A randomized double-blind placebo-controlled trial of low-dose interleukin-2 in relapsing-remitting multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is associated with regulatory T cells (Tregs) insufficiency while low-dose interleukin-2 (IL2LD) activates Tregs and reduces disease activity in autoimmune diseases. METHODS: We aimed at addressing whether IL2LD improved Tregs from MS patients. MS-IL2 was a single-center double-blind phase-2 study. Thirty patients (mean [SD] age 36.8 years [8.3], 16 female) with relapsing-remitting MS with new MRI lesions within 6 months before inclusion were randomly assigned in a 1:1 ratio to placebo or IL-2 at 1 million IU, daily for 5 days and then fortnightly for 6 months. The primary endpoint was change in Tregs at day-5. RESULTS: Unlike previous trials of IL2LD in more than 20 different autoimmune diseases, Tregs were not expanded at day-5 in IL2LD group, but only at day-15 (median [IQR] fold change from baseline: 1.26 [1.21-1.33] in IL2LD group; 1.01 [0.95-1.05] in placebo group, p < 0.001). At day-5, however, Tregs had acquired an activated phenotype (fold change of CD25 expression in Tregs: 2.17 [1.70-3.55] in IL2LD versus 0.97 [0.86-1.28] in placebo group, p < 0.0001). Regulator/effector T cells ratio remained elevated throughout treatment period in the IL2LD group (p < 0.001). Number of new active brain lesions and of relapses tended to be reduced in IL2LD treated patients, but the difference did not reach significance in this trial not powered to detect clinical efficacy. CONCLUSION: The effect of IL2LD on Tregs in MS patients was modest and delayed, compared to other auto-immune diseases. This, together with findings that Tregs improve remyelination in MS models and recent reports of IL2LD efficacy in amyotrophic lateral sclerosis, warrants larger studies of IL2LD in MS, notably with increased dosages and/or modified modalities of administration. TRIAL REGISTRATION INFORMATION: ClinicalTrials.gov: NCT02424396; EU Clinical trials Register: 2014-000088-42.

Serum tryptophan metabolites are associated with erosive hand osteoarthritis and pain: results from the DIGICOD cohort.

OBJECTIVE: To investigate host and gut-microbiota related Tryptophan metabolism in hand osteoarthritis (HOA). METHODS: The baseline serum concentration of 20 Tryptophan metabolites was measured in 416 HOA patients in a cross-sectional analysis of the DIGICOD cohort. Tryptophan metabolites levels, metabolite-ratios and metabolism pathway activation were compared between erosive (N = 141) and non-erosive HOA (N = 275) by multiple logistic regressions adjusted on age, BMI and sex. The association between Tryptophan metabolite levels and HOA symptoms was investigated by a Spearman's rank correlation analysis. RESULTS: Four serum Tryptophan metabolites, eight metabolite ratios and one metabolism pathway were associated with erosive HOA. Erosive HOA was negatively associated with Tryptophan (odds ratio (OR) = 0.41, 95% confidence interval [0.24-0.70]), indole-3-aldehyde (OR = 0.67 [0.51-0.90]) and 3-OH-anthranilic acid (OR = 1.32 [1.13-1.54]) and positively with 5-OH-Tryptophan levels (OR = 1.41 [1.13-1.77]). The pro-inflammatory kynurenine-indoleamine 2,3-dioxygenase pathway was upregulated in erosive HOA (OR = 1.60 [1.11-2.29]). Eleven metabolites were correlated with HOA symptoms and were mostly pain-related. Serotonin and N-acetyl serotonin levels were negatively correlated with number of tender joints. Indole-3-aldehyde level was negatively correlated and 3-OH-anthranilic acid, 3-OH-kynurenine and 5-OH-Tryptophan levels were positively correlated with number of patients-reported painful joints. Quinolinic acid and 3-OH-kynurenine levels correlated positively with AUSCAN pain. CONCLUSIONS: Tryptophan metabolites disturbance is associated with erosive HOA and pain and emphasize the role of low-grade inflammation and gut dysbiosis in HOA.

Specific Follicular Helper T Cell Signature in Takayasu Arteritis.

