Associate Professor, Medicine - Nephrology
Director of Basic Research, Division of Nephrology (2015 - Present)
Boards, Advisory Committees, Professional Organizations
American Society of Transplantation, Board of Directors (2018 - Present)
Federation of Clinical Immunology Societies, Secretary / Treasurer (2017 - Present)
BS, MIT, Biology (1989)
PhD, University of Pennsylvania School of Medicine, Immunology (1997)
MD, University of Pennsylvania School of Medicine, Medicine (1997)
Internship/Residency, University of Chicago Hospitals, Internal Medicine (2000)
Fellowship, University of Pennsylvania School of Medicine, Nephrology (2003)
Doctoral Dissertation Reader (AC)
The ONE Study: One Small Step for Patient Care, a Giant Leap for Cell Therapy
AMERICAN JOURNAL OF KIDNEY DISEASES
2021; 77 (2): 297–99
View details for Web of Science ID 000609881700018
Anti-HLA and anti-SARS-CoV2 Antibodies in Kidney Transplant Recipient with COVID-19.
Transplant international : official journal of the European Society for Organ Transplantation
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and its associated illness coronavirus disease 2019 (COVID-19) have severely affected organ transplant recipients, with all-cause mortality rates exceeding 20% (1, 2). Although no clear guidelines exist on how to adjust immunosuppression, most centers reduce anti-rejection drugs to facilitate the T and B cell response against the virus.
View details for DOI 10.1111/tri.13829
View details for PubMedID 33481306
- Aging Tregs need DCAFinating. Science immunology 2020; 5 (52)
- Sestrins teach old T cells new tricks. Science immunology 2020; 5 (47)
Development and Validation of a Multiplex, Bead-Based Assay to Detect Antibodies Directed Against SARS-CoV-2 Proteins.
Transplant recipients who develop COVID-19 may be at increased risk for morbidity and mortality. Determining the status of antibodies against SARS-CoV-2 in both candidates and recipients will be important to understand the epidemiology and clinical course of COVID-19 in this population. While there are multiple tests to detect antibodies to SARS-CoV-2, their performance is variable. Tests vary according to their platforms and the antigenic targets which make interpretation of the results challenging. Furthermore, for some assays, sensitivity and specificity is less than optimal. Additionally, currently available serological tests do not exclude the possibility that positive responses are due to cross reactive antibodies to community coronaviruses rather than SARS-CoV-2.This study describes the development and validation of a high throughput multiplex antibody detection assay.The multiplex assay has the capacity to identify, simultaneously, patient responses to 5 SARS-CoV-2 proteins, namely, the full spike protein, 3 individual domains of the spike protein (S1, S2 and receptor binding domain) and the nucleocapsid protein. The antibody response to the above proteins are SARS-CoV-2 specific, as antibodies against 4 common coronaviruses do not cross-react.This new assay provides a novel tool to interrogate the spectrum of immune responses to SAR-CoV-2 and is uniquely suitable for use in the transplant setting. Test configuration is essentially identical to the single antigen bead assays used in the majority of histocompatibility laboratories around the world and could easily be implemented into routine screening of transplant candidates and recipients.
View details for DOI 10.1097/TP.0000000000003524
View details for PubMedID 33273320
- The ONE Study: One Small Step for Patient Care, a Giant Leap for Cell Therapy. American journal of kidney diseases : the official journal of the National Kidney Foundation 2020
- To debug or not to debug, a question worth asking. Science immunology 2019; 4 (41)
Transplantation Alters Function and Clonality of Cytomegalovirus-Responsive T Cells
LIPPINCOTT WILLIAMS & WILKINS. 2019
View details for Web of Science ID 000511467800256
- Single cell immune profiling in transplantation research AMERICAN JOURNAL OF TRANSPLANTATION 2019; 19 (5): 1278–87
- Optimization of single-cell plate sorting for high throughput sequencing applications JOURNAL OF IMMUNOLOGICAL METHODS 2019; 466: 17–23
Single cell immune profiling in transplantation research.
American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons
Recently developed single-cell profiling technologies hold promise to provide new insights including analysis of population heterogeneity and linkage of antigen receptors with gene expression. These technologies produce complex data sets that require knowledge of bioinformatics for appropriate analysis. In this minireview, we discuss several single-cell immune profiling technologies for gene and protein expression, including cytometry by time-of-flight, RNA sequencing, and antigen receptor sequencing, as well as key considerations for analysis that apply to each. Because of the critical importance of data analysis for high parameter single cell analysis, we discuss essential factors in analysis of these data, including quality control, quantification, examples of methods for high dimensional analysis, immune repertoire analysis, and preparation of analysis pipelines. We provide examples of, and suggestions for, application of these innovative methods to transplantation research. This article is protected by copyright. All rights reserved.
View details for PubMedID 30768832
- Duel(ling) precursors in thymic Treg development. Science immunology 2019; 4 (36)
- Duel(ling) precursors in thymic Treg development. Science immunology 2019; 4 (36)
Optimization of single-cell plate sorting for high throughput sequencing applications.
