Allogeneic Hematopoietic Cell Transplantation (HCT) can cure hematologic malignancies through ben... more Allogeneic Hematopoietic Cell Transplantation (HCT) can cure hematologic malignancies through beneficial graft-v-leukemia (GVL) allo-immune responses, but its full potential is limited by graft-versus-host disease (GVHD). Recent studies demonstrate allogeneic antibodies develop against mHA encoded on the Y-chromosome, called H-Y antigens develop after sex-mismatch HCT in association with chronic GVHD and persistent disease remission. We hypothesize novel mHA can be serologically identified as targets of allogeneic antibody responses that develop after transplant and were absent pretransplant. Over 70,000 nonsynonymous single nucleotide polymorphisms (nsSNPs) encode polymorphic amino acid residues in human proteins that are potential mHA, and their analysis requires a novel high-throughput approach. ProtoArray™ (Invitrogen) displays five thousand full-length human proteins expressed as N-terminal GST-fusion proteins in a baculovirus system that are affinity purified under native conditions maintaining their cellular enzymatic activities/native conformations. These human antigens are printed in duplicate on nitrocellulose-coated glass slides. In order to determine if targets of allogeneic antibodies can be detected using microarray technology we tested three cGVHD patients for de novo Ab development after HCT using ProtoArrays™. Plasma from three male patients with AML was collected 1) prior to myeloablative HCT 2) from each HLA-identical sibling donor, and 3) one year after HCT. All three had developed extensive chronic GVHD. Plasma was diluted 1:150 and incubated on two microarrays providing replicate results. After washing, each microarray was incubated with anti-human IgG conjugated to Alexa Fluor 647 dye and detected. Negative controls include buffer, BSA, and GST, and their maximum fluorescent signals with these samples were 5,631 units with mean SD of 546. In contrast, influenza A protein is a positive control with fluorescent signal ranging 30,000–45,000 units. Correlation coefficients (R2 values) between duplicate slides ranged from 0.85 to 0.91. Data analysis was performed using Invitrogen’s Prospector Analyzer Software. Fluorescent intensity is measured for each “spot” and individual antigen results are reported as both flourescent signal intensity and a Z-score which is a measure of the intensity of a given signal relative to all of the other human protein targets reported in units of standard deviation. In order to identify human protein targets of allogeneic antibodies, each target antigen’s pretransplant Z-score was subtracted from his respective one year post-transplant Z-score yielding the “ΔZ” for that antigen. Using a conservative ΔZ of 0.1, these three patients developed new antibody responses for 67, 66, and 74 human proteins. Ninety-two percent of these proteins have known nsSNPs. No single protein was recognized by “new” antibodies in all three proteins, however polymorphic proteins Growth Arrest Specific-7 (GAS7), laminin A/C, and ribosomal protein S19 (RPS19) were recognized by two of the three patients after HCT. Results from plasma samples collected 3 and 6 months after HCT demonstrate the progression of alloimmune immune response with few new antibody responses at 3 months. Conclusion: Protein microarrays are an innovative, powerful tool for high-throughput global assessment of B cell alloimmunity after HCT. Microarray technology provides sufficient reproducibility for candidate mHA discovery. These novel mHA require validation by large clinically characterized patient samples.
Using both mixed lymphocyte reaction (MLR) and graft-versus-host reaction (GVHR) assays, investig... more Using both mixed lymphocyte reaction (MLR) and graft-versus-host reaction (GVHR) assays, investigations have been made on 60 multiparous mice of late pregnancy, derived from C57BL mating with SWI as well as C57BL mating with BGI. The results demonstrate the presence of suppressor cells both in uterus-draining-lymphnodes (DLN) and in spleen of allogeneic pregnant mice. The suppressive immune regulatory activity mediated by these cells might play an important role in the successful maintenance of fetus allograft. The possible mechanisms about the inducing of the changes in lymphocyte subpopulation are also studied in this paper.
