Effective antigen-specific immunotherapy in the marmoset model of multiple sclerosis.

Mature T cells initially respond to Ag by activation and expansion, but high and repeated doses of Ag cause programmed cell death and can suppress T cell-mediated diseases in rodents. We evaluated repeated systemic Ag administration in a marmoset model of experimental allergic encephalomyelitis that closely resembles the human disease multiple sclerosis. We found that treatment with MP4, a chimeric, recombinant polypeptide containing human myelin basic protein and human proteolipid protein epitopes, prevented clinical symptoms and did not exacerbate disease. CNS lesions were also reduced as assessed in vivo by magnetic resonance imaging. Thus, specific Ag-directed therapy can be effective and nontoxic in primates.

Antigen presentation determines the fate of the T memory response in vivo after sublethal gamma-irradiation.

The survival of memory T cells is critical to vaccination strategies for infectious diseases and cancer, whereas their elimination may be crucial for treatment of autoimmune states. We examined the consequences of gamma-irradiation, which induces apoptosis of memory T cells in vitro, on the memory response to MHC class I alloantigen in vivo. Sublethal gamma-irradiation of primed mice eliminated accelerated rejection of skin allografts but failed to induce tolerance. Accelerated rejection was restored in irradiated mice by infusion of bone marrow cells expressing the priming alloantigen on immunostimulatory APCs (dendritic cells), whereas the memory response was not restored by infusion of bone marrow cells expressing the priming alloantigen on nonstimulatory APCs (B cells). Strikingly, irradiated mice infused with nonstimulatory bone marrow APCs exhibited long-term survival or tolerance to skin grafts expressing the priming MHC class I alloantigen. The mechanism of tolerance in this setting is explored.

Serial MR imaging of experimental autoimmune encephalomyelitis induced by human white matter or by chimeric myelin-basic and proteolipid protein in the common marmoset.

BACKGROUND AND PURPOSE: Experimental autoimmune encephalomyelitis (EAE) in the marmoset was monitored by serial MR imaging to determine correlates to the natural-history MR studies in multiple sclerosis (MS). The relationships of MR-revealed lesions to clinical status and histopathologic findings were also explored. METHODS: We induced EAE by subcutaneous inoculation in two marmosets by human white matter (HWM) and in seven marmosets by MP4 (a chimeric recombinant fusion protein of myelin-basic and proteolipid protein) in adjuvant along with intravenous inactivated pertussis vaccine to facilitate the disease process. The HWM-inoculated animals were induced with Freund's adjuvant as the established model of marmoset EAE. The MP4-inoculated animals were induced with either Freund's incomplete adjuvant or TiterMax as part of a preclinical treatment trial. MR imaging was performed at 1.5 T at baseline, and repeated at 1- to 2-week intervals for a period of up to 16 weeks in six EAE-induced marmosets, and intermittently for up to 70 weeks in three EAE-induced and two control marmosets. Proton density- (PD-) and T2-weighted, pre- and postgadopentetate dimeglumine enhancement, T1-weighted, and magnetization transfer (MT) images were obtained. The brains were prepared for histologic evaluation of lesion distribution and counts, characterization of lesions as demyelinating or inflammatory, and histopathologic scoring. The clinical, MR, and pathologic scoring were done on grading systems, and correlated for evaluation. RESULTS: White matter (WM) changes after EAE induction were observed first at 9 days in the HWM-induced animals and at 2.5 weeks in the MP4-induced animals, with subsequent week-to-week fluctuations on PD- and T2-weighted images. Contrast-enhancing lesions were not observed in all animals. MR-revealed WM lesions correlated to histopathologic analysis of EAE lesions, measuring from 0.5 mm to 1.5 mm. The lesion count and extent of demyelination was greater in the HWM-induced animals than in the MP4-induced animals. Some MR-revealed lesions correlated directly to clinical symptoms, but the majority of lesions were clinically silent. CONCLUSION: On MR images, lesions in the EAE marmoset model were confined to the WM, and their development, resolution, distribution, and enhancing characteristics fluctuated over the duration of the study. The dynamic presentation of MR-revealed lesions confirms the parallels between EAE in the marmoset and relapsing-remitting MS. Clinical symptoms alone were not representative of ongoing pathologic brain lesions. Therefore, serial MR imaging serves as a very important adjunct to clinical and histologic surveillance of the development of new and the persistence of existing brain lesions in this animal model of MS.

