Immune regulation of transgene expression in the brain: B cells regulate an early phase of elimination of transgene expression from adenoviral vectors

Abstract

Cellular immune mechanisms that regulate viral gene expression within infected brain cells remain poorly understood. Previous work has shown that systemic immunization against adenovirus after vector delivery to the brain results in complete loss of brain cells infected by adenoviral vectors. Although T cells play an important role in this process, we demonstrate herein that B cells also significantly regulate transgene expression from the CNS. After the systemic immunization against adenovirus of animals injected via the brain with an adenoviral vector 30 days earlier, we uncovered substantial infiltration by CD19+ B cells of the area of the brain transduced by the virus. This suggests the involvement of B cells in the adaptive immune response-mediated loss of transduced cells from the brain. Confocal analysis of these brains demonstrated physical contacts between transduced brain cells and CD19+ cells. To test the hypothesis that B cells play a causal role in the loss of infected cells from the brain, we demonstrated that animals devoid of B cells were unable to eliminate transgene expression at early time points after immunization. This demonstrates that B cells play a necessary role in the loss of transgene expression at early, but not late, time points postimmunization. Thus, these data have important implications for our understanding of the role of B cells as immune effectors during the immune-mediated clearance of viral infections from the CNS, and also for understanding mechanisms operating in brain autoimmunity, as well as for the potential safety of clinical gene therapy for brain diseases.

FIG. 1

Qualitative and quantitative microscopy analysis of immune cell infiltration at time points postimmunization, using stereological quantification. (A-C) CD19⁺ B cells selectively infiltrate the area of the brain infected with adenoviral vectors. (A) Schematic view of the brain at the level of the striatum; the boxed area illustrates the injection site, which is shown in (B) (section immunoreacted for the marker transgene TK) and (C) (section immunoreacted for CD19⁺ B cells). str, striatum; ctx, neocortex; ec, external capsule. (D-F) Infiltration of CD19⁺ B cells in C57BL/6 mice injected with RAd-TK and immunized with RAd-HPRT 30 days later [(D) TK (green); (E) CD19⁺ (red); (F) merged], shown 30 days after the immunization analyzed by confocal microscopy. (G) Stereological quantification of CD19⁺ T cells in the brains of animals injected with RAd-TK and immunized with RAd-HPRT, shown in the absence of immunization (0 days) and 14 and 30 days postimmunization. ^**p < 0.01 versus controls.

FIG. 2

Qualitative and quantitative laser confocal microscopy analysis of CD19⁺ immune cell interactions with adenovirally transduced brain cells. (A-C) Quantitative measures of contacts between CD19⁺ T cells and transduced cells: (A) percentage of CD19⁺ cells contacting TK cells; (B) percentage of TK cells with CD19⁺ contacts; (C) average number of CD19⁺ T cell contacts per TK-expressing cell. This demonstrates that a minority (up to 20%) of CD19⁺ cells contact transduced cells; only 10% of transduced cells are contacted by a CD19⁺ cell; and on average only one CD19⁺ cell will contact a target transduced cell. (D) Projection image of a group of transduced neurons expressing TK (green), with closely apposed CD19⁺ cells (red). (E) Single contact in a 0.5-μm-thick optical confocal section (CD19⁺ [red]; TK [green]; DAPI [blue nuclei]), at 30 days postimmunization; thinner (0.5 μm) optical sections are shown to demonstrate actual contacts, which are indicated by the white arrows.

FIG. 3

Qualitative and quantitative analysis of TK expression in the CNS of animals devoid of B cells. (A) Immunohistochemistry images (insets, lower power) of TK transgene expression in the striatum of Igh-6^-/- mice. Left to right: No immunization, and 30, 60, 90, and 120 days postimmunization. TK expression persists 30 days after systemic RAd-HPRT immunization and clearly decreases to significantly lower levels by 90-120 days postimmunization. (B) Stereological quantification of TK-immunoreactive cells in the striatum of nonimmunized Igh-6^-/- mice (no-imm), or HPRT-immunized Igh-6^-/- mice at 30-120 days postimmunization. (C) Stereological quantification of TK-immunoreactive cells in the striatum of HPRT-immunized Igh-6^-/- mice compared with control HPRT-immunized C57BL/6 mice, both shown 30 d postimmunization. Notice that transgene expression has been eliminated in control immunized C57BL/6 mice, whereas there has been no decrease in the number of TK-immunoreactive cells in immunized B celldeficient Igh-6^-/- mice. (D) Stereological quantification of TK-immunoreactive cells in the striatum of C57BL/6 mice 120 days after sham immunization, showing continued transgene expression in the absence of immunization.