In vivo engineering of mobilized stem cell grafts with the immunomodulatory drug FTY720 for allogeneic transplantation.

The immunological attributes of stem cell grafts play an important role in the outcome of allogeneic stem cell transplants. Currently, ex vivo manipulation techniques such as bulk T-cell depletion or positive selection of CD34(+) cells are utilized to improve the immunological attributes of grafts and minimize the potential for graft-versus-host disease (GvHD). Here, we demonstrate a novel graft engineering technique, which utilizes the immunomodulatory drug FTY720 for in vivo depletion of naïve T (TN ) cells from donor G-CSF-mobilized grafts without ex vivo manipulation. We show that treatment of donor mice with FTY720 during mobilization depletes grafts of TN cells and prevents lethal GvHD following transplantation in a major mismatch setting. Importantly, both stem cells and NK cells are retained in the FTY720-treated grafts. FTY720 treatment does not negatively affect the engraftment potential of stem cells as demonstrated in our congenic transplants or the functionality of NK cells. In addition, potentially useful memory T cells may be retained in the graft. These findings suggest that FTY720 may be used to optimize the immunological attributes of G-CSF-mobilized grafts by removing potentially deleterious TN cells which can contribute to GvHD, and by retaining useful cells which can promote immunity in the recipient.

CD244 is expressed on dendritic cells and regulates their functions.

Signaling lymphocytic activation molecule (SLAM) receptors have an important role in the development of immune responses because of their roles, for exampe, in NK cell cytotoxicity and cytokine production by NK, T cells and myeloid cells. The SLAM receptor CD244 (2B4, SLAMf4) is expressed on a variety of immune cell types but most of its functions have been examined on NK and T cells. In the present study, we investigated expression and function of CD244 in murine subsets of dendritic cells (DCs). We report that all subsets of murine DCs examined expressed CD244, although the expression levels of CD244 varied between subsets. Splenic and resident mesenteric lymph node (MLN) DCs from CD244(-/-) mice expressed lower levels of CD86 and MHC class II compared with wild-type mice. Upon Toll-like receptor (TLR) stimulation, no differences in surface expression of these molecules were observed between DCs from CD244(-/-) and wild-type mice. However, splenic DCs from CD244(-/-) mice upon stimulation with TLR binding ligands lipopolysaccharide (LPS) and CpG produced significantly higher levels of pro-inflammatory cytokines. In addition, DCs from CD244(-/-) mice elicited increased NK cell activation in vitro. These data add CD244 to a growing list of immuno-modulatory receptors found on DCs.

GABAergic signaling is linked to a hypermigratory phenotype in dendritic cells infected by Toxoplasma gondii.

During acute infection in human and animal hosts, the obligate intracellular protozoan Toxoplasma gondii infects a variety of cell types, including leukocytes. Poised to respond to invading pathogens, dendritic cells (DC) may also be exploited by T. gondii for spread in the infected host. Here, we report that human and mouse myeloid DC possess functional γ-aminobutyric acid (GABA) receptors and the machinery for GABA biosynthesis and secretion. Shortly after T. gondii infection (genotypes I, II and III), DC responded with enhanced GABA secretion in vitro. We demonstrate that GABA activates GABA(A) receptor-mediated currents in T. gondii-infected DC, which exhibit a hypermigratory phenotype. Inhibition of GABA synthesis, transportation or GABA(A) receptor blockade in T. gondii-infected DC resulted in impaired transmigration capacity, motility and chemotactic response to CCL19 in vitro. Moreover, exogenous GABA or supernatant from infected DC restored the migration of infected DC in vitro. In a mouse model of toxoplasmosis, adoptive transfer of infected DC pre-treated with GABAergic inhibitors reduced parasite dissemination and parasite loads in target organs, e.g. the central nervous system. Altogether, we provide evidence that GABAergic signaling modulates the migratory properties of DC and that T. gondii likely makes use of this pathway for dissemination. The findings unveil that GABA, the principal inhibitory neurotransmitter in the brain, has activation functions in the immune system that may be hijacked by intracellular pathogens.

Rapid cytoskeleton remodelling in dendritic cells following invasion by Toxoplasma gondii coincides with the onset of a hypermigratory phenotype.

