Deciphering the maturation of tertiary lymphoid structures in cancer and inflammatory diseases of the digestive tract using imaging mass cytometry.

Persistent inflammation can promote the development of tertiary lymphoid structures (TLS) within tissues resembling secondary lymphoid organs (SLO) such as lymph nodes (LN). The composition of TLS across different organs and diseases could be of pathophysiological and medical interest. In this work, we compared TLS to SLO in cancers of the digestive tract and in inflammatory bowel diseases. Colorectal and gastric tissues with different inflammatory diseases and cancers from the department of pathology of CHU Brest were analyzed based on 39 markers using imaging mass cytometry (IMC). Unsupervised and supervised clustering analyses of IMC images were used to compare SLO and TLS. Unsupervised analyses tended to group TLS per patient but not per disease. Supervised analyses of IMC images revealed that LN had a more organized structure than TLS and non-encapsulated SLO Peyer's patches. TLS followed a maturation spectrum with close correlations between germinal center (GC) markers' evolution. The correlations between organizational and functional markers made relevant the previously proposed TLS division into three stages: lymphoid-aggregates (LA) (CD20+CD21-CD23-) had neither organization nor GC functionality, non-GC TLS (CD20+CD21+CD23-) were organized but lacked GC's functionality and GC-like TLS (CD20+CD21+CD23+) had GC's organization and functionality. This architectural and functional maturation grading of TLS pointed to differences across diseases. TLS architectural and functional maturation grading is accessible with few markers allowing future diagnostic, prognostic, and predictive studies on the value of TLS grading, quantification and location within pathological tissues in cancers and inflammatory diseases.

Identification of altered cell signaling pathways using proteomic profiling in stable and progressive chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is characterized by significant biologic and clinical heterogeneity. This study was designed to explore CLL B-cells' proteomic profile in order to identify biologic processes affected at an early stage and during disease evolution as stable or progressive. Purified B cells from 11 untreated CLL patients were tested at two time points by liquid chromatography-tandem mass spectrometry. Patients included in the study evolved to either progressive (n = 6) or stable disease (n = 5). First, at an early stage of the disease (Binet stage A), based on the relative abundance levels of 389 differentially expressed proteins (DEPs), samples were separated into stable and progressive clusters with the main differentiating factor being the RNA splicing pathway. Next, in order to test how the DEPs affect RNA splicing, a RNA-Seq study was conducted showing 4217 differentially spliced genes between the two clusters. Distinct longitudinal evolutions were observed with predominantly proteomic modifications in the stable CLL group and spliced genes in the progressive CLL group. Splicing events were shown to be six times more frequent in the progressive CLL group. The main aberrant biologic processes controlled by DEPs and spliced genes in the progressive group were cytoskeletal organization, Wnt/ß-catenin signaling, and mitochondrial and inositol phosphate metabolism with a downstream impact on CLL B-cell survival and migration. This study suggests that proteomic profiles at the early stage of CLL can discriminate progressive from stable disease and that RNA splicing dysregulation underlies CLL evolution, which opens new perspectives in terms of biomarkers and therapy.

The regulatory capacity of B cells directs the aggressiveness of CLL.

Chronic lymphocytic leukemia (CLL) is associated with abnormal T-cell responses responsible for defective anti-tumor activities. Intriguingly, CLL B cells share phenotypical characteristics with regulatory B (Breg) cells suggesting that they might negatively control the T-cell activation and immune responses. We elaborated an in vitro co-culture system with T cells to evaluate the Breg capacities of CLL B cells following innate Toll-like receptor 9 (TLR9) engagement. We demonstrated that B cells from half of the patients exhibited regulatory capacities, whilst B cells from the remaining patients were unable to develop a Breg function. The T cell sensitivities of all patients were normal suggesting that defective Breg activities were due to intrinsic CLL B cell deficiencies. Thus, TLR-dedicated gene assays highlighted differential signature of the TLR9 negative regulation pathway between the two groups of patients. Furthermore, correlations of the doubling time of lymphocytosis, the time to first treatment, the mutational status of IgVH and the Breg functions indicate that patients with efficient Breg activities have more aggressive CLL than patients with defective Breg cells. Our in vitro observations may open new approaches for adjusting therapeutic strategies targeting the Breg along with the evolution of the disease.

Combining cytogenetic and epigenetic approaches in chronic lymphocytic leukemia improves prognosis prediction for patients with isolated 13q deletion.

