MIG Working Group  PI
(Institution)
Study Title Collaborator
(Institution)
Project Summary Protocols Developed
(Brief Description)
Study Outcome / Publications
MIG GI  Ian McGowan
(U Pittsburgh)
Mucosal GI working group comparative flow cytometry project Peter Anton,
(UCLA);
Steve De Rosa,
(U Washington/
FHCRC)
The aim of this multi-stage, multi-center study was to generate and compare data on normative values for gut-associated lymphoid tissue (GALT) T-cell populations when highly standardized methods for mucosal tissue collection and flow cytometric analysis are adopted in clinical settings. The investigators characterized potential variance in normal T-cell population distributions and their functional attributes in the gut, with the goal of better understanding the experimental factors that may influence the normative ranges reported by different labs.  Identical protocols and reagents for enzymatic digestion and cell staining were used by each center with centralized data analysis performed at FHCRC.  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
In preparation for this study, methods for GALT isolation, flow cytometric staining and acquisition of samples on the flow cytometer were standardized and implemented in two laboratories (UCLA and U of Pittsburgh)  to analyze flex-sig biopsies collected from 20 HIV-negative males at each clinic.

GALT mucosal mononuclear cells (MMC) were isolated from rectal biopsies by enzymatic digestionand compared to matched peripheral blood mononuclear cells (PBMC). Cryopreserved PBMC from a single donor were supplied to both sites from a central repository for use as a quality control (qPBMC). Flow data from both sites were analyzed centrally by an independent analyst.  Ranges for both CD4+ and CD8+ T lymphocytes derived from the qPBMC samples, were equivalent at both UCLA and U of Pittsburgh. However, there were significant inter-site differences for the majority of PBMC and MMC phenotypic markers. The use of standardized protocols to collect, stain, and analyze MMC and PBMC, including centralized analysis of flow data, can reduce but not exclude data variability within multicenter studies. Moreover, while fluorescence minus one (FMO) controls were performed on all MMC and PBMC samples, because the assay was highly standardized, gating templates could be used for most experiments, supporting that FMO controls are not needed for each sample.

For detailed study findings and SOP (in online supplemental materials) please see McGowan et al., PLoS ONE 2015.
Ron Veazey
(Tulane U)
Optimization of cryopreservation techniques from intestinal tissue samples Dayong Gao,
(U Washington)
Study purpose is to optimize 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. Protocols for controlled freezing and thawing/cryoprotectant removal from NHP gut biopsies that will better preserve T cell viability and function Pilot studies that tested existing protocols to freeze PBMC or NHP embryos showed 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.

Subsequently, research aims evolved to focus on studies to  experimentally measure key intrinsic cellular/tissue parameters (e.g., membrane permeability to water or DMSO; intracellular ice formation (IIF) temperature) of NHP gut mucosal specimens in parallel to studies conducted with human mucosal samples (see below).

Please see Hughes et al., PLoS ONE 2016 for study findings
Paul Johnson
(Emory U)
Multi-center comparison of immunological phenomena at various sites along the GI tract in SIV-uninfected macaques Jason Brenchley,
(NIAID);
Ron Veazey,
(Tulane U)
This study evaluated variations in T cell populations and other key immune cell populations (DC; NK; B cell; APC) within biopsies taken at different locations of the GI tract from normal, healthy macaques housed at NIAID, Tulane, and Harvard.    T cell subsets were 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 could be compared to the same cell populations in matched PBMC and peripheral lymph node specimens. Data from each cohort of uninfected, normal animals will be compared across labs to determine extent of intrasite and intersite variability. Additionally, transcriptional profiling by microarray analysis was performed to compare gene expression signatures in sorted T cells, NK cells and B cells from the GI tract.   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) Development of six polychromatic flow panels is complete; panels were successfully implemented at three research laboratories for the characterization of major leukocyte populations present in the gut of healthy,  SIV-uninfected macaques (~7-10 animals per laboratory center).  Profiling of gene expression signatures in different sorted immune cell subpopulations isolated from the gut  is also complete and final statistical analyses are in progress.

Please contact Project PI for further detail and SOP distribution.
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);
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 determined 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 compared 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.   
The objectives of this multi-center clinical trial were to determine yields of viable leukocytes and their subtypes from non-invasive sampling of the female genital tract (CVL and cervical cytobrush) compared to cervical biopsies obtained from healthy women.  Methods were highly standardized between participating clinics (Seattle; Chicago; Nairobi; Cape Town) including sample collection methods (e.g., brand of cytobrush; biopsy forcep bite size) and timing (e.g., during the secretory phase of the menstrual cycle), cell isolation and staining procedures (e.g., identical reagent lots), flow cytometric methods (e.g., identical staining reagents and conditions; controls and TruCount beads) and centralized data analyses.  Key conclusions from this study include that: 1) among the three FGT sampling methods tested, while two endocervical cytobrushes yielded comparable numbers of viable leukocytes to one ectocervical biopsy, cytobrushes yielded more macrophages whereas biopsies yielded more T lymphocytes, potentially reflecting microanatomical differences; and 2) the use of highly standardized methods to collect, process and analyze mucosal cells makes it feasible to obtain consistent flow cytometric analyses of isolated genital cells from multiple study sites in the US and Africa. 

