Browsing by Subject "T cells"

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Analyzing infection-driven immune perturbations by quantitative IR-Seq
(2017-09-08) Wendel, Ben Shogo; Jiang, Ning Jenny; Georgiou, George; Maynard, Jennifer; Alper, Hal; Davis, Mark
Immune repertoire sequencing (IR-Seq) rapidly emerged with the advent of high-throughput sequencing as a means of characterizing the adaptive immune system. Early generations of IR-Seq were plagued by sequencing errors and low diversity coverage. We developed Molecular Identifier Clustering-based IR-Seq (MIDCIRS) to quantitatively and comprehensively measure the immune repertoire from a small amount of blood. We used naive B cells to formulate a general framework for IR-Seq experimental validation and quality control and showed that MIDCIRS can be applied to as few as 1,000 naive B cells with excellent diversity coverage. Using MIDCIRS, we studied the antibody repertoire response to acute malaria infection in young children. We found that the infant antibody repertoire is surprisingly competent at introducing somatic hypermutations (SHM) and diversifying B cell clonal lineages in response to malaria infection. Detailed analysis of memory B cell-containing lineages in malaria-experienced toddlers revealed that memory B cells further mutate upon malaria rechallenge. IgM-expressing memory B cells largely retain IgM expression upon rechallenge, but a subset class switch to IgG and IgA. Accurate antibody repertoire analysis requires not only accurate sequencing data, but also correct reference germline allele sequences. Mismatches between the reference sequence and an individual’s true germline sequence would be mistakenly counted as SHMs, inflating the SHM load and skewing the repertoire analysis. We developed a simple yet effect method for predicting novel germline allele sequences from antibody repertoire data and validating them via targeted sequencing of non-rearranged genomic DNA. HIV infection has a profound impact on the CD4⁺ T cell compartment, which can have a devastating effect on the adaptive immune system as a whole. Paradoxically, while peripheral CD4⁺ T cell counts drop with disease severity, T[subscript FH] cells show an inverse relationship. We found that these expanded T[subscript FH] cells exhibit a functionally restricted phenotype, which could contribute to ineffective antibody responses both to HIV and unrelated vaccines. Using MIDCIRS, we found that these expanded T[subscript FH] cells are enriched with HIV-specific sequences and show signs of antigen-driven convergent evolution, suggesting that HIV-specific T cells are selected and recruited into the T[subscript FH] compartment during infection.
Characterizing dynein in T cells
(2010-08) Tan, Sarah Youngsun; Poenie, Martin F.; Kenneth, Johnson; Theresa, O'Halloran; Ellen, Richie; Philip, Tucker
T cells play pivotal roles in the immune system and focused secretion of either cytokines or cytotoxic molecules toward its target is crucial for T cell functions. This directional secretion involves two critical steps: the movement of the microtubule organizing center (MTOC) up to the cell-cell contact site and the directed movement of secretory vesicles towards the MTOC. The minus end-directed microtubule motor protein dynein was previously shown in our studies and those of others to accumulate and anchor at the contact site where it then draws the MTOC up to the contact site. A variety of studies led to the suggestion that there were two functionally different pools of dynein in Jurkat cells, one a ring-like structure that pulled the MTOC to the contact site and the other one uniquely corresponding to the distribution of dynactin. This led to the hypothesis that the second pool of dynein drove vesicle transport. To address this possibility, we used siRNA to deplete the cell of dynactin. These studies showed that almost complete knockdown of dynactin (p150[superscript Glued]) had little effect on MTOC translocation but it also had little effect on a panel of Golgi vesicle markers, whose movement the literature suggested was dynein dependent. As an alternative, a Jurkat cell line expressing fluorescent CTLA4, a known marker for the secretory lysosomes was generated. CTLA4 accumulated at the contact site when Jurkat cells made contact with synthetic target cells. When we repeated the p150[superscript Glued] knockdown in these cells, we found that vesicle transport was blocked, whereas MTOC polarization remained normal. These studies suggest that dynein serves critical roles in both aspects of T cell effector function, the movement of the MTOC up to the cell-cell contact site and the movement of a special class of secretory vesicles up to the MTOC. By the combined processes of MTOC translocation and the minus end-directed movement of vesicles, T cells make it so that a concentrated pool of secretory vesicles are aimed to secrete locally only towards target cells. This ensures that the antigen-specificity of T cell activation is followed by a localized response aimed at the intended target cell.
