Prenatal Allospecific NK Cell Tolerance Hinges on Instructive Allorecognition through the Activating Receptor during Development.

Little is known about how the prenatal interaction between NK cells and alloantigens shapes the developing NK cell repertoire toward tolerance or immunity. Specifically, the effect on NK cell education arising from developmental corecognition of alloantigens by activating and inhibitory receptors with shared specificity is uncharacterized. Using a murine prenatal transplantation model, we examined the manner in which this seemingly conflicting input affects NK cell licensing and repertoire formation in mixed hematopoietic chimeras. We found that prenatal NK cell tolerance arose from the elimination of phenotypically hostile NK cells that express an allospecific activating receptor without coexpressing any allospecific inhibitory receptors. Importantly, the checkpoint for the system appeared to occur centrally within the bone marrow during the final stage of NK cell maturation and hinged on the instructive recognition of allogeneic ligand by the activating receptor rather than through the inhibitory receptor as classically proposed. Residual nondeleted hostile NK cells expressing only the activating receptor exhibited an immature, anergic phenotype, but retained the capacity to upregulate inhibitory receptor expression in peripheral sites. However, the potential for this adaptive change to occur was lost in developmentally mature chimeras. Collectively, these findings illuminate the intrinsic process in which developmental allorecognition through the activating receptor regulates the emergence of durable NK cell tolerance and establishes a new paradigm to fundamentally guide future investigations of prenatal NK cell-allospecific education.

Natural killer activity: early days, advances, and seminal observations.

This manuscript describes the early history of NK cell discovery, with emphasis on the events in the first decade of NK cell studies, 1972-1982. The authors highlight some of the earliest and most important observations that would later prove to be milestones in the study of NK cells and their activity.

IFN-gamma AU-rich element removal promotes chronic IFN-gamma expression and autoimmunity in mice.

We generated a mouse model with a 162 nt AU-rich element (ARE) region deletion in the 3' untranslated region (3'UTR) of the interferon-gamma (IFN-γ) gene that results in chronic circulating serum IFN-γ levels. Mice homozygous for the ARE deletion (ARE-Del) (-/-) present both serologic and cellular abnormalities typical of patients with systemic lupus erythematosus (SLE). ARE-Del(-/-) mice display increased numbers of pDCs in bone marrow and spleen. Addition of IFN-γ to Flt3-ligand (Flt3L) treated in vitro bone marrow cultures results in a 2-fold increase in pDCs with concurrent increases in IRF8 expression. Marginal zone B (MZB) cells and marginal zone macrophages (MZMs) are absent in ARE-Del(-/-) mice. ARE-Del(+/-) mice retain both MZB cells and MZMs and develop no or mild autoimmunity. However, low dose clodronate treatment in ARE-Del(+/-) mice specifically eliminates MZMs and promotes anti-DNA antibody development and glomerulonephritis. Our findings demonstrate the consequences of a chronic IFN-γ milieu on B220(+) cell types and in particular the impact of MZB cell loss on MZM function in autoimmunity. Furthermore, similarities between disease states in ARE-Del(-/-) mice and SLE patients suggest that IFN-γ may not only be a product of SLE but may be critical for disease onset and progression.

Systemic IL-12 administration alters hepatic dendritic cell stimulation capabilities.

The liver is an immunologically unique organ containing tolerogenic dendritic cells (DC) that maintain an immunosuppressive microenvironment. Although systemic IL-12 administration can improve responses to tumors, the effects of IL-12-based treatments on DC, in particular hepatic DC, remain incompletely understood. In this study, we demonstrate systemic IL-12 administration induces a 2-3 fold increase in conventional, but not plasmacytoid, DC subsets in the liver. Following IL-12 administration, hepatic DC became more phenotypically and functionally mature, resembling the function of splenic DC, but differed as compared to their splenic counterparts in the production of IL-12 following co-stimulation with toll-like receptor (TLR) agonists. Hepatic DCs from IL-12 treated mice acquired enhanced T cell proliferative capabilities similar to levels observed using splenic DCs. Furthermore, IL-12 administration preferentially increased hepatic T cell activation and IFNγ expression in the RENCA mouse model of renal cell carcinoma. Collectively, the data shows systemic IL-12 administration enables hepatic DCs to overcome at least some aspects of the inherently suppressive milieu of the hepatic environment that could have important implications for the design of IL-12-based immunotherapeutic strategies targeting hepatic malignancies and infections.

