Honors & Awards
Percy Cyril Garnham Award for best PhD student, London School of Hygiene and Tropical Medicine (2010)
Mass cytometry was used to investigate the effect of CMV reactivation on lymphocyte reconstitution in hematopoietic cell transplant patients. For eight transplant recipients (four CMV negative and four CMV positive), we studied PBMCs obtained 6 mo after unrelated donor hematopoietic cell transplantation (HCT). Forty cell-surface markers, distinguishing all major leukocyte populations in PBMC, were analyzed with mass cytometry. This group included 34 NK cell markers. Compared with healthy controls, transplant recipients had higher HLA-C expression on CD56(-)CD16(+) NK cells, B cells, CD33(bright) myeloid cells, and CD4CD8 T cells. The increase in HLA-C expression was greater for CMV-positive HCT recipients than for CMV negative recipients. Present in CMV-positive HCT recipients, but not in CMV-negative HCT recipients or controls, is a population of killer cell Ig-like receptor (KIR)-expressing CD8 T cells not previously described. These CD8 T cells coexpress CD56, CD57, and NKG2C. The HCT recipients also have a population of CD57(+)NKG2A(+) NK cells that preferentially express KIR2DL1. An inverse correlation was observed between the frequencies of CD57(+)NKG2C(+) NK cells and CD57(+)NKG2A(+) NK cells. Although CD57(+)NKG2A(+) NK cells are less abundant in CMV-positive recipients, their phenotype is of a more activated cell than the CD57(+)NKG2A(+) NK cells of controls and CMV-negative HCT recipients. These data demonstrate that HCT and CMV reactivation are associated with an increased expression of HLA-C. This could influence NK cell education during lymphocyte reconstitution. The increased inhibitory KIR expression by proliferating CMV-specific CD8 T cells suggests regulatory interactions between HLA-C and KIR might promote Graft-versus-Leukemia effects following transplantation.
View details for DOI 10.4049/jimmunol.1401990
View details for Web of Science ID 000362968500049
View details for PubMedID 26416275
Natural killer (NK) cells play critical roles in immune defense and reproduction, yet remain the most poorly understood major lymphocyte population. Because their activation is controlled by a variety of combinatorially expressed activating and inhibitory receptors, NK cell diversity and function are closely linked. To provide an unprecedented understanding of NK cell repertoire diversity, we used mass cytometry to simultaneously analyze 37 parameters, including 28 NK cell receptors, on peripheral blood NK cells from 5 sets of monozygotic twins and 12 unrelated donors of defined human leukocyte antigen (HLA) and killer cell immunoglobulin-like receptor (KIR) genotype. This analysis revealed a remarkable degree of NK cell diversity, with an estimated 6000 to 30,000 phenotypic populations within an individual and >100,000 phenotypes in the donor panel. Genetics largely determined inhibitory receptor expression, whereas activation receptor expression was heavily environmentally influenced. Therefore, NK cells may maintain self-tolerance through strictly regulated expression of inhibitory receptors while using adaptable expression patterns of activating and costimulatory receptors to respond to pathogens and tumors. These findings further suggest the possibility that discrete NK cell subpopulations could be harnessed for immunotherapeutic strategies in the settings of infection, reproduction, and transplantation.
View details for DOI 10.1126/scitranslmed.3006702
View details for PubMedID 24154599
Chimpanzees have orthologs of the six fixed, functional human MHC class I genes. But, in addition, the chimpanzee has a seventh functional gene, Patr-AL, which is not polymorphic but contributes substantially to population diversity by its presence on only 50% of MHC haplotypes. The ancestral AL gene emerged long before the separation of human and chimpanzee ancestors and then subsequently and specifically lost function during human evolution, but was maintained in chimpanzees. Patr-AL is an alloantigen that participates in negative and positive selection of the T cell repertoire. The three-dimensional structure and the peptide-binding repertoire of Patr-AL and HLA-A*02 are surprisingly similar. In contrast, the expression of these two molecules is very different, as shown using specific mAbs and polyclonal Abs made against Patr-AL. Peripheral blood cells and B cell lines express low levels of Patr-AL at the cell surface. Higher levels are seen for 221-cell transfectants expressing Patr-AL, but in these cells a large majority of Patr-AL molecules are retained in the early compartments of the secretory pathway: mainly the endoplasmic reticulum, but also cis-Golgi. Replacing the cytoplasmic tail of Patr-AL with that of HLA-A*02 increased the cell-surface expression of Patr-AL substantially. Four substitutions distinguish the Patr-AL and HLA-A*02 cytoplasmic tails. Systematic mutagenesis showed that each substitution contributes changes in cell-surface expression. The combination of residues present in Patr-AL appears unique, but each individual residue is present in other primate MHC class I molecules, notably MHC-E, the most ancient of the functional human MHC class I molecules.
