Unsaturated fatty acids (UFA) are crucial for T-cell effector functions, as they can affect the growth, differentiation, survival, and function of T cells. Nonetheless, the mechanisms by which UFA... Show moreUnsaturated fatty acids (UFA) are crucial for T-cell effector functions, as they can affect the growth, differentiation, survival, and function of T cells. Nonetheless, the mechanisms by which UFA affects T-cell behavior are ill-defined. Therefore, we analyzed the processing of oleic acid, a prominent UFA abundantly present in blood, adipocytes, and the fat pads surrounding lymph nodes, in CD4+ T cells. We found that exogenous oleic acid increases proliferation and enhances the calcium flux response upon CD3/CD28 activation. By using a variety of techniques, we found that the incorporation of oleic acid into membrane lipids, rather than regulation of cellular metabolism or TCR expression, is essential for its effects on CD4+ T cells. These results provide novel insights into the mechanism through which exogenous oleic acid enhances CD4+ T-cell function. Show less
Essen, M.F. van; Schlagwein, N.; Hoven, E.M.P. van den; Gijlswijk-Janssen, D.J. van; Lubbers, R.; Bos, R.M. van den; ... ; COMBAT Consortium 2023
Properdin, the only known positive regulator of the complement system, stabilizes the C3 convertase, thereby increasing its half-life. In contrast to most other complement factors, properdin is... Show moreProperdin, the only known positive regulator of the complement system, stabilizes the C3 convertase, thereby increasing its half-life. In contrast to most other complement factors, properdin is mainly produced extrahepatically by myeloid cells. Recent data suggest a role for properdin as a pattern recognition molecule. Here, we confirmed previous findings of properdin binding to different necrotic cells including Jurkat T cells. Binding can occur independent of C3, as demonstrated by HAP-1 C3 KO cells, excluding a role for endogenous C3. In view of the cellular source of properdin, interaction with myeloid cells was examined. Properdin bound to the surface of viable monocyte-derived pro- and anti-inflammatory macrophages, but not to DCs. Binding was demonstrated for purified properdin as well as fractionated P2, P3, and P4 properdin oligomers. Binding contributed to local complement activation as determined by C3 and C5b-9 deposition on the cell surfaces and seems a prerequisite for alternative pathway activation. Interaction of properdin with cell surfaces could be inhibited with the tick protein Salp20 and by different polysaccharides, depending on sulfation and chain length. These data identify properdin as a factor interacting with different cell surfaces, being either dead or alive, contributing to the local stimulation of complement activation. Show less
Huisman, W.; Gier, M. de; Hageman, L.; Shomuradova, A.S.; Leboux, D.A.T.; Amsen, D.; ... ; Jedema, I. 2022
Anti-viral T-cell responses are usually directed against a limited set of antigens, but often contain many T cells expressing different T-cell receptors (TCRs). Identical TCRs found within virus... Show moreAnti-viral T-cell responses are usually directed against a limited set of antigens, but often contain many T cells expressing different T-cell receptors (TCRs). Identical TCRs found within virus-specific T-cell populations in different individuals are known as public TCRs, but also TCRs highly-similar to these public TCRs, with only minor variations in amino acids on specific positions in the Complementary Determining Regions (CDRs), are frequently found. However, the degree of freedom at these positions was not clear. In this study, we used the HLA-A*02:01-restricted EBV-LMP2(FLY)-specific public TCR as model and modified the highly-variable position 5 of the CDR3 beta sequence with all 20 amino acids. Our results demonstrate that amino acids at this particular position in the CDR3 beta region of this TCR are completely inter-changeable, without loss of TCR function. We show that the inability to find certain variants in individuals is explained by their lower recombination probability rather than by steric hindrance. Show less
Koers, J.; Pollastro, S.; Tol, S.; Niewold, I.T.G.; Schouwenburg, P.A. van; Vries, N. de; Rispens, T. 2022