Host cell manipulation is an important feature of the obligate intracellular parasite Toxoplasma gondii. Recent reports have shown that the tachyzoite stages subvert dendritic cells (DC) as a conduit for dissemination (Trojan horse) during acute infection. To examine the cellular basis of these processes, we performed a detailed analysis of the early events following tachyzoite invasion of human monocyte-derived DC. We demonstrate that within minutes after tachyzoite penetration, profound morphological changes take place in DC that coincide with a migratory activation. Active parasite invasion of DC led to cytoskeletal actin redistribution with loss of adhesive podosome structures and redistribution of integrins (CD18 and CD11c), that concurred with the onset of DC hypermotility in vitro. Inhibition of parasite rhoptry secretion and invasion, but not inhibition of parasite or host cell protein synthesis, abrogated the onset of morphological changes and hypermotility in DC dose-dependently. Also, infected DC, but not by-stander DC, exhibited upregulation of C-C chemokine receptor 7 (CCR7). Yet, the onset of parasite-induced DC hypermotility preceded chemotactic migratory responsesin vitro. Collectively, present data reveal that invasion of DC by T. gondii initiates a series of regulated events, including rapid cytoskeleton rearrangements, hypermotility and chemotaxis, that promote the migratory activation of DC.

Selenite induces posttranscriptional blockade of HLA-E expression and sensitizes tumor cells to CD94/NKG2A-positive NK cells.

CD94/NKG2A is an inhibitory receptor that controls the activity of a large proportion of human NK cells following interactions with the nonclassical HLA class Ib molecule HLA-E expressed on target cells. In this study, we show that selenite (SeO(3)(2-)), an inorganic selenium compound, induces an almost complete loss of cell surface expression of HLA-E on tumor cells of various origins. Selenite abrogated the HLA-E expression at a posttranscriptional level, since selenite exposure led to a dose-dependent decrease in cellular HLA-E protein expression whereas the mRNA levels remained intact. The loss of HLA-E expression following selenite treatment was associated with decreased levels of intracellular free thiols in the tumor cells, suggesting that the reduced HLA-E protein synthesis was caused by oxidative stress. Indeed, HLA-E expression and the level of free thiols remained intact following treatment with selenomethionine, a selenium compound that does not generate oxidative stress. Loss of HLA-E expression, but not of total HLA class I expression, on tumor cells resulted in increased susceptibility to CD94/NK group 2A-positive NK cells. Our results suggest that selenite may be used to potentiate the anti-tumor cytotoxicity in settings of NK cell-based immunotherapies.

Long-lasting T-cell response to SARS-CoV-2 antigens after vaccination-a prospective cohort study of HCWs working with COVID-19 patients.

BACKGROUND: Vaccination against SARS-CoV-2 reduces the risk of hospitalisation and death, but vaccine-induced IgG antibodies against the spike protein (IgG S) decline over time. Less is known about the nature of the vaccine-induced T-cell response to SARS-CoV-2 antigens. METHODS: IgG antibodies against nucleocapsid protein (IgG N), IgG S, and T-cell response towards SARS-CoV-2 antigens were determined in samples taken between November 2020 and November 2021 from a cohort of healthcare workers at an Infectious Diseases Department. RT-PCR screening for SARS-CoV-2 was encouraged once every four weeks in addition to testing when symptomatic or identified through contact tracing. Vaccination data were collected at the end of the study. RESULTS: At inclusion, T-cell response to SARS-CoV-2 antigens was found in 10/15 (66.7%) of participants with a previous/current COVID-19 infection and in 9/54 (16.7%) of participants with no prior/current history of COVID-19 infection. All participants with complete follow-up (n = 59) received two doses of a SARS-CoV-2 vaccine during the study. All participants demonstrated detectable IgG (S) antibodies at the end of the study, in median 278 days (IQR 112) after the second vaccine dose. All but four participants displayed T-cell responses towards SARS-CoV-2 antigens. IgG S antibody levels correlated with time since the second vaccine dose. In addition, previous COVID-19 infection and the strength of the S1 T-cell response correlated with IgG S antibody levels. However, no correlation was demonstrated between the strength of the T-cell response and time since the second vaccine dose. CONCLUSION: COVID-19 vaccination induces robust T-cell responses that remain for at least nine months.

