Graft-versus-host disease (GVHD) is mediated by mature donor T cells contained in the hematopoietic stem cell graft. During the development of GVHD, signaling through a variety of costimulatory receptors plays an important role in allogeneic T cell responses. Even though delivery of costimulatory signals is a prerequisite for full activation of donor T cells in the phase of their interactions with host APCs, their involvement with GVHD might occur over multiple stages. Like many other aspects of GVHD, promise of therapeutic interventions with costimulatory pathways has been gleaned from preclinical models. In this review, I summarize some of the advances in roles of costimulatory molecules in GVHD pathophysiology and discuss preclinical approaches that warrant further exploration in the clinic, focusing on novel strategies to delete pathogenic T cells.
Animals ; Graft vs Host Disease/*immunology/metabolism/*therapy ; Hematopoietic Stem Cell Transplantation ; Humans ; T-Lymphocytes/immunology/metabolism ; Transplantation Immunology/immunology ; Transplantation, Homologous

BACKGROUNDAdoptive transfer of allogeneic tumor-specific T cells often results in severe graft-versus-host disease (GVHD). Here, we sought to maximize graft-versus-tumor and minimize GVHD by using haploidentical T cells in pre-irradiated B16-melanoma bearing mice.

METHODSC57BL/6 mice bearing B16-melanoma tumors were irradiated with 0, 5, or 7 Gy total body irradiation (TBI), or 7 Gy TBI plus bone marrow transplantation. Tumor areas were measured every 3 days to assess the influence of irradiation treatment on tumor regression. B16-melanoma bearing mice were irradiated with 7 Gy TBI; sera and spleens were harvested at days 1, 3, 5, 7, 9, 11, and 13 after irradiation. White blood cell levels were measured and transforming growth factor β1 (TGF-b1) and interleukin 10 (IL-10) levels in serum were detected using enzyme-linked immunosorbent assay (ELISA) kits. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry were performed to test TGF-b1, IL-10 and Foxp3 mRNA levels and the proportion of CD4+CD25+Foxp3+ T-regulatory cells (Tregs) in spleens. B16-melanoma bearing C57BL/6 mice were irradiated with 7 Gy TBI followed by syngeneic (Syn1/Syn2) or haploidentical (Hap1/Hap2), dendritic cell-induced cytotoxic T lymphocytes (DC-CTLs) treatment, tumor areas and system GVHD were observed every 3 days. Mice were killed 21 days after the DC-CTLs adoptive transfer; histologic analyses of eyes, skin, liver, lungs, and intestine were then performed.

RESULTSIrradiation with 7 Gy TBI on the B16-melanoma-bearing mice did not influence tumor regression compared to the control group; however, it down-regulated the proportion of Tregs in spleens and the TGF-b1 and IL-10 levels in sera and spleens, suggesting inhibition of autoimmunity and intervention of tumor microenvironment. Adoptive transfer of haploidentical DC-CTLs significantly inhibited B16-melanoma growth. GVHD assessment and histology analysis showed no significant difference among the groups.

CONCLUSIONAdoptive transfer of haploidentical tumor-specific T cells in irradiation-pretreated B16-melanoma bearing mice preserved antitumor capacity without causing a GVHD response.

Animals ; Enzyme-Linked Immunosorbent Assay ; Female ; Flow Cytometry ; Graft vs Host Disease ; Immunotherapy, Adoptive ; methods ; Male ; Melanoma, Experimental ; metabolism ; therapy ; Mice ; Mice, Inbred C57BL ; Real-Time Polymerase Chain Reaction ; T-Lymphocytes ; immunology ; T-Lymphocytes, Regulatory ; immunology

OBJECTIVETo study the molecular characteristics of CDR3 repertoires of T cell receptor beta chain variable region (TCRBV) of T lymphocytic clones in leukemia recipients after allogeneic hematopoietic stem cell transplantation ( allo-HSCT).

METHODSRT-PCR was used to amplify 24 subfamily genes of TCRBV from peripheral blood (PB) lymphocytes in twenty-four leukemia patients underwent three kinds of allo-HSCT and in five normal donors as control. The PCR products were further analyzed by genescan to evaluate the clonality of BV subfamily and characteristics of CDR3 and calculate usage rate of BV subfamily. The monoclonal bands which associated with GVHD and CMV infection were obtained by denaturation polyacrylamide gel electrophoresis and sequenced. Comparison of the sequences of TCRBV CDR3 with other CDR3 sequences which associated with GVHD or CMV infection was reported.

