Presence of T-cell subset abnormalities in newly diagnosed cases of multiple sclerosis and relationship with short-term clinical activity

Abnormalities of T-cell subsets in patients with multiple sclerosis are well known; in order to assess whether immunological abnormalities are relevant in the pathogenesis of the disease after its clinical onset, peripheral blood lymphocyte subsets (CD3⁺, CD4⁺, CD4⁺ CD45RA⁺, CD4⁺CD45RA^–, CD8⁺, CD8⁺CD57⁺, CD57⁺, CD25⁺) were analysed serially in 25 patients at the first clinical episode suggestive of inflammatory demyelinating disease and in an equal number of age- and sex-matched controls. During the follow-up period (12–18 months, mean 14) 6 of 25 patients presented new relapses: in this subgroup of patients, significant changes in CD4⁺ ratio (% CD4⁺CD45RA^–/%CD4⁺CD45RA^–) were detected in comparison both with healthy controls and with clinically stable patients. Patients clinically stable at follow-up did not display immunological abnormalities, regardless of the presence or absence of cerebrospinal fluid and/or magnetic resonance imaging alterations consistent with multiple sclerosis. These findings suggest a possible prognostic role of early T-cell subset imbalance in multiple sclerosis.     

The role of dendritic cells in multiple sclerosis

Although multiple sclerosis (MS) is a presumed T-cell-mediated disease, it is unclear what triggers the development of neuroantigen-specific T cells. Autoreactive CD4⁺ T cells are activated by antigen presenting cells; dendritic cells (DCs) are the primary antigen presenting cells directing T-cell functions and are, therefore, extremely important in directing the immune pathology characteristic of MS. Three important concepts have emerged regarding DCs in MS. First, DCs are present within the healthy central nervous system (CNS) in association with the cerebrospinal fluid space and microvasculature. Therefore, the potential for sampling of CNS antigens in similar fashion to other tissues and organs exists and likely plays an integral role in CNS immunity. The degree of involvement, as well as the source, of these CNS DCs has been addressed by several studies using the experimental autoimmune encephalomyelitis animal model. Second, DCs are found within MS lesions and have been shown to be functionally abnormal in patients with MS. Lastly, therapeutics directed at DCs could potentially be engineered for treatment in MS and in fact may already be involved in the mechanisms of current immunomodulatory therapies.     

The immune profile of multiple sclerosis: T-lymphocyte effects predominate over all other factors in cyclophosphamide-treated patients

It is widely believed that multiple sclerosis is a T-cell mediated autoimmune disease associated with abnormalities in immunoregulation. This large, prospective study evaluated the lymphocyte immunophenotypic profile of 246 MS patients, divided clinically into a remitting/relapsing group (n = 176) and a progressive group (n = 70), and compared their results to those of 117 healthy controls. All patients were found to have significantly elevated percentage and absolute numbers of IL2R⁺CD3⁺ cells as well as depressed percentages of CD45RA⁺CD4⁺ cells. However, when the factor of treatment with cyclophosphamide (CY) versus no treatment or treatment with other agents was used to group patients, dramatic declines in both percentages and absolute numbers of CD45RA ⁺ CD4⁺ cells were discovered. These declines were associated specifically with CY and could be explained by this factor independent of the clinical state of the patient. The effects were seen in patients undergoing current treatment or in those exposed to CY in the near or remote past. These findings highlight the confounding effect of specific treatments on the immune profile of MS patient groups and suggest that there may be important implications for cellular function and clinical outcome in these and other patient groups.     

Imbalance in T-cell and cytokine profiles in patients with relapsing-remitting multiple sclerosis