Identifying molecular targets for eliciting broadly virus-neutralizing antibodies is one of the k... more Identifying molecular targets for eliciting broadly virus-neutralizing antibodies is one of the key steps toward development of vaccines against emerging viral pathogens. Owing to genomic and somatic diversities among viral species, identifying protein targets for broad-spectrum virus neutralization is highly challenging even for the same virus, such as HIV-1. However, viruses rely on host glycosylation machineries to synthesize and express glycans and, thereby, may display common carbohydrate moieties. Thus, exploring glycan-binding profiles of broad-spectrum virus-neutralizing agents may provide key information to uncover the carbohydrate-based virus-neutralizing epitopes. In this study, we characterized two broadly HIV-neutralizing agents, human monoclonal antibody 2G12 and Galanthus nivalis lectin (GNA), for their viral targeting activities. Although these agents were known to be specific for oligomannosyl antigens, they differ strikingly in virus-binding activities. The former ...
Mechanosensory neurons (SNs) ofAplysiaform synapses in culture with some targets (L7), but not ot... more Mechanosensory neurons (SNs) ofAplysiaform synapses in culture with some targets (L7), but not others (L11), even when a SN is plated with both targets. We examined whether branch-specific net export of mRNA encoding synapse-specific molecules might contribute to branch-specific synapse formation. Single-cell RT-PCR was used to assay levels of mRNA encoding the SN-specific neuropeptide (sensorin A) and other transcripts in cell bodies and neuritic processes of SNs cultured alone or with synaptic targets. Some mRNAs are exported to neurites, but not others. Sensorin A mRNA is detected only in SN cell bodies and neurites, and expression levels correlate with the strength of the synaptic connections formed with L7 after 4 d in culture. After 4 d, more sensorin A transcripts are detected in SN neurites contacting L7 than in SN neurites contacting L11. The differential expression at 4 d is found even when a single SN contacts both targets simultaneously. By contrast, no significant diffe...
Macrophage-mediated programmed cell removal (PrCR) is a process essential for the clearance of un... more Macrophage-mediated programmed cell removal (PrCR) is a process essential for the clearance of unwanted (damaged, dysfunctional, aged, or harmful) cells. The detection and recognition of appropriate target cells by macrophages is a critical step for successful PrCR, but its molecular mechanisms have not been delineated. Here using the models of tissue turnover, cancer immunosurveillance, and hematopoietic stem cells, we show that unwanted cells such as aging neutrophils and living cancer cells are susceptible to "labeling" by secreted calreticulin (CRT) from macrophages, enabling their clearance through PrCR. Importantly, we identified asialoglycans on the target cells to which CRT binds to regulate PrCR, and the availability of such CRT-binding sites on cancer cells correlated with the prognosis of patients in various malignancies. Our study reveals a general mechanism of target cell recognition by macrophages, which is the key for the removal of unwanted cells by PrCR in...
Advances in experimental medicine and biology, 2017
We present here an experimental approach for exploring a new class of tumor biomarkers that are o... more We present here an experimental approach for exploring a new class of tumor biomarkers that are overexpressed by circulating tumor cells (CTCs) and are likely targetable in immunotherapy against tumor metastasis. Using carbohydrate microarrays, anti-tumor monoclonal antibodies (mAbs) were scanned against a large panel of carbohydrate antigens to identify potential tumor glycan markers. Subsequently, flow cytometry and fiber-optic array scanning technology (FAST) were applied to determine whether the identified targets are tumor-specific cell-surface markers and are, therefore, likely suitable for targeted immunotherapy. Finally, the tumor glycan-specific antibodies identified were validated using cancer patients' blood samples for their performance in CTC-detection and immunotyping analysis. In this article, identifying breast CTC-specific glycan markers and targeting mAbs serve as examples to illustrate this tumor biomarker discovery strategy.
