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Inhibition of costimulatory pathways for T-cell activation by 1,25-dihydroxyvitamin D3
Inhibition of Costimulatory Pathways for T-Cell Activation by 1,25-Dihydroxyvitamin D3 G. Penna and L. Adorini
D
ENDRITIC CELLS (DCs) are critical antigen-presenting cells (APCs) for the initiation of CD4⫹ T-cell responses.1,2 Induction of T-cell responses requires T-cell receptor activation and costimulatory interactions between DCs and T cells;1 in the absence of costimulation T cells become anergic.3 The two major costimulatory pathways for T-cell activation depend on the engagement of CD28 and CD154 on T cells by CD80/CD86 and CD40 on DCs, respectively.4,5 Once activated, T cells also express CD152, a CD28 homologue that binds to CD80/CD86 with higher affinity than CD28 itself and inhibits IL-2 production, IL-2 receptor expression, and cell cycle progression in activated T cells.6 Disruption of these costimulatory pathways by biological agents such as CD152-Ig and anti-CD154 mAb has been shown to be beneficial in autoimmune diseases and allograft rejection.4,5 A short treatment with CD152-Ig and antiCD154 mAb can induce tolerance to allografts in mice7 and, possibly, also in nonhuman primates.8,9 These promising results, along with the side effects induced by antiCD154 mAb in patients,10 have stimulated the search for low molecular weight compounds that can disrupt costimulatory pathways for T-cell activation. 1,25-dixydroxyvitamin D3 [1,25(OH)2D3], the biologically active metabolite of vitamin D3, is a secosteroid hormone that not only regulates bone and calcium/phosphate metabolism but also performs a number of other biological activities, including modulation of the immune response via specific receptors expressed in APC and activated T cells. Immunosuppression by 1,25(OH)2D3 and its analogues has been demonstrated in different models of autoimmune diseases and in experimental organ transplantation.11 We have recently shown that DCs are primary targets for the immunosuppressive effects of 1,25(OH)2D3 on T-cell activation. 1,25(OH)2D3 inhibits differentiation and maturation of DCs and modulates their activation and survival, leading to a profound modulation of T-cell phenotype and function.12 INHIBITION OF DENDRITIC CELL COSTIMULATORY MOLECULE EXPRESSION BY 1,25-(OH)2D3
Immature DCs obtained by a 7-day culture with GM-CSF and IL-4 can be induced to mature by incubation with LPS. DC maturation is accompanied by slight down-regulation of
CD1a, decreased expression of the mannose receptor, induction of the maturation marker CD83 (not shown), and up-regulation of HLA-DR, CD40, CD80, and CD86 molecules (Fig 1A). Addition of 1,25(OH)2D3 prevented the LPS-induced maturation of immature DCs, maintaining DCs at the immature stage characterized by high mannose receptor and low CD86 expression (Fig 1A). Compared to control mature DCs, addition of 1,25(OH)2D3 during maturation inhibited by over 50% the expression of MHC class II, CD40, CD80, and CD86 molecules, as determined by median fluorescence intensity (Fig 1A) and gave rise to DCs with reduced capacity to activate alloreactive CD4⫹ cells (Fig 1B). INDUCTION OF ALLOREACTIVE T-CELL HYPORESPONSIVENESS BY 1,25(OH)2D3-TREATED DENDRITIC CELLS
These results suggest that the ability of 1,25(OH)2D3 to decrease the expression of costimulatory molecules during DC maturation might contribute to its inhibitory effect on APC-dependent T-cell activation. Thus, we examined whether DCs matured in the presence of 1,25(OH)2D3 could modulate T-cell responsiveness. Coculture of alloreactive CD4⫹ T cells with 1,25(OH)2D3-treated DCs resulted in T-cell hyporesponsiveness, as demonstrated by their reduced IFN-␥ secretion upon restimulation by untreated, mature DCs in a secondary MLR assay (Fig 1C). Addition to cultures of exogenous IL-2 could not reverse the inhibition of IFN-␥ secretion. PROSPECTS
Inactivation of host DCs has been proposed recently to prevent graft-versus-host disease in allogeneic bone marrow transplantation,13 and a similar strategy is applicable to solid organ allografts.14 1,25(OH)2D3 inhibits DC differentiation and maturation, leading to marked down-regulation of MHC class II and costimulatory molecules. In addition, 1,25(OH)2D3 inhibits IL-12 while enhancing IL-10 production and promotes DC apoptosis.12 These effects could From the Roche Milano Ricerche, Milano, Italy. Address reprint requests to Luciano Adorini, Roche Milano Ricerche, Via Olgettina 58, I-20132 Milano, Italy.