Detecting calcium flux in T cells using a Bayesian model
(2015-08) Hu, Zicheng; Müller, Peter, 1963 August 9-; Ehrlich, Lauren
Upon antigen recognition, T cells are activated to carry out its effector functions. A hallmark of T cell activation is the dramatic increase of the intracellular calcium concentration (calcium influx). Indo-1 is a calcium indicator dye widely used to detect T cell activation events in in vitro assays. The use of Indo-1 to detect T cell activation events in live tissues remains a challenge, due to the high noise to signal ratio data generated. Here, we developed a Bayesian probabilistic model to identify T cell activation events from noisy Indo-1 data. The model was able to detect T cell activation events accurately from simulated data, as well as real biological data in which the time of T cell activation events are known. We then used the model to detect OTII T cells that are activated by dendritic cells in thymic medulla in Rip-OVAhi transgenic mouse. We found that dendritic cells contribute 60% of all T cell activations in the mouse model.
Endogenous MMTV Proviruses Induce Susceptibility to Both Viral and Bacterial Pathogens
(Public Library of Science, 2006-12-01) Bhadra, Sanchita; Lozano, Mary M; Payne, Shelley M; Dudley, Jaquelin P
Most inbred mice carry germline proviruses of the retrovirus, mouse mammary tumor virus (MMTV) (called Mtvs), which have multiple replication defects. A BALB/c congenic mouse strain lacking all endogenous Mtvs (Mtv-null) was resistant to MMTV oral and intraperitoneal infection and tumorigenesis compared to wild-type BALB/c mice. Infection of Mtv-null mice with an MMTV-related retrovirus, type B leukemogenic virus, also resulted in severely reduced viral loads and failure to induce T-cell lymphomas, indicating that resistance is not dependent on expression of a superantigen (Sag) encoded by exogenous MMTV. Resistance to MMTV in Mtv-null animals was not due to neutralizing antibodies. Further, Mtv-null mice were resistant to rapid mortality induced by intragastric inoculation of the Gram-negative bacterium, Vibrio cholerae, but susceptibility to Salmonella typhimurium was not significantly different from BALB/c mice. Susceptibility to both MMTV and V. cholerae was reconstituted by the presence of any one of three endogenous Mtvs located on different chromosomes and was associated with increased pathogen load. One of these endogenous proviruses is known to encode only Sag. Therefore, Mtv-encoded Sag appears to provide a unique genetic susceptibility to specific viruses and bacteria. Since human endogenous retroviruses also encode Sags, these studies have broad implications for pathogen-induced responses in mice and humans.
Enhanced Protection against Ebola Virus Mediated by an Improved Adenovirus-Based Vaccine
(Public Library of Science, 2009-04-23) Richardson, Jason S.; Yao, Michel K.; Tran, Kaylie N.; Croyle, Maria A.; Strong, James E.; Feldmann, Heinz; Kobinger, Gary P.
Background -- The Ebola virus is transmitted by direct contact with bodily fluids of infected individuals, eliciting death rates as high as 90% among infected humans. Currently, replication defective adenovirus-based Ebola vaccine is being studied in a phase I clinical trial. Another Ebola vaccine, based on an attenuated vesicular stomatitis virus has shown efficacy in post-exposure treatment of nonhuman primates to Ebola infection. In this report, we modified the common recombinant adenovirus serotype 5-based Ebola vaccine expressing the wild-type ZEBOV glycoprotein sequence from a CMV promoter (Ad-CMVZGP). The immune response elicited by this improved expression cassette vector (Ad-CAGoptZGP) and its ability to afford protection against lethal ZEBOV challenge in mice was compared to the standard Ad-CMVZGP vector. Methodology/Principal Findings -- Ad-CMVZGP was previously shown to protect mice, guinea pigs and nonhuman primates from an otherwise lethal challenge of Zaire ebolavirus. The antigenic expression cassette of this vector was improved through codon optimization, inclusion of a consensus Kozak sequence and reconfiguration of a CAG promoter (Ad-CAGoptZGP). Expression of GP from Ad-CAGoptZGP was substantially higher than from Ad-CMVZGP. Ad-CAGoptZGP significantly improved T and B cell responses at doses 10 to 100-fold lower than that needed with Ad-CMVZGP. Additionally, Ad-CAGoptZGP afforded full protections in mice against lethal challenge at a dose 100 times lower than the dose required for Ad-CMVZGP. Finally, Ad-CAGoptZGP induced full protection to mice when given 30 minutes post-challenge. Conclusions/Significance -- We describe an improved adenovirus-based Ebola vaccine capable of affording post-exposure protection against lethal challenge in mice. The molecular modifications of the new improved vaccine also translated in the induction of significantly enhanced immune responses and complete protection at a dose 100 times lower than with the previous generation adenovirus-based Ebola vaccine. Understanding and improving the molecular components of adenovirus-based vaccines can produce potent, optimized product, useful for vaccination and post-exposure therapy.
