Limits...
B-Cell Gene Therapy for Tolerance Induction: Host but Not Donor B-Cell Derived IL-10 is Necessary for Tolerance.

Su Y, Zhang AH, Noben-Trauth N, Scott DW - Front Microbiol (2011)

Bottom Line: We found that peptide-IgG transduced IL-10 KO B cells have the same effects as wt B cells in tolerance induction in an experimental autoimmune encephalomyelitis model.Moreover, we demonstrated that the tolerogenic effect of peptide-IgG B cells was completely abrogated in anti-IL-10 receptor antibody treated recipients.Taken together, our results suggest that tolerance induced by peptide-IgG B-cell gene therapy requires IL-10 from the host but not donor B cells.

View Article: PubMed Central - PubMed

Affiliation: Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine Baltimore, MD, USA.

ABSTRACT
Genetically modified B cells are excellent tolerogenic antigen-presenting cells (APCs) in multiple models of autoimmunity. However, the mechanisms of action are still not completely understood. In our models, we generate antigen-specific tolerogenic B cells by transducing naïve or primed B cells with an antigen-immunoglobulin G (peptide-IgG) construct. In order to be transduced, B cells require activation with mitogens such as LPS. We and others have found that LPS stimulation of B cells upregulates the production of IL-10, a key cytokine for maintaining immune tolerance. In the current study, we defined the role of B-cell produced IL-10 in tolerance induction by using IL-10 deficient B cells as donor APCs. We found that peptide-IgG transduced IL-10 KO B cells have the same effects as wt B cells in tolerance induction in an experimental autoimmune encephalomyelitis model. Moreover, we demonstrated that the tolerogenic effect of peptide-IgG B cells was completely abrogated in anti-IL-10 receptor antibody treated recipients. Taken together, our results suggest that tolerance induced by peptide-IgG B-cell gene therapy requires IL-10 from the host but not donor B cells. These data shed important insights into the mechanisms of tolerance induction mediated by B-cell gene therapy.

No MeSH data available.


Related in: MedlinePlus

Donor B cells from IL-10 KO mice are tolerogenic. (A) LPS-activated wt or IL-10 KO B cells were transduced with pOVA323–339–IgG or a mock control vector (PLP–IgG) and 1 × 107 B cells per mouse were injected i.p. into naïve Balb/c recipients. Seven days later, mice were immunized with 25 μg OVA protein emulsified in CFA via a hind footpad and the base of tail. Two weeks after immunization, sera were collected and assayed for total anti-OVA–IgG by the endpoint ELISA method (n = 4). (B) Animals were treated as described in Figure 1A. Mice were sacrificed 2 weeks after immunization and draining lymph nodes were removed for CD4+ T-cell proliferation assay. Five × 105 cells from draining LNs were seeded per well in 96-well plates in the presence of indicated concentrations of p323–339. After 48 h, cultures were pulsed with 1 μCi/well of [3H] thymidine (Amersham Life Sciences, Arlington Heights, IL, USA) and incubated for another 16–20 h. Cells were then harvested on glass fiber filters and T-cell proliferation to p323–339 was determined by [3H] thymidine incorporation. Values represent mean Δcpm (count per minute by subtraction of the background) ± SE for four animals. Data are representative of two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3139928&req=5

Figure 1: Donor B cells from IL-10 KO mice are tolerogenic. (A) LPS-activated wt or IL-10 KO B cells were transduced with pOVA323–339–IgG or a mock control vector (PLP–IgG) and 1 × 107 B cells per mouse were injected i.p. into naïve Balb/c recipients. Seven days later, mice were immunized with 25 μg OVA protein emulsified in CFA via a hind footpad and the base of tail. Two weeks after immunization, sera were collected and assayed for total anti-OVA–IgG by the endpoint ELISA method (n = 4). (B) Animals were treated as described in Figure 1A. Mice were sacrificed 2 weeks after immunization and draining lymph nodes were removed for CD4+ T-cell proliferation assay. Five × 105 cells from draining LNs were seeded per well in 96-well plates in the presence of indicated concentrations of p323–339. After 48 h, cultures were pulsed with 1 μCi/well of [3H] thymidine (Amersham Life Sciences, Arlington Heights, IL, USA) and incubated for another 16–20 h. Cells were then harvested on glass fiber filters and T-cell proliferation to p323–339 was determined by [3H] thymidine incorporation. Values represent mean Δcpm (count per minute by subtraction of the background) ± SE for four animals. Data are representative of two independent experiments.