High mannose type N-glycans are an important component of neutralizing epitopes on HIV-1 envelope... more High mannose type N-glycans are an important component of neutralizing epitopes on HIV-1 envelope glycoprotein gp120. They also serve as signals for protein folding, trafficking, and degradation in protein quality control. A number of lectins and antibodies recognize high-mannose type N-glycans, and glycan array technology has provided an avenue to probing these oligomannose-specific proteins. We describe in this paper a top-down chemoenzymatic approach to synthesizing a library of high-mannose N-glycans and related neoglycoproteins for glycan microarray analysis. The method involves the sequential enzymatic trimming of two readily available natural N-glycans, the Man9GlcNAc2Asn prepared from soybean flour and the sialoglycopeptide (SGP) isolated from chicken egg yolks, coupled with chromatographic separation to obtain a collection of a full range of natural high-mannose N-glycans. The Asn-linked N-glycans were conjugated to bovine serum albumin (BSA) to provide neoglycoproteins con...
Recognition of abnormal glycosylation in virtually every cancer type has raised great interest in... more Recognition of abnormal glycosylation in virtually every cancer type has raised great interest in exploration of the tumor glycome for biomarker discovery. Identifying glycan markers of circulating tumor cells (CTCs) represents a new development in tumor biomarker discovery. The aim of this study was to establish an experimental approach to enable rapid screening of CTCs for glycan marker identification and characterization. We applied carbohydrate microarrays and a high-speed fiber-optic array scanning technology (FAST scan) to explore potential glycan markers of breast CTCs (bCTCs) and targeting antibodies. An anti-tumor monoclonal antibody, HAE3-C1 (C1), was identified as a key immunological probe in this study. In our carbohydrate microarray analysis, C1 was found to be highly specific for an O-glycan cryptic epitope, gp(C1). Using FAST-scan technology, we established a procedure to quantify expression levels of gp(C1) in tumor cells. In blood samples from five stage IV metastatic breast cancer patients, the gp(C1) positive CTCs were detected in all subjects; ∼40% of bCTCs were strongly gp(C1) positive. Interestingly, CTCs from a triple-negative breast cancer patient with multiple sites of metastasis were predominantly gp(C1) positive (92.5%, 37/40 CTCs). Together we present here a practical approach to examine rare cell expression of glycan markers. Using this approach, we identified an O-core glyco-determinant gp(C1) as a potential immunological target of bCTCs. Given its bCTC-expression profile, this target warrants an extended investigation in a larger cohort of breast cancer patients.
Allogeneic Hematopoietic Cell Transplantation (HCT) can cure hematologic malignancies through ben... more Allogeneic Hematopoietic Cell Transplantation (HCT) can cure hematologic malignancies through beneficial graft-v-leukemia (GVL) allo-immune responses, but its full potential is limited by graft-versus-host disease (GVHD). Recent studies demonstrate allogeneic antibodies develop against mHA encoded on the Y-chromosome, called H-Y antigens develop after sex-mismatch HCT in association with chronic GVHD and persistent disease remission. We hypothesize novel mHA can be serologically identified as targets of allogeneic antibody responses that develop after transplant and were absent pretransplant. Over 70,000 nonsynonymous single nucleotide polymorphisms (nsSNPs) encode polymorphic amino acid residues in human proteins that are potential mHA, and their analysis requires a novel high-throughput approach. ProtoArray™ (Invitrogen) displays five thousand full-length human proteins expressed as N-terminal GST-fusion proteins in a baculovirus system that are affinity purified under native conditions maintaining their cellular enzymatic activities/native conformations. These human antigens are printed in duplicate on nitrocellulose-coated glass slides. In order to determine if targets of allogeneic antibodies can be detected using microarray technology we tested three cGVHD patients for de novo Ab development after HCT using ProtoArrays™. Plasma from three male patients with AML was collected 1) prior to myeloablative HCT 2) from each HLA-identical sibling donor, and 3) one year after HCT. All three had developed extensive chronic GVHD. Plasma was diluted 1:150 and incubated on two microarrays providing replicate results. After washing, each microarray was incubated with anti-human IgG conjugated to Alexa Fluor 647 dye and detected. Negative controls include buffer, BSA, and GST, and their maximum fluorescent signals with these samples were 5,631 units with mean SD of 546. In contrast, influenza A protein is a positive control with fluorescent signal ranging 30,000–45,000 units. Correlation coefficients (R2 values) between duplicate slides ranged from 0.85 to 0.91. Data analysis was performed using Invitrogen’s Prospector Analyzer Software. Fluorescent intensity is measured for each “spot” and individual antigen results are reported as both flourescent signal intensity and a Z-score which is a measure of the intensity of a given signal relative to all of the other human protein targets reported in units of standard deviation. In order to identify human protein targets of allogeneic antibodies, each target antigen’s pretransplant Z-score was subtracted from his respective one year post-transplant Z-score yielding the “ΔZ” for that antigen. Using a conservative ΔZ of 0.1, these three patients developed new antibody responses for 67, 66, and 74 human proteins. Ninety-two percent of these proteins have known nsSNPs. No single protein was recognized by “new” antibodies in all three proteins, however polymorphic proteins Growth Arrest Specific-7 (GAS7), laminin A/C, and ribosomal protein S19 (RPS19) were recognized by two of the three patients after HCT. Results from plasma samples collected 3 and 6 months after HCT demonstrate the progression of alloimmune immune response with few new antibody responses at 3 months. Conclusion: Protein microarrays are an innovative, powerful tool for high-throughput global assessment of B cell alloimmunity after HCT. Microarray technology provides sufficient reproducibility for candidate mHA discovery. These novel mHA require validation by large clinically characterized patient samples.
