Evaluation of treatment response in adults with relapsing MOG-Ab-associated disease

Background: Myelin oligodendrocyte glycoprotein antibodies (MOG-Ab) are related to several acquired demyelinating syndromes in adults, but the therapeutic approach is currently unclear. We aimed to describe the response to different therapeutic strategies in adult patients with relapsing MOG-Ab-associated disease. Methods: This is a retrospective study conducted in France and Spain including 125 relapsing MOG-Ab patients aged ≥ 18 years. First, we performed a survival analysis to investigate the relapse risk between treated and non-treated patients, performing a propensity score method based on the inverse probability of treatment weighting. Second, we assessed the annualised relapse rates (ARR), Expanded Disability Status Scale (EDSS) and visual acuity pre-treatment and on/end-treatment. Results: Median age at onset was 34.1 years (range 18.0-67.1), the female to male ratio was 1.2:1, and 96% were Caucasian. At 5 years, 84% (95% confidence interval [CI], 77.1-89.8) patients relapsed. At the last follow-up, 66 (52.8%) received maintenance therapy. Patients initiating immunosuppressants (azathioprine, mycophenolate mophetil [MMF], rituximab) were at lower risk of new relapse in comparison to non-treated patients (HR, 0.41; 95CI%, 0.20-0.82; p = 0.011). Mean ARR (standard deviation) was reduced from 1.05(1.20) to 0.43(0.79) with azathioprine (n = 11; p = 0.041), from 1.20(1.11) to 0.23(0.60) with MMF (n = 11; p = 0.033), and from 1.08(0.98) to 0.43(0.89) with rituximab (n = 26; p = 0.012). Other immunosuppressants (methotrexate/mitoxantrone/cyclophosphamide; n = 5), or multiple sclerosis disease-modifying drugs (MS-DMD; n = 9), were not associated with significantly reduced ARR. Higher rates of freedom of EDSS progression were observed with azathioprine, MMF or rituximab. Conclusion: In adults with relapsing MOG-Ab-associated disease, immunosuppressant therapy (azathioprine, MMF and rituximab) is associated with reduced risk of relapse and better disability outcomes. Such an effect was not found in the few patients treated with MS-DMD

DMTs and Covid-19 severity in MS: a pooled analysis from Italy and France

We evaluated the effect of DMTs on Covid-19 severity in patients with MS, with a pooled-analysis of two large cohorts from Italy and France. The association of baseline characteristics and DMTs with Covid-19 severity was assessed by multivariate ordinal-logistic models and pooled by a fixed-effect meta-analysis. 1066 patients with MS from Italy and 721 from France were included. In the multivariate model, anti-CD20 therapies were significantly associated (OR = 2.05, 95%CI = 1.39–3.02, p < 0.001) with Covid-19 severity, whereas interferon indicated a decreased risk (OR = 0.42, 95%CI = 0.18–0.99, p = 0.047). This pooled-analysis confirms an increased risk of severe Covid-19 in patients on anti-CD20 therapies and supports the protective role of interferon

Diminution de l'alloréactivité de cellules T cultivées ex vivo (étude in vitro des mécanismes et effet sur la reconstitution immunitaire in vivo dans un modèle de greffe allogénique de cellules souches hématopoïétiques)

