Natural Killer Lysis Receptor (NKLR)/NKLR-Ligand Matching as a Novel Approach for Enhancing Anti-Tumor Activity of Allogeneic NK Cells

Background

NK cells are key players in anti tumor immune response, which can be employed in cell-based therapeutic modalities. One of the suggested ways to amplify their anti tumor effect, especially in the field of stem cell transplantation, is by selecting donor/recipient mismatches in specific HLA, to reduce the inhibitory effect of killer Ig-like receptors (KIRs). Here we suggest an alternative approach for augmentation of anti tumor effect of allogeneic NK cells, which is founded on profile matching of donor NK lysis receptors (NKLR) phenotype with tumor lysis-ligands.

Methodology/Principal Findings

We show that an NKLR-mediated killing directly correlates with the NKLR expression intensity on NK cells. Considerable donor variability in the expression of CD16, NKp46, NKG2D and NKp30 on circulating NK cells, combined with the stability of phenotype in several independently performed tests over two months, indicates that NKLR-guided selection of donors is feasible. As a proof of concept, we show that melanoma cells are dominantly recognized by three NKLRs: NKG2D, NKp30 and NKp44. Notably, the expression of NKp30 on circulating NK cells among metastatic melanoma patients was significantly decreased, which diminishes their ability to kill melanoma cells. Ex vivo expansion of NK cells results not only in increased amount of cells but also in a consistently superior and predictable expression of NKG2D, NKp30 and NKp44. Moreover, expanded NK cultures with high expression of NKG2D or NKp30 were mostly derived from the corresponding NKG2D^high or NK30^high donors. These NK cultures subsequently displayed an improved cytotoxic activity against melanoma in a HLA/KIR-ligand mismatched setup, which was NKLR-dependent, as demonstrated with blocking anti-NKG2D antibodies.

Conclusions/Significance

NKLR/NKLR-ligand matching reproducibly elicits enhanced NK anti-tumor response. Common NKLR recognition patterns of tumors, as demonstrated here in melanoma, would allow implementation of this approach in solid malignancies and potentially in hematological malignancies, either independently or in adjunction to other modalities.     

Expression analysis of the ligands for the Natural Killer cell receptors NKp30 and NKp44

Background

The natural cytotoxicity receptors (NCR) are important to stimulate the activity of Natural Killer (NK) cells against transformed cells. Identification of NCR ligands and their level of expression on normal and neoplastic cells has important implications for the rational design of immunotherapy strategies for cancer.

Methodology/Principal Findings

Here we analyze the expression of NKp30 ligand and NKp44 ligand on 30 transformed or non-transformed cell lines of different origin. We find intracellular and surface expression of these two ligands on almost all cell lines tested. Expression of NKp30 and NKp44 ligands was variable and did not correlate with the origin of the cell line. Expression of NKp30 and NKp44 ligand correlated with NKp30 and NKp44-mediated NK cell lysis of tumor cells, respectively. The surface expression of NKp30 ligand and NKp44 ligand was sensitive to trypsin treatment and was reduced in cells arrested in G₂/M phase.

Conclusion/Significance

These data demonstrate the ubiquitous expression of the ligands for NKp30 and NKp44 and give an important insight into the regulation of these ligands.     

Combining Antiangiogenic Therapy with Adoptive Cell Immunotherapy Exerts Better Antitumor Effects in Non-Small Cell Lung Cancer Models

Introduction

Cytokine-induced killer cells (CIK cells) are a heterogeneous subset of ex-vivo expanded T lymphocytes which are characterized with a MHC-unrestricted tumor-killing activity and a mixed T-NK phenotype. Adoptive CIK cells transfer, one of the adoptive immunotherapy represents a promising nontoxic anticancer therapy. However, in clinical studies, the therapeutic activity of adoptive CIK cells transfer is not as efficient as anticipated. Possible explanations are that abnormal tumor vasculature and hypoxic tumor microenvironment could impede the infiltration and efficacy of lymphocytes. We hypothesized that antiangiogenesis therapy could improve the antitumor activity of CIK cells by normalizing tumor vasculature and modulating hypoxic tumor microenvironment.

Methods

We combined recombinant human endostatin (rh-endostatin) and CIK cells in the treatment of lung carcinoma murine models. Intravital microscopy, dynamic contrast enhanced magnetic resonance imaging, immunohistochemistry, and flow cytometry were used to investigate the tumor vasculature and hypoxic microenvironment as well as the infiltration of immune cells.

Results

Our results indicated that rh-endostatin synergized with adoptive CIK cells transfer to inhibit the growth of lung carcinoma. We found that rh-endostatin normalized tumor vasculature and reduced hypoxic area in the tumor microenvironment. Hypoxia significantly inhibited the proliferation, cytotoxicity and migration of CIK cells in vitro and impeded the homing of CIK cells into tumor parenchyma ex vivo. Furthermore, we found that treatment with rh-endostatin significantly increased the homing of CIK cells and decreased the accumulation of suppressive immune cells in the tumor tissue. In addition, combination therapy produced higher level of tumor-infiltration lymphocytes compared with other treatments.