Protective immunity induced by an inhaled SARS-CoV-2 subunit vaccine.

Targeting the site of infection is a promising strategy for improving vaccine effectivity. To date, licensed COVID-19 vaccines have been administered intramuscularly despite the fact that SARS-CoV-2 is a respiratory virus. Here, we aim to induce local protective mucosal immune responses with an inhaled subunit vaccine candidate, ISR52, based on the SARS-CoV-2 Spike S1 protein. When tested in a lethal challenge hACE2 transgenic SARS-CoV-2 mouse model, intranasal and intratracheal administration of ISR52 provided superior protection against severe infection, compared to the subcutaneous injection of the vaccine. Interestingly for a protein-based vaccine, inhaled ISR52 elicited both CD4 and CD8 T-cell Spike-specific responses that were maintained for at least 6 months in wild-type mice. Induced IgG and IgA responses cross-reacting with several SARS- CoV-2 variants of concern were detected in the lung and in serum and protected animals displayed neutralizing antibodies. Based on our results, we are developing ISR52 as a dry powder formulation for inhalation, that does not require cold-chain distribution or the use of needle administration, for evaluation in a Phase I/II clinical trial.

Migratory activation of primary cortical microglia upon infection with Toxoplasma gondii.

Disseminated toxoplasmosis in the central nervous system (CNS) is often accompanied by a lethal outcome. Studies with murine models of infection have focused on the role of systemic immunity in control of toxoplasmic encephalitis, while knowledge remains limited on the contributions of resident cells with immune functions in the CNS. In this study, the role of glial cells was addressed in the setting of recrudescent Toxoplasma infection in mice. Activated astrocytes and microglia were observed in the close vicinity of foci with replicating parasites in situ in the brain parenchyma. Toxoplasma gondii tachyzoites were allowed to infect primary microglia and astrocytes in vitro. Microglia were permissive to parasite replication, and infected microglia readily transmigrated across transwell membranes and cell monolayers. Thus, infected microglia, but not astrocytes, exhibited a hypermotility phenotype reminiscent of that recently described for infected dendritic cells. In contrast to gamma interferon-activated microglia, Toxoplasma-infected microglia did not upregulate major histocompatibility complex (MHC) class II molecules and the costimulatory molecule CD86. Yet Toxoplasma-infected microglia and astrocytes exhibited increased sensitivity to T cell-mediated killing, leading to rapid parasite transfer to effector T cells in vitro. We hypothesize that glial cells and T cells, besides their role in triggering antiparasite immunity, may also act as "Trojan horses," paradoxically facilitating dissemination of Toxoplasma within the CNS. To our knowledge, this constitutes the first report of migratory activation of a resident CNS cell by an intracellular parasite.

Impact of FASL-induced apoptosis in the elimination of tumor cells by NK cells.

The cytotoxic effector functions of NK cells are important for enabling the immune system to cope efficiently with infection and malignancy. Two major mechanisms of cytotoxicity are perforin/granzyme- and death receptor-mediated (e.g., FASL- or TRAIL-mediated) induction of cell death. Many studies, including studies in perforin-deficient animals, have led to the conclusion that perforin/granzyme-mediated induction of cell death is a principal pathway used by NK cells to eliminate virus-infected or transformed cells. However, death receptor-mediated apoptosis may also contribute to NK cell-mediated cytotoxicity, as revealed by more recent reports. In the present paper, we have reviewed current data on death receptor-mediated tumor cell apoptosis by NK cells with a particular emphasis on the role of NK cell FASL in the RMA/RMA-S tumor model.

Deletion of Wiskott-Aldrich syndrome protein triggers Rac2 activity and increased cross-presentation by dendritic cells.

