CD11b− Donor Dendritic Cells Enhance Donor T-Cells’ Th1 Polarization and Graft Versus Leukemia Activity in Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1252-1252 ◽  
Author(s):  
Jian-Ming Li ◽  
Kataryna A. Darlak ◽  
Ying Lu ◽  
Cynthia Giver ◽  
Wayne Harris ◽  
...  
Keyword(s):  
T Cells ◽  
Serum Levels ◽  
Hematopoietic Stem ◽  
Treatment Groups ◽  
Post Transplant ◽  
Tumor Bearing ◽  
Donor T Cells ◽  
Ifn Γ ◽  
Th1 Cytokines

Abstract Background: Based on a clinical association of donor plasmacytoid dendritic cell (DC) content with leukemia relapses after allogeneic BMT (Waller, Blood 2001), we have previously reported that CD11b− donor DC added to a graft containing FACS-purified hematopoietic stem cells (HSC) and T-cells enhanced interferon-γ (IFN-γ) production and GvL activity in MHC-mismatched allogeneic transplant mouse models (Li, Blood 2007). Objective: In this study, we studied the mechanisms whereby donor DC in the graft modulate donor T-cell activity and the graft-versus-leukemia (GvL) effect in MiHA (C3H.SW → C57BL/6J)- and MHC (C57BL/6J → B10.BR)- mismatched models of allogeneic hematopoietic stem cell transplantation (HSCT). Methods: Mice irradiated to 11 Gy received 5 × 104 log-phase viable MMB3.19 myeloid lymphoma cells via intraperitoneal injection or intravenous injection of 1 x 105 LBRM T-cell lymphoma cells one day before transplant. Allografts consisted of 5 × 104 FACS-purified donor BM CD11b− DC or CD11b+ DC plus 3 × 103 FACS-purified c-kit+ sca-1+ lineage− hematopoietic stem cells (HSC) in combination with either 3 × 105 T-cells, 3 × 105 CD8+ T-cells or no additional T-cells transplanted via tail vein. Graft-versus-host disease (GvHD) clinical scores (based on body weight loss, posture, skin, fur texture, activity) were recorded twice weekly in non-tumor bearing recipients. In vitro proliferation and cytotoxic activity of donor-derived T-cells against tumor targets was assessed by CFSE staining and a caspase flow cytometry assay (CyToxiLux PLUS) using donor T-cells harvested from recipients on day 34 and day 82 post transplant. Serum and intracellular Th1 cytokines (IFN-γ, IL-2, and TNF-α) and Th2 cytokines (IL-4, IL-5, and IL-10) from recipients’ peripheral blood and spleens day 3 and day 10 post-transplant was measured by ELISA and flow cytometry. IFN-γ direct killing of leukemia cells was tested by in vitro IFN-γ exposure. Results: In non-tumor bearing mice, recipients of all combinations of donor DC subsets, with and without donor T-cells had equivalent survival (75% – 85%) at 3 months post transplant without significant clinical signs of GvHD. Transplantation of tumor cells to recipients of HSC alone, HSC plus donor T-cells, or HSC plus T-cells and CD11b+ DC in the MiHA- and the MHC-mismatched transplant models led to 0% or 5% 3 month survival, respectively. Strikingly, tumor-bearing mice transplanted with CD11b− DC had significantly enhanced 3 month survival (35% in the MiHA-mismatched model and 45% in the MHCmismatched model) without increased GvHD (p<0.001). There was no significant difference in survival between mice that received HSC plus CD11b− DC and a mixture of CD4+ and CD8+ donor T-cells versus mice that received HSC plus CD11b− DC and only CD8+ donor T-cells. Donor T-cells harvested from recipients of CD11b− DC 34 days after transplant in the MiHA-mismatched model as well as 82 days after transplant in the MHC-mismatched model displayed increased cell proliferation following co-culture with irradiated hosttype splenocytes as a source of alloantigen compared with donor T-cells harvested from recipients of CD11b+ DC or recipients of HSC plus T-cells without donor DC. Leukemia cell killing was greater following incubation of purified donor T-cells recovered from recipients of CD11b− DC with tumor targets compared to T-cells recovered from other treatment groups. Recipients of CD11b− DC had higher serum levels of Th1 cytokines IFN-γ and IL-2 and higher number of Th1 positive donor T-cells compared with recipients of other treatment groups. In contrast, recipients of CD11b+ DC had higher serum levels of Th2 cytokines IL-4, IL-5, and IL-10 and higher number of Th2 positive donor T-cells. IFN-γ added to in vitro cultures with MMB3.19, and LBRM, had no direct cell killing effect. Conclusion: CD11b− donor DC enhanced Th1 polarization of donor T-cells and GvL without increasing GvHD. Donor CD8+ T-cells mediated tumor killing effect. CD11b+ donor DC enhanced Th2 polarization of donor CD4+ T-cells and led to limited GvHD and GvL.

