Immunomodulatory drugs stimulate natural killer‐cell function, alter cytokine production by dendritic cells, and inhibit angiogenesis enhancing the anti‐tumour activity of rituximab in vivo - Reddy - 2008 - British Journal of Haematology - Wiley Online Library Free Access Immunomodulatory drugs stimulate natural killer-cell function, alter cytokine production by dendritic cells, and inhibit angiogenesis enhancing the anti-tumour activity of rituximab invivo Nishitha Reddy, Departments of Medicine ImmunologySearch for more papers by this authorFrancisco J. Hernandez-Ilizaliturri, Departments of Medicine ImmunologySearch for more papers by this authorGeorge Deeb, ImmunologySearch for more papers by this authorMark Roth, ImmunologySearch for more papers by this authorMary Vaughn, PathologySearch for more papers by this authorJoy Knight, ImmunologySearch for more papers by this authorPaul Wallace, Flow Cytometry, Roswell Park Cancer Institute, Buffalo, NY, USASearch for more papers by this authorMyron S. Czuczman, Departments of Medicine ImmunologySearch for more papers by this author Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URLShare a linkShare onEmailFacebookTwitterLinked InRedditWechat Summary The immunomodulatory drugs (IMiDs) lenalidomide and actimid (also known as CC-4047) are thalidomide analogues which are more potent than their parental compound. In combination with rituximab, we have previously demonstrated that IMiDs have synergistic invivo anti-tumour activity in preclinical studies in a human lymphoma severe combined immunodeficiency mouse model. This report further explored the mechanisms by which IMiDs exert their anti-lymphoma effects. Following exposure of subcutaneous lymphoma tumours in murine models to IMiDs, there was a significant increase in the recruitment of natural killer (NK) cells to tumour sites. This increase in NK cells was mediated via stimulation of dendritic cells and modification of the cytokine microenvironment associated with an increase in monocyte chemotactic protein-1, tumour necrosis factor-α and interferon-γ and probably augmented rituximab-associated antibody-dependent cellular cytotoxicity. IMiDs also had significant anti-angiogenic effects in our B-cell lymphoma models. Thus, by modulation of the immune system mediated via dendritic cells and NK cells, changing the cytokine milieu, as well as by their anti-angiogenic effects, IMiDs in combination with rituximab resulted in augmented invivo anti-tumour effects against B-cell lymphoma. Our positive preclinical data adds additional support for the evaluation of IMiDs plus rituximab in patients with relapsed/refractory B-cell lymphoma. Immunomodulatory drugs (IMiDs) [i.e. lenalidomide and actimid (also known as CC-4047)] are thalidomide derivatives with augmented anti-tumour activity and safer toxicity profiles. Lenalidomide has shown promising anti-tumour activity in clinical studies involving patients with multiple myeloma, amyloidosis, chronic B-cell lymphocytic leukaemia (CLL) and B-cell lymphoma (List etal, 2005, 2006; Rajkumar etal, 2005; Chanan-Khan etal, 2006; Dispenzieri etal, 2007). Thalidomide and other IMiDs possess multiple mechanisms-of-action, affecting not only cancer cells, but also immune effector and stromal cells. In addition, IMiDs affect the tumour microenvironment by inhibiting angiogenesis, shifting cytokine production and activating immune-effector cells (Davies etal, 2001; Dredge etal, 2005). Recently, lenalidomide entered clinical trials for patients with relapsed B-cell lymphoproliferative disorders. The encouraging early clinical results stress the need to further understand its mechanisms-of-action against CLL and B-cell lymphoma in an attempt to more rationally design future clinical trials integrating these novel agents with other biological or chemotherapy agents. An area of increasing scientific interest is the capacity of these novel drugs to modulate the immune system. IMiDs are known to stimulate cytotoxic T cells and promote the activity of natural killer (NK) cells by increasing levels of cytokines, such as interleukin-2 (IL-2) (Davies etal, 2001; Lentzsch etal, 2003; LeBlanc etal, 2004; Hayashi etal, 2005). Other groups of investigators have demonstrated that in follicular lymphoma, the tumour microenvironment and the non-neoplastic immunological cells infiltrating the tumour bed are predictors of survival (Dave etal, 2004). Moreover, recent cDNA micro array gene profiling studies identified two distinct immunological gene signatures with opposite clinical responses to rituximab plus CHOP (cyclophosphamide, doxorubicin, prednisone and vincristine) immunochemotherapy (Harjunpaa etal, 2006). Over the last decade, rituximab-based therapies have improved the clinical outcome of B-cell lymphoma patients without significant additional toxicity (Coiffier etal, 2002; Feugier etal, 2005). While anti-tumour activity is observed with rituximab as a single agent, the quality and durability of responses are best when it is combined with systemic chemotherapy (Czuczman etal, 1999, 2004; Coiffier etal, 2002; Feugier etal, 2005). Current challenges in the treatment of B-cell lymphomas, include (i) developing targeted therapies that enhance rituximab activity while decreasing the amount of systemic