Ageing promotes early T follicular helper cell differentiation by modulating expression of RBPJ.

Ageing profoundly changes our immune system and is thought to be a driving factor in the morbidity and mortality associated with infectious disease in older people. We have previously shown that the impaired immunity to vaccination that occurs in aged individuals is partly attributed to the effect of age on T follicular helper (Tfh) cell formation. In this study, we examined how age intrinsically affects Tfh cell formation in both mice and humans. We show increased formation of Tfh precursors (pre-Tfh) but no associated increase in germinal centre (GC)-Tfh cells in aged mice, suggesting age-driven promotion of only early Tfh cell differentiation. Mechanistically, we show that ageing alters TCR signalling which drives expression of the Notch-associated transcription factor, RBPJ. Genetic or chemical modulation of RBPJ or Notch rescues this age-associated early Tfh cell differentiation, and increased intrinsic Notch activity recapitulates this phenomenon in younger mice. Our data offer mechanistic insight into the age-induced changes in T-cell activation that affects the differentiation and ultimately the function of effector T cells

GIMAP6 is required for T cell maintenance and efficient autophagy in mice

The GTPases of the immunity-associated proteins (GIMAP) GTPases are a family of proteins expressed strongly in the adaptive immune system. We have previously reported that in human cells one member of this family, GIMAP6, interacts with the ATG8 family member GABARAPL2, and is recruited to autophagosomes upon starvation, suggesting a role for GIMAP6 in the autophagic process. To study this possibility and the function of GIMAP6 in the immune system, we have established a mouse line in which the Gimap6 gene can be inactivated by Cre-mediated recombination. In mice bred to carry the CD2Cre transgene such that the Gimap6 gene was deleted within the T and B cell lineages there was a 50-70% reduction in peripheral CD4(+) and CD8(+) T cells. Analysis of splenocyte-derived proteins from these mice indicated increased levels of MAP1LC3B, particularly the lipidated LC3-II form, and S405-phosphorylation of SQSTM1. Electron microscopic measurements of Gimap6(-/-) CD4(+) T cells indicated an increased mitochondrial/cytoplasmic volume ratio and increased numbers of autophagosomes. These results are consistent with autophagic disruption in the cells. However, Gimap6(-/-) T cells were largely normal in character, could be effectively activated in vitro and supported T cell-dependent antibody production. Treatment in vitro of CD4(+) splenocytes from GIMAP6(fl/fl) ERT2Cre mice with 4-hydroxytamoxifen resulted in the disappearance of GIMAP6 within five days. In parallel, increased phosphorylation of SQSTM1 and TBK1 was observed. These results indicate a requirement for GIMAP6 in the maintenance of a normal peripheral adaptive immune system and a significant role for the protein in normal autophagic processes. Moreover, as GIMAP6 is expressed in a cell-selective manner, this indicates the potential existence of a cell-restricted mode of autophagic regulation

Integrative analyses of normal and neoplastic B cell differentiation at the single-cell level