© 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/01/$–see front matter PII S0041-1345(01)01957-1
Transplantation Proceedings, 33, 2083–2084 (2001)
2083
2084
PENNA AND ADORINI
Fig 1. Inhibition of costimulatory molecule expression and induction of T-cell hyporesponsiveness by 1,25(OH)2D3-treated DCs. Negatively selected CD14⫹ monocytes were cultured in medium supplemented with 800 U/mL GM-CSF and 1000 U/mL IL-4 to generate immature DCs. Maturation was induced by incubation of immature DCs with LPS (200 ng/mL) for 48 hours. (A) Inhibition of costimulatory molecule expression by DC matured in the presence of 10 nmol/L 1,25(OH)2D3. Stippled lines refer to isotype controls. The staining profile for the indicated surface molecules of immature (solid thin line) and mature (broken line) DCs and of DCs matured in the presence of 1,25(OH)2D3 (bold line) is shown. (B) After extensive washing, the indicated numbers of DCs (⫻103/well), generated as described above, were cocultured with CD4⫹ T cells (2 ⫻ 105/well) from a different donor. After 5 days, proliferation and IFN-␥ secretion were measured. Proliferative responses are shown as mean (⫾ SE) from triplicate cultures. iDC, immature DCs; mDC, mature DCs; mDC-D3, DCs matured in the presence of 1,25(OH)2D3. (C) Induction of T-cell hyporesponsiveness by 1,25(OH)2D3-treated DCs. Allogeneic CD4⫹ T cells (2 ⫻ 105/well) were cocultured with immature or mature DC populations (103 cells/well) or with DCs matured in the presence of 1,25(OH)2D3. T cells were recovered 36 hours after culture initiation, rested for 2 to 4 days in complete medium supplemented with 2 U/mL IL-2, and restimulated with mature untreated DCs, generated from the same donor used as a DC source for the first culture, with (right panel) or without (left panel) 100 U/mL IL-2. IFN-␥ secretion was measured 48 hours after restimulation. Bars represent the mean (⫾ SE) percent variation (n ⫽ 3) of IFN-␥ secretion compared to restimulation with untreated mature DCs.
contribute substantially to decreasing DC-dependent T-cell activation and could largely account for the immunosuppressive properties of 1,25(OH)2D3. We are currently examining whether a short-term administration of 1,25(OH)2D3 to islet allograft recipients may be able to induce long-lasting graft acceptance and transplantation tolerance via inhibition of host DCs. If this is the case, a 1,25(OH)2D3 analogue with enhanced immunosuppressive activity and reduced effects on the calcium/phosphate metabolism may lead to an effective prevention of human allograft rejection. REFERENCES 1. Banchereau J, Steinman R: Nature 392:245, 1998 2. Reid S, Penna G, Adorini L: Curr Opin Immunol 12:114, 2000
3. Schwartz RH: Science 248:1349, 1990 4. Lenschow DJ, Walunas TL, Bluestone JA: Annu Rev Immunol 14:233, 1996 5. Grewal I, Flavell R: Annu Rev Immunol 16:111, 1998 6. Walunas TL, Bakker CY, Bluestone JA: J Exp Med 183:2541, 1996 7. Larsen C, Elwood E, Alexander D, et al: Nature 381:434, 1996 8. Kenyon N, Chatzipetrou M, Masetti M, et al: Proc Natl Acad Sci USA 96:8132, 1999 9. Kirk A, Burkly L, Batty D, et al: Nature Med 6:686, 1999 10. Kawai T, Andrews D, Colvin RB, et al: Nat Med 6:114, 2000 11. Verstuyf A, Segaert S, Verlinden L, et al: Curr Opin Nephrol Hypertens 7:397, 1998 12. Penna G, Adorini L: J Immunol 164:2000 13. Shlomchik WD, Couzens MS, Tang CB, et al: Science 285:412, 1999 14. Thomson AW, Lu L: Transplantation 68:1, 1999
D
