Coblockade of the LFA1:ICAM and CD28/CTLA4:B7 pathways is a highly effective means of preventing acute lethal graft-versus-host disease induced by fully major histocompatibility complex-disparate donor grafts

We have developed an in vitro system in which C57BL/6 donor splenocytes are exposed to B10.BR host alloantigens in the context of deficient CD28:B7 signaling as a means of preventing graft-versus-host disease (GVHD). Although 54% to 82% of MLR alloresponse was inhibited by cytotoxic T-lymphocyte antigen 4 (CTLA4)-Ig treatment of host stimulator cells, treated splenocytes were still capable of causing GVHD when infused in vivo. By adding anti-leukocyte function antigen 1 (anti-LFA1) antibody to hCTLA4-Ig in vitro to coblock the LFA1:intercellular adhesion molecule (ICAM) signaling, splenic alloresponse was inhibited by > or = 89%, yet GVHD induction capabilities were retained. Because antigen-primed cells might be more susceptible to CD28:B7 blockade, we investigated whether hCTLA4-Ig alone, anti-LFA1 antibody alone, or the combination of both added to donor-antihost in vitro primed cells could reduce GVHD. To facilitate hyporesponsiveness induction and to block B7 and ICAM ligands that are upregulated during GVHD, these reagents were also administered to recipients post-BMT. We have shown that hCTLA4-Ig plus anti-LFA1 antibody is highly effective in preventing GVHD-induced lethality (88% to 100% of treated mice surviving versus 0% to 28% of controls surviving). For optimal prevention, both hCTLA4-Ig and anti-LFA1 must be used in vitro in the context of donor-antihost primed splenocytes and continued in vivo. This in vitro-in vivo combined approach was associated with donor engraftment, and recipients were not globally immunosuppressed. We conclude that blocking both the CD28/B7 and the LFA1:ICAM pathways are critical to effective GVHD prevention and may offer advantages to in vitro donor T-cell removal.

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Prevention of Transfusion-Associated Graft-Versus-Host Disease by Photochemical Treatment

Blood ◽

10.1182/blood.v93.9.3140 ◽

1999 ◽

Vol 93 (9) ◽

pp. 3140-3147 ◽

Cited By ~ 100

Author(s):

Joshua A. Grass ◽

Tamim Wafa ◽

Aaron Reames ◽

David Wages ◽

Laurence Corash ◽

...

Keyword(s):

T Cells ◽

Gamma Radiation ◽

Graft Versus Host Disease ◽

Clinical Signs ◽

Peripheral Blood Cell ◽

Control Group ◽

Host Disease ◽

Graft Versus Host

Abstract Photochemical treatment (PCT) with the psoralen S-59 and long wavelength ultraviolet light (UVA) inactivates high titers of contaminating viruses, bacteria, and leukocytes in human platelet concentrates. The present study evaluated the efficacy of PCT to prevent transfusion-associated graft-versus-host disease (TA-GVHD) in vivo using a well-characterized parent to F1 murine transfusion model. Recipient mice in four treatment groups were transfused with 108 splenic leukocytes. (1) Control group mice received syngeneic splenic leukocyte transfusions; (2) GVHD group mice received untreated allogeneic splenic leukocytes; (3) gamma radiation group mice received gamma irradiated (2,500 cGy) allogeneic splenic leukocytes; and (4) PCT group mice received allogeneic splenic leukocytes treated with 150 μmol/L S-59 and 2.1 J/cm2UVA. Multiple biological and clinical parameters were used to monitor the development of TA-GVHD in recipient mice over a 10-week posttransfusion observation period: peripheral blood cell levels, spleen size, engraftment by donor T cells, thymic cellularity, clinical signs of TA-GVHD (weight loss, activity, posture, fur texture, skin integrity), and histologic lesions of liver, spleen, bone marrow, and skin. Mice in the control group remained healthy and free of detectable disease. Mice in the GVHD group developed clinical and histological lesions of TA-GVHD, including pancytopenia, marked splenomegaly, wasting, engraftment with donor derived T cells, and thymic hypoplasia. In contrast, mice transfused with splenic leukocytes treated with (2,500 cGy) gamma radiation or 150 μmol/L S-59 and 2.1 J/cm2 UVA remained healthy and did not develop detectable TA-GVHD. Using an in vitro T-cell proliferation assay, greater than 105.1 murine T cells were inactivated by PCT. Therefore, in addition to inactivating high levels of pathogenic viruses and bacteria in PC, these data indicate that PCT is an effective alternative to gamma irradiation for prevention of TA-GVHD.

