Specific T-cell proliferation to myelin peptides in relapsing-remitting multiple sclerosis.

BACKGROUND: The identification of major immunogenic peptides in multiple sclerosis (MS) is of great importance for the development of antigen-specific therapies. Cellular reactivity against a selected mix of seven myelin peptides was evaluated in vitro. The evolution of this reactivity over time and its correlation with clinical variables was also analysed. MATERIAL AND METHODS: Forty-two patients with MS, 15 with other demyelinating diseases and 40 healthy donors (HD) were studied. Cell proliferation was measured by 3[H] thymidine incorporation into samples obtained at 0, 3, 6 and 12months of MS patient follow-up. RESULTS: A positive reaction to the peptide mix was detected in 31 of the 42 patients (74%), 12 of the 40 HD (30%) and 6 of the 15 (40%) patients with other demyelinating diseases. Patients with positive proliferation had greater disability (EDSS score, 3 [1-5.5] vs. 1.0[1-2], P=0.021), higher number of relapses (7±4.1 vs. 3±1.2, P<0.001) and shorter time since the last relapse (9±7.5 vs. 32±12.3months, P=0.036). After 12months of follow-up, cell reactivity was maintained in 33 patients (78%). CONCLUSION: A high percentage of patients exhibit a significant and maintained reactivity to myelin peptides over time. Therefore, this mix may be useful as a source of antigen in the development of protocols aimed at inducing specific tolerance in MS.

The transcription factor PU.1 is involved in macrophage proliferation.

PU.1 is a tissue-specific transcription factor that is expressed in cells of the hematopoietic lineage including macrophages, granulocytes, and B lymphocytes. Bone marrow-derived macrophages transfected with an antisense PU.1 expression construct or treated with antisense oligonucleotides showed a decrease in proliferation compared with controls. In contrast, bone marrow macrophages transfected with a sense PU.1 expression construct displayed enhanced macrophage colony-stimulating factor (M-CSF)-dependent proliferation. Interestingly, there was no effect of sense or antisense constructs of PU.1 on the proliferation of the M-CSF-independent cell line, suggesting that the response was M-CSF dependent. This was further supported by the finding that macrophages transfected with a sense or an antisense PU.1 construct showed, respectively, an increased or a reduced level of surface expression of receptors for M-CSF. The enhancement of proliferation seems to be selective for PU.1, since transfections with several other members of the ets family, including ets-2 and fli-1, had no effect. Various mutants of PU.1 were also tested for their ability to affect macrophage proliferation. A reduction in macrophage proliferation was found when cells were transfected with a construct in which the DNA-binding domain of PU.1 was expressed. The PEST (proline-, glutamic acid-, serine-, and threonine-rich region) sequence of the PU.1 protein, which is an important domain for protein-protein interactions in B cells, was found to have no influence on PU.1-enhanced macrophage proliferation when an expression construct containing PU.1 minus the PEST domain was transfected into bone marrow-derived macrophages. In vivo, PU.1 is phosphorylated on several serine residues. The transfection of plasmids containing PU.1 with mutations at each of five serines showed that only positions 41 and 45 are critical for enhanced macrophage proliferation. We conclude that PU.1 is necessary for the M-CSF-dependent proliferation of macrophages. One of the proliferation-relevant targets of this transcription factor could be the M-CSF receptor.

Dendritic cells pulsed with antigen-specific apoptotic bodies prevent experimental type 1 diabetes.

Dendritic cells (DCs) are powerful antigen-presenting cells capable of maintaining peripheral tolerance. The possibility to generate tolerogenic DCs opens new therapeutic approaches in the prevention or remission of autoimmunity. There is currently no treatment inducing long-term tolerance and remission in type 1 diabetes (T1D), a disease caused by autoimmunity towards beta cells. An ideal immunotherapy should inhibit the autoimmune attack, avoid systemic side effects and allow islet regeneration. Apoptotic cells--a source of autoantigens--are cleared rapidly by macrophages and DCs through an immunologically silent process that contributes to maintaining tolerance. Our aims were to prevent T1D and to evaluate the re-establishment of peripheral tolerance using autologous DCs pulsed in vitro with apoptotic bodies from beta cells. Immature DCs derived from bone marrow of non-obese diabetic (NOD) mice were obtained and pulsed with antigen-specific apoptotic bodies from the beta cell line NIT-1. Those DCs that phagocytosed apoptotic cells diminished the expression of co-stimulatory molecules CD40 and CD86 and reduced secretion of proinflammatory cytokines. Moreover, these cells were resistant to increase the expression of co-stimulatory molecules after lipopolysaccharide activation. The administration of these cells to NOD transgenic mice expressing interferon-beta in their insulin-producing cells, a model of accelerated autoimmune diabetes, decreased diabetes incidence significantly and correlated positively with insulitis reduction. DCs pulsed with apoptotic cells that express disease-associated antigens constitutes a promising strategy to prevent T1D.

