The effect of TCR Vβ8 peptide protection and therapy on T cell populations isolated from the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis

Vaccination or treatment of Lewis rats with TCR Vβ8 peptides can prevent or reverse the clinical signs of experimental autoimmune encephalomyelitis (EAE) which is mediated predominantly by Vβ8.2⁺ CD4⁺/CD45R lo T cells. However, rats protected or treated with Vβ8 peptides still developed histological lesions in the spinal cord (SC), even though they remained clinical well. We sought to discern phenotypic changes characteristic of these SC infiltrating lymphocytes. In particular, we focused on whether the immunoregulatory mechanism induced by TCR peptides caused a reduction of Vβ8.2⁺ T cells, or induced changes in CD45R lo or hi/CD4⁺ subpopulations that have been associated respectively with EAE induction or recovery. In the Vβ8 peptide vaccinated rats there was a dramatic decrease in the number of Vβ8.2⁺ T cells isolated from the SC early in disease. During the recovery phase, however, the number of Vβ8.2⁺ SC T cells was similar in protected and control groups; in contrast, there was a striking reduction in the number and size of CD45R hi/CD4⁺ T cells in the protected animals. In rats treated with Vβ8.2 peptide, no changes were observed in the number of SC Vβ8.2⁺ T cells or expression of Vβ8.2 message, but similar to vaccinated rats, there was a marked decrease in the number of CD45R hi/CD4⁺ T cells. These data suggest that vaccination with TCR peptides prevented the initial influx of encephalitogenic Vβ8.2⁺ T cells into the central nervous system (CNS), whereas treatment appeared to inactivate Vβ8.2⁺ T cells already present in the CNS. In both cases, TCR peptide-induced inhibition of the encephalitogenic T cells apparently preempted the need for CD45R hi/CD4⁺ T cells that may normally be necessary to resolve the disease.     

Modulation of EAE by vaccination with T cell receptor peptides: Vβ8 T cell receptor peptide-specific CD4 lymphocytes lack direct immunoregulatory activity

Resistance to experimental autoimmune encephalomyelitis (EAE) induced by vaccination with a peptide representing amino acids 39–59 of the rat T cell receptor (TCR) Vβ8 element has been ascribed to the induction of protective antibodies and T lymphocytes, both recognizing the Vβ8 TCR peptide (TCRP) as well as Vβ8 TCR-expressing encephalitogenic lymphocytes. In this study immunization with the Vβ8 TCR peptide conferred partial resistance to active induction of EAE in three of six rats. The immunoregulatory role of TCRP-specific T cells in resistance to EAE was investigated. In vitro, D4⁺ T cell lines reactive with the Vβ8 TCRP did not respond to encephalitogenic Vβ8 TCRP-bearing cell lines nor did they impair their MBP-induced activation. In vivo, activated TCRP-specific line cells did not ameliorate actively induced EAE. The beneficial effect of Vβ8 TCRP-vaccination on the course of EAE may be due to the induction of protective antibodies. Neither before, nor during or after EAE did we observe a cellular response to the Vβ8 TCRP in lymph nodes or spleens of MBP-immunized animals. Moreover, we were not able to established TCRP-specific T cell lines from EAE rats, but from all rats immunized with the TCRP. Our data do not support the assumption that Vβ8 TCRP-reactive CD4⁺ T cells are the population operative in resistance to EAE after recovery from disease.     

Direct demonstration of the infiltration of murine central nervous system by Pgp-1/CD44 CD45RB CD4 T cells that induce experimental allergic encephalomyelitis

