC-A plot, to include highly activated and cycling lymphocytes (15); 5) CD8 T cells. CD8 T cells were gated on the CD3 versus CD8 plot; 6) Refined singlets. A few remaining doublets composed by one cell sitting on top of another (so called “shadow” doublets) were excluded as Ki-67int/^- events having > 2n DNA content (16). This gating strategy was used as a base for the subsequent gates. (B) The following naïve/memory subsets of CD8 T cells were identified: CD45RA⁺ CCR ⁺ Naïve, CD45RA^- CCR7⁺ central memory (CM), CD45RA^- CCR7^- effector memory (EM), and CD45RA⁺ CCR7^- (EMRA). (C) Cell cycle phases of each naïve/memory CD8 T cell subset were defined on DNA-A vs Ki67-A plot as follows: cells in G₀ were identified as DNA 2n/Ki67^- (bottom left quadrant); cells in G₁ as DNA 2n/Ki67⁺ (upper left quadrant); cells in S-G2/M (or TDS cells) as DNA>2n/Ki67⁺ (top right quadrant). Unpublished data in relation to (16).

“HD PBMCs were stained with the viability dye eFluor 780 (eF780), the DNA dye Hoechst-33342, and fluorochrome conjugated mAbs against surface markers and Ki-67, as described (16). An example of flow cytometry analysis is shown. (A) Gating of viable single CD8 T cells in 6 steps: 1) DNA-A/-W singlets. Single cells having 2n≤ DNA content ≤4n were selected on the DNA-area (A) versus (vs) DNA-width (W) plot; 2) Time exclusion. Stable acquisition over time (seconds) was monitored on the time vs DNA-A plot and any events collected in case of pressure fluctuations were excluded; 3) Viable cells, no “dump”. Cells expressing CD4, CD14 and CD19, and dead cells were excluded; 4) FSC-A/SSC-A “relaxed” gate. A “relaxed” gate was used on the FSC-A vs SSC-A plot, to include highly activated and cycling lymphocytes (15); 5) CD8 T cells. CD8 T cells were gated on the CD3 versus CD8 plot; 6) Refined singlets. A few remaining doublets composed by one cell sitting on top of another (so called “shadow” doublets) were excluded as Ki-67^int/^- events having > 2n DNA content (16). This gating strategy was used as a base for the subsequent gates. (B) The following naïve/memory subsets of CD8 T cells were identified: CD45RA⁺ CCR7⁺ Naïve, CD45RA^- CCR7⁺ central memory (CM), CD45RA^- CCR7^- effector memory (EM), and CD45RA⁺ CCR7^- (EMRA). (C) Cell cycle phases of each naïve/memory CD8 T cell subset were defined on DNA-A vs Ki67-A plot as follows: cells in G₀ were identified as DNA 2n/Ki67^- (bottom left quadrant); cells in G₁ as DNA 2n/Ki67⁺ (upper left quadrant); cells in S-G₂/M (or T_DS cells) as DNA>2n/Ki67⁺ (top right quadrant). Unpublished data in relation to (16).”

In the original article, there was also a mistake in the legend for Supplementary Table 1 as published. The peptide- HLA-A*02 tetramer list was incorrectly formatted, there was missing information about numbers in the table (they represent average percentages); missing information about the number of mice (panel A) and number of human donors (panel B and C); and a missing citation of original references at the end. The corrected Supplementary Material File is linked below.

In the original article, there was also a mistake in Figure 1 as published. There was an incorrect y-axis label in panel A, third graph from left. The corrected Figure 1 appears below.

The authors apologize for these errors and state that they do not change the scientific conclusions of the article in any way. The original article has been updated.

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Supplementary Material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2021.756641/full#supplementary-material