An Organic CD4 Inhibitor Reduces the Clinical and Pathological Symptoms of Acute Experimental Allergic Encephalomyelitis

doi:10.1006/jaut.2001.0576, available online at http://www.idealibrary.com on

Journal of Autoimmunity (2002) 18, 169–179

An Organic CD4 Inhibitor Reduces the Clinical and Pathological Symptoms of Acute Experimental Allergic Encephalomyelitis Andrea E. Edling, Swati Choksi, Ziwei Huang and Robert Korngold Kimmel Cancer Institute, Jefferson Medical College, Philadelphia, PA

Received 9 August 2001 Accepted 27 August 2001 Key words: autoimmunity, CD4, EAE, T cells, therapeutics

CD4 + T cells have an important role in mediating the pathogenesis of many human and experimental autoimmune diseases including experimental allergic encephalomyelitis (EAE), a demyelinating animal model for multiple sclerosis (MS). We applied a computer screening approach to select a small organic molecule, TJU103, that would specifically inhibit autoreactive CD4 + T cells by disrupting the function of the CD4 molecule during activation. Upon studying the therapeutic effect of TJU103 in acute EAE, it was found that administration shortly before or after the onset of clinical symptoms reduced the severity of disease in both SJL and SWXJ-14 mouse models. In addition, TJU103 treatment could affect both in vivo responses to EAE rechallenge and secondary in vitro proliferation and cytokine production of T cells responding to proteolipid protein epitope 139–151 (PLPe). These results demonstrate the potential of the TJU103 organic inhibitor for future clinical application in CD4 + T cell-mediated diseases. © 2002 Elsevier Science Ltd

Introduction

that a surface binding pocket mainly formed by the CDR3 and CC′ loops allows for the formation of such oligomers, and a computer screening approach was used to select a small organic molecule, TJU103, based on its potential ability to bind this pocket on human CD4 [11]. Initial studies suggested that TJU103 could exhibit inhibitory activity in CD4 + T cell-mediated responses [11]. Murine EAE is an inflammatory paralytic autoimmune disease of the central nervous system that serves as a model for the human demyelinating disease MS, and has often been used to test and evaluate potential therapeutic agents. Studies have indicated that encephalitogenic CD4 + T cells play a role in both EAE and MS disease pathogenesis. The elimination of CD4 + T cells prevents the adoptive transfer of EAE in mice [12], and this subset predominates in the early lesions of both EAE and MS [13, 14]. Treatments for MS have included the use of general drug-induced immunosuppression, which involves an increased risk of susceptibility to infections and potential onset of neoplasia. Two current forms of therapy, IFN- [15, 16] and Copaxone [17] have been shown to reduce the severity and frequency of clinical exacerbations. However, these treatments do not specifically target the autoreactive CD4 + T cells. Treatment with mAb to CD4 has been successful in a number of autoimmune diseases including EAE. Anti-CD4 mAb therapy has the capacity to prevent and/or decrease the severity of EAE [18–20], however, this approach lacks Ag specificity, involves general

The CD4 molecule is a transmembrane glycoprotein expressed on the surface of helper T cells, with the extracellular segment consisting of tandem domains (D1–4) that share high homology with other members of the Ig superfamily [1]. Similar to the V
Ig chain, which uses three complementary determining regions (CDR)1–3 to contact antigen (Ag) [2], CD4D1 may use similar CDR-like structures to mediate protein– protein interactions on the surface of the T cell during activation. CD4 functions as a coreceptor for the T cell receptor (TCR), specific for Ag peptides bound by MHC class II molecules on the surface of Ag presenting cells. CD4 helps stabilize the molecular interaction by binding the 2 domain of MHC class II [3], and is also involved in signal transduction by the association of tyrosine kinase p56lck with its cytoplasmic tail [4]. The CD4/MHC class II interaction is critical for optimal CD4 + T cell activation [5], and involves the CD4D1 domain with surface contact sites consisting of the CDR2, CDR3 and CC′ loops of the  sheet structure [6, 7]. It has been proposed that oligomerization of CD4/MHC complexes may provide the necessary focused TCR and p56lck signaling for proper initiation of activation [8–11]. Furthermore, it was hypothesized Correspondence to: Robert Korngold, Kimmel Cancer Institute, Jefferson Medical College, 233 S. 10th Street, Philadelphia, PA 19107. Tel: 215-503-4552; Fax: 215-923-4153; E-mail: [email protected] 169 0896–8411/02/020169+11 $35.00/0