In experimental allergic encephalomyelitis (EAE), autoimmune T cells infiltrate the central nervous system (CNS) and initiate demyelinating pathology. We have used flow cytometry to directly analyse the migration to the CNS of MBP-reactive CD4⁺ T cells labelled with a lipophilic fluorescent dye (PKH2), in SJL/J mice with passively transferred EAE. Labelled cells constituted about 45% of the CNS CD4⁺ population at the time of EAE onset. Almost all (>90%) of the PKH2-labelled CD4⁺ T cells from EAE CNS were blasts and were α/β T cell receptor (TCR)⁺, CD44(Pgp-1)^high, and the majority were CD45RB^low. By contrast, most PKH2-labelled CD4⁺ T cells in lymph nodes, although CD44^high, were CD45RB^high cells. The cells that were transferred to induce EAE were essentially similar to antigen-primed lymph node cell populations, containing less than 15% CD44^high cells, and most of them were CD45RB^high. The CD44^high CD45RB^low phenotype is characteristic of memory/effector T cells that have been activated by antigen recognition. The difference in CD45RB expression between CNS and LN could therefore reflect differential exposure and/or response to antigen. Consistent with this, PKH2-labelled CD4⁺ cells isolated from the CNS were responsive to MBP in vitro, whereas PKH2⁺ CD4⁺ cells from lymph nodes showed almost undetectable responses. In control experiments in which ovalbumin (OVA)-reactive T cells were transferred, a small number of fluorescent-labelled CD4⁺ T cells were also detected in CNS, but there were very few blasts, and these remained CD45RB^high. These results argue for induction of the memory/effector phenotype of CD4⁺ cells, their selective retention in the CNS, as a consequence of antigen recognition.     

PECAM-1 (CD31) expression in the central nervous system and its role in experimental allergic encephalomyelitis in the rat

The role of T cell activation associated adhesion molecules on lymphocyte traffic and the initiation of inflammation has received considerable attention. This study, using a new monoclonal antibody (mAb) TLD-3A12, describes the distribution of PECAM-1 (CD31), an Ig supergene family adhesion molecule thought to be important in leukocyte transmigration during inflammation, in rat lymphoid organs and spinal cord. PECAM expression within the CNS is confined to endothelial cells of the blood brain barrier (BBB). Induction of inflammation within the CNS using the adoptive transfer of myelin reactive CD4⁺ T cells results in the de novo expression of immune adhesion and accessory molecules in the spinal cord, while the level of PECAM appeared only mildly increased. The distribution of PECAM on CNS endothelial cells became more diffuse during EAE induction, possibly the result of endothelial cell activation. In vitro studies demonstrate a partial inhibition of antigen-specific CD4⁴ T cell proliferation following anti-PECAM mAb treatment. Treatment of Lewis rats with TLD-3A12 antibody prior to T cell injection and throughout EAE induction does not result in a delay in the onset of clinical signs or weight loss, nor does it decrease the incidence and severity of disease. These data suggest that the expression of PECAM by CNS endothelial cells is not a requirement for the initiation of inflammation and clinical signs of EAE following the adoptive transfer of encephalitogenic lymphocytes. Thus, cells requiring PECAM-1 to migrate and perform their pathogenic functions are not critical to the development of rat EAE. © 1996 Wiley-Liss, Inc.     

Corticosteroid treatment of experimental autoimmune encephalomyelitis in the Lewis rat results in loss of Vβ8.2 and myelin basic protein-reactive cells from the spinal cord, with increased total T-cell apoptosis but reduced apoptosis of Vβ8.2 cells

We have studied the effects of corticosteroid treatment on the numbers of lymphocytes obtained from the spinal cords of Lewis rats with acute experimental autoimmune encephalomyelitis (EAE) induced by inoculation with myelin basic protein (MBP) and adjuvants. Flow cytometric studies showed that treatment with dexamethasone (4 mg/kg) 8–12 h prior to study on day 14 after inoculation resulted in a reduction in the numbers of CD5⁺, TCRαβ⁺ and Vβ8.2⁺ cells in the spinal cord. Limiting dilution analysis indicated that dexamethasone treatment 12 h prior to study on day 12 after inoculation reduced the frequencies of MBP-reactive and interleukin-2-responsive lymphocytes in the spinal cord to low levels, but reduced the frequency of concanavalin-A-responsive lymphocytes to a lesser extent. Using propidium iodide staining of nuclear chromatin we also studied lymphocyte apoptosis. Greater numbers of apoptotic cells were found in the cells extracted from the spinal cords of rats, examined on day 14, that had been treated 1–12 h previously with dexamethasone, than in saline-treated controls. This increased level of apoptosis was observed in the CD5⁺ and TCRαβ⁺ cell populations. At 1–4 h after dexamethasone treatment there was a reduction in the selective apoptosis of Vβ8.2⁺ cells that normally occurs during spontaneous recovery from EAE. Therefore apoptosis of Vβ38.2⁺ cells cannot explain the reduction in the numbers of Vβ8.2⁺ cells and MBP-reactive cells in the CNS after dexamethasone treatment. By 8–12 h after dexamethasone treatment the selectivity of the apoptotic process was restored. These studies suggest that a reduction in the number of T-lymphocytes in the central nervous system contributes to the beneficial effects of corticosteroids in EAE.     

