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Quantitative and qualitative characterization of expanded CD4+ T cell clones in rheumatoid arthritis patients.

Ishigaki K, Shoda H, Kochi Y, Yasui T, Kadono Y, Tanaka S, Fujio K, Yamamoto K - Sci Rep (2015)

Bottom Line: Our in-depth characterization of ECs in RA successfully demonstrated the presence of the specific immunological selection pressure, which determines the phenotype of ECs.Moreover, transcriptome tracking added novel aspects to the underlying sequential immune processes.Our approach may provide new insights into the pathophysiology of RA.

View Article: PubMed Central - PubMed

Affiliation: Department of Allergy and Rheumatology, Graduate School of Medicine, the University of Tokyo, Tokyo, Japan.

ABSTRACT
Rheumatoid arthritis (RA) is an autoimmune destructive arthritis associated with CD4(+) T cell-mediated immunity. Although expanded CD4(+) T cell clones (ECs) has already been confirmed, the detailed characteristics of ECs have not been elucidated in RA. Using combination of a single-cell analysis and next-generation sequencing (NGS) in TCR repertoire analysis, we here revealed the detailed nature of ECs by examining peripheral blood (PB) from 5 RA patients and synovium from 1 RA patient. When we intensively investigated the single-cell transcriptome of the most expanded clones in memory CD4(+) T cells (memory-mECs) in RA-PB, senescence-related transcripts were up-regulated, indicating circulating ECs were constantly stimulated. Tracking of the transcriptome shift within the same memory-mECs between PB and the synovium revealed the augmentations in senescence-related gene expression and the up-regulation of synovium-homing chemokine receptors in the synovium. Our in-depth characterization of ECs in RA successfully demonstrated the presence of the specific immunological selection pressure, which determines the phenotype of ECs. Moreover, transcriptome tracking added novel aspects to the underlying sequential immune processes. Our approach may provide new insights into the pathophysiology of RA.

No MeSH data available.


Related in: MedlinePlus

The non-Th1/Th17/Tfh subset in RA-PB contained the majority of expanded and synovium-infiltrating clones.The Th1, Th17, Tfh and non-Th1/Th17/Tfh subsets were sorted from 3 RA-PB and the NGS TCR repertoire analysis was performed. In order to assess the distribution of target clones within different subsets of CD4+ T cells, frequencies assessed by the TCR repertoire analysis (the total number of reads of target clones divided by the total number of reads with functional CDR3 sequences) were multiplied by the frequency of each CD4+ T cell subset within all CD4+ T cells assessed by FACS in order to correct for the subset size. (A) The distribution of the mECs in RA1 and RA2 (corresponding to C1.1, C2.1 in Fig. 1). (B) The distribution of ECs (clones with more than 0.2% of total number of reads with functional CDR3 sequences). (C) The distribution (within PB) of clones that were also detected in the synovium of the same patient.
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f3: The non-Th1/Th17/Tfh subset in RA-PB contained the majority of expanded and synovium-infiltrating clones.The Th1, Th17, Tfh and non-Th1/Th17/Tfh subsets were sorted from 3 RA-PB and the NGS TCR repertoire analysis was performed. In order to assess the distribution of target clones within different subsets of CD4+ T cells, frequencies assessed by the TCR repertoire analysis (the total number of reads of target clones divided by the total number of reads with functional CDR3 sequences) were multiplied by the frequency of each CD4+ T cell subset within all CD4+ T cells assessed by FACS in order to correct for the subset size. (A) The distribution of the mECs in RA1 and RA2 (corresponding to C1.1, C2.1 in Fig. 1). (B) The distribution of ECs (clones with more than 0.2% of total number of reads with functional CDR3 sequences). (C) The distribution (within PB) of clones that were also detected in the synovium of the same patient.

Mentions: We initially focused on the most expanded CD4+ T cell clones (mECs) that persistently expanded with the highest frequency as shown in Fig. 1 (C1.1 for RA1 and C2.1 for RA2). Both of these were the most frequently observed in the non-Th1/Th17/Tfh subsets (Fig. 3A). The distributions of target clones (Fig. 3A–C) were calculated based on the frequency assessed by the TCR repertoire analysis and the size of each subset assessed by FACS analysis (see details in the figure legend). We then examined the distribution of ECs (more than 0.2%) in the 4 subsets of memory CD4+ T cells and found that the majority of ECs were also detected in the non-Th1/Th17/Tfh subsets (Fig. 3B). We tracked synovial tissue-infiltrating CD4+ T cells and examined their localization in PB. A comparison of the PB and synovial tissue TCR repertoires from RA1 revealed that the non-Th1/Th17/Tfh subsets contained the majority of synovial tissue-infiltrating CD4+ T cells (Fig. 3C). The distinct clonality of ECs between naïve and memory CD4+ T cells and evident skewing of memory ECs toward the non-Th1/Th17/Tfh subsets collectively suggested that the expansion of ECs reflected a specific differentiation process rather than a non-specific generalized activation process.