Determinant spreading associated with demyelination in a nonhuman primate model of multiple sclerosis.

Definition of the immune process that causes demyelination in multiple sclerosis is essential to determine the feasibility of Ag-directed immunotherapy. Using the nonhuman primate, Callithrix jacchus jacchus (common marmoset), we show that immunization with myelin basic protein and proteolipid protein determinants results in clinical disease with significant demyelination. Demyelination was associated with spreading to myelin oligodendrocyte glycoprotein (MOG) determinants that generated anti-MOG serum Abs and Ig deposition in central nervous system white matter lesions. These data associate intermolecular "determinant spreading" with clinical autoimmune disease in primates and raise important issues for the pathogenesis and treatment of multiple sclerosis.

Treatment of experimental encephalomyelitis with a novel chimeric fusion protein of myelin basic protein and proteolipid protein.

It has been shown that peripheral T cell tolerance can be induced by systemic antigen administration. We have been interested in using this phenomenon to develop antigen-specific immunotherapies for T cell-mediated autoimmune diseases. In patients with the demyelinating disease multiple sclerosis (MS), multiple potentially autoantigenic epitopes have been identified on the two major proteins of the myelin sheath, myelin basic protein (MBP) and proteolipid protein (PLP). To generate a tolerogenic protein for the therapy of patients with MS, we have produced a protein fusion between the 21.5-kD isoform of MBP (MBP21.5) and a genetically engineered form of PLP (deltaPLP4). In this report, we describe the effects of treatment with this agent (MP4) on clinical disease in a murine model of demyelinating disease, experimental autoimmune encephalomyelitis (EAE). Treatment of SJL/J mice with MP4 after induction of EAE either by active immunization or by adoptive transfer of activated T cells completely prevented subsequent clinical paralysis. Importantly, the administration of MP4 completely suppressed the development of EAE initiated by the cotransfer of both MBP- and PLP-activated T cells. Prevention of clinical disease after the intravenous injection of MP4 was paralleled by the formation of long-lived functional peptide-MHC complexes in vivo, as well as by a significant reduction in both MBP- and PLP-specific T cell proliferative responses. Mice treated with MP4 were resistant to disease when rechallenged with an encephalitogenic PLP peptide emulsified in CFA, indicating that MP4 administration had a prolonged effect in vivo. Administration of MP4 was also found to markedly ameliorate the course of established clinical disease. Finally, MP4 therapy was equally efficacious in mice defective in Fas expression. These results support the conclusion that MP4 protein is highly effective in suppressing disease caused by multiple neuroantigen epitopes in experimentally induced demyelinating disease.

In vivo state of antiviral CTL precursors. Characterization of a cycling cell population containing CTL precursors in immune mice.

The in vivo state of CD8+ mouse memory CTL specific to lymphocytic choriomeningitis virus (LCMV) was characterized. During acute LCMV infection, the majority of the LCMV-specific CTL activity tested immediately ex vivo was mediated by CD8+ L-selectin- Mac-1+ CTL. The L-selectin- population of CD8+ cells elicited during acute infection also carried > 99% of the restimulatable CD8+ CTL precursors (CTLp) to LCMV, and these required added IL-2 for development into effectors in vitro. In contrast with the acute infection, most of the virus-specific CTLp in immune mice were L-selectin+. Examination of CD8+ T cells in LCMV-immune mice revealed that a L-selectin+ blast-size population of cycling CD8+ cells contained CTLp, which developed into effector CTL in the absence of added IL-2. These cells also expressed Mac-1 and IL-2R. Flow cytometric sorting for IL-2R+ and IL-2R- CD8+ cells in the immune animal revealed, by limiting dilution analysis, similar frequencies of CTLp in both populations. In bulk restimulation assays, the CD25+ CTLp did not require added IL-2 for their in vitro development into effectors, whereas the CD25- CTLp did. Hence, the different requirements for CTLp to effector development in vitro reflect qualitative differences in the in vivo state of the CTLp in the various subpopulations. LCMV-specific memory CTLp that did not require added IL-2 for differentiation were also found in the small-size, noncycling, CD8+L-selectin- cells. In contrast, the small-size, noncycling, CD8+L-selectin+, and CD8+IL-2R- populations also carried CTLp, but these required added IL-2 for development into effector CTL. Hence, T cell memory to LCMV is distributed among various lymphocyte subpopulations in immune animals, and the presence of an activated cycling cell component may account for the long-term perpetuation of antiviral immunologic memory.

