The cancer process as a type of immunocomplex hypersensibility involving C3b,natural killer cytotoxicity and antibody-dependent cell cytotoxicity: proposals for tumour immunotherapy and vaccine

I have previously assumed that stem tumour cells are ‘para-embryonal cells’ (PECs) poor or missing in major histocompatibility complex (MHC) antigens. PECs might induce adjoining differentiated hyperplastic cells to also express tumoral phenotype and properties, thus transforming them into ‘differentiated para-embryonal cells’ (DPECs), MHC endowed. In such a way, PECs, MHC-lacking, would be automatically surrounded by DPECs, MHC-endowed: this tumour organization was experimentally found by Cordon-Cardo et al in a variety of cancers. Now, I suggest that such a tumour histology might preferentially induce an anti-DPEC T cell immune response which, sparing PECs, might release increasing amounts of DPEC antigens in the peritumour site. DPEC antigens might increase synthesis of specific antibodies and subsequent immunocomplex formation at the peritumour site. Here, abundant immunocomplexes might react through their Fc pieces with CD16 receptors of antibody-dependent cell cytotoxicity (ADCC)-endowed immune cells (natural killer (NK) cells, macrophages, polymorphonuclear cells). These cells would thus be stimulated to secrete their lytic factors before and without their coming into contact with target tumour cells. On the other hand, abundant immunocomplexes at the peritumour site might massively activate the complement system, thus generating large amounts of C3b. C3b might react with CD11b receptors of NK cells, stimulating them to also secrete their lytic factors in an ectopic way at the peritumour site, thus impairing NK cytotoxicity. In such a way, in the absence of ADCC and NK cytotoxicity, a tumour cell enhancement might easily occur. In the light of these ideas, a strategy for antitumour immunotherapy and vaccine is then proposed.     

Post-thymectomy Murine Experimental Autoimmune Oophoritis Is Associated With Reduced Natural Killer Cell Activity

PROBLEM: Natural killer (NK) cells can influence the immune response by secreting potent lymphokines. It has been suggested that NK cells have a suppressive action on B cells, and that impaired NK cell activity may play a role in some types of autoimmunity. NK cell abnormalities have been reported in women with premature ovarian failure. We therefore examined NK cell activity during the development of murine experimental autoimmune oophoritis, which serves as a model for autoimmune ovarian failure in women. METHOD OF STUDY: Neonatally thymectomized and sham-operated C57B1/6 × A/J (B6A) mice were prepared and sacrificed at 4, 6, 8, and 10 weeks after surgery. Splenic NK cell activity was determined in groups of five or more mice by measuring the percent specific lysis of target YAC-1 lymphoma cells using a standard 4-hr chromium release cytotoxicity assay. The number of splenic NK cells in neonatally thymectomized and sham-operated animals was also compared using flow cytometry. In a subsequent experiment, interleukin 12 (IL-12; NK cell-stimulating factor) was administered to neonatal mice before neonatal thymectomy. RESULTS: Neonatally thymectomized mice with associated autoimmune oophoritis had a 75% reduction in the number of splenic NK cells, and 50% or greater reduction in splenic NK cell activity at 4, 6, and 8 weeks after surgery. IL-12 treatment before neonatal thymectomy maintained NK cell activity and was shown to ameliorate the associated autoimmune oophoritis. CONCLUSION: Murine post-thymectomy autoimmune oophoritis is associated with reduced NK cell number and impaired NK cell activity, and in these respects the model is similar to premature ovarian failure in women. Research to define the relationship between NK cell abnormalities and the mechanism of ovarian failure in this model might lend insight into the pathogenesis of premature ovarian failure in women.     

Recycling and reutilization of cytotoxic molecules,a new type of energy conservation of NK cells?

