ABSTRACT
BACKGROUND: Tumor relapse due to mutation in CD19 can hinder the efficacy of chimeric antigen receptor (CAR)-T cell therapy. Herein, we focused on lymphoma patients whose B cells exhibited a point mutation in CD19 of B cells after CAR-T cell infusion. METHODS: The CAR-T and CD19+ B cells from peripheral blood or bone marrow were assessed using flow cytometry. Genome sequencing was conducted to identify the molecular characteristics of CAR-T and CD19+ B cells from pre-relapse and postrelapse samples. CD19 in CARs comprising single chain fragments variable (scFV) antibody with FMC63 or 21D4 was constructed. The cytotoxic efficacy of CAR-T cells was also evaluated via in vitro and in vivo experiments. RESULTS: A patient with high-grade B cell lymphoma exhibited complete response, but the lymphoma relapsed in her left breast at 6 months after CD19 CAR (FMC63)-T cell infusion. A mutation was found in exon 3 of CD19 (p.163. R-L) in malignant B cells of the patient. In two lymphoma patients who exhibited resistance to CAR-T cell therapy, a mutation was detected in exon 3 of CD19 (p.174. L-V). Functional analysis revealed that FMC63 CAR-T cells exhibited antitumor ability against wild-type CD19+ cells but were unable to eradicate these two types of mutated CD19+ cells. Interestingly, 21D4 CAR-T cells were potentially capable of eradicating these mutated CD19+ cells and exhibiting high antitumor capacity against CD19+ cells with loss of exon 1, 2, or 3. CONCLUSIONS: These findings suggest that point mutation can facilitate immune escape from CAR-T cell therapy and that alternative CAR-T cells can effectively eradicate the mutated B cells, providing an individualized therapeutic approach for lymphoma patients showing relapse.
Subject(s)
Antigens, CD19/immunology , Cell- and Tissue-Based Therapy/methods , Immunotherapy, Adoptive/methods , Lymphoma, B-Cell/therapy , Point Mutation/immunology , Animals , Female , Humans , Male , Mice , Mice, SCID , Middle Aged , Young AdultABSTRACT
Therapeutic concepts exploiting tumor-specific antibodies are often established in pre-clinical xenograft models using immuno-deficient mice. More complex therapeutic paradigms, however, warrant the use of immuno-competent mice, that more accurately capture the relevant biology that is being exploited. These models require the use of (surrogate) mouse or rat antibodies to enable optimal interactions with murine effector molecules. Immunogenicity is furthermore decreased, allowing longer-term treatment. We recently described controlled Fab-arm exchange (cFAE) as an easy-to-use method for the generation of therapeutic human IgG1 bispecific antibodies (bsAb). To facilitate the investigation of dual-targeting concepts in immuno-competent mice, we now applied and optimized our method for the generation of murine bsAbs. We show that the optimized combinations of matched point-mutations enabled efficient generation of murine bsAbs for all subclasses studied (mouse IgG1, IgG2a and IgG2b; rat IgG1, IgG2a, IgG2b, and IgG2c). The mutations did not adversely affect the inherent effector functions or pharmacokinetic properties of the corresponding subclasses. Thus, cFAE can be used to efficiently generate (surrogate) mouse or rat bsAbs for pre-clinical evaluation in immuno-competent rodents.
Subject(s)
Antibodies, Bispecific/biosynthesis , Immunoglobulin G/immunology , Neoplasms/therapy , Animals , Antibodies, Bispecific/immunology , Humans , Immunoglobulin G/genetics , Immunoglobulin G/therapeutic use , Mice , Models, Animal , Neoplasms/genetics , Neoplasms/immunology , Point Mutation/genetics , Point Mutation/immunology , Rats , Xenograft Model Antitumor AssaysABSTRACT
The vaccine strains for live attenuated influenza vaccines (LAIVs) have cold-adapted, temperature-sensitive, and attenuated phenotypes, which are guaranteed by the presence of specific mutations from the master donor virus in their internal genes. In this study, we used mutant viruses of the pathogenic A/Puerto Rico/8/34 (H1N1) that contained ts-mutations in PB1 (K265N, V591I), PB2 (V478L), and PA (L28P, V341L) genes along and/or in different combinations to evaluate the impact of these mutations in the immune responses. Sequential addition of tested mutations resulted in the stepwise decrease in virus-specific serum and, to a lesser extent, mucosal antibody levels. We demonstrated strong positive correlation between virus attenuation (virus titer in lung) and antibody titers. The ts-mutations in PB1, PB2, and PA genes are mostly involved in the modulation of the humoral immunity, but also have a moderate effect on the cellular adaptive immune response.
Subject(s)
Immunity, Cellular , Influenza A Virus, H1N1 Subtype , Point Mutation/immunology , RNA-Dependent RNA Polymerase , Viral Proteins , Animals , Disease Models, Animal , Humans , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/immunology , Mice , Mice, Inbred CBA , Orthomyxoviridae Infections , RNA-Dependent RNA Polymerase/genetics , RNA-Dependent RNA Polymerase/immunology , Viral Proteins/genetics , Viral Proteins/immunologyABSTRACT
Various immunosuppressive factors are derived from polydnaviruses (PDVs) mutually symbiotic to some ichneumonid and braconid wasps. CrV1 was originally identified from a PDV called Cotesia rubecula bracovirus. CrV1 orthologs are reported in other Cotesia-associated PDVs, but not clearly understood in their physiological functions. This study determined a function of CrV1 encoded in Cotesia plutellae bracovirus (CpBV). CpBV-CrV1 is the largest molecule among the known CrV1s and is predicted to possess three coiled-coil motifs. It was constitutively expressed in parasitized host, Plutella xylostella. In vivo transient expression of CpBV-CrV1 significantly impaired hemocyte nodule formation. However, its specific RNA interference significantly recovered the immune response. Two point mutations (AlaâPro at 192nd and 196th positions) were designed to remove the main coiled-coil motif of CpBV-CrV1. When CpBV-CrV1 and the mutant CpBV-CrV1 were expressed in Sf9 cells, their proteins were synthesized and secreted into each culture medium. When each culture medium was overlaid on hemocytes of nonparasitized P. xylostella, an immunofluorescence assay showed that CpBV-CrV1 entered the hemocytes, but the mutant protein did not. The entered CpBV-CrV1 significantly inhibited hemocyte-spreading behavior by preventing F-actin formation. These results indicate that CpBV-CrV1 is an immunosuppressive factor of CpBV, in which its coiled-coil motif is essential.
Subject(s)
Amino Acid Motifs/genetics , Amino Acid Motifs/immunology , Hemocytes/virology , Immunity, Cellular/immunology , Mutagenesis/genetics , Point Mutation/genetics , Polydnaviridae/genetics , Amino Acid Sequence , Animals , Cell Line , Hemocytes/immunology , Host-Parasite Interactions/genetics , Host-Parasite Interactions/immunology , Larva/immunology , Larva/virology , Molecular Sequence Data , Mutagenesis/immunology , Point Mutation/immunology , Polydnaviridae/immunology , RNA Interference/immunology , Sequence Alignment , Sf9 Cells , Viral Proteins/genetics , Wasps/immunology , Wasps/virologyABSTRACT
Autosomal recessive mutations in UNC13D, the gene that encodes Munc13-4, are associated with familial hemophagocytic lymphohistiocytosis type 3 (FHL3). Munc13-4 expression is obligatory for exocytosis of lytic granules, facilitating cytotoxicity by T cells and natural killer (NK) cells. The mechanisms regulating Munc13-4 expression are unknown. Here, we report that Munc13-4 is highly expressed in differentiated human NK cells and effector CD8(+) T lymphocytes. A UNC13D c.118-308C>T mutation, causative of FHL3, disrupted binding of the ETS family member ELF1 to a conserved intronic sequence. This mutation impairs UNC13D intron 1 recruitment of STAT4 and the chromatin remodeling complex component BRG1, diminishing active histone modifications at the locus. The intronic sequence acted as an overall enhancer of Munc13-4 expression in cytotoxic lymphocytes in addition to representing an alternative promoter encoding a novel Munc13-4 isoform. Mechanistically, T cell receptor engagement facilitated STAT4-dependent Munc13-4 expression in naive CD8(+) T lymphocytes. Collectively, our data demonstrates how chromatin remodeling within an evolutionarily conserved regulatory element in intron 1 of UNC13D regulates the induction of Munc13-4 expression in cytotoxic lymphocytes and suggests that an alternative Munc13-4 isoform is required for lymphocyte cytotoxicity. Thus, mutations associated with primary immunodeficiencies may cause disease by disrupting transcription factor binding.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Immunologic Deficiency Syndromes/immunology , Killer Cells, Natural/immunology , Membrane Proteins/immunology , Point Mutation/immunology , Blotting, Western , CD8-Positive T-Lymphocytes/metabolism , Cells, Cultured , Cytotoxicity, Immunologic/genetics , Cytotoxicity, Immunologic/immunology , DNA Helicases/genetics , DNA Helicases/immunology , DNA Helicases/metabolism , Gene Expression Regulation/immunology , Humans , Immunologic Deficiency Syndromes/genetics , Immunologic Deficiency Syndromes/metabolism , Introns/genetics , Killer Cells, Natural/metabolism , Lymphohistiocytosis, Hemophagocytic/genetics , Lymphohistiocytosis, Hemophagocytic/immunology , Lymphohistiocytosis, Hemophagocytic/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/immunology , Nuclear Proteins/metabolism , Protein Binding/genetics , Protein Binding/immunology , Protein Isoforms/genetics , Protein Isoforms/immunology , Protein Isoforms/metabolism , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , STAT4 Transcription Factor/genetics , STAT4 Transcription Factor/immunology , STAT4 Transcription Factor/metabolism , Transcription Factors/genetics , Transcription Factors/immunology , Transcription Factors/metabolismABSTRACT
PURPOSE: Immunological and molecular evaluation of a patient presenting with recurrent infections caused by Streptococcus pneumoniae and low complement component 3 (C3) levels. METHODS: Immunological evaluation included complement components and immunoglobulin level quantification as well as number and function of T cells, B cells and neutrophils. Serotype-specific immunoglobulin G antibodies against S. pneumoniae capsular polysaccharides were quantified by ELISA in serum samples before and after vaccination with unconjugated polysaccharide vaccine. For the molecular analysis, genomic DNA from the patient and parents were isolated and all exons as well as exon-intron boundaries of the C3 gene were sequenced by Sanger sequencing. RESULTS: A 16-year-old male, born to consanguineous parents, presented with recurrent episodes of pneumonia caused by S. pneumoniae and bronchiectasis. The patient showed severely reduced C3 and immunoglobulin A levels, while the parents showed moderately reduced levels of C3. Mutational analysis revealed a novel, homozygous missense mutation in the C3 gene (c. C4554G, p. Cys1518Trp), substituting a highly conserved amino acid in the C345C domain of C3 and interrupting one of its disulfide bonds. Both parents were found to be carriers of the affected allele. Vaccination against S. pneumoniae resulted in considerable clinical improvement. CONCLUSIONS: We report a novel homozygous mutation in the C3 gene in a patient with concomitant selective IgA deficiency who presented with a marked clinical improvement after vaccination against S. pneumoniae. This observation underlines the notion that vaccination against this microorganism is an important strategy for treatment of PID patients, particularly those presenting with increased susceptibility to infections caused by this agent.
Subject(s)
Complement C3/genetics , IgA Deficiency/genetics , IgA Deficiency/immunology , Mutation, Missense , Adolescent , Bronchiectasis/complications , Bronchiectasis/genetics , Bronchiectasis/immunology , Child , Child, Preschool , Comorbidity , Complement C3/antagonists & inhibitors , Complement C3/biosynthesis , Female , Humans , IgA Deficiency/complications , Male , Mutation, Missense/genetics , Mutation, Missense/immunology , Pedigree , Pneumococcal Vaccines/therapeutic use , Pneumonia, Pneumococcal/genetics , Pneumonia, Pneumococcal/immunology , Pneumonia, Pneumococcal/prevention & control , Point Mutation/genetics , Point Mutation/immunology , Sequence Homology, Amino AcidABSTRACT
PURPOSE: Common Variable Immunodeficiency Disorder (CVID) is a complex disorder that predisposes patients to recurrent and severe infections. The C104R mutation in the transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) is the most frequent mutation identified in patients with CVID. We carried out a detailed immunological and molecular study in a family with a C104R mutation. METHODS: We have undertaken segregation analysis of a kindred with C104R mutations of the TACI gene. Detailed immunological and molecular investigations were carried out for this kindred and the clinical phenotype was compared to the genotype. RESULTS: Segregation analysis of our kindred showed that inheriting single or double copy of the C104R mutation does not consign an individual to CVID. All heterozygotes in the family were phenotypically different, ranging from asymptomatic to ill-health. A family member with a wild type TACI variant had CVID-related phenotype including IgA deficiency and type 1 diabetes. Interestingly, a family member with the homozygous C104R/C104R variant did not meet the criteria for CVID because he had excellent, albeit unsustained, vaccine responses to T cell dependent and T cell independent vaccine antigens despite profound hypogammaglobulinemia. CONCLUSION: The C104R mutation does not correlate with the clinical phenotypes in this family.
Subject(s)
Common Variable Immunodeficiency/genetics , Common Variable Immunodeficiency/immunology , Genetic Variation/immunology , Point Mutation , Transmembrane Activator and CAML Interactor Protein/genetics , Adult , Aged, 80 and over , Child , Chromosome Segregation/genetics , Chromosome Segregation/immunology , Common Variable Immunodeficiency/diagnosis , Female , Gene Dosage/genetics , Gene Dosage/immunology , Genetic Predisposition to Disease , Genotype , Humans , Immunophenotyping/methods , Male , Middle Aged , Pedigree , Point Mutation/genetics , Point Mutation/immunology , Transmembrane Activator and CAML Interactor Protein/metabolismABSTRACT
LPS is recognized by TLR4 and radioprotective 105 kDa in B cells. Susceptibility to LPS in murine B cells is most closely linked to the locus containing the TLR4 gene. However, the molecular mechanism underlying genetic control of LPS sensitivity by this locus has not been fully elucidated. In this study, we revealed that C57BL/6 (B6) B cells respond to mAb-induced, TLR4-specific signals stronger than BALB/c (BALB) B cells, as assessed by proliferation and upregulation of CD69 and CD86. In contrast, BALB B cells were not hyporesponsive to agonistic anti-radioprotective 105 kDa mAb or the TLR9 agonist CpG. Although the level of TLR4 mRNA in BALB B cells was comparable with that in B6 B cells, surface TLR4 expression in BALB B cells was lower than that in B6 B cells. This lower surface expression of BALB TLR4 was also observed when HEK293 and Ba/F3 cells were transfected with a BALB TLR4 expression construct. We identified a V254I mutation as the responsible single nucleotide polymorphism for lower surface expression of BALB TLR4. Furthermore, cotransfection of myeloid differentiation factor-2 increased BALB TLR4 expression, although it was still lower than B6 TLR4 expression. In concordance with reduced expression, Ba/F3 cells transfected with BALB TLR4 and myeloid differentiation factor-2 were hyporesponsive compared with those with B6 TLR4, as assessed by LPS-induced NF-κB activation. In conclusion, we revealed that LPS sensitivity is genetically controlled by the level of surface TLR4 expression on B cells. A V254I mutation accounts for the LPS hyporesponsive phenotype of BALB B cells.
Subject(s)
B-Lymphocyte Subsets/immunology , Lipopolysaccharides/genetics , Point Mutation/immunology , Toll-Like Receptor 4/antagonists & inhibitors , Toll-Like Receptor 4/biosynthesis , Animals , B-Lymphocyte Subsets/metabolism , Cell Line, Tumor , Cell Membrane/genetics , Cell Membrane/immunology , Cells, Cultured , HEK293 Cells , Humans , Immunophenotyping , Lipopolysaccharides/biosynthesis , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Rats , Rats, Wistar , Toll-Like Receptor 4/deficiencyABSTRACT
Through recognition of HLA class I, killer cell Ig-like receptors (KIR) modulate NK cell functions in human immunity and reproduction. Although a minority of HLA-A and -B allotypes are KIR ligands, HLA-C allotypes dominate this regulation, because they all carry either the C1 epitope recognized by KIR2DL2/3 or the C2 epitope recognized by KIR2DL1. The C1 epitope and C1-specific KIR evolved first, followed several million years later by the C2 epitope and C2-specific KIR. Strong, varying selection pressure on NK cell functions drove the diversification and divergence of hominid KIR, with six positions in the HLA class I binding site of KIR being targets for positive diversifying selection. Introducing each naturally occurring residue at these positions into KIR2DL1 and KIR2DL3 produced 38 point mutants that were tested for binding to 95 HLA- A, -B, and -C allotypes. Modulating specificity for HLA-C is position 44, whereas positions 71 and 131 control cross-reactivity with HLA-A*11:02. Dominating avidity modulation is position 70, with lesser contributions from positions 68 and 182. KIR2DL3 has lower avidity and broader specificity than KIR2DL1. Mutation could increase the avidity and change the specificity of KIR2DL3, whereas KIR2DL1 specificity was resistant to mutation, and its avidity could only be lowered. The contrasting inflexibility of KIR2DL1 and adaptability of KIR2DL3 fit with C2-specific KIR having evolved from C1-specific KIR, and not vice versa. Substitutions restricted to activating KIR all reduced the avidity of KIR2DL1 and KIR2DL3, further evidence that activating KIR function often becomes subject to selective attenuation.
Subject(s)
HLA-C Antigens/metabolism , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Point Mutation/immunology , Receptors, KIR2DL1/metabolism , Receptors, KIR2DL3/metabolism , Amino Acid Substitution/genetics , Amino Acid Substitution/immunology , Animals , Cell Line , Genetic Variation/genetics , Genetic Variation/immunology , HLA-C Antigens/genetics , Humans , Point Mutation/genetics , Protein Binding/genetics , Protein Binding/immunology , Protein Structure, Tertiary/genetics , Receptors, KIR2DL1/antagonists & inhibitors , Receptors, KIR2DL1/genetics , Receptors, KIR2DL3/antagonists & inhibitors , Receptors, KIR2DL3/geneticsABSTRACT
Multiple genetic events and subsequent clonal evolution drive carcinogenesis, making disease elimination with single-targeted drugs difficult. The multiplicity of gene mutations derived from clonal heterogeneity therefore represents an ideal setting for multiepitope tumor vaccination. Here, we used next generation sequencing exome resequencing to identify 962 nonsynonymous somatic point mutations in B16F10 murine melanoma cells, with 563 of those mutations in expressed genes. Potential driver mutations occurred in classical tumor suppressor genes and genes involved in proto-oncogenic signaling pathways that control cell proliferation, adhesion, migration, and apoptosis. Aim1 and Trrap mutations known to be altered in human melanoma were included among those found. The immunogenicity and specificity of 50 validated mutations was determined by immunizing mice with long peptides encoding the mutated epitopes. One-third of these peptides were found to be immunogenic, with 60% in this group eliciting immune responses directed preferentially against the mutated sequence as compared with the wild-type sequence. In tumor transplant models, peptide immunization conferred in vivo tumor control in protective and therapeutic settings, thereby qualifying mutated epitopes that include single amino acid substitutions as effective vaccines. Together, our findings provide a comprehensive picture of the mutanome of B16F10 melanoma which is used widely in immunotherapy studies. In addition, they offer insight into the extent of the immunogenicity of nonsynonymous base substitution mutations. Lastly, they argue that the use of deep sequencing to systematically analyze immunogenicity mutations may pave the way for individualized immunotherapy of cancer patients.
