Glass-supported lipid bilayers presenting freely diffusing proteins have served as a powerful too... more Glass-supported lipid bilayers presenting freely diffusing proteins have served as a powerful tool for studying cell-cell interfaces, in particular, T cell-antigen presenting cell (APC) interactions, using optical microscopy. Here we expand upon existing protocols and describe the preparation of liposomes by an extrusion method, and describe how this system can be used to study immune synapse formation by Jurkat cells. We also present a method for forming such lipid bilayers on silica beads for the study of signaling responses by population methods, such as western blotting, flow cytometry, and gene-expression analysis. Finally, we describe how to design and prepare transmembrane-anchored protein-laden liposomes, following expression in suspension CHO (CHOs) cells, a mammalian expression system alternative to insect and bacterial cell lines, which do not produce mammalian glycosylation patterns. Such transmembrane-anchored proteins may have many novel applications in cell biology and immunology.
TCR-dependent signaling events have been observed to occur in TCR microclusters. We found that so... more TCR-dependent signaling events have been observed to occur in TCR microclusters. We found that some TCR microclusters are present in unstimulated murine T cells, indicating that the mechanisms leading to microcluster formation do not require ligand binding. These pre-existing microclusters increase in absolute number following engagement by low-potency ligands. This increase is accompanied by an increase in cell spreading, with the result that the density of TCR microclusters on the surface of the T cell is not a strong function of ligand potency. In characterizing their composition, we observed a constant number of TCRs in a microcluster, constitutive exclusion of the phosphatase CD45, and preassociation with the signaling adapters linker for activation of T cells and growth factor receptor-bound protein 2. The existence of TCR microclusters prior to ligand binding in a state that is conducive for the initiation of downstream signaling could explain, in part, the rapid kinetics with which TCR signal transduction occurs.
The molecular mechanisms governing severe acute respiratory syndrome coronavirus-induced patholog... more The molecular mechanisms governing severe acute respiratory syndrome coronavirus-induced pathology are not fully understood. Virus infection and some individual viral proteins, including the 3a protein, induce apoptosis. However, the cellular targets leading to 3a protein-mediated apoptosis have not been fully characterized. This study showed that the 3a protein modulates the mitochondrial death pathway in two possible ways. Activation of caspase-8 through extrinsic signal(s) caused Bid activation. In the intrinsic pathway, there was activation of caspase-9 and cytochrome c release from the mitochondria. This was the result of increased Bax oligomerization and higher levels of p53 in 3a protein-expressing cells, which depended on the activation of p38 MAP kinase (MAPK) in these cells. For p38 activation and apoptosis induction, the 3a cytoplasmic domain was sufficient. In direct Annexin V staining assays, the 3a protein-expressing cells showed increased apoptosis that was attenuated with the p38 MAPK inhibitor SB203580. A block in nuclear translocation of the STAT3 transcription factor in cells expressing the 3a protein was also observed. These results have been used to present a model of 3a-mediated apoptosis.
Purpose of review To describe recent findings on the effect of HIV/SIV infection on lymph node vi... more Purpose of review To describe recent findings on the effect of HIV/SIV infection on lymph node viral and T-cell dynamics using imaging-based methodologies. Recent findings Chronic infection, particularly HIV/SIV, alters dramatically the microenvironment, immune cell frequency, distribution, function and tissue organization of secondary lymphoid tissues. These changes are not always reversible. Over the past few years, the implementation of advanced imaging protocols on human lymph node biopsies as well as on longitudinal lymphoid tissues samples from nonhuman primates (NHP) have provided a wealth of information on how local immune responses evolve over time in response to a persisting retroviral pathogen. Most of the information concerns cytotoxic and helper T cells and viral dynamics. In this review, we detail this information focusing on HIV/SIV infection. We also comment on the gaps that imaging technologies have bridged in our understanding and discuss the translational value of these new findings in the light of emerging therapeutic agendas. Summary Novel imaging platforms allow for dissecting the spatiotemporal dynamics of immune interactions further improving our understanding of the interplay between virus and host and providing important information for designing successful preventive and curative strategies.
Early secretion of IL-12 by mouse dendritic cells (DCs) instructs T cells to make IFN-g. However,... more Early secretion of IL-12 by mouse dendritic cells (DCs) instructs T cells to make IFN-g. However, only activated, but not naive T cells are able to license DCs for IL-12 production. We hypothesized that it might be due to different levels of CD40L expression on the surface of these cells, as CD40 signals are required for IL-12 production. Using quantitative cell-free systems incorporating CD40L in lipid bilayers combined with total internal reflection fluorescence microscopy and flow cytometry, we show that as low as ∼200 CD40L molecules/mm 2 in combination with IL-4 is sufficient to induce IL-12 production by DCs. Remarkably, CD40L alone is adequate to induce IL-23 secretion by DCs. Thus, although activated T cells have somewhat higher levels of CD40L, it is the combination of CD40L and the cytokines they secrete that licenses DCs and influences the effector class of the immune response.