Paradoxical Immunological Activity of Flagellin: A Novel Method to Reduce GvHD In Allogeneic HSCT Recipients.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1465-1465
Author(s):  
Mohammad S Hossain ◽  
Andrew T Gewirtz ◽  
John Roback ◽  
Edmund Waller
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Cell Transplant ◽  
Term Survival ◽  
Hematopoietic Stem ◽  
Allogeneic Hsct ◽  
Post Transplant ◽  
Donor T Cells ◽  
Ifn Γ

Abstract Abstract 1465 Background: Graft-vs-host disease (GvHD) is a major complication in allogeneic Hematopoietic Stem Cell Transplant (HSCT) recipients. Using flagellin, a bacterial protein that agonistically binds with TLR5, we previously reported that two doses of flagellin (before irradiation and after allogeneic HSCT with 5×106 splenocytes) in H-2K à H-2b model significantly reduced GvHD and had 67% long-term survival by 132 days post-transplant whereas control recipients had severe acute GvHD and 100% early mortality within 15 days post transplant. Here, we report the mechanism by which flagellin reduces GvHD using the same H-2K à H-2b HSCT murine model. Methods: 3×106 splenocytes and 5 ×106 T cell depleted bone marrow (BM) cells harvested from the congeneic H-2K donors were transplanted into lethally irradiated (11Gy) C57BL/6 (H-2b) recipients. 50-μg HPLC purified LPS-free flagellin diluted in ice-cold PBS were administered i.p 3 hours before irradiation and 24 hours after transplant. Control recipients were treated with PBS. Recipients (6/group) were sacrificed on day 4 and 10 after post transplant. Serum was collected to determine cytokines by ELISA and splenocytes were analyzed by FACS to determine immune cells phenotypes. Results: Although both Fla and PBS-treated recipients showed identical weight-loss within day 10 post transplant, surprisingly significantly lower numbers of cells/spleen were determined in the spleens of Fla recipients compared to control recipients [Fla 0.9 ± 0.1 (x106) vs PBS 1.7 ± 0.4(x106), p=0.002] on day 4 post transplant but not on day 10 [Fla 186.1 ± 35.1 (x106) vs PBS 151.2 ± 40.5(x106), p=0.43] post-transplant. We investigated the paradoxical immune response of flagellin on donor T cells on day 4-post transplant. First, we determined the numbers of donor spleen-derived Thy1.2+ T cells per spleen. The numbers of donor spleen-derived T cells per spleen were significantly lower in Fla recipients compared to PBS-treated recipients [Fla 0.005 ± 0.002 (x103) vs PBS 0.04 ± 0.03(x103), p=0.02]. Accordingly, donor spleen-derived both CD4 and CD8 T cells per spleen of Fla recipients were also found significantly lower compared to PBS-treated recipients (CD4, p=0.04; CD8, p= 0.003). The CD62L, a naïve and also markers for allo-reactive T cells that cause GvHD were found significantly lower in both CD4 and CD8 T cells (CD4, p= 0.03; CD8, p=0.003) and the inducible co-stimulatory molecule 1 (ICOS-1), another prominent T cells activation marker were also found significantly lower (CD4, p= 0.04; CD8, p=0.007) in Fla recipients compared to PBS-treated recipients. These lower immune phenotypes of donor T cells in Fla recipients may reduce the initiation of GvHD at the early time points of transplant. However, flagellin-induced reduction of donor T cells activity were not suppressed considerably as the numbers of donor spleen-derived CD4 T cells expressing activation markers such as CD25 {Fla 0.5 ± 0.2 (x103) vs PBS 4.5 ± 0.5 (x103), p=0.07} and CD69 {Fla 0.5 ± 0.2 (x103) vs PBS 6.5 ± 5.0 (x103), p=0.08} per spleen were not found significantly different. On the other hand, the numbers of CD8 T cells those expressed CD25 {Fla 0.2 ± 0.04 (x103) vs PBS 1.3 ± 0.7 (x103), p=0.01} and CD69 {Fla 0.4 ± 0.1 (x103) vs PBS 2.4 ± 1.7 (x103), p=0.03} per spleen were found significantly lower in Fla recipients compared to PBS-treated recipients. Although over 90% of donor spleen CD4 T cells of both Fla and PBS-treated recipients expressed PD-1, 24% and 32% respectively, expressed IFN-γ after vitro PMA stimulation. Similar results were found in case of CD8 T cells. The over expression of PD-1 in HSCT recipients, thus did not make the donor T cells exhausted as they expanded over 100 times within day 10 post transplant with the expression of PD-1. Although similar level of serum IFN-γ was determined between the Fla and PBS-treated recipients on day 10 post-transplant, IFN-γ level was found below detection limit on day 4 post-transplant. The serum levels of IL-1β and IL-10 were undetectable on both days 4 and 10 post-transplant. Serum LPS were found identical in both Fla and PBS-treated groups determined on day 4 and 10 days post transplant. Conclusion: Flagellin protected allogeneic HSCT recipients from lethal GvHD by reducing donor CD62L+ and ICOS-1+ T cells expansion and activation within 4 days post-transplant without compromising their normal immune responses. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Modifying a Xenogeneic Gvhd Model to Allow for Use of CRISPR-Treated Primary Human T Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4788-4788
Author(s):  
Archana Ramgopal ◽  
Darlene A. Monlish ◽  
Manda Ramsey ◽  
Craig Byersdorfer
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Biology ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Human T Cells ◽  
Donor T Cells ◽  
Ifn Γ

