Journal of Neuroimmunology 270 (2014) 95–97
Contents lists available at ScienceDirect
Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Short communication
Immunoglobulin GM and FcγRIIIa genotypes influence cytotoxicity of neuroblastoma cells Janardan P. Pandey ⁎, Aryan M. Namboodiri Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, SC 29425, USA
a r t i c l e
i n f o
Article history: Received 20 November 2013 Accepted 2 March 2014 Keywords: GM allotypes FcγR genotypes Neuroblastoma Anti-GD2 antibodies ADCC
a b s t r a c t Immunoglobulin GM (γ marker) allotypes are strongly associated with neuroblastoma, but the mechanism is not known. One mechanism could involve antibody-dependent cell-mediated cytotoxicity (ADCC) of neuroblastoma cells. Using an ADCC inhibition assay, we show that IgG1 expressing GM 3 +,1 −,2 − allotypes blocked all phenylalanine-expressing FcγRIIIa present on NK cells, resulting in total inhibition of anti-GD2 antibodymediated ADCC of GD2-overexpressing neuroblastoma cells. In contrast, the inhibitory effect of this protein was significantly lower when the NK cells were homozygous for the valine allele of FcγRIIIa (100 vs. 21%; p = 0.00004). These and other findings presented here could lead to a more effective immunotherapy of neuroblastoma. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Immunoglobulin GM allotypes are encoded by three very closely linked genes—immunoglobulin heavy chain G1 (IGHG1), IGHG2, and IGHG3—on chromosome 14q32. They are localized on the constant region of γ1, γ2, and γ3 chains (Pandey and Li, 2013). Using a hypothesis-driven candidate gene approach, a particular GM genotype—GM 1/a,3/f,5/b—was found to be strongly associated (p b 0.001) with susceptibility to neuroblastoma (Morell et al., 1977), the most common extracranial solid tumor of childhood that is often lethal. Mechanism underlying this association is not known. One mechanism could involve the contributions of these polymorphic determinants to the antibody-dependent cell-mediated cytotoxicity (ADCC) of neuroblastoma tumor cells. ADCC, which links the specific humoral responses to the vigorous innate cytotoxic effector responses, is a major host defense mechanism against tumors. IgG antibody mediated ADCC is triggered upon ligation of FcγR to the Fc of IgG molecules (Nimmerjahn and Ravetch, 2011). It follows that genetic variation in FcγR and Fc could contribute to the differences in the magnitude of ADCC. The majority of GM epitopes are present on the Fc portion of the IgG molecule. Thus, Fc of a particular GM genotype could preferentially bind to the FcγR of a particular genotype and influence tumor destruction through ADCC.
⁎ Corresponding author at: Department of Microbiology and Immunology, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425-2230, USA. Tel.: + 1 843 792 4360; fax: + 1 843 792 4882. E-mail address: [email protected] (J.P. Pandey).
http://dx.doi.org/10.1016/j.jneuroim.2014.03.003 0165-5728/© 2014 Elsevier B.V. All rights reserved.
We investigated the interactive effects of GM and FcγR alleles on the ADCC of cells from the human neuroblastoma cell line BE(2)-C, which overexpresses the disialoganglioside antigen GD2. We used three allotypically different IgG1 proteins to inhibit the ADCC of BE(2)-C cells mediated by the chimeric human–murine anti-GD2 monoclonal antibody ch14.18. In this ADCC inhibition assay, anti-GD2 ch14.18 antibodies bound to the target BE(2)-C cells compete with allotypically disparate IgG1 proteins for binding to the activating receptor FcγRIIIa expressed on natural killer (NK) cells. 2. Materials and methods 2.1. GM allotyping and IgG1 affinity purification Serum samples from healthy blood donors were allotyped for all four known IgG1 allotypes—GM 1/a, 2/x, 3/f, and 17/z—by a standard hemagglutination-inhibition assay (Schanfield and van Loghem, 1986). Allotypes 3 and 17 are expressed in the Fd, whereas 1 and 2 are expressed in the Fc, region of the γ1 chains. Total IgG from the pooled sera of subjects—10 expressing the GM 3+,1−,2−, 10 expressing GM 17+,1+,2+, and 10 expressing GM 17 +,1+,2− allotypes— was concentrated by ammonium sulfate fractionation. IgG1 proteins were isolated from the total IgG by subclass-specific affinity chromatography. 2.2. FcγRIIIa genotyping A change from nucleotide T to G in the FcγRIIIa gene results in amino acid change from phenylalanine (F) to valine (V) at position 158 in the
96
J.P. Pandey, A.M. Namboodiri / Journal of Neuroimmunology 270 (2014) 95–97
IgG binding domain of FcγRIIIa. Genotyping of FcγRIIIa alleles was performed by RT-PCR, using a pre-designed TaqMan® assay from Applied Biosystems Inc. (Foster City, CA).