Journal of immunological methods
Single cell sequencing has recently been applied to many immunological studies. Flow cytometric index sorting isolates cells for single cell sequencing with protein level data linked to sequences. However, successful sequencing of index sorted samples requires careful optimization of several sort parameters, including nozzle size, flow rate, threshold rate, and yield calculations. In this study, considerations and optimization data for each of these variables are presented. Our analysis focused on index sorting, but the findings can be applied to any plate sorting protocol. Minimization of flow rates and use of the 70 mum nozzle improved cell yields. Improvements in total read counts after sequencing were obtained by decreasing the threshold rate, or the number of cells processed per second. In addition, this technique provided linked protein and gene expression analysis of the cytokine interferon (IFN)gamma, demonstrating that on a single cell basis IFNgamma+ cells tend to express IFNG mRNA, and IFNgamma- cells do not. Through rigorous optimization and quality control, we have identified parameters important to plate sorting and recommend the use of the 70 mum nozzle and low flow and threshold rates for analysis of rare populations of human lymphocytes.
View details for PubMedID 30590019
The outstanding questions in transplantation: It's about time...
AMERICAN JOURNAL OF TRANSPLANTATION
2018; 18 (1): 271–72
View details for PubMedID 28758364
Inflammatory macrophage-associated 3-gene signature predicts subclinical allograft injury and graft survival.
2018; 3 (2)
Late allograft failure is characterized by cumulative subclinical insults manifesting over many years. Although immunomodulatory therapies targeting host T cells have improved short-term survival rates, rates of chronic allograft loss remain high. We hypothesized that other immune cell types may drive subclinical injury, ultimately leading to graft failure. We collected whole-genome transcriptome profiles from 15 independent cohorts composed of 1,697 biopsy samples to assess the association of an inflammatory macrophage polarization-specific gene signature with subclinical injury. We applied penalized regression to a subset of the data sets and identified a 3-gene inflammatory macrophage-derived signature. We validated discriminatory power of the 3-gene signature in 3 independent renal transplant data sets with mean AUC of 0.91. In a longitudinal cohort, the 3-gene signature strongly correlated with extent of injury and accurately predicted progression of subclinical injury 18 months before clinical manifestation. The 3-gene signature also stratified patients at high risk of graft failure as soon as 15 days after biopsy. We found that the 3-gene signature also distinguished acute rejection (AR) accurately in 3 heart transplant data sets but not in lung transplant. Overall, we identified a parsimonious signature capable of diagnosing AR, recognizing subclinical injury, and risk-stratifying renal transplant patients. Our results strongly suggest that inflammatory macrophages may be a viable therapeutic target to improve long-term outcomes for organ transplantation patients.
View details for PubMedID 29367465
- Expanding the Toolkit for the Study of Allospecific B and T Cell Responses TRANSPLANTATION 2017; 101 (11): 2661–62
T cells expand after solid organ transplantation in the absence of CMV disease.
American journal of transplantation
Cytomegalovirus (CMV) is a major cause of morbidity and mortality in solid-organ transplant recipients. Approximately 60% of adults are CMV seropositive indicating previous exposure. Following resolution of primary infection, CMV remains in a latent state. Reactivation is controlled by memory T cells in healthy individuals; transplant recipients have reduced memory T cell function due to chronic immunosuppressive therapies. In this study, CD8(+) T cell responses to CMV polypeptides IE-1 and pp65 were analyzed in sixteen CMV seropositive renal and cardiac transplant recipients longitudinally pre- and post-transplant. All patients received standard of care maintenance immunosuppression, antiviral prophylaxis and CMV viral load monitoring, with approximately half receiving T cell depleting induction therapy. The frequency of CMV-responsive CD8(+) T cells, defined by production of effector molecules in response to CMV peptides, increased during the course of a year post-transplant. The increase commenced after the completion of antiviral prophylaxis, and these T cells tended to be terminally differentiated effector cells. Based on this small cohort, these data suggest that even in the absence of disease, antigenic exposure may continually shape the CMV-responsive T cell population post-transplant. This article is protected by copyright. All rights reserved.
View details for DOI 10.1111/ajt.14227
View details for PubMedID 28199780
Virtual Global Transplant Laboratory Standard Operating Procedures for Blood Collection, PBMC Isolation, and Storage.
2016; 2 (9)
Research on human immune responses frequently involves the use of peripheral blood mononuclear cells (PBMC) immediately, or at significantly delayed timepoints, after collection. This requires PBMC isolation from whole blood and cryopreservation for some applications. It is important to standardize protocols for blood collection, PBMC isolation, cryopreservation, and thawing that maximize survival and functionality of PBMC at the time of analysis. This resource includes detailed protocols describing blood collection tubes, isolation of PBMC using a density gradient, cryopreservation of PBMC, and thawing of cells as well as preparation for functional assays. For each protocol, we include important considerations, such as timing, storage temperatures, and freezing rate. In addition, we provide alternatives so that researchers can make informed decisions in determining the optimal protocol for their application.
