The tetraspanin CD53 modulates responses from activating NK cell receptors, promoting LFA-1 activation and dampening NK cell effector functions

Abstract

NK cells express several tetraspanin proteins, which differentially modulate NK cell activities. The tetraspanin CD53 is expressed by all resting NK cells and was previously shown to decrease NK cell cytotoxicity upon ligation. Here, we show that CD53 ligation reduced degranulation of rat NK cells in response to tumour target cells, evoked redirected inhibition of killing of Fc-bearing targets, and reduced the IFN-γ response induced by plate-bound antibodies towards several activating NK cell receptors (Ly49s3, NKR-P1A, and NKp46). CD53 induced activation of the β2 integrin LFA-1, which was further enhanced upon co-stimulation with activating NK cell receptors. Concordant with a role for CD53 in increasing NK cell adhesiveness, CD53 ligation induced a strong homotypic adhesion between NK cells. Further, the proliferative capacity of NK cells to a suboptimal dose of IL-2 was enhanced by CD53 ligation. Taken together, these data suggest that CD53 may shift NK cell responses from effector functions towards a proliferation phase.

Figure 1. CD53 induces CD2-independent homotypic clustering of NK cells.

A) Purity of 12 day-old LAK cells, as determined by staining with anti-CD3 and anti-NKR-P1A. LAK cells were cultured for 2 h in the presence of soluble antibodies towards CD53, CD2, NKR-P1A, or an isotype control IgG. B) Cluster formation was photographed with a light microscope, or C) quantified by counting the number of free, non-aggregated cells in treated samples relative to cells cultured in medium using a hemocytometer. D and E) LAK cells were co-cultured for up to 2 h with antibodies towards CD53 and CD2 or with CD53 and isotype control antibody. Cluster formation was assessed qualitatively by microscopy (D), or quantitatively by hemocytometer (E) as described above. Results are presented as the mean±SEM of 3 independent experiments with samples in duplicates (n = 6). Comparisons within an experimental group were performed with the One-way analysis of variance (ANOVA) and a post hoc Tukey's multiple comparisons test to compare treated samples to untreated sample. *, p<0.05; **, p<0.005; ***, p<0.001.

Figure 2. Elevated LFA-1 activation in response to CD53 ligation, but normal target conjugation and reduced migratory response.

A) Enriched, primary NK cells, or B) LAK cells were pre-incubated with the indicated antibodies for 30 min on ice. Stimulation was induced for 5 min at 37°C in PBS supplemented with Ca²⁺ and Mg²⁺ with a crosslinking secondary F(ab)₂ anti-mouse antibody in the presence of a soluble rat ICAM-1/Fc-fusion protein. PBS with Mg²⁺/EGTA was used as positive control. Binding of ICAM-1 to NKR-P1A⁺CD3⁻ NK cells was assessed by flow cytometry. The data represents 3–5 independent experiments with samples in duplicates (n = 6–10), and are presented as the mean±SEM. Data were analysed using the two-tailed unpaired t-test. *, p<0.05; **, p<0.005.

Figure 3. CD53 ligation reduces NK cell degranulation and receptor-induced IFN-γ production.

A) Degranulation of primary NKR-P1A⁺CD3⁻ NK cells in response to YAC-1 target cells was assessed by flow cytometry. Lymphocytes and YAC-1 cells mixed 1∶1 were co-cultured for 4 h with anti-CD107a antibodies in combination with anti-CD53 or isotype control antibodies. B) Cytotoxicity of RNK-16 cells (open symbols) or LAK cells (filled symbols) against Fc⁻ YAC-1 target cells (left panel) or Fc⁺ P388D1 target cells (right panel) in the presence of anti-CD53 or isotype control antibodies were determined with a standard 4 h ⁵¹Cr release assay. Data represent the mean values of triplicates ± SEM from one representative experiment out of three. C) Conjugate formation between CFSE-stained RNK-16 cells and SNARF-1 stained YAC-1 target cells was assessed by flow cytometry. Percent NK cells in conjugates with target cells was calculated as described in Materials and methods. The data are representative of 3 independent experiments, and presented as the mean±SEM. D) Ig-depleted spleen cells were stimulated for 6 h in vitro by the indicated plate-bound antibodies or cytokines, and the proportion of IFN-γ positive cells was assessed by flow cytometry gating on NKR-P1A⁺CD3⁻ NK cells. The data represents 3 independent experiments (n = 3), and are presented as the mean ± SEM. n = 3. Data were analysed using the two-tailed unpaired t-test. *, p<0.05; **, p<0.005.

Figure 4. Increased proliferative activity of NK cells in response to CD53 ligation.

A) CFSE-labeled lymphocytes were cultured for 7 days with a sub-optimal dose of IL-2 in the presence of an anti-CD53 antibody or isotype control antibody at 10 µg/ml. The CFSE dilution profiles of NKR-P1A⁺CD3⁻ NK cells (upper panels) or CD3⁺NKR-P1A⁻ T cells (lower panels) were analyzed by flow cytometry. Data are representative of 3 independent experiments (n = 3). B) Flow cytometric analysis of CD53 expression by primary NK cells (upper panels) or T cells (lower panels) before and after culture in IL-2 for 24 h. NK cells were gated as NKR-P1A⁺CD3⁻ cells. Shown are stainings with anti-CD53 (solid line) and an isotype control antibody (grey line). n = 3.

Figure 5. CD53 enhances protein tyrosine phosphorylation induced by activating NK receptors, but not of the most proximal signalling molecules.

A) LAK cells were stimulated with Dynabeads co-coated with combinations of anti-Ly49s3, anti-CD53 or control antibodies for 1 min as indicated. Cell lysates were subjected to immunoblotting with an anti-phosphotyrosine antibody, and show enhanced tyrosine phosphorylation of bands at ∼40 and 160 kDa ∼in sampled co-ligated with CD53 and Ly49s3 antibodies. B) NKR-P1A⁺ spleen cells were positively selected with NKR-P1A-coated Dynabeads on ice, and stimulation was induced at 37°C in the presence of either anti-CD53 or isotype control antibodies for 1 or 5 min. Cell lysates were subjected to immunoblotting with anti-phosphotyrosine. The western blots are representative for 3 independent experiments, and show enhanced tyrosine phosphorylation of bands at ∼25, 40, and 75 kDa in samples co-ligated with CD53 and NKR-P1A antibodies. C) LAK cells were stimulated with Dynabeads co-coated with combinations of anti-Ly49s3, anti-CD53 or control antibodies for 1 min. Cell lysates were immunoprecipitated with antibodies to Syk, PI3K, Vav, or PKC-θ, then immunoblotted with either an anti-phosphotyrosine antibody or the immunoprecipitating antibody as control. The data are representative for 3 independent experiments.