Conclusions

Our results demonstrate that rh-endostatin improves the therapeutic effect of adoptive CIK cells therapy against lung carcinomas and unmask the mechanisms of the synergistic antitumor efficacy, providing a new rationale for combining antiangiogenesis therapy with immunotherapy in the treatment of lung cancer.     

NK Cell Terminal Differentiation: Correlated Stepwise Decrease of NKG2A and Acquisition of KIRs

Background

Terminal differentiation of NK cells is crucial in maintaining broad responsiveness to pathogens and discriminating normal cells from cells in distress. Although it is well established that KIRs, in conjunction with NKG2A, play a major role in the NK cell education that determines whether cells will end up competent or hyporesponsive, the events underlying the differentiation are still debated.

Methodology/Principal Findings

A combination of complementary approaches to assess the kinetics of the appearance of each subset during development allowed us to obtain new insights into these terminal stages of differentiation, characterising their gene expression profiles at a pan-genomic level, their distinct surface receptor patterns and their prototypic effector functions. The present study supports the hypothesis that CD56^dim cells derive from the CD56^bright subset and suggests that NK cell responsiveness is determined by persistent inhibitory signals received during their education. We report here the inverse correlation of NKG2A expression with KIR expression and explore whether this correlation bestows functional competence on NK cells. We show that CD56^dimNKG2A⁻KIR⁺ cells display the most differentiated phenotype associated to their unique ability to respond against HLA-E+ target cells. Importantly, after IL-12 + IL-18 stimulation, reacquisition of NKG2A strongly correlates with IFN-γ production in CD56^dimNKG2A⁻ NK cells.

Conclusions/Significance

Together, these findings call for the reclassification of mature human NK cells into distinct subsets and support a new model, in which the NK cell differentiation and functional fate are based on a stepwise decrease of NKG2A and acquisition of KIRs.     

Tumor growth decreases NK and B cells as well as common lymphoid progenitor

Background

It is well established that chronic tumor growth results in functional inactivation of T cells and NK cells. It is less clear, however, whether lymphopoeisis is affected by tumor growth.

Principal Findings

In our efforts of analyzing the impact of tumor growth on NK cell development, we observed a major reduction of NK cell numbers in mice bearing multiple lineages of tumor cells. The decrease in NK cell numbers was not due to increased apoptosis or decreased proliferation in the NK compartment. In addition, transgenic expression of IL-15 also failed to rescue the defective production of NK cells. Our systematic characterization of lymphopoeisis in tumor-bearing mice indicated that the number of the common lymphoid progenitor was significantly reduced in tumor-bearing mice.The number of B cells also decreased substantially in tumor bearing mice.

Conclusions and Significance

Our data reveal a novel mechanism for tumor evasion of host immunity and suggest a new interpretation for the altered myeloid and lymphoid ratio in tumor bearing hosts.     

A Novel CXCL10-Based GPI-Anchored Fusion Protein as Adjuvant in NK-Based Tumor Therapy

Background

Cellular therapy is a promising therapeutic strategy for malignant diseases. The efficacy of this therapy can be limited by poor infiltration of the tumor by immune effector cells. In particular, NK cell infiltration is often reduced relative to T cells. A novel class of fusion proteins was designed to enhance the recruitment of specific leukocyte subsets based on their expression of a given chemokine receptor. The proteins are composed of an N-terminal chemokine head, the mucin domain taken from the membrane-anchored chemokine CX3CL1, and a C-terminal glycosylphosphatidylinositol (GPI) membrane anchor replacing the normal transmembrane domain allowing integration of the proteins into cell membranes when injected into a solid tumor. The mucin domain in conjunction with the chemokine head acts to specifically recruit leukocytes expressing the corresponding chemokine receptor.

Methodology/Principal Findings

A fusion protein comprising a CXCL10 chemokine head (CXCL10-mucin-GPI) was used for proof of concept for this approach and expressed constitutively in Chinese Hamster Ovary cells. FPLC was used to purify proteins. The recombinant proteins efficiently integrated into cell membranes in a process dependent upon the GPI anchor and were able to activate the CXCR3 receptor on lymphocytes. Endothelial cells incubated with CXCL10-mucin-GPI efficiently recruited NK cells in vitro under conditions of physiologic flow, which was shown to be dependent on the presence of the mucin domain. Experiments conducted in vivo using established tumors in mice suggested a positive effect of CXCL10-mucin-GPI on the recruitment of NK cells.

