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Blood, 1 October 2007, Vol. 110, No. 7, pp. 2561-2564. Prepublished online as a Blood First Edition Paper on May 2, 2007; DOI 10.1182/blood-2007-01-070656. This Article Abstract Full Text (PDF) All Versions of this Article: blood-2007-01-070656v1 110/7/2561    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Hatjiharissi, E. Articles by Treon, S. P. Search for Related Content PubMed PubMed Citation Articles by Hatjiharissi, E. Articles by Treon, S. P. Related Collections Immunobiology Immunotherapy Brief Reports Clinical Trials and Observations Related Article in Blood Online Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  IMMUNOBIOLOGY Brief Report Increased natural killer cell expression of CD16, augmented binding and ADCC activity to rituximab among individuals expressing the FcRIIIa-158 V/V and V/F polymorphism Evdoxia Hatjiharissi1,3, Lian Xu1, Daniel Ditzel Santos1,2, Zachary R. Hunter1, Bryan T. Ciccarelli1, Sigitas Verselis2,4, Michael Modica2,4, Yang Cao1, Robert J. Manning1, Xavier Leleu1,2, Elizabeth A. Dimmock1, Alexandros Kortsaris3, Constantine Mitsiades2,5, Kenneth C. Anderson2,5, Edward A. Fox2,4, and Steven P. Treon1,2 1 Bing Center for Waldenstrom's Macroglobulinemia, Dana-Farber Cancer Institute, Boston, MA; 2 Harvard Medical School, Boston, MA; 3 School of Medicine, Democritus University of Thrace, Alexandroupolis, Greece; 4 Molecular Diagnostics Laboratory, Dana-Farber Cancer Institute, Boston, MA; and 5 Jerome Lipper Multiple Myeloma Center, Dana-Farber Cancer Institute, Boston, MA    Abstract Top Abstract Introduction Materials and methods Results and discussion Authorship References   The presence of valine (V) at position 158 of FcRllla (CD16) is known to improve clinical response to rituximab in indolent non-Hodgkin lymphoma (NHL). Little is known about the basic mechanisms for this observation. We examined natural killer (NK) cells from healthy donors representing the FcRIIIa-158 polymorphic subgroups (V/V, V/F, and F/F) for gene transcript and cell surface CD16 expression, rituximab binding, and rituximab-dependent NK cell-mediated cytotoxicity. We observed higher levels of FcRIIIa transcripts among individuals with the FcRIIIa-158 V/V versus V/F or F/F genotype (P < .001); increased cell surface CD16 expression by quantitative flow cytometry on NK cells from individuals expressing at least one valine at FcRIIIa-158 versus F/F (P = .029); as well as augmented rituximab binding and rituximab-mediated, antibody-dependent cellular cytotoxicity (ADCC). These results suggest that individuals expressing at least one valine at FcRIIIa-158 might, in part, have better clinical outcomes due to increased CD16 expression, rituximab binding, and rituximab-mediated ADCC.    Introduction Top Abstract Introduction Materials and methods Results and discussion Authorship References   Rituximab is a CD20-directed, IgG1-chimeric monoclonal antibody (mAb) used to treat patients with B-cell lymphomas and various autoimmune disorders. Both quantitative as well as qualitative differences in natural killer (NK) cell function may explain rituximab clinical activity. Higher circulating NK cell levels and responses to rituximab have been reported in patients with indolent non-Hodgkin lymphoma (NHL), suggesting that antibody-dependent cellular cytotoxicity (ADCC) enacted by NK cells may be a primary mechanism by which rituximab functions.1,2 Moreover, responses to rituximab may depend upon polymorphisms present in the FcRIIIa (CD16) receptor, a receptor mainly expressed on NK cells.3–5 Polymorphisms in position 48 and 158 of the FcRIIIa receptor expression have been reported to influence human IgG1 binding and ADCC activity.6–9 Polymorphisms at position 158 result in either valine (V) or phenylalanine (F) expression,6,8,9 the former of which is associated with increased depletion of peripheral blood B cells10 and response to rituximab in patients with indolent NHL3–5 but not chronic lymphocytic leukemia (CLL).11 At position 