Hydrodynamic delivery of human IL-15 cDNA increases murine natural killer cell recovery after syngeneic bone marrow transplantation.

Immune deficiency immediately following bone marrow transplantation (BMT) increases susceptibility to opportunistic infections as well as tumor relapse. Natural Killer (NK) cells play important roles in the resistance to virally infected and transformed cells. Interleukin (IL)-15 has been shown to be essential for NK cell development and survival. We administered human (h) IL-15 cDNA (pIL-15) via hydrodynamic delivery to murine recipients undergoing congenic BMT to determine its effects on NK cell reconstitution. Hydrodynamic pIL-15 delivery resulted in high levels of hIL-15 protein in the serum that lasted for several days and then quickly declined. The appearance of hIL-15 was followed by a significant increase of mature donor-derived NK cells within the bone marrow, spleens, and livers of the treated recipients. No accumulation of immature NK cell progenitors was observed. The NK cells from IL-15-treated recipients displayed an activated phenotype and were lytically active toward tumor targets in vitro to a similar degree as did those cells from recipients treated with control plasmid. This suggests that the predominant effect of IL-15 was a quantitative increase in total NK cell numbers and not qualitative changes in NK cell functions. No toxicities or adverse effects were observed. Studies performed in transplanted mice bearing renal carcinoma tumors demonstrated that this mode of hIL-15 gene delivery resulted in increased antitumor responses. These results support the use of cytokine gene transfer-based regimens as a platform to augment NK cell recovery after BMT.

mTOR kinase inhibitor AZD8055 enhances the immunotherapeutic activity of an agonist CD40 antibody in cancer treatment.

mTOR is a central mediator of cancer cell growth, but it also directs immune cell differentiation and function. On this basis, we had explored the hypothesis that mTOR inhibition can enhance cancer immunotherapy. Here, we report that a combination of αCD40 agonistic antibody and the ATP-competitive mTOR kinase inhibitory drug AZD8055 elicited synergistic antitumor responses in a model of metastatic renal cell carcinoma. In contrast to the well-established mTOR inhibitor rapamycin, AZD8055 increased the infiltration, activation, and proliferation of CD8(+) T cells and natural killer cells in liver metastatic foci when combined with the CD40 agonist. AZD8055/αCD40-treated mice also display an increased incidence of matured macrophages and dendritic cells compared with that achieved in mice by αCD40 or AZD8055 treatment alone. We found that the combination treatment also increased macrophage production of TNFα, which played an indispensable role in activation of the observed antitumor immune response. Levels of Th1 cytokines, including interleukin 12, IFN-γ, TNFα, and the Th1-associated chemokines RANTES, MIG, and IP-10 were each elevated significantly in the livers of mice treated with the combinatorial therapy versus individual treatments. Notably, the AZD8055/αCD40-induced antitumor response was abolished in IFN-γ(-/-) and CD40(-/-) mice, establishing the reliance of the combination therapy on host IFN-γ and CD40 expression. Our findings offer a preclinical proof of concept that, unlike rapamycin, the ATP-competitive mTOR kinase inhibitor AZD8055 can contribute with αCD40 treatment to trigger a restructuring of the tumor immune microenvironment to trigger regressions of an established metastatic cancer.

TCR-dependent and -independent activation underlie liver-specific regulation of NKT cells.

