Autoimmunity to antigens of the central nervous system is usually considered detrimental. T cells specific to a central nervous system self antigen, such as myelin basic protein, can indeed induce experimental autoimmune encephalomyelitis, but such T cells may nevertheless appear in the blood of healthy individuals. We show here that autoimmune T cells specific to myelin basic protein can protect injured central nervous system neurons from secondary degeneration. After a partial crush injury of the optic nerve, rats injected with activated anti-myelin basic protein T cells retained approximately 300% more retinal ganglion cells with functionally intact axons than did rats injected with activated T cells specific for other antigens. Electrophysiological analysis confirmed this finding and suggested that the neuroprotection could result from a transient reduction in energy requirements owing to a transient reduction in nerve activity. These findings indicate that T-cell autoimmunity in the central nervous system, under certain circumstances, can exert a beneficial effect by protecting injured neurons from the spread of damage.

IntroductionMesenchymal stem cells (MSCs) comprise an adult population that resides in many organs and exhibits multiple functions and phenotypes upon in vitro culture; MSCs can be induced to differentiate into mesodermal cell lineages, 1,2 support and regulate hematopoiesis, [3][4][5][6][7] regulate the stem-cell niche, [8][9][10][11][12] and may participate in the repair of tissue damage inflicted by normal wear and tear, injury, or disease. [13][14][15][16] MSCs comprise 0.01% to 0.001% of the bone marrow (BM)-nucleated cells and are obtained by expansion of the BM, plastic-adherent cell fraction. 1,[17][18][19][20][21] Under certain physiologic or experimental conditions, MSCs can be induced to differentiate in vitro into cells of the mesodermal lineage, specifically to osteocytes, adipocytes, chondrocytes, myocytes, tenocytes, myocardiocytes, and hematopoietic supportive stroma. 1,17,19,22 MSCs are an attractive cell-based therapy tool for developmental defects; degenerating diseases; and bone, cartilage, muscle, and other mesodermal tissue injuries. [23][24][25][26][27][28][29][30] Toll-like receptors (TLRs) are a class of molecules first discovered to play a role in body development 31 and later found to play a role in body maintenance. [32][33][34][35][36] The TLR family has been shown to be of importance in the innate immune system for the recognition of pathogen-associated molecular patterns (PAMPs) by immune cells, initiating a primary response toward invading pathogens and recruitment of the adaptive immune response. 32,[37][38][39][40][41][42][43][44][45][46][47][48][49] TLRs can be activated not only by pathogen components, but also by mammalian endogenous molecules such as heat-shock proteins and extracellular matrix breakdown products. [50][51][52] In the steady state, during the generation of immune cells, as well as under pathologic conditions, there are intimate interactions between lymphocyte populations and the organ stroma mesenchyme. These interactions regulate cell growth and differentiation and control cell functions. It is possible therefore that lymphocytes and the stromal mesenchyme share regulatory mechanisms. To test this possibility we aimed, in the present study, to examine the expression and possible regulatory functions of TLRs in mesenchymal cells.We explored the expression of TLRs by MSCs, the response of MSCs to known TLR activators, and the ability of a TLR-2 ligand to regulate MSC proliferation and differentiation. We show here that cultured MSCs express TLR molecules 1 to 8, but not TLR-9. Activation of MSCs by TLR ligands induced interleukin-6 (IL-6) secretion and nuclear factor B (NF-B) nuclear translocation. Pam3Cys, a prototypic ligand for TLR-2, induced proliferation of MSCs and regulated their differentiation. Relatively little is known about the signals that regulate MSC proliferation, differentiation, and development. 53,54 Our findings suggest that TLR signaling may play a role in restraining MSC differentiation and thus promote MSC renewal. Materials and methods ...

The isolation and propagation of functional antigen-specific lines of T lymphoblasts is described. These lines were found to recognize foreign or self antigens in association with accessory cells of syngeneic major histocompatibility complex genotype. Intravenous inoculation of a T cell reactive only against myelin basic protein led to development of clinical paralysis in syngeneic rats. Thus, it is possible to study biological function as well as antigen specificity using T cell lines.

