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- Materials and Methods
Dendritic cells (DC) are uniquely well-equipped, professional antigen (Ag)-presenting cells (APC) that can induce T cell immunity or tolerance [1, 2]. While immunogenic DC represent mature APC essential for stimulating naïve and memory T cell responses, tolerogenic DC include DC in the normal steady-state (immature DC), that can promote and maintain peripheral tolerance to auto- or allo-antigen. Mechanisms that underline DC tolerogenecity include: inducing effector T cell apoptosis, skewing of T cell polarization, promoting T cell anergy and generation of regulatory T cells (Treg) [1, 2]. Tolerogenic DC can be induced in vitro or in vivo by exposure to diverse immunosuppressive agents, that impact their phenotype and function, resulting in regulation of T cell immunity .
The mammalian target of rapamycin (mTOR) inhibitor, rapamycin (RAPA) is a macrocyclic triene with immunoregulatory properties [4-8]. While mTOR exists in two complexes, that is mTOR complex 1 (mTORC1) and mTORC2, RAPA mainly targets mTORC1, a highly conserved serine/threonine protein kinase, that controls cell responses to environmental cues [2, 9-11]. The ability of RAPA to inhibit myeloid DC differentiation, maturation and function has been studied extensively in vitro and in animal models [5, 9, 12, 13]. In murine systems, RAPA exerts a profound inhibitory effect on DC differentiation and function in vitro, impairing their phenotipic maturation in response to Toll-like receptor (TLR) or CD40 ligation, suppressing their T cell allostimulatory function and conferring the ability to induce allo-Ag-specific T cell hyporesponsiveness [5-7, 9, 12, 14]. In vivo, Taner et al.  first showed that recipient-derived RAPA-DC pulsed with donor allo-Ag and administered systemically before transplantation could prolong mouse heart allograft survival, while Turnquist et al.  found that a single infusion of similarly pulsed RAPA-DC, combined with a short course of low-dose RAPA, could promote transplant tolerance.
Human RAPA-DC generated from blood monocytes display a more immature phenotype than control (CTRL)-DC, suppress T cell proliferation and can induce Treg, depending on the protocol used and the timing of exposure to RAPA during DC differentiation [9, 15-17]. Paradoxically, however, following TLR ligation with LPS, both mouse and human RAPA-DC display enhanced IL-12p70 secretion, with concomitant decreased production of IL-10, thus acquiring a potential to promote activation of double positive Type-1/Type-2 T cell immunity, rather than immune regulation [14-16]. Moreover, when RAPA is administrated to transplant recipients, various adverse inflammatory reactions have been observed, including interstitial pneumonitis, increased incidence of acute cellular rejection, augmented pro-inflammatory cytokine production by myeloid cells and enhanced recall Ag-specific memory CD8+ T cell responses [18-20].
Notably, however, the impact of an inflammatory cytokine cocktail (ICC) IL-1β, IL-6, TNF-α plus IFN-γ on human RAPA-DC has not yet been examined. These two synergistic signals have been shown to direct human immature DC into Type-1 polarized DC (DC1) capable of a log increase in IL-12p70 production as compared to ICC alone, with subsequent strong downstream Type-1 T cell polarization . This pathway of DC maturation has clinical relevance for transplant recipients on RAPA maintenance therapy, since both viral infections and acute cellular rejection after organ transplantation trigger inflammation and Type-1 interferon (IFN)-γ release.
Natural killer (NK) cells are critical components of the innate immune response and play an important role in early defense against viral infections and tumor growth [22-25]. In response to IL-12p70, they release large amounts of IFN-γ, a cytokine that mediates ‘helper’ activity during generation of Type-1 T cell immunity, including immunity to viruses, bacteria, oncogenic transformed cells and allo-immunity [26-28]. IFN-γ release by NK cells also promotes DC maturation and differentiation of Type-1-polarized DC with enhanced ability to secrete IL-12p70, and further elevate Type-1 immunity [29, 30]. Since NK cells constitutively express functional IL-12R β1 and β2, they can readily respond to IL-12p70 and thus may represent an important target of augmented IL-12p70 produced by RAPA-DC exposed to inflammatory signals.