Natural killer (NK) cells play critical roles in defense against tumors and viral infections. They exert their cytotoxic functions through the secretion of granules containing cytotoxic molecules, such as perforin and granzymes. These cytotoxic molecules are stored within dual-functional organelles, known as secretory lysosomes. Target cell recognition induces the formation of an "immunological synapse", between the NK cell and its target, into which cytotoxic granules release their contents. However the post-exocytosis regulation of the process is still largely unknown. Recent research and the data accumulated therefrom lead to new hypotheses that suggest that, not unlike synaptic vesicle recycling in neuronal terminals, NK cells also recycle not just their secretory lysosome membranes but their correlated cytotoxic molecules (perforin and granzymes). The newly endocytosed vesicles are used to replenish the "reserve pool" of vesicles for continued NK cell serial killings. These hypotheses, if proved to be correct, will significantly improve our understanding of NK cell cytotoxicity mechanisms and might even suggest new NK cell-based therapies that rely on NK serial killing abilities for overcoming tumors.     

Roles and mechanism of natural killer cells in clinical and experimental transplantation

Natural killer (NK) cells are an important component of the innate immune response against intracellular pathogens and also play a role in the tissue inflammation associated with autoimmune diseases. Being a potent effector response, it must be well regulated in order to avoid unwanted destruction of normal tissues. Indeed, NK cells are unique in bearing both stimulatory and inhibitory receptor tolerance. The fine balance between activation and inhibition that decides their final action provides an opportunity for their possible modulation in specific therapeutic settings. The recent evidence implicating NK cells in promoting tolerance during allotransplantation is one such setting that has important implications for a successful transplant. This review provides an insight into NK cell biology and their involvement in allotransplant tolerance.     

The development of a bi-specific anti-CD161A x anti-tumor antibody for rat NK cell targeting

In order to improve the therapeutic efficacy of adoptive immunotherapy of cancer using IL-2-activated NK (A-NK) cells, we developed a bi-specific monoclonal antibody (BimAb) 3.2.3xCC52. One specificity of the BimAb (mAb 3.2.3) was directed against rat CD161A (NKR-P1A) which has been shown to be an activation structure on rat NK cells involved in lysis of target cells and cytokine secretion. The other specificity (mAb CC52) was directed against a tumor associated antigen on the rat colon adenocarcinoma cell line CC531. The hybridomas producing 3.2.3 and CC52 were fused, resulting in a quadroma producing the desired 3.2.3xCC52 BimAb. The hybridomas produced antibodies of different isotypes (IgG2b and IgG1 respectively) which enabled us to pre-select quadromas with a high likelihood for production of BimAb, through testing for the production of bi-isotypic antibodies. Production of functional BimAb by the selected quadromas was demonstrated in an assay showing enhanced conjugate formation between CD161A+ cells and CC531 tumor cells. We also tested the 3.2.3xCC52 BimAb for its capacity to enhance NK cell-mediated lysis of CC531 tumor cells in 4 h and 19 h 51Cr release assays; in a prolonged (2 day) tumor neutralization assay using a tetrazolium salt (MTT)-based assay; and in tests for apoptosis using Annexin V-FITC. Although this BimAb was not demonstrated to cause enhanced lysis of CC531 cells by CD161A+ effector cells in vitro, it might be a useful tool to enhance the number of NK cells at the tumor site and/or prolong contact between tumor cells and NK cells in vivo, thereby probably enhancing the therapeutic efficacy of NK cells.     

Acute myeloid leukemia impairs natural killer cells through the formation of a deficient cytotoxic immunological synapse

Acute myeloid leukemia (AML) cells are killed by allogeneic NK cells. However, autologous NK cells from AML patients express decreased levels of activating receptors, and show reduced cytotoxicity. Here, we investigated how interactions between NK and AML cells might cause loss of NK‐cell activity in patients. Our results show that AML cell lines and primary blasts alter the NK‐cell phenotype, reducing their cytotoxic potential upon prolonged contact. Downregulation of NK‐cell‐activating receptors was contact‐dependent and correlated with conjugate formation. Time‐lapse imaging of HL60 AML cell line and NK‐cell interactions showed a high proportion of noncytolytic contacts. Studies of NK‐cell immunological synapses revealed a defect in lytic synapse formation. Namely, despite correct F‐actin and LFA‐1 recruitment, polarization of lytic granules toward primary blasts or AML cell lines was reduced. The NK–AML cell line synapses showed impairment of CD3ζ recruitment. Attempts to correct these synapse defects by cytokine stimulation of NK cells improved conjugate formation, but not granule polarization. Pretreatment of AML cell lines with the immunomodulating molecule lenalidomide significantly enhanced granule polarization. We speculate that combining immunomodulatory drugs and cytokines could increase AML cell sensitivity to autologous NK cells and reinforce the activity of allogeneic NK cells in adoptive immunotherapy.     