chemotherapy used (e.g. especially in elderly patients with comorbid medical issues) and (ii) identifying and validating biomarkers of response to rituximab-based therapies that can be utilized to develop tailored treatment. Altering the microenvironment and recruiting immune-effector cells to tumour sites to enhance the activity of rituximab is a novel strategy to approach these scientific challenges. We previously published on the important role that the innate immune system (i.e. neutrophils and NK cells) has on the biological activity of rituximab. Then, we studied strategies by which to enhance neutrophil [i.e. granulocyte colony-stimulating factor (G-CSF) or granulocyte–macrophage colony-stimulating factor (GM-CSF)] or NK cells (i.e. via IMiDs) in an attempt to improve rituximab activity in human lymphoma preclinical models (Hernandez-Ilizaliturri etal, 2003, 2005a,b). Subsequently, we have shown that IMiDs are capable of enhancing rituximab anti-tumour activity in a lymphoma bearing severe combined immunodeficiency (SCID) mouse model (Hernandez-Ilizaliturri etal, 2005b). This present work further characterized the mechanisms by which IMiDs affect the tumour vasculature, modulate the cytokine milieu, and activate cells of the innate immune system leading to enhanced rituximab anti-tumour activity invivo. To our knowledge, this is the first report that seeks to address the mechanisms-of-action of IMiDs against B-cell lymphoma invivo. The following culture media were used: RPMI 1640 medium (Sigma Chemical, St Louis, MO, USA) and RPMI 1640 medium supplemented with 10% heat inactivated (60°C, 45 min) fetal bovine serum (FBS, Atlanta Biologicals, Norcross, GA, USA), 5 mmol/l HEPES, penicillin 100 U/ml and 100 μg/ml of streptomycin (Invitrogen Corp., Grand Island, NY, USA). ACK lysis buffer (0·5 M NH4CL, 10 mM KHCO3 and 0·1 nM Na2EDTA at pH 7·2–7·4) was used for removal of red blood cells from splenocytes. Sodium chromate51 (51Cr) (Perkin-Elmer Life Inc., Boston, MA, USA) and [3H] thymidine radioisotopes (Perkin-Elmer Life Inc., Boston, MA, USA) were utilized in functional assays assessing antibody-associated cytotoxicity and cell proliferation respectively. Triton X-100 was purchased from Sigma Chemical, St Louis, MO, USA. The Raji cell line is a well-characterized B-lymphoblastic cell line (phenotype: CD20+, CD19+ and CD22+) obtained from the American Tissue and Cell Collection (Manassas, VA, USA). Cells were cultured and maintained in RPMI media supplemented with 10% heat-inactivated FBS, 5 mmol/l HEPES, penicillin 100 U/ml and 100 μg/ml of streptomycin. Cultured cells were periodically tested for mycoplasma and other pathogenic contaminants. Lenalidomide (RevlimidTM) and actimid were obtained from Celgene Inc. (Warren, NJ, USA) and dissolved freshly in dimethyl sulfoxide (DMSO) to make a 10 mg/ml solution. The compounds were then diluted directly into the tissue culture media at the required concentrations. For invivo studies, IMiDs stock solutions were diluted in sterile 0·9% normal saline to a final concentration of 1 mg/ml. The final concentration of DMSO in all experiments was 0·01%, and all treatment conditions were compared with vehicle controls (DMSO 0·01%). Rituximab (BiogenIdec, San Diego, CA and Genentech Inc. San Francisco CA, USA) was obtained from the Roswell Park Cancer Institute (RPCI) Pharmacy Department at a stock concentration of 10 mg/ml. The antibody was dosed at 10 mg/kg and diluted in sterile phosphate-buffered saline (PBS; 200 μg/100 μl) for tail vein injection into SCID mice. Trastuzumab (Herceptin™, Genentech Inc., San Francisco, CA, USA) was used as an isotype control. For immunohistochemistry (IHC) studies, a rat anti-mouse CD31, an endothelial cell adhesion molecule, antibody and a rat anti-mouse CD49, a pan NK-cell marker, were obtained from BD Pharmingen (San Diego, CA, USA). An isotype-matched rat IgM was used as a negative control, BD Pharmingen (San Diego CA, USA). For phenotypic analysis of murine dendritic cells (DCs) an allophycocyanin-labelled rat anti-mouse CD11c and phycoerythrin (PE)-labelled major histocompatibility complex (MHC) class II (I-A/I-E), hamster anti-mouse IgG were obtained from BD Pharmingen (San Diego, CA, USA). For phenotypic analysis of lymphoma cells, purified PE-conjugated monoclonal mouse anti-human CD19 and CD22 were obtained from Caltag (Burlingame, CA, USA). PE-conjugated mouse anti-human CD20 and CD59 as well as Cy-Chrome-conjugated mouse anti-human CD55 monoclonal antibodies were purchased from BD Pharmingen Inc (San Diego, CA, USA). PE-conjugated mouse anti-human CD3 was utilized as isotype control (BD Pharmingen San Diego, CA, USA) and mouse IgG from Sigma (St Louis, MO, USA) was utilized to block Fc receptors. To study the invivo mechanisms-of-action of lenalidomide and actimid, several experiments were conducted using a lymphoma xenograft model as previously described (Hernandez-Ilizaliturri etal, 2003). For our experiments, we utilized 6- to 8-week-old SCID mice that were bred and maintained at the Department of Laboratory Animal Resources facility at RPCI. The experimental design was approved by the Institutional Animal Care and Use Committee at RPCI (Protocols M821 and P966). All animals were housed and maintained in laminar flow cabinets or