To test the concept of site-specific delivery of cytokines by transduced T cells, the murine model of EAE was used. An encephalitogenic, MBP-specific T cell hybridoma, G1.15H, was used in these preliminary studies due to its transduction efficiency. Adoptive transfer of the unmodified G1.15H to naive (SJL/J × PL/J)F1 mice verified its EAE-inducing, and thus CNS-homing, capability (Fig. ). It was decided to transduce this T cell line for expression of the IL-4 gene since this cytokine has been demonstrated to be a mediator in EAE regulation as evidenced by its role in the natural recovery from disease (13
), experimental protection from disease after oral tolerance induction (14
), and after immunization with altered peptide ligands of MBP (15
Figure 1 Adoptive transfer of EAE using the MBP-specific T cell hybridoma G1.15H. Three naive (PL/J × SJL/J)F1 mice were given 106 G1.15H cells intravenously on day 0. Each mouse (circles, squares, or triangles, respectively) was scored daily for clinical (more ...)
After transduction of G1.15H with an IL-4–encoding retrovirus and drug selection, the line was confirmed to secrete IL-4 by ELISA (Table ). Subsequent limiting dilution cloning of these transduced hybrids yielded individual lines secreting between 1 and 8 pg/ml IL-4/105 cells (Table ).
IL-4 and IL-10 Secreted by Cell Lines Used in this Study
10 d after MBP immunization to induce EAE, (SJL/J × PL/J)F1 mice were given transduced or control cells. Clinical EAE was ameliorated by adoptive transfer of 106 transduced T cells secreting high levels of IL-4 (line N5). Disease onset for the IL-4 treatment group was delayed by 2 d (P <0.01 Student's t test) and the average disease score was significantly lower (P <0.05) than an experimental group receiving cells transduced to express IL-10, or control mice receiving untransduced cells or PBS (Fig. ).
Figure 2 Adoptive transfer of T cells transduced to express IL-4 ameliorates MBP-induced EAE. Mice immunized to induce EAE were treated with T cells transduced to express IL-4 (circles) or IL-10 (triangles), untransduced T cells (diamonds), or PBS (squares), (more ...)
That amelioration of disease was due to local delivery of IL-4 was supported by several experimental approaches. In the first, mice that received T cells transduced to express IL-4 were bled at various time points after cell transfer, and serum IL-4 was determined by ELISA. No cytokine was detected in the serum until day 24 after transfer, well after initial recovery from disease. Serum IL-4 levels at this time were low, ranging from 1.19 pg/ml to 2.53 pg/ml (Table ). This finding indicates that disease remission was not due to high systemic levels of IL-4. In a second experimental approach, we verified the presence of transduced T cells in the CNS at the time of disease amelioration by testing spinal cord tissue from treated animals for retroviral-specific IL-4 expression by reverse transcriptase PCR analysis. Retroviral IL-4 transcription could be detected in the CNS of treated animals 15 d after transfer of transduced T cells (data not shown). The third experimental approach demonstrated that amelioration of disease was dependent on T cell homing to the CNS.
Serum Levels of IL-4 at Various Times after Transfer of T Cell Hybrids Transduced to Express IL-4
We reasoned that T cells transduced to express IL-4 that could not recognize CNS antigens would be ineffective at delivering IL-4 to the CNS. To test this hypothesis, additional transfer experiments were performed, using as controls, transduced T cells expressing IL-4 but lacking antigen specific TCR expression. In the first experiment, an IL-4– expressing transductant of the hybridoma fusion partner BW5147 was used as a control. Transfer of this cell line to MBP-immunized mice had no effect on the disease course, whereas transfer of an MBP-specific line, clone 4.9, secreting low levels of IL-4 (matched to the fusion partner control) had a significant therapeutic effect (P <0.05; data not presented). In the second experimental approach, a TCR negative variant of the IL-4–secreting disease ameliorating N5 clone was used as a control. This line had significantly less effect on the disease course (P <0.05) when compared to its TCR-expressing counterpart (Fig. ).
Figure 3 Amelioration of EAE is dependent upon TCR expression by IL-4–secreting T cells. MBP-immunized (PL/J × SJL/J)F1 mice (10 mice/group) were given 106 transduced TCR-positive, IL-4–secreting N5 cells (circles) or TCR-negative, IL-4–secreting (more ...)
The majority of approaches taken to control autoimmune disease result in deleterious side effects due to the systemic administration of antiinflammatory agents. Our findings indicate that the disease processes can be modulated by the transfer of MBP-specific T cells which have been retrovirally modified to express the antiinflammatory cytokine IL-4. Disease amelioration was due to local, rather than systemic, delivery of IL-4 as evidenced by the following points. (a) Serum levels of IL-4 were undetectable at the time of disease remission. (b) Retroviral-encoded IL-4 expression could be detected in the CNS of treated mice. (c) Disease amelioration was dependent upon antigen-specific T cell receptor expression on transduced T cells, indicating that T cell antigen recognition and presumably “trafficking” were necessary for delivery of cytokines.
The use of antigen-specific T cells, transduced to express regulatory cytokines, selectively target antiinflammatory molecules to the site of pathology represents a unique therapeutic approach to the treatment of autoimmune disease. T cells are advantageous since they are easily manipulated and expanded in tissue culture before reintroduction into the host. More importantly, the antigen specificity of T cells allows them to home to depots of antigen in the body, such as at inflammatory sites of autoimmune disease. This has been demonstrated in the murine model of EAE where MBP-specific T cells have been shown to traffic to the CNS, both during the induction phase of disease as well as during relapses in a relapsing-remitting model of EAE (16
). Results presented here demonstrate that a statistically significant benefit can be observed when mice, immunized to develop EAE, are given MBP-specific T cells retrovirally transduced to express IL-4.