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Autologous haematopoietic stem cell transplantation has been tried as one experimental strategy for the treatment of patients with aggressive multiple sclerosis refractory to other immunotherapies. The procedure is aimed at ablating and repopulating the immune repertoire by sequentially mobilizing and harvesting haematopoietic stem cells, administering an immunosuppressive conditioning regimen, and re-infusing the autologous haematopoietic cell product. 'Non-myeloablative' conditioning regimens to achieve lymphocytic ablation without marrow suppression have been proposed to improve safety and tolerability. One trial with non-myeloablative autologous haematopoietic stem cell transplantation reported clinical improvement and inflammatory stabilization in treated patients with highly active multiple sclerosis. The aim of the present study was to understand the changes in the reconstituted immune repertoire bearing potential relevance to its mode of action. Peripheral blood was obtained from 12 patients with multiple sclerosis participating in the aforementioned trial and longitudinally followed for 2 years. We examined the phenotype and function of peripheral blood lymphocytes by cell surface or intracellular staining and multi-colour fluorescence activated cell sorting alone or in combination with proliferation assays. During immune reconstitution post-transplantation we observed significant though transient increases in the proportion of CD4+ FoxP3+ T cells and CD56(high) natural killer cell subsets, which are cell subsets associated with immunoregulatory function. CD8+ CD57+ cytotoxic T cells were persistently increased after therapy and were able to suppress CD4+ T cell proliferation with variable potency. In contrast, a CD161(high) proinflammatory CD8+ T cell subset was depleted at all time-points post-transplantation. Phenotypic characterization revealed that the CD161(high)CD8+ T cells were mucosal-associated invariant T cells, a novel cell population originating in the gut mucosa but expressing the central nervous system-homing receptor CCR6. Detection of mucosal-associated invariant T cells in post-mortem multiple sclerosis brain white matter active lesions confirmed their involvement in the disease pathology. Intracellular cytokine staining demonstrated interferon γ and interleukin 17 production and lack of interleukin 10 production, a pro-inflammatory profile. Mucosal-associated invariant T cell frequency did not change in patients treated with interferon β; and was more depleted after autologous haematopoietic stem cell transplantation than in patients who had received high-dose cyclophosphamide (n = 7) or alemtuzumab (n = 21) treatment alone, suggesting an additive or synergistic effect of the conditioning regime components. We propose that a favourably modified balance of regulatory and pro-inflammatory lymphocytes underlies the suppression of central nervous system inflammation in patients with multiple sclerosis following non-myeloablative autologous haematopoietic stem cell transplantation with a conditioning regimen consisting of cyclophosphamide and alemtuzumab.

To identify differentially expressed genes in multiple sclerosis, microarrays were used in a stringent experimental setting-leukapheresis from disease-discordant monozygotic twins and gene expression profiling in CD4(+) and CD8(+) T-cell subsets. Disease-related differences emerged only in the CD8(+) T-cell subset. The five differentially expressed genes identified included killer cell lectin-like receptor subfamily B, member 1, also known as natural killer receptor protein 1a/CD161, presented by the International Multiple Sclerosis Genetics Consortium as one of the non-MHC candidate loci. Flow cytometric analysis on peripheral blood of healthy donors and patients with multiple sclerosis and rheumatoid arthritis confirmed an upregulation of CD161 at the protein level, showing also a significant excess of CD161(high)CD8(+) T cells in multiple sclerosis. This subset prevalently included chemokine (C-C motif) receptor 6(+), cytokine-producing, effector-memory T cells with proinflammatory profiles. It also included all circulating interleukin-17(+)CD8(+) T cells. In the CD161(high)CD8(+) subset, interleukin-12 facilitated proliferation and interferon-γ production, with CD161 acting as a co-stimulatory receptor. CD161(+)CD8(+)CD3(+) T cells producing interferon-γ were part of intralesional immune infiltrates and ectopic B cell follicles in autopsy multiple sclerosis brains. Variations of CD161 expression on CD8(+) T cells identify a subset of lymphocytes with proinflammatory characteristics that have not been previously reported in multiple sclerosis and are likely to contribute to disease immunopathology.