Table 2 Inhibition of ch14.18-mediated ADCC of BE(2)-C cells by NK cells expressing different FcγRIIIa genotypes in the presence of allotypically disparate IgG1 proteins. IgG1 genotype
FcγRIIIa genotype
Inhibition of ADCC (%)
p value
GM 3+,1−,2−
VV FF VV FF VV FF
20.62 ± 19.73 100.00 ± 0.00 60.88 ± 28.48 100.00 ± 0.00 15.28 ± 14.11 12.33±13.03
0.00004
2.3. Purification of NK cells Blood from volunteers homozygous for either V or F alleles of FcγRIIIa was collected in EDTA-coated vacutainer tubes (BD, Franklin Lakes, NJ). NK cells were isolated by affinity depletion of non-NK cells using a kit from Miltenyi Biotec (Auburn, CA).
GM 17+,1+,2+ GM 17+,1+,2−
0.011 0.69
2.4. Cell cytotoxicity by ADCC and ADCC inhibition This method is analogous, in principle, to that described by Macdonald et al. (1981). Human neuroblastoma cell line BE(2)-C was obtained from ATCC (Manassas, VA). Cells were cultured overnight in medium containing 10% FBS, harvested with trypsin-EDTA, and washed 3 times with RPMI-1640 containing 1% BSA. Cells were coated with 10 μg/ml of anti-GD2 antibody ch14.18 (NCI) for 30 min on ice and washed three times with RPMI 1640 containing 1% BSA. These coated cells (5 × 103) were further incubated with 25 μg/ml of aggregated human IgG1—expressing one of the three allotypic combinations—and 5 × 104 NK cells (1:10 ratio of target to effector cells) in a volume of 100 μl RPMI 1640 containing 1% FBS in 96-well plates overnight. The plates were then centrifuged and the supernatant was assayed for lactate dehydrogenase (LDH) activity, using the Cytotox96 kit from Promega Corporation (Madison, WI). Spontaneous LDH release—possibly due to killer-cell immunoglobulin-like receptor dependent cytotoxicity—from target cells incubated with NK cells was used as blank (negative control).
ðControl LDH activity–Test LDH activityÞ ADCC inhibition ð% Þ ¼ −−−−−−−−−−−−−−−−−−−−−− 100 ðControl LDH activityÞ
where Test consists of BE(2)-C target cells incubated with aggregated IgG1, ch14.18 anti-GD2 antibody and NK cells and Control (positive) consists of BE(2)-C cells incubated with ch14.18 antibody and NK cells. Standard deviations were obtained via 7 experimental replications. 2.5. Statistical analyses Student's unpaired t-test was used to compare the percentage of ADCC inhibition associated with GM-FcγRIIIa genotypic combinations. All tests were two-tailed, and the statistical significance was defined as p b 0.05. 3. Results and discussion The amino acid substitutions that characterize the four known human IgG1 allotypes are shown in Table 1. As shown in Table 2, at a concentration of 25 μg/ml, IgG1 expressing the GM 3 +,1 −,2 − allotypes blocked all F-expressing FcγRIIIa present on the NK cells, resulting in 100% inhibition of ch14.18-mediated ADCC of BE(2)-C cells. In contrast, the inhibitory effect of this protein was significantly lower when the NK cells were homozygous
Table 1 Amino acid substitutions characterizing IgG1 allotypes. IgG1 genotype
CH1 214
CH3 356
CH3 358
CH3 431
GM 3+,1−,2− GM 17+,1+,2+ GM 17+,1+,2−
Arg Lys Lys
Glu Asp Asp
Met Leu Leu
Ala Gly Ala
for the V allele of FcγRIIIa (100 vs. 21%; p = 0.00004). A similar trend was evident for the IgG1 proteins expressing the GM 17 +,1 +,2 + allotypes: Inhibitory effect being significantly higher in the presence of NK cells expressing the F allele of FcγRIIIa as compared to those expressing the V variant of this receptor (100 vs. 61%; p = 0.011). IgG1 proteins that lacked the GM 2 determinant, but expressed the GM 1 and 17 alleles, had similar (low) inhibitory effect when the NK cells were homozygous for either allele of FcγRIIIa (15 vs. 12%; p = 0.69). These results show distinct epistatic contributions of GM and FcγRIIIa alleles to the ADCC of neuroblastoma cells. They also suggest that a naturally occurring anti-GD2 IgG1 antibody that expresses the GM 2 determinant in the Fc region could be efficient in mediating ADCC of neuroblastoma cells in the presence of both major (F) and minor (V) variants of FcγRIIIa. Although its inhibitory effect on the anti-GD2-mediated ADCC of neuroblastoma cells was lower