NK Cell Infiltration Research Articles

Unusual case of metachronous EBV‐associated B‐cell and NK/T‐cell lymphoma mimicking polymyositis‐diagnostic challenges and pitfalls

Unusual case of metachronous EBV‐associated B‐cell and NK/T‐cell lymphoma mimicking polymyositis‐diagnostic challenges and pitfalls

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

BackgroundCellular 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 FindingsA 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.ConclusionsThe 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.

Increased Numbers of Monocyte-Derived Dendritic Cells during Successful Tumor Immunotherapy with Immune-Activating Agents

Local treatment with selected TLR ligands or bacteria such as bacillus Calmette-Guérin increases antitumor immune responses and delays tumor growth. It is thought that these treatments may act by activating tumor-associated dendritic cells (DCs), thereby supporting the induction of antitumor immune responses. However, common parameters of successful immune activation have not been identified. We used mouse models to compare treatments with different immune-activating agents for the ability to delay tumor growth, improve priming of tumor-specific T cells, and induce early cytokine production and DC activation. Treatment with polyinosinic-polycytidylic acid or a combination of monosodium urate crystals and Mycobacterium smegmatis was effective at delaying the growth of s.c. B16 melanomas, orthotopic 4T1 mammary carcinomas, and reducing 4T1 lung metastases. In contrast, LPS, monosodium urate crystals, or M. smegmatis alone had no activity. Effective treatments required both NK1.1(+) and CD8(+) cells, and resulted in increased T cell priming and the infiltration of NK cells and CD8(+) T cells in tumors. Unexpectedly, both effective and ineffective treatments increased DC numbers and the expression of costimulatory molecules in the tumor-draining lymph node. However, only effective treatments induced the rapid appearance of a population of monocyte-derived DCs in the draining lymph node, early release of IL-12p70 and IFN-γ, and low IL-10 in the serum. These results suggest that the activation of existing DC subsets is not sufficient for the induction of antitumor immune responses, whereas early induction of Th1 cytokines and monocyte-derived DCs are features of successful activation of antitumor immunity.

218: Mechanisms of lytic HSV-1 clearance during primary infection of the trigeminal ganglia

Herpes simplex virus type 1 (HSV-1) is a prevalent human pathogen worldwide. HSV-1 remains lytic and resists entry into latency within neurons of the trigeminal ganglia (TG) in the absence of type I interferon (IFN). However, the immunologic mechanisms involved in clearing lytic virus in the TG during primary infection remain poorly defined. HSV-1 is able to develop progeny virus during primary infection in the TG, suggesting that the establishment of HSV-1 latency is not simply due to infection of a non-permissive neuron. To address this issue, the immune response to HSV-1 in the TG was examined in a murine model of ocular infection. Viral titer and leukocyte infiltration in TG were surveyed in bone marrow (BM) chimeras generated from C57BL/6 wildtype (WT) mice and mice deficient in the type I IFN receptor alpha chain (CD118 −/− ) to determine the contribution of type I IFN signaling in resident and BM-derived cells. Data show that both resident and BM-derived cells contribute to type I IFN-mediated clearance of lytic virus within the TG. Moreover, HSV-1 titers correlated inversely with infiltrating NK cells, CD8 + T cells and HSV-specific CD8 + T cells. These results were supported by the observation that gBT-I.1 TCR transgenic mice in which CD8 + T cells are specific for the immunodominant epitope of HSV-1 glycoprotein B have significantly less virus in the TG compared to WT following infection. CD8 T cell depletion at the site of infection showed a trend towards increased virus in the TG and systemic depletion is currently being pursued. Infection of WT and STING-deficient mouse TG in situ revealed that the innate sensing pathway critical for production of type I IFN in peripheral tissues is dispensable in the TG. Understanding the immune response to lytic HSV-1 infection in the TG is critical for rational prophylactic HSV-1 vaccine design.

