Notch signalling regulates cytokine production by CD8+ and CD4+ T cells.

The Notch signalling pathway regulates several aspects of cellular differentiation such as T lineage commitment and effector functions on peripheral T cells; however, there is limited information regarding Notch receptor expression on different T cell subsets and the putative role of the different receptors on T cell effector function. Here, we studied the protein expression of Notch receptors on murine T cells in vitro and in vivo and analysed the role of the Notch pathway in cytokine production by CD4+ and CD8+ T cells. We found that resting CD4+ and CD8+ T cells do not express Notch receptors, but they upregulate Notch 1 and Notch 2 shortly after in vitro and in vivo activation. Using a γ-secretase inhibitor, which blocks Notch signalling through all Notch receptors, we demonstrated that the Notch pathway regulates IL-10 production by CD4+ T cells and IFN-γ and IL-17 production by CD8+ T cells. These results suggest that Notch 1 and 2 are expressed by CD4+ and CD8+ T cells and represent the putative Notch receptors that regulate effector functions and cytokine production by these cells.

Nanosecond pulsed electric field (nsPEF) and vaccines: a novel technique for the inactivation of SARS-CoV-2 and other viruses?

Since the beginning of 2020, worldwide attention has been being focussed on SARS-CoV-2, the second strain of the severe acute respiratory syndrome virus. Although advances in vaccine technology have been made, particularly considering the advent of mRNA vaccines, up to date, no single antigen design can ensure optimal immune response. Therefore, new technologies must be tested as to their ability to further improve vaccines. Nanosecond Pulsed Electric Field (nsPEF) is one such method showing great promise in different biomedical and industrial fields, including the fight against COVID-19. Of note, available research shows that nsPEF directly damages the cell's DNA, so it is critical to determine if this technology could be able to fragment either viral DNA or RNA so as to be used as a novel technology to produce inactivated pathogenic agents that may, in turn, be used for the production of vaccines. Considering the available evidence, we propose that nsPEF may be used to produce inactivated SARS-CoV-2 viruses that may in turn be used to produce novel vaccines, as another tool to address 20 the current COVID-19 pandemic.Key MessagesViral inactivation by using pulsed electric fields in the nanosecond frequency.DNA fragmentation by a Nanosecond Pulsed Electric Field (nsPEF).Opportunity to apply new technologies in vaccine development.

Differential regulation of Notch ligands in dendritic cells upon interaction with T helper cells.

The Notch signalling pathway has recently been linked to T helper 1 (Th1)/T helper 2 (Th2) cell polarization via a mechanism involving differential expression of Notch ligands, Delta-like and Jagged, in antigen-presenting cells. However, whether stimuli other than pathogen-derived factors are involved in the regulation of Notch ligand expression in dendritic cells (DCs) remains unknown. Here, we address the effect of T helper cells (Th1 and Th2) on Delta-like 4 and Jagged 2 expression in bone marrow-derived DCs. We demonstrate that both Th1 and Th2 cells induce Delta-like 4 mRNA expression in DCs, in a process that is, in part, mediated by CD40 signalling. In contrast, only Th2 cells induce a significant increase in Jagged 2 mRNA levels in DCs. Additionally, we show that IL-4, a hallmark Th2 cytokine, plays a role in Jagged 2 expression, as evidenced by the fact that cholera toxin, a Th2-promoting stimulus, induces Jagged 2 mRNA expression in DCs only in the presence of IL-4. Finally, we demonstrate that DCs also express Notch 1 and that this expression is downregulated by IL-4. These data suggest that Notch ligands are differentially regulated in DCs: Delta-like 4 is regulated by T helper cells and by pathogen-derived Th1 stimuli, whereas Jagged 2 is regulated by Th2 cells and pathogen-derived Th2-promoting stimuli. Based on our results, we propose that the positive feedback loop that Th2 cells exert on T cell polarization may involve the induction of Jagged 2 expression in DCs.

Alemtuzumab induction in kidney transplantation: clinical results and impact on T-regulatory cells.

