Fractalkine-induced MFG-E8 leads to enhanced apoptotic cell clearance by macrophages

Clearance of apoptotic cells is crucial to maintain cellular function under normal and pathological conditions. We have recently shown that administration of immature dendritic cell-derived exosomes to septic animals promotes phagocytosis of apoptotic cells and improves survival by providing milk fat globule epidermal growth factor-factor VIII (MFG-E8). MFG-E8 acts as an opsonin for apoptotic cells to be engulfed by phagocytosis. In the present study we investigated whether the CX(3)C-chemokine fractalkine (CX(3)CL1) promotes apoptotic cell clearance through the induction of MFG-E8 in peritoneal macrophages. Cultured rat peritoneal macrophages (pMphi) and RAW264.7 macrophages were stimulated with LPS and CX(3)CL1. MFG-E8 expression was assessed by Western blot, cytokine secretion was assessed by ELISA, and phagocytosis of apoptotic thymocytes was determined by microscopy. For in vivo studies, cecal ligation and puncture (CLP) was used to induce sepsis in rats and mice. LPS significantly decreased MFG-E8 levels and phagocytosis of apoptotic cells, whereas CX(3)CL1 induced MFG-E8 expression in both nonstimulated and LPS-stimulated pMphi, without affecting TNF-alpha and IL-6 release. Anti-MFG-E8 blocking antibodies completely abrogated the prophagocytic effect of CX(3)CL1. Twenty hours after the induction of sepsis in rats via CLP, plasma CX(3)CL1 levels as well as MFG-E8 production in peritoneal macrophages decreased by 21% and 56%, respectively. Administration of CX(3)CL1 on the other hand induced MFG-E8 and prevented tissue injury. We conclude that CX(3)CL1 induces MFG-E8 in vitro and in vivo and enhances clearance of apoptotic cells in an MFG-E8-dependent manner. These findings suggest a possible novel treatment for patients in sepsis.     

Sphingolipid-mediated inhibition of apoptotic cell clearance by alveolar macrophages

A decreased clearance of apoptotic cells (efferocytosis) by alveolar macrophages (AM) may contribute to inflammation in emphysema. The up-regulation of ceramides in response to cigarette smoking (CS) has been linked to AM accumulation and increased detection of apoptotic alveolar epithelial and endothelial cells in lung parenchyma. We hypothesized that ceramides inhibit the AM phagocytosis of apoptotic cells. Release of endogenous ceramides via sphingomyelinase or exogenous ceramide treatments dose-dependently impaired apoptotic Jurkat cell phagocytosis by primary rat or human AM, irrespective of the molecular species of ceramide. Similarly, in vivo augmentation of lung ceramides via intratracheal instillation in rats significantly decreased the engulfment of instilled target apoptotic thymocytes by resident AM. The mechanism of ceramide-induced efferocytosis impairment was dependent on generation of sphingosine via ceramidase. Sphingosine treatment recapitulated the effects of ceramide, dose-dependently inhibiting apoptotic cell clearance. The effect of ceramide on efferocytosis was associated with decreased membrane ruffle formation and attenuated Rac1 plasma membrane recruitment. Constitutively active Rac1 overexpression rescued AM efferocytosis against the effects of ceramide. CS exposure significantly increased AM ceramides and recapitulated the effect of ceramides on Rac1 membrane recruitment in a sphingosine-dependent manner. Importantly, CS profoundly inhibited AM efferocytosis via ceramide-dependent sphingosine production. These results suggest that excessive lung ceramides may amplify lung injury in emphysema by causing both apoptosis of structural cells and inhibition of their clearance by AM.     

Functional complement C1q abnormality leads to impaired immune complexes and apoptotic cell clearance

C1q plays a key role in apoptotic cell and immune complex removal. Its absence contributes to the loss of tolerance toward self structures and development of autoimmunity. C1q deficiencies are extremely rare and are associated with complete lack of C1q or with secretion of surrogate C1q fragments. To our knowledge, we report the first case of a functional C1q abnormality, associated with the presence of a normal C1q molecule. Homozygous GlyB63Ser mutation was found in a patient suffering from lupus with neurologic manifestations and multiple infections. The GlyB63Ser C1q bound to Igs, pentraxins, LPSs, and apoptotic cells, similarly to C1q from healthy donors. However, the interaction of C1r(2)C1s(2) and C1 complex formation was abolished, preventing further complement activation and opsonization by C3. The mutation is located between LysB(61) and LysB(65) of C1q, suggested to form the C1r binding site. Our data infer that the binding of C1q to apoptotic cells in humans is insufficient to assure self-tolerance. The opsonization capacity of C4 and C3 fragments has to be intact to fight infections and to prevent autoimmunity.     