In past years ex vivo and in vivo experimental approaches involving human naive B cells have proven fundamental for elucidation of mechanisms promoting B cell differentiation in both health and... Show moreIn past years ex vivo and in vivo experimental approaches involving human naive B cells have proven fundamental for elucidation of mechanisms promoting B cell differentiation in both health and disease. For such studies, it is paramount that isolation strategies yield a population of bona fide naive B cells, i.e., B cells that are phenotypically and functionally naive, clonally non-expanded, and have non-mutated BCR variable regions. In this study different combinations of common as well as recently identified B cell markers were compared to isolate naive B cells from human peripheral blood. High-throughput BCR sequencing was performed to analyze levels of somatic hypermutation and clonal expansion. Additionally, contamination from mature mutated B cells intrinsic to each cell-sorting strategy was evaluated and how this impacts the purity of obtained populations. Our results show that current naive B cell isolation strategies harbor contamination from non-naive B cells, and use of CD27-IgD(+) is adequate but can be improved by including markers for CD45RB glycosylation and IgM. The finetuning of naive B cell classification provided herein will harmonize research lines using naive B cells, and will improve B cell profiling during health and disease, e.g. during diagnosis, treatment, and vaccination strategies. Show less
The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and... Show moreThe third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples and respective applications of flow cytometry in the context of a variety of autoimmune diseases, cancers as well as acute and chronic infectious diseases such as COVID-19. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers. Show less
Rumpret, M.; Richthofen, H.J. von; Linden, M. van der; Westerlaken, G.H.A.; Ormeno, C.T.; Low, T.Y.; ... ; Meyaard, L. 2021
Signal inhibitory receptor on leukocytes-1 (SIRL-1) is an inhibitory receptor with a hitherto unknown ligand, and is expressed on human monocytes and neutrophils. SIRL-1 inhibits myeloid effector... Show moreSignal inhibitory receptor on leukocytes-1 (SIRL-1) is an inhibitory receptor with a hitherto unknown ligand, and is expressed on human monocytes and neutrophils. SIRL-1 inhibits myeloid effector functions such as reactive oxygen species (ROS) production. In this study, we identify S100 proteins as SIRL-1 ligands. S100 proteins are composed of two calcium-binding domains. Various S100 proteins are damage-associated molecular patterns (DAMPs) released from damaged cells, after which they initiate inflammation by ligating activating receptors on immune cells. We now show that the inhibitory SIRL-1 recognizes individual calcium-binding domains of all tested S100 proteins. Blocking SIRL-1 on human neutrophils enhanced S100 protein S100A6-induced ROS production, showing that S100A6 suppresses neutrophil ROS production via SIRL-1. Taken together, SIRL-1 is an inhibitory receptor recognizing the S100 protein family of DAMPs. This may help limit tissue damage induced by activated neutrophils. Show less
Borst, J.; Busselaar, J.; Bosma, D.M.T.; Ossendorp, F. 2021
Immunotherapy targeting the Programmed Death (PD-1) receptor/ligand (L) "checkpoint" rapidly gains ground in the treatment of many cancer types. To increase treatment scope and efficacy, predictive... Show moreImmunotherapy targeting the Programmed Death (PD-1) receptor/ligand (L) "checkpoint" rapidly gains ground in the treatment of many cancer types. To increase treatment scope and efficacy, predictive biomarkers and rational selection of co-treatments are required. To meet these demands, we must understand PD-1 function in detail. We here outline recent insights into the regulation of the CD8(+) T cell response by PD-1. The prevailing view has been that blockade of PD-1/ligand (L) interaction "reinvigorates" cytotoxic T lymphocytes (CTL) that were rendered dysfunctional in the tumor microenvironment (TME). However, this review stresses that tumors continuously communicate with adjacent draining lymph nodes (LNs) and that the PD-1 checkpoint also operates during T cell priming. We clarify the role of the PD-(L)1 system at the T cell/DC interface, where it regulates T cell receptor (TCR) signaling and CD28 costimulation and thus controls activation of tumor-specific T cells. We also highlight the importance of CD4(+) T cell help during priming, which allows DCs to provide other costimulatory and cytokine signals required for optimal CTL differentiation and likely avoidance of a dysfunctional state. Therefore, we pose that PD-(L)1 blockade should exploit LN function and be combined with "help" signals to optimize CTL efficacy. Show less