The fate of invariant NKT (iNKT) cells following activation remains controversial and unclear. We systemically examined how iNKT cells are regulated following TCR-dependent and -independent activation with α-galactosylceramide (αGC) or IL-18 plus IL-12, respectively. Our studies reveal activation by αGC or IL-18 plus IL-12 induced transient depletion of iNKT cells exclusively in the liver that was independent of caspase 3-mediated apoptosis. The loss of iNKT cells was followed by repopulation and expansion of phenotypically distinct cells via different mechanisms. Liver iNKT cell expansion following αGC, but not IL-18 plus IL-12, treatment required an intact spleen and IFN-γ. Additionally, IL-18 plus IL-12 induced a more prolonged expansion of liver iNKT cells compared with αGC. iNKT cells that repopulate the liver following αGC had higher levels of suppressive receptors PD-1 and Lag3, whereas those that repopulate the liver following IL-18 plus IL-12 had increased levels of TCR and ICOS. In contrast to acute treatment that caused a transient loss of iNKT cells, chronic αGC or IL-18 plus IL-12 treatment caused long-term systemic loss requiring an intact thymus for repopulation of the liver. This report reveals a previously undefined role for the liver in the depletion of activated iNKT cells. Additionally, TCR-dependent and -independent activation differentially regulate iNKT cell distribution and phenotype. These results provide new insights for understanding how iNKT cells are systemically regulated following activation.

In vivo regulation of experimental autoimmune encephalomyelitis by NK cells: alteration of primary adaptive responses.

Innate immune responses provide the host with its first line of defense against infections. Signals generated by subsets of lymphocytes, including NK cells, NKT cells, and APC during this early host response determine the nature of downstream adaptive immune responses. In the present study, we have examined the role of innate NK cells in an autoimmune model through the use of primary immunization with the myelin oligodendrocyte glycoprotein peptide to induce experimental autoimmune encephalomyelitis (EAE). Our studies have shown that in vivo depletion of NK cells can affect the adaptive immune responses, because NK cells were found to regulate the degree of clinical paralysis and to alter immune adaptive responses to the myelin oligodendrocyte glycoprotein peptide. The requirement for NK cells was reflected by changes in the T cell responses and diminished clinical disease seen in mice treated with anti-NK1.1, anti-asialo GM1, and selected Ly49 subtype-depleted mice. In addition to alteration in T cell responses, the maturational status of dendritic cells in lymph nodes was altered both quantitatively and qualitatively. Finally, examination of TCR Vbeta usage of the brain lymphocytes from EAE mice indicated a spectra-type change in receptor expression in NK- depleted mice as compared with non-NK-depleted EAE mice. These findings further establish a recently postulated link between NK cells and the generation of autoreactive T cells.

Modulation of lymphocyte function with inhibitory CD2: loss of NK and NKT cells.

Analysis of the NK cell developmental pathway suggests that CD2 expression may be important in regulating NK maturation. To test this hypothesis, we developed mice containing only an inhibitory CD2 molecule by linking the extracellular domain of CD2 to an intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM) motif. Mice containing the CD2 Tg(ITIM) transgene, introduced into a CD2 KO background, have no morphologically detectable lymph nodes, although development of the thymus appears normal. In addition, these mice had major loss of both NK and NKT subsets in peripheral organs, while T and B cell frequencies were intact. Expression of CD2 was low on T cells and lacking on B cells and functional defects were observed in these populations. NKT cells expressing CD4 were absent, while the CD8+ and double negative NKT cells were retained. Small subsets of NK cells were detected but expression of CD2 on these cells was very low or absent, and their maturation was impaired. Based on the phenotype described here, we believe that these mice represent a unique model to study lymphoid organ and lymphocyte development.

Enhanced antitumor response by divergent modulation of natural killer and natural killer T cells in the liver.

The use of interleukin-18 (IL-18) together with IL-12 induced high levels of IFN-gamma in tumor-bearing mice and regression of liver tumors that was abolished in IFN-gamma((-/-)) mice. Natural killer (NK) and NKT cells were the major producers of IFN-gamma in the livers of mice treated with IL-18 and/or IL-12. Liver NK cells were significantly increased by treatment with IL-18/IL-12, whereas the degree of liver NKT cell TCR detection was diminished by this treatment. Reduction of NK cells with anti-asGM1 decreased the antitumor activity of IL-18/IL-12 therapy and revealed NK cells to be an important component for tumor regression in the liver. In contrast, the antitumor effects of both IL-18 and IL-12 were further increased in CD1d((-/-)) mice, which lack NKT cells. Our data, therefore, show that the antitumor activity induced in mice by IL-18/IL-12 is NK and IFN-gamma dependent and is able to overcome an endogenous immunosuppressive effect of NKT cells in the liver microenvironment. These results suggest that immunotherapeutic approaches that enhance NK cell function while eliminating or altering NKT cells could be effective in the treatment of cancer in the liver.

