Immunotherapy of autoimmune encephalomyelitis with redirected CD4⁺CD25⁺ T lymphocytes

CD4⁺CD25⁺ regulatory T cells potently suppress immune responses.¹  When therapeutically administered, even antigen nonspecific CD4⁺CD25⁺ T cells can prevent or treat immunopathology in several disease models.^2-8  Yet, infusions of nonspecific regulatory cells would be expected to have poor specificity and low potency. Indeed, some studies have found that the administration of antigen-specific regulatory T cells substantially increases therapeutic activity.^9,10 

Considering the low precursor frequency of regulatory T cells, identifying, isolating, and expanding antigen-specific cells for immunotherapy would be technically challenging. As an alternative, we have developed a gene-therapeutic approach that specifically redirects regulatory T cells against pathologic T cells. Therapeutic cells are transgenically modified with a chimeric antigen–major histocompatibility complex (MHC)–ζ receptor.^11-13  The extracellular antigen-MHC serves as a bait, engaging the T-cell receptor (TCR) of potentially pathologic antigen-specific T cells. This engagement generates an immunoreceptor tyrosine-based activation motif (ITAM)–mediated signal through the chimeric receptors'cytoplasmic ζ chain.

We hypothesized that the chimeric receptors, by enforcing the interaction between a CD4⁺CD25⁺ regulatory T cell and its target, should antigen-specifically focus CD4⁺CD25⁺ T-regulatory activity against autoreactive T cells. We previously showed that T cells modified with a chimeric receptor that genetically links the 89-101 epitope of the myelin basic protein (MBP) with its IA^s-restricting MHC and the cytoplasmic domain of TCR-ζ (receptor-modified T cells [RMTCs]) specifically recognize MBP89-101–reactive T cells in vitro and in vivo.¹²  We test here whether the MBP89-101-IA^s-ζ receptor can redirect CD4⁺CD25⁺ T cells against autoreactive MBP-specific T cells and thereby treat experimental allergic encephalomyelitis (EAE).

Female SJL/J mice (Jackson Laboratory, Bar Harbor, ME) were age matched. 25S89P (MBP89-101-IA^s-ζ transgenic [Tg]) mice have been described elsewhere.^12,14 

MBP89-101 (VHFFKNIVTPRTP) was synthesized and purified to more than 90% purity by the St Jude Hartwell Center.

Anti-CD4 (RM4-5), anti-CD25 (7D4), and anti-CD28 antibodies were obtained from Pharmingen (San Diego, CA). 2C11 (anti-CD3ϵ; gift of M. Blackman, Trudeau Institute, Saranac Lake, NY) was purified from hybridoma supernatants. Sorting was performed with a Mo-Flo hi-speed cell sorter (DAKO, Fort Collins, CO).

Disease was induced and scored on a 0 to 5 scale as previously described.^12,15 

Proliferation was measured after a 72-hour culture by ³H-thymidine (³H-TdR) incorporation. Cytokines were measured after a 48-hour culture by enzyme-linked immunosorbent assay (ELISA) or Bio-Plex (Bio-Rad, Hercules, CA).

Approximately 5% of splenic and lymph node (LN) CD4⁺ T cells from MBP89-101-IA^s-ζ Tg mice^12,14  express the CD25 activation marker (data not shown). Both Tg and non-Tg CD4⁺CD25⁺ T cells showed limited proliferation themselves in response to stimulation, a characteristic of regulatory T cells (Figure 1A). The Tg and non-Tg cells also equivalently suppressed the proliferation of naive T cells. Therefore, the suppressive activity of CD4⁺CD25⁺ T cells is unaffected by the chimeric receptor transgene.

We previously showed that unsorted MBP89-101-IA^s-ζ RMTCs are specifically stimulated by MBP89-101–reactive 6F11 T cells.¹²  To verify that cognate TCR could similarly stimulate sorted CD4⁺CD25⁺ RMTCs, we measured cytokine production by them or CD4⁺CD25^- RMTCs in response to 6F11 T cells or control anti-CD3ϵ antibody (Figure 1B). The pattern of cytokines produced by Tg or non-Tg CD4⁺CD25⁺ T cells to control anti-CD3ϵ was identical, as was that produced by Tg or non-Tg CD4⁺CD25^- T cells. Therefore, the chimeric receptor did not significantly impact the T-cell cytokine profile. As expected, cytokine production differed between CD4⁺CD25⁺ and CD4⁺CD25^- T cells, with CD4⁺CD25⁺ cells producing similar amounts of interleukin 4 (IL-4), reduced interferon γ (IFN-γ), and increased amounts of IL-10 and transforming growth factor β (TGF-β) compared with CD4⁺CD25^- cells. Most importantly, whereas non-Tg cells failed to produce cytokines in response to 6F11 stimulation, Tg cells produced quantities of each cytokine comparable to that induced by anti-CD3ϵ. This demonstrates that the chimeric receptor serves as an effective surrogate for the TCR in CD4⁺CD25⁺ T cells, can engage MBP89-101–specific T cells, and can redirect RMTC function.