ENDRITIC CELLS (DCs) are critical antigen-presenting cells (APCs) for the initiation of CD4⫹ T-cell responses.1,2 Induction of T-cell responses requires T-cell receptor activation and costimulatory interactions between DCs and T cells;1 in the absence of costimulation T cells become anergic.3 The two major costimulatory pathways for T-cell activation depend on the engagement of CD28 and CD154 on T cells by CD80/CD86 and CD40 on DCs, respectively.4,5 Once activated, T cells also express CD152, a CD28 homologue that binds to CD80/CD86 with higher affinity than CD28 itself and inhibits IL-2 production, IL-2 receptor expression, and cell cycle progression in activated T cells.6 Disruption of these costimulatory pathways by biological agents such as CD152-Ig and anti-CD154 mAb has been shown to be beneficial in autoimmune diseases and allograft rejection.4,5 A short treatment with CD152-Ig and antiCD154 mAb can induce tolerance to allografts in mice7 and, possibly, also in nonhuman primates.8,9 These promising results, along with the side effects induced by antiCD154 mAb in patients,10 have stimulated the search for low molecular weight compounds that can disrupt costimulatory pathways for T-cell activation. 1,25-dixydroxyvitamin D3 [1,25(OH)2D3], the biologically active metabolite of vitamin D3, is a secosteroid hormone that not only regulates bone and calcium/phosphate metabolism but also performs a number of other biological activities, including modulation of the immune response via specific receptors expressed in APC and activated T cells. Immunosuppression by 1,25(OH)2D3 and its analogues has been demonstrated in different models of autoimmune diseases and in experimental organ transplantation.11 We have recently shown that DCs are primary targets for the immunosuppressive effects of 1,25(OH)2D3 on T-cell activation. 1,25(OH)2D3 inhibits differentiation and maturation of DCs and modulates their activation and survival, leading to a profound modulation of T-cell phenotype and function.12 INHIBITION OF DENDRITIC CELL COSTIMULATORY MOLECULE EXPRESSION BY 1,25-(OH)2D3
Immature DCs obtained by a 7-day culture with GM-CSF and IL-4 can be induced to mature by incubation with LPS. DC maturation is accompanied by slight down-regulation of
CD1a, decreased expression of the mannose receptor, induction of the maturation marker CD83 (not shown), and up-regulation of HLA-DR, CD40, CD80, and CD86 molecules (Fig 1A). Addition of 1,25(OH)2D3 prevented the LPS-induced maturation of immature DCs, maintaining DCs at the immature stage characterized by high mannose receptor and low CD86 expression (Fig 1A). Compared to control mature DCs, addition of 1,25(OH)2D3 during maturation inhibited by over 50% the expression of MHC class II, CD40, CD80, and CD86 molecules, as determined by median fluorescence intensity (Fig 1A) and gave rise to DCs with reduced capacity to activate alloreactive CD4⫹ cells (Fig 1B). INDUCTION OF ALLOREACTIVE T-CELL HYPORESPONSIVENESS BY 1,25(OH)2D3-TREATED DENDRITIC CELLS
These results suggest that the ability of 1,25(OH)2D3 to decrease the expression of costimulatory molecules during DC maturation might contribute to its inhibitory effect on APC-dependent T-cell activation. Thus, we examined whether DCs matured in the presence of 1,25(OH)2D3 could modulate T-cell responsiveness. Coculture of alloreactive CD4⫹ T cells with 1,25(OH)2D3-treated DCs resulted in T-cell hyporesponsiveness, as demonstrated by their reduced IFN-␥ secretion upon restimulation by untreated, mature DCs in a secondary MLR assay (Fig 1C). Addition to cultures of exogenous IL-2 could not reverse the inhibition of IFN-␥ secretion. PROSPECTS
Inactivation of host DCs has been proposed recently to prevent graft-versus-host disease in allogeneic bone marrow transplantation,13 and a similar strategy is applicable to solid organ allografts.14 1,25(OH)2D3 inhibits DC differentiation and maturation, leading to marked down-regulation of MHC class II and costimulatory molecules. In addition, 1,25(OH)2D3 inhibits IL-12 while enhancing IL-10 production and promotes DC apoptosis.12 These effects could From the Roche Milano Ricerche, Milano, Italy. Address reprint requests to Luciano Adorini, Roche Milano Ricerche, Via Olgettina 58, I-20132 Milano, Italy.