NK cells use NKG2D to recognize a mouse renal cancer (Renca), yet require intercellular adhesion molecule-1 expression on the tumor cells for optimal perforin-dependent effector function.

The NKG2D receptor on NK cells can recognize a variety of ligands on the tumor cell surface. Using a mouse renal cancer (Renca), we show that NKG2D recognition by NK cells was crucial for their ability to limit tumor metastases in vivo in both liver and lungs using perforin-dependent effector mechanisms. However, for the R331 cell line established from Renca, NKG2D recognition and perforin-dependent lysis played no role in controlling liver metastases. R331 cells were also more resistant to perforin-dependent lysis by NK cells in vitro. We therefore used these phenotypic differences between Renca and R331 to further investigate the crucial receptor:ligand interactions required for triggering lytic effector functions of NK cells. Reconstitution of R331 cells with ICAM-1, but not Rae-1gamma, restored NKG2D-mediated, perforin-dependent lysis. Interestingly, R331 cells were efficiently lysed by NK cells using death ligand-mediated apoptosis. This death ligand-mediated killing did not depend on NKG2D recognition of its ligands on tumor cells. This result suggests that the intracellular signaling in NK cells required for perforin and death ligand-mediated lysis of tumor target cell are quite distinct, and activation of both of these antitumor lytic effector functions of NK cells could improve therapeutic benefits for certain tumors.

Glucocorticoid amplifies IL-2-dependent expansion of functional FoxP3(+)CD4(+)CD25(+) T regulatory cells in vivo and enhances their capacity to suppress EAE.

IL-2 is crucial for the production of CD4(+)CD25(+) T regulatory (Treg) cells while important for the generation of effective T cell-mediated immunity. How to exploit the capacity of IL-2 to expand Treg cells, while restraining activation of T effector (Teff) cells, is an important and unanswered therapeutic question. Dexamethasone (Dex), a synthetic glucocorticoid steroid, has been reported to suppress IL-2-mediated activation of Teff cells and increase the proportion of Treg cells. Thus, we hypothesized that glucocorticoids may be useful as costimulants to amplify IL-2-mediated selective expansion of Treg cells. We show in this study that short-term simultaneous administration of Dex and IL-2 markedly expanded functional suppressive Foxp3(+)CD4(+)CD25(+) T cells in murine peripheral lymphoid tissues. In a myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (EAE) mouse model, we observed that splenic CD4(+)CD25(+) T cells failed to suppress the proliferation of CD4(+)CD25(-) T cells. Pretreatment with Dex/IL-2 remarkably increased the proportion of CD4(+)FoxP3(+) cells and partially restored the function of splenic CD4(+)CD25(+) T cells, and inhibited the development of EAE. Therefore, the combination of glucocorticoid and IL-2, two currently used therapeutics, may provide a novel approach for the treatment of autoimmune diseases, transplant rejection and graft-vs.-host disease.

IL-18 as critical co-stimulatory molecules in modulating the immune response of ITAM bearing lymphocytes.

NK cells responses are controlled by inhibitory and activating cell surface receptors. Inhibitory receptors serve to moderate NK activity by dampening cytokine release and cytotoxicity if activating ligands are also triggered by interaction with their receptors on the NK cells. This dampening effect is critical to prevent wide scale self destruction. These studies have focused on cytokine signaling in the context of an environment where ITAM bearing receptors are triggered on NK, NKT and T cells. Our findings indicate that cytokines are an important co-stimulatory signal that is needed to maximize the host innate immune response.

Pertussis toxin as an adjuvant suppresses the number and function of CD4+CD25+ T regulatory cells.

