Numerical Modelling Of The V-J Combinations Of The T Cell Receptor TRA/TRD Locus

T-Cell antigen Receptor (TR) repertoire is generated through rearrangements of V and J genes encoding α and β chains. The quantification and frequency for every V-J combination during ontogeny and development of the immune system remain to be precisely established. We have addressed this issue by building a model able to account for Vα-Jα gene rearrangements during thymus development of mice. So we developed a numerical model on the whole TRA/TRD locus, based on experimental data, to estimate how Vα and Jα genes become accessible to rearrangements. The progressive opening of the locus to V-J gene recombinations is modeled through windows of accessibility of different sizes and with different speeds of progression. Furthermore, the possibility of successive secondary V-J rearrangements was included in the modelling. The model points out some unbalanced V-J associations resulting from a preferential access to gene rearrangements and from a non-uniform partition of the accessibility of the J genes, depending on their location in the locus. The model shows that 3 to 4 successive rearrangements are sufficient to explain the use of all the V and J genes of the locus. Finally, the model provides information on both the kinetics of rearrangements and frequencies of each V-J associations. The model accounts for the essential features of the observed rearrangements on the TRA/TRD locus and may provide a reference for the repertoire of the V-J combinatorial diversity

Numerical Modelling Of The V-J Combinations Of The T Cell Receptor TRA/TRD Locus

International audienceLymphocytes of the immune system ensure the body defense by the expression of receptors which are specific of targets, termed antigens. Each lymphocyte, deriving from the same original clone, expresses the same unique receptor. To achieve the production of receptors covering the wide variety of antigens, lymphocytes use a specialized genetic mechanism consisting of gene rearrangements. For instance, the genes encoding the receptor of the alpha chain of the T lymphocyte receptor (TRA) spread over a 1500 Kb genetic region which includes around 100 V genes, 60 J genes, and a single C gene. To constitute a functional alpha chain, one of the V and one of the J genes rearrange together to form a single exon. The precise definition of these V-J combinations is essential to understand the repertoire of TRA. We have developed a numerical model simulating all of the V-J combinations of TRA, fitting the available experimental observations obtained from the analysis of TRA in T lymphocytes of the thymus and the blood. Our model gives new insights on the rules controlling the use of V and J genes in providing a dynamic estimation of the total V-J combinations

Fine characterization of intrahepatic NK cells expressing natural killer receptors in chronic hepatitis B and C.: NK cells in chronic hepatitis C and hepatitis B

International audienceBACKGROUND/AIMS: The fate of intrahepatic NK cell subsets in the course of HCV and HBV infections is not clearly understood. METHODS: Blood and intrahepatic CD56(+) NK cell subsets (expressing NKG2A, CD158a,h or CD158b,j receptors) from HCV or HBV patients were quantified by flow cytometry and localized by immunohistochemistry in liver biopsies. RESULTS: A significant reduction in NK cell frequency and a quantitative imbalance between CD56(bright) and CD56(dim) subsets were observed in chronic HCV patients as compared to HBV patients, underlining that the inflammatory environment is not the only cause of these phenomena. The proportions of intrahepatic NK cells expressing either NKG2A, and/or CD158a,h, CD158b,j differed significantly between HCV and HBV patients. A higher frequency of perforin among intrahepatic CD56(+)CD3(-) cells was observed in HCV compared to HBV patients. Double immunohistochemical staining showed that CD56(+)CD3(-) cells were localized within necrotic areas. Immune monitoring of circulating CD56 subsets revealed that CD3(-)CD56(bright)NKG2A(+) and CD3(-)CD56(dim)NKG2A(+) cells were positively correlated with the necroinflammatory score and inversely correlated with viral load, respectively, in HCV patients. CONCLUSIONS: HCV and HBV affect NK cell subsets according to the status of the diseases, especially CD3(-)CD56(dim)NKG2A(+) and CD3(-)CD56(bright)NKG2A(+) cells, may be of interest for disease monitoring

Flow diagram for the sequential windowing model algorithm.

<p>Flow diagram for the sequential windowing model algorithm.</p

J region use by V14: comparison between experimental and simulation data.

*<p>Frequencies of rearrangements of V14 genes were calculated from <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1000682#pcbi-1000682-g002" target="_blank">Figure 2</a> in Aude-Garcia <i>et al.</i><a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1000682#pcbi.1000682-AudeGarcia1" target="_blank">[10]</a>, for the combinations with three J panels, corresponding to series of J genes scattered along the J region.</p>#<p>Frequencies of rearrangements of V14 genes and of all V genes were calculated from modelling data for the combinations with same series of J genes.</p

Density and RSS scores of the J genes.

<p>Values for the density (open squares) and RSS scores (dark circles) were calculated, as described in methods, for each J gene from the four previous and next genes. X axis represents the J genes, the Y axis the density or the RSS score for all J genes.</p

Validation of the modelling approach: analysis of the V and J region uses.

<p>(A) V region utilization: the X axis represents the V region in Kb. The Y axis shows the V gene percentage utilization in simulation. The simulated data sets have been normalized in order to be compared according to the following formula The fixed parameters of the simulation were as follow, one million of alpha chains, ongoing 1 to 4 rearrangements with opening speeds of 18 Kb/h and 1.03 Kb/h for the V and the J region respectively; (B) J region utilization: the X axis represents the J region in Kb; (C) and (D) Amplitude of J region utilization by opposite V genes, V1 (distal) and V21 (proximal). The X axis represents experimental quantification on 9 J genes. The Y axis shows the experimental utilization frequency of 9 J genes by the V1 and V21 genes. (E) and (F) Amplitude of J region utilization in the model. The X axis represents the J genes. The Y axis shows the model frequency utilization by each J genes. (G) and (H) Superposition of experimental and simulated data for the 9 J genes. The X axis represents experimental quantification on 9 J genes. V and J regions utilization from simulated data are similar to experimental data obtained from <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1000682#pcbi.1000682-Pasqual1" target="_blank">[9]</a>.</p

J locus accessibility: J genes seen rearranged to V19 during ontogeny.

§<p>Thymus from Fetal Day 18 (F18) to Day of birth (D0); data are analyzed from <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1000682#pcbi-1000682-g005" target="_blank">Figure 5</a> in Pasqual et al. <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1000682#pcbi.1000682-Pasqual1" target="_blank">[9]</a>.</p>#<p>Length of the DNA sequence corresponding to the J opening.</p>*<p>For F20, the opening speed has been estimated between 375 bp/h to 750 pb/h depending on the offset of maxima 12 hours (9000 bp/24 h or 9000 bp/12 h).</p

Quantification of the J region use by the V14 family.

<p>254 V14 rearrangements were cloned from T lymphocytes, the V14 members and J genes were determined by sequencing <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1000682#pcbi.1000682-AudeGarcia1" target="_blank">[10]</a>. (A) Profile of the J use by the six members of the V14 family. The two arrows indicate the localization of the two Hot Spots. (B) Profile of the three members the nearest from J genes and (C) J use by the most 5′ V14 members.</p

Model interface and results.

<p>(A) The main user interface window of the simulation program, (B) 2D representation of the rearrangement frequencies, (C) 3D representation of the rearrangement frequencies over all V and J gene associations.</p