Immunoglobulin GM and KM allotypes are associated with antibody responses to Pseudomonas aeruginosa antigens in chronically infected cystic fibrosis patients.

BACKGROUND: Chronic infection with Pseudomonas aeruginosa (P. aeruginosa) is a leading cause of death in patients with cystic fibrosis (CF). Immunobiology of P. aeruginosa infection is complex and not well understood. Chronically infected CF patients generate high levels of antibodies to P. aeruginosa, but this response does not lead to clinical improvement. Therefore, additional studies aimed at identification and understanding of the host factors that influence naturally occurring immune responses to P. aeruginosa are needed. In this investigation, we evaluated the contribution of immunoglobulin GM (γ marker) and KM (κ marker) allotypes to the antibody responses to P. aeruginosa lipopolysaccharide (LPS) O1, O6, O11, and alginate antigens and the broadly-conserved surface polysaccharide expressed by many microbial pathogens, poly-N-acetyl-D-glucosamine (PNAG), in 58 chronically infected CF patients. METHODS: IgG1 markers GM 3 and 17 and IgG2 markers GM 23- and 23+ were determined by a pre-designed TaqMan® genotyping assay. The κ chain determinants KM 1 and 3 were characterized by PCR-RFLP. Antibodies to the LPS O antigens, alginate, and PNAG were measured by an ELISA. RESULTS: Several significant associations were noted with KM alleles. Particular KM 1/3 genotypes were individually and epistatically (with GM 3/17) associated with the level of IgG antibodies to O1, O11, alginate, and PNAG antigens. CONCLUSIONS: Immunoglobulin GM and KM genotypes influence the magnitude of humoral immunity to LPS O, alginate, and PNAG antigens. These results, if confirmed in a larger study population, will be helpful in devising novel immunotherapeutic approaches against P. aeruginosa.

IGHG, IGKC, and FCGR genes and endogenous antibody responses to GARP in patients with breast cancer and matched controls.

Glycoprotein-A repetitions predominant (GARP) is a transmembrane protein that is highly expressed in breast cancer. Its overexpression correlates with worse survival, and antibodies to GARP appear to play a protective role in a mouse model. No large-scale studies of immunity to GARP in humans have yet been undertaken. In this investigation, using a large multiethnic cohort (1738 subjects), we aimed to determine whether the magnitude of anti-GARP antibody responsiveness was significantly different in patients with breast cancer from that in matched healthy controls. We also investigated whether the allelic variation at the immunoglobulin GM (γ marker), KM (κ marker), and Fcγ receptor (FcγR) loci contributed to the interindividual variability in anti-GARP IgG antibody levels. A combined analysis of all subjects showed that levels of anti-GARP antibodies were significantly higher in patients with breast cancer than in healthy controls (mean ±â€¯SD: 7.4 ±â€¯3.5 vs. 6.9 ±â€¯3.5 absorbance units per mL (AU/µL), p < 0.0001). In the two populations with the largest sample size, the probability of breast cancer generally increases as anti-GARP antibody levels increase. Several significant individual and epistatic effects of GM, KM, and FcγR genotypes on anti-GARP antibody responsiveness were noted in both patients and controls. These results, if confirmed by independent investigations, will aid in devising personalized GARP-based immunotherapeutic strategies against breast cancer and other GARP-overexpressing malignancies.

Human Immunoglobulin Heavy Gamma Chain Polymorphisms: Molecular Confirmation Of Proteomic Assessment.

Immunoglobulin G (IgG) proteins are known for the huge diversity of the variable domains of their heavy and light chains, aimed at protecting each individual against foreign antigens. The IgG also harbor specific polymorphism concentrated in the CH2 and CH3-CHS constant regions located on the Fc fragment of their heavy chains. But this individual particularity relies only on a few amino acids among which some could make accurate sequence determination a challenge for mass spectrometry-based techniques.The purpose of the study was to bring a molecular validation of proteomic results by the sequencing of encoding DNA fragments. It was performed using ten individual samples (DNA and sera) selected on the basis of their Gm (gamma marker) allotype polymorphism in order to cover the main immunoglobulin heavy gamma (IGHG) gene diversity. Gm allotypes, reflecting part of this diversity, were determined by a serological method. On its side, the IGH locus comprises four functional IGHG genes totalizing 34 alleles and encoding the four IgG subclasses. The genomic study focused on the nucleotide polymorphism of the CH2 and CH3-CHS exons and of the intron. Despite strong sequence identity, four pairs of specific gene amplification primers could be designed. Additional primers were identified to perform the subsequent sequencing. The nucleotide sequences obtained were first assigned to a specific IGHG gene, and then IGHG alleles were deduced using a home-made decision tree reading of the nucleotide sequences. IGHG amino acid (AA) alleles were determined by mass spectrometry. Identical results were found at 95% between alleles identified by proteomics and those deduced from genomics. These results validate the proteomic approach which could be used for diagnostic purposes, namely for a mother-and-child differential IGHG detection in a context of suspicion of congenital infection.