Loss rather than downregulation of CD4 T cells as a mechanism for remission from experimental allergic encephalomyelitis

SJL/J mice recover from clinical signs of experimental allergic encephalomyelitis (EAE) 2 to 3 days following the onset of the initial attack. The immunoregulatory events that induce clinical recovery are not well understood. In this paper we have compared the activation state of the T cells infiltrating the central nervous system (CNS) in symptomatic and remitted mice. We isolated mononuclear cells from the CNS at various time points during the course of EAE and used flow cytometry to describe the kinetics of CNS infiltration by CD45⁺, CD2⁺, CD3⁺, TCRαβ⁺, CD4⁺ cells. There was a 30-fold reduction in the number of CNS CD4⁺ T cells in remitted mice 10 days following the initial attack. More than 60% of CNS CD4⁺ cells were of a CD44^high, CD45RB^low memory/effector phenotype both in active EAE, peak EAE and in remission, contrast to lymph nodes where this phenotype never constituted more than 17%. The proportion of CD8⁺ T cells was not increased in remitted mice, and we detected no TCRγδ⁺ cells within the CNS. Our findings demonstrate an overt loss of CD4⁺ T cells from the CNS and the maintenance of an activated state by T cells within the CNS and during remission from EAE. This argues against downregulation of T cell function as a mechanism for remission.     

Leukocyte infiltration into spinal cord of EAE mice is attenuated by removal of endothelial leptin signaling

Leptin, a pleiotropic adipokine, crosses the blood-brain barrier (BBB) and blood–spinal cord barrier (BSCB) from the periphery and facilitates experimental autoimmune encephalomyelitis (EAE). EAE induces dynamic changes of leptin receptors in enriched brain and spinal cord microvessels, leading to further questions about the potential roles of endothelial leptin signaling in EAE progression. In endothelial leptin receptor specific knockout (ELKO) mice, there were lower EAE behavioral scores in the early phase of the disorder, better preserved BSCB function shown by reduced uptake of sodium fluorescein and leukocyte infiltration into the spinal cord. Flow cytometry showed that the ELKO mutation decreased the number of CD3 and CD45 cells in the spinal cord, although immune cell profiles in peripheral organs were unchanged. Not only were CD4⁺ and CD8⁺ T lymphocytes reduced, there were also lower numbers of CD11b⁺Gr1⁺ granulocytes in the spinal cord of ELKO mice. In enriched microvessels from the spinal cord of the ELKO mice, the decreased expression of mRNAs for a few tight junction proteins was less pronounced in ELKO than WT mice, as was the elevation of mRNA for CCL5, CXCL9, IFN-γ, and TNF-α. Altogether, ELKO mice show reduced inflammation at the level of the BSCB, less leukocyte infiltration, and better preserved tight junction protein expression and BBB function than WT mice after EAE. Although leptin concentrations were high in ELKO mice and microvascular leptin receptors show an initial elevation before inhibition during the course of EAE, removal of leptin signaling helped to reduce disease burden. We conclude that endothelial leptin signaling exacerbates BBB dysfunction to worsen EAE.     