in presence of the V variant (61%), it was significantly higher than that of the two IgG1 proteins that lack the GM 2 determinant (61 vs. 21%, p = 0.01; 61 vs. 15%; p = 0.004). ADCC is unlikely to be the only mechanism responsible for the GM gene involvement in neuroblastoma. To gain further mechanistic insights, possible epistatic contributions of all 18 serologically determined GM specificities and FcγRIIa and FcγRIIIa alleles in ADCC and other Fc-mediated effector functions (e.g. complement-dependent cytotoxicity and phagocytosis) need to be investigated. GM genes could also be involved in neuroblastoma via their modulating influence on the immunoevasion strategies employed by cytomegalovirus (Namboodiri and Pandey, 2011; Pandey, 2013), a common herpesvirus that has been implicated in the etiopathogenesis of this malignancy (Wolmer-Solberg et al., 2013). Involvement of GM 2 in the ADCC of neuroblastoma cells suggests a putative mechanism underlying the racial disparity in neuroblastoma survival. A higher proportion of African American children than European American children present with high-risk disease and have worse survival (Henderson et al., 2011). The GM 2 allele is extremely rare in people of African descent, which could adversely affect their ADCC-mediated immunosurveillance against neuroblastoma tumors. A trans-population analysis identified a common single-nucleotide polymorphism (SNP) within sperm associated antigen 16 to be responsible for the ethnic disparities in survival (Gamazon et al., 2013). This analysis, however, did not include GM variants, as it selected only those riskconferring genes that were identified by the genome-wide association studies (GWAS) of neuroblastoma. GM alleles, although common within a racial group, are usually not evaluated by GWAS. The IGHG gene segments harboring these determinants are highly homologous and apparently not amenable to the high throughput genotyping technology used in GWAS. Because these genes were not typed in the HapMap or the 1000 Genomes projects, they cannot be imputed or tagged (through linkage disequilibrium) by any SNPs that are included in the genotyping platforms. This could be one reason why the highly significant GM-neuroblastoma association (Morell et al., 1977) has neither been confirmed nor refuted by the GWAS of this malignancy.
J.P. Pandey, A.M. Namboodiri / Journal of Neuroimmunology 270 (2014) 95–97
Immunotherapy—using a chimeric monoclonal anti-GD2 antibody— appears to be the most effective treatment for patients with high-risk neuroblastoma. This treatment, however, is not effective in all patients; furthermore, it is effective only when combined with toxic cytokines such as interleukin-2, which augment the level of ADCC (Simon et al., 2004; Yu et al., 2010). None of the anti-GD2 antibodies currently in clinical trials expresses the GM 2 allotype. Results presented here would suggest that the inclusion of this variant in the Fc region of these antibodies could improve their clinical efficacy. A great deal of effort is currently being directed at engineering Fc variants with optimized affinity for activating and inhibiting FcγRs (Lazar et al., 2006; Weiner et al., 2010). Along with these efforts, evaluation of the role of naturally occurring Fc (GM) variants—that have been maintained through various selective forces during our evolutionary history—in ADCC and other immunosurveillance mechanisms is warranted. Acknowledgments We are grateful to the National Cancer Institute and United Therapeutics Corporation for providing the ch14.18 antibody. References Gamazon, E.R., Pinto, N., Konkashbaev, A., Im, H.K., Diskin, S.J., London, W.B., Maris, J.M., Dolan, M.E., Cox, N.J., Cohn, S.L., 2013. Trans-population analysis of genetic mechanisms of ethnic disparities in neuroblastoma survival. J. Natl. Cancer Inst. 105, 302–309. Henderson, T.O., Bhatia, S., Pinto, N., London, W.B., McGrady, P., Crotty, C., Sun, C.L., Cohn, S.L., 2011. Racial and ethnic disparities in risk and survival in children with neuroblastoma: a Children's Oncology Group study. J. Clin. Oncol. 29, 76–82.
