Limits...
Lower Pre-Treatment T Cell Activation in Early- and Late-Onset Tuberculosis-Associated Immune Reconstitution Inflammatory Syndrome.

Goovaerts O, Jennes W, Massinga-Loembé M, Ondoa P, Ceulemans A, Vereecken C, Worodria W, Mayanja-Kizza H, Colebunders R, Kestens L, TB-IRIS Study Gro - PLoS ONE (2015)

Bottom Line: CD8+ T cell activation before ART was decreased in both early-onset (77% vs. 82%, p = 0.014) and late-onset (71% vs. 83%, p = 0.012) TB-IRIS patients compared to non-IRIS controls.During late-onset, but not early-onset TB-IRIS, we observed a skewing from memory to terminal effector CD4+ and CD8+ T cell populations (p≤0.028).Our data provide evidence of reduced CD8+ T cell activation before ART as a common predisposing factor of early- and late-onset TB-IRIS.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium; Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium.

ABSTRACT

Background: Tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is an inflammatory complication in HIV-TB co-infected patients receiving antiretroviral therapy (ART). The role of disturbed T cell reconstitution in TB-IRIS is not well understood. We investigated T cell activation and maturation profiles in patients who developed TB-IRIS at different intervals during ART.

Methods: Twenty-two HIV-TB patients who developed early-onset TB-IRIS and 10 who developed late-onset TB-IRIS were matched for age, sex and CD4 count to equal numbers of HIV-TB patients who did not develop TB-IRIS. Flow cytometry analysis was performed on fresh blood, drawn before and after ART initiation and during TB-IRIS events. T cell activation and maturation was measured on CD4+ and CD8+ T cells using CD45RO, CD38, HLA-DR, CCR7 and CD27 antibodies.

Results: CD8+ T cell activation before ART was decreased in both early-onset (77% vs. 82%, p = 0.014) and late-onset (71% vs. 83%, p = 0.012) TB-IRIS patients compared to non-IRIS controls. After ART initiation, the observed differences in T cell activation disappeared. During late-onset, but not early-onset TB-IRIS, we observed a skewing from memory to terminal effector CD4+ and CD8+ T cell populations (p≤0.028).

Conclusion: Our data provide evidence of reduced CD8+ T cell activation before ART as a common predisposing factor of early- and late-onset TB-IRIS. The occurrence of TB-IRIS itself was not marked by an over-activated CD8+ T cell compartment. Late- but not early-onset TB-IRIS was characterized by a more terminally differentiated T cell phenotype.

No MeSH data available.


Related in: MedlinePlus

Gating strategy for CD8+ lymphocytes.These plots represent the gating strategy used for flowcytometry analysis of blood samples; A, lymphocytes were gated on a forward scatter area versus side scatter area dotplot; B, CD8+ bright lymphocytes were gated for further determination of subpopulations; C, CD45RO- and CD45RO+ events were gated to determine naïve and memory subpopulations of CD8+ bright lymphocytes; D, activation of memory CD8+ T cells was determined by determining the percentage of CD38 and HLA-DR double positive CD8+ memory lymphocytes. Maturational T cell subpopulations were identified by analysing the (co-)expression of CCR7 and CD27 within E, naïve and F, memory CD8+ bright lymphocytes.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4514632&req=5

pone.0133924.g001: Gating strategy for CD8+ lymphocytes.These plots represent the gating strategy used for flowcytometry analysis of blood samples; A, lymphocytes were gated on a forward scatter area versus side scatter area dotplot; B, CD8+ bright lymphocytes were gated for further determination of subpopulations; C, CD45RO- and CD45RO+ events were gated to determine naïve and memory subpopulations of CD8+ bright lymphocytes; D, activation of memory CD8+ T cells was determined by determining the percentage of CD38 and HLA-DR double positive CD8+ memory lymphocytes. Maturational T cell subpopulations were identified by analysing the (co-)expression of CCR7 and CD27 within E, naïve and F, memory CD8+ bright lymphocytes.