48, polymorphisms of the FcRIIIa receptor result in expression of either leucine, arginine, or histidine, the first of which is linked to FcRIIIa-158F and the latter 2 with the FcRIIIa-158V polymorphisms.5,8,9 However, the binding of IgG1 to FcRIIIa appears to occur independently of position 48 polymorphisms most likely on the basis of tight genetic linkage to FcRIIIa-158 polymorphisms.5,8 Genetic linkage between polymorphisms in FcRIIa (CD32), a receptor also implicated in predicting rituximab clinical response, and FcRIIIa has recently been demonstrated by us and points to the primacy of FcRIIIa-158 polymorphisms in predicting rituximab response.12 While these studies suggest that variable responses to rituximab among FcRIIIa-158 polymorphic groups are likely the result of qualitative (ie, antibody affinity) differences, the possibility that quantitative differences in cell surface CD16 expression, rituximab binding, and ADCC activity have not been addressed. As such, we sought to delineate differences in FcRIIIa gene expression, cell surface CD16 expression, rituximab binding, and rituximab-dependent ADCC activity in NK cells isolated from healthy individuals representing the 3 FcRIIIa-158 polymorphic subgroups (V/V, V/F, and F/F).    Materials and methods Top Abstract Introduction Materials and methods Results and discussion Authorship References   FcRIIIa-158 genotype analysis We analyzed the genotype of 52 unrelated healthy individuals by sequencing exon 4 of the FcRIIIa gene. FcRIIIa-158 polymorphisms were determined by allele-specific reverse transcription polymerase chain reaction (RT-PCR) and direct sequencing of genomic DNA, as we previously described.5 Genomic DNA was extracted from peripheral blood using a DNA isolation kit (Qiagen, Valencia, CA). The study was approved by the Dana Farber Cancer Institute's Institutional Review Board, and written consent was obtained from each donor in accordance with the Declaration of Helsinki. Cell isolation and culture Peripheral blood mononuclear cells (PBMNCs) were isolated using Ficoll-Paque (Amersham, Uppsala, Sweden). NK cells were selected from PBMNCs using the NK-cell isolation kit II (Miltenyi, Auburn, CA) resulting in more than 95% purity (CD3–/CD56+). ARH-77 and Daudi cells were cultured as previously described. RT-PCR analysis FcRIIIa gene expression was determined by quantitative real-time RT-PCR (Applied Biosystems, Foster City, CA). RNA was extracted from NK cells. Primer sequences were as follows: FcRllla sense (5'-CCAAAAGCCACACTCAAAGAC-3') and antisense (5'-ACCCAGGTGGAAAGAATGATG-3'); TaqMan probe (5'-AACATCACCATCACTCAAGGTTTGG-3'). The quantity of FcRIIIa mRNA in each sample was normalized to the relative quantity of HR-18S. Quantitative flow cytometry CD16 receptors were quantified using the QuantiBRITE system. NK cells (2 x 105) were stained with 5 µL (0.287 mg/mL) of anti-CD16 PE bead-conjugated mAb for 20 minutes at 4°C (BD Biosciences, San Jose, CA). After incubation, NK cells were washed twice and resuspended in 1x PBS. Prior to each analysis, the flow cytometer was calibrated by QuantiBRITE PE calibration beads. CD16 receptors were assessed by gating 104 (CD3–CD56+) cells. Samples were analyzed using CellQuest software (BD Biosciences). Rituximab binding to NK cells Rituximab (Genentech BioOncology, San Francisco, CA) binding was determined using an indirect method as previously described,7 using an anti-CD16 (3G8 clone) mAb. NK cells (2 x 105) were incubated with rituximab at concentrations of 10, 50, 100, and 200 µg/mL for 30 minutes at 4°C, followed by incubation with anti-CD16 FITC and anti-CD56 PE mAbs at 4°C for 20 minutes. After incubation, NK cells were washed with PBS and analyzed by flow cytometry. CD16 median fluorescence intensity (MFI) was determined by gating on CD3-CD56+ lymphocytes. Rituximab binding was defined as percentage of inhibition for binding of anti-CD16 mAb and calculated as follows: [(MFI without rituximab) – (MFI with rituximab)] x 100/(MFI without rituximab). ADCC assays ADCC