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. 2000 Jul;106(2):245-52.
doi: 10.1172/JCI9168.

Retroviral Gene Therapy With an Immunoglobulin-Antigen Fusion Construct Protects From Experimental Autoimmune Uveitis

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Free PMC article

Retroviral Gene Therapy With an Immunoglobulin-Antigen Fusion Construct Protects From Experimental Autoimmune Uveitis

R K Agarwal et al. J Clin Invest. .
Free PMC article

Abstract

Immunoglobulins can serve as tolerogenic carriers for antigens, and B cells can function as tolerogenic antigen-presenting cells. We used this principle to design a strategy for gene therapy of experimental autoimmune uveitis, a cell-mediated autoimmune disease model for human uveitis induced with the uveitogenic interphotoreceptor retinoid-binding protein (IRBP). A retroviral vector was constructed containing a major uveitogenic IRBP epitope in frame with mouse IgG1 heavy chain. This construct was used to transduce peripheral B cells, which were infused into syngeneic recipients. A single infusion of transduced cells, 10 days before uveitogenic challenge, protected mice from clinical disease induced with the epitope or with the native IRBP protein. Protected mice had reduced antigen-specific responses, but showed no evidence for a classic Th1/Th2 response shift or for generalized anergy. Protection was not transferable, arguing against a mechanism dependent on regulatory cells. Importantly, the treatment was protective when initiated 7 days after uveitogenic immunization or concurrently with adoptive transfer of primed uveitogenic T cells. We suggest that this form of gene therapy can induce epitope-specific protection not only in naive, but also in already primed recipients, thus providing a protocol for treatment of established autoimmunity.

Figures

Figure 1
Figure 1
Structure of tolerogenic mIRBP161-180-IgG (a) and control cI 12-26-IgG (b) constructs. LTR, long terminal repeat; Ψ, packaging signal; NeoR, neomycin resistance gene; P&E, promoter and enhancer.
Figure 2
Figure 2
EAU scores in recipients of LPS blasts transduced with mIRBP161-180-IgG, infused 8–10 days before uveitogenic challenge with (a) murine 161-180 or human 161-180, or (b) human peptide or whole IRBP. The incidence (number of positive out of total mice) is shown within the bars. The data were compiled from five experiments.
Figure 3
Figure 3
Ocular histology of peptide-immunized mice that had been pretreated with either the LPS-stimulated B cells transduced with the mock control construct (a) or with the tolerogenic construct (b), compared with retina of naive mice (c). Eyes were processed for histology 21 days after uveitogenic immunization. Shown are results after immunization with human peptide. Ocular pathology of mice challenged with the murine construct was essentially identical.
Figure 4
Figure 4
Lymphocyte proliferation to murine 161-180 epitope in protected mice. Shown is an average of two identical experiments. Counts were normalized to mock control at 30 μM peptide after background subtraction (100%) to compensate for interexperiment variation. (Actual 100% values for spleen and lymph node, respectively, were 31,650 and 42,900 cpm, with background of 7,000 cpm). The EAU scores of these mice are shown in Figure 2.
Figure 5
Figure 5
IL-2, IFN-γ, and IL-10 production by spleen cells of tolerized mice to 30 μM peptide. Shown are IFN-γ and IL-10 production as assayed 21 days after immunization (average of three experiments) and IL-2 as assayed only 10 days after immunization (single experiment). Values are normalized against the mock control (100%). Lower level of detectability in picograms per milliliter was 26 for IL-2, 30 for IFN-γ, and 10 for IL-10. Actual 100% values in picograms per milliliter were 3,600 for IL-2, 910 for IFN-γ, and 56 for IL-10. The pattern of IFN-γ and IL-10 responses on day 10 was the same as on day 21, though the absolute amounts secreted were higher. Lymph node cytokine responses were essentially identical to spleen responses.
Figure 6
Figure 6
Humoral response to m161-180 in tolerized mice. The bars show average IgG2a and IgG1 titers of 20 mice compiled from four repeat experiments. The IgG1/IgG2a ratio shown at the top was calculated as an average of individual Ig isotype ratios.
Figure 7
Figure 7
EAU (a) and DTH (b) scores in recipients of B cell–depleted splenocytes. (a) EAU scores in recipients of cells from donors who received infusion of LPS blasts transduced with the protective or mock control retroviral constructs. Mice were challenged with the human peptide 24 hours after the infusion and scored for EAU on day 21. (b) DTH scores to IRBP in recipients of spleen cells prepared by the same method from IRBP-immunized or from naive donors (positive control). Recipients were challenged with 10 μg of IRBP into the ear pinna 72 hours after transfer. DTH scores were read after 48 hours.
Figure 8
Figure 8
EAU scores in primed recipients of LPS blasts, infused 7 days after uveitogenic challenge with h161-180. The incidence (number of positive out of total mice) is shown within the bars.

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