Abstract Background: Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a curative treatment for high-risk leukemia, immunodeficiencies, and bone marrow failure syndromes. Therapeutic use of alloHSCT remains limited by acute graft-versus-host disease (GVHD), where activated donor T cells attack and destroy host tissues. We have previously shown that GVHD-causing T cells increase activation of AMP-activated protein kinase (AMPK), a cellular energy sensor, and that T cell-specific ablation of AMPK in murine models decreases GVHD severity. To study human T cell biology, we modified a previous xenogeneic model. Current models transplant whole peripheral blood mononuclear cells (PBMCs) from healthy human donors into lightly irradiated immunodeficient NOD-scid IL2Rgamma null (NSG) mice. However, in our hands, CRISPR-treatment of primary human T cells requires up to 10 days of culturing to obtain sufficient cells for transplantation. Therefore, prior to assessing GVHD severity using AMPK-deficient human T cells, we optimized the xenogeneic GVHD model to allow time for the manipulation and subsequent injection of CRISPR-engineered cells. Aim: To develop a xenogeneic model compatible with CRISPR/Cas9 manipulated T cells to determine whether changes in human T cells decrease GVHD severity similar to what is seen using murine T cells. Results: We first demonstrated that expanded T cells alone cause minimal xenogeneic GVHD, but that disease could be significantly facilitated with addition of non-T cell antigen presenting cells (APCs). Transplanting T cells plus non-T cell APCs increased numbers of human CD45+CD3+ T cells recovered on day 25 post-transplant (Figure 1A-B) and elevated levels of human interferon (IFN)-γ (Figure 1C). Liver sections from recipients of T cells + APCs subjectively had more perivascular infiltrates than mice receiving T cells alone (data not shown). Additionally, as seen in murine T cells, xenogeneic human T cells increased fatty acid oxidation, additional evidence that our model recapitulates the murine findings (Figure 1D). We next wished to fix the number of co-administered APCs and optimize the number of activated T cells to reliably reproduce xenogeneic GVHD without inducing overt toxicity. To accomplish this goal, we performed xenogeneic transplants with serially decreasing numbers of expanded human T cells (starting with 6×10 6/recipient) and administered 1×10 6 APCs in all cohorts. We also trialed inclusion of recently thawed non-T cell APCs in place of freshly derived cells. Reassuringly, the number of human CD45+CD3+ cells recovered on day 25 post-transplant remained proportional to the number of cells injected (Figure 2A-B), as did levels of human IFN-γ (detected by serum ELISA (Figure 2C). These data indicate that robust xenogeneic GVHD can be induced with as few as 2×10 6 expanded T cells and 1×10 6 autologous APCs, with a concomitant increase in GVHD-associated proinflammatory cytokines. Importantly, these data also demonstrate that 1×10 6 APCs are sufficient to cause reproducibly severe disease and may be recovered from a cryopreserved source. Finally, we wished to extend the assessment of GVHD severity following administration of varying doses of donor T cells. Serum from recipient mice on day 25 post-transplant was analyzed for the production of murine-derived cytokines via a LEGENDplex assay. Of 13 cytokines tested, both murine MCP-1 and TNF-alpha were proportional to the number of T cells injected (Figure 3A-B), with the MCP-1 result confirmed by ELISA (data not shown). Thus, both MCP-1 and TNF-α, cytokines commonly implicated in acute GVHD pathogenesis, provide additional host-derived soluble factors that can be utilized to quantitate the severity of GVHD in our modified xenogeneic model. Expression of these proteins will serve as valuable biomarkers in the assessment of xenogeneic GVHD using CRISPR-treated cells. Conclusions: We have successfully adapted a xenogeneic model of GVHD using in vitro expanded T cells and cryopreserved APCs, thereby allowing for expanded testing of genetically manipulated human T cells in an in vivo model. Future studies will compare the necessity of genes in human donor T cells using CRISPR-mediated gene editing and compare CRISPR techniques with novel pharmacological inhibition using this modified xenogeneic approach. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals LYG1 Deficiency Attenuates the Severity of Acute Graft-Versus-Host Disease via Skewing Allogeneic T Cells Polarization Towards Treg Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Huihui Liu ◽  
Zhengyu Yu ◽  
Bo Tang ◽  
Shengchao Miao ◽  
Chenchen Qin ◽  
...  
Keyword(s):  
T Cells ◽  
Graft Versus Host Disease ◽  
Foxp3 Expression ◽  
Hematopoietic Stem ◽  
Host Disease ◽  
Graft Versus Host ◽  
Donor T Cells ◽  
Ifn Γ ◽  
Acute Graft

Acute graft-versus-host disease (aGVHD) is a lethal complication after allogeneic hematopoietic stem cell transplantation. The mechanism involves the recognition of host antigens by donor-derived T cells which induces augmented response of alloreactive T cells. In this study, we characterized the role of a previously identified novel classical secretory protein with antitumor function-LYG1 (Lysozyme G-like 1), in aGVHD. LYG1 deficiency reduced the activation of CD4+ T cells and Th1 ratio, but increased Treg ratio in vitro by MLR assay. By using major MHC mismatched aGVHD model, LYG1 deficiency in donor T cells or CD4+ T cells attenuated aGVHD severity, inhibited CD4+ T cells activation and IFN-γ expression, promoted FoxP3 expression, suppressed CXCL9 and CXCL10 expression, restrained allogeneic CD4+ T cells infiltrating in target organs. The function of LYG1 in aGVHD was also confirmed using haploidentical transplant model. Furthermore, administration of recombinant human LYG1 protein intraperitoneally aggravated aGVHD by promoting IFN-γ production and inhibiting FoxP3 expression. The effect of rhLYG1 could partially be abrogated with the absence of IFN-γ. Furthermore, LYG1 deficiency in donor T cells preserved graft-versus-tumor response. In summary, our results indicate LYG1 regulates aGVHD by the alloreactivity of CD4+ T cells and the balance of Th1 and Treg differentiation of allogeneic CD4+ T cells, targeting LYG1 maybe a novel therapeutic strategy for preventing aGVHD.

scholarly journals Impact of Different T Cell Depletion Protocols on Immune Reconstitution Following Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 43-44
Author(s):  
Amandine Pradier ◽  
Adrien Petitpas ◽  
Anne-Claire Mamez ◽  
Federica Giannotti ◽  
Sarah Morin ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Reconstitution ◽  
Cell Depletion ◽  
Unrelated Donor ◽  
T Cell Depletion ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
The Impact

