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Efficient and versatile manipulation of the peripheral CD4+ T-cell compartment by antigen targeting to DNGR-1/CLEC9A.

Joffre OP, Sancho D, Zelenay S, Keller AM, Reis e Sousa C - Eur. J. Immunol. (2010)

Bottom Line: Notably, distinct adjuvants allowed qualitative modulation of CD4+ T-cell behavior: poly I:C induced a strong IL-12-independent Th1 response, whereas curdlan led to the priming of Th17 cells.Thus, antigen targeting to DNGR-1 is a versatile approach for inducing functionally distinct CD4+ T-cell responses.Given the restricted pattern of expression of DNGR-1 across species, this strategy could prove useful for developing immunotherapy protocols in humans.

View Article: PubMed Central - PubMed

Affiliation: Immunobiology Laboratory, Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, London, UK.

ABSTRACT
DC NK lectin group receptor-1 (DNGR-1, also known as CLEC9A) is a C-type lectin receptor expressed by mouse CD8alpha+ DC and by their putative equivalents in human. DNGR-1 senses necrosis and regulates CD8+ T-cell cross-priming to dead-cell-associated antigens. In addition, DNGR-1 is a target for selective in vivo delivery of antigens to DC and the induction of CD8+ T-cell and Ab responses. In this study, we evaluated whether DNGR-1 targeting can be additionally used to manipulate antigen-specific CD8+ T lymphocytes. Injection of small amounts of antigen-coupled anti-DNGR-1 mAb into mice promoted MHC class II antigen presentation selectively by CD8alpha+ DC. In the steady state, this was sufficient to induce proliferation of antigen-specific naïve CD4+ T cells and to drive their differentiation into Foxp3+ regulatory lymphocytes. Co-administration of adjuvants prevented this induction of tolerance and promoted immunity. Notably, distinct adjuvants allowed qualitative modulation of CD4+ T-cell behavior: poly I:C induced a strong IL-12-independent Th1 response, whereas curdlan led to the priming of Th17 cells. Thus, antigen targeting to DNGR-1 is a versatile approach for inducing functionally distinct CD4+ T-cell responses. Given the restricted pattern of expression of DNGR-1 across species, this strategy could prove useful for developing immunotherapy protocols in humans.

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DNGR-1 targeting leads to limited antigen-specific Ab production in the absence of adjuvant WT (A–C) or DNGR-1 KO (B) mice were injected with 0.5 μg (A, B) or with the indicated amounts (C) of anti-DNGR-1 or isotype-matched control mAb in the presence/absence of 40 μg of poly I:C. Anti-rat IgG serum Ab titers were determined (A, B) at the indicated time points or (C) 20 days after immunization. Data are the mean±SD of three mice from one experiment representative of two.
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fig03: DNGR-1 targeting leads to limited antigen-specific Ab production in the absence of adjuvant WT (A–C) or DNGR-1 KO (B) mice were injected with 0.5 μg (A, B) or with the indicated amounts (C) of anti-DNGR-1 or isotype-matched control mAb in the presence/absence of 40 μg of poly I:C. Anti-rat IgG serum Ab titers were determined (A, B) at the indicated time points or (C) 20 days after immunization. Data are the mean±SD of three mice from one experiment representative of two.