Enzyme-mediated methylthioadenosine depletion as a novel immune checkpoint therapy
(2021-05) Gjuka, Donjeta; Georgiou, George; Stone, Everett Monroe, 1971-; Maynard, Jennifer; Jiang, Ning; Ehrlich, Lauren
Methylthioadenosine phosphorylase (MTAP) is an enzyme that is homozygously deleted in approximately 15% of all human cancer types. MTAP deletion has been correlated with cancer progression, poor patient survival, and resistance to immune checkpoint therapies. Frequent MTAP loss in cancer leads to the accumulation of its substrate 5’-deoxy-5’-methylthioadenosine (MTA), which exerts potent immunosuppressive effects in T cells. We hypothesize that accumulated MTA in the tumor microenvironment of MTAP-deficient tumors induces an immunomodulatory effect on surrounding T cells and engenders tumor tolerance. In this dissertation, we explore the therapeutic efficacy of enzyme-mediated MTA depletion in several MTAP-deficient tumor models. Our results indicate that treating MTAP-deficient tumors with MTA-degrading enzymes can drastically suppress tumor growth. Moreover, our in vivo studies and immunophenotyping experiments suggest that the therapeutic benefit of MTA-depletion is mediated by CD8 T cells that infiltrate the tumor microenvironment. Additionally, we tested the combined therapeutic effects of MTA depletion with immune checkpoint therapy (e.g., anti-PD1 or anti-CTLA4) and observed a potent synergistic effect. Furthermore, we investigate MTA’s mechanism of action on T cells. Due to MTA’s structural similarities with adenosine, we examined putative signaling of MTA through adenosine receptors. Our findings show that adenosine signaling is not the main pathway that MTA utilizes to suppress T cells. Instead, our proteomics analysis of T cells incubated with MTA suggest that MTA inhibits T cells, at least in part, via the differential expression of methyltransferase/demethylase enzymes, upregulation of proapoptotic proteins, and inhibition of proteins crucial for TCR activation and cytokine signaling. In this work, we also discuss the engineering and optimization of the MTAP enzyme for therapeutic use. Collectively, this dissertation elucidates the suppression mechanism of MTA on T cells and demonstrates the potential of utilizing MTA-depletion therapy as a biomarker-driven (namely, MTAP status) immunotherapeutic modality.
Flanking Residues Are Central to DO11.10 T Cell Hybridoma Stimulation by Ovalbumin 323–339
(Public Library of Science, 2012-10-23) Roy, Benjamin M.; Zhukov, Dmitriy V.; Maynard, Jennifer A.
T cell activation requires formation of a tri-molecular interaction between a major histocompatibility complex (MHC), peptide, and T cell receptor. In a common model system, the ovalbumin epitope 323–339 binds the murine class II MHC, I-Ad, in at least three distinct registers. The DO11.10 T cell recognizes the least stable of these, as determined by peptide-MHC dissociation rates. Using exogenous peptides and peptide insertions into a carrier protein in combination with IL-2 secretion assays, we show that the alternate registers do not competitively inhibit display of the active register four. In contrast, this weakly binding register is stabilized by the presence of N-terminal flanking residues active in MHC binding. The DO11.10 hybridoma is sensitive to the presence of specific wild-type residues extending to at least the P-3 peptide position. Transfer of the P-4 to P-2 flanking residues to a hen egg lysozyme epitope also presented by I-Ad increases the activity of that epitope substantially. These results illustrate the inherent complexity in delineating the interaction of multiple registers based on traditional thermodynamic measurements and demonstrate the potential of flanking residue modification for increasing the activity of weakly bound epitopes. The latter technique represents an alternative to substitution of anchor residues within a weakly bound register, which we show can significantly decrease the activity of the epitope to a responding T cell.
