Halina Offner

Antibodies against I-A and I-E determinants inhibit the activation and function of encephalitogenic T-lymphocyte lines☆

Two monoclonal antibodies, OX-6 and OX-17, were used to evaluate respectively the roles of I-A and I-E major histocompatibility complex Class II gene products in the in vitro activation and subsequent function in recipient rats of encephalitogenic T-cell lines. Activation of the T-cell lines with guinea pig myelin basic protein (GP-BP) presented by accessory cells (APC) resulted in an increase in the number of blast cells in culture and was reflected by increased uptake of [3H]thymidine [( 3H]Tdy). The number of blasts recovered and [3H]Tdy uptake during activation was reduced drastically in the presence of OX-6, but to a much lesser extent in the presence of OX-17. OX-6 but not OX-17 appeared to block T-cell activation primarily by inhibiting APC function, since preincubation of APC but not T cells with OX-6 before stimulation resulted in complete inhibition of the cultures. After activation, the BP-1 T-cell line or D-9 clone transferred severe paralysis to normal recipient rats. Recipients of OX-6-treated BP-1 or D-9 T cells exhibited very mild or no signs, whereas recipients of OX-17-treated cells developed only slightly less severe experimental autoimmune encephalomyelitis (EAE) than recipients of untreated encephalitogenic control cultures. In contrast, treatment with OX-17 but not OX-6 reduced the ability of BP-reactive T cells to transfer delayed-type hypersensitivity reactions. Dermal testing with GP-BP in the ears of recipient rats just prior to onset of clinical signs decreased significantly the clinical intensity of EAE induced by activated BP-reactive T cells, but increased the clinical scores in rats which received unstimulated or OX-6-treated T cells. This potentiating effect of GP-BP was due most likely to the presentation of processed antigen to circulating BP-reactive T cells by APC in the ear. These results suggest that both the I-A and I-E gene products may contribute to the activation and subsequent function of encephalitogenic T cells, perhaps through separate mechanisms.

The autoreactive T cell population in experimental allergic encephalomyelitis: T cell receptor β-chain rearrangements☆

T cell receptor beta-chain gene rearrangements were examined in myelin basic protein (MBP)-reactive T cell lines and hybridomas from Lewis (Lew) rats. Nearly 75% of the cloned hybrids were specific for the major encephalitogenic determinant residues 68-88 of guinea pig (GP) MBP; three fine specificities could be distinguished. Southern blot analysis of receptor beta-chain genes revealed polyclonality and shared rearrangements not seen in non-68-88-specific clones. Generation of short-term, encephalitogenic Lew T cell lines revealed rearrangements shared with the 68-88-specific hybrids, indicating that the hybrids were representative of the whole antigen-specific population and suggesting restricted V beta gene usage within this polyclonal population.

Specific ganglioside binding to receptor sites on T lymphocytes that couple to ganglioside-induced decrease of CD4 expression

The binding of different gangliosides to rat T-helper lymphocytes was characterized under conditions that decrease CD4 expression on different mammalian T-helper lymphocytes. Saturation binding by monosialylated [3H]-GM1 to rat T-lymphocytes was time- and temperature-dependent, had a dissociation constant (KD) of 2.2 +/- 1.4 microM and a binding capacity near 2 fmoles/cell. Competitive inhibition of [3H]-GM1 binding demonstrated a structural-activity related to the number of unconstrained sialic acid moieties on GM1-congeneric gangliosides. A comparison between the results of these binding studies and ganglioside-induced decrease of CD4 expression demonstrated that every aspect of [3H]-GM1 binding concurs with ganglioside modulation of CD4 expression. It is concluded that the specific decrease of CD4 expression induced by pretreatment with gangliosides involves the initial process of ganglioside binding to specific sites on CD4+ T-helper lymphocytes.

Transmembrane signalling associated with ganglioside-induced CD4 modulation

Ganglioside (GM1) treatment of CD4+ human CEM lymphoma cells stimulated transient phosphoinositide (PI) breakdown, production of inositol phosphates (IP), protein phosphorylation and rapid decrease of CD4 surface expression. A comparison between the actions of GM1 and other agents that affect these signal transduction pathways demonstrated a distinct mechanism for GM1-induced decrease of CD4. GM1 stimulated both phospholipase C activity and protein phosphorylation but had no effect on either cellular cAMP levels or tyrosine kinase activity. Phorbol myristate acetate (PMA) stimulated protein phosphorylation and caused a significant decrease in surface display of CD4. Both of these processes were blocked by pretreating cells with the protein kinase C (PKC) inhibitor H7. These results demonstrate that GM1 stimulates PI turnover and induces a rapid decrease of CD4 surface expression by processes that do not activate adenylate cyclase or tyrosine kinase. They further demonstrate that the mechanism for GM1-induced decrease of CD4 is distinct from the CD4 internalization processes mediated by PKC activity.

Protection against experimental encephalomyelitis: Idiotypic autoregulation induced by a non-encephalitogenic T cell clone expressing a cross-reactive T cell receptor V gene

The recovery process in experimental autoimmune encephalomyelitis (EAE) in Lewis rats is characterized by an increasing diversity of T cell clones directed at secondary epitopes of myelin basic protein. Of particular interest, residues 55 to 69 of guinea pig basic protein could induce protection against EAE. A nonencephalitogenic T cell clone, C455-69, that was specific for this epitope transferred protection against both active and passive EAE. Clone C4 was found to express V beta 8.6 in its Ag receptor, and residues 39 to 59 of the TCR V beta 8.6 sequence were found to be highly crossreactive with the corresponding residues 39 to 59 of TCR V beta 8.2, which is known to induce protective anti-idiotypic T cells and antibodies. Like the TCR V beta 8.2 peptide, the V beta 8.6 sequence induced autoregulation and provided effective treatment of established EAE. Thus, the EAE-protective effect of the guinea pig basic protein 55-69 sequence was most likely mediated by T cell clones such as C4 that could efficiently induce anti-TCR immunity directed at a cross-reactive regulatory idiotope.