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Systematic comparison of hUC-MSCs at various passages reveals the variations of signatures and therapeutic effect on acute graft-versus-host disease

Stem Cell Research & Therapy ◽

10.1186/s13287-019-1478-4 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 10

Author(s):

Qinjun Zhao ◽

Leisheng Zhang ◽

Yimeng Wei ◽

Hao Yu ◽

Linglin Zou ◽

...

Keyword(s):

Cell Proliferation ◽

Therapeutic Effect ◽

Graft Versus Host Disease ◽

Cytokine Secretion ◽

Differentiation Potential ◽

Host Disease ◽

Graft Versus Host ◽

Acute Graft

Abstract Background Mesenchymal stem cells are heterogenous populations with hematopoietic supporting and immunomodulating capacities. Enormous studies have focused on their preclinical or clinical therapeutic effects, yet the systematic study of continuous in vitro passages on signatures and functions of UC-MSCs at both the cellular and molecular levels is still lacking. Methods In this study, to systematically evaluate the biological properties of MSCs at various passages, we analyzed biomarker expression, cell proliferation and apoptosis, chromosome karyotype, and tri-lineage differentiation potential. Subsequently, we took advantage of whole-exome sequencing to compare the somatic hypermutation of hUC-MSCs at P3, P6, and P15 including SNV and INDEL mutations. In addition, to explore the safety of the abovementioned hUC-MSCs, we performed metabolic pathway enrichment analysis and in vivo transplantation analysis. Furthermore, we cocultured the abovementioned hUC-MSCs with UCB-CD34+ HSCs to evaluate their hematopoietic supporting capacity in vitro. Finally, we transplanted the cells into acute graft-versus-host disease (aGVHD) mice to further evaluate their therapeutic effect in vivo. Results The hUC-MSCs at P3, P6, and P15 showed similar morphology, biomarker expression, and cytokine secretion. hUC-MSCs at P15 had advantages on adipogenic differentiation and some cytokine secretion such as IL-6 and VEGF, with disadvantages on cell proliferation, apoptosis, and osteogenic and chondrogenic differentiation potential. Based on the SNP data of 334,378 exons and bioinformatic analyses, we found the somatic point mutations could be divided into 96 subsets and formed 30 kinds of signatures but did not show correlation with risk of tumorigenesis, which was confirmed by the in vivo transplantation experiments. However, hUC-MSCs at P15 showed impaired hematologic supporting effect in vitro and declined therapeutic effect on aGVHD in vivo. Conclusions In this study, we systematically evaluated the biological and genetic properties of hUC-MSCs at various passages. Our findings have provided new references for safety and effectiveness assessments, which will provide overwhelming evidence for the safety of hUC-MSCs after continuous in vitro passages both at the cellular and molecular levels for the first time. Taken together, our studies could help understand the controversial effects of disease treatment and benefit the clinical research of UC-MSCs.

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Pretreatment of murine donor grafts with L-leucyl-L-leucine methyl ester: elimination of graft-versus-host disease without detrimental effects on engraftment

Blood ◽

10.1182/blood.v75.3.798.798 ◽

1990 ◽

Vol 75 (3) ◽

pp. 798-805 ◽

Cited By ~ 14

Author(s):

BR Blazar ◽

DL Thiele ◽

DA Vallera

Keyword(s):

