Biology subjects

Spaapen, R. M.

Publications and source records attributed to Spaapen, R. M..

9 recordsLinked to original sources

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

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↗

bioRxiv2020

The SPPL3-defined glycosphingolipid repertoire regulates immune responses by improving HLA class I access

HLA class I (HLA-I) drives immune responses by presenting antigen-derived peptides to cognate CD8+ T cells. This process is often hijacked by tumors and pathogens for immune evasion. Since therapeutic options for restoring HLA-I antigen presentation are limited, we aimed to identify new HLA-I pathway targets. By iterative genome-wide screens we uncovered that the cell surface glycosphingolipid (GSL) repertoire determines effective HLA-I antigen presentation. We show that absence of the protease SPPL3 augments B3GNT5 enzyme activity, resulting in upregulated levels of surface (neo)lacto-series GSLs. These GSLs sterically impede molecular interactions with HLA-I and diminish CD8+ T cell activation. In accordance, a disturbed SPPL3-B3GNT5 pathway in glioma associates with decreased patient survival. Importantly, we show that this immunomodulatory effect can be reversed through GSL synthesis inhibition using clinically approved drugs. Overall, our study identifies a GSL signature that functionally inhibits antigen presentation and represents a potential therapeutic target in cancer, infection and autoimmunity.

immunology↗

bioRxiv2020

Healthy cells functionally present TAP-independent SSR1 peptides: implications for selection of clinically relevant antigens

Tumors with an impaired transporter associated with antigen processing (TAP) present several ER-derived self-antigens on HLA class I (HLA-I) which are absent on healthy cells. Selection of such TAP-independent antigens for T cell-based immunotherapy should include analysis of their expression on healthy cells to prevent therapy-induced adverse toxicities. However, it is unknown how the absence of clinically relevant antigens on healthy cells needs to be validated. Here we monitored TAP-independent antigen presentation on various healthy cells using a new toolbox consisting of a T cell clone recognizing a TAP-independent SSR1-derived antigen. We found that most but not all healthy cells present this antigen under normal and inflammatory conditions, indicating that TAP-independent antigen presentation is a variable phenomenon. Our data emphasize the necessity of extensive testing of a wide variety of healthy cell types to define clinically relevant TAP-independent antigens that can be safely targeted by immunotherapy.

immunology↗

bioRxiv2020

T cells expanded from renal cell carcinoma are tumor-reactive but fail to produce IFN-γ, TNF-α or IL-2

Metastatic renal cell carcinoma (RCC) has a poor prognosis. Recent advances have shown beneficial responses to immune checkpoint inhibitors, such as anti-PD-1 or anti-PD-L1 antibodies. As only a subset of RCC patients respond, alternative strategies should be explored. Patients refractory to anti-PD-1 therapy may benefit from autologous tumor infiltrating lymphocyte (TIL) therapy. Even though efficient TIL expansion was reported from RCC lesions, it is not well established how many RCC TIL products are tumor-reactive, how well they produce pro-inflammatory cytokines in response to autologous tumors, and whether their response correlates with the presence of specific immune cells in the tumor lesions. We here compared the immune infiltrate composition of RCC lesions with that of autologous kidney tissue of 18 RCC patients. T cell infiltrates were increased in the tumor lesions, and CD8+ T cell infiltrates were primarily of effector memory phenotype. Nine out of 16 (56%) tested TIL products we generated were tumor-reactive, as defined by CD137 upregulation after exposure to autologous tumor digest. Tumor reactivity was found in particular in TIL products originating from tumors with a high percentage of infiltrated T cells compared to autologous kidney, and coincided with increased ex vivo CD25 expression on CD8+ T cells. Importantly, although TIL products had the capacity to produce the key effector cytokines IFN-{gamma}, TNF- or IL-2, they failed to do so in response to autologous tumor digests. In conclusion, TIL products from RCC lesions contain tumor-reactive T cells. Their lack of tumor-specific cytokine production requires further investigation of immunosuppressive factors in RCC and subsequent optimization of RCC-derived TIL culture conditions.

immunology↗

bioRxiv2020

PAKC: A novel Panel of HLA class I Antigen presentation machinery Knockout Cells from the same genetic origin

With the emergence of immunotherapies, the understanding of functional HLA class I antigen presentation to T cells is more relevant than ever. Current knowledge on antigen presentation is based on decades of research in a wide variety of cell types with varying antigen presentation machinery (APM) expression patterns, proteomes and HLA haplotypes. This diversity complicates the establishment of individual APM contributions to antigen generation, selection and presentation. Therefore, we generated a novel Panel of APM Knockout Cell lines (PAKC) from the same genetic origin. After CRISPR/Cas9 genome-editing of ten individual APM components in a human cell line, we derived clonal cell lines and confirmed their knockout status and phenotype. We then show how PAKC will accelerate research on the functional interplay between APM components and their role in antigen generation and presentation. This will lead to improved understanding of peptide-specific T cell responses in infection, cancer and autoimmunity. Key pointsO_LIWe generated a panel of cell lines to study HLA class I antigen presentation C_LIO_LIWe show how this will spark research in infection, tumor biology and autoimmunity C_LI

immunology↗

bioRxiv2018

Polyfunctional tumor-reactive T cells are effectively expanded from non-small cell lung cancers, and correlate with an immune-engaged T cell profile in situ

Non-small cell lung cancer (NSCLC) is the second most prevalent type of cancer. With the current treatment regimens, the mortality rate remains high. Therefore, better therapeutic approaches are necessary. NSCLCs generally possess many genetic mutations and are well infiltrated by T cells (TIL), making TIL therapy an attractive option. Here we show that T cells from treatment naive, stage I-IVa NSCLC tumors can effectively be isolated and expanded, with similar efficiency as from normal lung tissue. Importantly, 76% (13/17) of tested TIL products isolates from NSCLC lesions exhibited clear reactivity against primary tumor digests, with 0.5%-30% of T cells producing the inflammatory cytokine Interferon (IFN)-{gamma}. Both CD4+ and CD8+ T cells displayed tumor reactivity. The cytokine production correlated well with CD137 and CD40L expression. Furthermore, almost half (7/17) of the TIL products contained polyfunctional T cells that produced Tumor Necrosis Factor (TNF)- and/or IL-2 in addition to IFN-{gamma}, a hallmark of effective immune responses. Tumor-reactivity in the TIL products correlated with high percentages of CD103+CD69+CD8+ T cell infiltrates in the tumor lesions, with PD-1hiCD4+ T cells, and with FoxP3+CD25+CD4+ regulatory T cell infiltrates, suggesting that the composition of T cell infiltrates may predict the level of tumor reactivity. In conclusion, the effective generation of tumor-reactive and polyfunctional TIL products implies that TIL therapy will be a successful treatment regimen for NSCLC patients.

immunology↗