Encephalitogenic T lymphocytes develop from SJL/J hematopoietic cells transplanted into severe combined immimodeficient ( SCID) mice

Previously, we constructed chimeras by injecting hematopoietic cells from experimental autoimmune encephalomyelitis (EAE)-susceptible SJL (H-2^S) strain mice into severe combined immunodeficient (SCID) C.B-17scid /scid (H-2^d) mice. These SCID mouse-SJL mouse hematopoietic cell chimeras developed passive EAE following adoptive transfer of PLP S139–151-specific SJL T lymphocyte line cells, but were resistant to active EAE induced by primary immunization with PLP S139–151. In order to gain an understanding of the encephalitogenic potential of transplanted hematopoietic progenitors in SCID mouse-SJL mouse chimeras, we attempted to induce EAE in hematopoietic chimeras constructed with or without an additional SJL fetal thymus implant. Chimeras with the thymus implant were susceptible to passive and active EAE while chimeras without the thymus implant were susceptible to passive but not active EAE. Encephalitogenic, CD4⁺, TCR⁺ T lymphocytes were selected in vitro from PLP S139-151-immunized, thymus-implanted chimeras. These results showed that hematopoietic SJL progenitors developed into antigen-presenting accessory cells and immunocompetent encephalitogenic T lymphocytes following transplantation into SCID mice. The development of primary immune reactivity depended on a fetal thymus implant for expression in SCID mouse-SJL mouse chimeras.     

Inflammatory cells, microglia and MHC class II antigen-positive cells in the spinal cord of Lewis rats with acute and chronic relapsing experimental autoimmune encephalomyelitis

Chronic relapsing experimental autoimmune encephalomyelitis (CR-EAE) was induced in Lewis rats by inoculation with guinea pig spinal cord and adjuvants and treatment with low dose cyclosporin A (CsA). Acute EAE was induced by the same method without CsA treatment. Immunocytochemistry and flow cytometry were used to assess inflammatory cells and MHC class II (Ia) antigen expression in the central nervous system of these rats. The inflammatory infiltrate was composed mainly of CD4⁺ T cells and macrophages, and αß T cells constituted about 65% of the CD2⁺ T cells. After recovery from acute EAE and during the first remission of CR-EAE, the number of T cells was significantly less than in the preceding episodes. The number of T cells was higher in the second episode of CR-EAE than in the first remission. Throughout the course of CR-EAE, the majority of the CD2⁺ T cells were CD45RC⁻. The ratio of IL-2R⁺ cells to CD2⁺ cells ranged from 10.5 to 24.0%. The ratio of CD4⁺ T cells to B cells was lower in the later episodes of CR-EAE than in the first episode. Ia antigen was expressed on filtrating round cells at all stages of CR-EAE and on microglial cells (identified by dendritic morphology) with increasing intensity throughout the course of CR-EAE. With flow cytometry, the number of Ia⁺ cells obtained from the spinal cord rose throughout the course of CR-EAE. The number of FSC^lowOX1^low cells, which we consider represent microglia, also increased during the course of CR-EAE.     

Expression of CD45RC and Ia antigen in the spinal cord in acute experimental allergic encephalomyelitis: an immunocytochemical and flow cytometric study.

We performed immunocytochemical studies to analyze the inflammatory infiltrate and major histocompatibility complex class II (Ia) antigen expression in the spinal cord of Lewis rats with acute experimental allergic encephalomyelitis (EAE) induced by inoculation with myelin basic protein and adjuvants. Using antibodies to lymphocyte markers and other monoclonal antibodies we found that during clinical episodes the inflammatory infiltrate was chiefly composed of T lymphocytes and macrophages. The majority of cells in the inflammatory infiltrate were stained by the W3/25 antibody to CD4 and a proportion was stained by OX22 which labels the high molecular weight form of the leucocyte common antigen (CD45RC). CD8+ T cells were sparse and B cells were not detected. There was minimal staining with the OX39 antibody to the interleukin-2 receptor. Presumptive microglia, identified by their dendritic morphology, expressed Ia antigen during the clinical episodes and after recovery. The prominence of Ia antigen expression after recovery could indicate that this Ia expression was associated with downregulation of the encephalitogenic immune response. We also performed flow cytometry studies on cells extracted from the spinal cord of rats before and during attacks of EAE. With flow cytometry, we found that in established disease a mean of 83(SD, 23)% of CD2+ cells were CD4+, and a mean of 27(SD, 12)% of CD2+ cells were CD45RC+. In rats sampled on the first day of signs, a mean of 43(SD, 22)% of CD2+ cells were CD45RC+. In the cells extracted from the spinal cord of rats with established disease a mean of 47(SD, 32)% of macrophages were CD45RC+. Our study has combined an immunocytochemical assessment of tissue sections with quantitative flow cytometry assessment of cells extracted from the spinal cord of rats with acute EAE. We have shown that the majority of T lymphocytes in the spinal cord are CD45RC-. We have also found prominent Ia expression on dendritic cells in acute EAE and after clinical recovery.     

