Biology subjects

van Ham, S. M.

Publications and source records attributed to van Ham, S. M..

6 recordsLinked to original sources

bioRxiv2024

Characterization of a heterogenous activated B cell compartment arising early after antigen exposure preceding long-lived memory B cell formation

Once formed, plasma cells and memory B cells (MBCs) are difficult to eradicate, posing a problem in the context of unwanted antibody responses. Characterizing early B cell differentiation stages after antigen encounter is thus crucial to target and prevent unwanted antibody formation. Here, we unravelled in-depth antigen-specific B cell responses longitudinally after SARS-CoV-2 mRNA vaccination in healthy individuals using multiparameter spectral flow cytometry. The early antigen-specific B cell response was dominated by spike-specific IgG+ CD27+ CD71+ activated B cells (ActBCs), previously assigned as germinal center-derived and DN2 extrafollicular B cells. Within the early IgG+ ActBC compartment, six distinct clusters were identified with specific contraction dynamics, whereby some of these clusters were more closely related to pre-ASCs and others more to long-lived MBCs. Some of the highly contracting ActBC clusters expressed CD11c, a marker previously used to define atypical B cells. The transient presence of different ActBC clusters could also be observed in total B cells when gated in an antigen- independent manner. Our results thus delineate the early stages of the antigen-specific B cell response, with a further dissection of the CD71+ ActBC compartment. Detection of ActBC clusters early after antigen encounter in total B cells opens avenues for future evaluation of their potential to serve as a proxy for antigen-reactive B cells in autoimmunity or other unwanted B cell responses.

immunology↗

bioRxiv2024

nsGSLs on tumors impair anti-tumor immune responses by OT-I T cells in vitro and support tumor growth in vivo

Tumors often evolve to engage numerous strategies to circumvent detection by the immune system. Our group recently discovered elevated neolacto-series glycosphingolipids (nsGSL) surface levels as a possible immune evasion mechanism of tumors. We demonstrated a direct disruption of both innate and adaptive anti-tumor immunity in vitro when expression of nsGSLs was upregulated on established target cells. It remains unclear however, whether in vivo nsGSLs play an active role in tumor development and can aid tumors in evading immune responses. To investigate whether nsGSLs facilitate tumor progression in vivo, we first established a murine model system using MC38-OVA cell lines with varying cell surface levels of nsGSLs. In vitro analysis revealed reduced MHC-I accessibility on tumor cells with elevated nsGSLs profiles, leading to diminished activation of OVA-specific OT-I T cells as evidenced by decreased expression of CD25, CD69, and production of IFN{gamma}, which subsequently resulted in decreased tumor cell death. Subsequent in vivo experiments investigating tumor outgrowth after engraftment of subcutaneously injected MC38-OVA cell lines with low or high cell surface levels of nsGSLs demonstrated better growth of nsGSL-rich tumor cells compared to nsGSL-poor tumors which could be controlled. Together these results suggest that nsGSLs expressed by tumors can facilitate immune evasion and subsequent tumor progression. These data pave the way to explore whether targeting of the GSL pathway with specific inhibitors could be advantageous as a therapy against tumors with high nsGSL levels.

immunology↗

bioRxiv2024

Tumor-expressed SPPL3 supports innate anti-tumor immune responses.

The development of an effective anti-tumor response relies on the synergistic actions of various immune cells that recognize tumor cells via distinct receptors. Tumors, however, often manipulate receptor-ligand interactions to evade recognition by the immune system. Recently, we highlighted the role of neolacto-series glycosphingolipids (nsGSLs), produced by the enzyme {beta}1,3-N-acetylglucosaminyltransferase 5 (B3GNT5), in tumor immune escape. We previously demonstrated that loss of signal peptidase like 3 (SPPL3), an inhibitor of B3GNT5, results in elevated levels of nsGSLs and impairs CD8 T cell activation. The impact of loss of SPPL3 and an elevated nsGSL profile in tumor cells on innate immune recognition remains to be elucidated. This study investigates the anti-tumor efficacy of neutrophils, NK cells, and {gamma}{delta} T cells on tumor cells lacking SPPL3. Our findings demonstrate that SPPL3-deficient target cells are less susceptible to trogocytosis by neutrophils and killing by NK cells and {gamma}{delta} T cells. Mechanistically, SPPL3 influences trogocytosis and {gamma}{delta} T cell instigated killing through modulation of nsGSL expression while SPPL3-mediated reduced killing by NK cells is nsGSL-independent. The nsGSL-dependent SPPL3 sensitivity depends on the proximity of surface receptor domains to the cell membrane and the affinity of receptor-ligand interactions as shown with various sets of defined antibodies. Thus, SPPL3 expression by tumor cells alters crosstalk with immune cells through the receptor-ligand interactome thereby driving escape not only from adaptive but also from innate immunity. These data underline the importance of investigating a potential synergism of GSL synthesis inhibitors with current immune cell activating immunotherapies.

immunology↗

bioRxiv2023

Deep profiling of antigen-specific B cells from different pathogens identifies novel compartments in the IgG memory B cell and antibody-secreting cell lineages