View details for PubMedID 27795993
Differentially Expressed Gene Transcripts Using RNA Sequencing from the Blood of Immunosuppressed Kidney Allograft Recipients
2015; 10 (5)
We performed RNA sequencing (RNAseq) on peripheral blood mononuclear cells (PBMCs) to identify differentially expressed gene transcripts (DEGs) after kidney transplantation and after the start of immunosuppressive drugs. RNAseq is superior to microarray to determine DEGs because it's not limited to available probes, has increased sensitivity, and detects alternative and previously unknown transcripts. DEGs were determined in 32 adult kidney recipients, without clinical acute rejection (AR), treated with antibody induction, calcineurin inhibitor, mycophenolate, with and without steroids. Blood was obtained pre-transplant (baseline), week 1, months 3 and 6 post-transplant. PBMCs were isolated, RNA extracted and gene expression measured using RNAseq. Principal components (PCs) were computed using a surrogate variable approach. DEGs post-transplant were identified by controlling false discovery rate (FDR) at < 0.01 with at least a 2 fold change in expression from pre-transplant. The top 5 DEGs with higher levels of transcripts in blood at week 1 were TOMM40L, TMEM205, OLFM4, MMP8, and OSBPL9 compared to baseline. The top 5 DEGs with lower levels at week 1 post-transplant were IL7R, KLRC3, CD3E, CD3D, and KLRC2 (Striking Image) compared to baseline. The top pathways from genes with lower levels at 1 week post-transplant compared to baseline, were T cell receptor signaling and iCOS-iCOSL signaling while the top pathways from genes with higher levels than baseline were axonal guidance signaling and LXR/RXR activation. Gene expression signatures at month 3 were similar to week 1. DEGs at 6 months post-transplant create a different gene signature than week 1 or month 3 post-transplant. RNAseq analysis identified more DEGs with lower than higher levels in blood compared to baseline at week 1 and month 3. The number of DEGs decreased with time post-transplant. Further investigations to determine the specific lymphocyte(s) responsible for differential gene expression may be important in selecting and personalizing immune suppressant drugs and may lead to targeted therapies.
View details for DOI 10.1371/journal.pone.0125045
View details for PubMedID 25946140
Regulatory T Cells Require TCR Signaling for Their Suppressive Function
JOURNAL OF IMMUNOLOGY
2015; 194 (9): 4362-4370
Regulatory T cells (Tregs) are a subset of CD4(+) T cells that maintain immune tolerance in part by their ability to inhibit the proliferation of conventional CD4(+) T cells (Tconvs). The role of the TCR and the downstream signaling pathways required for this suppressive function of Tregs are not fully understood. To yield insight into how TCR-mediated signals influence Treg suppressive function, we assessed the ability of Tregs with altered TCR-mediated signaling capacity to inhibit Tconv proliferation. Mature Tregs deficient in Src homology 2 domain containing leukocyte protein of 76 kDa (SLP-76), an adaptor protein that nucleates the proximal signaling complex downstream of the TCR, were unable to inhibit Tconv proliferation, suggesting that TCR signaling is required for Treg suppressive function. Moreover, Tregs with defective phospholipase C γ (PLCγ) activation due to a Y145F mutation of SLP-76 were also defective in their suppressive function. Conversely, enhancement of diacylglycerol-mediated signaling downstream of PLCγ by genetic ablation of a negative regulator of diacylglycerol kinase ζ increased the suppressive ability of Tregs. Because SLP-76 is also important for integrin activation and signaling, we tested the role of integrin activation in Treg-mediated suppression. Tregs lacking the adaptor proteins adhesion and degranulation promoting adapter protein or CT10 regulator of kinase/CT10 regulator of kinase-like, which are required for TCR-mediated integrin activation, inhibited Tconv proliferation to a similar extent as wild-type Tregs. Together, these data suggest that TCR-mediated PLCγ activation, but not integrin activation, is required for Tregs to inhibit Tconv proliferation.
View details for DOI 10.4049/jimmunol.1402384
View details for Web of Science ID 000353727400034
View details for PubMedID 25821220
AST Cutting Edge of Transplantation 2013 Meeting Report: A Comprehensive Look at B Cells and Antibodies in Transplantation
AMERICAN JOURNAL OF TRANSPLANTATION
2014; 14 (3): 524-530
Antibody-mediated rejection (ABMR) represents a significant clinical challenge for solid organ transplantation. Mechanistic understanding of ABMR is incomplete and diagnostic accuracy for ABMR is limited, and as a result, targeted treatment remains elusive and new treatment modalities are difficult to validate. Three hundred twenty-six participants from 15 countries met for the first Cutting Edge of Transplantation (CEOT) symposium organized by the American Society of Transplantation (AST) in Chandler, Arizona, February 14-16, 2013. During the 3-day interactive symposium, presentations, moderated poster sessions and round table discussions addressed cutting edge knowledge of B and plasma cell biology, mechanisms of antibody-mediated tissue injury, advances and limitations in ABMR diagnostics, as well as current and potential new treatment options for ABMR. The outcome of the meeting identified the following unmet needs for: (a) improved understanding of the regulation of B cell maturation and antibody response to enable targeted therapies; (b) more precise diagnostics of ABMR, including molecular pathology, risk stratification by sensitive antibody testing and monitoring of treatment effects; and (c) innovative multicenter trial designs that enhance observational power, in particular, in assessing synergistic multimodality therapies with reduced toxicities.
View details for DOI 10.1111/ajt.12593
View details for Web of Science ID 000331783200007
View details for PubMedID 24674597
T-Cell Costimulatory Blockade in Organ Transplantation
COLD SPRING HARBOR PERSPECTIVES IN MEDICINE
2013; 3 (12)
Before it became possible to derive T-cell lines and clones, initial experimentation on the activation requirements of T lymphocytes was performed on transformed cell lines, such as Jurkat. These studies, although technically correct, proved misleading as most transformed T cells can be activated by stimulation of the clonotypic T-cell receptor (TCR) alone. In contrast, once it became possible to study nontransformed T cells, it quickly became clear that TCR stimulation by itself is insufficient for optimal activation of naïve T cells, but in fact, induces a state of anergy. It then became clear that functional activation of T cells requires not only recognition of major histocompatibility complex (MHC) and peptide by the TCR, but also requires ligation of costimulatory receptors expressed on the cell surface.