Background: Both defective DNA methylation and active DNA demethylation processes are emerging as important risk factors in chronic lymphocytic leukemia (CLL). However, associations between 5-cytosine epigenetic markers and the most frequent chromosomal abnormalities detected in CLL remain to be established. Methods: CLL patients were retrospectively classified into a cytogenetic low-risk group (isolated 13q deletion), an intermediate-risk group (normal karyotype or trisomy 12), and a high-risk group (11q deletion, 17p deletion, or complex karyotype [≥ 3 breakpoints]). The two 5-cytosine derivatives, 5-methylcytosine (5-mCyt) and 5-hydroxymethylcytosine (5-hmCyt), were tested by ELISA (n = 60), while real-time quantitative PCR was used for determining transcriptional expression levels of DNMT and TET (n = 24). Results: By using global DNA methylation/demethylation levels, in the low-risk disease group, two subgroups with significantly different clinical outcomes have been identified (median treatment-free survival [TFS] 45 versus > 120 months for 5-mCyt, p = 0.0008, and 63 versus > 120 months for 5-hmCyt, p = 0.04). A defective 5-mCyt status was further associated with a higher percentage of 13q deleted nuclei (> 80%), thus suggesting an acquired process. When considering the cytogenetic intermediate/high-risk disease groups, an association of 5-mCyt status with lymphocytosis (p = 0.0008) and the lymphocyte doubling time (p = 0.04) but not with TFS was observed, as well as a reduction of DNMT3A, TET1, and TET2 transcripts. Conclusions: Combining cytogenetic studies with 5-mCyt assessment adds accuracy to CLL patients' prognoses and particularly for those with 13q deletion as a sole cytogenetic abnormality.

Alterations in DNA methylation/demethylation intermediates predict clinical outcome in chronic lymphocytic leukemia.

Cytosine derivative dysregulations represent important epigenetic modifications whose impact on the clinical outcome in chronic lymphocytic leukemia (CLL) is incompletely understood. Hence, global levels of 5-methylcytosine (5-mCyt), 5-hydroxymethylcytosine (5-hmCyt), 5-carboxylcytosine (5-CaCyt) and 5-hydroxymethyluracil were tested in purified B cells from CLL patients (n = 55) and controls (n = 17). The DNA methylation 'writers' (DNA methyltransferases [DNMT1/3A/3B]), 'readers' (methyl-CpG-binding domain [MBD2/4]), 'editors' (ten-eleven translocation [TET1/2/3]) and 'modulators' (SAT1) were also evaluated. Accordingly, patients were stratified into three subgroups. First, a subgroup with a global deficit in cytosine derivatives characterized by hyperlymphocytosis, reduced median progression free survival (PFS = 52 months) and shorter treatment free survival (TFS = 112 months) was identified. In this subgroup, major epigenetic modifications were highlighted including a reduction of 5-mCyt, 5-hmCyt, 5-CaCyt associated with DNMT3A, MBD2/4 and TET1/2 downregulation. Second, the cytosine derivative analysis revealed a subgroup with a partial deficit (PFS = 84, TFS = 120 months), mainly affecting DNA demethylation (5-hmCyt reduction, SAT1 induction). Third, a subgroup epigenetically similar to controls was identified (PFS and TFS > 120 months). The prognostic impact of stratifying CLL patients within three epigenetic subgroups was confirmed in a validation cohort. In conclusion, our results suggest that dysregulations of cytosine derivative regulators represent major events acquired during CLL progression and are independent from IGHV mutational status.

Cell-specific epigenome-wide DNA methylation profile in long-term cultured minor salivary gland epithelial cells from patients with Sjögren's syndrome.

OBJECTIVES: The aetiology of primary Sjögren's syndrome (pSS), also referred to as autoimmune epithelitis, is incompletely understood but includes an epigenetic contribution. Accordingly, the aim of this study was to investigate DNA methylation in salivary gland epithelial cells (SGEC), and to compare results with those publicly available from pSS B and T cells. METHODS: Long-term cultured SGEC were selected to conduct an epigenome-wide association study (EWAS) in patients with pSS with comparison to controls using the HumanMethylation 450 K array from Illumina. RESULTS: The analysis of differentially methylated CpG (DMC) uncovered 4662 positions corresponding to 2560 genes, and 575 genes with two or more DMC sites (DMCs), in SGEC as compared with controls. Further analysis highlighted an important proportion of interferon-regulated genes (61%), the calcium pathway (hypomethylated) and the Wnt pathway (hypermethylated). When comparing SGEC with pSS T and/or B cell results, an important overlap was observed with respect to differentially methylated genes (38.8%) and pSS risk factors (71.4%), although such assertion was not true when comparing DMCs. CONCLUSIONS: This study conducted in SGEC emphasises the role of DNA methylation in pSS pathogenesis and supports the necessity to conduct pure cell analysis for future EWAS studies when analysing salivary glands from patients with pSS.

Human endogenous retrovirus group E and its involvement in diseases.