micro isolator units and provided with sterilized food and water. Our laboratory facility has been certified by the American Association for Accreditation of Laboratory Animal Care and in accordance with current regulation and standards of the United States Department of Agriculture and the United States Department of Health and Human Services. Exvivo co-stimulation of murine NK cells with IMiDs in the presence of murine DCs To study the mechanisms by which IMiDs enhance NK-cell mediated rituximab-associated antibody-dependant cellular cytotoxic antibody-dependant cellular cytotoxic (ADCC), we performed various co-stimulation exvivo studies. Murine DCs were generated as previously described (Inaba etal, 1992). Briefly, 6-week-old SCID mice were euthanized and stem cells were extracted from their femoral bones by saline flushing. Stem cells were collected in sterile flasks and cultured for 72 h in the presence of murine granulocyte–macrophage colony-stimulating factor (GM-CSF, 20 nmol/l) and IL-2 (10 μ/ml). Subsequently, lenalidomide (5 μg/ml), actimid (5 μg/ml) or DMSO (control, 0·01%) was added to the flask containing murine haematopoietic cells and cultured for an additional 48 h. The haematopoietic cells were examined by phase contrast microscopy for the development of DCs morphologic features. DC phenotype was confirmed by flow cytometric analysis. Established mature murine DCs were then co-cultured with NK-cell rich splenocytes isolated from SCID mice as previously described (Kruisbeek, 2000). NK cells were allowed to interact with DCs cells in the presence of IL-2/GM-CSF and lenalidomide, actimid or DMSO for five additional days at 37°C, 5% CO2. Co-stimulated NK cells were ultimately used in standard ADCC assays against rituximab or isotype coated 51Cr-labelled Raji cells at an effector: target ratio of 40:1. A 2 × 105 Raji cells were labelled with 51Cr by incubating for 1 h at 37°C with 3·7 MBq 51Cr, then washed three times in PBS and resuspended to a final concentration of 1 × 105 cells/ml. Cells were then seeded in 96-well plates (1 × 104 cells/well) and exposed in triplicates to rituximab, or trastuzumab at 10 μg/ml final concentration and splenocytes co-stimulated with either lenalidomide, actimid or DMSO at a effector:target ratio of 40:1. Cells were incubated at 37°C and 5% CO2 for 6 h (final volume adjusted to 200 μl). Upon completion, 100 μl of the cell supernatant was collected and the cell lysis quantification was determined using a gamma-counter. The percentage of specific lysis was calculated as follows: %lysis = [(test sample release − background release)/(maximum release − background release)] × 100. Capture and detection antibody pairs directed against different non-competing epitopes of their respective cytokine and recombinant protein standards for murine IL-2, IL-4, and monocyte chemotactic protein-1 (MCP-1), GM-CSF, were purchased from R&D Systems (Minneapolis, MN, USA). Antibodies to these cytokines were covalently coupled to Multi-Analyte carboxylated microspheres (Luminex Corp., Austin, TX, USA) through free amino groups with carbodimide and N-hydroxysuccinimide according to the manufacturer’s directions. Antibodies and standards for interferon-γ (IFN-γ), IL-10, tumour necrosis factor-α (TNF-α), and IL-12 were purchased from Invitrogen (Carlsbad, CA, USA) coupled to beads by the manufacturer. The Luminex cytokine assays were performed in 96-well microtitre plates (Multiscreen HV plates; Millipore, Billerica, MA, USA) with polyvinylidene difluoride membranes using a Tecan Genesis liquid handling robot (Research Triangle Park, NC, USA) for all dilutions, reagent additions and manipulations of the microtiter plate. Bead sets, coated with capture antibody were diluted in assay solvent, pooled and 1000 beads from each set were added per well. Recombinant protein standards were titrated using threefold dilutions in diluent. Samples and standards were added to wells containing beads. The plates were incubated at ambient temperature on a rocker, and then washed twice with wash buffer using a vacuum manifold to aspirate. Biotinylated detection antibodies to each cytokine were added next and the plates were incubated and washed as before. Finally, PE-conjugated streptavidin (Invitrogen) was added to each well and the plates were incubated and washed. The beads were resuspended in 150 μl of wash buffer and analysed on a Luminex 100 (Luminex Corp.). Blank values were subtracted from all readings. Using BeadView Software (Millipore) a log regression curve was calculated using the bead mean fluorescence intensity (MFI) values versus concentration of recombinant protein standard. Points deviating from the best fit line, i.e. below detection limits or above saturation, were excluded from the curve. Sample cytokine concentrations were calculated from their beads MFIs by interpolating the resulting best fit line. Invivo assessment of microvessel density changes following treatment with IMiDs in lymphoma-bearing SCID mice Thalidomide has antiangiogenic properties, a mechanism-of-action that has been also demonstrated in the new IMiDs (D’Amato etal, 1994; Dredge etal, 2002). We studied the ability of IMiDs to inhibit angiogenesis invivo. For this experiment, 6- to 8-week-old SCID mice were inoculated subcutaneously