Mentions: Injection of anti-DNGR-1 mAb did not lead to any detectable activation of splenic CD8α+ DC (not shown). Nevertheless, we evaluated whether antigen targeting to DNGR-1 could lead to CD4+ T-cell priming in the absence of adjuvant, as recently suggested 17. To avoid any contribution from memory or Treg, we transferred sorted naïve OT-II lymphocytes into B6 mice. One day later, the mice were injected with 0.5 μg of OVA323–339-coupled anti-DNGR-1 mAb with or without 40 μg of poly I:C, a TLR3 and RIG-I/MDA5 agonist recently described as the most potent Th1-promoting adjuvant in experiments of antigen targeting to DEC205 23. In the absence of poly I:C, we observed CD4+ T-cell expansion but no detectable differentiation into Th1, Th2 or Th17 cells (Fig. 2B and C and data not shown). Consistent with the absence of immunity in these conditions, the mice did not develop a strong Ab response to rat IgG following anti-DNGR-1 injection (Fig. 3A). Low titers of anti-rat antibodies were detected only when injecting a higher dose of anti-DNGR-1 mAb (Fig. 3C), matching the one used in a previous report 17. However, the anti-rat IgG response seen with anti-DNGR-1 alone was dwarfed by that which could be induced by co-administration of poly I:C (Fig. 3C). Poly I:C additionally led to increased expression of MHC class II and co-stimulatory molecules on CD8α+ DC (not shown) and strongly boosted the expansion of OT-II cells induced by the Ab–antigen complex (Fig. 2B, D, E). Notably, it also induced robust differentiation of naïve T cells into Th1 effectors, as shown by IFN-γ staining after acute ex vivo restimulation with OVA323–339 peptide (Fig. 2B, C, E). Demonstrating the specificity of the targeting, no T-cell expansion, Th1 priming or anti-rat IgG response was observed when an isotype-matched control mAb was used (Fig. 2B–D and 3A) or when anti-DNGR-1 conjugates were injected into clec9aegfp/egfp (“DNGR-1 knockout”; DNGR-1 KO) mice (Fig. 2E and 3B). Th1 differentiation could also be induced with other adjuvants such as anti-CD40 mAb or CpG-containing DNA oligonucleotides (not shown) and could be reproduced in a different adoptive transfer model (Supporting Information Fig. 3). Finally, although CD8α+ DC can produce IL-12 in response to innate stimuli, such as poly I:C, identical Th1 responses were seen in WT and IL-12 p40 KO hosts (Supporting Information Fig. 3), confirming that Th1 priming to antigens presented by CD8α+ DC is not dependent on IL-12p70 or IL-23 10.


Efficient and versatile manipulation of the peripheral CD4+ T-cell compartment by antigen targeting to DNGR-1/CLEC9A.

Joffre OP, Sancho D, Zelenay S, Keller AM, Reis e Sousa C - Eur. J. Immunol. (2010)

DNGR-1 targeting leads to limited antigen-specific Ab production in the absence of adjuvant WT (A–C) or DNGR-1 KO (B) mice were injected with 0.5 μg (A, B) or with the indicated amounts (C) of anti-DNGR-1 or isotype-matched control mAb in the presence/absence of 40 μg of poly I:C. Anti-rat IgG serum Ab titers were determined (A, B) at the indicated time points or (C) 20 days after immunization. Data are the mean±SD of three mice from one experiment representative of two.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3064981&req=5