The Ig superfamily protein glycoprotein A33 (GPA33) has been implicated in immune dysregulation, but little is known about its expression in the immune compartment. Here, we comprehensively... Show moreThe Ig superfamily protein glycoprotein A33 (GPA33) has been implicated in immune dysregulation, but little is known about its expression in the immune compartment. Here, we comprehensively determined GPA33 expression patterns on human blood leukocyte subsets, using mass and flow cytometry. We found that GPA33 was expressed on fractions of B, dendritic, natural killer and innate lymphoid cells. Most prominent expression was found in the CD4(+) T cell compartment. Naive and CXCR5(+) regulatory T cells were GPA33(high), and naive conventional CD4(+) T cells expressed intermediate GPA33 levels. The expression pattern of GPA33 identified functional heterogeneity within the CD4(+) central memory T cell (Tcm) population. GPA33(+) CD4(+) Tcm cells were fully undifferentiated, bona fide Tcm cells that lack immediate effector function, whereas GPA33(-) Tcm cells exhibited rapid effector functions and may represent an early stage of differentiation into effector/effector memory T cells before loss of CD62L. Expression of GPA33 in conventional CD4(+) T cells suggests a role in localization and/or preservation of an undifferentiated state. These results form a basis to study the function of GPA33 and show it to be a useful marker to discriminate between different cellular subsets, especially in the CD4(+) T cell lineage. Show less
In recent years there have been major advances in our understanding of the role of free fatty acids (FAs) and their metabolism in shaping the functional properties of macrophages and DCs. This... Show moreIn recent years there have been major advances in our understanding of the role of free fatty acids (FAs) and their metabolism in shaping the functional properties of macrophages and DCs. This review presents the most recent insights into how cell intrinsic FA metabolism controls DC and macrophage function, as well as the current evidence of the importance of various exogenous FAs (such as polyunsaturated FAs and their oxidation products-prostaglandins, leukotrienes, and proresolving lipid mediators) in affecting DC and macrophage biology, by modulating their metabolic properties. Finally, we explore whether targeted modulation of FA metabolism of myeloid cells to steer their function could hold promise in therapeutic settings. Show less
Ho, N.I.; Camps, M.G.M.; Verdoes, M.; Munz, C.; Ossendorp, F. 2021
Autophagy has been reported to be involved in supporting antigen cross-presentation by dendritic cells (DCs). We have shown that DCs have the ability to store antigen for a prolonged time in... Show moreAutophagy has been reported to be involved in supporting antigen cross-presentation by dendritic cells (DCs). We have shown that DCs have the ability to store antigen for a prolonged time in endolysosomal compartments and thereby sustain MHCI antigen cross-presentation to CD8(+) T cells. In the current study, we investigated the role of autophagy in long-term antigen presentation. We show that the autophagy machinery has a negative impact on storage of antigen in DCs. Atg5(-/-)DCs which are deficient in autophagy or DCs treated with common autophagy inhibitors showed enhanced antigen storage and antigen cross-presentation. This augmented antigen cross-presentation effect is independent of altered proteasome enzyme activity or MHCI surface expression on DCs. We visualized that the storage compartments are in close proximity to LC3 positive autophagosomes. Our results indicate that autophagosomes disrupt antigen storage in DCs and thereby regulate long-term MHCI cross-presentation. Show less
A single model system for integrative studies on multiple facets of antigen presentation is lacking. PAKC is a novel panel of ten cell lines knocked out for individual components of the HLA class I... Show moreA single model system for integrative studies on multiple facets of antigen presentation is lacking. PAKC is a novel panel of ten cell lines knocked out for individual components of the HLA class I antigen presentation pathway. PAKC will accelerate HLA-I research in the fields of oncology, infectiology, and autoimmunity. Show less
