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Characterisation of the CD161+ CD4+ T cell population in HIV and MTB infected individuals.

Human Immunodeficiency Virus (HIV) infection is characterized by immune dysfunction

that predisposes infected individuals to opportunistic infections such as Mycobacterium

Tuberculosis (MTB). The result of this is an exacerbation of HIV-TB related deaths

annually. Therefore there is an imperative need for HIV-TB focused research that aims to

identify immunological factors that are involved in the control of MTB and HIV in both

mono- and co-infected individuals. The CD161+ CD4+ T cell subset is linked to a distinct

phenotypic and functional profile. Importantly, these CD161+ T cells may act as an

important component of immunological defense and provide protection in infected tissues.

CD161+ CD4+ T cells have also been identified as the precursor population of Th17 cells

and it has been previously reported that reduction of CD161+ CD4+ T cells during HIV

infection may limit Th17 reconstitution (Prendergast et al., 2010). This may ultimately

contribute to impairment of mucosal immunity leading to the acquisition of opportunistic

infections such as MTB and disease progression in HIV infected individuals. Our study

aimed to comprehensively characterise the impact of HIV and MTB infection on the

CD161+ CD4+ T cell subset and to assess the frequency, phenotype and function of these

cells. The study also aimed to correlate the longitudinal variation in frequency, phenotype

and function with markers of HIV disease progression.

Methods

The frequency, phenotype and function of the CD161+ CD4+ T cell subset was measured

by flow cytometry. For the frequency and phenotypic assessment, whole blood was

collected from HIV negative and HIV/MTB mono and co-infected subjects (n = 17 per

patient group). Whole blood was surface stained with antibodies specific to CD3, CD4,

CD8, CD161 and chemokine receptors CD103, CCR6, CXCR4, CCR5 and CXCR6. The

percentage positive expression of CD161 on CD4+ T cells and chemokine receptor

expression was measured. The functional assessment of CD161+ CD4+ T cells involved

PBMC stimulation with antigenic stimulant, phorbol 12-myristate 13-acetate (PMA) and

ionomycin or ESAT-6/CFP-10, GAG, TB10.4 and Ag85a followed by intracellular

cytokine staining for IFN-γ, IL-17A, IL-22 and TNF-α. A subgroup of HIV negative

(frequency and phenotype, n = 10, function n = 7) and HIV mono-infected subjects

(frequency and phenotype, n = 10, function n = 7) were longitudinally followed to assess

variations in the frequency, phenotype and function of CD161+ CD4+ T cells over time.

Results

The CD161+ CD4+ T cell subset demonstrated high-level expression of chemokine

receptors CCR5, CCR6, CXCR4 and low-level expression of CD103 and CXCR6. The

subset also demonstrated the ability to produce cytokines IFN-γ, IL17A, IL-22 and TNF-α

in healthy subjects. Analysis of HIV infected samples revealed a significant reduction in

the frequency of the CD161+ CD4+ subset (median = 06.86%, p < 0.0001) compared to

that of healthy individuals (median = 14.75%). Correlation of the subset frequency to

markers of disease progression revealed a positive trend to CD4 count (r = 0.2590,

p = 0.0787) and a significant negative correlation to viral load (r = -0.3522, p = 0.0152).

Unlike with HIV infection, no significant changes in CD161+ CD4+ T cell frequency was

observed in individuals with LTBI (mono- or HIV co-infected) or active TB disease

compared to that of the healthy patient group. However, the exception to this was HIV

infected individuals with active TB disease (co-infected) (median = 03.80%, p < 0.0001).

Decreased CCR6 expression on CD161+ CD4+ T cells was observed in HIV monoinfected

(p = 0.0065) and HIV infected individuals with active TB disease (p = 0.007). No

functional changes were observed in both the HIV and MTB mono- and co-infected

cohorts following non-specific stimulation. An interesting positive trend in correlation

between IFN-γ production and CD4 count (r = 0.2727, p = 0.0733) was demonstrated with

a significant negative correlation between IFN-γ production and viral load observed

following non-specific antigenic stimulation (r = -0.3705, p = 0.0133). CD161+ CD4+ T

cells demonstrated antigen-specific T cell responses to peptides ESAT-6/CFP-10, TB10.4,

Ag85a and GAG in a small proportion of 69 study participants with variable ranges in

magnitude of the responses observed. The longitudinal assessment of CD161+ CD4+ T

cell frequency and phenotype demonstrated low-level proportion of CD4+ T cells

expressing CD161 and CCR6 expression longitudinally maintained in HIV mono-infected

compared to that of healthy individuals.

Conclusion

The phenotypic and functional profile of the CD161+ CD4+ T cell population indicates

that it may be an important component of immunological defense that may provide

mucosal defense and protection at epithelial sites and tissues e.g. expression of tissue

homing markers like CCR6 and the production of cytokines such as IL-17A and IL-22

(important in mucosal immunity). HIV infection is associated with a reduced frequency of

CD161+ CD4+ T cells. The correlation between CD161+ CD4+ T cell frequency and

markers of disease progression suggests that the observed low-level frequency in HIV

infected individuals may in part be a result of non-specific HIV-mediated depletion of

CD4+ T cells. However, lower levels of CD161+ CD4+ T cells in HIV infected individuals

could also be a result of naturally lower levels being present in individuals prior to

infection, thereby making these individuals more susceptible to HIV infection. The

significantly reduced levels of CCR6 expression on CD161+ CD4+ T cells in HIV monoinfected

individuals may also be an indication of cell subset migration to gut associated

lymphoid tissue (GALT, target site of HIV replication) during HIV infection. Given their

potential role in mediating signals that are essential for immune responses to microbes and

microbial products, migration of CCR6+ CD161+ CD4+ T cells to target sites of HIV

infection could serve as a protective measure in the fight against HIV infection. Although

there were no observable changes in the functional capacity of the CD161+ CD4+ T subset

in HIV infection, we believe that the reduction in frequency may contribute to HIV disease

progression and susceptibility to opportunistic infections such as MTB or active TB

disease. Unlike with HIV infection, infection with MTB appeared to have no significant

impact on CD161+ CD4+ T cells as there were no observable differences in frequency or

the functional capacity of the cell subset following PMA stimulation. However, MTB and

HIV antigen-specific responses were observed in a small proportion of the total 69 subjects

tested. This therefore indicates that a subset of CD161+ CD4+ T cells may act in an HIV

and MTB-specific manner. Additional MTB and HIV-specific responses may be present in

this CD161+ CD4+ population and may only be identified through stimulation with

additional antigenic targets.

Further investigation of CD161+ CD4+ T cells should be performed at the actual sites of

infection to investigate if CD161+ CD4+ T cells are concentrated at sites of disease. Also

it may be important to investigate the polyfunctionality of CD161+ CD4+ T cells to

understand the multifunctional capacity of the cell subset in providing immunological

defense to pathogens such as HIV and MTB. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2012.

Identiferoai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ukzn/oai:http://researchspace.ukzn.ac.za:10413/9642
Date January 2012
CreatorsGovender, Pamla.
ContributorsKasprowicz, Victoria.
Source SetsSouth African National ETD Portal
Languageen_ZA
Detected LanguageEnglish
TypeThesis

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