Transcription factors that regulate monocyte/macrophage differentiation.

Although all the cells in an organism contain the same genetic information, differences in the cell phenotype arise from the expression of lineage-specific genes. During myelopoiesis, external differentiating signals regulate the expression of a set of transcription factors. The combined action of these transcription factors subsequently determines the expression of myeloid-specific genes and the generation of monocytes and macrophages. In particular, the transcription factor PU.1 has a critical role in this process. We review the contribution of several transcription factors to the control of macrophage development.

Dendritic cells can be successfully generated from CD34+ cord blood cells in the presence of autologous cord blood plasma.

Dendritic cells (DCs) are currently being considered as adjuvants in immunotherapy. Depending on their source and culture conditions, they show different features and maturation states. Dendritic cells can be generated from monocytes and CD34+ haematopoietic stem cells, from both adult and cord blood. Here, we report the generation of mature DCs from enriched CD34+ cord blood (CB) cells using autologous cord blood plasma (ACBP) as a source of serum proteins and factors. In the presence of ACBP, CD34+ cells proliferated and differentiated resulting in a population of cells with a dendritic phenotype as assessed by morphology and flow cytometry analyses. The DC population obtained using ACBP showed higher levels of HLA class II molecules, co-stimulatory molecules including CD40, CD80 or CD86, and the dendritic cell marker CD83, compared with those generated in adult blood serum (ABS). Furthermore, the DCs generated in the presence of ACBP were more potent stimulatory cells in the mixed lymphocyte:dendritic cell reactions (MLDCR), compared to cells generated in ABS. Similar results were obtained using homologous cord blood plasma (HCBP). These results show that ACBP can support the generation of DCs from CD34+ progenitor cells when only GM-CSF and TNFalpha are used as differentiating cytokines.

Myelin peptides in multiple sclerosis.

The development of specific therapies for organ-specific autoimmune diseases requires the identification of relevant immunogenic epitopes, recognized by both pathogenic T cells and autoantibodies. Here, we review the most relevant studies focused in the identification of peptides in multiple sclerosis (MS) and the distinct T cell reactivity induced in patients compared to controls. Only a few studies reported significant differences in terms of T cell reactivity to them. The current knowledge on this issue, and the diagnostic and therapeutic possibilities opened by the identification of pathogenic MS epitopes are discussed in this paper.

Treatment of monocytes with interleukin (IL)-12 plus IL-18 stimulates survival, differentiation and the production of CXC chemokine ligands (CXCL)8, CXCL9 and CXCL10.

During inflammation, interleukin (IL)-12 and IL-18 are produced by macrophages and other cell types such as neutrophils (IL-12), keratinocytes and damaged endothelial cells (IL-18). To explore the role of IL-12 and IL-18 in inflammatory innate immune responses we investigated their impact on human peripheral blood monocytes and mature bronchoalveolar lavage (BAL) macrophages. IL-12 and IL-18 together, but not alone, prevented spontaneous apoptosis of cultured monocytes, promoted monocyte clustering and subsequent differentiation into macrophages. These morphological changes were accompanied by increased secretion of CXC chemokine ligands (CXCL)9, CXCL10 (up to 100-fold, P < 0.001) and CXCL8 (up to 10-fold, P < 0.001) but not CCL3, CCL4 or CCL5. Mature macrophages (from BALs) expressed high basal levels of CXCL8, that were no modified upon stimulation with IL-12 and IL-18. In contrast, the basal production of CXCL9 and CXCL10 by BALs was increased by 10-fold (P < 0.001) in the presence of either IL-12 or IL-18 alone and by 50-fold in the presence of both cytokines. In conclusion, our results indicate a relevant role for IL-12 and IL-18 in the activation and resolution of inflammatory immune responses, by increasing the survival of monocytes and by inducing the production of chemokines. In particular, those that may regulate angiogenesis and promote the recruitment of monocytes, activated T cells (CXCL9 and CXCL10) and granulocytes (CXCL8).

