T lymphocyte inhibitory/exhaustion marker expression in chronic HIV-1 infection and the impact of TB co-infection

Golden, Maxine (2016-03)

Thesis (MSc)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: Introduction Chronic HIV-1 infection is driven by inflammation and immune activation which ultimately induces T lymphocyte exhaustion. Co-infection with TB is problematic as the HIV-impaired immune system is unable to effectively contain the TB, and in turn the TB disease promotes further immune activation and exhaustion through additional antigenic burden. In the current two component study, a total of 101 HIV-1 infected, 25 HIV-1-TB co-infected and 52 uninfected control individuals were included from the Cape Town peri-urban region. A cross sectional investigation of expression of markers of immune activation (CD38 and HLA-DR), exhaustion/inhibition (PD-1, Tim-3, LAG-3, 2B4) and apoptosis (CD95) was investigated on both CD4+ and CD8+ T cells. Functional proliferative responsiveness of T cells was also assessed. Materials and Methods Flow cytometry (both 4–colour and 10-colour) was used to determine expression of phenotypic markers using both fresh whole blood (pilot study) and PBMC (main study). CD4+ and CD8+ T lymphocyte populations of all study groups were stained for all the markers and evaluated for positive expression or co-expression. A functional proliferation assay (αCD3 and αCD28 stimulation) was conducted using CFSE-staining on the HIV-1+ samples. Impact of blocking Tim-3 and PD-1 pathways was evaluated. Results Chronic HIV-1 infection was accompanied by a significant increase in CD38 (p<0.0001), CD95 (p<0.01), PD-1 (p<0.01) and Tim-3 (p<0.01) expression on CD8+ T cells (pilot study). TB co-infection led to significantly elevated expression of CD38, CD95 and Tim-3, but not PD-1 (all p<0.05). CD4+ T cells displayed decreased expression of all of these markers in the infected groups, except for Tim-3, which was consistently <5%. In the main study CD8+ T cell-associated CD38, CD95, and PD-1 displayed a similar trend, with significant higher expression in the infected groups (p<0.0001, p<0.05, and p<0.0001, respectively), In contrast to the pilot study, Tim-3 expression was consistently <10%, with no difference between the groups. The novel marker 2B4 showed high level baseline expression (median 59.2%) which was significantly increased in the HIV and HIV/TB groups (69.6% and 75.1% respectively, p=0.025). LAG-3 was poorly expressed. Co-expression of PD-1 and 2B4 as well as CD95 and CD38 was also significantly increased (p<0.0001 for both). Discussion Increased immune activation and exhaustion was evident in the infected groups in both studies. PD-1 and 2B4 were both strongly expressed on CD8+ T cells and were significantly up-regulated in infected groups. PD-1 correlated positively with Tim-3 and LAG-3, and 2B4 with LAG-3, (both p<0.01) indicating that they are useful as biomarkers of exhaustion. Although significantly elevated exhaustion markers were observed in the TB co-infected setting in the pilot study, this did not reflect in the main study (except for CD95, 2B4). This suggests that immune dysfunction is mainly driven by HIV-1 infection alone. Short-term anti-TB therapy may also have had a restorative impact on the exhaustive marker expression. Differences in expression patterns using fresh whole blood vs. PBMC (especially Tim-3) warrant further investigation. Blocking Tim-3 and PD-1 expression on CD4+ and CD8+ T cells did not appear to have beneficial effects on T cell proliferation.

AFRIKAANSE OPSOMMING: Inleiding Kroniese MIV-1 infeksie word gedryf deur inflammasie en immuun-aktivering wat lei tot T limfosiet uitputting. Mede-infeksie met TB is veral problematies omdat die immuunstelsel nie die infeksie kan oorkom nie, wat lei tot die verdere aftakeling van die immuun stelsel. Tydens die tweeledige studie ‘n totaal van 178 individue was ondersoek wat bestaan het uit die volgende: 101 MIV-1 positiewe individue, 25 MIV-1-TB mede-geïnfekteerde individue en 52 kontrole individue. Individue wat deel gevorm het van die studie groep was afkomstig van Kaapstad se buitestedelike areas. Die studie het die uitdrukking van merkers van immuun- aktivering (CD38 en HLA-DR), onderdrukking/uitputting (PD-1, Tim-3, LAG-3, 2B4) en seldood (CD95) ondersoek op CD 4+ en CD8+ selle. Die proliferatiewe reaksie van T selle was ook geassesseer. Materiale en Metodes Vloeisitometrie (4-kleur en 10-kleur) was gebruik om die uitdrukking van fenotipiese merkers te ondersoek in beide vars heelbloed en PBMS. CD4+ en CD8+ T limfosiet populasies was ondersoek deur immuun merkers te kleur en daarna te evalueer vir positiewe uitdrukking of ko-uitdrukking. ‘n Funksionele proliferasie toets (αCD3 en αCD28 stimulasie) was uitgevoer op die HIV-1+ monsters deur middel van CFSE-kleuring. Die impak van Tim-3 en PD-1 paaie was geëvalueer. Resultate Kroniese MIV-1 infeksie het gepaard gegaan met ‘n verhoogde- CD38 (p<0.0001), CD95 (p<0.01), PD-1 (p<0.01) en Tim-3 (p<0.01) uitdrukking op CD8+ T selle. TB mede-infeksie het gelei tot ‘n verhoogde uitdrukking van CD38, CD95 en Tim-3, maar nie PD-1 (p<0.05) nie. CD4+ T selle in geïnfekteerde groepe het ‘n verlaagde uitdrukking van alle immuun-merkers getoon, behalwe vir Tim-3 (<5%). Die hoof studie het bevind at CD8+ T sel-geassosieerde CD38, CD95, en PD-1 ‘n soortgelyke patroon vertoon het, met verhoogde uitdrukking in geïnfekteerde groepe (p<0.0001, p<0.05, en p<0.0001, onderskeidelik). In kontras met die loodsstudie was uitdrukking van Tim-3 konstant <10%, met geen verskille tussen studie groepe. Die nuwe merker 2B4 het ‘n verhoogde basislyn uitdrukking getoon (mediaan 59.2%) wat beduidend verhoog was in die HIV-1+ en HIV-TB groepe (69.6% en 75.1% onderskeidelik, p=0.025). LAG-3 was swak uitgedruk. Ko-uitdrukking van PD-1 en 2B4 sowel as CD95 en CD38 was beduidend verhoog (p<0.0001). Bespreking Verhoogde immuun-aktivering en uitputting was opmerklik in geïnfekteerde groepe in beide studies. PD-1 en 2B4 was albei sterk uitgedruk op CD8+ T selle en was beduidend opgereguleer in geïnfekteerde groepe. PD-1 is positief gekorreleer met Tim-3 en LAG-3, en 2B4 met LAG-3, (beide p<0.01), wat toon dat hul gebruik kan word as biomerkers van uitputting. Alhoewel uitputting merkers beduidend verhoog was in die TB ko-geinfekteerde individue in die loodsstudie, was dit nie gereflekteer in die hoof studie nie (behalwe vir CD95 en 2B4). Dit kan ‘n indikasie wees dat immuunstelsel aftakeling gedryf word deur MIV-1 infeksie. Korttermyn anti-TB medikasie mag ook ‘n restoratiewe impak tot gevolg gehad het. Verskille in die uitdrukking van merkers tussen vars bloed en PBMS dui daarop dat verdere navorsing benodig word. Onderdrukking van Tim-3 en PD-1 uitdrukking op CD4+ en CD8+ T selle het geen voordelige effek gehad op T sel proliferering nie.

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