Human Allergen-Specific IgG Subclass Antibodies Measured Using ImmunoCAP Technology.

BACKGROUND: Knowledge of human IgG subclass antibody responses to various allergens has been hampered by a lack of reliable standardized assays. The aim here was to develop quantitative immunoassays for human IgG1, IgG2, and IgG3 antibodies using ImmunoCAP® technology and to evaluate their application. METHODS: Enzyme conjugates with isotype-specific monoclonal antibodies and calibrators composed of purified myeloma paraproteins were developed for each assay and used together with other standardized assay reagents for the Phadia® 100 instrument. The calibrators were adjusted to the international reference preparation IRP 67/86. The assays were characterized and used together with other standard ImmunoCAP assays to measure antibodies to various allergens in preliminary studies. RESULTS: The new assays had limits of quantitation of 1.0 (IgG1), 4.6 (IgG2), and 0.04 mgA/L (IgG3), and coefficients of variation of <20%. Only some minor cross-reactivity with IgG2 was observed for the specific IgG1 assay. The specific IgG2 assay showed a bias for the allotype G2m(23) and compensation factors were used to adjust the measured concentrations accordingly. Preliminary studies indicated a strong and stable IgG4 antibody response to ß-lactoglobulin in healthy individuals, a high IgG1 and even higher IgG2 antibody response to house dust mite in sensitized and nonsensitized subjects, and a mixed IgG subclass response to venom allergens in allergic patients with increasing IgG4 antibody levels during venom immunotherapy. CONCLUSIONS: The new research assays are valuable tools for immunological studies, enabling the characterization of antibody profiles using a standardized approach, and facilitating data interpretation and the comparison of results across studies.

Genetic markers of immunoglobulin G and immunity to cytomegalovirus in patients with breast cancer.

Human cytomegalovirus (CMV), a ubiquitous herpesvirus, has been implicated in the etiology of breast cancer. It is clear that not all people exposed to CMV are equally likely to develop this malignancy, implying the presence of host genetic factors that might modulate the cancer-spurring properties of the virus. CMV has evolved sophisticated strategies for evading host immunosurveillance. One strategy involves encoding decoy Fcγ receptors (FcγR) that thwart the Fcγ-mediated effector functions, such as antibody-dependent cellular cytotoxicity. In this investigation, using an enzyme-linked immunosorbent assay (ELISA), we aimed to determine whether the decoy FcγR encoded by the CMV gene RL13 binds differentially to anti-CMV antibodies expressing different immunoglobulin GM (γ marker) allotypes, genetic markers of immunoglobulin G (IgG). Results of our ELISA binding studies showed that the absorbance values for the binding of the viral FcγR to the GM 17-expressing IgG antibodies were significantly higher than for the GM 3-expressing antibodies (0.60 vs. 0.36; p=0.0019). These findings provide mechanistic insights into the modulating role played by the genetic variants of IgG in the generation of immunity to CMV in patients with breast cancer.

Endogenous antibody responsiveness to epidermal growth factor receptor is associated with immunoglobulin allotypes and overall survival of patients with glioblastoma.

