IL-18-based combinatorial adjuvants promote the intranodal production of CCL19 by NK cells and dendritic cells of cancer patients

Abstract

The effective accumulation and interaction of mature dendritic cells (DCs) and naïve T cells within lymph nodes (LNs), which are driven by the CCR7-CCL19/CCL21 chemokine axis, are critical for the induction of adaptive T-cell immunity. Human natural killer (NK) cells activated by interleukin (IL)-18 exhibit a unique 'helper' activity in promoting productive DC-T cell interactions, inducing DC maturation and shifting DC-primed T-cell responses toward a T_H1 polarization. Here, we demonstrate that such IL-18-activated 'helper' NK cells uniquely stimulate DCs to produce high levels of CCL19 through tumor necrosis factor α (TNFα) and interferon γ (IFNγ), a process that relies on secondary NK-cell activation by additional inflammatory signals including IFNα, IL-15, IL-12 and IL-2. DCs activated by helper NK cells not only promote the efficient CCR7-mediated recruitment of naïve CD8⁺ T cells, but also stimulate their expansion and expression of granzyme B. Using an ex vivo explant culture system based on LNs isolated from colorectal cancer patients, we found that CCL19 is upregulated in human tumor-associated lymphoid tissues treated with helper NK cell-stimulating factors. Our findings demonstrate the ability of 2 signal-activated helper NK cells to promote the production of the DC- and naïve/memory T cell-attracting chemokine CCL19 in LNs, and provide a rationale for the therapeutic application of IL-18-containing 'combinatorial adjuvants' to facilitate the induction of antitumor immune responses.

Keywords: CCL19; IL-18; adjuvant; colorectal cancer; dendritic cell; lymph node; natural killer cell.

Figure 1. IL-18-activated NK cells stimulate DCs to produce CCL19. (A) CCL19 levels in supernatants from DCs cultured for 48 h, alone or together with autologous NK cells (1:2 NK cell:DC ratio), in the presence of interleukin (IL)-2, IL-18 and/or interferon α (IFNα) (left); or for 24 h in the presence of CD40 ligand (CD40L)-expressing cells upon previous co-culture with NK cells (right). Data are reported as means ± SD of samples from 6 independent individuals. (B) CCL19 levels in supernatants from DCs cultured for 48 h together with autologous NK cells (1:2 NK cell:DC ratio) in the presence of IL-18, IL-1β, and/or IFNα. Data, which are representative of 1 out of 2 independent experiments yielding similar results, are reported as means ± SD of triplicate cultures. (C) NK cells were pre-treated or not for 24 h with IL-2 or IL-18, washed, and re-plated with autologous DCs, alone or in the presence of IFNα, IL-15, IL-12, IL-2, or IL-18. CCL19 levels were analyzed by ELISA 48 h after the administration of the secondary stimulus. Data, which are representative of 1 out of 3 independent experiments yielding similar results, are reported as means ± SD of triplicate cultures. ***P < 0.001, **P < 0.01, as compared with the indicated samples or all samples when not specified.

Figure 2. The ability of helper NK cells to stimulate the secretion of CCL19 by DCs depends on TNFα and IFNγ. (A) CCL19 levels in supernatants from DCs cultured for 48 h, alone or together with autologous NK cells (1:2 NK cell:DC ratio), in the presence or in the absence of interleukin (IL)-18 plus interferon (IFN)α and soluble tumor necrosis factor α (TNFα) receptor 1 (sTNFR1) or IFNγ receptor 1 (sIFNγR1) decoys (left); or for 24 h in the presence of sTNFR1 and sIFNγR1 decoys and CD40 ligand (CD40L)-expressing cells upon previous co-culture with NK cells (right). Data, which are representative of 1 out of 3 independent experiments yielding similar results, are reported as means ± SD of triplicate cultures. (B) CCL19 levels in supernatants from DCs cultured for 48 h together with NK cells activated by IL-18 and IFNα, in the presence of TNFα- or IFNγ-blocking antibodies or isotype-matched control antibodies (left); or for 24 h in the presence of TNFα- or IFNγ-blocking antibodies or isotype-matched control antibodies and CD40 ligand (CD40L)-expressing cells upon previous co-culture with NK cells (right). Data, which are representative of 1 out of 2 independent experiments yielding similar results, are reported as means ± SD of triplicate cultures. ***P < 0.001, as compared with the indicated samples or all samples when not specified. < depicts levels that were below the limit of detection of the assay.

Figure 3. NK cell-activated DCs recruit naïve T cells and promote their expansion and functional differentiation. (A) Migration of naïve CD8⁺ T cells toward supernatants from DCs cultured for 48 h, alone or together with autologous NK cells (1:2 NK cell:DC ratio), in the presence or in the absence of interleukin (IL)-18 plus interferon α (IFNα). Data are reported as means ± SD of samples from 4 independent individuals. (B) Migration of naïve CD8⁺ T cells pretreated or not with an anti-CCR7 antibody toward supernatants from DCs cultured for 48 h together with autologous NK cells activated by IL-18 and IFNα. Data are reported as means ± SD of samples from 4 independent individuals. (C) Total number (left) or number of proliferating granzyme B (GzmB)⁺ CD8⁺ T cells (right) observed upon the co-culture of migrating T cells with DCs 7 d after migration. Data are reported as means ± SD of triplicate cultures. ***P < 0.001, *P < 0.05, as compared with all samples.

Figure 4. IL-18-based combinatorial adjuvants drive CCL19 production in human tumor-associated lymph nodes. (A) Expression of tumor necrosis factor α (TNFα), interferon γ (IFNγ), and CCL19 in lymph nodes from colorectal cancer patients, upon exposure or not to interleukin (IL)-18 plus interferon α (IFNα) for 24 h. Normalized gene expression data, which are representative of 1 out of 3 independent experiments yielding similar results, are reported as means ± SD of triplicate lymph node cultures. (B) Expression of the CCL19-coding mRNA in tissues (left) and CCL19 in supernatants (right) of lymph node cultures established with biopsy material from colorectal cancer patients, upon exposure or not to IL-18 plus IFNα for 24 h. Data are reported as means of samples from 3 independent patients. **P < 0.01, *P < 0.05, as compared with the indicated samples.