Wiskott-Aldrich syndrome (WAS) is caused by loss-of-function mutations in the WASp gene. Decreased cellular responses in WASp-deficient cells have been interpreted to mean that WASp directly regulates these responses in WASp-sufficient cells. Here, we identify an exception to this concept and show that WASp-deficient dendritic cells have increased activation of Rac2 that support cross-presentation to CD8(+) T cells. Using two different skin pathology models, WASp-deficient mice show an accumulation of dendritic cells in the skin and increased expansion of IFNγ-producing CD8(+) T cells in the draining lymph node and spleen. Specific deletion of WASp in dendritic cells leads to marked expansion of CD8(+) T cells at the expense of CD4(+) T cells. WASp-deficient dendritic cells induce increased cross-presentation to CD8(+) T cells by activating Rac2 that maintains a near neutral pH of phagosomes. Our data reveals an intricate balance between activation of WASp and Rac2 signalling pathways in dendritic cells.

Natural killer and dendritic cell contact in lesional atopic dermatitis skin--Malassezia-influenced cell interaction.

The regulation of dendritic cells is far from fully understood. Interestingly, several recent reports have suggested a role for natural killer cells in affecting dendritic cell maturation and function upon direct contact between the cells. It is not known if this interaction takes place also in vivo, or if a potential interaction of natural killer cells and dendritic cells would be affected by allergen exposure of the dendritic cells. The yeast Malassezia can act as an allergen in atopic eczema/dermatitis syndrome, and induce maturation of dendritic cells. Our aims were to study the distribution of natural killer cells in the skin from atopic eczema/dermatitis syndrome patients with the emphasis on possible natural killer cell-dendritic cell interaction, and to assess whether the interaction of Malassezia with dendritic cells would affect subsequent interaction between dendritic cells and natural killer cells. A few scattered natural killer (CD56+/CD3-) cells were found in the dermis of healthy individuals and in nonlesional skin from atopic eczema/dermatitis syndrome patients. In lesional skin and in biopsies from Malassezia atopy-patch-test-positive skin, however, natural killer cells were differentially distributed and for the first time we could show close contact between natural killer cells and CD1a+ dendritic cells. Dendritic cells preincubated with Malassezia became less susceptible to natural-killer-cell-induced cell death, suggesting a direct effect imposed by Malassezia upon interaction of dendritic cells with natural killer cells. These findings indicate that natural killer cells and dendritic cells can interact in the skin and that Malassezia affects the interaction between natural killer cells and dendritic cells. Our data suggest that natural killer cells may play a role in regulating dendritic cells in atopic eczema/dermatitis syndrome.

Gelsolin-independent podosome formation in dendritic cells.

Podosomes, important structures for adhesion and extracellular matrix degradation, are claimed to be involved in cell migration. In addition, podosomes are also reported to be of importance in tissue remodelling, e.g., in osteoclast-mediated bone resorption. Podosomes are highly dynamic actin-filament scaffolds onto which proteins important for their function, such as matrix metallo-proteases and integrins, attach. The dynamics of the podosomes require the action of many proteins regulating actin assembly and disassembly. One such protein, gelsolin, which associates to podosomes, has been reported to be important for podosome formation and function in osteoclasts. However, podosome-like structures have been reported in gelsolin-deficient dendritic cells, but the identity of these structures was not confirmed, and their dynamics and function was not investigated. Like many other cells, dendritic cells of the immune system also form matrix degrading podosomes. In the present study, we show that dendritic cells form podosomes independently of gelsolin, that there are no major alterations in their dynamics of formation and disassembly, and that they exhibit matrix-degrading function. Furthermore, we found that gelsolin is not required for TLR4-induced podosome disassembly. Thus, the actin cytoskeleton of podosomes involved in dendritic cell extracellular matrix degradation appears to be regulated differently than the cytoskeleton in podosomes of osteoclasts mediating bone resorption.

Dendritic cell function at low physiological temperature.