The orf3a (also called X1 or U274) gene is the largest unique open reading frame in the severe ac... more The orf3a (also called X1 or U274) gene is the largest unique open reading frame in the severe acute respiratory syndrome coronavirus genome and has been proposed to encode a protein with three transmembrane domains and a large cytoplasmic domain. Recent work has suggested that the 3a protein may play a structural role in the viral life cycle, although the mechanisms for this remain uncharacterized. Here, the expression of the 3a protein in various in vitro systems is shown, it has been localized to the Golgi region and its membrane topology in transfected cells has been confirmed. Three potential caveolin-1-binding sites were reported to be present in the 3a protein. By using various biochemical, biophysical and genetic techniques, interaction of the 3a protein with caveolin-1 is demonstrated. Any one of the potential sites in the 3a protein was sufficient for this interaction. These results are discussed with respect to the possible roles of the 3a protein in the viral life cycle.
The interaction of T cells with antigen-presenting cells is the hallmark of adaptive immunity. In... more The interaction of T cells with antigen-presenting cells is the hallmark of adaptive immunity. In vitro studies have described the formation of an immunological synapse between these cells, and intra-vital imaging has described in great detail the dynamics of these interactions. The immunological synapse has become a paradigm to study signals exchanged between the two cells. A wealth of information has been amassed regarding the localization of signalling molecules, their kinetics and the transcription factors they activate. We continue to discover mechanisms that cause receptors and signalling molecules to compartmentalize in the cell; however, the emerging challenge lies in understanding how the immunological synapse contributes to differentiation. Here, we review some of the transcription factors activated downstream of T-cell receptor signalling and discuss mechanisms by which antigen dose and affinity may influence differentiation. Antigen affinity might change the kind of transcription factors that are activated whereas antigen dose is likely to influence the temporal dynamics of the transcription factors. The immunological synapse is therefore likely to influence differentiation by modulating the trafficking of transcription factors and by promoting asymmetric cell division, an emerging concept.
Background What determines the development of germinal centers (GCs) in lymph nodes (LNs) early a... more Background What determines the development of germinal centers (GCs) in lymph nodes (LNs) early after HIV/SIV infection is likely to be crucial for developing neutralizing antibodies and B cell memory. Materials and Methods Rhesus macaques were inoculated i.v. with either SIVmac239 (n=2) or iMac239ΔD385 (iMacΔD) (n=4), a CD4-independent variant of SIVmac239 lacking a CD4 binding site (J. Virol. 90:4966, 2016). LNs from different time points were analyzed using polychromatic flow cytometry and multiplexed confocal imaging. Results In contrast to SIVmac239, iMacΔD infection was associated with relative preservation of total and CD28hiCD95hi memory CD4 T cells, consistent with its compromised capacity to target CD4 T cells. Imaging analysis revealed accelerated formation of mature, polarized GCs in iMacΔD compared to SIVmac239 infected animals, a profile associated with i) increased frequencies of PD-1hiBcl-6hi CD4 T cells, ii) higher levels of IL-10 and IL-21 within the follicles, iii) increased frequency of PH2AX+ (a required factor for isotype switching) B cells, iv) lower levels of Fas-L in the follicular areas, associated with lower TUNEL (cell death) positivity and v) lower accumulation of CD163+ cells (monocytes/macrophages; a surrogate of local inflammation) in proximity to follicles. The observed GC dynamics associate with earlier virus-specific antibody responses in iMacΔD compared to SIVmac239. Conclusions Our data indicate that CD4 tropism of SIV greatly affects the development of GC reactions early after infection. The iMacΔD model could provide critical information on how GC reactions can be manipulated to augment the generation of neutralizing antibodies and B cell memory.
Induction of immediate early genes (like Fos, Jun) in response to T cell receptor stimulation wit... more Induction of immediate early genes (like Fos, Jun) in response to T cell receptor stimulation with different MHC-peptide (MHC-p) dose and potency is not well understood. Our aim is to study the kinetics of c-Fos induction in T cells in response to antigen dose and potency. Our model system is AND TCR transgenic mice, whose T cells are specific for moth Cytochrome-C (MCC) peptide. We study the impact of MHC-p potency using altered peptide ligands (APLs). To study c-Fos induction we crossed AND mice to c-Fos-EGFP transgenic mice that express a fusion protein of c-Fos and EGFP driven by the Fos promoter. We observed that upon stimulation of TCR, expression of c-Fos starts after ~30 min, peaks at 3hrs and plateaus for another 2hrs. Strong agonists induce c-Fos even without co-stimulatory molecules where as weak agonists fail to do so. Co-stimulation enhances the percentage of cells responding as well as the level of c-Fos expression. Highering or lowering the peptide dose reduces the ex...