Primary alloproliferative TH1 response induced by immature plasmacytoid dendritic cells in collaboration with myeloid DCs.

The role played by dendritic cell (DC) subsets in the immune response to alloantigens is not well defined. In vitro experiments have extensively shown that freshly isolated myeloid (M)DCs induce a strong T lymphocyte proliferation whereas plasmacytoid (P)DCs do not, unless activated by CD40 ligation. The aim of these studies was to explore whether the interplay among PDCs, MDCs and T cells modulates alloresponse. Freshly isolated MDCs and PDCs were merged in different proportions and used as antigen presenting cells (APCs) in mixed lymphocyte cultures (MLC). As described, isolated PDCs only induced a mild alloresponse, while MDCs were potent inducers of alloproliferation. Unexpectedly, when PDCs were merged with even low numbers of MDCs (down to 100 cells) and used as APCs, a potent Th1 cell proliferation was detected. Survival and maturation of PDCs was increased in these MLC conditions, which could partially explain the magnitude of the T-cell response. Interestingly, the proportion of IFNgamma-producing cells generated in such cultures was higher compared to MDC-stimulated cultures. These data suggest that the interaction between both DC subsets is determinant to generate a potent Th1 response, at least in an allogeneic situation, and may be relevant to the outcome of allogeneic stem cell transplantation.

Sustained expression of CD154 (CD40L) and proinflammatory cytokine production by alloantigen-stimulated umbilical cord blood T cells.

Recent data suggests that graft-versus-host disease (GVHD) is initiated by host APCs. Blockade of CD40:CD154 interactions between APCs and T cells in vivo induces T cell tolerance to host alloantigen and dramatically reduces GVHD. Because allogeneic cord blood (CB) transplantation results in a lower incidence and severity of acute GVHD compared with bone marrow transplantation, we have investigated whether CB T cells can express CD154 in response to stimulation by allogeneic monocyte-derived dendritic cells (MDDC) and have used 5- (and 6-)carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling in combination with intracellular cytokine analysis to assess the proliferation and cytokine profiles of alloantigen-responsive cells. CB T cells stimulated with allogeneic MDDC showed stronger proliferation than adult blood T cells. Surface CD154 expression was detected in the actively dividing CFSElow populations of both the CD4+ and CD4- subsets and was brightest in cells that had divided the most. Assessment of supernatants from MDDC-stimulated CB and adult blood T cells showed no significant difference in the levels of either IFN-gamma or TNF-alpha, but CB T cell supernatants did show a significant lack of detectable IL-2. Intracellular cytokine analysis revealed that dividing CB T cells had been primed to produce IFN-gamma, TNF-alpha, and IL-2 on restimulation. Further phenotype analysis showed that 75% of CB T cells producing IFN-gamma were CD8+. These data suggest that MDDC-stimulated CB T cells express functional CD154 and provide enough costimulation for dendritic cells to prime naive CD8+ CB T cells and induce type 1 cytokine production.

Repression of I-A beta gene expression by the transcription factor PU.1.

The PU.1 protein is an ets-related transcription factor that is expressed in macrophages and B lymphocytes. We present evidence that PU.1 binds to the promoter of the I-A beta gene, i.e. a PU box located next to the Y box. Transfection of PU.1 in B lymphocytes or in interferon-gamma-treated macrophages represses I-A beta gene expression. The inhibitory effect of PU.1 was obtained with the DNA binding domain of the protein, but not with the activation domain. Using the gel shift retardation assay we found that in vitro transcribed/translated NF-YA and NF-YB bind to the Y box of the I-A beta promoter. When PU.1 was added to the assay, a supershifted DNA band was found, indicating that PU.1 and NFY proteins bind to the same DNA molecule. We conclude that I-A beta gene expression is repressed by PU.1 binding to the PU box domain.

Identification of both myeloid CD11c+ and lymphoid CD11c- dendritic cell subsets in cord blood.