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Lazar, G.A., Dang, W., Karki, S., Vafa, O., Peng, J.S., Hyun, L., Chan, C., Chung, H.S., Eivazi, A., Yoder, S.C., Vielmetter, J., Carmichael, D.F., Hayes, R.J., Dahiyat, B.I., 2006. Engineered antibody Fc variants with enhanced effector function. Proc. Natl. Acad. Sci. U. S. A. 103, 4005–4010. MacDonald, I.M., Dumble, L.J., Jack, I., Clunie, G.I., 1981. Inhibition of whole blood antibody dependent cellular cytotoxicity by heat aggregated human IgG. J. Immunol. Methods 46, 69–76. Morell, A., Scherz, R., Käser, H., Skvaril, F., 1977. Evidence for an association between uncommon Gm phenotypes and neuroblastoma. Lancet 1, 23–24. Namboodiri, A.M., Pandey, J.P., 2011. The human cytomegalovirus TRL11/IRL11-encoded FcγR binds differentially to allelic variants of immunoglobulin G1. Arch. Virol. 156, 907–910. Nimmerjahn, F., Ravetch, J.V., 2011. FcγRs in health and disease. Curr. Top. Microbiol. Immunol. 350, 105–125. Pandey, J.P., 2013. Immunoglobulin GM allotypes as effect modifiers of cytomegalovirusspurred neuroblastoma. Cancer Epidemiol. Biomarkers Prev. http://dx.doi.org/10. 1158/1055-9965.EPI-13-0612. Pandey, J.P., Li, Z., 2013. The forgotten tale of immunoglobulin allotypes in cancer risk and treatment. Exp. Hematol. Oncol. 2, 6. Schanfield, M.S., van Loghem, E., 1986. Human immunoglobulin allotypes. In: Weir, D.M. (Ed.), Handbook of Experimental Immunology. Blackwell, Boston (94.118.16). Simon, T., Hero, B., Faldum, A., Handgretinger, R., Schrappe, M., Niethammer, D., Berthold, F., 2004. Consolidation treatment with chimeric anti-GD2-antibody ch14.18 in children older than 1 year with metastatic neuroblastoma. J. Clin. Oncol. 22, 3549–3557. Weiner, L.M., Surana, R., Wang, S., 2010. Monoclonal antibodies: versatile platforms for cancer immunotherapy. Nat. Rev. Immunol. 10, 317–327. Wolmer-Solberg, N., Baryawno, N., Rahbar, A., Fuchs, D., Odeberg, J., Taher, C., Wilhelmi, V., Milosevic, J., Mohammad, A.A., Martinsson, T., 2013. Frequent detection of human cytomegalovirus in neuroblastoma: a novel therapeutic target? Int. J. Cancer 133, 2351–2361. Yu, A.L., Gilman, A.L., Ozkaynak, M.F., London, W.B., Kreissman, S.G., Chen, H.X., Smith, M., Anderson, B., Villablanca, J.G., Matthay, K.K., Shimada, H., Grupp, S.A., Seeger, R., Reynolds, C.P., Buxton, A., Reisfeld, R.A., Gillies, S.D., Cohn, S.L., Maris, J.M., Sondel, P.M., Children's Oncology Group, 2010. Anti-GD2 antibody with GM-CSF, interleukin-2, and isotretinoin for neuroblastoma. N. Engl. J. Med. 363, 1324–1334.
