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. 2012 Jan 16;209(1):77-91.
doi: 10.1084/jem.20110675. Epub 2011 Dec 19.

Loss of the signaling adaptor TRAF1 causes CD8+ T cell dysregulation during human and murine chronic infection

Chao Wang  1 , Ann J McPherson, R Brad Jones, Kim S Kawamura, Gloria H Y Lin, Philipp A Lang, Thanuja Ambagala, Marc Pellegrini, Thomas Calzascia, Nasra Aidarus, Alisha R Elford, Feng Yun Yue, Elisabeth Kremmer, Colin M Kovacs, Erika Benko, Cecile Tremblay, Jean-Pierre Routy, Nicole F Bernard, Mario A Ostrowski, Pamela S Ohashi, Tania H Watts
Affiliations

Loss of the signaling adaptor TRAF1 causes CD8+ T cell dysregulation during human and murine chronic infection

Chao Wang et al. J Exp Med. .

Abstract

The signaling adaptor TNFR-associated factor 1 (TRAF1) is specifically lost from virus-specific CD8 T cells during the chronic phase of infection with HIV in humans or lymphocytic choriomeningitis virus (LCMV) clone 13 in mice. In contrast, TRAF1 is maintained at higher levels in virus-specific T cells of HIV controllers or after acute LCMV infection. TRAF1 expression negatively correlates with programmed death 1 expression and HIV load and knockdown of TRAF1 in CD8 T cells from viral controllers results in decreased HIV suppression ex vivo. Consistent with the desensitization of the TRAF1-binding co-stimulatory receptor 4-1BB, 4-1BBL-deficient mice have defects in viral control early, but not late, in chronic infection. TGFβ induces the posttranslational loss of TRAF1, whereas IL-7 restores TRAF1 levels. A combination treatment with IL-7 and agonist anti-4-1BB antibody at 3 wk after LCMV clone 13 infection expands T cells and reduces viral load in a TRAF1-dependent manner. Moreover, transfer of TRAF1(+) but not TRAF1(-) memory T cells at the chronic stage of infection reduces viral load. These findings identify TRAF1 as a potential biomarker of HIV-specific CD8 T cell fitness during the chronic phase of disease and a target for therapy.