Introduction Allogeneic hematopoietic stem cell transplantation (HSCT) is a well-established therapeutic modality for a variety of hematological malignancies and congenital disorders. One of the major complications of the procedure is graft-versus-host-disease (GVHD) initiated by T cells co-administered with the graft. Removal of donor T cells from the graft is a widely employed and effective strategy to prevent GVHD, although its impact on post-transplant immune reconstitution might significantly affect anti-tumor and anti-infectious responses. Several approaches of T cell depletion (TCD) exist, including in vivo depletion using anti-thymocyte globulin (ATG) and/or post-transplant cyclophosphamide (PTCy) as well as in vitro manipulation of the graft. In this work, we analyzed the impact of different T cell depletion strategies on immune reconstitution after allogeneic HSCT. Methods We retrospectively analysed data from 168 patients transplanted between 2015 and 2019 at Geneva University Hospitals. In our center, several methods for TCD are being used, alone or in combination: 1) In vivo T cell depletion using ATG (ATG-Thymoglobulin 7.5 mg/kg or ATG-Fresenius 25 mg/kg); 2) in vitro partial T cell depletion (pTCD) of the graft obtained through in vitro incubation with alemtuzumab (Campath [Genzyme Corporation, Cambridge, MA]), washed before infusion and administered at day 0, followed on day +1 by an add-back of unmanipulated grafts containing about 100 × 106/kg donor T cells. The procedure is followed by donor lymphocyte infusions at incremental doses starting with 1 × 106 CD3/kg at 3 months to all patients who had received pTCD grafts with RIC in the absence of GVHD; 3) post-transplant cyclophosphamide (PTCy; 50 mg/kg) on days 3 and 4 post-HSCT. Absolute counts of CD3, CD4, CD8, CD19 and NK cells measured by flow cytometry during the first year after allogeneic HSCT were analyzed. Measures obtained from patients with mixed donor chimerism or after therapeutic DLI were excluded from the analysis. Cell numbers during time were compared using mixed-effects linear models depending on the TCD. Multivariable analysis was performed taking into account the impact of clinical factors differing between patients groups (patient's age, donor type and conditioning). Results ATG was administered to 77 (46%) patients, 15 (9%) patients received a pTCD graft and 26 (15%) patients received a combination of both ATG and pTCD graft. 24 (14%) patients were treated with PTCy and 26 (15%) patients received a T replete graft. 60% of patients had a reduced intensity conditioning (RIC). 48 (29%) patients received grafts from a sibling identical donor, 94 (56%) from a matched unrelated donor, 13 (8%) from mismatched unrelated donor and 13 (8%) received haploidentical grafts. TCD protocols had no significant impact on CD3 or CD8 T cell reconstitution during the first year post-HSCT (Figure 1). Conversely, CD4 T cells recovery was affected by the ATG/pTCD combination (coefficient ± SE: -67±28, p=0.019) when compared to the T cell replete group (Figure 1). Analysis of data censored for acute or chronic GVHD requiring treatment or relapse revealed a delay of CD4 T cell reconstitution in the ATG and/or pTCD treated groups on (ATG:-79±27, p=0.004; pTCD:-100±43, p=0.022; ATG/pTCD:-110±33, p<0.001). Interestingly, pTCD alone or in combination with ATG resulted in a better reconstitution of NK cells compared to T replete group (pTCD: 152±45, p<0.001; ATG/pTCD: 94±36, p=0.009; Figure 1). A similar effect of pTCD was also observed for B cells (pTCD: 170±48, p<.001; ATG/pTCD: 127±38, p<.001). The effect of pTCD on NK was confirmed when data were censored for GVHD and relapse (pTCD: 132±60, p=0.028; ATG/pTCD: 106±47, p=0.023) while only ATG/pTCD retained a significant impact on B cells (102±49, p=0.037). The use of PTCy did not affect T, NK or B cell reconstitution when compared to the T cell replete group. Conclusion Our results indicate that all TCD protocols with the only exception of PTCy are associated with a delayed recovery of CD4 T cells whereas pTCD of the graft, alone or in combination with ATG, significantly improves NK and B cell reconstitution. Figure 1 Disclosures No relevant conflicts of interest to declare.

Donor DC Subsets Polarize Donor T-Cell Immune Responses in Allogeneic BMT.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 573-573
Author(s):  
Jian-Ming Li ◽  
Cynthia Giver ◽  
Doug McMillan ◽  
Wayne Harris ◽  
David L. Jaye ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Peripheral Blood ◽  
Immune Reconstitution ◽  
Hematopoietic Cell ◽  
Post Transplant ◽  
Donor T Cells ◽  
Ifn Γ