Here, we show for the first time, that human monocyte-derived RAPA-DC exposed to ICC + IFN-γ display a mature phenotype and markedly enhanced secretion of both IL-12p70 and IL-27, associated with low production of IL-10. When co-cultured with allogeneic peripheral blood mononuclear cells (PBMC), ICC + IFN-γ matured (m)RAPA-DC deliver stimulatory cell-to-cell contact-dependent signals that target NK and T cells to augment downstream Type-1 immunity in allo-reactive CD8+ and bystander CD4+ and CD8+ T cells. In addition, mRAPA-DC cytokines IL-12p70 and IL-27 appear to instruct NK cells to exert either ‘regulatory’ or ‘stimulatory’ effects on IFN-γ production by allogeneic T cells. These findings have important implications for further understanding the impact of mTOR modulation on human allogeneic DC-NK-T cell interactions associated with inflammatory events, and for evaluation of RAPA as an immune regulatory drug in clinical transplantation.
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- Materials and Methods
A goal of our laboratory is to define the molecular and functional pathways in DC that are impacted by mTOR inhibition. Here we tested for the first time the responsiveness of human RAPA-DC generated from a cohort of healthy individuals to an ICC that mimic factors encountered by immature DC in peripheral tissues during viral infections or episodes of transplant rejection in patients on RAPA therapy. We incorporated IFN-γ in the inflammatory cocktail as a second signal for DC maturation because of its role in these inflammatory disorders, and since it has been shown that IFN-γ has the unique capacity to prime human DC into DC1 to produce high levels of IL-12p70 [38, 39]. Using this cytokine cocktail, our phenotypic analyses showed that RAPA-DC could up-regulate HLA-DR, CD40 and CD86 to the same levels as those expressed on mature CTRL-DC. In addition, when compared to CTRL-DC, immature or mature RAPA-DC exhibited low expression of PD-L1 (B7-H1), a PD-1 ligand that negatively regulates T cell activation and can promote peripheral tolerance . Other reports have demonstrated a similar capacity of mTORi to modulate the phenotype of monocyte-derived DC. Although the timing of exposure of these APC to RAPA has varied between studies, the overall conclusion is that human RAPA-DC is not resistant to maturation.
Recent rodent and human studies have shown that RAPA-DC have a unique cytokine production profile upon stimulation with TLR agonists (e.g. lipopolysaccharide), that is increased secretion of IL-12 due to inhibited GSK-3, while IL-10 is reduced [9, 41]. Other reports have shown that mTORi controls IL-12p40 production by human monocyte-derived DC after TLR-dependent and -independent stimulation, due to enhanced NF-κB activity [14, 15]. Here we demonstrate that, when RAPA-DC are exposed to ICC + IFN-γ, not only IL-12p40 but also IL-12p35 is up-regulated, resulting in increased bioactive IL-12p70 secretion, paralleled by decreased IL-10. In addition, for the first time we provide direct evidence that exposure to inflammatory cytokines including IFN-γ elicits significantly increased IL-27 secretion by mRAPA-DC compared to mCTRL-DC. These findings may have important clinical relevance, since DC-derived IL-12p70 is regarded as the key cytokine for induction of Type-1 immunity (e.g. IFN-γ) in both NK cells and CD8+ effector T cells . In addition, IL-27 is also a member of the IL-12 cytokine family, which acts synergistically with IL-12p70 to promote Type-1 responses [34, 42]. IL-27 also has a nonredundant role in selectively increasing T cell proliferation and cytotoxicity [34, 42]. However, IL-27 can also exert an inhibitory effect on Th1, Th2 and Th17 cells in pathogen-induced disease models, due to its ability to induce IL-10 production by T cells [43, 44]. Our data show that soluble IL-27 renders NK cells to produce IL-10, whereas cell-to-cell contact plus IL-27 may trigger NK to also produce IFN-γ and decrease IL-10. Thus, the role of IL-27 is more complex, due to its concomitant stimulatory and/or regulatory effects making such observations worthy of future investigation.
Based on the complex helper/regulatory cytokine and co-stimulatory/co-regulatory molecule expression profile, we postulated that mRAPA-DC would have unique abilities to integrate and deliver heterogeneous cell-to-cell contact (signal 1 and 2) and IL-12/IL-27 (signal 3) signals that are critically required for instructing allogeneic NK to modulate T cells to mount IFN-γ responses. In addition, the lower IL-10 secretion by RAPA-DC may favor Type-1 T cell polarization since IL-10 cross-regulates IL-12p70 and vice versa . Similar to early IFN-γ ‘helper’ responses that facilitate the initiation and maintenance of Type-1 anti-viral or anti-cancer T cell immunity [28, 37, 46] those mediated by NK and memory T cells stimulated by mRAPA-DC appear to also modulate Type-1 allo-immunity. Although these responses were always higher than those induced by mCTRL-DC, they varied in strength between healthy individuals.