Using both mixed lymphocyte reaction (MLR) and graft-versus-host reaction (GVHR) assays, investig... more Using both mixed lymphocyte reaction (MLR) and graft-versus-host reaction (GVHR) assays, investigations have been made on 60 multiparous mice of late pregnancy, derived from C57BL mating with SWI as well as C57BL mating with BGI. The results demonstrate the presence of suppressor cells both in uterus-draining-lymphnodes (DLN) and in spleen of allogeneic pregnant mice. The suppressive immune regulatory activity mediated by these cells might play an important role in the successful maintenance of fetus allograft. The possible mechanisms about the inducing of the changes in lymphocyte subpopulation are also studied in this paper.
Identifying molecular targets for eliciting broadly virus-neutralizing antibodies is one of the k... more Identifying molecular targets for eliciting broadly virus-neutralizing antibodies is one of the key steps toward development of vaccines against emerging viral pathogens. Owing to genomic and somatic diversities among viral species, identifying protein targets for broad-spectrum virus neutralization is highly challenging even for the same virus, such as HIV-1. However, viruses rely on host glycosylation machineries to synthesize and express glycans and, thereby, may display common carbohydrate moieties. Thus, exploring glycan-binding profiles of broad-spectrum virus-neutralizing agents may provide key information to uncover the carbohydrate-based virus-neutralizing epitopes. In this study, we characterized two broadly HIV-neutralizing agents, human monoclonal antibody 2G12 and Galanthus nivalis lectin (GNA), for their viral targeting activities. Although these agents were known to be specific for oligomannosyl antigens, they differ strikingly in virus-binding activities. The former ...
Mechanosensory neurons (SNs) ofAplysiaform synapses in culture with some targets (L7), but not ot... more Mechanosensory neurons (SNs) ofAplysiaform synapses in culture with some targets (L7), but not others (L11), even when a SN is plated with both targets. We examined whether branch-specific net export of mRNA encoding synapse-specific molecules might contribute to branch-specific synapse formation. Single-cell RT-PCR was used to assay levels of mRNA encoding the SN-specific neuropeptide (sensorin A) and other transcripts in cell bodies and neuritic processes of SNs cultured alone or with synaptic targets. Some mRNAs are exported to neurites, but not others. Sensorin A mRNA is detected only in SN cell bodies and neurites, and expression levels correlate with the strength of the synaptic connections formed with L7 after 4 d in culture. After 4 d, more sensorin A transcripts are detected in SN neurites contacting L7 than in SN neurites contacting L11. The differential expression at 4 d is found even when a single SN contacts both targets simultaneously. By contrast, no significant diffe...