Bone Marrow ◽

T Cell ◽

Methyl Ester ◽

Graft Versus Host Disease ◽

Host Cells ◽

Host Disease ◽

Murine Bone Marrow ◽

Graft Versus Host

Abstract Incubation of murine bone marrow and splenocytes with the dipeptide methyl ester, L-leucyl-L-leucine methyl ester (Leu-Leu-OMe), which results in the selective depletion of cytotoxic T cells and their precursors, natural killer cells, and monocytes, completely protected 30 recipients of fully allogeneic donor grafts from lethal graft-versus- host disease (GVHD). These results were comparable with those obtained in 30 recipients of anti-Thy 1.2 plus complement (C')-treated donor marrow. However, in contrast to antibody- and C'-dependent T-cell depletion, which reduces the level of donor cell engraftment in our model system, we did not observe such effects using Leu-Leu-OMe marrow pretreatment. As compared with the 24 H-2 typed recipients of anti-Thy 1.2 + C'-treated donor grafts, the 29 H-2 typed recipients of Leu-Leu- OMe-treated donor grafts had significantly (P less than .001) higher percentages of donor cells (mean = 93% v 74%) and significantly (P less than .001) lower percentages of host cells (mean = 6% v 15%) posttransplantation. In vitro limiting dilution assay (LDA) was performed to assess the comparative efficacy of cytolytic T-lymphocyte (CTL) precursor depletion by Leu-Leu-OMe or anti-Thy 1.2 + C' pretreatment. We observed greater levels of CTL precursor depletion in Leu-Leu-OMe treated as compared with anti-Thy 1.2 + C'-treated bone marrow plus spleen cells (BMS) obtained from nontransplanted mice. This suggests that the in vivo results cannot simply be attributed to a less efficacious functional inactivation of cytolytic T-cell precursors by Leu-Leu-OMe treatment as compared with anti-Thy 1.2 + C' treatment. Immunoreconstitution was similar in recipients of Leu-Leu-OMe-treated grafts and anti-Thy 1.2 + C'-treated grafts 100 days posttransplant. In our opinion, Leu-Leu-OMe marrow pretreatment deserves further investigation as a methodology to achieve GVHD prevention without significantly reducing the propensity toward host cell repopulation.

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The role of granzyme B in murine models of acute graft-versus-host disease and graft rejection

Blood ◽

10.1182/blood.v87.4.1232.bloodjournal8741232 ◽

1996 ◽

Vol 87 (4) ◽

pp. 1232-1237 ◽

Cited By ~ 95

Author(s):

TA Graubert ◽

JH Russell ◽

TJ Ley

Keyword(s):

Nk Cells ◽

Graft Versus Host Disease ◽

Graft Rejection ◽

Granzyme B ◽

Cytotoxic Lymphocytes ◽

Host Disease ◽

Graft Versus Host ◽

Hybrid Resistance

A complete molecular description of the syndromes of graft-versus-host disease (GVHD) and graft rejection could have a significant impact on clinical bone marrow transplantation. Recent in vitro experiments (Heusel et al, Cell 76:977, 1994 and Shresta et al, Proc Natl Acad Sci USA 92:5679, 1995) have shown that the putative mediators of these two syndromes, cytotoxic lymphocytes (CTL) and natural killer (NK) cells, respectively, initiate a program of cell death (apoptosis) in susceptible target tissues in a manner critically dependent on the serine protease Granzyme B (gzm B). In the present study, we have analyzed the phenotype of gzm B-deficient mice using experimental transplant models designed to isolate their CD8+ CTL, CD4+ CTL, and NK compartments. We found a significant impairment in class I-dependent GVHD mediated by gzm B -/- CD8+ CTL, whereas class II-dependent GVHD was not altered using gzm B -/- CD4+ effectors. In a hybrid resistance model, gzm B -/- hosts rejected haplo-identical marrow grafts as efficiently as did their wild-type littermates. This result is surprising in light of a severe defect in the ability of gzm B -/- NK cells to induce apoptosis in susceptible targets in vitro. These in vivo data define significant role for gzm B in cytotoxicity mediated by CD8+ CTL, but not by CD4+ CTL. Furthermore, these results do not support a model of hybrid resistance in which NK cells play a pivotal role.