Une modulation de l'alloréactivité des cellules T présentes dans un greffon de cellules souches hématopoïétiques (CSH) peut être obtenue par transfert par voie rétrovirale d'un gène "suicide" dans les lymphocytes T (LT) du donneur. Le transfert de gène nécessite notamment une période de 12 jours de culture ex vivo de cellules mononucléées (CMN) activées par des anticorps (Acs) CD3 solubles en présence d'Interleukine (IL)-2 qui est en partie responsable d'une diminution de l'alloréactivité des cellules cultivées (appelées Co). Les hypothèses d'induction d'anergie, de délétion des cellules alloréactives au cours de l'expansion ex vivo ou de mort induite par activation au cours de la reconnaissance de l'allo-antigène, testées pour expliquer cette diminution d'alloréactivité, ont été invalidées (résultats non publiés). L'augmentation d'expression de marqueurs des cellules T régulatrices au cours de l'expansion ex vivo nous a incités à tester l'hypothèse selon laquelle une expansion préférentielle des cellules T régulatrices pourrait être à l'origine de la diminution d'alloréactivité des Co. Les résultats obtenus n'ont pas permis de mettre en évidence une activité suppressive liés à la présence de cellules T régulatrices (Montcuquet N et al. Immunology 2008) et suggèrent que la diminution d'alloréactivité résulte d'un épuisement fonctionnel des LT. - Dans un second temps nous avons cherché à améliorer les conditions de culture afin de maintenir cette alloréactivité. L'activation des CMN a été réalisée par des Acs soluble CD3 ou par des Acs CD3 et CD28 co-immobilisés sur billes, puis les cellules ont été cultivées en présence d'IL-2, d'IL-7 ou d'IL-15. Nous avons observé que le fait de remplacer la stimulation CD3 par une co-stimulation CD3/CD28 conduit à une alloréactivité similaire in vitro mais augmente la prolifération cellulaire et l'alloréactivité testée in vivo dans un modèle xénogénique de réaction du greffon contre l'hôte (GvH). Par ailleurs, le fait de remplacer l'IL-2 par l'IL-7 mais pas par l'IL-15, ou de diminuer les concentrations d'IL-2 et d'IL-7 utilisées lors de la culture, améliore l'alloréactivité des Co, au détriment cependant d'une expansion cellulaire plus faible (Mercier-Letondal P et al. Cytotherapy 2008). Ces résultats posent les bases de futurs protocoles de production de cellules T génétiquement modifiées conservant leur alloréactivité en vue d'obtenir un effet anti-leucémique maximal en situation d'allogreffe hématopoïétique. - La culture ex vivo de CMN entraîne non seulement une diminution d'alloréactivité, mais aussi des modifications phénotypiques se traduisant par l'acquisition par les Co d'un phénotype de LT mémoires. Or, les LT mémoires ont une alloréactivité plus faible que des LT naïfs en termes d'induction de GvH mais sont capables d'accélérer la reconstitution immunitaire. Nous avons pu montrer que, de façon similaire, des LT cultivés issus de splénocytes murins étaient capables d'améliorer la reconstitution immunitaire au même titre que des LT mémoires frais dans un modèle murin d'allogreffe de CSH par un effet indépendant de l'alloréactivité. - Ces résultats posent les bases d'un possible développement des LT cultivés comme nouvel outil de thérapie cellulaire permettant d'accélérer la reconstitution immunitaire dans des contextes de greffe où celle-ci est lente (greffe de sang de cordon, greffe haplo-identique...).We have demonstrated previously that retroviral-mediated transfer of a suicide gene into bone marrow (BM) donor T cells allows an efficient control of graft-versus-host disease (GvHD) after allogeneic BM transplantation. However, 12 days of ex vivo culture is required for the production of sufficient gene-modified cells (GMC). This process requires both CD3 monoclonal antibody (MAb) activation and interleukin-2 (IL-2) expansion resulting in a diminution of expanded cells alloreactivity (termed Co). This phenomena is independent of anergy, clonaI deletion during expansion or apoptosis inducing cell death during mixed lymphocyte reaction. - Here, we demonstrate that this phenomena is the result of T-cell functional exhaustion and not due to anypreferential expansion of regulatory T cells in the culture (Montcuquet N et al. Immunology 2008). - Our approach was then to try to improve the ex vivo culture conditions in order to maintain alloreactivity. Peripheral blood mononuclear cells were activated with soluble CD3 MAb or CD3 and CD28 MAb co-immobilized on beads and expanded for 12 days in the presence of IL-2, IL- 7 or IL-15 before analysis of alloreactivity and phenotype. Replacing the CD3 MAb by CD3/CD28 beads led to similar in vitro alloreactivity but improved also expansion and in vivo alloreactivity of GMC. Replacing the IL-2 with IL- 7, but not IL-15, or decreasing IL-2 or IL- 7 concentrations, improved the in vitro alloreactivity of expanded cells but with lower expansion. Indeed, the alloreactivity of expanded cells was negatively correlated with cell expansion and positively correlated with CD4/CD8 ratio and CD8 expression levels (Mercier-Letondal P, Montcuquet N et al. Cytotherapy 2008). - ln a mouse model of Graft-versus-host Disease (GvHD) induction, memory T-cells are less allogeneic than naïve T cells. Memory T-cells also improve immune reconstitution. We examined the potential of expanded T-cells to improve immune reconstitution in the absence of GvHD. Indeed, expanded splenocytes can have the same effects as fresh memory T-cells. - Taken together these results should be useful in designing GMC therapy protocols where alloreactivity is maintained and co-administrated expanded T-cells offer a new cell therapy product.BESANCON-BU Médecine pharmacie (250562102) / SudocSudocFranceF