Abstract 58: Signal Transducer and Activation of Transcription (STAT) 4 Deficiency Reduces Adipose Tissue Inflammation and Atherosclerosis

Adipose tissue (AT) inflammation is key for the development of insulin resistance (IR) and contributes to progression of atherosclerosis. Previous work from our group showed that STAT4 deficiency is protective for IR in obesity and reduces atherosclerosis. However, the mechanisms are not completely understood. In this study we examined the effect of STAT4 deficiency on peri-vascular and visceral AT inflammation in atherosclerosis. To this purpose we: i) generated STAT4-deficient apo E-deficient ( Stat-4 -/- Apoe -/- ) mice and, ii) used a novel small molecule inhibitor of the IL12/STAT4 pathway (Cpd9) in Apoe -/- mice. Compared to Apoe -/- controls, Stat-4 -/- Apoe -/- female mice fed Western diet (WD) for 12 weeks showed a ~40% reduction in the thoracic aorta lesion area. Peri-aortic AT immune cells analyzed by flow cytometry showed a significant (p<0.001) and prominent decrease in CD45+CD3+ (3-fold), CD3+CD8+ (10-fold) and Nk46+ (9-fold) cells in Stat-4 -/- Apoe -/- vs. Apoe -/- mice. Also, the percentage of CD206+/F4/80+ macrophages was increased by ~8.5-fold, while the CD45+/F4/80+ cells were reduced by ~5-fold in the Stat-4 -/- Apoe -/- mice (p<0.01). In addition gene expression of TNFα and IL12p40 were both reduced by 3.5 and 6-fold, respectively in Stat-4 -/- Apoe -/- , while PPARγ expression was increased by 2-fold (p<0.05). Peri-gonadal adipose tissue showed similar reduction in CD8+ and NK cell infiltration in STAT4 deficient mice but no differences were found in macrophage numbers or phenotype. The short-term effects of IL12/STAT4 inhibition on AT inflammation were examined in Apoe -/- mice on 7 days WD alone or with oral Cpd9 treatment (0.3mmol/kg). Gene array analysis showed between 2.5- and 6-fold reduction of several cytokines and chemokines such as Ccl2, Ccl12, Cxcl13. Also, adiponectin as well and Bmp2, 4 and 7 were 4- to 15-fold reduced, suggesting reduced inflammation and possibly adipogenesis. Vegfa expression was one of the 4 up-regulated genes by 2.5-fold in Cpd9 treated mice. Collectively, data show that STAT4 reduction has early beneficial effects on AT inflammation and that peri-aortic AT inflammation is dramatically reduced. Thus targeting STAT4 may be a novel approach to treat atherosclerosis by reducing peri-vascular AT inflammation.

Tumor microenvironment induces selective migration of specific subsets of natural killer cells and inhibits their cytolytic function. (P2039)

Abstract Cytolytic function of NK cells plays an important role to regulate the cancer growth. We hypothesized that inflamed tumor microenvironment regulates the migration of specific subset of NK cells and inhibits their cytolytic function. To investigate the affect of tumor microenvironment on NK cell migration and function, B16F10 melanoma cells (1 X 106 cells/mouse) were injected (s.c.) into C57BL/6 mice. Depletion of NK cells showed 3-4 fold increased tumor growth compared to control IgG treated mice. Tumor transplanted (day 10) and naïve mice did not showed any significant difference in percentage and absolute number of CD11b+CD27+, CD11b+CD27- or CD11b-CD27+ NK cells in spleen and lymph nodes. However, CD11b+CD27+CD3-NK1.1+ cells were selectively recruited into the tumor. At day 5, increase CD31+ endothelial cell staining were observed. qRT-PCR analysis of tumor associated endothelial cells (CD31+CD45- cells) showed increased CCL2, CCL21, CXCL10, and CXCL12 mRNA expression compared to splenic blood endothelial cells. Tumor infiltrating NK cells (day 13) showed increased expression of CCR2, CXCR4, IL-6, FasL mRNA expression, and decreased perforin, IFNg and IL-21 expression compared to splenic NK subsets. These results demonstrate that inflamed tumor microenvironment selectively recruits a specific subset of NK cell and inhibit their cytolytic function. This suggests that modulating tumor microenvironment may be key to boost the NK cell function to control cancer progression.