We observed a remarkable reduction in the frequency and immunosuppressive activity of splenic CD4+CD25+ T cells in C57BL/6 mice with MOG33-55-induced experimental autoimmune encephalomyelitis (EAE). Our study revealed that pertussis toxin (PTx), one component of the immunogen used to induce murine EAE, was responsible for down-regulating splenic CD4+CD25+ cells. Treatment of normal BALB/c mice with PTx in vivo reduced the frequency, suppressive activity and FoxP3 expression by splenic CD4+CD25+ T cells. However, PTx treatment did not alter the expression of characteristic phenotypic markers (CD45RB, CD103, GITR and CTLA-4) and did not increase the expression of CD44 and CD69 by the residual splenic and lymph node CD4+CD25+ T cells. This property of PTx was attributable to its ADP-ribosyltransferase activity. PTx did not inhibit suppressive activity of purified CD4+CD25+ T regulatory (Treg) cells in vitro, but did so in vivo, presumably due to an indirect effect. Although the exact molecular target of PTx that reduces Treg activity remains to be defined, our data suggests that alteration of both distribution and function of splenic immunocytes should play a role. This study concludes that an underlying cause for the immunological adjuvanticity of PTx is down-regulation of Treg cell number and function.

Regulation of ITAM-positive receptors: role of IL-12 and IL-18.

Our previous studies have identified mechanisms by which cytokine production, blocked by Ly49G2 receptor cross-linking, can be overridden. In this study we analyzed the regulation of other ITAM-positive receptor signaling on NK, NKT, and T cells and characterized the biochemical pathways involved in this signaling. Our studies demonstrate that cross-linking of NKG2D and NK1.1 results in a synergistic NK IFN-gamma response when combined with IL-12 or IL-18. Examination of NKT- and T-cell responses demonstrated that cross-linking of NKG2D and CD3 resulted in potent synergy when combined with IL-12 and, to a lesser degree, with IL-18. We have now found that both the p38 MAP kinase and the ERK-dependent signal transduction pathways are required for the synergistic response. Further mechanistic examination of the synergy indicated a potent up-regulation of total IFN-gamma mRNA in the nuclear and the cytoplasmic compartment, but mRNA half-life was not affected. Fifteen minutes of IL-12 pretreatment was sufficient to result in maximal synergistic activation, indicating that the response of the cells to the IL-12 signal was rapid and immediate. Thus, our data demonstrate that multiple convergent signals maximize the innate immune response by triggering complementary biochemical signaling pathways.

In vivo hydrodynamic delivery of cDNA encoding IL-2: rapid, sustained redistribution, activation of mouse NK cells, and therapeutic potential in the absence of NKT cells.

In the present study, we have tested the ability of hydrodynamically delivered IL-2 cDNA to modulate the number and function of murine leukocyte subsets in different organs and in mice of different genetic backgrounds, and we have evaluated effects of this mode of gene delivery on established murine tumor metastases. Hydrodynamic administration of the IL-2 gene resulted in the rapid and transient production of up to 160 ng/ml IL-2 in the serum. The appearance of IL-2 was followed by transient production of IFN-gamma and a dramatic and sustained increase in NK cell numbers and NK-mediated cytolytic activity in liver and spleen leukocytes. In addition, significant increases in other lymphocyte subpopulations (e.g., NKT, T, and B cells) that are known to be responsive to IL-2 were observed following IL-2 cDNA plasmid delivery. Finally, hydrodynamic delivery of only 4 mug of the IL-2 plasmid to mice bearing established lung and liver metastases was as effective in inhibiting progression of metastases as was the administration of large amounts (100,000 IU/twice daily) of IL-2 protein. Studies performed in mice bearing metastatic renal cell tumors demonstrated that the IL-2 cDNA plasmid was an effective treatment against liver metastasis and moderately effective against lung metastasis. Collectively, these results demonstrate that hydrodynamic delivery of relatively small amounts of IL-2 cDNA provides a simple and inexpensive method to increase the numbers of NK and NKT cells, to induce the biological effects of IL-2 in vivo for use in combination with other biological agents, and for studies of its antitumor activity.

Rapid conversion of effector mechanisms from NK to T cells during virus-induced lysis of allogeneic implants in vivo.