A better understanding of the bifurcation of human B cell differentiation into memory B cells (MBC) and antibody-secreting cells (ASC) and identification of MBC and ASC precursors is crucial to optimize vaccination strategies or block undesired antibody responses. To unravel the dynamics of antigen-induced B cell responses, we compared circulating B cells reactive to SARS-CoV-2 (Spike, RBD and Nucleocapsid) in COVID-19 convalescent individuals to B cells specific to Influenza-HA, RSV-F and TT, induced much longer ago. High-dimensional spectral flow cytometry indicated that the decision point between ASC- and MBC-formation lies in the CD43+CD71+IgG+ Activated B cell compartment, showing properties indicative of recent germinal center activity and recent antigen encounter. Within this Activated B cells compartment, CD86+ B cells exhibited close phenotypical similarity with ASC, while CD86- B cells were closely related to IgG+ MBCs. Additionally, different activation stages of the IgG+ MBC compartment could be further elucidated. The expression of CD73 and CD24, regulators of survival and cellular metabolic quiescence, discerned activated MBCs from resting MBCs. Activated MBCs (CD73-CD24lo) exhibited phenotypical similarities with CD86- IgG+ Activated B cells and were restricted to SARS-CoV-2 specificities, contrasting with the resting MBC compartment (CD73-/CD24hi) that exclusively encompassed antigen-specific B cells established long ago. Overall, these findings identify novel stages for IgG+ MBC and ASC formation and bring us closer in defining the decision point for MBC or ASC differentiation. ImportanceIn this study, researchers aimed to better understand human B cell differentiation and their role in establishing long-lived humoral immunity. Using high-dimensional flow cytometry, they studied B cells reactive to three SARS-CoV-2 antigens in individuals convalescent for COVID-19, and compared their phenotypes to B cells reactive to three distinct protein antigens derived from vaccines or viruses encountered months to decades before. Their findings showed that Activated B cells reflect recent germinal center graduates that may have diverse fates; with some feeding the pool of antibody-secreting cells and others fueling the resting memory B cell compartment. Activated B cells gradually differentiate into resting memory B cells through an activated MBC phase. Increased expression of the cellular metabolic regulators CD73 and CD24 in resting memory B cells distinguishes them from the activated memory B cells phase, and is likely involved in sustaining a durable memory of humoral immunity. These findings are crucial for the development of vaccines that provide lifelong protection and may show potential to define reactive B cells in diseases where the cognate-antigen is still unknown such as in autoimmunity, cancers, or novel viral outbreaks.

immunology↗

bioRxiv2023

Isolation and expansion of pure and functional γδ T cells

{gamma}{delta} T cells are important components of the immune system due to their ability to elicit a fast and strong response against infected and transformed cells. Because they can specifically and effectively kill target cells in an MHC independent fashion, there is great interest to utilize these cells in anti-tumor therapies where antigen presentation may be hampered. Since only a small fraction of T cells in the blood or tumor tissue are {gamma}{delta} T cells, they require extensive expansion to allow for fundamental, preclinical and ex vivo research. Although expansion protocols can be successful, most are based on depletion of other cell types rather than {gamma}{delta} T cell specific isolation, resulting in unpredictable purity of the isolated fraction. Moreover, the primary focus only lies with expansion of V{delta}2+ T cells, while V{delta}1+ T cells likewise have anti-tumor potential. Here, we investigated whether {gamma}{delta} T cells directly isolated from blood could be efficiently expanded while maintaining function. {gamma}{delta} T cell subsets were isolated using MACS separation, followed by FACS sorting, yielding >99% pure {gamma}{delta} T cells. Isolated V{delta}1+ and V{delta}2+ T cells could effectively expand immediately after isolation or upon freeze/thawing and reached expansion ratios between 200 to 2000-fold starting from varying numbers using cytokine supported feeder stimulations. After expansion, potential effector functions of {gamma}{delta} T cells were demonstrated by IFN-{gamma}, TNF- and granzyme B production upon PMA/ionomycin stimulation and effective killing capacity of multiple tumor cell lines was confirmed in killing assays. In conclusion, pure {gamma}{delta} T cells can productively be expanded while maintaining their anti-tumor effector functions against tumor cells. Moreover, {gamma}{delta} T cells could be expanded from low starting numbers suggesting that this protocol may even allow for expansion of cells extracted from tumor biopsies.

immunology↗

bioRxiv2020

Soluble FAS Ligand directs human memory B cells towards antibody-secreting cells

Differentiation of antigen-specific B cells into class-switched, high affinity antibody-secreting cells provides protection against invading pathogens but is undesired when antibodies target self-tissues in autoimmunity, beneficial non-self blood transfusion products or therapeutic proteins. Essential T cell factors have been uncovered that regulate T cell-dependent B cell differentiation. We performed a screen using a secreted protein library to identify novel factors that promote this process and may be used to combat undesired antibody formation. We tested the differentiating capacity of 756 secreted proteins on human naive or memory B cell differentiation in a setting with suboptimal T cell help in vitro (suboptimal CD40L and IL-21). High-throughput flow cytometry screening and validation revealed that type I IFNs and soluble FAS ligand (sFASL) induce plasmablast differentiation in memory B cells. Furthermore, sFASL induces robust secretion of IgG1 and IgG4 antibodies, indicative of functional plasma cell differentiation. Our data suggest a mechanistic connection between elevated sFASL levels and the induction of autoreactive antibodies, providing a potential therapeutic target in autoimmunity. Indeed, the modulators identified in this secretome screen are associated with systemic lupus erythematosus and may also be relevant in other autoimmune diseases and allergy.

immunology↗