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. 2013 Mar;43(3):734-46.
doi: 10.1002/eji.201242914. Epub 2013 Jan 18.

Dendritic cell modification as a route to inhibiting corneal graft rejection by the indirect pathway of allorecognition

Adnan Khan  1 , Hongmei Fu, Lee Aun Tan, Jennifer E Harper, Sven C Beutelspacher, Daniel F P Larkin, Giovanna Lombardi, Myra O McClure, Andrew J T George
Affiliations
Free PMC article

Dendritic cell modification as a route to inhibiting corneal graft rejection by the indirect pathway of allorecognition

Adnan Khan et al. Eur J Immunol. 2013 Mar.
Free PMC article

Abstract

Dendritic cell (DC) modification is a potential strategy to induce clinical transplantation tolerance. We compared two DC modification strategies to inhibit allogeneic T-cell proliferation. In the first strategy, murine DCs were transduced with a lentiviral vector expressing CTLA4-KDEL, a fusion protein that prevents surface CD80/86 expression by retaining the co-stimulatory molecules within the ER. In the second approach, DCs were transduced to express the tryptophan-catabolising enzyme IDO. CTLA4-KDEL-expressing DCs induced anergy in alloreactive T cells and generated both CD4(+) CD25(+) and CD4(+) CD25(-) Treg cells (with direct and indirect donor allospecificity and capacity for linked suppression) both in vitro and in vivo. In contrast, T-cell unresponsiveness induced by IDO(+) DCs lacked donor specificity. In the absence of any immunosuppressive treatment, i.v. administration of CTLA4-KDEL-expressing DCs resulted in long-term survival of corneal allografts only when the DCs were capable of indirect presentation of alloantigen. This study demonstrates the therapeutic potential of CTLA4-KDEL-expressing DCs in tolerance induction.