The CD94/NKG2 complex is expressed on T and NK lymphocytes. CD94 molecules covalently associate to activating or inhibitory NKG2 molecules, and their expression finely tunes cell responses. Human γδ T cells express several NKRs. Expression of these receptors is confined to the cytolytic Vδ2 subset, which coexpresses the FcγRIII CD16 and CD45RA and has been defined as Vγ9Vδ2 T(EMRA) cells. We show that the CD94/NKG2C complex, associated with KARAP/DAP12, is fully functional in γδ T cells, as determined by measuring IFN-γ production, T cell proliferation, and cytolytic activity by γδ lymphocytes. In contrast, NKG2A expression was found on all γδ T cell memory subsets, suggesting a crucial role of the inhibitory signal provided by this receptor on γδ T cell responses. Moreover, we found Vγ9Vδ2 T(EMRA), NK, and CD8+ αβ T cells coexpressing NKG2A and NKG2C receptors. Functional experiments showed that the inhibitory signal mediated by the NKG2A receptor prevails when double-positive cells are activated. Finally, NKG2A expression on γδ LDGL correlates with asymptomatic pathology, even in the presence of NKG2C coexpression, whereas in symptomatic patients affected by severe disease, the inhibitory NKG2A receptor is absent, and a variety of activatory NKRs was found. We propose that the silent behavior of γδ cells in LDGL patients is a result of effective inhibitory HLA class I receptors.

Forkhead box P3 (FoxP3) is a transcription factor whose expression characterizes regulatory T cells (T(reg)), but it is also present on activated T cells, thus hindering correct T(reg) identification. Using classical markers for T(reg) recognition, discordant results were found in terms of T(reg) expansion during active tuberculosis (TB) disease. Recently CD39 has been shown to be an accurate marker for T(reg) detection. The objectives of this study were: (i) to identify T(reg) expressing CD39 in patients with TB and to compare the results with those obtained by the standard phenotypic markers; (ii) to evaluate if T(reg) are expanded in vitro by exogenous interleukin (IL)-2 or by antigen-specific stimulation; and (iii) to characterize T(reg) function on the modulation of antigen-specific responses. We enrolled 13 patients with pulmonary TB and 12 healthy controls. T(reg) were evaluated by flow cytometry ex vivo and after antigen-specific in vitro stimulation using CD25, FoxP3, CD127 and CD39 markers. Results indicate that CD39(+) cells within the CD4(+)CD25(high) cells have T(reg) properties (absence of interferon-gamma production and transforming growth factor-beta1 release upon stimulation). Ex vivo analysis did not show significant differences between TB patients and controls of T(reg) by classical or novel markers. In contrast, a significantly higher percentage of T(reg) was found in TB patients after antigen-specific stimulation both in the presence or absence of IL-2. Depletion of CD39(+) T(reg) increased RD1-specific responses significantly. In conclusion, CD39 is an appropriate marker for T(reg) identification in TB. These results can be useful for future studies to monitor Mycobacterium tuberculosis-specific response during TB.

The immunomodulatory activities of human mesenchymal stem cells (MSCs) provide a rational basis for their application in the treatment of immune-mediated diseases, such as graft versus host disease and multiple sclerosis. The effects of MSCs on invariant natural killer T (iNKT) and gammadelta T cells, both involved in the pathogenesis of autoimmune diseases, are unknown. Here, we investigated the effects of MSCs on in vitro expansion of these unconventional T-cell populations. MSCs inhibited iNKT (Valpha24(+)Vbeta11(+)) and gammadelta T (Vdelta2(+)) cell expansion from peripheral blood mononuclear cells in both cell-to-cell contact and transwell systems. Such inhibition was partially counteracted by indomethacin, a prostaglandin E(2) inhibitor. Block of indoleamine 2,3-deoxygenase and transforming growth factor beta1 did not affect Valpha24(+)Vbeta11(+) and Vdelta2(+) cell expansion. MSCs inhibited interferon-gamma production by activated Valpha24(+)Vbeta11(+) and impaired CD3-mediated proliferation of activated Valpha24(+)Vbeta11(+) and Vdelta2(+) T cells, without affecting their cytotoxic potential. MSCs did not inhibit antigen processing/presentation by activated Vdelta2(+) T cells to CD4(+) T cells. In contrast, MSCs were lysed by activated Vdelta2(+) T cells through a T-cell receptor-dependent mechanism. These results are translationally relevant in view of the increasing interest in MSC-based therapy of autoimmune diseases.

