Phenotypic and functional characterization of cytotoxic T lymphocytes in HIV-1 infected South African adults

Pillay, Santhoshan Thiagaraj (2010-12)

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Thesis (MScMedSc (Pathology. Medical Virology))--University of Stellenbosch, 2010.

Thesis

ENGLISH ABSTRACT: In just 25 years since the first reported cases in 1981, the number of Human Immunodeficiency virus (HIV) infected people has risen to 65 million, and over 25 million have died of acquired immunodeficiency syndrome (AIDS). Sub-Saharan Africa accounts for 67% of all people living with HIV and 72% of deaths in this region were AIDS related. Tuberculosis (TB) is one of the most common opportunistic infections in AIDS patients, particularly in developing countries, where 60 - 70% of TB cases occur in HIV-1-infected persons. HIV-1 is a high risk factor for the development of TB, the reactivation of a latent Mycobacterium tuberculosis infection and also progressive TB. CD8+ Cytotoxic T Lymphocytes (CTL) are pivotal in the host immune response to HIV infection. CTL are associated with resolution of acute infection and with reduction in viral load. Studies in macaques and humans indicate the importance of CTL in the control of HIV infection, where reduction in CD8+ T cell number has been correlated with progression to AIDS. The current study was a cross-sectional descriptive study of CD8+ T cells of HIV+ adult South Africans with and without TB co-infection (TB disease). The cohort consisted of anti-retroviral therapy (ART) naive patients and all CTL analyses were carried out on peripheral blood mononuclear cells (PBMCs). A total of 60 South African adults from the Western Cape were utilized in this study, including 15 healthy controls; 30 HIV+TB-individuals and 15 HIV+TB+ individuals. Expression of phenotypic, activation and functional markers were investigated by flow cytometry with the use of fluorochomeconjugated antibodies. The markers examined included the novel activation marker CD137, the CTL associated markers Perforin, Granzyme A, CD107a/b, Fas (CD95), and FasL (CD95L), intracellular cytokines IFN-y and TNF-a and the chronic HIV CTL dysfunction marker PD-1. HIV infection alone was associated with increased baseline expression of TNF-a, Perforin, Granzyme A, PD-1, Fas (CD95), and FasL (CD95L), but not CD137(4-1BB) or IFN-y as compared to uninfected controls. TB co-infection resulted in further increased baseline expression of TNF-a, perforin, PD-1, FasL (CD95L), as well as increased IFN-y. HIV-1 antigen (gag)-specific stimulation in vitro indicated that in HIV infection was associated with antigen-specific upregulation of activation and cytotoxicity markers CD137, IFN-y, TNF-a, Fas, FasL and CD107a/b. In TB co-infection a reduction in antigen-specific degranulation (CD107a/b up-regulation) and also Fas and FasL expression was observed. TB co-infection (in the form of active pulmonary TB) reduced antigen-specific CTL functional activity, but simultaneously there was an association with increased baseline PD-1 expression and also cytolytic marker expression (Fas, FasL, TNF-a). These cytolytic markers could be involved in non-antigen-specific bystander target cell death. The expression of the co-stimulatory molecule CD137 appeared to correlate with interferon-y production and levels of degranulation, confirming its usefulness as a putative surrogate marker of functional responsiveness. These data indicate that in addition to impacting on CD4 T cell function, TB co-infection leads to higher baseline expression of CTL-associated markers, but to dysfunctional antigen-specific CTL responses.

