Retroviral gene therapy with an immunoglobulin-antigen fusion construct protects from experimental autoimmune uveitis (original) (raw)
Recipients of LPS blasts transduced with antigen-IgG construct are protected from EAU. Zambidis et al. (6) reported that infusion of retrovirally transduced cells containing a construct encoding a dominant epitope of phage λ repressor protein in frame with an IgG heavy chain (cI 12-26-IgG) led to long-term suppression of the immune response to that epitope. To adapt this methodology to immunotherapy of autoimmune disease, the murine 161-180 epitope of IRBP was engineered into the same vector (m161-180-IgG) (Figure 1).
LPS-stimulated B cells prepared from spleens of naive B10.RIII mice were transduced with m161-180-IgG vector or the control cI 12-26-IgG vector. Recipients of 30–40 million transduced cells were challenged after 8–10 days for induction of EAU by immunization with the murine or the human homologue of peptide 161-180 (designated as m161-180 or h161-180, respectively). Note that whereas treatment was always with the autologous murine epitope, uveitogenic challenge was either with the murine or with the human homologue (for reasons stated in Methods). Histopathology of eyes obtained 21 days after immunization, when disease in controls is at its peak, showed that the single infusion of transduced B cells afforded highly significant protection from disease, whether induced by challenge with the autologous murine or the homologous human epitope (Figure 2a). Many of the treated mice remained completely free of disease. Importantly, mice immunized with the native IRBP molecule were also significantly protected, indicating that tolerance to dominant epitope can afford protection against the whole multiepitope protein (Figure 2b). Typical EAU histopathology in mice that received cells transduced with the tolerogenic versus control vectors is shown in Figure 3. In experiments not shown here, animals challenged with peptide as late as 2 months after the tolerogenic infusion were still protected (data not shown).
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.
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.
To find out whether packaging cells carried over with the transduced B cells might have contributed to the protection, their percentage in the tolerizing inoculum was assessed by staining cytocentrifuged preparations with Giemsa and by performing a differential count of lymphocytes versus fibroblasts. Contamination by packaging cells was found to be 3–5%. Two million p161-180-IgG retrovirus-producing irradiated packaging cells (equivalent to 6% contamination) infused into recipient mice had no protective effect whatsoever against peptide challenge, confirming that tolerogenesis was caused by the transduced B-cell blasts.
Cell-mediated and humoral responses in treated mice. Mice infused with transduced LPS blasts as above were challenged for DTH with the immunizing peptide on day 19. Mice that were functionally protected from EAU (whose disease scores are shown in Figure 2) had moderately, albeit significantly, reduced DTH responses (P < 0.002, data not shown). Draining lymph node and spleen cells collected from these mice on day 21 were tested for proliferation against the immunizing peptides (Figure 4). Mice challenged with the murine peptide had significantly and reproducibly depressed proliferative responses and exhibited a dose-response shift over approximately 1.5 logs of antigen concentration. The effect on in vitro proliferation to human peptide was less pronounced, which probably reflects the fact that the tolerizing peptide and the immunizing peptide are not identical (data not shown).
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.
Antigen-specific cytokine responses were examined 10 and 21 days after immunization. We chose to study responses only of mice immunized with the murine 161-180 homologue so that any cross-reactive responses would not obscure specific hyporesponsiveness. Type 1/proinflammatory cytokines IL-2, IFN-γ, and TNF-α and type 2/anti-inflammatory cytokines IL-4, IL-5, IL-10, and TGFβ1 were assayed by ELISA in 48-hour supernatants of cells stimulated with the immunizing peptide. Diminished cytokine responses resembling the effects on DTH and lymphocyte proliferation were seen for IL-2, IFN-γ, and IL-10 (Figure 5). TNF-α secretion by spleen and lymph node cells of the protected group was half that of the control group on day 10 (but not on day 21), and several other cytokines, including TGF-β, did not exhibit consistent differences (data not shown).
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.
Antigen-specific IgG1 and IgG2a antibody responses were assayed in individual sera collected from recipients of protective or mock control cells 21 days after a uveitogenic challenge with mouse 161-180 peptide. Because the switch factor for IgG1 is IL-4 and the switch factor for IgG2a is IFN-γ, these antibody isotypes can also serve as a readout of the Th1 or Th2 bias of the antigen-specific response. Thus, skewing of the IgG1/IgG2a ratio would be indicative of an immune deviation, whereas an overall reduction in both isotypes would indicate a mechanism unrelated to the Th1/Th2 balance. Isotype-specific ELISA showed that, on average, both IgG1 and IgG2a anti-IRBP antibody titers in protected mice were reduced to half the values of controls, although individual titers were variable (Figure 6). Recipients of 161-180-IgG–transduced cells showed a trend toward higher IgG1/IgG2a ratios than the control group. Because of the large individual variability, however, the difference between the group averages did not attain statistical significance (t test).
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.
Protection from EAU is not transferable. We next wished to examine whether spleens of mice that received the protective treatment contain regulatory cells that could adoptively transfer protection to untreated recipients. Donor mice were given LPS-stimulated B cells transduced with the protective or control retroviral construct. After 10 days, when the animals would normally be challenged for EAU induction, their spleens were removed and depleted of B220+ cells by immunomagnetic beads (resulting in < 0.5% residual B cells) to minimize carryover of transduced B cells that could be tolerogenic APCs in the adoptive transfer recipient. Recipients infused with an equivalent of two B cell–depleted donor spleens were challenged for EAU development. Recipients of splenocytes from donors who received the protective treatment developed EAU scores equivalent to scores developed by recipients of control splenocytes, suggesting absence of regulatory cells in the transferred population (Figure 7a). Because this interpretation is based on a negative result, a positive control was used to show that a measurable immune function could be successfully transferred under these conditions. In a parallel experiment, an equivalent number of B cell–depleted splenocytes were able to transfer a DTH response from IRBP-immunized donors to naive recipients (Figure 7b).
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.
Induction of protection in primed recipients. For an immunotherapy strategy to be clinically relevant, it must be effective in an already primed subject. To test whether this kind of retroviral gene therapy would be able to ameliorate disease if administered after priming, we infused LPS blasts transduced with the protective or the control constructs into recipients that had been immunized with human 161-180 seven days earlier.
Clinical onset of EAU in this model normally occurs between 9 to 12 days. To confirm that on day 7 the animals have primed cells that are functional in terms of disease, we extracted spleen and lymph node cells from animals immunized with human 161-180 seven days before, and subjected them to a standard 3-day in vitro activation before adoptive transfer into naive recipients. Recipients of 40 × 106 cells developed EAU with a mean score of 3, which is directly comparable to scores seen routinely with recipients of an equivalent number of cells extracted 2–3 weeks after immunization.
A single infusion of gene-transduced cells, which was highly effective in protecting preimmune animals, was ineffective in ameliorating disease in primed recipients. However, three consecutive infusions, given every other day, were highly effective in reducing EAU scores (Figure 8). Furthermore, a single infusion was able to reduce EAU elicited by adoptive transfer of primed T cells. Thus, six of six mice given 30 × 106 m161-180-IgG–transduced B cells and challenged 12 hours later by adoptive transfer of uveitogenic T cells from donors immunized with peptide 161-180 were completely protected. In contrast, three of six recipients of mock control T cells and three of five naive recipients developed EAU. This treatment strategy can, therefore, be protective in a situation where primed effector cells have already been generated.
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.