IgM promotes the clearance of small particles and apoptotic microparticles by macrophages

Background

Antibodies are often involved in enhancing particle clearance by macrophages. Although the mechanisms of antibody-dependent phagocytosis have been studied for IgG in greater detail, very little is known about IgM-mediated clearance. It has been generally considered that IgM does not support phagocytosis. Recent studies indicate that natural IgM is important to clear microbes and other bioparticles, and that shape is critical to particle uptake by macrophages; however, the relevance of IgM and particle size in their clearance remains unclear. Here we show that IgM has a size-dependent effect on clearance.

Methodology/Principal Findings

We used antibody-opsonized sheep red blood cells, different size beads and apoptotic cells to determine the effect of human and mouse IgM on phagocytosis by mouse alveolar macrophages. Our microscopy (light, epifluorescence, confocal) and flow cytometry data show that IgM greatly enhances the clearance of small particles (about 1–2 micron) by these macrophages. There is an inverse relationship between IgM-mediated clearance by macrophages and the particle size; however, macrophages bind and internalize many different size particles coated with IgG. We also show that IgM avidly binds to small size late apoptotic cells or bodies (2–5 micron) and apoptotic microparticles (<2 µm) released from dying cells. IgM also promotes the binding and uptake of microparticle-coated beads.

Conclusions/Significance

Therefore, while the shape of the particles is important for non-opsonized particle uptake, the particle size matters for antibody-mediated clearance by macrophages. IgM particularly promotes the clearance of small size particles. This finding may have wider implications in IgM-mediated clearing of antigens, microbial pathogens and dying cells by the host.     

Follicular dendritic cell secreted protein (FDC-SP) regulates germinal center and antibody responses

We previously identified follicular dendritic cell secreted protein (FDC-SP), a small secreted protein of unknown function expressed in human tonsillar germinal centers (GC). To assess potential in vivo activities of FDC-SP, transgenic mice were generated to constitutively express FDC-SP in lymphoid tissues. FDC-SP transgenic mice show relatively normal development of immune cell populations, with the exception of a small increase in mature follicular B cells, and normal lymphoid tissue architecture. Upon immunization with a T-dependent Ag, FDC-SP transgenic mice were capable of producing an Ag-specific Ab; however, the titers of Ag-specific IgG2a and IgE were significantly reduced. GC responses after immunization were markedly diminished, with transgenic mice showing decreased numbers and sizes of GCs but normal development of follicular dendritic cell networks and normal positioning of GCs. FDC-SP transgenic mice also showed reduced production of Ag-specific IgG3 Ab after immunization with a type II T-independent Ag, suggesting that the FDC-SP can also regulate the induction of B cell responses outside the GC. Purified FDC-SP transgenic B cells function normally in vitro, with the exception of blunted chemotaxis responses to CXCL12 and CXCL13. FDC-SP can induce the chemotaxis of CD40-stimulated nontransgenic B cells and can significantly enhance B cell migration in combination with chemokines, indicating that FDC-SP may function in part by regulating B cell chemotaxis. These results provide the first evidence for immunomodulatory activities of FDC-SP and implicate this molecule as a regulator of B cell responses.     

Enhanced apoptotic cell clearance capacity and B cell survival factor production by IL-10-activated macrophages: implications for Burkitt's lymphoma

