Single-Cell Sequencing of T cell Receptors: A Perspective on the Technological Development and Translational Application.

T cells recognize peptides bound to major histocompatibility complex (MHC) class I and class II molecules at the cell surface. This recognition is accomplished by the expression of T cell receptors (TCR) which are required to be diverse and adaptable in order to accommodate the various and vast number of antigens presented on the MHCs. Thus, determining TCR repertoires of effector T cells is necessary to understand the immunological process in responding to cancer progression, infection, and autoimmune development. Furthermore, understanding the TCR repertoires will provide a solid framework to predict and test the antigen which is more critical in autoimmunity. However, it has been a technical challenge to sequence the TCRs and provide a conceptual context in correlation to the vast number of TCR repertoires in the immunological system. The exploding field of single-cell sequencing has changed how the repertoires are being investigated and analyzed. In this review, we focus on the biology of TCRs, TCR signaling and its implication in autoimmunity. We discuss important methods in bulk sequencing of many cells. Lastly, we explore the most pertinent platforms in single-cell sequencing and its application in autoimmunity.

Therapeutic Antibody Discovery in Infectious Diseases Using Single-Cell Analysis.

Since the discovery of mouse hybridoma technology by Kohler and Milstein in 1975, significant progress has been made in monoclonal antibody production. Advances in B cell immortalization and phage display technologies have generated a myriad of valuable monoclonal antibodies for diagnosis and treatment. Technological breakthroughs in various fields of 'omics have shed crucial insights into cellular heterogeneity of a biological system in which the functional individuality of a single cell must be considered. Based on this important concept, remarkable discoveries in single-cell analysis have made in identifying and isolating functional B cells that produce beneficial therapeutic monoclonal antibodies. In this review, we will discuss three traditional methods of antibody discovery. Recent technological platforms for single-cell antibody discovery will be reviewed. We will discuss the application of the single-cell analysis in finding therapeutic antibodies for human immunodeficiency virus and emerging Zika arbovirus.

Multicenter Evaluation of Whole-Blood Epstein-Barr Viral Load Standardization Using the WHO International Standard.

The first WHO international standard for Epstein-Barr virus (EBV) (WHO EBV standard) for nucleic acid amplification technology (NAT)-based assays was commercialized in January 2012 by the National Institute for Biological Standards and Control. In the study reported here, we compared whole-blood EBV DNA load (EDL) results from 12 French laboratories for seven samples (Quality Controls for Molecular Diagnostics 2013 proficiency panel) in order to determine whether expression in international units reduces interlaboratory variability in whole-blood EDLs. Each testing laboratory used a conversion factor to convert EDL results from copies per milliliter to international units per milliliter. This conversion factor was calculated from the WHO EBV standard according to the protocol described in this study (nine laboratories) or the recommendations of the PCR kit suppliers (three laboratories). The interlaboratory variability in whole-blood EDL results was reduced after standardization of the results using the WHO EBV standard. For the seven samples tested, standard deviations (SD) ranged from 0.41 to 0.55 when the results were expressed in log copies per milliliter, whereas the SD ranged from 0.17 to 0.32 when results were given in log international units per milliliter. Comparing the variance data (F test), we showed that the dispersion of whole-blood EDL results was significantly lower when they were expressed in log international units per milliliter (P < 0.001 for six of seven samples and P < 0.05 for one sample with a low mean EDL of 2.62 log IU/ml). This study showed that the use of the WHO EBV standard could improve the homogeneity of whole-blood EDL results between laboratories as well as the monitoring of patients at high risk of posttransplant lymphoproliferative disorders or other EBV-associated diseases.

Methylation of Epstein-Barr virus Rta promoter in EBV primary infection, reactivation and lymphoproliferation.

During Epstein-Barr virus (EBV) latency, the EBV genome is largely silenced by methylation. This silencing is overturned during the switch to the lytic cycle. A key event is the production of the viral protein Zta which binds to three Zta-response elements (ZRE) from the Rta promoter (Rp), two of which (ZRE2 and ZRE3) include three CpG motifs methylated in the latent genome. The bisulphite pyrosequencing reaction was used to quantify the methylation of ZRE2, ZRE3a, and ZRE3b in EBV-positive cell lines and in ex vivo samples of EBV-related diseases, in order to assess whether the level of methylation in these ZREs could provide additional information to viral DNA load and serology in the characterization of EBV-associated diseases. In PBMC from two patients with infectious mononucleosis, over time Rp became increasingly methylated whereas EBV load decreased. In tonsil from patients with chronic tonsillitis, the methylation was less than in EBV-associated tumors, regardless of the viral load. This was even more striking when only the ZRE3a and ZRE3b were considered since some samples presented unbalanced profiles on ZRE2. EBV reactivation in cell culture showed that the reduction in the overall level of methylation was closely related to the production of unmethylated virions. Thus, an assessment of the level of methylation may help to better characterize EBV replication in PBMC and in biopsies with high EBV load, during infectious mononucleosis and EBV-associated cancers. J. Med. Virol. 88:1814-1820, 2016. © 2016 Wiley Periodicals, Inc.