Contents lists available at ScienceDirect
Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Short communication
Immunoglobulin GM and FcγRIIIa genotypes influence cytotoxicity of neuroblastoma cells Janardan P. Pandey ⁎, Aryan M. Namboodiri Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, SC 29425, USA
a r t i c l e
i n f o
Article history: Received 20 November 2013 Accepted 2 March 2014 Keywords: GM allotypes FcγR genotypes Neuroblastoma Anti-GD2 antibodies ADCC
a b s t r a c t Immunoglobulin GM (γ marker) allotypes are strongly associated with neuroblastoma, but the mechanism is not known. One mechanism could involve antibody-dependent cell-mediated cytotoxicity (ADCC) of neuroblastoma cells. Using an ADCC inhibition assay, we show that IgG1 expressing GM 3 +,1 −,2 − allotypes blocked all phenylalanine-expressing FcγRIIIa present on NK cells, resulting in total inhibition of anti-GD2 antibodymediated ADCC of GD2-overexpressing neuroblastoma cells. In contrast, the inhibitory effect of this protein was significantly lower when the NK cells were homozygous for the valine allele of FcγRIIIa (100 vs. 21%; p = 0.00004). These and other findings presented here could lead to a more effective immunotherapy of neuroblastoma. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Immunoglobulin GM allotypes are encoded by three very closely linked genes—immunoglobulin heavy chain G1 (IGHG1), IGHG2, and IGHG3—on chromosome 14q32. They are localized on the constant region of γ1, γ2, and γ3 chains (Pandey and Li, 2013). Using a hypothesis-driven candidate gene approach, a particular GM genotype—GM 1/a,3/f,5/b—was found to be strongly associated (p b 0.001) with susceptibility to neuroblastoma (Morell et al., 1977), the most common extracranial solid tumor of childhood that is often lethal. Mechanism underlying this association is not known. One mechanism could involve the contributions of these polymorphic determinants to the antibody-dependent cell-mediated cytotoxicity (ADCC) of neuroblastoma tumor cells. ADCC, which links the specific humoral responses to the vigorous innate cytotoxic effector responses, is a major host defense mechanism against tumors. IgG antibody mediated ADCC is triggered upon ligation of FcγR to the Fc of IgG molecules (Nimmerjahn and Ravetch, 2011). It follows that genetic variation in FcγR and Fc could contribute to the differences in the magnitude of ADCC. The majority of GM epitopes are present on the Fc portion of the IgG molecule. Thus, Fc of a particular GM genotype could preferentially bind to the FcγR of a particular genotype and influence tumor destruction through ADCC.
⁎ Corresponding author at: Department of Microbiology and Immunology, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425-2230, USA. Tel.: + 1 843 792 4360; fax: + 1 843 792 4882. E-mail address: [email protected] (J.P. Pandey).
http://dx.doi.org/10.1016/j.jneuroim.2014.03.003 0165-5728/© 2014 Elsevier B.V. All rights reserved.
We investigated the interactive effects of GM and FcγR alleles on the ADCC of cells from the human neuroblastoma cell line BE(2)-C, which overexpresses the disialoganglioside antigen GD2. We used three allotypically different IgG1 proteins to inhibit the ADCC of BE(2)-C cells mediated by the chimeric human–murine anti-GD2 monoclonal antibody ch14.18. In this ADCC inhibition assay, anti-GD2 ch14.18 antibodies bound to the target BE(2)-C cells compete with allotypically disparate IgG1 proteins for binding to the activating receptor FcγRIIIa expressed on natural killer (NK) cells. 2. Materials and methods 2.1. GM allotyping and IgG1 affinity purification Serum samples from healthy blood donors were allotyped for all four known IgG1 allotypes—GM 1/a, 2/x, 3/f, and 17/z—by a standard hemagglutination-inhibition assay (Schanfield and van Loghem, 1986). Allotypes 3 and 17 are expressed in the Fd, whereas 1 and 2 are expressed in the Fc, region of the γ1 chains. Total IgG from the pooled sera of subjects—10 expressing the GM 3+,1−,2−, 10 expressing GM 17+,1+,2+, and 10 expressing GM 17 +,1+,2− allotypes— was concentrated by ammonium sulfate fractionation. IgG1 proteins were isolated from the total IgG by subclass-specific affinity chromatography. 2.2. FcγRIIIa genotyping A change from nucleotide T to G in the FcγRIIIa gene results in amino acid change from phenylalanine (F) to valine (V) at position 158 in the
96
J.P. Pandey, A.M. Namboodiri / Journal of Neuroimmunology 270 (2014) 95–97
IgG binding domain of FcγRIIIa. Genotyping of FcγRIIIa alleles was performed by RT-PCR, using a pre-designed TaqMan® assay from Applied Biosystems Inc. (Foster City, CA).