Vaccinia virus protein K7 is a virulence factor that alters the acute immune response to infection

Vaccinia virus (VACV) encodes many proteins that antagonize the innate immune system including a family of intracellular proteins with a B-cell lymphoma (Bcl)-2-like structure. One of these Bcl-2 proteins called K7 binds Toll-like receptor-adaptor proteins and the DEAD-box RNA helicase DDX3 and thereby inhibits the activation of NF-κB and interferon regulatory factor 3. However, the contribution of K7 to virus virulence is not known. Here a VACV lacking the K7R gene (vΔK7) was constructed and compared with control viruses that included a plaque purified wt (vK7), a revertant with the K7R gene reinserted (vK7-rev) and a frame-shifted virus in which the translational initiation codon was mutated to prevent K7 protein expression (vK7-fs). Data presented show that loss of K7 does not affect virus replication in cell culture or in vivo; however, viruses lacking the K7 protein were less virulent than controls in murine intradermal (i.d.) and intranasal (i.n.) infection models and there was an altered acute immune response to infection. In the i.d. model, vΔK7 induced smaller lesions than controls, and after i.n. infection vΔK7 induced a reduced weight loss and signs of illness, and more rapid clearance of virus from infected tissue. Concomitantly, the intrapulmonary innate immune response to infection with vΔK7 showed increased infiltration of NK cells and CD8+ T-cells, enhanced MHC class II expression by macrophages, and enhanced cytolysis of target cells by NK cells and VACV-specific CD8+ T-cells. Thus protein K7 is a virulence factor that affects the acute immune response to infection.

Growth inhibition of different human colorectal cancer xenografts after a single intravenous injection of oncolytic vaccinia virus GLV-1h68

BackgroundDespite availability of efficient treatment regimens for early stage colorectal cancer, treatment regimens for late stage colorectal cancer are generally not effective and thus need improvement. Oncolytic virotherapy using replication-competent vaccinia virus (VACV) strains is a promising new strategy for therapy of a variety of human cancers.MethodsOncolytic efficacy of replication-competent vaccinia virus GLV-1h68 was analyzed in both, cell cultures and subcutaneous xenograft tumor models.ResultsIn this study we demonstrated for the first time that the replication-competent recombinant VACV GLV-1h68 efficiently infected, replicated in, and subsequently lysed various human colorectal cancer lines (Colo 205, HCT-15, HCT-116, HT-29, and SW-620) derived from patients at all four stages of disease. Additionally, in tumor xenograft models in athymic nude mice, a single injection of intravenously administered GLV-1h68 significantly inhibited tumor growth of two different human colorectal cell line tumors (Duke’s type A-stage HCT-116 and Duke’s type C-stage SW-620), significantly improving survival compared to untreated mice. Expression of the viral marker gene ruc-gfp allowed for real-time analysis of the virus infection in cell cultures and in mice. GLV-1h68 treatment was well-tolerated in all animals and viral replication was confined to the tumor. GLV-1h68 treatment elicited a significant up-regulation of murine immune-related antigens like IFN-γ, IP-10, MCP-1, MCP-3, MCP-5, RANTES and TNF-γ and a greater infiltration of macrophages and NK cells in tumors as compared to untreated controls.ConclusionThe anti-tumor activity observed against colorectal cancer cells in these studies was a result of direct viral oncolysis by GLV-1h68 and inflammation-mediated innate immune responses. The therapeutic effects occurred in tumors regardless of the stage of disease from which the cells were derived. Thus, the recombinant vaccinia virus GLV-1h68 has the potential to treat colorectal cancers independently of the stage of progression.