Mentions: Fresh whole blood was collected and processed locally within 6 hours of collection. Whole blood was stained with fluorescently labelled antibodies, lysed, washed with PBS and fixed with 1% paraformaldehyde in PBS before measuring with a FACSCalibur four-color flow cytometer (Becton Dickinson (BD)). Three antibody panels were used to determine lymphocyte activation and maturation; panel 1: CD45RO-FITC (Dako), CD38-PE (BD), CD8-PerCP (BD), HLA-DR-APC (BD); panel 2: CD45RO-FITC, CCR7-PE (BD-pharmingen), CD8-PerCP, CD27-APC (eBioscience); panel 3: CD45RO-FITC, CCR7-PE, CD4-PerCP (BD), CD27-APC. Data were analysed with FlowJo software (v9.7 Tree Star), using the following gating strategy. First, lymphocytes were gated on a forward scatter area versus side scatter area dotplot (Fig 1). Next, CD4+ bright and CD8+ bright lymphocytes were gated for further determination of subpopulations. Activation of memory CD8+ T cells (panel 1) was determined by gating CD8+ bright lymphocytes for CD45RO+ events and subsequently determining the percentage of CD38 and HLA-DR double positive CD8+ memory lymphocytes (Fig 1). Maturational T cell subpopulations were identified by gating CD8+ bright (panel 2) or CD4+ bright (panel 3) lymphocytes for CD45RO+ and CD45RO- events and subsequently analysing the (co-)expression of CCR7 and CD27, as described previously (Figs 1 and 2) [28,29]. Analysed subpopulations were expressed as frequencies of total CD8+ bright or CD4+ bright lymphocytes and included: naïve (Tn; CD45RO-CCR7+CD27+), central memory (Tcm; CD45RO+CCR7+CD27+), effector memory (Tem; CD45RO+CCR7-CD27+), terminal effector memory (Ttem; CD45RO+CCR7-CD27-), early effector (Tearly eff; CD45RO-CCR7-CD27+) and effector (Teff; CD45RO-CCR7-CD27-) T cells.


Lower Pre-Treatment T Cell Activation in Early- and Late-Onset Tuberculosis-Associated Immune Reconstitution Inflammatory Syndrome.

Goovaerts O, Jennes W, Massinga-Loembé M, Ondoa P, Ceulemans A, Vereecken C, Worodria W, Mayanja-Kizza H, Colebunders R, Kestens L, TB-IRIS Study Gro - PLoS ONE (2015)

Gating strategy for CD8+ lymphocytes.These plots represent the gating strategy used for flowcytometry analysis of blood samples; A, lymphocytes were gated on a forward scatter area versus side scatter area dotplot; B, CD8+ bright lymphocytes were gated for further determination of subpopulations; C, CD45RO- and CD45RO+ events were gated to determine naïve and memory subpopulations of CD8+ bright lymphocytes; D, activation of memory CD8+ T cells was determined by determining the percentage of CD38 and HLA-DR double positive CD8+ memory lymphocytes. Maturational T cell subpopulations were identified by analysing the (co-)expression of CCR7 and CD27 within E, naïve and F, memory CD8+ bright lymphocytes.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4514632&req=5

