High expression of CD161 on CD8+ T cells is associated with a population of cells thought to play a role in mucosal immunity. We wished to investigate this subset in an HIV and Mycobacterium ...tuberculosis (MTB) endemic African setting.
A flow cytometric approach was used to assess the frequency and phenotype of CD161++CD8+ T cells. 80 individuals were recruited for cross-sectional analysis: controls (n = 18), latent MTB infection (LTBI) only (n = 16), pulmonary tuberculosis (TB) only (n = 8), HIV only (n = 13), HIV and LTBI co-infection (n = 15) and HIV and TB co-infection (n = 10). The impact of acute HIV infection was assessed in 5 individuals recruited within 3 weeks of infection. The frequency of CD161++CD8+ T cells was assessed prior to and during antiretroviral therapy (ART) in 14 HIV-positive patients.
CD161++CD8+ T cells expressed high levels of the HIV co-receptor CCR5, the tissue-homing marker CCR6, and the Mucosal-Associated Invariant T (MAIT) cell TCR Vα7.2. Acute and chronic HIV were associated with lower frequencies of CD161++CD8+ T cells, which did not correlate with CD4 count or HIV viral load. ART was not associated with an increase in CD161++CD8+ T cell frequency. There was a trend towards lower levels of CD161++CD8+ T cells in HIV-negative individuals with active and latent TB. In those co-infected with HIV and TB, CD161++CD8+ T cells were found at low levels similar to those seen in HIV mono-infection.
The frequencies and phenotype of CD161++CD8+ T cells in this South African cohort are comparable to those published in European and US cohorts. Low-levels of this population were associated with acute and chronic HIV infection. Lower levels of the tissue-trophic CD161++ CD8+ T cell population may contribute to weakened mucosal immune defense, making HIV-infected subjects more susceptible to pulmonary and gastrointestinal infections and detrimentally impacting on host defense against TB.
The ability of antigen-specific T cells to simultaneously produce multiple cytokines is thought to correlate with the functional capacity and efficacy of T cells. These 'polyfunctional' T cells have ...been associated with control of HIV. We aimed to assess the impact of co-infection with Mycobacterium tuberculosis (MTB) on HIV-specific CD8+ and CD4+ T cell function. We assessed T cell functionality in 34 South African adults by investigating the IFN-y, IL-2, TNF-α, IL-21 and IL-17 cytokine secretion capacity, using polychromatic flow cytometry, following HIV Gag-specific stimulation of peripheral blood mononuclear cells. We show that MTB is associated with lower HIV-specific T cell function in co-infected as compared to HIV mono-infected individuals. This decline in function was greatest in co-infection with active Tuberculosis (TB) compared to co-infection with latent MTB (LTBI), suggesting that mycobacterial load may contribute to this loss of function. The described impact of MTB on HIV-specific T cell function may be a mechanism for increased HIV disease progression in co-infected subjects as functionally impaired T cells may be less able to control HIV.
Here we describe the development and validation of a highly sensitive assay of antigen-specific IFN-γ production using real time quantitative PCR (qPCR) for two reporters--monokine-induced by IFN-γ ...(MIG) and the IFN-γ inducible protein-10 (IP10). We developed and validated the assay and applied it to the detection of CMV, HIV and Mycobacterium tuberculosis (MTB) specific responses, in a cohort of HIV co-infected patients. We compared the sensitivity of this assay to that of the ex vivo RD1 (ESAT-6 and CFP-10)-specific IFN-γ Elispot assay. We observed a clear quantitative correlation between the two assays (P<0.001). Our assay proved to be a sensitive assay for the detection of MTB-specific T cells, could be performed on whole blood samples of fingerprick (50 uL) volumes, and was not affected by HIV-mediated immunosuppression. This assay platform is potentially of utility in diagnosis of infection in this and other clinical settings.
Monitoring of latent Mycobacterium tuberculosis infection may prevent disease. We tested an ESAT-6 and CFP-10-specific IFN-γ Elispot assay (RD1-Elispot) on 163 HIV-infected individuals living in a ...TB-endemic setting. An RD1-Elispot was performed every 3 months for a period of 3-21 months. 62% of RD1-Elispot negative individuals were positive by cultured Elispot. Fluctuations in T cell response were observed with rates of change ranging from -150 to +153 spot-forming cells (SFC)/200,000 PBMC in a 3-month period. To validate these responses we used an RD1-specific real time quantitative PCR assay for monokine-induced by IFN-γ (MIG) and IFN-γ inducible protein-10 (IP10) (MIG: r=0.6527, p=0.0114; IP-10: r=0.6967, p=0.0056; IP-10+MIG: r=0.7055, p=0.0048). During follow-up 30 individuals were placed on ARVs and 4 progressed to active TB. Fluctuations in SFC did not correlate with CD4 count, viral load, treatment initiation, or progression to active TB. The RD1-Elispot appears to have limited value in this setting.
This study aimed to assess how Mycobacterium tuberculosis (MTB) coinfection alters the impact of interleukin-10 in chronic HIV infection.