View details for DOI 10.1101/cshperspect.a015537
View details for Web of Science ID 000328277800002
View details for PubMedID 24296352
An obligate cell-intrinsic function for CD28 in Tregs
JOURNAL OF CLINICAL INVESTIGATION
2013; 123 (2): 580-593
Tregs expressing the transcription factor FOXP3 are critical for immune homeostasis. The costimulatory molecule CD28 is required for optimal activation and function of naive T cells; however, its role in Treg function has been difficult to dissect, as CD28 is required for thymic Treg development, and blockade of CD28-ligand interactions has confounding effects in trans on nonregulatory cells. To address this question, we created Treg-specific Cd28 conditional knockout mice. Despite the presence of normal numbers of FOXP3+ cells, these animals accumulated large numbers of activated T cells, developed severe autoimmunity that primarily affected the skin and lungs, and failed to appropriately resolve induced experimental allergic encephalomyelitis. This in vivo functional impairment was accompanied by dampened expression of CTLA-4, PD-1, and CCR6. Disease occurrence was not due to subversion of Cd28-deficient Tregs into pathogenic cells, as complementation with normal Tregs prevented disease occurrence. Interestingly, in these "competitive" environments, Cd28-deficient Tregs exhibited a pronounced proliferative/survival disadvantage. These data demonstrate clear postmaturational roles for CD28 in FOXP3+ Tregs and provide mechanisms which we believe to be novel to explain how interruption of CD28-ligand interactions may enhance immune responses independent of effects on thymic development or on other cell types.
View details for DOI 10.1172/JCI65013
View details for Web of Science ID 000314553600015
View details for PubMedID 23281398
Homeostatic Division Is Not Necessary for Antigen-Specific CD4(+) Memory T Cell Persistence
JOURNAL OF IMMUNOLOGY
2012; 189 (7): 3378-3385
CD4(+) memory T cells are generated in response to infection or vaccination, provide protection to the host against reinfection, and persist through a combination of enhanced survival and slow homeostatic turnover. We used timed deletion of the TCR-signaling adaptor molecule Src homology 2 domain-containing phosphoprotein of 76 kDa (SLP-76) with MHC:peptide tetramers to study the requirements for tonic TCR signals in the maintenance of polyclonal Ag-specific CD4(+) memory T cells. SLP-76-deficient I-A(b):gp61 cells are unable to rapidly generate effector cytokines or proliferate in response to secondary infection. In mice infected with lymphocytic choriomeningitis virus (LCMV) or Listeria monocytogenes expressing the LCMV gp61-80 peptide, SLP-76-deficient I-A(b):gp61(+) cells exhibit reduced division, similar to that seen in in vitro-generated CD44(hi) and endogenous CD4(+)CD44(hi) cells. Competitive bone marrow chimera experiments demonstrated that the decrease in homeostatic turnover in the absence of SLP-76 is a cell-intrinsic process. Surprisingly, despite the reduction in turnover, I-A(b):gp61(+) Ag-specific memory cells persist in normal numbers for >30 wk after LCMV infection in the absence of SLP-76. These data suggest the independent maintenance of a population of Ag-specific CD4(+) memory T cells in the absence of SLP-76 and normal levels of homeostatic division.
View details for DOI 10.4049/jimmunol.1201583
View details for Web of Science ID 000309164300013
View details for PubMedID 22956580
Foxp4 Is Dispensable for T Cell Development, but Required for Robust Recall Responses
2012; 7 (8)
Transcription factors regulate T cell fates at every stage of development and differentiation. Members of the Foxp family of forkhead transcription factors are essential for normal T lineage development; Foxp3 is required for T regulatory cell generation and function, and Foxp1 is necessary for generation and maintenance of naïve T cells. Foxp4, an additional member of the Foxp family, is highly homologous to Foxp1 and has been shown to dimerize with other Foxp proteins. We report the initial characterization of Foxp4 in T lymphocytes. Foxp4 is expressed in both thymocytes and peripheral CD4(+) and CD8(+) T cells. We used a CD4Cre mediated approach to evaluate the cell autonomous role for Foxp4 in murine T lymphocytes. T cell development, peripheral cellularity and cell surface phenotype are normal in the absence of Foxp4. Furthermore, Foxp3(+) T regulatory cells develop normally in Foxp4 deficient animals and naïve Foxp4 deficient CD4 T cells can differentiate to inducible T regulatory cells in vitro. In wild-type T cells, expression of Foxp4 increases following activation, but deletion of Foxp4 does not affect T cell proliferative responses or in vitro effector T cell differentiation. In vivo, despite effective control of Toxoplasma gondii and acute lymphocytic choriomeningitis virus infections, effector cytokine production during antigen specific recall responses are reduced in the absence of Foxp4. We conclude that Foxp4 is dispensable for T cell development, but necessary for normal T cell cytokine recall responses to antigen following pathogenic infection.