with 4 × 106 Raji cells in the right flank, tumours were allowed to engraft and grow until they reached a volume of at least 1·0 cm3 (day 0). Lymphoma-bearing SCID mice were treated with either lenalidomide (0·5 mg/kg), actimid (0·5 mg/kg) or DMSO (0·1%) administered i.p on days +3, +4, +8, +9, +13, +14, +18 and +19. Twenty-four hours after the last IMiD or control dose, animals were killed, tumour tissues isolated, fixed in 10% Zinc fixative (BD Pharmingen, San Diego CA, USA) for 24 h, and transferred to formalin for IHC. Paraffin-embedded sections were cut at 5 μm, placed on charged slides and dried at 60°C for 1 h. No antigen retrieval was necessary. Slides were then placed on the DAKO autostainer and run with PBS/T (1% Tween) wash and blocked in casein 0·03% for 30 min. Blood vessels were stained using a rat anti-mouse anti CD31 antibody (10 μg/ml) for 1 h. An isotype-matched control (10 μg/ml rat IgG) was used on a duplicate slide in place of the primary antibody as a negative control. A PBS/T wash was followed by the biotinylated secondary goat anti-rat Ig polyclonal antibody (BD Pharmingen, San Diego CA, USA) for 30 min. A PBS/T wash was followed by the streptavidin complex for another 30 min. PBS/T wash was used as a wash and the chromagen diaminobenzidene (DAB) was applied for 5 min. The slides were then counterstained with haematoxylin and dehydrated. Microvessel density was calculated by the RPCI Pathology Department. The tumour tissue sections were scanned at low power (100×) to determine the areas that were highest in microvascular density. After choosing the areas of interest, five low-power fields (LPF) (100×) were used to count the number of microvascular spaces, defined as closed circular or linear, straight or branched, spaces outlined completely by CD31 stain, using an Olympus light microscope BX45 (Olympus America Inc., Center Valley, PA, USA). Each experiment was performed in triplicate. In addition to studying the effects of IMiDs on angiogenesis, we studied their capacity to recruit NK cells into tumour beds. Tumours obtained from Raji-bearing SCID mice (as described above) were submitted for IHC to assess the number and pattern of NK-cell infiltration into the lymphoma bed following IMiD therapy compared with control. NK cells were detected by IHC using a rat-anti mouse CD49b antibody. Zinc-fixed paraffin blocks were cut at 5 μm, placed on charged slides and dried in a 60°C oven for 1 h. Room temperature slides were deparaffinized in three changes of xylene and rehydrated using graded alcohols. Endogenous peroxidase was quenched with aqueous 3% H2O2 for 20 min and washed with PBS/T. No antigen retrieval was required. The slides were then placed in a humidified chamber and blocked with PBS/T-casein 0·03% for 30 min. The primary antibody CD49b/PAN-NK (R-PE) (diluted to 10 μg/ml) was applied to slides and left on overnight at 4°C. An isotype-matched control (10 μg/ml rat IgM) was used on a duplicate slide in place of the primary antibody as a negative control. In the morning a PBS/T wash was followed by rabbit versus rat (DAKO) for 30 min. A PBS/T wash was followed by the rabbit Envision+ reagent (DAKO) for 30 min. This combination of reagents allowed a polymer, such as the Envision+, to be used with the rat primary even though it was made for use with a rabbit antibody. PBS/T was used as a wash and the chromagen DAB (DAKO, Carpentaria CA) was applied for 5 min (colour reaction product – brown). The slides are then counterstained with hematoxylin, and dehydrated. The differences in the amount of NK cells infiltrating the tumour bed, percentage of CD49 positive mononuclear cells out of total cells including those of the tumour, and the pattern of NK infiltration was evaluated and reported. Changes in CD20 surface expression has been observed in lymphoma cell lines and CLL cells exposed to specific cytokines (Sivaraman etal, 2000). We investigated whether an upregulation of surface CD20 expression following IMiDs exposure could explain the increase in rituximab-associated ADCC upon IMiD exposure. For these experiments, Raji cells were exposed to lenalidomide (5 μg/ml), actimid (5 μg/ml) or DMSO (control, 0·1%) for 24 and 48 h. Characterization of the phenotypic profile changes of the Raji cell line was performed with a fluorescence-activated cell sorter (FACS) using a FAC Star Plus (Becton Dickinson, Franklin Lakes, NJ, USA) flow cytometer. B-cell cluster designated (CD) antigen phenotype was determined by direct immunofluorescence using several monoclonal antibodies. Co-stimulation of murine splenocytes (NK cells) with DCs in the presence of lenalidomide or actimid enhance rituximab-associated ADCCInvitro culture of murine haematopoietic cells with IL-2 and GM-CSF resulted in successful generation of DCs as demonstrated by contrast microscopy (Fig1A). Subsequently, invitro exposure of DCs to lenalidomide or actimid resulted in maturation and activation of DCs as demonstrated by the expression of MHC-class II and CD11c antigens by flow cytometric analysis when compared with DMSO controls (Fig1B). Invitro exposure of stem cells to immunomodulatory drugs (IMiD’s) results in the generation and activation of murine dendritic cells (DCs). Murine stem cells were harvested from the femur of 6- to 8-week-old