11 Serological antigen selection of phage displayed colorectal tumour cDNA libraries

ABSTRACTArtificial insemination with cryopreserved sperm is a major assisted reproductive technology in many species. In horses, as in humans, insemination with cryopreserved sperm is associated with lower pregnancy rates than those for fresh sperm, however, direct effects of sperm cryopreservation on the development of resulting embryos are largely unexplored. The aim of this study was to investigate differences in gene expression between embryos resulting from fertilization with fresh or cryopreserved sperm. Embryos were obtained at 8, 10 or 12 days after ovulation from mares inseminated post-ovulation on successive cycles with either fresh sperm or frozen-thawed sperm from the same stallion, providing matched embryo pairs at each day. RNA was isolated from two matched pairs (4 embryos) for each day, and cDNA libraries were built and sequenced. Significant differences in transcripts per kilobase million (TPM) were determined using (i) genes for which the expression difference between treatments was higher than 99% of that in the random case (P < 0.01), and (ii) genes for which the fold change was ≥ 2, to avoid expression bias in selection of the candidate genes. Molecular pathways were explored using the DAVID webserver, followed by network analyses using STRING, with a threshold of 0.700 for positive interactions. The transcriptional profile of embryos obtained with frozen-thawed sperm differed significantly from that for embryos derived from fresh sperm on all days, showing significant down-regulation of genes involved in biological pathways related to oxidative phosphorylation, DNA binding, DNA replication, and immune response. Many genes with reduced expression were orthologs of genes known to be embryonic lethal in mice. This study, for the first time, provides evidence of altered transcription in embryos resulting from fertilization with cryopreserved spermatozoa in any species. As sperm cryopreservation is commonly used in many species, including human, the effect of this intervention on expression of developmentally important genes in resulting embryos warrants attention.

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Antigen Selection of Multiple Myeloma Clonogenic B Cells as Evidenced by VH and VL Gene Mutations

Blood ◽

10.1182/blood.v90.3.1334.1334_1334_1334 ◽

1997 ◽

Vol 90 (3) ◽

pp. 1334-1334

Author(s):

Christos Kosmas ◽

Kostas Stamatopoulos ◽

Dimitris Loukopoulos

Keyword(s):

Multiple Myeloma ◽

B Cells ◽

Gene Mutations ◽

Selection Of ◽

Antigen Selection

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Somatic diversification and selection of immunoglobulin heavy and light chain variable region genes in IgG+ CD5+ chronic lymphocytic leukemia B cells.

Journal of Experimental Medicine ◽

10.1084/jem.181.4.1507 ◽

1995 ◽

Vol 181 (4) ◽

pp. 1507-1517 ◽

Cited By ~ 97

Author(s):

S Hashimoto ◽

M Dono ◽

M Wakai ◽

S L Allen ◽

S M Lichtman ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Gene Segment ◽

Variable Region ◽

Lymphocytic Leukemia ◽

Somatic Diversification ◽

V Region ◽

Variable Region Genes ◽

Selection Of ◽

Antigen Selection

Chronic lymphocytic leukemia (CLL) is characterized by the clonal expansion of CD5-expressing B lymphocytes. Most studies have found that these leukemic CD5+ B cells, like their normal counterparts, use immunoglobulin (Ig) variable (V) region genes that exhibit minimal, if any, somatic diversity. These and other observations have suggested that CD5+ B cells may be incapable of generating Ig V gene diversity, and therefore may not be able to develop higher affinity binding sites that could be selected by antigen. However, most of the studies of CLL and normal CD5+ B cells have focused on IgM-producing cells. Since somatic mutations are most often seen in B cells that have undergone an isotype class switch, we analyzed the Ig heavy (H) and light (L) chain variable region genes of seven IgG+CD5+ CLL B cells to determine if somatic diversification and antigen selection had occurred. The data derived provide evidence for skewed use, somatic diversification, and antigenic selection of the Ig V region genes. Nonrandom use of both H and L chain V region genes was manifested by an overrepresentation of VH4 and VKI family genes and the underrepresentation of the JH4 gene segment. Furthermore, VH4 gene use was restricted to only two family members (4.21 and 4.18). In four of the seven cases, the VH and VL genes displayed > or = 5% difference from the most homologous known germline counterparts. Polymerase chain reaction and Southern blot analyses performed in two of these patients demonstrated that their unique VH CDR2 and adjacent sequences were not present in their germline DNA. In addition, a significant level of diversity was seen in the rearranged DJH segments and at the VL-JL junctions of every patient that occurred both at the time of recombination and subsequently. The localization of replacement changes to complementarity determining regions of some patients suggested that antigen selection had occurred. Furthermore, the mutations identified in the VH and VL genes of each individual patient were strikingly similar, both in number and location. Collectively, the data indicate that a subset of CD5+ CLL B cells can display Ig V region gene mutations. In addition, they are consistent with the notions that in some cases antigen selection of these mutations may have occurred, and that antigen stimulation may be a promoting factor in the evolution of certain CLL clones.