We assessed plasma cytokine levels (interleukin-10, ...interferon-γ, tumor necrosis factor-α, interleukin-2, interleukin-6 and interleukin-13) in 82 individuals presenting with HIV monoinfection, HIV-LTBI (latent MTB infection) coinfection or HIV-TB (active tuberculosis) coinfection. We also assessed the influence of MTB on the functional impact of interleukin-10 receptor alpha (interleukin-10Rα) blockade on HIV and MTB-specific CD4(+) T cells.
Plasma cytokine levels were measured by high sensitivity Luminex. We used an ex-vivo interleukin-10Rα blockade assay to assess if functional enhancement of HIV and MTB-specific CD4(+) T cells was possible following a 48-h stimulation with HIV gag or pooled ESAT-6 (6 kDa early secretory antigenic target) and CFP-10 (10-kDa culture filtrate protein) peptides. Cell supernatant was collected 48 h after stimulation and the cytokine profile was measured by Luminex.
Plasma interleukin-10 levels were elevated in HIV-TB as compared with HIV monoinfection (P < 0.05) and HIV-LTBI (P < 0.05). Plasma interleukin-10 levels correlated to HIV viral load in HIV monoinfection (P = 0.016) and HIV-LTBI (P = 0.042), but not HIV-TB. Ex-vivo blockade of interleukin-10Rα significantly enhanced MTB and HIV-specific CD4(+) T-cell function in HIV-LTBI individuals but not in HIV-TB individuals.
Tuberculosis disrupts the correlation between interleukin-10 and markers of HIV disease progression. In addition, HIV-TB is associated with a more inflammatory cytokine milieu compared with HIV monoinfection. Interestingly, interleukin-10Rα blockade can enhance both HIV and MTB-specific T-cell function in HIV-LTBI, but not in HIV-TB coinfection.
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.
High expression of CD161 on CD8+ T cells is associated with a population of cells thought to play a role in mucosal immunity. We wished to investigate this subset in an HIV and Mycobacterium ...tuberculosis (MTB) endemic African setting. A flow cytometric approach was used to assess the frequency and phenotype of CD161++CD8+ T cells. 80 individuals were recruited for cross-sectional analysis: controls (n = 13), latent MTB infection (LTBI) only (n = 14), pulmonary tuberculosis (TB) only (n = 9), HIV only (n = 16), HIV and LTBI co-infection (n = 13) and HIV and TB co-infection (n = 15). The impact of acute HIV infection was assessed in 5 individuals recruited within 3 weeks of infection. The frequency of CD161++CD8+ T cells was assessed prior to and during antiretroviral therapy (ART) in 14 HIV-positive patients. CD161++CD8+ T cells expressed high levels of the HIV co-receptor CCR5, the tissue-homing marker CCR6, and the Mucosal-Associated Invariant T (MAIT) cell TCR Valpha7.2. Acute and chronic HIV were associated with lower frequencies of CD161++CD8+ T cells, which did not correlate with CD4 count or HIV viral load. ART was not associated with an increase in CD161++CD8+ T cell frequency. There was a trend towards lower levels of CD161++CD8+ T cells in HIV-negative individuals with active and latent TB. In those co-infected with HIV and TB, CD161++CD8+ T cells were found at low levels similar to those seen in HIV mono-infection. The frequencies and phenotype of CD161++CD8+ T cells in this South African cohort are comparable to those published in European and US cohorts. Low-levels of this population were associated with acute and chronic HIV infection. Lower levels of the tissue-trophic CD161++ CD8+ T cell population may contribute to weakened mucosal immune defense, making HIV-infected subjects more susceptible to pulmonary and gastrointestinal infections and detrimentally impacting on host defense against TB.