View details for DOI 10.4049/jimmunol.1500397
View details for PubMedID 26371256
Cytomegalovirus (CMV) reactivates in >30% of CMV-seropositive patients after allogeneic hematopoietic cell transplantation (HCT). Previously, we reported an increase of natural killer (NK) cells expressing NKG2C, CD57, and inhibitory killer cell immunoglobulin-like receptors (KIRs) in response to CMV reactivation after HCT. These NK cells persist after the resolution of infection and display "adaptive" or memory properties. Despite these findings, the differential impact of persistent/inactive versus reactivated CMV on NK versus T cell maturation after HCT from different graft sources has not been defined. We compared the phenotype of NK and T cells from 292 recipients of allogeneic sibling (n = 118) or umbilical cord blood (UCB; n = 174) grafts based on recipient pretransplantation CMV serostatus and post-HCT CMV reactivation. This cohort was utilized to evaluate CMV-dependent increases in KIR-expressing NK cells exhibiting an adaptive phenotype (NKG2C(+)CD57(+)). Compared with CMV-seronegative recipients, those who reactivated CMV had the highest adaptive cell frequencies, whereas intermediate frequencies were observed in CMV-seropositive recipients harboring persistent/nonreplicating CMV. The same effect was observed in T cells and CD56(+) T cells. These adaptive lymphocyte subsets were increased in CMV-seropositive recipients of sibling but not UCB grafts and were correlated with lower rates of CMV reactivation (sibling 33% versus UCB 51%; P < .01). These data suggest that persistent/nonreplicating recipient CMV induces rapid production of adaptive NK and T cells from mature cells from sibling but not UCB grafts. These adaptive lymphocytes are associated with protection from CMV reactivation.
View details for DOI 10.1016/j.bbmt.2015.05.025
View details for PubMedID 26055301
NK cells are responsible for recognizing and killing transformed, stressed, and infected cells. They recognize a set of non-Ag-specific features termed "altered self" through combinatorial signals from activating and inhibitory receptors. These NKRs are also expressed on CD4(+) and CD8(+) T cells, B cells, and monocytes, although a comprehensive inventory of NKR expression patterns across leukocyte lineages has never been performed. Using mass cytometry, we found that NKR expression patterns distinguish cell lineages in human peripheral blood. In individuals with high levels of CD57, indicative of a mature immune repertoire, NKRs are more likely to be expressed on non-NK cells, especially CD8(+) T cells. Mature NK and CD8(+) T cell populations show increased diversity of NKR surface expression patterns, but with distinct determinants: mature NK cells acquire primarily inhibitory receptors, whereas CD8(+) T cells attain a specific subset of both activating and inhibitory receptors, potentially imbuing them with a distinct functional role. Concurrently, monocytes show decreased expression of the generalized inhibitory receptor leukocyte Ig-like receptor subfamily b member 1, consistent with an increased activation threshold. Therefore, NKR expression is coordinately regulated as the immune system matures, resulting in the transfer of "altered self" recognition potential among leukocyte lineages. This likely reduces Ag specificity in the mature human immune system, and implies that vaccines and therapeutics that engage both its innate and adaptive branches may be more effective in the settings of aging and chronic infection.