Purpose of review To describe recent findings on the effect of HIV/SIV infection on lymph node vi... more Purpose of review To describe recent findings on the effect of HIV/SIV infection on lymph node viral and T-cell dynamics using imaging-based methodologies. Recent findings Chronic infection, particularly HIV/SIV, alters dramatically the microenvironment, immune cell frequency, distribution, function and tissue organization of secondary lymphoid tissues. These changes are not always reversible. Over the past few years, the implementation of advanced imaging protocols on human lymph node biopsies as well as on longitudinal lymphoid tissues samples from nonhuman primates (NHP) have provided a wealth of information on how local immune responses evolve over time in response to a persisting retroviral pathogen. Most of the information concerns cytotoxic and helper T cells and viral dynamics. In this review, we detail this information focusing on HIV/SIV infection. We also comment on the gaps that imaging technologies have bridged in our understanding and discuss the translational value of these new findings in the light of emerging therapeutic agendas. Summary Novel imaging platforms allow for dissecting the spatiotemporal dynamics of immune interactions further improving our understanding of the interplay between virus and host and providing important information for designing successful preventive and curative strategies.
Peptide immunogens provide an approach to focus antibody responses to specific neutralizing sites... more Peptide immunogens provide an approach to focus antibody responses to specific neutralizing sites on the HIV envelope protein (Env) trimer or on other pathogens. However, the physical characteristics of peptide immunogens can limit their pharmacokinetic and immunological properties. Here, we have designed synthetic "star" nanoparticles based on biocompatible N-[(2-hydroxypropyl)methacrylamide] (HPMA)-based polymer arms extending from a poly(amidoamine) (PAMAM) dendrimer core. In mice, these star nanoparticles trafficked to lymph nodes (LNs) by 4 hours following vaccination, where they were taken up by subcapsular macrophages and then resident dendritic cells (DCs). Immunogenicity optimization studies revealed a correlation of immunogen density with antibody titers. Furthermore, the co-delivery of Env variable loop 3 (V3) and T-helper peptides induced titers that were 2 logs higher than if the peptides were given in separate nanoparticles. Finally, we performed a nonhuman primate (NHP) study using a V3 glycopeptide minimal immunogen that was structurally optimized to be recognized by Env V3/glycan broadly neutralizing antibodies (bnAbs). When administered with a potent Toll-like receptor (TLR) 7/8 agonist adjuvant, these nanoparticles elicited high antibody binding titers to the V3 site. Similar to human V3/ glycan bnAbs, certain monoclonal antibodies (mAbs) elicited by this vaccine were glycan dependent or targeted the GDIR peptide motif. To improve affinity to native Env trimer affinity, nonhuman primates (NHPs) were boosted with various SOSIP Env proteins; however, significant neutralization was not observed. Taken together, this study provides a new
Early secretion of IL-12 by mouse dendritic cells (DCs) instructs T cells to make IFN-γ. However,... more Early secretion of IL-12 by mouse dendritic cells (DCs) instructs T cells to make IFN-γ. However, only activated, but not naive T cells are able to license DCs for IL-12 production. We hypothesized that it might be due to different levels of CD40L expression on the surface of these cells, as CD40 signals are required for IL-12 production. Using quantitative cell-free systems incorporating CD40L in lipid bilayers combined with total internal reflection fluorescence microscopy and flow cytometry, we show that as low as ∼200 CD40L molecules/μm2 in combination with IL-4 is sufficient to induce IL-12 production by DCs. Remarkably, CD40L alone is adequate to induce IL-23 secretion by DCs. Thus, although activated T cells have somewhat higher levels of CD40L, it is the combination of CD40L and the cytokines they secrete that licenses DCs and influences the effector class of the immune response.
Glass‐supported lipid bilayers presenting freely diffusing proteins have served as a powerful too... more Glass‐supported lipid bilayers presenting freely diffusing proteins have served as a powerful tool for studying cell‐cell interfaces, in particular, T cell–antigen presenting cell (APC) interactions, using optical microscopy. Here we expand upon existing protocols and describe the preparation of liposomes by an extrusion method, and describe how this system can be used to study immune synapse formation by Jurkat cells. We also present a method for forming such lipid bilayers on silica beads for the study of signaling responses by population methods, such as western blotting, flow cytometry, and gene‐expression analysis. Finally, we describe how to design and prepare transmembrane‐anchored protein‐laden liposomes, following expression in suspension CHO (CHOs) cells, a mammalian expression system alternative to insect and bacterial cell lines, which do not produce mammalian glycosylation patterns. Such transmembrane‐anchored proteins may have many novel applications in cell biology an...