Quantitative and qualitative characterization of expanded CD4+ T cell clones in rheumatoid arthritis patients.

Ishigaki K, Shoda H, Kochi Y, Yasui T, Kadono Y, Tanaka S, Fujio K, Yamamoto K - Sci Rep (2015)

The non-Th1/Th17/Tfh subset in RA-PB contained the majority of expanded and synovium-infiltrating clones.The Th1, Th17, Tfh and non-Th1/Th17/Tfh subsets were sorted from 3 RA-PB and the NGS TCR repertoire analysis was performed. In order to assess the distribution of target clones within different subsets of CD4+ T cells, frequencies assessed by the TCR repertoire analysis (the total number of reads of target clones divided by the total number of reads with functional CDR3 sequences) were multiplied by the frequency of each CD4+ T cell subset within all CD4+ T cells assessed by FACS in order to correct for the subset size. (A) The distribution of the mECs in RA1 and RA2 (corresponding to C1.1, C2.1 in Fig. 1). (B) The distribution of ECs (clones with more than 0.2% of total number of reads with functional CDR3 sequences). (C) The distribution (within PB) of clones that were also detected in the synovium of the same patient.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4542667&req=5

f3: The non-Th1/Th17/Tfh subset in RA-PB contained the majority of expanded and synovium-infiltrating clones.The Th1, Th17, Tfh and non-Th1/Th17/Tfh subsets were sorted from 3 RA-PB and the NGS TCR repertoire analysis was performed. In order to assess the distribution of target clones within different subsets of CD4+ T cells, frequencies assessed by the TCR repertoire analysis (the total number of reads of target clones divided by the total number of reads with functional CDR3 sequences) were multiplied by the frequency of each CD4+ T cell subset within all CD4+ T cells assessed by FACS in order to correct for the subset size. (A) The distribution of the mECs in RA1 and RA2 (corresponding to C1.1, C2.1 in Fig. 1). (B) The distribution of ECs (clones with more than 0.2% of total number of reads with functional CDR3 sequences). (C) The distribution (within PB) of clones that were also detected in the synovium of the same patient.
Mentions: We initially focused on the most expanded CD4+ T cell clones (mECs) that persistently expanded with the highest frequency as shown in Fig. 1 (C1.1 for RA1 and C2.1 for RA2). Both of these were the most frequently observed in the non-Th1/Th17/Tfh subsets (Fig. 3A). The distributions of target clones (Fig. 3A–C) were calculated based on the frequency assessed by the TCR repertoire analysis and the size of each subset assessed by FACS analysis (see details in the figure legend). We then examined the distribution of ECs (more than 0.2%) in the 4 subsets of memory CD4+ T cells and found that the majority of ECs were also detected in the non-Th1/Th17/Tfh subsets (Fig. 3B). We tracked synovial tissue-infiltrating CD4+ T cells and examined their localization in PB. A comparison of the PB and synovial tissue TCR repertoires from RA1 revealed that the non-Th1/Th17/Tfh subsets contained the majority of synovial tissue-infiltrating CD4+ T cells (Fig. 3C). The distinct clonality of ECs between naïve and memory CD4+ T cells and evident skewing of memory ECs toward the non-Th1/Th17/Tfh subsets collectively suggested that the expansion of ECs reflected a specific differentiation process rather than a non-specific generalized activation process.

Bottom Line: Our in-depth characterization of ECs in RA successfully demonstrated the presence of the specific immunological selection pressure, which determines the phenotype of ECs.Moreover, transcriptome tracking added novel aspects to the underlying sequential immune processes.Our approach may provide new insights into the pathophysiology of RA.

View Article: PubMed Central - PubMed

Affiliation: Department of Allergy and Rheumatology, Graduate School of Medicine, the University of Tokyo, Tokyo, Japan.

ABSTRACT
Rheumatoid arthritis (RA) is an autoimmune destructive arthritis associated with CD4(+) T cell-mediated immunity. Although expanded CD4(+) T cell clones (ECs) has already been confirmed, the detailed characteristics of ECs have not been elucidated in RA. Using combination of a single-cell analysis and next-generation sequencing (NGS) in TCR repertoire analysis, we here revealed the detailed nature of ECs by examining peripheral blood (PB) from 5 RA patients and synovium from 1 RA patient. When we intensively investigated the single-cell transcriptome of the most expanded clones in memory CD4(+) T cells (memory-mECs) in RA-PB, senescence-related transcripts were up-regulated, indicating circulating ECs were constantly stimulated. Tracking of the transcriptome shift within the same memory-mECs between PB and the synovium revealed the augmentations in senescence-related gene expression and the up-regulation of synovium-homing chemokine receptors in the synovium. Our in-depth characterization of ECs in RA successfully demonstrated the presence of the specific immunological selection pressure, which determines the phenotype of ECs. Moreover, transcriptome tracking added novel aspects to the underlying sequential immune processes. Our approach may provide new insights into the pathophysiology of RA.

No MeSH data available.


Related in: MedlinePlus