Follicular lymphoma regulatory T-cell origin and function.

Introduction: Follicular Lymphoma (FL) results from the malignant transformation of germinal center (GC) B cells. FL B cells display recurrent and diverse genetic alterations, some of them favoring their direct interaction with their cell microenvironment, including follicular helper T cells (Tfh). Although FL-Tfh key role is well-documented, the impact of their regulatory counterpart, the follicular regulatory T cell (Tfr) compartment, is still sparse. Methods: The aim of this study was to characterize FL-Tfr phenotype by cytometry, gene expression profile, FL-Tfr origin by transcriptomic analysis, and functionality by in vitro assays. Results: CD4+CXCR5+CD25hiICOS+ FL-Tfr displayed a regulatory program that is close to classical regulatory T cell (Treg) program, at the transcriptomic and methylome levels. Accordingly, Tfr imprinting stigmata were found on FL-Tfh and FL-B cells, compared to their physiological counterparts. In addition, FL-Tfr co-culture with autologous FL-Tfh or cytotoxic FL-CD8+ T cells inhibited their proliferation in vitro. Finally, although FL-Tfr shared many characteristics with Treg, TCR sequencing analyses demonstrated that part of them derived from precursors shared with FL-Tfh. Discussion: Altogether, these findings uncover the role and origin of a Tfr subset in FL niche and may be useful for lymphomagenesis knowledge and therapeutic management.

Innovative cellular therapies for autoimmune diseases: expert-based position statement and clinical practice recommendations from the EBMT practice harmonization and guidelines committee.

Autoimmune diseases (ADs) are characterized by loss of immune tolerance, high chronicity, with substantial morbidity and mortality, despite conventional immunosuppression (IS) or targeted disease modifying therapies (DMTs), which usually require repeated administration. Recently, novel cellular therapies (CT), including mesenchymal stromal cells (MSC), Chimeric Antigen Receptors T cells (CART) and regulatory T cells (Tregs), have been successfully adopted in ADs. An international expert panel of the European Society for Blood and Marrow Transplantation and the International Society for the Cell and Gene Therapy, reviewed all available evidence, based on the current literature and expert practices, on use of MSC, CART and Tregs, in AD patients with rheumatological, neurological, and gastroenterological indications. Expert-based consensus and recommendations for best practice and quality of patient care were developed to support clinicians, scientists, and their multidisciplinary teams, as well as patients and care providers and will be regularly updated.

Cell cross talk within the lymphoma tumor microenvironment: follicular lymphoma as a paradigm.

ABSTRACT: Follicular lymphoma (FL) is an indolent yet incurable germinal center B-cell lymphoma retaining a characteristic follicular architecture. FL tumor B cells are highly dependent on direct and indirect interactions with a specific and complex tumor microenvironment (TME). Recently, great progress has been made in describing the heterogeneity and dynamics of the FL TME and in depicting how tumor clonal and functional heterogeneity rely on the integration of TME-related signals. Specifically, the FL TME is enriched for exhausted cytotoxic T cells, immunosuppressive regulatory T cells of various origins, and follicular helper T cells overexpressing B-cell and TME reprogramming factors. FL stromal cells have also emerged as crucial determinants of tumor growth and remodeling, with a key role in the deregulation of chemokines and extracellular matrix composition. Finally, tumor-associated macrophages play a dual function, contributing to FL cell phagocytosis and FL cell survival through long-lasting B-cell receptor activation. The resulting tumor-permissive niches show additional layers of site-to-site and kinetic heterogeneity, which raise questions about the niche of FL-committed precursor cells supporting early lymphomagenesis, clonal evolution, relapse, and transformation. In turn, FL B-cell genetic and nongenetic determinants drive the reprogramming of FL immune and stromal TME. Therefore, offering a functional picture of the dynamic cross talk between FL cells and TME holds the promise of identifying the mechanisms of therapy resistance, stratifying patients, and developing new therapeutic approaches capable of eradicating FL disease in its different ecosystems.