Mentions: Our laboratory has successfully applied the B-cell based peptide–IgG gene therapy protocol in multiple animal models for autoimmunity such as type 1 diabetes, EAE, experimental autoimmune uveitis, and also in a mouse model for hemophilia A. In this gene therapy protocol, B cells have to be activated before retroviral transduction as only dividing cells can be effectively transduced with the peptide–IgG construct. We routinely use LPS to stimulate B cells. Other B-cell mitogens such as anti-IgM and anti-CD40 have similar effects as LPS on B cells in term of tolerance induction. Surprisingly, although it could activate B cells effectively, unmethylated CpG DNA stimulation results in B cells losing their tolerogenic phenotype (Lei et al., 2005). We systematically compared the phenotypic changes of B cells upon LPS and CpG stimulation. Compared to the resting B cells, although both LPS and CpG B-cell blasts have upregulated expression of IL-10 at protein and messenger RNA levels, LPS B cells produce much more IL-10 than CpG B cells (Skupsky et al., 2007). These findings, together with the contradict reports from the literature, led us to ask whether IL-10 is critical for tolerance induction through our B-cell delivered gene therapy protocol. Therefore, in the current study, we first studied the role of B-cell derived IL-10 in our gene therapy system by using H-2d IL-10 KO mice as B-cell donors. We transduced LPS-activated B cells with a retroviral construct encoding an immunodominant epitope 323–339 fragment of ovalbumin (OVA) and IgG1 heavy chain (pOVA323–339–IgG) or a mock control (phospholipid protein, PLP). As expected, mice that received pOVA323–339–IgG transduced wt B cells exhibited reduced anti-OVA antibody. Surprisingly, pOVA323–339–IgG transduced IL-10 KO B cells also induced a significant reduction of antibody to OVA (Figure 1). This suggests that IL-10 KO B cells have normal antigen-presenting function and more importantly, that IL-10 produced by donor B cells is not required for tolerance induction. In addition, both pOVA323–339–IgG transduced wt or IL-10 KO B cells significantly reduced the T-cell response to pOVA323-339 in recipients. We confirmed these results by using H-2b IL-10 KO B cells as donors to induce tolerance to OVA protein in C57BL/6 mice (data not shown).


B-Cell Gene Therapy for Tolerance Induction: Host but Not Donor B-Cell Derived IL-10 is Necessary for Tolerance.

Su Y, Zhang AH, Noben-Trauth N, Scott DW - Front Microbiol (2011)

Donor B cells from IL-10 KO mice are tolerogenic. (A) LPS-activated wt or IL-10 KO B cells were transduced with pOVA323–339–IgG or a mock control vector (PLP–IgG) and 1 × 107 B cells per mouse were injected i.p. into naïve Balb/c recipients. Seven days later, mice were immunized with 25 μg OVA protein emulsified in CFA via a hind footpad and the base of tail. Two weeks after immunization, sera were collected and assayed for total anti-OVA–IgG by the endpoint ELISA method (n = 4). (B) Animals were treated as described in Figure 1A. Mice were sacrificed 2 weeks after immunization and draining lymph nodes were removed for CD4+ T-cell proliferation assay. Five × 105 cells from draining LNs were seeded per well in 96-well plates in the presence of indicated concentrations of p323–339. After 48 h, cultures were pulsed with 1 μCi/well of [3H] thymidine (Amersham Life Sciences, Arlington Heights, IL, USA) and incubated for another 16–20 h. Cells were then harvested on glass fiber filters and T-cell proliferation to p323–339 was determined by [3H] thymidine incorporation. Values represent mean Δcpm (count per minute by subtraction of the background) ± SE for four animals. Data are representative of two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3139928&req=5

