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A transcriptome-based model of central memory CD4 T cell death in HIV infection

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ABSTRACT

Background: Human central memory CD4 T cells are characterized by their capacity of proliferation and differentiation into effector memory CD4 T cells. Homeostasis of central memory CD4 T cells is considered a key factor sustaining the asymptomatic stage of Human Immunodeficiency Virus type 1 (HIV-1) infection, while progression to acquired immunodeficiency syndrome is imputed to central memory CD4 T cells homeostatic failure. We investigated if central memory CD4 T cells from patients with HIV-1 infection have a gene expression profile impeding proliferation and survival, despite their activated state.

Methods: Using gene expression microarrays, we analyzed mRNA expression patterns in naive, central memory, and effector memory CD4 T cells from healthy controls, and naive and central memory CD4 T cells from patients with HIV-1 infection. Differentially expressed genes, defined by Log2 Fold Change (FC) ≥ /0.5/ and Log (odds) > 0, were used in pathway enrichment analyses.

Results: Central memory CD4 T cells from patients and controls showed comparable expression of differentiation-related genes, ruling out an effector-like differentiation of central memory CD4 T cells in HIV infection. However, 210 genes were differentially expressed in central memory CD4 T cells from patients compared with those from controls. Expression of 75 of these genes was validated by semi quantitative RT-PCR, and independently reproduced enrichment results from this gene expression signature. The results of functional enrichment analysis indicated movement to cell cycle phases G1 and S (increased CCNE1, MKI67, IL12RB2, ADAM9, decreased FGF9, etc.), but also arrest in G2/M (increased CHK1, RBBP8, KIF11, etc.). Unexpectedly, the results also suggested decreased apoptosis (increased CSTA, NFKBIA, decreased RNASEL, etc.). Results also suggested increased IL-1β, IFN-γ, TNF, and RANTES (CCR5) activity upstream of the central memory CD4 T cells signature, consistent with the demonstrated milieu in HIV infection.

Conclusions: Our findings support a model where progressive loss of central memory CD4 T cells in chronic HIV-1 infection is driven by increased cell cycle entry followed by mitotic arrest, leading to a non-apoptotic death pathway without actual proliferation, possibly contributing to increased turnover.

Electronic supplementary material: The online version of this article (doi:10.1186/s12864-016-3308-8) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

Unique TCM cell signature in HIV infection. Differential expression was defined as Log2 of fold change (Log FC) ≥ /0.5/, and Log (odds) > 0. a Pairwise comparisons of samples of CD4 T cells subpopulations from HIV+ and HIV¯ groups indicated by arrows a and d. Number of genes differentially expressed in each comparison are shown. Blue circles, controls’ samples; red squares, HIV+ patients’ samples. b Venn diagram of sets of differentially expressed genes. Each pairwise comparison is depicted by a colored oval. The number of differentially expressed genes found in more than one comparison appear in the intersections. c Heat map displaying a two-way unsupervised hierarchical clustering of 210 differentially expressed gens distinguishing HIV+ patients’ TCM cells (red bar) and controls’ TCM cells (blue bar), grouped in dendrograms. Each column represents an independent sample (biological replica) of each subpopulation, numbered 1 to 3. Each row corresponds to a differentially expressed gene
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Fig2: Unique TCM cell signature in HIV infection. Differential expression was defined as Log2 of fold change (Log FC) ≥ /0.5/, and Log (odds) > 0. a Pairwise comparisons of samples of CD4 T cells subpopulations from HIV+ and HIV¯ groups indicated by arrows a and d. Number of genes differentially expressed in each comparison are shown. Blue circles, controls’ samples; red squares, HIV+ patients’ samples. b Venn diagram of sets of differentially expressed genes. Each pairwise comparison is depicted by a colored oval. The number of differentially expressed genes found in more than one comparison appear in the intersections. c Heat map displaying a two-way unsupervised hierarchical clustering of 210 differentially expressed gens distinguishing HIV+ patients’ TCM cells (red bar) and controls’ TCM cells (blue bar), grouped in dendrograms. Each column represents an independent sample (biological replica) of each subpopulation, numbered 1 to 3. Each row corresponds to a differentially expressed gene