Modeling the crosstalk between malignant B cells and their microenvironment in B-cell lymphomas: challenges and opportunities.

B-cell lymphomas are a group of heterogeneous neoplasms resulting from the clonal expansion of mature B cells arrested at various stages of differentiation. Specifically, two lymphoma subtypes arise from germinal centers (GCs), namely follicular lymphoma (FL) and GC B-cell diffuse large B-cell lymphoma (GCB-DLBCL). In addition to recent advances in describing the genetic landscape of FL and GCB-DLBCL, tumor microenvironment (TME) has progressively emerged as a central determinant of early lymphomagenesis, subclonal evolution, and late progression/transformation. The lymphoma-supportive niche integrates a dynamic and coordinated network of immune and stromal cells defining microarchitecture and mechanical constraints and regulating tumor cell migration, survival, proliferation, and immune escape. Several questions are still unsolved regarding the interplay between lymphoma B cells and their TME, including the mechanisms supporting these bidirectional interactions, the impact of the kinetic and spatial heterogeneity of the tumor niche on B-cell heterogeneity, and how individual genetic alterations can trigger both B-cell intrinsic and B-cell extrinsic signals driving the reprogramming of non-malignant cells. Finally, it is not clear whether these interactions might promote resistance to treatment or, conversely, offer valuable therapeutic opportunities. A major challenge in addressing these questions is the lack of relevant models integrating tumor cells with specific genetic hits, non-malignant cells with adequate functional properties and organization, extracellular matrix, and biomechanical forces. We propose here an overview of the 3D in vitro models, xenograft approaches, and genetically-engineered mouse models recently developed to study GC B-cell lymphomas with a specific focus on the pros and cons of each strategy in understanding B-cell lymphomagenesis and evaluating new therapeutic strategies.

Axicabtagene ciloleucel as second-line therapy in large B cell lymphoma ineligible for autologous stem cell transplantation: a phase 2 trial.

Axicabtagene ciloleucel (axi-cel) demonstrated superior efficacy compared to standard of care as second-line therapy in patients with high-risk relapsed/refractory (R/R) large B cell lymphoma (LBCL) considered eligible for autologous stem cell transplantation (ASCT); however, in clinical practice, roughly half of patients with R/R LBCL are deemed unsuitable candidates for ASCT. The efficacy of axi-cel remains to be ascertained in transplant-ineligible patients. ALYCANTE, an open-label, phase 2 study, evaluated axi-cel as a second-line therapy in 62 patients with R/R LBCL who were considered ineligible for ASCT. The primary end point was investigator-assessed complete metabolic response at 3 months from the axi-cel infusion. Key secondary end points included progression-free survival, overall survival and safety. The study met its primary end point with a complete metabolic response of 71.0% (95% confidence interval, 58.1-81.8%) at 3 months. With a median follow-up of 12.0 months (range, 2.1-17.9), median progression-free survival was 11.8 months (95% confidence interval, 8.4-not reached) and overall survival was not reached. There was no unexpected toxicity. Grade 3-4 cytokine release syndrome and neurologic events occurred in 8.1% and 14.5% of patients, respectively. These results support axi-cel as second-line therapy in patients with R/R LBCL ineligible for ASCT. ClinicalTrials.gov Identifier: NCT04531046 .

Phenotypic and proteomic analysis of plasma extracellular vesicles highlights them as potential biomarkers of primary Sjögren syndrome.