Alemtuzumab (ALT), a humanized monoclonal anti-CD52 antibody, was introduced in solid organ transplantation as an induction agent. ALT associated with anticalcineurins has provided a low incidence of acute rejection episodes (ARE) and potential tolerogenic properties. We analyzed the clinical outcomes and effects on peripheral Treg of renal transplant recipients treated with ALT. Six-month data on kidney alone or kidney combined with pancreas or liver patients treated with ALT and tacrolimus (TAC) in standard doses were compared with those on renal transplant recipients of similar demography who were not treated with ALT. We evaluated patient and graft survivals, ARE incidence, hematological parameters, renal function, adverse events, and CD4+CD25+FoxP3+ T cells in peripheral blood. Demographics of recipients, donors, and transplants were similar in both groups. Mean HLA mismatch was slightly greater among ALT-treated patients (3.5 vs 2.5). No combined transplantation was performed in the ALT-untreated group. Patient and graft survivals were 100% without rejection or serious infections in both groups. ALT-treated recipients showed anemia and leukopenia in 3 patients as well as severe lymphopenia in 5 recipients, who partially recovered on day 90. Final mean plasma creatinine was 1.4 mg/dL, while calculated creatinine clearance was approximately 65 mL/min in both groups. Mean Treg cell percentage was higher among ALT-treated recipients than the comparative group or healthy controls (P < .05). In conclusion, renal transplantation results obtained using ALT with rigorous immunosuppressive therapy were excellent; serious adverse events and acute rejection were absent. The effect of the increased proportion of Treg cells must be evaluated with longer observation.

Coexpression of two fibronectin receptors, VLA-4 and VLA-5, by immature human erythroblastic precursor cells.

Human erythroblastic precursor cells adhere to fibronectin (Fn) but the exact nature of the receptors mediating this interaction has not been characterized. In this study, we report data showing that immature human erythroblasts express the integrins VLA-4 and VLA-5 and that both these molecules act as fibronectin receptors on these cells. We have recently demonstrated that adhesion to Fn of purified human CFU-E and their immediate progeny preproerythroblasts was inhibited by antibodies directed against the human fibronectin receptor (VLA-5). Here we have extended those results and characterized by immunoprecipitation with specific antibodies the integrins expressed on surface-labeled normal human immature erythroblasts. A polyclonal antibody recognizing the common VLA beta 1 subunit yielded two polypeptides of 120 and 160 kD. Our data further demonstrate that the polypeptide of 160 kD contains alpha subunits corresponding to both alpha 4 and alpha 5. Thus, erythroblast lysates prepared in 0.3% CHAPS and immunoprecipitated with antibodies which specifically recognize the alpha 4 subunit showed a heterodimer with peptides of 120 (beta 1) and 160 kD (alpha 4) and the additional peptides of 70 and 80 kD which usually coprecipitate with the alpha 4 chain. On the other hand, specific anti-alpha 5 antibodies immunoprecipitated an alpha 5/beta 1 complex with peptides of 120 and 160 kD which under reducing conditions migrated as a single band of 130 kD. Similar experiments performed with an erythroleukemic cell line (KU 812) showed that these cells also coexpress both the VLA-4 and VLA-5 members of the integrin family. Furthermore, monoclonal antibodies recognizing the VLA alpha 4 chain blocked the adhesion of immature erythroblasts to Fn-coated surfaces, thus demonstrating that, as VLA-5, VLA-4 is also a functional Fn receptor on these cells.

Generation of dendritic cells with regulatory properties.

Dendritic cells (DCs) are professional antigen presenting cells with the ability to induce and regulate an immune response. DCs that capture and present antigen under noninflammatory conditions maintain an immature phenotype and acquire tolerogenic properties. These DCs generate regulatory T lymphocytes that potentiate tolerogenic responses. Here we developed a method for the generation of immature murine DCs able to process and present a specific antigen in a tolerogenic context. Immature DCs were prepared from bone marrow precursors after differentiation with granulocyte-macrophage colony-stimulating factor (GM-CSF) in the presence of vitamin D(3) and characterized by their low expression of major histocompatibility complex class (MHC) II and CD86 molecules. Purified phagosomes containing either MHC II molecules or ovalbumin were used to deliver antigens to immature DCs. More than 80% of the DCs captured the phagosomes, while maintaining a low expression of maturation markers and showing basal levels of secretion of activating cytokines such as interleukin (IL)-2 and IL-12. Treatment of the immature DCs with lipopolysaccharides (LPS) increased IL-10 secretion, in agreement with their anti-inflammatory and immune regulatory properties. Cocultures of transgenic OT-II T lymphocytes with the immature DCs carrying OVA-phagosomes succeeded in generating a subpopulation of regulatory T lymphocytes characterized by the expression of CD4, CD25, CD62L, and Foxp3. Taken together, our results suggest that vitamin D(3) generates immune tolerance through the modulation of DC phenotype and could be useful to induce tolerance to allotransplants.