Multiple sclerosis (MS) is characterized by autoimmune attack leading to demyelination of the white matter in the central nervous system with devastating clinical consequences. Several immune-mediated destruction mechanisms were previously proposed including different T-cell subsets but complex view on immune system function in patients with MS is missing. In the present study, T-lymphocyte populations and pro-inflammatory as well as suppressive cytokine profiles were evaluated in detail in previously untreated patients with relapsing-remitting MS (RRMS). CD4⁺ and CD8⁺ naïve, central memory (Tcm), effector memory (Tem), terminal effector memory (Ttem), CD4⁺ regulatory T-cells (Treg) and CD8⁺ T-suppressor cells (Ts) were analysed using flow cytometry, and levels of ten plasma cytokines were determined using fluorescent bead-based immunoassay. We evaluated two groups of RRMS with minor (n = 33) and major (n = 25) clinical impairment and compared them with healthy controls (n = 40) in order to detect any correlation between severity of MS clinical symptoms and immune disturbances. Significant differences were noted in CD4⁺CD45RA⁺CCR7⁺ naïve T-cells, CD4⁺CD45RO⁺CCR7^? and CD8⁺CD45RO⁺CCR7^? Tem cells, while no differences were recognized in Tcm, Ttem, Treg and Ts cells in RRMS patients. Nine out of ten studied cytokines were disturbed in plasma samples of patients with RRMS. In conclusion, we demonstrate complex immune dysbalances in untreated MS patients.     

Human CD8 TCR-αβ and TCR-γδ cells modulate autologous autoreactive neuroantigen-specific CD4 T-cells by different mechanisms

To investigate the regulatory interactions among autologous T-cells during the course of multiple sclerosis (MS), proteolipid protein peptide-specific CD4⁺ T-cell clones (TCCs) were irradiated and used as immunogens to stimulate purified populations of autologous CD8⁺ TCR-αβ⁺ and TCR-γδ⁺ T-cells isolated from the peripheral blood of MS patients, patients with other non-inflammatory neurological diseases, and healthy blood donors. The resulting blasts were expanded in the presence of hIL-2 and then cloned by limiting dilution. Two different groups of CD8⁺ TCCs were revealed. A first group of CD8⁺ TCCs recognized autologous CD4⁺ T-cells based in their TCRVβ structures (anti-idiotypic responsiveness). A second group of CD8⁺ TCCs recognized Ag activated autologous CD4⁺ TCCs irrespective of their Ag specificity or TCRVβ expression (anti-ergotypic responsiveness). Both groups showed MHC class I restricted cytotoxicity against CD4⁺ T-cells and were able to secrete IFN-γ, TNFα/β and TGF-β. TCR-γδ⁺ TCCs isolated in response to stimulation with autologous peptide-specific CD4⁺ TCCs showed only anti-ergotypic cytotoxicity, which was not inhibited by anti-MHC class Ia monoclonal antibodies. Moreover, they were able to secrete IFN-γ and TNFα/β, but not TGF-β. These data demonstrate that regulatory mechanisms among human autologous T-cells can be mediated by cytolytic interactions or by the release of specific cytokines. Furthermore, they provide evidence that CD8⁺ TCR-αβ⁺ and TCR-γδ⁺ cells differ in their patterns of recognition and in their abilities to modulate the immune response mediated by autologous autoreactive CD4⁺ T-cells.     

A study of human T-cell lines generated from multiple sclerosis patients and controls by stimulation with peptides of myelin basic protein

We generated T-cell lines from the peripheral blood of controls and of patients with multiple sclerosis (MS) by stimulation with overlapping synthetic peptides representing the entire sequences of all four isoforms of human myelin basic protein (MBP). The T-cell lines reacted to a wide range of epitopes in the major isoforms of MBP and to epitopes that were present only in the minor isoforms. Many MS patients and controls had T-cells responding to one or more cryptic MBP epitopes, as indicated by the generation of a peptide-specific T-cell line(s)by stimulation with synthetic peptides but not by stimulation with whole MBP. About one-third of the peptide-generated lines were cytotoxic. Although we have shown that this technique of peptide stimulation is effective in generating human antiviral cytotoxic CD8⁺ T-cell lines, all the cytotoxic MBP-specific lines generated by this method were predominantly CD4⁺. Our study did not reveal any significant differences, between MS patients and controls, in reactivity to epitopes within any of the isoforms of MBP.     