View details for DOI 10.1371/journal.pone.0042273
View details for Web of Science ID 000307500100010
View details for PubMedID 22912696
Cutting Edge: IL-2 Signals Determine the Degree of TCR Signaling Necessary To Support Regulatory T Cell Proliferation In Vivo
JOURNAL OF IMMUNOLOGY
2012; 189 (1): 28-32
To ensure immune tolerance, regulatory T cell (Treg) numbers must be maintained by cell division. This process has been thought to be strictly dependent on the Treg TCR interacting with MHC class II. In this study, we report that Treg division does not absolutely require cell-autonomous TCR signaling in vivo, depending on the degree of IL-2-mediated stimulation provided. At steady state IL-2 levels, Tregs require cell-autonomous TCR signaling to divide. However, when given exogenous IL-2 or when STAT5 is selectively activated in Tregs, Treg division can occur independently of MHC class II and TCR signaling. Thus, depending on the amount of IL-2R stimulation, a wide range of TCR signals supports Treg division, which may contribute to preservation of a diverse repertoire of Treg TCR specificities. These findings also have therapeutic implications, as TCR signaling by Tregs may not be required when using IL-2 to increase Treg numbers for treatment of inflammatory disorders.
View details for DOI 10.4049/jimmunol.1200507
View details for Web of Science ID 000305636000005
View details for PubMedID 22623329
Site-specific accumulation of recently activated CD4+Foxp3+ regulatory T cells following adoptive transfer
EUROPEAN JOURNAL OF IMMUNOLOGY
2012; 42 (6): 1429-1435
CD4(+) Foxp3(+) regulatory T (Treg) cells are required for the maintenance of self-tolerance, as demonstrated by profound autoimmunity in mice and humans with inactivating Foxp3 mutations. Recent studies demonstrate that Treg cells are anatomically compartmentalized within secondary lymphoid organs based on their TCR repertoire and specific organ-protective function; however, whether this reflects differential homing or in situ selection is not known. Here, using Foxp3-GFP reporter mice, we have examined the ability of polyclonal Treg cells from cervical LNs to return to their site-of-origin following adoptive transfer to nonlymphopenic congenic recipients. We find that bulk cervical LN Treg cells do not home directly to cervical LNs but rather accumulate site specifically over time following transfer. Site-specific enrichment is both more rapid and more pronounced among a population of recently activated (CD69(+) ) Treg cells. These data suggest that compartmentalization of Treg cells within secondary lymphoid organs may be governed by antigen recognition and implicate CD69 as a potential marker of recently activated Treg cells recognizing locally expressed antigens.
View details for DOI 10.1002/eji.201142286
View details for Web of Science ID 000304997800008
View details for PubMedID 22678899
- Oral ivermectin as an unexpected initiator of CreT2-mediated deletion in T cells NATURE IMMUNOLOGY 2012; 13 (3): 197-198
Probucol ameliorates renal and metabolic sequelae of primary CoQ deficiency in Pdss2 mutant mice
EMBO MOLECULAR MEDICINE
2011; 3 (7): 410-427
Therapy of mitochondrial respiratory chain diseases is complicated by limited understanding of cellular mechanisms that cause the widely variable clinical findings. Here, we show that focal segmental glomerulopathy-like kidney disease in Pdss2 mutant animals with primary coenzyme Q (CoQ) deficiency is significantly ameliorated by oral treatment with probucol (1% w/w). Preventative effects in missense mutant mice are similar whether fed probucol from weaning or for 3 weeks prior to typical nephritis onset. Furthermore, treating symptomatic animals for 2 weeks with probucol significantly reduces albuminuria. Probucol has a more pronounced health benefit than high-dose CoQ(10) supplementation and uniquely restores CoQ(9) content in mutant kidney. Probucol substantially mitigates transcriptional alterations across many intermediary metabolic domains, including peroxisome proliferator-activated receptor (PPAR) pathway signaling. Probucol's beneficial effects on the renal and metabolic manifestations of Pdss2 disease occur despite modest induction of oxidant stress and appear independent of its hypolipidemic effects. Rather, decreased CoQ(9) content and altered PPAR pathway signaling appear, respectively, to orchestrate the glomerular and global metabolic consequences of primary CoQ deficiency, which are both preventable and treatable with oral probucol therapy.
View details for DOI 10.1002/emmm.201100149
View details for Web of Science ID 000293277400005
View details for PubMedID 21567994
Conditional deletion of SLP-76 in mature T cells abrogates peripheral immune responses
EUROPEAN JOURNAL OF IMMUNOLOGY
2011; 41 (7): 2064-2073
The adaptor protein Src homology 2 domain-containing leukocyte-specific protein of 76 kDa (SLP-76) is central to the organization of intracellular signaling downstream of the T-cell receptor (TCR). Evaluation of its role in mature, primary T cells has been hampered by developmental defects that occur in the absence of WT SLP-76 protein in thymocytes. Here, we show that following tamoxifen-regulated conditional deletion of SLP-76, mature, antigen-inexperienced T cells maintain normal TCR surface expression but fail to transduce TCR-generated signals. Conditionally deficient T cells fail to proliferate in response to antigenic stimulation or a lymphopenic environment. Mice with induced deletion of SLP-76 are resistant to induction of the CD4+ T-cell-mediated autoimmune disease experimental autoimmune encephalomyelitis. Altogether, our findings demonstrate the critical role of SLP-76-mediated signaling in initiating T-cell-directed immune responses both in vitro and in vivo and highlight the ability to analyze signaling processes in mature T cells in the absence of developmental defects.