TCR-dependent signaling events have been observed to occur in TCR microclusters. We found that so... more TCR-dependent signaling events have been observed to occur in TCR microclusters. We found that some TCR microclusters are present in unstimulated murine T cells, indicating that the mechanisms leading to microcluster formation do not require ligand binding. These pre-existing microclusters increase in absolute number following engagement by low-potency ligands. This increase is accompanied by an increase in cell spreading, with the result that the density of TCR microclusters on the surface of the T cell is not a strong function of ligand potency. In characterizing their composition, we observed a constant number of TCRs in a microcluster, constitutive exclusion of the phosphatase CD45, and preassociation with the signaling adapters linker for activation of T cells and growth factor receptor-bound protein 2. The existence of TCR microclusters prior to ligand binding in a state that is conducive for the initiation of downstream signaling could explain, in part, the rapid kinetics wit...
SummaryThe interaction of T cells with antigen‐presenting cells is the hallmark of adaptive immun... more SummaryThe interaction of T cells with antigen‐presenting cells is the hallmark of adaptive immunity. In vitro studies have described the formation of an immunological synapse between these cells, and intra‐vital imaging has described in great detail the dynamics of these interactions. The immunological synapse has become a paradigm to study signals exchanged between the two cells. A wealth of information has been amassed regarding the localization of signalling molecules, their kinetics and the transcription factors they activate. We continue to discover mechanisms that cause receptors and signalling molecules to compartmentalize in the cell; however, the emerging challenge lies in understanding how the immunological synapse contributes to differentiation. Here, we review some of the transcription factors activated downstream of T‐cell receptor signalling and discuss mechanisms by which antigen dose and affinity may influence differentiation. Antigen affinity might change the kind ...
Background What determines the development of germinal centers (GCs) in lymph nodes (LNs) early a... more Background What determines the development of germinal centers (GCs) in lymph nodes (LNs) early after HIV/SIV infection is likely to be crucial for developing neutralizing antibodies and B cell memory. Materials and Methods Rhesus macaques were inoculated i.v. with either SIVmac239 (n=2) or iMac239ΔD385 (iMacΔD) (n=4), a CD4-independent variant of SIVmac239 lacking a CD4 binding site (J. Virol. 90:4966, 2016). LNs from different time points were analyzed using polychromatic flow cytometry and multiplexed confocal imaging. Results In contrast to SIVmac239, iMacΔD infection was associated with relative preservation of total and CD28hiCD95hi memory CD4 T cells, consistent with its compromised capacity to target CD4 T cells. Imaging analysis revealed accelerated formation of mature, polarized GCs in iMacΔD compared to SIVmac239 infected animals, a profile associated with i) increased frequencies of PD-1hiBcl-6hi CD4 T cells, ii) higher levels of IL-10 and IL-21 within the follicles, iii...
Aim: Because the highly pathogenic SARS-CoV-2 is newly introduced to humans, we aimed to understa... more Aim: Because the highly pathogenic SARS-CoV-2 is newly introduced to humans, we aimed to understand the unique features of its genome and proteins, crucial for high transmissibility and disease severity. Materials & methods: The available genome and protein sequences of SARS-CoV-2 with known human and nonhuman CoV were analyzed using multiple sequence alignment programs. Results: Our analysis revealed some unique mutations in SARS-CoV-2 spike, ORF1a/b, ORF3a/3b and ORF8. The most interesting ones were in the spike angiotensin-converting enzyme 2 receptor binding-motif and generation of a furin-like cleavage site as well as deletions of ORF3a ‘diacidic motif’ and the entire ORF3b. Conclusion: Our data suggest that SARS-CoV-2 has diverged from SARS-CoV-1 but is most close to bat-SL-CoV. Unique mutations in spike and ORF3a/b proteins strongly endorse its adaptive evolution, enhanced infectivity and severe pathogenesis in humans.
The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is reported to cause apoptosis of in... more The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is reported to cause apoptosis of infected cells and several of its proteins including the 3a accessory protein, are pro-apoptotic. Since the 3a protein localizes to the endoplasmic reticulum (ER)-Golgi compartment, its role in causing ER stress was investigated in transiently transfected cells. Cells expressing the 3a proteins showed ER stress based on activation of genes for the ER chaperones GRP78 and GRP94. Since ER stress can cause differential modulation of the unfolded protein response (UPR), which includes the inositol-requiring enzyme 1 (IRE-1), activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) pathways, these were individually tested in 3a-expressing cells. Only the PERK pathway was found to be activated in 3a-expressing cells based on (1) increased phosphorylation of eukaryotic initiation factor 2 alpha (eIF2a) and inhibitory effects of a dominant-negative form of eIF2a on GRP78 promoter act...