Burkitt's lymphoma (BL) is typified by frequent tumor cell apoptosis and significant macrophage infiltration. Since BL cells have an inherent tendency to undergo apoptosis at a high rate, we reasoned that macrophages in BL are functionally enhanced in at least two activities that have implications for tumor pathogenesis: 1) engulfment of apoptotic cells, an anti-inflammatory process known to suppress immune responses, and 2) production of BL cell survival factors that limit the extent of tumor cell apoptosis. In this study, we show that the microenvironment of BL is rich in the pleiotropic cytokine IL-10, which can be produced by both tumor cells and macrophages, and that IL-10-activated human macrophages have enhanced capacity to engulf apoptotic cells in vitro. This was found to be dependent on the macrophage tethering receptor of apoptotic cells, CD14. Furthermore, IL-10-activated macrophages were found to produce markedly higher levels of the B cell survival factor, B cell-activating factor of the TNF family/B lymphocyte stimulator (BAFF/BLyS) than macrophages matured in the absence of IL-10. Coculture of macrophages with BL cells further enhanced BAFF secretion. Significantly, we show that enhancement of BL cell survival by IL-10-activated macrophages is mediated by a BAFF-dependent component and that BAFF is produced at high levels by tumor-associated macrophages in situ. These results indicate that macrophages, regulated by IL-10, have the potential to promote BL pathogenesis, first, through suppression of antitumor immunity following enhanced engulfment of apoptotic tumor cells and, second, through increased production of tumor cell growth/survival factors.     

Inhibition of adenosine deaminase leads to enhanced antibody responses in the mouse

Inhibition of adenosine deaminase activity leads to decreased cellular immunity. The effect of deoxycoformycin (DCF), a potent inhibitor of adenosine deaminase, on the ability of mouse spleen cells to generate antibody responses in vitro has been examined. With either continuous exposure to or pretreatment of the cells with deoxycoformycin, there was a decrease in cell survival and an increase in antibody-producing cells in the surviving cell population. To identify the cell population most susceptible to the inhibitor, the spleen was separated into B-cell, and T-cell, and macrophage components and each population was pretreated with deoxycoformycin before combination with its complementary treated or untreated population. Deoxycoformycin pretreatment had no effect on macrophages or B cells; however, pretreatment of the T cells resulted in increased antibody responses. When T cells and B cells were both pretreated and combined, there was a synergistic increase in the antibody response. In addition, supernatants from cultures in which both B cells and T cells had been pretreated with DCF were capable of enhancing antibody responses in cultures containing DCF-treated T cells. Though adenosine was increased in the stimulatory culture supernatants, adenosine alone did not enhance antibody responses in either untreated or DCF-treated cultures.     

Novel role of ICAM3 and LFA-1 in the clearance of apoptotic neutrophils by human macrophages

Apoptotic cells express eat-me signals which are recognized by several receptors mainly on professional phagocytes of the mononuclear phagocyte system. This “engulfment synapse” can define a safe and effective clearance of apoptotic cells in order to maintain tissue homeostasis in the entire body. We show that the expression of four genes related to apoptotic cell clearance is strongly up-regulated in human macrophages 30 min after administration of apoptotic neutrophils. Out of these the significant role of the up-regulated intercellular adhesion molecule 3 (ICAM3) in phagocytosis of apoptotic neutrophils could be demonstrated in macrophages by gene silencing as well as treatment with blocking antibodies. Blocking ICAM3 on the surface of apoptotic neutrophils also resulted in their decreased uptake which confirmed its role as an eat-me signal expressed by apoptotic cells. In macrophages but not in neutrophils silencing and blocking integrin alphaL and beta2 components of lymphocyte function-associated antigen 1 (LFA-1), which can strongly bind ICAM3, resulted in a decreased phagocytosis of apoptotic cells indicating its possible role to recognize ICAM3 on the surface of apoptotic neutrophils. Finally, we report that engulfing portals formed in macrophages during phagocytosis are characterized by accumulation of ICAM3, integrin alphaL and beta2 which show co-localization on the surface of phagocytes. Furthermore, their simultaneous knock-down in macrophages resulted in a marked deficiency in phagocytosis and a slight decrease in the anti-inflammatory effect of apoptotic neutrophils. We propose that ICAM3 and LFA-1 act as recognition receptors in the phagocytosis portals of macrophages for engulfment of apoptotic neutrophils.     

Impaired clearance and enhanced pulmonary inflammatory/fibrotic response to carbon nanotubes in myeloperoxidase-deficient mice

Advancement of biomedical applications of carbonaceous nanomaterials is hampered by their biopersistence and pro-inflammatory action in vivo. Here, we used myeloperoxidase knockout B6.129X1-MPO (MPO k/o) mice and showed that oxidation and clearance of single walled carbon nanotubes (SWCNT) from the lungs of these animals after pharyngeal aspiration was markedly less effective whereas the inflammatory response was more robust than in wild-type C57Bl/6 mice. Our results provide direct evidence for the participation of MPO - one of the key-orchestrators of inflammatory response - in the in vivo pulmonary oxidative biodegradation of SWCNT and suggest new ways to control the biopersistence of nanomaterials through genetic or pharmacological manipulations.     