Abstract IA25: Immune cells promote metastasis and control dormancy

Abstract While metastasis is the main cause of death by cancer, the nature, location and polarization of immune cells that infiltrate the primary tumor often constitute an independent predictor of cancer patient survival. In addition, several studies have shown that tumor-infiltrating immune cells correlate with clinical responses to treatments, including chemo-, radio- and targeted therapies. However, the underlying mechanisms of these correlations are still poorly understood. More efficient stratification of cancer patients based on immune cell infiltration could help in disease management and development of novel therapeutic strategies. Using a spontaneous mouse model of uveal melanoma, we showed that myeloid-derived suppressor cells (MDSC) preferentially accumulate in the primary tumor under the influence of CXCR2 ligands. These MDSC are known to suppress the immune response. In addition, they provide growth and angiogenic factors which promote cancer cell proliferation. Our study showed that granulocytic MDSC also promote cancer cell dissemination and metastasis by inducing epithelial-mesenchymal transition (EMT) in cancer cells. Accordingly, depletion of MDSC reduces cancer cell dissemination and metastasis 1. This novel role of MDSC in promoting metastasis likely explains their negative effect on survival. Indeed, primary tumors infiltrated with granulocytic cells correlate with increased risk of metastasis, accelerated progression and shorter survival in several types of human cancer. In the same mouse model, we observed that cancer cells start to disseminate at 3 weeks of age but macroscopic metastases only develop after 3-6 months. CD8+ T cells play a crucial role in controlling the dormancy of disseminated cancer cells since CD8+ T cell depletion accelerates the development of visceral metastases and shortens mouse survival. Surprisingly however, CD8+ T cell depletion has no effect on the development and growth of cutaneous metastases. In fact, CD8+ T cells poorly infiltrate cutaneous tumors. CD8+ T cell infiltration is regulated by chemokines, especially CXCR3 ligands and CCL5. By overexpressing these chemokines in cutaneous tumors, we were able to restore CD8+ T cell infiltration. We have identified several chemotherapeutic drugs that induce CXCR3 ligands and CCL5 expression in melanoma cell lines. Systemic treatment of tumor-bearing mice with these chemotherapies also induces intratumoral expression of these chemokines and promotes T cell infiltration into cutaneous tumors. Interestingly, in a cohort of melanoma patients treated with dacarbazine, T cell infiltration correlates with chemokine expression after chemotherapy. Moreover, chemotherapy augments chemokine expression and T cell infiltration only in chemotherapy-responsive tumors. Increased intratumoral expression of these chemokines after chemotherapy correlated with prolonged patient survival 2. In patients with hepatocellular carcinoma, CXCR3 ligands and CCL5 also control T and NK cell infiltration. In this cancer however, pro-inflammatory cytokines and TLR ligands induce intratumoral expression of these chemokines. Taken together, these data indicate that intratumoral chemokines control immune cell infiltration. Accordingly, different chemokines attract different types of immune cells, thereby shaping either a protective or a progressive environment. This network of chemokines and immune cells may be context-dependent and vary with the type of cancer and tumor location. Further identification of the signals that regulate chemokine expression in tumors may facilitate the prediction of disease evolution and response to therapy. Rational manipulation of these signals should help improve the efficacy of existing and future anti-cancer therapies. 1. Toh, B., et al. 2011. Mesenchymal transition and dissemination of cancer cells is driven by myeloid-derived suppressor cells infiltrating the primary tumor. PLoS Biol. 9: e1001162. 2. Hong, M., et al. 2011. Chemotherapy induces intratumoral expression of chemokines in cutaneous melanoma, favoring T-cell infiltration and tumor control. Cancer Res. 71: 6997-7009. Citation Format: Benjamin Toh, Valerie Chew, Muly Tham, Jean-Pierre Abastado. Immune cells promote metastasis and control dormancy. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr IA25.

Lack of MICA Expression Predicts a Worse Prognosis in Patients with Bladder Cancer

Background: Bladder and lung cancer are among the ten most common cancers in both genders. The NKG2D receptor and one of its ligands, MICA, are associated with smoking and susceptibility to both chronic obstructive pulmonary disease and lung cancer. Objective: We hypothesized that NKG2D-MICA system was associated with other smoking-related epithelial cancers such as bladder cancer. Design, Setting, and Participants: 70 cases of primary non-muscle invasive bladder cancer were screened for the MICA expression and CD8+, CD4+ and NK cell infiltration. Most patients (n = 55, 78.6%) were current or former smokers. Measurements: Tissue microarray (TMA) technology was chosen to evaluate MICA and tumor infiltrating lymphocytes in samples with confirmed bladder cancer. Kaplan-Meier curves and univariate Cox analysis was used to assess relapse, all-cancer mortality and specific bladder cancer mortality. Results and Limitations: MICA was expressed in most cancer specimens examined (i.e., 70%). Relapse of bladder cancer was not associated with the status of MICA expression (log rank p = 0.1123). Nevertheless, a significant association existed between high MICA expression and bladder cancer mortality (HR = 0.25; CI95% = 0.06 - 0.97). Tumor infiltrating CD4+ and CD8+ lymphocytes were found in the majority (64%) of samples. Cells expressing the NKG2D receptor were found in only 3% of the samples. There was no linear function between NKG2D+ cells and number or ratio of CD4+ and CD8+ TIL. Conclusions: MICA is expressed in a significant proportion of bladder carcinomas. MICA expression associates with significant survival advantages in the face of both all-cancer and bladder cancer. The NKG2D-MICA system could represent a common mechanism involved in the immunopathology and natural history of bladder neoplasms.