Glycerol-3-phosphate acyltransferase regulates T cell effector function and metabolism
(2013-08) Faris, Robert Allen, Jr.; Jolly, Christopher A.
The aged T cell is characterized by decreased responsiveness to stimulation. Aging is associated with reduced membrane glycerophospholipid (GPL) to cholesterol ratios so it is interesting that deletion of mitochondrial glycerol-3-phosphate acyltransferase-1 which catalyzes the first step in de novo GPL synthesis induces an aged T cell phenotype in otherwise healthy mice. GPAT-1 could regulate T cell function through three possible mechanisms: maintenance of membrane GPL ratios and membrane based signaling, providing a specific substrate for downstream signaling, or direct regulation of cellular metabolism. Therefore, the goal of this project was to determine whether these mechanisms contribute to the dysfunctional T cell phenotype observed with decreased GPAT-1 activity. T cell stimulation requires significant upregulation of metabolic processes to drive clonal expansion and cytokine production. T cell dysfunction in GPAT-1 knockout mice may be partially explained by altered metabolic function. We found that GPAT-1 KO T cells have significantly reduced basal respiration rates and spare respiratory capacity which is not compensated for by increased glycolytic metabolism suggesting an inherent metabolic defect in GPAT-1 KO T cells. To better understand mechanistically how GPAT-1 regulates T cell function we moved into the Jurkat T cell line and found that shRNA mediated knockdown of the human isoform of GPAT-1 (GPAM) recapitulated key aspects of the dysfunctional T cell phenotype we observed in the mouse including highly significant reductions in IL-2 production and altered membrane GPL to cholesterol ratios. Phosphatidic acid addition was not capable of rescuing these deficiencies suggesting that GPAT-1/GPAM activity is required for proper T cell function. This was the first time that GPAT-1 activity has been shown to be important for T cell function in a non-murine model system and strongly suggests that GPAT-1/GPAM deficiency regulates T cell function at the cellular level. We further demonstrate that phosphorylation of ZAP-70 a proximal effector of T cell activation is significantly reduced in GPAM knock down Jurkat T cells, suggesting that membrane based signaling is dysfunctional. Taken together these data suggest that GPAT-1 is necessary for regulating cellular energy demands in T cells and essential for optimal T cell activation following stimulation.
Glycomic approaches to understanding HIV-1 budding in T cells
(2008-12) Krishnamoorthy, Lakshmipriya, 1978-; Mahal, Lara K.
The causative agent of AIDS (acquired immune deficiency syndrome), HIV (human immunodeficiency virus), is one of the most extensively studied pathogens in modern history. The virus has multiple mechanisms of persisting in the host including evading host immune response. Since HIV-1 depends heavily on the host machinery for various aspects of its life cycle, unraveling the complex interplay between the host and HIV-1 could provide new clues to therapeutic avenues. In T cells, HIV assembles and subsequently buds through the plasma membrane incorporating host derived proteins and lipids in the viral envelope. HIV is thought to utilize a pre-existing mechanism for the budding of normal cellular vesicles called microvesicles to exit host cells. The evidence for this theory comes from reports of similarities between HIV and microvesicles observed for a small subset of proteins and lipids, leading to controversies about its validity. To further test this hypothesis, we utilized lectin microarrays to obtain a comprehensive glycomic profile of HIV and microvesicles derived from a panel of T cell lines. Glycosylation is critical to protein sorting and has a crucial role in HIV-1 biology, making it an ideal marker to compare the particles and the host cell membrane. We observed similar glycomic profiles for HIV-1 and microvesicles strongly suggesting an analogous mode of egress. Glycosylation of both particles seems to vary based on the parent cell line, providing additional evidence for this hypothesis. Microvesicles are involved in immune response modulation; hence the incorporation of microvesicular proteins could influence interactions of HIV with the immune system. The differences in glycosylation between these two particles could be potentially explained by the heavily glycosylated viral envelope glycoprotein. I also demonstrated that these vesicles bud from particular glycan enriched domains of the plasma membrane. Additionally, this work sheds light on the potential mode of interaction between galectin, an immune lectin and HIV-1. This work strongly argues for a conserved mechanism of exocytosis for both particles and sets the stage for examining the role of glycosylation in trafficking of proteins to the sites of microvesicular and viral budding.