The subarachnoid space as a site for precursor T cell proliferation and effector T cell selection in experimental autoimmune encephalomyelitis

To characterize the phenotype of inflammatory cells in the central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE), Lewis rats were immunized with guinea pig myelin basic protein and frozen sections of the spinal cord with EAE were examined immunohistochemically using a panel of monoclonal antibodies against T cells and adhesion molecules. In addition, double immunostaining was performed with glial and T cells markers to examine the interaction between infiltrating T cells and reactive brain cells during the course of EAE. In the early stage of EAE, inflammatory cells first appeared in the subarachnoid space (SAS) and infiltrated the subpial region. The majority of inflammatory cells in SAS expressed TCRaβ and either CD4 or CD8 molecules. However, only CD4⁺ T cells infiltrated the parenchyma while the majority of CD8⁺ cells remained in SAS. A similar differential localization of T cells was observed with regard to CD45RC molecules. Inflammatory cells in SAS consisted of both CD45RC⁺ and CD45RC^- population, while those in the parenchyma were largely CD45RC˜. With regard to adhesion molecules, the leptomeninges constitutively expressed fibronectin (FN) and intercellular adhesion molecule 1 (ICAM-1). Most SAS inflammatory cells expressed very late activation antigen 4 (VLA-4) and, to lesser extent, lymphocyte function-associated antigen 1 (LFA-1) in the early stage of EAE. On the other hand, parenchyma! infiltrating cells expressed LFA-1 more strongly in the peak stage. Double staining for Vβ8.2 TCR and microglia demonstrated an increase in the number of microglia together with morphological changes into rod-shape cells in the vicinity of infiltrating T cells. Furthermore, these cells expressed adhesion molecules, such as LFA-1, ICAM-1 and CD4. These findings suggest that VLA-4/FN and LFA-1/ICAM-1 interactions between infiltrating cells and brain cells may be involved in the early and peak stages of EAE. Phenotype switching occurring in the process of inflammatory cell infiltration may be regulated by these adhesion molecules and factor(s) provided by the parenchyma, possibly by microglia.     

Preferential increase of IL-2R+ CD4+ T cells and CD45RB- CD4+ T cells in the central nervous system in experimental allergic encephalomyelitis.

We examined lymphocytes isolated from the spinal cord (SC), peripheral blood (PB) and lymph nodes (LN) draining the immunization site of Lewis rats with acute experimental allergic encephalomyelitis (EAE). Cells were analysed for T cell subset markers CD4 (mAb W3/25) and CD8 (mAb OX8), for IL-2R (mAb OX39), and for high molecular mass leukocyte common antigen (LCA, CD45RB) expression (mAb OX22). T cells expressing high (CD45RB+) or low (CD45RB-) molecular mass LCA are of different maturational stages and/or separate lineages. CD4+ T cells were more predominant in SC than in PB and LN; CD8+ T cells were scarce in SC but common in PB and LN. Activated CD4+ T cells (IL-2R+) were common in the SC and LN but infrequent in blood. CD4+ T cells that were CD45RB+ were scarce in the SC. In contrast, the majority of CD4+ T cells in the PB and LN were CD45RB+. The preferential accumulation of IL-2R+ CD4+ T cells and of CD45RB- CD4+ T cells in the central nervous system (CNS) indicates that a selective mechanism directs cell egress into CNS lesions in EAE.     