Compared with the stable core temperature, the skin temperature is lower and varies depending on ambient temperature and convection conditions. The function of DCs, which are plentiful in the skin at lower physiological temperatures, has not been reported. We show that DC performed some functions normally at 28°C, including phagocytosis and macropinocytosis. TLR-4 signaling via MAPK pathways was delayed at 28°C but reached normal levels, which may explain the observed slower kinetics of stimulated macropinocytosis and TNF production. TLR-4-induced NO production was compromised severely at 28°C. Collagen degradation and migration through matrigel-coated transwell inserts were decreased, but no effect on podosome number or DC migration through noncoated transwell filters was seen. Lowering the temperature differentially regulated functions associated with the role of DCs in adaptive immunity. LPS-induced up-regulation of CD86 was normal; however, CD40 up-regulation was suppressed after TLR-4 stimulation at 28°C. Nonactivated DC processed and presented antigen on MHC class II equally well at 28°C and 37°C. However, DCs that were loaded with antigens and stimulated with TLR ligand at 28°C were poor at activating T cells at 37°C compared with DCs that were activated and loaded with antigen at 37°C.

Regulation of perforin-independent NK cell-mediated cytotoxicity.

Natural killer (NK) cells have been thought to depend largely on perforin-mediated mechanisms for the induction of cell death in targets. However, this view has more recently been challenged. It is now clear that NK cells are capable of using death ligands like Fas ligand (FasL) or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to induce cytotoxicity. Still, relatively little is known about the control of these "perforin-independent" cell death eliciting reactions, for example, the regulation of FasL expression on NK cells. In the present study, we confirm the ability of NK cells to mediate target cytotoxicity in the absence of perforin, in vivo and in vitro. We show that the induction of perforin-independent NK cell-mediated cell death is prevented by inhibiting signals mediated by MHC class I recognition. Furthermore, we demonstrate in vitro that cross-linking of the activation receptor NK1.1 on NK cells leads to the up-regulation of FasL on the cell surface. However, simultaneous engagement of an MHC class I binding inhibitory receptor prevents the externalization of FasL. These results provide a mechanistic explanation for theMHC class I-dependent regulation of perforin-independent cytotoxicity.

Efficient antigen presentation of soluble, but not particulate, antigen in the absence of Wiskott-Aldrich syndrome protein.

B cells and dendritic cells, lacking functional Wiskott-Aldrich syndrome protein (WASP), have aberrant formation of membrane protrusions. We hypothesized that protrusions may play a role in antigen presentation, and consequently, that impaired antigen presentation may be an underlying factor of the immune deficiency in patients with Wiskott-Aldrich syndrome. In this paper, we investigated the antigen presentation capacity of B cells and dendritic cells from WASP knockout mice, using soluble and particulate antigen, to CD4+ T cells from T-cell receptor transgenic DO11.10 mice. As antigen we used soluble ovalbumin (OVA), a peptide thereof (amino acids 323-339) or bacteria expressing OVA. We found that WASP-deficient B cells and dendritic cells efficiently processed and presented soluble OVA protein as well as its peptide in vitro, inducing proliferation and cytokine production from CD4+ T cells. Antigen presentation of soluble protein was efficient also in vivo, because immunization of WASP-deficient mice with OVA elicited proliferation of transferred, fluorescent-labelled, CD4+ T cells. Although we could detect uptake of bacteria in dendritic cells, processing and presentation of bacterial-expressed OVA was impaired in WASP-deficient dendritic cells. In conclusion, our data suggest that WASP is not needed for processing and presentation of soluble antigen, but that efficient presentation of particulate antigen require WASP.

Variable expression of Toll-like receptor in murine innate and adaptive immune cell lines.

Pattern recognition receptors (PRR) play an important roll in immediate responses to different conserved molecules produced by microbes. In this paper we describe the cloning of the mouse homolog of Toll-like receptor (TLR) 3, and present an analysis of the expression of this gene in innate and adaptive immune cell lines. We also performed a broad expression study on these cells of other TLR, including TLR family members whose expression pattern is not known, i.e. TLR7. The analysis was done in order to understand, and possibly predict, how innate and adaptive immune cells respond to microbial pattern antigens. This first large-scale analysis of immune cell TLR expression in the mouse reveals that cells of the innate immune system express a broader number of TLR than cells of the adaptive immune system, supporting preconceptions concerning the hierarchy of immune cells involved in direct pathogen recognition. Additionally, the expression of TLR transcripts by mast cells, neutrophils and microglial cells observed here suggests that pathogen-associated molecular pattern molecules could induce activation of these cells through TLR. Finally, the mouse homolog of human TLR3 identified here may, like its human counterpart, be an exceptional TLR molecule due to its lack of a conserved proline residue seen to be involved in existing TLR signaling capabilities found in other TLR family members.