OBJECTIVE: Our aim was to compare transcriptome and phenotype profiles of CD4+ T cells and CD19+ B cells in patients with Takayasu arteritis (TAK), patients with giant cell arteritis (GCA), and healthy donors. METHODS: Gene expression analyses, flow cytometry immunophenotyping, T cell receptor (TCR) gene sequencing, and functional assessments of cells from peripheral blood and arterial lesions from TAK patients, GCA patients, and healthy donors were performed. RESULTS: Among the most significantly dysregulated genes in CD4+ T cells of TAK patients compared to GCA patients (n = 720 genes) and in CD4+ T cells of TAK patients compared to healthy donors (n = 1,447 genes), we identified a follicular helper T (Tfh) cell signature, which included CXCR5, CCR6, and CCL20 genes, that was transcriptionally up-regulated in TAK patients. Phenotypically, there was an increase in CD4+CXCR5+CCR6+CXCR3- Tfh17 cells in TAK patients that was associated with a significant enrichment of CD19+ B cell activation. Functionally, Tfh cells helped B cells to proliferate, differentiate into memory cells, and secrete IgG antibodies. Maturation of B cells was inhibited by JAK inhibitors. Locally, in areas of arterial inflammation, we found a higher proportion of tertiary lymphoid structures comprised CD4+, CXCR5+, programmed death 1+, and CD20+ cells in TAK patients compared to GCA patients. CD4+CXCR5+ T cells in the aortas of TAK patients had an oligoclonal α/ß TCR repertoire. CONCLUSION: We established the presence of a specific Tfh cell signature in both circulating and aorta-infiltrating CD4+ T cells from TAK patients. The cooperation of Tfh cells and B cells might be critical in the occurrence of vascular inflammation in patients with TAK.

Pregnant type 1 diabetes women with rises in C-peptide display higher levels of regulatory T cells: A pilot study.

AIM: During pregnancy of type 1 diabetes (T1D) women, a C peptide rise has been described, which mechanism is unclear. In T1D, a defect of regulatory T cells (Tregs) and its major controlling cytokine, interleukin-2 (IL2), is observed. METHODS: Evolution of clinical, immunological (Treg (CD4+CD25hiCD127-/loFoxp3+ measured by flow cytometry and IL2 measured by luminex xMAP technology) and diabetes parameters (insulin dose per day, HbA1C, glycaemia, C peptide) was evaluated in 13 T1D women during the three trimesters of pregnancy and post-partum (PP, within 6 months) in a monocentric pilot study. Immunological parameters were compared with those of a healthy pregnant cohort (QuTe). RESULTS: An improvement of beta cell function (C peptide rise and/or a decrease of insulin dose-adjusted A1c index that estimate individual exogenous insulin need) was observed in seven women (group 1) whereas the six others (group 2) did not display any positive response to pregnancy. A higher level of Tregs and IL2 was observed in group 1 compared to group 2 during pregnancy and at PP for Tregs level. However, compared to the healthy cohort, T1D women displayed a Treg deficiency CONCLUSION: This pilot study highlights that higher level of Tregs and IL2 seem to allow improvement of endogenous insulin secretion of T1D women during pregnancy.

The regulatory dendritic cell marker C1q is a potent inhibitor of allergic inflammation.

The complement subunit C1q was recently identified as a marker for monocyte-derived regulatory dendritic cells supporting the differentiation of interleukin (IL)-10-secreting CD4+ T cells with a suppressive activity. Furthermore, C1q expression is upregulated in peripheral blood mononuclear cells of allergic patients in the course of successful allergen immunotherapy. Herein, we investigated a potential direct role of C1q in downregulating allergic inflammation. In mice with ovalbumin (OVA) or birch pollen (BP)-induced allergic asthma, C1q is as efficacious as dexamethasone to reduce both airway hyperresponsiveness (AHR), eosinophil, and ILC2 infiltrates in bronchoalveolar lavages, as well as allergen-specific T helper 2 cells in the lungs. Administration of C1q does not expand IL-10+/Foxp3+ regulatory T cells in the lungs, spleen, or in the blood. Depletion of plasmacytoid dendritic cells (pDCs) abrogates the capacity of C1q to reduce AHR and eosinophilic infiltrates in OVA-sensitized mice. Also C1q treatment inhibits the activation of human and mouse pDCs by CpGs, thereby demonstrating a critical role for pDCs in the anti-inflammatory activity of C1q. We conclude that regulatory dendritic cells can mediate a potent direct anti-inflammatory activity via the expression and/or secretion of molecules such as C1q, independently of their capacity to expand the pool of regulatory T cells.

