Research ArticleNK CELLS

Clonal expansion and compartmentalized maintenance of rhesus macaque NK cell subsets

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Science Immunology  02 Nov 2018:
Vol. 3, Issue 29, eaat9781
DOI: 10.1126/sciimmunol.aat9781
  • Fig. 1 Clonal composition of PB NK cells.

    (A) FACS analysis of CD3CD20CD14 PBMCs for ZH33 at 12 months and ZG66 at 17 months after transplantation, with sorting gates for CD56+CD16 and CD56CD16+ NK cell subpopulations shown. (B) Pairwise Pearson correlation coefficients between fractional abundances of all barcodes for CD56+CD16 and CD56CD16+ NK cells from 6 months through the most recent follow-up [r values, P values, and 95% confidence intervals (CIs) in table S1]. (C) Shannon diversity indices for all barcode contributions to T, B, Gr, CD56+CD16 NK, and CD56CD16+ NK cells over time. Table S3 gives the number of barcodes above threshold for each sample. † indicates that the 30-month ZH33 CD56CD16+ sample was additionally sorted to be CD8α+NKG2+.

  • Fig. 2 Clonal bias of CD56CD16+ NK cell subpopulations.

    (A) Heat maps of the natural log fractional abundance of the highest-contributing clones defined as all barcodes present as a top 10 highest-contributing barcode in at least one of the samples, mapped over all samples for each animal. Corresponding percentage contributions in table S2. Each row corresponds to one unique barcode; * indicates that the barcode is one of the top 10 for that sample. Heat maps are organized by unsupervised hierarchical clustering of Euclidean distances between barcodes’ log fractional abundances, with relative contribution shown via color gradient. (B) Highly biased CD56CD16+ NK cell clone contributions over time. A barcode is defined as highly biased at a time point if it meets the following conditions: (i) >1% contribution to CD56CD16+ cells and (ii) >10-fold abundance in contribution to CD56CD16+ cells compared with T, B, Gr, and CD56+CD16 cells. y axes show the fraction of all barcode reads in CD56CD16+ cells contributed by the “highly biased” clones. Each color represents the contribution of a single clone; the same color at different time points indicates the same clone. The 30-month ZH33 CD56CD16+ sample was additionally sorted to be CD8α+NKG2+.

  • Fig. 3 Cluster tracking of barcodes with similar kinetic behavior over time.

    Heat maps (left) show the log fractional contribution to hematopoiesis of the highest-contributing clones (top 10 in each sample, as defined in Fig. 2A) organized by unsupervised hierarchical clustering in each cell type over time for animal ZH33. The hierarchical tree in each cell type was cut at a level to retrieve clear clusters via visual inspection of the dendrograms and heat maps. The clusters so defined are designated by color bars on the left of the heat maps. The fractional abundances of each clone in these clusters are averaged for all clones in the cluster and plotted over time (right), with the shaded ribbons around each line representing the SEM of the average fractional abundance. Line colors match the colors of the clusters in the corresponding heat map. (A) Gr, (B) CD56+CD16 NK cells, (C) CD56CD16+ NK cells, and (D) T cells.

  • Fig. 4 Clonal segregation of CD16+NKG2+ NK cells based on KIR3DL01 expression and/or SIV-Gag GY9/Mamu-A1*002 tetramer staining.

    (A) NK cell surface receptor subpopulations of CD3NKG2+ NK cells from RM PB (n = 9) detected by SIV-Gag GY9/Mamu-A1*002 tetramer and anti-KIR2D (NKVFS1) staining. (B) FACS plots of SIV-Gag GY9/Mamu-A1*002 tetramer and anti-KIR2D staining of PB CD3CD16+NKG2+ NK cells from barcoded RMs. (C) Top 10 barcoded clone heat maps for bulk CD16+NKG2+ NK cells and sorted NK cell subpopulations based on anti-KIR2D and tetramer staining for the time points and animals shown in (B). Distinct clonal distribution within different NK cell subpopulations distinguished by the presence and/or absence of staining is shown. (D) Pearson correlation coefficients comparing pairwise fractional contributions between NK KIR subpopulations from four monkeys same as in (C). The color scale for r values is shown on the right (r values, P values, and 95% CIs in table S1).

  • Fig. 5 In vivo depletion of CD16+ cells.

    FACS plots and NK cell subset absolute concentrations (A) and Ki-67+ percentage of PB NK cell subsets (B) before and after anti-CD16–depleting antibody treatment (50 mg/kg) of ZH33 32 months after transplant. (C) Top 15 clones’ heat map shows the log fractional abundance of the T, B, Gr, CD16bright, CD16dim, CD56+, and total PBMC samples over time before and after antibody treatment. Depletion and regeneration of individual day −7 CD56CD16+ highly biased NK cell clones (numbered in red; defined in Fig. 2) are shown (left). The fractional contributions of these highly biased clones decreased on day 4 versus day 0 (P = 0.002, paired t test). (D) Pearson correlation between clonal contributions before and after anti-CD16 (r values, P values, and 95% CIs in table S1). (E) Stacked bar plots of relative clonal contributions to CD56CD16bright NK cells from CD16+ highly biased clones versus multilineage or clones biased toward other lineages before and after anti-CD16. NK cell–biased clones defined as >10-fold abundant in contribution to CD56CD16+ cells compared with T, B, Gr, and CD56+CD16 cells regardless of clone size. Gray bars: non-CD16bright NK cell–biased clones’ contributions; CD16bright NK cell–biased clones’ contributions are shown in colors reflecting time point of appearance, and each individual clone’s contribution is delineated by lines and stacked to create bars.