Highly purified sarcoplasmic reticulum vesicles are devoid of Ca2+-independent ('basal') ATPase activity.

On solubilization with Triton X-100 of sarcoplasmic reticulum vesicles isolated by differential centrifugation, the Ca2+-ATPase is selectively extracted while approximately half of the initial Mg2+-, or 'basal', ATPase remain in the Triton X-100 insoluble residue. The insoluble fraction, which does not contain the 100 000 dalton polypeptide of the Ca2+-ATPase, contains high levels of cytochrome c oxidase. Furthermore, its Mg2+-ATPase activity is inhibited by specific inhibitors of mitochondrial ATPase, indicating that the 'basal' ATPase separated from the Ca2+-ATPase by detergent extraction originates from mitochondrial contaminants. To minimize mitochondrial contamination, sarcoplasmic reticulum vesicles were fractionated by sedimentation in discontinuous sucrose density gradients into four fractions: heavy, intermediate and light, comprising among them 90-95% of the initial sarcoplasmic reticulum protein, and a very light fraction, which contains high levels of Mg2+-ATPase. Only the heavy, intermediate and light fractions originate from sarcoplasmic reticulum; the very light fraction is of surface membrane origin. Each fraction of sarcoplasmic reticulum origin was incubated with calcium phosphate in the presence of ATP and the loaded fractions were separated from the unloaded fractions by sedimentation in discontinuous sucrose density gradients. It was found that vesicles from the intermediate fraction had, after loading, minimal amounts of mitochondrial and surface membrane contamination, and displayed little or no Ca2+-independent basal ATPase activity. This shows conclusively that the basal ATPase is not an intrinsic enzymatic activity of the sarcoplasmic reticulum membrane, but probably originates from variable amounts of mitochondrial and surface membrane contamination in sarcoplasmic reticulum preparations isolated by conventional procedures.

The human SRY protein is present in fetal and adult Sertoli cells and germ cells.

Sex determination in mammals is controlled by the Y chromosome located SRY gene. Despite recent advances towards understanding the mechanisms that regulate sex determination in mammals, the expression profile of the SRY protein in human tissues is unknown. To localize the SRY protein and determine its cellular distribution, we prepared monoclonal antibodies (mAb) against the recombinant SRY protein. One antibody, LSRY1.1, recognizes a SRY-specific epitope and was used to localize the protein in different cells and tissues. The mAb recognizes a protein of 27 kDa in total lysates of HeLa SRYB3 cells. Immunocytochemical staining showed a nuclear localization of the protein. Immunohistochemical studies performed on gonadal tissue of a fetus, a one month-old boy and an adult man, demonstrated the presence of SRY protein in the nucleus of Sertoli and germ cells. In addition two 46,XX SRY(+) males had the SRY protein in their gonadal tissues. All other samples were negative, including all female tissue studied and the testis of a 46,XX SRY(-) male. The presence of SRY protein in fetal and adult gonadal tissues including germ cells suggests that SRY may have other male-specific functions in addition to sex determinism.

Lymphocyte adhesion to endothelium derived from human lymphoid tissue.

