When killers become thieves: Trogocytosed PD-1 inhibits NK cells in cancer

Abstract

Trogocytosis modulates immune responses, with still unclear underlying molecular mechanisms. Using leukemia mouse models, we found that lymphocytes perform trogocytosis at high rates with tumor cells. While performing trogocytosis, both Natural Killer (NK) and CD8⁺ T cells acquire the checkpoint receptor PD-1 from leukemia cells. In vitro and in vivo investigation revealed that PD-1 on the surface of NK cells, rather than being endogenously expressed, was derived entirely from leukemia cells in a SLAM receptor-dependent fashion. PD-1 acquired via trogocytosis actively suppressed NK cell antitumor immunity. PD-1 trogocytosis was corroborated in patients with clonal plasma cell disorders, where NK cells that stained for PD-1 also stained for tumor cell markers. Our results, in addition to shedding light on a previously unappreciated mechanism underlying the presence of PD-1 on NK and cytotoxic T cells, reveal the immunoregulatory effect of membrane transfer occurring when immune cells contact tumor cells.

Fig. 1.. Lymphocytes acquire PD-1 and Thy-1.1 from RMA cancer cells.

(A) RMA cells (red) were transduced with a retroviral vector encoding Thy-1.1 to generate RMA-Thy1.1 (blue), and then PD-1 was knocked out by CRISPR-Cas9 to generate RMA-Pdcd1^−/−Thy1.1 (purple). A representative flow cytometry staining depicting PD-1 and Thy-1.1 expression is shown. (B and C) Splenocytes from Pdcd1^+/+ or Pdcd1^−/− littermates were incubated with RMA-Thy1.1 or RMA-Pdcd1^−/−Thy1.1. After 3 days, cells were stained with Thy-1.1 and PD-1 antibodies. NK cells were gated as singlets/live-NK1.1⁺NKp46⁺DX5⁺ events. CD8⁺ T cells were gated as singlets/live-CD3⁺CD8⁺ events and B cells as singlets/live-CD19⁺. (D) NK cells isolated from Pdcd1^+/+ or Pdcd1^−/− littermates were incubated with RMA-Thy1.1 or RMA-Pdcd1^−/−Thy1.1. After 3 days, cells were stained with Thy-1.1 and PD-1 antibodies. The experiment depicted is representative of 15 performed with similar results.

Fig. 2.. NK cells and T cells acquire PD-1 from C1498 cancer cells.

(A) C1498 cells were stained with PD-1 antibody or isotype control. (B) PD-1⁺ cells (in blue) were flow-sorted and, after 2 weeks in culture, stained for PD-1, alongside parental C1498 cells. (C) Splenocytes from Pdcd1^+/+ or Pdcd1^−/− littermates were cultured with C1498 or PD-1⁺ C1498 cells for 3 days and then stained with PD-1 antibodies. The experiment depicted is representative of three performed with similar results. The numbers in the graphs refer to the percentage of PD-1⁺ cells. (D) Splenic NK cells isolated from Pdcd1^−/− mice were cocultured with C1498 or PD-1⁺ C1498 cells, or without tumor cells as a control, for 24 or 72 hours and stained for PD-1. The experiment depicted is representative of six performed with similar results. The numbers in the graphs refer to the percentage of PD-1⁺ cells.

Fig. 3.. Trogocytosis is responsible for intercellular transfer of PD-1 from tumor to NK cells.

(A and B) Splenic NK cells isolated from a Pdcd1^−/− mouse were cocultured for 24 hours with RMA cells separated or not by a transwell semipermeable membrane before staining for PD-1 and Thy-1.1. The experiment depicted is representative of four performed with similar results. Cumulative analysis is shown in (B) (n = 3), where the statistical analysis was performed with a one-way ANOVA with multiple comparisons, ****P < 0.0001. (C and D) Splenic NK cells isolated from a Pdcd1^−/− mouse were cocultured for 24 hours with RMA cells or with medium conditioned for 3 days by RMA cells and then stained for PD-1 and Thy-1.1. The experiment depicted is representative of three performed with similar results. Cumulative analysis is shown in (D) (n = 3), where the statistical analysis was performed with a one-way ANOVA with multiple comparisons, *P < 0.05. (E) Splenic NK cells isolated from Pdcd1^−/− mice were pretreated with sodium azide or antimycin A and then cocultured for 1 hour with RMA cells and stained for PD-1 and Thy-1.1. Three independent experiments are plotted. Statistical analysis with one-way ANOVA with repeated measurements. (F) Splenic NK cells were isolated from Pdcd1^−/− mice and treated for 3 days with rhIL-2. NK cells were then pretreated with latrunculin and cocultured with RMA-Thy1.1 cells. Three independent experiments are plotted. Statistical analysis with two-tailed paired Student’s t test. (G) NK cells were incubated with RMA cells prelabeled with CellVue for 24 hours. CellVue and PD-1 staining on NK cells is depicted on the left and right, respectively. Numbers in the histogram flow plot refer to the frequency of PD-1⁺ cells.

