Patricia A. Nelson

Aquaporin 4-Specific T Cells in Neuromyelitis Optica Exhibit a Th17 Bias and Recognize Clostridium ABC Transporter

Aquaporin 4 (AQP4)‐specific autoantibodies in neuromyelitis optica (NMO) are immunoglobulin (Ig)G1, a T cell‐dependent Ig subclass, indicating that AQP4‐specific T cells participate in NMO pathogenesis. Our goal was to identify and characterize AQP4‐specific T cells in NMO patients and healthy controls (HC).

Laquinimod, a quinoline-3-carboxamide, induces type II myeloid cells that modulate central nervous system autoimmunity.

Laquinimod is a novel oral drug that is currently being evaluated for the treatment of relapsing-remitting (RR) multiple sclerosis (MS). Using the animal model for multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), we examined how laquinimod promotes immune modulation. Oral laquinimod treatment reversed established RR-EAE and was associated with reduced central nervous system (CNS) inflammation, decreased Th1 and Th17 responses, and an increase in regulatory T cells (Treg). In vivo laquinimod treatment inhibited donor myelin-specific T cells from transferring EAE to naive recipient mice. In vivo laquinimod treatment altered subpopulations of myeloid antigen presenting cells (APC) that included a decrease in CD11c+CD11b+CD4+ dendritic cells (DC) and an elevation of CD11bhiGr1hi monocytes. CD11b+ cells from these mice exhibited an anti-inflammatory type II phenotype characterized by reduced STAT1 phosphorylation, decreased production of IL-6, IL-12/23 and TNF, and increased IL-10. In adoptive transfer, donor type II monocytes from laquinimod-treated mice suppressed clinical and histologic disease in recipients with established EAE. As effects were observed in both APC and T cell compartments, we examined whether T cell immune modulation occurred as a direct effect of laquinimod on T cells, or as a consequence of altered APC function. Inhibition of Th1 and Th17 differentiation was observed only when type II monocytes or DC from laquinimod-treated mice were used as APC, regardless of whether myelin-specific T cells were obtained from laquinimod-treated or untreated mice. Thus, laquinimod modulates adaptive T cell immune responses via its effects on cells of the innate immune system, and may not influence T cells directly.

Immunodominant T Cell Determinants of Aquaporin-4, the Autoantigen Associated with Neuromyelitis Optica

Autoantibodies that target the water channel aquaporin-4 (AQP4) in neuromyelitis optica (NMO) are IgG1, a T cell-dependent Ig subclass. However, a role for AQP4-specific T cells in this CNS inflammatory disease is not known. To evaluate their potential role in CNS autoimmunity, we have identified and characterized T cells that respond to AQP4 in C57BL/6 and SJL/J mice, two strains that are commonly studied in models of CNS inflammatory diseases. Mice were immunized with either overlapping peptides or intact hAQP4 protein encompassing the entire 323 amino acid sequence. T cell determinants identified from examination of the AQP4 peptide (p) library were located within AQP4 p21-40, p91-110, p101-120, p166-180, p231-250 and p261-280 in C57BL/6 mice, and within p11-30, p21-40, p101-120, p126-140 and p261-280 in SJL/J mice. AQP4-specific T cells were CD4+ and MHC II-restricted. In recall responses to immunization with intact AQP4, T cells responded primarily to p21-40, indicating this region contains the immunodominant T cell epitope(s) for both strains. AQP4 p21-40-primed T cells secreted both IFN-γ and IL-17. The core immunodominant AQP4 21-40 T cell determinant was mapped to residues 24-35 in C57BL/6 mice and 23-35 in SJL/J mice. Our identification of the AQP4 T cell determinants and characterization of its immunodominant determinant should permit investigators to evaluate the role of AQP4-specific T cells in vivo and to develop AQP4-targeted murine NMO models.

Effect of oral beta interferon on subsequent immune responsiveness.

Oral administration of myelin antigens reduces the incidence and severity of EAE in rat and mouse models and decreases the frequency of MBP-reactive cells and the frequency of attacks in some patients with multiple sclerosis. Low-dose oral tolerance has been shown to be mediated by Th2-type regulatory cells that secrete TGFbeta and IL-4/IL-10. Adjuvants and cytokines may modulate oral tolerance. The addition of betaIFN to the experimental therapy regimen, either orally or by intraperitoneal injection, has been shown to enhance the suppressive effects of oral myelin antigens when either are fed the suboptimal dosing regimen to suppress EAE. The current studies were conducted to elucidate the mechanism of the observed in vivo synergy of betaIFN and antigen feeding. Analysis of the in vitro proliferative response and cytokine production by lymphocytes from fed animals in response to specific antigen in culture shows that the synergistic effect may be related to both independent suppression of the immune response by oral betaIFN and enhanced production of TGFbeta and IL-4/IL-10. There was an unexpected increase in the production of gammaIFN by lymphocytes in vitro after three doses of oral betaIFN in vivo. These observations have important implications for the use of cytokines to modulate oral tolerance.

