MIG Working Group	PI (Institution)	Study Title	Collaborator (Institution)	Project Summary	Protocols in Development (Brief Description)	Study Update June, 2011
MIG GI	Ian McGowan (U Pittsburgh)	Mucosal GI working group comparative flow cytometry project	Peter Anton, (UCLA) and Steve De Rosa, (U Washington/ FHCRC)	The aim of this research is to collect normative data values for GALT T cell populations analyzed by FACS. This study will characterize potential variance in normal T-cell population distributions and their functional attributes, as well as improve understanding of experimental factors that may influence normative ranges reported by different labs. Stage 1 involves the statistical comparison of historical T-cell phenotypic data from HIV-negative male subjects from several clinical centers including FHCRC, U Pittsburgh, UCLA, UC Davis, and NIH VRC. Stage 2 includes the development and standardization of a consensus flow panel of phenotypic markers for GALT T cells that will be collected and analyzed at UCLA and U Pittsburgh, with centralized data analysis at FHCRC. Identical protocols for enzymatic digestion and cell staining will be used by each center. Stage 3 comprises a cross-center implementation to determine variability between different clinical centers using the same protocols and reagents.	Consensus protocol for flow cytometric analysis of GALT T cell phenotype (MMC isolation from flex-sig gut biopsies;  three, 8-color staining panels to evaluate: 1) T cell memory, 2) T cell activation,  3) T cell polyfunctionality)	The results of Stage 1 statistical analyses established the need for standardized approaches to minimize variance between different clinical centers in the analysis and reporting of GALT T-cell parameters. There are significant differences between sites in percent of cells expressing a particular phenotype marker for CD3, CD4, CD8 and CD4/CCR5. Development of the Stage 2 consensus protocol for flow cytometric analyses of GALT T cells is complete with participant enrollments expected to begin at UCLA and U Pittsburgh this month and continue for 6-9 mos.
	Ron Veazey (Tulane U)	Optimization of cryopreservation techniques from intestinal tissue samples	Dayong Gao, (U Washington)	This project is focused on optimizing methods for viably freezing and thawing gut mucosal cells and intestinal tissue explants using a non-human primate model. This study will examine whether intestinal cells and tissues can be viably preserved by direct comparison to fresh samples in phenotypic, viability, and immune function assays. Study will determine whether storage of cells for short terms (3 months) in -80 C freezers and in serum free media affects the results of phenotypic and functional assays on intestinal lymphocytes or jejunum/colon/rectum organ cultures. Primary objective is to define an optimal, practical, and economical method to preserve the viability and function of intestinal cell samples so that preservation and shipping methods can be standardized for mucosal tissue sampling in HIV vaccine research.	Protocols for controlled freezing and thawing/cryoprotectant removal from NHP gut biopsies that will better preserve T cell viability and function	Pilot studies testing existing protocols to freeze PBMC or NHP embryos have shown limited success preserving T cell viability in NHP gut mucosal tissues frozen as punch biopsies, although decreasing the size of the biopsies improves recovery, presumably by allowing better tissue penetration of DMSO. Future studies will experimentally measure key intrinsic cellular/tissue parameters (e.g., membrane permeability to water or DMSO; intracellular ice formation (IIF) temperature) of gut mucosal specimens and use biophysical principles to predict optimal cryopreservation protocols for subsequent verification experimentally.
	Paul Johnson (Harvard U)	Multi-center comparison of immunological phenomena at various sites along the GI tract in SIV-uninfected and -infected macaques	Jason Brenchley, (NIAID) and Ron Veazey, (Tulane U)	This study will determine variations in CD4 T cell depletion within biopsies taken at different locations of the GI tract from SIV+ and SIV- macaques housed at NIAID, Tulane, and Harvard.    T cell subsets will be determined by FACS evaluation of CD3, CD4, CD8, CD28, CD95, CD45, CCR7, and CCR5 markers, so that frequencies of naïve and memory CD4 and CD8 T cells and the frequencies of CCR5+ T cells at each intestinal site can be compared to the same cell populations in matched PBMC and peripheral lymph node specimens. Additionally transcriptional profiling by microarray analysis will be performed to compare gene expression signatures at different sites along the GI tract. Data from each cohort of infected and uninfected animals will be compared across each lab site to determine extent of intrasite and intersite variability that exists.	Consensus protocol for flow cytometric analysis of major leukocyte populations in gut mucosa; (multiple polychromatic flow panels to quantitate: 1) CD4 T cell, 2) CD8 T cell, 3) Dendritic cell, 4) NK cell, 5) B cell, and 6) APC populations)	Selection of the polychromatic flow consensus panels is nearly complete; subsequent collection and analysis of NHP specimens is expected to continue for 5-6 mos.