AFRIKAANSE OPSOMMING: Slegs vyf en twintig jaar na die eerste berigte van die menslike immuniteitsgebrekvirus (MIV) in 1981, het die getal MIV-geinfekteerde individue gestyg tot 65 miljoen en het meer as 25 miljoen mense alreeds gesterf aan die verworwe immuniteitsgebrek sindroom (VIGS). Sub Sahara Afrika maak 67% uit van alle HIV gevalle en het `n MIVverwante doodsyfer van 72%. Een van die algemeenste opportunistiese infeksies in VIGS pasiente is Tuberkulose (TB). In ontwikkelende lande, veral, kom 60-70% van TB gevalle voor in MIV-1 geinfekteerde individue. MIV-1 is `n hoe risiko faktor vir die ontwikkeling van TB, die heraktivering van latente Mycobacterium tuberculosis infeksie en progressiewe TB. Die CD8+ sitotoksiese T Limfosiete (STL) se immuun reaksie teen `n MIV infeksie is noodsaaklik en word geassosieer met `n resolusie van die akute infeksie en `n afname in viruslading. Studies in die mens en macaque het getoon dat sitotoksiese T limfosiete belangrik is vir die beheer van MIV infeksies aangesien die afname in CD8+ sel getalle korreleer met die verloop tot VIGS. Hierdie deursnit-beskrywende studie het die CD8+ T selle van MIV+ volwasse Suid-Afrikaners, met of sonder`n TB mede-infeksie, ondersoek. STL analise is gedoen op die perifere bloed mono-nuklere selle (PBMS) van pasiente wat geen teen-retrovirale terapie (TRT) ontvang het nie. `n Totaal van sestig Suid-Afrikaanse volwassenes van die Wes-Kaap het deelgeneem aan die studie wat 15 gesonde kontroles; 30 MIV+TBen 15 MIV+TB+ individue ingesluit het. Die uitdrukking van fenotipiese, aktiverings en funksionele merkers is ondersoek deur middel van vloeisitometrie en fluorochroomgekonjugeerde teenliggaampies. Laasgenoemde het ingesluit die nuwe aktiversingsmerker CD 137, die STL geassosieerde merkers Perforien en Gransiem A, CD 107a/b, Fas (CD95) en FasL (CD95L), intrasellulere sitokiene IFN-y en TNF-a en PD-1, die merker vir chroniese MIV CTL disfunksie. Daar is gevind dat `n TB mede-infeksie (in die vorm van aktiewe pulmonere TB) die antigeen-spesifieke STL funksie verlaag en terselftertyd `n verhoging in die uitdrukking van PD-1 en sitolitiese merkers (Fas, FasL, TNF-a) bewerkstellig. Hierdie sitolitiese basislyn merkers is moontlik betrokke by die dood van nie-antigeen-spesifieke omstander teiken selle. Die uitdrukking van die mede-stimulatoriese molekule CD 137 blyk om te korreleer met die produksie van STL IFN-y en die vlakke van degranulasie. Dit bevestig die merker se bruikbaarheid as `n gewaande surrogaat merker vir funksionele reaksies. Die data toon verder dat `n TB mede-infeksie nie net `n effek het op die CD4 T sel funksie nie, dit lei ook tot `n verhoogde basislyn uitdrukking van STLgeassosieerde merkers, maar met disfunksionele antigeen-spesifieke STL reaksies. Hierdie studie het bepaal dat `n MIV infeksie verbind word met `n toename in die basislyn uitdrukking van TNF-a, Perforien, Gransiem A, PD-1, Fas (CD95) en FasL (CD95L). Dit is egter nie die geval wanneer die uitdrukking van CD 137 (4-1BB) of IFN-y vergelyk word met nie-geinfekteerde kontroles. `n TB mede-infeksie het `n verdere toename in die uitdrukking van TNF-a, Perforien, PD-1, FasL (CD95L) getoon, asook `n verhoging in IFN-y vanaf die basislyn. In vitro MIV-1 antigeen (gag)-spesifieke stimulasies het aangedui dat `n MIV infeksie met die antigeen-spesifieke op-regulasie van aktiverings en sitotoksiese merkers CD137, IFN-y, TNF-a, Fas, FasL en CD107a/b geassosieer word. In `n TB mede-infeksie, is `n verlaging van antigeen-spesifieke degranulasie (CD 107a/b op-regulasie) asook die uitdrukking van Fas en FasL waargeneem.

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