Flow Cytometric Analysis of Gut Biopsies. Ian McGowan MD PhD FRCP University of Pittsburgh Pittsburgh, PA USA
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1 Flow Cytometric Analysis of Gut Biopsies Ian McGowan MD PhD FRCP University of Pittsburgh Pittsburgh, PA USA
2 Overview GALT flow cytometry GALT flow data from completed studies MTN studies Non-MTN studies Lessons learned and questions for the future
3 GALT Flow Cytometry
4 GALT Flow Cytometry GALT flow cytometry increasingly used to characterize mucosal cell populations in: HIV pathogenesis studies Evaluation of HIV vaccine responses PreP studies Non-HIV related fields such as inflammatory bowel disease research Primary approach is to collect biopsies and to mechanically/enzymatically disassociate into single cells
5 GALT Flow Data
6 GALT Flow Data (1) HPTN-056 HVTN-MIG study No intervention No intervention RMP-01 MTN-017 UC781 gel (Phase 1) TFV gel/oral (Phase 2) RMP-02 / MTN-006 CHARM-03 TFV gel & oral (Phase 1) Maraviroc gel & oral (Phase 1) MTN-007 HPTN-069 TFV gel (Phase 1) CHARM-01 Oral TFV, MVC, FTC (Phase 2) TFV gel (Phase 1)
7 GALT Flow Data (2) ACTG 5330 (Multi-center) The effect of isotretinoin on immune activation among HIV-1-infected subjects with incomplete CD4+ T cell recovery Dipyridamole (DP) study (Single center) The effect of DP on HIV-associated immune activation and inflammation ACTG 5341s (Multi-center) Size and decay of HIV-1 reservoirs in tissues
8 HPTN-056 Population Panel HIV-negative (N=8) HIV-positive (N=8) CD3, CD4, CD8, CD45, Center(s) Single Sampling Colon 10 cm and 30 cm HLA-DR, CD38, CXCR4, CCR5, DC- SIGN CD19 CD16, CD56 BL, +2/52 +4/52 McGowan et al. JAIDS 2007
9 HPTN-056 ICC Scores ICC Score CD3 CD4 CD8 CD19 CD16/56 CD4/CCR5 CD4/CXCR4 CD4/CD38 CD8/CD38 CD4/HLA-DR % MMC Phenotype McGowan et al. JAIDS 2007
10 RMP-01 Population HIV-negative (N=36) Center(s) Single Sampling Colon 10 cm and 30 cm BL, post single dose, and post seven doses Products (1:1:1) UC781 gel (0.1%) UC781 gel (0.25%) HEC placebo Panel CD4, CD8, CD45, HLA-DR, CD38 CXCR4, CCR5 Anton PA et al. PLoS One 2011
11 RMP-01 Single dose UC781(0.1%) vs HEC (p >0.05) UC781 (0.25%) vs. HEC (p >0.05) 7 day exposure UC781(0.1%) vs HEC (p >0.05) UC781 (0.25%) vs. HEC CCR5 RFI on CD4 (p = 0.025) CCR5/CXCR4 on CD4 (p = 0.020) Anton PA et al. PLoS One 2011
12 RMP-02 / MTN-006 Population HIV-negative (N=18) Center(s) 2 sites Samples shipped to UCLA for analysis Sampling Colon (15 cm) BL, post single dose, and post seven doses Products (2:1) TFV gel (1%) HEC placebo Panel CD4, CD8, CD45, HLA-DR, CD38 CXCR4, CCR5 Anton PA et al. AIDS Res Hum Retroviruses 2012
13 RMP-02 / MTN-006 Oral Single 7 day P values CD3+ on CD CD4+ on CD CD8+ on CD CD38+ on CD CD38 RFI on CD HLA- DR+ on CD HLA- DR+ CD38+ on CD CCR5+ on CD CCR5 RFI on CD Anton PA et al. AIDS Res Hum Retroviruses 2012
14 MTN-007 Population HIV-negative (N=60) Center(s) 3 centers Samples shipped to Pittsburgh Sampling Anoscopic Flex sig (15 cm) BL, post SD, and post 7D Products (1:1:1:1) TFV gel (1.0%) N9 gel (2.0%) HEC placebo No Rx Panel CD3, CD4, CD8, CD45, CD69 CXCR4, CCR5 McGowan I et al. PLoS One 2013
15 MTN-007 7doses of TFV gel versus HEC gel associated with a significant increase in CD45+/CD3+ cells isolated from Flex sig biopsies (57% versus 42.8%; p = 0.04) McGowan I et al. PLoS One 2013
16 CHARM-01 Population Products (crossover) HIV-negative (N=14) TFV gel (1.0%) Center(s) RG TFV gel (1.0%) 2 centers RS TFV gel (1.0%) Samples shipped to Pittsburgh Sampling Panel CD3, CD4, CD8, CD45, Flex sig (15 cm) CD69 BL, and post 7D of each formulation CXCR4, CCR5 McGowan I et al. PLoS One 2015
17 McGowan I et al. PLoS One 2015 CHARM-01
18 HVTN-MIG Feasibility of multi-site flow cytometric processing of GALT samples Clinical sites UCLA (N = 18) Pittsburgh (N = 17) Samples PBMC, qpbmc, MMC Flow analysis Fred Hutchinson CRC McGowan I et al. PLoS One 2015
19 MIG Panel
20 McGowan I et al. PLoS One 2015 qpbmc Data
21 McGowan I et al. PLoS One 2015 MMC Data
22 MIG Summary Ranges for qpbmc CD4/CD8 equivalent at both sites Significant differences for the majority of other parameters Standardized protocols can reduce but not eliminate variability between sites HSV seropositivity influences T cell phenotype FMO not routinely required
23 Lessons Learned
24 Lessons Learned GALT flow cytometry is challenging and variable Cryopreservation and centralized analysis may provide more stable data Unclear whether flow cytometry routinely required in microbicide studies Further data may clarify the situation MTN-017, HPTN-069, CHARM-03
25 Future Questions
26 Future Questions What studies require flow cytometry? What panels should be included? Multi-center studies Should sample processing/staining and/or analysis be centralized? Could cryopreservation of tissue samples or isolated MMC lead to more robust data?
27 Acknowledgements The Microbicide Trials Network is funded by the National Institute of Allergy and Infectious Diseases (UM1AI068633, UM1AI068615, UM1AI106707), with co-funding from the National Institute of Child Health and Human Development and the National Institute of Mental Health, all components of the U.S. National Institutes of Health.
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