View details for DOI 10.4049/jimmunol.1401821
View details for PubMedID 25288567
This chapter describes a protocol to assess activation of human NK cells following in vitro stimulation with malaria-infected red blood cells. Activation is assessed by flow cytometry, staining for cell surface expression of CD69 and accumulation of intracellular IFN-?. Procedures are described for in vitro propagation and purification of Plasmodium falciparum parasites, separation of peripheral blood mononuclear cells from heparinized blood by density centrifugation, in vitro culture of PBMC and for staining and analysis of PBMC by flow cytometry. Some examples of typical FACS plots are shown.
View details for PubMedID 22990797
RTS,S/AS01, a vaccine targeting pre-erythrocytic stages of Plasmodium falciparum, is undergoing clinical trials. We report an analysis of cellular immune response to component Ags of RTS,S-hepatitis B surface Ag (HBs) and P. falciparum circumsporozoite (CS) protein-among Tanzanian children in a phase IIb RTS,S/AS01(E) trial. RTS,S/AS01 (E) vaccinees make stronger T cell IFN-?, CD69, and CD25 responses to HBs peptides than do controls, indicating that RTS,S boosts pre-existing HBs responses. T cell CD69 and CD25 responses to CS and CS-specific secreted IL-2 were augmented by RTS,S vaccination. Importantly, more than 50% of peptide-induced IFN-?(+) lymphocytes were NK cells, and the magnitude of the NK cell CD69 response to HBs peptides correlated with secreted IL-2 concentration. CD69 and CD25 expression and IL-2 secretion may represent sensitive markers of RTS,S-induced, CS-specific T cells. The potential for T cell-derived IL-2 to augment NK cell activation in RTS,S-vaccinated individuals, and the relevance of this for protection, needs to be explored further.
View details for DOI 10.4049/jimmunol.1102710
View details for Web of Science ID 000303634300038
View details for PubMedID 22504653
Subsets of NK cells can have distinct functions. Here, we report that >25% of human peripheral blood NK cells express HLA-DR after culture with IL-2. This can be driven by an expansion of a small subset of NK cells expressing HLA-DR, in contrast to previous assumptions that HLA-DR is upregulated on previously negative cells. HLA-DR-expressing NK cells showed enhanced degranulation to susceptible target cells and expressed chemokine receptor CXCR3, which facilitated their enrichment following exposure to CXCL11/I-TAC. Suggesting HLA-DR-expressing NK cells have an important role in an immune response, stimulation of PBMCs with Mycobacterium bovis BCG (BCG) triggered expansion of this subset. Importantly, the magnitude of an individual's NK cell IFN-? response triggered by BCG was associated with the initial frequency of HLA-DR-expressing NK cells in PBMCs. More directly indicating the importance of HLA-DR-expressing NK cells, enriching the frequency of this subset in PBMCs substantially augmented the IFN-? response to BCG. Thus, HLA-DR expression marks a distinct subset of NK cells, present at low frequency in circulating blood but readily expanded by IL-2, that can play an important role during immune responses to BCG.
View details for DOI 10.1002/eji.201041180
View details for Web of Science ID 000293131200013
View details for PubMedID 21491418
Trachomatous trichiasis (TT) caused by repeated or chronic ocular infection with Chlamydia trachomatis is the result of a pro-fibrotic ocular immune response. At the conjunctiva, the increased expression of both inflammatory (IL1B, TNF) and regulatory cytokines (IL10) have been associated with adverse clinical outcomes. We measured in vitro immune responses of peripheral blood to a number of chlamydial antigens. Peripheral blood effector cells (CD4, CD69, IFN?, IL-10) and regulatory cells (CD4, CD25, FOXP3, CTLA4/GITR) were readily stimulated by C. trachomatis antigens but neither the magnitude (frequency or stimulation index) or the breadth and amount of cytokines produced in vitro [IL-5, IL-10, IL-12 (p70), IL-13, IFN?, and TNF?] were significantly different between TT cases and their non-diseased controls. Interestingly we observed that CD4+ T cells account for <50% of the IFN? positive cells induced following stimulation. Further investigation in individuals selected from communities where exposure to ocular infection with C. trachomatis is endemic indicated that CD3-CD56+ (classical natural killer cells) were a major early source of IFN? production in response to C. trachomatis elementary body stimulation and that the magnitude of this response increased with age. Future efforts to unravel the contribution of the adaptive immune response to conjunctival fibrosis should focus on the early events following infection and the interaction with innate immune mediated mechanisms of inflammation in the conjunctiva.