Conclusions

The results suggest enhanced recruitment of NK cells by CXCL10-mucin-GPI. This class of fusion proteins represents a novel adjuvant in cellular immunotherapy. The underlying concept of a chemokine head fused to the mucin domain and a GPI anchor signal sequence may be expanded into a broader family of reagents that will allow targeted recruitment of cells in various settings.     

Susceptibility of Human Melanoma Cells to Autologous Natural Killer (NK) Cell Killing: HLA-Related Effector Mechanisms and Role of Unlicensed NK Cells

Background

Despite Natural Killer (NK) cells were originally defined as effectors of spontaneous cytotoxicity against tumors, extremely limited information is so far available in humans on their capability of killing cancer cells in an autologous setting.

Methodology/Principal Findings

We have established a series of primary melanoma cell lines from surgically resected specimens and here showed that human melanoma cells were highly susceptible to lysis by activated autologous NK cells. A variety of NK cell activating receptors were involved in killing: particularly, DNAM-1 and NKp46 were the most frequently involved. Since self HLA class I molecules normally play a protective role from NK cell-mediated attack, we analyzed HLA class I expression on melanomas in comparison to autologous lymphocytes. We found that melanoma cells presented specific allelic losses in 50% of the patients analyzed. In addition, CD107a degranulation assays applied to NK cells expressing a single inhibitory receptor, revealed that, even when expressed, specific HLA class I molecules are present on melanoma cell surface in amount often insufficient to inhibit NK cell cytotoxicity. Remarkably, upon activation, also the so called “unlicensed” NK cells, i.e. NK cells not expressing inhibitory receptor specific for self HLA class I molecules, acquired the capability of efficiently killing autologous melanoma cells, thus additionally contributing to the lysis by a mechanism independent of HLA class I expression on melanoma cells.

Conclusions/Significance

We have investigated in details the mechanisms controlling the recognition and lysis of melanoma cells by autologous NK cells. In these autologous settings, we demonstrated an efficient in vitro killing upon NK cell activation by mechanisms that may be related or not to abnormalities of HLA class I expression on melanoma cells. These findings should be taken into account in the design of novel immunotherapy approaches against melanoma.     

Natural killer cell mediated missing-self recognition can protect mice from primary chronic myeloid leukemia in vivo

Background

Natural Killer (NK) cells are thought to protect from residual leukemic cells in patients receiving stem cell transplantation. However, multiple retrospective analyses of patient data have yielded conflicting conclusions regarding a putative role of NK cells and the essential NK cell recognition events mediating a protective effect against leukemia. Further, a NK cell mediated protective effect against primary leukemia in vivo has not been shown directly.

Methodology/Principal Findings

Here we addressed whether NK cells have the potential to control chronic myeloid leukemia (CML) arising based on the transplantation of BCR-ABL1 oncogene expressing primary bone marrow precursor cells into lethally irradiated recipient mice. These analyses identified missing-self recognition as the only NK cell-mediated recognition strategy, which is able to significantly protect from the development of CML disease in vivo.

Conclusion

Our data provide a proof of principle that NK cells can control primary leukemic cells in vivo. Since the presence of NK cells reduced the abundance of leukemia propagating cancer stem cells, the data raise the possibility that NK cell recognition has the potential to cure CML, which may be difficult using small molecule BCR-ABL1 inhibitors. Finally, our findings validate approaches to treat leukemia using antibody-based blockade of self-specific inhibitory MHC class I receptors.     

Myeloid suppressor cell depletion augments antitumor activity in lung cancer

Background

Myeloid derived suppressor cells (MDSC) are important regulators of immune responses. We evaluated the mechanistic role of MDSC depletion on antigen presenting cell (APC), NK, T cell activities and therapeutic vaccination responses in murine models of lung cancer.

Principal Findings

Individual antibody mediated depletion of MDSC (anti-Gr1 or anti-Ly6G) enhanced the antitumor activity against lung cancer. In comparison to controls, MDSC depletion enhanced the APC activity and increased the frequency and activity of the NK and T cell effectors in the tumor. Compared to controls, the anti-Gr1 or anti-Ly6G treatment led to increased: (i) CD8 T cells, (ii) NK cells, (iii) CD8 T or NK intracytoplasmic expression of IFNγ, perforin and granzyme (iv) CD3 T cells expressing the activation marker CD107a and CXCR3, (v) reduced CD8 T cell IL-10 production in the tumors (vi) reduced tumor angiogenic (VEGF, CXCL2, CXCL5, and Angiopoietin1&2) but enhanced anti-angiogenic (CXCL9 and CXCL10) expression and (vii) reduced tumor staining of endothelial marker Meca 32. Immunocytochemistry of tumor sections showed reduced Gr1 expressing cells with increased CD3 T cell infiltrates in the anti-Gr1 or anti-Ly6G groups. MDSC depletion led to a marked inhibition in tumor growth, enhanced tumor cell apoptosis and reduced migration of the tumors from the primary site to the lung compared to controls. Therapeutic vaccination responses were enhanced in vivo following MDSC depletion with 50% of treated mice completely eradicating established tumors. Treated mice that rejected their primary tumors acquired immunological memory against a secondary tumor challenge. The remaining 50% of mice in this group had 20 fold reductions in tumor burden compared to controls.