Sjögren syndrome (SjS) is an autoimmune disease characterized by the destruction of the exocrine gland epithelia, causing a dryness of mucosa called sicca symptoms, and whose main life-threatening complication is lymphoma. There is a need for new biomarkers in this disease, notably diagnostic biomarkers for patients with genuine sicca symptoms that do not meet current criteria, and prognostic biomarkers for patients at risk of lymphoma. Plasma extracellular vesicles (EVs) are promising biomarker candidates in several diseases, but their potential has not yet been explored in SjS. In this proof-of-concept study, we characterized EVs from primary SjS patients (pSS, n=12) at the phenotypic and proteomic levels, compared to EVs from healthy donor (HD, n=8) and systemic lupus erythematosus patients (SLE, n=12). Specific plasma EVs subpopulations, derived from neutrophils, endothelial, and epithelial cells, were found increased in pSS. We also identified a pSS proteomic signature in plasma EVs, including neutrophil-, epithelial-, and endothelial-related proteins, such as integrin alpha M (ITGAM), olfactomedin-4 (OLFM4), Ras-related protein RAB10, and CD36. Overall, our results support the relevance of plasma EVs as biomarkers in SjS.

An International Society for Cell and Gene Therapy Mesenchymal Stromal Cells (MSC) Committee perspectives on International Standards Organization/Technical Committee 276 Biobanking Standards for bone marrow-MSCs and umbilical cord tissue-derived MSCs for research purposes.

The rapidly growing field of mesenchymal stromal cell (MSC) basic and translational research requires standardization of terminology and functional characterization. The International Standards Organization's (ISO) Technical Committee (TC) on Biotechnology, working with extensive input from the International Society for Cells and Gene Therapy (ISCT), has recently published ISO standardization documents that are focused on biobanking of MSCs from two tissue sources, Wharton's Jelly, MSC(WJ) and Bone Marrow, MSC(M)), for research and development purposes and development. This manuscript explains the path towards the consensus on the following two documents: the Technical Standard ISO/TS 22859 for MSC(WJ) and the full ISO Standard 24651 for MSC(M) biobanking. The ISO standardization documents are aligned with ISCT's MSC committee position and recommendations on nomenclature because there was active input and incorporation of ISCT MSC committee recommendations in the development of these standards. The ISO standardization documents contain both requirements and recommendations for functional characterization of MSC(WJ) and MSC(M) using a matrix of assays. Importantly, the ISO standardization documents have a carefully defined scope and are meant for research use of culture expanded MSC(WJ) and MSC(M). The ISO standardization documents can be updated in a revision process and will be systematically reviewed after 3-5 years as scientific insights grow. They represent international consensus on MSC identity, definition, and characterization; are rigorous in detailing multivariate characterization of MSCs and represent an evolving-but-important first step in standardization of MSC biobanking and characterization for research use and development.

Functional specialization of short-lived and long-lived macrophage subsets in human tonsils.

Macrophages play a central role in tissue homeostasis and host defense. However, the properties of human macrophages in non-diseased tissues remain poorly understood. Here, we characterized human tonsil macrophages and identified three subsets with distinct phenotype, transcriptome, life cycle, and function. CD36hi macrophages were related to monocytes, while CD36lo macrophages showed features of embryonic origin and CD36int macrophages had a mixed profile. scRNA-seq on non-human primate tonsils showed that monocyte recruitment did not pre-exist an immune challenge. Functionally, CD36hi macrophages were specialized for stimulating T follicular helper cells, by producing Activin A. Combining reconstruction of ligand-receptor interactions and functional assays, we identified stromal cell-derived TNF-α as an inducer of Activin A secretion. However, only CD36hi macrophages were primed for Activin A expression, via the activity of IRF1. Our results provide insight into the heterogeneity of human lymphoid organ macrophages and show that tonsil CD36hi macrophage specialization is the result of both intrinsic features and interaction with stromal cells.

Niche-expressed Galectin-1 is involved in pre-B acute lymphoblastic leukemia relapse through pre-B cell receptor activation.