BACKGROUND: Immunoglobulin γ marker (GM) and κ marker (KM) allotypes, hereditary antigenic determinants of γ and κ chains, respectively, have been shown to be associated with immunity to a variety of self and nonself antigens, but their possible contribution to immunity to the tumor-associated antigens epidermal growth factor receptor (EGFR) and EGFR variant (v)III has not been evaluated. The aim of the present investigation was to determine whether the interindividual variation in endogenous antibody responsiveness to EGFR and EGFRvIII is associated with particular GM, KM, and Fcγ receptor (FcγR) genotypes and whether antibody levels were associated with the overall survival of patients with glioblastoma. METHODS: A total of 126 Caucasian participants with glioblastoma were genotyped for several GM, KM, and FcγR alleles and characterized for IgG antibodies to EGFR and EGFRvIII antigens. RESULTS: The anti-EGFR antibody levels associated with GM 3/3 homozygotes and GM 3/17 heterozygotes were similar (15.9 vs 16.4 arbitrary units [AU]/µL) and significantly lower than those associated with GM 17/17 homozygotes (19.6 AU/µL; nominal P = .007). Participants homozygous for the GM 21 allele also had significantly higher levels of anti-EGFR antibodies than GM 5/5 homozygotes and GM 5/21 heterozygotes (20.1 vs 16.0 and 16.3 AU/µL; nominal P = .005). Similar associations were found with immune responsiveness to EGFRvIII. Higher anti-EGFR and anti-EGFRvIII antibody levels were associated with enhanced overall survival (16 vs 11 mo, nominal P = .038 and 20 vs 11 mo, nominal P = .004, respectively). CONCLUSIONS: GM allotypes contribute to humoral immunity to EGFR in glioblastoma.

Allotype analysis to determine the origin of cytomegalovirus immunoglobulin-G after allogeneic stem cell transplantation.

BACKGROUND: Cytomegalovirus (CMV) reactivation still remains a major problem following allogeneic hematopoietic stem cell transplantation (HSCT). PATIENTS AND METHODS: In this study, we analyzed an immunoglobulin allotype, IgG1m(f), in CMV-seropositive HSCT recipients and their donors to distinguish donor-derived antibody from recipient-derived antibody. Eight donor-recipient pairs were informative regarding the appearance of donor-derived immunoglobulin-G (IgG), as the recipients were homozygous null for the IgG1m(f) allotype and the donors were IgG1m(f) positive. In these patients, total IgG, IgM, and allotype-specific IgG against CMV were measured by enzyme-linked immunosorbent assay. All subjects were monitored for at least 9 months after HSCT with (n = 5) or without (n = 3) CMV reactivation. RESULTS: Donor-derived CMV IgG tended to be elevated earlier in patients with CMV-seropositive donors than in those with CMV-seronegative donors. In 1 patient with a CMV-negative donor, donor-derived CMV IgG was not detected until late CMV reactivation. In 3 patients without CMV reactivation, donor-derived CMV IgG was also elevated within 1-6 months after HSCT. CONCLUSION: In conclusion, the CMV serostatus of the donor may be related to the timing of the appearance of donor-derived CMV IgG and the reconstitution of humoral immunity against CMV, regardless of the CMV antigenemia level after HSCT.

Immunoglobulin GM and FcγRIIIa genotypes influence cytotoxicity of neuroblastoma cells.

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 antibody-mediated 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.

Genetic variants of immunoglobulin γ and κ chains influence humoral immunity to the cancer-testis antigen XAGE-1b (GAGED2a) in patients with non-small cell lung cancer.

GM (γ marker) allotypes, genetic variants of immunoglobulin γ chains, have been reported to be associated strongly with susceptibility to lung cancer, but the mechanism(s) underlying this association is not known. One mechanism could involve their contribution to humoral immunity to lung tumour-associated antigens. In this study, we aimed to determine whether particular GM and KM (κ marker) allotypes were associated with antibody responsiveness to XAGE-1b, a highly immunogenic lung tumour-associated cancer-testis antigen. Sera from 89 patients with non-small cell lung cancer (NSCLC) were allotyped for eight GM and two KM determinants and characterized for antibodies to a synthetic XAGE-1b protein. The distribution of various GM phenotypes was significantly different between XAGE-1b antibody-positive and -negative patients (P = 0·023), as well as in the subgroup of XAGE-1b antigen-positive advanced NSCLC (P = 0·007). None of the patients with the GM 1,17 21 phenotype was positive for the XAGE-1b antibody. In patients with antigen-positive advanced disease, the prevalence of GM 1,2,17 21 was significantly higher in the antibody-positive group than in those who lacked the XAGE-1b antibody (P = 0·026). This phenotype also interacted with a particular KM phenotype: subjects with GM 1,2,17 21 and KM 3,3 phenotypes were almost four times (odds ratio = 3·8) as likely to be positive for the XAGE-1b antibody as the subjects who lacked these phenotypes. This is the first report presenting evidence for the involvement of immunoglobulin allotypes in immunity to a cancer-testis antigen, which has important implications for XAGE-1b-based immunotherapeutic interventions in lung adenocarcinoma.