Identification and characterization of the T-cell-specific enhancer of type B leukemogenic virus
(2003) Mertz, Jennifer Andrea; Dudley, Jaquelin
Mouse mammary tumor virus (MMTV) is a retrovirus that causes mammary adenocarcinomas and T-cell lymphomas. T-cell lymphomas induced by MMTV have additional proviral integrations that invariably have alterations in the long terminal repeats (LTRs). Type B leukemogenic virus (TBLV) is a thymotropic strain of MMTV that induces rapidly appearing T-cell tumors in mice. TBLV is highly related to MMTV except that TBLV LTRs have a deletion of negative regulatory elements and a multimerization of sequences flanking the deletion. In this study, the multimerized sequences from the TBLV LTR were identified as a novel T-cell-specific enhancer that can act on the TBLV promoter as well as thymidine kinase and c-myc promoters. Substitution mutagenesis of the enhancer identified a critical region that contains binding sites for RUNX1, NFkB and two unknown factors, NF-A and NF-B. RUNX1, NF-A and NF-B all positively regulate TBLV enhancer activity. However, the role of NF-kB in enhancer function is unclear since it does not appear to contribute to the activity of the TBLV LTR in T cells. Additionally, expression of NF-kB antagonizes the positive effect of RUNX1 expression on the TBLV LTR in non-T cells. NF-A appears to be the major contributor to enhancer function and is a lymphoidrestricted factor. The TBLV enhancer also contains functional glucocorticoid receptor (GR) binding sites; however GR binding is not necessary for enhancer activity in T cells. A c-Myb binding site overlaps the GR binding site and expression of c-Myb in non-T cells activates transcription from the LTR. Characterization of TBLV enhancers in proviruses integrated near c-myc revealed that the number of enhancer elements varied, but the most clonal tumors had proviruses containing four enhancer elements. Reporter gene assays showed that LTRs containing four enhancer elements were less effective at activating transcription from either the TBLV or c-myc promoters. In vivo passage of tumor cells in immunocompetent mice revealed a selection for proviral integrations near c-myc with four enhancer elements. Since c-myc overexpression often leads to apoptosis, these results suggested that selection for clonal growth occurred in tumor cells that had modest c-myc overexpression after TBLV insertion to prevent apoptosis.
Importance of mitochodrial [i.e. mitochondrial] glycerol-3-phosphate acyltranferase [i.e. acyltransferase] in T-lymphocyte function and aging
(2005) Collison, Lauren West; Jolly, Christopher A.
Aging is associated with immune dysfunction, characterized by reduced Tlymphocyte proliferation ex vivo. One potential mechanism for the reduced proliferation in aged T-lymphocytes is a decreased ratio of membrane glycerophospholipid to cholesterol. Extracting membrane cholesterol does not completely restore T-lymphocyte proliferation, therefore, the goal of this project was to test the novel hypothesis that aged T-lymphocyte proliferative capacity may be reduced, in part, due to suppressed phosphatidic acid (PA) biosynthesis. We show that stimulation and recombinant acylCoA binding protein (ACBP) increase mitochondrial glycerol-3-phosphate acyltransferase (mtGPAT) activity in rat splenic T-lymphocytes and that this effect is blunted in aged T-lymphocytes. Consequently, we wanted to determine the mechanism by which mtGPAT activity is regulated in young and old T-lymphocytes. We show that aged T-lymphocyte mtGPAT activity is not increased by ex vivo stimulation or in vitro phosphorylation with casein kinase 2 (CK2) and protein kinase C theta (PKC θ) as is seen in young T-lymphocytes. A second isoform of mtGPAT, mtGPAT2, has been identified, however, its expression is undetectable in T-lymphocytes, suggesting that at least mtGPAT 1 is responsive to phosphorylation in vitro. Other factors that might influence mtGPAT activity such as reduced mtGPAT protein levels, gene expression or alterations in the soluble acyl-CoA pool were not affected by age or stimulation indicating that posttranslational modification may be the primary mechanism by which mtGPAT activity is regulated in T-lymphocytes. Because aging is associated with both reduced proliferation and reduced mtGPAT1 activity, we hypothesized that young mtGPAT1 knockout mice would have altered glycerophospholipid levels and reduced proliferation ex vivo. We show that mtGPAT1 knockout T-lymphocytes exhibit similar changes seen in aged T-lymphocytes including reduced splenic T-lymphocyte IL-2 secretion and subsequent proliferation, decreased ratio of membrane glycerophospholipid to cholesterol, changes in thymic development, and increased cell death following Tlymphocyte activation. Taken together, these data suggest a novel mechanism by which mtGPAT1 regulates young T-lymphocyte proliferation and indicate that mtGPAT1 may serve as a key target in the age-dependent decline in T-lymphocyte proliferation.