Significance

Our data demonstrate that targeting MDSC can improve antitumor immune responses suggesting a broad applicability of combined immune based approaches against cancer. This multifaceted approach may prove useful against tumors where MDSC play a role in tumor immune evasion.     

Renal transplant immunosuppression impairs natural killer cell function in vitro and in vivo

Background

Despite an increasing awareness of the importance of innate immunity, the roles of natural killer (NK) cells in transplant rejection and antiviral and cancer immunity during immunosuppression have not been clearly defined.

Methods

To address this issue we have developed a quantitative assay of NK cell function that can be used on clinical samples and have studied the influence of immunosuppression on NK cell function. NK cell degranulation and intracellular interferon (IFN)-γ production were determined by flow cytometry of peripheral blood samples.

Results

Overnight ex vivo treatment of peripheral blood cells from healthy controls with ciclosporin or tacrolimus inhibited NK cell degranulation and IFN-γ production in a dose-dependent manner. A similar impairment of function was seen in NK cells from patients treated in vivo with calcineurin inhibitors. In the early post-transplant period, there was a variable reduction of NK cell counts after treatment with alemtuzumab and basiliximab.

Conclusions

The functional inhibition of NK cells in early transplant patients coincides with the period of maximum susceptibility to viral infections. The ability to assay NK cell function in clinical samples allows assessment of the impact of immunosuppression on these effector cells. This information may be helpful in guiding the titration of immunosuppression in the clinical setting.     

The ADMR Receptor Mediates the Effects of Adrenomedullin on Pancreatic Cancer Cells and on Cells of the Tumor Microenvironment

Background

Adrenomedullin (AM) is highly expressed in pancreatic cancer and stimulates pancreatic cancer cells leading to increased tumor growth and metastasis. The current study examines the role of specific AM receptors on tumor and cells resembling the tumor microenvironment (human pancreatic stellate - HPSC, human umbilical vein – HUVEC and mouse lung endothelial cells - MLEC).

Methods and Findings

AM receptors ADMR and CRLR were present in HPSC, HUVEC and MLECs while PDAC cells possessed only ADMR receptors as assessed by RT-PCR and western blotting. All cell lines expressed and secreted AM as indicated by ELISA. The growth of each of the cell lines was stimulated by exogenous AM and inhibited by the antagonist AMA. AM also stimulated in vitro angiogenesis assessed by polygon formation of endothelial cell lines. SiRNA-mediated silencing of ADMR, but not CRLR, reduced basal growth of all cells examined and reduced polygon formation of endothelial cells in vitro. Orthotopic tumors developed with shADMR bearing cancer cells had dramatically reduced primary tumor volume (>90%) and lung and liver metastasis compared to shControl bearing cells. To validate ADMR as a potential therapeutic target, in vivo studies were conducted using neutral nanoliposomes to systemically deliver human siRNA to ADMR to silence human cancer cells and mouse siRNA to ADMR to silence mouse tumor stromal cells. Systemic silencing of both human and mouse ADMR had no obvious adverse effects but strongly reduced tumor development.

Conclusion

ADMR mediates the stimulatory effects of AM on cancer cells and on endothelial and stellate cells within the tumor microenvironment. These data support the further development of ADMR as a useful target treatment of pancreatic cancer.     

A One Year Follow-Up Study of Natural Killer and Dendritic Cells Activities in Multiple Sclerosis Patients Receiving Glatiramer Acetate (GA)

Background

Multiple sclerosis (MS) is a chronic inflammatory, demyelinating and neurodegenerative disease. It is thought to be mediated by CD4⁺ Th1/Th17 cells. More recently, cells of the innate immune system such as dendritic cells (DCs) and natural killer (NK) cells have been in focus. Glatiramer acetate (GA) is an approved drug for treating MS patients.

Methodology/Principal Findings

In the current study we examined the activities of NK and DCs in nine relapsing remitting MS patients for up to one year after initiation of GA treatment. We observed that NK cells isolated from most of these patients have increased cytotoxic activity against K562 cells. Further analysis showed that the same NK cells lysed both autologous immature (i) and mature (m) DCs. In most patients this increased activity was correlated with increased NK cell activating cytotoxicity receptors such as NKp30, NKp44, NKp46 and NKG2D, and reduced expression of the inhibitory molecule CD158 on the surface of these NK cells. The expression of HLA-DR was increased on iDCs and mDCs in the majority of the patients, but no consistency was observed for the expression of HLA-I or HLA-E. Also, the co-stimulatory receptors CD80, CD83 or CD86 expression was down-regulated on iDCs and mDCs in most cases. Further, the expression of CCR6 was increased on mDCs at later time points of therapy (between 32–48 weeks).

