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. 2023 Aug 11;2(1):kyad012.
doi: 10.1093/discim/kyad012. eCollection 2023.

CD56bright natural killer cells preferentially kill proliferating CD4+ T cells

Mercede Lee  1 , Charles J M Bell  1 , Arcadio Rubio Garcia  1 , Leila Godfrey  1 , Marcin Pekalski  1 , Linda S Wicker  1 , John A Todd  1 , Ricardo C Ferreira  1
Affiliations

CD56bright natural killer cells preferentially kill proliferating CD4+ T cells

Mercede Lee et al. Discov Immunol. .

Abstract

Human CD56br natural killer (NK) cells represent a small subset of CD56+ NK cells in circulation and are largely tissue-resident. The frequency and number of CD56br NK cells in blood has been shown to increase following administration of low-dose IL-2 (LD-IL2), a therapy aimed to specifically expand CD4+ regulatory T cells (Tregs). Given the potential clinical application of LD-IL-2 immunotherapy across several immune diseases, including the autoimmune disease type 1 diabetes, a better understanding of the functional consequences of this expansion is urgently needed. In this study, we developed an in vitro co-culture assay with activated CD4+ T cells to measure NK cell killing efficiency. We show that CD56br and CD56dim NK cells show similar efficiency at killing activated CD4+ conventional T (Tconv) and Treg cell subsets. However, in contrast to CD56dim cells, CD56br NK cells preferentially target highly proliferative cells. We hypothesize that CD56br NK cells have an immunoregulatory role through the elimination of proliferating autoreactive CD4+ Tconv cells that have escaped Treg suppression. These results have implications for the interpretation of current and future trials of LD-IL-2 by providing evidence for a new, possibly beneficial immunomodulatory mechanism of LD-IL-2-expanded CD56br NK cells.

Keywords: CD4+ T cells; CD56br NK cells; Natural killer (NK) cells; in vitro NK killing assay; low-dose IL-2 immunotherapy.

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Conflict of interest statement

J.A.T. is a member of the science advisory boards of GSK, Precion, Qlife, and Vesalius Therapeutics M.L. was supported by a grant from Bristol Myers Squibb (BMS 2338). The remaining authors declare no competing interests.