Figure 1: Donor B cells from IL-10 KO mice are tolerogenic. (A) LPS-activated wt or IL-10 KO B cells were transduced with pOVA323–339–IgG or a mock control vector (PLP–IgG) and 1 × 107 B cells per mouse were injected i.p. into naïve Balb/c recipients. Seven days later, mice were immunized with 25 μg OVA protein emulsified in CFA via a hind footpad and the base of tail. Two weeks after immunization, sera were collected and assayed for total anti-OVA–IgG by the endpoint ELISA method (n = 4). (B) Animals were treated as described in Figure 1A. Mice were sacrificed 2 weeks after immunization and draining lymph nodes were removed for CD4+ T-cell proliferation assay. Five × 105 cells from draining LNs were seeded per well in 96-well plates in the presence of indicated concentrations of p323–339. After 48 h, cultures were pulsed with 1 μCi/well of [3H] thymidine (Amersham Life Sciences, Arlington Heights, IL, USA) and incubated for another 16–20 h. Cells were then harvested on glass fiber filters and T-cell proliferation to p323–339 was determined by [3H] thymidine incorporation. Values represent mean Δcpm (count per minute by subtraction of the background) ± SE for four animals. Data are representative of two independent experiments.
Mentions: Our laboratory has successfully applied the B-cell based peptide–IgG gene therapy protocol in multiple animal models for autoimmunity such as type 1 diabetes, EAE, experimental autoimmune uveitis, and also in a mouse model for hemophilia A. In this gene therapy protocol, B cells have to be activated before retroviral transduction as only dividing cells can be effectively transduced with the peptide–IgG construct. We routinely use LPS to stimulate B cells. Other B-cell mitogens such as anti-IgM and anti-CD40 have similar effects as LPS on B cells in term of tolerance induction. Surprisingly, although it could activate B cells effectively, unmethylated CpG DNA stimulation results in B cells losing their tolerogenic phenotype (Lei et al., 2005). We systematically compared the phenotypic changes of B cells upon LPS and CpG stimulation. Compared to the resting B cells, although both LPS and CpG B-cell blasts have upregulated expression of IL-10 at protein and messenger RNA levels, LPS B cells produce much more IL-10 than CpG B cells (Skupsky et al., 2007). These findings, together with the contradict reports from the literature, led us to ask whether IL-10 is critical for tolerance induction through our B-cell delivered gene therapy protocol. Therefore, in the current study, we first studied the role of B-cell derived IL-10 in our gene therapy system by using H-2d IL-10 KO mice as B-cell donors. We transduced LPS-activated B cells with a retroviral construct encoding an immunodominant epitope 323–339 fragment of ovalbumin (OVA) and IgG1 heavy chain (pOVA323–339–IgG) or a mock control (phospholipid protein, PLP). As expected, mice that received pOVA323–339–IgG transduced wt B cells exhibited reduced anti-OVA antibody. Surprisingly, pOVA323–339–IgG transduced IL-10 KO B cells also induced a significant reduction of antibody to OVA (Figure 1). This suggests that IL-10 KO B cells have normal antigen-presenting function and more importantly, that IL-10 produced by donor B cells is not required for tolerance induction. In addition, both pOVA323–339–IgG transduced wt or IL-10 KO B cells significantly reduced the T-cell response to pOVA323-339 in recipients. We confirmed these results by using H-2b IL-10 KO B cells as donors to induce tolerance to OVA protein in C57BL/6 mice (data not shown).

Bottom Line: We found that peptide-IgG transduced IL-10 KO B cells have the same effects as wt B cells in tolerance induction in an experimental autoimmune encephalomyelitis model.Moreover, we demonstrated that the tolerogenic effect of peptide-IgG B cells was completely abrogated in anti-IL-10 receptor antibody treated recipients.Taken together, our results suggest that tolerance induced by peptide-IgG B-cell gene therapy requires IL-10 from the host but not donor B cells.

View Article: PubMed Central - PubMed

Affiliation: Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine Baltimore, MD, USA.

ABSTRACT
Genetically modified B cells are excellent tolerogenic antigen-presenting cells (APCs) in multiple models of autoimmunity. However, the mechanisms of action are still not completely understood. In our models, we generate antigen-specific tolerogenic B cells by transducing naïve or primed B cells with an antigen-immunoglobulin G (peptide-IgG) construct. In order to be transduced, B cells require activation with mitogens such as LPS. We and others have found that LPS stimulation of B cells upregulates the production of IL-10, a key cytokine for maintaining immune tolerance. In the current study, we defined the role of B-cell produced IL-10 in tolerance induction by using IL-10 deficient B cells as donor APCs. We found that peptide-IgG transduced IL-10 KO B cells have the same effects as wt B cells in tolerance induction in an experimental autoimmune encephalomyelitis model. Moreover, we demonstrated that the tolerogenic effect of peptide-IgG B cells was completely abrogated in anti-IL-10 receptor antibody treated recipients. Taken together, our results suggest that tolerance induced by peptide-IgG B-cell gene therapy requires IL-10 from the host but not donor B cells. These data shed important insights into the mechanisms of tolerance induction mediated by B-cell gene therapy.

No MeSH data available.


Related in: MedlinePlus