CD14-dependent clearance of apoptotic cells by human macrophages: the role of phosphatidylserine

Apoptotic-cell clearance is dependent on several macrophage surface molecules, including CD14. Phosphatidylserine (PS) becomes externalised during apoptosis and participates in the clearance process through its ability to bind to a novel receptor, PS-R. CD14 has the proven ability to bind phospholipids and may function as an alternative receptor for the externalised PS of apoptotic cells. Here we demonstrate that CD14 does not function preferentially as a PS receptor in apoptotic-cell clearance. Compared with phosphatidylcholine and phosphatidylethanolamine, PS was the least active phospholipid binding to human monocyte-derived macrophages and showed no specificity for soluble or membrane-anchored CD14. Significantly, PS-containing liposomes failed to inhibit CD14-dependent uptake of apoptotic cells by macrophages. PS exposure was, however, found to be insufficient for either CD14-dependent or CD14-independent apoptotic-cell uptake by phagocytes. The additional features that enable apoptotic-cell clearance are derived from mechanisms that can be divorced temporally from those responsible for the morphological features of apoptosis.     

Increased radiation toxicity by enhanced apoptotic clearance of HL-60 cells in the presence of the pentapeptide thymopentin, which selectively binds to apoptotic cells

Radiotoxic insult to cells is associated with genetic instability and heritable damage [Mutat. Res. 517 (2002) 173]. A strengthened response to such insult by enhanced apoptotic clearance, which would be associated with anti-inflammatory [Nature 390 (1997) 350; Nature 407 (2000) 784] and anti-necrotic intercellular signaling [Nature 418 (2002) 191], has been previously reported. The pentapeptide thymopentin (TP5) improves immunological parameters in cancer patients following radiotherapy without clinically observable side effects. We assessed the effects of TP5 on human promyeloid leukemia HL-60 cells exposed to therapeutic (2Gy) doses of X-rays. We observed an increased accumulation of cells in the G2/M phase of the cell cycle after irradiation when treated with TP5. However, TP5 had no effect on the cell cycle distribution of non-irradiated HL-60 cells. Additionally, TP5 treatment of irradiated cells increased the number of cells undergoing apoptosis. Furthermore, TP5 was found to selectively bind to apoptotic cells. These findings represent a promising and novel approach employing TP5-mediated modulation of cellular radiation response to augment both clinical gain in radiation oncology and safety measures for radiation protection.     

磷脂酰丝氨酸外翻和磷脂氧化在凋亡细胞被吞噬细胞清除过程中的协作效应

为探讨磷脂酰丝氨酸(phosphatidylserine,PS)外翻和磷脂氧化在凋亡细胞被吞噬细胞清除中的作用,用脂质体整合的方法将不同的磷脂整合到红细胞上或用N-乙酰马来酰胺(N-ethylmaleimide,NEM)预处理红细胞然后整合磷脂,制备含不同凋亡信号的红细胞模型,测定巨噬细胞对整合不同磷脂信号红细胞的结合率和吞噬率。结果表明,单独整合PS或用NEM处理造成PS外翻,可显著性提高巨噬细胞对红细胞的结合率,但对吞噬率没有影响;同时整合PS和氧化磷脂(氧化PS或氧化磷脂酰胆碱(phosphatidylcholine,PC)),或用NEM处理造成PS外翻后再整合氧化PS或氧化PC,不仅可显著提高巨噬细胞对红细胞的结合率,而且可显著性提高吞噬率。这些结果提示PS外翻可能参与了巨噬细胞对凋亡细胞的结合,而磷脂氧化可能启动了巨噬细胞对凋亡细胞的吞噬,二者协作才可能完成巨噬细胞对凋亡细胞的清除。     

Pulmonary infection with influenza A virus induces site-specific germinal center and T follicular helper cell responses