Glass-supported lipid bilayers presenting freely diffusing proteins have served as a powerful too... more Glass-supported lipid bilayers presenting freely diffusing proteins have served as a powerful tool for studying cell-cell interfaces, in particular, T cell-antigen presenting cell (APC) interactions, using optical microscopy. Here we expand upon existing protocols and describe the preparation of liposomes by an extrusion method, and describe how this system can be used to study immune synapse formation by Jurkat cells. We also present a method for forming such lipid bilayers on silica beads for the study of signaling responses by population methods, such as western blotting, flow cytometry, and gene-expression analysis. Finally, we describe how to design and prepare transmembrane-anchored protein-laden liposomes, following expression in suspension CHO (CHOs) cells, a mammalian expression system alternative to insect and bacterial cell lines, which do not produce mammalian glycosylation patterns. Such transmembrane-anchored proteins may have many novel applications in cell biology and immunology.
TCR-dependent signaling events have been observed to occur in TCR microclusters. We found that so... more TCR-dependent signaling events have been observed to occur in TCR microclusters. We found that some TCR microclusters are present in unstimulated murine T cells, indicating that the mechanisms leading to microcluster formation do not require ligand binding. These pre-existing microclusters increase in absolute number following engagement by low-potency ligands. This increase is accompanied by an increase in cell spreading, with the result that the density of TCR microclusters on the surface of the T cell is not a strong function of ligand potency. In characterizing their composition, we observed a constant number of TCRs in a microcluster, constitutive exclusion of the phosphatase CD45, and preassociation with the signaling adapters linker for activation of T cells and growth factor receptor-bound protein 2. The existence of TCR microclusters prior to ligand binding in a state that is conducive for the initiation of downstream signaling could explain, in part, the rapid kinetics with which TCR signal transduction occurs.
The molecular mechanisms governing severe acute respiratory syndrome coronavirus-induced patholog... more The molecular mechanisms governing severe acute respiratory syndrome coronavirus-induced pathology are not fully understood. Virus infection and some individual viral proteins, including the 3a protein, induce apoptosis. However, the cellular targets leading to 3a protein-mediated apoptosis have not been fully characterized. This study showed that the 3a protein modulates the mitochondrial death pathway in two possible ways. Activation of caspase-8 through extrinsic signal(s) caused Bid activation. In the intrinsic pathway, there was activation of caspase-9 and cytochrome c release from the mitochondria. This was the result of increased Bax oligomerization and higher levels of p53 in 3a protein-expressing cells, which depended on the activation of p38 MAP kinase (MAPK) in these cells. For p38 activation and apoptosis induction, the 3a cytoplasmic domain was sufficient. In direct Annexin V staining assays, the 3a protein-expressing cells showed increased apoptosis that was attenuated with the p38 MAPK inhibitor SB203580. A block in nuclear translocation of the STAT3 transcription factor in cells expressing the 3a protein was also observed. These results have been used to present a model of 3a-mediated apoptosis.
Purpose of review To describe recent findings on the effect of HIV/SIV infection on lymph node vi... more Purpose of review To describe recent findings on the effect of HIV/SIV infection on lymph node viral and T-cell dynamics using imaging-based methodologies. Recent findings Chronic infection, particularly HIV/SIV, alters dramatically the microenvironment, immune cell frequency, distribution, function and tissue organization of secondary lymphoid tissues. These changes are not always reversible. Over the past few years, the implementation of advanced imaging protocols on human lymph node biopsies as well as on longitudinal lymphoid tissues samples from nonhuman primates (NHP) have provided a wealth of information on how local immune responses evolve over time in response to a persisting retroviral pathogen. Most of the information concerns cytotoxic and helper T cells and viral dynamics. In this review, we detail this information focusing on HIV/SIV infection. We also comment on the gaps that imaging technologies have bridged in our understanding and discuss the translational value of these new findings in the light of emerging therapeutic agendas. Summary Novel imaging platforms allow for dissecting the spatiotemporal dynamics of immune interactions further improving our understanding of the interplay between virus and host and providing important information for designing successful preventive and curative strategies.
Early secretion of IL-12 by mouse dendritic cells (DCs) instructs T cells to make IFN-g. However,... more Early secretion of IL-12 by mouse dendritic cells (DCs) instructs T cells to make IFN-g. However, only activated, but not naive T cells are able to license DCs for IL-12 production. We hypothesized that it might be due to different levels of CD40L expression on the surface of these cells, as CD40 signals are required for IL-12 production. Using quantitative cell-free systems incorporating CD40L in lipid bilayers combined with total internal reflection fluorescence microscopy and flow cytometry, we show that as low as ∼200 CD40L molecules/mm 2 in combination with IL-4 is sufficient to induce IL-12 production by DCs. Remarkably, CD40L alone is adequate to induce IL-23 secretion by DCs. Thus, although activated T cells have somewhat higher levels of CD40L, it is the combination of CD40L and the cytokines they secrete that licenses DCs and influences the effector class of the immune response.