B-cell acute lymphoblastic leukemia (B-ALL) reflects the malignant counterpart of developing B cells in the bone marrow (BM). Despite tremendous progress in B-ALL treatment, the overall survival of adults at diagnosis and patients at all ages after relapse remains poor. Galectin-1 (GAL1) expressed by BM supportive niches delivers proliferation signals to normal pre-B cells through interaction with the pre-B cell receptor (pre-BCR). Here, we asked whether GAL1 gives non-cell autonomous signals to pre-BCR+ pre-B ALL, in addition to cell-autonomous signals linked to genetic alterations. In syngeneic and patient-derived xenograft (PDX) murine models, murine and human pre-B ALL development is influenced by GAL1 produced by BM niches through pre-BCR-dependent signals, similarly to normal pre-B cells. Furthermore, targeting pre-BCR signaling together with cell-autonomous oncogenic pathways in pre-B ALL PDX improved treatment response. Our results show that non-cell autonomous signals transmitted by BM niches represent promising targets to improve B-ALL patient survival.

Randomized Phase III Trial Evaluating Subcutaneous Rituximab for the First-Line Treatment of Low-Tumor Burden Follicular Lymphoma: Results of a LYSA Study.

PURPOSE: Rituximab improves progression-free survival (PFS) and time to next treatment (TTNT) when compared with the watch and wait strategy for patients with low-tumor burden follicular lymphoma (FL). Prolonged rituximab maintenance did not prolong TTNT, whereas it raises concerns about resources use and patient adhesion. Our aim was then to investigate the use of short rituximab maintenance using the subcutaneous (SC) route in patients with low-tumor burden FL. METHODS: Patients with histologically confirmed CD20+ low-tumor burden FL were randomly assigned to receive either rituximab, 375 mg/m2 once daily on D1, D8, D15, and D22, intravenous route (IV, control arm), or rituximab, 375 mg/m2, on day 1 (D1), IV followed by rituximab 1,400 mg total dose, SC once daily on D8, D15, and D22, with maintenance at months 3 (M3), M5, M7, and M9 (experimental arm). The primary end point was PFS. Secondary end points included safety, overall response rates, TTNT, and overall survival (OS). RESULTS: Two hundred two patients with low-tumor burden FL were randomly assigned to the experimental (n = 100) or control arm (n = 102). The primary end point was met: the 4-year PFS was 58.1% (95% CI, 47.5 to 67.4) and 41.2% (95% CI, 30.6 to 51.6) in experimental and control arms, respectively (hazard ratio, 0.585 [0.393 to 0.871]; P = .0076). Complete response (CR) rates were 59.0% (95% CI, 48.7 to 68.7) in the experimental arm and 36.3% (95% CI, 27.0 to 46.4) in the control arm (P = .001). TTNT and OS were not significantly different. CR was associated with longer PFS and TTNT. High rituximab exposure during the first three months was independently associated with higher CR, PFS, and TTNT. CONCLUSION: SC rituximab improves PFS for patients with low-tumor burden FL when used in induction followed by short maintenance. High rituximab exposure during the first 3 months after treatment initiation is, however, the only parameter influencing patient outcomes.

Regulatory B Cells Contribute to the Clinical Response After Bone Marrow-Derived Mesenchymal Stromal Cell Infusion in Patients With Systemic Sclerosis.

Mesenchymal stromal cells (MSCs) have recently emerged as an interesting therapeutic approach for patients with progressive systemic sclerosis (SSc), a rare and life-threatening orphan autoimmune disease. Whereas MSC immunomodulatory potential is considered as a central mechanism for their clinical benefit, very few data are available on the impact of MSCs on immune cell subsets in vivo. In the current extended study of a phase I/II clinical trial exploring the injection of a single dose of allogeneic bone marrow-MSCs (alloBM-MSCs) in patients with severe SSc (NCT02213705), we performed a longitudinal in-depth characterization of circulating immune cells in 19 MSC-treated patients, including 14 responders and 5 non-responders. By a combination of flow cytometry and transcriptomic analyses, we highlighted an increase in circulating CD24hiCD27posCD38lo/neg memory B cells, the main IL-10-producing regulatory B cell (Breg) subset, and an upregulation of IL10 expression in ex-vivo purified B cells, specifically in responder patients, early after the alloBM-MSC infusion. In addition, a deeper alteration of the B-cell compartment before alloBM-MSC treatment, including a higher expression of profibrotic cytokines IL6 and TGFß by sorted B cells was associated with a non-responder clinical status. Finally, BM-MSCs were able to directly upregulate IL-10 production in activated B cells in vitro. These data suggest that cytokine-producing B cells, in particular Breg, are pivotal effectors of BM-MSC therapeutic activity in SSc. Their quantification as activity biomarkers in MSC potency assays and patient selection criteria may be considered to reach optimal clinical benefit when designing MSC-based clinical trials.