fig03: DNGR-1 targeting leads to limited antigen-specific Ab production in the absence of adjuvant WT (A–C) or DNGR-1 KO (B) mice were injected with 0.5 μg (A, B) or with the indicated amounts (C) of anti-DNGR-1 or isotype-matched control mAb in the presence/absence of 40 μg of poly I:C. Anti-rat IgG serum Ab titers were determined (A, B) at the indicated time points or (C) 20 days after immunization. Data are the mean±SD of three mice from one experiment representative of two.
Mentions: Injection of anti-DNGR-1 mAb did not lead to any detectable activation of splenic CD8α+ DC (not shown). Nevertheless, we evaluated whether antigen targeting to DNGR-1 could lead to CD4+ T-cell priming in the absence of adjuvant, as recently suggested 17. To avoid any contribution from memory or Treg, we transferred sorted naïve OT-II lymphocytes into B6 mice. One day later, the mice were injected with 0.5 μg of OVA323–339-coupled anti-DNGR-1 mAb with or without 40 μg of poly I:C, a TLR3 and RIG-I/MDA5 agonist recently described as the most potent Th1-promoting adjuvant in experiments of antigen targeting to DEC205 23. In the absence of poly I:C, we observed CD4+ T-cell expansion but no detectable differentiation into Th1, Th2 or Th17 cells (Fig. 2B and C and data not shown). Consistent with the absence of immunity in these conditions, the mice did not develop a strong Ab response to rat IgG following anti-DNGR-1 injection (Fig. 3A). Low titers of anti-rat antibodies were detected only when injecting a higher dose of anti-DNGR-1 mAb (Fig. 3C), matching the one used in a previous report 17. However, the anti-rat IgG response seen with anti-DNGR-1 alone was dwarfed by that which could be induced by co-administration of poly I:C (Fig. 3C). Poly I:C additionally led to increased expression of MHC class II and co-stimulatory molecules on CD8α+ DC (not shown) and strongly boosted the expansion of OT-II cells induced by the Ab–antigen complex (Fig. 2B, D, E). Notably, it also induced robust differentiation of naïve T cells into Th1 effectors, as shown by IFN-γ staining after acute ex vivo restimulation with OVA323–339 peptide (Fig. 2B, C, E). Demonstrating the specificity of the targeting, no T-cell expansion, Th1 priming or anti-rat IgG response was observed when an isotype-matched control mAb was used (Fig. 2B–D and 3A) or when anti-DNGR-1 conjugates were injected into clec9aegfp/egfp (“DNGR-1 knockout”; DNGR-1 KO) mice (Fig. 2E and 3B). Th1 differentiation could also be induced with other adjuvants such as anti-CD40 mAb or CpG-containing DNA oligonucleotides (not shown) and could be reproduced in a different adoptive transfer model (Supporting Information Fig. 3). Finally, although CD8α+ DC can produce IL-12 in response to innate stimuli, such as poly I:C, identical Th1 responses were seen in WT and IL-12 p40 KO hosts (Supporting Information Fig. 3), confirming that Th1 priming to antigens presented by CD8α+ DC is not dependent on IL-12p70 or IL-23 10.

Bottom Line: Notably, distinct adjuvants allowed qualitative modulation of CD4+ T-cell behavior: poly I:C induced a strong IL-12-independent Th1 response, whereas curdlan led to the priming of Th17 cells.Thus, antigen targeting to DNGR-1 is a versatile approach for inducing functionally distinct CD4+ T-cell responses.Given the restricted pattern of expression of DNGR-1 across species, this strategy could prove useful for developing immunotherapy protocols in humans.

View Article: PubMed Central - PubMed

Affiliation: Immunobiology Laboratory, Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, London, UK.

ABSTRACT
DC NK lectin group receptor-1 (DNGR-1, also known as CLEC9A) is a C-type lectin receptor expressed by mouse CD8alpha+ DC and by their putative equivalents in human. DNGR-1 senses necrosis and regulates CD8+ T-cell cross-priming to dead-cell-associated antigens. In addition, DNGR-1 is a target for selective in vivo delivery of antigens to DC and the induction of CD8+ T-cell and Ab responses. In this study, we evaluated whether DNGR-1 targeting can be additionally used to manipulate antigen-specific CD8+ T lymphocytes. Injection of small amounts of antigen-coupled anti-DNGR-1 mAb into mice promoted MHC class II antigen presentation selectively by CD8alpha+ DC. In the steady state, this was sufficient to induce proliferation of antigen-specific naïve CD4+ T cells and to drive their differentiation into Foxp3+ regulatory lymphocytes. Co-administration of adjuvants prevented this induction of tolerance and promoted immunity. Notably, distinct adjuvants allowed qualitative modulation of CD4+ T-cell behavior: poly I:C induced a strong IL-12-independent Th1 response, whereas curdlan led to the priming of Th17 cells. Thus, antigen targeting to DNGR-1 is a versatile approach for inducing functionally distinct CD4+ T-cell responses. Given the restricted pattern of expression of DNGR-1 across species, this strategy could prove useful for developing immunotherapy protocols in humans.

Show MeSH
Related in: MedlinePlus