The ability of cannabinoids to modulate both inflammatory and degenerative neuronal damage prompted investigations on the potential benefits of such compounds in multiple sclerosis (MS) and in animal models of this disorder. Here we measured endocannabinoid levels, metabolism and binding, and physiological activities in 26 patients with MS (17 females, aged 19-43 years), 25 healthy controls and in mice with experimental autoimmune encephalomyelitis (EAE), a preclinical model of MS. Our results show that MS and EAE are associated with significant alterations of the endocannabinoid system. We found that anandamide (AEA), but not 2-arachidonoylglycerol (2-AG), was increased in the CSF of relapsing MS patients. AEA concentrations were also higher in peripheral lymphocytes of these patients, an effect associated with increased synthesis and reduced degradation of this endocannabinoid. Increased synthesis, reduced degradation, and increased levels of AEA were also detected in the brains of EAE mice in the acute phase of the disease, possibly accounting for its anti-excitotoxic action in this disorder. Accordingly, neurophysiological recordings from single neurons confirmed that excitatory transmission in EAE slices is inhibited by CB1 receptor activation, while inhibitory transmission is not. Our study suggests that targeting the endocannabinoid system might be useful for the treatment of MS.

Autoreactive CD4+ and CD8+ T cells directed against CNS autoantigens may play a role in the development of multiple sclerosis (MS). Identical twins share the same genetic background but not the TCR repertoire that is shaped by the encounter with self or foreign antigens. To gain insights into the interplay between MS and T cell repertoire, peripheral blood CD4+ and CD8+ T lymphocytes and their CCR7+/CCR7- subsets from five pairs of identical twins (four discordant and one concordant for MS; none of which had taken disease-modifying therapy) were compared by TCR beta-chain (TCRB) complementary-determining region 3 (CDR3) spectratyping. CD4+ T cells generally showed a Gaussian distribution, whereas CD8+ T cells exhibited subject-specific, widely skewed TCR spectratypes. There was no correlation between CD8+ T cell oligoclonality and disease. Sequencing of predominant spectratype expansions revealed shared TCRB-CDR3 motifs when comparing inter- and/or intrapair twin members. In many cases, these sequences were homologous to published TCRs, specific for viruses implicated in MS pathogenesis, CNS autoantigens, or copaxone [glatiramer acetate (GA)], implying the occurrence of naturally GA-responding CD8+ T cells. It is notable that these expanded T cell clones with putative pathogenic or regulatory properties were present in the affected as well as in the healthy subject, thus suggesting the existence of a "MS predisposing trait" shared by co-twins discordant for MS.

The recruitment of lymphocytes across the blood brain barrier (BBB) is mediated by adhesion molecules and chemokines. The expression of activation markers and of chemokine receptors on T cells homing to the nervous system (NS) may help define their functional state. In the cerebrospinal fluid (CSF) of subjects with inflammatory neurological diseases (IND), including multiple sclerosis, we observed an increased number of T cells coexpressing CXCR3 and CCR5 as well as T cells with a CD45RO+ CCR7+ CD27+ memory phenotype. A subset of CCR7+ T cells coexpressed CXCR3 and CCR5. We also detected an increased number of interferon-gamma-producing T cells in the CSF compared with peripheral blood, mostly but not exclusively in the CD45RO+ CCR7- CD27- compartment. T helper 1 (Th1) clones, established from the CSF of individuals with IND and from a healthy subject, similarly migrated to CXCL10, CXCL12, and CCL5. CXCL10, CXCL12, and CCL19 were increased in the CSF of individuals with neuroinflammation. These findings suggest that CSF is enriched in Th1-polarized memory T cells capable of differentiating into effector cells upon antigen encounter. These cells are recruited into the CSF by inducible chemokines. Thus, CSF represents a transitional station for T cells trafficking to and from the NS.

Vgamma9Vdelta2 T lymphocytes are broadly reactive against various intracellular pathogens and display both lytic and proliferative responses to human immunodeficiency virus (HIV)-infected cells. HIV infection of peripheral blood mononuclear cell cultures led to absolute increases in Vgamma9Vdelta2 T cells accompanied by decreased p24 levels. Strong gammadelta T cell activation with nonpeptidic mycobacterial phosphoantigens (TUBAg1 extract or synthetic isopentenyl pyrophosphate) resulted in potent inhibition of HIV replication through soluble released factors. Subsequent analyses showed that phosphoantigen-activated gammadelta T cells produced substantial amounts of beta-chemokines (macrophage inflammatory protein [MIP]-1alpha, MIP-1beta, and regulated-on-activation, normal T-cell-expressed and -secreted beta-chemokine [RANTES]), which represent the natural ligand for the CCR5 HIV coreceptor. Accordingly, anti-beta-chemokine antibodies neutralized the inhibition of monocytotropic HIV strains by gammadelta T cell-released factors. Moreover, a T-tropic HIV strain using the CXCR4 coreceptor for virus entry was potently inhibited. Together, these data reveal that phosphoantigen-activated gammadelta T cells are an important source of CC chemokines and may suppress HIV replication through cell-released antiviral factors.