The impact of HLA class I and EBV latency-II antigen-specific CD8(+) T cells on the pathogenesis of EBV(+) Hodgkin lymphoma.

In 40% of cases of classical Hodgkin lymphoma (cHL), Epstein-Barr virus (EBV) latency-II antigens [EBV nuclear antigen 1 (EBNA1)/latent membrane protein (LMP)1/LMP2A] are present (EBV(+) cHL) in the malignant cells and antigen presentation is intact. Previous studies have shown consistently that HLA-A*02 is protective in EBV(+) cHL, yet its role in disease pathogenesis is unknown. To explore the basis for this observation, gene expression was assessed in 33 cHL nodes. Interestingly, CD8 and LMP2A expression were correlated strongly and, for a given LMP2A level, CD8 was elevated markedly in HLA-A*02(-) versus HLA-A*02(+) EBV(+) cHL patients, suggesting that LMP2A-specific CD8(+) T cell anti-tumoral immunity may be relatively ineffective in HLA-A*02(-) EBV(+) cHL. To ascertain the impact of HLA class I on EBV latency antigen-specific immunodominance, we used a stepwise functional T cell approach. In newly diagnosed EBV(+) cHL, the magnitude of ex-vivo LMP1/2A-specific CD8(+) T cell responses was elevated in HLA-A*02(+) patients. Furthermore, in a controlled in-vitro assay, LMP2A-specific CD8(+) T cells from healthy HLA-A*02 heterozygotes expanded to a greater extent with HLA-A*02-restricted compared to non-HLA-A*02-restricted cell lines. In an extensive analysis of HLA class I-restricted immunity, immunodominant EBNA3A/3B/3C-specific CD8(+) T cell responses were stimulated by numerous HLA class I molecules, whereas the subdominant LMP1/2A-specific responses were confined largely to HLA-A*02. Our results demonstrate that HLA-A*02 mediates a modest, but none the less stronger, EBV-specific CD8(+) T cell response than non-HLA-A*02 alleles, an effect confined to EBV latency-II antigens. Thus, the protective effect of HLA-A*02 against EBV(+) cHL is not a surrogate association, but reflects the impact of HLA class I on EBV latency-II antigen-specific CD8(+) T cell hierarchies.

A mine is a terrible thing to waste: high content, single cell technologies for comprehensive immune analysis.

In recent years, an incredible variety of single cell technologies have become available to analyze immune responses. These technologies include polychromatic flow cytometry, mass cytometry, highly multiplexed single cell qPCR, RNA sequencing, microtools, and high-resolution imaging. In this article, we review these platforms, describing their power and limitations for comprehensive analysis of the immune system. We relate the properties of these technologies to the various cellular states relevant to an immune response, in order to address which technologies are most appropriate for which settings.

Characterization of circulating T cells specific for tumor-associated antigens in melanoma patients.

We identified circulating CD8+ T-cell populations specific for the tumor-associated antigens (TAAs) MART-1 (27-35) or tyrosinase (368-376) in six of eleven patients with metastatic melanoma using peptide/HLA-A*0201 tetramers. These TAA-specific populations were of two phenotypically distinct types: one, typical for memory/effector T cells; the other, a previously undescribed phenotype expressing both naive and effector cell markers. This latter type represented more than 2% of the total CD8+ T cells in one patient, permitting detailed phenotypic and functional analysis. Although these cells have many of the hallmarks of effector T cells, they were functionally unresponsive, unable to directly lyse melanoma target cells or produce cytokines in response to mitogens. In contrast, CD8+ T cells from the same patient were able to lyse EBV-pulsed target cells and showed robust allogeneic responses. Thus, the clonally expanded TAA-specific population seems to have been selectively rendered anergic in vivo. Peptide stimulation of the TAA-specific T-cell populations in other patients failed to induce substantial upregulation of CD69 expression, indicating that these cells may also have functional defects, leading to blunted activation responses. These data demonstrate that systemic TAA-specific T-cell responses can develop de novo in cancer patients, but that antigen-specific unresponsiveness may explain why such cells are unable to control tumor growth.

Publishing flow cytometry data.