Periosteal skeletal stem cells can migrate into the bone marrow and support hematopoiesis after injury.

Functional stromal cells are known to support bone marrow regeneration after chemotherapy or radiation-induced injury to prevent prolonged myelosuppression. However, it is not known how stromal cells within the bone marrow are regenerated after injury. We have utilized a whole bone transplantation model that mimics the initial bone marrow necrosis and fatty infiltration that is seen after bone marrow injury and subsequent recovery. We demonstrate that periosteal skeletal stem cells (P-SSCs) can migrate into the bone marrow and contribute to stromal regeneration and hematopoietic recovery. Once in the bone marrow, P-SSCs are phenotypically and functionally reprogrammed into bone marrow mesenchymal stem cells (BM-MSCs), expressing high levels of hematopoietic stem cell (HSC) niche factors, such as Cxcl12 and Kitl. Additionally, our results further indicate that P-SSCs are more resistant to acute stress than BM-MSCs. Here, we report a new function of P-SSCs, highlighting their major plasticity and the role of the periosteum as a potential source of BM-MSCs following acute bone marrow injury.

Citrulline enteral administration markedly reduces immunosuppressive extrafollicular plasma cell differentiation in a preclinical model of sepsis.

The sustained immunosuppression associated with severe sepsis favors an increased susceptibility to secondary infections and remains incompletely understood. Plasmablast and plasma cell subsets, whose primary function is to secrete antibodies, have emerged as important suppressive populations that expand during sepsis. In particular, sepsis supports CD39hi plasmablast metabolic reprogramming associated with adenosine-mediated suppressive activity. Arginine deficiency has been linked to an increased risk of secondary infections in sepsis. Overcoming arginine shortage by citrulline administration efficiently improves sepsis-induced immunosuppression and secondary infections in the cecal ligation and puncture murine model. Here, we aimed to determine the impact of citrulline administration on B cell suppressive responses in sepsis. We demonstrate that restoring arginine bioavailability through citrulline administration markedly reduces the dominant extrafollicular B cell response, decreasing the immunosuppressive LAG3+ and CD39+ plasma cell populations, and restoring splenic follicles. At the molecular level, the IRF4/MYC-mediated B cell reprogramming required for extrafollicular plasma cell differentiation is shunted in the splenic B cells of mice fed with citrulline. Our study reveals a prominent impact of nutrition on B cell responses and plasma cell differentiation and further supports the development of citrulline-based clinical studies to prevent sepsis-associated immune dysfunction.

MONOCYTIC MYELOID-DERIVED SUPPRESSOR CELL EXPANSION AFTER CARDIAC SURGERY WITH CARDIOPULMONARY BYPASS INDUCES LYMPHOCYTE DYSFUNCTION.

