Ping Zhu

TCR spectratyping revealed T lymphocytes associated with graft-versus-host disease after allogeneic hematopoietic stem cell transplantation

Clonal expansion of T cells after allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been observed, but their characteristics remain to be fully elucidated. We report here that CD8+ T cells were the dominant T lymphocytes seen and T-cell repertoire diversity decreased dramatically during the first 3 months after allo-HSCT. Patients with GVHD grade II – IV had significantly lower T-cell repertoire diversity compared with non-GVHD patients. TCR beta variable gene (TCRBV) subfamily 8, 5.1, 5.2, 4, and 13 were the five most frequently expanded subfamilies among these patients. Among the 49 over-expanded clones identified, clonotype “TCR3-5” and “TCR18-5” were isolated from four patients with HLA-A2 allele and skin GVHD. Their frequencies correlated well with skin symptoms (i.e. rash). Moreover, they were detected in donors but not detected in recipients before transplantation. Lastly, three common TCRBV CDR3 motifs shared by T cells related with GVHD were discovered: TGDS, GLAG, and GGG. These findings suggest that TCR spectratyping is helpful for revealing GVHD-related T cells and may have utility in early diagnosis.

CYP2B6 gene single nucleotide polymorphisms and leukemia susceptibility

CYP2B6 is a highly variable and polymorphic cytochrome P450 enzyme which plays a vital role in the degradation of some endogenous metabolites, xenobiotics, and harmful compounds. The 516G>T single nucleotide polymorphism (SNP) in exon 4 of CYP2B6 gene may change CYP2B6 enzyme activity and the gene expression in the liver. Carcinogens’ failure to be degraded by CYP2B6 may cause DNA injury and cancer. Here, we aimed to evaluate the association between genotype or allele of CYP2B6 516G>T SNP and acute leukemia and myelodysplastic syndrome (MDS). We recruited 300 patients including 164 cases of acute myeloid leukemia (AML), 96 cases of acute lymphoblastic leukemia (ALL, including 17 cases of T-ALL and 79 cases of B-ALL), 40 cases of MDS, as well as 348 unrelated umbilical cord blood as the controls. Karyotype analysis and multiplex reverse transcription-polymerase chain reaction (RT-PCR) was performed to determine different recurrent genetic abnormalities in these cases. Genotype of CYP2B6 516G>T SNP was determined by allele-specific primers PCR, and confirmed by gel electrophoresis and sequencing. The GT and GT + TT genotype frequencies of c.516G>T SNP were higher in ALL (37.5% and 42.7%, respectively, P < 0.01), and AML (37.2% and 40.9%, respectively, P < 0.01) than in control (23.9% and 25.9%, respectively). In the subtypes of acute leukemias, the GT + TT genotype frequency was significantly higher in AML with recurrent genetic abnormalities (41.7%, p < 0.05), in AML-NOS (40.6%, p < 0.01), in acute monoblastic and monocytic leukemia (48.3%, p < 0.01), and in T-ALL (70.6%, p < 0.01) as compared with those in the controls. The frequency of CYP2B6 516 T allele was higher in AML (22.3%, p < 0.01) and ALL (24.0%, p < 0.01) compared with cord blood (13.9%). In different types of acute leukemias, CYP2B6 516 T allele frequency was significantly higher in AML with AML1-ETO (19.2%, p < 0.05), AML-NOS (22.7%, p < 0.01), acute monoblastic and monocytic leukemia (25.9%, p < 0.01), and T-ALL (38.2%, p < 0.01). MDS was unrelated to the genotype and allele frequencies of c.516G>T SNP in CYP2B6. T allele of CYP2B6 516G>T SNP may be one of the risk factors predisposing to the pathogenesis of a majority of ALL and AML, but has no relationship with B-ALL and leukemia with or without chromosome abnormalities.

Patterns of T-cell reconstitution by assessment of T-cell receptor excision circle and T-cell receptor clonal repertoire after allogeneic hematopoietic stem cell transplantation in leukemia patients – a study in Chinese patients

Objective:  Successful allogeneic hematopoietic stem cell transplantation (HSCT) requires reconstitution normal T‐cell immunity. Measurement of T‐cell receptor excision circles (TRECs) and T‐cell receptor β (TCRBV) CDR3 repertoire is a means of quantifying recent thymic T‐cell production and reflecting antigen‐specific T‐cell clones proliferation.

An improved design of PCR primers for detection of human T cell receptor β chain repertoire

Analysis of T cell receptor β variable region (TCRBV) gene rearrangement is useful for clonal assessment of abnormal T cell proliferations in various diseases. However, most primer panels previously used can only amplify the third complementarity-determining region. Following IMGT database we established a panel of primers, which can amplify entire sequences of all functional TCRBV families. To confirm the usefulness of this panel of primers, we detected different TCRBV repertoires. In 15 healthy donors, most of the BV families were expressed and appeared as a Gaussian distribution. 13 acute myeloid leukemia patients showed monoclonal or oligoclonal changes of BV15 family, and some of them also had monoclonal or oligoclonal expansion of BV2, BV4, BV6 or BV13 families. In one patient after allo-hematopoietic stem cell transplantation, monoclonal proliferation of BV10 family occurred during graft-versus-host disease. In conclusion, this panel of primers improves our abilities to analyze TCRBV repertoire changes in related diseases.