Tetraspanins displayed in retrovirus-derived virus-like particles and their immunogenicity.

Virus-like particles (VLPs) are a particular subset of subunit vaccines which are currently explored as safer alternatives to live attenuated or inactivated vaccines. VLPs derived from retrovirus (retroVLPs) are commonly used as scaffolds for vaccine candidates due to their ability to incorporate heterologous envelope proteins. Pseudotyping retroVLPs is however not a selective process therefore, host cellular proteins such as tetraspanins are also included in the membrane. The contribution of these host-proteins to retrovirus immunogenicity remains unclear. In this work, human cells silenced and not silenced for tetraspanin CD81 were used to produce CD81(-) or CD81(+) retroVLPs. We first analyzed mice immune response against human CD81. Despite effective silencing of CD81 in retroVLP producing cells, both humoral and cellular immune responses showed persistent anti-CD81 immunogenicity, suggesting cross reactivity to related antigens. We thus compared the incorporation of related tetraspanins in retroVLPs and showed that decreased CD81 incorporation in CD81(-) retro-VLPs is compensated by an increased incorporation of CD9 and CD63 tetraspanins. These results highlight the dynamic nature of host-derived proteins incorporation in retroVLPs membrane, which should be considered when retrovirus-based biopharmaceuticals are produced in xenogeneic cells.

C-terminal cleavage of human Foxp3 at a proprotein convertase motif abrogates its suppressive function.

Foxp3 plays a critical role in the development and function of regulatory T cells (Tregs). Differences in translational and post-translational processing of murine and human Foxp3 have been recently reported. Human Foxp3 exists as four isoforms generated by alternative splicing. Mouse Foxp3 only exists as one isoform, but can be proteolytically cleaved by N-terminal and/or C-terminal proprotein convertase subtilisin/kexins (PCSKs). Here, we show by transcriptome analysis that the proprotein convertases PCSK7, PCSK5 and Furin are present in human CD4(+) T cells with different expression patterns. Notably, after in vitro activation, only PCSK7 and Furin are expressed in Tregs and T effector cells (Teffs), with overexpression of PCSK7 in Tregs compared to Teffs. Human Foxp3 protein displays specific motifs that can be potentially cleaved by convertases. Consequently, we transduced human CD4(+) cells with Foxp3-expressing lentiviral vectors and assessed the generation of proteolytically cleaved Foxp3 forms by Western blot. Three different Foxp3 forms were detected, indicating that human Foxp3 can also be subjected to proteolytic cleavage at the N-terminal and C-terminal ends. These results prompted us to assess the suppressive activity associated with each forms. We observed that full length and N-cleaved Foxp3-transduced CD4(+) T cells similarly suppressed the in vitro proliferation of Teffs. However, the C-cleaved or N&C-cleaved Foxp3 forms afforded almost no suppressive function, indicating a crucial role of the human Foxp3 C-terminal region in Tregs suppressive activity, in marked contrast with the report of a superior suppressive activity for the C-cleaved murine Foxp3 compared to the full length.

Th1 and Th17 cytokines drive inflammation in Takayasu arteritis.

OBJECTIVE: Takayasu arteritis (TAK) is a large-vessel vasculitis that induces damage to the aorta and its branches. Glucocorticoids remain the gold standard of therapy for TAK. The nature of the T cells driving vascular inflammation and the effects of glucocorticoids on the systemic components of TAK are not understood. The aim of this study was to analyze T cell homeostasis and cytokine production in peripheral blood and inflammatory lesions of the aorta in patients with TAK. METHODS: T cell homeostasis and cytokine production in peripheral blood and inflammatory lesions of the aorta were analyzed using Luminex analysis, flow cytometry, and immunohistochemical analysis. The study included 41 patients fulfilling the American College of Rheumatology 1990 criteria for the classification of TAK (17 patients with active TAK and 24 patients with disease in remission), 30 patients with giant cell arteritis and 39 patients with Behçet's disease (disease controls), and 20 age- and sex-matched healthy control subjects. RESULTS: We observed a marked increase in the expression of Th1 and Th17 cells, which correlated with TAK disease activity. The addition of serum from patients with active TAK to sorted CD4+ T cells from healthy donors in culture medium induced significant production of interferon-γ (IFNγ) and interleukin-17A (IL-17A). We demonstrated the presence of IFNγ-, IL-6-, and IL-17A-producing T cells in vascular inflammatory infiltrates in patients with TAK. Corticosteroid therapy was associated with decreased levels of circulating Th1 cytokines in corticosteroid-treated patients with TAK compared with steroid-free patients with TAK (for IL-2, mean ± SD 5,079 ± 5,300 versus 7,359 ± 3,197 pg/ml; for IFNγ, 2,592 ± 3,072 versus 8,393 ± 3,392 pg/ml; for tumor necrosis factor α, 847 ± 724 versus 1,491 ± 392 pg/ml). However, glucocorticoids had essentially no effect on the frequency of Th17 cytokines (IL-1 receptor, IL-17, and IL-23). CONCLUSION: The Th17 and Th1 pathways contribute to the systemic and vascular manifestations of TAK. Glucocorticoid treatment suppresses Th1 cytokines but spares Th17 cytokines in patients with TAK.