experiments were performed using NK cells as effectors cells. To avoid killer cell immunoglobulin-like receptor (KIR) dependent ADCC, HLA class I expressing (ARH-77) and nonexpressing (Daudi) cell lines (both of which are CD20+) were used as a target cells. Cells were incubated with 10 µg/mL rituximab and human IgG1 (control) for 1 hour, washed twice, and cocultured (5 x 103 per well) in varying ratios with effector cells for 4 hours at 37°C with 5% CO2. A colorimetric-based lactate dehydrogenase (LDH) assay (CytoTox 96; Promega, Madison, WI) was used and cytotoxicity calculated according to manufacturer's instructions. Statistical analysis Differences among polymorphic groups were compared by the Kruskal-Wallis and Mann-Whitney tests. The correlations between gene expression and CD16 receptors were assessed using the Pearson correlation coefficient.    Results and discussion Top Abstract Introduction Materials and methods Results and discussion Authorship References   We first analyzed the gene expression of FcRIIIa by performing real-time RT-PCR analysis for 13 donors whose genotyping demonstrated FcRIIIa-158 V/V (n = 4), V/F (n = 4), and F/F (n = 5). Individuals with the FcRIIIa-158 V/V genotype expressed higher FcRIIIa transcript levels versus those individuals with the FcRIIIa V/F and F/F genotype (P < .001). However, no significant difference in FcRIIIa transcripts was observed between individuals with the FcRIIIa V/F and F/F genotypes (Figure 1A). This observation is particularly intriguing in view of the fact that the absolute number of CD16 receptors per NK cell was significantly higher in donors who expressed at least one valine at FcRIIIa-158 (ie, were either V/V or V/F) versus F/F (P = .029; Figure 1B). The basis for these discordant findings between FcRIIIa transcript expression and cell surface protein levels among individuals with FcRIIIa-V/F remains to be clarified but may reflect relative differences in transcript or protein stability and/or recycling of CD16 at the cell surface imposed by the expression of valine. View larger version (8K): [in this window] [in a new window]   Figure 1. FcRIIIa transcript expression on NK cells for 13 donors. Transcript expression was assayed by real-time RT-PCR analysis (A), and cell surface CD16 expression by quantitative flow cytometry (B), on NK cells for donors whose genotyping demonstrated FcRIIIa-158 V/V (n = 4), V/F (n = 4), and F/F (n = 5). P is less than .001 for transcript levels for individuals with FcRIIIa-158 V/V versus V/F or F/F genotype. P equals .029 for CD16 expression for individuals expressing at least one valine at FcRIIIa-158 versus F/F. Values represent means plus or minus SE.   We next evaluated the functional implications of FcRIIIa-158 polymorphisms by studying rituximab binding and rituximab-dependent NK cell-mediated cytotoxicity. Both rituximab binding (Figure 2A) as well as rituximab-mediated ADCC activity by NK cells (Figure 2B,C) increased with the presence of at least one valine, in comparison with the donors' homozygous for phenylalanine (V/V> V/F> F/F), at all concentrations (10 to 200 µg/mL) of rituximab studied. The cytotoxicity induced by the control (10 µg huIgG1) was less than 10% (data not shown). These results are unlikely to be explained by KIR mismatching because HLA class I expressing (ARH-77) and nonexpressing (Daudi) cell lines were used as target cells. Independent of the FcRIIIa-158 polymorphic subgroup, rituximab-mediated ADCC activity was observed to correlate with the number of cell surface CD16 receptors (Figure 2D). These results are in agreement with previous studies demonstrating increased binding of IgG1-class antibodies, including rituximab among individuals expressing valine at FcRIIIa-158,6–9,13 and suggest that the expression level of cell surface CD16 may also contribute to augmented rituximab binding and ADCC activity in addition to possible differences in binding affinity. View larger version (9K): [in this window] [in a new window]   Figure 2. Characterization of NK-cell RTX-binding and RTX-mediated ADCC activity by NK cells against Daudi and ARH-77 CD20+ B cells. RTX-mediated ADCC activity by NK cells against (A) ARH-77 and (B) Daudi CD20+ B cells was assayed for 9 donors whose genotyping demonstrated FcRIIIa-158 V/V (n = 3), V/F (n = 3), and F/F (n = 3) and (C) NK-cell RTX binding. (D) The correlation between cell-surface CD16 receptors and RTX-dependent ADCC activity for these individuals. Values represent means plus or minus SE.   