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immunosuppression, and can lead to immunoreactivity to the mAb, limiting its use. An alternative approach that could provide specific inhibition of the activated autoreactive CD4 + T cells in EAE would be to use TJU103 to disrupt the oligomerization of CD4/ MHC class II complexes. In this study we examined the range of efficacy of TJU103 in EAE, and found that it significantly reduced the severity of acute disease in both the SJL and SWXJ-14 mouse models of EAE, when administered before the onset of clinical symptoms. TJU103 also resulted in decreased mononuclear cell infiltration in brain and spinal cord tissue samples. Furthermore, TJU103 downregulated both Th1 and Th2 cytokine profiles in response to PLPe in vitro, and exhibited inhibitory effects in vivo, both after the appearance of EAE symptoms and upon rechallenge with Ag.

Induction of EAE For the induction of EAE, SJL or SXWJ-14 mice were immunized, s.c., at two sites in the flank on d0 and 7 with either 100 g PLPe or 1 mg mouse spinal cord homogenate (MSCH), prepared from SJL mice, as previously described [25]. Ag was prepared in a 1:1 ratio of PBS with CFA/H37Ra (Difco, Detroit, MI). Mice were evaluated daily for clinical symptoms of disease and scored in blinded fashion, based on the following criteria: 0, no clinical symptoms; 1, flaccid tail and/or hind limb weakness; 2, flaccid tail with moderate hind limb weakness; 3, severe hind limb and mild forelimb weakness; 4, total hind limb paralysis and moderate forelimb weakness; 5, quadriplegia and/or moribund. For rechallenge responses, SJL mice that had developed acute EAE were allowed to go into remission, and were given another s.c. inoculation of PLPe/CFA on d50.

Materials and Methods Proliferation assays Mice Female SJL/J (H2 ) mice were purchased from the NCI/Frederick Cancer Research and Developmental Center (Frederick, MD). Female SWXJ-14/Bm mice were purchased from the Jackson Laboratory (Bar Harbor, ME). All mice were housed in a pathogen-free environment and used for experiments between the ages of 7–10 weeks. Animal care and use was in compliance with institutional guidelines. TCR-HA SFE transgenic mice were obtained from Dr A. Caton (Wistar Institute, Philadelphia, PA), and backcrossed to BALB/c mice to generate heterozygous mice for use in the experiments. The SFE transgenic mice express TCR V4/8.2, which is specific for the MHC class II I-Ed–restricted p111–119 epitope of influenza hemagglutinin (HA), and allows for a strong CD4 + T cell response [21]. TCR transgene expression was monitored by PCR analysis from tail snip DNA. LCMV-P14/rag2 TCR transgenic mice were obtained from the Emerging Models Program of Taconic (Germantown, NY). These mice are homozygous for expression of TCR V2/8.1, which is specific for the MHC class I H2Db–restricted GP33-41 P14 epitope of LCMV, and almost all peripheral T cells are CD8 + and responsive to the antigen [22].

Lymph node (LN) T cells from experimental groups were analyzed in 96-well U-bottom microtiter plates at 2.5×105 cells/well in 100 l RPMI 1640 with 10% FCS (GIBCO, Grand Island, NY), 2 mM L-glutamine, 50 IU/ml penicillin and streptomycin, and 0.05 mM 2-ME. Irradiated (20 Gy) syngeneic SJL splenocytes (5×105 cells/well; 50 l) were added along with either PLPe or KLH (50 g/ml; 25 l), and TJU103 (12.5 M; 0.33% DMSO). For T cells from transgenic mice, either LN CD4 + T cells (5×104 cells/well) from SFE or CD8 + T cells (1×104 cells/well) from LCMV transgenic mice were plated along with irradiated (20 Gy) syngeneic splenocytes (1×105 or 2×104 cells, respectively; 50 l/ well) in the presence of either p111–119 or GP33–41 antigenic peptides (10 g/well), as appropriate. AntiCD4 (GK1.5) and anti-CD8 (2.43) mAb were used as respective positive controls for inhibition. For all analyses, wells with cells but no Ag served as background controls. Plates were incubated at 37°C, 7% CO2 for 2–4 d and then pulsed for 8 h with [3H]-TdR (1 Ci/well, 25 l). Cells were harvested onto glass fiber filters with a TomTec cell harvester and the level of incorporated radioactivity was determined by a Wallac Beta-plate liquid scintillation counter. Results were expressed as the mean cpm incorporation from triplicate wells.