Expansion of autoreactive unresponsive CD21-/low B cells in Sjögren's syndrome-associated lymphoproliferation.

OBJECTIVE: Primary Sjögren's syndrome (SS) is an autoimmune disease associated with a high risk of developing non-Hodgkin's lymphoma. This study was undertaken to determine the nature of B cells driving lymphoproliferation in primary SS. METHODS: B cell subsets and function were analyzed in peripheral blood from 66 adult patients with primary SS (including 14 patients with B cell lymphoproliferative disease [LPD]) and 30 healthy donors, using flow cytometry, calcium mobilization, and gene array analysis. The reactivity of recombinant antibodies isolated from single B cells from patients with primary SS and LPD was tested using an enzyme-linked immunosorbent assay. RESULTS: We observed an expansion of an unusual CD21-/low B cell population that correlated with lymphoproliferation in patients with primary SS. A majority of CD21-/low B cells from patients with primary SS expressed autoreactive antibodies, which recognized nuclear and cytoplasmic structures. These B cells belonged to the memory compartment, since their Ig genes were mutated. They were unable to induce calcium flux, become activated, or proliferate in response to B cell receptor and/or CD40 triggering, suggesting that these autoreactive B cells may be anergic. However, CD21-/low B cells from patients with primary SS remained responsive to Toll-like receptor (TLR) stimulation. Molecules specifically expressed in CD21-/low B cells that are likely to induce their unresponsive stage were detected in gene array analyses. CONCLUSION: Patients with primary SS who display high frequencies of autoreactive and unresponsive CD21-/low B cells are susceptible to developing lymphoproliferation. These cells remain in peripheral blood controlled by functional anergy instead of being eliminated, and chronic antigenic stimulation through TLR stimulation may create a favorable environment for breaking tolerance and activating these cells.

Down-regulation of CD81 tetraspanin in human cells producing retroviral-based particles: tailoring vector composition.

Retroviral-derived biopharmaceuticals (RV) target numerous therapeutic applications, from gene therapy to virus-like particle (rVLP)-based vaccines. During particle formation, beside the pseudotyped envelope proteins, RV can incorporate proteins derived from the virus producer cells (VPC). This may be detrimental by reducing the amounts of the pseudotyped envelope and/or by incorporating protein capable of inducing immune responses when non-human VPC are used. Manipulating the repertoire of VPC proteins integrated onto the vector structure is an underexplored territory and should provide valuable insights on potential targets to improve vector pharmacokinetic and pharmacodynamic properties. In this work, human HEK 293 cells producing retrovirus-like particles (rVLPs) and infectious RV vectors were used to prove the concept of customizing RV composition by manipulating cellular protein content. The tetraspanin CD81 was chosen since it is significantly incorporated in the RV membrane, conferring to the vector significant immunogenicity when used in mice. RNA interference-mediated by shRNA lentiviral vector transduction was efficiently used to silence CD81 expression (up to 99%) and the rVLPs produced by knocked-down cells lack CD81. Silenced clones were analyzed for cell proliferation, morphological changes, susceptibility to oxidative stress conditions, and rVLP productivities. The results showed that the down-regulation of VPC proteins requires close monitoring for possible side effects on cellular production performance. Yet, they confirm that it is possible to change the composition of host-derived immunogens in RV by altering cellular protein content with no detriment for vector productivity and titers. This constitutes an important manipulation tool in vaccinology--by exploiting the potential adjuvant effect of VPC proteins or using them as fusion agents to other proteins of interest to be exposed on the vector membrane--and in gene therapy, by reducing the immunogenicity of RV-based vector and enhancing in vivo half-life. Such tools can also be applied to lentiviral or other enveloped viral vectors.

