Abstract: The thymic-dependent pathway that involves generation of new naive T cells from donor-derived precursor cells accounts for the more durable reconstitution of the T-cell compartment and generates a more diverse TCR repertoire. Thymic function and production of recent thymic emigrants (RTEs) may be directly evaluated through the quantification, by real-time polymerase chain reaction (PCR), of the T-cell receptor excision circles (TRECs). Following hematopoietic stem cell transplant (HSCT), there is a prolonged period of profound immune deficiency, which continues for years after HSCT. The factors that inhibit thymic function may include age, graft-versus-host disease (GVHD), and direct thymic damage from chemoradiotherapy. GVHD after HSCT also leads to thymic insufficiency, possibly by direct attack on the thymic stroma by allogeneic effector cells. The aim of our study is to analysis T cell receptor excision circles (TRECs) in patients with GVHD after allogeneic stem cell transplantation. We used real-time polymerase chain reaction (PCR) to quantify SjTRECs in 12 patients with GVHD(9 males, 3 females; median age 32 years old), who underwent HLA-matching sibling BMT and/or peripheral blood stem cell transplantation (PBSCT) at our department. Quantitative detection of sjTRECs in DNA of peripheral blood mononuclear cells from 13 normal individualals. The median value of sjTRECs copies P1 000 PBMCs was 4.37±3.64 in normal indiviuals. However, the decreased levels of TRECs were most profound in the group of patients with active chronic GVHD at the time of study. it was 0.26±0.22 copies P1 000 PBMCs in 12 patients with GVHD (P 〈 0. 00007). We conclude that measurement of sjTREC may provide an important tool for predicting thymus-dependent T-cell reconstitution in GVHD patients after transplantation.

Abstract: Introduction The myelodysplastic syndromes (MDS) comprise a heterogeneous group of clonal hematopoietic stem cell disorders, while, immunological abnormalities are frequently observed in patients with MDS[1]. Several reports [2,3] revealed that about 10% of MDS patients have clinical autoimmune disorders like skin vasculitis, rheumatic disease, or autoimmune hemolytic anemia. Furthermore, serological immunological abnormalities like hyper- or hypogammaglobulinemia, positivities of antinuclear antibody, positivities of direct Coombs test, or inverted CD4/8 ratios were found in 18–65% of patients with MDS. Recently immunosuppressive therapies including prednisolone, antithymocyte globulin, and cyclosporin A (CsA) are used to treat cytopenia in some patients with MDS. We reported four patients with MDS. Rearrangements of the TCR-beta genes were seen in these patients using RT-PCR and Genescan analysis (CDR 3 length analysis). Also they had skewed TCR usages using TCR repertoire analysis. Methods Two patients with refractory anemia(RA), two with refractory anemia with blasts(RAEB). four males from 41to 68 yearsold. Complementarity determining region 3(CDR3) of TCR Vβ with 24 variable region gene was amplified in peripheral blood mononuclear cells, which were drawn from five patients with myelodysplastic syndromes (MDS) using RT-PCR, to observe the expression of TCR Vβreceptoroire T cells, the PCR products were further analyzed by genescan to evaluating clonality of T cells (CDR 3 length analysis), and compare results with age-matched healthy donors and patients with graft versus host disease(GVHD). Results We found a significantly higher number of skewed Vb profiles in the MDS and GVHD patients compared with donors. In peripheral blood T cells, Only 2-11 Vb subfamily T cells could be identified in MDS patinets, clonal expansion T cells could be found in Vb1, 3,13, 14 and 21 subfamilies. Disussion In this study, we evaluated the total T-cell repertoire of 4 MDS patients using gendscan analysis to look for evidence of T-lymphocyte clonality. This analysis showed that all 4 patients exhibited extensive skewing of their TCR spectratypes, suggesting clonal or oligoclonal T-cell expansions. Epperson [10]also reported the same results. As we know, acute and chronic graft-versus-host disease(GVHD) is a major complication after allogeneic bone marrow transplantation. GVHD is mediated by T cells that are derived from the BM graft. In this study, we compared the results of GVHD patients with that of MDS patients, and found that these two groups patients show TCR Vb skew distribution and clonal expansion. These findings provide further evidence that T cell mediated immune processes are a feature of MDS.

