Optimisation of a whole blood flow cytometry assay to aid in the diagnosis of tuberculosis by detecting intracellular cytokines released by CD4+ T-cells

Snyders, Candice Irene (2016-03)

Thesis (MSc)--Stellenbosch University, 2017

Thesis

ENGLISH ABSTRACT : Background: South Africa (SA) sees 8 million new Tuberculosis (TB) cases each year and has a significant problem with Human Immunodeficiency Virus (HIV) and TB co-infection. Latent TB infection (LTBI) is described in persons infected with mycobacterium tuberculosis (M.tb) but shows no signs and symptoms of active disease. HIV+ individuals with LTBI can develop active TB infection more readily than that of HIV- individuals. Gold standard methods for diagnosing active disease have been criticized for, among other things, their lengthy turnaround times. Currently there is no gold standard for the diagnosis of LTBI. Flow cytometry allows one to measure cytokine responses in CD4+ T-cells following overnight stimulation with TB antigens ESAT-6 and CFP-10 (E/C). Studying these cytokine expression patterns will make it possible to classify patients into active disease vs. LTBI. Methods:A total of 18 TB+ patients which included 6 HIV+ patients, were recruited from Tygerberg Hospital, Western Cape. A whole blood no-centrifuge intracellular flow cytometry assay was optimised to study the cytokine expression patterns in CD4+ T-cells that have been stimulated with TB antigens and Staphylococcus Enterotoxin B (SEB), following an 18 hour overnight incubation. CD3+CD4+ T-cells were delineated into the following subsets: naïve (TN) (CD45RO-CD27+ ), central memory (TCM) (CD45RO+CD27+ ),effector memory (TEM)(CD45RO+CD27- ) and terminally differentiated effector memory cells (TDEM) (CD45RO-CD27- ). The expression patterns and effect of stimulation on cytokines IFN-γ and TNF-α as well as T-cell exhaustion marker TIM3, was assessed. Results: This study has demonstrated higher levels of IFN-γ expression in the control group compared to that of the TB positive patients (median %IFN-γ 2.960 ± 3.51 versus patient 2.370 ± 2.07; p=0.2800). TNF-α had higher expression in the patient group compared to the control subjects (median %TNF-α 2.415 ± 2.60 versus control 1.340 ± 1.86; p=0.1729). Dual expression of cytokines was almost similar in the two groups (control median % IFN-γ + TNF-α + 0.5400 ± 0.36 versus patient 0.8550 ± 0.60; p=0.3961). TIM3 expression was not significantly different between the four T-cell subsets (median TN 0.0750 ± 1.89, TCM 0.3400 ± 4.28, TEM 0.0850 ± 2.73, TDEM 0.1600 ± 1.93; p= 0.5877). When comparing the subset distribution in the patient group, TN cells were the most abundant (median 47.48 ± 20.96) followed by TEM cells (median 21.92 ± 13.25), TDEM cells (median 13.02 ± 20.13) and finally TCM cells (median 11.51 ± 8.62). These results showed a significant difference in expression between the four groups (p=<0.0001). Conclusion: Through careful titration of antibodies and relevant optimisation steps, we established a flow cytometry assay that may be used to study cytokine patterns in TB patients. The increased TNF-α only expression in the patient group is suggestive of active TB and the increased IFN-γ in the control group could indicate BCG vaccination. TIM3 would be a useful marker in a larger HIV+ cohort of patients as this will allow identification of functionally exhausted T-cells. In SA, HIV prevalence is rising and this assay proves its suitability by using minimal volumes of whole blood rather than sputum. By generating intracellular cytokine profiles one would be able to distinguish between active and LTBI which would aid in treatment management of patients.