The orf3a (also called X1 or U274) gene is the largest unique open reading frame in the severe ac... more The orf3a (also called X1 or U274) gene is the largest unique open reading frame in the severe acute respiratory syndrome coronavirus genome and has been proposed to encode a protein with three transmembrane domains and a large cytoplasmic domain. Recent work has suggested that the 3a protein may play a structural role in the viral life cycle, although the mechanisms for this remain uncharacterized. Here, the expression of the 3a protein in various in vitro systems is shown, it has been localized to the Golgi region and its membrane topology in transfected cells has been confirmed. Three potential caveolin-1-binding sites were reported to be present in the 3a protein. By using various biochemical, biophysical and genetic techniques, interaction of the 3a protein with caveolin-1 is demonstrated. Any one of the potential sites in the 3a protein was sufficient for this interaction. These results are discussed with respect to the possible roles of the 3a protein in the viral life cycle.
The interaction of T cells with antigen-presenting cells is the hallmark of adaptive immunity. In... more The interaction of T cells with antigen-presenting cells is the hallmark of adaptive immunity. In vitro studies have described the formation of an immunological synapse between these cells, and intra-vital imaging has described in great detail the dynamics of these interactions. The immunological synapse has become a paradigm to study signals exchanged between the two cells. A wealth of information has been amassed regarding the localization of signalling molecules, their kinetics and the transcription factors they activate. We continue to discover mechanisms that cause receptors and signalling molecules to compartmentalize in the cell; however, the emerging challenge lies in understanding how the immunological synapse contributes to differentiation. Here, we review some of the transcription factors activated downstream of T-cell receptor signalling and discuss mechanisms by which antigen dose and affinity may influence differentiation. Antigen affinity might change the kind of transcription factors that are activated whereas antigen dose is likely to influence the temporal dynamics of the transcription factors. The immunological synapse is therefore likely to influence differentiation by modulating the trafficking of transcription factors and by promoting asymmetric cell division, an emerging concept.
Background What determines the development of germinal centers (GCs) in lymph nodes (LNs) early a... more Background What determines the development of germinal centers (GCs) in lymph nodes (LNs) early after HIV/SIV infection is likely to be crucial for developing neutralizing antibodies and B cell memory. Materials and Methods Rhesus macaques were inoculated i.v. with either SIVmac239 (n=2) or iMac239ΔD385 (iMacΔD) (n=4), a CD4-independent variant of SIVmac239 lacking a CD4 binding site (J. Virol. 90:4966, 2016). LNs from different time points were analyzed using polychromatic flow cytometry and multiplexed confocal imaging. Results In contrast to SIVmac239, iMacΔD infection was associated with relative preservation of total and CD28hiCD95hi memory CD4 T cells, consistent with its compromised capacity to target CD4 T cells. Imaging analysis revealed accelerated formation of mature, polarized GCs in iMacΔD compared to SIVmac239 infected animals, a profile associated with i) increased frequencies of PD-1hiBcl-6hi CD4 T cells, ii) higher levels of IL-10 and IL-21 within the follicles, iii) increased frequency of PH2AX+ (a required factor for isotype switching) B cells, iv) lower levels of Fas-L in the follicular areas, associated with lower TUNEL (cell death) positivity and v) lower accumulation of CD163+ cells (monocytes/macrophages; a surrogate of local inflammation) in proximity to follicles. The observed GC dynamics associate with earlier virus-specific antibody responses in iMacΔD compared to SIVmac239. Conclusions Our data indicate that CD4 tropism of SIV greatly affects the development of GC reactions early after infection. The iMacΔD model could provide critical information on how GC reactions can be manipulated to augment the generation of neutralizing antibodies and B cell memory.
Induction of immediate early genes (like Fos, Jun) in response to T cell receptor stimulation wit... more Induction of immediate early genes (like Fos, Jun) in response to T cell receptor stimulation with different MHC-peptide (MHC-p) dose and potency is not well understood. Our aim is to study the kinetics of c-Fos induction in T cells in response to antigen dose and potency. Our model system is AND TCR transgenic mice, whose T cells are specific for moth Cytochrome-C (MCC) peptide. We study the impact of MHC-p potency using altered peptide ligands (APLs). To study c-Fos induction we crossed AND mice to c-Fos-EGFP transgenic mice that express a fusion protein of c-Fos and EGFP driven by the Fos promoter. We observed that upon stimulation of TCR, expression of c-Fos starts after ~30 min, peaks at 3hrs and plateaus for another 2hrs. Strong agonists induce c-Fos even without co-stimulatory molecules where as weak agonists fail to do so. Co-stimulation enhances the percentage of cells responding as well as the level of c-Fos expression. Highering or lowering the peptide dose reduces the ex...