severe combined immunodeficiency mice and cultured with granulocyte–macrophages colony-forming factor (20 nmol/l) and interleukin-2 (10 μg/ml) for 72 h. Generation of DCs was determined by contrast microscopy (A). DCs were subsequently co-cultured with dimethyl sulfoxide, lenalidomide or actimid for two additional days. DC maturation and activation by flow cytometric studies staining for CD11c and class II antigen, were observed in IMiD-cultured cells as compared with control exposed DCs (B). Upon co-stimulation of murine NK cells with IMiD’s and dendritic cells, there was a significant enhancement of rituximab-dependant cellular cytotoxicity compared with the DMSO-treated murine NK cells. The mean percentage of rituximab-induced specific cell lyses for DMSO control co-stimulated NK cells was 7·7 ± 1·6% in contrast to lenalidomide co-stimulated NK cells 15·3 ± 4·3%, P = 0·007. A lesser degree of augmented ADCC was seen in actimid co-stimulated NK cells at the dose and schedule of actimed utilized (Fig2). Co-stimulation of murine splenocytes with immunomodulatory drug activated dentritic cells (DCs) enhanced rituximab-mediated invitro antibody dependent cellular cytotoxicity (ADCC) (A). DCs generated by invitro exposure to granulocyte–macrophages colony-forming factor/interleukin-2 followed by either dimethyl sulfoxide, lenalidomide or actimid were co-cultured with freshly isolated murine splenocytes [serving as a source of natural killer (NK) cells]. Co-cultured NK cells subsequently were used in standardized Cr51 release assays to assess the capacity of rituximab to effectively trigger ADCC in Raji cells. No improvement in rituximab-mediated ADCC was observed when NK cells were cultured with lenalidomide or actimid in the absence of DCs (B). PMBC, peripheral blood mononuclear cells. Moreover, splenocytes co-cultured in the presence of IL-2/GM-CSF and, lenalidomide or actimid in the absence of DCs did not induce augmented rituximab-mediated ADCC when compared with controls (data not shown). Our data suggest that DCs are necessary for the effects of IMiDs on NK cells mediated rituximab-associated ADCC. To further study potential mechanisms by which IMiDs enhance rituximab-associated ADCC, we studied changes in cytokine production by DCs/haematopoietic cells when exposed to either invitro lenalidomide or actimid. Upon co-stimulating dendritic cells with IMiDs, the concentration of various cytokines (see Materials and methods) from the supernatant was measured by flow cytometry using the luminex assay. Invitro exposure of DCs to lenalidomide resulted in an increase in mouse (m)-IFN-γ (P = 0·04), m-TNF-α (P = 0·004) and m-MCP-1 (P = 0·002) as compared with control-incubated DCs (Fig3). Of interest, INF-γ levels were significantly more elevated following invitro exposure to lenalidomide, mean value of 18·9 ± 9·1 pg/ml or actimid, mean value of 14·5 ± 2·3 pg/ml when compared with DMSO, mean value of 2·3 ± 1·4 pg/ml (P = 0·014 lenalidomide versus DMSO and P = 0·004 in the actimid versus DMSO-treated groups). Invitro exposure of dentritic cells (DCs) to lenalidomide or activid results in significant changes in the cytokine milieu. Murine DCs exposed invitro to immunomodulatory drugs secreted higher amounts of interferon-γ (A), tumour necrosis factor-α (B), and monocyte chemotactic protein-1 (C) as compared with dimethyl sulfoxide exposed DCs. Murine cytokines were quantified using a luminex cytokine assay. Results presented are the mean of triplicate experiments with standard error bars. In addition, there was also a significant elevation of TNF-α levels in the supernatant of DCs exposed to actimid (mean = 537·3 ± 336·2 pg/ml) or to lenalidomide (mean = 496·0 ± 233·2 pg/ml) when compared with DMSO-exposed cells (mean = 35·0 ± 8·9 pg/ml; P = 0·024 and P = 0·013 respectively). Furthermore, there was a significant increase in MCP-1 levels in the supernatant of DCs exposed to lenalidomide (mean 3947 ± 97·7 pg/ml) or actimid (mean 3440 ± 399·8 pg/ml) when compared with control-exposed DC supernatants (mean 353 ± 192·5 pg/ml; P   0·001 and P = 0·001 respectively) (Fig3). No changes in the levels of other cytokines (i.e. IL-2, IL-4, GM-CSF, IL-10 and IL-12) were observed in IMiD-stimulated DCs as compared with DMSO controls (data not shown). The peripheral part of the tissue with the greatest microvessel density was used to count the vessels, using CD31 staining as a surrogate of angiogenesis. The mean microvessel density in the placebo group was 109 vessels/LPF ±4·58 standard error of the mean (SEM) and in mice treated with lenalidomide, the mean micro-vessel density (MVD) was 49·6 vessels/LPF ±7·17 SEM P = 0·009 (Fig4). Similar results were seen in mice treated with actimid, where the mean MVD was 80·66 vessels/LPF ±6·56 SEM, P = 0·005 (Fig4). There was no difference in the observed pattern of distribution of vessels among treated and untreated tumour samples. Treatment invivo with lenalidomide results in a significant decrease of angiogenesis in lymphoma xenografts. Six- to 8-week-old severe combined immunodeficiency mice were inoculated with 4 × 106 Raji cells subcutaneously and subsequently treated with dimethyl sulfoxide, lenalidomide or actimid. 