Immunoglobulin GM allotypes as effect modifiers of cytomegalovirus-spurred neuroblastoma.

An uncommon immunoglobulin GM (γ marker) genotype has been reported to be strongly associated with susceptibility to neuroblastoma, but the mechanism(s) underlying this association is not known. Increasing evidence implicates human cytomegalovirus (HCMV) in the pathogenesis of neuroblastoma. HCMV has evolved a large repertoire of sophisticated strategies to evade host immunosurveillance. Particular GM alleles modulate an immunoevasion strategy of HCMV and contribute to humoral immunity to HCMV epitopes, attributes that provide possible mechanistic explanations for their involvement in the etiopathogenesis of neuroblastoma and explain, at least partially, why a common virus causes/spurs an uncommon cancer.

Suggestive evidence that Fc variants of IgG2 and FcγRIIa loci interact to contribute to the risk of prostate cancer.

GM and KM allotypes-hereditary antigenic variants of immunoglobulin γ and κ chains, respectively-and the genetic variants of activating Fcγ receptors (FcγR) are associated with the immunobiology of several malignant diseases, but their role in susceptibility to prostate cancer has not been examined. This investigation aimed to determine whether these genes-individually or in particular epistatic combinations-contribute to the risk of prostate cancer. We genotyped DNA from 200 Caucasian patients with prostate cancer and 185 healthy controls (matched for age, race, gender, and geography) for several GM, KM, FcγRIIa, and FcγRIIIa alleles by molecular methods. None of the genotypes by itself was associated with the risk of prostate cancer. However, particular alleles at GM 23 and FcγRIIa loci interactively contributed to the risk of this malignancy (p = 0.031), the odds ratios ranging from 0.44 in subjects homozygous for the GM 23- allele at the IgG2 locus and for the histidine allele at the FcγRIIa locus to 2.86 in subjects homozygous for the GM 23+ allele at the IgG2 locus and the histidine allele at the FcγRIIa locus. To our knowledge, this is the first report implicating GM and FcγR loci as risk/protective factors for prostate cancer. Additional, independent, studies are warranted to confirm and extend these findings.

IgG and FcγR genotypes and humoral immunity to mucin 1 in prostate cancer.

Immunoglobulin GM and KM allotypes-hereditary antigenic determinants of γ and κ chains, respectively-and Fcγ receptor IIa (FcγRIIa) and FcγRIIIa genes are associated with the immunobiology of several malignant diseases, but their role in humoral immunity to the tumor-associated antigen mucin 1 (MUC1) in prostate cancer has not been examined. This investigation aimed to determine whether these genes-individually or in particular epistatic combinations-contribute to the inter-individual variability in the magnitude of antibody responsiveness to MUC1 in patients with prostate cancer. We genotyped DNA from 127 Caucasian American (CA) and 76 African American (AA) patients with histologically verified adenocarcinoma of the prostate for several GM, KM, FcγRIIa, and FcγRIIIa alleles by molecular methods. We also quantitated antibodies to MUC1 in the plasma from these patients by ELISA. In CA patients, homozygosity for the valine allele at the FcγRIIIa locus was significantly associated with low antibody responsiveness to MUC1 (p=0.029). In AA patients, the KM 1/3 heterozygotes had significantly higher anti-MUC1 antibody levels than 1/1 and 3/3 homozygotes (p=0.044). These results, the first to implicate FcγRIIIa and KM loci in humoral immunity to MUC1 in prostate cancer, might be relevant to MUC1-based immunotherapy of this malignancy.

Allotype analysis to distinguish the origin of varicella-zoster virus immunoglobulin G after allogeneic stem cell transplantation.