Mentions: We asked if differential gene expression by patients’ and controls’ TCM cells reflected greater differentiation of patients’ cells (towards effector stages) [23, 24]. Using the criteria defined in methods (Log2FC ≥ /0.5/ and Log (odds) > 0) we looked in the whole transcriptome for all differentially expressed genes in the following pair-wise comparisons of CD4 T cell subpopulations from controls: TCM vs. TN, TEM vs. TCM, and TEM vs. TN (arrows a, b and c in Fig. 2a). The resulting 1858 differentially expressed genes are subsequently referred to as differentiation-related genes (corresponding to subpopulations in distinct stages of differentiation). We performed an unsupervised 2-way hierarchical clustering analysis of these 1858 differentiation-related genes (Fig. 1b, and Additional file 3). TN and TCM cells from patients grouped with their counterparts from controls (Fig. 1b). Samples of a same subpopulation were assigned to a same node (green circles 1, 2, and 3 on Fig. 1b), regardless of their HIV status. The expression of the differentiation-related genes progressively decreased or increased in the order of linear differentiation (TN → TCM → TEM ), agreeing with previous reports [40–43] (Fig. 1c). For instance, LEF1, ACTN1, FOXP1, IL6ST and CERS6 reportedly undergoing down-regulation in naive T cells after antigen recognition and differentiation [44–48], along with TAF4B, appeared progressively down regulated when samples were ordered according to the linear model of peripheral differentiation (Fig. 1c, left panel). These changes agree with previous reports [42]. Conversely, differentiation and effector function-associated transcripts, like EOMES, TBX21 (t-bet), PRDM1 (Blimp-1) [49, 50], GZMA and PRF1 [51, 52], were gradually increased in the same order (Fig. 1c right panel). A same pattern was followed by the expression of KLRG1,an indicator of replicative senescence [53, 54] (Fig. 1c). TBX21 (t-bet) was the only gene with increased expression in TCM cells from patients, compared with controls (p = 0.003), which, along with the increased expression of IL12R e IL18R, suggests a Th1-skewed response driven by HIV infection. A Th1-skewed response was also predicted by Ingenuity Canonical Pathway analysis (See Additional file 4). Thus, TCM cells from patients did not seem to be more differentiated than their counterparts from controls, but appeared polarized to Th1 functions.Fig. 2


A transcriptome-based model of central memory CD4 T cell death in HIV infection
Unique TCM cell signature in HIV infection. Differential expression was defined as Log2 of fold change (Log FC) ≥ /0.5/, and Log (odds) > 0. a Pairwise comparisons of samples of CD4 T cells subpopulations from HIV+ and HIV¯ groups indicated by arrows a and d. Number of genes differentially expressed in each comparison are shown. Blue circles, controls’ samples; red squares, HIV+ patients’ samples. b Venn diagram of sets of differentially expressed genes. Each pairwise comparison is depicted by a colored oval. The number of differentially expressed genes found in more than one comparison appear in the intersections. c Heat map displaying a two-way unsupervised hierarchical clustering of 210 differentially expressed gens distinguishing HIV+ patients’ TCM cells (red bar) and controls’ TCM cells (blue bar), grouped in dendrograms. Each column represents an independent sample (biological replica) of each subpopulation, numbered 1 to 3. Each row corresponds to a differentially expressed gene
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Fig2: Unique TCM cell signature in HIV infection. Differential expression was defined as Log2 of fold change (Log FC) ≥ /0.5/, and Log (odds) > 0. a Pairwise comparisons of samples of CD4 T cells subpopulations from HIV+ and HIV¯ groups indicated by arrows a and d. Number of genes differentially expressed in each comparison are shown. Blue circles, controls’ samples; red squares, HIV+ patients’ samples. b Venn diagram of sets of differentially expressed genes. Each pairwise comparison is depicted by a colored oval. The number of differentially expressed genes found in more than one comparison appear in the intersections. c Heat map displaying a two-way unsupervised hierarchical clustering of 210 differentially expressed gens distinguishing HIV+ patients’ TCM cells (red bar) and controls’ TCM cells (blue bar), grouped in dendrograms. Each column represents an independent sample (biological replica) of each subpopulation, numbered 1 to 3. Each row corresponds to a differentially expressed gene
Mentions: We asked if differential gene expression by patients’ and controls’ TCM cells reflected greater differentiation of patients’ cells (towards effector stages) [23, 24]. Using the criteria defined in methods (Log2FC ≥ /0.5/ and Log (odds) > 0) we looked in the whole transcriptome for all differentially expressed genes in the following pair-wise comparisons of CD4 T cell subpopulations from controls: TCM vs. TN, TEM vs. TCM, and TEM vs. TN (arrows a, b and c in Fig. 2a). The resulting 1858 differentially expressed genes are subsequently referred to as differentiation-related genes (corresponding to subpopulations in distinct stages of differentiation). We performed an unsupervised 2-way hierarchical clustering analysis of these 1858 differentiation-related genes (Fig. 1b, and Additional file 3). TN and TCM cells from patients grouped with their counterparts from controls (Fig. 1b). Samples of a same subpopulation were assigned to a same node (green circles 1, 2, and 3 on Fig. 1b), regardless of their HIV status. The expression of the differentiation-related genes progressively decreased or increased in the order of linear differentiation (TN → TCM → TEM ), agreeing with previous reports [40–43] (Fig. 1c). For instance, LEF1, ACTN1, FOXP1, IL6ST and CERS6 reportedly undergoing down-regulation in naive T cells after antigen recognition and differentiation [44–48], along with TAF4B, appeared progressively down regulated when samples were ordered according to the linear model of peripheral differentiation (Fig. 1c, left panel). These changes agree with previous reports [42]. Conversely, differentiation and effector function-associated transcripts, like EOMES, TBX21 (t-bet), PRDM1 (Blimp-1) [49, 50], GZMA and PRF1 [51, 52], were gradually increased in the same order (Fig. 1c right panel). A same pattern was followed by the expression of KLRG1,an indicator of replicative senescence [53, 54] (Fig. 1c). TBX21 (t-bet) was the only gene with increased expression in TCM cells from patients, compared with controls (p = 0.003), which, along with the increased expression of IL12R e IL18R, suggests a Th1-skewed response driven by HIV infection. A Th1-skewed response was also predicted by Ingenuity Canonical Pathway analysis (See Additional file 4). Thus, TCM cells from patients did not seem to be more differentiated than their counterparts from controls, but appeared polarized to Th1 functions.Fig. 2