To define the clinical efficacy of the CD4⁺CD25⁺ RMTCs, we tested their ability to prevent EAE induced by MBP89-101 peptide. We found limited and inconsistent effects of doses of up to 3 × 10⁶ non-Tg CD4⁺CD25⁺ T cells, which would be expected to roughly double to triple the total circulating regulatory T-cell number (data not shown). Partial disease suppression has been observed in 2 other EAE models using similar regulatory cell doses, suggesting that this represents an approximate threshold for disease abatement.^2,3  In contrast to the limited effects of nonspecific regulatory T cells, 1 × 10⁶ CD4⁺CD25⁺ RMTCs consistently and potently suppressed EAE (Figure 2A).

Suppression did not result from MBP89-101-IA^s-ζ chimeric receptor expression alone. Adoptive transfer of as many as 1 × 10⁷ CD4⁺CD25^- RMTCs failed to influence disease outcome (data not shown). In contrast, as few as 2.5 × 10⁵ CD4⁺CD25⁺ RMTCs diminished EAE (P < .05 d30-45 vs saline), and disease suppression was nearly complete at doses of 5 × 10⁵ or more cells (P < .05 vs saline, Figure 2B). Suppression was antigen specific and prophylaxis with MBP89-101-IA^s-ζ CD4⁺CD25⁺ RMTCs could not prevent EAE induced with a different autoantigen, proteolipid protein (PLP) 139-151 (Rajshekar Alli and T.L.G., unpublished observations, September 2004).

Unlike disease prevention, in which RMTCs may act against naive T cells, disease treatment requires effectiveness after T-cell activation. We therefore treated at day 11, corresponding with the development of early disease symptoms (Figure 2C). The CD4⁺CD25⁺ RMTCs, in contrast to antigen-nonspecific CD4⁺CD25⁺ T cells, efficiently suppressed the incipient autoreactive T-cell response at this time.

We hypothesized that the RMTCs acted directly on differentiated effector T cells, rather than by influencing their priming and development. To test this, we induced EAE by the adoptive transfer of mature pathologic effector T cells. CD4⁺CD25⁺ RMTCs, but not control cell populations, were highly effective in blocking adoptively transferred EAE (Figure 2D) and can therefore directly suppress pathologic T-cell function.

The specificity of pathologic T cells in EAE diversifies with time from the inducing MBP89-101 antigen to include additional autoantigenic epitopes, particularly PLP139-151 and PLP178-191.¹⁶  This epitope spread helps sustain the autoimmune pathology. Responses to both of these PLP epitopes were detected 31 days after immunization.¹²  We delayed RMTC treatment until day 31 to test their effectiveness after epitope spread. Whereas non-Tg CD4⁺CD25⁺ T cells and CD4⁺CD25^- RMTCs remained ineffective at this late time, CD4⁺CD25⁺ RMTCs ameliorated disease (Figure 2E). Clinical disease scores returned toward baseline. Mortality decreased; 5 of 9 mice receiving saline treatment and 5 of 9 receiving control non-Tg CD4⁺CD25⁺ cells succumbed to disease, whereas 8 of 8 receiving CD4⁺CD25⁺ RMTCs survived. Remission was sustained for more than 80 days (Figure 2E and data not shown).

CD4⁺CD25⁺ T cells require close contact with their targets and must be activated to suppress. Our chimeric receptors facilitate this, luring self-specific target cells and stimulating the CD4⁺CD25⁺ RMTCs. Clinical translation of RMTCs will be challenging and require an improved understanding of the epitope dynamics and pathogenesis of human autoimmune diseases, and of how regulatory RMTCs may best be applied. Nevertheless, acquiring requisite numbers of immunotherapeutic RMTCs for clinical application should be achievable. We have transduced small numbers of CD4⁺CD25⁺ T cells with retroviral vectors, and have shown that they remain functional when expanded in vitro¹⁷  (Christine Duthoit and T.L.G., unpublished data, August 2001). Cotransduction of chimeric receptors and FoxP3, which has been shown to confer regulatory activity, into CD4⁺CD25^- T cells may generate similar regulatory cell populations.^4,8 

Critically, the RMTCs ameliorated EAE even after the pathologic T-cell response disseminated to include specificities not directly targeted by the CD4⁺CD25⁺ RMTCs. Due to the complexity of the pathologic T-cell repertoire at the time of intervention in autoimmune diseases, this capacity for bystander T-cell inhibition will be essential for this or similar approaches to ultimately achieve clinical success.

We thank Richard Cross, Jennifer Hoffrage, and Dick Ashmun for assistance with flow cytometric sorting, and Janet Gatewood for assistance with Bio-Plex cytokine analysis.