Abstract Introduction: Impaired or inappropriate immune reconstitution after allogeneic bone marrow transplantation (BMT) can lead to infection, graft-versus-host disease (GvHD) and leukemia relapse. We have previously reported that BM contains two populations of dendritic cell (DC) subsets, CD11b+ DC and CD11b− DC, and that CD11b depleted donor BM promoted increased donor T-cell chimerism and increased graft-versus-leukemia (GvL) activity in C57BL/6 → B10BR transplants [BBMT, 2004, 10: 540]. To explore the mechanism by which CD11b-depletion improved allo-reactivity, we performed allogeneic hematopoietic cell transplants using defined populations of donor stem cells, DCs, and T-cells in a MHC mis-matched BMT model. Methods: We transplanted FACS purified populations of 50,000 GFP+ CD11b- DC or CD11b+ DC in combination with 5,000 FACS purified Lin- Sca-1+ c-kit+ hematopoietic stem cells (HSC) and 300,000 or 1,000,000 congenic spleen T-cells from C57BL/6 donors into C57BL/6[H-2Kb], B10BR[H-2Kk] and PL/J[H-2Ku] recipients. Proliferation of CFSE stained donor T-cells was measured at 72 hours post-transplant. FACS cytometric bead array and intracellular cytokine staining measured serum and intracellular cytokines in donor T-cells. Results: The initial proliferation and Ki-67 expression of CFSE labeled donor T-cells in allogeneic recipients were much higher than in syngeneic recipients (homeostatic proliferation). Confocal microscopy showed co-localization of donor DC subsets with donor T-cells in the recipient spleens at 3 and 10 days post-transplant. In the allogeneic transplant settings, donor T-cells co-transplanted with CD11b- DC showed increased IFN-γ synthesis at 3 and 10 days post-transplant compared to donor T-cells co-transplanted with HSC plus CD11b+ DC or HSC alone. Increased proliferation of donor T-cells led to increased donor T-cell chimerism at day 10, 30, 60, and day105 post-transplant among recipients of CD11b- DC compared to recipients of HSC alone or HSC plus CD11b+ DC (Figure 1). Transplantation of spleen T-cells and CD11b- DC did not increase GvHD, but was associated with full donor chimerism. In contrast, transplantation of allogeneic CD11b+ DC led to persistence and expansion of residual host T-cells (Figure 2), increased numbers of donor CD4+CD25++Foxp3+ T-cells, and higher serum level of IL-10 supporting early post-transplant expansion of donor T regulatory cells (Treg). Conclusions: Donor CD11b- DC promoted immune reconstitution by polarizing donor T-cells to Th1 immune responses associated with increased IFN-γ synthesis and donor T-cell proliferation, while donor CD11b+ DC suppressed immune reconstitution by inhibiting donor T-cell allogeneic immune responses. These data support a novel paradigm for the regulation of post-transplant immunity and suggest clinical methods to test the hypothesis that manipulation of the DC content of a hematopoietic cell allograft regulates post transplant immunity in the clinical setting. Figure 1. Donor Spleen Derived T-cells in Peripheral Blood [* p<0.05, v.s. recipients of HSC plus CD11b(+)DC and spleen T-cells] Figure 1. Donor Spleen Derived T-cells in Peripheral Blood [* p<0.05, v.s. recipients of HSC plus CD11b(+)DC and spleen T-cells] Figure 2. Host Derived T-cells in Peripheral Blood [* p<0.05, v.s. recipients of HSC plus CD11b(-)DC and spleen T-cells] Figure 2. Host Derived T-cells in Peripheral Blood [* p<0.05, v.s. recipients of HSC plus CD11b(-)DC and spleen T-cells]

OP9-DL1 Cell Line Supports the Development of Phenotypically and Functionally Mature Tcrαβ And Tcrγδ T Cells, through Both Conventional and Aberrant Developmental Pathways

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2902-2902
Author(s):  
Tessa C. C. Kerre ◽  
Greet Verstichel ◽  
Stefanie Van Coppernolle ◽  
Imke Velghe ◽  
Frank Timmermans ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Selection Process ◽  
Receptor Activation ◽  
Notch Receptor ◽  
Activated T Cells ◽  
Hematopoietic Stem ◽  
Ifn Γ

Abstract In vitro generation of mature T cells from human hematopoietic stem and progenitor cells (HSPC) could fulfill two existing needs. First, it could enhance and quicken T cell immune reconstitution after stem cell transplantation, which is very slow and generates a skewed TCR repertoire. Second, by generation of tumour antigen specific T cells it could provide an efficient therapy for numerous malignancies and could enhance GVT effect in the context of allogeneic SCT, without aggravating GVHD. T cells can be generated from human HSPC by culturing them on the murine stromal cell line OP9-transduced with the Notch ligand Delta-like-1 (OP9-DL1). Notch receptor activation is essential for T cell development. However, it is unclear whether Notch activation is sufficient for end maturation into functionally and phenotypically mature TCR positive cells. It was shown that human CD34+ cells cultured on OP9-DL1 differentiate to T cells which can proliferate and produce interferon-g upon polyclonal stimulation. The nature of the mature cells generated in these cultures, however, has not been well studied. CD34+ HSPC from postnatal thymus (PNT) or cord blood were cocultured with OP9-DL1, in the presence of the cytokines Flt-3L (5 ng/ml), SCF (2.5 ng/ml) and IL-7 (5 ng/ml). Every 3–5 days cells were harvested and transferred to fresh OP9-DL1 cells. At repetitive timepoints, an aliquot of the cells was analysed phenotypically. In some experiments, IL-15 was added to the culture. For some experiments, cells harvested from OP9-DL1 at the timepoint mature T cells were observed (usually about d 40 of culture), were transferred to feeder cells, consisting of JY cell line (5.104 cells/ml irradiated with 50 Gy and PBMC (5.105/ml irradiated with 40 Gy), in the presence of PHA (1 mg/ml). After 7 days, IL-2 (50 IU/ml) was added to the culture. Every 14 days, cells were restimulated with new feeders (irradiated JY and PBMC) and new addition of PHA. After 3 weeks of stimulation cells were stimulated overnight with 15 ng/ml PMA and 1500 ng/ml ionomycin, and 18 hours later cells were checked for intracellular presence of cytokines. We investigated whether the T cell population generated in these cultures contains mature cells with the characteristics of TCRγδ cells and of positively selected CD8 or CD4 single positive (SP) TCRαβ cells as observed in the human thymus. We found that under the described conditions, HSPC mature into CD1-CD27+ phenotypically mature T cells, with the TCRγδ fraction maturing faster and more efficiently compared to the TCRαβ fraction. Consistent with a mature phenotype, TCRγδ cells were mostly CD8αα or double negative (DN). No mature CD4 SP TCRαβ cells were observed and the mature CD8 SP cells co-expressed variable ratios of CD8αβ and CD8αα dimers, suggesting that these cells are not conventional positively selected TCRαβ cells. In support of this hypothesis, both mature CD1- TCRαβ and TCRγδ cells expressed the IL2Rβ receptor consitutively and both populations proliferated on IL-15 without prior antigen stimulation, CD8αα (TCRαβ and TCRγδ) cells being the most IL-15 responsive. Mature activated T cells secreted IFN-γ and TNFα, little or no IL-2 and IL-4, with no difference observed between TCRαβ and TCRγδ cells. These data suggest that CD8 TCRαβ cells generated in these cultures are unconventional CD8 cells possibly maturated through agonist selection. However, when cells harvested after 40 days of culture on OP9-DL1 were stimulated with PHA and IL-2 for 3 weeks, conventional appearing CD8αβ cells emerged, with a cytokine production profile similar to that of thymic CD8αβ TCRαβ T cells, with the majority of cells secreting IFN-γ and IL-2. We can conclude from these data that OP9-DL1 supports the development of both unconventional and conventional CD8+ TCRαβ cells, of which the generation and selection process are currently being investigated. Also the in vitro anti-tumor capacities of both populations need to be addressed.