Complementary Roles of Nod2 in Hematopoietic and Nonhematopoietic Cells in Preventing Gut Barrier Dysfunction Dependent on MLCK Activity

Background: Crohn's disease (CD) pathogenesis is multifactorial involving genetic and environmental factors. Loss of function mutations in the nucleotide oligomerization domain 2 (NOD2) gene are the main genetic risk factor for CD. Like patients with CD, Nod2(KO) mice are characterized by an enhanced Th1 immune response and a defective mucosal barrier function evidenced by increased intestinal permeability. We previously showed that the latter is related to hematopoietic Nod2 deficiency. Our aim was to explore the mechanisms by which Nod2 expressed in the hematopoietic and in the nonhematopoietic compartments interplay to control epithelial paracellular permeability. Methods: Depletion of CD4(+) T cells in Nod2(KO) mice and treatments with inhibitors were conducted in chimeric mice transplanted with bone marrow cells from Nod2-deficient donors into Nod2-sufficient recipients or vice versa. Caco-2 cells overexpressing a NOD2 gene which did or did not include a CD-associated polymorphism were treated with inhibitors or siRNAs and cocultured with hematopoietic cells from Peyer's patches. Results: In vivo and in vitro Nod2 in hematopoietic cells regulates epithelial paracellular permeability through cytokine production influencing myosin light chain kinase (MLCK) activity. Indeed, tumor necrosis factor-a and interferon-g secretion by CD4(+) T cells upregulated expression and activity of epithelial MLCK leading to increased epithelial tight junction opening. When stimulated by muramyl dipeptide, Nod2 in the nonhematopoietic compartment normalized the permeability and T-cell cytokine secretion and regulated MLCK activity. This MLCK regulation is mediated by TAK1 and RICK-dependent mechanisms. Conclusions: Our study demonstrates how hematopoietic and nonhematopoietic Nod2 regulate intestinal barrier function, improving our knowledge on the mechanisms involved in CD pathogenesis

Beyond 40 fluorescent probes for deep phenotyping of blood mononuclear cells, using spectral technology

International audienceThe analytical capability of flow cytometry is crucial for differentiating the growing number of cell subsets found in human blood. This is important for accurate immunophenotyping of patients with few cells and a large number of parameters to monitor. Here, we present a 43-parameter panel to analyze peripheral blood mononuclear cells from healthy individuals using 41 fluorescence-labelled monoclonal antibodies, an autofluorescent channel, and a viability dye. We demonstrate minimal population distortions that lead to optimized population identification and reproducible results. We have applied an advanced approach in panel design, in selection of sample acquisition parameters and in data analysis. Appropriate autofluorescence identification and integration in the unmixing matrix, allowed for resolution of unspecific signals and increased dimensionality. Addition of one laser without assigned fluorochrome resulted in decreased fluorescence spill over and improved discrimination of cell subsets. It also increased the staining index when autofluorescence was integrated in the matrix. We conclude that spectral flow cytometry is a highly valuable tool for high-end immunophenotyping, and that fine-tuning of major experimental steps is key for taking advantage of its full capacity

CD3+ CD20+ cells may be an artifact of flow cytometry.

International audienc

Alloreactivity of ex vivo-expanded T cells is correlated with expansion and CD4/CD8 ratio.

International audienceBackground We have demonstrated previously that retroviral-mediated transfer of a suicide gene into bone marrow (BM) donor T cells allows an efficient control of graft-versus-host disease (GvHD) after allogeneic BM transplantation. However, the 12 days of ex vivo culture required for the production of gene-modified cells (GMC), including soluble CD3 monoclonal antibody (MAb)-mediated activation and expansion with interleukin (IL)-2, induced a decrease of GMC alloreactivity and a reversal of their CD4/CD8 ratio. Improving the culture protocol in order to maintain the highest alloreactivity is of critical importance in obtaining an optimal graft-versus-leukemia (GvL) effect. Methods Peripheral blood mononuclear cells were activated with soluble CD3 MAb or CD3 and CD28 MAb co-immobilized on beads and expanded for 12 days in the presence of IL-2, IL-7 or IL-15 before analysis of alloreactivity and phenotype. Results Replacing the CD3 MAb by CD3/CD28 beads led to similar in vitro alloreactivity but improved the expansion and in vivo alloreactivity of GMC. Replacing the IL-2 with IL-7, but not IL-15, or decreasing IL-2 or IL-7 concentrations, improved the in vitro alloreactivity of expanded cells but was associated with lower expansion. Indeed, the alloreactivity of expanded cells was negatively correlated with cell expansion and positively correlated with CD4/CD8 ratio and CD8 expression level. Discussion Quantitative (i.e. low CD4/CD8 ratio) and qualitative (e.g. low CD8 expression) defects may account for the decreased alloreactivity of GMC. Using CD3/CD28 beads and/or IL-7 is more beneficial than CD3 MAb and IL-2 for preventing perturbations of the alloreactivity and phenotype of GMC

