Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016;1(2):260-290.
doi: 10.20411/pai.v1i2.129.

Reconstitution of Peripheral T Cells by Tissue-Derived CCR4+ Central Memory Cells Following HIV-1 Antiretroviral Therapy

Affiliations
Free PMC article

Reconstitution of Peripheral T Cells by Tissue-Derived CCR4+ Central Memory Cells Following HIV-1 Antiretroviral Therapy

Yolanda D Mahnke et al. Pathog Immun. .
Free PMC article

Abstract

Background: Highly active antiretroviral therapy induces clinical benefits to HIV-1 infected individuals, which can be striking in those with progressive disease. Improved survival and decreased incidence of opportunistic infections go hand in hand with a suppression of the plasma viral load, an increase in peripheral CD4+ T-cell counts, as well as a reduction in the activation status of both CD4+ and CD8+ T cells.

Methods: We investigated T-cell dynamics during ART by polychromatic flow cytometry in total as well as in HIV-1-specific CD4+ and CD8+ T cells in patients with advanced disease. We also measured gene expression by single cell transcriptomics to assess functional state.

Results: The cytokine pattern of HIV-specific CD8+ T cells was not altered after ART, though their magnitude decreased significantly as the plasma viral load was suppressed to undetectable levels. Importantly, while CD4+ T cell numbers increased substantially during the first year, the population did not normalize: the increases were largely due to expansion of mucosal-derived CCR4+ CD4+ TCM; transcriptomic analysis revealed that these are not classical Th2-type cells.

Conclusion: The apparent long-term normalization of CD4+ T-cell numbers following ART does not comprise a normal balance of functionally distinct cells, but results in a dramatic Th2 shift of the reconstituting immune system.

Keywords: Cytokines; Immune reconstitution; Polarization; T helper subsets.

Conflict of interest statement

CONFLICTS The authors declare no competing interests.