Donor BM Dendritic Cells Expressing IDO Limit Graft-Versus-Host Disease After Allogeneic Bone Marrow Transplant.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1331-1331
Author(s):  
Ying Lu ◽  
Wayne Harris ◽  
Jian-Ming Li ◽  
Edmund K. Waller
Keyword(s):  
T Cells ◽  
T Cell ◽  
Marrow Transplant ◽  
Allogeneic Bone ◽  
Transplant Recipients ◽  
Hematopoietic Stem ◽  
Graft Versus Host ◽  
Donor T Cells ◽  
Ifn Γ ◽  
Th1 Polarization

Abstract Abstract 1331 Poster Board I-353 Background In contrast to the essential role of host dendritic cells (DC) in the initiation of graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) reactions, less is known about the effects of donor DC on T cells in these processes. We have previously reported that adding donor BM plasmacytoid DC (pDC) progenitors to a murine graft composed of purified hematopoietic stem cells (HSC) and T-cells increased donor activation and Th1 polarization leading to enhanced GVL activity without increasing GVHD (Li et al. 2007 Blood 110:2181), while larger numbers of human donor pDC were associated with less GVL activity following allogeneic bone marrow transplant (BMT) (Waller et al. 2001 Blood 97:2948). To explore the dissociation of GVHD from GVL we tested the hypothesis that activation of donor T-cells by donor pDC leads to reciprocal induction of indoleamine 2,3-dioxygenase (IDO) expression and immune counter-regulatory activity by donor DC that limits donor T-cell allo-reactivity. Methods pDC precursors were purified by high-speed FACS from un-stimulated BM harvested from wild type (WT) and IDO knock-out (IKO) mice. T-cell proliferation and immune polarization in response to indirect antigen presentation by syngenic DC was measured in mixed lymphocyte reaction (MLR) and by recovery of CFSE-labeled donor T-cells from allogeneic transplant recipients. IDO expression in DC was measured by FACS and intracellular staining using pDC from IKO BM as a negative staining control. FACS-purified 5 × 104 pDC either from WT mice or from IKO mice in combination with 3 × 103 c-kit+ Sca-1+ hematopoietic stem cells (HSC) and 3 × 105 T-cells were transplanted in MHC mismatched C57BL/6→B10.BR model following lethal irradiation. Results FACS-purified lineage−CD11cloCD11b− pDC expressed B220 (72%), CD90 (51%), and CD317 (PDCA-1) (93%), had low levels of MHC-II, partial expression of CD4, and lacked expression of CD24, CD80, CD86 and NK cell or granulocytic markers. IDO expression in purified pDC was up regulated by IFN-γ produced by syngenic T-cells in vitro in one-way MLR. In vivo proliferation of CFSE-labeled donor T-cells was enhanced in mice that received pDC from either WT or IKO mice. Co-transplantation of IKO pDC led to higher proliferation rates of CD8+ T-cells but not CD4+ T-cells compared with the proliferation of corresponding donor T-cell subset co-transplanted with WT DC. The incidence and severity of GVHD (weight loss and GVHD score) were markedly increased in recipients receiving pDC from IKO mice as compared with mice receiving WT pDC. The enhanced GVL activity of donor T-cells induced by transplanted donor WT pDC was abolished when IKO pDC were transplanted into tumor-bearing recipients. Transplanting WT donor pDC led to larger numbers of donor-derived CD4+CD25+Foxp3+ T-reg cells in the spleens of transplant recipients compared with mice receiving IKO pDC (p<0.01) in combination with purified HSC and T-cells. Conclusions Taken together, our data suggest IDO expression in pDC as a critical downstream event that inhibits continued T-cell activation and GVHD. We propose a feedback model in which donor pDC initially induce Th1 polarization of activated donor CD8+ T-cells that secret high levels of IFN-γ. IDO expressed by donor pDC in response to local IFN-γ subsequently induces a counter-regulatory effect including the generation of T-reg and down-modulation of CD8+ T-cell allo-reactivity and proliferation, limiting GVHD while preserving the GVL activity of donor T-cells. Disclosures No relevant conflicts of interest to declare.