Human endogenous retrovirus group E (HERV-E) elements are stably integrated into the human genome, transmitted vertically in a Mendelian manner, and are endowed with transcriptional activity as alternative promoters or enhancers. Such effects are under the control of the proviral long terminal repeats (LTR) that are organized into three HERV-E phylogenetic subgroups, namely LTR2, LTR2B, and LTR2C. Moreover, HERV-E expression is tissue-specific, and silenced by epigenetic constraints that may be disrupted in cancer, autoimmunity, and human placentation. Interest in HERV-E with regard to these conditions has been stimulated further by concerns regarding the capacity of HERV-E elements to modify the expression of neighboring genes and/or to produce retroviral proteins, including immunosuppressive env peptides, which in turn may induce (auto)-antibody (Ab) production. Finally, better understanding of HERV-E elements may have clinical applications for prevention, diagnosis, prognosis, and therapy.

DNA methylation modulates HRES1/p28 expression in B cells from patients with Lupus.

Systemic lupus erythematosus (SLE) disease is an autoimmune disease of unknown aetiology that affects predominantly women of child bearing age. Since previous studies, including ours, have demonstrated that CD4+ T cells and B cells from SLE patients are defective in their ability to methylate their DNA upon antigen stimulation, the aim of this study was to investigate whether DNA demethylation affects the transcription of HRES-1 in B cells. HRES-1 is the prototype of Human Endogenous Retrovirus (HERV) overexpressed in SLE. We have observed that SLE B cells were characterized by their incapacity to methylate the HRES-1 promoter, both in unstimulated and in anti-IgM stimulated B cells. In turn, HRES-1/p28 expression was increased in SLE B cells after B cell receptor engagement, but not in controls. In SLE B cells the Erk/DNMT1 pathway was defective. In addition, blocking the autocrine-loop of IL-6 in SLE B cells with an anti-IL-6 receptor monoclonal antibody restores DNA methylation and control of HRES-1/p28 expression became effective. As a consequence, a better understanding of HERV dysregulation in SLE reinforces our comprehension of the disease and opens new therapeutic perspectives.

Rationale for treating primary Sjögren's syndrome patients with an anti-CD6 monoclonal antibody (Itolizumab).

CD6 is a cell surface receptor expressed on the majority of T cells and a subset of B cells. When expressed, CD6 contributes to lymphocyte activation through its extracellular domain 1, while adhesion and cellular migration are related to the extracellular scavenger receptor cysteine-rich domain (SRCR-D)-3 of CD6. Itolizumab, clone T1h, is a newly developed humanized IgG1 monoclonal antibody that targets CD6 SRCR-D1 and blocks immune activation. Itolizumab has been proposed to be effective in autoimmune diseases such as rheumatoid arthritis, Sjögren's syndrome and multiple sclerosis. In Sjögren's syndrome, the utilization of itolizumab as therapeutic option is reinforced by our recent observation that ALCAM, the CD6 ligand, is overexpressed and that CD6-positive T and B cells are detected within salivary glands from Sjögren's syndrome patients. In this study, itolizumab-positive target cells were characterized within both peripheral blood and salivary glands in order to provide rational for anti-CD6 treatment in Sjögren's syndrome.

Epigenetic dysregulation in salivary glands from patients with primary Sjögren's syndrome may be ascribed to infiltrating B cells.

Sjögren's syndrome (SS) is an autoimmune exocrinopathy characterized by an epithelium injury with dense lymphocytic infiltrates, mainly composed of activated T and B cells. Present at the interface of genetic and environmental risk factors, DNA methylation is suspected to play a key role in SS. To clarify this point, global DNA methylation was tested within salivary gland epithelial cells (SGEC), peripheral T cells and B cells from SS patients. Global DNA methylation was reduced in SGEC from SS patients, while no difference was observed in T and B cells. SGEC demethylation in SS patients was associated with a 7-fold decrease in DNA methyl transferase (DNMT) 1 and a 2-fold increase in Gadd45-alpha expression. The other DNA methylation/demethylation partners, tested by real time PCR (DNMT3a/b, PCNA, UHRF1, MBD2, and MBD4), were not different. Interestingly, SGEC demethylation may be attributed in part to the infiltrating B cells as suspected in patients treated with anti-CD20 antibodies to deplete B cells. Such hypothesis was confirmed using co-culture experiments with human salivary gland cells and B cells. Furthermore, B cell-mediated DNA demethylation could be ascribed to an alteration of the PKC delta/ERK/DNMT1 pathway. As a consequence, part of the SGEC dysfunction in SS may be linked to epigenetic modifications, thus opening new therapeutic perspectives in SS.

Epigenetics and Sjögren's syndrome.