Conclusions/Significance

Our results are the first showing the effects of GA treatment on NK cells in MS patients, which may impact future use of this and other drugs to treat this disease.     

Cytotoxic T cells expressing the co-stimulatory receptor NKG2 D are increased in cigarette smoking and COPD

Background

A suggested role for T cells in COPD pathogenesis is based on associations between increased lung cytotoxic T lymphocyte (CD8⁺) numbers and airflow limitation. CD69 is an early T cell activation marker. Natural Killer cell group 2 D (NKG2D) receptors are co-stimulatory molecules induced on CD8⁺ T cells upon activation. The activating function of NKG2 D is triggered by binding to MHC class 1 chain-related (MIC) molecules A and B, expressed on surface of stressed epithelial cells. The aim of this study was to evaluate the expression of MIC A and B in the bronchial epithelium and NKG2 D and CD69 on BAL lymphocytes in subjects with COPD, compared to smokers with normal lung function and healthy never-smokers.

Methods

Bronchoscopy with airway lavages and endobronchial mucosal biopsy sampling was performed in 35 patients with COPD, 21 healthy never-smokers and 16 smokers with normal lung function. Biopsies were immunohistochemically stained and BAL lymphocyte subsets were determined using flow cytometry.

Results

Epithelial CD3⁺ lymphocytes in bronchial biopsies were increased in both smokers with normal lung function and in COPD patients, compared to never-smokers. Epithelial CD8⁺ lymphocyte numbers were higher in the COPD group compared to never-smoking controls. Among gated CD3⁺cells in BAL, the percentage of CD8⁺ NKG2D⁺ cells was enhanced in patients with COPD and smokers with normal lung function, compared to never-smokers. The percentage of CD8⁺ CD69⁺ cells and cell surface expression of CD69 were enhanced in patients with COPD and smokers with normal lung function, compared to never-smokers. No changes in the expression of MIC A or MIC B in the airway epithelium could be detected between the groups, whereas significantly decreased soluble MICB was detected in bronchial wash from smokers with normal lung function, compared to never-smokers.

Conclusions

In COPD, we found increased numbers of cytotoxic T cells in both bronchial epithelium and airway lumen. Further, the proportions of CD69- and NKG2D-expressing cytotoxic T cells in BAL fluid were enhanced in both subjects with COPD and smokers with normal lung function and increased expression of CD69 was found on CD8⁺ cells, indicating the cigarette smoke exposure-induced expansion of activated cytotoxic T cells, which potentially can respond to stressed epithelial cells.     

Mouse natural killer (NK) cells express the nerve growth factor receptor TrkA, which is dynamically regulated

Background

Nerve growth factor (NGF) is a neurotrophin crucial for the development and survival of neurons. It also acts on cells of the immune system which express the NGF receptors TrkA and p75^NTR and can be produced by them. However, mouse NK cells have not yet been studied in this context.

Methodology/Principal Findings

We used cell culture, flow cytometry, confocal microscopy and ELISA assays to investigate the expression of NGF receptors by NK cells and their secretion of NGF. We show that resting NK cells express TrkA and that the expression is different on NK cell subpopulations defined by the relative presence of CD27 and CD11b. Expression of TrkA is dramatically increased in IL-2-activated NK cells. The p75^NTR is expressed only on a very low percentage of NK cells. Functionally, NGF moderately inhibits NK cell degranulation, but does not influence proliferation or cytokine production. NK cells do not produce NGF.

Conclusions/Significance

We demonstrate for the first time that mouse NK cells express the NGF receptor TrkA and that this expression is dynamically regulated.     

Cyclophosphamide Chemotherapy Sensitizes Tumor Cells to TRAIL-Dependent CD8 T Cell-Mediated Immune Attack Resulting in Suppression of Tumor Growth

Background

Anti-cancer chemotherapy can be simultaneously lymphodepleting and immunostimulatory. Pre-clinical models clearly demonstrate that chemotherapy can synergize with immunotherapy, raising the question how the immune system can be mobilized to generate anti-tumor immune responses in the context of chemotherapy.

Methods and Findings

We used a mouse model of malignant mesothelioma, AB1-HA, to investigate T cell-dependent tumor resolution after chemotherapy. Established AB1-HA tumors were cured by a single dose of cyclophosphamide in a CD8 T cell- and NK cell-dependent manner. This treatment was associated with an IFN-α/β response and a profound negative impact on the anti-tumor and total CD8 T cell responses. Despite this negative effect, CD8 T cells were essential for curative responses. The important effector molecules used by the anti-tumor immune response included IFN-γ and TRAIL. The importance of TRAIL was supported by experiments in nude mice where the lack of functional T cells could be compensated by agonistic anti-TRAIL-receptor (DR5) antibodies.