Seasonal H1N1 2007 influenza virus infection is associated with elevated pre-exposure antibody titers to the 2009 pandemic influenza A (H1N1) virus.

The new influenza strain detected in humans in April 2009 has caused the first influenza pandemic of the 21st century. A cross-reactive antibody response, in which antibodies against seasonal H1N1 viruses neutralized the 2009 pandemic influenza A (H1N1) virus (2009 pH1N1), was detected among individuals aged >60 years. However, factors other than age associated with such a cross-reactive antibody response are poorly documented. Our objective was to examine factors potentially associated with elevated pre-exposure viro-neutralization and hemagglutination-inhibition antibody titers against the 2009 pH1N1. We also studied factors associated with antibody titers against the 2007 seasonal H1N1 virus. One hundred subjects participating in an influenza cohort were selected. Sera collected in 2008 were analysed using hemagglutination inhibition and viro-neutralization assays for the 2009 pH1N1 virus and the 2007 seasonal H1N1 virus. Viro-neutralization results were explored using a linear mixed-effect model and hemagglutination-inhibition results using linear-regression models for interval-censored data. Elevated antibody titers against 2009 pH1N1 were associated with seasonal 2007 H1N1 infection (viro-neutralization, p 0.006; hemagglutination-inhibition, p 0.018). Elevated antibody titers were also associated with age in the viro-neutralization assay (p <0.0001). Seasonal 2007 H1N1 infection is an independent predictor of elevated pre-exposure antibody titers against 2009 pH1N1 and may have contributed to lowering the burden of the 2009 pH1N1 pandemic.

Relevance of an academic GMP Pan-European vector infra-structure (PEVI).

In the past 5 years, European investigators have played a major role in the development of clinical gene therapy. The provision of substantial funds by some individual member states to construct GMP facilities makes it an opportune time to network available gene therapy GMP facilities at an EU level. The integrated coordination of GMP production facilities and human skills for advanced gene and genetically-modified (GM) cell therapy, can dramatically enhance academic-led "First-in-man" gene therapy trials. Once proof of efficacy is gathered, technology can be transferred to the private sector which will take over further development taking advantage of knowledge and know-how. Complex technical challenges require existing production facilities to adapt to emerging technologies in a coordinated manner. These include a mandatory requirement for the highest quality of production translating gene-transfer technologies with pharmaceutical-grade GMP processes to the clinic. A consensus has emerged on the directions and priorities to adopt, applying to advanced technologies with improved efficacy and safety profiles, in particular AAV, lentivirus-based and oncolytic vectors. Translating cutting-edge research into "First-in-man" trials require that pre-normative research is conducted which aims to develop standard assays, processes and candidate reference materials. This research will help harmonise practices and quality in the production of GMP vector lots and GM-cells. In gathering critical expertise in Europe and establish conditions for interoperability, the PEVI infrastructure will contribute to the demands of the advanced therapy medicinal products* regulation and to both health and quality of life of EU-citizens.

Antigen quality determines the efficiency of antitumor immune responses generated in the absence of regulatory T cells.

The observation that depletion or inhibition of regulatory T cells (Tregs) unleashes efficient antitumor effector immune responses that can lead to tumor eradication in mice has opened new perspectives for the development of cancer immunotherapy. The quality and overall efficiency of the effector immune responses induced in the absence of Tregs seem to depend on multiple factors that determine the result of a battle involving effector T cells (Teffs), Tregs and tumor cells. In this study, we investigated the quality of tumor-associated antigens (TAAs) as one such factor. We show that the presence of a strong dominant antigen is required for the induction of effector responses capable of tumor eradication in the absence of Tregs. The sole addition of a dominant antigen on tumor cells does not change tumor growth in unmanipulated mice, but improves tumor eradication rate from a few to almost 100% in the absence of Tregs. This eradication can be shown to result from the recruitment and activation of specific Teffs recognizing this antigen. We also show that the presence of such dominant antigens has the side effect of restricting the breadth of the immune response to other TAAs, which could favor the generation of escape mutant by tumor editing. Taken together, our results highlight the potential, and some requirements for cancer immunotherapy based on Treg depletion. They also show that, ultimately, tumor fate depends on multiple factors that should all be taken into consideration for the design of more efficient immunotherapy.