Effect of IFN-ß therapy on the frequency and function of CD4+CD25+ regulatory T cells and Foxp3 gene expression in relapsing–remitting multiple sclerosis (RRMS): A preliminary study

Interferon-ß (IFN-ß) is an immunomodulatory drug of choice to control relapsing–remitting multiple sclerosis (RR-MS), although its function is still unclear. A reduced suppressive function of CD4⁺CD25⁺ regulatory T cells (T_reg) has been shown in RR-MS patients. In this study, to understand the effect of IFN-ß on CD4⁺CD25⁺ regulatory T cells, we analyzed the frequency and function of these cells and Foxp3 gene expression before and after treatment.We evaluated the frequency and function of CD4⁺CD25⁺Foxp3⁺ regulatory T cells by flow cytometry and co-culture inhibition test respectively and gene expression of Foxp3 by real-time PCR in a longitudinal follow-up study in 18 relapsing–remitting MS patients. Our data revealed that IFN-ß significantly improved frequency and suppressive function of T_reg cells (P < 0.05) without any significant effect on gene expression of Foxp3 after 6 months. The results of the present study indicate that IFN-ß therapy in some of patients with RR-MS may restore function of regulatory T cells and control the unchecked immune cascade activity. Larger longitudinal studies on more MS patients are required to confirm our findings.     

Neuroprotection and neurogeneration in MS and its animal model EAE effected by glatiramer acetate

It is by now established that multiple sclerosis (MS) is not simply an autoimmune disease and that in addition to inflammation and demyelination, axonal injury and neuronal loss underlie the accumulation of disability and the disease progression. Specific treatment strategies should thus target the injury sites at the central nervous system (CNS) to interfere with both neuroinflammation and neurodegeneration. Glatiramer acetate (GA Copaxone^®, Copolymer 1), an approved drug for the treatment of multiple sclerosis, was shown earlier to act as an anti-inflammatory and immunomodulatory agent. In this mini-review its effect on neuroprotection, neurogenesis and on the remyelination process is delineated in the EAE model. The plausible mechanism underlying this multifactorial effect is the induction of GA-reactive T-cells in the periphery and their infiltration into the CNS, where they release immunomodulatory cytokines and neurotrophic factors in the injury site, suggesting a direct linkage to its therapeutic effect in both EAE and MS.     

Immune Mechanisms Underlying the Beneficial Effects of Autologous Hematopoietic Stem Cell Transplantation in Multiple Sclerosis

A recent phase I/II clinical trial drew serious attention to the therapeutic potential of autologous hematopoietic stem cell transplantation (AHSCT) in multiple sclerosis. However, questions were raised as to whether these beneficial effects should be attributed to the newly reconstituted immune system per se, or to the lymphoablative conditioning regimen-induced immunosuppression, given that T-cell depleting combinational drug therapies were used in the study. We discuss here the possibility that both AHSCT and T-cell depleting therapies may re-program alternatively the immune system, and why transplantation of CD34⁺ hematopoietic stem cells may offer AHSCT a possible advantage regarding long-term remission.     

Characterization of a unique T-cell clone established from a patient with HAM/TSP which recognized HTLV-I-infected T-cell antigens as well as spinal cord tissue antigens

Five T-cell clones reactive to autologous HTLV-I-infected T-cells (KODA-TV) were established from peripheral blood lymphocytes of a HAM/TSP patient (KODA) by the limiting dilution method. All the clones showed CD3⁺, CD4⁺ and CD25⁺ surface markers and expressed αβ⁺ T-cell receptors to recognize KODA-TV antigens. One of the five T-cell clones (KODA-408) was infected with HTLV-I but the remaining four clones (KODA-400, 404, 405 and 409) were free of HTLV-I infection. KODA-408 recognized both KODA-TV and spinal cord antigens, the latter being extracted from autopsy tissues of a HTLV-I seronegative donor. KODA-408 did not recognize either alloantigens of peripheral blood mononuclear cells extracted from unrelated HTLV-I seronegative donors or purified human myelin basic protein. KODA-408 T-cell clone produced a considerable amount of TNF-aβ, IFN-γ, and IL-6. The CDR3 motif of KODA-408 T-cell receptor showed a unique sequence CASSAGQS of Vβ8-Dβ-Jβ1.5. These results indicated that HAM/TSP CD4⁺ T-cells were polyclonally activated by HTLV-I infection and antigenic stimulation. The T-cell repertoire shaped by HTLV-I infection included T-cells which recognized HTLV-I-infected T-cell antigens as well as spinal cord antigen in particular.     