View details for DOI 10.3389/fmicb.2011.00010
View details for PubMedID 21747780
Natural killer (NK) cells are large granular lymphocytes that express a diverse array of germline encoded inhibitory and activating receptors for MHC Class I and Class I-like molecules, classical co-stimulatory ligands, and cytokines. The ability of NK cells to be very rapidly activated by inflammatory cytokines, to secrete effector cytokines, and to kill infected or stressed host cells, suggests that they may be among the very early responders during infection. Recent studies have also identified a small number of pathogen-derived ligands that can bind to NK cell surface receptors and directly induce their activation. Here we review recent studies that have begun to elucidate the various pathways by which viral, bacterial, and parasite pathogens activate NK cells. We also consider two emerging themes of NK cell-pathogen interactions, namely their contribution to adaptive immune responses and their potential to take on regulatory and immunomodulatory functions.
View details for DOI 10.3389/fimmu.2011.00088
View details for PubMedID 22566877
We characterized vaccine-induced cellular responses to rabies virus in naive adult volunteers. Contrary to current paradigms, we observed potent and prolonged in vitro NK cell cytokine production and degranulation responses after restimulation of PBMCs with inactivated rabies virus in vaccinated, but not in unvaccinated, individuals. This "recall" NK cell response was absolutely dependent on Ag-specific IL-2 from CD45RO(+) CD4(+) T cells as well as IL-12 and IL-18 from accessory cells. Importantly, NK cells represented over 70% of all IFN-gamma-secreting and degranulating cells in the first 12-18 h after virus rechallenge indicating they may be required for rapid control of infection after vaccination. Activation of NK cells may be a critical function of IL-2-secreting effector memory T cells. Although IL-2-dependent postvaccination NK cell activation has been reported previously, this is the first time the magnitude of this effect and its contribution to the overall vaccine-induced response has been appreciated and the mechanisms of NK activation postvaccination have been elucidated. Our data will allow standard protocols for evaluating vaccine-induced immunity to be adapted to assess NK cell effector responses.
View details for DOI 10.4049/jimmunol.1000844
View details for Web of Science ID 000281122600023
View details for PubMedID 20679529
Rapid cell-mediated immune responses, characterized by production of proinflammatory cytokines, such as IFN-gamma, can inhibit intraerythrocytic replication of malaria parasites and thereby prevent onset of clinical malaria. In this study, we have characterized the kinetics and cellular sources of the very early IFN-gamma response to Plasmodium falciparum-infected RBCs among human PBMCs. We find that NK cells dominate the early (12-18 h) IFN-gamma response, that NK cells and T cells contribute equally to the response at 24 h, and that T cells increasingly dominate the response from 48 h onward. We also find that although gammadelta T cells can produce IFN-gamma in response to P. falciparum-infected RBCs, they are greatly outnumbered by alphabeta T cells, and thus, the majority of the IFN-gamma(+) T cells are alphabeta T cells and not gammadelta T cells; gammadelta T cells are, however, an important source of TNF. We have previously shown that NK cell responses to P. falciparum-infected RBCs require cytokine and contact-dependent signals from myeloid accessory cells. In this study, we demonstrate that NK cell IFN-gamma responses to P. falciparum-infected RBCs are also crucially dependent on IL-2 secreted by CD4(+) T cells in an MHC class II-dependent manner, indicating that the innate response to infection actually relies upon complex interactions between NK cells, T cells, and accessory cells. We conclude that activation of NK cells may be a critical function of IL-2-secreting CD4(+) T cells and that standard protocols for evaluation of Ag-specific immune responses need to be adapted to include assessment of NK cell activation as well as T cell-derived IL-2.
View details for DOI 10.4049/jimmunol.1000106
View details for Web of Science ID 000278439600016
View details for PubMedID 20427769
This chapter describes a protocol to assess activation of human NK cells following in vitro stimulation with malaria-infected red blood cells. Activation is assessed by flow cytometry, staining for cell surface expression of CD69 and accumulation of intracellular IFN-gamma. Procedures are described for in vitro propagation and purification of Plasmodium falciparum parasites, separation of peripheral blood mononuclear cells from heparinised blood by density centrifugation, in vitro culture of PBMC and for staining and analysis of PBMC by flow cytometry. Some examples of typical FACS plots are shown.
