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The Advances of regulatory dendritic cells in The Advances of regulatory dendritic cells in GVHD and GVL after allogeneic bone marrow GVHD and GVL after allogeneic bone marrow

transplantationtransplantation

Yihong Huang, Kailin Xu ( 黄一虹，徐林开 )

Institute of HematologyThe Affiliated Hospital of Xuzhou Medical College

（徐州医学院血液病研究所）

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Ⅱ. Dendritic cells and regulation of GVHD and GVL

Ⅰ . The Immunological Basis for GVHD and GVL

Ⅲ. New Exploration to Modulate DCs in Our Lab

The classical Model of the Immunopathogenesis of GVHD Described by Three Interlinked Phases

Pro-inflammatory environment

Donor T cell proliferation after encountering activated

recipient APCs bearing alloantigen

Alloreactive cell migration to target tissues and resulting in

tissue damage in the form of GVHD

Ferrara JT, et al. Stem Cells. 1996,14:473-489.

Ferrara JT, et al. Semin Hematol.2006,43:3-10

Immunopathogenesis of Alloreactive Response and Development of GVHD

Immunopathogenesis of Alloreactive Response and Development of GVHD

New Approaches to Separate GVHD and

GVL Effects

Novel approaches to modify the pro-inflammatory environment

New exploration to modulate donor T cell priming and expansion

New ways to manipulate donor T cell migration to target tissues

Novel approaches to modify the pro-inflammatory environment

New exploration to modulate donor T cell priming and expansion

New ways to manipulate donor T cell migration to target tissues

APC involved

Pan B, Xu KL et al. Cytokine.2014, 68(2):69-75 Li ZY, Xu KL et al. Hematology. 2013,18(6):352-359 Zhao K, Xu KL et al. Immunobiology. 2013;218:1505-13Zeng LY, Xu KL et al. Int J Hematol. 2012,95(2):189-97

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Ⅱ. DCs and regulation of GVHD and GVL

2. The role of DCs in GVHD and GVL

3. Manipulated DCs for prevention or treatment of GVHD

4. Generation of tolDCs for prevention or treatment of GVHD

5. Cell therapies that target DCs in vivo

6. Active clinical trials that target/impact DCs

1. Overview of DCs subsets

Overview of DC subsetsBased on the source of DC Based on the function of DC

Mature DCs(mDCs): enhance T cell immune response;

ImmatureDCs (iDCs): involved in the induction of peripheral T cell tolerance under steady state conditions ,but likely change into mature under inflammatory conditions;

Regulatory DCs (rDCs)/Tolerogenic DCs(tolDCs): established DCs with potent immunoregulatory property, to impair the functions of alloreactive/xeno reactive T cells.

Elizabeth O. Stenger et al. Blood. 2012;119(22):5088-5103

The Important role of Dendritic Cells in GVHD and GVL

Acute GVHD can be induced by either host or donor APCs, whereas

host APCs are required for the initiation of aGVHD and donor APCs

amplify the process.

Dendritic cells(DCs) as professional APCs, have been well recognized

for their role in the induction of GVHD and GVL responses .

Traditional therapies have targeted T cells, yet immunostimulatory

DCs are critical in the pathogenesis of GVHD.

Zhao K, Xu KL et al. J Interferon Cytokine Res. 2014,34(9):707-15 . Pan B, Xu KL et al. Clinical Immunology (2014) 150, 101–8.Sang W, Xu KL et al. Immunol Lett . 2011,136(2):194-202 .

The role of DCs in GVHD

Initial tissue injury may trigger DCs within tissues and induce DCs to migrate to draining lymph nodes;

Migrating DCs “trap” autoreactive T cells , where they induce T cell activation and may “imprint” a homing phenotype that permits selective trafficking of T cells to GVHD target organs;

DCs within inflamed tissues may act to amplify the developing GVHD by providing further priming signals to T cells.