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Characterization of in vitro antimurine thymocyte globulin–induced regulatory T cells that inhibit graft-versus-host disease in vivo

Blood ◽

10.1182/blood-2007-08-106526 ◽

2008 ◽

Vol 111 (3) ◽

pp. 1726-1734 ◽

Cited By ~ 36

Author(s):

Melanie C. Ruzek ◽

James S. Waire ◽

Deborah Hopkins ◽

Gina LaCorcia ◽

Jennifer Sullivan ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Graft Versus Host Disease ◽

Antithymocyte Globulin ◽

Mrna Level ◽

Regulatory Cells ◽

Host Disease ◽

Graft Versus Host

Abstract Antithymocyte/antilymphocyte globulins are polyclonal antihuman T-cell antibodies used clinically to treat acute transplant rejection. These reagents deplete T cells, but a rabbit antihuman thymocyte globulin has also been shown to induce regulatory T cells in vitro. To examine whether antithymocyte globulin–induced regulatory cells might be functional in vivo, we generated a corresponding rabbit antimurine thymocyte globulin (mATG) and tested its ability to induce regulatory cells in vitro and whether those cells can inhibit acute graft-versus-host disease (GVHD) in vivo upon adoptive transfer. In vitro, mATG induces a population of CD4+CD25+ T cells that express several cell surface molecules representative of regulatory T cells. These cells do not express Foxp3 at either the protein or mRNA level, but do show suppressive function both in vitro and in vivo when adoptively transferred into a model of GVHD. These results demonstrate that in a murine system, antithymocyte globulin induces cells with suppressive activity that also function in vivo to protect against acute GVHD. Thus, in both murine and human systems, antithymocyte globulins not only deplete T cells, but also appear to generate regulatory cells. The in vitro generation of regulatory cells by anti-thymocyte globulins could provide ad-ditional therapeutic modalities for immune-mediated disease.

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Host γd T cells Exacerbate Experimental Acute Graft-Versus-Host Disease through Activation of Host Antigen Presenting Cells.

Blood ◽

10.1182/blood.v104.11.3045.3045 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3045-3045

Author(s):

Yoshinobu Maeda ◽

Pavan Reddy ◽

Chen Liu ◽

D. Keith Bishop ◽

James L.M. Ferrara

Keyword(s):

T Cells ◽

T Cell ◽

Mhc Class Ii ◽

Graft Versus Host Disease ◽

Serum Levels ◽

Host Disease ◽

Graft Versus Host ◽

Full Intensity

Abstract Large numbers of T cells bearing γd T cell receptors are present in graft-versus-host disease (GVHD) target tissues. We investigated the potential role of host γd T cells during acute GVHD in a well-characterized GVHD model following full intensity conditioning (11 Gy TBI). BM and spleen T cells from BALB/c (H2d) donors were transplanted into wild type (wt) B6, aß T cell deficient B6 (aß −/−) or γd T cell deficient B6 (γd −/−) hosts. γd −/− hosts demonstrated significantly better day 35 survival (85%) than wt (40%) or aß−/− hosts (18%) (P<0.05). Reconstitution of γd −/− B6 hosts with B6 type γd T cells 24 hr prior to BMT restored lethal GVHD (50 % day 35 survival). In vivo, γd −/− B6 hosts demonstrated at least a five fold reduction in donor T cell expansion and cytokine production. In vitro, T cells proliferated less when co-cultured with allogeneic γd −/− dendritic cells (DCs) than with wt DCs (40,127 ± 1634 vs. 72,503 ± 1296, P<0.05). BM-derived DCs cultured with γd T cells caused greater proliferation of allogeneic T cells than DCs cultured with aß T cells (15.1 ± 21 x 104 vs. 5.1 ± 1.2 x 104, P<0.05). We next tested the effect of γd T cells on host DCs in vivo using a model system in which only the DCs injected prior to BMT expressed the alloantigen that stimulated the GVHD reaction. MHC Class II −/− B6 mice that had been depleted of γd T cells were given 11 Gy TBI and injected one day prior to BMT with B6 DCs that had been co-cultured either with γd T cells or with medium. On day 0 both groups of recipient mice were injected with BM plus splenic T cells from allogeneic bm12 donors. On day +5, CD4+ donor T cells expanded four times more in recipients of DCs co-cultured with γd T cells than in recipients of control DCs and serum levels of TNF-a were significantly higher (36.7 + 6.8 vs. 21.3 + 3.7 pg/ml, P<0.05). Together these data demonstrate that γd T cells amplify the stimulatory function of host DCs and increase the severity of GVHD, suggesting that a new therapeutic target for the prevention of the major BMT toxicity.

