In vitro activities of GMCSF-MOG
The main question addressed in this study was whether murine GMCSF-NAg TTV could block disease in murine models of chronic EAE, including the chronic progressive model of EAE in C57BL/6 mice and the relapsing-remitting model of EAE in SJL mice. Murine fusion proteins were derived which consisted of the murine GM-CSF as the N-terminal domain and either the MOG35-55 epitope or the PLP139-151 epitope as the C-terminal domain. We first tested whether the bioactivity of GM-CSF was altered by the C-terminal addition of the NAg domain. The bioassays were based on the use of C57BL/6 bone marrow cells (Figure ) and the FDC-P1 cell line (Figure ). These assays revealed that the GM-CSF activity of GMCSF-MOG and GMCSF-PLP was essentially equipotent with murine GM-CSF and rat GM-CSF. Independently-derived preparations of each fusion protein were tested to verify reliability of the fusion protein preparations. Sample to sample variation of protein preparations was minimal. The conclusion was that neither the MOG35-55 nor an alternative PLP139-151 C-terminus interfered with the activity of the GM-CSF domain. These and other experiments revealed that mouse and rat GM-CSF proteins were equally cross-reactive on both mouse and rat indicator cells (data not shown).
Figure 1 The NAg domain of murine GMCSF-NAg TTV did not interfere with cytokine bioactivity. Designated concentrations (x-axis) of murine GM-CSF, rat GM-CSF, murine GMCSF-MOG, or murine GMCSF-PLP were cultured with C57BL/6 bone marrow cells (100,000 cells per (more ...)
Previous studies also showed that rat GMCSF-NAg TTV were potent antigens in vitro [15
]. The enhanced antigenic potency of GMCSF-NAg TTV was due to high affinity interactions of the cytokine domain with the respective cytokine receptors on APC which targeted the tethered NAg to the APC surface for enhanced presentation of NAg. As shown in Figure , the rat GMCSF-NAg TTV was approximately 1000-fold more potent than the guinea pig (GP) myelin basic protein (MBP) GP69-88 peptide when cultured with splenic APC and the RsL.11 T cell clone or the rat 1B3 or 1E2 T cell hybrids. The murine GMCSF-MOG protein also potently enhanced the recognition of the covalently tethered MOG35-55 peptide when assayed in the presence of rat splenic APC and a rat T cell line specific for MOG35-55 (Figure ). GMCSF-MOG was at least 1000-fold more potent as an antigen compared to MOG35-55 and 10,000 fold more potent than the extracellular IgV domain of rat MOG which contains the verbatim MOG35-55 sequence. At concentrations of 10 pM (10-11
M) to 1 uM (10-6
M), the stimulatory activity of GMCSF-MOG reflected a broad bell-shaped curve that varied from 43 to 88 (x 103
) cpm whereas MOG35-55 was substantially less potent but at high concentrations (100 nM) stimulated a more robust T cell response (130 × 103
cpm). A recombinant macaca IgV-MOG protein which contained two differences (rat/macaca S42P and K55R) did not stimulate this T cell line. GM-CSF alone did not stimulate proliferative activity (data not shown).
Figure 2 The GM-CSF domain of murine GMCSF-MOG potentiated the antigenic recognition of MOG35-55 by rat T cells. (A) The rat T cell clone RsL.11 or the rat 1E2 or 1B3 T cell hybrids were cultured with Lewis rat splenic APC and designated concentrations (x-axis) (more ...)
GMCSF-MOG pre-treatment prevented a subsequent phase of EAE
To assess whether GMCSF-MOG could prevent EAE, the TTV was administered as a pre-treatment regimen (2 nmole dose subcutaneously in saline) at 3, 2, and 1 weeks before encephalitogenic challenge (Table and Figure ). In experiments 1 and 2 combined, the TTV pretreatment prevented EAE in 12 of 13 mice for an incidence of 7.7%. That is, twelve mice had a maximal score of zero (no disease) and one mouse exhibited severe EAE (maximal score of 4.0). In contrast, mice pretreated with MOG35-55 or saline exhibited an incidence of 100% and 93.8% respectively (Table ). In the MOG-35-55 pretreated groups, 14 of 16 exhibited severe EAE (≥ 4.0 maximal score) whereas 2 mice exhibited mild EAE (scores of 1.0). In the saline-pretreated groups, 15 of 16 mice had scores of 4.0 whereas one mouse did not exhibit EAE. Thus, the GMCSF-MOG pretreatment reduced the mean cumulative score, mean maximal score, and the mean number of days with severe EAE. The GMCSF-MOG prevented the development of EAE in a majority of mice over a prolonged period of 40-50 days (Figure ). GMCSF-MOG also prevented the weight loss associated with EAE whereas mice pretreated with MOG35-55 or saline had a sustained loss of body weight (Figure ). These data indicate that GMCSF-MOG is an active tolerogen in the C57BL/6 mouse model of EAE.