Purpose of review To describe recent findings on the effect of HIV/SIV infection on lymph node vi... more Purpose of review To describe recent findings on the effect of HIV/SIV infection on lymph node viral and T-cell dynamics using imaging-based methodologies. Recent findings Chronic infection, particularly HIV/SIV, alters dramatically the microenvironment, immune cell frequency, distribution, function and tissue organization of secondary lymphoid tissues. These changes are not always reversible. Over the past few years, the implementation of advanced imaging protocols on human lymph node biopsies as well as on longitudinal lymphoid tissues samples from nonhuman primates (NHP) have provided a wealth of information on how local immune responses evolve over time in response to a persisting retroviral pathogen. Most of the information concerns cytotoxic and helper T cells and viral dynamics. In this review, we detail this information focusing on HIV/SIV infection. We also comment on the gaps that imaging technologies have bridged in our understanding and discuss the translational value of these new findings in the light of emerging therapeutic agendas. Summary Novel imaging platforms allow for dissecting the spatiotemporal dynamics of immune interactions further improving our understanding of the interplay between virus and host and providing important information for designing successful preventive and curative strategies.
Peptide immunogens provide an approach to focus antibody responses to specific neutralizing sites... more Peptide immunogens provide an approach to focus antibody responses to specific neutralizing sites on the HIV envelope protein (Env) trimer or on other pathogens. However, the physical characteristics of peptide immunogens can limit their pharmacokinetic and immunological properties. Here, we have designed synthetic "star" nanoparticles based on biocompatible N-[(2-hydroxypropyl)methacrylamide] (HPMA)-based polymer arms extending from a poly(amidoamine) (PAMAM) dendrimer core. In mice, these star nanoparticles trafficked to lymph nodes (LNs) by 4 hours following vaccination, where they were taken up by subcapsular macrophages and then resident dendritic cells (DCs). Immunogenicity optimization studies revealed a correlation of immunogen density with antibody titers. Furthermore, the co-delivery of Env variable loop 3 (V3) and T-helper peptides induced titers that were 2 logs higher than if the peptides were given in separate nanoparticles. Finally, we performed a nonhuman primate (NHP) study using a V3 glycopeptide minimal immunogen that was structurally optimized to be recognized by Env V3/glycan broadly neutralizing antibodies (bnAbs). When administered with a potent Toll-like receptor (TLR) 7/8 agonist adjuvant, these nanoparticles elicited high antibody binding titers to the V3 site. Similar to human V3/ glycan bnAbs, certain monoclonal antibodies (mAbs) elicited by this vaccine were glycan dependent or targeted the GDIR peptide motif. To improve affinity to native Env trimer affinity, nonhuman primates (NHPs) were boosted with various SOSIP Env proteins; however, significant neutralization was not observed. Taken together, this study provides a new
Early secretion of IL-12 by mouse dendritic cells (DCs) instructs T cells to make IFN-γ. However,... more Early secretion of IL-12 by mouse dendritic cells (DCs) instructs T cells to make IFN-γ. However, only activated, but not naive T cells are able to license DCs for IL-12 production. We hypothesized that it might be due to different levels of CD40L expression on the surface of these cells, as CD40 signals are required for IL-12 production. Using quantitative cell-free systems incorporating CD40L in lipid bilayers combined with total internal reflection fluorescence microscopy and flow cytometry, we show that as low as ∼200 CD40L molecules/μm2 in combination with IL-4 is sufficient to induce IL-12 production by DCs. Remarkably, CD40L alone is adequate to induce IL-23 secretion by DCs. Thus, although activated T cells have somewhat higher levels of CD40L, it is the combination of CD40L and the cytokines they secrete that licenses DCs and influences the effector class of the immune response.
Glass‐supported lipid bilayers presenting freely diffusing proteins have served as a powerful too... more Glass‐supported lipid bilayers presenting freely diffusing proteins have served as a powerful tool for studying cell‐cell interfaces, in particular, T cell–antigen presenting cell (APC) interactions, using optical microscopy. Here we expand upon existing protocols and describe the preparation of liposomes by an extrusion method, and describe how this system can be used to study immune synapse formation by Jurkat cells. We also present a method for forming such lipid bilayers on silica beads for the study of signaling responses by population methods, such as western blotting, flow cytometry, and gene‐expression analysis. Finally, we describe how to design and prepare transmembrane‐anchored protein‐laden liposomes, following expression in suspension CHO (CHOs) cells, a mammalian expression system alternative to insect and bacterial cell lines, which do not produce mammalian glycosylation patterns. Such transmembrane‐anchored proteins may have many novel applications in cell biology an...