Heat-shock proteins as activators of the innate immune system.

Peptides bound or linked to heat-shock proteins (HSPs) of microbial or mammalian origin have been shown to elicit potent antigen-specific immunity. Some members of the HSP family, such as hsp60, hsp70, hsp90 and gp96, are able also to stimulate cells of the innate immune system directly and thus, act as 'danger'-signaling molecules. This effect is independent of HSP-associated peptides and, in many respects, resembles the effect of lipopolysaccharide (LPS). Here, we discuss the similarities between the responses to HSPs and LPS and also, emphasize that care must be taken when working with preparations of HSPs in experimental settings and interpreting experimental data.

Processing and degradation of exogenous prion protein by CD11c(+) myeloid dendritic cells in vitro.

The immune system plays an important role in facilitating the spread of prion infections from the periphery to the central nervous system. CD11c(+) myeloid dendritic cells (DC) could, due to their subepithelial location and their migratory capacity, be early targets for prion infection and contribute to the spread of infection. In order to analyze mechanisms by which these cells may affect prion propagation, we studied in vitro the effect of exposing such DC to scrapie-infected GT1-1 cells, which produce the scrapie prion protein PrP(Sc). In this system, the DC efficiently engulfed the infected GT1-1 cells. Unexpectedly, PrP(Sc), which is generally resistant to protease digestion, was processed and rapidly degraded. Based on this observation we speculate that CD11c(+) DC may play a dual role in prion infections: on one hand they may facilitate neuroinvasion by transfer of the infectious agent as suggested from in vivo studies, but on the other hand they may protect against the infection by causing an efficient degradation of PrP(Sc). Thus, the migrating and highly proteolytic CD11c(+) myeloid DC may affect the balance between propagation and clearance of PrP(Sc) in the organism.

Tumor necrosis factor receptor 1-mediated signaling is required for skin cancer development induced by NF-kappaB inhibition.

NF-kappaB signaling plays an important role in skin development and epidermal growth control. Moreover, inhibition of NF-kappaB signaling in murine epidermal keratinocytes in vivo, by expression of a keratin 5 (K5)-directed superrepressor form of inhibitor of NF-kappaB (IkappaBalpha), results in an inflammatory response characterized by a massive dermal infiltration of neutrophils, epidermal hyperplasia, and a rapid development of aneuploid squamous cell carcinomas (SCC). We now show that by crossing K5-IkappaBalpha mice onto a tumor necrosis factor receptor 1(Tnfr1)-null background, both the inflammatory and the tumorigenic responses are blocked. The specificity of the block is illustrated by the fact that K5-IkappaBalpha mice lacking the IL-1 receptor type 1 (Il1r1) develop inflammation and squamous cell carcinomas. Reconstitution of lethally irradiated K5-IkappaBalpha/Tnfr1(-/-) mice with Tnfr1(+/-) bone-marrow does not induce the inflammatory or the tumorigenic phenotype, indicating a critical dependence on Tnfr1-mediated signaling in skin cells or nonimmune cells. Our results suggest a critical role of local Tnfr1-mediated signaling and associated inflammatory response cooperating with repressed keratinocyte NF-kappaB signaling in driving skin cancer development.

Enhanced dendritic cell antigen capture via toll-like receptor-induced actin remodeling.

Microbial products are sensed through Toll-like receptors (TLRs) and trigger a program of dendritic cell (DC) maturation that enables DCs to activate T cells. Although an accepted hallmark of this response is eventual down-regulation of DC endocytic capacity, we show that TLR ligands first acutely stimulate antigen macropinocytosis, leading to enhanced presentation on class I and class II major histocompatibility complex molecules. Simultaneously, actin-rich podosomes disappear, which suggests a coordinated redeployment of actin to fuel endocytosis. These reciprocal changes are transient and require p38 and extracellular signal-regulated kinase activation. Thus, the DC actin cytoskeleton can be rapidly mobilized in response to innate immune stimuli to enhance antigen capture and presentation.