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Chronic inflammatory demyelinating polyneuropathy (CIDP) is a debilitating condition caused by autoimmune demyelination of peripheral nerves. CIDP is associated with increased IL-10, a cytokine with well-described anti-inflammatory effects. However, the role of IL-10 in CIDP is unclear. In this study, we demonstrate that IL-10 paradoxically exacerbates autoimmunity against peripheral nerves. In IL-10-deficient mice, protection from neuropathy was associated with an accrual of highly activated CD4 + T cells in draining lymph nodes and absence of infiltrating immune cells in peripheral nerves. Accumulated CD4 + T cells in draining lymph nodes of IL-10-deficient mice expressed lower sphingosine-1-phosphate receptor 1 ( S1pr1 ), a protein important in lymphocyte egress. Additionally, IL-10 stimulation in vitro induced S1pr1 expression in lymph node cells in a STAT3-dependent manner. Together, these results delineate a novel mechanism in which IL-10-induced STAT3 increases S1pr1 expression and CD4 + T cell migration to accelerate T cell-mediated destruction of peripheral nerves., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Chronic inflammatory demyelinating polyneuropathy results from autoimmune destruction of the peripheral nervous system and is a component of the multiorgan autoimmunity syndrome that results from Aire gene mutations in humans. In parallel, peripheral nervous system autoimmunity resembling chronic inflammatory demyelinating polyneuropathy develops spontaneously in NOD mice with a partial loss of Aire function (NOD.Aire(GW/+) mice) and is a T cell-mediated disease. In this study, we analyze how key aspects of T cell activation and function modulate disease development in Aire-deficient mice. We show that genetic ablation of the Th1 cytokine IFN-γ completely prevents clinical and electrophysiological evidence of neuropathy in NOD.Aire(GW/+) mice. IFN-γ deficiency is associated with absence of immune infiltration and decreased expression of the T cell chemoattractant IP-10 in sciatic nerves. Thus, IFN-γ is absolutely required for the development of autoimmune peripheral neuropathy in NOD.Aire(GW/+) mice. Because IFN-γ secretion is enhanced by B7-CD28 costimulation of T cells, we sought to determine the effects of these costimulatory molecules on neuropathy development. Surprisingly, B7-2 deficiency accelerated neuropathy development in NOD.Aire(GW/+) mice, and Ab blockade of both B7-1 and B7-2 resulted in fulminant, early-onset neuropathy. Thus, in contrast to IFN-γ, B7-2 alone and B7-1/B7-2 in combination function to ameliorate neuropathy development in NOD.Aire(GW/+) mice. Together, these findings reveal distinct and opposing effects of the T cell costimulatory pathway and IFN-γ production on the pathogenesis of autoimmune peripheral neuropathy.

CD8+ cells inhibiting the response of CD4+ cells exist in rodents, recognizing epitopes unique to a CD4+ clone (Ids) or expressed by all activated CD4+ cell (ergotypes). Stimulation of CD8+ cells recognizing ergotypes shared by all Ag-activated CD4+ cells would be useful for treatment of diseases involving undesirable CD4+ responses to ill defined Ags, such as many autoimmune diseases and allergies. As a first step toward demonstrating the existence of anti-ergotype CD8+ immunoregulatory cells in humans, we investigated here whether CD8+ cells recognizing Ag-activated CD4+ cells exist in autoimmune and healthy humans. CD4+ cells specific for human muscle acetylcholine receptor, tetanus, or diphtheria toxoids were propagated from patients with myasthenia gravis patients and healthy controls. Ag-activated CD4+ cells were irradiated and used as Ag to test the response of CD(4+)-depleted CD(8+)-enriched PBMC (CD8+ PBMC) from myasthenic patients and controls and to propagate short-term CD8+ cell lines from CD8+ PBMC. In both patients and controls CD8+ PBMC and CD8+ lines responded vigorously to autologous Ag-activated CD4+ cells. The CD8+ lines responded equally well to the Ag-activated CD4+ cells of different Ag specificity, suggesting that they recognized CD4+ ergotypes. They did not seem to respond to CD4+ cells activated by PHA. The CD8+ cells recognized class I-restricted epitopes, as their response to activated CD4+ cells was suppressed by anti-class I Ab. CD8+ cells recognizing Ag-activated CD4+ were present cells in the controls for 5 to 12 wk after immunization. In myasthenic patients, CD8+ cells recognizing activated anti-acetylcholine receptor CD4+ cells seemed to be always present.