Identification of CD8+CD25+Foxp3+ suppressive T cells in colorectal cancer tissue.

BACKGROUND: The antitumoral immune response is one determinant of colorectal cancer (CRC) outcome. Recent work suggests that Foxp3(+)CD25(+)CD4(+) regulatory T cells (T4reg) might hamper effective immunosurveillance of emerging cancer cells and impede effective immune responses to established tumours. In this descriptive study, we analysed blood and tissue regulatory T cell populations in patients with CRC. METHODS: Blood and tissue regulatory Foxp3(+) T cells from 40 patients with CRC were compared to regulatory Foxp3(+) T cells from normal colonic tissue and from blood of 26 healthy volunteers. Flow cytometry was used to quantify and phenotype all Foxp3(+) T cell populations. Correlations were sought with the tumour stage and with micro-invasive status. The suppressive capacity of regulatory Foxp3(+) T cells was assessed by their effect on CD4(+)CD25(-) T cell proliferation in vitro and by their capacity to inhibit cytokine production by conventional T cells. RESULTS: We found a significant increase of CD8(+)CD25(+)Foxp3(+) cells (T8reg) in blood and CRC tissue; their phenotype was close to that of T4reg. T8reg cells infiltrating CRC were activated, as suggested by increased cytoxic T lymphocyte-associated antigen-4, glucocorticoid-induced tumour necrosis factor-related protein, and transforming growth factor (TGF)beta1 expression compared to T8reg from normal autologous colonic tissue. Moreover, T8reg were able to suppress CD4(+)CD25(-) T cell proliferation and Th1 cytokine production ex vivo, demonstrating that tumour-infiltrating T8reg have strong suppressive capacities. T8reg numbers correlated with the tumour stage and with micro-invasive status. Finally, interleukin 6 and TGF beta 1 synergistically induced the generation of CD8(+)CD25(+)Foxp3(+) T cells ex vivo. CONCLUSIONS: We have identified a new regulatory T cell population (CD8(+)Foxp3(+)) in colorectal tumours. After isolation from cancer tissue these CD8(+)Foxp3(+) cells demonstrated strong immunosuppressive properties in vitro. These data suggest that these cells may contribute to tumoral immune escape and disease progression.

The B lymphocyte stimulator receptor-ligand system in hepatitis C virus-induced B cell clonal disorders.

OBJECTIVE: The study aim was to examine the B lymphocyte stimulator (BLyS) receptor-ligand system in hepatitis C virus (HCV)-induced B lymphocyte clonal disorders. METHODS: 94 patients with chronic HCV (including 35 with HCV+ mixed cryoglobulinaemia (MC)-vasculitis and nine with HCV+ B cell non-Hodgkin's lymphoma (B-NHL)) and 15 healthy volunteers were included. RESULTS: A twofold serum BLyS increase was associated with HCV-induced MC-vasculitis, and a threefold increase with HCV-induced B-NHL, compared with patients that were HCV+, but without vasculitis, or healthy controls (p<0.05). Lower membrane BLyS expression in HCV-induced MC-vasculitis was observed. CD19+ BLyS binding and BLyS receptor 3 (BR3) staining showed a stepwise decrease with highest values in healthy controls and who were HCV+ without MC, and lowest in B-NHL (p<0.05, p<0.0001, respectively) with a further decrease in VH1-69+ clonal B cells. BLyS anti-apoptotic effects were maintained despite this decrease in BR3 staining. Complete clinical remission after antiviral treatment was associated with a decrease in serum BLyS, and an increase in BR3 staining. Rituximab treatment was associated with a fivefold increase in serum BLyS (p<0.001), mirroring the depletion of CD19+ cells. BR3 staining in repopulating B cells was significantly decreased (p<0.005). CONCLUSIONS: The BLyS ligand-receptor activity is increased in HCV-induced B cell clonal disorders, indicating a possible role for treatment targeting the BLyS receptor-ligand system.

[Neoretinal antigen expression: a comparison of anatomical and clinical features of a murine uveoretinitis model]. / Expressions oculaires d'un néoantigène rétinien: confrontation anatomoclinique dans un nouveau modèle murin d'uvéorétinite.