For detailed study findings and SOP (in online supplemental materials) please see McKinnon et al., PLoS ONE 2014.
Florian Hladik
(U Washington/
FHCRC)
Rational design of an optimal cryopreservation protocol for human cervicovaginal cells and tissues Dayong Gao,
(U Washington); Charlene Dezzutti, (U Pittsburgh);  Leonid Margolis (NIH)
The overall goal of this research is (a) to improve understanding of the cryobiology of immune cells (especially T lymphocytes and antigen-presenting cells such as macrophages and dendritic cells) from genital secretions and genital mucosal tissues in women, and (b) to develop optimal techniques for cryopreservation of these immune cells and mucosal tissues to meet the urgent and increasing needs to assess HIV vaccine and microbicide efficacy.
  
Protocols for controlled freezing and thawing/cryoprotectant removal from female genital tract tissues and mucosal mononuclear cells that will better preserve immune cell viability and function Experiments are complete with freshly isolated and sorted CD3+ T cells and CD14+ macrophages from human vaginal mucosa to determine cell-type specific parameters including the osmotically inactive cell volume (Vb) and cell membrane permeability to water (Lp) and cryoprotectants (Ps). For detailed study findings and SOP (in online supplemental materials) please see Hughes et al., PLoS ONE 2016.

Further experiments to determine whether optimal cryopreservation conditions for pure populations of mucosal immune cells can be successfully applied to improve post-freeze-thaw viability of intact tissue biopsies for subsequent functional analyses in
ex vivo FGT tissue explant assays are likewise complete and a manuscript is in progress. Please contact the study PI for further information.
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);
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. Research protocols have been developed and specimen collection and processing through single-cell functional assays is complete for initial cohort of 20, HIV-uninfected women as well as for second comparison cohort of 20 HIV-infected women on cART whose symptoms are well-controlled. Major platform advances have likewise been realized to move these assays and data analyses closer to implementation in clinical studies (see Yamanaka et al., Trends Immunol 2013; Gierahn et al., J Proteome Res 2014).
Blake Ball
(U Manitoba)
Comparative proteomic analysis of mucosal samples from the female genital tract  Adam Burgener
(U Manitoba);
Rick Novak,
(U Illinois, Chicago); Kristina Broliden (Karolinska Institutet)
This project cataloged and quantitatively compared 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) were cataloged. Likewise, the proteomes of FGT tissues from paired samples of endocervix, ectocervix, and endometrium from hysterectomy specimens obtained from 7 HIV-uninfected women were cataloged. Finally, individual paired specimens of mucosal secretions collected by cervical sponge or by CVL from 10 healthy women were analyzed for differential protein expression. Research protocols for mass-spectrometry-based proteome analyses of FGT mucosal secretions and tissues.
Pilot studies demonstrated the feasibility of identifying up to 3500 distinct proteins in mucosal tissue samples from FGT; up to ~500 proteins in mucosal secretions.

Proteomic surveys of FGT tissues are complete and results support that there is anatomical compartmentalization of immune factor expression within the FGT (see Burgener et al., J Virol 2013).

Proteomic surveys of mucosal secretions collected by CVL or cervical sponge and differential protein expression analysis of individual paired mucosal secretion samples are complete. Results demonstrate that there are significant differences in types and levels of immune factors and inflammatory mediators collected by CVL and cervical sponge sampling of mucosal secretions and argue for the inclusion of both sampling types for a more complete picture of mucosal responses
(see Birse at al., PLoS ONE 2013)

Rafick Sekaly
(Case Western)
Development of a systems biology platform for the assessment of mucosal innate and adaptive immunity Mark Cameron (Case Western);  Susan Cu-Uvin (Brown U) This project compared various mucosal sampling methodologies using transcriptional profiling to evaluate the homeostatic mucosal immune responses in healthy women. This study was 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 include RNA integrity using the Agilent platform, gene array on the Illumina platform, multiplex PCR and full bioinformatic analysis of all data sets.   Research protocols for collection and archiving of mucosal specimens for RNA microarray analysis.

SOP for RNA extraction and purification from cervical cytobrush and CVL samples.

Research protocols were developed and successfully implemented to identify functional genomic signatures unique to the genital tract in healthy women at three phases of the reproductive cycle. While preliminary paired analyses on 10-15 complete time-point data sets (depending on the sample type) identified gene expression differences between different phases of the menstrual cycle and confirmed that these gene signatures are unique to the FGT (i.e., not observed in peripheral blood), these data were statistically underpowered to make definitive conclusions.  Therefore, with additional Gates Foundation support, the study was  expanded to a complete dataset of 21 to 27 subjects, which confirmed the preliminary finding of genital tract-specific gene expression signatures that are generally enriched in epithelial cell, complement, cell-adhesion and pro-inflammatory cytokine signaling pathways and further delineated that certain pro-inflammatory genes are differentially expressed at different phases of the menstrual cycle and between different sampling techniques (CVL vs. cytobrush).

Please contact Project PI for further detail and SOP distribution.