Efficient priming of protein antigen-specific human CD4(+) T cells by monocyte-derived dendritic cells

Dendritic cells (DCs) have the unique ability to initiate an immune response in vivo by capturing antigens (Ags) in peripheral tissues and migrating to secondary lymphoid organs, where they sensitize naive CD4(+) T cells. To mimic this process in vitro, previous studies have shown that DCs directly isolated from peripheral blood can be used to elicit primary responses to neoantigens (neoAgs). In other studies, when monocyte-derived DCs have been utilized to sensitize total CD4(+) T cells in vitro, only secondary proliferation to neoAgs could be elicited. In the present study, the relative abilities of CD40 ligation, protein kinase C activation, and culture in tumor necrosis factor alpha (TNF-alpha) to induce functional and phenotypic maturation of human DCs from monocyte precursors were compared. Optimal TNF-alpha-induced maturation of DCs required a prolonged 4-day culture. It was then found that loading immature DCs with the neoAgs keyhole limpet hemocyanin or human immunodeficiency virus-1 p24 gag prior to TNF-alpha-induced maturation, rather than after maturation, was crucial to sensitize CD4(+) T cells to new Ags. This primary proliferation to neoAgs was initiated from the CD4(+) CD45RA(+) naive T-cell population. Finally, it was found that monocyte-derived DCs acquired the ability to secrete interleukin-12 p70, after contact with Ag-specific T cells. The ability to prime and expand Ag-specific CD4(+) T cells ex vivo to neoAgs in serum-free conditions has potential application for cellular vaccination and adoptive immunotherapy.     

Human invariant Valpha24+ natural killer T cells acquire regulatory functions by interacting with IL-10-treated dendritic cells

Glycolipid-reactive Valpha24(+) invariant natural killer T (iNKT) cells have been implicated in regulating a variety of immune responses and in the induction of immunologic tolerance. Activation of iNKT cells requires interaction with professional antigen-presenting cells, such as dendritic cells (DCs). We have investigated the capacity of distinct DC subsets to modulate iNKT cell functions. We demonstrate that tolerogenic DCs (tolDCs), generated by treatment of monocyte-derived DC with interleukin (IL)-10, induced regulatory functions in human iNKT cells. tolDCs, compared with immunogenic DCs, had reduced capacity to induce iNKT-cell proliferation, but these cells produced large amounts of IL-10 and acquired an anergic phenotype. These anergic Valpha24(+) iNKT cells were able to potently inhibit allogeneic CD4(+) T-cell proliferation in vitro. Furthermore, the anergic Valpha24(+) iNKT cells could suppress DC maturation in vitro. We conclude that the interaction of iNKT cells with tolDCs plays an important role in the immune regulatory network, which might be exploited for therapeutic purposes.     

Hepatitis C virus-infected hepatocytes extrinsically modulate dendritic cell maturation to activate T cells and natural killer cells

Dendritic cell maturation critically modulates antiviral immune responses, and facilitates viral clearance. Hepatitis C virus (HCV) is characterized by its high predisposition to persistent infection. Here, we examined the immune response of human monocyte-derived dendritic cells (MoDCs) to the JFH1 strain of HCV, which can efficiently replicate in cell culture. However, neither HCV RNA replication nor antigen production was detected in MoDCs inoculated with JFH1. None of the indicators of HCV interacting with MoDCs we evaluated were affected, including expression of maturation markers (CD80, 83, 86), cytokines (interleukin-6 and interferon-beta), the mixed lymphocyte reaction, and natural killer (NK) cell cytotoxicity. Strikingly, MoDCs matured by phagocytosing extrinsically-infected vesicles containing HCV-derived double-stranded RNA (dsRNA). When MoDCs were cocultured with HCV-infected apoptotic Huh7.5.1 hepatic cells, there was increased CD86 expression and interleukin-6 and interferon-beta production in MoDCs, which were characterized by the potential to activate NK cells and induce CD4+ T cells into the T helper 1 type. Lipid raft-dependent phagocytosis of HCV-infected apoptotic vesicles containing dsRNA was indispensable to MoDC maturation. Colocalization of dsRNA with Toll-like receptor 3 (TLR3) in phagosomes suggested the importance of TLR3 signaling in the MoDC response against HCV. CONCLUSION: The JFH1 strain does not directly stimulate MoDCs to activate T cells and NK cells, but phagocytosing HCV-infected apoptotic cells and their interaction with the TLR3 pathway in MoDCs plays a critical role in MoDC maturation and reciprocal activation of T and NK cells.     