In vitro and in vivo characterization of Fc-receptor expression and function on various immune cell subsets
(2018-08-01) Charab, Wissam; Georgiou, George; Maynard, Jennifer A; Jiang, Ning J; Alper, Hal S; Tucker, Haley O; Ehrlich, Lauren I
Over the years, the standard care in cancer treatment has changed from surgery, chemotherapy, and/or radiotherapy alone to include the promising field of immunotherapy. Cancer immunotherapy enhances a patient's own immune system to eradicate tumors and/or confer protection from recurrence. The potential of immune complexes (ICs) directly influencing T cells has long been debated, with conflicting reports on T cell expression of Fc[gamma]Rs. Fc[gamma]Rs are inhibiting or activating immune cell receptors that interact with antibodies and can determine the overall outcome of an immune response. Using various methods, we show that several human CD4 and CD8 T cell subsets express Fc[gamma]RII and Fc[gamma]RIII and that IgG ICs suppress T cell proliferation and cytotoxicity. Using an engineered IgG Fc that only binds Fc[gamma]RI but not Fc[gamma]RII/III, we demonstrate that these effects are Fc[gamma]R-mediated. Specifically, whereas memory T cell proliferation was more resistant, naïve T cell proliferation was strongly inhibited by IgG ICs. Nevertheless, IgG ICs significantly diminished effector T cell cytotoxicity. Moreover, we also show that T cells from virally infected and cancer patients display higher levels of surface Fc[gamma]RII/III relative to healthy controls. Finally, human data was followed up with in vitro and in vivo mouse data to demonstrate that murine T cells also express functional Fc[gamma]Rs that can attenuate calcium flux induced by TCR signaling. Confirming that IgG ICs can directly inhibit T cells is essential for the successful design of efficacious and safe therapeutics. Furthermore, we also demonstrate the utility of Fc-engineered antibodies to both enhance and unravel mechanistic insights of immune effector functions like dendritic cell cross presentation or macrophage phagocytosis.
Limits on Replenishment of the Resting CD4+ T Cell Reservoir for HIV in Patients on HAART
(Public Library of Science, 2007-03-31) Sedaghat, Ahmad R; Siliciano, Janet D; Brennan, Timothy P; Wilke, Claus O; Siliciano, Robert F
Whereas cells productively infected with human immunodeficiency virus type 1 (HIV-1) decay rapidly in the setting of highly active antiretroviral therapy (HAART), latently infected resting CD4+ T cells decay very slowly, persisting for the lifetime of the patient and thus forming a stable reservoir for HIV-1. It has been suggested that the stability of the latent reservoir is due to low-level viral replication that continuously replenishes the reservoir despite HAART. Here, we offer the first quantitative study to our knowledge of inflow of newly infected cells into the latent reservoir due to viral replication in the setting of HAART. We make use of a previous observation that in some patients on HAART, the residual viremia is dominated by a predominant plasma clone (PPC) of HIV-1 not found in the latent reservoir. The unique sequence of the PPC serves as a functional label for new entries into the reservoir. We employ a simple mathematical model for the dynamics of the latent reservoir to constrain the inflow rate to between 0 and as few as 70 cells per day. The magnitude of the maximum daily inflow rate is small compared to the size of the latent reservoir, and therefore any inflow that occurs in patients on HAART is unlikely to significantly influence the decay rate of the reservoir. These results suggest that the stability of the latent reservoir is unlikely to arise from ongoing replication during HAART. Thus, intensification of standard HAART regimens should have minimal effects on the decay of the latent reservoir.