HLA and KIR polymorphisms affect NK-cell anti-tumor activity

Killer cell immunoglobulin-like receptors (KIRs) and their cognate human leukocyte antigen (HLA) ligands are key to the maintenance of natural killer (NK) cell tolerance. The gene complexes encoding both KIRs and HLA ligands are extremely polymorphic. Because the extent of NK cell inhibition varies with the allelic forms expressed, NK cell tolerance can be broken more easily in some individuals than in others. This explains why particular combinations of KIR and HLA genes are associated with an increased risk of autoimmune diseases or with more efficient antiviral responses. Breaking of NK cell tolerance might be prerequisite to kill leukemic blasts. At present, there are ample indications that NK cells can eradicate acute myeloid leukemia blasts in patients with a favorable combination of HLA and KIR genes. Selecting these individuals for clinical trials should give insight into the feasibility of anti-tumor therapy mediated through NK cells.     

TIGIT expression levels on human NK cells correlate with functional heterogeneity among healthy individuals

Human NK cells display extensive phenotypic and functional heterogeneity among healthy individuals, but the mechanism responsible for this variation is still largely unknown. Here, we show that a novel immune receptor, T‐cell immunoglobulin and ITIM domain (TIGIT), is expressed preferentially on human NK cells but shows wide variation in its expression levels among healthy individuals. We found that the TIGIT expression level is related to the phenotypic and functional heterogeneity of NK cells, and that NK cells from healthy individuals can be divided into three categories according to TIGIT expression. NK cells with low levels of TIGIT expression show higher cytokine secretion capability, degranulation activity, and cytotoxic potential than NK cells with high levels of TIGIT expression. Blockade of the TIGIT pathway significantly increased NK‐cell function, particularly in NK cells with high levels of TIGIT expression. We further observed that the TIGIT expression level was inversely correlated with the IFN‐γ secretion capability of NK cells in patients with cancers and autoimmune diseases. Importantly, we propose a novel mechanism that links TIGIT expression with NK‐cell functional heterogeneity, and this mechanism might partially explain why individuals have different susceptibilities to infection, autoimmune disease, and cancer.     

NK cell receptor imbalance and NK cell dysfunction in HBV infection and hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is currently the third leading cause of cancer mortality and a common poor-prognosis malignancy due to postoperative recurrence and metastasis. There is a significant correlation between chronic hepatitis B virus (HBV) infection and hepatocarcinogenesis. As the first line of host defense against viral infections and tumors, natural killer (NK) cells express a large number of immune recognition receptors (NK receptors (NKRs)) to recognize ligands on hepatocytes, liver sinusoidal endothelial cells, stellate cells and Kupffer cells, which maintain the balance between immune response and immune tolerance of NK cells. Unfortunately, the percentage and absolute number of liver NK cells decrease significantly during the development and progression of HCC. The abnormal expression of NK cell receptors and dysfunction of liver NK cells contribute to the progression of chronic HBV infection and HCC and are significantly associated with poor prognosis for liver cancer. In this review, we focus on the role of NK cell receptors in anti-tumor immune responses in HCC, particularly HBV-related HCC. We discuss specifically how tumor cells evade attack from NK cells and how emerging understanding of NKRs may aid the development of novel treatments for HCC. Novel mono- and combination therapeutic strategies that target the NK cell receptor–ligand system may potentially lead to successful and effective immunotherapy in HCC.     

