Wednesday, December 13, 2023 9 AM to 3 PM (Pacific Time)
UW SLU F106/F107 and Zoom (University of Washington South Lake Union Campus) (Zoom link, registration required)
Goal: Collaborating in small groups to develop new analytical insights from 4 published bulk and single-cell RNAseq datasets that profile NHP host gene expression during Mtb infection and BCG vaccination.
Datasets: PMIDs: 37097292, 35483355, 37267955, 37390827
Program: Agenda is here. Short talks to introduce datasets, workshop tutorials on analysis techniques, and open-ended hack time to plan and execute analyses.
Breakfast included starting at 8:30am, lunch included at noon, happy hour snacks included starting at 3pm.
Registration: Online registration using Zoom
SEATRAC is dedicated to providing a harassment-free learning experience for everyone regardless of gender, gender identity and expression, sexual orientation, disability, physical appearance, body size, race, age or religion. We do not tolerate harassment of participants in any form. Please reference this code of conduct adopted by Ocean Hackweek that we will use for our TB Hackday.
Ocean Hackweek Code of Conduct
Link to aggregated datasets on Figshare
Foreman et al., 2023: bulk RNAseq from sorted cells in NHP Mtb challenge model
Gideon et al., 2022: scRNAseq from granulomas in NHP Mtb challenge model
Darrah et al., 2023: scRNAseq from BAL in NHP Mtb challenge (BCG route, correlates of protection)
Liu et al., 2023: whole-blood bulk RNAseq from NHP Mtb challenge (BCG route and IV BCG dose, should match Darrah et al. study)
NOTE: For the Gideon et al. and Darrah et al. single-cell datasets there is also a pseudo-bulk dataset that was created by summing raw counts across cells within a sample and an annotated cell type. Cell annotations from the original data were used for the summation. The resulting CSV contains counts for each sample, cell type and gene.
Foreman TW, Nelson CE, Sallin MA, Kauffman KD, Sakai S, Otaizo-Carrasquero F, Myers TG, Barber DL. CD30 co-stimulation drives differentiation of protective T cells during Mycobacterium tuberculosis infection. J Exp Med. 2023 Aug 7;220(8):e20222090. doi: 10.1084/jem.20222090. Epub 2023 Apr 25. PMID: 37097292; PMCID: PMC10130742.
PDF: https://rupress.org/jem/article-pdf/220/8/e20222090/1451373/jem_20222090.pdf; (github)
MS: https://rupress.org/jem/article/220/8/e20222090/214054/CD30-co-stimulation-drives-differentiation-of
DATA: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE227653 (M mulatta data)
https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE228114 for the superset that contains both the NHP and mouse data for that study
https://github.com/FredHutch/seatrac-hackday-2023/tree/main/foreman_etal
DATA SUMMARY: N=4 Rhesus macaques, Mtb. challenge (40–80 CFU of Mtb-Erdman-mCherry and euthanized 6–7 wk after infection); bulk RNAseq from FACS sorted T cells; bulk RNAseq count data is stored for CD4 and CD8 T cells in units of transcript per million (TPM).
Gideon HP, Hughes TK, Tzouanas CN, Wadsworth MH 2nd, Tu AA, Gierahn TM, Peters JM, Hopkins FF, Wei JR, Kummerlowe C, Grant NL, Nargan K, Phuah JY, Borish HJ, Maiello P, White AG, Winchell CG, Nyquist SK, Ganchua SKC, Myers A, Patel KV, Ameel CL, Cochran CT, Ibrahim S, Tomko JA, Frye LJ, Rosenberg JM, Shih A, Chao M, Klein E, Scanga CA, Ordovas-Montanes J, Berger B, Mattila JT, Madansein R, Love JC, Lin PL, Leslie A, Behar SM, Bryson B, Flynn JL, Fortune SM, Shalek AK. Multimodal profiling of lung granulomas in macaques reveals cellular correlates of tuberculosis control. Immunity. 2022 May 10;55(5):827-846.e10. doi: 10.1016/j.immuni.2022.04.004. Epub 2022 Apr 27. PMID: 35483355; PMCID: PMC9122264.
PDF: github MS: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9122264/
"We have performed high-throughput single-cell mRNA sequencing on 6 granulomas from 2 non-human primates at 4 weeks post infection with M. tuberculosis to understand cellular and molecular factors associated with bacterial control (n = 10,006 cells)."