RESULTS2 approximately 19 months after transplantation, there were 6 approximately 14 BV subfamilies expressed and the polyclonal expression reached 33% in nine patients underwent haploidentical bone marrow transplantation(HI-BMT). In five patients underwent matched unrelated peripheral blood stem cell transplantation ( MU-PBSCT), there were 10 approximately 15 BV subfamilies expressed of which 45% were poly-clones. In 10 patients underwent matched sibling bone marrow transplantation(MS-BMT), 10 approximately 16 BV subfamilies were expressed and more than 48% of them were poly-clones. Monoclones and oligo-clones existed in 24 BV subfamilies but no common one monoclone BV subfamilies was found. Immune reconstitution in patients underwent HI-BMT was later than that in other two groups. In 2 patients TCRBV was detected in 2m and 3m after allo-HSCT and found that there was a tendency of increasing usage of BV subfamilies and increasing expression of CDR3 polymorphism. Twenty three TCRBV CDR3 molecules associated with GVHD and CMV infection were compared each other by bioinformatics and found that different cases of the same BV subfamilies may share similarity in amino acid motif, while in different BV subfamilies none appeared to share the same amino acid motif.

CONCLUSIONIn 1.5 years after allo-HSCT, the usage of TCRBV subfamilies still restricted. Immune reconstitution in patients underwent HI-BMT was later than that in other two groups. TCRBV CDR3 molecules associated with GVHD and CMV infection showed that different cases of the same BV subfamilies may share similarity in amino acid motif, while in different BV subfamilies none of clones appeared to share the same amino acid motif.

Adolescent ; Adult ; Base Sequence ; Child ; Complementarity Determining Regions ; genetics ; metabolism ; Cytomegalovirus Infections ; immunology ; Female ; Graft vs Host Disease ; immunology ; Hematopoietic Stem Cell Transplantation ; Humans ; Leukemia ; immunology ; therapy ; Male ; Middle Aged ; Molecular Sequence Data ; Postoperative Period ; Receptors, Antigen, T-Cell, alpha-beta ; genetics ; metabolism ; T-Lymphocytes ; immunology ; metabolism ; Transplantation, Homologous

Although mesenchymal stem cells (MSCs) are increasingly used to treat graft-versus-host disease (GVHD), their immune regulatory mechanism in the process is elusive. The present study aimed to investigate the curative effect of third-party umbilical cord blood-derived human MSCs (UCB-hMSCs) on GVHD patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and their immune regulatory mechanism. Twenty-four refractory GVHD patients after allo-HSCT were treated with UCB-hMSCs. Immune cells including T lymphocyte subsets, NK cells, Treg cells and dendritic cells (DCs) and cytokines including interleukin-17 (IL-17) and tumor necrosis factor-alpha (TNF-α) were monitored before and after MSCs transfusion. The results showed that the symptoms of GVHD were alleviated significantly without increased relapse of primary disease and transplant-related complications after MSCs transfusion. The number of CD3(+), CD3(+)CD4(+) and CD3(+)CD8(+) cells decreased significantly, and that of NK cells remained unchanged, whereas the number of CD4(+) and CD8(+) Tregs increased and reached a peak at 4 weeks; the number of mature DCs, and the levels of TNF-α and IL-17 decreased and reached a trough at 2 weeks. It was concluded that MSCs ameliorate GVHD and spare GVL effect via immunoregulations.
Adolescent ; Adult ; Cord Blood Stem Cell Transplantation ; methods ; Cytokines ; metabolism ; Dendritic Cells ; metabolism ; Female ; Graft vs Host Disease ; immunology ; therapy ; Hematopoietic Stem Cell Transplantation ; adverse effects ; Humans ; Immunomodulation ; Killer Cells, Natural ; metabolism ; Male ; T-Lymphocyte Subsets ; metabolism ; Transplantation, Homologous ; adverse effects ; Young Adult

