Evidence For Tcell Mediated Autoimmunity In Ms

Interest in a potential autoimmune explanation for MS led to a search for T-cells reactive to myelin antigens. These antigens include MBP, myelin associated glyco-protein (MAG), myelin oligodendrocyte glycoprotein (MOG), and proteolipid protein (PLP). Increased frequencies of PLP-reactive T-cells are reported in both blood and CSF of MS patients compared to controls (64,65). However, it has been shown that healthy controls harbor T-cells in their peripheral blood also reactive to MBP, MAG, and MOG in frequencies similar to MS patients (66). Thus, investigators have sought to demonstrate differences between MS patients and controls in the fine specificity, functional state, and activation state of these myelin-reactive T-cells.

The fine specificity of a TCR denotes its specific recognition of an antigen epitope. MBP83-99 has been cited as the human immunodominant sequence within MBP (67), but T-cells reactive to this epitope have been identified in healthy controls as well as MS patients (68). Likewise, investigation into the fine specificity for T-cell recognition of MOG has yielded no clear distinction between MS patients and healthy controls, including recognition of the immunodominant regions of MOG (a.a. 11-30) (69). This same region has proven encephalitogenic in the EAE model (70). Pelfrey et al. (71) found that T-cells from MS patients and healthy controls responded to many different epitopes of PLP, scattered throughout the molecule. However, MS patients responded to four times more peptide sequences of PLP than controls, and they had 11 times higher numbers of PLP peptide-specific IFN-y-pro-ducing cells than controls.

T-cells from MS patients and controls also differ in cytokine-secreting profile upon activation. MHC Il-restricted CD4+ T-cells that manufacture IFN-y, IL-2, lym-photoxin, and TNF-a are defined as Th1 cells and may be thought of as "pro-inflammatory" cells promoting disease in MS. Functions of Th1 cytokines include immune cell activation and induction of adhesion molecule expression, recruitment of additional immune cells, and perhaps direct mediation of myelin damage. T-cells producing IL-4, IL-5, IL-10, and IL-13 are termed Th2 cells and promote antibody-mediated, immune complex, and allergic disorders. In the context of MS, these cells are considered "anti-inflammatory" and antagonistic to the effects of Thl cells (72,73).

In reality, human T-cells do not strictly conform to the dichotomous cytokine expression patterns of Thl and Th2-cells as seen in mice and it is an oversimplification to consider these as "pro-" and "anti-inflammatory," respectively. Some studies have suggested a tendency for myelin-reactive T-cells in MS patients toward the Thl phenotype. For example, Correale et al. (74) found that T-cell clones to PLP generated during acute MS attacks were skewed toward Thl phenotypes. During disease quiescence, clones showed Th0, Thl, and Th2 phenotypes. Several investigators have reported increased expression of the chemokine receptor CCR5, characteristic of Thl cells, and its corresponding chemokines in the CSF and CNS tissues from MS patients (75-77).

Hellings et al. (78) demonstrated a temporal association between clinical disease activity and antimyelin T-cell responses. Earlier studies of a limited number of MS patients also suggested such an association (79). Soderstrom et al. (80) observed increased levels of T-cells recognizing MBP, PLP, and myelin associated glycoprotein in peripheral blood and CSF of untreated MS patients, but did not observe an association of T-cell responses with disease activity. Hellings found a number of immune changes coincident in some instances with the detection of active lesions by MRI or with clinical exacerbations. These changes included an increase in myelin-reactive IFN-y secreting T-cells, clonally expanded myelin-reactive T-cells, elevated pro-inflammatory and decreased anti-inflammatory cytokine production, upregulation of ICAM-l, and highly increased serum soluble VCAM-l.

Clearly, the mere presence of myelin-reactive T-cells in the periphery is not sufficient to cause MS. It has been reasoned that if myelin specific T-cells caused MS, these cells would show signs of prior activation. Several different lines of investigation have shown that in many MS patients myelin reactive T-cells have been previously activated. Zhang et al. examined whether peripheral blood-derived mye-lin-reactive T-cells in MS patients existed in a different state of activation compared with healthy controls. Activated T-cells, but not resting T-cells, express IL-2 receptors. In an in vitro study, no difference in the frequency of MBP or PLP-reactive CD4+ T-cells was found after primary antigen stimulation between RRMS patients and normal controls. However, when cells were first cultured with recombinant IL-2 to enrich IL-2 receptor positive cells prior to stimulation with antigen, the frequency of MBP and PLP-reactive T-cells was higher in MS patient cell lines than in controls. In CSF samples, MBP-reactive T-cells were recovered from MS patients but not from controls. In the CSF, IL-2 stimulation yielded MBP-reactive cells more than l0-fold higher in paired blood samples (8l) indicating that these activated MBP-specific T-cells entered the CNS.