Transplantation with Lewis bone marrow induces the reinstatement of cocaine-seeking behavior in male F344 resistant rats

One of the main challenges to understand drug addiction is defining the biological mechanisms that underlie individual differences in recidivism. Studies of these mechanisms have mainly focused on the brain, yet we demonstrate here a significant influence of the peripheral immune system on this phenomenon. Lewis (LEW) and Fischer 344 (F344) rats have different immunological profiles and they display a distinct vulnerability to the reinforcing effects of cocaine, with F344 more resistant to reinstate cocaine-seeking behavior. Bone marrow from male LEW and F344 rats was transferred to male F344 rats (F344/LEW-BM and F344/F344-BM, respectively), and these rats were trained to self-administer cocaine over 21 days. Following extinction, these animals received a sub-threshold primer dose of cocaine to evaluate reinstatement. F344/LEW-BM but not F344/F344-BM rats reinstated cocaine-seeking behavior, in conjunction with changes in their peripheral immune cell populations to a profile that corresponded to that of the LEW donors. After cocaine exposure, higher CD4⁺ T-cells and lower CD4⁺CD25⁺ T-cells levels were observed in F344/LEW-BM rats referred to control, and the splenic expression of Il-17a, Tgf-β, Tlr-2, Tlr-4 and Il-1β was altered in both groups. We propose that peripheral T-cells respond to cocaine, with CD4⁺ T-cells in particular undergoing Th17 polarization and generating long-term memory, these cells releasing mediators that trigger central mechanisms to induce reinstatement after a second encounter. This immune response may explain the high rates of recidivism observed despite long periods of detoxification, shedding light on the mechanisms underlying the vulnerability and resilience of specific individuals, and opening new perspectives for personalized medicine in the treatment of relapse.     

Role of perforin secretion from CD8+ T-cells in neuronal cytotoxicity in multiple sclerosis

Objectives: Multiple sclerosis (MS) is the most prevalent autoimmune disease of the central nervous system, and is characterized by inflammation and myelin damage. The immune system initiates the autoimmune response, although the mechanisms of neuronal damage have not been elucidated. The purpose of the present study was to investigate autoreactive CD4+ and CD8⁺ T lymphocytes, in conjunction with other inflammatory cells and cytokines in active MS lesions.

Methods: EAE animal models was established by plantar injections of MBP (200 μg per rat). Purified CD4+ or CD8+ T-cells were isolated from heparinized peripheral blood (EAE animals and control animals) via negative selection. To examine effects of presence of autoreactive CD4+ and CD8+ T lymphocytes, we carried out ELISA, Western blot analysis and TUNEL. In addition, we examined the direct effects of various factors on neuronal cell death using MTT assay.

Results: The data revealed that CD8+ T-cells were more toxic to neurons compared to CD4+ T-cells, in both the MBP and EAE conditions. Bax was greater increased when neurons were co-cultured with CD8+ T-cells in the MBP group. There is a significant increase in IL-17 secretion by CD4+ T-cells in both the MBP group and EAE group. Neuronal viability were affected by Perforin (1.5 μg/mL).

Conclusion: The present study extends previous research by demonstrating the role of CD8+ T-cells in MS and supports perforin secretion by CD8+ T-cells as a potential therapeutic factor. Furthermore, we determined that CD4⁺ T-cells can enhance CD8⁺ T-cell neuronal cytotoxicity via induction of intense inflammation.     

Increased T-cell Reactivity and Elevated Levels of CD8<Superscript>+</Superscript> Memory T-cells in Alzheimer’s Disease-patients and T-cell Hyporeactivity in an Alzheimer’s Disease-mouse Model: Implications for Immunotherapy

Neuroinflammation is observed in neurodegenerative diseases like Alzheimer’s disease (AD). However, a little is known about the mechanisms of neural-immune interactions. The involvement of peripheral T-cell function in AD is still far from clear, though it plays an important role in immunotherapy. The aim of this study was to determine peripheral T-cell reactivity in AD patients and in an AD mouse model. Mitogenic activation via ligation of the T-cell receptor (TCR) with PHA-L was measured in T lymphocytes from AD patients and Thy1(APP_751SL) × HMG(PS1_M146L)-transgenic mice (APP × PS1). In order to uncover failures in TCR signaling, the TCR was also bypassed by PMA and ionomycin treatment. All patients were sporadic late onset cases and the transgenic mice expressed no mutant APP in lymphocytes, so that direct interactions of mutant APP on T-cell function can be excluded. CD4⁺ and CD8⁺ T-cell showed increased reactivity (tyrosine phosphorylation, CD69 expression, and proliferation) in AD, while APP × PS1 transgenic mice displayed hyporeactive CD8⁺ T-cells after TCR ligation. Increased levels of CD8⁺ T memory cells and down regulation of CD8 receptor were found in AD and the animal model. Anergic TCR uncoupling was associated with loss of MAPK signaling (p38, ERK1 and ERK2) in APP × PS1. Our data implicate the generation of reactive memory T-cell in AD and of anergic memory T-cells in transgenic mice and should be taken into concern when designing immunotherapy.     