PURPOSE: Uveitis is an inflammation involving the retina. The antigens targeted by the experimental models are located in the pigmentary epithelium-photoreceptor complex. To gain insights into the variations in topographic expression of the antigen in the retina, we studied a new mouse model. MATERIAL: and methods: Stable retinal expression of the influenza virus hemagglutinin (HA) was obtained after intravitreal or subretinal injection of recombinant adeno-associated virus carrying HA (AAV-HA). One month later, we transferred HA-specific T cells, followed by a subcutaneous immunization of the cognate antigen emulsified in CFA. The animals were clinically examined with a slit lamp biomicroscope. Infiltration of donor cells was detected by immunostaining on retina flatmounts with anti-Thy-1.1 antibody, and infiltrating cells were studied using FACS analysis. RESULTS: Whatever the location of the HA expression, intraocular inflammation was clinically and histologically detected in all animals, between 10 and 15 days after immunization with HA. Lesions were identified with histopathological analysis. The ocular infiltrate was mostly composed of macrophages and HA-specific T cells in different proportions. CONCLUSIONS: The topographic variations of targeted ocular antigens do not seem to modify the development of inflammatory reactions in our model. By targeting different antigen-presenting cells, ocular infiltrating cells are different.

Transient depletion of dividing T lymphocytes in mice induces the emergence of regulatory T cells and dominant tolerance to islet allografts.

We previously showed that transient depletion of dividing T cells at the time of an allogeneic transplantation induces long-term tolerance to the allograft. Here we investigated the role of homeostatic perturbation and regulatory T cells (Treg) in such tolerance. Transient depletion of dividing T cells was induced at the time of an allogeneic pancreatic islets graft, by administration of ganciclovir for 14 days, into diabetic transgenic mice expressing a thymidine kinase (TK) conditional suicide gene in T cells. Allograft tolerance was obtained in 63% of treated mice. It was not due to global immunosuppression, permanent deletion or anergy of donor-alloantigens specific T cells but to a dominant tolerance process since lymphocytes from tolerant mice could transfer tolerance to naïve allografted recipients. The transient depletion of dividing T cells induces a 2- to 3-fold increase in the proportion of CD4(+)CD25(+)Foxp3(+) Treg, within 3 weeks that persisted only in allograft-bearing mice but not in nongrafted mice. Tolerance with similar increased proportion of Treg cells was also obtained after a cytostatic hydroxyurea treatment in normal mice. Thus, the transient depletion of dividing T cells represents a novel means of immuno-intervention based on disturbance of T-cell homeostasis and subsequent increase in Treg proportion.

T cell repertoire in patients with stable scleroderma.

At onset of systemic sclerosis (SSc), T cells have been found to oligoclonally expand in the skin, presumably in response to auto-antigens, but the T cell repertoire has not been evaluated at a later stage. To determine whether a perpetuating immune response contributes to the pathogenesis of stable SSc, the T cell repertoire was analysed in patients with diffuse (d) or limited (l) SSc, and compared to patients with primary Raynaud's phenomenon (RP) or healthy volunteers (Ctrl). The T cell repertoire (total, CD4 or CD8 sorted blood T cells) was analysed by qualitative and quantitative immunoscope (14 BV families analysed) in 11 untreated dSSc and 11 untreated lSSc, 10 RP and 11 Ctrl. To better detect in vivo activated cells, repertoire analysis was also performed on sorted CD4 T cells after in vitro culture with IL-2. In parallel, 6 skin biopsies from SSc patients were analysed. After 7-8 years of disease evolution, SSc patients did not show detectable clonal T cell expansions in the skin, even after tentative expansion from the biopsy with IL-2. Total T cell, sorted CD4 and CD8 T cell repertoires from the blood of patients with SSc did not show significant perturbation as compared to patients with RP and Ctrl. After IL-2 culture for 7 days, blood CD4 T cells from the patients did not preferentially expand as compared to RP and Ctrl. These findings suggest that antigen-driven immune responses may play a lesser role in established SSc than at disease onset.

Long-term survival after gene therapy for a recurrent glioblastoma.

A patient presenting with a recurrent glioblastoma (GBM) survived 3 years after suicide gene therapy and finally died of a disseminated breast cancer with no indication of tumor recurrence on MRI. Postmortem analysis showed no evidence of recurrence of the GBM, neither near the initial tumor localization nor in any other area of the brain. Such an evolution is unusual in the course of this disease and may suggest in this particular case a cure of the GBM.