T-cells that have been activated previously do not require B7 costimulation of CD28 for reactivation. Thus, another method of demonstrating prior activation of myelin-reactive T-cells is to quantify the number that do not require costimulation for activation. Using cell transfectants expressing MHC-IIDR2 alone or cotransfected with human B7-1 or -2, to present the immunodominant MBP85-99 to purified CD4+ T-cells from DR2+ RRMS patients and controls, Scholz et al. (82) observed that cells from control subjects did not expand in response to the MBP85-99 in the absence of costimulation, but MBP-reactive T-cells from MS patients were activated without B7 costimulation. Lovett-Racke et al. (83) had a similar rationale when using anti-CD28 antibodies to block costimulation by B7 molecules. In their studies, MBP-reactive T-cell expansion was inhibited by blockade of the CD28 : B7 interaction in normal individuals but not in MS patients.

Another marker of previous activation and proliferation in T-cells is genetic mutation. T-cells that have proliferated previously can develop mutations in the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene; these cells may then be isolated by exposure to 6-thioguanine, which is toxic to nonmutated cells. Allegretta et al. (84). identified MBP and MBP-peptide specific HPRT mutant T-cells from the peripheral blood of MS patients but not from controls. Later experiments did find HPRT mutants in control blood, but to a lesser degree than in MS patients (85,86). Trotter et al. (87) found significantly more HPRT mutant T-cell lines in MS patients than controls, and in addition some of these mutated T-cell lines recognized multiple epitopes of PLP. During a clinical exacerbation, HPRT-mutant lines derived from one MS patient recognized the specific PLP178-191 peptide. These PLP178-191 reactive mutant T-cell lines were not detected during remission.

Wulff et al. (88) took another approach to explore the previous exposure of T-cells to myelin antigens as indirect evidence for autoimmune pathogenesis of MS. They exploited their finding that human effector memory T-cells express high levels of the voltage-gated Kvl.3 channel, whereas naive and central memory T-cells express far lower levels. T-cells reactive with MOG, MBP, or PLP from MS patients expressed far more Kvl.3 channels per cell than T-cells reactive with these antigens from control subjects. In contrast, the level of Kvl.3 channels in GAD65-reactive T-cells, insulin-reactive T-cells, and the vast majority of ovalbumin-reactive T-cells derived from MS patients was low and not higher than that for controls. Mitogen-reactive T-cells from MS patients and controls had similar levels of Kvl.3 channels per cell, suggesting that the general level of effector memory T-cells in MS patients was similar to that of the controls. Taken together, data from the studies discussed in the preceding paragraphs strongly indicate that MS patients harbor more previously activated memory T-cells directed against myelin antigens than do control subjects.

Despite the varied studies, indicating that T-cells reactive with myelin antigens are more frequently activated or previously activated in MS patients than controls, it should be recognized that T-cell activation may be secondary to the liberation of myelin antigens that occurs with myelin damage. One manner in which MS might be proven to be autoimmune would entail specific deletion of myelin-directed T-cells in MS patients, followed by sustained demonstration of disease remission. These cell populations have been selectively deleted in vivo by vaccination with autologous myelin-reactive T-cells harvested from CSF, but to date no blinded results demonstrating clinical efficacy have been published (89,90).

Strong evidence that the pathogenesis of MS is autoimmune derived from an attempt to induce anergy into myelin-reactive T-cells in MS patients with the hope that this would be beneficial. The therapy, known as altered peptide ligand therapy, involves altering several amino acids within an antigenic peptide capable of activating T-cells. Alterations within the TCR contact regions can lead to T-cell inactivation when the altered peptide is presented to the T-cell. When Bielekova et al. (91) treated MS patients with a high dose of an altered peptide ligand of the major immunogenic epitope of human MBP83-99, instead of inducing anergy, 3 of 8 patients experienced expansion of their myelin-directed T-cells anywhere from 10-fold to 300-fold. All the three patients had a dramatic increase in MRI contrast-enhancing lesions and all three had clinical relapses. The results of this trial, which was halted early, directly link disease activity with an enhanced T-cell response to an autoantigen (MBP). This therapy is still undergoing investigation, but with modifications including lowered dose.

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