Amelioration of autoimmune reactions by antigen-induced apoptosis of T cells.

We have shown that T cells vigorously cycling in response to growth lymphokines are driven into apoptosis by potent TCR restimulation. This process, termed propriocidal regulation, appears to be a normal feedback inhibitory mechanism to prevent excessive T cell proliferation and lymphokine production. Exposure of T cells to repeated high dose antigen treatments creates the conditions just described by activating T cells, and stimulating the production of growth lymphokines and their receptors. High growth lymphokine levels induced by the large amount of antigen present, stimulate vigorous cycling. The continued presence of high antigen levels subjects the cycling T cells to strong TCR restimulation as they enter the vulnerable S phase, inducing apoptosis in T cells responsive to the administered antigen. Thus, simple, repetitive, intravenous administration of high dose antigen may be used to delete potentially destructive clones of T cells, resulting in a state of peripheral tolerance. This has obvious therapeutic potential in disorders where the elimination of pathogenic T cell clones could be beneficial. We have described in EAE, an animal model for MS, that high dose MBP therapy is effective in preventing CNS pathology and the onset of disease as well as reducing the severity of the clinical symptoms of established EAE. We are currently involved in expanding this approach to other animal models of autoimmunity and graft rejection, as well as refining the immunotherapy in the EAE model with the objective of developing a clinical therapy for human demyelinating disease.

CD11b (Mac-1): a marker for CD8+ cytotoxic T cell activation and memory in virus infection.

We have found that CD11b, a cell surface integrin of macrophages, granulocytes, and NK cells, is expressed by a subset of CD8+ T cells that include both the active virus-specific CTL and the virus-specific memory CTL populations. CD8+CD11b+ cells comprise less than 3% of naive mouse splenocytes, but after lymphocytic choriomeningitis virus (LCMV) infection increase by 9- to 12-fold by the peak (day 8) of the virus-specific CTL response. Depletion of day-8 splenocytes with anti-Mac-1 and C' or enrichment by sorting for CD11b+ or CD8+CD11b+ spleen cells demonstrated that LCMV-specific CTL are CD11b+. The CD11b+ subpopulation also contained the bulk of the IL-2-responsive CD8+ cells. MEL-14, a homing marker down-regulated on activated T cells, was down-regulated on the majority of CD8+ cells that became CD11b+. Less than 1% of LCMV-immune splenic lymphocytes expressed CD11b. Antibody and C' depletion of this population severely impaired the ability of immune splenocytes to respond to in vitro secondary stimulation with LCMV-infected peritoneal macrophages, but did not affect the generation of a primary allospecific CTL response in MLC. Mixing of CD8-depleted and CD11b-depleted LCMV-immune splenocytes failed to restore the ability of these cells to mount a virus-specific memory CTL response, indicating that a cell coexpressing CD8 and CD11b is essential for this response. As determined by limiting dilution analysis, the precursors for the LCMV-specific memory CTL response were enriched in the CD11b+ population of LCMV-immune splenocytes. CD11b stained far fewer CD8+ splenocytes from naive mice than did CD44 (Pgp-1), and among immune splenocytes it identified a small subpopulation of CD44hi cells, indicating that CD11b may be the best single marker available for discriminating between naive and memory CD8+ T cells.

