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IL-12hi Rapamycin-Conditioned Dendritic Cells Mediate IFN-γ-Dependent and Fas-Supported Apoptosis of Alloreactive CD4+ T Cells and Inhibit Graft-Versus-Host Disease

Track: Poster Abstracts
Saturday, March 1, 2014, 6:45 PM-7:45 PM
Longhorn Hall E (Exhibit Level 1) (Gaylord Texan)
Elizabeth Stenger, MD , Children's Healthcare of Atlanta, Atlanta, GA
Brian Rosborough, PhD , Starzl Transplant Institute, University of Pittsburgh, Pittsburgh, PA
Lisa Mathews , Starzl Transplant Institute, University of Pittsburgh, Pittsburgh, PA
Hui Hui Ma, MD PhD , Columbia University, New York, NY
Markus Y Mapara, MD, PhD , Columbia University, New York, NY
Angus W. Thomson, PhD , Starzl Transplant Institute, University of Pittsburgh, Pittsburgh, PA
Heth Turnquist, PhD , Starzl Transplant Institute, University of Pittsburgh, Pittsburgh, PA

Rapamycin inhibits mTOR, a crucial immune regulator. RAPA-conditioned DC (RAPA-DC) enrich for Treg and induce alloreactive T cell apoptosis. They promote experimental allograft survival, yet secrete increased IL-12, crucial for generation of IFN-γ+CD4+ T cells. IFN-γ is also pro-apoptotic and IL-12-driven IFN-γ inhibits GVHD.  We hypothesized that IL-12hi RAPA-DC would facilitate IFN-γ-mediated alloreactive T cell apoptosis and, that unlike control (CTR)-DC, would suppress GVHD.

DC were generated from wt, IFN-γ-receptor (R)-/-, or IL-12p40-/- C57BL/6 or BALB/c BM in 7 d culture ± RAPA (10 ng/ml).  After LPS (100 ng/ml) or no stimulation for 18 h DC were co-cultured with allogeneic CD4+ T cells in 5 d MLR ± anti-IFN-γ Ab.  T cells were examined by flow for apoptosis (Annexin V+7-AAD+), CD4 and Fas [CD95], and intracellular IFN-γ and cleaved caspase 8.  Capacity of syngeneic DC to prevent GVHD was assessed in irradiated BALB/c mice reconstituted with 5x106 T cell-depleted B6 BM on d0. Recipients received 106 BALB/c DC and 106 B6 T cells on d1.

Compared to CTR-DC, RAPA-DC enhanced T cell apoptosis (12.7±2.3% vs 7.3±0.4%; +LPS: 28.5±4.4% vs 15.4±2.9%; p<0.05).  Neutralization of IFN-γ in LPS-treated RAPA-DC cultures decreased apoptosis to that of LPS-treated CTR-DC (18.7±1.0% vs 15.4±2.9%; NS).  Both RAPA-DC groups increased IFN-γ production by T cells compared to CTR (Fig 1).  IFN-γ-R-/- T cells exposed to LPS-stim RAPA-DC showed decreased apoptosis compared to wt T cells (15.6±3.6% vs 4.9±1.1%; p<0.05) (Fig 2).  DC responses to IFN-γ did not mediate the function of RAPA-DC (12.4±7.6% [IFN-γ-R-/- DC] vs 10.5±7.6% [wt]; +LPS: 27.2±11.9% vs 30.1±16.3%; NS).  LPS-stim IL-12p40-/- RAPA-DC induced lower levels of apoptosis compared to wt DC (16.9±8.9% vs 20.8±10.0%; p<0.05), which was further reduced with anti-IFN-γ (12.0±6.1%; p<0.05).  Compared to LPS-stim CTR-DC, IL-12hi RAPA-DC increased T cell Fas and cleaved caspase 8 (Fig 1).  Whereas CTR-DC accelerated mortality from GVHD, LPS-treated RAPA-DC significantly prolonged survival (Fig 3).

Increased apoptosis of alloreactive T cells induced by LPS-stim IL-12hi RAPA-DC is mediated via IFN-γ and partly via increased IL-12. Thus enhanced production of IL-12, the principal inducer of IFN-γ, may support the capacity of RAPA-DC to prolong experimental allograft survival and inhibit GVHD.  IL-12hi human RAPA-DC, generated with endotoxin-free, synthetic TLR4 agonists, may allow exploitation of both IL-12 and regulatory DC to suppress GVHD.

DC

%CD4+IFNγ+

%CD4+Fas+

%CD4+ Cleaved caspase 8+

d 1

d 2

d 1

d 2

d 1

d2

CTR

3.2±0.8

3.6±2.1

1.3±0.6

1.9±1.5

9.2±4.7

6.2±3.9

CTR-LPS

6.1±1.2

6.0±3.1

3.5±1.0

3.1±1.6

10.6±4.0

8.7±4.2

RAPA

10.6±0.9*

10.5±5.9*

3.3±0.8*

4.1±1.5*

14.2±5.3*

11.4±5.6*

RAPA-LPS

10.6±2.1*

10.1±3.4*

7.1±1.4*

6.6±2.4

14.2±2.4

10.9±6.6

*p<0.05 compared to CTR

*denotes p<0.05

*denotes p<0.05 compared to untreated GVHD

1Median survival

Disclosures:
Nothing To Disclose
See more of: Poster Session 2: GVH/GVL
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