Background The continued advance of antibiotic resistance threatens the treatment and control of many infectious diseases. This is exemplified by the largest global outbreak of extensively ...drug-resistant (XDR) tuberculosis (TB) identified in Tugela Ferry, KwaZulu-Natal, South Africa, in 2005 that continues today. It is unclear whether the emergence of XDR-TB in KwaZulu-Natal was due to recent inadequacies in TB control in conjunction with HIV or other factors. Understanding the origins of drug resistance in this fatal outbreak of XDR will inform the control and prevention of drug-resistant TB in other settings. In this study, we used whole genome sequencing and dating analysis to determine if XDR-TB had emerged recently or had ancient antecedents. Methods and Findings We performed whole genome sequencing and drug susceptibility testing on 337 clinical isolates of Mycobacterium tuberculosis collected in KwaZulu-Natal from 2008 to 2013, in addition to three historical isolates, collected from patients in the same province and including an isolate from the 2005 Tugela Ferry XDR outbreak, a multidrug-resistant (MDR) isolate from 1994, and a pansusceptible isolate from 1995. We utilized an array of whole genome comparative techniques to assess the relatedness among strains, to establish the order of acquisition of drug resistance mutations, including the timing of acquisitions leading to XDR-TB in the LAM4 spoligotype, and to calculate the number of independent evolutionary emergences of MDR and XDR. Our sequencing and analysis revealed a 50-member clone of XDR M. tuberculosis that was highly related to the Tugela Ferry XDR outbreak strain. We estimated that mutations conferring isoniazid and streptomycin resistance in this clone were acquired 50 y prior to the Tugela Ferry outbreak (katG S315T isoniazid; gidB 130 bp deletion streptomycin; 1957 95% highest posterior density (HPD): 1937-1971), with the subsequent emergence of MDR and XDR occurring 20 y (rpoB L452P rifampicin; pncA 1 bp insertion pyrazinamide; 1984 95% HPD: 1974-1992) and 10 y (rpoB D435G rifampicin; rrs 1400 kanamycin; gyrA A90V ofloxacin; 1995 95% HPD: 1988-1999) prior to the outbreak, respectively. We observed frequent de novo evolution of MDR and XDR, with 56 and nine independent evolutionary events, respectively. Isoniazid resistance evolved before rifampicin resistance 46 times, whereas rifampicin resistance evolved prior to isoniazid only twice. We identified additional putative compensatory mutations to rifampicin in this dataset. One major limitation of this study is that the conclusions with respect to ordering and timing of acquisition of mutations may not represent universal patterns of drug resistance emergence in other areas of the globe. Conclusions In the first whole genome-based analysis of the emergence of drug resistance among clinical isolates of M. tuberculosis, we show that the ancestral precursor of the LAM4 XDR outbreak strain in Tugela Ferry gained mutations to first-line drugs at the beginning of the antibiotic era. Subsequent accumulation of stepwise resistance mutations, occurring over decades and prior to the explosion of HIV in this region, yielded MDR and XDR, permitting the emergence of compensatory mutations. Our results suggest that drug-resistant strains circulating today reflect not only vulnerabilities of current TB control efforts but also those that date back 50 y. In drug-resistant TB, isoniazid resistance was overwhelmingly the initial resistance mutation to be acquired, which would not be detected by current rapid molecular diagnostics employed in South Africa that assess only rifampicin resistance.
The ability of antigen-specific T cells to simultaneously produce multiple cytokines is thought to correlate with the functional capacity and efficacy of T cells. These 'polyfunctional' T cells have ...been associated with control of HIV. We aimed to assess the impact of co-infection with Mycobacterium tuberculosis (MTB) on HIV-specific CD8+ and CD4+ T cell function. We assessed T cell functionality in 34 South African adults by investigating the IFN-y, IL-2, TNF- alpha , IL-21 and IL-17 cytokine secretion capacity, using polychromatic flow cytometry, following HIV Gag-specific stimulation of peripheral blood mononuclear cells. We show that MTB is associated with lower HIV-specific T cell function in co-infected as compared to HIV mono-infected individuals. This decline in function was greatest in co-infection with active Tuberculosis (TB) compared to co-infection with latent MTB (LTBI), suggesting that mycobacterial load may contribute to this loss of function. The described impact of MTB on HIV-specific T cell function may be a mechanism for increased HIV disease progression in co-infected subjects as functionally impaired T cells may be less able to control HIV.
Background High expression of CD161 on CD8+ T cells is associated with a population of cells thought to play a role in mucosal immunity. We wished to investigate this subset in an HIV and ...Mycobacterium tuberculosis (MTB) endemic African setting. Methods A flow cytometric approach was used to assess the frequency and phenotype of CD161++CD8+ T cells. 80 individuals were recruited for cross-sectional analysis: controls (n = 18), latent MTB infection (LTBI) only (n = 16), pulmonary tuberculosis (TB) only (n = 8), HIV only (n = 13), HIV and LTBI co-infection (n = 15) and HIV and TB co-infection (n = 10). The impact of acute HIV infection was assessed in 5 individuals recruited within 3 weeks of infection. The frequency of CD161++CD8+ T cells was assessed prior to and during antiretroviral therapy (ART) in 14 HIV-positive patients. Results CD161++CD8+ T cells expressed high levels of the HIV co-receptor CCR5, the tissue-homing marker CCR6, and the Mucosal-Associated Invariant T (MAIT) cell TCR V alpha 7.2. Acute and chronic HIV were associated with lower frequencies of CD161++CD8+ T cells, which did not correlate with CD4 count or HIV viral load. ART was not associated with an increase in CD161++CD8+ T cell frequency. There was a trend towards lower levels of CD161++CD8+ T cells in HIV-negative individuals with active and latent TB. In those co-infected with HIV and TB, CD161++CD8+ T cells were found at low levels similar to those seen in HIV mono-infection. Conclusions The frequencies and phenotype of CD161++CD8+ T cells in this South African cohort are comparable to those published in European and US cohorts. Low-levels of this population were associated with acute and chronic HIV infection. Lower levels of the tissue-trophic CD161++ CD8+ T cell population may contribute to weakened mucosal immune defense, making HIV-infected subjects more susceptible to pulmonary and gastrointestinal infections and detrimentally impacting on host defense against TB.
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