Cellular measurements by flow cytometric analysis constitute an important step toward understanding individual attributes within a population of cells. Assessing individual cells within a population by protein expression using fluorescently labeled antibodies and other fluorescent probes can identify cellular patterns. The technology for accurately identifying subtle changes in protein expression within a population of cells using a vast array of technology has resulted in controversy and questions regarding reproducibility, which can be explained at least in part by the absence of standard methods to facilitate comparison of flow cytometric data. The complexity of technological advancements and the need for improvements in biological resolution results in the generation of complex data that demands the use of minimum standards for their publication. Herein we present a summarized view for the inclusion of consistent flow cytometric experimental information as supplemental data. Four major points, experimental and sample information, data acquisition, analysis, and presentation are emphasized. Together, these guidelines will facilitate the review and publication of flow cytometry data that provide an accurate foundation for ongoing studies with this evolving technology.

CD4 T cell subsets in the mucosa are CD28+Ki-67-HLA-DR-CD69+ but show differential infection based on alpha4beta7 receptor expression during acute SIV infection.

BACKGROUND: CD4 T cell depletion in the mucosa has been well documented during acute HIV and SIV infections. The demonstration the HIV/SIVcan use the alpha4beta7 receptor for viral entry suggests that these viruses selectively target CD4 T cells in the mucosa that express high levels of alpha4beta7 receptor. METHODS: Mucosal samples obtained from SIV infected rhesus macaques during the early phase of infection were used for immunophenotypic analysis. CD4 T cell subsets were sorted based on the expression of beta7 and CD95 to quantify the level of SIV infection in different subsets of CD4 T cells. Changes in IL-17, IL-21, IL-23 and TGFbeta mRNA expression was determined using Taqman PCR. RESULTS: CD4 T cells in the mucosa were found to harbor two major population of cells; -25% of CD4 T cells expressed the alpha4(+)beta7(hi) phenotype, whereas the rest of the 75% expressed an alpha4(+)beta7(int) phenotype. Both the subsets were predominantly CD28(+)Ki-67(-)HLA-DR(-) but CD69(+), and expressed detectable levels of CCR5 on their surface. Interestingly, however, alpha4(+)beta7(hi)CD4 T cells were found to harbor more SIV than the alpha4(+)beta7(int) subsets at day 10 pi. Early infection was associated with a dramatic increase in the expression of IL-17, and IL-17 promoting cytokines IL-21, IL-23, and TGFbeta that stayed high even after the loss of mucosal CD4 T cells. CONCLUSIONS: Our results suggest that the differential expression of the alpha4beta7 receptor contributes to the differences in the extent of infection in CD4 T cell subsets in the mucosa. Early infection is associated dysregulation of the IL-17 network in mucosal tissues involves other non-Th-17 cells that likely contributes to the pro-inflammatory environment in the mucosa during acute stages of SIV infection.

Recombinant HIV-1 vaccine candidates based on replication-defective flavivirus vector.

Multiple approaches utilizing viral and DNA vectors have shown promise in the development of an effective vaccine against HIV. In this study, an alternative replication-defective flavivirus vector, RepliVax (RV), was evaluated for the delivery of HIV-1 immunogens. Recombinant RV-HIV viruses were engineered to stably express clade C virus Gag and Env (gp120TM) proteins and propagated in Vero helper cells. RV-based vectors enabled efficient expression and correct maturation of Gag and gp120TM proteins, were apathogenic in a sensitive suckling mouse neurovirulence test, and were similar in immunogenicity to recombinant poxvirus NYVAC-HIV vectors in homologous or heterologous prime-boost combinations in mice. In a pilot NHP study, immunogenicity of RV-HIV viruses used as a prime or boost for DNA or NYVAC candidates was compared to a DNA prime/NYVAC boost benchmark scheme when administered together with adjuvanted gp120 protein. Similar neutralizing antibody titers, binding IgG titers measured against a broad panel of Env and Gag antigens, and ADCC responses were observed in the groups throughout the course of the study, and T cell responses were elicited. The entire data demonstrate that RV vectors have the potential as novel HIV-1 vaccine components for use in combination with other promising candidates to develop new effective vaccination strategies.

Altered representation of naive and memory CD8 T cell subsets in HIV-infected children.