The inflammatory cytokine IL-18 induces self-reactive innate antibody responses regulated by natural killer T cells

Inflammatory responses initiate rapid production of IL-1 family cytokines, including IL-18. This cytokine is produced at high levels in inflammatory diseases, including allergy and autoimmunity, and is known to induce IgE production in mice. Here we provide evidence that IL-18 is directly coupled to induction of self-reactive IgM and IgG antibody responses and recruitment of innate B2 B cells residing in the marginal zone of the spleen. Moreover, the data suggest that the B-cell activation occurs predominantly in splenic extrafollicular plasma cell foci and is regulated by natural killer T (NKT) cells that prevent formation of mature germinal centers. We also find evidence that NKT cells control this type of B-cell activation via cytotoxicity mediated by both the perforin and CD95/CD178 pathways. Thus, NKT cells regulate innate antibody responses initiated by an inflammatory stimulus, suggesting a general mechanism that regulates B-cell behavior in inflammation and autoreactivity.     

Human TSLP promotes CD40 ligand-induced IL-12 production by myeloid dendritic cells but maintains their Th2 priming potential

Interleukin-4 (IL-4), a major T-helper type 2 (Th2) cytokine, primes dendritic cells (DCs) for IL-12 production, suggesting a negative feedback loop to prevent dysregulated Th2 inflammation, such as allergy. We previously showed that human thymic stromal lymphopoietin (TSLP), highly expressed by keratinocytes of atopic dermatitis, activates CD11c(+) DCs to induce the differentiation of naive CD4(+) and CD8(+) T cells into proallergic effectors. Here we show that TSLP primes DCs to produce large amounts of IL-12 after CD40 ligand stimulation, similar to IL-4 priming of DCs. In contrast to IL-4 priming, DCs activated with TSLP and CD40 ligand induce the differentiation of naive CD4(+) T cells into effectors producing both Th1 and Th2 cytokines, a unique profile that is reminiscent of the late phase of allergy. Thus, TSLP is a major regulatory cytokine for IL-12 production by DCs, and TSLP-activated DCs could promote the persistence of Th2 inflammation even in the presence of IL-12-inducing signals.     

Production of interferons by dendritic cells, plasmacytoid cells, natural killer cells, and interferon-producing killer dendritic cells.

The capacity of mouse spleen conventional dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs) to produce interferon-gamma (IFN-gamma) or IFN-alpha was assessed, and compared with that of natural killer (NK) cells and the recently identified interferon-producing killer dendritic cells (IKDCs), both of which are frequent contaminants in DC preparations. Fully developed cDCs or pDCs, if free of NK cells or IKDCs, showed little capacity for IFN-gamma production. However, an early developmental form of the CD4-8+ cDC subtype, and the Ly6C- Ly49Q- pDC subtype, both were able to produce moderate amounts of IFN-gamma, although less than IKDCs. In response to toll-like receptor 9 stimuli, both the Ly6C+ Ly49Q+ and the Ly6C- Ly49Q- pDC subtypes were effective producers of IFN-alpha. However, IKDCs, which efficiently produced IFN-gamma and showed immediate cytotoxicity on NK target cells, did not produce IFN-alpha under these conditions.     