Varicella-zoster virus (VZV) reactivation is a frequent complication after allogeneic hematopoietic stem cell transplantation (HSCT). Although previous studies have revealed that cellular immunity is important for suppressing reactivation, the role of humoral immunity against VZV has been poorly evaluated. We analyzed inherited polymorphisms in the immunoglobulin G (IgG) heavy chain constant regions of 50 HSCT recipient-donor pairs to distinguish donor-derived and recipient-derived antibodies. Twelve pairs were informative regarding the origin of IgG, since either the donors (n = 3) or recipients (n = 9) were homozygous null for the IgG1m(f) allotype. In these 9 homozygous-null recipients, allotype-specific IgG against VZV were measured by enzyme-linked immunosorbent assay and compared with measles-IgG. All 9 homozygous-null recipients were monitored for more than 1 year after HSCT, with (n = 4, localized zoster) or without (n = 5) clinical VZV disease. In 3 patients with VZV disease, donor-derived IgG against VZV was elevated between 500 to 700 days after HSCT after the episode of VZV disease. In 1 patient who suffered from VZV disease just before HSCT, donor-derived VZV IgG was elevated within 3 months after HSCT. On the other hand, 2 patients who received reduced-intensity conditioning (RIC) transplantation from an IgG1m(f) null donor maintained recipient-derived IgG against VZV for more than 1 year, whereas it was decreased within 3 months in 1 recipient who received conventional conditioning. In conclusion, the production of anti-VZV IgG by recipient plasma cells persists long after RIC. In patients without symptomatic VZV reactivation, donor-derived anti-VZV IgG did not reach titers comparable to those measured in healthy virus carriers.

Racially restricted contribution of immunoglobulin Fcγ and Fcγ receptor genotypes to humoral immunity to human epidermal growth factor receptor 2 in breast cancer.

Tumour-associated antigen human epidermal growth factor receptor 2 (HER2) is over-expressed in 25-30% of breast cancer patients and is associated with poor prognosis. Naturally occurring anti-HER2 antibody responses have been described in patients with HER2 over-expressing tumours. There is significant interindividual variability in antibody responsiveness, but the host genetic factors responsible for this variability are poorly understood. The aim of the present investigation was to determine whether immunoglobulin genetic markers [GM (genetic determinants of γ chains)] and Fcγ receptor (FcγR) alleles contribute to the magnitude of natural antibody responsiveness to HER2 in patients with breast cancer. A total of 855 breast cancer patients from Japan and Brazil were genotyped for several GM and FcγR alleles. They were also characterized for immunoglobulin (Ig)G antibodies to HER2. In white subjects (n = 263), GM 23-carriers had higher levels of anti-HER2 antibodies than non-carriers of this allele (p = 0·004). At the GM 5/21 locus, the homozygotes for the GM 5 allele had higher levels of anti-HER2 antibodies than the other two genotypes (P = 0·0067). In black subjects (n = 42), FcγRIIa-histidine/histidine homozygotes and FcγRIIIa-phenylalanine/valine heterozygotes were associated with high antibody responses (P = 0·0071 and 0·0275, respectively). FcγR genotypes in white subjects and GM genotypes in black subjects were not associated with anti-HER2 antibody responses. No significant associations were found in other study groups. These racially restricted contributions of GM and FcγR genotypes to humoral immunity to HER2 have potential implications for immunotherapy of breast cancer.

Epistatic interactions between Fc (GM) and FcγR genes and the host control of human immunodeficiency virus replication.

Host genetic factors are thought to contribute to the interindividual differences in the control of human immunodeficiency virus (HIV) replication. The aim of the present investigation was to determine whether genes encoding GM and KM allotypes-genetic markers of immunoglobulin γ and κ chains, respectively-and those encoding Fcγ receptor (FcγR) IIa and IIIa are associated with the host control of HIV replication. A case-control design was employed among HIV-infected subjects, with a group that spontaneously controlled HIV replication ("controllers") as cases (n = 73) and those who did not control replication as controls (n = 100). Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism, direct DNA sequencing, and TaqMan genotyping assays. In Caucasian Americans, certain combinations of FcγR and GM genotypes were differentially distributed between controllers and noncontrollers. Among the noncarriers of the FcγRIIa arginine allele, GM21 noncarriers had over 7-fold greater odds of being controllers than the carriers of this allele (odds ratio [OR] = 7.47). These GM determinants also interacted with FcγRIIIa alleles. Among the carriers of the FcγRIIIa valine allele, GM21 noncarriers had over 3-fold greater odds of being controllers than the carriers of this allele (OR = 3.26). These results demonstrate epistatic interactions of genes on chromosomes 14 (GM) and 1 (FcγR) in influencing the control of HIV replication.

Bovine herpesvirus type 1 (BHV-1) UL49.5 luminal domain residues 30 to 32 are critical for MHC-I down-regulation in virus-infected cells.