Evidence for a missed signal to the CD8+ cells in CSF of Multiple Sclerosis patients

Peripheral blood (PB) and cerebrospinal fluid (CSF) lymphocyte subpopulations, defined by various T-cell specific monoclonal antibodies and flow cytometry, were analysed in 44 relapsing remitting multiple sclerosis (RRMS) patients (including 21 subjects in the acute phase and 23 in the stable phase), 40 chronic-progressive multiple sclerosis (CPMS) patients, and 24 patients with other neurological diseases (OND), in order to verify the presence of any abnormality in the lymphocyte subset pattern. A significant increase in the total number of T-lymphocytes and the CD4+ subpopulation was found in the PB of the MS patients in comparison with the OND group. Moreover, a not statistically significant increase in CD4⁺ cells was observed in the CSF of MS patients. A statistically significant increase was also found in the CD4⁺ Leu 8⁺ (suppressor inducer) cells in the CSF of all of the MS groups. Finally, the CD8⁺ (suppressor/cytotoxic) cell levels, were significantly lower in the CSF of CPMS and stable RMS patients than in the CSF of the OND patients. As a whole, our data suggest that the immunosuppressive deficit that seems to be a constant finding in MS is not due to a decrease in suppressor inducer cell levels, as previously suggested, but may be caused by a missed or altered signal from the suppressor inducer to CD8⁺ suppressor cells.     

Evidence for a missed signal to the CD8+ cells in CSF of Multiple Sclerosis patients

Peripheral blood (PB) and cerebrospinal fluid (CSF) lymphocyte subpopulations, defined by various T-cell specific monoclonal antibodies and flow cytometry, were analysed in 44 relapsing remitting multiple sclerosis (RRMS) patients (including 21 subjects in the acute phase and 23 in the stable phase), 40 chronic-progressive multiple sclerosis (CPMS) patients, and 24 patients with other neurological diseases (OND), in order to verify the presence of any abnormality in the lymphocyte subset pattern. A significant increase in the total number of T-lymphocytes and the CD4+ subpopulation was found in the PB of the MS patients in comparison with the OND group. Moreover, a not statistically significant increase in CD4⁺ cells was observed in the CSF of MS patients. A statistically significant increase was also found in the CD4⁺ Leu 8⁺ (suppressor inducer) cells in the CSF of all of the MS groups. Finally, the CD8⁺ (suppressor/cytotoxic) cell levels, were significantly lower in the CSF of CPMS and stable RMS patients than in the CSF of the OND patients. As a whole, our data suggest that the immunosuppressive deficit that seems to be a constant finding in MS is not due to a decrease in suppressor inducer cell levels, as previously suggested, but may be caused by a missed or altered signal from the suppressor inducer to CD8⁺ suppressor cells.This Work was partially supported by an IRCCS Current Research Grant 1994.     

Oligodendrocyte lysis by CD4+ T cells independent of tumor necrosis factor

The capacity of human CD4⁺ T cells to lyse heterologous human oligodendrocytes in an 18-hour chromium 51–release assay was compared to that of systemic blood-derived macrophages and central nervous system–derived microglia. CD4⁺ T cells, activated with either phytohemagglutinin, anti-CD3 antibody, or antigen (myelin basic protein), could induce lysis of the oligodendrocytes whereas macrophages and microglia, activated with interferon-γ and lipopolysaccharide, could not. The CD4⁺ T-cell effect was not inhibited with an anti–tumor necrosis factor-α–neutralizing antibody. Both the CD4⁺ T cells and the macrophages could induce lysis of tumor necrosis factor–sensitive rodent cell lines, Wehi 164, and L929; these effects were inhibited with anti–tumor necrosis factor antibody. Pretreatment of the CD4⁺ T cells with cyclosporine or mitomycin C did not inhibit oligodendrocyte lysis. These results indicate that at least in vitro, CD4⁺ T cells can induce a form of oligodendrocyte injury that is not reproduced by macrophages or microglia or by tumor necrosis factor. The non–major histocompatibility complex (MHC)–restricted injury of oligodendrocytes induced by both myelin antigen–reactive and mitogen-stimulated T cells may provide a basis whereby cytotoxic CD4⁺ T cells could interact with a target cell that does not express MHC class II molecules. Our results suggest that immune-mediated oligodendrocyte/myelin injury, as is postulated to occur in the disease multiple sclerosis, may involve multiple effector mechanisms.     