Distinct roles of IL-12 and IL-15 in human natural killer cell activation by dendritic cells from secondary lymphoid organs

Dendritic cells (DCs) are known to induce the growth and function of natural killer (NK) cells. Here, we address the capacity of DCs to interact with NK cells in human lymphoid organs and identify the role of specific DC-derived cytokines. We demonstrate that DCs colocalize with NK cells in the T cell areas of lymph nodes. In culture, DCs from either blood or spleen primarily stimulate the CD56(bright)CD16- NK cell subset, which is enriched in secondary lymphoid tissues. Blocking of IL-12 abolished DC-induced IFN-gamma secretion by NK cells, whereas membrane-bound IL-15 on DCs was essential for NK cell proliferation and survival. Maturation by CD40 ligation promoted the highest IL-15 surface presentation on DCs and led to the strongest NK cell proliferation induced by DCs. These results identify secondary lymphoid organs as a potential DC/NK cell interaction site and identify the distinct roles for DC-derived IL-12 and IL-15 in NK cell activation.     

Intravenous immunoglobulins suppress T-cell priming by modulating the bidirectional interaction between dendritic cells and natural killer cells

The modes of action of intravenous immunoglobulins (IVIgs) in exerting their immunomodulatory properties are broad and not fully understood. IVIgs can modulate the function of various immune cells, including suppressing the capacity of dendritic cells (DCs) to stimulate T cells. In the present study, we showed that DCs matured in the presence of IVIgs (IVIg-DCs) activated NK cells, and increased their interferon- production and degranulation. The activated NK cells induced apoptosis of the majority of IVIg-DCs. In consequence, only in the presence of NK cells, IVIg-DCs were 4-fold impaired in their T-cell priming capacity. This was due to NK-cell–mediated antibody-dependent cellular cytotoxicity (ADCC) to IVIg-DCs, probably induced by IgG multimers, which could be abrogated by blockade of CD16 on NK cells. Furthermore, IVIg-DCs down-regulated the expression of NKp30 and KIR receptors, and induced the generation of CD56^brightCD16^–CCR7⁺ lymph node–type NK cells. Our results identify a novel pathway, in which IVIgs induce ADCC of mature DCs by NK cells, which downsizes the antigen-presenting pool and inhibits T-cell priming. By influencing the interaction between DCs and NK cells, IVIgs modulate the ability of the innate immunity to trigger T-cell activation, a mechanism that can "cool down" the immune system at times of activation.      

Escape of monocyte-derived dendritic cells of HIV-1 infected individuals from natural killer cell-mediated lysis

OBJECTIVE: To verify whether the in vitro sensitivity of immature dendritic cells (iDC) to lysis by autologous natural killer (NK) cells from HIV-infected individuals might be correlated with HIV disease progression. DESIGN: Both dendritic cells (DC) and interlekin (IL)-2 activated NK cells were obtained from 13 HIV-infected individuals early after seroconversion and not receiving highly active antiretroviral therapy (HAART) and from 14 individuals with chronic HIV infection under HAART. The rate of NK cell-mediated killing of autologous iDC was correlated with classical parameters of HIV evolution. METHODS: Peripheral blood monocytes obtained from the Ficoll-derived leukocyte fraction after adherence to plastic were stimulated with granulocyte-macrophage colony stimulating factor plus IL-4 to induce their differentiation into iDC to be used as target cells in a standard 4-h cytotoxicity assay. A fraction of autologous leukocytes was stimulated with IL-2 to induce activation of NK cells to be used as effector cells. RESULTS: During early HIV infection the extent of ex vivo lysis of monocyte-derived DC by activated autologous NK cells was inversely and directly correlated with the levels of viraemia and with the percentage of circulating CD4 T cells, respectively. In contrast, the capacity of NK cells to kill iDC was lost independently of the levels of plasma viraemia or the concurrence of HAART in chronically infected individuals. Addition of exogenous HIV Tat during the cytotoxicity assay inhibited NK cell-mediated lysis of DC. CONCLUSIONS: NK cell-mediated immune surveillance against infected DC may be effective only during early HIV infection and may not be restored by HAART.     

