Persistent Clonal Expansion of Vδ3 and Vδ4 T Cells in T-ALL Case with Minimal Residual Disease after HSCT
Authors: Xu Ling , Weng Jianyu , Huang Xin , Chen Shaohua , Geng Suxia , Yang Lijian , Wu Suijing , Huang Suming , Xin Du , Li Yangqiu 
Published: October 2019

Abstract
Analysis of diversity and clonality is an important tool in monitoring reconstitution of the immune system and identificating specific immune reaction clones involved in hematological malignancy individuals who accepted hematological stem cell transplantation (HSCT). We have previously reported the evolution of malignant and reactive γδ+ T cell clones in a case with relapse T-ALL before and after allogeneic stem cell transplantation (allo-SCT) (from 4 weeks to 108 weeks), relapse was discovered with the same TCR Vδ5+ T cell clone at 100 weeks after transplantation, and the patient underwent chemotherapy over the next two months and achieved again remission with minimal residual disease (MRD). We sequentially monitored the clinical and laboratorial features of this T-ALL case. The patient was received three times donor lymphocytes infusion (DLI) at 117, 120 and 124 weeks after HSCT. In 178 weeks after transplantation, the patient underwent relapse again, however, in this time the patient underwent III grade chronic graft versus host disease (cGvHD). We continued to analyze the samples at different time points after HSCT and DLI, the leukemic Vδ5+ T cell clone was detected in most samples, while this clone could not be detected in donor sample (Figure 1). Moreover, we found that a monoclonally expanded TCR Vδ4 which we reported before and it remained detectable in all samples post transplantation, interesting same monoclonal Vδ4 clone was identified in sample from donor which was collected at time for DLI. The sequence of Vδ4 was confirmed as same TCR rearrangement: Vδ4Dδ3Jδ1, and the expression level of Vδ4 was significantly increased at the time of disease relapse, the Vδ4 expression level in samples from PBMC and bone marrow at different time point was showed in figure 2. The monoclone Vδ4 from donor was thought as specifically expanded for unknown antigen (maybe for virus or leukemia) and may possess cytotoxicity in donor, however, the persistent expansion of Vδ4 in receptor was thought that might be responsible for leukemic cells, virus infection or cGvHD, it remains an opne question. Because patient underwent III grade cGvHD, and whether this Vδ4+ T cell clone was mainly associated with cGvHD, it is needed further investigation. In addition, the clonally expanded Vδ3 with same size was also detected at time before HSCT, and most time points after HSCT, while it could not be identified in donor sample (Figure 3). Obviously, this expanded Vδ3+ T cell clone originated from patient, and it is possible that this T cell clone possesssed a specific anti-leukemia function, because it appeared at the time with minimal residual disease, however, the function of clonally expanded Vδ3 is needed further confirmation. And it is needed also to analyze the clonality of TCR Vα and Vβ subfamilies. In conclusion, dynamically monitoring TCR repertoire and combining the characteristic of clinical status might benefit for characterizing the malignant clonal evolution, monitoring the specific T cell clone for graft-versus-leukemia (GvL) or GvHD effect and designing specific therapeutic strategies in T-ALL.
Competing interests
The authors declare that they have no competing interests.
Grants: This study was supported by grants from the National Natural Science Foundation of China (Nos. 91129720 and 81270604), the Collaborated grant for HK-Macao-TW of the Ministry of Science and Technology (2012DFH30060), the Guangdong Science & Technology Project (No. 2012B050600023), the Fundamental Research Funds for the Central Universities (No. 21610603, 21612116).
Figure 1. The CDR3 spectratyping of the TCR Vγ and Vδ subfamily T cells in peripheral blood from donor and recipient at different time points after allo-HSCT. Figure 1. The CDR3 spectratyping of the TCR Vγ and Vδ subfamily T cells in peripheral blood from donor and recipient at different time points after allo-HSCT.
Figure 2. The expression level of the TCR Vδ4 gene in recipient at different time points post-HSCT: Red dots showing the expression level of TCRVδ4 (TCDV4) gene in PBMC at time points of 117 W, 120 W, 135 W, 142 W, 146 W, 178 W, 190 W and 200 W post-HSCT. Blue triangles showing the expression level of TCRDV4 genes in bone marrow (BM) at time points of 129.5 W, 178 W, 190 W and 200 W post-HSCT. Figure 2. The expression level of the TCR Vδ4 gene in recipient at different time points post-HSCT: Red dots showing the expression level of TCRVδ4 (TCDV4) gene in PBMC at time points of 117 W, 120 W, 135 W, 142 W, 146 W, 178 W, 190 W and 200 W post-HSCT. Blue triangles showing the expression level of TCRDV4 genes in bone marrow (BM) at time points of 129.5 W, 178 W, 190 W and 200 W post-HSCT.
Figure 3. The The CDR3 spectratyping of Vδ3 T cells in the samples of donor and recipient at different time points post-HSCT. The red boxes indicated the clonally expanded Vδ3 T cell clone with 526 bp which contained the same sequence confirmed by PCR product direct nucleotide sequencing. Figure 3. The The CDR3 spectratyping of Vδ3 T cells in the samples of donor and recipient at different time points post-HSCT. The red boxes indicated the clonally expanded Vδ3 T cell clone with 526 bp which contained the same sequence confirmed by PCR product direct nucleotide sequencing.

Disclosures
Xu: National Natural Science Foundation of China (Nos. 91129720 and 81270604), the Collaborated grant for HK-Macao-TW of the Ministry of Science and Technology (2012DFH30060), the Guangdong Science & Technology Project (No. 2012B050600023): Research Funding; Fundamental Research Funds for the Central Universities (No. 21610603, 21612116): Research Funding. Li:Fundamental Research Funds for the Central Universities (No. 21610603, 21612116). : Research Funding; National Natural Science Foundation of China (Nos. 91129720 and 81270604), the Collaborated grant for HK-Macao-TW of the Ministry of Science and Technology (2012DFH30060), the Guangdong Science & Technology Project (No. 2012B050600023): Research Funding.