Altered phenotype of HLA-G expressing trophoblast and decidual natural killer cells in pathological pregnancies

BACKGROUND: The interaction between decidual natural killer (NK) cells and alloantigens expressed on fetal trophoblast cells are thought to be essential for successful implantation and placentation. Consequently, a disturbed interaction during the first trimester of pregnancy might well lead to a subsequent pregnancy failure. METHODS: We investigated the expression of HLA-G and NK cell markers in tissue sections from recurrent miscarriage (n = 9) and ectopic tubal pregnancies (n = 5), and two hysterectomy specimens of healthy pregnancy as well as decidual biopsies (n = 9) were used as controls. RESULTS: We show in normal pregnancy not only a decrease, but also a morphological change in CD56+ NK cells upon interaction with HLA-G-expressing trophoblasts. The cells appear to be transitioning from a blast-like (activation) state into a state of apoptosis. The number of CD16+ NK cells was low. In contrast, in recurrent miscarriage tissue a sustained NK cell marker expression of both CD56 and CD16 was paralleled by a decreased expression of HLA-G. No morphological changes from the blast-like stage were apparent. Finally, in ectopic pregnancies HLA-G expression in the absence of decidual NK cells was associated with a disturbed trophoblast differentiation. CONCLUSIONS: In pathological pregnancies we show an in-situ altered phenotype of trophoblast and NK cells.     

Education of hyporesponsive NK cells by cytokines

NK‐cell tolerance to self is mediated via engagement of inhibitory receptors by cognate MHC molecules. This event is critical for NK‐cell education to achieve functional competence. Thus, NK cells expressing self‐MHC‐specific inhibitory receptors are responsive to activating stimuli while those lacking such receptors are hyporesponsive. Nevertheless, the mechanisms underlying NK‐cell education are still poorly understood. Here, we show that after stimulation with cytokines, hyporesponsive NK cells acquire stable expression of killer Ig‐like receptors (KIR) as reflected by DNA hypomethylation of their KIR locus. Remarkably, only hyporesponsive NK cells that acquire KIR in the presence of their cognate MHC molecule gain functional competence and this process can occur in the absence of any accessory cells. Acquisition of competence does not result in autoreactivity, since acquired KIR are functional and therefore able to inhibit NK‐cell cytotoxicity. Our data demonstrate that competent NK cells can be generated by cytokine stimulation, suggesting that NK‐cell education might not only be an early event which takes place during NK‐cell development but might also occur in the periphery during an immune response.     

Malassezia enhances natural killer cell-induced dendritic cell maturation

Human natural killer (NK) cells can induce cell death in autologous dendritic cells (DCs), though an interaction between these two cell types can also lead to a reciprocal activation. We have recently shown cell contact between NK cells and DCs in vivo, in Malassezia-induced lesional skin of patients with atopic eczema, where the yeast acts as an allergen although it is part of the normal skin microflora. Here we characterize the interaction of human NK cells and monocyte-derived DCs (MDDCs) by using an in vitro system where short-term activated polyclonal NK cells are cocultured with autologous, immature, Malassezia-stimulated or lipopolysaccharide-matured MDDCs. We found that the number of CD83(+) MDDCs increased in the immature and Malassezia-stimulated MDDCs upon coculture with NK cells, while an increased number of CD86(+) cells was detected in the Malassezia-stimulated MDDCs. NK cell-MDDC interaction induced the production of interleukin-8 (IL-8). In conclusion, our results imply that NK cells provide maturation signals and may play a role in inducing IL-8 production in DCs. Furthermore, the increased expression of CD86 on Malassezia-stimulated MDDCs might have a function in subsequent T-cell activation by DCs, and indicate a role for NK cell-DC interaction in modulating the immune responses to microbial stimuli.     