View details for DOI 10.1002/eji.201040809
View details for Web of Science ID 000293131200027
View details for PubMedID 21469089
T cell receptor signal strength in T-reg and iNKT cell development demonstrated by a novel fluorescent reporter mouse
JOURNAL OF EXPERIMENTAL MEDICINE
2011; 208 (6): 1279-1289
The ability of antigen receptors to engage self-ligands with varying affinity is crucial for lymphocyte development. To further explore this concept, we generated transgenic mice expressing GFP from the immediate early gene Nr4a1 (Nur77) locus. GFP was up-regulated in lymphocytes by antigen receptor stimulation but not by inflammatory stimuli. In T cells, GFP was induced during positive selection, required major histocompatibility complex for maintenance, and directly correlated with the strength of T cell receptor (TCR) stimulus. Thus, our results define a novel tool for studying antigen receptor activation in vivo. Using this model, we show that regulatory T cells (T(reg) cells) and invariant NKT cells (iNKT cells) perceived stronger TCR signals than conventional T cells during development. However, although T(reg) cells continued to perceive strong TCR signals in the periphery, iNKT cells did not. Finally, we show that T(reg) cell progenitors compete for recognition of rare stimulatory TCR self-ligands.
View details for DOI 10.1084/jem.20110308
View details for Web of Science ID 000291424200014
View details for PubMedID 21606508
Antiviral memory CD8 T-cell differentiation, maintenance, and secondary expansion occur independently of MyD88
2011; 117 (11): 3123-3130
Inflammatory signals induced during infection regulate T-cell expansion, differentiation, and memory formation. Toll-like receptors (TLRs) are inflammatory mediators that allow innate immune cells to recognize and respond to invading pathogens. In addition to their role in innate immune cells, we have found that signals delivered through the TLR adapter protein myeloid differentiation protein 88 (MyD88) play a critical, T cell-intrinsic role in supporting the survival and accumulation of antigen-specific effector cells after acute viral infection. However, the importance of MyD88-dependent signals in regulating the generation and maintenance of memory T cells remained unclear. To address this, we used a novel, inducible knockout system to examine whether MyD88 is required for optimal memory CD8 T-cell generation and responses after lymphocytic choriomeningitis virus infection. We show that whereas MyD88 is critical for initial T-cell expansion, it is not required for the subsequent differentiation and stable maintenance of a memory T-cell population. Furthermore, in contrast to naive CD8 T cells, memory CD8 T cells do not depend on MyD88 for their secondary expansion. Our findings clarify the importance of MyD88 during distinct phases of the antiviral T-cell response and establish differential dependence on MyD88 signaling as a novel characteristic that distinguishes naive from memory CD8 T cells.
View details for DOI 10.1182/blood-2010-11-318485
View details for Web of Science ID 000288496300022
View details for PubMedID 21233312
Loss of tonic T-cell receptor signals alters the generation but not the persistence of CD8(+) memory T cells
2010; 116 (25): 5560-5570
The requirements for tonic T-cell receptor (TCR) signaling in CD8(+) memory T-cell generation and homeostasis are poorly defined. The SRC homology 2 (SH2)-domain-containing leukocyte protein of 76 kDa (SLP-76) is critical for proximal TCR-generated signaling. We used temporally mediated deletion of SLP-76 to interrupt tonic and activating TCR signals after clearance of the lymphocytic choriomeningitis virus (LCMV). SLP-76-dependent signals are required during the contraction phase of the immune response for the normal generation of CD8 memory precursor cells. Conversely, LCMV-specific memory CD8 T cells generated in the presence of SLP-76 and then acutely deprived of TCR-mediated signals persist in vivo in normal numbers for more than 40 weeks. Tonic TCR signals are not required for the transition of the memory pool toward a central memory phenotype, but the absence of SLP-76 during memory homeostasis substantially alters the kinetics. Our data are consistent with a model in which tonic TCR signals are required at multiple stages of differentiation, but are dispensable for memory CD8 T-cell persistence.
View details for DOI 10.1182/blood-2010-06-292458
View details for Web of Science ID 000285383900019
View details for PubMedID 20884806
Ablation of SLP-76 signaling after T cell priming generates memory CD4 T cells impaired in steady-state and cytokine-driven homeostasis
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2010; 107 (2): 827-831
The intracellular signaling mechanisms regulating the generation and long-term persistence of memory T cells in vivo remain unclear. In this study, we used mouse models with conditional deletion of the key T cell receptor (TCR)-coupled adaptor molecule SH2-domain-containing phosphoprotein of 76 kDa (SLP-76), to analyze signaling mechanisms for memory CD4 T cell generation, maintenance, and homeostasis. We found that ablation of SLP-76 expression after T cell priming did not inhibit generation of phenotypic effector or memory CD4 T cells; however, the resultant SLP-76-deficient memory CD4 T cells could not produce recall cytokines in response to TCR-mediated stimulation and showed decreased persistence in vivo. In addition, SLP-76-deficient memory CD4 T cells exhibited reduced steady-state homeostasis and were impaired in their ability to homeostatically expand in vivo in response to the gamma(c) cytokine IL-7, despite intact proximal signaling through the IL-7R-coupled JAK3/STAT5 pathway. Direct in vivo deletion of SLP-76 in polyclonal memory CD4 T cells likewise led to impaired steady-state homeostasis as well as impaired homeostatic responses to IL-7. Our findings demonstrate a dominant role for SLP-76-dependent TCR signals in regulating turnover and perpetuation of memory CD4 T cells and their responses to homeostatic cytokines, with implications for the selective survival of memory CD4 T cells following pathogen exposure, vaccination, and aging.