Ex vivo perfusion of HLA‐E/CD46 transgenic pig limbs with human blood: evaluation of NK cell recruitment

In vitro, interactions between human NK cells and porcine endothelial cells (pEC) are characterized by NK cell recruitment and cytotoxicity.NK cells play a role in allo‐ and xenotransplantation due to the incompatibility of the MHC class I molecules expressed on transplanted cells and tissues on one hand, and the MHC class I specific inhibitory NK receptors of the recipient on the other hand. In this context we published previously that: (i) the expression of human HLA‐E on pEC partially protects from polyclonal huNK cytotoxicity and completely protects from killing by NKG2Abright NK clones in vitro; but does not affect the adhesion of huNK cells to pEC or the heteroconjugate formation between huNK and porcine cells (1, 2); (ii) lymphoblasts and pEC derived from HLA‐E/human beta2microglobulin transgenic pigs are effectively protected against huNK cell‐mediated cytotoxicity and inhibit the secretion of IFN‐gamma by co‐cultured huNK cells in vitro, depending on CD94/NKG2A expression on the NK cells (3); (iii) HLA‐G expression on pEC protects partially against direct huNK cytotoxicity but not against ADCC (4); (iv) HLA‐G expression inhibits rolling adhesion of activated huNK cells on pEC (5, 6); (v) HLA‐Cw3 expression on pEC protects against NK cell‐mediated cytotoxicity (7), HLA‐Cw4 reduced NK cell adhesion and cytotoxicity; HLA‐Cw4 and HLA‐Cw3 co‐expression is not sufficient to completely overcome NK cytotoxicity via recognition of the KIR CD158a (KIR2DL1) and CD158b (KIR2DL2/3) receptors (8).The aim of this study was to evaluate NK cell recruitment and infiltration of porcine tissues using an ex vivo perfusion system of HLA‐E transgenic pig legs with human blood. Over the past year, a pilot study has been set up in order to establish an experimental protocol that would allow for the study and the evaluation of the rejection mechanisms occurring during xenotransplantation and especially following reperfusion of pig organs with human blood (9). Amputated pig legs were perfused with heparinised human blood over a period of 12h. Blood samples were collected at several time points and blood cell populations were analyzed by flow cytometry (FACS) analysis.Two wild type and 2 HLA‐E/CD46 transgenic pig legs were perfused ex vivo. A strong diminution of NK cells was observed after 7 h of perfusion in both the wild type and the transgenic legs. However, this decrease occurred earlier in the wild type leg than in the transgenic. In addition, the apparition of a SLA‐I+ cells population of pig origin of higher granularity than the lymphocyte population over time was observed. We hypothesized that these cells could be pig endothelial cells detached from the vessel wall due to xenorejection, or alternatively originating from the pig bone marrow. These results confirmed previous in vitro studies demonstrating that pEC damage was mediated by human NK cells. Moreover, the expression of HLA‐E/CD46 provided a partial protection with regard to NK cell recruitment and tissue infiltration.From these preliminary experiments the following conclusions were drawn:Pig leg perfusion with human blood was feasible for up to 12 h with stable perfusion parameters thus extending the observation time compared to previously described pig kidney, lung and beating heart perfusion systems. pH and potassium were maintained at normal levels and muscle stimulation resulted in contraction throughout the entire perfusion. Thus the pig leg reperfusion system appears to be a stable and optimized system to study rejection mechanisms over several hours in the pig‐to‐human xenotransplantation setting.This system allows for the study of the dynamics of the different cell populations in the blood. Indeed, a strong diminution of NK, NKT and T cells was observed.In addition, this system enables to study the apparition of cells of pig origin in the circulating blood.In conclusion, this system represents a powerful tool to study the basic molecular mechanisms taking place in a setting of xenotransplantation, and in particular to evaluate the protection from early cell‐mediated rejection mechanisms in tissues originating from transgenic pigs.