24 h after the last dose of immunomodulatory drugs or control, tumours were harvested and blood vessels were stained using a rat anti-mouse CD31 antibody (A–C). Blinded evaluation of micro-vessel density (MVD) was quantified and reported as vessels/low power field (LPF) (D). Images are scanned at 100×. Invivo exposure to either lenalidosmide or actimid results in changes in the number and pattern of NK-cell infiltration into the tumour bed of Raji-bearing SCID miceInvivo therapy of lymphoma-bearing SCID mice with lenalidomide resulted in both: (i) an increase in the infiltration of; and, (ii) changes in the patter of NK-cell infiltration. IHC to detect CD49b-expressing cells (murine NK cells) demonstrated that animals exposed to lenalidomide (50%) or actimid (42·5%) recruited more NK cells within the tumour bed as compared with DMSO-treated animals (30%, P = 0·015) (Fig5A). Notably, there was also a difference in the pattern of NK-cell infiltration between DMSO- and IMiD-treated animals. Tumours obtained from animals treated with lenalidomide and actimid consistently showed a central infiltration of NK cells and DMSO-exposed animals limited the infiltration of NK cells to the periphery of the tumour (Fig5B). Differences in the number and pattern of natural killer (NK) cells infiltrating the tumour bed of lymphoma-bearing severe combined immunodeficiency (SCID) mice treated with lenalidomide or actimid. Six- to 8-week-old SCID mice were inoculated with 4 × 106 Raji cells subcutaneously and subsequently treated with dimethyl sulfoxide (DMSO), lenalidomide or actimid. 24 h after the last dose of immunomodulatory drugs (IMiDs) or control, tumours were harvested and NK cells were stained using a rat anti-mouse CD49 antibody. IMiDs therapy resulted in an increase in the number of NK cells (A) into the tumour bed and interestingly, an increase in the trafficking of NK cells from the periphery into the centre of the tumour when compared with DMSO-treated animals (B and C). In an attempt to more fully understand the mechanism(s) by which IMiDs potentiate the effects of rituximab, we investigated whether lenalidomide or actimid modulated CD20 expression. To this end, Raji cells were exposed to IMiDs, at a drug concentration of 10μg/ml for 5 d. We did not observe any significant change in the CD20 antigen as measured by flow cytometric analysis. We therefore concluded that the anti-tumour effect of IMiDs is not mediated through upregulation of CD20 antigen (data not shown). The introduction of chimeric/humanized monoclonal antibodies (e.g. rituximab) has revolutionized the treatment of cancer, and in particular, B-cell non-Hodgkin lymphoma (NHL). The mechanisms-of-action of monoclonal antibodies are complex and varied and depend not only on the host immune status, but also on the expression/function of the targeted antigen, and the type of constant region of the specific monoclonal antibody. Approximately 60% of previously treated indolent lymphoma patients do not respond to a second course of rituximab after an initial response. The mechanism that governs responses to rituximab are not well defined but probably are multifactorial and include: in the degree of CD20 expression by tumour cells, inter-patient pharmacokinetics and pharmacodynamics of rituximab, phenotypic changes in tumour cells upon selective pressure leading to acquired rituximab resistance and/or status of the immune effector cells (i.e. FcγRIII genetic polymorphisms). It has been demonstrated in various preclinical models that induction of ADCC is one of the most important invivo mechanisms-of-action of rituximab. A possible mechanism by which the activity of rituximab can be enhanced is by activating the immune cells and improving rituximab-mediated ADCC. Resistance that is related to changes in tumour cells or in immune responses may be mitigated by the use of novel agents, such as IMiDs. Our group has previously demonstrated that IMiDs act synergistically with rituximab and prolong survival of human lymphoma-bearing SCID mice. We also demonstrated that this is mediated by activation of NK cells, and that invivo depletion of NK cells results in a complete loss of this synergistic activity (Hernandez-Ilizaliturri etal, 2005b). This interesting observation led us to further investigate the role of NK cells and the possibility of other mechanisms-of-action of IMiDs in NHL. As shown previously by other investigators, IMiDs have direct anti-myeloma effects and can overcome resistance to conventional therapy (Hideshima etal, 2000). It was demonstrated that in multiple myeloma, IMiDs increase the NK-cell pool by triggering the release of IL-2 secretion by T cells, an event mediated primarily by the PI-3 kinase signalling pathway (Hayashi etal, 2005). There is evidence to support that lenalidomide enhances the expansion of NK and T cells in patients with multiple myeloma and that cells activated in the presence of lenalidomide secrete IFN-γ (Chang etal, 2006). The current study further evaluated the mechanism(s) by which the invivo activity of rituximab is enhanced by IMiDs. Our data strongly suggests that both lenalidomide and actimid improve rituximab activity not by modulating (i.e. increasing) the expression of CD20, but by expanding, activating and trafficking NK cells into the tumour bed and thereby facilitating a more efficient ADCC. The