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Partial injury to the spinal cord can propagate itself, sometimes leading to paralysis attributable to degeneration of initially undamaged neurons. We demonstrated recently that autoimmune T cells directed against the CNS antigen myelin basic protein (MBP) reduce degeneration after optic nerve crush injury in rats. Here we show that not only transfer of T cells but also active immunization with MBP promotes recovery from spinal cord injury. Anesthetized adult Lewis rats subjected to spinal cord contusion at T7 or T9, using the New York University impactor, were injected systemically with anti-MBP T cells at the time of contusion or 1 week later. Another group of rats was immunized, 1 week before contusion, with MBP emulsified in incomplete Freund's adjuvant (IFA). Functional recovery was assessed in a randomized, double-blinded manner, using the open-field behavioral test of Basso, Beattie, and Bresnahan. The functional outcome of contusion at T7 differed from that at T9 (2.9 Ϯ 0.4, n ϭ 25, compared with 8.3 Ϯ 0.4, n ϭ 12; p Ͻ 0.003). In both cases, a single T cell treatment resulted in significantly better recovery than that observed in control rats treated with T cells directed against the nonself antigen ovalbumin. Delayed treatment with T cells (1 week after contusion) resulted in significantly better recovery (7.0 Ϯ 1; n ϭ 6) than that observed in control rats treated with PBS (2.0 Ϯ 0.8; n ϭ 6; p Ͻ 0.01; nonparametric ANOVA). Rats immunized with MBP obtained a recovery score of 6.1 Ϯ 0.8 (n ϭ 6) compared with a score of 3.0 Ϯ 0.8 (n ϭ 5; p Ͻ 0.05) in control rats injected with PBS in IFA. Morphometric analysis, immunohistochemical staining, and diffusion anisotropy magnetic resonance imaging showed that the behavioral outcome was correlated with tissue preservation. The results suggest that T cell-mediated immune activity, achieved by either adoptive transfer or active immunization, enhances recovery from spinal cord injury by conferring effective neuroprotection. The autoimmune T cells, once reactivated at the lesion site through recognition of their specific antigen, are a potential source of various protective factors whose production is locally regulated.

Insulin-dependent diabetes mellitus is caused by autoimmune destruction of the insulin-producing beta cells resident in the pancreatic islets. We recently discovered that the pathogenesis of diabetes in NOD strain mice was associated with T-cell reactivity to an antigen cross-reactive with a mycobacterial 65-kDa heat shock protein. To identify peptide epitopes critical to the insulin-dependent diabetes mellitus of NOD mice, we studied the specificities of helper T-cell clones capable ofcausing hyperglycemia and diabetes. We now report the identification of a functionally important peptide within the sequence ofthe human variant ofthe 65-kDa heat shock protein molecule. T-cell clones recognizing this peptide mediate insulitis and hyperglycemia. Alternatively, the T cells can be attenuated and used as therapeutic T-cell vaccines to abort the diabetogenic process. Moreover, administration of the peptide itself to NOD mice can also down-regulate immunity to the 65-kDa heat shock protein and prevent the development of diabetes. Thus, T-cefl vaccination and specific peptide therapy are feasible in spontaneous autoimmune diabetes.Insulin-dependent diabetes mellitus (IDDM) of both humans and NOD strain mice becomes clinically overt after most of the beta cells in the islets have been destroyed by an autoimmune process (1). The destruction of the beta cells seems to be caused by autoimmune T cells (2-4) that may recognize a processed peptide antigen presented by a major histocompatibility complex (MHC) class II molecule (5).To identify a peptide epitope important in the IDDM of NOD mice, we investigated the antigen specificity recognized by diabetogenic T-cell clones responding to the 65-kDa heat shock protein (hsp65) of Mycobacterium tuberculosis (MT-hsp65). We earlier reported that diabetogenic T cells recognized an epitope on this molecule (6). We now report that the target epitope is present in the sequence ofthe human hsp65 (H-hsp65) molecule, that the T cells responding to this epitope can therapeutically vaccinate mice against IDDM, and that the peptide epitope itself can be used to treat the disease. MATERIALS AND METHODSMice. E. Leiter (The Jackson Laboratory) kindly supplied breeding nuclei of the spontaneously diabetic NOD/Lt (NOD) strain and of the nondiabetic NON.H-2NOD strain. The NON.H_2NoD mice were in their 11th backcross generation and were congenic at the H-2 complex with the NOD mice.Antigens. Recombinant H-hsp65, recombinant MT-hsp65, recombinant mycobacterial 70-kDa heat shock protein (hsp70), and control Escherichia coli antigen were prepared as described (6-8). Control E. coli were transfected with the pEX2 plasmid that did not contain the hsp65 genes. The H-hsp65 gene was the gracious gift of Richard A. Young (Massachusetts Institute of Technology, Cambridge). Peptides p277 and p278 were synthesized by Ora Goldberg (Biological Services Laboratory ofthe Weizmann Institute of Science) with an automated synthesizer and were purified on a Biogel p-4 column (50 x 1.5 cm; Bio-Rad). The sequence...