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TJU103, N-(3-indoylmethylene)-isonicotinic hydrazone, was selected for study based on ligand docking analysis of a surface binding pocket of human CD4D1, as previously described [23]. TJU103 was reconstituted in PBS/0.25% DMSO (500 g/ml). PLPe (HSLGKWLGHPDKF; [24]), HA p110–119 (SFERFEIFPK), and LCMV-P14 GP33-41 (KAVYNFATCG) peptides were synthesized and purified by the KCI Protein Facility, and keyhole limpet hemocyanin (KLH) was purchased from Sigma (St Louis, MO).

Three mice from each of the experimental groups were sacrificed on days 17 and 30 post-EAE induction, and perfused via cardiac puncture with PBS (4°C) and 4% paraformaldehyde. Brains and spinal cords were removed, processed for paraffin embedding, sectioned (6 m), and stained with hematoxylin and eosin. Histology was blindly scored according to the following criteria: 0, normal, no inflammatory cells; 1, mild meningitis with some perivascular cuffing; 2, moderate meningitis and perivascular cuffing; 3, moderately

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severe meningitis and perivascular cuffing; 4, severe meningitis and extensive perivascular cuffing. For spinal cord analysis, four distinct regional sections from each mouse in each group were scored and the mean calculated.

Flow cytometric analysis The phenotype of LN and splenic populations from experimental mice were analyzed by flow cytometry. Cells (5×105/well) were incubated with mAb (25 l; 1:100 dilution; 30 min; 4°C), washed 2× with PBS, fixed with 1% paraformaldehyde (100 l; 15 min), washed, and resuspended in PBS (100 l) for analysis by a Profile II flow cytometer (Coulter, Hialeah, FL). All FITC-conjugated mAb were purchased from PharMingen (San Diego, CA) and were directed to either Thy1.2 (30-H12), CD4 (RM4-5), CD8 (53-6.7), or B220 (RA3-6B2). Isotype-matched rat IgG2a served as a negative control.

Priming with KLH SJL mice were primed with 100 g of KLH in CFA/ H37Ra, s.c. in the hind footpads 4 weeks prior to EAE induction. Draining LN (popliteal and inguinal) were harvested 18 days later, pooled from four mice per group, and analyzed for their proliferative responsiveness to KLH.

ELISPOT analysis The frequency of antigen-stimulated cytokine producing cells was determined by utilizing an enzymelinked immunospot assay (ELISPOT), as previously described [26]. Draining LN (popliteal and inguinal) were harvested from SWXJ-14 mice on day 18 postEAE induction, and CD4 + T cells were enriched using an anti-CD8 mAb (2.43, ATCC TIB-210; 1:100) in the presence of guinea pig complement (1:5 dilution of a stock generated in our laboratory). CD4 + T cells were stimulated with PLPe (50 g/ml; T25 flasks; 24 h; 37°C), washed, and plated (2.5×105 cells/well) in 96-well nitrocellulose-backed microtiter plates (Millipore, Bedford, MA) that had been precoated overnight with either purified rat anti-mouse IL-2 (JES6-1A12; 25 g/ml), IL-4 (BVD4-1D11; 10 g/ml), or IFN- (R46A2; 25 g/ml) mAb. After incubation for 18 h at 37°C, biotinylated rat anti-mouse mAb to IL-2 (JES65H4; 2 g/ml), IL-4 (BVD6-24G2; 0.6 g/ml), or IFN- (XMG1.2; 4 g/ml) were added for 2 h. All mAb were obtained from PharMingen. Plates were developed with an alkaline phosphatase-labeled protein streptavidin (2 h; 1:500; Southern Biotech, Birmingham, AL), and Sigma Fast BCIP/NBT tablets used for color spot detection. The mean number of spots were calculated from triplicate wells in each experimental group.