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Donor Dual TCR T Cells Preferentially Expand and Mediate Pathologic Alloreactivity in Acute Graft Versus Host Disease

Blood ◽

10.1182/blood.v118.21.1972.1972 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1972-1972

Author(s):

Gerald P. Morris ◽

Geoffrey L Uy ◽

David L Donermeyer ◽

Paul M Allen ◽

John F. DiPersio

Keyword(s):

T Cells ◽

T Cell ◽

Graft Versus Host Disease ◽

Peripheral Blood ◽

Thymic Selection ◽

Cell Repertoire ◽

Host Disease ◽

Graft Versus Host

Abstract Abstract 1972 The nature of the T cell repertoire mediating pathologic in vivo alloreactivity is an important question for understanding the development of acute graft-versus-host disease (aGvHD) following clinical allogeneic transplantation. We have previously demonstrated that the small proportion of T cells that naturally express 2 T cell receptors (TCR) as a consequence of incomplete TCRa allelic exclusion during thymic development contribute disproportionately to the alloreactive T cell repertoire, both in vitro and in vivo in a mouse model of graft versus host disease (GvHD) (J. Immunol., 182:6639, 2009). Here, we extend these findings to human biology, examining dual TCR T cells from healthy volunteer donors (n = 12) and patients who have undergone allogeneic hematopoietic stem cell transplantation (HSCT) (n = 19). Peripheral blood was collected at day 30 post-HSCT or at the time of presentation with symptomatic acute GvHD. Dual TCR T cells were measured in peripheral blood by pair-wise staining with 3 commercially-available and 2 novel TCRa mAbs. Dual TCR T cells were consistently and significantly expanded in patients with symptomatic aGvHD, representing 5.3±3.8 % of peripheral T cells, compared to 1.7±0.8 % of T cells in healthy controls (p < 0.005) (Figure 1). There was no correlation between dual TCR T cell frequency and GvHD severity. Furthermore, sequential analysis of peripheral blood in 2 patients demonstrated expansion of dual TCR T cells concurrent with the development of aGvHD (Figure 2). Dual TCR T cells from patients with symptomatic aGvHD demonstrated increased expression of CD69 as compared to T cells expressing a single TCR, indicative of preferential activation of dual TCR T cells during aGvHD. Similarly, dual TCR T cells isolated from patients with symptomatic aGvHD demonstrate increased production of IFN-g ex vivo, indicative of the ability to mediate pathogenic alloreactive responses. Dual TCR T cell clones isolated from healthy donors and patients post-HSCT by single cell FACS sorting demonstrate alloreactive responses against a range of allogeneic cell lines in vitro. We propose that the increased alloreactivity of dual TCR T cells results from the less stringent thymic selection for secondary TCR, and thus provides a link between thymic selection, the TCR repertoire, and alloreactivity. These findings may lead to simple ways of phenotypically identifying specific T cells predisposed to inducing aGvHD for subsequent examination of T cell repertoires and functional studies. Furthermore, these data suggest that dual TCR T cells represent a potential predictive biomarker for aGvHD and a potential target for selective T cell depletion in HSCT. Disclosures: No relevant conflicts of interest to declare.

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Endothelial microparticles carrying hedgehog-interacting protein induce continuous endothelial damage in the pathogenesis of acute graft-versus-host disease

AJP Cell Physiology ◽

10.1152/ajpcell.00372.2015 ◽

2016 ◽

Vol 310 (10) ◽

pp. C821-C835 ◽

Cited By ~ 17

Author(s):

Di-min Nie ◽

Qiu-ling Wu ◽

Peng Zheng ◽

Ping Chen ◽

Ran Zhang ◽

...