NKG2D ligand expression in glomeruli correlates with renal NK cell infiltration in the MRL mouse model

NKG2D ligand expression in glomeruli correlates with renal NK cell infiltration in the MRL mouse model

Anti-tumor immune responses of tumor-associated macrophages via toll-like receptor 4 triggered by cationic polymers

Agonists of toll-like receptors (TLRs) are potential therapeutic reagents for cancer immunotherapy. Cationic polymers such as polyethyleneimine (PEI) with nucleic acid drug delivery capability are approved for use in clinical trials, and recent reports indicate that these cationic polymers have significant immunological activity mediated by TLRs. In the present study, we demonstrated that cationic polymers such as PEI and cationic dextran could reverse tumor-associated macrophages (TAMs) polarization and promote IL-12 expression both in vitro and in vivo. The stimulatory role of cationic polymers was remarkably attenuated in TAMs pre-treated with TLR-4 blocking antibody or TAMs from TLR-4 knockout mice. Additionally, these cationic polymers exerted direct tumoricidal activity by promoting Th 1 and NK cell infiltration, suppressing tumor angiogenesis and prolonging the survival of sarcoma-bearing wild-type. These phenomena were abrogated in TLR-4 knockout mice, suggesting that the immune stimulation was primarily mediated by TLR-4. In conclusion, these results demonstrated that cationic polymers could transform the immunotolerogenic phenotype of TAMs through TLR-4 signaling, thereby promoting therapeutic anti-tumor immunity. Our present study suggests a new class of drugs as a candidate for future cancer immunotherapy.

IL-2- or IL-15-activated NK cells enhance Cetuximab-mediated activity against triple-negative breast cancer in xenografts and in breast cancer patients

Triple-negative breast cancer (TNBC) patients do not benefit from target-specific treatments and is associated with a high relapse rate. Epidermal growth factor receptor is frequently expressed in TNBC and is a candidate for new therapies. In this work, we studied Cetuximab-mediated immune activity by NK cells. Thirteen activating/inhibitory receptors were examined on peripheral blood and tumor infiltrating NK cells. NK-cell functionality was evaluated using as effectors tumor-modulated NK cells and NK cells from patients. We evaluated the treatment with Cetuximab plus IL-2 or IL-15 in vivo in TNBC xenografts. Tumor NK-cells receptor profile showed upregulation of inhibitory receptors and downregulation of activating ones. Tumor-modulated NK cells were less cytotoxic. They could perform antibody-dependent cellular cytotoxicity (ADCC) triggered by Cetuximab, although impaired, it could still be restored by stimulation with IL-2 or IL-15. Patients with advanced disease displayed diminished levels of ADCC compared to healthy volunteers. ADCC was restored and potentiated with both cytokines, which were also effective in enhancing the therapeutic activity of Cetuximab in vivo. The combination of Cetuximab with IL-15 and IL-2 may be considered an attractive therapeutic approach to enhance the clinical efficacy of Cetuximab in TNBC.

Altered phenotype in peripheral blood and tumor-associated NK cells from colorectal cancer patients

Despite NK cells being originally identified because of their ability to kill tumor cells in vitro, only limited information is available on NK cells infiltration of malignant tumors, especially in humans. NK cells infiltrating human colorectal carcinomas (CRCs) were analyzed to identify their potential protective role in an antitumor immune response. The expression and function of relevant molecules were analyzed from different sources, comparing tumor-associated NK cells (TANKs) with autologous peripheral blood NK cells (PB-NKs) from CRC patients-the latter in comparison with PB-NKs from normal donors. TANKs displayed a profound alteration of their phenotype with a drastic reduction of NK cell receptor expression. Co-culture experiments showed that CRC cells produce modulation in NK phenotype and functionality. Moreover, PB-NKs from CRC patients also exhibited an altered phenotype and profound defects in the ability to activate degranulation and IFN-γ production. For the first time, TANK and PB-NK cells from CRC patients have been characterized. It is shown that they are not capable of producing relevant cytokines and degranulate. Taken together, our results suggest that NK cells from CRC patients present alterations of phenotype and function therefore supporting the progression of cancer.