Figures

Figure 1:
Figure 1:
Assessing the proliferative capacity of the CD56+ NK cell subsets in vitro. (A) Sorting strategy for the isolation of the four assessed immune cell subsets: (i) CD3–CD56br NK cells (CD56br NK); (ii) CD3–CD56dim NK cells (CD56dim NK); (iii) CD3+CD4+CD25–/low (Tconv); and (iv) CD3+CD4+CD127lowCD25hi (Treg). Plots represent an illustrative example from the sorting of cryopreserved PBMCs from four healthy donors. (B) Representative histograms depicting the expression of CD56, CD127, CD16, and CD25 in sorted CD56br (light green) and CD56dim (dark green) NK cells at day 0. (C) Representative histograms depicting the proliferative capacity of CD56br (light green) and CD56dim (dark green) NK cells after 3 (left panels) or 7 (right panels) days of culture in the presence or absence of 100 IU/ml IL-2. (D) Representative histograms depicting the proliferative capacity of CD4+ Tconvs and Tregs after 3 (left panel) or 7 (right panel) days of culture in the presence of 100 IU/ml IL-2 and TCR stimulation with αCD3/CD28 beads (1 bead: 3 T cells). (E and F) Bar plots depict the distribution (mean ± SEM) of the recovered cell yields (defined as the percentage of plated cells harvested after the initial 3 or 7 days in vitro culture period) in both CD56+ NK cell (E) and CD4+ T cell (F) subsets. CD4+ Tconvs (depicted by triangles) and Tregs (depicted by inverted triangles) isolated from the same donor are depicted using the same colour.
Figure 2:
Figure 2:
Development of a CD56br and CD56dim NK cell in vitro killing assay. (A) Gating strategy for the quantification of dead responder T cells from the in vitro killing assays. Killing assays were performed using T and NK cells sorted from freshly isolated PBMCs from 14 healthy donors. (B) Scatter plot depicts the frequency of specific killing of CD4+ Tconvs (red) and Tregs (blue). Frequency of specific killing was determined as the relative proportion of dead responder T cells after co-culture with CD56br (left panel) and CD56dim (right panel) NK cells, after subtracting the relative proportion of background spontaneous cell death, estimated in both responder T cell subsets (Tconvs and Tregs) incubated in the absence of NK cells (see Methods). Individuals where the initial stimulation period was 2.5 days (n = 10) or 3.5 days (n = 4) are depicted by squares or triangles, respectively. Densities depict the mean posterior frequency of cell death values in each group estimated by beta regression, with an annotation for the median and a 95% credible interval. CD4+ Tconv and Treg cells isolated from the same donor are identified by a connecting line. (C) Densities depict the effect size distribution (measured as fold change) for the observed relative rates of specific killing in CD4+ Tconv (red) and Treg (blue) by CD56br (bottom panel) and CD56dim (top panel) NK cells. (D) Scatter plots depicts the percentage of spontaneous cell death of CD4+ Tconv (red) and Treg (blue) cells. The frequency of spontaneous cell death was assessed in each donor by culturing each T cell subset in the absence of CD56+ NK cells. Data were stratified according to whether the CD4+ T cells subsets were stimulation for an initial period of 2.5 (depicted by squares) or 3.5 (depicted by triangles) days. (E) Densities depict the effect size distribution (measured as fold change) for the observed relative rates of spontaneous cell death at days 2.5 and 3.5 in CD4+ Tconv (bottom panel) and Treg (top panel) cells.
Figure 3:
Figure 3:
Assessing the proliferative capacity of CD4+ T cells in vitro. (A) Representative histograms depicting the proliferative capacity of CD4+ Tconv and Treg cells after 2.5 or 3.5 days of in vitro culture in the presence of 100 IU/ml IL-2 and TCR stimulation with αCD3/CD28 beads (1 bead: 3 T cells). The respective cell generation (Gn) are shown in each histogram. G0 represents undivided CD4+ T cells. Data were generated from freshly isolated PBMCs from 14 healthy donors (n = 10 and n = 4 for day 2.5 and day 3.5, respectively). (B) Histograms depict the frequency (mean ± SEM) of CD4+ Tconv (red) and Treg (blue) cells at each generation of cell division after 2.5 (B) and 3.5 (C) days of stimulation with αCD3/CD28 beads and 100 IU/ml IL-2, followed by an additional 4 h of co-culture in the presence or absence of the respective CD56+ NK cell subset. The reported frequencies of proliferating CD4+ Tconvs and Tregs for each donor at each generation represent the average of the three co-culture conditions assessed in this study: (i) co-culture with CD56br NK cells; (ii) co-culture with CD56dim NK cells; and (iii) cultured alone in the absence of NK cells (background control to assess spontaneous cell death). The T cell proliferation rates were not affected by the different co-culture conditions, and were therefore averaged to increase the number of T cells assessed. CD4+ Tconvs (depicted by triangles) and Tregs (depicted by inverted triangles) isolated from the same donor are depicted using the same colour. (D) Scatter plots depict the distribution (mean ± SEM) of the observed DI for CD4+ Tconv and Treg cells after co-culture at 2.5 (left panel) or 3.5 (right panel) days of stimulation. (E) Same as for D, but depicting the PI of the cells. P values were calculated using two-tailed paired t-tests. DI denotes the average number of cell divisions undergone by a cell in the original population and PI denotes the average number of divisions among the cells that have divided at least once. n.s., P value not significant.
Figure 4:
Figure 4:
CD56br NK cells preferentially kill highly proliferative CD4+ T cell subsets. (A) Representative dot plots depicting the gating strategy used to define the frequency of T cell death among highly (prolif.high; orange) and lowly (prolif.low; teal) proliferative CD4+ T cells co-cultured with CD56br (left panels) and CD56dim (right panels)NK cells. The relative frequency of cell death was calculated in each proliferative group and takes the background levels of spontaneous CD4+ T cell death (CD4+ T cells co-cultured without NK cells) into account (see Methods). Data were generated from freshly isolated PBMCs from 14 healthy donors for day 2.5 (n = 10; squares) and day 3.5 (n = 4; triangles), respectively. (B and C) Scatter plots depict the relative frequency of cell death in CD4+ Tconv (B) and Treg (C) cells from the prolif.high and prolif.low proliferation groups after co-culture with CD56br NK cells. (D and E) Same as B and C but for CD4+ Tconvs (D) and Tregs (E) co-cultured with CD56dim NK cells. Densities depict the mean posterior frequency of cell death values in each group estimated by Beta regression, with an annotation for the median and a 95% credible interval. (F) Densities depict the effect size distribution (measured as fold change) for the observed relative rates of specific killing in highly and lowly proliferative for each of the CD4+ responder T cell subsets by either CD56br (top panels) or CD56dim (bottom panels) NK cells.
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