The importance of interleukin (IL)-33 in promoting effective antiviral immune responses is evident, yet the critical cellular sources of IL-33 in homeostasis and infection are largely unknown. In... Show moreThe importance of interleukin (IL)-33 in promoting effective antiviral immune responses is evident, yet the critical cellular sources of IL-33 in homeostasis and infection are largely unknown. In this issue of the European Journal of Immunology, Aparicio-Domingo et al. [Eur. J. Immunol. 2021. 51: XX-XX] explore the main source of IL-33 expression in lymph nodes (LNs) and dissect its role in LN homeostasis and antiviral adaptive immune response. The authors reveal that fibroblastic reticular cells and lymphatic endothelial cells are both producing IL-33 in steady-state LNs. Remarkably, however, by using cell-type specific deletion approaches, the authors demonstrate that exclusively fibroblastic reticular cells, and not lymphatic endothelial cells, are the critical cellular source for promoting antiviral CD8(+) T-cell responses upon infection. These findings provide an important insight into the role of specific LN stromal cell subsets as potent modulators of antiviral immunity. Show less
B cell targeting therapies are effective in various autoimmune diseases, among others rheumatoid arthritis, pemphigus vulgaris, and systemic lupus erythematosus. Given these successes, it is... Show moreB cell targeting therapies are effective in various autoimmune diseases, among others rheumatoid arthritis, pemphigus vulgaris, and systemic lupus erythematosus. Given these successes, it is evident that B cells are central orchestrators in the processes leading to the signs and symptoms hallmarking many human autoimmune diseases. The pathways provoking the generation of such autoreactive B cells or mechanisms preventing their induction in health are, however, poorly explored. Nevertheless, such information is crucial for the development of preventative/curative interventions aiming to permanently deplete- or prohibit the emergence of autoreactive B cells. Hence, this review will focus on how B cell tolerance might be breached, and which checkpoints are at play preventing the arousal of autoreactive B cells in human. Especially antigen presentation by follicular dendritic cells, somatic hypermutation, and cross-reactivity to the microbiome/environment could operate as actors playing pivotal roles in the induction of B cell-mediated humoral autoimmunity. Moreover, we highlight the human autoimmune disease rheumatoid arthritis as a prototype where autoreactive B cells combine several mechanisms to overcome peripheral B cell checkpoints. Show less
RAG complexes recognise (cryptic) RSS sites both in and outside immunoglobulin sites. Excision circles may be reinserted into V(D)J rearrangements as long templated insertions to diversify the... Show moreRAG complexes recognise (cryptic) RSS sites both in and outside immunoglobulin sites. Excision circles may be reinserted into V(D)J rearrangements as long templated insertions to diversify the adaptive immune repertoire. We show that such VDJ with templated insertions are incidentally found in the repertoire of healthy donors. Show less
Invariant natural killer T cells (iNKT) constitute up to 50% of liver lymphocytes and contribute to immunosurveillance as well as pathogenesis of the liver. Systemic activation of iNKT cells... Show moreInvariant natural killer T cells (iNKT) constitute up to 50% of liver lymphocytes and contribute to immunosurveillance as well as pathogenesis of the liver. Systemic activation of iNKT cells induces acute immune-mediated liver injury. However, how tissue damage events regulate iNKT cell function and homeostasis remains unclear. We found that specifically tissue-resident iNKT cells in liver and spleen express the tissue-damage receptor P2RX7 and the P2RX7-activating ectoenzyme ARTC2. P2RX7 expression restricted formation of iNKT cells in the liver suggesting that liver iNKT cells are actively restrained under homeostatic conditions. Deliberate activation of P2RX7 in vivo by exogenous NAD resulted in a nearly complete iNKT cell ablation in liver and spleen in a P2RX7-dependent manner. Tissue damage generated by acetaminophen-induced liver injury reduced the number of iNKT cells in the liver. The tissue-damage-induced iNKT cell depletion was driven by P2RX7 and localized to the site of injury, as iNKT cells in the spleen remained intact. The depleted liver iNKT cells reconstituted only slowly compared to other lymphocytes such as regulatory T cells. These findings suggest that tissue-damage-mediated depletion of iNKT cells acts as a feedback mechanism to limit iNKT cell-induced pathology resulting in the establishment of a tolerogenic environment. Show less