"We have performed high-throughput single-cell mRNA sequencing on 26 granulomas from 4 non-human primates at 10 weeks post infection with M. tuberculosis to understand cellular and molecular factors associated with bacterial control (n = 109,584 cells)"
DATA 4 WKS: Alexandria; Github; Figshare
Single-cell data is on Figshare
DATA SUMMARY: single-cell RNAseq (SeqWell); 6 granulomas from 2 NHP at 4 wks and 26 granulomas from 4 NHP at 10 weeks post-infection (includes CFU per granuloma)
Darrah PA, Zeppa JJ, Wang C, Irvine EB, Bucsan AN, Rodgers MA, Pokkali S, Hackney JA, Kamath M, White AG, Borish HJ, Frye LJ, Tomko J, Kracinovsky K, Lin PL, Klein E, Scanga CA, Alter G, Fortune SM, Lauffenburger DA, Flynn JL, Seder RA, Maiello P, Roederer M. Airway T cells are a correlate of i.v. Bacille Calmette-Guerin-mediated protection against tuberculosis in rhesus macaques. Cell Host Microbe. 2023 Jun 14;31(6):962-977.e8. doi: 10.1016/j.chom.2023.05.006. Epub 2023 Jun 1. PMID: 37267955; PMCID: PMC10355173.
PDF: https://www.nature.com/articles/s41586-019-1817-8.pdf; (github)
MS: https://www.nature.com/articles/s41586-019-1817-8
DATA: https://singlecell.broadinstitute.org/single_cell/study/SCP796/prevention-of-mycobacterium-tuberculosis-infection-and-disease-in-nonhuman-primates-following-intravenous-bcg-vaccination?scpbr=the-alexandria-project Single-cell data is on Figshare
DATA SUMMARY: single-cell; n=15 Rhesus macaques with BCG vaccination and Mtb challenge; 3 individuals per group for AE, IDhigh, IDlow, IV, Naïve-controls; BAL collected at Weeks 13 and 25 prior to challenge at Week 26; stimulated and unstimulated conditions; 60 samples, 1000 – 5000 cells each.
Mycobacterium tuberculosis (Mtb) is the leading cause of death from infection worldwide. Intradermal (ID) vaccination with BCG has variable efficacy against pulmonary tuberculosis, the major cause of mortality and disease transmission. Here we show that the route and dose of BCG vaccination alters circulating and lung resident T cells and subsequent protection against Mtb challenge in nonhuman primates (NHP). NHP immunized with BCG by the intravenous (IV) route induced substantially higher antigen-specific CD4 (Th1 or Th17) and CD8 responses in blood, spleen, bronchoalveolar lavage (BAL), and lung lymph nodes compared to the same BCG dose administered by ID or aerosol (AE) routes. Moreover, IV immunization was the only route that induced a high frequency of antigen-specific tissue resident T cells in lung parenchyma. Six months after BCG vaccination, NHP were challenged with virulent Mtb. Strikingly, 9 of 10 NHP that received BCG IV were highly protected, with 6 NHP showing no detectable infection as determined by PET CT imaging, mycobacterial growth, pathology, granuloma formation, or de novo immune responses to Mtb-specific antigens. The finding that BCG IV prevents or significantly limits Mtb infection in NHP has important implications for vaccine development and provides a model for determining immune correlates and mechanisms of protection against TB.
Liu YE, Darrah PA, Zeppa JJ, Kamath M, Laboune F, Douek DC, Maiello P, Roederer M, Flynn JL, Seder RA, Khatri P. Blood transcriptional correlates of BCG-induced protection against tuberculosis in rhesus macaques. Cell Rep Med. 2023 Jul 18;4(7):101096. doi: 10.1016/j.xcrm.2023.101096. Epub 2023 Jun 29. PMID: 37390827; PMCID: PMC10394165.
PDF: github MS: https://www.sciencedirect.com/science/article/pii/S266637912300215X?via%3Dihub#sec4.1
DATA IV BCG DOSE STUDY (167 samples, 34 NHP): https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE218270
DATA BCG ROUTE STUDY (144 samples, 36 NHP): https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE218157
DATA SUMMARY: Darah et al. IV BCG dose and BCG route study matching; Pre BCG and Day 2, Wk 4, Wk 12 for IV BCG dose study; Pre, Day 2, Wk2 and Wk12 for route study; bulk RNAseq from whole blood; includes protection data for each individual NHP