In vivo priming of T cells against cryptic determinants by dendritic cells exposed to interleukin 6 and native antigen

T cells recognizing poorly displayed self determinants escape tolerance mechanisms and persist in the adult repertoire. The process by which these T cells are primed is not clear, but once activated, they can cause autoimmunity. Here, we show that dendritic cells treated with interleukin 6 (IL-6) process and present determinants from a model native antigen in a qualitatively altered hierarchy, activating T cells in vitro and in vivo against determinants that were previously cryptic because of poor display. IL-6 does not induce conventional maturation of dendritic cells but alters the pH of peripheral, early endosomal compartments and renders the cells more susceptible to killing by chloroquine. Acidification of endosomes by ouabain mimics the effect of IL-6 and allows processing of the same cryptic determinant. These results suggest that cytokines such as IL-6 could initiate and help to propagate an autoimmune disease process by differentiating dendritic cells into a state distinct from that induced by normal maturation.     

IL-15 availability conditions homeostasis of peripheral natural killer T cells

Steady-state numbers of peripheral lymphocyte are tightly controlled. For conventional T cells, signals delivered through the interaction of the T cell receptor (TCR) with antigen-loaded MHC molecules are required for the peripheral survival of naive T cells and for their homeostatic expansion in lymphopenic hosts. Cytokines, including IL-7, are also essential for survival of peripheral naive T cells. CD1d-restricted, V alpha 14(+) natural killer (NK)-T cells are a specialized autoreactive T subset with immunoregulatory activity. The relative roles of TCR engagement and cytokine signaling in the peripheral homeostasis of V alpha 14(+) NK-T cells were investigated. After adoptive transfer, the survival and expansion of peripheral V alpha 14(+) NK-T cells was independent of CD1d expression in the host. In contrast, IL-15 (but not IL-7) was required for maintenance of peripheral CD1d-reactive V alpha 14(+) T cells. Comparison of V alpha 14(+) T cell transfers into NK-proficient vs. deficient hosts suggests that NK-T cells and NK cells compete for peripheral resources. Our results indicate that IL-15 maintains the homeostasis of peripheral V alpha 14(+) NK-T cells. In contrast, TCR "tickling" of NK-T cells, if it occurs under steady-state conditions, does not by itself provide a sufficient signal for their peripheral survival.     

Liver injury due to sequential activation of natural killer cells and natural killer T cells by carrageenan

BACKGROUND/AIMS: Carrageenan is a high molecular weight polysaccharide and is widely used as a food additive for the solidification of plant oils and the thickening of many beverages. It is known that acute toxicity of carrageenan is possibly induced by the activation of phagocytic cells. We investigated other effects of carrageenan on lymphocytes in this study. METHODS: Carrageenan was intraperitoneally injected once into mice and phenotypic and functional characterizations were conducted in various immune organs. RESULTS: Natural killer (NK) cells were prominently activated in the liver, lungs, and spleen. A time-kinetic study showed sequential activation of NK and natural killer T (NKT) cells in the liver on days 3-10 after the injection. In parallel with the activation of NK and NKT cells in number, NK and NKT cytotoxicities were augmented. At this time, liver injury was induced, accompanied by massive hepatic necrosis and the elevation of transaminases. The in vivo elimination of NK cells reduced the liver injury induced by carrageenan. Direct binding of carrageenan onto NK cells was also demonstrated. Such a binding then induced a subsequent production of IFN gamma. Perforin molecules of NK cells were responsible for this liver injury. CONCLUSIONS: These results suggest that not only phagocytic cells but also primitive lymphocyte (mainly NK cells) subsets might be important targets for the acute toxicity of carrageenan.     

Activation of natural killer cells with interleukin 2 (IL-2) and IL-12 increases perforin binding and subsequent lysis of tumour cells