There is growing evidence that epigenetics, the study of heritable changes in gene expression that do not involve mutations in the DNA itself, may play an essential role in autoimmune diseases (AID). In Sjögren's syndrome (SS), a chronic AID characterized by an epithelis of the exocrine glands, epigenetic studies have focused on three mechanisms: DNA methylation and its consequences including human endogenous retrovirus (HERV) expression; microRNA expression; and protein post-translational modifications associated with autoantibody production. Although in its infancy, comprehension of the epigenetic (dys)regulation in SS may help us to understand: why SS affects predominantly middle-aged women; why genetically predisposed individuals develop SS but not others; why flare-ups occur; why treatment responses differ between patients; and why some patients develop lymphoma. From these studies will arise a better comprehension of the pathophysiology of SS as well as development of new diagnostic and prognostic biomarkers, and novel therapeutics for prevention and perhaps early intervention.

Aberrant expression of CD6 on B-cell subsets from patients with Sjögren's syndrome.

CD6 is one of a pair of related genes encoding CD5-associated receptors on all T cells and a subset of B cells. The current availability of "T1h", a humanized anti-CD6 monoclonal antibody for B cell-mediated autoimmune disorders revives analysis of the B-cell subset expression of CD6, particularly in primary Sjögren's syndrome (SS). Refined phenotype of B-lymphocytes peripheral blood (PB), bone marrow and tonsils revealed that the overlap between the expression of CD6 is less close to that of CD5 than currently acknowledged. In contrast to CD5, CD6 is absent on transitional B cells, while present on mature and memory B cells. Interestingly, the PB proportion of CD6(+) B cells is decreased in patients with primary SS, as opposed to those with rheumatoid arthritis. The reduction in primary SS does not result from the shedding of CD6 from the membrane of B cells, but from the lowering of memory B lymphocytes. It may result from the ability of CD6 to make transmigration of CD27(+) memory B cells into the salivary glands (SGs) easier. Consistent with this view is our finding that CD166 (one of the ligands for CD6) is highly expressed on epithelial cells of patients' SGs. This study is relevant in that the humanized T1h anti-CD6 becomes an alternative to anti-CD20 for treatment of primary SS.

IL-10 production by B cells expressing CD5 with the alternative exon 1B.

B lymphocytes are divided into two subpopulations, B1 and B2 cells based on expression of the T cell-associated protein CD5. Natural B1 cells are further divided into B1a cells that express CD5 on their membrane and B1b cells that do not but share most other biological characteristics of B1a cells. Recent studies from our laboratory have revealed, in humans, the existence of two alternative isoforms of the CD5 protein. A cell surface CD5 isoform which uses exon 1A (E1A) of the gene in B1a cells, and an intracellular isoform which uses exon 1B (E1B) mainly in human B1b cells. Indeed, the protein isoform encoded by transcripts containing E1B lack the leader peptide and is, thus, retained in the cytoplasm of B cells. The restriction of interleukin (IL)-10 to B1 lymphocytes in the mouse raises the possibility that the human CD5-E1B-expressing B cells produce IL-10. This prediction was confirmed in the CD5 negative Jok-1 B cells transfected with cDNA for either isoforms resulted in high level IL-10 production. Our data indicate that E1B-CD5-expressing B cells have the capacity to interfere with the immune response through their ability to produce high levels of IL-10.

IL-6 modulates CD5 expression in B cells from patients with lupus by regulating DNA methylation.

B lymphocytes from patients with systemic lupus erythematosus (SLE) are characterized by reduced expression levels of membrane CD5. Recent studies from our laboratory have revealed that the level of membrane CD5 is determined by the relative level of two alternative CD5 isoforms; CD5-E1A, which is expressed on the membrane, and CD5-E1B, which is retained in the cytoplasm. Using bisulfite sequencing and methylation-sensitive endonuclease assays we show that the promoter for the alternative CD5-E1B isoform is demethylated in B cells from patients with SLE but not in healthy controls. We go on to show that differential methylation is more pronounced following BCR engagement. As a result of this demethylation, CD5-E1B mRNA is transcribed at the expense of CD5-E1A mRNA transcription. We provide further evidence that production of high IL-6 levels by SLE B cells abrogates the ability of SLE B cells to induce DNA methyl transferase (DNMT1) and then to methylate DNA, an effect that is reversed in the presence of a blocking Ab to the IL-6 receptor. The pattern of demethylation of CpG islands in the CD5-E1B promoter in SLE B cells is similar to those in B cells from healthy controls stimulated in the presence of IL-6, or treated with the methylation inhibitor PD98059. The study reveals that engagement of the BCR with constitutive IL-6 down-regulates the level of membrane CD5, which negatively regulates BCR signaling, in SLE B cells. This altered signaling could, in turn, promote the activation and expansion of autoreactive B cells in SLE patients.