The results of these studies may help to explain augmented responses to rituximab observed with the addition of certain agents known to up-regulate CD16, including one study wherein the addition of IL-2 to rituximab appeared to selectively result in clinical responses among rituximab-refractory patients expressing FcRIIIa-158 F/F.14 Further exploration of agents aimed at augmenting CD16 expression, particularly in context with newer CD20-directed mAb-bearing enhanced Fc binding and ADCC activity,15,16 may lead to improved responses among patients with indolent NHL, including for those individuals expressing FcRIIIa-158 F/F. In summary, the results of these studies suggest that individuals expressing at least one valine at FcRIIIa-158 might in part have better clinical outcomes due to increased CD16 expression.    Authorship Top Abstract Introduction Materials and methods Results and discussion Authorship References   Contribution: E.H. designed the study, performed research, analyzed the data, and wrote the first draft of the manuscript; L.X., designed, performed, and analyzed experiments in molecular biology; D.D.S, performed various pertinent research; Z.R.H., collected samples and analyzed the data; B.T.C. performed various pertinent research; S.V. and M.M. designed, performed, and analyzed experiments in molecular biology; Y.C. performed various pertinent research; R.J.M. collected samples and analyed the data; X.L. performed various pertinent research; E.A.D. collected samples and analyzed the data; A.K., C.M., and K.C.A. contributed advice to the design and interpretation of the study; E.A.F. designed, performed, and analyzed experiments in molecular biology; and S.P.T. designed the study, oversaw the experiments, and wrote the final draft of the manuscript. Conflict-of-interest disclosure: The authors declare no competing financial interests. Correspondence: Steven P. Treon, Bing Center for Waldenstrom's Macroglobulinemia, Dana-Farber Cancer Institute, M548, 44 Binney St, Boston, MA 02115; e-mail:steven_treon{at}dfci.harvard.edu.    Acknowledgments   This work was funded by the Peter and Helen Bing Fund for Waldenstrom macroglobulinemia, the Bailey Family Fund at the Dana-Farber Cancer Institute, and a National Institutes of Health Career Development Award (K23CA087977-03) (S.P.T.).    Footnotes   Submitted January 30, 2007; accepted April 23, 2007. Prepublished online as Blood First Edition Paper, May 2, 2007 DOI: 10.1182/blood-2007-01-070656 An Inside Blood analysis of this article appears at the front of this issue. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.    References Top Abstract Introduction Materials and methods Results and discussion Authorship References   Janakiraman N, McLaughlin P, White CA, et al. Rituximab: correlation between effector cells and clinical activity in NHL [abstract]. Blood 1998; 92:337a Abstract 1384. Gluck A, Hurst D, Yuen A, et al. Phase I studies of interleukin (IL)-2 and rituximab in B-cell non-Hodgkin's lymphoma: IL-2 mediated natural killer cell expansion correlations with clinical response. Clin Cancer Res 2004; 10:2253–2264.[Abstract/Free Full Text] Cartron G, Dacheux L, Salles G, et al. Therapeutic activity of humanized anti-CD20 monoclonal antibody and polymorphism in IgG Fc receptor Fcgamma RIIIA gene. Blood 2002; 99:754–758.[Abstract/Free Full Text] Weng WK and Levy R. Two immunoglobulin G Fc receptor polymorphisms independently predict response to rituximab in patients with follicular lymphoma. J Clin Oncol 2003; 21:3940–3947.