View details for DOI 10.1073/pnas.0908126107
View details for Web of Science ID 000273559300057
View details for PubMedID 20080760
Conditional gene expression: A new tool for the transplantologist
AMERICAN JOURNAL OF TRANSPLANTATION
2007; 7 (4): 733-740
The ability to generate genetically manipulated mice has revolutionized the study of development, cell biology, immunobiology and transplantation. Conventional gene targeting approaches lead to inactivation of the target gene in all tissues. This approach often has unintended consequences, such as embryonic lethality, which preclude studying the originally intended tissue. Newer approaches allowing conditional gene expression in a tissue-specific or temporally controlled fashion have the advantage of normal development with gene deletion only in the desired tissues. While nuances to these techniques continue to be developed, the underlying concepts remain consistent. This minireview focuses on the use of conditional gene targeting in mice using the Cre-loxP system and drug inducible gene expression using the tetracycline system.
View details for DOI 10.1111/j.1600-6143.2006.01685.x
View details for Web of Science ID 000245151500002
View details for PubMedID 17391118
Conditional deletion reveals a cell-autonomous requirement of SLP-76 for thymocyte selection
JOURNAL OF EXPERIMENTAL MEDICINE
2005; 202 (7): 893-900
The SH2 domain containing leukocyte phosphoprotein of 76 kD (SLP-76) is critical for pre-TCR-mediated maturation to the CD4+CD8+ double positive (DP) stage in the thymus. The absolute block in SLP-76null mice at the CD4-CD8-CD44-CD25+ (double-negative 3, DN3) stage has hindered our understanding of the role of this adaptor in alphabeta TCR-mediated signal transduction in primary thymocytes and peripheral T lymphocytes. To evaluate the requirements for SLP-76 in these events, we used a cre-loxP approach to generate mice that conditionally delete SLP-76 after the DN3 checkpoint. These mice develop DP thymocytes that express the alphabeta TCR on the surface, but lack SLP-76 at the genomic DNA and protein levels. The DP compartment has reduced cellularity in young mice and fails to undergo positive selection to CD4+ or CD8+ single positive (SP) cells in vivo or activation-induced cell death in vitro. A small number of CD4+SP thymocytes are generated, but these cells fail to flux calcium in response to an alphabeta TCR-generated signal. Peripheral T cells are reduced in number, lack SLP-76 protein, and have an abnormal surface phenotype. These studies show for the first time that SLP-76 is required for signal transduction through the mature alphabeta TCR in primary cells of the T lineage.
View details for DOI 10.1084/jem.20051128
View details for Web of Science ID 000232619000003
View details for PubMedID 16186188
Role of EGR1 in regulation of stimulus-dependent CD44 transcription in B lymphocyte
MOLECULAR AND CELLULAR BIOLOGY
1996; 16 (5): 2283-2294
The immediate-early gene egr-1 encodes a transcription factor (EGR1) that links B-cell antigen receptor (BCR) signals to downstream activation events through the regulation of previously unidentified target genes. Here we identify the gene encoding the lymphocyte homing and migration protein CD44 as a target of EGR1 regulation in B cells. BCR-induced increases in CD44 mRNA expression and transcription levels are shown to occur in EGR1-expressing but not in nonexpressing subclones of the B-cell line WEHI-231. Kinetics of egr-1 transcription and the appearance of nuclear EGR1 protein precede CD44 induction and occur within 30 min after stimulation in the EGR1-expressing subclone. A single EGR1 binding motif is demonstrated at bp -301 of the human CD44 promoter. Cotransfection of a CD44 promoter-chloramphenicol acetyltransferase reporter construct with an egr-1 expression vector resulted in a 6.5- to 8.5-fold induction of transcriptional activity relative to an empty expression vector. The EGR1 binding motif was shown to be necessary for stimulus-induced expression of a CD44 promoter-chloramphenicol acetyltransferase reporter construct in nontransformed B lymphocytes and was required for transactivation by an EGR1 expression vector in a B-cell line. These studies identify EGR1 as an intermediary linking BCR-derived signals to the induction of CD44. The relevance of these molecular events to BCR signal transduction and antigen-stimulated B-cell-mediated immune responses is discussed.