IL-23 modulated myelin-specific T cells induce EAE via an IFNγ driven, IL-17 independent pathway

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease of the central nervous system (CNS) mediated by myelin-reactive CD4⁺ T cells. An unresolved issue that has important clinical implications concerns the cytokines produced by myelin-reactive T cells that determine their pathogenicity. Initially, IL-12 polarized, IFNγ producing Th1 cells were thought to be essential for the development of EAE. More recently, IL-23 polarized, IL-17 producing Th17 cells have been highlighted as critical encephalitogenic effectors. There is growing evidence that parallel autoimmune pathways can result in common clinical and histopathological endpoints. In the current study, we describe a form of EAE induced by the transfer of IL-23 modulated CD4⁺ T cells into IL-17 receptor (IL-17R) deficient hosts. We found that IL-23 stimulates myelin-reactive T cells to produce both IFNγ and IL-17. Surprisingly, in this model the development of EAE is IFNγ dependent. Our findings illustrate a novel mechanism by which IL-23 promotes encephalitogenicity and they further expand the spectrum of autoreactive T cells capable of mediating inflammatory demyelinating disease of the CNS.     

OX-40 antibody enhances for autoantigen specific Vβ8.2+ T cells within the spinal cord of lewis rats with autoimmune encephalomyelitis

The Vβ8.2 T cell receptor (TCR) component is the predominant Vβ gene product associated with antigen specific CD4⁺ T cell response to the major encephalitogenic epitope of myelin basic protein (MBP) in Lewis rats. Lewis rats were actively immunized with MBP in complete Freund's adjuvant and the Vβ8.2 positive and negative cells were analyzed for IFN-γ mRNA production and OX-40 cell surface expression during the onset of EAE. The Vβ8.2⁺ T cells isolated from the spinal cord produced the majority of mRNA for IFN-γ and also showed a marked enhancement for OX-40 expression compared to Vβ8.2⁺ T cells isolated from the lymph nodes. Only a fraction of IL-2 receptor positive T cells examined ex vivo from the inflammatory compartments co-expressed the OX-40 antigen. These results suggested that OX-40 cell surface expression could be used to identify and isolate the most recently activated T cells ex vivo. OX-40⁺ T cells isolated from the spinal cord were highly enriched for the Vβ8.2 T cell receptor component compared to OX-40^? or unsorted spinal cord lymphocytes. OX-40⁺ T cells isolated from the spinal cord had an enhanced response to MBP, whereas OX-40⁺ cells isolated from the lymph nodes responded to both MBP and purified protein derivative. These data suggest that activated T cells can be isolated and characterized with the OX-40 antibody which only respond to the antigens present at the local site. The data also imply that isolation of OX-40⁺ T cells will be useful in identifying Vβ biases and autoantigen specific cells within inflamed tissues even when the antigen specificity is unknown. © 1996 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America

     

Therapeutic potential of experimental autoimmune encephalomyelitis by Fasudil,a Rho kinase inhibitor

The migration of aberrant inflammatory cells into the central nervous system plays an important role in the pathogenesis of demyelinating diseases potentially through the Rho/Rho‐kinase (Rock) pathway, but direct evidence from human and animal models remains inadequate. Here we further confirm that Fasudil, a selective Rock inhibitor, has therapeutic potential in a mouse model of myelin oligodendrocyte glycoprotein (MOG)‐induced experimental autoimmune encephalomyelitis (EAE). The results show that Fasudil decreased the development of EAE in C57BL/6 mice. Immunohistochemistry disclosed that expression of Rock‐II in the perivascular spaces and vascular endothelial cells of spleens, spinal cords, and brains was elevated in EAE and was inhibited in the Fasudil‐treated group. T‐cell proliferation specific to MOG_35–55 was markedly reduced, together with a significant down‐regulation of interleukin (IL)‐17, IL‐6, and MCP‐1. In contrast, secretion of IL‐4 was increased, and IL‐10 was slightly elevated. There were no differences in the percentages of CD4⁺CD25⁺, CD8⁺CD28^?, and CD8⁺CD122⁺ in mononuclear cells. Histological staining disclosed a marked decrease of inflammatory cells in spinal cord and brain of Fasudil‐treated mice. These results, together with previous studies showing the inhibitory effect of Fasudil on T‐cell migration, might expand its clinical application as a new therapy for multiple sclerosis by decreasing cell migration and regulating immune balance. © 2010 Wiley‐Liss, Inc.     