Dans le centre germinatif (GC) les lymphocytes B (LB) activés suivent une dynamique fine aboutissant à la différenciation en LB mémoires ou en plasmocytes (PC) à longue durée de vie. La différenciation en PC est cruciale pour une protection immunitaire hautement spécifique et durable, mais les facteurs régulant ce processus sont mal connus. Le lymphome folliculaire (FL) est issu d’une transformation maligne de LB du GC. Il a récemment été démontré que les LB du FL ne sont pas bloqués à un stade GC, suggérant leur plasticité. Une hétérogénéité intra-patient des LB du FL a également été décrite mais la dynamique fonctionnelle de ces cellules reste toujours inconnue. L’hétérogénéité fonctionnelle et la diversité clonale des LB normaux et néoplasiques rend cruciale l’utilisation de méthodes à l’échelle de la cellule unique. Nous avons donc développé le FB5P-seq, une méthode de RNA-seq intégrant le phénotype, le transcriptome et la séquence du récepteur B de chaque cellule unique. Chez des patients FL, nos analyses suggèrent que les LB néoplasiques sont plastiques et détournent le processus physiologique de différenciation du GC en formant un continuum d’états transitionnels.L’étude de la différenciation en PC a fait l’objet du développement d’outils originaux pour l’obtention de données préliminaires. Chez l’Homme nous avons étudié les signatures d’activation des LB du GC in vitro. Chez la souris, nous avons développé un modèle pour cartographier le destin in vivo des LB du GC, notamment lors de leur différenciation en PC.L’ensemble de ces travaux apporte une vision nouvelle sur la dynamique du FL ainsi que des méthodes novatrices pour étudier la différenciation en PC.High-affinity antibodies mostly arise from germinal centers (GC). Within the GC, activated B cells follow tightly regulated dynamics resulting in their differentiation into memory B cells or long-lived plasma cells (PC). PC differentiation is crucial for highly specific long-term immune protection. Yet, the mechanisms underlying the regulation of this process are still poorly understood. Follicular lymphoma (FL) originates from GC-experienced B cells. It was recently shown that FL B cells are not blocked in a GC B cell state, suggesting their plasticity. It was also described that FL B cells display intratumoral heterogeneity. However, FL B cell dynamics remain unknown. The functional and clonal diversity in B cells make single-cell analyses crucial to study the biological and neoplastic processes involving these cells. We therefore developed FB5P-seq, a single-cell RNA-seq method for integrative analyses of gene expression, surface phenotype and B cell receptor sequence. In FL patients, our single-cell analyses revealed that FL B cells hijack the physiological GC differentiation process through a continuum of transitional cell states sustained by extrinsic signals delivered by tumor infiltrating T cells. The study of PC differentiation process led us to develop innovative experimental tools which we used to produce preliminary data. In humans, we studied gene expression signatures triggered by in vitro activation of GC B cells. In mice, we developed an in vivo model to map the fate of GC B cells, particularly their differentiation into PC. Altogether, our data shed new light on FL B cell dynamics and provide original approaches to study PC differentiation

Heterogeneity of germinal center B cells: New insights from single‐cell studies

International audienc

FB5P-seq: FACS-Based 5-Prime End Single-Cell RNA-seq for Integrative Analysis of Transcriptome and Antigen Receptor Repertoire in B and T Cells

International audienceSingle-cell RNA sequencing (scRNA-seq) allows the identification, characterization, and quantification of cell types in a tissue. When focused on B and T cells of the adaptive immune system, scRNA-seq carries the potential to track the clonal lineage of each analyzed cell through the unique rearranged sequence of its antigen receptor (BCR or TCR, respectively) and link it to the functional state inferred from transcriptome analysis. Here we introduce FB5P-seq, a FACS-based 5′-end scRNA-seq method for cost-effective, integrative analysis of transcriptome and paired BCR or TCR repertoire in phenotypically defined B and T cell subsets. We describe in detail the experimental workflow and provide a robust bioinformatics pipeline for computing gene count matricesand reconstructing repertoire sequences from FB5P-seq data. We further present two applications of FB5P-seq for the analysis of human tonsil B cell subsets and peripheral blood antigen-specific CD4T cells. We believe th at our novel integrative scRNA-seq method will be a valuable option to study rare adap tive immune cell subsets in immunology research

Single-Cell and Spatial Analyses Characterize Distinct Subsets of Malignant T Cells in Angioimmunoblastic T Cell Lymphoma