A Microchip CD4 Counting Method for HIV Monitoring in Resource-Poor Settings
(Public Library of Science, 2005-07-19) Rodriguez, William R; Christodoulides, Nicolaos; Floriano, Pierre N; Graham, Susan; Mohanty, Sanghamitra; Dixon, Meredith; Hsiang, Mina; Peter, Trevor; Zavahir, Shabnam; Thior, Ibou; Romanovicz, Dwight; Bernard, Bruce; Goodey, Adrian P; Walker, Bruce D; McDevitt, John T
Background -- More than 35 million people in developing countries are living with HIV infection. An enormous global effort is now underway to bring antiretroviral treatment to at least 3 million of those infected. While drug prices have dropped considerably, the cost and technical complexity of laboratory tests essential for the management of HIV disease, such as CD4 cell counts, remain prohibitive. New, simple, and affordable methods for measuring CD4 cells that can be implemented in resource-scarce settings are urgently needed. Methods and Findings -- Here we describe the development of a prototype for a simple, rapid, and affordable method for counting CD4 lymphocytes. Microliter volumes of blood without further sample preparation are stained with fluorescent antibodies, captured on a membrane within a miniaturized flow cell and imaged through microscope optics with the type of charge-coupled device developed for digital camera technology. An associated computer algorithm converts the raw digital image into absolute CD4 counts and CD4 percentages in real time. The accuracy of this prototype system was validated through testing in the United States and Botswana, and showed close agreement with standard flow cytometry (r = 0.95) over a range of absolute CD4 counts, and the ability to discriminate clinically relevant CD4 count thresholds with high sensitivity and specificity. Conclusion -- Advances in the adaptation of new technologies to biomedical detection systems, such as the one described here, promise to make complex diagnostics for HIV and other infectious diseases a practical global reality.
Notch ligand functionalized microheads for T cell differentiation of stem cells
(2007-12) Taqvi, Sabia Zehra, 1980-; Roy, Krishnendu
In recent years, great advances have been made in the field of stem cell differentiation. Seminal insights in the area of developmental biology and tissue regeneration have made ex vivo differentiated cells a realistic alternative for transplantation applications. The recent application of these murine-based insights to human systems has paved new paths in autoimmune disease, chemotherapy, and immuno-deficiency research. Such strides would eliminate the hurdles associated with adoptive transfer including limited availability of transplantable cells, site morbidity, difficulties in cell isolation and expansion lag time. Current approaches in ex vivo hematopoiesis and T cell differentiation have begun to explore the effects of biomaterials on differentiation efficiency. These approaches, however, have not fully studied the quantitative effects of biomaterials and their properties on hematopoietic and T cell differentiation generation. Our goal was to design biomaterials whose properties could be tailored to improve differentiation efficiencies in T cell differentiation. Our work is dedicated to fabricating and characterizing Notch ligand functionalized microbeads for T cell differentiation applications. Our work has shown stable functionalization of Notch ligands on microbeads that can be quantitatively varied to achieve optimal Notch signaling. We have also demonstrated limited cellular toxicity and effective Notch signaling upon exposure to Notch ligand functionalized beads. Finally, we have successfully differentiated T cell progenitors from hematopoietic stem cells using the functionalized microbeads. As a side study, we have fabricated and characterized polymeric PLA scaffolds that were systematically varied and studied for their effects on hematopoietic differentiation efficiency. Insights gained from these studies should provide a better understanding of the microenvironmental signals in hematopoiesis and aid in the development of efficient technologies for the production of hematopoietic progenitors and T cells for therapeutic applications.
The role of ADAP in T cell MTOC polarization
(2006) Combs, Jeffrey Howard; Poenie, Martin F.
Binding of T cells to antigen-presenting cells (APCs) leads to the formation of the immunological synapse, translocation of the microtubule-organizing center (MTOC) to the synapse, and focused secretion of effector molecules. Translocation of the MTOC towards the synapse is essential for guiding the microtubule-dependent movement of secretory granules to the secretory zone at the synapse. Although MTOC translocation is essential for T cell effector function, the mechanism of MTOC polarization is still unknown. Here, data are presented that provide insight into the mechanisms of this event. It is shown that upon activation, ADAP forms a ring at the synapse that marks the site where microtubules interact with the cortex. The ADAP ring colocalizes with dynein and the dynein binding proteins β-catenin and PLAC-24. In Jurkat T cells, when ADAP expression is reduced by antisense morpholino oligonucleotides, MTOC translocation is blocked and dynein fails to localize at the synapse. These results suggest the involvement of ADAP in a mechanism that links signaling through the TCR to translocation of the MTOC.