Subject(s)
Cancer Vaccines/therapeutic use , Exome , Melanoma, Experimental/therapy , Point Mutation/immunology , Animals , Cancer Vaccines/classification , Cancer Vaccines/genetics , Cancer Vaccines/immunology , Cell Line, Tumor , Epitopes/genetics , Female , Melanoma, Experimental/genetics , Melanoma, Experimental/immunology , Mice , Mice, Inbred C57BL , Sequence Analysis, DNA , Vaccination , Vaccines, Subunit/therapeutic useABSTRACT
BACKGROUND: Alloantibodies against human neutrophil antigen-3 (HNA-3) are responsible for the fatalities reported in transfusion-related acute lung injury. Consequently, reliable detection of these alloantibodies is mandatory to improve blood transfusion safety. In this study, we developed stable cell lines for the detection of HNA-3 antibodies. STUDY DESIGN AND METHODS: HEK293T were transfected with HNA-3a or HNA-3b constructs and sorted by flow cytometry according to high surface expression. Transfected cells were tested with sera containing HNA-3 antibodies in flow cytometry and antibody capture assay (ACA). The results were compared with granulocyte agglutination test and granulocyte immunofluorescence test. RESULTS: In flow cytometry, 12 of 14 HNA-3a sera reacted specifically with HNA-3aa cells. One serum sample showed positive reaction with HNA-3bb cells. All HNA-3b sera recognized HNA-3bb cells. No reaction was observed with broad reactive antibodies against HLA Class I. In ACA, all HNA-3a sera (12/12) showed positive reactivity with HNA-3aa cells with no cross-reactivity with HNA-3bb cells. Again, all HNA-3b sera reacted with HNA-3bb cells only. Furthermore, genotyping of 249 individuals detected a new HNA-3 allele caused by a nucleotide substitution C>T at Position 457 leading to L(153)F mutation in choline transporter-like protein-2. This mutation impairs polymerase chain reaction with sequence-specific primers based HNA-3a typing. However, analysis with cells expressing F(153) isoform showed that this mutation did not alter the binding of HNA-3 antibodies. CONCLUSIONS: This study demonstrated that HEK293T cells expressing stable recombinant HNA-3 are suitable for the detection of HNA-3 alloantibodies allowing reliable screening of blood products.
Subject(s)
Blood Component Transfusion/standards , Flow Cytometry/methods , Isoantibodies/immunology , Isoantibodies/isolation & purification , Isoantigens/genetics , Isoantigens/immunology , Acute Lung Injury/etiology , Acute Lung Injury/immunology , Acute Lung Injury/prevention & control , Agglutination Tests/methods , Blood Component Transfusion/adverse effects , Flow Cytometry/standards , Fluorescent Antibody Technique/methods , Genotype , HEK293 Cells , Humans , Mass Screening/methods , Mass Screening/standards , Membrane Glycoproteins/genetics , Membrane Glycoproteins/immunology , Membrane Transport Proteins/genetics , Membrane Transport Proteins/immunology , Point Mutation/immunology , Recombinant Proteins/genetics , Recombinant Proteins/immunology , TransfectionABSTRACT
Brain metastases (BM) are frequent and carry a dismal prognosis. BRAF V600E mutations are found in a broad range of tumor types and specific inhibitors targeting BRAF V600E protein exist. We analyzed tumoral BRAF V600E-mutant protein expression using the novel mutation-specific antibody VE1 in a series of 1,120 tumor specimens (885 BM, 157 primary tumors, 78 extra-cranial metastases) of 874 BM patients. In 85 cases, we performed validation of immunohistochemical results by BRAF exon 15 gene sequencing. BRAF V600E protein was expressed in BM of 42/76 (55.3%) melanomas, 1/15 (6.7%) ovarian cancers, 4/72 (5.5%) colorectal cancers, 1/355 (0.3%) lung cancers, 2/6 thyroid cancers and 1/2 choriocarcinomas. BRAF V600E expression showed high intra-tumoral homogeneity and was similar in different tumor manifestations of individual patients. VE1 immunohistochemistry and BRAF exon 15 sequencing were congruent in 68/70 (97.1%) cases, but VE1 immunostaining identified small BRAF V600E expressing tumor cell aggregates in 10 cases with inconclusive genetic results. Melanoma patients with BRAF V600E mutant protein expressing tumors were significantly younger at diagnosis of the primary tumor and at operation of BM than patients with non-mutated tumors. In conclusion, expression of BRAF V600E mutant protein occurs in approximately 6% of BM and is consistent in different tumor manifestations of the same patient. Thus, BRAF V600E inhibiting therapies seem feasible in selected BM patients. Immunohistochemical visualization of V600E-mutant BRAF protein is a promising tool for patient stratification. An integrated approach combining both, VE1 immunohistochemistry and genetic analysis may increase the diagnostic accuracy of BRAF mutation analysis.
Subject(s)
Brain Neoplasms/genetics , Brain Neoplasms/secondary , Genetic Predisposition to Disease/genetics , Point Mutation , Proto-Oncogene Proteins B-raf/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Brain Neoplasms/pathology , Female , Glutamic Acid/genetics , HEK293 Cells , Humans , Immunohistochemistry/methods , Male , Middle Aged , Point Mutation/genetics , Point Mutation/immunology , Proto-Oncogene Proteins B-raf/biosynthesis , Proto-Oncogene Proteins B-raf/immunology , Valine/genetics , Young AdultABSTRACT
TLR7 is the mammalian receptor for ssRNA and some nucleotide-like small molecules. We have generated a mouse by N-nitrose-N'-ethyl urea mutagenesis in which threonine 68 of TLR7 was substituted with isoleucine. Cells bearing this mutant TLR7 lost the sensitivity to the small-molecule TLR7 agonist resiquimod, hence the name TLR7(rsq1). In this work, we report the characterization of this mutant protein. Similar to the wild-type counterpart, TLR7(rsq1) localizes to the endoplasmic reticulum and is expressed at normal levels in both primary cells and reconstituted 293T cells. In addition to small-molecule TLR7 agonists, TLR7(rsq1) fails to be activated by ssRNA. Whole-transcriptome analysis demonstrates that TLR7 is the exclusive and indispensable receptor for both classes of ligands, consistent with the fact that both ligands induce highly similar transcriptional signatures in TLR7(wt/wt) splenocytes. Thus, TLR7(rsq1) is a bona fide phenocopy of the TLR7 null mouse. Because TLR7(rsq1) binds to ssRNA, our studies imply that the N-terminal portion of TLR7 triggers a yet to be identified event on TLR7. TLR7(rsq1) mice might represent a valuable tool to help elucidate novel aspects of TLR7 biology.