Pathogenic and non-pathogenic T lymphocytes specific for the encephalitogenic epitope of myelin basic protein: Functional characteristics and vaccination properties

Activated CD4⁺ T lymphocytes specific for myelin basic protein (MBP) can cause experimental autoimmune encephalomyelitis (EAE) upon their inoculation into syngeneic recipients. In Lewis rats, most of the pathogenic T cell clones that develop following immunization with MBP are reactive against the 72–84 amino acid sequence of MBP, the major encephalitogenic region for Lewis rats. In this study, some MBP-specific T cell clones were found to be non-pathogenic, in spite of their strong reactivity against the encephalitogenic epitope. One of these non-pathogenic clones, designated Znp, and an encephalitogenic clone, Z1a-p, were derived from Z1a encephalitogenic line cells. These subclones were compared for epitope specificity, T cell receptor variable gene expression and for various functional activities, in order to delineate properties crucialof pathogenicity. The Z1a-p and Znp cells expressed comparable levels of the T cell receptor genes and shared strong reactivity against the 72–84 epitope of MBP. The pathogenic Z1a-p cells displayed MBP-specific cytolytic activity in vitro, provided an in-vivo ‘help’ for elicitation of MBP-specific antibodies, mediated a delayed type hypersensitivity (DTH) response to MBP, caused EAE and vaccinated against the disease, thus demonstrating that a single CD4⁺ T cell clone is capable of eliciting various functions. The non-pathogenic Znp cells could also carry out most of these various functions, but failed to mediate a DTH response to MBP in normal animals. However, when inoculated into sublethally (650 R) irradiated syngeneic recipients, the Znp cells became highly pathogenic and mediated DTH response to MBP. Local irradiation of the recipient facilitated a DTH response to MBP in the irradiated ear, indicating that Znp cells are equipped with the effector mechanisms required for pathogenicity, and that their failure to cause disease may be accounted for by their inability to migrate into extravascular target tissue. Similar data were obtained with an independently isolated non-pathogenic clone, LB-3, specific for the encephalitogenic epitope of MBP. The ability of these non-pathogenic cells to vaccinate against EAE mediated by pathogenic cells raises the possibility that such non-pathogenic cells may play a role in triggering downregulation of pathogenic T cells.     

Temporal kinetics of CD8+CD28+ and CD8+CD28− T lymphocytes in the injured rat spinal cord

This study aims to explore the temporal changes of cytotoxic CD8⁺CD28⁺ and regulatory CD8⁺ CD28⁻ T‐cell subsets in the lesion microenvironment after spinal cord injury (SCI) in rats, by combination of immunohistochemistry (IHC) and flow cytometry (FCM). In the sham‐opened spinal cord, few CD8⁺ T cells were found. After SCI, the CD8⁺ T cells were detected at one day post‐injury (dpi), then markedly increased and were significantly higher at 3, 7, and 14 dpi compared with one dpi (p < 0.01), the highest being seven dpi. In CD8⁺ T cells, more than 90% were CD28⁺, and there were only small part of CD28⁻ ( < 10%). After 14 days, the infiltrated CD8⁺ T cells were significantly decreased, and few could be found in good condition at 21 and 28 dpi. Annexin V and propidium iodide (PI) staining showed that the percentages of apoptotic/necrotic CD8⁺ cells at 14 dpi and 21 dpi were significantly higher than those of the other early time‐points (p < 0.01). These results indicate that CD8⁺ T cells could rapidly infiltrate into the injured spinal cords and survive two weeks, however, cytotoxic CD8⁺ T cells were dominant. Therefore, two weeks after injury might be the “time window” for treating SCI by prolonging survival times and increasing the fraction of CD8⁺ regulatory T‐cells. © 2016 Wiley Periodicals, Inc.     