Macrophage-mediated programmed cell removal (PrCR) is a process essential for the clearance of un... more Macrophage-mediated programmed cell removal (PrCR) is a process essential for the clearance of unwanted (damaged, dysfunctional, aged, or harmful) cells. The detection and recognition of appropriate target cells by macrophages is a critical step for successful PrCR, but its molecular mechanisms have not been delineated. Here using the models of tissue turnover, cancer immunosurveillance, and hematopoietic stem cells, we show that unwanted cells such as aging neutrophils and living cancer cells are susceptible to "labeling" by secreted calreticulin (CRT) from macrophages, enabling their clearance through PrCR. Importantly, we identified asialoglycans on the target cells to which CRT binds to regulate PrCR, and the availability of such CRT-binding sites on cancer cells correlated with the prognosis of patients in various malignancies. Our study reveals a general mechanism of target cell recognition by macrophages, which is the key for the removal of unwanted cells by PrCR in...
Advances in experimental medicine and biology, 2017
We present here an experimental approach for exploring a new class of tumor biomarkers that are o... more We present here an experimental approach for exploring a new class of tumor biomarkers that are overexpressed by circulating tumor cells (CTCs) and are likely targetable in immunotherapy against tumor metastasis. Using carbohydrate microarrays, anti-tumor monoclonal antibodies (mAbs) were scanned against a large panel of carbohydrate antigens to identify potential tumor glycan markers. Subsequently, flow cytometry and fiber-optic array scanning technology (FAST) were applied to determine whether the identified targets are tumor-specific cell-surface markers and are, therefore, likely suitable for targeted immunotherapy. Finally, the tumor glycan-specific antibodies identified were validated using cancer patients' blood samples for their performance in CTC-detection and immunotyping analysis. In this article, identifying breast CTC-specific glycan markers and targeting mAbs serve as examples to illustrate this tumor biomarker discovery strategy.
High mannose type N-glycans are an important component of neutralizing epitopes on HIV-1 envelope... more High mannose type N-glycans are an important component of neutralizing epitopes on HIV-1 envelope glycoprotein gp120. They also serve as signals for protein folding, trafficking, and degradation in protein quality control. A number of lectins and antibodies recognize high-mannose type N-glycans, and glycan array technology has provided an avenue to probing these oligomannose-specific proteins. We describe in this paper a top-down chemoenzymatic approach to synthesizing a library of high-mannose N-glycans and related neoglycoproteins for glycan microarray analysis. The method involves the sequential enzymatic trimming of two readily available natural N-glycans, the Man9GlcNAc2Asn prepared from soybean flour and the sialoglycopeptide (SGP) isolated from chicken egg yolks, coupled with chromatographic separation to obtain a collection of a full range of natural high-mannose N-glycans. The Asn-linked N-glycans were conjugated to bovine serum albumin (BSA) to provide neoglycoproteins con...
Recognition of abnormal glycosylation in virtually every cancer type has raised great interest in... more Recognition of abnormal glycosylation in virtually every cancer type has raised great interest in exploration of the tumor glycome for biomarker discovery. Identifying glycan markers of circulating tumor cells (CTCs) represents a new development in tumor biomarker discovery. The aim of this study was to establish an experimental approach to enable rapid screening of CTCs for glycan marker identification and characterization. We applied carbohydrate microarrays and a high-speed fiber-optic array scanning technology (FAST scan) to explore potential glycan markers of breast CTCs (bCTCs) and targeting antibodies. An anti-tumor monoclonal antibody, HAE3-C1 (C1), was identified as a key immunological probe in this study. In our carbohydrate microarray analysis, C1 was found to be highly specific for an O-glycan cryptic epitope, gp(C1). Using FAST-scan technology, we established a procedure to quantify expression levels of gp(C1) in tumor cells. In blood samples from five stage IV metastatic breast cancer patients, the gp(C1) positive CTCs were detected in all subjects; ∼40% of bCTCs were strongly gp(C1) positive. Interestingly, CTCs from a triple-negative breast cancer patient with multiple sites of metastasis were predominantly gp(C1) positive (92.5%, 37/40 CTCs). Together we present here a practical approach to examine rare cell expression of glycan markers. Using this approach, we identified an O-core glyco-determinant gp(C1) as a potential immunological target of bCTCs. Given its bCTC-expression profile, this target warrants an extended investigation in a larger cohort of breast cancer patients.
Uploads
Papers by Denong Wang