Of mice and men: different functions of the murine and human 2B4 (CD244) receptor on NK cells

2B4 was initially discovered on murine NK cells and T cells displaying non-MHC dependent cytotoxicity. Human 2B4 was cloned based on sequence homology with mouse 2B4. Recent evidence suggests that the function of this receptor might be different in the two species. Human 2B4 activates NK cell cytotoxicity and interferon gamma production when engaged by CD48, its ligand, on target cells. This activating function of human 2B4 requires recruitment of the SH2 domain containing molecule, SLAM-associated protein or SAP. In the absence of SAP in human NK cells, as occurs in immature NK cells or NK cells from X-linked lymphoproliferative disorder (XLPD) patients, human 2B4 acts as an inhibitory receptor. In contrast, in vitro and in vivo studies using 2B4-deficient mice suggest that the major function of mouse 2B4 is to inhibit murine NK cell functions when triggered by CD48 on target cells, although there are reports of activating function of murine 2B4. This inhibitory function of murine 2B4 is mediated by EAT-2, ERT and possibly other phosphatases like SHP-1 and SHIP. 2B4-SAP interaction in mouse NK cells might be a low affinity one and might not be physiologically relevant considering the inhibitory function of 2B4. This suggests that mouse and human 2B4 diverged functionally with the evolution of greater affinity between 2B4 and SAP in the human species. We speculate that evolutionary pressure from viral infections, possibly EBV, might have led to the emergence of this association and activating function of 2B4 in humans.     

Cord blood stem cell-generated KIR+NK cells effectively target leukemia cell lines

Acute lymphoid (ALL) and myeloid leukemia (AML) are known to be invasive and highly lethal hematological malignancies. Because current treatments are insufficient and have a variety of side effects, researchers are looking for new and more effective therapeutic methods. Interestingly, ongoing efforts to find the best approach to optimize NK cell anti-leukemia potential shed light on the successful treatment of cancer. Mature KIR⁺NK cells ability to remove HLA Class-I deficient cells has been exploited in cancer immunotherapy. Here, we generated KIR⁺NK cells from cord blood stem cells using IL-2 and IL-15 cytokines. Our finding underlined the importance of KIR expression in the cytotoxic function of NK cells. Taken together, this study presented an effective in vitro method for the expansion and differentiation of KIR⁺NK cells using cytokines without any feeder cells. Furthermore, the presented culture condition could be useful for the generation of mature and pure NK cells from limited numbers of CD34⁺ cord blood cells and might be used as a novel method to improve the current state of cancer therapy.     

The biology of natural killer cells during sepsis

Natural killer (NK) cells are large granular lymphocytes largely recognized for their importance in tumour surveillance and the host response to viral infections. However, as the major innate lymphocyte population, NK cells also coordinate early responses to bacterial infections by amplifying the antimicrobial functions of myeloid cells, especially macrophages, by production of interferon‐γ (IFN‐γ). Alternatively, excessive NK cell activation and IFN‐γ production can amplify the systemic inflammatory response during sepsis resulting in increased physiological dysfunction and organ injury. Our understanding of NK cell biology during bacterial infections and sepsis is mostly derived from studies performed in mice. Human studies have demonstrated a correlation between altered NK cell functions and outcomes during sepsis. However, mechanistic understanding of NK cell function during human sepsis is limited. In this review, we will review the current understanding of NK cell biology during sepsis and discuss the challenges associated with modulating NK cell function during sepsis for therapeutic benefit.     

Bone marrow versus thymic pathways of natural killer cell development

Summary:  Understanding natural killer (NK) cell developmental pathways is crucial for harnessing the potential therapeutic benefits of this specialized lymphocyte subset. The bone marrow (BM) plays a major role in NK cell development, providing the appropriate environmental cues for NK cell commitment and subsequent NK cell differentiation. Nevertheless, the molecular signals provided in this context remain enigmatic. It is widely assumed that BM seeds the periphery with NK cells. However, the precise origins of NK cells found in lymphoid organs and tissues are not defined. Recently, we found that thymic NK cells bear molecular markers and functional attributes that distinguish them from most peripheral NK cells. We find that NK cells are actively exported from the thymus to the periphery, suggesting that thymus-derived NK cells may have unique roles both intrathymically and in secondary lymphoid organs. Here we compare the properties of thymic NK cells with properties of other NK cell subsets that have been identified in the mouse. We propose that heterogeneity in NK cell function can be achieved through distinct thymic and bone marrow pathways of NK cell development.     