Dopamine favors expansion of glucocorticoid-resistant IL-17-producing T cells in multiple sclerosis

Dopamine (DA) is a neurotransmitter produced mainly in the central nervous system (CNS) that has immunomodulatory actions on T cells. As the multiple sclerosis (MS) has long been regarded as an autoimmune disease of CNS mediated by T cells, the objective of this study was to evaluate the impact of DA on in vitro functional status of T cells from relapsing–remitting (RR)–MS patients. Peripheral T-cells from RR–MS patients were activated by mitogens and cell proliferation and cytokine production were assayed by [³H]-thymidine uptake and ELISA, respectively. Our results demonstrated that DA enhanced in vitro T cell proliferation and Th17-related cytokines in MS-derived cell cultures. In addition, this catecholamine reduced Treg-related cytokines (IL-10 and TGF-β) release by activated CD4⁺ T cells. These DA-induced effects on T cells were mainly dependent on IL-6 production by both polyclonally-activated CD4⁺ T cells and LPS-stimulated monocytes. Furthermore, the production of IL-17 and IL-6 by MS-derived T cells was directly related with neurological disability (EDSS score), and the release of these cytokines was less sensitive to glucocorticoid inhibition in MS patients than in control group, mainly after DA addition. In conclusion, our data suggest that DA amplifies glucocorticoid-resistant Th17 phenotype in MS patients, and this phenomenon could be, at least in part, due to its ability to induce IL-6 production by monocytes and CD4⁺ T cells.     

Administration of monthly pulses of cyclophosphamide in multiple sclerosis patients: Delayed recovery of several immune parameters following discontinuation of long-term cyclophosphamide treatment

Recovery of various components of the immune system was followed in eight patients with multiple sclerosis who had received monthly pulses of cyclophosphamide (CY) for approximately one year. CD8 cell numbers and NK and ADCC functions recovered in 1–2 months; B cells and FcR⁺ cell numbers recovered in 2–4 months. The recovery of CD4 cells and total T cell numbers, CD4/CD8 ratio and proliferative responses to PHA took more than 4 months. The impaired proliferation was not attributable to low IL-2 receptor expression. Once immunosuppression has been achieved pulse administration of CY at 2- to 4-month intervals may be feasible for long-term maintenance treatment.     

Murine T-lymphocytes express vasoactive intestinal peptide receptor 1 (VIP-R1) mRNA

Vasoactive intestinal peptide (VIP), a neuropeptide present in primary and secondary lymphoid organs has been previously reported to inhibit IL-2 and IL-4 production, as well as the proliferation of mitogen- or antigen-stimulated T-cells. Binding studies suggested that the immunoregulatory effects of VIP are mediated through specific VIP-binding sites present on lymphocyte subpopulations. Here we report on the expression of VIP-R1 mRNA in various murine lymphocyte subpopulations. By using RT-PCR, RNase protection assay, cDNA cloning, and sequence analysis, we show that stimulated and unstimulated murine spleen cells, thymocytes, CD4⁺ and CD8⁺ T-cells express VIP-R1. The VIP-R1 fragment amplified from murine brain, thymocytes, spleen cells and CD4⁺ T-cells share identical nucleotide sequences, and a high degree of homology with the corresponding nonlymphoid rat and human VIP-R1 sequences. The expression of VIP-R1 in thymocytes and peripheral lymphocytes, and especially in the CD4⁺ T-cell subset supports the idea that VIP produced or released locally in the lymphoid microenvironment could directly affect cytokine production and proliferation of T-lymphocytes.