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1.
Front Immunol ; 9: 1962, 2018.
Article in English | MEDLINE | ID: mdl-30214446

ABSTRACT

Among Immunotherapeutic approaches for cancer treatment, the adoptive transfer of antigen specific T cells is still a relevant approach, that could have higher efficacy when further combined with immune check-point blockade. A high number of adoptive transfer trials have been performed in metastatic melanoma, due to its high immunogenic potential, either with polyclonal TIL or antigen-specific polyclonal populations. In this setting, the extensive characterization of T cell functions and receptor diversity of infused polyclonal T cells is required, notably for monitoring purposes. We developed a clinical grade procedure for the selection and amplification of polyclonal CD8 T cells, specific for two shared and widely expressed melanoma antigens: Melan-A and MELOE-1. This procedure is currently used in a clinical trial for HLA-A2 metastatic melanoma patients. In this study, we characterized the T-cell diversity (T-cell repertoire) of such T cell populations using a new RNAseq strategy. We first assessed the added-value of TCR receptor sequencing, in terms of sensitivity and specificity, by direct comparison with cytometry analysis of the T cell populations labeled with anti-Vß-specific antibodies. Results from these analyzes also confirmed specific features already reported for Melan-A and MELOE-1 specific T cell repertoires in terms of V-alpha recurrence usage, on a very high number of T cell clonotypes. Furthermore, these analyses also revealed undescribed features, such as the recurrence of a specific motif in the CDR3α region for MELOE-1 specific T cell repertoire. Finally, the analysis of a large number of T cell clonotypes originating from various patients revealed the existence of public CDR3α and ß clonotypes for Melan-A and MELOE-1 specific T cells. In conclusion, this method of high throughput TCR sequencing is a reliable and powerful approach to deeply characterize polyclonal T cell repertoires, and to reveal specific features of a given TCR repertoire, that would be useful for immune follow-up of cancer patients treated by immunotherapeutic approaches.


Subject(s)
Adoptive Transfer , Antigens, Neoplasm , High-Throughput Nucleotide Sequencing , MART-1 Antigen , Melanoma , Neoplasm Proteins , T-Lymphocytes/immunology , Antigens, Neoplasm/genetics , Antigens, Neoplasm/immunology , Female , Humans , MART-1 Antigen/genetics , MART-1 Antigen/immunology , Male , Melanoma/genetics , Melanoma/immunology , Melanoma/pathology , Melanoma/therapy , Neoplasm Proteins/genetics , Neoplasm Proteins/immunology , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/immunology , T-Lymphocytes/pathology
2.
J Virol ; 82(13): 6566-75, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18448535

ABSTRACT

APOBEC3 proteins are cytidine deaminases which help defend cells against retroviral infections. One antiviral mechanism involves deaminating dC residues in minus-strand DNA during reverse transcription, resulting in G-to-A mutations in the coding strand. We investigated the effects of mouse APOBEC3 (mA3) and human APOBEC3G (hA3G) upon Moloney murine leukemia virus (MLV). We find that mA3 inactivates MLV but is significantly less effective against MLV than is hA3G. In contrast, mA3 is as potent against human immunodeficiency virus type 1 (HIV-1, lacking the protective Vif protein) as is hA3G. The two APOBEC3 proteins are packaged to similar extents in MLV particles. Dose-response profiles imply that a single APOBEC3 molecule (or oligomer) is sufficient to inactivate an MLV particle. The inactivation of MLV by mA3 and hA3G is accompanied by relatively small reductions in the amount of viral DNA in infected cells. Although hA3G induces significant levels of G-to-A mutations in both MLV and HIV DNAs, and mA3 induces these mutations in HIV DNA, no such mutations were detected in DNA synthesized by MLV inactivated by mA3. Thus, MLV has apparently evolved to partially resist the antiviral effects of mA3 and to totally resist the ability of mA3 to induce G-to-A mutation in viral DNA. Unlike the resistance of HIV-1 and human T-cell leukemia virus type 1 to hA3G, the resistance of MLV to mA3 is not mediated by the exclusion of APOBEC from the virus particle. The nature of its resistance and the mechanism of inactivation of MLV by mA3 are completely unknown.


