Lack of T-cell-mediated IL-2 and TNFα production is linked to decreased CD58 expression in intestinal tissue during acute simian immunodeficiency virus infection

Abstract

For an effective T-cell activation and response, co-stimulation is required in addition to the antigen-specific signal from their antigen receptors. The CD2/CD58 interaction is considered as one of the most important T-cell co-stimulatory pathways for T-cell activation and proliferation, and its role in regulating intestinal T-cell function in acute and chronic SIV -infected macaques is poorly documented. Here, we demonstrated a significant reduction of CD58 expression in both T- and B-cell populations during acute SIV infection along with high plasma viral load and a loss of intestinal CD4⁺ T cells compared to SIV-uninfected control macaques. The reduction of CD58 expression in T cells was correlated with the reduced expression of T-cell-mediated IL-2 and TNFα production. Together, these results indicate that reduction in the CD2/CD58 interaction pathway in mucosal lymphocytes might play a crucial role in mucosal T-cell dysfunction during acute SIV/HIV infection.

Human immunodeficiency virus type 1 (HIV-1) infection causes a progressive impairment of the immune system characterized by massive CD4⁺ T-cell loss, CD8⁺ T-cell expansion and sustained immune activation and inflammation [1, 2]. For effective T-cell activation and response, co-stimulation is required in addition to the antigen-specific signal from their antigen receptors. The first signal for T-cell activation is initiated by interaction of the peptide/major histocompatibility complex and T-cell receptors (TCR). The second signal of T-cell activation, proliferation, and expansion come from many co-stimulatory molecules [3]. The well-characterized co-stimulatory pathway for T-cell activation is the T-cell surface receptor CD28, which binds to the activated antigen-presenting cells (APCs) surface molecules such as B7-1 (CD80) and B7-2 (CD86) [4]. Several other proteins that are structurally related to B7-1 and B7-2 or CD28 were identified and found to be responsible for T-cell activation. Co-stimulatory receptor ICOS (inducible co-stimulatory, CD278) was found to bind with the ICOS-ligand (CD275) expressed by dendritic cells (DCs), macrophages and B cells. Similarly, co-stimulatory receptors CTLA-4 and PD-1 expressed in naïve and activated T cells, respectively, play an important role in inducing T-cell activation by binding to their contemporary ligands such as B7-2 expressed by DCs, macrophages and B cells, and PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273) expressed by DCs, macrophages, B cells, endothelial cells, epithelial cells and tumour cells, respectively. CTLA-4 and PD-1 are considered as inhibitory receptors of the CD28 family and are key players in balancing T-cell activation. CD2 (LFA-2) also plays an important role in T-cell activation, both as a signal transducer and an intracellular adhesion molecule, where the major ligand of CD2 is CD48 in mice and CD58 (LFA-3) in human DCs and other APCs [5, 6]. The CD2/CD58 interaction is considered as one of the key T-cell co-stimulatory pathways for T-cell activation and proliferation [3, 7], and has been shown to alter T-cell-mediated cytokine production [8]. Intestinal lamina propria (LP) T cells were reported to be highly responsive to a stimulus delivered by the CD2 pathway compared to the conventional CD3/TCR pathway [9, 10]. A recent report has also shown that CD2/CD58 is the primary co-stimulatory pathway for effector CD8⁺CD28^– T-cell proliferation and activation [7]. CD2/CD58 interaction was also found to be a key regulator for early activation and function of adaptive NKG2C⁺CD57⁺ NK cells in human cytomegalovirus (HCMV) infection [11]. However, these studies are limited to peripheral blood, with few data on how the expression of CD2/CD58 in intestinal LP T and B-cells is affected during acute and chronic SIV infection, and their impact on T-cell-produced cytokines, despite the fact that intestinal lamina propria lymphocytes (LPLs) are considered a major site of viral replication and CD4⁺T-cell depletion [12]. Here, we have quantified CD2 and CD58 expression in LPL T and B cells in SIV-uninfected control and SIV-infected rhesus macaques (RMs), and correlated this with the cytokines produced by functional T cells to determine whether the expression of those important co-stimulatory pathways has any impact on T-cell-mediated cytokine expression. We have also performed a mechanistic study showing the dependence of cytokine production on CD58 expression in T cells.

The infectious SIV_MAC251 strain, SIV_MAC251-CX1, generated by infecting continuous CEM×174 cells with SIV_MAC251, was used for RM infection. Single-genome amplification sequencing analysis of the full-length Env gene showed 1.5 % of genetic diversity compared to molecular clone SIV_MAC239 stock used for non-human primate studies [13, 14]. In this study, 19 adult female Indian RMs (Macaca mulatta) were selected and shown negative for SIV, HIV-2, type-D retrovirus and simian T-cell leukaemia virus 1 infection. Eight animals were infected intravaginally once with 500 TCID₅₀ of SIV_MAC251-CX1, a known infectious dose for RM as reported previously [1, 15, 16], and were then divided equally into two groups (acute and chronic). Four animals were not SIV challenged and therefore used as naïve, uninfected controls. Plasma and jejunal tissues were collected at 21 days post-SIV infection from the acute group, and at 309–437 days post-SIV infection from the chronic group at the time of necropsy. Plasma and jejunal tissues were also collected from SIV-uninfected control animals at the time of necropsy. Depo-Provera (medroxyprogesterone acetate) is a derivative of progesterone that causes thinning of vaginal epithelial layers, rendering the vaginal mucosa more susceptible to SIV infection [17, 18]. In our study female macaques were treated with Depo-Provera (30 mg intramuscularly) 28 days prior to SIV exposure, to facilitate SIV infection by a single vaginal injection [19]. Peripheral blood mononuclear cells (PBMCs) isolated from an additional seven control, SIV-uninfected RMs were used for an in vitro CD58 blocking assay. All RMs were housed at the Tulane National Primate Research Center (TNPRC) as per the regulations of the American Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC), under the full care of TNPRC veterinarians in accordance with the standards incorporated in the Guide for the Care and Use of Laboratory Animals. All experiments were reviewed and approved by the Tulane Institutional Animal Care and Use Committee. Plasma viral RNA was quantified by quantitative RT-PCR assay for SIV-infected macaques [12]. The lower limit of RNA detection for RT-PCR assays was 60 SIV-RNA copies ml^–1 of plasma. Jejunal samples (6–10 cm in length) were collected in ice-cold HBSS and processed by cutting into small pieces after removal of unwanted debris and fat. Finally, LPLs were isolated as described previously [20, 21]. All cells were washed twice and re-suspended in complete RPMI-1640 medium containing 10 % fetal bovine serum (FBS) before staining. All cells were >90 % viable according to the trypan blue dye exclusion method.

Next, we performed flow cytometry staining where cells were adjusted to 1×10⁶ cells per tube. For cell phenotyping, cells were first stained with live/dead stain (Invitrogen), washed with wash buffer (PBS containing 0.1 %BSA) and then stained with appropriately diluted and directly conjugated anti-CD2 (RPA-2.10, BD Biosciences), anti-CD3 (SP34-2, BD Biosciences) anti-CD4 (L200, BD Biosciences), anti-CD8 (SK1, BD Biosciences), anti-CD20 (2H7, BD Biosciences), anti-CD45 (D058-1283, BD Biosciences), anti-CD58 (1C3, BD Biosciences) and anti-HLA-DR (Mamu-DR, G46-6, BD Biosciences) monoclonal antibodies (MAbs), and incubated for 30 min at room temperature in the dark as previously described [21, 22]. Finally, fixed and stained cells were processed through a BD LSRII Flow Cytometer (BD Bioscience) and data acquisition was performed. Whole blood samples from uninfected control RMs were used for fluorochrome compensations. At least 100 000 events were collected from each sample by gating on live cells, and data were analysed using FlowJo software (TreeStar Inc.) (Fig. S1, available in the online version of this article).

All macaques had a high peak viral load within 14–21 days after challenge. Chronically infected animals maintained a viral set point (ranging 1020–636 181 viral RNA copies ml⁻¹ of plasma) at the time of necropsy. Chronically infected animals had significantly lower viral load compared to acute animals (Fig. 1a). There was a statistically significant reduction in jejunal LP CD4⁺ T cells (mean 47.1 % in uninfected control versus 12.8 % in SIV acute) in acute infection, and numbers remained low during chronic SIV infection as reported previously (Fig. 1b) [12, 20, 23]. Early loss of CD4 interferes with the maintenance of effective CD8⁺ T-cell responses, where, CD8⁺ T cells are thought to be a key player in generating antiviral immunity and early antigen presentation of viral epitopes for rapid clearance of HIV/SIV-infected cells [24, 25]. Statistical analysis of data was performed using GraphPad Prism Version 7 (GraphPad Software, USA). Unpaired Mann–Whitney t-testing was done for inter-group comparison. For multiple group comparisons, ANOVA with Bonferroni’s post hoc test was performed. P<0.05 was considered statistically significant.

Fig. 1.

(a) Plasma viral loads with mean (± standard error) in macaques during acute and chronic phase of SIV_MAC251 infection, as determined by RT-PCR (n=4). (b) Percentages of jejunal LPL CD4⁺ T cells with mean (± standard error) in control uninfected, acute and chronic SIV_MAC251-infected macaques (n=4). Percentage expression of CD2, CD58, Mamu-DR⁺CD2⁺and Mamu-DR⁺CD58⁺ with mean (± standard error) is shown for jejunal LPL CD4⁺ (c–f) and CD8⁺ (g–j) T cells in control uninfected, acute and chronic SIV-infected macaques. Percentage of jejunal LPL CD20⁺ B cells (k), as well as the expression of CD2 (l) and CD58 (m) in CD20⁺ B cells with mean (± standard error), are shown. Asterisks indicate statistically significant differences between groups (P<0.05).

The impact of SIV infection on the distribution of CD2 and CD58 expression in CD4 and CD8 T cells, as well as CD20⁺ B cells, was measured in jejunal LPLs (Fig. 1c–m). The majority of T cells expressed CD2, and there was no statistically significant difference in CD2 expression among SIV-uninfected control, SIV acute and SIV chronic infected RMs in CD4⁺ T cells (Fig. 1c). Similar to CD2, CD58 was also expressed in the majority of T cells and its expression in CD4⁺ T cells decreased significantly in acute infection compared to uninfected control animals (Fig. 1d). In chronic infection, CD58 expression increased in CD4⁺ T cells compared to acute infection time points. The median fluorescence intensity (MFI) of CD58 expression was also analysed in both normal and SIV-infected RMs, but the difference among groups was not statistically significant (Fig. S2a). Mamu-DR, a late activation marker which is expressed on the surface of antigen-activated CD4 and CD8 T cells [26, 27], was used to determine the activation of CD2 and CD58⁺ T cells in normal and SIV-infected macaques. A significant upregulation of Mamu-DR expression was detected in CD2 or CD58-expressing CD4⁺ T cells in chronic infection compared to uninfected control and acute SIV infection (Fig. 1e–f).

Similar to CD4⁺ T cells, there was no statistically significant difference in CD2 expression among SIV-uninfected control, SIV acute and SIV chronic infected RMs in CD8⁺T cells (Fig. 1g). The expression of CD58 in CD8⁺ T cells decreased significantly in acute infection compared to uninfected control animals (Fig. 1h). In chronic infection, CD58 expression in CD8⁺ T cells increased significantly compared to acute infection time points. CD58 MFI values in CD8⁺ T cells were also lower in chronic infection, but no differences in MFI values among any of the animal groups were statistically significant (Fig. S2b, available in the online version of this article). Similar to CD4, a significant upregulation of Mamu-DR expression was also shown in CD2- or CD58-expressing CD8⁺ T cells in chronic infection compared to acute SIV infection (Fig. 1i–j).

Depletion of jejunal LP CD20⁺ B cells was also detected in all acutely infected RMs (mean 13.5 % in uninfected control versus 5.4 % in acute), and the numbers returned to near normal in chronic infection (mean value 11.4 %, Fig. 1k) as previously observed in PBMC [22]. CD2 expression was lower in B cells (ranging 6.4–38.7 %), with no decrease in expression during acute infection despite the reduction in B-cell population. CD2 expression in B cells decreased in chronic SIV infection (Fig. 1l), but this is not statistically significant. CD58 (mean 98.2 %) expression was much higher than CD2 in B cells in uninfected control RMs, and the expression of CD58 was significantly decreased in B cells from both acute and chronic infected groups (Fig. 1m). HIV infection also downregulated CD48 expression in primary human peripheral blood T-cell blasts, suggesting the role of CD48 (or CD58) expression in regulating NK cell cytotoxicity [28]. Neutralization of CD58 also inhibited CD8⁺ T-cell functions in eliminating motile HIV-infected targets within the 3D collagen matrix culture model, and demonstrated that efficient motile target lysis is dependent on the interaction of co-stimulatory molecules [29]. Reduction in CD58 expression was also detected in peripheral monocytes 1 day after in vitro infection by Mycobacterium avium where low CD58 expression was thought to be responsible for the reduced proliferation of antigen-independent autologous CD4⁺ T cells [30].

To determine the distribution of CD58 expression in jejunal tissues, tissue sections (approximately 8–10 µm from OCT frozen tissue) were processed for immunofluorescence staining using a representative animal from each group. After thawing, slides were fixed in 2 % paraformaldehyde solution, washed several times to remove traces of formaldehyde and immunofluoresence staining was performed as described previously [21, 31]. Nuclear staining was performed with ToPro-3 (1 uM, Invitrogen). Finally, the sections were mounted using Prolong Gold antifade medium (Invitrogen) and stored at 4 °C for imaging. Tissue sections were stained with one or combination of primary antibodies (Table S1, available in the online version of this article). Confocal imaging was performed with a TCS SP2 confocal laser scanning microscope (Leica, Wetzlar, Germany) equipped with an argon-krypton laser, a krypton laser and a helium-neon laser. Negative controls were performed either by omitting the primary antibody or using isotype control. ImageJ 1.43 u (NIH, USA) and Adobe Photoshop CS5 Extended (ver. 12.0×64, Adobe Systems CA, USA) were used to assign colours to the channels collected and to measure MFI for each fluorochrome. CD58 was expressed by multiple cell types in intestinal tissue. We assessed the source of CD58 in RM jejunum by multi-label confocal microscopy. T cells (CD3⁺), monocytes/macrophages (CD68⁺), B-/plasma cells (CD79a⁺), macrophages (HAM56⁺) and activated cells (Mamu-DR⁺) expressed CD58 in tissues (Fig. 2a–e). Rhesus jejunum maintained a CD58-rich environment and CD58⁺ cells were distributed among LPLs and the intra-epithelial region (Fig. 2a–e). Similar to CD58, CD2⁺ cells were predominantly distributed in LPLs and the majority of cells were positive for T cells (Fig. 2f). Reduction in CD58 expression was shown in jejunum from a representative SIV-infected acute animal (MFI, 2.58; Fig. 2g) compared to SIV-infected chronic (MFI, 5.5; Fig. 2h) and SIV-uninfected control RMs (MFI, 5.76; Fig. 2i).

Fig. 2.

Representative expression of CD58 in SIV-uninfected, control jejunal tissue by multi-label confocal microscopy in multiple cell types including CD3⁺ T cells (a), CD68⁺ monocytes/histiocytes (b), CD79a⁺ B-/plasma cells (c), Ham56⁺ macrophages (d) and Mamu-DR⁺ B-/late-activated cells (e) is shown (scale bar 20 µm). CD2-positive cells are also positive for CD3 and are shown in white arrows (f). Insets in each panel show the pattern of CD2/CD58 expression in conjunction with other cellular markers. Reduction in CD58 expression is shown for a representative SIV-infected acute animal (g), compared to SIV-infected chronic (h) and SIV-uninfected control RMs (i). The labels on each image are indicated on the y-axis.

We then quantified cytokine production in T cells and correlated this with the CD2 and CD58 expression in tissues. To examine cytokine production, a cytokine flow cytometry assay was conducted on freshly isolated samples to detect either CD4⁺ or CD8⁺ T cells producing cytokines (IFNγ, TNFα, IL-2 and IL-17), in the presence of Brefeldin A (BfA) and stimulated for 6 h with PMA/Ionomycin according to methods previously described [1, 22, 23]. Briefly jejunal LPLs were stained with live/dead stain and surface-stained with directly conjugated MAbs to CD3, CD4 and CD8. Cells were washed, fixed and permeabilized using Cytofix/Cytoperm (BD Biosciences), washed twice in perm buffer (BD Biosciences), stained with MAbs to IFNγ (B27), TNFα (Mab11), IL-2 (MQ1-17H12) (BD Biosciences) and IL-17A (eBio64CAP17, Invitrogen) at room temperature for 25 min, fixed and data were then acquired on a BD LSRII instrument. To quantify cytokine (IFNγ, TNFα, IL-2 and IL-17A) responses, a minimum of 50 000 CD3⁺CD8⁺ T cells were acquired and analysed by gating on singlets and lymphocytes, followed by live cells and then on CD3⁺ T cells. CD3⁺ T cells were further gated to define CD4⁺ and CD8⁺ T-cells, which were further analysed for the presence of IFNγ-, TNFα-, IL-2- and IL-17A-positive cells. Positive staining for each cytokine was determined by fluorescence minus one control tubes. Early loss of IL-17A-positive cells was detected in both CD4 and CD8 T cells during acute infection, and remained low during chronic infection (P<0.05, Fig. 3a–c), which is in agreement with previous reports [32, 33]. Similarly, the levels of IL-2- and TNFα-positive CD4+ and CD8+ T cells were also lower during acute SIV infection compared to control RMs (Fig. 3b–c), as previously reported by us [12]. The production of IL-2 and TNFα increased again in both CD4⁺ and CD8⁺ T cells in the chronic phase. No change was noted in IFNγ production in the CD4⁺ population in acute and chronic infected RMs compared to control RMs. However, CD8⁺ cells produced increased levels of IFNγ during both acute and chronic infection, suggesting a compensatory function to combat SIV infection (Fig. 3b–c). Nonparametric Spearman’s rank correlation coefficient between percentage change in CD58 expression and percentages of IL-2 or TNFα cytokines in both CD4⁺ and CD8⁺ T cells from control and SIV-infected RMs indicated a highly significant positive correlation between the changes observed in IL-2 and TNFα levels and CD58 expression (P<0.05, Fig. 3d–e). Despite increased IFNγ production in CD8⁺ T cells during both acute and chronic infection, as well as a concomitant significant reduction in IL-17A⁺ T cells, no significant positive or negative correlation between either IFNγ- or IL-17A-positive cells and CD58 expression was detected (P>0.05). Similarly, no significant correlation was detected between IL-2-, TNFα- or IFNγ or IL-17A-positive cells and CD2 expression in CD4⁺ or CD8⁺ T cells and CD2 or CD58 expression in B cells. Although early cytokine-mediated immune responses can completely eliminate a viral infection [34, 35], SIV and HIV have several means of evading adaptive immune responses [12, 36]. This experimental design does not address other cytokine/chemokine changes that might have a correlation with CD2/CD58 population in the gut and other time points beyond the acute (21 days) and chronic (309–437 days) stages of SIV infection.

Fig. 3.

(a) Representative contour plot showing loss of IL-17A-positive cells from both CD4⁺ and CD8⁺ T cells in acute SIV infection compared to uninfected control animals. The percentages of TNFα- and/or IL-17A-positive cells are shown in each quadrant. Percentages of IFNγ, IL-17A, IL-2 and TNFα expression with mean (± standard error) in jejunal LPL CD4⁺ (b) and CD8⁺ (c) T cells from SIV-uninfected control and acute and chronic SIV_MAC251-infected RMs are shown (n=4). Cells were gated on singlets and lymphocytes, followed by live cells and then on CD3⁺ T cells and subsequently on CD3⁺CD4⁺ and CD3⁺CD8⁺ T-cell subsets. CD3⁺CD4⁺ or CD3⁺CD8⁺ T cells were further analysed for the presence of IFNγ-, IL-2-, IL-17A- and TNFα-positive cells using Flowjo software. All values were subtracted from medium control values. Spearman’s rank correlation coefficient of determination between IL-2 and CD58 percentages (d) and TNFα and CD58 percentages (e) is shown for all uninfected, control and SIV_MAC251-infected macaques, where a positive correlation was detected with the expression of CD58, IL-2 and TNFα by both CD4⁺ and CD8⁺ T cells.

To demonstrate the role of CD58 in regulating IL-2 and TNFα production in T cells, PBMC from seven SIV-uninfected, normal RMs were used for a CD58 blocking experiment where isolated PBMCs were stimulated with either Staphyloccal Enterotoxin B (SEB, a T-cell mitogen, at 1 µg ml⁻¹ final concentration) or SEB⁺anti-CD58 MAb (at 5 µg ml⁻¹ final concentration) along with media control (RPMI-1640+10 % FBS). After 6 h of stimulation in the presence of BfA, cells were stained with conjugated live/dead, anti-CD3, anti-CD4 and anti-CD8 MAbs, and then an intracellular stain with anti-IL-2 and TNFα MAbs as reported previously [1, 22, 23]. Cells were run through a BD Fortessa flow cytometer and data acquisition was performed. IL-2 production was significantly reduced in anti-CD58 MAb⁺SEB-treated CD4 (mean, 0.31 % versus 1.10 %) and CD8 (mean, 0.05 % versus 0.19 %) T cells compared to SEB-treated T cells (Fig. 4a, b). Similarly, a significant reduction in TNFα production was also evident in anti-CD58 MAb⁺SEB-treated CD4 (mean, 1.54 % versus 4.05 %) and CD8 (mean, 1.65 % versus 3.27 %) T cells compared to SEB-treated T-cells (Fig. 4a, b). Our in vitro CD58 MAb blocking experiment performed in PBMC isolated from normal, SIV-uninfected RMs may not represent the responses detected in SIV-infected RMs. However, a significant loss of CD58 expression in T cells in acute SIV-infected macaques, and the anti-CD58 MAb blocking experiment in PBMC culture from normal, SIV-uninfected RMs, suggest that CD58 may have an important role in regulating the early induction of IL-2 and TNFα in controlling SIV/HIV infection during the acute phase. CD2/CD58 interaction in human intra-epithelial lymphocytes is responsible for TNFα production [37]. Blocking of CD2–CD48 interaction by anti-CD48 MAb treatment in an in vitro splenocyte culture inhibited IL-2 and TNFα production from anti-CD3-activated T cells [38]. CD58 blockade also resulted in diminished IL-2 and TNFα production by adaptive human NK cells in response to HCMV-infected cells [11]. Blockage of CD48 expression in mice demonstrated suppression of cell-mediated immunity as well as defects in CD4⁺ T-cell activation [39, 40]. Knockdown of CD2 and CD48 signalling in PBMC T cells demonstrated reduction in IL-2 production due to defective signalling to the TCR/CD3 complex [41].

Fig. 4.

(a) Representative contour plots showing percentages of IL-2 and TNFα expression in CD4 T cells in PBMC following treatment with either SEB (Staphylococcal enterotoxin B) or SEB⁺anti-CD58 antibody (MAb) for 6 h in the presence of Brefeldin A (BfA). The levels of cytokine-positive cells are shown at top right of the gated boxes. FSC denotes forward scatter on the X-axis. (b) Bar diagrams (mean ± standard error) of IL-2 and TNFα cytokines in either SEB or SEB⁺anti-CD58 MAb-treated cultures are shown for both CD4 and CD8⁺T cells in PBMC from seven independent experiments. All values were subtracted from medium control values. The differences in cytokine responses between two treatment groups in both CD4 and CD8 T cells are statistically significant (P<0.05).

In conclusion, we determined a novel correlation between CD58 and cytokine production in our in vivo SIV-infected macaque study, demonstrating that the downregulation of CD58 expression in both intestinal CD4⁺ and CD8⁺ T cells during acute infection may play a key role in reducing IL-2 and TNFα production, thereby potentially compromising the ability of mucosal T cells to combat SIV infection.