Figures

Figure 1.
Figure 1.
Longitudinal analysis of HIV-1 Gag-specific cytokine production by T cells and the phenotype of cytokine-producing cells. The effect of ART on HIV-1 Gag-reactive CD4+ (A-E) and CD8+ T cells (F-J) was determined in longitudinal PBMC samples of HIV-1+ patients. (A, F) Total response magnitude, measured by production of IFN-γ, IL-2, or TNF. (B, G) Cytokine pattern. Relative proportion of total HIV-1 Gag-reactive cells producing each possible combination of the cytokines measured. Black arcs indicate all IL-2 (B) or IFN-γ (G) producing cells. (C, H) Actual frequency of cells producing only IFN-γ, IL-2, or TNF, or any combination thereof. Potential phenotypic alterations occurring due to ART were explored in HIV-1 Gag-reactive CD4+ (D, E) and CD8+ T cells (I, J) in longitudinal PBMC samples of HIV-1+ patients. (D, I) Differentiation state. T-cell differentiation subsets of cytokine-positive cells were defined by expression of CD45RO (“RO”), CCR7 (“R7”), and CD27 (“27”). Differentiation indices (DI; medians and interquartile ranges) are indicated below each pie. (E, J) Phenotype. The frequency of cytokine-positive cells expressing differentiation markers (CD7, CD28, CD31, CD57, CD127) or inhibitory receptors (PD-1, TIM-3) was determined. Graphs show interquartile ranges, median bars, as well as individual data points. All time-points were compared to corresponding pre-ART measurements: *P <0 .01, **P < 0.001, ***P < 0.0001.
Figure 2.
Figure 2.
ART-induced change towards less differentiated CD4+ T cells. PBMC were sampled before ART, and after 1, 3, 6, and 12 months of ART. (A) The differentiation pattern was investigated in CD4+ T cells. Subsets were defined by expression of CD45RO (“RO”), CCR7 (“R7”) and CD27 (“27”). TNV–naïve; TCM–central memory; TTM–transitional memory; TEM–effector memory; TTE–terminal effector. TCM*, TTM*, and TTE* are populations not classically discussed in the literature, but arise by this gating scheme; their activation phenotype and cytokine potential most closely resemble that of TCM, TTM, and TTE, respectively, hence their nomenclature. Differentiation indices (DI; medians and interquartile ranges) are indicated. The change in frequency over the course of treatment relative to pre-ART levels (B, D), as well as absolute cell count (C, E) of TNV (B, C) and TCM (D, E), and total frequency of late-differentiation (TEM, TTE*, and TTE) (F) CD4+ T-cells are shown. (G) Pre-ART PVL was plotted against pre-ART late-differentiation (TEM, TTE*, and TTE) CD4+ T cells. Graphs show development in individual patients, as well as medians and interquar-tile ranges. Corresponding interquartile ranges in healthy donors are shown where applicable (orange). All time-points were compared to corresponding pre-ART measurements: *P < 0.01, **P < 0.001, ***P < 0.0001.
Figure 3.
Figure 3.
Reversal of CD4+ and T-cell activation during ART. Phenotypic characteristics of CD4+ T cells were analyzed by polychromatic flow cytometry in PBMC sampled before ART, as well as after 1, 3, 6, and 12 months of ART. (A) T-cell differentiation and subtypes; RTE–recent thymic emigrants. (B) Markers of activation; GrB–Granzyme B. (C) Mean fluorescence intensity of CD38. (D) Inhibitory receptors. Graphs show interquartile ranges, median bars, as well as individual data points. Orange areas represent the inter-quartile ranges of corresponding measurements in healthy individuals. All time-points were compared to corresponding pre-ART measurements: *P < 0.01, **P < 0.001, ***P < 0.0001. i.d.–insufficient data.
Figure 4.
Figure 4.
Early appearance of activated CCR4+ TCM in peripheral blood during ART. PBMC samples taken pre-ART and after one month of ART (mo1) were analyzed to investigate co-expression of CCR4, HLA DR, ICOS, and PD-1 on CD4+ T cells, as well as whether these phenotypes coincide with the TCM subset. (A) Frequency of CCR4+ cells within HLA-DR+, ICOS+, and PD-1+ cells. (B) Frequency of HLA-DR, ICOS, or PD-1 expressing CCR4+ cells. Expression of Th subset-defining chemokine receptors on CCR4+ (C) or non-naïve (D) CD4+ T cells. (E) Differentiation pattern of CCR4+ cells. Differentiation indices (DI; medians and interquartile ranges) are indicated below each pie. (F) Expression of Th subset-defining chemokine receptors on TCM cells. (G) Proportion of CCR4+ TCM and CCR4 TCM over time. (H) Expression of the proliferation marker Ki67 in CCR4+ TCM and CCR4 TCM over time. Bar graphs show interquartile ranges, median bars, as well as individual data points. The interquartile range of given phenotypes (orange areas in bar charts) or average distribution patterns (pie charts) in healthy donors are shown. Mo1 measurements were compared to corresponding pre-ART values: *P < 0.01, **P < 0.001, ***P < 0.0001.
Figure 5.
Figure 5.
The gene expression profile of CCR4+ TCM is different from that of Th2-like cells. PBMC from healthy donors, as well as cells isolated before or after 1 or 12 months of ART from HIV-1-infected adults were stained with the “sorting” panel (Supplementary Table 1). Subsets of CD4+ T cells were sorted as indicated in Supplementary Figure 4 and their gene expression profiles determined by multi-parametric quantitative RT-PCR. (A) Th2-associated and (B) other cytokine genes were compared in Th2-like, CCR4+ TCM, and Th1-like cells isolated from healthy donors. CCR4+ TCM from HIV-1+ patients after 1 month of ART were compared to their counterparts from healthy donors in respect to expression of (C) Th2-associated or (D) other cytokine genes. (E) The expression profile of cytokine genes was investigated in nonnaïve cells. Relative expression in HIV-1-infected individuals before ART, after 1 month of ART, or 1 year of ART was compared to that in healthy donors. (F) The overall gene expression pattern of CCR4+ TCM, CCR4+ TCM, CCR4 TCM, and CCR4 TCM cells was compared to that of Th1- or Th2-like cells sorted from healthy donors. Their calculated “Th-ness” is expressed as a point between those two extremes. Bar graphs show interquartile ranges, median bars, as well as individual data points. Statistically significant differences are indicated: *P < 0.01, **P < 0.001, ***P < 0.0001.
