Simplified Monitoring of CD4 T-Cell Receptor Repertoire Provides Effective Management Strategies in the Patients with Persistent or Recurrent Graft-Versus-Host Disease
Authors: Kuroda Rie , Mase Shintaro , Maeba Hideaki , Araki Raita , Ikawa Yasuhiro , Fujiki Toshihiro , Fukuda Masaki , Koizumi Shoichi , Yachie Akihiro , Nishimura Ryosei 
Published: October 2019

Abstract
T-cell receptor (TCR) repertoire has been studied regarding graft-versus-host disease (GVHD), graft-versus-tumor effect, and immunological recovery in hematopoietic stem cell transplantation (HSCT) as well as autoimmune diseases. It is unclear yet whether a TCR restriction is a useful marker for a decision on courses of treatment for persistent or recurrent GVHD, such as increase or tapering of immunosuppressive agents. Ninety-four peripheral blood samples obtained from 42 HSCT recipients (39 surviving more than one year and 3 surviving more than 6 months) and 30 healthy volunteers were analyzed. The relative TCR Vb usage of CD4+ and CD8+ T-cells was investigated using a panel of monoclonal antibodies specific for 21 different Vb regions by flow cytometry. A valuable describing the degree of Vb-repertoire restriction was defined, namely cumulative deviation index (CDI) calculated as shown in figure 1; the higher number of CDI indicated the more restricted pattern of TCR repertoire usage. CDI of TCR repertoire in CD4 T-cells 6 months later after HSCT was much higher in the patients with persistent or recurrent GVHD treated with steroids, compared with the patients without persistent or recurrent GVHD (p<0.05 as shown in Figure 2A). On the other hand, any specific findings were not obtained from CDI in CD8 T-cells. Interestingly, much more restriction of TCR repertoire in CD4 T-cells was observed in the patients who could not stop steroids in the future, compared with the patients who could discontinue them in the near future (p<0.05 as shown in Figure 2B). Next when combined with representative T-cell activation marker such as HLA-DR, we have found that immunosuppressive therapy could be discontinued finally when TCR repertoire in CD4 T-cells not CD8 recovered and maintained normal ranges even if HLA-DR were highly expressed (>=30% on CD4). Instead, when CDI of TCR repertoire in CD4 maintained relatively high level even if HLA-DR were below 30% on CD4, complete discontinuation of immunosuppressive agents was quite difficult. Finally we studied the correlation between TCR repertoire and the frequency of regulatory T-cells (Tregs). Some patients clearly demonstrated that the frequency of Tregs increased over time in concert with recovering of CD4 T-cell repertoire. However we were not able to show this trend in all patients, probably due to the administration of calcineurin inhibitors, which negatively affect the reconstitution of Tregs compared with those of conventional T-cells. Although much more detailed analysis of T-cell repertoire could be done recently by utilizing high-throughput sequencing, our method based on flow cytometry has a cost advantage and are easy to monitor the patients for immunological recovery. In conclusion, monitoring of TCR repertoire restriction in CD4 T-cells but not CD8 provides useful information for managements of the patients with prolonged GVHD. Special attention should be paid to the patients with strong restriction of TCR repertoire in CD4 T-cells.
Figure 1 Figure 1. Figure 2 Figure 2.

Disclosures
No relevant conflicts of interest to declare.