Subject(s)
Cytidine Deaminase/metabolism , Cytosine Deaminase/metabolism , DNA, Viral/genetics , Moloney murine leukemia virus/metabolism , Virus Inactivation , APOBEC Deaminases , Animals , Base Sequence , Cell Line , Cytidine Deaminase/genetics , Cytosine Deaminase/genetics , DNA Primers/genetics , Humans , Immunoblotting , Mice , Molecular Sequence Data , Moloney murine leukemia virus/genetics , Mutation/genetics , Sequence Analysis, DNA
3.
J Virol ; 81(12): 6623-31, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17392359

ABSTRACT

Assembly of retrovirus particles normally entails the selective encapsidation of viral genomic RNA. However, in the absence of packageable viral RNA, assembly is still efficient, and the released virus-like particles (termed "Psi-" particles) still contain roughly normal amounts of RNA. We have proposed that cellular mRNAs replace the genome in Psi- particles. We have now analyzed the mRNA content of Psi- and Psi+ murine leukemia virus (MLV) particles using both microarray analysis and real-time reverse transcription-PCR. The majority of mRNA species present in the virus-producing cells were also detected in Psi- particles. Remarkably, nearly all of them were packaged nonselectively; that is, their representation in the particles was simply proportional to their representation in the cells. However, a small number of low-abundance mRNAs were greatly enriched in the particles. In fact, one mRNA species was enriched to the same degree as Psi+ genomic RNA. Similar results were obtained with particles formed from the human immunodeficiency virus type 1 (HIV-1) Gag protein, and the same mRNAs were enriched in MLV and HIV-1 particles. The levels of individual cellular mRNAs were approximately 5- to 10-fold higher in Psi- than in Psi+ MLV particles, in agreement with the idea that they are replacing viral RNA in the former. In contrast, signal recognition particle RNA was present at the same level in Psi- and Psi+ particles; a minor fraction of this RNA was weakly associated with genomic RNA in Psi+ MLV particles.


Subject(s)
HIV/metabolism , Leukemia Virus, Murine/genetics , Retroviridae/genetics , Animals , Blotting, Northern , Blotting, Western , DNA Primers/chemistry , Gene Products, gag/metabolism , Humans , Mice , Oligonucleotide Array Sequence Analysis , RNA, Viral/metabolism , Retroviridae/metabolism , Ribonuclease H/chemistry , Transfection , Virus Assembly
4.
J Biol Chem ; 278(37): 35292-8, 2003 Sep 12.
Article in English | MEDLINE | ID: mdl-12824173

ABSTRACT

PMR1 is the yeast secretory pathway pump responsible for high affinity transport of Mn2+ and Ca2+ into the Golgi, where these ions are sequestered and effectively removed from the cytoplasm. Phenotypic growth assays allow for convenient screening of side chains important for Ca2+ and Mn2+ transport. Earlier we demonstrated that mutant Q783A at the cytoplasmic interface of M6 could transport Ca2+, but not Mn2+. Scanning mutagenesis of side chains proximal to residue Gln-783 in membrane helices M2, M4, M5, and M6 revealed additional residues near the cytoplasmic interface, notably Leu-341 (M5), Phe-738 (M5), and Leu-785 (M6) that are sensitive to substitution. Importantly, we obtained evidence for a packing interaction between Val-335 in M4 and Gln-783 in M6 that is critical for Mn2+ transport. Thus, mutant V335G mimics the Mn2+ transport defect of Q783A and mutant V335I can effectively suppress the Mn2+-defective phenotype of Q783A. These changes in ion selectivity were confirmed by cation-dependent ATP hydrolysis using purified enzyme. Other substitutions at these sites are tolerated individually, but not in combination. Exchange of side chains at 335 and 783 also results in ion selectivity defects, suggesting that the packing interaction may be conformation-sensitive. Homology models of M4, M5, and M6 of PMR1 have been generated, based on the structures of the sarcoplasmic reticulum Ca2+-ATPase. The models are supported by data from mutagenesis and reveal that Gln-783 and Val-335 show conformation-sensitive packing at the cytoplasmic interface. We suggest that this region may constitute a gate for access of Mn2+ ions.


Subject(s)
Calcium-Transporting ATPases/chemistry , Golgi Apparatus/enzymology , Molecular Chaperones/chemistry , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/enzymology , Amino Acid Sequence , Animals , Binding Sites , Calcium-Transporting ATPases/genetics , Calcium-Transporting ATPases/metabolism , Genes, Reporter , Kinetics , Models, Molecular , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Molecular Sequence Data , Mutagenesis, Site-Directed , Plasmids , Protein Structure, Secondary , Rabbits , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Sequence Alignment , Sequence Homology, Amino Acid
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