View details for DOI 10.1007/978-1-60761-362-6_29
View details for PubMedID 20033658
A novel assay to assess antigen-specific cytokine release from stimulated CD8(+) T cells derived from the mucosal and peripheral blood compartments has been developed and standardized using the influenza A virus matrix protein (MP) peptide, GILGFVFTL. This technology is based on the capacity for the human leucocyte antigen (HLA)-A2:Ig dimeric protein to stimulate CD8(+) T cells in a major histocompatibility complex (MHC) class I-restricted fashion without the necessity for antigen presenting cells (APC). This assay has been optimized utilizing a 9-amino acid residue (9mer) peptide, the optimal peptide length for presenting an epitope to CD8(+) T cells. Compared to existing assays, this more sensitive and specific methodology requires fewer cells, enabling easier and more accurate monitoring of the CD8(+) T-cell response in biological compartments, such as the mucosa during the course of viral infection and may be utilized to assess epitope-specific CD8(+) T-cell responses in vaccine trials.
View details for DOI 10.1111/j.1365-3083.2009.02317.x
View details for Web of Science ID 000271251500002
View details for PubMedID 19874545
IFN-gamma emanating from NK cells is an important component of innate defense against infection. In this study, we demonstrate that, following in vitro stimulation of human peripheral blood NK cells with a variety of microbial ligands, CD56(dim) as well as CD56(bright) NK cells contribute to the overall NK cell IFN-gamma response with, for most cell donors, IFN-gamma(+) CD56(dim) NK cells outnumbering IFN-gamma(+) CD56(bright) NK cells. We also observe that the magnitude of the human NK IFN-gamma response to microbial ligands varies between individuals; that the antimicrobial response of CD56(bright), but not CD56(dim), NK cells is highly correlated with that of myeloid accessory cells; and that the ratio of IFN-gamma(+) CD56(dim) to IFN-gamma(+) CD56(bright) NK cells following microbial stimulation differs between individuals but remains constant for a given donor over time. Furthermore, ratios of IFN-gamma(+) CD56(dim) to IFN-gamma(+) CD56(bright) NK cells for different microbial stimuli are highly correlated and the relative response of CD56(dim) and CD56(bright) NK cells is highly significantly associated with killer Ig-like receptor (KIR) genotype. These data reveal an influence of KIR genotype, possibly mediated via NK cell education, on the ability of NK cells to respond to nonviral infections and have implications for genetic regulation of susceptibility to infection in humans.
View details for DOI 10.4049/jimmunol.0804224
View details for Web of Science ID 000265899800061
View details for PubMedID 19414796
NKT cells are a subset of lymphocytes that share features of T cells and NK cells and bridge the innate and adaptive immune responses. They are able to be infected by HIV, but their function in HIV-infected individuals is not known. NKT cell percentage and function was measured in individuals with acute HIV infection before and 1 year into highly active anti-retroviral therapy (HAART). This study demonstrates that percentages of both CD161+ NKT cells and CD161+, CD4+ NKT cells decline within the first few months after HIV-1 infection, but initiating therapy during the acute infection period can prevent a further decline in these NKT cell subsets during the first year. NKT cell function is also impaired during early HIV infection, but significantly improved by effective treatment with HAART. Finally, preservation of NKT cell function may be important in HIV-infected individuals, as NKT cells display an anti-HIV-1 activity in vitro, mediated by IFN-gamma secretion.
View details for DOI 10.1093/intimm/dxm055
View details for Web of Science ID 000250681500005
View details for PubMedID 17702988
Two novel hybrid molecules 3-O-sulfo-alpha/beta-galactosylceramide 3 and 4, which are derived from an immunostimulatory agent alpha-GalCer 1 and self-glycolipid ligand sulfatide 2, were designed and synthesized. Compound 3 was shown to efficiently stimulate human NKT cells to secret IL-4 and IFN-gamma, with activities similar to 1, suggesting that modification of the 3''-OH position of the galactose moiety with sulfate has no significant effect on NKT cell stimulation. As a comparison, the beta-isomer 4 has no affinity to NKT cells, which demonstrates that the alpha-glycosidic bond of galactosylceramide is crucial to the NKT cells activation.