Protection from influenza A virus (IAV) challenge requires switched, high affinity Abs derived from long-lived memory B cells and plasma cells. These B cell subsets are generated in germinal centers (GCs), hallmark structures of T helper cell-driven B cell immunity. A full understanding of the GC reaction after respiratory IAV infection is lacking, as is the characterization of T follicular helper (T(FH)) cells that support GCs. Here, GC B cell and T(FH) cell responses were studied in mice following pulmonary challenge with IAV. Marked GC reactions were induced in draining lymph nodes (dLNs), lung, spleen and nasal-associated lymphoid tissue (NALT), although the magnitude and kinetics of the response was site-specific. Examination of switching within GCs demonstrated IgG2(+) cells to compose the largest fraction in dLNs, lung and spleen. IgA(+) GC B cells were infrequent in these sites, but composed a significant subset of the switched GC population in NALT. Further experiments demonstrated splenectomized mice to withstand a lethal recall challenge, suggesting the spleen to be unnecessary for long-term protection in spite of strong GC responses in this organ. Final studies showed that T(FH) cell numbers were highest in dLNs and spleen, and peaked in all sites prior to the height of the GC reaction. T(FH) cells purified from dLNs generated IL-21 and IFNγ upon activation, although CD4(+)CXCR5(-) T effector cells produced higher levels of all cytokines. Collectively, these findings reveal respiratory IAV infection to induce strong T helper cell-driven B cell responses in various organs, with each site displaying unique attributes.     

Modification of low density lipoproteins by polymorphonuclear cell elastase leads to enhanced uptake by human monocyte-derived macrophages via the low density lipoprotein receptor pathway

In previous studies we reported that polymorphonuclear cell (PMN) elastase cleaves apoB-100 of human plasma low density lipoprotein (LDL) into seven or eight large Mr fragments (1, Polacek, D., R.E. Byrne, G.M. Fless, and A.M. Scanu. 1986. J. Biol. Chem. 261: 2057-2063). In the present studies we examined the interaction of native and elastase-digested LDL (ED-LDL) with primary cultures of human monocyte-derived macrophages (HMD-M). For this purpose LDL was digested with purified PMN elastase, re-isolated by ultracentrifugation at d 1.063 g/ml to remove the enzyme, and radiolabeled with 125I. At all LDL concentrations in the medium, the degradation of 125I-labeled ED-LDL was 1.5- to 2.5-fold greater than that of 125I-labeled native LDL, and for both lipoproteins species it was further enhanced by prior incubation of the cells in autologous lipoprotein-deficient serum (ALPDS). ED-LDL incubated with HMD-M in a medium containing [14C]oleate stimulated cholesteryl [14C]oleate formation 2- to 3-fold more than native LDL. In competitive degradation experiments, unlabeled ED-LDL did not inhibit the degradation of 125I-labeled acetylated LDL, whereas it caused a 90% inhibition of the degradation of 125I-labeled native LDL. At 4 degrees C, the binding of both 125I-labeled native and 125I-labeled ED-LDL was specific and of a high affinity. At saturation (Bmax), the binding of 125I-labeled ED-LDL was 2-fold higher (68 ng/mg cell protein) than that of 125I-labeled native LDL (31 ng/mg), with Kd values of 6.5 x 10(-8) M and 2.1 x 10(-8) M, respectively. A possible explanation of the binding data was provided by electrophoretic analyses suggesting that ED-LDL was twice the size of native LDL and thus potentially capable of delivering proportionately more cholesterol to the cells. Taken together, the results indicate that 1) digestion of LDL by purified PMN elastase results in a greater mass of ED-LDL (relative to native LDL) being degraded per unit time by HMD-M; 2) uptake of ED-LDL occurs via the LDL receptor; and 3) LDL digested by PMN elastase undergoes a physical change that may be responsible for its unique interactions with HMD-M. We speculate that if this process were to occur in vivo during an inflammatory process, macrophages could acquire excess cholesterol and be transformed into foam cells which are considered to be precursors of the atherosclerotic process.     