Fig. 4.. SLAM receptors are essential for trogocytosis.

(A) Splenic NK cells isolated from a Pdcd1^−/− mouse were cultured for 3 days, and then PD-L2 and PD-L1 expression was analyzed by flow cytometry. Representative of three experiments performed with similar results. (B and C) NK cells were incubated with RMA cells in the presence of a PD-L1 blocking antibody or an isotype control for 24 hours, before being stained for PD-1 and PD-L1. As additional controls, NK cells were (i) cocultured with RMA without adding any antibody or (ii) cultured alone without adding tumor cells. The experiment depicted is representative of three performed. The numbers in the flow histograms represent the geometric mean fluorescence intensity (MFI) of PD-L1 or PD-1. (D and E) NK cells were isolated from the spleen of Pdcd1^−/−Ncr1^+/CreCd274^fl/fl (D) or Cd274^−/− (E) mice (or control littermates, in red) and cocultured for 1 or 3 days with RMA or C1498 tumor cells, when PD-1 and Thy-1.1 staining was assessed by flow cytometry. (F) Splenocytes from SLAM-deficient mice or control littermates were cocultured for 3 days with RMA or C1498 cells. PD-1 and Thy-1.1 staining on NK cells was then assessed by flow cytometry. Three independent experiments are depicted, n = 9. Statistical analysis with two-tailed unpaired Student’s t test. ***P < 0.001. (G) SLAM-sufficient (CD45.1) or SLAM-deficient (CD45.2) NK cells were negatively isolated and cultured with rhIL-2 for 3 days. NK cells were then mixed in a 1:1 ratio (competitive condition) or not (noncompetitive condition) and cocultured with RMA-Thy1.1 tumor cells. PD-1 staining on NK cells is shown from two different experiments, n = 6. Statistical analysis with two-tailed Student’s t test, unpaired for the noncompetitive condition and paired for the competitive condition. **P < 0.01.

Fig. 5.. Intratumoral lymphocytes acquire PD-1 from tumor cells.

(A to D) Pdcd1^+/+ or Pdcd1^−/− mice were injected with RMA or RMA-Pdcd1^−/− tumors. PD-1 and Thy-1.1 staining was assessed by flow cytometry on tumor-infiltrating NK and T cells (gated as in Fig. 1, B and C, respectively). The experiment shown is representative of five performed with similar results. (E) RMA or RMA-Thy1.1 cells were injected in either flank of a Pdcd1^−/− mouse. PD-1 and Thy-1.1 staining was analyzed in intratumoral or splenic NK cells. The experiment depicted is representative of three performed, n = 9 for PD-1 staining and n = 4 for Thy-1.1 staining. The three experiments are pooled in (F). (G) Expression of Sca-1 and CD69 was analyzed on Pdcd1^−/− NK cells infiltrating RMA-Thy1.1 tumors, stratifying NK cells for PD-1 staining. Representative of two experiments performed, n = 5. Statistical analysis with two-tailed paired Student’s t test; *P < 0.05. (H) SLAM-deficient mice or control littermates were injected with RMA-Thy1.1 cells. PD-1 staining was determined on tumor-infiltrating NK cells. Data depict two experiments performed, n = 6. Statistical analysis with two-tailed paired Student’s t test; **P < 0.01; ***P < 0.001.

Fig. 6.. PD-1 blockade is effective in Pdcd1^−/− mice when NK cells are present and tumor cells express PD-1.

(A) PD-1 and PD-L1 expression in RMA-S-Pdl1 or RMA-S-Pdl1-Pdcd1^−/− cells was analyzed by flow cytometry. (B to H) In all experiments, the indicated cell lines were resuspended in Matrigel and injected, alone or mixed with different PD-1 blocking or control antibody. Tumor growth was assessed over time, and data were analyzed with two-way ANOVA. (B) n = 6 per group; the experiment depicted is representative of two performed with similar results. (C) n = 6 per group; the experiment depicted is representative of two performed with similar results. (D) n = 4 per group; the experiment depicted is representative of two performed with similar results. (E) n = 6 per group; the experiment depicted is representative of three performed with similar results. (F) n = at least 5 per group; the experiment depicted is representative of two performed with similar results. (G) n = 5 per group; the experiment depicted is representative of two performed with similar results. (H) n = 5 per group; the experiment depicted is representative of two performed with similar results. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 7.. NK cells costain for CD138 and PD-1 in the BM of patients with clonal plasma cell disorders.

The BM aspirates of 28 patients with clonal plasma cell disorders were analyzed by flow cytometry. The frequency of NK cells staining for either, neither, or both CD138 and PD-1 is depicted.