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. 2010 Dec 20;207(13):2869-81.
doi: 10.1084/jem.20100090. Epub 2010 Nov 29.

Preferential infection and depletion of Mycobacterium tuberculosis-specific CD4 T cells after HIV-1 infection

Christof Geldmacher  1 , Njabulo Ngwenyama, Alexandra Schuetz, Constantinos Petrovas, Klaus Reither, Edwin J Heeregrave, Joseph P Casazza, David R Ambrozak, Mark Louder, William Ampofo, Georgios Pollakis, Brenna Hill, Erica Sanga, Elmar Saathoff, Leonard Maboko, Mario Roederer, William A Paxton, Michael Hoelscher, Richard A Koup
Affiliations

Preferential infection and depletion of Mycobacterium tuberculosis-specific CD4 T cells after HIV-1 infection

Christof Geldmacher et al. J Exp Med. .

Abstract

HIV-1 infection results in the progressive loss of CD4 T cells. In this study, we address how different pathogen-specific CD4 T cells are affected by HIV infection and the cellular parameters involved. We found striking differences in the depletion rates between CD4 T cells to two common opportunistic pathogens, cytomegalovirus (CMV) and Mycobacterium tuberculosis (MTB). CMV-specific CD4 T cells persisted after HIV infection, whereas MTB-specific CD4 T cells were depleted rapidly. CMV-specific CD4 T cells expressed a mature phenotype and produced very little IL-2, but large amounts of MIP-1β. In contrast, MTB-specific CD4 T cells were less mature, and most produced IL-2 but not MIP-1β. Staphylococcal enterotoxin B-stimulated IL-2-producing cells were more susceptible to HIV infection in vitro than MIP-1β-producing cells. Moreover, IL-2 production was associated with expression of CD25, and neutralization of IL-2 completely abrogated productive HIV infection in vitro. HIV DNA was found to be most abundant in IL-2-producing cells, and least abundant in MIP-1β-producing MTB-specific CD4 T cells from HIV-infected subjects with active tuberculosis. These data support the hypothesis that differences in function affect the susceptibility of pathogen-specific CD4 T cells to HIV infection and depletion in vivo, providing a potential mechanism to explain the rapid loss of MTB-specific CD4 T cells after HIV infection.