Human T-lymphotropic virus type I-associated myelopathy and tax gene expression in CD4+ T lymphocytes

Infection by human T-lymphotropic virus type I (HTLV-I) is associated with adult T-cell leukemia and a slowly progressive disease of the central nervous system (CNS), HTLV-I-associated myelopathy/tropical spastic paraparesis, characterized pathologically by inflammation and white matter degeneration in the spinal cord. One of the explanations for the tissue destruction is that HTLV-I infects cells in the CNS, or HTLV-I-infected CD4⁺ T lymphocytes enter the CNS, and this drives local expansion of virus-specific CD8⁺ cytotoxic T lymphocytes, which along with cytokines cause the pathological changes. Because both in the circulation and in the cerebrospinal fluid, CD8⁺ cytotoxic T lymphocytes are primarily reactive to the product of the HTLV-I tax gene, we sought evidence of expression of this gene within cells in the inflammatory lesions. After using double-label in situ hybridization techniques, we now report definitive localization of HTLV-I tax gene expression in CD4⁺ T lymphocytes in areas of inflammation and white matter destruction. These findings lend support to a hypothetical scheme of neuropathogenesis in which HTLV-I tax gene expression provokes and sustains an immunopathological process that progressively destroys myelin and axons in the spinal cord.     

Enhanced response to antigen within lymph nodes of SJL/J mice that were protected against experimental allergic encephalomyelitis by T cell vaccination

The effects of T cell vaccination on peripheral immune responsiveness are not yet fully understood. We have induced resistance to rat spinal cord homogenate (RSCH)-induced experimental allergic encephalomyelitis (EAE) in SJL/J mice by vaccination with four T cell lines (RZ8, RZ15, RZ16, and A51) which were reactive to myelin basic protein (MBP) but not to proteolipid protein (PLP). The effect was relatively neuroantigen-specific since vaccination with ovalbumin (OVA)-reactive and alloantigen-specific cells did not prevent EAE induction. Alloantigen-reactive cells reduced the rate of relapse. The number of central nervous system (CNS) infiltrates and mean clinical EAE scores were significantly reduced. This is the first report demonstrating T cell vaccination in the SJL/J mouse, a strain in which PLP is the predominant encephalitogen in RSCH. The vaccinating cells were of the memory/effector (CD44^high, CD45RB^low) surface phenotype. We examined the effect of T cell vaccination on lymph node T cell proliferative responses to MBP, encephalitogenic peptides of PLP and MBP, OVA and anti-CD3. With the exception of polyclonal cytokine responses to anti-CD3, which remained unchanged, vaccination led to a 5–10-fold augmentation in all, including background, responses. By comparison with lymph node cell (LNC) responses from naive mice and mice primed with OVA, it appeared that T cell vaccination restored cellular activation levels which had been depleted in peripheral lymphoid tissues of unvaccinated animals with EAE.     

Treatment of PL/J mice with the superantigen, staphylococcal enterotoxin B, prevents development of experimental allergic encephalomyelitis

Experimental allergic encephalomyelitis (EAE), an antigen induced autoimmune disease, is mediated by Vβ8⁺ CD4⁺ T cells in PL/J mice after injection with the autoantigen, myelin basic protein (MBP). Recently the superantigen, staphylococcal enterotoxin B (SEB), has been shown to peripherally anergize and delete T cells in a Vβ specific manner. By treatment of PL/J mice with SEB, we have been able to protect PL/J mice from the development of EAE. Two-color FACS analysis of the spleens of SEB treated mice showed depletion of Vβ8⁺ CD4⁺ T cells. Consistent with this observation, spleen cells of SEB treated mice that did not show signs of EAE could not be stimulated in vitro with SEB but did respond to SEA. Thus, Vβ specific superantigens may prove to be a preventive therapy for autoimmune diseases mediated by Vβ specific T lymphocytes.