The development of an efficient immune response depends on the capacity of antigen-specific lymphocytes to migrate into secondary lymphoid organs. The first step in the process of lymphocyte extravasation involves lymphocyte binding to the vascular endothelium. Although several adhesion receptors have been implicated in the migration of lymphocytes to inflamed tissue, their role in the extravasation of these cells to normal lymphoid organs is not yet clearly established. The involvement of adhesion molecules in lymphocyte entrance to secondary lymphoid organs can be better assessed in an in vitro system using endothelial cells in culture. Here we report on the isolation and culture of a homogeneous population of adherent cells of endothelial origin derived from human tonsils (TEC) and on adhesion studies performed with these cells. Beginning from primary cultures of human tonsils, we isolated a population of cells that we show by FACScan analysis to present the intracellular endothelial cell marker Von Willebrand factor and LVAP-2, a surface molecule present in venules from lymphoid organs. The cells are negative for FDC, IDC and macrophage markers. They express ICAM-1, VCAM-1 and CD40 both constitutively and in inducible forms and are induced by IFN-gamma to express major histocompatibility complex class II antigens. As opposed to endothelial cells from human umbilical cord (HUVEC), they do not need to be activated by cytokines to bind lymphoid cells via VLA-4. The mAb HP2/1 directed to the integrin VLA-4 blocks adhesion of Ramos and Daudi cells to tumor necrosis factor alpha (TNF-alpha)-treated HUVEC and to untreated TEC but not of tonsil-derived MNC. On the other hand, an anti-VCAM-1 antibody that blocks adhesion of Ramos and Daudi cells to TNF-alpha-treated HUVEC, does not block adhesion of these cells to TEC, suggesting the presence on the tonsillar endothelial cells of a ligand for VLA-4 different from VCAM-1. We show here that this ligand is not fibronectin.

Spleen-derived stromal cells. Adhesion molecules expression and lymphocyte adhesion to reticular cells.

The final steps of lymphocyte differentiation occur in secondary lymphoid organs where B and T lymphocytes interact with the lymphoid microenvironment. Although numerous studies describe the interactions of murine lymphocytes with dendritic, follicular and other antigen presenting cells, little is known on the interactions between lymphocytes and reticular cells, an important cellular component of spleen stroma. In this work we describe the culturing of complete murine spleen stromas and of two cell lines, Sp-1 and Sp-2, identified as of possible reticular origin, and describe the adhesive interactions between murine lymphocytes and human lymphoid cells with murine spleen stromal cells. FACS analysis indicates that the Sp-1 cell line shows a single cell type expressing VCAM-1 and CD44 constitutively. They do not express any of the markers described for follicular cells, interdigitating cells, macrophages or endothelial cells. Our data suggests that these cells represent a population of spleen reticular cells. The Sp-2 cell line shows two phenotypically different cell types that grow in association. FACS analysis demonstrates that both cell types express VCAM-1 and CD44 constitutively, but that they can be differentiated by the expression of CD11b and FcR. These data suggest that the Sp-2 cell line is composed of one type of stromal cell growing over an adherent layer of reticular cells. Furthermore, analysis of the non-B non-T cell fraction prepared from murine spleen shows that approximately 30% of these cells correspond to the CD44/VCAM-1 double positive cells. Murine B and T cells adhere to the complete stromas and to Sp-1 and Sp-2 cell lines. Activation of B cells with LPS had no effect on binding while binding of T cells to complete stromas increased up to threefold after Con-A treatment. Adhesion of human lymphoblastoid Daudi cells to complete spleen stromas is blocked by an anti-(murine) VCAM-1 antibody but not by an antibody to the (human) integrin alpha 4 subunit, while adhesion to the Sp-1 and Sp-2 stromas is blocked by antibodies against both molecules. Also, adhesion of Ramos cells to Sp-2 stromas is inhibited by antibodies to the integrin alpha 4 subunit and to murine VCAM-1. Antibodies to other adhesion receptors such as the integrin beta 2 subunit, ICAM-1 or CD44 have no effect on human cell binding to these stromas. Our results suggest that we have isolated a fraction of splenic reticular cells and that these cells can be cultured as a distinct cell line. The finding that these cells express CD44 and VCAM-1 constitutively and use some of these molecules for lymphocyte binding suggests that spleen reticular cells may be involved in the regulation of normal lymphocyte traffic through the spleen.

Alzheimer's disease: microtubule-associated proteins 2 (MAP 2) are not components of paired helical filaments.