Abstract: Objective In order to analyze the relationship between the content of T-cell receptor excision DNA circles (TRECs) and BCR-ABL mRNA levels, evaluating the prognostic significance of thymic recent output function monitoring in patients with chronic myelogenous leukemia (CML). Methods Quantitative detection of TRECs and BCR-ABL fusion gene transcripts in peripheral blood from 15 CML patients were preformed using real-time PCR technique. And the TREC-number was related to the number of T-cells by determination of the number of CD3-positive cells. The change of BCR-ABL level in 6 CML patients was followed-up for two years. Results There was no significant correlation between TRECs and BCR-ABL mRNA in peripheral blood from CML patients at the first diagnose. Patients who had higher TRECs at diagnosis had a larger reduction of BCR-ABL level after 2 years of follow-up. While in 2 patients who underwent haemopoietic stem cell transplantation(HSCT), BCR-ABL in one patient with higher pre-transplantation TRECs value became undetectable with three consecutive detections during the first year post transplantation, but low level of BCR-ABL could be identified in the other patient. Conclusion High thymic output function in CML patients could be helpful for anti-residual CML cells.

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.

Abstract: Immunological phenotypes and differentiation statuses commonly decide the T cell function and anti-tumor ability. However, little is known about these alterations in CML patients. Method Here, we investigated the immunologic phenotypes (CD38/CD69/HLA-DR/CD28/CD57/BTLA/TIGIT/PD-1) of T subsets (TN, TCM, TEM, and TEMRA) in peripheral blood (PB) and bone marrow (BM) from de novo CML patients (DN-CML), patients who achieved a molecular response (MR) and those who failed to achieve an MR (TKI-F) after tyrosine kinase inhibitor (TKI) treatment using multicolor flow cytometry. Results CD38 or HLA-DR positive PB CD8+TN and TCM cells decreased in the DN-CML patients and this was further decreased in TKI-F patients. Meanwhile, the level of PD-1 elevated in CD8+ TEM and TEMRA cells from PB in all groups. Among BM sample, the level of HLA-DR+CD8+TCM cells significantly decreased in all groups and CD8+TEMRA cells from TKI-F patients exhibited increased level of TIGIT and CD8+ tissue-residual T cells (TRM) from DN-CML patients expressed a higher level of PD-1 and TIGIT. Lastly, we found a significantly decreased proportion of CD86+ dendritic cells (DCs) and an imbalanced CD80/CD86 in the PB and BM of DN-CML patients, which may impair the activation of T cells. Conclusion In summary, early differentiated TN and TCM cells from CML patients may remain in an inadequate activation state, particularly for TKI-F patients. And effector T cells (TEM, TEMRA and TRM) may be dysfunctional due to the expression of PD-1 and TIGIT in CML patients. Meanwhile, DCs cells exhibited the impairment of costimulatory molecule expression in DN-CML patients. Those factors may jointly contribute to the immune escape in CML patients.

