VJ Segment Usage of TCR-Beta Repertoire in Monozygotic Cystic Fibrosis Twins

Abstract

Sixteen monozygotic cystic fibrosis (CF) twin pairs of whom 14 pairs were homozygous for the most common p.Phe508del CFTR mutation were selected from the European Cystic Fibrosis Twin and Sibling Study Cohort. The monozygotic twins were examined in their T cell receptor (TCR) repertoire in peripheral blood by amplicon sequencing of the CDR3 variable region of the ß-chain. The recruitment of TCR J and V genes for recombination and selection in the thymus showed a strong genetic influence in the CF twin cohort as indicated by the shortest Jensen-Shannon distance to the twin individual. Exceptions were the clinically most discordant and/or most severely affected twin pairs where clonal expansion probably caused by recurrent pulmonary infections overshadowed the impact of the identical genomic blueprint. In general the Simpson clonality was low indicating that the population of TCRß clonotypes of the CF twins was dominated by the naïve T-cell repertoire. Intrapair sharing of clonotypes was significantly more frequent among monozygotic CF twins than among pairs of unrelated CF patients. Complete nucleotide sequence identity was observed in about 0.11% of CDR3 sequences which partially should represent persisting fetal clones derived from the same progenitor T cells. Complete amino acid sequence identity was noted in 0.59% of clonotypes. Of the nearly 40,000 frequent amino acid clonotypes shared by at least two twin siblings 99.8% were already known within the immuneACCESS database and only 73 had yet not been detected indicating that the CDR3ß repertoire of CF children and adolescents does not carry a disease-specific signature but rather shares public clones with that of the non-CF community. Clonotypes shared within twin pairs and between unrelated CF siblings were highly abundant among healthy non-CF people, less represented in individuals with infectious disease and uncommon in patients with cancer. This subset of shared CF clonotypes defines CDR3 amino acid sequences that are more common in health than in disease.

Keywords: CDR3; T cell receptor repertoire; TCRB; VJ usage; cyctic fibrosis; immunotyping; twins.

Figure 1

Clinical data of the 16 CF twin pairs discerned by their sample number ID in the European Cystic Fibrosis Twin and Sibling Study (see Table 1). The figure shows the age of the 32 monozygotic twins and their ranks within the 1,060 participants of the European Cystic Fibrosis Twin and Sibling Study for the clinical parameters “Weight-for-height” (left) and “FEVPerc” (middle) at the day of blood sampling. A low rank number indicates a severe outcome. Patients had to be older than 6 years to perform a robust FEV1 measurement. The delta rank (right) indicates the intrapair discordance of the twin pairs. A low delta rank indicates a comparable disease manifestation of the twins. In contrast, large delta values indicate divergent clinical phenotypes.

Figure 2

Correlation of productive Simpson clonality with age. Productive clonality is plotted for each twin. Twin pairs are connected with a black line. The gray dots indicate 590 healthy PBMC controls (36, 37) and the blue line indicates the trend of the controls. As expected, the clonality increases with age. Of the twins only the severely affected patients are above the trend line indicating that the CF samples are comparable to those from healthy controls.

Figure 3

Proportion of shared TCRß CDR3 clonotypes in monozygotic CF twins at the nucleotide sequence (A) and amino acid sequence (B) levels. The abscissa indicates the numbers of raw sequencing reads (A) and in-frame coding reads derived peptides (B) of the twin's sample with the lower number of reads that were taken as the reference for the calculation of the proportion of shared clonotypes. The dots indicate the proportion of CDR3 sequences shared within the 16 CF twin pairs (red) or between 480 arbitrary pairs of unrelated CF twins generated from the same data-sets (gray). Please note the different scales of the ordinate in (A,B).

Figure 4

Comparison of intra- and inter-pair clonality difference. The absolute difference in clonality was calculated for each related (n = 13) and unrelated twin pair (n = 312). The three severely affected outlier pairs 180, 244, and 294 were excluded from the analysis. A non-significant trend was observed that twin pairs show a lower clonality difference than unrelated patient samples.

Figure 5

Comparison of VJ-usage in related und unrelated twin pairs. The boxplot presentation shows the Jensen-Shannon divergence (–42) for the comparison of each sample to the twin sibling and to the 30 unrelated twins for the (A) V-segment and (B) J-segment usage of the CDR3. Red dots represent the Jensen-Shannon divergence to the twin sibling. The gray background areas indicate the reference values for healthy unrelated twins (upper gray panel) and related healthy twins (lower gray panel) (21). In case of the J-segment, the reference panels overlap indicated by the dark gray area.

Figure 6

Frequency comparison of V-segment usage of the distant twin pair 180. The boxplots show pairwise comparisons of both (A) twin 180_A and (B) twin 180_B to the twin sibling and the 30 unrelated twins for all V-segment groups. Red dots symbolize the comparison to the 180 twin sibling. Values above 1 indicate an overrepresentation of the frequency of the respective V-segment family in the investigated twin sibling. If no clonotypes were found in one of the “other” samples the value was fixed to 10 to allow a calculation and to take into account that the V-segment family occurs in the 180_A or 180_B sample, respectively.

Figure 7

Frequency distribution of amino acid clonotypes present in two or more CF siblings. The frequency distribution of shared amino acid clonotypes (n = 39,812) shows that the majority is found in two samples. However, there are also public clonotypes which are present in more than 50% of the twins. In this presentation we did not distinguish between related and unrelated twin pairs.

Figure 8

Presence of frequent amino acid clonotypes of CF siblings in the samples deposited in the immuneACCESS database. 39,812 amino acid clonotypes were detected in at least two samples from CF siblings. (A) 1,384 clonotypes exclusively identified in a related twin pair were found in an only low number of database samples. (B) 3,430 clonotypes were detected in at least one related CF twin pair plus CF singletons. These public clones are very frequent within the immuneACCESS database. (C) 34,998 clonotypes were only detected in unrelated CF siblings. The majority of these clonotypes is only present with low frequency in the immuneACCESS database. (D) The Kaplan-Meier plot shows that clonotypes present in twins and at least one independent sample are more frequently found in the immuneACCESS database (black line). In contrast, clonotypes only present in twins or in unrelated samples are comparably rare.

Figure 9

Frequency distribution of shared CF amino acid clonotypes in samples of healthy humans (A), patients with infectious disease (B) and patients with cancer (C) deposited in the immuneACCESS database.