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Figures

Figure 1.
Figure 1.
MTB- and CMV-specific CD4 T cells are lost at different rates after HIV infection. (A) The frequency of MTB-specific (black, 5 PPD responding subjects, who remained TB asymptomatic) and CMV-specific memory CD4 T cell responses (gray; n = 6 CMV responding subjects who remained CMV disease free) at 6–12 mo after HIV seroconversion as the percentage of baseline response detected at 3 mo before the first HIV-seropositive follow up in latently infected subjects. The frequency of MTB- or CMV-specific CD4 T cell responses in chronically HIV-infected subjects (median time since HIV infection >3 yr; n = 17) and a HIV− control group (n = 17) is shown in B and C, respectively. The limit of detection is indicated. Memory CD4 T cells were defined by expression of CD27 and CD45RO. IFN-γ+ memory CD4 T cells were detected after in vitro stimulation of PBMCs with PPD or whole inactivated CMV virus by intracellular cytokine staining. Statistical analysis was performed using the Mann-Whitney test.
Figure 2.
Figure 2.
Differences in cellular maturation between MTB- and CMV-specific CD4 T cells are diminished during active TB disease and are associated with diametrically opposed production of IL-2 and MIP-1β. (A) Surface expression of the maturation markers CD27, CD45RO, and CD57 on total CD4 T cells (black) is shown for representative subjects as a density plot overlay. MTB-specific CD4 T cells in the absence (blue, HIV−) or presence (green, HIV+) of active TB disease and for CMV-specific CD4 T cells (red). The percentage of CD27+CD45RO+, CD27−CD45RO+, and CD57+ subsets within MTB- and CMV-specific CD4 T cells from HIV− subjects (blue, n = 17) and HIV+ subjects with active TB (green, n = 11; bottom). (B) Flow cytometric analysis of IFN-γ, IL-2, MIP-1β, and TNF production within pathogen-specific CD4 T cells. PBMCs from subjects with latent MTB infection (defined by positive response to region of difference 1 [RD1] antigens) or from HIV+ subjects with active TB were stimulated with MTB-antigens (PPD or a mix of PPD and RD1 peptide pools) or CMV whole antigen. The pie charts show the fraction of cells with 1, 2, 3, or 4 functions. The color-coded circles indicate the proportion of the 4, 3, 2, and 1 functional responses response that are contributed by the single cytokines IFN-γ (red), IL-2 (blue), Mip-1β (green), and TNF (pink). The fraction of cells that produce MIP-1β (right top) or IL-2 (bottom right) is shown as percentage of total cytokine-positive CD4 T cells. The median is indicated. Further delineation of the 16 different possible cytokine combinations is shown (bottom). (C) Intracellular staining of IL-2 (y axis) and MIP-1β (x axis) within CD27+CD45RO+, CD27−CD45RO+CD57−, and CD27−CD45RO+CD57+ CD4 T cells after stimulation with PPD or CMV is shown for one representative subject (left) and for all studied responses further delineated by IL-2 and MIP-1β production and presented as percentage of total cytokine-positive response (right). Statistical analysis was performed using the Mann-Whitney test (***, P < 0.0005; **, P < 0.005; *, P < 0.05).
Figure 3.
Figure 3.
HIV infection is characterized by increased fractions of MIP-1β+ and decreased fractions of IL-2+ CMV-specific CD4 T cells. (left) The fraction of IFN-γ+, TNF+, MIP-1β+, or IL-2+ among total cytokine positive CD4 T cells (y axis) is shown for HIV− (n = 11) and HIV+ subjects (n = 16). (right) The fraction of IL-2+MIP-1β+, IL-2+MIP-1β−, and IL-2−MIP-1β+ among total cytokine-positive CD4 T cells. PBMCs were stimulated overnight with whole inactivated CMV and the background (unstimulated control) was subtracted. Statistical analysis was performed using the Mann-Whitney test.
Figure 4.
Figure 4.
Productive HIV-1 infection of SEB-responding CD4 T cells is inhibited by in vitro neutralization of IL-2. (A) Productive HIV infection of CD4 T cells was analyzed after gating on CD4low T cells and staining for HIV-p24 (y axis). Shown are dot plots from samples stimulated in the presence or absence of 1 µM AZT. (B) Dot plots from samples stimulated in the presence (bottom) or absence (top) of a neutralizing anti–IL-2 antibody comparing CFSE-fluorescence intensity (left), surface CD25 (middle), and CCR5 (right) with HIV-p24 staining. PBMC samples were stimulated for 24 h with SEB and then infected for another 24 h with the CCR5 tropic HIV-1 strain BAL.
Figure 5.
Figure 5.
The capacity to secrete IL-2 by antigen-specific CD4 T cells is associated with increased CD25 expression. (A) Representative histogram of the CD25 expression on cytokine-negative, MIP-1β+IL-2−, and MIP-1β−IL-2+ CD4 T cells after 6 h of SEB stimulation of PBMCs. (B) The fraction of CD25+ CD4 T cells among the same subsets (n = 7). Statistical analysis was performed using the Mann-Whitney test.
Figure 6.
Figure 6.
In vitro HIV infection of cytokine-expressing cells. (A) Representative histograms of the HIV p24 staining from CD4 T cells delineated by intracellular staining for MIP-1β, IFN-γ, and IL-2 and (B) the fraction of p24+ CD4 T cells delineated by intracellular staining for MIP-1β, IFN-γ, and IL-2 from six independent experiments. PBMC samples were stimulated for 24h with SEB and then infected for another 24 h with the CCR5 tropic HIV-1 strain BAL. Productive HIV infection of CD4 T cells was analyzed after gating on CD4low T cells and staining for HIV-p24.
Figure 7.
Figure 7.
In vivo HIV gag DNA in MTB-specific CD4 T cells. (A) Gating/sorting strategy used to sort different memory CD4 T cell populations delineated by IFN-γ, IL-2, and MIP-1β production. MIP-1β− memory CD4 T cells were further delineated into IFNγ+IL-2+TNF+, IFNγ+IL-2−TNF+, and cytokine-negative memory CD4 T cells. The HIV gag DNA/10,000 cells determined within these populations is indicated. (B) The ratio of HIV gag copies/10,000 cells detected within cytokine-positive to cytokine-negative memory T cells from 16 cytokine-positive CD4 T cell populations sorted from 10 HIV+ subjects (different symbols) with active TB. (C) The mean number of gag copies/10,000 cells detected in MTB-specific CD4 T cells or total memory CD4 T cells is shown for each subject. Memory CD4 T cells were defined by expression of CD45RO and CD27. The PBMCs were stimulated overnight with a mix of PPD and RD1 peptide pools and further analyzed as described in the Materials and methods section. Gag DNA within different CD4 T cell populations of the same subject was quantified during the same RT-PCR run. The statistical analysis in C was performed using the Wilcoxon-rank-matched pairs test.
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