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Statistical analysis For calculating the mean EAE severity scores, data were pooled from at least two experiments. Wilcoxin non-parametric signed rank analysis was used to determine significance between groups at individual timepoints post-EAE induction. Student’s T-test was used for comparison of in vitro results from proliferation, histological, and ELISPOT analyses.

Results TJU103 inhibits murine CD4 + , but not CD8 + T cell responses In order to demonstrate the cross-species effectiveness and specificity of TJU103 for murine CD4 + T cells in vitro, it was added to antigen-stimulated cultures of LN T cells from either SFE (responding to MHC class II-restricted p111–119) or LCMV-P14 (responding to MHC class I-restricted GP33-41) TCR / transgenic mice. The presence of TJU103 (50 M) in the culture significantly inhibited proliferation of the SFE CD4 + T cell response (49% reduction, P<0.01 and equivalent to that found with GK1.5 anti-CD4 mAb treatment; Figure 1A), whereas the compound had no effect upon the LCMV-P14 CD8 + T cell response (Figure 1B).

TJU103 efficacy in the SJL EAE model EAE was induced in SJL mice by inoculation of PLPe (100 g s.c.) in CFA emulsion on d0 and 7, and TJU103 was administered on day 12 (100 g i.v.), before clinical symptoms developed. This treatment significantly reduced (P<0.04; days 15–24; Figure 2A) the mean EAE severity score in contrast to untreated or 0.25% DMSO-treated mice (used as solvent). At the peak of disease on day 16, the untreated and DMSO-treated mice reached a maximum mean±SEM severity of 2.1±0.2 and 2.5±0.3, respectively, in contrast to a maximum of 1.0±0.2 on day 20 for the TJU103-treated group. In addition, the overall incidence of disease was reduced from 100% in control groups to 60% in the TJU103-treated group (Figure 2B).

TJU103 effect on in vitro secondary T cell responses to PLPe SJL mice were sensitized with PLPe/CFA (100 g s.c.) in the flank, draining LN harvested on day 7, and in vitro T cell proliferative responses to PLPe (50 g/ml) analyzed in the presence of 12.5 M TJU103. The compound significantly inhibited the response (by 46%; P≤0.05), compared to either the untreated or DMSO-treated T cells (Figure 2C). Thus, TJU103 was capable of directly affecting the in vitro secondary T cell response to PLPe.

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Figure 1. TJU103 inhibits murine CD4 + , but not CD8 + T cell responses. LN CD4 + T cells from SFE transgenic mice (A) or LN CD8 + T cells from LCMV transgenic mice (B) were plated with irradiated syngeneic splenocytes in the presence of either p111–119 or GP33-41 peptides, respectively, as described in Materials and Methods. Cultures were pulsed with [3H]-TdR and harvested on day 2. Anti-CD4 (GK1.5) and anti-CD8 (2.43) mAb were used as appropriate positive controls for inhibition.

Histologic evaluation of disease Brain and spinal cord tissue sections from TJU103treated or control mice, taken on days 17 and 30 post-EAE induction with MSCH, were scored for pathological signs of disease. On day 17, the untreated mice (clinical score of 1.8±0.2) exhibited moderately severe meningitis with perivascular cuffing throughout the spinal cord and brain (Figure 3A,B), resulting in mean pathological scores of 2.8±0.3 and 2.5±0.3, respectively. In contrast, TJU103-treated mice (clinical score of 0.7±0.4) exhibited only mild meningitis throughout the spinal cord and brain (Figure 3C,D), with mean pathological scores of 0.9±0.4 and 0.7±0.3, respectively. DMSO-treated mice (clinical score of 1.3±0.2) exhibited pathological symptoms of disease in the spinal cord (2.4±0.2) and brain (2.0±1.0) equivalent to the untreated group (P>0.05). D30 evaluation of histology in the spinal cord and brain rendered similar results (data not shown). Therefore, in addition to the capacity of TJU103 to reduce the clinical severity of disease, it was also able to inhibit disease at the pathological level.

