Bachelor of Science, Stanford University, BIO-BSH (2009)
Bachelor of Science, Stanford University, HSTRY-MIN (2009)
Doctor of Philosophy, University of Washington (2014)
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 DOI 10.1111/ajt.15316
View details for PubMedID 30768832
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 DOI 10.1016/j.jim.2018.12.006
View details for PubMedID 30590019
- The outstanding questions in transplantation: It's about time... AMERICAN JOURNAL OF TRANSPLANTATION 2018; 18 (1): 271–72
- 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
View details for PubMedCentralID PMC5068195
Caught Off Center: Rethinking the Requirements for Antibody Affinity Maturation
2015; 43 (1): 5-6
Antibody affinity maturation involves selective survival of high affinity B cells and is thought to require the germinal center (GC) microenvironment. In this issue of Immunity, Di Niro et al. (2015) challenge this view, showing that low affinity B cells initiate Salmonella responses and affinity mature outside of GCs.
View details for DOI 10.1016/j.immuni.2015.07.002
View details for Web of Science ID 000360101100003
View details for PubMedID 26200006
ICOS and Bcl6-dependent pathways maintain a CD4 T cell population with memory-like properties during tuberculosis
JOURNAL OF EXPERIMENTAL MEDICINE
2015; 212 (5): 715-728
Immune control of persistent infection with Mycobacterium tuberculosis (Mtb) requires a sustained pathogen-specific CD4 T cell response; however, the molecular pathways governing the generation and maintenance of Mtb protective CD4 T cells are poorly understood. Using MHCII tetramers, we show that Mtb-specific CD4 T cells are subject to ongoing antigenic stimulation. Despite this chronic stimulation, a subset of PD-1(+) cells is maintained within the lung parenchyma during tuberculosis (TB). When transferred into uninfected animals, these cells persist, mount a robust recall response, and provide superior protection to Mtb rechallenge when compared to terminally differentiated Th1 cells that reside preferentially in the lung-associated vasculature. The PD-1(+) cells share features with memory CD4 T cells in that their generation and maintenance requires intrinsic Bcl6 and intrinsic ICOS expression. Thus, the molecular pathways required to maintain Mtb-specific CD4 T cells during ongoing infection are similar to those that maintain memory CD4 T cells in scenarios of antigen deprivation. These results suggest that vaccination strategies targeting the ICOS and Bcl6 pathways in CD4 T cells may provide new avenues to prevent TB.
View details for DOI 10.1084/jem.20141518
View details for Web of Science ID 000353898100013
View details for PubMedID 25918344
Receptor revision in CD4 T cells is influenced by follicular helper T cell formation and germinal-center interactions
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2014; 111 (15): 5652-5657
Peripheral CD4 T cells in Vβ5 transgenic (Tg) C57BL/6J mice undergo tolerance to an endogenous superantigen encoded by mouse mammary tumor virus 8 (Mtv-8) by either deletion or T-cell receptor (TCR) revision. Revision is a process by which surface expression of the Vβ5(+) TCR is down-regulated in response to Mtv-8 and recombination activating genes are expressed to drive rearrangement of the endogenous TCRβ locus, effecting cell rescue through the expression of a newly generated, non-self-reactive TCR. In an effort to identify the microenvironment in which revision takes place, we show here that the proportion of T follicular helper cells (Tfh) and production of high-affinity antibody during a primary response are increased in Vβ5 Tg mice in an Mtv-8-dependent manner. Revising T cells have a Tfh-like surface phenotype and transcription factor profile, with elevated expression of B-cell leukemia/lymphoma 6 (Bcl-6), CXC chemokine receptor 5, programmed death-1, and other Tfh-associated markers. Efficient revision requires Bcl-6 and is inhibited by B lymphocyte-induced maturation protein-1. Revision completes less efficiently in the absence of signaling lymphocytic activation molecule-associated protein although initiation proceeds normally. These data indicate that Tfh formation is required for the initiation of revision and germinal-center interactions for its completion. The germinal center is known to provide a confined space in which B-cell antigen receptors undergo selection. Our data extend the impact of this selective microenvironment into the arena of T cells, suggesting that this fluid structure also provides a regulatory environment in which TCR revision can safely take place.
View details for DOI 10.1073/pnas.1321803111
View details for Web of Science ID 000334288600054
View details for PubMedID 24706795
Recent thymic emigrants are preferentially incorporated only into the depleted T-cell pool
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2011; 108 (13): 5366-5371
Recent thymic emigrants (RTEs) are the youngest subset of peripheral T cells, and they differ functionally and phenotypically from the rest of the naïve T-cell pool. RTEs are present in the peripheral T-cell pool throughout life but are the most common subset of T cells in neonates and adults recovering from lymphoablation. Using a murine model to study the homeostasis of RTEs, we show that under lymphoreplete conditions, RTEs are at a competitive disadvantage to already established mature naïve (MN) T cells. This disadvantage may be caused by a defect in survival, because RTEs may transduce homeostatic signals inefficiently, and their ability to survive is enhanced with increased expression of IL-7 receptor or B-cell lymphoma 2 (Bcl-2). Conversely, under lymphopenic conditions, enhanced proliferation by RTEs allows them to out-compete their MN T-cell counterparts. These results suggest that in times of need, such as in neonates or lymphopenic adults, RTEs perform well to fill the gaps in the peripheral T-cell pool, but when the periphery already is full, many RTEs are not incorporated into the pool of recirculating lymphocytes.
View details for DOI 10.1073/pnas.1015286108
View details for Web of Science ID 000288894800046
View details for PubMedID 21402911