  • Fig. 6 In vivo depletion of NK cells with anti–IL-15.

    (A) Schematic of anti–IL-15 treatment. ZH33 received anti–IL-15 mAb on day 0 (20 mg/kg) and on days 14 and 28 (10 mg/kg). (B) Top and middle rows show NK cell subsets based on NKG2, CD16, and CD56 expression in CD3CD14CD20 PBMCs; bottom row shows CD95 and CD28 expression on CD3+ T cells. (C) Absolute PB concentration of NK and T cell subpopulations. (D) Heat map of the top 50 most abundant clones in CD16+ NK, CD56+ NK, T, B, mono, and Gr samples over time. Depleted and newly arising clones of different bias types are designated on the left of the heat map (defined per criteria in Fig. 2). Newly biased T clones are defined as non–NK cell–biased clones contributing to T cells at a ratio of 10:1 compared with fractional contribution to any other cell type, including CD56+CD16 NK, CD56CD16+ NK, B, or Gr cells at day 362. CD56+CD16 NK samples from days 167 and 341 were not obtained. (E) Stacked bar plots of relative contributions to CD56CD16+ NK cells from CD56CD16+ NK cell–biased clones versus multilineage or clones biased toward other lineages before and after anti–IL-15, using the same criteria defined in Fig. 5E. Each individual clone’s contribution is delineated by lines and stacked to create the bars, with colors designating clone type. NK cell–biased clones defined as >10-fold abundant in contribution to CD56CD16+ cells compared with T, B, Gr, and CD56+CD16 NK cells regardless of clone size.

  • Fig. 7 Model for NK cell homeostasis and regeneration.

    (A) Long-term repopulating stem cells (LT-HSCs), short-term repopulating progenitors (ST-HPCs), or NK cell restricted progenitors may be lentivirally barcoded. Each may give rise to mature CD16+NK cells, containing barcodes corresponding to the parental progenitors (red for those derived from ST-HPCs and blue for those derived from LT-HSCs). A few of these mature NK cells expand to large clonal populations, maintaining receptor expression and barcodes of the originating NK cells and persisting for months to years. ST-HPCs disappear, explaining lack of overlap of ST-HPC–derived NK cells with other hematopoietic lineages, including CD56+CD16 immature NK cells. (B) Upon anti-CD16 antibody depletion of CD56CD16bright NK cells, CD56CD16dimNK cells proliferate and regenerate the mature NK cell pool without significant contributions from CD56+CD16NK cells; thus, the populations remain clonally distinct. (C) After prolonged depletion of all NK cells with anti–IL-15, many expanded clones disappear, to be replaced by a different set of expanded NK cell clones upon return of IL-15. Some of the new expanded NK cell clones have barcodes overlapping multiple lineages because they develop from mature NK cells that have arisen from LT-HSCs after anti–IL-15 treatment.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/3/29/eaat9781/DC1

    Fig. S1. Barcoded CD34+ HSPC monkey transplantation parameters and hematopoietic lineage cells FACS sort gating strategy.

    Fig. S2. Phenotype of NK cell populations in RM blood, marrow, and LNs.

    Fig. S3. Bias of all clones toward T, B, Gr, CD56+CD16 NK, and CD56CD16+ NK fractions.

    Fig. S4. Cluster tracking of barcodes with similar kinetic behavior over time for ZG66 and ZJ31.

    Fig. S5. Individual clonal tracking of CD56CD16+-biased clones in ZH33.

    Fig. S6. Epigenetic profile and functionality of NKG2+CD56CD16+ NK cells.

    Fig. S7. The effect of in vivo depletion of CD16+ NK cells on other lineages and Ki-67 expression on RM PB NK cell subsets.

    Material and Methods

    Table S1. Pearson correlation coefficient r values, P values, and 95% confidence intervals for the correlation coefficient.

    Table S2. Corresponding percentage contributions to the samples presented in Fig. 2A.

    Table S3. The number of barcodes above threshold for each sample from ZH33, ZG66, and ZJ31 in Figs. 1 and 2.

    Table S4. Primer sequence information.

    Table S5. Antibody information.

    Data file S1.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Barcoded CD34+ HSPC monkey transplantation parameters and hematopoietic lineage cells FACS sort gating strategy.
    • Fig. S2. Phenotype of NK cell populations in RM blood, marrow, and LNs.
    • Fig. S3. Bias of all clones toward T, B, Gr, CD56+CD16 NK, and CD56CD16+ NK fractions.
    • Fig. S4. Cluster tracking of barcodes with similar kinetic behavior over time for ZG66 and ZJ31.
    • Fig. S5. Individual clonal tracking of CD56CD16+-biased clones in ZH33.
    • Fig. S6. Epigenetic profile and functionality of NKG2+CD56CD16+ NK cells.
    • Fig. S7. The effect of in vivo depletion of CD16+ NK cells on other lineages and Ki-67 expression on RM PB NK cell subsets.
    • Material and Methods

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Pearson correlation coefficient r values, P values, and 95% confidence intervals for the correlation coefficient.
    • Table S2 (Microsoft Excel format). Corresponding percentage contributions to the samples presented in Fig. 2A.
    • Table S3 (Microsoft Excel format). The number of barcodes above threshold for each sample from ZH33, ZG66, and ZJ31 in Figs. 1 and 2.
    • Table S4 (Microsoft Excel format). Primer sequence information.
    • Table S5 (Microsoft Excel format). Antibody information.
    • Data file S1 (.zip format).

    Files in this Data Supplement:

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