TJU103 efficacy in the SWXJ-14 EAE model SWXJ-14 mice develop a very severe form of acute EAE following inoculation on days 0 and 7 with MSCH/CFA (1 mg s.c.; Figure 4A). Nevertheless, a single injection of TJU103 (100 g i.v.) on day 10 resulted in significant reduction of disease severity (P≤0.04; day 16, 19–22), with a peak mean EAE score of 1.0±0.6, in comparison to the untreated (3.1±0.4) and DMSO-treated (2.6±0.6) groups. TJU103 treatment also reduced the incidence of EAE with a maximum of 33.3% during the peak time of disease compared to 100% and 66.7% for the untreated or DMSOtreated groups, respectively (Figure 4B). In addition, T cells from PLPe/CFA-inoculated SWXJ-14 mice (days

0 and 7; s.c.), were isolated on day 18 and restimulated with PLPe in vitro. T cells from untreated or DMSOtreated mice exhibited a strong proliferative response to Ag, whereas responses from TJU103-treated mice were significantly reduced (by 76.9%; Figure 4C). Thus, in this model, TJU103 treatment in vivo was capable of affecting the later development of secondary T cell responses to specific Ag in vitro.

TJU103 effect during active EAE phase To determine whether TJU103 treatment could affect the further development of EAE once clinical symptoms had emerged, EAE was induced in SJL mice with PLPe. On day 13, mice exhibiting an EAE clinical score of 1.0 were selected, randomly regrouped, and administered either TJU103 (100 g i.v.), 0.25% DMSO, or left untreated. The untreated and DMSO-treated mice progressed to a maximum mean severity score of 2.0±0.4 and 1.6±0.3, respectively (Figure 5). In contrast, the TJU103-treated mice never progressed past the 1.0 level and by day 15, dropped to significantly lower levels of disease severity (P<0.04; days 15–18), demonstrating its capacity to inhibit responses during the effector phase.

TJU103 effect during in vivo PLPe rechallenge Acute EAE was induced in SJL mice with PLPe/CFA (100 g; day 0, 7) and allowed to go into remission. On day 50, the mice were rechallenged with PLPe/CFA, and the next day were treated with either TJU103 (100 g, i.v.), DMSO (0.25%), or left untreated. The control groups exhibited a typical secondary response with more rapid kinetics and a higher level of EAE severity, reaching peak mean scores of 3.2±0.3 (untreated) and 2.8±0.4 (DMSO) (Figure 6A). TJU103treated mice had a significant reduction in the peak

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mean severity score to 2.0±0.4 (P≤0.04 on days 6, 7, 15–19, 22–43). The majority of individual scores of mice from the control groups were consistently higher than the TJU103-treated group at progressive time points post-rechallenge (Figure 6B). For example, on day 12, 41.6% (untreated) and 33.3% (DMSO) of mice reached the 4.0 severity level, compared to only 8.3% of the TJU103 group. These results suggested that TJU103 was effective during an EAE secondary T cell response.

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One advantage of TJU103 is that it should specifically affect only the small fraction of CD4 + T cells that are activated by antigen in its presence. To test this hypothesis, EAE was induced in SJL mice with PLPe/ CFA (day 0, 7), and treated with TJU103 (100 g i.v.) or DMSO (0.25%) on day 12. The next day, lymphoid cell subsets (Thy 1.2 + , CD4 + , CD8 + , and B220 + ) were analyzed by flow cytometry, and no significant quantitative differences were found (Figure 7A). Next, to examine the antigen specificity of TJU103 inhibition, SJL mice were initially primed with KLH (100 g s.c. via footpads), 4 weeks later EAE was induced by PLPe/CFA challenge, and on day 12 TJU103 (100 g i.v.) administered. On day 18, in vitro proliferative responses to 50 g/ml of PLP or KLH were assessed. There were no significant differences in responsiveness to KLH (P>0.6) between untreated, DMSO, and TJU103 treatment groups, whereas the latter exhibited at least 50% inhibition of proliferative responses to PLPe (Figure 7B). These results demonstrated that TJU103 can affect specific CD4 + T cells during an active antigenic exposure, but does not inhibit potential memory responses to recall antigens.