MIG GU	Florian Hladik (U Washington/ FHCRC)	Cervicovaginal specimen collection for evaluation of immune responses: Standardization and comparative assessment of sampling techniques	Jo-Ann Passmore (U Cape Town), Rick Novak, (U Illinois, Chicago), Rupert Kaul, (U Toronto), Alan Landay, (Rush U), and Blake Ball, (U Manitoba)	Isolating sufficient numbers of immune cells from the female genital mucosa is the critical limiting step to perform meaningful assays of cell-mediated immunity. Genital immune cells can be isolated from cervicovaginal lavage (CVL), cervical swab, cervical cytobrush, menstrual cup and mucosal biopsy specimens. Non-invasive procedures are preferable to taking biopsies because they are better tolerated by study participants and have lower rates of side effects. However, it is unknown whether the number and functional phenotype of cells obtained from noninvasive specimens are equivalent to those isolated from biopsies, which is the focus of this study. Aim 1 will determine yields of viable leukocytes and their subtypes from non-invasive cervicovaginal lavage and cervical cytobrush specimens using standardized collection and processing protocols at clinics in Seattle, Chicago, Cape Town and Nairobi . Aim 2 will compare the yields of viable leukocytes and their subtypes between non-invasive cervical cytobrush specimens and ectocervical biopsies.	Standardized protocol for collection of cervicovaginal mucosal specimens; (CVL, cervical cytobrush, endocervical biopsy) Compare protocols for isolation of lymphocytes from mucosal biopsies; (among three different cell isolation methods) Standardized protocol for polychromatic flow analysis to quantitate primary leukocyte subpopulations present in  FGT specimens from HIV-uninfected women.	Aim 1 non-invasive collection and analysis of cervical specimens from 15 healthy women is complete at Chicago, well underway at Seattle and Cape Town and soon to begin at Nairobi.  Data will be re-analyzed centrally at FHCRC when all sites have completed Aim 1. Comparison of cell isolation methods from mucosal biopsies is in progress at Seattle in order to develop optimized protocol for processing Aim 2 biopsies.
MIG Systems Biology	Chris Love (MIT)	Integrated single-cell assays for multidimensional analysis of HIV-specific mucosal cellular responses and intercellular network mapping	Barbara Shacklett (UC Davis), Ruth Greenblatt (UCSF), and Susan Cu-Uvin (Brown U)	The goal of this project is to develop isolation and assay methods to evaluate intercellular cytokine networks using novel, multidimensional single-cell analyses of immune cells derived from mucosal samples of limited size.  These studies will include an analysis of the quality of functional responses measured from immune cells extracted from a variety of mucosal specimen types (both GI and GU) and transported by various means using a proprietary single-cell assay platform (Aim #1), and a systematic series of experiments on a collection of HIV+ and HIV- samples (Aim #2)	Research protocols for high content, single-cell  analyses of immune function in mucosal specimens from a variety of collection types and tissue sources.	Pilot studies have demonstrated feasibilty of evaluating polyfunctionality of T cell populations from cervical cytobrushes transported O/N across US.  Research protocol is in development prior to collecting specimens from initial cohort of HIV-uninfected women, in order to evaluate the biological variability of selected functional endpoints in mucosal specimens.
	Blake Ball (U Manitoba)	Comparative proteomic analysis of mucosal samples from the female genital tract	Rick Novak, (U Illinois, Chicago), and Kristina Broliden (Karolinska Institutet)	The goal of this project is to catalog and quantitatively compare the presence of immune correlates of mucosal protection against HIV in different compartments of the female genital tract (FGT) using the tools of mass spectrometry. Several cytokines and chemokines identified as potential HIV inhibitory factors (e.g., RANTES, SLPI) are released by lymphocytes and other immune cells, while others (e.g.,cystatins, serpins) are produced both in epithelial and immune cells, making their cellular origin uncertain. Further, the relative amount of production of these factors by cells in the cervix vs by cells of the vaginal vault may vary considerably, given physiological differences between these sites.  Accordingly, the proteomes of mucosal secretions released by the cervix (captured by cervical sponge) or released from the vaginal vault (captured by CVL) will be cataloged. Likewise, the proteomes of FGT tissues from paired samples of endocervix, ectocervix, and endometrium from hysterectomy specimens obtained from HIV-uninfected women will be cataloged. Individual paired specimens of mucosal secretions collected by cervical sponge or by CVL from 30 healthy women will be analyzed for differential protein expression.	Research protocols for mass-spectrometry-based proteome analyses of FGT mucosal secretions and tissues.	Pilot studies demonstrated feasibility of identifying up to 2000 distinct proteins in mucosal tissue samples from FGT; up to ~500 proteins in mucosal secretions. Proteomic surveys of FGT tissues are complete and data analysis is ongoing. Proteomic surveys of mucosal secretions collected by CVL or cervical sponge and differential protein expression analysis of individual paired mucosal secretion samples in progress, with estimated completion by August, 2011.
	Rafick Sekaly (VGTI-Florida)	Development of a systems biology platform for the assessment of mucosal innate and adaptive immunity	Susan Cu-Uvin (Brown U)	The goal of this project is to compare various mucosal sampling methodologies using transcriptional profiling to evaluate the homeostatic mucosal immune responses in healthy women. This study will initially be performed using non-invasive specimens obtained by CVL and cervical cytobrush, collected at three time points within the menstrual cycle, one of which overlaps with the collection time point being used by the multi-site comparative study collaboration of Florian Hladik. Mucosal samples will be compared to samples obtained from the peripheral blood to determine the correlation (if any) between peripheral and localized responses. Assessments will include RNA integrity using the Agilent platform, gene array on the Illumina platform, multiplex PCR and full bioinformatic analysis of all data sets. The same mucosal samples will be evaluated in parallel in FACS-based phenotypic assays to collect complementary data on the distribution of leukocyte populations within the sample to help identify the contribution of each cell subset to observed gene expression signatures.	Research protocols for collection and archiving of mucosal specimens for RNA microarray analysis.	IRB recently approved mucosal specimen collection protocols; application for subawards in progress at NIH. Study enrollment expected to open later this summer.