CD8 T cells are divided into naive and memory subsets according to both function and phenotype. In HIV-negative children, the naive subset is present at high frequencies, whereas memory cells are virtually absent. Previous studies have shown that the overall number of CD8 T cells does not decrease in HIV-infected children. In studies here, we use multiparameter flow cytometry to distinguish naive from memory CD8 T cells based on expression of CD11a, CD45RA, and CD62L. With this methodology, we show that within the CD8 T cell population, the naive subset decreases markedly (HIV+ vs. HIV-, 190 vs. 370 cells/microliter; P < or = 0.003), and that there is a reciprocal increase in memory cells, such that the total CD8 T cell counts remained unchanged (800 vs. 860 cells/microliter; P < or = 0.76). In addition, we show that for HIV-infected children, the naive CD8 T cell and total CD4 T cell counts correlate (chi 2 P < or = 0.001). This correlated loss suggests that the loss of naive CD8 T cells in HIV infection may contribute to the defects in cell-mediated immunity which become progressively worse as the HIV disease progresses and CD4 counts decrease.

CD8 naive T cell counts decrease progressively in HIV-infected adults.

We show here that CD8 naive T cells are depleted during the asymptomatic stage of HIV infection. Although overall CD8 T cell numbers are increased during this stage, the naive CD8 T cells are progressively lost and fall in parallel with overall CD4 T cell counts. In addition, we show that naive CD4 T cells are preferentially lost as total CD4 cell counts fall. These findings, presented here for adults, and in the accompanying study for children, represent the first demonstration that HIV disease involves the loss of both CD4 T cells and CD8 T cells. Furthermore, they provide a new insight into the mechanisms underlying the immunodeficiency of HIV-infected individuals, since naive T cells are required for all new T cell-mediated immune responses. Studies presented here also show that the well-known increase in total CD8 counts in most HIV-infected individuals is primarily due to an expansion of memory cells. Thus, memory CD8 T cells comprise over 80% of the T cells in PBMC from individuals with < 200 CD4/microliter, whereas they comprise roughly 15% in uninfected individuals. Since the naive and memory subsets have very different functional activities, this altered naive/memory T cell representation has significant consequences for the interpretation of data from in vitro functional studies.

HIV does not replicate in naive CD4 T cells stimulated with CD3/CD28.

In this report, we demonstrate that the T cell tropic strain of HIV, LAI, does not replicate in naive CD4 T cells stimulated by cross-linking CD3 and CD28. In contrast, LAI replicates well in memory CD4 T cells stimulated in the same way. Unlike this physiologically relevant stimulation, PHA stimulates productive LAI replication in both naive and memory T cells. These studies were conducted with highly purified (FACS-isolated) subsets of CD4 T cells identified by expression of both CD45RA and CD62L. Remixing of purified T cells showed that naive T cells do not suppress LAI replication in memory T cells and that memory T cells do not restore LAI expression in naive T cells. The suppression of productive LAI replication in naive T cells is not due to differential expression of viral coreceptors, nor is it due to inhibition of activation of the important HIV transcription factors, nuclear factor-kappaB and activator protein-1. The inherent resistance of naive T cells to productive HIV infection, coupled with their proliferative advantage as demonstrated here, provides a sound basis for proposed clinical therapies using ex vivo expansion and reinfusion of CD4 T cells from HIV-infected adults.

ET-18-OCH3 inhibits nuclear factor-kappa B activation by 12-O-tetradecanoylphorbol-13-acetate but not by tumor necrosis factor-alpha or interleukin 1 alpha.