Keyword(s):

Graft Versus Host Disease ◽

Endothelial Damage ◽

Endothelial Microparticles ◽

Interacting Protein ◽

Host Disease ◽

Graft Versus Host ◽

Shh Pathway ◽

Acute Graft

Accumulating evidence suggests that endothelial microparticles (EMPs), a marker of endothelial damage, are elevated in acute graft-versus-host disease (aGVHD), and that endothelial damage is implicated in the pathogenesis of aGVHD, but the mechanisms remain elusive. In this study, we detected the plasma EMP levels and endothelial damage in patients and mice with aGVHD in vivo and then examined the effects of EMPs derived from injured endothelial cells (ECs) on endothelial damage and the role of hedgehog-interacting protein (HHIP) carried by EMPs in these effects in vitro. Our results showed that EMPs were persistently increased in the early posttransplantation phase in patients and mice with aGVHD. Meanwhile, endothelial damage was continuous in aGVHD mice, but was temporary in non-aGVHD mice after transplantation. In vitro, EMPs induced endothelial damage, including increased EC apoptosis, enhanced reactive oxygen species, decreased nitric oxide production and impaired angiogenic activity. Enhanced expression of HHIP, an antagonist for the Sonic hedgehog (SHH) signaling pathway, was observed in patients and mice with aGVHD and EMPs from injured ECs. The endothelial damage induced by EMPs was reversed when the HHIP incorporated into EMPs was silenced with an HHIP small interfering RNA or inhibited with the SHH pathway agonist, Smoothened agonist. This work supports a feasible vicious cycle in which EMPs generated during endothelial injury, in turn, aggravate endothelial damage by carrying HHIP into target ECs, contributing to the continuously deteriorating endothelial damage in the development of aGVHD. EMPs harboring HHIP would represent a potential therapeutic target for aGVHD.

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G-CSF as immune regulator in T cells expressing the G-CSF receptor: implications for transplantation and autoimmune diseases

Blood ◽

10.1182/blood-2002-04-1200 ◽

2003 ◽

Vol 102 (2) ◽

pp. 734-739 ◽

Cited By ~ 130

Author(s):

Anke Franzke ◽

Wenji Piao ◽

Jörg Lauber ◽

Patricia Gatzlaff ◽

Christian Könecke ◽

...

Keyword(s):

T Cells ◽

Stem Cell ◽

Autoimmune Diseases ◽

Graft Versus Host Disease ◽

Bone Marrow Failure ◽

Host Disease ◽

Graft Versus Host ◽

Acute Graft

Abstract Results from experimental models, in vitro studies, and clinical data indicate that granulocyte colony-stimulating factor (G-CSF) stimulation alters T-cell function and induces Th2 immune responses. The immune modulatory effect of G-CSF on T cells results in an unexpected low incidence of acute graft-versus-host disease in peripheral stem cell transplantation. However, the underlying mechanism for the reduced reactivity and/or alloreactivity of T cells upon G-CSF treatment is still unknown. In contrast to the general belief that G-CSF acts exclusively on T cells via monocytes and dendritic cells, our results clearly show the expression of the G-CSF receptor in class I– and II– restricted T cells at the single-cell level both in vivo and in vitro. Kinetic studies demonstrate the induction and functional activity of the G-CSF receptor in T cells upon G-CSF exposure. Expression profiling of T cells from G-CSF–treated stem cell donors allowed identification of several immune modulatory genes, which are regulated upon G-CSF administration in vivo (eg, LFA1-α, ISGF3-γ) and that are likely responsible for the reduced reactivity and/or alloreactivity. Most importantly, the induction of GATA-3, the master transcription factor for a Th2 immune response, could be demonstrated in T cells upon G-CSF treatment in vivo accompanied by an increase of spontaneous interleukin-4 secretion. Hence, G-CSF is a strong immune regulator of T cells and a promising therapeutic tool in acute graft-versus-host disease as well as in conditions associated with Th1/Th2 imbalance, such as bone marrow failure syndromes and autoimmune diseases.

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