Conclusion

The data support a model in which chemotherapy sensitizes tumor cells for T cell-, and possibly NK cell-, mediated apoptosis. A key role of tumor cell sensitization to immune attack is supported by the role of TRAIL in tumor resolution and explains the paradox of successful CD8 T cell-dependent anti-tumor responses in the absence of CD8 T cell expansion.     

Shed NKG2D ligand boosts NK cell immunity

Ligands for natural killer (NK) cell activating receptors can be released from tumor cells and are believed to promote tumor growth by acting as decoys for effector lymphocytes. In a recent paper published in Science, Deng et al. report another scenario in which a shed form of the MULT1 mouse NKG2D ligand boosts NK cell functions.Natural killer (NK) cells are cytolytic and cytokine-producing lymphocytes of the innate immune system that participate in the control of tumor growth and microbial infections¹. NK cell effector activities are tightly controlled by a fine balance of inhibitory and activating signals delivered by surface receptors. Activating receptors can recognize two types of ligands, self-molecules encoded by the host''s own genome whose expression is upregulated upon cellular stress, or exogenous molecules produced by microbes during infection. NKG2D, one of the best characterized activating receptor expressed by NK and T cells, binds to several different ligands in human and mouse². NKG2D ligands are poorly expressed on the vast majority of normal cell surfaces, but are upregulated on tumor and virus-infected cells. In addition, NKG2D ligands can be released by both surface cleavage and exosome excretion. It has been reported that shed ligands can block tumor cell recognition by effector cells by preventing NKG2D interaction with its ligands³. However, several reports do not correlate the presence of soluble ligands with decreased NKG2D expression nor functional activities.Deng et al.⁴ focused their analysis on the NKG2D mouse ligand MULT1, which is commonly overexpressed on primary tumor cells. They first showed that MULT1-transduced fibroblast can cleave MULT1 from the plasma membrane, resulting in a released shed form in the supernatant. Shed MULT1 is of high affinity to NKG2D (∼13 nM) similar to recombinant MULT1. They further reveal the presence of shed MULT1 in the serum of mice developing spontaneous MULT1⁺ tumors. Interestingly, the authors detected a very high concentration of shed MULT1 in the sera of Apoe^−/− mice exhibiting severe atherosclerosis and liver inflammation. Given that these autoimmune injuries observed in this mouse model depend on NKG2D activity⁵, it was unlikely that shed MULT1 exert an inhibitory effect on immunity.Surprisingly, the authors further showed that mouse tumor cells engineered to release a secreted form of MULT1 (secMULT1) similar to the shed MULT1 were rejected when injected into syngenic mice. Tumor rejection is dependent on NK cells as cells grow in NK but not in CD8⁺ T cell-depleted host and requires NKG2D. Importantly, the controlled release of secMULT1 from tumors harboring inducible secMULT1 promotes tumor rejection. To rule out the possibility that tumor cell rejection was due to intrinsic modifications of tumor cells, the author monitored the rejection of a mixture of 9:1 secMULT1⁻: secMULT1⁺ tumor cells and showed an improved antitumoral effect on both secMULT1⁺ and, importantly, secMULT1⁻ tumors. In addition, direct intratumoral injection of recombinant MULT1 promotes tumor rejection. These results suggested that soluble MULT1 mobilizes or activates anti-tumor effector cells. Deng et al. further reported increased frequencies of cytotoxic and IFN-γ-secreting NK cells associated with secMULT1⁺ tumors as compared to control tumor cells. Altogether, these data suggest that a shed NKG2D ligand can promote tumor rejection by boosting NK cell effector functions.Shed MULT1 could crosslink NKG2D and thus activate NK cells. However, shed and secMULT1 are monomeric molecules similar to the recombinant MULT1 which fails to activate NK cells in vitro. Formation of multivalent structures in vivo was not detected. In addition, whereas the transmembrane form of MULT1 can activate NK cells by crosslinking NKG2D and induces NKG2D downregulation, soluble MULT1 upregulates NKG2D on the NK cell surface. This upregulation is probably due do a decreased downregulation of NKG2D surface expression because no increase in NKG2D mRNA or protein was observed. Based on these findings, the authors hypothesized that NKG2D ligands expressed on non-tumor host cell membrane continuously engage NKG2D on NK cells, leading to NKG2D downregulation and NK cell desensitization, whereas soluble MULT1 blocks these interactions to increase NK cell responsiveness (Figure 1). Along this line, NK cells from mutant mice genetically deficient for the NKG2D ligand expressed by tumor-associated myeloid cells are not desensitized.Open in a separate windowFigure 1Tumor-associated cells express NKG2DL which can desensitize NK cells. Tumor shedding of MULT1 delivers soluble MULT1 that outcompetes for NKG2D binding and prevents NK cell desensitization. Boosted NK cell functions lead to improved tumor cell rejection by other activating receptors.The induction of cell desensitization by a frequent or even constant stimulation is a very common mechanism across living objects. Regarding NK cells, another example of tuning via desensitization resides in the impact of the long lasting absence of MHC class I molecules in their environment. Indeed, NK cells are hyporesponsive in a MHC-I-deficient host⁶. There are accumulating data indicating that in the absence of engagement of inhibitory receptors for MHC class I molecules, NK cells get desensitized due to their chronic interaction with endogenous stimulating ligands⁷. Indeed, in the absence of engagement of this inhibitory pathway, NK cell activation would be unleashed⁸. This scenario is supported by a series of in vitro and in vivo experiments in which NK cells are desensitized following chronic exposure to stimulatory molecules expressed at the surface of interacting cells^9,10. Thus, the induction of MHC class I downregulation or NKG2D ligand upregulation boosts NK cell function, whereas the sustained lack of MHC class I or expression of NKG2D ligands impairs NK cell reactivity. This tuning of immune response as a function of the speed of change of the stimuli detected by lymphocytes is at the center of the recently proposed Discontinuity Theory¹¹.Finally, consistent with their findings with secMULT1 but somewhat counter-intuitively, Deng et al. also show that NKG2D receptor deficiency or blockade using anti-NKG2D monoclonal antibodies mimics the effect of soluble MULT1. Indeed, in both conditions, NK cell effector functions are boosted, resulting in improved tumor rejection. Similarly, blocking other NK activating receptors, such as NKp46, may also lead to NK cell desensitization¹². Checkpoint inhibitory receptors are revolutionizing the treatment of cancers by inhibiting the inhibitory receptors. The findings reported by Deng et al. together with earlier results propose alternative strategies of cancer treatment using antibodies that are directed against activating receptors. In the case of NKG2D, the chronic engagement of NK cells with membrane-bound NKG2D ligand affects not only NKG2D-dependent but also NKG2D-independent signaling pathways⁹. The blockade of NKG2D desensitization by antibodies directed against NKG2D should thus also boost NK cell activation via other pathways, such as antibody-dependent cell cytotoxicity. However, the precise identification of the ligand-receptor pair involved in the control of tumors by NK cells will be a limiting factor to these innovative therapeutic approaches. Indeed, antibodies against activating receptors should be designed to boost NK cell reactivity but should not block the recognition of the tumors by NK cells. Finally, as the tuning of NK cell reactivity by soluble NKG2D ligands depends on their affinity for NKG2D, the pre-clinical development of this new class of drug candidates might reveal novel pharmacokinetics and the pharmacodynamics guidelines.     