Vaccination with BV8S2 protein amplifies TCR specific regulation and protection against experimental autoimmune encephalomyelitis in TCR BV8S2 transgenic mice

TCR determinants overexpressed by autopathogenic Th1 cells can naturally induce a second set of TCR-specific regulatory T cells. We addressed the question of whether immune regulation could be induced naturally in a genetically restricted model in which a major portion of TCR-specific regulatory T cells expressed the same target TCR BV8S2 chain as the pathogenic T cells specific for myelin basic protein (MBP). We found vigorous T cell responses to BV8S2 determinants in naive mice that could be further potentiated by vaccination with heterologous BV8S2 proteins, resulting in the selective inhibition of MBP-specific Th1 cells and protection against experimental encephalomyelitis. Moreover, coculture with BV8S2-specific T cells or their supernatants reduced proliferation, IFN-gamma secretion, and encephalitogenic activity of MBP-specific T cells. These results suggest that immune regulation occurs through a nondeletional cytokine-driven suppressive mechanism.

Androgen treatment of encephalitogenic T-cells alters cytokine production and reduces the severity of experimental autoimmune encephalomyelitis

4 4 9 increased Expression of the Thl-associated Chemokinc Receptors, C X C R 3 and CCR5, by T Cells D u r i n g Progress ive Mult ip le Sclerosis K.E. Balashov, Brigham & Womens Hospital, USA, W.W. Hancock, Letd:oS ite, lnc., USA, S.J. Khoury, H.L. Weiner, Brigham & Women s Hospital, USA 4 5 2 Androgen Treatment of Encephalitogenic T-cells Al te rs Cytok ine Produc t ion and Reduces the Severity of Exper imen ta l A u t o i m m u n e Encephalomyelitis B.F. Bebo. Jr.. J.C. Schuster, A.A. Vandenbark, H. Offimr, Oregon Health Sciences Univ., Portland, USA

TCR peptide-specific human TH2 cells inhibit BP-specific TH1 effector cells

Multiple sclerosis patients boosted intradermally with low doses of TCR CDW peptides developed significantly increased frequencies of TCR peptide specific T cells, with concomitant reduction in frequencies of BP-reactive T cells. T cell clones obtained from TCR peptide-responsive donors produced lymphokine message and protein for IL-4, IL-5, IL-lo, and in some cases TGF-0, but not for IFN-y or IL-2 after stimulation with specific TCR peptide, indicating a ThWike response. Supernatants from these clones inhibited proliferation and Thl lymphokines (IFN-7 and IL-2) of BP-specific clones from the same or heterologous donors in a dose-dependent fashion, indicating the presence of soluble regulatory factors. This inhibition could be abrogated predominantly by anti-IL-10 and in some cases anti-TGF-/3 antibodies, implicating these lymphokines in regulation. The release of soluble inhibitory factors by TCR peptide specific clones suggest regulation of target as well as local bystander T effector cells. These demonstrate an in vitro regulatory mechanism that may account for the apparent in viva regulation induced by TCR peptide therapy. FURTHER ORSERVAIIONS ON CELL ADHFSION MOLECULES IN CIRCULATING AND CSF LYMFHOCYTRS IN MS

Immunoregulatory determinants on rat TCR Vβ8.2.

Antineulrophil cytoplasmic antibodies (ANCA) have been detected in patients with Wegener granulomatosis, various forms of necrotizing vasculitis and other autoimmune diseases such as connective tissue diseases, inflammatory bowel disease and autoimmune liver disease. The humoral immune response seems to play a role in the pathogenesis of MS. The blood brain barrier and particulary the endothelial cell may be a potential target for this immune response. The possible expression of proteinase-3 (target for C-ANCA) and of myeloperoxidase (target for P-ANCA) at the surface of activated endothelial cells might allow these antibodies to injure the endothelium. It is from this standpoint that we have investigated the prevalence of ANCA in the sera of 50 consecutive patients with clinically definite MS (28 women, 22 men; mean age 35.1 years; mean time from disease’s onset 6 years; 27 relapsing-remitting and 23 chronic progressive). ANCA were investigateg by indirect immunefluorescence test and confirm by ELBA. Three out of 50 (6%) had a positive titre of ANCA although at low levels (one P-ANCA, two C-ANCA). No correlation between ANCA and any clinical parameter was found. ANCA do not seem to play a role in the pathogenesis of MS.

TCR Peptide Therapy in Autoimmunity

T cell-mediated autoimmunity may be viewed as the selection and expansion of “self” reactive clones that are not deleted during thymic maturation. The thymic negative selection process presumably does not function because organ specific autoantigens are virtually absent, and one may assume that potentially pernicious T cells are continually replenished from stem cells throughout the life of an individual. The presence of autoreactive T cells in the circulation of clinically normal individuals indicates that natural mechanisms effectively regulate these T cells. The goal of this manuscript is to describe and evaluate one such regulatory mechanism directed at epitopes found on germline T cell receptor V region sequences.