Flagellin, a TLR5 Agonist, Reduces GvHD in Allogeneic HSCT Recipients While Enhancing Anti-Viral Immunity: A Novel Therapeutic Approach

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 144-144
Author(s):  
Mohammad S Hossain ◽  
David L Jaye ◽  
Brian P Pollack ◽  
Alton B Farr ◽  
John Roback ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Cd8 T Cells ◽  
Viral Immunity ◽  
Hematopoietic Stem ◽  
Allogeneic Hsct ◽  
Post Transplant ◽  
Radiation Chimeras ◽  
Donor T Cells

Abstract Abstract 144 In MHC-mismatched allogeneic hematopoietic stem cell transplantation (allo-HSCT), host antigen specific donor T cells mediate acute and chronic graft-versus-host disease (GvHD). Based upon the radio-protective effects of flagellin, a TLR5 agonist protein (∼50 kDa) extracted from bacterial flagella, we reasoned that flagellin might modulate donor T cells immune responses toward host antigens, reduce GvHD, and improve immune responses to CMV infection in experimental models of allogeneic HSCT. Two 50mg/mouse i.p doses of highly purified flagellin were administered 3 hrs before irradiation and 24 hrs after allo-HSCT in H-2b ^ CB6F1 and H-2k ^ B6 models. GvHD scores were obtained with weekly clinical examination and with histological scoring of intestine, colon, liver and skin at necropsy. Flagellin treatment successfully protected allo-HSCT recipients from acute and chronic GvHDs after transplantation of 5×106 splenocytes and 5×106 T cell depleted (TCD) BM, and significantly increased survival compared to PBS-treated control recipients. Reduced acute GvHD was associated with significant reduction of a) early post-transplant proliferation of donor CD4+ and CD8+ T cells measured by Ki67 and CFSE staining, b) fewer CD62L+, CD69+, CD25+, ICOS-1+ and PD-1+ donor CD4+ and CD8+ T cells compared with the PBS-treated control recipients. Decreased numbers of activated and proliferating donor T cells were associated with significantly reduced pro-inflammatory serum IFN-g, TNF-a, and IL-6 on days 4–10 post transplant in flagellin-treated recipients compared with the PBS-treated recipients. Interestingly, both flagellin-treated recipients and PBS-treated recipients had over 99% donor T cell chimerism at 2 months post transplant. Moreover, MCMV infection on 100+ days post-transplant flagellin-treated mice significantly enhanced anti-viral immunity, including more donor MCMV-peptide-tetramer+ CD8+ T cells in the blood (p<0.05), and less MCMV in the liver on day 10 post infection (p<0.02) compared with the PBS-treated control recipients. Overall immune reconstitution after flagellin-treatment was robust and associated with larger numbers of CD4+CD25+foxp3+ regulatory T cells in the thymus. To further define the role of flagellin-TLR5 agonistic interactions in the reduction of GvHD, we next generated B6 ^ TLR5 KO (KO) and KOB^6 radiation chimeras by transplanting 10 × 106 BM cells from wild-type (WT) B6 or TLR5 KO donors into the congenic CD45.1+ B6 or KO recipients conditioned with 11Gy (5.5Gyx2) TBI. The radiation chimeras were irradiated again with 9.0Gy (4.5Gy × 2) on 60 days after the first transplant and transplanted with 3 × 106 splenocytes and 5 × 106 TCD BM from H-2K congenic donors. Two 50mg doses of flagellin were administered 3 hrs before irradiation and 24 hrs after HSCT. All flagellin-treated B6 ^ B6 radiation chimeras survived with only 12% weight-loss by 80 days post transplant compared with 50% survival among recipients of flagellin-treated B6 ^ KO and 40% survival among KO ^ B6 radiation chimeras. All flagellin-treated KO^ KO and PBS-treated radiation chimeras died within 65 days post transplant. These data suggested that interaction of flagellin with the TLR5 expressing host gut epithelium and donor hematopoietic cells are both required for the maximum protective effect of this TLR5 agonist on GvHD in allogeneic HSCT recipients. Together our data demonstrate that peritransplant administration of flagellin effectively controls acute and chronic GvHD while preserving enhanced post-transplant donor anti-opportunistic immunity. Since flagellin has been found to be safe for use in humans as vaccine adjuvant in a number of clinical trials, the clinical use of flagellin in the setting of allogeneic HSCT is of interest. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Host Reactive Donor T Cells Are Associated With Lung Injury After Experimental Allogeneic Bone Marrow Transplantation

Blood ◽  
1998 ◽  
Vol 92 (7) ◽  
pp. 2571-2580 ◽  
Author(s):  
Kenneth R. Cooke ◽  
Werner Krenger ◽  
Geoff Hill ◽  
Thomas R. Martin ◽  
Lester Kobzik ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Bone Marrow Transplantation ◽  
Lung Injury ◽  
Allogeneic Bone Marrow Transplantation ◽  
Allogeneic Bone ◽  
Allogeneic Bmt ◽  
Donor T Cells ◽  
Ifn Γ