Vaccination with immunoglobulin frame region-derived nonapeptide elicits cellular immune response against lymphoma in human leukocyte antigen-A2.1 transgenic mice

The epitope in the frame region (FR) of the immunoglobulin heavy chain variable region (IgHV) is a potential target for lymphoma immunotherapy. Our previous work identified a FR-derived nonapeptide (QLVQSGAEV) capable of in vitro eliciting anti-lymphoma specific cytotoxic T lymphocytes (CTLs) in lymphocytes from human leukocyte antigen (HLA)-A2.1 donors.  Here we used HLA-A2.1 transgenic mice and SCID (severe combined immunodeficiency) mice to confirm the ability of these specific CTLs against lymphomas. We immunized transgenic mice with nonapeptide conjugated to the adjuvant of PADRE (pan HLA DR-binding epitope). The specificity of the elicited CTLs from the immunized transgenic mice was identified by enzyme-linked immunospot (ELISpot) assay and pentamer staining. The elicited CTLs specifically attacked lymphomas with surface IgHV1 in vitro. Adoptive transfer of the CTLs to SCID mice loaded with QLVQSGAEV(+)/HLA-A2.1(+) lymphoma cells effectively inhibited tumor growth. Our results indicate that the relatively constant epitope in the IgHV FR may be useful for immunotherapy of lymphomas with the same IgHV subfamily.

Genetic variant spectrum in 265 Chinese patients with hemophagocytic lymphohistiocytosis: Molecular analyses of PRF1, UNC13D, STX11, STXBP2, SH2D1A, and XIAP.

Hemophagocytic lymphohistiocytosis (HLH) is a rare life-threatening hyperinflammatory disease. This study aimed to investigate the frequencies and distributions of inherited variants in PRF1, UNC13D, STX11, STXBP2, SH2D1A, and XIAP genes in Chinese patients with HLH. A total of 265 patients diagnosed with HLH from January 2010 to December 2016 were recruited and analyzed for the six genes. Genetic variants were observed in 87 (32.83%) patients. 36 (13.58%) exhibited variants in UNC13D, 18 (6.79%) exhibited PRF1 variants, 10 (3.77%) had variants in XIAP, 9 (3.40%) exhibited variants in STXBP2, 6 (2.26%) carried variants in SH2D1A, 1 (0.38%) had STX11 variant, and 7 (2.64%) exhibited digenic variants. Monoallelic variants were the most common, which accounted for 49.43% of all cases with variants. All variants were confirmed to be germline derived. The present study describes a distinct variant spectrum in Chinese patients with HLH, whereby UNC13D is the most frequently mutated gene with missense variants that are the most common molecular defects. The variant profile of Chinese HLH patients is quite different from that of Western cohorts but similar to that of Korean patients, yet showing its own uniqueness. This racial difference shows the role of genetic background in the occurrence of HLH.

Donor lymphocytes may acquire cytolytic specificity to donor's engrafted hematopoietic cells after a hematopoietic stem cell allograft resulting in marrow failure.

BACKGROUND Secondary rejection sometimes occurs after engraftment of allogeneic hematopoietic stem cell transplantation (allo-HSCT), which results in marrow failure. To clear possible reasons for BM failure, we observed a patient with acute myeloblastic leukemia who died of hematopoietic failure one year after apparently successful allo-HSCT. METHODS The patient was a 44 year old male. Allo-HSCT was successful after 40 days, and 100% of marrow cells were of donors origin. Graft-versus-host disease (GVHD) began at the 60th day with involvement of multiple organs. He died of marrow failure on the 360th day after allo-HSCT. RESULTS We identified the origin of the patients lymphocytes by a donors specific HLA locus, and a dominant T-cell clone by spectratyping of the TCRVB subfamily on T-cells. The patients dominant CD8+ cells were separated by magnetic beads and incubated with donors cells or patients leukemic cells to evaluate their cytolytic specificity. TCRalpha and TCRbeta cDNAs were cloned from the dominant CD8+ T-cells, transfected into Jurkat cells, and characterized the cytolytic specificity of the transfected Jurkat cells. In the period of 60 to 120th day after allo-HSCT, blood CD3+CD8+ cytotoxic T lymphocytes (CTLs) gradually increased and fluctuated in the range of 60 to 90%, CD3+CD4+ cells fluctuated in the range of 5 to 18%, and CD4+CD25+ cells accounted for between 3 to 13%. Spectratyping of the 24 TCRVbeta subfamilies in blood lymphocytes demonstrated a dominant TCRVbeta13.1 clone with HLA-A*0201 of donor origin. The dominant CD8+ cells separated by magnetic beads showed cytolytic specificity to donors blood mononuclear cells (BMCs), but not to patients fibroblasts. Jurkat cells containing the cDNAs of TCRalpha and TCRbeta chains cloned from the dominant CD8+ cell clone had cytolytic activity against donors BMCs, patients leukemic cells, and BMCs from an unrelated subject accounting for 43%, 15%, and 0.42%, respectively. The dominant lymphocyte clone of donors origin with CD3+CD8+ TCRVbeta13.1 markers in the patient may have been determinant in the hematopoiesis failure. CONCLUSIONS Donors lymphocytes changed in the recipient, acquiring the cytolytic specificity to donors hematopoietic cells and presumably leading to marrow failure.