View details for DOI 10.1016/j.bmc.2005.02.018
View details for Web of Science ID 000228254000019
View details for PubMedID 15781400
Rapid progression to AIDS after acute HIV-1 infection, though uncommon, has been noted, as has the transmission of multidrug resistant viruses. Here, we describe a patient in whom these two factors arose concomitantly and assess the effects.We did a case study of a patient with HIV-1 seroconversion. We genotyped the virus and host genetic markers by PCR and nucleotide sequencing. To ascertain the drug susceptibility of our patient's HIV-1 we did phenotypic studies with the PhenoSense assay. We assessed viral coreceptor use via syncytium formation in vitro and with a modified PhenoSense assay.Our patient seems to have been recently infected by a viral variant of HIV-1 resistant to multiple classes of antiretroviral drugs. Furthermore, his virus population is dual tropic for cells that express CCR5 or CXCR4 coreceptor. The infection has resulted in progression to symptomatic AIDS in 4-20 months.The intersection of multidrug resistance and rapid development of AIDS in this patient is of concern, especially in view of his case history, which includes high-risk sexual contacts and use of metamfetamine. The public health ramifications of such a case are great.
View details for Web of Science ID 000227731800027
View details for PubMedID 15781098
The CD1 family of proteins binds self and foreign glycolipids for presentation to CD1-restricted T cells. To identify previously uncharacterized active CD1 ligands, especially those of microbial origin, numerous glycolipids were synthesized and tested for their ability to stimulate mouse and human natural killer T (NKT) cells. They included analogs of the well known NKT cell agonist alpha-galactosyl ceramide (alpha-GalCer), bacterial glycolipids, and variations of the self-glycolipid, sulfatide. Bacterial glycolipids, alpha-galacturonosyl-ceramides from Sphingomonas wittichii, although structurally similar to alpha-GalCer, have significant differences in the sugar head group as well as the ceramide portion. The Sphingomonas glycosphingolipids (GSLs) and sulfatide variants were shown to activate human NKT cells as measured by IL-4 and IFN-gamma secretion. Moreover, CD1d-dimer staining revealed human NKT cell reactivity toward these GSLs and to the sulfatides in a fashion comparable with alpha-GalCer. Because alpha-GalCer is a marine-sponge-derived ligand, our study here shows that bacterium-derived antigens are also able to stimulate mouse and human NKT cells.
View details for DOI 10.1073/pnas.0408696102
View details for Web of Science ID 000226877300020
View details for PubMedID 15665086
Given its population of CCR5-expressing, immunologically activated CD4(+) T cells, the gastrointestinal (GI) mucosa is uniquely susceptible to human immunodeficiency virus (HIV)-1 infection. We undertook this study to assess whether a preferential depletion of mucosal CD4(+) T cells would be observed in HIV-1-infected subjects during the primary infection period, to examine the anatomic subcompartment from which these cells are depleted, and to examine whether suppressive highly active antiretroviral therapy could result in complete immune reconstitution in the mucosal compartment. Our results demonstrate that a significant and preferential depletion of mucosal CD4(+) T cells compared with peripheral blood CD4(+) T cells is seen during primary HIV-1 infection. CD4(+) T cell loss predominated in the effector subcompartment of the GI mucosa, in distinction to the inductive compartment, where HIV-1 RNA was present. Cross-sectional analysis of a cohort of primary HIV-1 infection subjects showed that although chronic suppression of HIV-1 permits near-complete immune recovery of the peripheral blood CD4(+) T cell population, a significantly greater CD4(+) T cell loss remains in the GI mucosa, despite up to 5 yr of fully suppressive therapy. Given the importance of the mucosal compartment in HIV-1 pathogenesis, further study to elucidate the significance of the changes observed here is critical.
View details for DOI 10.1084/jem.20041196
View details for Web of Science ID 000224105500007
View details for PubMedID 15365095