Enhanced capacity of females for early interferon-gamma production by natural killer-like cells following stimulation by staphylococcal enterotoxin A.

Staphylococcal enterotoxin A (SEA) induced the production of interferon-gamma (IFN-gamma) by spleen cells from ICR Swiss mice during the first 24 h of culture. Splenocytes from females produced higher levels of IFN-gamma than did those from males at 8, 12, and 16 h. By 20 h after SEA stimulation, IFN-gamma production by spleen cells from males was similar to that of females. The cell types involved in IFN-gamma production in this SEA/spleen cell system were analyzed by depletion studies. Removal of Thy-1+ cells by panning prevented production of IFN-gamma in the 24 h after SEA stimulation. In vivo depletion of asialo GM1+ (AGM1+) cells prevented production of IFN-gamma through 16 h of culture with SEA, but permitted a modest IFN-gamma response at 20 h that was similar in magnitude in both sexes. Following removal of L3T4+ and Lyt-2+ cells by panning, IFN-gamma production was detected at 12 h after SEA stimulation and maintained through 24 h of culture with cells from females producing higher levels of IFN-gamma. These data suggest that male ICR Swiss mice are deficient in the activity of Thy-1+, AGM1+, L3T4-, and Lyt-2- cells in the early (8-16 h) production of IFN-gamma following SEA stimulation of spleen cells.

AGM1+ spleen cells contain gamma interferon (IFN-gamma) gene transcripts in the early, sex-dependent production of IFN-gamma after picornavirus infection.

Encephalomyocarditis D variant (EMCV-D)-infected spleen cell cultures prepared from diabetes-resistant ICR Swiss female mice produce more gamma interferon (IFN-gamma) activity over a 24-h period than do spleen cell cultures from diabetes-susceptible male mice of this strain. Pretreatment of mice with anti-asialo GM1 eliminates early in vitro IFN-gamma production from 4 to 16 h postinfection (p.i.) and reduces IFN-gamma production from 16 to 24 h p.i. In this study, depletion of spleen cells with anti-Thy-1 by panning greatly reduced IFN-gamma activity in EMCV-D-infected spleen cell cultures throughout a 24-h period. Populations of asialo GM1 (AGM1), L3T4, and Lyt-2-positive cells were isolated from cells harvested at 9 h p.i. from EMCV-D-infected spleen cell cultures by a modified panning technique on polystyrene microscope slides. By in situ hybridization with a [35S]dATP-labeled IFN-gamma cDNA probe, only the AGM1-bearing cells were found to contain detectable IFN-gamma gene transcripts. An AGM1+, Thy-1+ natural killer-like cell is the probable producer of the early, sex-dependent IFN-gamma activity in this system.

Sex-dependent, early cytokine production by NK-like spleen cells following infection with the D variant of encephalomyocarditis virus (EMCV-D).

Spleen cell cultures from diabetes-resistant ICR Swiss females exhibited an increase in expression of Ia antigens 24 hours post-infection (PI) with EMCV-D while comparable spleen cell cultures from diabetes-susceptible males of this strain did not exhibit this increase in Ia antigens expression. A monoclonal antibody specific for mouse interferon-gamma (IFN gamma) eliminated this increase in Ia antigens expression. Interferon-gamma (IFN gamma) and interleukin 2 (IL-2) production by EMCV-D-infected spleen cell cultures were monitored at 4-hour intervals for 24 hours. Female spleen cells produced IFN gamma earlier (less than 16 hours PI) and in greater amounts than did comparably treated male spleen cells. Addition of a monoclonal rat anti-mouse IL-2 to virus-infected cultures did not significantly affect the early (less than 16 hours PI) production of IFN gamma by spleen cells of females. Treatment of the spleen cell donors with rabbit anti-asialo GM1 (AAGM1) abolished early production of IFN gamma in virus-infected female spleen cell cultures and reduced the early IL-2 production by infected male and female cells. These results suggest that an NK-like cell is responsible for the early female IFN gamma production; this may be a factor in the resistance of female ICR Swiss mice to EMCV-D-induced diabetes.