We propagated from myasthenia gravis (MG) patients by stimulation in vitro with synthetic sequences (alpha 48-67, alpha 304-322, gamma 75-94 and gamma 321-340) of the human muscle acetylcholine receptor (AChR), CD4+ lines against four 20-residue sequence regions of the AChR alpha and gamma subunits that are recognized by Th cells of most MG patients. Most lines secreted IL-2 and not IL-4, suggesting that they comprise Th1 cells. For three lines we verified that, as reported previously, AChR epitopes are presented by DR molecules: their response to the relevant peptide was abolished by anti-DR Abs. The DR molecules presenting AChR epitopes were identified by testing the response of the lines to the relevant peptide, using APC from donors homozygous for the different DR alleles of the line. We tested the lines with single residue-substituted analogues of the epitope sequence. The results of these experiments indicated that the lines were polyclonal and recognized overlapping epitopes. Their response was abolished by some substitutions, identifying residues common to all epitopes within a given region, whereas other substitutions reduced but did not obliterate the response, indicating residues included in some but not all epitopes recognized by the line. Comparison of the residues involved in epitope formation for different lines supported the conclusion that within the 20-residue immunodominant regions investigated here, the same sequence segment is involved in formation of epitopes, even in DR-discordant patients.

Autoimmune T cell lines specific for muscle nicotinic acetylcholine receptor (AChR) were propagated from the blood of three myasthenia gravis patients by the use of a pool of synthetic peptides (delta-pool) corresponding to the complete sequence of the delta-subunit of human muscle AChR. Propagation of AChR-specific T cell lines was attempted unsuccessfully from four other myasthenia gravis patients and from four healthy controls. The lines had CD3+, CD4+, CD8- phenotype, strongly recognized the delta-pool, and cross-reacted vigorously with non-denatured AChR purified from mammalian muscle. They did not cross-react detectably with pools of similar overlapping synthetic peptides corresponding to the complete sequences of the alpha- and gamma-subunits of human muscle AChR. The sequence segments of the delta-subunit that contain T epitopes were identified by investigating the response of the three CD4+ T cell lines to the individual synthetic peptides forming the delta-pool. Each line had an individual pattern of peptide recognition. Although no immunodominant region, recognized in association with different DR haplotypes, could be identified, the sequence segments most strongly recognized by the CD4+ T cell lines were clustered within residues 121-290. One of the peptides more strongly recognized by the T cells corresponded to a sequence segment with high predicted propensity to form an amphipathic alpha-helix, a structural motif proposed to be typical of T epitopes.

Acetylcholine receptor-(AcChR) specific T cell lines were propagated from the PBL of six myasthenia gravis (MG) patients by the use of a pool of synthetic peptides (alpha-pool) corresponding to the complete sequence of the alpha-subunit of the human AcChR. All the lines had CD4+ phenotype and strongly recognized the alpha-pool. Four lines cross-reacted with native Torpedo AcChR. Five lines showed, at certain stages of their propagation, some degree of reactivity to autologous or DR-matched APC. One of the CD4+ T lines was challenged with each one of the peptides present in the alpha-pool. Several peptides, corresponding to the sequence segments 48-67, 101-120, 304-322, 320-337, and 419-437 of the human alpha-subunit were recognized, indicating that different epitopes and multiple T cell clones are involved in the recognition of the autoantigen in MG. Human AcChR-specific CD4+ T cell lines will be useful to identify the repertoire of epitopes recognized by the autoreactive Th cells in MG, to investigate the TCR genes utilized by autoreactive Th cells and to develop specific immunosuppressive treatments using anti-T cell vaccination.

The production of antinicotinic acetylcholine receptor (AcChR) antibodies in myasthenia gravis (MG) is modulated by specific Th (CD4+) lymphocytes that can recognize epitopes on the denatured AcChR alpha subunit. Thirty-two overlapping synthetic peptides corresponding to the complete sequence of human AcChR alpha subunit were used to investigate the anti-alpha subunit response of unselected lymphocytes and of CD8(+)-depleted, CD4(+)-enriched lymphocytes from the blood of nine MG patients and from four healthy controls. One subject was a newly diagnosed MG patient that was tested three times after the development of the disease. An anti-AcChR response of the CD4(+)-enriched cells was present that could be detected only after removal of the CD8+ population and that seems to be related to the clinical conditions of the patient. The high basal rate of the cell proliferation of the unselected unstimulated blood lymphocytes and the normal basal rate observed for the CD8(+)-depleted population suggested the presence of activated CD8+ cells. The study of surface markers of the T cells confirmed the existence of activated CD8+ and CD4+ cells in numbers correlated with the severity of the disease and the results of the in vitro response of the T cells. The anti-AcChR activity of the CD4+ cells in MG may be a useful marker of the activity of the disease and it seems to be influenced by activated CD8+ cells present in the patients' blood.