Figure 6.
Figure 6.
CCR4+ TCM appear to be released from peripheral tissue sites upon ART initiation. PBMC from healthy donors, as well as cells isolated before or after 1 or 12 months of ART from HIV-1-infected adults were stained with the “sorting” panel (Supplementary Table 1). Subsets of CD4+ T cells were sorted as indicated in Supplementary Figure 4 and their gene expression profiles determined by multi-parametric quantitative RT-PCR. (A) CD103 expression on Th2-like, CCR4+ TCM, and Th1-like cells isolated after 1 month of antiretroviral therapy. (B) Expression of CD103 in CCR4+ TCM, Th2-, and Th1-like cells from healthy donor PBMC (n = 9), and longitudinal samples from HIV-1 patients (n = 12). Bar graphs show interquartile ranges, median bars, as well as individual data points. Statistically significant differences are indicated: *P < 0.01, **P < 0.001, ***P < 0.0001.
Supplementary Figure 1.
Supplementary Figure 1.
Suppression of viral loads and recovery of T-cell counts on ART. (A) The plasma viral load was determined by measuring HIV-1 RNA at each sampling time-point. The detection threshold was 50 copies/ml (indicated by the broken line), with rare exceptions of 100 or 500 copies/ ml. The CD4/CD8 ratio (B), and number of CD4+ T-cells (C) or CD8+ T-cells/μl (D) in peripheral blood are shown. Bars illustrate median values, while boxes show the inter-quartile range. Healthy ranges are indicated in orange (generated from 288 healthy donor PBMC processed in the testing laboratory). All time-points were compared to corresponding pre-ART measurements: * P < 0.01, ** P < 0.001, *** P < 0.0001.
Supplementary Figure 2.
Supplementary Figure 2.
ART-induced change towards less differentiated CD8+ T cells. PBMC were sampled before, as well as after 1, 3, 6, and 12 months of ART. The distribution of differentiation stages (A) and frequency of individual differentiation subsets (B) was investigated in CD8+ T cells. T-cell differentiation subsets were defined by expression of CD45RO (“RO”), CCR7 (“R7”) and CD27 (“27”). Differentiation indices (DI; medians and interquartile ranges) are indicated next to each pie. The average T-cell differentiation profile as well as the interquartile range of the differentiation indices in healthy donors are shown. (C) Markers of Tcell differentiation. (D) Inhibitory receptors. (E) Markers of activation; GrB– Granzyme B. (F) Mean fluorescence intensity of CD38. Graphs show interquartile ranges, median bars, as well as individual data points. Orange areas represent the interquartile ranges of corresponding measurements in healthy individuals. All timepoints were compared to corresponding pre-ART measurements: * P < 0.01, ** P < 0.001, *** P < 0.0001.
Supplementary Figure 3.
Supplementary Figure 3.
Chemokine receptor staining and definition of Th subsets. Gates for the expression of CCR4, CCR6, CCR10, and CXCR3 were defined within total CD4+ T cells. Th subsets were identified by the resulting co-expression pattern, following the gating scheme published in OMIP-017[57].
Supplementary Figure 4.
Supplementary Figure 4.
T-cell subsets sorted for gene expression analysis. PBMC were stimulated with SEB for 3 hours, before sorting seven CD4+ T-cell subsets from SEB-reactive cells as indicated in this gating scheme. After identifying live CD3+ cells within singlet, aggregate-negative lymphocytes, non-naive CD4+ T cells were selected by excluding CD45RO CCR7+ cells. Within these, T cells expressing TCRVβ12, -Vβ14 or -Vβ17 are known to react to SEB, so FITC-conjugated Abs were used for these three TCR-Vβ chains. Within such SEB-reactive cells, CCR4+ and CCR4− cells were selected and individually gated for TCM (CD45RO+ CCR7+ CD27+) or TCM subsets. Further, total SEB-reactive CD4+ T cells, as well as stringently gated Th1- (CCR4, CCR6, CCR10, CXCR3+) and Th2-like cells (CCR4+ CCR6 CCR10 CXCR3) were also sorted.
Supplementary Figure 5.
Supplementary Figure 5.
CCR4+ TCM resemble Th2-like cells in respect to their expression of Th1 associated genes. PBMC from healthy donors, as well as cells isolated before or after 1 or 12 months of ART from HIV-1-infected adults were stained with the “sorting” panel (Supplementary Table 1). Subsets of CD4+ T cells were sorted as indicated in Supplementary Figure 4 and their gene expression profiles determined by multi-parametric quantitative RT-PCR. (A) Th1-associated genes were compared in Th2-like, CCR4+ TCM, and Th1-like cells isolated from healthy donors. (B) CCR4+ TCM from HIV-1+ patients after 1 month of ART were compared to their counterparts from healthy donors in respect to expression of Th1-associated genes. Bar graphs show interquartile ranges, median bars, as well as individual data points. Statistically significant differences are indicated: * P < 0.01, ** P < 0.001, *** P < 0.0001.
Supplementary Figure 6.
Supplementary Figure 6.
Migration marker expression on CD4+ T cells. The expression of migration markers on the cell surface of CD4+ T cells was investigated by flow cytometry. (A) Dot plots show the expression of CD103, CCR9, integrin β7, CD49d, and CD11a on CD4+ T-cells from two different patients after 1 month of ART. (B) CD103 and CCR9 expression pre-ART and after 1 month of ART compared to healthy donors. (C) CD49d, CD11a, and integrin β7 expression pre-ART and after 1 month of ART compared to healthy donors. Bar graphs show interquartile ranges, median bars, as well as individual data points. Due to the small sample size, no statistical comparison was performed.