DCs can influence development of GVHD at multiple levels:

Ronjon Chakraverty et al. Blood. 2007;110:9-17

The role of DCs in GVL

Host DCs are required for full GVL effects, although donor DCs can initiate GVL activity when low burden of tumor are present..

Depletion of donor BM CD11b myeloid cells in an experimental model enhanced survival of recipients with tumor;

Addition of precursor pDCs, augmented GVL without increase in GVHD;

Increased graft pDC is associated with relapse and decreased patient overall survival (OS).

Li JM et al .Biol Blood Marrow Transplant. 2004;10(8):540-551.

Li JM et al. Immunol. 2009;183(12):7799-7809.

Rajasekar R et al.Biol Blood MarrowTransplant. 2010;16(6):854-860.

Manipulated DCs for prevention or treatment of GVHD

DCs may be targeted using antibodies to cell surface molecules or can be manipulated in vitro to skew their function to suppress GVHD.

Pharmacologic interventions Biologic interventions Vaccination strategies

Manipulated DCs in HSCTTable 1. Impact of interventional stategies on DCs and outcome in experimental HSCT


Manipulated DCs in HSCTTable 2. Overview of DC vaccination trials after allo-HSCt

Maud Plantinga et al. Frontiers in immunology. 2014,5:218

Generation of tolDCs for prevention or treatment of GVHD

Immature or maturation-resistant tolDCs can promote tolerance in experimental organ and HSCT, while still protecting against leukemia relapse.

DCs can be manipulated in vitro to produce tolDCs:

• Cytokine growth factors• Genetic engineering• Phamacological mediators

Generation of tolerogenic DC in vitro

Usage of tolDC therapy to inbibit GVHD in miceTable 3. Summary of tolDC used in allo-HSCT


Mesenchymal stem cells (MSCs): have shown promise in the prevention and treatment of GVHD.

• Human MSCs impair DC maturation and induce T-cell hyporesponsiveness.

• TolDCs generated by coculture of DCs with human MSCs (MSC-DCs) induce Ag specific Treg via activation of the Notch pathway.

Cell therapies that target DCs in vivo

Myeloid-derived suppressor cells (MDSCs) : modulate both innate and adaptive immunity with immunosuppressive properties.•MDSCs inhibit the differentiation and maturation of DCs.

•MDSCs were more potent inhibitors of MHC-mismatched GVHD.

Perla Filippini et al. Expert Rev. Clin. Immunol. 2014,10(10), 1357–1374

Chen W, Xu KL et al. Immunol Lett. 2012,143(2):161-9.Chen W, Xu KL et al. Cell Biochem Biophys. 2013, 67:1181–1191 .

Cell therapies that target DCs in vivo

Regulatory T cells(Treg):

• Treg therapy supports tolerance through effects on DCs, while DCs

also control the number and function of Treg.

• Human Treg can abrogate GVHD via induction of PD-L1 expression

on conditioned DCs and on effector T cells.

• Ag-specific Treg can be induced and expanded by monDCs in an

IDO-dependent manner.

Cao J, Xu KL et al. Leuk Res. 2010,34(10): 1374-82Chen C, Xu KL et al. Transplant Proc. 2011,43(5): 2041-8 .Perla Filippini et al. Expert Rev. Clin. Immunol. 2014,10(10), 1357–1374

DC based methods of transplant tolerence

Adrian E. Morelli et al. Nature review immunology. 2007,7:610-321

There are numerous open clinical trials for the prevention

or treatment of GVHD currently studying pharmacologic or

biologic interventions and cellular therapies that target or

impact DCs.

Active clinical trials that target/impact DCs

Table 4. Active clinical trials using interventions that target/ impact DCs


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1. Effects of regulatory dendritic cells on GVHD and GVL in mice after

allogeneic bone marrow transplantation

2. Separation of GVHD and GVL effects in mouse allo-HSCT model by

engineered DCs

Part 1:

Effects of regulatory dendritic cells on GVHD and GVL in mice after

allogeneic bone marrow transplantation

Generation of inducible DCreg

A, imDC ； B, mDC ； C, DCreg ； D, LPS stimulated regDC

Figure 1. The cell morphological observation of DC subsets

Compared with mDCs, shorter synapsis was shown in DCreg; and rare wrinkles were observed in DCreg.