Table 2 Inhibition of ch14.18-mediated ADCC of BE(2)-C cells by NK cells expressing different FcγRIIIa genotypes in the presence of allotypically disparate IgG1 proteins. IgG1 genotype
FcγRIIIa genotype
Inhibition of ADCC (%)
p value
GM 3+,1−,2−
VV FF VV FF VV FF
20.62 ± 19.73 100.00 ± 0.00 60.88 ± 28.48 100.00 ± 0.00 15.28 ± 14.11 12.33±13.03
0.00004
2.3. Purification of NK cells Blood from volunteers homozygous for either V or F alleles of FcγRIIIa was collected in EDTA-coated vacutainer tubes (BD, Franklin Lakes, NJ). NK cells were isolated by affinity depletion of non-NK cells using a kit from Miltenyi Biotec (Auburn, CA).
GM 17+,1+,2+ GM 17+,1+,2−
0.011 0.69
2.4. Cell cytotoxicity by ADCC and ADCC inhibition This method is analogous, in principle, to that described by Macdonald et al. (1981). Human neuroblastoma cell line BE(2)-C was obtained from ATCC (Manassas, VA). Cells were cultured overnight in medium containing 10% FBS, harvested with trypsin-EDTA, and washed 3 times with RPMI-1640 containing 1% BSA. Cells were coated with 10 μg/ml of anti-GD2 antibody ch14.18 (NCI) for 30 min on ice and washed three times with RPMI 1640 containing 1% BSA. These coated cells (5 × 103) were further incubated with 25 μg/ml of aggregated human IgG1—expressing one of the three allotypic combinations—and 5 × 104 NK cells (1:10 ratio of target to effector cells) in a volume of 100 μl RPMI 1640 containing 1% FBS in 96-well plates overnight. The plates were then centrifuged and the supernatant was assayed for lactate dehydrogenase (LDH) activity, using the Cytotox96 kit from Promega Corporation (Madison, WI). Spontaneous LDH release—possibly due to killer-cell immunoglobulin-like receptor dependent cytotoxicity—from target cells incubated with NK cells was used as blank (negative control).
ðControl LDH activity–Test LDH activityÞ ADCC inhibition ð% Þ ¼ −−−−−−−−−−−−−−−−−−−−−− 100 ðControl LDH activityÞ
where Test consists of BE(2)-C target cells incubated with aggregated IgG1, ch14.18 anti-GD2 antibody and NK cells and Control (positive) consists of BE(2)-C cells incubated with ch14.18 antibody and NK cells. Standard deviations were obtained via 7 experimental replications. 2.5. Statistical analyses Student's unpaired t-test was used to compare the percentage of ADCC inhibition associated with GM-FcγRIIIa genotypic combinations. All tests were two-tailed, and the statistical significance was defined as p b 0.05. 3. Results and discussion The amino acid substitutions that characterize the four known human IgG1 allotypes are shown in Table 1. As shown in Table 2, at a concentration of 25 μg/ml, IgG1 expressing the GM 3 +,1 −,2 − allotypes blocked all F-expressing FcγRIIIa present on the NK cells, resulting in 100% inhibition of ch14.18-mediated ADCC of BE(2)-C cells. In contrast, the inhibitory effect of this protein was significantly lower when the NK cells were homozygous
Table 1 Amino acid substitutions characterizing IgG1 allotypes. IgG1 genotype
CH1 214
CH3 356
CH3 358
CH3 431
GM 3+,1−,2− GM 17+,1+,2+ GM 17+,1+,2−
Arg Lys Lys
Glu Asp Asp
Met Leu Leu
Ala Gly Ala
for the V allele of FcγRIIIa (100 vs. 21%; p = 0.00004). A similar trend was evident for the IgG1 proteins expressing the GM 17 +,1 +,2 + allotypes: Inhibitory effect being significantly higher in the presence of NK cells expressing the F allele of FcγRIIIa as compared to those expressing the V variant of this receptor (100 vs. 61%; p = 0.011). IgG1 proteins that lacked the GM 2 determinant, but expressed the GM 1 and 17 alleles, had similar (low) inhibitory effect when the NK cells were homozygous for either allele of FcγRIIIa (15 vs. 12%; p = 0.69). These results show distinct epistatic contributions of GM and FcγRIIIa alleles to the ADCC of neuroblastoma cells. They also suggest that a naturally occurring anti-GD2 IgG1 antibody that expresses the GM 2 determinant in the Fc region could be efficient in mediating ADCC of neuroblastoma cells in the presence of both major (F) and minor (V) variants of FcγRIIIa. Although its inhibitory effect on the anti-GD2-mediated ADCC of neuroblastoma cells was lower in presence of the V variant (61%), it was significantly higher than that of the two IgG1 proteins that lack the GM 2 determinant (61 vs. 21%, p = 0.01; 61 vs. 15%; p = 0.004). ADCC is unlikely to be the only mechanism responsible for the GM gene involvement in neuroblastoma. To gain further mechanistic insights, possible epistatic contributions of all 18 serologically determined GM specificities and FcγRIIa and FcγRIIIa alleles in ADCC and other Fc-mediated effector functions (e.g. complement-dependent cytotoxicity and phagocytosis) need to be investigated. GM genes could also be involved in neuroblastoma via their modulating influence on the immunoevasion strategies employed by cytomegalovirus (Namboodiri and Pandey, 2011; Pandey, 2013), a common herpesvirus that has been implicated in the etiopathogenesis of this malignancy (Wolmer-Solberg et al., 2013). Involvement of GM 2 in the ADCC of neuroblastoma cells suggests a putative mechanism underlying the racial disparity in neuroblastoma survival. A higher proportion of African American children than European American children present with high-risk disease and have worse survival (Henderson et al., 2011). The GM 2 allele is extremely rare in people of African descent, which could adversely affect their ADCC-mediated immunosurveillance against neuroblastoma tumors. A trans-population analysis identified a common single-nucleotide polymorphism (SNP) within sperm associated antigen 16 to be responsible for the ethnic disparities in survival (Gamazon et al., 2013). This analysis, however, did not include GM variants, as it selected only those riskconferring genes that were identified by the genome-wide association studies (GWAS) of neuroblastoma. GM alleles, although common within a racial group, are usually not evaluated by GWAS. The IGHG gene segments harboring these determinants are highly homologous and apparently not amenable to the high throughput genotyping technology used in GWAS. Because these genes were not typed in the HapMap or the 1000 Genomes projects, they cannot be imputed or tagged (through linkage disequilibrium) by any SNPs that are included in the genotyping platforms. This could be one reason why the highly significant GM-neuroblastoma association (Morell et al., 1977) has neither been confirmed nor refuted by the GWAS of this malignancy.
J.P. Pandey, A.M. Namboodiri / Journal of Neuroimmunology 270 (2014) 95–97
Immunotherapy—using a chimeric monoclonal anti-GD2 antibody— appears to be the most effective treatment for patients with high-risk neuroblastoma. This treatment, however, is not effective in all patients; furthermore, it is effective only when combined with toxic cytokines such as interleukin-2, which augment the level of ADCC (Simon et al., 2004; Yu et al., 2010). None of the anti-GD2 antibodies currently in clinical trials expresses the GM 2 allotype. Results presented here would suggest that the inclusion of this variant in the Fc region of these antibodies could improve their clinical efficacy. A great deal of effort is currently being directed at engineering Fc variants with optimized affinity for activating and inhibiting FcγRs (Lazar et al., 2006; Weiner et al., 2010). Along with these efforts, evaluation of the role of naturally occurring Fc (GM) variants—that have been maintained through various selective forces during our evolutionary history—in ADCC and other immunosurveillance mechanisms is warranted. Acknowledgments We are grateful to the National Cancer Institute and United Therapeutics Corporation for providing the ch14.18 antibody. References Gamazon, E.R., Pinto, N., Konkashbaev, A., Im, H.K., Diskin, S.J., London, W.B., Maris, J.M., Dolan, M.E., Cox, N.J., Cohn, S.L., 2013. Trans-population analysis of genetic mechanisms of ethnic disparities in neuroblastoma survival. J. Natl. Cancer Inst. 105, 302–309. Henderson, T.O., Bhatia, S., Pinto, N., London, W.B., McGrady, P., Crotty, C., Sun, C.L., Cohn, S.L., 2011. Racial and ethnic disparities in risk and survival in children with neuroblastoma: a Children's Oncology Group study. J. Clin. Oncol. 29, 76–82.
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