Noninfectious lung injury is common after allogeneic bone marrow transplantation (BMT), but its association with acute graft-versus-host disease (GVHD) is unclear. Using a murine BMT system where donor and host differ by multiple minor histocompatibility (H) antigens, we investigated the nature of lung injury and its relationship both to systemic GVHD and host-reactive donor T cells. Lethally irradiated CBA hosts received syngeneic BMT or allogeneic (B10.BR) T-cell–depleted (TCD) bone marrow (BM) with and without the addition of T cells. Six weeks after BMT, significant pulmonary histopathology was observed in animals receiving allogeneic BMT compared with syngeneic controls. Lung damage was greater in mice that received allogeneic T cells and developed GVHD, but it was also detectable after TCD BMT when signs of clinical and histologic acute GVHD were absent. In each setting, lung injury was associated with significant alterations in pulmonary function. Mature, donor (Vβ6+and Vβ3+) T cells were significantly increased in the broncho-alveolar lavage (BAL) fluid of all allogeneic BMT recipients compared with syngeneic controls, and these cells proliferated and produced interferon-γ (IFN-γ) to host antigens in vitro. These in vitro responses correlated with increased IFN-γ and tumor necrosis factor-α (TNF-α) in the BAL fluid. We conclude that alloreactive donor lymphocytes are associated with lung injury in this allogeneic BMT model. The expansion of these cells in the BAL fluid and their ability to respond to host antigens even when systemic tolerance has been established (ie, the absence of clinical GVHD) suggest that the lung may serve as a sanctuary site for these host reactive donor T cells. These findings may have important implications with regard to the evaluation and treatment of pulmonary dysfunction after allogeneic BMT even when clinical GVHD is absent.

Flagellin, a TLR5 Agonist, Down-Regulate CD62L on Donor T Cells and Limit GvHD in Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3521-3521
Author(s):  
Mohammad Hossain ◽  
Andrew T Gewirtz ◽  
John D Roback ◽  
Edmund K. Waller
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Immune Reconstitution ◽  
Major Complication ◽  
Hematopoietic Stem ◽  
Allogeneic Hsct ◽  
Post Transplant ◽  
Donor T Cells

Abstract Bacground: Graft-vs-host disease (GvHD) is a major complication in allogeneic Hematopoietic Stem Cell Transplant (HSCT) recipients. Flagellin is a bacterial protein and a TLR5 agonist that showed diverse immunological responses in both human and animal including both activation of dendritic cells and immuno-suppression. We recently observed that prophylactic use of flagellin protected allogeneic HSCT recipient from GvHD without affecting host immune reconstitution. Acute GvHD has been reported to be mediated by allo-reactive CD62L+ T cells, and over 80% of murine naïve splenic CD4+ and CD8+ T cells express CD62L. In order to test the effect of flagellin on GvHD mediated by the CD62L+ CD4+ and CD62L+CD8+ donor T cells, we investigated clinical manifestation of GvHD as well as the in vivo expression of CD62L on donor T cells in flagellin treated versus control treated allogeneic HSCT recipients. Methods: We established a parent →F1 MHC major mismatched model (C57BL/6 → C57BL/6 × BALB/c) for allogeneic HSCT for which GvHD is the major complication. Recipient mice received 5 × 10^6 T cell depleted (TCD) bone marrow cells and 5×10^6 or 10×10^6 CFSE labeled donor splenocytes from naïve C57Bl/6 congenic donors. 50 μg flagellin per recipient was administered intraperitoneally 3 hours before irradiation and 24 hours after allogeneic HSCT (treated). CB6F1 recipients that received no flagellin (untreated) and recipients of syngeneic HSCT were used as control. Recipients were sacrificed on day 66+ transplant and the numbers of CD62L+ T cells and foxp3+CD4+CD25+ T cells were determined by FACS. Recipients of CFSE treated donor splenocytes were sacrificed on day 4 post HSCT, splenocytes were harvested and analyzed for CD62L expression on T cell subsets undergone in vivo cell division by Flow cytometry. 5 mice were used per group. Results: Flagellin treated recipients did not have GvHD and had no mortality. Untreated control recipients had 87% survival at 30 days post transplant and had signs of chronic GvHD. While total cell number and also donor spleen- and BM-derived CD4+ and CD8+ T cells per spleen in untreated recipients were significantly lower compared to flagellin treated recipients (p=0.0006) on day 66 post transplant, persistent of donor spleen-derived CD62L+CD4+ T cells and CD62L+CD8+ T cells per spleen were not significantly different (p=0.13 and p=0.07, respectively). Moreover, higher number of foxp3+CD25+CD4+ regulatory T cells were found in the spleen and thymus in treated recipients compared to untreated recipients. Within day 4 post transplant, the number of CD4+ T cells per spleen of treated and untreated recipients increased significantly compared to syngeneic recipients (p=0.001 and p=0.03, respectively). Although equivalent numbers of CD62L+CD4+ T cells were observed in both treated and untreated recipients (p=0.3), significantly increased numbers of CD62L+CD8+ T cells was found in treated recipients compare to untreated recipients (p=0.02). Moreover, significantly higher numbers of divided (far left CFSE staining population) CD62L+CD4+ and CD62L+CD8+ T cells were found in recipients of treated splenocytes within day 4 post transplant followed by down regulation of CD62L surface marker compared to untreated recipients (p=0.02 and p=0.01, respectively). Conclusion: Flagellin treated recipients had limited GvHD and had rapid increased divided CD4+CD62L+ T cells followed by CD62L-ve activated CD4+ T cells per spleen in treated recipients compared to untreated recipients may be one of the major affect mediated by flagellin. Flagellin-TLR5 receptor agonistic effect may reduce production of biological factor(s) essential to generate allo-reactive T cells or directly stimulate CD62L+CD4+ and CD62L+CD8+ T cells in different activation status other than allo-reactive T cells; maintain a balanced immune reconstitution in lymphoid organs by producing regulatory T cells through their thymus. Therefore, use of flagellin may be a novel therapeutic approach to treat blood cancer patients with allogeneic HSCT without GvHD and toxicity.

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