GMCSF-MOG prevented a subsequent bout of EAE induced by challenge with MOG35-55 in CFA
Figure 3 The murine GMCSF-MOG TTV induced tolerance and prevented a subsequent episode of EAE. C57BL/6 mice were treated with 2 nmoles of GMCSF-MOG, 2 nmoles of MOG35-55, or saline on days -21, -14, and -7 before active challenge on day 0 with 200 ug of MOG35-55 (more ...)
As previously shown for rat GMCSF-NAg TTV, the murine GMCSF-MOG TTV required physical linkage of GM-CSF and NAg domains for tolerogenic activity (Table and Figure ). Mice pretreated with GMCSF-MOG were largely protected from subsequent EAE. Only 3 of 16 mice pretreated with the TTV showed EAE, and the course was mild (Table experiments 1 and 2 combined). Maximal scores for these mice were 2.0, 2.0, and 0.5. In contrast, mice pretreated with a mixture of GM-CSF and NAg had severe paralytic EAE. In this group, 15 of 16 mice had a maximal score of 4.0 whereas one mouse had a maximal score of 2.0. Mice pre-treated with GM-CSF did not differ in severity or time-course from saline-treated mice (Figure ) (note that the GM-CSF treatment group was not performed in experiment 2; Figure ). In this group, all mice had maximal scores of 3.5 (2 mice) or 4.0 (6 mice). Mice pre-treated with MOG35-55 had a slightly delayed onset but otherwise exhibited a full paralytic course of EAE (14 mice had maximal scores of ≥ 3.5 whereas 2 mice had scores of 0.5 and 2.0). Mice pre-treated with saline had severe EAE (14 of 16 had scores ranging from 3.0 to 5.0 whereas the two additional mice had scores of 2.0). Unlike the other pre-treatment groups, GMCSF-MOG prevented the EAE-associated loss of body weight (Figure ), and the covalent linkage of GMCSF with MOG35-55 was required for maintenance of normal body weight.
GMCSF-MOG required physically-linked cytokine and NAg domains for tolerance induction
Figure 4 Induction of MOG-specific tolerance required covalent linkage of the GM-CSF and MOG domains. C57BL/6 mice were treated with GMCSF-MOG, a mixture of murine GM-CSF and MOG35-55, the MOG35-55 peptide, GM-CSF, or saline subcutaneously on days -21, -14, and (more ...)
Mice successfully treated with GMCSF-MOG that had a clinical score of 0 did not exhibit histological evidence of EAE whereas EAE-afflicted control mice had abundant focal lesions of the CNS. These CNS lesions were marked by perivascular infiltration of mononuclear cells in white matter of the spinal cord (Figure ). Overall, pretreatment with the GMCSF-MOG reduced EAE incidence, the cumulative score, the maximal disease score, weight loss, and the mean number of days that mice were afflicted by severe EAE. Due to the requirement for linked cytokine and NAg domains, GMCSF-MOG appeared to target the covalently-tethered NAg to APC in vivo as part of the tolerogenic mechanism. Overall, these data indicate that GMCSF-NAg TTV mediate antigen-targeting in both mice (Table ) and rats [15
Figure 5 GMCSF-MOG TTV prevented histological signs of EAE. Shown are representative histological sections from mice from Figure 4, experiment 2. Mice from control groups that were afflicted with severe EAE had perivascular infiltrations of mononuclear cells typical (more ...)
GMCSF-PLP pre-treatment prevented a subsequent phase of EAE
Given that GMCSF-NAg TTV were able to inhibit both monophasic (Lewis rats) and chronic progressive (C57BL/6 mice) models of EAE, an important question was whether GMCSF-NAg could inhibit relapsing-remitting EAE. Thus, GMCSF-PLP TTV was tested for tolerogenic activity in SJL mice (Table and Figure ). Mice were administered 2 nmoles GMCSF-PLP, 2 nmoles PLP139-151, or saline on days -21, -14, and -7 and then were challenged with 200 ug PLP139-151 in Complete Freund's Adjuvant (CFA) on day 0. One TTV-treated mouse exhibited EAE (incidence of 1/8, score of 1.0 for a total of 1 day) whereas mice pre-treated with either saline or PLP139-151 exhibited protracted, relapsing-remitting EAE. These data provide evidence that GMCSF-NAg TTV were effective in a second murine model of EAE.