Bovine herpesvirus type 1 (BHV-1) U(L)49.5 inhibits transporter associated with antigen processing (TAP) and down-regulates cell-surface expression of major histocompatibility complex (MHC) class I molecules to promote immune evasion. We have constructed a BHV-1 U(L)49.5 cytoplasmic tail (CT) null and several U(L)49.5 luminal domain mutants in the backbone of wild-type BHV-1 or BHV-1 U(L)49.5 CT- null viruses and determined their relative TAP mediated peptide transport inhibition and MHC-1 down-regulation properties compared with BHV-1 wt. Based on our results, the U(L)49.5 luminal domain residues 30-32 and U(L)49.5 CT residues, together, promote efficient TAP inhibition and MHC-I down-regulation functions. In vitro, BHV-1 U(L)49.5 Δ30-32 CT-null virus growth property was similar to that of BHV-1 wt and like the wt U(L)49.5, the mutant U(L)49.5 was incorporated in the virion envelope and it formed a complex with gM in the infected cells.

Differential inhibition of trastuzumab- and cetuximab-induced cytotoxicity of cancer cells by immunoglobulin G1 expressing different GM allotypes.

Antibody-dependent cell-mediated cytotoxicity (ADCC), which links the innate and the adaptive arms of immunity, is a major host immunosurveillance mechanism against tumours, as well as the leading mechanism underlying the clinical efficacy of therapeutic antibodies such as cetuximab and trastuzumab, which target tumour antigens, human epidermal growth factor receptor (HER)1 and HER2, respectively. Immunoglobulin (Ig)G antibody-mediated ADCC is triggered upon ligation of Fcγ receptor (FcγR) to the Fc region of IgG molecules. It follows that genetic variation in FcγR and Fc could contribute to the differences in the magnitude of ADCC. Genetic variation in FcγR is known to contribute to the differences in the magnitude of ADCC, but the contribution of natural genetic variation in Fc, GM allotypes, in this interaction has hitherto not been investigated. Using an ADCC inhibition assay, we show that IgG1 expressing the GM 3+, 1-, 2- allotypes was equally effective in inhibiting cetuximab- and trastuzumab-mediated ADCC of respective target cells, in the presence of natural killer (NK) cells expressing either valine or phenylalanine allele of FcγRIIIa. In contrast, IgG1 expressing the allelic GM 17+, 1+, 2+ allotypes was significantly more effective in inhibiting the ADCC - mediated by both monoclonal antibodies - when NK cells expressed the valine, rather than the phenylalanine, allele of FcγRIIIa. These findings have important implications for engineering antibodies (with human γ1 constant region) against malignancies characterized by the over-expression of tumour antigens HER1 and HER2 - especially for patients who, because of their FcγRIIIa genotype, are unlikely to benefit from the currently available therapeutics.

The human G1m1 allotype associates with CD4+ T-cell responsiveness to a highly conserved IgG1 constant region peptide and confers an asparaginyl endopeptidase cleavage site.

The human G1m1 allotype comprises two amino acids, D12 and L14, in the CH3 domain of IGHG1. Although the G1m1 allotype is prevalent in human populations, ~40% of Caucasiods are homozygous for the nG1m1 allotype corresponding to E12 and M14. Peptides derived from the G1m1 region were tested for their ability to induce CD4+ T-cell proliferative responses in vitro. A peptide immediately downstream from the G1m1 sequence was recognized by CD4+ T cells in a large percentage of donors (peptide CH315-29). CD4+ T-cell proliferative responses to CH315-29 were found at an increased frequency in nG1m1 homozygous donors. Homozygous nG1m1 donors possessing the HLA-DRB1*07 allele displayed the highest magnitudes of proliferation. CD4+ T cells from donors homozygous for nG1m1 proliferated to G1m1-carrying Fc-fragment proteins, whereas CD4+ T cells from G1m1 homozygous donors did not. The G1m1 sequence creates an enzymatic cleavage site for asparaginyl endopeptidase in vitro. Proteolytic activity at D12 may allow the presentation of the CH315-29 peptide, which in turn may result in the establishment of tolerance to this peptide in G1m1-positive donors. Homozygous nG1m1 patients may be more likely to develop CD4+ T-cell-mediated immune responses to therapeutic antibodies carrying the G1m1 allotype.