Melanoma cells become resistant to NK‐cell‐mediated killing when exposed to NK‐cell numbers compatible with NK‐cell infiltration in the tumor

During the past few years, a number of studies reported that different melanoma cell lines could be extensively lysed in vitro by IL-2-activated NK cells at appropriate effector/target ratios. Here, we show, by histological evaluation of different melanoma lesions, that NK/target-cell ratios compatible with those allowing efficient melanoma cell killing in vitro are hardly reached at the tumor site. We then investigated the outcome of cocultures established at low NK/melanoma cell ratios. After initial NK-mediated lysis, residual melanoma cells acquired resistance to IL-2-activated NK cells. This reflected primarily an increased expression, on melanoma cells, of classical and nonclassical HLA class I molecules, accompanied by a partial downregulation of NKG2D-ligands, and was dependent on NK-mediated IFN-γ release. Consistently, melanoma lesions showed a higher HLA class I expression on tumor cells that were proximal to infiltrating NK cells. In long-term cocultures, the "protective phenotype" acquired by melanoma cells was lost over time. However, this phenomenon was counteracted by downregulation of relevant activating receptors in cocultured NK cells. Analysis of different NK-cell-activating cytokines indicated that IL-15 can partially overcome this novel tumor escape mechanism suggesting that IL-15, rather than IL-2, may be eligible for NK-cell-based immunotherapy.

Densely Granulated Murine NK Cells Eradicate Large Solid Tumors

Natural killer (NK) cells inhibit early stages of tumor formation, recurrence, and metastasis. Here, we show that NK cells can also eradicate large solid tumors. Eradication depended on the massive infiltration of proliferating NK cells due to interleukin 15 (IL-15) released and presented by the cancer cells in the tumor microenvironment. Infiltrating NK cells had the striking morphologic feature of being densely loaded with periodic acid-Schiff-positive, diastase-resistant granules, resembling uterine NK cells. Perforin-mediated killing by these densely granulated NK cells was essential for tumor eradication. Expression of the IL-15 receptor α on cancer cells was needed to efficiently induce granulated NK cells, and expression on host stromal cells was essential to prevent tumor relapse after near complete destruction. These results indicate that IL-15 released at the cancer site induces highly activated NK cells that lead to eradication of large solid tumors.

Abstract LB-220: Translational research with RG7160 (GA201) leads to a phase II clinical study in combination with FOLFIRI in 2nd line metastatic colorectal cancer (mCRC)

Abstract GA201 is a novel dual-acting humanized, engineered IgG1 anti-EGFR mAb designed to enhance ADCC in combination with signaling inhibition. Superior efficacy was demonstrated versus cetuximab in orthotopic CRC xenograft models. Preclinical data indicated an increase in macrophages (4-5 fold) and NK cells (2-3 fold) infiltration in tumors treated with GA201 compared to cetuximab. In a phase I clinical study objective responses and long lasting disease stabilizations were observed. A marked reduction in circulating NK cells and an increased infiltration of immune cells into skin rash was seen. Preliminary evidence of the enhanced ADCC capacity of GA201 was investigated in 25 third line patients with KRAS-mutant mCRC. Best overall response was SD in 40% of patients and median OS was 9.4 months. Reduction in peripheral NK cells and regulatory T-cells was observed. Comparison of pre- and post-treatment tumour biopsies revealed that tumour-infiltrating immune cells, in particular CD68+ and CD3+ cells increased overall. EGFR-membrane staining in baseline tumour biopsies was markedly higher than in the corresponding archival tumour specimens (median H-score 52 vs 3, respectively), which might reflect technical, but most importantly, biological variability. Based on these results and the commitment to investigate the tumor immune infiltration profile as a potential predictive biomarker, a fresh tumor biopsy at baseline was mandated in the ongoing randomized phase II trial (GAIN-C). This compares GA201 plus irinotecan, infusional fluorouracil and leucovorin (FOLFIRI) with FOLFIRI alone in KRAS-mutant 2nd line mCRC patients and with cetuximab plus FOLFIRI in KRAS-wild type patients (n=160). Primary objective is PFS. The fresh tumor biopsy will be centrally analyzed for EGFR and KRAS. A comprehensive biomarker program was implemented to investigate potential predictive biomarkers, with an emphasis on the real time tumor immune-infiltration status. The safety run-in phase with 36 patients was completed. Most common treatment related AEs on GA201 arms vs cetuximab vs. chemotherapy included (≥ grade 3): rash (33 vs. 17 vs. 0%), IRR (10 vs. 0 vs. 0%), diarrhea (17 vs. 0 vs. 11%), fatigue/asthenia (0 vs. 17 vs. 0%), hypomagnesaemia (17 vs. 0 vs. 0%), stomatitis (17 vs. 0 vs. 0%) and vomiting/nausea (6 vs. 0 vs. 0%). So far, no patient discontinued due to EGFR-related skin toxicity. Immunological profiling is ongoing. Extensive research efforts, including a fresh tumor biopsy in second line mCRC patients, are taken to understand the immunological mechanism of action of GA201 and to select and test (ph. II) and validate (ph. III) potential predictive biomarkers. To guide personalized cancer immunotherapies there is a clear need for reliable biomarkers which would necessitate a tumor assessment directly prior to the therapy and immune monitoring throughout treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-220. doi:1538-7445.AM2012-LB-220