Dynamics of Epstein-Barr viral load after hematopoietic stem cell transplantation and effect of preemptive rituximab therapy.

BACKGROUND: Epstein-Barr virus (EBV) displays oncogenic properties, particularly in the immunocompromised host. Notably, hematopoietic stem cell transplantation (HSCT) recipients with a detectable blood EBV viral load (BEBVL) are considered at higher risk of post-transplant lymphoproliferative diseases (PTLD). Therefore, BEBVL is monitored after HSCT, and preemptive rituximab may be used in patients with high values. However, little is known about post-HSCT BEBVL dynamics, and the threshold that should lead to anti-CD20 therapy is poorly defined. METHODS: We retrospectively analyzed the post-HSCT BEBVL of 332 adult HSCT recipients in our center from 2005 to 2013, including the effect of rituximab. RESULTS: Detection of BEBVL >100, 1000, 5000, 10 000, and 50 000 copies/mL occurred in, respectively, 77.7%, 69.6%, 37.0%, 27.1%, and 7.5% of the patients after a respective median time of 9, 14, 15, 16, and 14 weeks. No BEBVL threshold was associated with an overall survival difference. Seventy-eight patients received rituximab, with a BEBVL decrease in most. Among patients with detectable BEBVL, long-term survival did not differ in rituximab treated and non-treated, except for patients with BEBVL ≥50 000. Only one case of PTLD was observed. CONCLUSIONS: BEBVL is frequently detectable after HSCT, but suggests no strong association with survival. Preemptive rituximab therapy threshold remains to be defined.

Comparison of commercial extraction systems and PCR assays for quantification of Epstein-Barr virus DNA load in whole blood.

The automation of DNA extraction and the use of commercial quantitative real-time PCR assays could help obtain more reliable results for the quantification of Epstein-Barr virus DNA loads (EBV VL). This study compared two automated extraction platforms and two commercial PCRs for measurement of EBV VL in 10 EBV specimens from Quality Control for Molecular Diagnostics (QCMD) and in 200 whole-blood (WB) specimens from transplant (n = 137) and nontransplant (n = 63) patients. The WB specimens were extracted using the QIAcube or MagNA Pure instrument; VL were quantified with the EBV R-gene quantification kit (Argene) or the artus EBV RG PCR kit (Qiagen) on the Rotor-Gene 6000 real-time analyzer; and the results were compared with those of a laboratory-developed PCR. DNA was extracted from the QCMD specimens by use of the QIAamp DNA minikit and was quantified by the three PCR assays. The extraction platforms and the PCR assays showed good correlation (R, >0.9; P, <0.0001), but as many as 10% discordant results were observed, mostly for low viral loads (<3 log(10) copies/ml), and standard deviations reached as high as 0.49 log(10) copy/ml. In WB but not in QCMD samples, Argene PCR tended to give higher VL values than artus PCR or the laboratory-developed PCR (mean difference for the 200 WB VL, -0.42 or -0.36, respectively). In conclusion, the two automated extraction platforms and the two PCRs provided reliable and comparable VL results, but differences greater than 0.5 log(10) copy/ml remained between the two commercial PCRs after common DNA extraction.

Evolution of EBV seroprevalence and primary infection age in a French hospital and a city laboratory network, 2000-2016.

BACKGROUND: According to rare studies, the age at EBV primary infection (PI) has recently risen in some developed countries. A later age at infection is generally considered a risk factor for severe EBV PI, although few studies exist on this subject. Our investigation aimed to determine whether EBV seroprevalence and EBV PI epidemiology have evolved in France, and to what extent age and infection intensity (regarding biological parameters) are correlated. METHODS AND FINDINGS: We conducted a retrospective study of the following EBV serological tests databases: tests carried out at Grenoble University Hospital (2000-2016) (n = 53,553); and tests carried out by a network of city laboratories in Grenoble area (2008-2015) (n = 27,485). The hospital population showed a continuous, significant decrease in EBV seroprevalence over the studied period for patients aged 20 and over (p<0.01). The seroprevalence also decreased for different age classes (<10, 15-19, 20-30, and 30-40 years old) over the periods 2001-2005, 2006-2010, and 2011-2015. Consistently, the age at PI was significantly higher in the years 2008-2015 than in the years 2001-2007 (15.6±12.0 vs. 13.7±11.0; p = 0.03). The city laboratory population showed the same trend of decreasing seroprevalence (p = 0.06); no significant variations in age at PI were observed. The age at PI was positively correlated with ASAT, ALAT, γGT, and bilirubin blood levels (p<0.01) and negatively correlated with platelet counts (p<0.05). CONCLUSION: In the last 15 years, the age at EBV PI has increased, whereas seroprevalence has decreased. Moreover, our findings confirm the positive correlation between age and biological abnormalities. Taken together, these results suggest that the incidence of severe EBV PI will increase in the future.