Mechanisms of failed apoptotic cell clearance by phagocyte subsets in cardiovascular disease

Recent evidence in humans indicate that defective phagocytic clearance of dying cells is linked to progression of advanced atherosclerotic lesions, the precursor to atherothrombosis, ischemic heart disease, and leading cause of death in the industrialized world. During atherogenesis, apoptotic cell turnover in the vascular wall is counterbalanced by neighboring phagocytes with high clearance efficiency, thereby limiting cellularity and maintaining lesion integrity. However, as lesions mature, phagocytic removal of apoptotic cells (efferocytosis) becomes defective, leading to secondary necrosis, expansion of plaque necrotic cores, and susceptibility to rupture. Recent genetic causation studies in experimental rodents have implicated key molecular regulators of efferocytosis in atherosclerotic progression. These include MER tyrosine kinase (MERTK), milk fat globule-EGF factor 8 (MFGE8), and complement C1q. At the cellular level, atheromata are infiltrated by a heterogenous population of professional phagocytes, comprised of monocytes, differentiated macrophages, and CD11c⁺ dendritic-like cells. Each cell type is characterized by disparate clearance efficiencies and varying activities of key phagocytic signaling molecules. It is in this context that we outline a working model whereby plaque necrosis and destabilization is jointly promoted by (1) direct inhibition of core phagocytic signaling pathways and (2) expansion of phagocyte subsets with poor clearance capacity. Towards identifying targets for promoting efficient apoptotic cell clearance and resolving inflammation in atherosclerosis and during ischemic heart disease and post myocardial infarction, this review will discuss potential in vivo suppressors of efferocytosis at each stage of clearance and how these putative interventional targets may differentially affect uptake at the level of vascular phagocyte subsets.     

Induction of apoptotic cell death leads to the development of bacterial rot caused by Pseudomonas cichorii

Pseudomonas cichorii is the major causal agent of bacterial rot of lettuce. Collapse and browning symptoms were observed in lettuce leaf tissue from 15 to 24 h after inoculation (HAI) with P. cichorii; superoxide anion generation was detected at 1 to 6 HAI; and cell death was induced at 6 HAI, reaching a maximum at approximately 9 and 12 HAI. Heterochromatin condensation and DNA laddering also were observed within 3 HAI. Pharmacological studies showed that induction of cell death and DNA laddering was closely associated with de novo protein synthesis, protein kinase, intracellular reactive oxygen species, DNase, serine protease, and caspase III-like protease. Moreover, chemicals, which inhibited the induction of cell death and DNA laddering, also suppressed the development of disease symptoms. These results suggest that apoptotic cell death might be closely associated with the development of bacterial rot caused by P. cichorii.     

SOCS3 deletion in B cells alters cytokine responses and germinal center output

B cell behavior is fine-tuned by internal regulatory mechanisms and external cues such as cytokines and chemokines. Suppressor of cytokine signaling 3 (SOCS3) is a key regulator of STAT3-dependent cytokine responses in many cell types and has been reported to inhibit CXCL12-induced retention of immature B cells in the bone marrow. Using mice with SOCS3 exclusively deleted in the B cell lineage (Socs3(Δ/Δ)mb1cre(+)), we analyzed the role of SOCS3 in the response of these cells to CXCL12 and the STAT3-inducing cytokines IL-6 and IL-21. Our findings refute a B cell-intrinsic role for SOCS3 in B cell development, because SOCS3 deletion in the B lineage did not affect B cell populations in naive mice. SOCS3 was strongly induced in B cells stimulated with IL-21 and in plasma cells exposed to IL-6. Its deletion permitted excessive and prolonged STAT3 signaling following IL-6 stimulation of plasma cells and, in a T cell-dependent immunization model, reduced the number of germinal center B cells formed and altered the production of Ag-specific IgM and IgE. These data demonstrate a novel regulatory signal transduction circuit in plasma cells, providing, to our knowledge, the first evidence of how these long-lived, sessile cells respond to the external signals that mediate their longevity.     

Cutting edge: CD8+ T cell priming in the absence of NK cells leads to enhanced memory responses

It is uncertain whether NK cells modulate T cell memory differentiation. By using a genetic model that allows the selective depletion of NK cells, we show in this study that NK cells shape CD8(+) T cell fate by killing recently activated CD8(+) T cells in an NKG2D- and perforin-dependent manner. In the absence of NK cells, the differentiation of CD8(+) T cells is strongly biased toward a central memory T cell phenotype. Although, on a per-cell basis, memory CD8(+) T cells generated in the presence or the absence of NK cells have similar functional features and recall capabilities, NK cell deletion resulted in a significantly higher number of memory Ag-specific CD8(+) T cells, leading to more effective control of tumors carrying model Ags. The enhanced memory responses induced by the transient deletion of NK cells may provide a rational basis for the design of new vaccination strategies.