Malignant glioma cells use MHC class II transactivator (CIITA) promoters III and IV to direct IFN‐γ‐inducible CIITA expression and can function as nonprofessional antigen presenting cells in endocytic processing and CD4+ T‐cell activation

Malignant gliomas (MGs), lethal human central nervous system (CNS) neoplasms, contain tumor infiltrating lymphocytes (TIL). Although MHC class II molecules are frequently detected on MG cells, suggesting that they may be capable of antigen (Ag) presentation to CD4+ T cells, deficiencies in CD4+ T‐cell activation are associated with these nonimmunogenic tumors. We evaluated regulation of the MHC class II transactivator (CIITA), the key intermediate that controls class II expression, in MG cells and tested whether MG cells could process native Ag. After interferon‐γ (IFN‐γ) stimulation, MG cells upregulated CIITA and class II molecules. IFN‐γ‐inducible CIITA expression in MG cells, as well as primary human astrocytes, was directed by two CIITA promoters, pIV, the promoter for IFN‐γ‐inducible CIITA expression in nonprofessional antigen‐presenting cells (APC), and pIII, the promoter that directs constitutive CIITA expression in B cells. Both pIII and pIV directed CIITA transcription in vivo in MGs and ex vivo in IFN‐γ‐activated primary MG cultures. We also demonstrate for the first time that MG cells can process native Ag for presentation to CD4+ MHC class II‐restricted Th1 cells, indicating that MG cells can serve as nonprofessional APC. CIITA may be a key target to modulate MHC class II expression, which could augment immunogenicity, Ag presentation, and CD4+ T‐cell activation in MG therapy. GLIA 36:391–405, 2001.

Immunodominant T-cell epitopes of MOG reside in its transmembrane and cytoplasmic domains in EAE

Objective: Studies evaluating T-cell recognition of myelin oligodendrocyte glycoprotein (MOG) in multiple sclerosis (MS) and its model, experimental autoimmune encephalomyelitis (EAE), have focused mostly on its 117 amino acid (aa) extracellular domain, especially peptide (p) 35-55. We characterized T-cell responses to the entire 218 aa MOG sequence, including its transmembrane and cytoplasmic domains. Methods: T-cell recognition in mice was examined using overlapping peptides and intact full-length mouse MOG. EAE was evaluated by peptide immunization and by adoptive transfer of MOG epitope-specific T cells. Frequency of epitope-specific T cells was examined by ELISPOT. Results: Three T-cell determinants of MOG were discovered in its transmembrane and cytoplasmic domains, p119–132, p181–195, and p186–200. Transmembrane MOG p119-132 induced clinical EAE, CNS inflammation, and demyelination as potently as p35-55 in C57BL/6 mice and other H-2b strains. p119-128 contained its minimal encephalitogenic epitope. p119-132 did not cause disease in EAE-susceptible non-H-2b strains, including Biozzi, NOD, and PL/J. MOG p119-132–specific T cells produced Th1 and Th17 cytokines and transferred EAE to wild-type recipient mice. After immunization with full-length MOG, a significantly higher frequency of MOG-reactive T cells responded to p119-132 than to p35-55, demonstrating that p119-132 is an immunodominant encephalitogenic epitope. MOG p181-195 did not cause EAE, and MOG p181-195–specific T cells could not transfer EAE into wild-type or highly susceptible T- and B-cell–deficient mice. Conclusions: Transmembrane and cytoplasmic domains of MOG contain immunodominant T-cell epitopes in EAE. A CNS autoantigen can also contain nonpathogenic stimulatory T-cell epitopes. Recognition that a myelin antigen contains multiple encephalitogenic and nonencephalitogenic determinants may have implications for therapeutic development in MS.

Tolerance checkpoint bypass permits emergence of pathogenic T cells to neuromyelitis optica autoantigen aquaporin-4.

Significance Neuromyelitis optica (NMO) is a CNS autoimmune demyelinating disease involving aquaporin-4 (AQP4)-specific IgG1, a T-cell–dependent antibody subclass. The role of T cells in NMO is unclear. We evaluated AQP4-specific T cells in WT and AQP4−/− mice. AQP4 epitopes identified in WT mice were not pathogenic. AQP4 peptide (p) 135–153 and p201–220 elicited robust T-cell responses in AQP4−/− but not WT, mice. T-cell receptor repertoire utilization for these determinants in AQP4−/− mice was unique. Donor AQP4−/− p135–153- or p201–220-specific Th17 cells entered the CNS of recipient WT mice and induced CNS autoimmunity. Our findings indicate pathogenic AQP4-specific T cells are normally restrained by central tolerance, which could be relevant to understanding the origin of pathogenic T cells in NMO. Aquaporin-4 (AQP4)-specific T cells are expanded in neuromyelitis optica (NMO) patients and exhibit Th17 polarization. However, their pathogenic role in CNS autoimmune inflammatory disease is unclear. Although multiple AQP4 T-cell epitopes have been identified in WT C57BL/6 mice, we observed that neither immunization with those determinants nor transfer of donor T cells targeting them caused CNS autoimmune disease in recipient mice. In contrast, robust proliferation was observed following immunization of AQP4-deficient (AQP4−/−) mice with AQP4 peptide (p) 135–153 or p201–220, peptides predicted to contain I-Ab–restricted T-cell epitopes but not identified in WT mice. In comparison with WT mice, AQP4−/− mice used unique T-cell receptor repertoires for recognition of these two AQP4 epitopes. Donor T cells specific for either determinant from AQP4−/−, but not WT, mice induced paralysis in recipient WT and B-cell–deficient mice. AQP4-specific Th17-polarized cells induced more severe disease than Th1-polarized cells. Clinical signs were associated with opticospinal infiltrates of T cells and monocytes. Fluorescent-labeled donor T cells were detected in CNS lesions. Visual system involvement was evident by changes in optical coherence tomography. Fine mapping of AQP4 p201–220 and p135–153 epitopes identified peptides within p201–220 but not p135–153, which induced clinical disease in 40% of WT mice by direct immunization. Our results provide a foundation to evaluate how AQP4-specific T cells contribute to AQP4-targeted CNS autoimmunity (ATCA) and suggest that pathogenic AQP4-specific T-cell responses are normally restrained by central tolerance, which may be relevant to understanding development of AQP4-reactive T cells in NMO.