effect on NK cells by IMiDs observed in our murine system appears to be dependant on the presence of dendritic cells and the cytokines secreted by these cells. The effects of IMiDs on splenocytes, which are rich in NK cells, are lost in the absence of DCs, suggesting that IMiDs may act as co-stimulatory molecules to NK cells via activation of DCs. It is important to notice differences in the anti-tumour activity between revlimid and actimid when combined with rituximab invitro and invivo. In our previous report (Hernandez-Ilizaliturri etal, 2005b), actimid enhanced the activity of rituximab to a greater extent than revlimid in our murine lymphoma xenograft model. In the present work, revlimid appears to be more effective in enhancing the activity of rituximab-associated ADCC invitro. Differences noted could be the results of variability observed in the types of models used (invivo versus invitro) or to differences in pharmacokinetics between revlimid and actimid in SCID mice. Similar to that study, which has been demonstrated by other investigators, IMiDs can affect the cytokine milieu. Measurement of cytokines in the supernatant of dendritic cells co-cultured with IMiDs, demonstrated an increase in INFγ, TNF-α and MCP-1. Thalidomide analogues exert a specific pattern of cytokine secretion by peripheral blood mononuclear cells and were noted to be potent inhibitors of TNF-α (Corral etal, 1999). We demonstrate an increase in the secretion of TNF-α by the DCs stimulated by IMiDs. This appears to be a protective mechanism of the host immune response. Others have demonstrated an increase in INFγ, as measured in the plasma level of patients treated with IMiDs (Davies etal, 2001). Cytokines play an important role in localizing normal or malignant B cells within tissues (Husson etal, 2001). The chemokines secreted by follicular dendritic cells in humans appear to modulate chemotaxis. In particular, MCP-1 aids in migration of neutrophils, macrophages, T- and B-cell lymphocytes, and NK cells (Matsushima etal, 1989; Allavena etal, 1994; Carr etal, 1996). Besides, chemotaxis, these cytokines also are capable of augmenting ADCC of cytotoxic T-cell lymphocytes and NK cells (Taub etal, 1996). Dave etal (2004), demonstrated the importance of tumour microenvironment and that in follicular lymphomas, the non-malignant immune cells appeared to correlate with prognostic significance. Natural killer cells are one of the primarily cells that aid in the innate immune system. Various strategies to improve antibody-based therapy have been proposed. We specifically studied the role of IMiDs and its effects on DCs and NK cells using a human lymphoma-bearing SCID mouse model. Our data suggests that a major mechanism-of-action of IMiDs is activation of the innate immune system (NK cells). Lenalidomide and actimid, via their interaction on dendritic cells and NK cells plays a key role in enhancing the anti-tumour effects of rituximab. In addition to their effects on DCs and NK cells, we also demonstrated that IMiDs can inhibit invivo angiogenesis, which further underscores the complexity of the biological activity of these agents. In summary, agents such as lenalidomide and actimid largely exert their actions by altering the tumour microenvironment rather than exhibiting direct apoptotic effects on the tumour cell itself in our lymphoma preclinical model. Direct extrapolation of our preclinical results to primary B-cell lymphoma is difficult. It is possible that differential direct anti-tumour activity by IMiDs may exist in different subtypes of NHL. This is an important point to understand as IMiD monotherapy will probably not result in sustained activity. Durable responses are more likely to occur when IMiDs are combined with monoclonal antibodies or other agents. A novel strategy is to combine IMiDs and rituximab in the treatment of CD20-positive neoplasms. This report focused on the importance of understanding the mechanisms-of-action of IMiDs and will help in the design of future clinical trials using these novel agents in the setting of recurrent/refractory B-cell NHL.Sozzani, S. & Mantovani, A. (1994) Induction of natural killer cell migration by monocyte chemotactic protein-1, -2 and -3. European Journal of Immunology, 24, 3233– 3236.Alon, R. & Springer, T.A. (1996) The C-C chemokine MCP-1 differentially modulates the avidity of beta 1 and beta 2 integrins on T lymphocytes. Immunity, 4, 179– 187.Starostik, P. & Czuczman, M.S. (2006) Clinical efficacy of lenalidomide in patients with relapsed or refractory chronic lymphocytic leukemia: results of a phase II study. Journal of Clinical Oncology, 24, 5343– 5349.Jagannath, S. & Dhodapkar, M.V. (2006) Enhancement of ligand-dependent activation of human natural killer T cells by lenalidomide: therapeutic implications. Blood, 108, 618– 621.Lederlin, P. & Gisselbrecht, C. (2002) CHOP chemotherapy plus rituximab compared with CHOP alone in elderly patients with diffuse large-B-cell lymphoma. New England Journal of Medicine, 346, 235– 242.Stirling, D.I. & Kaplan, G. (1999) Differential cytokine modulation and T cell activation by two distinct classes of thalidomide analogues that are potent inhibitors of TNF-alpha. Journal of Immunology, 163, 380– 386.Dallaire, B. & Varns, C. (1999) Treatment of