Subject(s)
Point Mutation/immunology , Signal Transduction/genetics , Signal Transduction/immunology , Toll-Like Receptor 7/genetics , Toll-Like Receptor 7/metabolism , Animals , Cell Line , Cells, Cultured , HEK293 Cells , Humans , Imidazoles/pharmacology , Ligands , Membrane Glycoproteins/deficiency , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Mutagenesis, Site-Directed , Protein Binding/drug effects , Protein Binding/genetics , Protein Binding/immunology , Signal Transduction/drug effects , Toll-Like Receptor 7/deficiencyABSTRACT
Biological therapies, even humanized mAbs, may induce antiglobulin responses that impair efficacy. We tested a novel strategy to induce tolerance to a therapeutic mAb. Twenty patients with relapsing-remitting multiple sclerosis received an initial cycle of alemtuzumab (Campath-1H), up to 120 mg over 5 d, preceded by 500 mg SM3. This Ab differs from alemtuzumab by a single point mutation and is designed not to bind to cells. Twelve months later, they received a second cycle of alemtuzumab, up to 72 mg over 3 d. One month after that, 4 of 19 (21%) patients had detectable serum anti-alemtuzumab Abs compared with 145 of 197 (74%) patients who received two cycles of alemtuzumab without SM3 in the phase 2 CAMMS223 trial (p < 0.001). The efficacy and safety profile of alemtuzumab was unaffected by SM3 pretreatment. Long-lasting "high-zone" tolerance to a biological therapy may be induced by pretreatment with a high i.v. dose of a drug variant, altered to reduce target-binding.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Antibodies, Neoplasm/therapeutic use , Immune Tolerance , Multiple Sclerosis, Relapsing-Remitting/immunology , Multiple Sclerosis, Relapsing-Remitting/therapy , Adolescent , Adult , Alemtuzumab , Amino Acid Substitution/genetics , Amino Acid Substitution/immunology , Antibodies, Anti-Idiotypic/biosynthesis , Antibodies, Anti-Idiotypic/blood , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal, Humanized , Antibodies, Neoplasm/genetics , Antibodies, Neoplasm/immunology , Dose-Response Relationship, Immunologic , Drug Administration Schedule , Female , Humans , Magnetic Resonance Imaging , Male , Multiple Sclerosis, Relapsing-Remitting/diagnosis , Pilot Projects , Point Mutation/genetics , Point Mutation/immunology , Randomized Controlled Trials as Topic , Treatment Outcome , Young AdultABSTRACT
Signals elicited by TLRs following the detection of microbes are integrated and diversified by a group of four cytoplasmic adaptor molecules featuring an evolutionarily conserved Toll/IL-1R signaling domain. Single nucleotide polymorphisms (SNPs) in TLRs and their adaptor molecules have been shown to influence susceptibility to a range of infectious and other diseases. The adaptor MyD88 adaptor-like (Mal)/Toll/IL-1R-containing adaptor protein is involved in TLR2 and 4 signal transduction by recruiting another adaptor molecule, MyD88, to the plasma membrane. In this study, we used naturally occurring variants of Mal as tools to study the molecular biology of Mal in more detail in cellular model systems and to thereby identify functionally interesting variants whose corresponding nonsynonymous SNPs might be of further epidemiological interest. Of seven reported variants for Mal, we found Mal D96N associated with reduced NF-kappaB signaling and cytokine production after overexpression in HEK293 and Huh-7 cells. The D96N mutation prevented Mal from recruiting its signaling partner MyD88 to the plasma membrane and altered posttranslational modification of Mal. These findings led us to investigate the frequency of heterozygosity for the corresponding SNP rs8177400 in a Caucasian case-control study on the etiology of lymphoma, a disease in which TLRs have been implicated. Although rs8177400 did not modify lymphoma risk in general, its frequency of heterozygosity was accurately determined to 0.97%. Our data add rs8177400 (D96N) to the list of functionally important variants of Mal and warrant further research into its immunological, epidemiological, and diagnostic relevance.