DataSheet_1_Beyond 40 fluorescent probes for deep phenotyping of blood mononuclear cells, using spectral technology.pdf

The analytical capability of flow cytometry is crucial for differentiating the growing number of cell subsets found in human blood. This is important for accurate immunophenotyping of patients with few cells and a large number of parameters to monitor. Here, we present a 43-parameter panel to analyze peripheral blood mononuclear cells from healthy individuals using 41 fluorescence-labelled monoclonal antibodies, an autofluorescent channel, and a viability dye. We demonstrate minimal population distortions that lead to optimized population identification and reproducible results. We have applied an advanced approach in panel design, in selection of sample acquisition parameters and in data analysis. Appropriate autofluorescence identification and integration in the unmixing matrix, allowed for resolution of unspecific signals and increased dimensionality. Addition of one laser without assigned fluorochrome resulted in decreased fluorescence spill over and improved discrimination of cell subsets. It also increased the staining index when autofluorescence was integrated in the matrix. We conclude that spectral flow cytometry is a highly valuable tool for high-end immunophenotyping, and that fine-tuning of major experimental steps is key for taking advantage of its full capacity.</p

DataSheet_2_Beyond 40 fluorescent probes for deep phenotyping of blood mononuclear cells, using spectral technology.pdf

The analytical capability of flow cytometry is crucial for differentiating the growing number of cell subsets found in human blood. This is important for accurate immunophenotyping of patients with few cells and a large number of parameters to monitor. Here, we present a 43-parameter panel to analyze peripheral blood mononuclear cells from healthy individuals using 41 fluorescence-labelled monoclonal antibodies, an autofluorescent channel, and a viability dye. We demonstrate minimal population distortions that lead to optimized population identification and reproducible results. We have applied an advanced approach in panel design, in selection of sample acquisition parameters and in data analysis. Appropriate autofluorescence identification and integration in the unmixing matrix, allowed for resolution of unspecific signals and increased dimensionality. Addition of one laser without assigned fluorochrome resulted in decreased fluorescence spill over and improved discrimination of cell subsets. It also increased the staining index when autofluorescence was integrated in the matrix. We conclude that spectral flow cytometry is a highly valuable tool for high-end immunophenotyping, and that fine-tuning of major experimental steps is key for taking advantage of its full capacity.</p

Transcriptome of retrovirally transduced CD8+ lymphocytes: influence of cell activation, transgene integration, and selection process.

International audienceA suicide gene introduced by retroviral means can allow in vivo control of alloreactivity mediated by donor gene-modified T cells (GMTC) after allogeneic hematopoietic stem cell transplantation. The present study establishes the transcriptomic profile of GMTC prepared according to the GMTC production process used in our clinical trial (activation/selection methods, CD3/NeoR), which was previously demonstrated to induce phenotypical and functional alterations. This transcriptomic profile was compared with that of GMTC prepared by a novel process (CD3-CD28/DeltaNGFR-MACS) that limits alterations. Using a human pan-genomic microarray and GeneSpring software, we determined the gene expression profiles of CD8+ T cells from four healthy donors before and after the different steps required for gene modification. This analysis revealed that the gene expression pattern of GMTC is affected mainly by the activation step. Specific analysis of GMTC production processes showed that DeltaNGFR-MACS selection combined with CD3-CD28 activation limits the aberrant expression of genes involved in immunological functions and apoptotic pathways. Furthermore, our results indicate a limited risk of oncogenesis associated with retroviral-mediated gene transfer in CD8+ cells, a lower perturbation of the cell cycle regulation pathway after CD3-CD28 activation than after CD3 activation, and no significant involvement of the DeltaNGFR transduction signaling pathway when DeltaNGFR is used for selection. Moreover, genes that might be targeted to limit T cell functional alterations after ex vivo manipulation and culture were identified. These findings should be relevant to further adoptive T cell immunotherapy trials using ex vivo-expanded, gene-modified or unmodified T cells