TCR-dependent signaling events have been observed to occur in TCR microclusters. We found that so... more TCR-dependent signaling events have been observed to occur in TCR microclusters. We found that some TCR microclusters are present in unstimulated murine T cells, indicating that the mechanisms leading to microcluster formation do not require ligand binding. These pre-existing microclusters increase in absolute number following engagement by low-potency ligands. This increase is accompanied by an increase in cell spreading, with the result that the density of TCR microclusters on the surface of the T cell is not a strong function of ligand potency. In characterizing their composition, we observed a constant number of TCRs in a microcluster, constitutive exclusion of the phosphatase CD45, and preassociation with the signaling adapters linker for activation of T cells and growth factor receptor-bound protein 2. The existence of TCR microclusters prior to ligand binding in a state that is conducive for the initiation of downstream signaling could explain, in part, the rapid kinetics wit...
SummaryThe interaction of T cells with antigen‐presenting cells is the hallmark of adaptive immun... more SummaryThe interaction of T cells with antigen‐presenting cells is the hallmark of adaptive immunity. In vitro studies have described the formation of an immunological synapse between these cells, and intra‐vital imaging has described in great detail the dynamics of these interactions. The immunological synapse has become a paradigm to study signals exchanged between the two cells. A wealth of information has been amassed regarding the localization of signalling molecules, their kinetics and the transcription factors they activate. We continue to discover mechanisms that cause receptors and signalling molecules to compartmentalize in the cell; however, the emerging challenge lies in understanding how the immunological synapse contributes to differentiation. Here, we review some of the transcription factors activated downstream of T‐cell receptor signalling and discuss mechanisms by which antigen dose and affinity may influence differentiation. Antigen affinity might change the kind ...
Background What determines the development of germinal centers (GCs) in lymph nodes (LNs) early a... more Background What determines the development of germinal centers (GCs) in lymph nodes (LNs) early after HIV/SIV infection is likely to be crucial for developing neutralizing antibodies and B cell memory. Materials and Methods Rhesus macaques were inoculated i.v. with either SIVmac239 (n=2) or iMac239ΔD385 (iMacΔD) (n=4), a CD4-independent variant of SIVmac239 lacking a CD4 binding site (J. Virol. 90:4966, 2016). LNs from different time points were analyzed using polychromatic flow cytometry and multiplexed confocal imaging. Results In contrast to SIVmac239, iMacΔD infection was associated with relative preservation of total and CD28hiCD95hi memory CD4 T cells, consistent with its compromised capacity to target CD4 T cells. Imaging analysis revealed accelerated formation of mature, polarized GCs in iMacΔD compared to SIVmac239 infected animals, a profile associated with i) increased frequencies of PD-1hiBcl-6hi CD4 T cells, ii) higher levels of IL-10 and IL-21 within the follicles, iii...
Aim: Because the highly pathogenic SARS-CoV-2 is newly introduced to humans, we aimed to understa... more Aim: Because the highly pathogenic SARS-CoV-2 is newly introduced to humans, we aimed to understand the unique features of its genome and proteins, crucial for high transmissibility and disease severity. Materials & methods: The available genome and protein sequences of SARS-CoV-2 with known human and nonhuman CoV were analyzed using multiple sequence alignment programs. Results: Our analysis revealed some unique mutations in SARS-CoV-2 spike, ORF1a/b, ORF3a/3b and ORF8. The most interesting ones were in the spike angiotensin-converting enzyme 2 receptor binding-motif and generation of a furin-like cleavage site as well as deletions of ORF3a ‘diacidic motif’ and the entire ORF3b. Conclusion: Our data suggest that SARS-CoV-2 has diverged from SARS-CoV-1 but is most close to bat-SL-CoV. Unique mutations in spike and ORF3a/b proteins strongly endorse its adaptive evolution, enhanced infectivity and severe pathogenesis in humans.
The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is reported to cause apoptosis of in... more The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is reported to cause apoptosis of infected cells and several of its proteins including the 3a accessory protein, are pro-apoptotic. Since the 3a protein localizes to the endoplasmic reticulum (ER)-Golgi compartment, its role in causing ER stress was investigated in transiently transfected cells. Cells expressing the 3a proteins showed ER stress based on activation of genes for the ER chaperones GRP78 and GRP94. Since ER stress can cause differential modulation of the unfolded protein response (UPR), which includes the inositol-requiring enzyme 1 (IRE-1), activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) pathways, these were individually tested in 3a-expressing cells. Only the PERK pathway was found to be activated in 3a-expressing cells based on (1) increased phosphorylation of eukaryotic initiation factor 2 alpha (eIF2a) and inhibitory effects of a dominant-negative form of eIF2a on GRP78 promoter act...
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Papers by Kartika Padhan