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Affinity selection of cDNA libraries by λ phage surface display

Gene ◽

10.1016/s0378-1119(00)00348-6 ◽

2000 ◽

Vol 256 (1-2) ◽

pp. 229-236 ◽

Cited By ~ 22

Author(s):

Mikio Niwa ◽

Hiroko Maruyama ◽

Takashi Fujimoto ◽

Kazuhiro Dohi ◽

Ichi N Maruyama

Keyword(s):

Surface Display ◽

Cdna Libraries ◽

Affinity Selection ◽

Selection Of

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Genetic and Structural Evidence for Antigen Selection of Anti-DNA Antibodies

Annual Review of Immunology ◽

10.1146/annurev.iy.12.040194.002415 ◽

1994 ◽

Vol 12 (1) ◽

pp. 487-520 ◽

Cited By ~ 338

Author(s):

M Z Radic ◽

M Weigert

Keyword(s):

Dna Antibodies ◽

Selection Of ◽

Antigen Selection

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T Cell Receptor Gene Repertoire Restriction in Chronic Lymphocytic Leukemia with Stereotyped IGHV4–34/IGKV2–30 Antigen Receptors

Blood ◽

10.1182/blood.v120.21.3908.3908 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3908-3908 ◽

Cited By ~ 1

Author(s):

Anna Vardi ◽

Andreas Agathangelidis ◽

Evangelia Stalika ◽

Millaray Marincevic ◽

Maria Karypidou ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Receptor ◽

Clonal Selection ◽

Cell Receptor ◽

Lymphocytic Leukemia ◽

Gene Repertoire ◽

Time Points ◽

Selection Of ◽

Antigen Selection

Abstract Abstract 3908 Chronic lymphocytic leukemia (CLL) exhibits a remarkably skewed immunoglobulin (IG) gene repertoire mainly evident in the existence of subsets of patients with quasi-identical IGs in their B cell receptors (BcRs), collectively accounting for one-third of CLL patients. BcR stereotypy is strongly suggestive of clonal selection by a restricted set of antigens. However, it is not yet clear at which phase of clonal evolution these antigens act, or whether the stimulation is persistent. Furthermore, the possible role of antigens in the selection and activation of cognate T lymphocytes remains obscure yet highly relevant, given recent data about T cell interactions with CLL B cells and their tolerized behavior. Here, we analyzed the repertoire of T cell receptor β chain genes (TRB) in CLL expressing stereotyped IGHV4–34/IGKV2–30 BcR IGs (subset #4), which exhibit a series of immunogenetic features, such as pronounced intraclonal diversification of IG genes, suggestive of ongoing interactions with (auto)antigens. Furthermore, subset #4 CLL cells have distinctive functional responses to BcR and/or Toll-like receptor triggering, rendering this subset a paradigmatic example for seeking evidence of antigen selection also within the T cell population. We analyzed 18 peripheral blood samples of 12 untreated subset #4 patients (samples from different time points were analyzed in 4 cases). No case had evidence of infection at sampling. PCR amplicons for TRBV-TRBD-TRBJ gene rearrangements (BIOMED2 protocol) were subcloned by transformation into E. coli/TOP10F bacteria and randomly chosen individual colonies were subjected to Sanger sequencing. Only productive rearrangements (n=320, ranging from 14–52/case) were analyzed. All cases were found to carry clusters of identical rearrangements (≥2) corresponding to distinct clonotypes; the number of expanded clonotypes/case ranged from 1–13 (median 5). The relative frequency of each clonotype/case was determined by dividing the number of the corresponding identical sequences by the total number of subcloned sequences analyzed. The frequency of the most expanded (immunodominant) clonotype/case ranged from 8.1–70.4%. Collectively, the frequency of all expanded clonotypes/case ranged from 29.7–93.3%. In 2/4 cases that were analyzed at different time points, at least one clonotype was found to persist. Importantly, cluster analysis of the TRB CDR3 sequences of all cases identified ‘public’ clonotypes: 2 identical clonotypes (TRBV15*02/TRBD1*01/TRBJ2–2*01 and TRBV30*01/TRBD1*01/TRBJ2–2*01) each shared by a pairs of different patients and a highly similar clonotype shared by an additional pair of patients. In conclusion, the present study provides clear evidence of repertoire skewing among T cells in CLL patients belonging to subset #4, strongly supporting antigen selection. The finding of ‘public’ clonotypes raises the possibility that shared antigenic epitopes may be relevant for clonal selection of T cells in different subset #4 cases. Whether the antigens that drive T cell repertoire restriction are identical/related to those implicated in the selection of CLL progenitors of subset #4 or even the malignant cells themselves or whether they are tumor-associated antigens remains to be clarified. Disclosures: No relevant conflicts of interest to declare.