The study was aimed to investigate the association of FOXP3 gene expression in donor grafts with acute graft-versus-host disease after HLA-identical sibling allogeneic hematopoietic stem cell transplantation. Twenty-six donor grafts (peripheral blood or bone marrow) and their respective clinical characteristics were evaluated. Flow cytometry analysis was performed to assess the percentage of CD4+CD25+ and CD4+CD25(high) T cells in cord blood, healthy controls' peripheral blood and donor grafts. Relative transcripts of FOXP3 mRNA were determined by real-time quantitative reverse transcription -polymerase chain reaction with beta2-MG as the internal control gene. The specificity of FOXP3 and beta2-MG amplifications was confirmed by analyzing the dissociation curves and electrophoresis of the target amplicon. The results showed that the CD4+CD25+ T cells in peripheral blood, peripheral blood stem cell (PBSC) or BM grafts exhibited a continuous and primarily low expression of CD25 and the frequencies of CD4+CD25+ T and CD4+CD25(high) T in CD4+ T cells were (48.5 +/- 16.3)% and (9.6 +/- 2.5)%, (42.1 +/- 14.7)% and (13.1 +/- 4.2)%, (43.4 +/- 9.6)% and (14.6 +/- 4.5)%, respectively. There was no significant difference in the frequencies and absolute numbers of CD4+CD25(high) T cells between patients with aGVHD and patients without aGVHD (P > 0.05). The plot of log transfused cDNA amount versus DeltaCt had a slope of 0.0826 which indicated approximately equal efficiency of FOXP3 and beta2-MG amplifications in real-time PCR. The specificities of amplification were confirmed by analyzing the dissociation curves and electrophoresis of PCR products with the values of Tm 86.5 degrees C and 82.3 degrees C, respectively. The relative transcripts of FOXP3 in PBSC grafts of recipients without aGVHD were 318%high as those with aGVHD (median of 41.0 x 10(-5) and 12.9 x 10(-5), respectively) (P = 0.03). No significant difference was found in other related variables for GVHD. It is concluded that coexpression of CD4 and CD25 may be insufficient to identify regulatory T cells; FOXP3 mRNA expression may be specifically quantified with real-time quantitative RT-PCR using SYBR Green I chemistry. FOXP3 mRNA expression in donor grafts is significantly low in patients with aGVHD compared with patients without aGVHD. It indicated that the expression level of FOXP3 mRNA may be one of the useful indicators for in predicting aGVHD.
Adolescent ; Adult ; Female ; Forkhead Transcription Factors ; biosynthesis ; genetics ; Gene Expression Regulation, Neoplastic ; genetics ; Graft vs Host Disease ; genetics ; metabolism ; Hematologic Neoplasms ; therapy ; Hematopoietic Stem Cell Transplantation ; adverse effects ; Hematopoietic Stem Cells ; immunology ; Humans ; Male ; Middle Aged ; RNA, Messenger ; biosynthesis ; genetics ; T-Lymphocytes, Regulatory ; immunology

Acute graft-versus-host disease (aGVHD) is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the mechanisms of aGVHD are not well understood. We aim to investigate the roles of the three angiogenic factors: angiopoietin-1 (Ang-1), Ang-2 and vascular endothelial growth factor (VEGF) in the development of aGVHD. Twenty-one patients who underwent allo-HSCT were included in our study. The dynamic changes of Ang-1, Ang-2 and VEGF were monitored in patients before and after allo-HSCT. In vitro, endothelial cells (ECs) were treated with TNF-β in the presence or absence of Ang-1, and then the Ang-2 level in the cell culture medium and the tubule formation by ECs were evaluated. After allo-HSCT, Ang-1, Ang-2 and VEGF all exhibited significant variation, suggesting these factors might be involved in the endothelial damage in transplantation. Patients with aGVHD had lower Ang-1 level at day 7 but higher Ang-2 level at day 21 than those without aGVHD, implying that Ang-1 may play a protective role in early phase yet Ang-2 is a promotion factor to aGVHD. In vitro, TNF-β promoted the release of Ang-2 by ECs and impaired tubule formation of ECs, which were both weakened by Ang-1, suggesting that Ang-1 may play a protective role in aGVHD by influencing the secretion of Ang-2, consistent with our in vivo tests. It is concluded that monitoring changes of these factors following allo-HSCT might help to identify patients at a high risk for aGVHD.
Acute Disease ; Adolescent ; Adult ; Angiogenesis Inducing Agents ; immunology ; metabolism ; pharmacology ; Angiopoietin-1 ; genetics ; immunology ; pharmacology ; Angiopoietin-2 ; genetics ; immunology ; pharmacology ; Antineoplastic Agents ; therapeutic use ; Female ; Gene Expression Regulation, Neoplastic ; Graft vs Host Disease ; genetics ; immunology ; pathology ; Hematopoietic Stem Cell Transplantation ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; immunology ; Humans ; Leukemia, Myeloid ; genetics ; immunology ; pathology ; therapy ; Lymphoma, Non-Hodgkin ; genetics ; immunology ; pathology ; therapy ; Male ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; immunology ; pathology ; therapy ; Retrospective Studies ; Signal Transduction ; Transplantation, Homologous ; Tumor Necrosis Factor-alpha ; pharmacology ; Vascular Endothelial Growth Factor A ; genetics ; immunology