patients with low-grade B-cell lymphoma with the combination of chimeric anti-CD20 monoclonal antibody and CHOP chemotherapy. Journal of Clinical Oncology, 17, 268– 276.Berlfein, J. & Grillo-Lopez, A.J. (2004) Prolonged clinical and molecular remission in patients with low-grade or follicular non-Hodgkin’s lymphoma treated with rituximab plus CHOP chemotherapy: 9-year follow-up. Journal of Clinical Oncology, 22, 4711– 4716.Folkman, J. (1994) Thalidomide is an inhibitor of angiogenesis. Proceedings of the National Academy of Sciences of the United States of America, 91, 4082– 4085.Chiorazzi, M. & Staudt, L.M. (2004) Prediction of survival in follicular lymphoma based on molecular features of tumor-infiltrating immune cells. New England Journal of Medicine, 351, 2159– 2169.Stirling, D.I. & Anderson, K.C. (2001) Thalidomide and immunomodulatory derivatives augment natural killer cell cytotoxicity in multiple myeloma. Blood, 98, 210– 216.Kabat, B. & Gertz, M.A. (2007) The activity of lenalidomide with or without dexamethasone in patients with primary systemic amyloidosis. Blood, 109, 465– 470.Stirling, D. & Dalgleish, A.G. (2002) Novel thalidomide analogues display anti-angiogenic activity independently of immunomodulatory effects. British Journal of Cancer, 87, 1166– 1172.Dalgleish, A.G. & Bartlett, J.B. (2005) Orally administered lenalidomide (CC-5013) is anti-angiogenic in vivo and inhibits endothelial cell migration and Akt phosphorylation in vitro. Microvascular Research, 69, 56– 63.Reyes, F. & Coiffier, B. (2005) Long-term results of the R-CHOP study in the treatment of elderly patients with diffuse large B-cell lymphoma: a study by the Groupe d’Etude des Lymphomes de l’Adulte. Journal of Clinical Oncology, 23, 4117– 4126.Meri, S. & Leppa, S. (2006) Differential gene expression in non-malignant tumour microenvironment is associated with outcome in follicular lymphoma patients treated with rituximab and CHOP. British Journal Haematology, 135, 33– 42.Richardson, P. & Anderson, K.C. (2005) Molecular mechanisms whereby immunomodulatory drugs activate natural killer cells: clinical application. British Journal Haematology, 128, 192– 203.Repasky, E. & Czuczman, M.S. (2003) Neutrophils contribute to the biological antitumor activity of rituximab in a non-Hodgkin’s lymphoma severe combined immunodeficiency mouse model. Clinical Cancer Research, 9, 5866– 5873.Repasky, E.A. & Czuczman, M.S. (2005a) Concurrent administration of granulocyte colony-stimulating factor or granulocyte-monocyte colony-stimulating factor enhances the biological activity of rituximab in a severe combined immunodeficiency mouse lymphoma model. Leukaemia and Lymphoma, 46, 1775– 1784.Ottman, E. & Czuczman, M.S. (2005b) Immunomodulatory drug CC-5013 or CC-4047 and rituximab enhance antitumor activity in a severe combined immunodeficient mouse lymphoma model. Clinical Cancer Research, 11, 5984– 5992.Stirling, D.I. & Anderson, K.C. (2000) Thalidomide and its analogs overcome drug resistance of human multiple myeloma cells to conventional therapy. Blood, 96, 2943– 2950.Schultze, J. & Freedman, A.S. (2001) MCP-1 modulates chemotaxis by follicular lymphoma cells. British Journal Haematology, 115, 554– 562.Muramatsu, S. & Steinman, R.M. (1992) Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. Journal of Experimental Medicine, 176, 1693– 1702. Kruisbeek, A.M. (2000) Isolation of murine natural killer cells. Current Protocols in Immunology, (Suppl. 39), 3·1·1– 3·1·5.Anderson, K.C. & Munshi, N.C. (2004) Immunomodulatory drug costimulates T cells via the B7-CD28 pathway. Blood, 103, 1787– 1790.Stirling, D.I. & Anderson, K.C. (2003) Immunomodulatory analogs of thalidomide inhibit growth of Hs Sultan cells and angiogenesis in vivo. Leukemia, 17, 41– 44.Knight, R. & Zeldis, J.B. (2005) Efficacy of lenalidomide in myelodysplastic syndromes. New England Journal of Medicine, 352, 549– 557.Zeldis, J. & Knight, R. (2006) Lenalidomide in the myelodysplastic syndrome with chromosome 5q deletion. New England Journal of Medicine, 355, 1456– 1465.DuBois, G.C. & Oppenheim, J.J. (1989) Purification and characterization of a novel monocyte chemotactic and activating factor produced by a human myelomonocytic cell line. Journal of Experimental Medicine, 169, 1485– 1490.Witzig, T.E. & Gertz, M.A. (2005) Combination therapy with lenalidomide plus dexamethasone (Rev/Dex) for newly diagnosed myeloma. Blood, 106, 4050– 4053.O’Brien, T. & Preisler, H.D. (2000) Effect of interferon-alpha on CD20 antigen expression of B-cell chronic lymphocytic leukemia. Cytokines Cellular Molecular Therapy, 6, 81– 87.Longo, D.L. & Murphy, W.J. (1996) Beta chemokines costimulate lymphocyte cytolysis, proliferation, and lymphokine production. Journal of Leukocyte Biology, 59, 81– 89. The full text of this article hosted at iucr.org is unavailable due to technical difficulties. Please check your email for instructions on resetting your password. If you do not receive an email within 10 minutes, your email address may not be registered, and you may need to create a new Wiley Online Library account. Can\'t sign in? Forgot your username? Enter your email address below and we will send you your username If the address matches an existing account you will receive an email with instructions to retrieve your username