Comparison of Immunity in Mice Cured of Primary/Metastatic Growth of EMT6 or 4THM Breast Cancer by Chemotherapy or Immunotherapy

Purpose

We have compared cure from local/metastatic tumor growth in BALB/c mice receiving EMT6 or the poorly immunogenic, highly metastatic 4THM, breast cancer cells following manipulation of immunosuppressive CD200:CD200R interactions or conventional chemotherapy.

Methods

We reported previously that EMT6 tumors are cured in CD200R1KO mice following surgical resection and immunization with irradiated EMT6 cells and CpG oligodeoxynucleotide (CpG), while wild-type (WT) animals developed pulmonary and liver metastases within 30 days of surgery. We report growth and metastasis of both EMT6 and a highly metastatic 4THM tumor in WT mice receiving iv infusions of Fab anti-CD200R1 along with CpG/tumor cell immunization. Metastasis was followed both macroscopically (lung/liver nodules) and microscopically by cloning tumor cells at limiting dilution in vitro from draining lymph nodes (DLN) harvested at surgery. We compared these results with local/metastatic tumor growth in mice receiving 4 courses of combination treatment with anti-VEGF and paclitaxel.

Results

In WT mice receiving Fab anti-CD200R, no tumor cells are detectable following immunotherapy, and CD4+ cells produced increased TNFα/IL-2/IFNγ on stimulation with EMT6 in vitro. No long-term cure was seen following surgery/immunotherapy of 4THM, with both microscopic (tumors in DLN at limiting dilution) and macroscopic metastases present within 14 d of surgery. Chemotherapy attenuated growth/metastases in 4THM tumor-bearers and produced a decline in lung/liver metastases, with no detectable DLN metastases in EMT6 tumor-bearing mice-these latter mice nevertheless showed no significantly increased cytokine production after restimulation with EMT6 in vitro. EMT6 mice receiving immunotherapy were resistant to subsequent re-challenge with EMT6 tumor cells, but not those receiving curative chemotherapy. Anti-CD4 treatment caused tumor recurrence after immunotherapy, but produced no apparent effect in either EMT6 or 4THM tumor bearers after chemotherapy treatment.