International audienceAbstract Introduction Angioimmunoblastic T-cell lymphoma (AITL) is the most frequent of nodal peripheral T-cell lymphomas. AITL results from the transformation of T follicular helper (T FH) cells and is characterized by chemo-resistance and poor survival (5-year OS around 30%). Recent data from prospective clinical trials suggest that disease outcome may be impacted by factors other than genomic features, such as the tumor microenvironment (TME) and overall intra-tumoral heterogeneity. Our understanding of AITL intra-tumoral genetic, transcriptional and functional heterogeneity is limited because most molecular data generated so far have come from bulk analyses. Single-cell RNA sequencing (scRNA-seq) enables fine characterization of cell types and functional cell states. When focused on T or B cells, 5'-end scRNA-seq also yields the TCR or BCR sequences that allow tracking clonally related cells. Here we studied the intra-tumor heterogeneity of AITL tumors using integrative scRNA-seq. Methods We analyzed lymph node live cell suspensions from AITL patients (n=10) using droplet-based 10x Genomics 5'-end scRNA-seq. Malignant T cells from 4 AITL samples were also analyzed by FACS index sorting and plate-based 5'-end scRNA-seq to link cell surface phenotype and gene expression profile. We identified malignant T cell clones by intersecting the gene expression and TCR sequencing data, and performed separate focused analyses of TME subsets and malignant T cells. We compared subsets of malignant T cells from all patients using marker gene-based metaclustering to identify AITL T cell states conserved across patients. We explored the genetic heterogeneity of malignant T cells by mapping RHOA G17V mutations and inferring copy number variation (CNV) subclones from scRNA-seq data. In select cases, we performed in situ analysis by immunohistochemistry (IHC) or spatial transcriptomics to characterize the spatial distribution of malignant T cell subsets identified by scRNA-seq. Results Based on gene expression, malignant T cells grouped in patient-specific clusters, while non-malignant T, B and myeloid TME cells from all patients clustered by cell type or cell state. Among TME cells, we identified 7 subsets of B cells (including activated B cells, plasma cells, and one patient-specific monoclonal B cell proliferation), 6 subsets of myeloid cells (including macrophages, conventional and plasmacytoid dendritic cells), and 8 subsets of non-malignant T cells (including activated cytotoxic T lymphocytes (CTL) with clonal expansions). Patient-specific malignant T cells were heterogeneous and divided into several gene-expression based clusters. Metaclustering of malignant T cell subsets identified T central memory (T CM)-like and T FH-like states in 10/10 samples. We also identified in 3/10 samples clusters of CTL-like malignant T cells expressing characteristic marker genes (including NKG7, GNLY, GZMK, PRF1). We observed an intra-sample continuum of gene expression states from quiescent T CM-like to proliferating T FH-like states. T FH-like cells were larger in size and expressed higher levels of surface PD1 and ICOS than T CM-like and CTL-like subsets. We detected the RHOA G17V mutation in malignant T cells of 4/4 mutated cases, with no evidence of subclonal heterogeneity for that mutation. We detected clonal and subclonal CNV in most AITL malignant T cells. CTL-like states were enriched in specific CNV subclones, but the T CM-like to T FH-like continuum was observed in all CNV subclones, suggesting that functional plasticity and subclonal genetic evolution may occur independently. In situ staining of markers for T FH-like (PD1, ICOS, CD200) and CTL-like (GZMK, GZMA) cells showed that T FH-like and CTL-like cells occupied distinct tissue niches within the tumor. In spatial transcriptomics analysis, T FH-like cells mapped to follicular dendritic cell (FDC)-rich areas, while T CM-like cells were associated with T-zone reticular cells. Conclusions Our analyses recapitulate known characteristics of AITL TME, and uncover previously unrecognized heterogeneity among malignant T cells across multiple patients. The distinct gene expression programs, phenotypes, genetics, and locations of T FH-like, T CM-like and CTL-like states suggest that AITL malignant T cells undergo significant functional plasticity and genetic divergence, which could influence response to therapy and overall clinical course. Figure 1 Figure 1. Disclosures Lemonnier: Institut Roche: Research Funding; Gilead: Other: travel grant. Gaulard: Gilead: Consultancy; Innate Pharma: Research Funding; Sanofi: Research Funding; Alderaan: Research Funding; Takeda: Consultancy, Honoraria. Milpied: Institut Roche: Research Funding; Innate Pharma: Research Funding; Bristol Myers Squibb: Research Funding

Effect on the expression of selected proteins of Gimap6 ablation in vitro from GIMAP6^fl/flERT2Cre CD4⁺ T cells.