Viral infections can strongly stimulate both NK cell and allospecific CD8 T cell responses, and these same effector cells can lyse allogeneic cell lines in vitro. However, the impact of viral infections on the effector systems mediating rejection of allogeneic tissues in vivo has not been fully explored. Using in vivo cytotoxicity assays, we evaluated the effector systems mediating the rejection of CFSE-labeled allogeneic splenocytes after an infection of C57BL/6 (B6) mice with lymphocytic choriomeningitis virus. Naive B6 mice predominantly used a NK cell-effector mechanism to reject allogeneic splenocytes because they rejected BALB/C (H2(d)) splenocytes but not CBA (H2(k)) splenocytes, and the rejection was prevented by immunodepletion of NK1.1(+) or Ly49D(+) NK cells. This rapid and efficient in vivo cytotoxicity assay recapitulated the specificity of NK cell-mediated rejection seen in longer duration in vivo assays. However, as early as 1 day after infection with lymphocytic choriomeningitis virus, a CD8 T cell-dependent mechanism participated in the rejection process and a broader range of tissue haplotypes (e.g., H2(k)) was susceptible. The CD8 T cell-mediated in vivo rejection process was vigorous at a time postinfection (day 3) when NK cell effector functions are peaking, indicating that the effector systems used in vivo differed from those observed with in vitro assays measuring the killing of allogeneic cells. This rapid generation of allospecific CTL activity during a viral infection preceded the peak of viral epitope-specific T cell responses, as detected by in vivo or in vitro cytotoxicity assays.

Mouse Ly49 NK receptors: balancing activation and inhibition.

Previous studies from numerous laboratories have demonstrated that inhibitory class I binding NK receptors dominate functional interactions in vitro. Our previous studies have shown that in addition to lysis, a major consequence of triggering the murine activating NK receptor Ly49D is the expression of cytokines and chemokines. We have recently shown that the activating Ly49D murine NK cell receptor can potently synergize during co-stimulation with IL-12 and IL-18 for selective production of IFN-gamma. Activation both in vitro and in vivo and synergistic production of IFN-gamma by Ly49D expressing NK cells results from cytokine stimulation combined with co-receptor ligation. In addition, IL-12 is capable of overriding the inhibitory receptor blockade for cytokine production, both in vitro and in vivo. Our current studies will expand this finding of IL-12 synergy to other receptors in the NK repertoire and evaluate potential biochemical mechanisms involved in this synergy. These findings place NK cells and their activating Ly49 receptors as important initiators of microbial, antiviral and anti-tumor immunity and provide a mechanism for the release of activating Ly49 receptors from an inhibitory receptor blockade. Discussion of how activation of the innate immune system provides important initiators of adaptive immune responses by receptor cross-linking and cytokine co-receptor engagement will ensue.

Peroxisome proliferator-activated receptor-gamma and its ligands attenuate biologic functions of human natural killer cells.

Interferon-gamma (IFN-gamma) production and cytolytic activity are 2 major biologic functions of natural killer (NK) cells that are important for innate immunity. We demonstrate here that these functions are compromised in human NK cells treated with peroxisome proliferator-activated-gamma (PPAR-gamma) ligands via both PPAR-gamma-dependent and -independent pathways due to variation in PPAR-gamma expression. In PPAR-gamma-null NK cells, 15-deoxy-Delta(12,14) prostaglandin J(2) (15d-PGJ(2)), a natural PPAR-gamma ligand, reduces IFN-gamma production that can be reversed by MG132 and/or chloroquine, and it inhibits cytolytic activity of NK cells through reduction of both conjugate formation and CD69 expression. In PPARgamma-positive NK cells, PPAR-gamma activation by 15d-PGJ(2) and ciglitazone (a synthetic ligand) leads to reduction in both mRNA and protein levels of IFN-gamma. Overexpression of PPAR-gamma in PPAR-gamma-null NK cells reduces IFN-gamma gene expression. However, PPAR-gamma expression and activation has no effect on NK cell cytolytic activity. In addition, 15d-PGJ(2) but not ciglitazone reduces expression of CD69 in human NK cells, whereas CD44 expression is not affected. These results reveal novel pathways regulating NK cell biologic functions and provide a basis for the design of therapeutic agents that can regulate the function of NK cells within the innate immune response.