Prevention of colorectal cancer liver metastasis by exploiting liver immunity via chitosan-TPP/nanoparticles formulated with IL-12

The development of effective therapies for the prevention of colorectal cancer (CRC) liver metastasis is of great importance. Recently, chitosan (CS) nanoparticles have been utilized as carriers of interluekin-12 (IL-12) administered locally to deliver therapeutic proteins and genes. In this study, we encapsulated IL-12 by incorporation using tripolyphosphate (TPP) as the coacervated crosslinking agent to form CS-TPP/IL-12 nanoparticles. We further characterized the association efficiency, rate of release, liver-targeting, and toxicity, which were predominantly dependent on the factors of particle size, zeta potential, pH of solution, and whether or not modified with TPP. Systemic delivery of CS-TPP/IL-12 nanoparticles significantly reduced the number and volume of CRC liver metastasis foci compared to the CS-TPP treated mouse group. Although delivery of IL-12 alone also inhibited the number of CRC liver metastasis observed, further study of the change in hepatic metastasis volume demonstrated no significant differences between the groups treated with CS-TPP or IL-12 alone. Mechanistically, CS-TPP nanoparticles blocked the toxicity of IL-12 and induced infiltration of NK cells and some T cells, which are most likely the effector cells that mediate tumor metastasis inhibition during CS-TPP/IL-12 immunotherapy. The results obtained from this study demonstrate the potential benefit of using chitosan modification technology as a cytokine delivery system for the successful prevention of CRC liver metastasis by exploiting liver immunity.

Modification of Chemokine Pathways and Immune Cell Infiltration as a Novel Therapeutic Approach in Liver Inflammation and Fibrosis

Despite increasing knowledge about molecular pathways in pathogenesis of chronic liver disease, selective therapeutic options are scarce, especially in advanced diseases characterized by scarring of the liver (termed fibrosis) or even complete cirrhosis. Sustained hepatic inflammation as a result to various types of injury (e.g., hepatitis C, nonalcoholic steatohepatitis) is generally accepted to represent the key prerequisite for fibrogenesis. Liver inflammation is characterized by an activation of distinct chemokine pathways in the liver and the circulation allowing distinct immune cell populations to enter the liver via sinusoids and postsinusoidal venules. Recent investigations have shed light on the intimate interactions between the fibrogenic hepatic stellate cell (HSC) and infiltrating immune cells, which fundamentally drive liver scarring. Experimental fibrosis and inflammation models have demonstrated that disruption of chemokine pathways such as CCL2 (MCP-1) or its receptor CCR2, CCL5 (RANTES) or CCR1 / CCR5 and others may efficiently prevent collagen deposition, by targeting monocytes and macrophages, T-cell populations or NKT cells. However, immigration of certain mononuclear cells may even be beneficial in the course of fibrosis. Infiltrating NK cells and monocyte-derived macrophage subsets can promote resolution of extracellular matrix. This emphasizes that hepatic fibrosis is not a unidirectional process, but can be reverted up to a certain point. The present review aims at summarizing the contribution of immune cell infiltration as well as related chemokine systems to experimental liver fibrosis and will discuss possible therapeutic applications in humans, with a special emphasis on the monocyte/macrophage lineage and their related chemokine pathways.