Dendritic cells (DCs) are the most potent antigen-presenting cells described to date. In human peripheral blood, both myeloid and lymphoid subsets of DCs have been identified. In contrast, cord blood (CB) DCs have recently been described as being exclusively of the immature CD11c- lymphoid DC subset. Using an alternative method of enrichment, based on a negative selection system, both lymphoid (HLA-DR+ CD123+++ CD11c- CD33-) and myeloid (HLA-DR++ CD123+ CD11c+ CD33+) DCs were identified in CB. Although the majority of CB DCs showed a lymphoid phenotype, a significant number of CD11c+ myeloid DCs (25.6% +/- 14.5%, n = 13) were also present. Other markers, such as CD80 and CD83, were negative in both subsets. Analyses of the allostimulatory capacity of both subsets showed that freshly isolated CB lymphoid DCs failed to induce a potent allostimulation of naive CB T cells. These features are therefore consistent with previous work reporting an immature phenotype for lymphoid DCs in adult blood. The significance of the inverted CD11c+/CD11c- ratio observed in CB DCs (1:3) with respect to adult blood DCs (3:1) remains to be explained.

Differential up-regulation of HLA-DM, invariant chain, and CD83 on myeloid and plasmacytoid dendritic cells from peripheral blood.

Two main dendritic cell (DC) subsets have been described in peripheral blood, the myeloid subset or DC1 that is characterized by the presence of CD11c and the plasmacytoid subset or DC2 negative for this marker. The two subsets may perform different functions and have been defined as immunogenic (the myeloid subset) or tolerogenic (the plasmacytoid subset). The expression of human leukocyte antigen (HLA)-DM molecules, which act as peptide editors in the antigen presentation process, was studied in freshly isolated plasmacytoid and myeloid DCs from peripheral blood. The expression of the invariant chain (Ii), the major histocompatibility complex class II (MHC-II) : class II-associated Ii peptide (CLIP) complex, and CD83 was also investigated. The results showed that intracellular expression of HLA-DM and the Ii was significantly higher in the plasmacytoid than in the myeloid DC subset. In contrast, a higher fraction of cell expressing MHC-II : CLIP complex was found in the myeloid than in the plasmacytoid DC subpopulation. CD83 was not detected in any of these two subsets. Following culture of these cells with interleukin-3 (IL-3), tumor necrosis factor-alpha (TNFalpha) and/or heat shock protein-70 (HSP-70), the expression of intracellular HLA-DM was up-regulated in the myeloid DCs to levels similar to those found in the plasmacytoid DCs, whilst the Ii was down-regulated in the plasmacytoid subset to similar levels to those expressed in the myeloid DCs. In addition, CD83 was up-regulated in the myeloid (CD11c+) but not in the plasmacytoid (CD11c-) DCs. The expression pattern of these antigen-processing molecules could be related to the immaturity and function attributed to these DC subsets.

Differential expression of the cytokine receptors for human interleukin (IL)-12 and IL-18 on lymphocytes of both CD45RA and CD45RO phenotype from tonsils, cord and adult peripheral blood.

The objective of this study was to demonstrate the variable expression of cytokine receptors on naive versus memory human CD4+ T cell subpopulations in tonsillar tissue, cord blood and adult blood. We prove that the receptors for both interleukin (IL)-12 and IL-18 are expressed exclusively on memory T cells. This observation was seen not only on the CD45RO+ memory T cells but also on a significant percentage of the CD45RA+, CD62L-, CD27- and CCR7- populations. Furthermore, CD45RA+ CD62L+, CD27+ or CCR7+ CD4+ T cells that expressed IL-12Rbeta1 and IL-18Ralpha did not express CD31, a marker for recent thymic emigrants. We reveal that cord blood lymphocytes do not express IL-12Rbeta1 whereas IL-18Ralpha expression was detected at low levels. Importantly, the IL-12Rbeta2 signalling chain, which is absent in all resting T cells, was up-regulated in both CD45RA+ and CD45RO+ T cells as a result of stimulation with anti-CD3 and anti-CD28 in vitro. This observed up-regulation was, however, restricted to 80% of the total CD4+ population. Finally, a very small proportion of the CD4+ CD45RO+ tonsillar T cells expressed the IL-12 and IL-18 receptors, thereby establishing the differential expression of these receptors between peripheral and tonsillar memory T cell subpopulations.