© 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
0041-1345/01/$–see front matter PII S0041-1345(01)01957-1
Transplantation Proceedings, 33, 2083–2084 (2001)
2083
2084
PENNA AND ADORINI
Fig 1. Inhibition of costimulatory molecule expression and induction of T-cell hyporesponsiveness by 1,25(OH)2D3-treated DCs. Negatively selected CD14⫹ monocytes were cultured in medium supplemented with 800 U/mL GM-CSF and 1000 U/mL IL-4 to generate immature DCs. Maturation was induced by incubation of immature DCs with LPS (200 ng/mL) for 48 hours. (A) Inhibition of costimulatory molecule expression by DC matured in the presence of 10 nmol/L 1,25(OH)2D3. Stippled lines refer to isotype controls. The staining profile for the indicated surface molecules of immature (solid thin line) and mature (broken line) DCs and of DCs matured in the presence of 1,25(OH)2D3 (bold line) is shown. (B) After extensive washing, the indicated numbers of DCs (⫻103/well), generated as described above, were cocultured with CD4⫹ T cells (2 ⫻ 105/well) from a different donor. After 5 days, proliferation and IFN-␥ secretion were measured. Proliferative responses are shown as mean (⫾ SE) from triplicate cultures. iDC, immature DCs; mDC, mature DCs; mDC-D3, DCs matured in the presence of 1,25(OH)2D3. (C) Induction of T-cell hyporesponsiveness by 1,25(OH)2D3-treated DCs. Allogeneic CD4⫹ T cells (2 ⫻ 105/well) were cocultured with immature or mature DC populations (103 cells/well) or with DCs matured in the presence of 1,25(OH)2D3. T cells were recovered 36 hours after culture initiation, rested for 2 to 4 days in complete medium supplemented with 2 U/mL IL-2, and restimulated with mature untreated DCs, generated from the same donor used as a DC source for the first culture, with (right panel) or without (left panel) 100 U/mL IL-2. IFN-␥ secretion was measured 48 hours after restimulation. Bars represent the mean (⫾ SE) percent variation (n ⫽ 3) of IFN-␥ secretion compared to restimulation with untreated mature DCs.
contribute substantially to decreasing DC-dependent T-cell activation and could largely account for the immunosuppressive properties of 1,25(OH)2D3. We are currently examining whether a short-term administration of 1,25(OH)2D3 to islet allograft recipients may be able to induce long-lasting graft acceptance and transplantation tolerance via inhibition of host DCs. If this is the case, a 1,25(OH)2D3 analogue with enhanced immunosuppressive activity and reduced effects on the calcium/phosphate metabolism may lead to an effective prevention of human allograft rejection. REFERENCES 1. Banchereau J, Steinman R: Nature 392:245, 1998 2. Reid S, Penna G, Adorini L: Curr Opin Immunol 12:114, 2000
3. Schwartz RH: Science 248:1349, 1990 4. Lenschow DJ, Walunas TL, Bluestone JA: Annu Rev Immunol 14:233, 1996 5. Grewal I, Flavell R: Annu Rev Immunol 16:111, 1998 6. Walunas TL, Bakker CY, Bluestone JA: J Exp Med 183:2541, 1996 7. Larsen C, Elwood E, Alexander D, et al: Nature 381:434, 1996 8. Kenyon N, Chatzipetrou M, Masetti M, et al: Proc Natl Acad Sci USA 96:8132, 1999 9. Kirk A, Burkly L, Batty D, et al: Nature Med 6:686, 1999 10. Kawai T, Andrews D, Colvin RB, et al: Nat Med 6:114, 2000 11. Verstuyf A, Segaert S, Verlinden L, et al: Curr Opin Nephrol Hypertens 7:397, 1998 12. Penna G, Adorini L: J Immunol 164:2000 13. Shlomchik WD, Couzens MS, Tang CB, et al: Science 285:412, 1999 14. Thomson AW, Lu L: Transplantation 68:1, 1999