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Figure 1
Figure 1
DC transduction with EIAV-CTLA4-KDEL or EIAV-IDO. BM-derived BALB/c DCs were transduced with EIAV on day 6 of culture prior to LPS stimulation on day 8. (A) DC transduction efficiency was assessed by GFP expression using flow cytometry 72 h after transduction with EIAV-GFP or EIAV-CTLA4-KDEL (control). Expression of the (B) 20 kDa CTLA4-KDEL protein and (C) 45 kDa IDO protein in DC lysates was determined by western blotting 72 h after transduction with EIAV-CTLA-KDEL or EIAV-IDO, respectively, and compared with expression in EIAV-GFP-transduced or untransduced DCs and, in the case of IDO, murine placenta (positive control). Expression of the 42 kDa β-actin housekeeping protein was measured as a loading control. (D) Flow cytometry histograms show the surface expression of CD80, CD86, ICOSL, and CD40 on untransduced DCs, and DCs 72 h after transduction with EIAV-CTLA4-KDEL or EIAV-IDO (control). The results shown in (A–D) are representative of three independent experiments. (E–F) Untransduced, immature DCs were treated on day 7 with either IFN-γ or CTLA4-Ig. The DC culture media was supplemented with L-tryptophan on day 6 (final concentration, 245 μM), followed by LPS stimulation on day 8. (E) DCs were harvested on day 9 for quantitative PCR analysis to assess IDO mRNA expression. (F) IDO activity was assessed by a kynurenine assay using DC culture supernatants that were either supplemented with tryptophan or unsupplemented. Results are shown as the mean ± SD of triplicate wells and are representative of three independent experiments performed. * p < 0.05, two-tailed t-test.
Figure 2
Figure 2
Inhibition of allogeneic T-cell proliferation after DC expression of CTLA4-KDEL or IDO. BALB/c (H-2d) DCs were transduced on day 6 of culture with EIAV-GFP (control), EIAV-CTLA4-KDEL or EIAV-IDO and stimulated with LPS on day 8 of culture. All DC populations were cultured on day 9 with fully MHC-disparate, spleen-derived C3H/He CD4+ T cells. Where indicated, 250 μM 1-methyl-D,L-tryptophan (1-MT), in combination with excess tryptophan, was added to the medium at the start of coculture. (A) Increasing numbers of both EIAV transduced and untransduced DC populations (0–105) were co-cultured with C3H/He-derived CD4+ T cells. Proliferation of CD4+ T cells was detected by thymidine incorporation on day 5 of the MLR, and the results are shown as the mean ± SD of triplicate wells and are representative of three independent experiments. *p < 0.05, two-tailed t-test. (B) Expression of the 52 kDa cyclin E protein and p27Kip1 protein (and the 42 kDa β-actin protein), in the lysates of CD4+ T cells incubated with either the transduced or untransduced DCs described was determined by western blotting on day 4 of co-culture. Data shown are representative of three independent experiments performed.
Figure 3
Figure 3
Induction of T-cell anergy and regulation in vitro with indirect donor allospecificity. CBK (H-2k + Kb) DCs were transduced with either EIAV-GFP (control), EIAV-CTLA4-KDEL or EIAV-IDO, followed by stimulation with LPS. The transduced (and untransduced) DCs were subsequently incubated in vitro with CBA/Ca-derived CD4+ T cells. After 10 days, T cells were harvested and rechallenged in vitro with (A) donor CBK DCs or (B) third-party B10.A (H-2k + Dd) DCs, and CD4+ T-cell proliferation was assessed by thymidine incorporation on days 3, 5 and 7. (C–D) Culture supernatant from the donor DC rechallenge assay was harvested for detection of the immunoregulatory cytokines (C) TGF-β and (D) IL-10 by ELISA. (E–F) In addition, after 10 days of the primary DC-CD4+ T-cell coculture, T cells were also harvested, irradiated and added (0–105 CD4+ T cells) to a primary MLR between freshly isolated CBA CD4+ T cells and (E) donor CBK DCs or (F) third-party B10.A DCs. T-cell proliferation was assessed by thymidine incorporation after 5 days. All results are shown as the mean ± SD of triplicate wells and are representative of three independent experiments performed. *p < 0.05, two-tailed t-test.
Figure 4
Figure 4
Induction of T-cell anergy in vivo with indirect donor allospecificity and production of immunoregulatory cytokines. 2.5 × 106 CBK DCs (either untransduced, or transduced with EIAV-GFP, EIAV-CTLA4-KDEL or EIAV-IDO) were injected i.v. into CBA/Ca mice. (A–D) After 10 days, CD4+ T cells purified from the spleens of injected mice were rechallenged in vitro with (A) donor CBK DCs or (B) third-party B10.A DCs, and CD4+ T-cell proliferation was assessed by thymidine incorporation on days 3, 5 and 7. The purified CD4+ T cells were also rechallenged with (C) donor CBK DCs or (D) third-party B10.A DCs in the presence of 100 U/mL exogenous IL-2. (E–F) Culture supernatant from donor DC rechallenge assays (including those from CD4+CD25+ T-cell-depleted rechallenge assays) was harvested for detection of the immunoregulatory cytokines (E) TGF-β and (F) IL-10 by ELISA. All results are shown as the mean ± SD of triplicate wells and are representative of three independent experiments performed. *p < 0.05, two-tailed t-test.
Figure 5
Figure 5
Generation of Treg cells in vivo with indirect donor allospecificity and capacity for linked suppression. 2.5 × 106 CBK DCs (either untransduced, or transduced with EIAV-GFP (control), EIAV-CTLA4-KDEL or EIAV-IDO) were injected i.v. into C3H/He mice. (A–B) After 10 days, CD4+CD25+ T cells purified from the spleens were irradiated and added (0–105 CD4+CD25+ T cells) to a primary MLR between freshly isolated CBA/Ca-derived CD4+ T cells and (A) donor CBK DCs or (B) third-party B10.A DCs. T-cell proliferation was assessed by thymidine incorporation after 5 days. (C–D) CD4+ T cells purified from the spleens of injected mice were also rechallenged in vitro with (C) (BALB/c × C57BL/6)F1 DCs expressing only third-party MHC or (D) (C57BL/6 × CBA/Ca)F1 DCs expressing both donor (Kb presented in the context of I-Ak/I-Ek) and third-party MHC, and CD4+ T-cell proliferation was assessed by thymidine incorporation on days 3, 5 and 7. (E–F) Rechallenge assays were repeated using CD4+ T cells that were depleted of CD4+CD25+ T cells prior to rechallenge with (E) (BALB/c × C57BL/6)F1 DCs or (F) (C57BL/6 × CBA/Ca)F1 DCs. (G–H) In addition, CD4+CD25+ T cells purified from the spleens of injected mice were irradiated and added (0–105 CD4+CD25+ T cells) to new primary MLRs between freshly isolated CBA/Ca-derived CD4+ T cells and (G) (BALB/c × C57BL/6)F1 DCs or (H) (C57BL/6 × CBA/Ca)F1 DCs and T-cell proliferation assessed by thymidine incorporation after 5 days. All results are shown as the mean ± SD of triplicate wells and are representative of three independent experiments performed. *p < 0.05, two-tailed t-test.
Figure 6
Figure 6
Enhanced survival of equine infectious anaemia virus (EIAV)-transduced DCs in vivo. 1 × 107 CFSE-labelled C3H DCs (either untransduced, or transduced with EIAV CTLA4-KDEL or EIAV OVA) were injected intravenously into female syngeneic mice. The spleens from these mice were harvested on days 3 and 8 post-injection and the total number of CFSE-labelled DCs present assessed by flow cytometric analysis. Each symbol represents an individual animal and bars represent the mean ± SD; data shown are representative of three independent experiments performed. *p < 0.05, two-tailed t-test.
Figure 7
Figure 7
Prolongation of corneal graft survival following administration of modified DCs. BALB/c mice were either untreated (n = 6) or given 2.5 × 106 C3H/He DCs intravenously. The DCs were either untransduced (n = 5), or transduced 72 h earlier ex vivo with equine infectious anaemia virus (EIAV)-GFP (control, n = 7), EIAV-IDO (n = 7) or EIAV-CTLA4-KDEL (n = 8). Ten days later, the BALB/c mice received a complete MHC-disparate C3H/He corneal graft or a syngeneic BALB/c graft (n = 6). (A) Data were plotted using the Kaplan–Meyer method and differences in graft survival were analysed using a log-rank test. (B) Corneal graft opacity scores were plotted and statistical differences between CTLA4-KDEL- and GFP (control)-expressing DCs calculated using the Mann–Whitney U test. * p < 0.008. (C) Using the same protocol, BALB/c mice were either untreated (n = 4) or given 2.5 × 106 (CBA × BALB/c)F1 DCs intravenously. The DCs were either untransduced (n = 5), or transduced 72 h earlier ex vivo with EIAV-GFP (n = 5), EIAV-IDO (n = 5) or EIAV-CTLA4-KDEL (n = 5). 10 days later, BALB/c mice received a complete MHC-disparate CBA/Ca corneal graft or a syngeneic BALB/c graft (n = 6). Data were analysed as above. (D) Corneal graft opacity scores were plotted and statistical differences between CTLA4-KDEL- or IDO-expressing DCs and GFP-expressing DCs calculated using the Mann–Whitney U Test. *p < 0.008. Data shown are representative of three independent experiments performed.
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