Human NK cells directly recognize Mycobacterium bovis via TLR2 and acquire the ability to kill monocyte-derived DC

NK cells are important players of the early innate defense against various pathogens. In this study, we investigated the interaction between human NK cells and Mycobacterium bovis [bacille Calmette-Guérin (BCG)] and we determined whether and how such an interaction might impact on NK cell activation, cytokine production and cytotoxicity. We show that highly purified NK cells, upon short-term co-culture with BCG, expressed activation markers including CD69 and CD25. Moreover, these NK cells released IFN-gamma and tumor necrosis factor-alpha and killed more efficiently different targets including monocyte-derived immature dendritic cell. All these functions were strongly up-regulated in the presence of exogenous IL-12. Although more efficient responses were detected in NK cell populations displaying an NCR(bright) phenotype, no direct evidence of an involvement of triggering NK receptors in BCG recognition could be obtained. On the other hand, anti-toll-like receptor (TLR)2 mAb inhibited NK cell responses to BCG, suggesting that NK cells may express a functional TLR2, which plays a role in their mechanism of direct BCG recognition. Taken together, these data suggest that BCG, by inducing simultaneous activation of NK and antigen-presenting cells via their 'shared' TLR2, can promote efficient bidirectional NK-dendritic cell interactions necessary for subsequent priming of T(h)1 responses.     

Tracking in vivo dynamics of NK cells transferred in patients undergoing stem cell transplantation

Haploidentical stem cell transplantation (haploSCT) offers an alternative treatment option for advanced leukemia patients lacking a HLA‐compatible donor. Transfer of NK cells represents a promising therapeutic option in combination with SCT, as NK cells can promote graft versus leukemia with low risk of GVH disease. In this study, we show results from a phase I/II trial in which 24 acute myeloid leukemia patients underwent haploSCT in combination with early transfer of unmodified NK cells and observed a promising 2‐year overall survival rate of 37%. By performing immunomonitoring and subsequent principal component analysis, we tracked donor NK‐cell dynamics in the patients and distinguished between NK cells reconstituting from CD34⁺ precursors, giving rise over time to a continuum of multiple differentiation stages, and adoptively transferred NK cells. Transferred NK cells displayed a mature phenotype and proliferated in vivo during the early days after haploSCT even in the absence of exogenous IL‐2 administration. Moreover, we identified the NK‐cell phenotype associated with in vivo expansion. Thus, our study indicates a promising path for adoptive transfer of unmodified NK cells in the treatment of high‐risk acute myeloid leukemia.     

Emerging role for NK cells in the pathogenesis of inflammatory arthropathies

Natural killer (NK) cells are large, granular lymphocytes devoted to the defense against microbial agents and cancer cells, traditionally recognized as an important arm of the innate immunity, even if more recent data underpin a role also in the responses of acquired immunity. Several studies have led to ascertain that NK cells are involved in the pathogenesis of many immune-mediated diseases, where they may exert both protective and pathogenic roles. In particular, the CD56(bright) NK cell subset, showing immunoregulatory properties, has been found to accumulate in tissue sites of inflammation, such as the skin lesions in psoriatic patients and the synovial membrane in rheumatoid arthritis (RA) patients. In this latter disease, while data on the number of NK cells are still controversial among the different studies, more consensuses exist on the impaired activity of these cells. In another group of inflammatory arthritides, the spondyloarthropathies (SpA), the presence of peculiar allotypes of the killer cell immunoglobulin-like receptors (KIR) superfamily, coding for molecules expressed on NK cells, seems to modulate the susceptibility to this group of diseases, especially ankylosing spondylitis and psoriatic arthritis. Interestingly, in vitro studies showed that NK cells of patients with inflammatory arthropathies might produce pro-inflammatory Th1 cytokines; furthermore, they are involved in bone damage, interact and activate different cell types such as monocytes, dendritic cells and resident fibroblast-like synoviocites cells, thus creating and/or maintaining the inflammatory response. Certainly, these features encourage more research on the role of NK cells in the pathogenesis of inflammatory arthropathies, which could be essential to define potential new therapeutic strategies.