AFRIKAANSE OPSOMMING : Agtergrond: Suid-Afrika (SA) het 8000000 nuwe Tuberkulose (TB) gevalle elke jaar en het 'n groot probleem met MIV en TB mede-infeksie. Latente TB-infeksie (LTBI) word beskryf in persone wat besmet is met Mycobacterium tuberculosis (M.tb), maar toon geen tekens en simptome van 'n aktiewe siekte. MIV+ individue met LTBI kan aktiewe TB-infeksie meer geredelik as dié van MIVindividue ontwikkel. Goud standaard metodes vir die diagnose van aktiewe siekte is gekritiseer vir, onder andere, hul lang omkeertye. Daar is tans geen goue standaard vir die diagnose van LTBI. Vloeisitometrie laat mens toe om sitokien uitdrukking in CD4+ T-selle te meet na oornag stimulasie met TB antigene ESAT-6 en CFP-10 (E / C). Die bestudering van hierdie sitokien uitdrukking patrone sal dit moontlik maak om pasiënte te klassifiseer as aktiewe siekte of LTBI. Metodes: 'n Totaal van 18 TB pasiënte wat 6 MIV+ pasiënte insluit, is gewerf uit die Tygerberg-hospitaal, Wes-Kaap. 'N Heel bloedgeen-centrifuge intrasellulêre vloeisitometrie toets is geoptimaliseer om die sitokien uitdrukking patrone in CD4+ Tselle wat reeds gestimuleer word met TB antigeneen Staphylococcus enterotoksien B (SEB), na aanleiding van 'n 18 uur oornag inkubasie te bestudeer. CD3+CD4+ Tselle is afgebaken in die volgende onderafdelings: naïef (TN) (CD45RO-CD27+ ), sentralegeheue (TCM) (CD45RO+CD27+ ), effektorgeheue (TEM) (CD45RO+CD27- ) enterminaal gedifferensieerde effektorgeheueselle (TDEM) (CD45RO-CD27- ). Die uitdrukkings patrone en effek van stimulasie op sitokiene IFN-γ en TNF-α asook Tseluitputtingmerker TIM3, is bestudeer. Resultate: Hierdie studie het hoër vlakke van IFN-γ uitdrukking getoon in die kontrole groep in vergelyking met dié van die TB-positiewe pasiënte (gemiddelde% IFN-gamma 2,960 ± 3,51 vs pasiënt 2,370 ± 2,07; p = 0,2800). TNF-α het hoëruitdrukking in die pasiëntgroep in vergelyking met die kontrole kandidate (mediaan% TNF-α 2,415 ± 2,60 teen beheer 1,340 ± 1,86; p = 0,1729). Dubbele uitdrukking van sitokiene was amper soortgelyk in die twee groepe (kontrole mediaan% IFN-γ + TNF-α + 0,5400 ± 0,36 teen pasiënt 0,8550 ± 0,60; p = 0,3961). TIM3 uitdrukking was nie beduidend anders in die vier T-sel deel versamelings nie (mediaan TN 0,0750 ± 1.89, TCM 0,3400 ± 4.28, TEM 0,0850 ± 2,73, TDEM 0,1600 ± 1,93; p = 0,5877). Wanneer die vergelyking van die subset verspreiding in die pasiëntgroep gedoen was, het TNselle die meeste voorgekom (mediaan 47,48 ± 20,96) gevolg deur TEM selle (mediaan 21,92 ± 13,25), TDEM selle (mediaan 13,02 ± 20,13) en uiteindelik TCM selle (mediaan 11,51 ± 8,62 ). Hierdie resultate toon 'n beduidende verskil in die uitdrukking tussen die vier groepe (p = <0,0001). Gevolgtrekking: Deur versigtige titrasie van teenliggaampies, ens het ons gestig 'n vloeisitometrietoets wat gebruik kan word om sitokien patrone te studeer in TBpasiënte. Die verhoogde enkele TNF-α uitdrukking in die pasiëntgroep is n aanduiding van aktiewe TB en die verhoogde IFN-γ in die kontrolegroep kan moontlike BCG inenting aan dui. TIM3 sou 'n nuttige merker in 'n groter MIV+ kohort van pasiënte wees, want dit sal die identifisering van funksioneel uitgepute T-selle identifiseer. In SA, is die voorkoms van MIV stygend en hierdie toets bewys sy geskiktheid deur die gebruik van minimale volumes van volbloed, eerder as sputum. Deur die opwekking van intrasellulêre sitokien profiele sou 'n mens in staat wees om te onderskei tussen aktiewe en LTBI wat sou help met die behandeling van pasiënte.

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