Mario Roederer

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.

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.

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.

8 color, 10-parameter flow cytometry to elucidate complex leukocyte heterogeneity.

We developed the chemistry, instrumentation, and software technologies needed to measure, simultaneously and independently, eight different fluorescent molecules on individual cells. Conjugation of these fluorochromes to monoclonal antibodies is straightforward; all immunofluorescence staining is accomplished with direct stains only. We built a hybrid flow cytometer with eight fluorescence detectors and two light scatter channels, with excitation provided by three spatially separated laser beams emitting at 407 nm, 488 nm, and 595 nm. The fluorescence compensation required to make the data orthogonal is of sufficient complexity that it cannot be performed manually; thus, we use software to compensate the data post hoc, based on data collected from singly stained compensation control samples. In this report, we evaluate the 8 color staining technology. Of the seven fluorochromes other than fluorescein, six have a useful brightness at least as great as fluorescein. Three of the fluorochromes (phycoerythrin, allophycocyanin, and the Cy5 resonance energy tandem of phycoerythrin) are considerably brighter than fluorescein and are useful for detecting antigens expressed at low levels. Finally, we show the power and utility of the 8 color, 10-parameter technology using staining experiments on both human and murine immune systems.

CD4 and CD8 T cells with high intracellular glutathione levels are selectively lost as the HIV infection progresses

Maintenance of intracellular glutathione (GSH) levels has been implicated in blocking cytokine-stimulated HIV replication in vitro, in both acute and latent infection models. We demonstrate here that subsets of human peripheral blood mononuclear cells differ substantially in mean GSH levels, as measured on a cell-by-cell basis with the fluorescence-activated cell sorter (FACS): B cells have the lowest GSH levels; T cells are intermediate; and monocytes and macrophages have the highest levels. Furthermore, GSH levels subdivide the CD4 and CD8 T cell subsets into two classes each: high- and low-GSH cells, which cannot be distinguished by cell size or by currently known surface markers. Significantly, the high-GSH T cells are selectively depleted early during the HIV infection, and are effectively missing in all ARC and AIDS patients.

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.

Data exploration, quality control and testing in single-cell qPCR-based gene expression experiments

Motivation: Cell populations are never truly homogeneous; individual cells exist in biochemical states that define functional differences between them. New technology based on microfluidic arrays combined with multiplexed quantitative polymerase chain reactions now enables high-throughput single-cell gene expression measurement, allowing assessment of cellular heterogeneity. However, few analytic tools have been developed specifically for the statistical and analytical challenges of single-cell quantitative polymerase chain reactions data. Results: We present a statistical framework for the exploration, quality control and analysis of single-cell gene expression data from microfluidic arrays. We assess accuracy and within-sample heterogeneity of single-cell expression and develop quality control criteria to filter unreliable cell measurements. We propose a statistical model accounting for the fact that genes at the single-cell level can be on (and a continuous expression measure is recorded) or dichotomously off (and the recorded expression is zero). Based on this model, we derive a combined likelihood ratio test for differential expression that incorporates both the discrete and continuous components. Using an experiment that examines treatment-specific changes in expression, we show that this combined test is more powerful than either the continuous or dichotomous component in isolation, or a t-test on the zero-inflated data. Although developed for measurements from a specific platform (Fluidigm), these tools are generalizable to other multi-parametric measures over large numbers of events. Availability: All results presented here were obtained using the SingleCellAssay R package available on GitHub (http://github.com/RGLab/SingleCellAssay). Contact: rgottard@fhcrc.org Supplementary information: Supplementary data are available at Bioinformatics online.

Use of Escherichiu coli (E. coli) lacZ (β-Galactosidase) as a Reporter Gene.

Our understanding of the molecular mechanisms that govern gene expression has been facilitated by the ability to introduce recombinant DNA molecules into heterologous cellular systems both in vitro and in vivo. One approach to defining DNA sequences important in the regulation of gene expression is to place controlling elements (e.g., promoter/enhancer sequences) upstream of a DNA coding sequence, introduce these constructs into transgenic animals or cells in culture, and analyze the levels of gene product produced by the introduced construct. Ideally, such a reporter gene should encode a product that is stable, innocuous to the cell or organism in which it is being expressed, and should be readily detectable, even when present in small quantities.

Cy7PE and Cy7APC: Bright new probes for immunofluorescence

We demonstrate the utility of indotricarbocyanine (Cy7) conjugates of the phycobiliproteins phycoerythrin (PE) and allophycocyanin (APC) in flow cytometry. This is the first demonstration of the use of an APC tandem dye for fluorescence measurements. These resonance energy transfer tandem dyes can be excited by the phycobiliprotein-specific excitation wavelengths and fluoresce at wavelengths above 780 nm. The tandem dyes, when conjugated to antibodies, are suitable for flow cytometry and other immunofluorescence applications. These conjugates are easily detectable above the very low autofluorescence in this part of the spectrum. Indeed, the Cy7-conjugated PE tandem (Cy7PE) has a brightness (fluorescence signal over cellular autofluorescence) comparable to that of fluorescein, and the Cy7APC tandem has a brightness comparable to that of APC. These tandems are also easily distinguished from other commonly used fluorophores, making them suitable for high-order multiparametric analysis. We show an example of six-color immunofluorescence analysis by flow cytometry, simultaneously measuring fluorescences from fluorescein, PE, Cy5PE, Texas red, APC, and Cy7APC.

Genetic immunization in the lung induces potent local and systemic immune responses

Successful vaccination against respiratory infections requires elicitation of high levels of potent and durable humoral and cellular responses in the lower airways. To accomplish this goal, we used a fine aerosol that targets the entire lung surface through normal respiration to deliver replication-incompetent recombinant adenoviral vectors expressing gene products from several infectious pathogens. We show that this regimen induced remarkably high and stable lung T-cell responses in nonhuman primates and that it also generated systemic and respiratory tract humoral responses of both IgA and IgG isotypes. Moreover, strong immunogenicity was achieved even in animals with preexisting antiadenoviral immunity, overcoming a critical hurdle to the use of these vectors in humans, who commonly are immune to adenoviruses. The immunogenicity profile elicited with this regimen, which is distinct from either intramuscular or intranasal delivery, has highly desirable properties for protection against respiratory pathogens. We show that it can be used repeatedly to generate mucosal humoral, CD4, and CD8 T-cell responses and as such may be applicable to other mucosally transmitted pathogens such as HIV. Indeed, in a lethal challenge model, we show that aerosolized recombinant adenoviral immunization completely protects ferrets against H5N1 highly pathogenic avian influenza virus. Thus, genetic immunization in the lung offers a powerful platform approach to generating protective immune responses against respiratory pathogens.