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Figure 2. TJU103 effect in SJL EAE model. SJL mice were induced for EAE with PLPe/CFA (100 g s.c.; day 0, 7), and treated with TJU103 (100 g i.v.; n=13), 0,25% DMSO (n=4), or left untreated (n=13) on day 12. (A) Data represents the mean±SEM severity scores and (B) the percentage incidence for each experimental group; data pooled from three experiments. (C) Draining LN T cells were analyzed on day 7 from PLPe/CFA primed mice for secondary in vitro responses to PLPe (50 g/ml), in the presence of TJU103 (12.5 M). The data represent mean±SEM cpm [3H]-Tdr incorporation of triplicate cultures on day 4 of incubation. —h—, untreated; – –d– –, DMSO; · · ·s· · ·, TJU103.

SWXJ-14 mice were induced for EAE with PLPe/CFA (100 g s.c.; day 0, 7), and either treated with TJU103 (100 g i.v.), 0.25% DMSO, or left untreated on day 10. CD4 + T cells from draining LN (popliteal and inguinal) were isolated on day 18, stimulated for 24 h with PLPe (50 g/ml) in vitro, and incubated for an additional 18 h on IL-2, IL-4, or IFN- precoated 96-well plates for a standard ELISPOT analysis. The TJU103-treated group exhibited a significant reduction in the frequency of IL-2, IL-4, and IFN- producing CD4 + T cells, in comparison to either the untreated or DMSO control groups (Table 1). For example, the reciprocal frequency of IL-2 secreting cells in the TJU103 group was 83.3×10 −3, compared to 2.1×10 −3 for the untreated and 3.7×10 −3 for the DMSO groups. Similar results were found for IL-4 and IFN-. Thus, TJU103 decreased equally the frequency of both Th1 and Th2-type PLPe-specific cytokine secreting cells and had no polarizing effect on the T cell response.

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Figure 3. Effect of TJU103 on the pathology of EAE in SJL mice. EAE was induced in SJL mice with MSCH/CFA (1 mg s.c.; days 0, 7) and treated with TJU103 (100 g i.v.) on day 12. Histological sections of spinal cord and brain tissue on day 17 post-EAE induction were stained with hematoxylin/eosin. Representative stained spinal cord (20×) and brain (10×) sections are shown from untreated (A and B, respectively) and TJU103-treated (C and D) groups (Bar, 0.1 mm).

Discussion We have demonstrated in these studies that a single injection of TJU103 was able to reduce the severity of acute EAE in both the SJL and SWXJ-14 mouse models, the latter of which develops a particularly severe form of disease. In addition, the level of pathological involvement in the brain and spinal cord of SJL mice correlated with the reduction in EAE symptomatic severity. It was also determined that TJU103 caused a significant reduction in the secondary PLPestimulated proliferation of T cells, whether it was administered in vivo during priming or in the incubation culture, itself. Amongst the immunotherapeutic strategies designed to inhibit development of EAE, some have tried to disrupt the TCR/MHC class II/Ag complex interaction with the CD4 molecule [27, 28], while others have targeted the autoreactive CD4 + T cells, themselves, with mAb approaches [18–20]. Although anti-CD4 mAb therapy has been shown to prevent the onset and reduce the clinical symptoms of EAE, it runs the risk of inducing a general immunosuppressive state and is limited by the immunogenicity of the mAb. In addition, it was found that clinical use of human chimeric anti-CD4 mAb did not inhibit activated CD4 + Th1 T cells in MS patients [29]. Other more promising studies with non-depleting anti-CD4 mAb have indicated potential efficacy in

preclinical autoimmune [30, 31] and transplantation models [32–35], and in clinical trials [36, 37]. Although still unclear, the mechanism of tolerance induction by these mAb may be similar to the inhibitor activity found with TJU103, in disrupting the T cell activation process. However, these mAb would still have the disadvantages related to immunogenicity, in addition to the lack of possible oral delivery. As an alternative approach, TJU103 was developed to antagonize putative CD4 oligomerization of CD4/ MHC/TCR/Ag complexes, and to thereby disrupt activation of encephalitogenic CD4 + T cells. This nonpeptidic organic molecule may function similarly to CD4D1 peptide analogs which have been found previously to inhibit acute EAE [38, 39], but is more stable due to its lower susceptibility to enzymatic degradation, and it more readily affords itself accessibility to chemical modifications. The advantages of such a small molecular compound would thus include potential availability (either by itself or after modification) for oral administration and the lack of immunogenicity. In these experiments, TJU103 was solubilized in DMSO and used at a maximum concentration of 0.33%, far below any observed toxicity levels of 4% [40]. DMSO control groups were included in all experiments, and it was clear that TJU103 effects were not due to a depletion of T cells, based upon yields and