Conclusion

Immunotherapy, but not chemotherapy, enhances CD4⁺ immunity and affords long-term control of breast cancer growth and resistance to new tumor foci.     

Inhibition of miR-191 contributes to radiation-resistance of two lung cancer cell lines by altering autophagy activity

Background

Lung cancer is the leading cause of cancer-related morbidity and mortality all over the world. Surgery resection, radiotherapy, chemotherapy, immunotherapy and combined treatments have been discovered and well established for treatments. However, low survival rate of five years after clinical treatments mainly due to recurrence of stress-resistant cancer cells calls for better understanding and new ideas. Our project aimed to understand the forming process of stress resistant lung cancer cells after radiotherapy.

Methods

Two classic non-small cell lung cancer (NSCLC) cell lines A549 and H1299 initially were radiated with a ¹³⁷Cs gamma-ray source with doses ranging from 0 to 12 Gy to generate radiation-resistant cancer cells. 8 Gy of radiation was regard as a standard dosage since it provides effective killing as well as good amount of survivals. The expression levels of autophagy-related proteins including Beclin-1, LC3-II and p62 were studied and measured by both western blot and quantitative real-time polymerase chain reaction (real-time RT-PCR).

Results

Increased Beclin-1, LC3-II and decreased p62 have been observed in radiation-resistant cells indicating elevated autophagy level. Decreased miR-191 in radiation-resistant cells performed by Taqman qRT-PCR also has been seen. Two binding sites between Beclin-1 and miR-191 suggest potential association between.

Conclusions

It is reasonable to speculate that inhibition of miR-191 expression in lung cancer cells would contribute to the establishment of radiation-resistant cells via mediating cellular autophagy. Therefore, miR-191 is a potential target for therapy in treating radiation-resistant lung cancer.     

Changes to the Natural Killer Cell Repertoire after Therapeutic Hepatitis B DNA Vaccination

Background

Improvements to the outcome of adaptive immune responses could be achieved by inducing specific natural killer (NK) cell subsets which can cooperate with dendritic cells to select efficient T cell responses. We previously reported the induction or reactivation of T cell responses in chronic hepatitis B patients vaccinated with a DNA encoding hepatitis B envelope proteins during a phase I clinical trial.

Methodology/Principal Findings

In this study, we examined changes in the peripheral NK cell populations occurring during this vaccine trial using flow cytometry analysis. Despite a constant number of NK cells in the periphery, a significant increase in the CD56^bright population was observed after each vaccination and during the follow up. Among the 13 different NK cell markers studied by flow cytometry analysis, the expression of CD244 and NKG2D increased significantly in the CD56^bright NK population. The ex vivo CD107a expression by CD56^bright NK cells progressively increased in the vaccinated patients to reach levels that were significantly higher compared to chronically HBV-infected controls. Furthermore, modifications to the percentage of the CD56^bright NK cell population were correlated with HBV-specific T cell responses detected by the ELISPOT assay.

Conclusions/Significance

These changes in the CD56^bright population may suggest a NK helper effect on T cell adaptive responses. Activation of the innate and adaptive arms of the immune system by DNA immunization may be of particular importance to the efficacy of therapeutic interventions in a context of chronic infections.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00988767","term_id":"NCT00988767"}}NCT00988767     

The Immune System Strikes Back: Cellular Immune Responses against Indoleamine 2,3-dioxygenase

Background

The enzyme indoleamine 2,3-dioxygenase (IDO) exerts an well established immunosuppressive function in cancer. IDO is expressed within the tumor itself as well as in antigen-presenting cells in tumor-draining lymph nodes, where it promotes the establishment of peripheral immune tolerance to tumor antigens. In the present study, we tested the notion whether IDO itself may be subject to immune responses.

Methods and Findings

The presence of naturally occurring IDO-specific CD8 T cells in cancer patients was determined by MHC/peptide stainings as well as ELISPOT. Antigen specific cytotoxic T lymphocytes (CTL) from the peripheral blood of cancer patients were cloned and expanded. The functional capacity of the established CTL clones was examined by chrome release assays. The study unveiled spontaneous cytotoxic T-cell reactivity against IDO in peripheral blood as well as in the tumor microenvironment of different cancer patients. We demonstrate that these IDO reactive T cells are indeed peptide specific, cytotoxic effector cells. Hence, IDO reactive T cells are able to recognize and kill tumor cells including directly isolated AML blasts as well as IDO-expressing dendritic cells, i.e. one of the major immune suppressive cell populations.

Conclusion

IDO may serve as an important and widely applicable target for anti-cancer immunotherapeutic strategies. Furthermore, as emerging evidence suggests that IDO constitutes a significant counter-regulatory mechanism induced by pro-inflammatory signals, IDO-based immunotherapy holds the promise to boost anti-cancer immunotherapy in general.