Subject(s)
Genetic Variation/immunology , Membrane Glycoproteins/deficiency , Membrane Glycoproteins/genetics , Myeloid Differentiation Factor 88 , Protein Processing, Post-Translational/immunology , Receptors, Interleukin-1/deficiency , Receptors, Interleukin-1/genetics , Signal Transduction/genetics , Signal Transduction/immunology , Adolescent , Adult , Aged , Aged, 80 and over , Asparagine/genetics , Aspartic Acid/genetics , Case-Control Studies , Cell Line, Transformed , Cell Line, Tumor , Genetic Predisposition to Disease/epidemiology , Humans , Immunophenotyping , Membrane Glycoproteins/physiology , Middle Aged , Multicenter Studies as Topic , Myeloid Differentiation Factor 88/physiology , NF-kappa B/metabolism , Point Mutation/immunology , Protein Processing, Post-Translational/genetics , Protein Transport/genetics , Protein Transport/immunology , Receptors, Interleukin-1/physiologyABSTRACT
Over the past decade, increasing attention has been focused on the contribution of naturally occurring mutations in the hepatitis B virus (HBV) genome to the clinical course of the chronic infection. The aim of this study was to investigate the effect of the HBV pre-core mutation G1896A on the expression of HLA class II molecules and the core protein of hepatitis B in liver biopsies of chronic hepatitis B (CHB) infection. In 30 HBeAg-negative CHB patients the pre-core region of the HBV genome was amplified and sequenced to determine the presence of the mutation G1896A. Liver biopsies were scored based on the Histology Activity Index (HAI) system and immunohistochemistry (IHC) was performed to study the expression of HLA class II molecules on the antigen-presenting cells and the core protein in hepatocytes. We found that 19 of the 30 patients (63%) harbored the G1896A mutation. Compared to the patients without this mutation, those with G1896A had lower HAI scores (5.0 +/- 2.8 versus 7.9 +/- 4, p = 0.03). The study of the expression of HLA-II molecules in our patients revealed that subjects with the G1896A mutation had lower expression of HLA-II compared to wild-type infected subjects (1.87 +/- 0.6 versus 3.27 +/- 1.5, p < 0.01). Core protein expression was present in four patients (13.3%) who had higher HBV DNA levels than patients without core protein expression (3.81 +/- 0.78 versus 2.02 +/- 0.16, p < 0.001). These results suggest that the G1896A pre-core mutation may directly interfere with antigen presentation, most likely by decreasing the availability of HLA class II molecules, and this may result in less aggressive liver disease in HBeAg-negative CHB infection.
Subject(s)
DNA, Viral/genetics , Genes, MHC Class II/physiology , Hepatitis B virus/genetics , Hepatitis B, Chronic/immunology , Point Mutation/immunology , Adult , Antigens, Viral/biosynthesis , Antigens, Viral/genetics , Down-Regulation , Female , Gene Expression Regulation, Viral , Genome, Viral , Hepatitis B virus/immunology , Hepatitis B virus/metabolism , Hepatitis B, Chronic/metabolism , Hepatitis B, Chronic/pathology , Hepatitis B, Chronic/virology , Humans , Immune Evasion , Liver/pathology , Male , Middle AgedABSTRACT
Genetic mutations frequently observed in human follicular lymphoma (FL) B-cells result in aberrant expression of the anti-apoptotic protein bcl-2 and surface immunoglobulins (Igs) which display one or more novel variable (V) region N-glycosylation motifs. In the present study, we develop a simulation model of the germinal center (GC) to explore how these mutations might influence the emergence and clonal expansion of key mutants which provoke FL development. The simulations employ a stochastic method for calculating the cellular dynamics, which incorporates actual IgV region sequences and a simplified hypermutation scheme. We first bring our simulations into agreement with experimental data for well-characterized normal and bcl-2(+) anti-hapten GC responses in mice to provide a model for understanding how bcl-2 expression leads to permissive selection and memory cell differentiation of weakly competitive B-cells. However, as bcl-2 expression in the GC alone is thought to be insufficient for FL development, we next monitor simulated IgV region mutations to determine the emergence times of key mutants displaying aberrant N-glycosylation motifs recurrently observed in human FL IgV regions. Simulations of 26 germline V(H) gene segments indicate that particular IgV regions have a dynamical selective advantage by virtue of the speed with which one or more of their key sites can generate N-glycosylation motifs upon hypermutation. Separate calculations attribute the high occurrence frequency of such IgV regions in FL to an ability to produce key mutants at a fast enough rate to overcome stochastic processes in the GC that hinder clonal expansion. Altogether, these simulations characterize three pathways for FL maturation through positively selected N-glycosylations, namely, via one of two key sites within germline V(H) region gene segments, or via a site in the third heavy chain complementarity-determining region (CDR-H3) that is generated from VDJ recombination.