Abstract: A previous study has demonstrated a significant decrease in the TCRζ gene expression level in chronic myeloid leukemia (CML); thus, we further investigated the expression of TCRζ-regulating factors, the distribution of the TCRζ 3' untranslated region (3'-UTR) splice variants, and the expression level and correlation of the alternative splicing factor/splicing factor 2 (ASF/SF-2), FcεRIγ and ZAP-70 genes. TCRζ 3'-UTR splice variants were identified in peripheral blood mononuclear cells (PBMCs) from 14 healthy individuals, 40 patients with CML and 22 patients with CML in complete remission (CML-CR) by RT-PCR. The expression level of the TCRζ, FcεRIγ, ASF/SF-2 and ZAP-70 genes was analyzed by real-time quantitative PCR. While the expression of TCRζ gene in the CML group was significantly lower than that in the healthy individual and CML-CR groups, a significantly higher expression of the FceRIγ and ASF/SF-2 genes was found in the CML group. Two types of splicing forms were detected in all of the healthy individual CML-CR cases: wild type (WT) TCRζ 3'-UTR and alternatively splieced (AS) TCRζ 3'-UTR which have been alternatively splieced in the WT TCRζ 3'-UTR . However, 35% of the CML cases contained only the wild type TCRζ 3'-UTR isoform. Based on the TCRζ 3'-UTR isoform expression characteristic, we divided the patients with CML into two subgroups: the WT + AS - CML group, containing patients that express only the wild type TCRζ 3'-UTR, and the WT + AS + CML group, which contained patients that expressed two TCRζ 3'-UTR isoforms. A significantly different ASF/SF-2 and FcεRIγ gene expression pattern was found between the WT + AS - and WT + AS + CML groups. We concluded that defective TCRζ expression may be characterized in the WT + AS - and WT + AS + CML subgroups by the different gene expression pattern. The overexpression of ASF/SF2, which alternatively splices the TCRζ 3’-UTR, is thought to participate in feedback regulation. The characteristics of TCRζ 3'-UTR alternative splicing may be a novel immunological marker for the evaluation of the CML immune status.

Abstract: Introduction The extensive diversity of the mature T-cell receptor(TCR) is determined primarily by the complementarity-determining regions (CDR3) of the TCR. The CDR3 of both TCRα and TCRβ genes is generated by extensive rearrangement and fusion between the V,D,and J segments and by random insertion and deletion of junctional nucleotides, which yields final products that are quite heterogeneous in size. As a result of these gene rearrangements, each T cell has a unique TCR and the diversity of the T-cell repertoire at any specific time can be characterized by the examination of CDR3 within that population. Using CDR3 spectratying technique, normal individuals demonstrate a highly diverse and polyclonal The aim of our study was to evaluate to investigate restricted expansion of TCR Vβ gene repertoire and the reconstitution of T cell receptor repertoire following allogeneic hematopoietic stem cell transplantation. Methods Patients Ten patients(9 males, 1 females; median age 31 years,range18–45) with 6 chronic myeloid leukemia-chronic phase and 4 cases of acute myelogenous lenkemia(CR1) who underwent HLA-matching sibling or unrelated BMT and/or peripheral blood stem cell transplantation (PBSCT) at our department between July 1999 and May 2000 were considered evaluable restricted expansion of TCR Vβ gene repertoire, the reconstitution of T cell receptor repertoire and oligoclonal T Cell Expansion in Chronic Graft-Versus-Host Disease. RT-PCR and Genes scan analysis (CDR 3 length analysis). Results Only 2-18Vβ genes were found in samples from these ten patients within one year, and there are different distribution in different patients. TCR repertoire complexity was abnormal in all patients, parts of the genes were expansion and part of them were suppressed. Samples from 9 patients with GVHD show V β3 in 7 cases, V β 8 and V β 23 in 6 patients. The results of genescan show that the PCR production of peripheral blood samples from these patients disply oligoclonal. Only 5–22Vβ subfamily T cells were found in samples from these patients whose transplantation more than one year. TCR repertoire complexity was abnormal in all patients. Discussion Following allogeneic BMT, regeneration of T-cell populations with a diverse repertoire can occure by at least two mechanisms: One mechanism is a thymic-dependent pathway, which presumably involves both negative and positive selection and recapitulates fetal ontogeny. Alternatively, regeneration of peripheral T cells may occur through thymic-independent mechanisms. All patients had marked abnormalities in their spectratypes, only 5-22Vβ subfamily T cells were found in samples from these patients, most of it was influenced after transplant, although the number of circulating CD3+ T lymphocytes in these patients have restored at normal lever by flow cytometic analysis, but the CD4+ T cell subset returned slowly in these patients resulting in an inversion of the normal CD4/CD8 ratio for more than 1 year after tuansplantation. Therefore, the analysis of TCRVβ subfamily is a usuaful methods and techniques for monitoring immune reconstitution after transplant.