Natural killer (NK) cells can lyse a variety of different tumour cells by exocytosis of perforin, subsequent binding of perforin to the target cell membrane and formation of lytic pores. Some tumour cells, however, are resistant to cellular cytotoxicity. Using the NK-resistant tumour cell lines ML-2, MONOMAC-1, RPMI and L540Cy, we demonstrated that activation of NK cells with interleukin 2 (IL-2) and IL-12 resulted in significant lysis of these tumour targets. To investigate the underlying mechanisms, we isolated the cytotoxic granules from non-activated and IL-2-/IL-12-activated NK cells and compared the killing of K562 leukaemia cells (sensitive to NK cell-mediated lysis) and ML-2 leukaemia cells (resistant to NK cell-mediated lysis). In contrast to K562 cells, which were easily killed by NK-cell granules, ML-2 cells were resistant to granules from non-activated NK cells. However, granules from NK cells activated with IL-2 and IL-12 were able to induce significant tumour cell lysis. Cell death of both K562 and ML-2 cells by granules from activated NK cells was completely blocked by anti-perforin antibodies, indicating that perforin mainly accounts for the lysis induced by NK granules. Comparing granules from non-activated and IL-2-/IL-12-activated NK cells, the increased cell death of ML-2 cells was caused by an improved binding of perforin to the target cell membrane. Functional assays, however, indicated that the differences in perforin binding were not as a result of an augmented production of perforin by activated NK cells. We conclude that activation of NK cells results in an increased binding of perforin and subsequent lysis of tumour cells.     

Contribution of natural killer cells to inhibition of angiogenesis by interleukin-12

Interleukin-12 (IL-12) inhibits angiogenesis in vivo by inducing interferon-gamma (IFN-gamma) and other downstream mediators. Here, we report that neutralization of natural killer (NK) cell function with antibodies to either asialo GM1 or NK 1.1 reversed IL-12 inhibition of basic fibroblast growth factor (bFGF)-induced angiogenesis in athymic mice. By immunohistochemistry, those sites where bFGF-induced neovascularization was inhibited by IL-12 displayed accumulation of NK cells and the presence of IP-10-positive cells. Based on expression of the cytolytic mediators perforin and granzyme B, the NK cells were locally activated. Experimental Burkitt lymphomas treated locally with IL-12 displayed tumor tissue necrosis, vascular damage, and NK-cell infiltration surrounding small vessels. After activation in vitro with IL-12, NK cells from nude mice became strongly cytotoxic for primary cultures of syngeneic aortic endothelial cells. Cytotoxicity was neutralized by antibodies to IFN-gamma. These results document that NK cells are required mediators of angiogenesis inhibition by IL-12, and provide evidence that NK-cell cytotoxicity of endothelial cells is a potential mechanism by which IL-12 can suppress neovascularization.     

Upregulation of natural killer cells functions underlies the efficacy of intratumorally injected dendritic cells engineered to produce interleukin-12

OBJECTIVE: Injection of dendritic cells (DC) engineered with recombinant adenoviral vectors to produce interleukin-12 (IL-12) inside experimental murine tumors frequently achieves complete regressions. In such a system the function of CD8(+) T cells has been shown to be an absolute requirement, in contrast to observations made upon depletion of CD4(+) T cells, which minimally affected the outcome. The aim of this work was to study the possible involvement of natural killer (NK) cells in this setting. MATERIALS, METHODS, AND RESULTS: Depletions with anti-AsialoGM1 antiserum showed only a small decrease in the proportion of complete regressions obtained that correlated with induction of NK activities in lymphatic tissues into which DC migrate, whereas combined depletions of CD4(+) and NK cells completely eliminated the antitumor effects. Likewise in vivo neutralization of interferon-gamma (IFN-gamma) also eliminated those therapeutic effects. Trying to define the cellular role played by NK cells in vivo, it was observed that injection of cultured DC inside the spleen of T- and B-cell-deficient (Rag1(-/-)) mice induced upregulation of NK activity only if DC had been adenovirally engineered to produce IL-12. In addition, identically transfected fibroblasts also activated NK cells, indicating that IL-12 transfection was the unique requirement. Equivalent human DC only activated in vitro the cytolytic and cytokine-secreting functions of autologous NK cells if transfected to express human IL-12. CONCLUSIONS: Overall, these results point out an important role played by NK cell activation in the potent immunotherapeutic effects elicited by intratumoral injection of IL-12--secreting DC and that NK activation under these conditions is mainly, if not only, dependent on IL-12.