Increased IFN-γ production by allogeneic CD4+ and CD8+ T cells was observed in 5-day MLR co-cultures with mRAPA-DC compared to mCTRL-DC. This observation contrasts with results reported by others, who have found either a significant decrease or no difference in IFN-γ release by T cells when stimulated by allogeneic mRAPA-DC [16, 17]. These discrepancies may result from differences in RAPA-DC generation and maturation protocols implemented by different investigators. Interestingly, none of these studies measured levels of IL-12p70 production by mRAPA-DC. Our data demonstrate augmented Type-1 responses by allogeneic CD4+ and CD8+ T cells. Notably, we have found that mRAPA-DC generated ex vivo from kidney transplant recipients on RAPA monotherapy significantly augment IFN-γ secretion by allogeneic CD4+ and CD8+ T cells (Macedo et al., unpublished observations), consistent with our in vitro finding. Furthermore, the Type-1 polarization of mRAPA-DC-allogeneic PBMC 5 days co-cultures was highly dependent on cell-to-cell contact while soluble factors only minimally interfered with IFN-γ secretion by CD4+ or CD8+ T cells. These data suggest that DC/T/NK cell interactions may stabilize the immunologic synapse during late T cell activation [46, 47], thus, making it difficult to interfere with the action of soluble factors. Moreover, among the proliferating (allo-reactive) CFSEdim T cells, only CD8+ T cells exhibited significantly higher IFN-γ secretion following mRAPA-DC stimulation compared to mCTRL-DC stimulation. This difference may be due to the preferential role of IL-27 in priming naïve CD8+ T cells rather than CD4+T cells into Type-1 effectors in this setting. Conversely, mRAPA-DC may only be able to reactivate bystander memory CD8+ T cells specific to recall Ag (pathogen-specific) rather than to prime allo-reactive CD4+ and CD8+ T cell responses, as reported [20, 47]. Thus, the increased IFN-γ production observed within CFSEdim proliferating CD8+ T cells may represent reactivation of heterologous, anti-viral memory CD8+ T cells that cross-react with human MHC class I allo-Ags rather than de novo priming of allo-reactive CD8+ T cells [32, 48].
Our data show, for the first time, that human NK cells are targets of mRAPA-DC stimulation in vitro. This finding is consistent with that of Brouard et al. , who showed that the peripheral blood transcriptional profile induced by RAPA monotherapy in stable kidney transplant patients was dominated by pro-inflammatory features of innate immune cells, including NK cells. The present observations further reveal that mRAPA-DC instruct allogeneic NK cells to convey either stimulatory or regulatory signals to allogeneic T depending on the responder/stimulator combination pairs. In literature, the precise role of human NK cells in organ transplantation is unclear. NK cells were shown to integrate complex stimulatory (NKp46, NKp30, NKG2D) and inhibitory (KIRs, CD94/NKG2A) signals combined with the release of diverse cytokines [49, 50]. In general, NK cells are considered rapid initiators of a pro-inflammatory milieu that promotes the licensing of DC and T cells into Type-1-polarized effectors, able to mediate acute or/and chronic allograft injury . Recent findings, however, have indicated that NK cells can also promote allograft tolerance, with DC and T cells serving as targets of NK cell killing as a result of ‘missing self’ [49-51]. While here we report the ability of human mRAPA-DC to instruct NK cells to produce increased IFN-γ, our findings add to the list of potential roles for NK (i) triggering receptors which further promote Type-1 help to allogeneic T cells in certain individuals and (ii) release of regulatory cytokine(s) that regulates allogeneic T cells by lowering their IFN-γ production in others. This effect may be mediated by IL-10, since our results and recently published data have shown IL-27 to induce IL-10 production by NK cells . Therefore future studies are required to identify which NK regulatory cytokine(s) and/or activating receptors are relevant during the complex DC/NK/T cell interactions in the setting of allo-recognition.
In conclusion, we have characterized the phenotype and cytokine secretion profile of human mRAPA-DC generated as the result of exposure to inflammatory cytokines including IFN-γ. We demonstrated their marked influence on both allo-reactive NK and T cell functions. These data reveal both immune stimulatory and regulatory properties of NK cells stimulated by mRAPA-DC, similar to the dichotomous functions proposed for NK cells in relation to transplant outcome. In total, out findings indicate that clinical mTORi use in transplantation may be complicated by conferring DC a novel capacity to drive NK cell support of Type-1 immunity, especially in patients where NK cells serve a stimulatory function, or lack regulatory properties.