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Antigen Selection of Anti-DSG1 Autoantibodies During and Before the Onset of Endemic Pemphigus Foliaceus

Journal of Investigative Dermatology ◽

10.1038/jid.2009.184 ◽

2009 ◽

Vol 129 (12) ◽

pp. 2823-2834 ◽

Cited By ~ 15

Author(s):

Ye Qian ◽

Stephen H. Clarke ◽

Valeria Aoki ◽

Gunter Hans-Filhio ◽

Evandro A. Rivitti ◽

...

Keyword(s):

Pemphigus Foliaceus ◽

Endemic Pemphigus Foliaceus ◽

Selection Of ◽

Antigen Selection

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Vaccinomics and adversomics as new trends in vaccinology

Postępy Higieny i Medycyny Doświadczalnej ◽

10.5604/01.3001.0010.7616 ◽

2017 ◽

Vol 71 (0) ◽

pp. 0-0 ◽

Cited By ~ 1

Author(s):

Anna Lutyńska ◽

Aleksandra Gołoś ◽

Ewa Augustynowicz ◽

Beata Orzechowska

Keyword(s):

Single Gene ◽

Vaccine Antigen ◽

Selection Strategy ◽

Selection Strategies ◽

Effective Immune Response ◽

Vaccine Antigens ◽

Widespread Availability ◽

Selection Of ◽

Antigen Selection

Currently used vaccines have been developed based on experimental pre-clinical and clinical trials. Although the widespread availability of vaccines is one of the greatest achievements in public health, the selection of antigens capable of inducing an effective immune response has not been successful for some pathogens to date. Searching for and detecting a relationship between genes or whole genome sequences and the level of immunization response has opened the second “golden age” of vaccinology and led to the development of two new branches: vaccinomics and adversomics. Vaccinomics is a combination of pharmacogenetics, which defines the correlation between single gene polymorphism and immunization response and pharmacogenomics, which characterizes the correlation between genome sequence polymorphism, immunogenicity induced by the vaccine. Adversomics is aimed at developing a strategy for reducing the risk of adverse events by diagnosing potentially high-risk individuals and using modified vaccines. The assumptions and achievements of vaccinomics, initiated by Georg Poland, have influenced the development of a new vaccine antigen selection strategy. This strategy consists of selecting the optimal antigen after characterizing the genetic and epigenetic determinants of the immune system components of all candidate vaccine antigens. Taking into account the role of variability, not only in the pathogen but also in the host in antigen, selection strategies may significantly improve the efficiency of the newly developed vaccines and vaccines currently in use after their respective modifications.

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