In Alzheimer's disease, the most characteristic neuropathological changes are the formation of neurofibrillary tangles (NFT) and neuritic plaques (NP) characterized by the presence of bundles of paired helical filaments (PHF) that accumulate in the degenerating neurites and neuronal cell bodies. Although the protein composition of the PHF is ill-defined, a number of microtubule-associated proteins have been implicated in these lesions. Here we report results with an antiserum monospecific for the microtubule-associated protein MAP 2 which does not cross-react with any other microtubular protein. Immunostaining with this antibody of sections from an Alzheimer's brain show a strong reactivity with NFT but no reactivity at the level of the NP. On the other hand, immunostaining of Alzheimer's brain sections with another antibody specific for the microtubule-associated protein tau shows strong staining of PHF on both NFT and NP. These findings confirm the presence of the tau proteins in the PHF and strongly suggest that MAP 2 may not be a main structural component of the PHF. Labelling of NFT with the anti-MAP 2 antiserum suggests a non-specific binding of MAP 2 to the PHF during the process of NFT formation.

Functional consequences of immune cell adhesion to endothelial cells.

Research regarding the interactions between the endothelium and immune cells has undergone a significant expansion during the past decade. Major shifts of emphasis have been the norm, from the production of a detail catalog of the cell surface receptors and counter-receptors acting at the interface between the vascular endothelium and circulating cells to a more mechanistic account of leukocyte/endothelium interactions. The past five years has seen new, groundbreaking developments in the field, with exiting studies aimed at understanding the functional consequences of the direct contact of endothelial cells and leukocytes. Based on early work to be discussed below, new data on local chemokine production and cell-to-cell contacts, attempt to clarify the physiopathological significance of these events. The exceptional anatomical arrangement of endothelial cells insures a permanent contact of the endothelium with leukocytes, an event likely to result in cellular signals originating from direct cell contact or through the action of soluble factors produced by endothelial cells or immune cells. As we will discuss, current evidence supports the idea that endothelial cells present at vascular endothelium as well as at specialized high endothelial venules, play not only a critical role in the homing and recruitment of immune cells but that it can also influence the outcome of the immune response. Additionally, new evidence clearly corroborates the idea that B and T lymphocytes as well as NK cells can modulate endothelial cell function.

A flow cytometric procedure for the quantification of cell adhesion in complex mixtures of cells.

We present a simple non-radioactive cytometry-based assay that permits the simultaneous quantitation of cell adhesion of distinct subsets of cells contained in a mixture without any previous fractionation. The procedure is simple and highly reproducible and has the advantage of confining the quantitation of cell adhesion to live cells only. This new approach is based on counting the absolute number of cells. This is done by adding known numbers of distinguishable beads to the cell suspension and counting beads and cells in a cytometer. Quantitation of adhesion is accomplished by counting each subpopulation of cells before and after the adhesive process. To illustrate this methodology we determined adhesion of Ramos cells to monolayers of endothelial cells and its inhibition by specific antibodies. Also, we determined adhesion to endothelial cells of B lymphocytes and subsets of T lymphocytes present in a preparation of unfractionated human mononuclear cells. The results presented here demonstrate that the new assay has the required properties to be used in the quantitation of cell adhesion.

Cyclosporine preconditions dendritic cells during differentiation and reduces IL-2 and IL-12 production following activation: a potential tolerogenic effect.

The mode of action of cyclosporine (CsA) has been ascribed to its capacity to inhibit IL-2 and IFNgamma production by T cells, two cytokines implicated in allograft rejection. Recently, it has been reported that upon activation, dendritic cells (DCs) exhibit transient production of IL-2, a property that appears to be related to their capacity to initiate immune responses. On the other hand, DCs can generate signals determining Th1/Th2 polarizing effects, an effect that can drastically influence the outcome of organ transplant. The purpose of the present study was to investigate the effect of CsA on cytokine production by immature and mature DCs. DC precursors from mouse bone marrow were induced to differentiate by incubation with GM-CSF for 5 days followed by activation with LPS for 4 hours. CsA was added at different times during this process. Our results show that when CsA is added during the differentiation period following activation with LPS, IL-2 and IL-12 secretion are significantly reduced without affecting the evolution of the DC. Conversely, CsA had no effect when added during the LPS activation period. These results show that CsA affects DCs before they receive the final activation stimulus, preconditioning them to antigen stimulation. This preconditioning of DCs by calcineurin-inhibiting drugs conceptually integrates the mode of action of CsA with the tolerogenic and T-cell polarization function ascribed to DCs. These results may be especially meaningful for the future design of immunosuppressive protocols.