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flow cytometric analysis of lymphocyte populations (Figure 7A). Although TJU103 reduced the clinical and pathological symptoms of disease during acute EAE, evaluating the efficacy of the inhibitor during active and secondary disease is much more relevant for potential application to MS. Since treatment only begins after clinical symptoms of disease have been established in

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MS patients, activated or memory T cells would likely be involved. In this study, a single injection of TJU103 was administered when mice first developed symptoms of acute EAE, and it prevented mice from progressing to more severe levels of disease. In this regard, it has been demonstrated that activated and memory CD4 + T cells upregulate surface CD4 expression, suggesting that it may still play an important role in reactivation [41]. The capacity of TJU103 to diminish secondary EAE responses when administered 24 h after d50 PLPe rechallenge (Figure 6) further demonstrated its translational therapeutic potential. Unlike other forms of immunotherapy that result in general immunosuppression, TJU103 was able to specifically effect only activated CD4 + T cells during the time in which it was present. There was no evidence of depletion of any lymphoid cell population in the lymph nodes or spleen, and particularly no diminution of the CD4 + T cell compartment in PLPeprimed SJL mice. Yet, although T cells from TJU103-

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Figure 4. TJU103 effect in SWXJ-14 EAE model. SWXJ-14 mice were induced for EAE with MSCH/CFA (1 mg s.c.; days 0, 7), and treated with TJU103 (100 g i.v.) or 0.25% DMSO on day 10 or left untreated. (A) The mean±SEM severity scores and (B) % incidence are represented, with data pooled from two experiments (n=10). (C) SWXJ-14 mice were inoculated with PLPe (100 g s.c.; days 0, 7) and treated with TJU103 on d12. Draining LN T cells were pooled from four mice per group on day 18, and analyzed for in vitro responses to PLPe (50 g/ml). The data represents mean±SEM cpm [3H]-TdR incorporation of triplicate wells on day 3 of incubation. —h—, untreated; – –d– –, DMSO; · · ·s· · ·, TJU103.

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Figure 6. TJU103 effect at time of PLPe rechallenge. EAE was induced in SJL mice with PLPe/CFA (100 g s.c.; days 0, 7), rechallenged on day 50 with the same Ag, and treated with TJU103 (100 g i.v.) or 0.25% DMSO 1 day later. (A) Severity scores are represented as the mean±SEM (n=12) (—h—, untreated; – –d– –, DMSO; · · ·s· · ·, TJU103), and (B) individual scores on days 6, 12, 19, and 38 (untreated j, DMSO m, and TJU103 s).

treated mice exhibited decreased responsiveness in vitro to PLPe-restimulation, there was no loss of memory T cell responses to KLH, another recall Ag that was not present during the in vivo exposure to the inhibitor. These experimental results demonstrate a distinct advantage of the TJU103 approach compared to other more non-specific forms of immunological intervention for EAE and MS. Hypothetically, TJU103 could be used to clear out autoreactive encephalitogenic T cells that are persistently being activated in an MS patient, and if administered over a short period of time, it can do so without endangering the remaining vast immune repertoire necessary for protection against infectious agents. Further studies on residual anti-microbial activity after treatment with TJU103 is warranted, considering the clinical problems that