Subject(s)
Germinal Center/pathology , Lymphoma, Follicular/etiology , Lymphoma, Follicular/pathology , Models, Biological , Algorithms , Animals , Apoptosis/genetics , Apoptosis/immunology , B-Lymphocytes/metabolism , B-Lymphocytes/pathology , Cell Count , Cell Differentiation/genetics , Cell Differentiation/immunology , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/immunology , Complementarity Determining Regions/genetics , Computer Simulation , Gene Rearrangement, B-Lymphocyte, Heavy Chain/genetics , Germinal Center/immunology , Glycosylation , Humans , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Variable Region/genetics , Lymphoma, Follicular/genetics , Mice , Point Mutation/genetics , Point Mutation/immunology , Probability , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Receptors, Antigen, B-Cell/genetics , Somatic Hypermutation, Immunoglobulin/genetics , Stochastic ProcessesABSTRACT
Regulatory T (T(reg)) cells expressing forkhead box P3 (Foxp3) arise during thymic selection among thymocytes with modestly self-reactive T cell receptors. In vitro studies suggest Foxp3 can also be induced among peripheral CD4(+) T cells in a cytokine dependent manner. T(reg) cells of thymic or peripheral origin may serve different functions in vivo, but both populations are phenotypically indistinguishable in wild-type mice. Here we show that mice with a Carma1 point mutation lack thymic CD4(+)Foxp3(+) T(reg) cells and demonstrate a cell-intrinsic requirement for CARMA1 in thymic Foxp3 induction. However, peripheral Carma1-deficient T(reg) cells could be generated and expanded in vitro in response to the cytokines transforming growth factor beta (TGFbeta) and interleukin-2 (IL-2). In vivo, a small peripheral T(reg) pool existed that was enriched at mucosal sites and could expand systemically after infection with mouse cytomegalovirus (MCMV). Our data provide genetic evidence for two distinct mechanisms controlling regulatory T cell lineage commitment. Furthermore, we show that peripheral T(reg) cells are a dynamic population that may expand to limit immunopathology or promote chronic infection.
Subject(s)
CARD Signaling Adaptor Proteins/genetics , Cytokines/genetics , Cytomegalovirus Infections/immunology , Forkhead Transcription Factors/immunology , Point Mutation , T-Lymphocytes, Regulatory/physiology , Thymus Gland/immunology , Animals , Autoimmune Diseases/genetics , Autoimmune Diseases/immunology , CARD Signaling Adaptor Proteins/immunology , Cytomegalovirus Infections/genetics , Gene Expression Regulation , Interleukin-2/genetics , Mice , Point Mutation/immunology , Receptors, Antigen, T-Cell/genetics , Signal Transduction , Thymus Gland/cytology , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/immunologyABSTRACT
BACKGROUND: Anaphylaxis after Hymenoptera stings has been reported in subjects with mastocytosis, but few data exist regarding disease prevalence in populations allergic to these insects. OBJECTIVE: The incidence of clonal mast cell (MC) disorders in subjects with both systemic reactions to Hymenoptera stings and increased serum baseline tryptase (sBT) levels was assessed by using bone marrow (BM) aspirates and biopsy specimens. METHODS: Subjects with a history of a systemic reaction caused by a Hymenoptera sting underwent the standard diagnostic work-up for Hymenoptera allergy, and sBT levels were measured. Subjects with an increased sBT level had BM evaluation that included histology/cytology, flow cytometry, and detection of KIT mutations. RESULTS: Forty-four (11.6%) of 379 subjects with systemic reactions had increased sBT levels (>11.4 ng/mL), and 31 (70.5%) of these had a history of anaphylaxis. Thirty-four subjects with increased sBT levels underwent a BM analysis. Histology detected diagnostic or subdiagnostic MC infiltrates in 22 (65%) of 34 patients. Abnormal MCs were identified by means of flow cytometry and cytology in 26 (78.8%) of 33 and 20 (58.8%) of 34 subjects, respectively. A KIT mutation was detected in 17 (54.8%) of 31 subjects. The diagnosis was indolent systemic mastocytosis in 21 (61.7%) of 34 subjects and monoclonal MC activation syndrome in 9 (26.5%) of 34 subjects. All subjects with anaphylaxis had one of those 2 disorders. CONCLUSION: The concomitant presence of systemic reactions (especially anaphylaxis) after Hymenoptera stings and increased sBT levels strongly suggests that a BM examination is indicated for the diagnosis of clonal MC disease.
Subject(s)
Anaphylaxis/etiology , Bone Marrow/pathology , Hymenoptera , Insect Bites and Stings/complications , Mast Cells/pathology , Mastocytosis/epidemiology , Tryptases/blood , Adolescent , Adult , Aged , Animals , Child , Clone Cells/pathology , Female , Humans , Incidence , Male , Mastocytosis/enzymology , Mastocytosis/pathology , Middle Aged , Point Mutation/genetics , Point Mutation/immunology , Proto-Oncogene Proteins c-kit/genetics , Proto-Oncogene Proteins c-kit/immunology , Proto-Oncogene Proteins c-kit/metabolism , Skin Tests , Young AdultABSTRACT
The cooperative activity of protein tyrosine kinases and phosphatases plays a central role in regulation of T cell receptor (TCR) signal strength. Perturbing this balance, and thus the threshold for TCR signals, has profound impacts on T cell development and function. We previously generated mice containing a point mutation in the juxtamembrane wedge of the receptor-like protein tyrosine phosphatase CD45. Demonstrating the critical negative regulatory function of the wedge, the CD45 E613R (WEDGE) mutation led to a lymphoproliferative disorder (LPD) and a lupus-like autoimmune syndrome. Using genetic, cellular, and biochemical approaches, we now demonstrate that the CD45 wedge influences T cell development and function. Consistent with increased TCR signal strength, WEDGE mice have augmented positive selection and enhanced sensitivity to the CD4-mediated disease experimental autoimmune encephalitis (EAE). These correspond with hyperresponsive calcium and pERK responses to TCR stimulation in thymocytes, but surprisingly, not in peripheral T cells, where these responses are actually depressed. Together, the data support a role for the CD45 wedge in regulation of T cell responses in vivo and suggest that its effects depend on cellular context.