Insulin-dependent diabetes mellitus is caused by autoimmune destruction of the insulin-producing beta cells of the pancreas. The results described here indicate that a beta-cell target antigen in non-obese diabetic (NOD/Lt) mice is a molecule cross-reactive with the 65-kDa heat shock protein (hsp65) of Mycobacterium tuberculosis. The onset of beta-cell destruction is associated with the spontaneous development of anti-hsp65 T lymphocytes. Subsequently hsp65 cross-reactive antigen becomes detectable in the sera of the prediabetic mice and some weeks later anti-hsp65 antibodies, anti-insulin antibodies, and anti-idiotypic antibodies to insulin antibodies become detectable. The hsp65-cross-reactive antigen, the autoantibodies, and the T-cell reactivity then decline with the development of overt insulin-dependent diabetes. The importance of hsp65 in the pathogenesis of insulin-dependent diabetes was confirmed by the ability of clones of anti-hsp65 T cells to cause insulitis and hyperglycemia in young NOD/Lt mice.Moreover, hsp65 antigen could be used either to induce diabetes or to vaccinate against diabetes, depending on the form of its administration to prediabetic NOD/Lt mice. Other antigens such as the 70-kDa heat shock protein (hsp7O) had no effect on the development of diabetes.Type 1 or insulin-dependent diabetes mellitus (IDDM) is caused in most cases by autoimmune destruction of the insulin-producing beta cells resident in the islets of the pancreas (1). It is thought that once the autoimmune process takes root, it progresses relentlessly without causing symptoms until the number of beta cells irreversibly destroyed is so large, perhaps 90% of the beta-cell mass, that the individual suffers a derangement in glucose homeostasis and requires an exogenous supply of insulin to sustain life.The non-obese diabetic (NOD/Lt) mouse is a useful experimental model of IDDM (1). NOD/Lt mice spontaneously develop inflammation of the islets, insulitis, beginning at 4-6 weeks of age which progresses to overt IDDM at 4-5 months of age. Autoimmune T lymphocytes would seem to be the cause of beta-cell destruction because IDDM can be adoptively transferred to very young prediabetic NOD/Lt mice with T lymphocytes from older mice (2).Identification of target antigens recognized in the pathogenesis of IDDM is important for at least two reasons: specific antigens would facilitate the early diagnosis of preclinical IDDM and they might be used to abort the destructive autoimmune process through modification of the autoimmune response. For example, copolymer 1 (COP 1), a synthetic peptide immunologically cross-reactive with myelin basic protein has been used to alter the course of multiple sclerosis (3).We now show that a beta-cell antigen cross-reactive with a 65-kDa heat shock protein (hsp65) of Mycobacterium tuberculosis, termed hsp65 cross-reactive (hsp65-CR) antigen, is involved in the pathogenesis of NOD/Lt mouse IDDM. MATERIALS AND METHODSMice. The breeding nucleus of NOD/Lt mice was a gift of E. Leiter (Jackson Laborat...