Delivery of alloantigens via apoptotic cells generates dendritic cells with an immature tolerogenic phenotype.

BACKGROUND: Dendritic cells (DCs) are professional antigen-presenting cells able to induce immunity or tolerance. The interactions of immature DCs with naive T lymphocytes induce peripheral tolerance through mechanisms that include anergy or deletion of lymphocytes or the generation of regulatory T cells. Because of the central role of DCs in the immune response, they are potential targets for the induction of experimental tolerance. Thus, the generation of immature (tolerogenic) DCs able to capture and present alloantigens to T cells represents an important aim in our efforts to achieve better transplant acceptance. METHODS: In this work, we generated immature DCs by using vitamin D(3) (VD3) during the process of DC differentiation. RESULTS: The VD3-DCs showed an immature phenotype characterized by a low expression of major histocompatibility complex antigens of class II, CD86, and CD80 molecules and the secretion of a tolerogenic cytokine pattern. Furthermore, we showed that VD3-DCs phagocytose apoptotic allogeneic cells efficiently without inducing DC maturation or activation. Most important, our experiments demonstrated that mice treated with VD3 produce immature DCs in vivo, and that DCs from VD3-treated mice immunized with allogeneic apoptotic cells maintained their tolerogenic phenotype. CONCLUSION: Our results show that allogeneic apoptotic cells in combination with VD3 generate DCs with tolerogenic characteristics that could be used to induce tolerance towards alloantigens.

Retinoic acid generates regulatory T cells in experimental transplantation.

Regulatory T cells play a key role to inhibit effector lymphocytes, avoid, autoimmunity, and restrain allogeneic immunity. Retinoic acid is an important cofactor that stimulates the generation and expansion of regulatory T cells. Naive T cells, coincubated with allogeneic antigen-presenting cells and retinoic acid, in conjunction with transforming growth factor (TGF) ß and interleukin (IL) 2, generated allogeneic regulatory T cells de novo. These cells were able to inhibit skin rejection in adoptive transfer experiments. The generation of regulatory T cells ex vivo with retinoic acid, TGF-ß, and IL-2 represents a new step toward specific regulation of allogeneic immune responses.

Dendritic cells and B cells cooperate in the generation of CD4(+)CD25(+)FOXP3(+) allogeneic T cells.

BACKGROUND: CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) play an essential role in immune tolerance, suppressing responses against self-antigens. Additionally, Treg play an important role in maintaining immunosuppression to alloantigens as well as to other antigens. It is well known that in the gut, a subset of dendritic cells produces retinoic acid (RA), which together with transforming growth factor (TGF-beta) is able to differentiate naïve T cells into Treg. The aim of this study was to establish the role of antigen-presenting cells (APC) in the differentiation of allogeneic Tregs under the effect of RA and TGF-beta. METHODS: Splenic CD4(+)CD25(-) naïve T cells from C57BL/6 mice were co-cultured with splenic CD11c-enriched APC from Balb/c mice in the presence of TGF-beta, RA, and interleukin (IL-2). After 6 days of culture, cells were analyzed for the expression of Foxp3 by flow cytometry. Additionally, we investigated the role of B cells and dendritic cells (DCs) and their stimulatory capacity in the generation of Tregs. RESULTS: Our results showed that co-culture of naive T cells with the appropriate level of stimulation by APC in the presence of TGF-beta, RA, and IL-2 provided a new powerful approach to generate allogeneic Treg cells. We demonstrated that although B cells and DCs can generate Tregs by themselves, a mixure of both APC improved their capacity to efficiently generate Tregs. Also, we observed that although the addition of IL-2 to the cultures was not crucial to generate Tregs, it was required to optimize their expansion and cell survival.