Surface molecules expression on DCs

mDCimDC DCreg

Co-stimulatory molecules were down regulated in DCreg

Figure 2. FACS was used to detected the molecules of expressed on DCs

Mice survival after transplantation

The first death in the allo-BMT group was found on 10 d, and all mice died within 25 d;

Most of the mice in the imDC group died within 21 d, while 20% of the mice were still alive at 30 d;

The first death in the DCreg group was found on 19 d, while 46.7% of the mice were still alive at 30 d.

Figure 3. The survival rate of mice with allo-BMT

Chen C, Xu KL. Leukemia Research, 2014 , 38(12):1460-8

Effects of DCreg on pathological changes in GVHD tissues after transplantation

Liver cell edema, focal necrosis, lymphocyte infiltration at the portal area, intestinal epithelial cells necrosis, significant inflammatory cell infiltration in the pulmonary vessels in the allo-BMT and imDC groups;

Mice in the DCreg group, we found only mild or even no pathological damage in target organs.

Normal allo-BMT imDC DCreg

Liver

Gut

Lung

Figure 4. Target organs were detected after HE staining

Pathological GVHD score was low in DCreg group

Groups Liver Intestine

Lung

allo-BMT 6.33±0.58 7.00±1.00 6.67±0.58

imDC 4.67±0.58* 4.67±0.58* 2.33±1.16*

DCreg 3.00±1.00*# 2.67±0.58*# 2.00±1.00*#

Table 1. GVHD pathologic scoring of liver, small intestine, and lung in mice after transplantation (n = 3, )sx ±

*P ＜ 0.05 vs allo-BMT group; #P ＜ 0.05 vs imDC group

Cytokine levels in plasma

IFN-γ levels increased significantly in the allo-BMT group, whereas low level of that was detected in DCreg group.

IL-10 as immune surppressor cytokine increased obviously in Dcreg mice.

Figure 5. cytokines from allo-BMT mice were detected by ELISA

Part 2:

Suppression of graft-vs-host disease and retention of graft-vs- leukemia reaction by murine genetic engineering dendritic cells after bone marrow transplantation

Establishment of engineered DCs

Lentivirus-mediated expression of soluble tumor necrosis factor

receptor 1 (sTNFR1) converted immature DC were established in

vitro, named sTNFR-DC;

PXZ9-DC were used as negetive vector control;

Mice survival following transplantation

Mice from the leukemia model group began to die after 13 days, and all died due to leukemia within 18 days;

allo-BMT group began to die after 8 days, and all died by 18 days

Most of the pXZ9-imDC group mice died within 21 days

sTNFR1-imDC group mice died starting at 15 day, and 40% of mice survived up to 30 days

Figure 6. The survival rate of mice after allo-BMT

Pan B, Xu KL. Immunol Lett . 2012,142(1-2):48-54

Evaluation of GVHD

High GVHD clinical score were evaluated in allo-BMT mice , whereas injected sTNFR-DC mice showed slight GVHD score.

More severe pathological damage were observed in allo-BMT group, but mild changes were shown in sTNFR-DC mice.

Skin

Liver

Gut

Allo-BMT sTNFR1-DC PXZ9-DC

Figure 6. The clinical score and pathological changes in mice

Cytokine levels in plasma

IFN-γ levels increased significantly in the allo-BMT group and PXZ9-DC group, whereas low level of IFN-γ was detected in sTNFR-DC group.

IL-4 which was considered as immune surppressor cytokine increased obviously in sTNFR-DC group.

Figure 7. Cytokine expression in mice after allo-BMT

Prospective

Synergistic therapies or those that

target both T and DC cells may be

more effective.

The injection site, dose and frequency

of the tolDCs induced in vitro should be

carefully considered.

Potential DC-based therapies for GVHD ：

Thank You for Your Attention

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