GMCSF-PLP prevented a subsequent bout of EAE induced by challenge with PLP139-151 in CFA
Figure 6 The murine GMCSF-PLP TTV induced tolerance and prevented a subsequent episode of EAE. SJL mice were treated with 2 nmoles of GMCSF-PLP (n = 8 each group), 2 nmoles of PLP139-151, or saline on days -21, -14, and -7 before active challenge on day 0 (200 (more ...)
GMCSF-PLP was also a more potent as an antigen than the synthetic PLP139-151 peptide (Figure ). The potency enhancement attributed to the covalent attachment with GM-CSF was approximately 10-fold and was independent of whether APC were obtained from the spleen (Figure ) or thymus (Figure ). GMCSF-MOG was also more potent than MOG35-55 for stimulation of a murine MOG-specific T cell line whereas GM-CSF did not stimulate proliferation (Figure ). The potency enhancement was evident over a wide concentration range and extended into the low picomolar range as was evident when the data were re-plotted on a logarithmic y-axis (Figure ). As noted previously (Figure ), the same murine GMCSF-MOG TTV showed an approximate 1000-fold potency enhancement when tested in a rat T cell system.
Figure 7 The murine GMCSF-PLP and GMCSF-MOG TTV targeted NAg to APC for enhanced antigen presentation. A PLP139-151-specific T cell line (A-B) or MOG35-55-specific T cell line (C-D) was cultured with irradiated SJL splenocytes (A), irradiated SJL thymocytes (B), (more ...)
GMCSF-MOG was a therapeutic that inhibited the effector phase of an encephalitogenic attack
A central question was whether the GMCSF-MOG TTV can be used as an intervention in chronic EAE (Table and Figure ). Treatment was started on day 13 when the initial clinical signs of EAE first appeared in mice. The incidence of EAE was 1 of 8 mice for each group on day 13 (score was 1.0 for each mouse). Additional treatments were administered on days 15, 17, and 20. Administration of GMCSF-MOG halted development of EAE in a majority of mice whereas mice treated with MOG35-55 progressed to severe EAE. Of the GMCSF-MOG TTV-treated mice, 2 of 8 exhibited EAE, and one had clinical signs before initiation of treatment. Only 1 of 8 mice had a brief episode of severe EAE (score of 2 for a total of 2 days). The main conclusion was that the GMCSF-MOG TTV effectively stopped the progression of EAE. The rank order for inhibition of EAE was: GMCSF-MOG > MOG35-55 > saline. Compared to the saline-treated group, mice treated with MOG35-55 also had less severe EAE, although GMCSF-MOG had superior inhibitory efficacy compared to MOG35-55. These data indicate that GMCSF-MOG, and to a lesser extent, MOG35-55, inhibited the effector phase of EAE.
The GMCSF-MOG TTV was a therapeutic treatment that blocked progression of actively-induced EAE
Figure 8 GMCSF-MOG was an effective intervention that prevented progression of EAE. Mice were immunized on day 0 with 200 ug MOG35-55 in CFA plus Pertussis toxin (200 ng i.p.) on days 0 and 2. Treatment was initiated when the first mice began showing clinical (more ...)
GMCSF-MOG was also tested as a therapeutic agent in an alternative model (Table and Figure ). EAE was passively-induced by adoptive transfer of encephalitogenic T cells, and then, after onset of clinical signs, three treatments were administered on days 9, 11, and 14. Because all mice had EAE before the initiation of treatment, the incidence of EAE in all groups was 100%. Following partial recovery from the initial passive bout of EAE, mice were actively challenged with MOG35-55 in CFA on day 42 but were not given any additional treatment with TTV. The GMCSF-MOG TTV, when administered on days 9, 11, and 14, blunted the initial bout of passive EAE, inhibited residual disease, and attenuated the subsequent bout of active EAE. Overall, these data show that therapeutic administration of TTV during the initial passively-induced bout of EAE had a pronounced inhibitory effect on the subsequent active induction of EAE as measured by both cumulative and maximal disease scores. These data indicate that tolerogenic activity can be initiated in peripheral lymphoid tissues despite ongoing inflammation in the CNS.
The GMCSF-MOG TTV was an intervention that reversed an established course of chronic EAE
Figure 9 GMCSF-MOG promoted tolerogenic activity despite ongoing inflammation in the CNS. Passive EAE was induced in C57BL/6 mice by adoptive transfer of activated MOG35-55-specific T cells on day 0 and by injection of Pertussis toxin on days 0 and 2. Mice were (more ...)