pone.0133924.g001: Gating strategy for CD8+ lymphocytes.These plots represent the gating strategy used for flowcytometry analysis of blood samples; A, lymphocytes were gated on a forward scatter area versus side scatter area dotplot; B, CD8+ bright lymphocytes were gated for further determination of subpopulations; C, CD45RO- and CD45RO+ events were gated to determine naïve and memory subpopulations of CD8+ bright lymphocytes; D, activation of memory CD8+ T cells was determined by determining the percentage of CD38 and HLA-DR double positive CD8+ memory lymphocytes. Maturational T cell subpopulations were identified by analysing the (co-)expression of CCR7 and CD27 within E, naïve and F, memory CD8+ bright lymphocytes.
Mentions: Fresh whole blood was collected and processed locally within 6 hours of collection. Whole blood was stained with fluorescently labelled antibodies, lysed, washed with PBS and fixed with 1% paraformaldehyde in PBS before measuring with a FACSCalibur four-color flow cytometer (Becton Dickinson (BD)). Three antibody panels were used to determine lymphocyte activation and maturation; panel 1: CD45RO-FITC (Dako), CD38-PE (BD), CD8-PerCP (BD), HLA-DR-APC (BD); panel 2: CD45RO-FITC, CCR7-PE (BD-pharmingen), CD8-PerCP, CD27-APC (eBioscience); panel 3: CD45RO-FITC, CCR7-PE, CD4-PerCP (BD), CD27-APC. Data were analysed with FlowJo software (v9.7 Tree Star), using the following gating strategy. First, lymphocytes were gated on a forward scatter area versus side scatter area dotplot (Fig 1). Next, CD4+ bright and CD8+ bright lymphocytes were gated for further determination of subpopulations. Activation of memory CD8+ T cells (panel 1) was determined by gating CD8+ bright lymphocytes for CD45RO+ events and subsequently determining the percentage of CD38 and HLA-DR double positive CD8+ memory lymphocytes (Fig 1). Maturational T cell subpopulations were identified by gating CD8+ bright (panel 2) or CD4+ bright (panel 3) lymphocytes for CD45RO+ and CD45RO- events and subsequently analysing the (co-)expression of CCR7 and CD27, as described previously (Figs 1 and 2) [28,29]. Analysed subpopulations were expressed as frequencies of total CD8+ bright or CD4+ bright lymphocytes and included: naïve (Tn; CD45RO-CCR7+CD27+), central memory (Tcm; CD45RO+CCR7+CD27+), effector memory (Tem; CD45RO+CCR7-CD27+), terminal effector memory (Ttem; CD45RO+CCR7-CD27-), early effector (Tearly eff; CD45RO-CCR7-CD27+) and effector (Teff; CD45RO-CCR7-CD27-) T cells.

Bottom Line: CD8+ T cell activation before ART was decreased in both early-onset (77% vs. 82%, p = 0.014) and late-onset (71% vs. 83%, p = 0.012) TB-IRIS patients compared to non-IRIS controls.During late-onset, but not early-onset TB-IRIS, we observed a skewing from memory to terminal effector CD4+ and CD8+ T cell populations (p≤0.028).Our data provide evidence of reduced CD8+ T cell activation before ART as a common predisposing factor of early- and late-onset TB-IRIS.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium; Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium.

ABSTRACT

Background: Tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is an inflammatory complication in HIV-TB co-infected patients receiving antiretroviral therapy (ART). The role of disturbed T cell reconstitution in TB-IRIS is not well understood. We investigated T cell activation and maturation profiles in patients who developed TB-IRIS at different intervals during ART.

Methods: Twenty-two HIV-TB patients who developed early-onset TB-IRIS and 10 who developed late-onset TB-IRIS were matched for age, sex and CD4 count to equal numbers of HIV-TB patients who did not develop TB-IRIS. Flow cytometry analysis was performed on fresh blood, drawn before and after ART initiation and during TB-IRIS events. T cell activation and maturation was measured on CD4+ and CD8+ T cells using CD45RO, CD38, HLA-DR, CCR7 and CD27 antibodies.

Results: CD8+ T cell activation before ART was decreased in both early-onset (77% vs. 82%, p = 0.014) and late-onset (71% vs. 83%, p = 0.012) TB-IRIS patients compared to non-IRIS controls. After ART initiation, the observed differences in T cell activation disappeared. During late-onset, but not early-onset TB-IRIS, we observed a skewing from memory to terminal effector CD4+ and CD8+ T cell populations (p≤0.028).

Conclusion: Our data provide evidence of reduced CD8+ T cell activation before ART as a common predisposing factor of early- and late-onset TB-IRIS. The occurrence of TB-IRIS itself was not marked by an over-activated CD8+ T cell compartment. Late- but not early-onset TB-IRIS was characterized by a more terminally differentiated T cell phenotype.

No MeSH data available.


Related in: MedlinePlus