1-O-Octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) is a synthetic diether phospholipid that is competitive with phosphatidylserine binding to the regulatory domain of protein kinase C (PKC). Our previous studies indicate that the selective inhibition of tumor cell growth by ET-18-OCH3 may be due to altered signal transduction mechanisms, including the inhibition of PKC. To further define the mechanism of action of ET-18-OCH3, we have used it to study the role of PKC in regulation of the transcription factor NF-kappa B, which is activated by diverse stimuli. In the 293.27.2 human kidney cell line, as in hematopoietic cells of all lineages, NF-kappa B is stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 alpha (IL-1 alpha). The response to either TNF-alpha or IL-1 alpha is synergistically enhanced by TPA. However, the regulatory mechanisms and signal transduction systems responsible for NF-kappa B activation in response to these different stimuli have not been determined in detail. We have used ET-18-OCH3 and auranofin, which inhibit PKC by different mechanisms, to assess the role of PKC in NF-kappa B activation. ET-18-OCH3 markedly inhibits TPA-induced NF-kappa B activation, as measured by HIV long terminal repeat-directed expression of beta-galactosidase. The IC50 for inhibition by ET-18-OCH3 is approximately 2 microM, a noncytotoxic concentration. Inhibition of TPA-induced NF-kappa B activation was dependent upon preincubation with ET-18-OCH3, and the drug was active at approximately 2 mol% of total cellular phospholipid. ET-18-OCH3 did not inhibit NF-kappa B activation by either TNF-alpha or IL-1 alpha, indicating that there are multiple distinct signal transduction pathways leading to activation of NF-kappa B. We have confirmed these results using auranofin, an antirheumatic drug that is a specific PKC inhibitor interacting with the catalytic domain. Like ET-18-OCH3, auranofin blocked NF-kappa B activation by TPA but not by TNF-alpha or IL-1 alpha. Also like the ether lipid, auranofin only partially blocked the synergy exhibited by TPA and TNF-alpha. To confirm the role of NF-kappa B in this response, we measured NF-kappa B by electrophoretic mobility shift assay. Both ET-18-OCH3 and auranofin inhibited cellular induction of the active NF-kappa B complex in response to TPA but not in response to TNF-alpha.(ABSTRACT TRUNCATED AT 400 WORDS)

Vdelta1 and Vdelta2 gammadelta T cells express distinct surface markers and might be developmentally distinct lineages.

We report here that the two major types of gammadelta T cells found in human blood, Vdelta1 and Vdelta2, were found to have markedly different phenotypes. Vdelta2 cells had a phenotype typical of most alphabeta T cells in blood; i.e., they were CD5(+), CD28(+), and CD57(-). In contrast, Vdelta1 cells tended to be CD5(-/dull), CD28(-), and CD57(+). Furthermore, although Vdelta1 T cells appeared to be "naive" in that they were CD45RA(+), they were CD62L(-) and on stimulation uniformly produced interferon-gamma, indicating that they are in fact memory/effector cells. This phenotype for Vdelta1 cells was similar to that of intestinal intraepithelial lymphocytes, a subset that can develop in the absence of the thymus. We suggest that the Vdelta1 and Vdelta2 T cell subsets represent distinct lineages with different developmental pathways. The disruption of the supply of normal, thymus-derived T cells in HIV-infected individuals might be responsible for the shift in the Vdelta2/Vdelta1 ratio that occurs in the blood of individuals with HIV disease.

Effect of confluence on endocytosis by 3T3 fibroblasts: increased rate of pinocytosis and accumulation of residual bodies.

We have compared lysosomal enzyme distributions on density gradients and rates of transport of endocytic markers for actively-growing and confluent cells. While it has been previously established that mammalian cells accumulate lysosomal enzymes during quiescence, we show that this accumulation is predominantly in residual bodies (p greater than 1.12 g/ml) rather than in dense lysosomes (p = 1.08-1.10 g/ml) and does not represent a change in the endosomal and lysosomal enzyme content. The accumulation is not caused by a change in the rate of production of dense lysosomes, since the rate of transfer of epidermal growth factor (EGF) from light to dense compartments is the same between confluent and subconfluent cells. Confluent cultures have a higher rate of initial pinocytosis, and a higher rate of retroendocytosis and/or recycling, causing a net lower rate of accumulation of fluid-phase material. The accumulation of residual bodies in confluent cultures may be caused by a lower rate of exocytosis of their contents and/or a lack of dilution by cell division. The data indicate that the impact of culture confluence must be carefully assessed in experiments designed to analyze endocytic pathways.

Endosomes can undergo an ATP-dependent density increase in the absence of dense lysosomes.

On the basis of evidence that lysosomal enzymes and membrane proteins are present in endosomes, we have previously suggested that the production of lysosomes involves maturation rather than vesicle fusion (Roederer, M., R. Bowser, R. F. Murphy, J. Cell. Physiol. 131, 200-209 (1987)). Since the appearance of endocytosed material in lysosomes is associated with an increase in buoyant density from that of endosomes, a prediction of the model is that endosomes should be capable of undergoing such an increase in vitro. We observe that under appropriate conditions, isolated endosomes containing [125I]EGF can undergo an increase in density in vitro to that of dense lysosomes, mimicking the density change which occurs in vivo. This occurs in the absence of dense lysosomes with which to fuse. The density increase requires ATP and can be efficiently inhibited in vitro by the presence of benzylamine, suggesting that vesicular acidification is required. Since low pH has previously been shown to induce formation of a matrix by lysosomal enzymes in vitro (Buckmaster, M. J., A. L. Ferris, B. Storrie, Biochem. J. 249, 921-923 (1988)), we propose that a mechanism by which endosomes and/or lysosomes increase their density is a low pH induced aggregation of vesicle contents which decreases the osmotic pressure inside the vesicle. Together with previous data, the results provide highly suggestive evidence that the pathway to lysosomes includes a maturation of the postsorting compartment into what has classically been termed a lysosome.

CD20 expression is increased on B lymphocytes from HIV-infected individuals.

In studies presented here, we show that expression of the pan B cell marker CD20 is markedly increased on B lymphocytes from HIV-infected individuals and that this increase tends to be greater in individuals with more advanced disease. By using multiparameter FACS analyses to quantitate surface density of CD20 and intracellular glutathione (GSH) levels simultaneously, we further show that the distribution of intracellular glutathione (GSH) levels in B cells of HIV-infected individuals is more heterogeneous than in uninfected controls. Finally, we show that the intracellular GSH levels correlate with CD20 expression on a per-cell basis in all infected individuals. These findings suggest that CD20 expression, which can be precisely measured, may prove to be a useful surrogate marker for monitoring HIV infection.

A practical approach to multicolor flow cytometry for immunophenotyping.

Through a series of novel developments in flow cytometry hardware, software, and dye-chemistry it is now possible to simultaneously measure up to 11 distinct fluorescences and two scattered light parameters on each cell. Such advanced multicolor systems have a number of advantages over current two- and three-color flow cytometric measurements. They provide a large amount of novel information for each sample studied, an exquisitely accurate quantitation of even rare cell populations, and allow identification and characterization of novel cell subsets. In particular, this technology is proving crucial to identifying functionally homogeneous subsets of cells within the enormously complex immune system; such identification and enumeration is important for understanding disease pathogenesis. However, multicolor flow cytometry comes with a new and sometimes difficult set of technical problems that must be overcome by users to derive meaningful results. In this manuscript, we describe the basic aspects of multicolor flow cytometry, including the technical hurdles and artefacts that may occur, and provide some suggestions for how to best overcome these hurdles. While inspired by the 11-color technology that we currently use, these principles apply to all flow cytometric experiments in which more than one fluorescent dye is used.

Direct functional analysis of epitope-specific CD8+ T cells in peripheral blood.

The functional status of virus-specific CD8+ T cells is important for the outcome and the immunopathogenesis of viral infections. We have developed an assay for the direct functional analysis of antigen-specific CD8+ T cells, which does not require prolonged in vitro cultivation and amplification of T cells. Whole blood samples were incubated with peptide antigens for <5 h, followed by staining with peptide-MHC tetramers to identify epitope-specific T cells. The cells were also stained for the activation marker CD69 or for the production of cytokines such as interferon-gamma (IFNgamma) or tumor necrosis factor-alpha (TNFalpha). With the combined staining with tetramer and antibodies to CD69 or cytokines the number of antigen-specific CD8+ T cells as well as the functional response of each individual cell to the cognate antigen can be determined in a single experiment. Virus-specific CD8+ T cells that are nonfunctional, as well as those that are functional under the same stimulating conditions can be simultaneously detected with this assay, which is not possible by using other T-cell functional assays including cytotoxicity assay, intracellular cytokine staining, and enzyme-linked immunospot (ELISPOT) assay.