[Abstract/Free Full Text] Treon SP, Hansen M, Branagan AR, et al. Polymorphisms in FcRIIIA (CD16) receptor expression are associated with clinical responses to rituximab in Waldenstrom's macroglobulinemia. J Clin Oncol 2005; 23:474–481.[Abstract/Free Full Text] Vance BA, Huizinga TWJ, Wardwell K, et al. Binding of monomeric human IgG defines an expression polymorphism of FcRIII on large granular lymphocyte/natural killer cells. J Immunol 1993; 151:6429–6439.[Abstract] Wu J, Edberg JC, Redecha PB, et al. A novel polymorphism of FcRIIIA (CD16) alters receptor function and predisposes to autoimmune disease. J Clin Invest 1997; 100:1059–1070.[Medline] [Order article via Infotrieve] Koene HR, Kleijer M, Alga J, et al. FcRIII-158V/F polymorphism influences the binding of IgG by natural killer cell FcRIIIA, independently of the FcRIIIA-48L/R/H phenotype. Blood 1997; 90:1109–1114.[Abstract/Free Full Text] de Haas M, Koene HR, Kleijer M, et al. Fc receptor type IIIA polymorphism influences the binding of human IgG by NK cell FcRIIIA. J Immunol 1996; 156:2948–2955.[Abstract] Anolik JH, Campbell D, Felgar RE, et al. The relationship of FcRIIIA genotype to degree of B cell depletion by rituximab in the treatment of systemic lupus erythematosus. Arthritis Rheum 2003; 48:455–459.[CrossRef][Medline] [Order article via Infotrieve] Farag SS, Flinn IW, Modali R, et al. FcRIIIA and FcRIIA polymorphisms do not predict response to rituximab in B-cell chronic lymphocytic leukemia. Blood 2004; 301:1472–1474. Hatjiharissi E, Hansen M, Verselis S, et al. Polymorphisms in FcRIIIA are genetically linked to FcRIIA and may account for the primary predictive role ascribed to polymorphisms in FcRIIIA-158 in determining rituximab responses. Clin Lymphoma Myeloma 2007; 7:286–290.[Medline] [Order article via Infotrieve] Dall'Ozzo S, Tartas S, Paintaud G, et al. Rituximab-dependent cytotoxicity by natural killer cells: influence of FCGR3A polymorphism on the concentration-effect relationship. Cancer Res 2004; 64:4664–4669.[Abstract/Free Full Text] Khan KD, Emmanouilides C, Benson DM Jr, et al. A phase 2 study of rituximab in combination with recombinant interleukin-2 for rituximab-refractory indolent non-Hodgkin's lymphoma. Clin Cancer Res 2006; 12:7046–7053.[Abstract/Free Full Text] Shields RL, Namenuk AK, Hong K, et al. High resolution mapping of the binding site on human IgG1 for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn and design of IgG1 variants with improved binding to Fc gamma R. J Biol Chem 2001; 276:6591–6604.[Abstract/Free Full Text] Bowles JA, Wang SY, Link BK, et al. Anti-CD20 monoclonal antibody with enhanced affinity for CD16 activates NK cells at lower concentrations and more effectively than rituximab. Blood 2006; 108:2648–2654.[Abstract/Free Full Text] CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? Related Article in Blood Online: FcRIIIa role in rituximab efficacy Julie M. Roda and John C. Byrd Blood 2007 110: 2220. [Full Text] [PDF] This article has been cited by other articles: J D Canete, B Suarez, M V Hernandez, R Sanmarti, I Rego, R Celis, C Moll, J A Pinto, F J Blanco, and F Lozano Influence of variants of Fc{gamma} receptors IIA and IIIA on the American College of Rheumatology and European League Against Rheumatism responses to anti-tumour necrosis factor {alpha} therapy in rheumatoid arthritis Ann Rheum Dis, October 1, 2009; 68(10): 1547 - 1552. [Abstract] [Full Text] [PDF] P. M. Cardarelli, M.-C. 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[Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) All Versions of this Article: blood-2007-01-070656v1 110/7/2561    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Hatjiharissi, E. Articles by Treon, S. P. Search for Related Content PubMed PubMed Citation Articles by Hatjiharissi, E. Articles by Treon, S. P. Related Collections Immunobiology Immunotherapy Brief Reports Clinical Trials and Observations Related Article in Blood Online Social Bookmarking                   What's this?

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