View details for Web of Science ID A1996UG29900042
View details for PubMedID 8628295
Transcriptional regulation of the Icam-1 gene in antigen receptor- and phorbol ester-stimulated B lymphocytes: Role for transcription factor EGR1
JOURNAL OF EXPERIMENTAL MEDICINE
1996; 183 (4): 1747-1759
Intercellular adhesion molecule (ICAM) 1/CD54 plays an important role in T cell dependent B cell activation and for function of B lymphocytes as antigen-presenting cells. ICAM-1 expression is upregulated as a consequence of B lymphocyte antigen receptor (BCR) signaling, thereby serving to render antigen-stimulated B cells more receptive to T cell-mediated costimulatory signals. We have investigated BCR-induced expression of the Icam-1 gene in primary B cells and B cell lines and have found it to be dependent on BCR-induced expression of the transcription factor EGR1. Icam-1 transcription, induced by BCR cross-linking or bypassing the BCR with phorbol ester, is absent in a B cell line in which the EGR1-encoding gene (egr-1) is methylated and not expressed. A potential EGR1-binding site was located at -701 bp upstream of the murine Icam-1 gene transcription start site and shown by electrophoretic mobility shift assay to bind to murine EGR1. Mutation of this site in the context of 1.1 kb of the Icam-1 promoter significantly abrogated transcriptional induction by phorbol ester and anti-mu stimulation in primary B cells. A direct effect of EGR1 on the Icam-1 promoter is suggested by the ability of EGR1 expressed from an SV40-driven expression vector transactivate the wild-type Icam-1 promoter, whereas mutation of the EGR1 mutation of the EGR1 binding motif at -701 bp markedly compromises this induction. These data identify EGR1 as a signaling intermediate in BCR-stimulated B cell functional responses, specifically linking BCR signal transduction to induction of the Icam-1 gene. Furthermore, similar findings for BCR-induced CD44 gene induction (Maltzman, J.S., J.A. Carman, and J.G. Monroe. 1996. Role of EGR1 in regulation of stimulus-dependent CD44 transcription in B lymphocytes. Mol. Cell. Biol. In press) suggest that EGR1 may be an important signaling molecule for regulating levels of migration and adhesion molecules during humoral immune responses.
View details for Web of Science ID A1996UH14400048
View details for PubMedID 8666932
MOLECULAR EVIDENCE FOR THE EXPRESSION OF NICOTINIC ACETYLCHOLINE-RECEPTOR ALPHA-CHAIN IN MOUSE THYMUS
JOURNAL OF IMMUNOLOGY
1992; 148 (10): 3105-3109
The presence and structure of nicotinic acetylcholine receptor (nAChR) in the thymus has been a subject of interest for many years because of its possible role in the pathogenesis of the autoimmune disease myasthenia gravis. Using the polymerase chain reaction with primers specific for the alpha-chain of nAChR (nAChR-alpha), an 880-bp homologous band was found after amplification of cDNA prepared from mouse thymus, thymic medullary and cortical epithelial cell lines, but not from thymocytes or kidney. Sequencing of the polymerase chain reaction product from the thymus and thymic medullary and cortical epithelial lines showed identity with skeletal muscle nAChR-alpha over the region examined. This region includes the domains of the molecule on which B cell and T cell autoantigenic targets have been described. No evidence was found in mouse tissue for the exon 3A, which has been described in human muscle and the human rhabdomyosarcoma cell line TE671. Our results provide evidence at the RNA level for the expression of the nAChR-alpha on stromal cells but not on thymocytes in normal murine thymus and are consistent with a role for intrathymic autoantigen expression in the pathogenesis of myasthenia gravis.
View details for Web of Science ID A1992HU68600019
View details for PubMedID 1578136
EXPRESSION OF THE V-BETA-5.1 GENE BY MURINE PERIPHERAL T-CELLS IS CONTROLLED BY MHC GENES AND SKEWED TO THE CD8+ SUBSET
JOURNAL OF IMMUNOLOGY
1990; 144 (3): 844-848
We have examined the usage of the V beta 5.1 region in peripheral T cell populations of several mouse strains by measuring V beta 5.1 transcript levels. The results show that V beta 5.1 is expressed predominantly by CD8+ T cells in mice of several MHC haplotypes. This finding suggests that V beta 5.1 may confer restriction to class 1 molecules present in these strains. Interestingly, however, T cells expressing V beta 5.1, like those expressing V beta 17a and V beta 11, are clonally deleted from both CD4 and CD8 subsets in mice that express MHC IE molecules. The latter result implies that V beta 5.1 confers reactivity to a class 2 molecule (IE). These results are discussed in terms of positive selection and clonal deletion. Finally we provide evidence that genes in the K or A regions of the MHC also control V beta 5.1 usage.
View details for Web of Science ID A1990CL02700011
View details for PubMedID 1967275
POSITIVE SELECTION DETERMINES T-CELL RECEPTOR V-BETA-14 GENE USAGE BY CD8+ T-CELLS
JOURNAL OF EXPERIMENTAL MEDICINE
1989; 170 (1): 135-143
We report here a mAb, 14-2, reactive with TCRs that include V beta 14. The frequency of V beta 14+ T cells varies with CD4 and CD8 subset and is controlled by the H-2 genes. Thus CD8+ T cells from H-2b mice include approximately 2.3% V beta 14+ T cells while CD8+ T cells from mice expressing K kappa include greater than 8% V beta 14+ T cells. In all strains examined, 7-8% of CD4+ T cells express V beta 14. The frequent usage of V beta 14 in CD8+ T cells of K kappa-expressing mice is a result of preferential positive selection of V beta 14+ CD8+ T cells as demonstrated by analysis of radiation chimeras. These studies demonstrate that H-2-dependent positive selection occurs in unmanipulated mice. Furthermore, the results imply that positive selection, and possibly H-2 restriction, can be strongly influenced by a V beta domain, with some independence from the beta-junctional sequence and alpha chain.
View details for Web of Science ID A1989AE68500010
View details for PubMedID 2501444