Studies of the regulatory function of L2a in mouse CD8 gene expression
(2006) Yao, Xin; Tucker, Philip W.
The TCR coreceptors CD4 and CD8 are crucial for thymocyte development and effector function of T cells. L2a was identified as a cis-acting DNA element putatively involved in CD8 expression. The L2a element has the properties of a nuclear matrix attachment region (MAR). It interacts with two MAR-binding proteins, SATB1 and CDP/Cux, through separated AT-rich regions, L and S. L2a mutants with an increased inter-LS region have decreased CDP/Cux binding, suggesting that both sites are required for binding at the same time. Upon binding of SATB1, these L2a mutants display altered DNase I hypersensitivity (DH) in the inter-LS region. A palindromic DNA 12-mer proximal to the S site was found to alter interactions between L2a and its binding proteins, and two 12-mer binding proteins have been identified. Transgenic studies suggested that L2a is potential silencer for regulating CD8 expression. Transgenes driven by the L2a-containing DH cluster II and an enhancer E8I showed no reporter expression in thymic subsets or in peripheral splenocytes or in intraepitheal lymphocytes (IELs). Deletion of L2a resulted in robust reporter expression, even in the DP population. A small fraction (1~5%) of the L2a-containing transgenic CD8SP thymocytes and peripheral T cells “escaped” L2a-silencing, suggesting that compensatory mechanisms can overcome silencing during transition to the CD8SP stage. Crossing this transgene onto a SATB1 knockdown background decreased the escape rate, indicating that SATB1 is involved in re-starting silenced CD8 expression. Knock-in studies were carried out to further investigate the function of L2a. The M1 mutant knock-in mice, which have altered binding sites that abolish SATB1 interaction, showed no significant changes in CD8 expression. Knock-in mice in which the entire L2a element was deleted (ΔL2a) showed modestly increased CD8 levels in CD8SP thymocytes, peripheral CD8 T cells, and IELs. These effects are indicative of the consequences of losing a potential CD8 silencer, but their modest magnitudes suggest that other compensatory mechanisms suppress L2a function in the germline. Finally, targeted deletion of L2a resulted in significantly decreased CD8αα expression on splenic dendritic cells, implicating an unsuspected regulatory role for L2a in the lineage development of this myeloid sub-population.
T-cell activation by ethanol: a possible mechanism for immunosuppression
(2007-12) Naqvi, Hassan Raza, 1976-; Poenie, Martin F.
Alcohol abuse has been commonly associated with enhanced susceptibility to pathogens. Studies on the effects of ethanol on the immune system are complicated by a lack of consensus on whether ethanol activates, inhibits or has no effect on immune cells. We present data showing that acute exposure of T cells to ethanol elicits responses that broadly parallel responses seen in normally stimulated T cells such as the formation of the immune synapse, polarization of the microtubule organizing center (MTOC) to the synapse and tyrosine phosphorylation of signaling proteins as seen when the T cell Receptor (TcR) engages antigen-MHC. However, incomplete activation of the T cell signaling program leads to unresponsive or anergic T cells. Our data suggests the hypothesis that ethanol can activate T cells in a manner that leads to anergy. We have found that ethanol triggers calcium signaling and this has provided one of the primary tools for analyzing the effects of ethanol on T cells. Ethanol induced calcium transients are dose-dependent and are comparable to those triggered by low doses of anti-TcR antibody. This is important because it allows us to compare ethanol dependent signaling to that normally triggered through stimulation of the T cell receptors. Analysis of the calcium signaling pathway indicates that ethanol-stimulated calcium transients depend on calcium entry and are likely due to opening of CRAC type calcium channels. The observed calcium transients go a long way towards explaining how ethanol may stimulate T cells and provides a mechanism for immune suppression through the observed translocation of NF-AT in ethanol pulsed cells. The translocation of NF-AT is particularly important because of reports that it plays a crucial role in triggering anergy and immunosuppression. Taken together, these data can help explain how ethanol can both activate T cells and cause immunosuppression.