View Article: PubMed Central - PubMed

ABSTRACT

Background: Human central memory CD4 T cells are characterized by their capacity of proliferation and differentiation into effector memory CD4 T cells. Homeostasis of central memory CD4 T cells is considered a key factor sustaining the asymptomatic stage of Human Immunodeficiency Virus type 1 (HIV-1) infection, while progression to acquired immunodeficiency syndrome is imputed to central memory CD4 T cells homeostatic failure. We investigated if central memory CD4 T cells from patients with HIV-1 infection have a gene expression profile impeding proliferation and survival, despite their activated state.

Methods: Using gene expression microarrays, we analyzed mRNA expression patterns in naive, central memory, and effector memory CD4 T cells from healthy controls, and naive and central memory CD4 T cells from patients with HIV-1 infection. Differentially expressed genes, defined by Log2 Fold Change (FC) ≥ /0.5/ and Log (odds) > 0, were used in pathway enrichment analyses.

Results: Central memory CD4 T cells from patients and controls showed comparable expression of differentiation-related genes, ruling out an effector-like differentiation of central memory CD4 T cells in HIV infection. However, 210 genes were differentially expressed in central memory CD4 T cells from patients compared with those from controls. Expression of 75 of these genes was validated by semi quantitative RT-PCR, and independently reproduced enrichment results from this gene expression signature. The results of functional enrichment analysis indicated movement to cell cycle phases G1 and S (increased CCNE1, MKI67, IL12RB2, ADAM9, decreased FGF9, etc.), but also arrest in G2/M (increased CHK1, RBBP8, KIF11, etc.). Unexpectedly, the results also suggested decreased apoptosis (increased CSTA, NFKBIA, decreased RNASEL, etc.). Results also suggested increased IL-1β, IFN-γ, TNF, and RANTES (CCR5) activity upstream of the central memory CD4 T cells signature, consistent with the demonstrated milieu in HIV infection.

Conclusions: Our findings support a model where progressive loss of central memory CD4 T cells in chronic HIV-1 infection is driven by increased cell cycle entry followed by mitotic arrest, leading to a non-apoptotic death pathway without actual proliferation, possibly contributing to increased turnover.

Electronic supplementary material: The online version of this article (doi:10.1186/s12864-016-3308-8) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus