α4β7+ CD4+ Effector/Effector Memory T Cells Differentiate into Productively and Latently Infected Central Memory T Cells by Transforming Growth Factor β1 during HIV-1 Infection

Abstract

HIV-1 transmission occurs mainly through mucosal tissues. During mucosal transmission, HIV-1 preferentially infects α₄β₇⁺ gut-homing CCR7^- CD4⁺ effector/effector memory T cells (T_EM) and results in massive depletion of these cells and other subsets of T_EM in gut-associated lymphoid tissues. However, besides being eliminated by HIV-1, the role of T_EM during the early stage of infection remains inconclusive. Here, using in vitro-induced α₄β₇⁺ gut-homing T_EM (α₄β₇⁺ T_EM), we found that α₄β₇⁺ T_EM differentiated into CCR7⁺ CD4⁺ central memory T cells (T_CM). This differentiation was HIV-1 independent but was inhibited by SB431542, a specific transforming growth factor β (TGF-β) receptor I kinase inhibitor. Consistently, T_EM-to-T_CM differentiation was observed in α₄β₇⁺ T_EM stimulated with TGF-β1 (TGF-β). The T_CM properties of the TGF-β-induced T_EM-derived T_CM (α₄β₇⁺ T_CM) were confirmed by their enhanced CCL19 chemotaxis and the downregulation of surface CCR7 upon T cell activation in vitro Importantly, the effect of TGF-β on T_CM differentiation also held in T_EM directly isolated from peripheral blood. To investigate the significance of the TGF-β-dependent T_EM-to-T_CM differentiation in HIV/AIDS pathogenesis, we observed that both productively and latently infected α₄β₇⁺ T_CM could differentiate from α₄β₇⁺ T_EM in the presence of TGF-β during HIV-1 infection. Collectively, this study not only provides a new insight for the plasticity of T_EM but also suggests that the TGF-β-dependent T_EM-to-T_CM differentiation is a previously unrecognized mechanism for the formation of latently infected T_CM after HIV-1 infection.IMPORTANCE HIV-1 is the causative agent of HIV/AIDS, which has led to millions of deaths in the past 30 years. Although the implementation of highly active antiretroviral therapy has remarkably reduced the HIV-1-related morbidity and mortality, HIV-1 is not eradicated in treated patients due to the presence of latent reservoirs. Besides, the pathogenesis in CD4 T cells early after infection still remains elusive. Immediately after HIV-1 mucosal infection, CD4 T cells are preferentially infected and depleted. However, in addition to being depleted, the other roles of the CD4 T cells, especially the effector/effector memory T cells (T_EM), in disease progression are not completely understood. The significance of this study is in revealing a novel mechanism for the formation of latently HIV-1-infected central memory CD4 T cells, a major latent reservoir from CD4 T_EM after infection. Our findings suggest previously unrecognized roles of CD4 T_EM in HIV-1 pathogenesis.

Keywords: CCR7; CD4 T cells; HIV-1; HIV-1 latent infection; central memory CD4 T cells; effector/effector memory CD4 T cells.

FIG 1

Characteristics of α₄β₇⁺ _MEMT generated from in vitro allogeneic T cell activation. (A) The schematic diagram illustrates the in vitro allogeneic T cell activation method used for the generation of α₄β₇⁺ _MEMT and α₄β₇⁺ T_EM. (B) Percentage of α4⁺ and β7⁺ α₄β₇⁺ _MEMT on day 8 (donors = 14). (C) α₄β₇⁺ _MEMT were incubated with 10 μg/ml MAdCAM-1–Fc for 30 min at 4°C. The percentage of MAdCAM-1–Fc-bound α₄β₇⁺ _MEMT was quantified by flow cytometry (donors = 3). (D) The expression of β7 and CCR5 on the CD45RO⁺ CD4 T cells generated by either the conventional PHA–IL-2 method or the in vitro allogeneic T cell activation method mentioned above was measured by flow cytometry. (i) Percentage of β7⁺ CCR5⁺ cells in PHA–IL-2-activated CD4 T cells and α₄β₇⁺ _MEMT (donors = 8). (ii) MFI of CCR5 expression on PHA–IL-2-activated CD4 T cells and α₄β₇⁺ _MEMT (donors = 8). Purified CD4 T cells treated with IL-2, IL-15, and RA were cultured in the presence or absence of gamma-irradiated RPMI8866 cells for 6 days. The phenotypes of the cells were analyzed by flow cytometry before CD45RO⁺ cell enrichment. (E) Representative histograms show the expression of CD45RO on CD4 T cells and the expression of α4, β7, and CCR5 on CD45RO⁺ CD4 T cells (donors = 4). The shaded histogram represents the isotype control. (F) (i) Percentage of CD45RO⁺ CD4 T cells cultured in the presence or absence of gamma-irradiated RPMI8866 cells (donors = 4). (ii and iii) Percentage (ii) and MFI (iii) of α4, β7, and CCR5 on CD45RO⁺ CD4 T cells cultured in the presence or absence of gamma-irradiated RPMI8866 cells (donors = 4). Data are expressed as the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 2

Characteristics of α₄β₇⁺ T_EM isolated from α₄β₇⁺ _MEMT. Purified CD4 T cells were cultured in the presence of gamma-irradiated RPMI8866 cells plus IL-2, IL-15, and RA. The phenotypes of the cells were analyzed by flow cytometry on day 6, day 8, and day 11 after CD45RO⁺ cell enrichment. (A) Representative flow cytometric analysis (donors = 8) shows the expression of CD45RO and CCR7 on allogeneic activated CD4 T cells. (B) Bar charts illustrate the kinetic changes in the percentage of CD45RO⁺ CCR7⁺ T_CM (i) and CD45RO⁺ CCR7⁻ T_EM (ii) in allogeneic activated CD4 T cells (donors = 8). The phenotypes of α₄β₇⁺ T_EM isolated from α₄β₇⁺ _MEMT on day 11 were analyzed by flow cytometry. (C) Percentage of α4⁺ and β7⁺ α₄β₇⁺ T_EM on day 11 (donors = 10). (D) Representative flow cytometric analysis (donors = 10) shows the expression of CD45RO, CCR7, integrin β7, and CCR5 on α₄β₇⁺ T_EM. The positive cells were defined using the corresponding isotype controls. SSC, side scatter; FSC, forward scatter. Data are expressed as the mean ± SEM. **, P < 0.01; ***, P < 0.001.

FIG 3

Presence of p24⁺ α₄β₇⁺ T_CM in HIV-1-infected α₄β₇⁺ T_EM. α₄β₇⁺ T_EM were infected with HIV-1_BJZS7 in the presence or absence of 1 μM maraviroc (Mara). The phenotypes of the cells were analyzed by flow cytometry. (A) Representative results show the expression of p24 versus CD4 (i) or CCR5 (ii) on CD3⁺ CD8⁻ T cells 6 days after infection (donors = 5). (B) (i) Representative flow cytometric analysis shows that CCR7 expression on uninfected and infected α₄β₇⁺ T_EM was upregulated at 6 days after infection (donors = 5). (ii) Percentage of p24⁺ α₄β₇⁺ T_EM and p24⁺ α₄β₇⁺ T_CM in CD3⁺ CD8⁻ T cells (donors = 5). (iii) Representative histograms show the expression of CCR5 on uninfected, p24⁻, and p24⁺ α₄β₇⁺ T_CM (CCR7⁺) or α₄β₇⁺ T_EM (CCR7⁻) (donors = 5). The shaded histogram represents the isotype control. Data are expressed as the mean ± SEM. **, P < 0.01.

FIG 4

TGF-β induces the differentiation of α₄β₇⁺ T_CM from α₄β₇⁺ T_EM. (A) α₄β₇⁺ T_EM were isolated and cultured in the presence of the indicated cytokines for 7 days. The results show the percentage of CD3⁺ CD8⁻ α₄β₇⁺ T_CM on day 7 (donors = 5). (B) α₄β₇⁺ T_EM were isolated and cultured in the presence of anti-IFN-γ (20 μg/ml) blocking antibody. The results show the percentage of CD3⁺ CD8⁻ α₄β₇⁺ T_CM on day 7 (donors = 4). The corresponding isotype antibody was used as a control. (C) A representative histogram shows the expression of TGF-βRII on CD3⁺ CD8⁻ CD4⁺ α₄β₇⁺ T_EM on the day of α₄β₇⁺ T_EM isolation (donors = 8). (D) Gating strategy used to identify the CD3⁺ CD4⁺ CD45RA⁻ α₄β₇⁺ T_CM and CD3⁺ CD4⁺ CD45RA⁻ CCR7⁺ T_CM in the assays whose results are shown in panels E and Fi and ii. A representative flow cytometric analysis shows the expression of CCR7 on α₄β₇⁺ T_EM stimulated with 1 ng/ml TGF-β for 7 days. The positive cells were defined using the corresponding isotype controls. (E) α₄β₇⁺ T_EM were isolated and cultured in the presence of 1 ng/ml or 10 ng/ml TGF-β. The effect of TGF-β on the induction of CD3⁺ CD4⁺ CD45RA⁻ α₄β₇⁺ T_CM from α₄β₇⁺ T_EM was determined on day 0 (before TGF-β stimulation), day 1, and day 7 during TGF-β coculture (donors = 8). (F) CCR7⁻ CD4 T cells directly isolated from peripheral blood were treated with TGF-β either in the presence (donors = 6) (i) or in the absence (donors = 6) (ii) of IL-2 (10 ng/ml) and IL-15 (20 ng/ml). The effect of TGF-β on the induction of CD3⁺ CD4⁺ CD45RA⁻ CCR7⁺ T_CM was determined on days 0 (before TGF-β stimulation), 1, 3, and 7. (G) Correlation between the percentage of LAP⁺ CD3⁺ α₄β₇⁺ T_EM immediately after α₄β₇⁺ T_EM isolation and the percentage of CD3⁺ CD8⁻ CD4⁺ α₄β₇⁺ T_CM after an additional 7 days of culture (donors = 9). (H) α₄β₇⁺ T_EM were isolated and cultured in the presence of the TGF-βRI inhibitor SB431542 for 7 days. The results show the percentage of CD3⁺ CD8⁻ CD4⁺ α₄β₇⁺ T_CM on day 7 (donors = 9). Dimethyl sulfoxide (DMSO) at the same concentration as the relative inhibitor concentration was used as the control. (I) Percentage of viable cells in the presence of 1 μM SB431542 (donors = 7), determined by a trypan blue exclusion assay. DMSO at the same concentration as the relative inhibitor concentration was used as the control. Cell counting was performed by use of a Countess automated cell counter. Data are expressed as the mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant.

FIG 5

Central memory T cell properties of α₄β₇⁺ T_CM. (A) α₄β₇⁺ T_EM either untreated or stimulated with TGF-β for 7 days were tested for CCL19 chemotaxis in vitro, and the number of cells (normalized to 1 × 10⁶/ml) that migrated to the CCL19-containing medium was determined (donors = 6). Data are expressed as the mean ± SEM. **, P < 0.01; ***, P < 0.001. (B) α₄β₇⁺ T_EM cultured in IL-2, IL-15, and TGF-β for 7 days were stimulated under the indicated conditions. A representative histogram shows the expression of CCR7 on CD3⁺ CD4⁺ CD45RA⁻ α₄β₇⁺ T_EM 3 days after stimulation (donors = 6).

FIG 6

Effect of TGF-β on α4 and β7 expression on α₄β₇⁺ T_EM and binding toward MAdCAM-1. α₄β₇⁺ T_EM were isolated and cultured in the presence of 1 ng/ml or 10 ng/ml TGF-β for 7 days. (A) The effect of TGF-β on the percentage (i) and the MFI (ii) of α4 and β7 expression on CD4⁺ CD45RO⁺ T cells was determined on day 7 (donors = 5). (B) The effect of TGF-β on the total percentage of MAdCAM-1–Fc binding cells and the binding of MAdCAM-1–Fc on α₄β₇⁺ T_EM and α₄β₇⁺ T_CM was determined on day 7 (donors = 3). Data are expressed as the mean ± SEM. *, P < 0.05; **, P < 0.01.

FIG 7

Changes of CD62L expression on α₄β₇⁺ T_EM and T_EM directly isolated from peripheral blood in the presence or absence of TGF-β. (A) Representative flow cytometric analysis shows the expression of CCR7 and CD62L on the isolated α₄β₇⁺ T_EM (day 0) and the α₄β₇⁺ T_EM stimulated with 1 ng/ml TGF-β for 7 days (day 7) (donors = 8). (B and C) Representative flow cytometric analysis shows the expression of CCR7 and CD62L on CD3⁺ CD4⁺ CD45RA⁻ CCR7⁻ T_EM directly isolated from peripheral blood on day 0 and on day 7 after stimulation with 1 ng/ml TGF-β in the presence (donors = 6) (B) or absence (donors = 6) (C) of IL-2 (10 ng/ml) and IL-15 (20 ng/ml). The positive cells were defined using the corresponding isotype controls.

FIG 8

CD62L expression in different memory T cell subsets of purified CD4 T cells. Representative flow cytometric analysis shows the expression of CD62L on CD3⁺ CD4⁺ CD45RA⁻ CCR7⁺ T_CM and CD3⁺ CD4⁺ CD45RA⁻ CCR7⁻ T_EM in CD4 T cells purified from peripheral blood (donors = 5). The positive cells were defined using the corresponding isotype controls.

FIG 9

α₄β₇⁺ T_EM and α₄β₇⁺ T_CM harbor productively and latently infected HIV-1. α₄β₇⁺ T_EM were infected with DuoFluo_JRFL, and the results were analyzed by flow cytometry at 6 days after infection. (A) A representative flow cytometric analysis shows that CCR7 is upregulated in the presence of TGF-β after infection. The presence of productively infected (GFP⁺ and mCherry⁺ GFP⁺) and latently infected (mCherry⁺ GFP⁻) α₄β₇⁺ T_EM (CCR7⁻) and α₄β₇⁺ T_CM (CCR7⁺) was also demonstrated (donors = 9). (B) Percentage of productively infected (GFP⁺ and mCherry⁺ GFP⁺) (i) and latently infected (mCherry⁺ GFP⁻) (ii) α₄β₇⁺ T_EM (CCR7⁻) and α₄β₇⁺ T_CM (CCR7⁺) (donors = 9). Data are expressed as the mean ± SEM. *, P < 0.05; ***, P < 0.001.