ABSTRACT: Cardiac surgery with cardiopulmonary bypass (CPB) is associated with an immune paresis that predisposes to the development of postoperative infections and sepsis. Among factors responsible for CPB-induced immunosuppression, circulating myeloid-derived suppressor cells (MDSCs) have been found to induce early lymphocyte apoptosis and lymphocyte proliferation inhibition. However, the mechanisms involved are not fully understood. In this study, we found that the main lymphocyte subsets decreased significantly 24 h after cardiac surgery with CBP. As expected, cardiac surgery with CPB induced a monocytic MDSC expansion associated with an increased T-cell apoptosis and decreased proliferation capacity. Noteworthy, granulocytic MDSCs remain stable. Myeloid-derived suppressor cell depletion restored the ability of T-cell to proliferate ex vivo . After CPB, indoleamine 2,3-dioxygenase activity and IL-10 plasma level were increased such as programmed death-ligand 1 monocytic expression, whereas plasma level of arginine significantly decreased. Neither the inhibition of indoleamine 2,3-dioxygenase activity nor the use of anti-programmed death-ligand 1 or anti-IL-10 blocking antibody restored the ability of T-cell to proliferate ex vivo . Only arginine supplementation restored partially the ability of T-cell to proliferate.

Distinct B-Cell Specific Transcriptional Contexts of the BCL2 Oncogene Impact Pre-Malignant Development in Mouse Models.

Upregulated expression of the anti-apoptotic BCL2 oncogene is a common feature of various types of B-cell malignancies, from lymphoma to leukemia or myeloma. It is currently unclear how the various patterns of deregulation observed in pathology eventually impact the phenotype of malignant B cells and their microenvironment. Follicular lymphoma (FL) is the most common non-Hodgkin lymphoma arising from malignant germinal center (GC) B-cells, and its major hallmark is the t(14:18) translocation occurring in B cell progenitors and placing the BCL2 gene under the control of the immunoglobulin heavy chain locus regulatory region (IgH 3'RR), thus exposing it to constitutive expression and hypermutation. Translocation of BCL2 onto Ig light chain genes, BCL2 gene amplification, and other mechanisms yielding BCL2 over-expression are, in contrast, rare in FL and rather promote other types of B-cell lymphoma, leukemia, or multiple myeloma. In order to assess the impact of distinct BCL2 deregulation patterns on B-cell fate, two mouse models were designed that associated BCL2 and its full P1-P2 promoter region to either the IgH 3'RR, within a "3'RR-BCL2" transgene mimicking the situation seen in FL, or an Ig light chain locus context, through knock-in insertion at the Igκ locus ("Igκ-BCL2" model). While linkage to the IgH 3' RR mostly yielded expression in GC B-cells, the Igκ-driven up-regulation culminated in plasmablasts and plasma cells, boosting the plasma cell in-flow and the accumulation of long-lived plasma cells. These data demonstrate that the timing and level of BCL2 deregulation are crucial for the behavior of B cells inside GC, an observation that could strongly impact the lymphomagenesis process triggered by secondary genetic hits.

FOSL2 truncating variants in the last exon cause a neurodevelopmental disorder with scalp and enamel defects.

PURPOSE: We aimed to investigate the molecular basis of a novel recognizable neurodevelopmental syndrome with scalp and enamel anomalies caused by truncating variants in the last exon of the gene FOSL2, encoding a subunit of the AP-1 complex. METHODS: Exome sequencing was used to identify genetic variants in all cases, recruited through Matchmaker exchange. Gene expression in blood was analyzed using reverse transcription polymerase chain reaction. In vitro coimmunoprecipitation and proteasome inhibition assays in transfected HEK293 cells were performed to explore protein and AP-1 complex stability. RESULTS: We identified 11 individuals from 10 families with mostly de novo truncating FOSL2 variants sharing a strikingly similar phenotype characterized by prenatal growth retardation, localized cutis scalp aplasia with or without skull defects, neurodevelopmental delay with autism spectrum disorder, enamel hypoplasia, and congenital cataracts. Mutant FOSL2 messenger RNAs escaped nonsense-mediated messenger RNA decay. Truncated FOSL2 interacts with c-JUN, thus mutated AP-1 complexes could be formed. CONCLUSION: Truncating variants in the last exon of FOSL2 associate a distinct clinical phenotype by altering the regulatory degradation of the AP-1 complex. These findings reveal a new role for FOSL2 in human pathology.