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CD56bright natural killer cells are present in human lymph nodes and are activated by T cell–derived IL-2: a potential new link between adaptive and innate immunity

Todd A. Fehniger, Megan A. Cooper, Gerard J. Nuovo, Marina Cella, Fabio Facchetti, Marco Colonna and Michael A. Caligiuri

Article Figures & Data

Figures

  • Fig. 1.

    CD56bright NK cells are present in human lymph nodes.

    (A) CD56+ and CD94+ lymphocytes are present in the paracortical T-cell areas of normal human lymph nodes. Low-power (× 40, left) and high-power (× 100, right) photomicrographs of normal human lymph nodes stained for CD56 (top panels) and CD94 (bottom panels). Results are representative of 2 reactive lymph nodes. (B) CD56+CD3 lymphocytes were observed in the parafollicular T-cell areas of human lymph nodes. Low-power (× 40, left) and high-power (× 100, right) photomicrographs of normal human lymph nodes evaluated for CD56 expression using in situ RT-PCR (top panels). High-power photomicrographs of similar sections from lymph nodes colabeled with anti-CD3 mAb and CD56 via in situ PCR demonstrating that the CD56+ cells lack CD3 expression (bottom panels). Results are representative of at least 4 different experiments. (C) Distinct populations of CD56brightCD3 NK cells were observed via flow cytometry in normal human lymph nodes. Flow cytometric density plots are shown gated on lymphocytes stained with mAbs reactive to CD56 and CD3 (left), CD56 and CD16 (middle), and CD56 and CD94 (right). Percentage of lymphocytes in each boxed region is shown. Results are representative of 3 independent experiments on at least 3 normal human lymph nodes.

  • Fig. 2.

    Low concentrations of IL-2 acting through the high-affinity IL-2 receptor costimulate CD56bright NK cell secretion of IFN-γ.

    (A) Sorted CD56bright NK cells were cultured for 72 hours with medium alone, IL-2 (10 pM, 2.3 IU/mL), IL-12 (10 U/mL), or IL-2 plus IL-12, and cell culture supernatants were then assayed for human IFN-γ protein production (top panel). Prior to the addition of cytokines, cells were preincubated for one hour with saturating concentrations of an anti-CD25 mAb (selectively blocking the α subunit of the high-affinity IL-2R) or an isotype control mAb (bottom panel). As a negative control, CD56dim NK cells do not express the high-affinity IL-2R and fail to produce IFN-γ. (B) Sorted CD56bright NK cells were cultured with various concentrations of IL-2 with or without 10 U/mL IL-12 (top panel) or various concentrations of IL-12 with or without 10 pM IL-2 (bottom panel). Cell culture supernatants were harvested at 72 hours and assayed for IFN-γ protein by ELISA. Results represent the means ± SEMs of replicate wells, expressed in pg/mL, and are representative of at least 3 separate experiments.

  • Fig. 3.

    CD56bright NK cells stimulated with low concentrations of IL-2 and IL-12 rapidly accumulate IFN-γ transcript followed by IFN-γ protein secretion.

    Sorted CD56bright (●) and CD56dim (▴) NK cells were cultured for 1, 3, 6, 12, and 24 hours with IL-2 (10 pM) plus IL-12 (10 U/mL). At each time point, cells and supernatants were collected and IFN-γ gene expression (left panel) and protein levels (right panel) were assessed. Results represent the means ± SEMs of replicate wells, expressed as fold increase in gene expression or ng/mL, and are representative of 4 separate experiments.

  • Fig. 4.

    CD56bright NK cells can use T cell–derived IL-2 to costimulate IFN-γ production when cocultured in vitro.

    Sorted NK cell subsets were cocultured with a tetanus toxoid–specific T-cell clone (T) and peptide-pulsed autologous APCs plus IL-12 in the presence of neutralizing anti–IL-2 (α-IL-2) or control (Cntrl) Abs. After 48 hours cell-free culture supernatants were harvested and assayed for IFN-γ production by ELISA. The T-cell clone used does not produce IFN-γ, and thus all IFN-γ is NK cell–derived (data not shown). As a positive control, NK cells were also cultured with rIL-2 (10 pM) plus IL-12 in the presence of α-IL-2 or Cntrl Abs. Results shown are the means ± SEMs of replicate wells and summarize 6 independent experiments. *P < .03.

  • Fig. 5.

    Schema of early and late immunoregulatory functions of the CD56bright NK cell subset.

    Early during the innate immune response, CD56bright NK cells are costimulated with monokines (eg, IL-12 plus IL-18, IL-15, IL-1β) to produce IFN-γ. Later, CD56bright NK cells may use T cell–derived IL-2 and APC-derived IL-12 to synergistically induce IFN-γ, which may then influence the developing adaptive immune response. TCR indicates T-cell receptor.