Similar articles

See all similar articles

Cited by 4 articles

References

    1. Autran B, Carcelain G, Li TS, Blanc C, Mathez D, Tubiana R, Katlama C, Debre P, Leibowitch J. Positive effects of combined antiretroviral therapy on CD4+ T cell homeostasis and function in advanced HIV disease. Science. 1997;277(5322):112–6. PubMed PMID: 9204894. - PubMed
    1. Guihot A, Bourgarit A, Carcelain G, Autran B. Immune reconstitution after a decade of combined antiretroviral therapies for human immunodeficiency virus. Trends Immunol. 2011;32(3):131–7. PubMed PMID: 21317040. doi: 10.1016/j.it.2010.12.002 - DOI - PubMed
    1. Pakker NG, Notermans DW, de Boer RJ, Roos MT, de Wolf F, Hill A, Leonard JM, Danner SA, Miedema F, Schellekens PT. Biphasic kinetics of peripheral blood T cells after triple combination therapy in HIV-1 infection: a composite of redistribution and proliferation. Nat Med. 1998;4(2):208–14. PubMed PMID: 9461195. - PubMed
    1. Bucy RP, Hockett RD, Derdeyn CA, Saag MS, Squires K, Sillers M, Mitsuyasu RT, Kilby JM. Initial increase in blood CD4(+) lymphocytes after HIV antiretroviral therapy reflects redistribution from lymphoid tissues. J Clin Invest. 1999;103(10):1391–8. PubMed PMID: 10330421. Pubmed Central PMCID: 408455. doi: 10.1172/JCI5863 - DOI - PMC - PubMed
    1. Steffens CM, Smith KY, Landay A, Shott S, Truckenbrod A, Russert M, Al-Harthi L. T cell receptor excision circle (TREC) content following maximum HIV suppression is equivalent in HIV-infected and HIV-uninfected individuals. AIDS. 2001;15(14):1757–64. PubMed PMID: 11579236. - PubMed

LinkOut - more resources

Feedback