The helminth Schistosoma mansoni (S. mansoni) induces a network of regulatory immune cells, including interleukin (IL)-10-producing regulatory B cells (Bregs). However, the signals required for the... Show moreThe helminth Schistosoma mansoni (S. mansoni) induces a network of regulatory immune cells, including interleukin (IL)-10-producing regulatory B cells (Bregs). However, the signals required for the development and activation of Bregs are not well characterized. Recent reports suggest that helminths induce type I interferons (IFN-I), and that IFN-I drive the development of Bregs in humans. We therefore assessed the role of IFN-I in the induction of Bregs by S. mansoni. Mice chronically infected with S. mansoni or i.v. injected with S. mansoni soluble egg antigen (SEA) developed a systemic IFN-I signature. Recombinant IFN-alpha enhanced IL-10 production by Bregs stimulated with S. mansoni SEA in vitro, while not activating Bregs by itself. IFN-I signaling also supported ex vivo IL-10 production by SEA-primed Bregs but was dispensable for activation of S. mansoni egg-induced Bregs in vivo. These data indicate that although IFN-I can serve as a coactivator for Breg IL-10 production, they are unlikely to participate in the development of Bregs in response to S. mansoni eggs. Show less
BM has been put forward as a major reservoir for memory CD8(+) T cells. In order to fulfill that function, BM should "store" memory CD8(+) T cells, which in biological terms would require these ... Show moreBM has been put forward as a major reservoir for memory CD8(+) T cells. In order to fulfill that function, BM should "store" memory CD8(+) T cells, which in biological terms would require these "stored" memory cells to be in disequilibrium with the circulatory pool. This issue is a matter of ongoing debate. Here, we unequivocally demonstrate that murine and human BM harbors a population of tissue-resident memory CD8(+) T (T-RM) cells. These cells develop against various pathogens, independently of BM infection or local antigen recognition. BM CD8(+) T-RM cells share a transcriptional program with resident lymphoid cells in other tissues; they are polyfunctional cytokine producers and dependent on IL-15, Blimp-1, and Hobit. CD8(+) T-RM cells reside in the BM parenchyma, but are in close contact with the circulation. Moreover, this pool of resident T cells is not size-restricted and expands upon peripheral antigenic re-challenge. This works extends the role of the BM in the maintenance of CD8(+) T cell memory to include the preservation of an expandable reservoir of functional, non-recirculating memory CD8(+) T cells, which develop in response to a large variety of peripheral antigens. Show less
Infection of C57BL/6 mice with lymphocytic choriomeningitis virus (LCMV) strain Armstrong (Arm) induces an acute infection with rapid virus clearance by CD8(+) T cells independently of CD4(+) T... Show moreInfection of C57BL/6 mice with lymphocytic choriomeningitis virus (LCMV) strain Armstrong (Arm) induces an acute infection with rapid virus clearance by CD8(+) T cells independently of CD4(+) T cell help. Residual viral antigen may, however, persist for a prolonged time. Here, we demonstrate that mice that had been transiently depleted of CD4(+) T cells during acute LCMV Arm infection generated high levels of virus-specific IgG antibodies (Ab) after viral clearance. Robust induction of LCMV-specific IgG after transient CD4(+) T cell depletion was dependent on Fc gamma receptors but not on the complement receptors CD21/CD35. In contrast to the potent production of LCMV-specific IgG, the generation of LCMV-specific isotype-switched memory B cells after transient CD4(+) T cell depletion was considerably reduced. Moreover, mice depleted of CD4(+) T cells during acute infection were strongly impaired in generating a secondary LCMV-specific B cell response upon LCMV rechallenge. In conclusion, our data indicate that LCMV antigen depots after viral clearance were capable of inducing high levels of virus-specific IgG. They failed, however, to induce robust virus-specific B cell memory revealing a previously unappreciated dichotomy of specific Ab production and memory cell formation after priming with residual antigen. Show less