<p>Panel A) Enriched CD4⁺ cell fractions were prepared from spleens of ERT2Cre or GIMAP6^fl/flERT2Cre mice. The cells were maintained in vitro for five days in the presence or absence of 200 nM 4HT. Cell lysates were prepared and analysed by SDS polyacrylamide gel electrophoresis with the indicated antibodies. Panel B) Enriched CD4⁺ cell fractions were prepared from spleens of GIMAP6^fl/flERT2Cre mice. The cells were maintained in vitro in the presence or absence of 4HT for the indicated times before preparation of cell lysates and western analysis with the indicated antibodies. The electrophoretic mobilities of marker proteins are indicated. Panel C) Enriched CD4⁺ cell fractions were prepared and incubated with or without 200 nM 4HT for 4 days. TBK1 inhibitor BX795 (1 μM) was then added to the cells as indicated and incubation continued for 6h. Cell lysates were prepared and proteins analysed by western blotting. In all panels, the electrophoretic mobility of marker proteins is indicated. All results were confirmed in at least two experiments.</p

The reduced number of peripheral CD4⁺ T cells in GIMAP6^fl/flCD2Cre mice reflects an increase in apoptosis.

<p>A) Splenocytes isolated from GIMAP6^fl/fl and GIMAP6^fl/flCD2Cre animals were stained with fluorescent antibodies to CD4 and CD24. The left-hand panel shows the distribution of CD24 staining on CD4⁺ cells from single mice of the two genotypes (GIMAP6^fl/fl–dotted line; GIMAP6^fl/flCD2Cre–solid line). The right-hand panel shows the median fluorescence intensity of CD24 staining of CD4⁺ cells. n = 4; **P<0.01. B) Freshly isolated splenocytes were stained with an APC-conjugated antibody to CD4 and PE- conjugated annexin V. The cells were then washed and stained with DAPI before flow analysis. The two dot-plots show typical staining patterns for a control (C) and knockout (KO) animal. The quadrants are as follows: Q1 –annexin V^- DAPI⁺; Q2 –annexin V⁺ DAPI⁺; Q3 –annexin V⁺ DAPI^-; Q4 –annexin V^- DAPI^-. The right-hand panel summarises the percentage of annexin V⁺ cells as a percentage of total DAPI^- CD4⁺ T cells for five animals in each group. ns P>0.05; ** P<0.01. C) CD4⁺ enriched naive splenocytes from GIMAP6^fl/fl and GIMAP6^fl/flCD2Cre mice were activated by maintenance on anti-CD3 antibody-coated plates in medium containing anti-CD28 and IL2 or were maintained in medium without activation as described in the Materials and Methods section. After 24 h cells were harvested and stained as in (B). The percentage of CD4⁺ T cells in each quadrant is indicated. n = 3 for each group; ns P>0.05; ** P<0.01.</p

GIMAP6 relocalises to autophagosomes on cell starvation.

<p>HEK293 cells stably expressing an N-terminally myc-tagged variant of mouse GIMAP6 were either left untreated or were treated with starvation buffer for 90 minutes. They were then fixed and stained with either (A) a mixture of rat monoclonal antibodies to mouse GIMAP6 and a rabbit polyclonal antibody to MAP1LC3B, or (B) the same mixture of rat monoclonal antibodies to GIMAP6 and a rabbit polyclonal antibody to SQSTM1, each followed by fluorochrome-conjugated secondary antibodies. GIMAP6 is shown false-coloured green and MAP1LC3B or SQSTM1, red. Scale bar represents 10 μm.</p

Activation of CD4⁺ splenocytes from GIMAP6^fl/flCD2Cre mice is comparable with those from GIMAP6^fl/fl mice.

<p>A) CD4⁺ enriched naïve splenocytes from GIMAP6^fl/fl and GIMAP6^fl/flCD2Cre mice were activated by maintenance on anti-CD3 antibody-coated plates in medium containing anti-CD28 and IL2 or were maintained in medium without activation. After 72h, cells were stained to measure expression of CD25, CD62L and CD44 to assess the degree of activation. On the histograms dotted lines indicate naïve cells and solid lines, activated cells. B) The median fluorescence intensities of the individual stains from three mice in each group is presented graphically. Unpaired 2-tailed Student’s t-test P values ns p>0.05 ***p<0.001; ****p<0.0001. The experiment was performed twice.</p