3

2

1

0

None

PLP139–151 In vitro stimulation

KLH

Figure 7. PLPe-specific TJU103 inhibition in vivo. (A) SJL mice were induced for EAE with PLPe/CFA (100 g s.c.; days 0, 7), and treated with TJU103 (100 g i.v.) or 0.25% DMSO on day 12. The next day, LN cells were analyzed by flow cytometry for lineage-specific surface markers: Thy 1.2, CD4, CD8, and B220. The values expressed are the mean % fluorescent-positive cells in each group (n=3). Similar results were found for splenocytes (not shown). (B) SJL mice were primed with KLH/CFA (100 g s.c.) 4 weeks prior to EAE induction with PLPe/CFA (100 g s.c., days 0, 7), and treated with TJU103 (100 g i.v.) or 0.25% DMSO on day 12. In vitro LN T cell responses (pooled from 4 mice per group) to either PLPe or KLH (50 g/ml) were measured on day 18. Data are representative of two experiments and values are mean±SE [3H]-TdR incorporation of triplicate cultures on d3 of incubation. j, untreated; , TJU103; , DMSO.

have developed in the past after use of other CD4 targeted approaches [42]. The inhibitory mechanism by which TJU103 was able to affect the immune response in EAE has not been completely established. One possibility is a switch in the polarization of the cytokine profile in EAE, which is dominated by Th1 production of IFN and TNF during the initiation of inflammation and peak of tissue damage in the CNS [43]. In contrast, Th2-type IL-4 and IL-10 dominate during the recovery phase of EAE [44]. Therefore, a decrease in the immune response in EAE may be induced by a reduction of the Th1 cytokine response, an increase of

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177

Table 1. Frequency of PLPe-specific cytokine producing CD4 + T cells in acute EAE Experimental group

Untreated Untreated+PLPe TJU103+PLPe DMSO+PLPe

potential candidate for treatment of MS and other CD4 + T cell-mediated autoimmune diseases.

Reciprocal frequency (×10 −3) IL-2

IL-4

IFN

Acknowledgements

192.3 2.1 83.3 3.7

833.3 83.3 ∝ 58.1

250.0 108.7 833.3 50.0

We thank the KCI Peptide Facility for the production of peptides, and the excellent technical assistance of David Dicker for flow cytometric analysis. This research was supported by funds provided by the KCI Translational Research Committee and NIH grant NS-34928.

EAE was induced in SWXJ-14 mice with PLPe/CFA (100 g s.c., days 0, 7), and mice treated with TJU103 (100 g; i.v.) or 0.25% DMSO on day 10. CD4 + T cell responses to PLPe (50 g/ml) were analyzed by ELISPOT. The mean of positive blue spots was counted in triplicate cultures for each group and the frequency calculated for 2.5×105 cells/well.

the Th2 response, or induction of a switch from a Th1 response to a Th2 response. However, the results of the ELISPOT analysis (Figure 6) suggested that the frequency of both Th1 and Th2-type PLPe-specific CD4 + T cells were equivalently reduced. Therefore, TJU103 may function through other means of inhibition, perhaps via anergy induction or programmed cell death (i.e. apoptosis). Our preliminary results in the SFE / TCR transgenic mouse model suggest that in the presence of TJU103 and specific antigen, CD4 + T cells undergo an enhanced level of apoptosis in vivo. Optimal activation of CD4 + T cells, resulting in cytokine production, proliferation, and effector functions, involves the interaction of the TCR with antigen bound to MHC class II, together with the necessary costimulatory signals provided by the APC. CD4 functions as a coreceptor that binds to the 2 domain of the MHC class II molecule [3], and is critical for optimal T cell activation [5]. Several recent studies have indicated that CD4 may form a dimer or oligomer, which may play an important role in the stabilization of the CD4/MHC class II interaction and the initiation of T cell activation [8–10]. The CDR3 region of CD4D1 is thought to be involved in this oligomerization [45, 46], and TJU103 was modeled to interact with the structural binding pocket formed between the CDR3 and CC′ loop regions [11]. By disrupting oligomerization of the CD4/MHC class II/TCR complex, TJU103 is proposed to interfere with the signal transduction involved in T cell activation and, as a consequence leads to programmed cell death or anergy of the antigen-stimulated CD4 + T cell. We are currently investigating the precise molecular mechanism by which TJU103 affects CD4 T cellmediated immune responses. In conclusion, TJU103, a small non-peptidic organic molecule selected by a computer screening approach, specifically regulates CD4 + T cell immune responses, and represents a novel approach to immunotherapy. The results in this study suggest that TJU103 is an effective therapeutic for the CD4 + T cell-mediated autoimmune response in the EAE model system. In light of these observations, TJU103 becomes a

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