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Figures

Figure 1.
Figure 1.
Loss of TRAF1 protein from HIV-specific T cells during chronic HIV infection. Freshly thawed PBMCs from HIV-infected individuals were stained for CD8, CD3, TRAF1, HIV-tetramer, and CD38 (a-d), and flow cytometry data were analyzed with the gating strategy described in Fig. S1. (a) Frequency of TRAF1+ HIV-specific CD8 T cells in recently or chronically infected donors or viral controllers (groups defined in Table S1). (top) Summary of the frequency of TRAF1+ HIV-tetramer+ cells for all donors analyze with each symbol representing a single epitope, with one to two epitopes per donor. Statistical analysis was performed by one-way ANOVA. (bottom) Representative histograms from each group, with shaded panels indicating FMO controls, open histograms indicating TRAF1 staining on CD3+CD8+tetramer+ T cells. Note: some donors showed bimodal staining for TRAF1 on their HIV-specific T cells. (b) Correlation between the frequency of TRAF1+ HIV-specific T cells and the level of activation as measured by the frequency of CD38+ HIV-specific T cells. Both viral controllers and chronic progressors are included in this analysis. Statistical analysis was performed using linear regression (P = 0.009). (c) TRAF1 expression in HIV-specific T cells using longitudinal samples from three donors with five HIV-specific epitopes. (top) Representative TRAF1 staining during early and later time point within the same donor (shown for two donors); (bottom) Summary plot with each donor represented by a different symbol and filled and open symbols distinguishing two epitopes within the same donor. FMO controls are indicated in the shaded histograms. (d) Frequency of TRAF1+ T cells of total CD8 T cells for the three groups of donors. (e) TRAF1 expression in total CD8+CD45RA− T cells before and after activation. Each symbol represents an individual donor. PBMCs were labeled with CFSE and stimulated with 1 µg/ml of anti-CD3 and 10 µg/ml of anti-CD28 for 6 d, and then stained for CD3, CD8, and TRAF1. The frequency of TRAF1+ T cells on the CFSE low (divided) CD8+CD45RA− population is reported for the stimulated cells (right axis). For the unstimulated cells, the frequency of TRAF1+ in the CD8+CD45RA− T cell population is reported (left axis). VC, viral controller.
Figure 2.
Figure 2.
TRAF1 expression negatively correlates with PD-1 levels and viral load. PBMCs from HIV-infected individuals were stained for CD3, CD8, HIV-tetramer, PD-1, and TRAF1. (a, top) Percentage of PD-1+ HIV-specific CD8 T cells is plotted against percentage of TRAF1+ HIV-specific CD8 T cells from the same chronically HIV-infected donors, with each symbol representing a single epitope from an individual donor. (bottom) Representative FACS plots of PD-1 and TRAF1 expression gated on HIV-tetramer+ CD8 T cells from three representative donors. Note: if we remove the extreme value point for the one donor with low PD-1 and high TRAF1, the correlation becomes r = −0.48, with a P value of 0.038. (b) Percentage of TRAF1 expression in HIV-specific CD8 T cells from the chronic stage of HIV infection, including both chronically infected donors and viral controllers, is plotted against log viral load recorded for each donor in Table S1. Each data point represents a single donor.
Figure 3.
Figure 3.
TRAF1 is required for HIV-specific CD8 T cell responses. (a) CD8 and CD4 T cells were separately purified from HIV+ or HIV− donors. CD4 T cells were infected with a primary isolate of HIV and at 48 h, co-cultured with their autologous CD8 T cells that had been transfected with either TRAF1-specific siRNA or a control scrambled RNA (ctrl). Irradiated autologous PBMCs were added as a source of APC. The frequency of Gag+ T cells was measured 5–7 d later by flow cytometry using CD3-, CD8-, and GAG-specific antibodies to assess the proportion of infected CD4 T cells. As CD4 is down-regulated on the infected cells, the absence of CD8 is used to determine the CD4 T cell population. Representative flow cytometry plots are shown in Fig. S2. (left and middle) representative suppression curves (based on three to five replicates at each effector-to-target ratio for each donor and representative of three viral controllers and a healthy uninfected control). Statistical significance was determined by linear regression of percentage of Gag+ T cells against log (effector: target ratio) using GraphPad (Prism) software. (right) Representative Western blot analysis of TRAF1 levels after knockdown, determined at 48 h after activation. (b). (left and middle) Viral suppression assay performed as in a. Bim-specific siRNA, TRAF1-specific siRNA, or both, or control scrambled RNA were used to transfect CD8 T cells from two viral controllers. CD8 T cells were plated at a ratio of 1:1 with infected CD4 T cells as in a. Open symbols on the right of each panel indicate the percentage of Gag expression in the CD4 T cells if no CD8 T cells were added at all. Cells were harvested for analysis of percentage of Gag+ CD4 T cells (CD8− T cells) after 7 d of co-culture. Statistical analysis was performed using one-way ANOVA. (right) Representative Western blot analysis of Bim levels after knockdown, determined at 48 h after activation. (c) Purified CD8 T cells from viral controllers were nucleofected with either control RNA or TRAF1 siRNA and incubated with autologous monocytes that had been pulsed with control or HIV peptide and pretreated with replication defective adenovirus expressing 4-1BBL or CD80 at a multiplicity of infection of 200, as previously described (Bukczynski et al., 2004). 8 d later, the cells were harvested for FACS analysis. (top left) Representative Western blot analysis of TRAF1 levels at the end of the 8-d culture. (bottom) Representative FACS plots. (top right) Summary plot for three experiments with cells from two different donors, shown as the number of HIV-tetramer+ CD8 T cells recovered in the TRAF1 siRNA-transfected population over those transfected with control RNA after stimulation with HIV peptide-pulsed 4-1BBL or CD80-expressing monocytes.
Figure 4.
Figure 4.
TRAF1 protein is selectively lost from antigen-specific CD8 T cells during chronic, but not acute, LCMV infection. Mice were infected with either acutely infecting LCMV Armstrong (A) or chronically infecting clone 13 (C). Tetramer+ (gp33+ gp276+ NP396+) CD8 T cells were sorted on day 7 and 21 after infection, and the pooled purified tetramer+ T cells were subjected to either Western blot analysis or semiquantitative RT-PCR analysis. (a) Representative blots of TRAF1, TRAF2, and actin protein levels (left) and summary of TRAF1 and TRAF2 levels normalized to actin for tetramer+ T cells isolated from individual mice at the times indicated for the two different viral infections (right). Total CD8 T cells were sorted from uninfected mice (U) as a control. (b) mRNA levels of TRAF1 relative to Actin on the sorted T cells for the indicated viruses and times of infection. Data are representative of three (for protein) or two (for mRNA) independent mouse experiments. Right side of the gel in (b) indicates a titration of the template, indicating nonsaturation of the signal.
Figure 5.
Figure 5.
4-1BBL is required for viral control early but has limited impact late in LCMV clone 13 infection. (a) Analysis of WT versus 4-1BBL−/− mice 8 d after LCMV clone 13 infection. (top) Percentage of LCMV-tetramer+ CD8 T cells in blood. Each data point represents one mouse. Data are pooled from three independent experiments. (bottom) Viral titer as measured in lung and kidney. Data are representative of two independent experiments. (b) Kinetic analysis of the frequencies of NP396-specific T cells in the blood of WT and 4-1BBL−/− mice after LCMV clone 13 infection. Note that the day 8 data from this experiment were included in the compiled day 8 data in panel a. (c) Analysis of WT versus 4-1BBL−/− mice 60 d after LCMV clone 13 infection. (left) Percentage of GP33-tetramer+ CD8 T cells in spleen. Data are pooled from two independent experiments. (right) Viral titers in kidney. Data are representative of two independent experiments.
Figure 6.
Figure 6.
TGFβ regulation of TRAF1 levels in T cells. (a) Mice were infected with LCMV clone 13 for 21 d. 200 µg/mouse of anti-TGFβ1 or control antibody was injected i.p. on day 21. On day 24, CD8 T cells were purified from total splenocytes, stained, and sorted for tetramer+PD-1+ CD8 T cells (GP33 + GP276 + NP396) and subjected to Western blot analysis. Left panel shows a representative blot, and the right panel shows the summary of results with each symbol representing a single mouse from the same experiment. A similar increase in TRAF1 upon TGFβ blockade was obtained in two additional smaller experiments. (b) Splenocytes from OT-1 mice were stimulated with SIINFEKL peptide for 36 h with or without TGFβ. Live CD8 T cells were purified and lysed for Western blotting for TRAF1, TRAF2, and Actin. Data are representative of 3 experiments. (c) Splenocytes from OT-1 mice were stimulated with SIINFEKL peptide for 20 h with or without TGFβ, followed by addition of 1 µg/ml of CHX in the continued presence of TGFβ. Note that if TGFβ was added at the same time as CHX, without the pretreatment, it did not cause loss of TRAF1 (not depicted). Cells were harvested at 0, 1, 2, 3, and 4 h after drug treatment. Live CD8 T cells were purified, and then subjected to Western blot. Data are representative of two experiments. (d) Same as in c, except that 25 nM of chloroquine (Clq) or 25 nM of lactacystin (Lac) was added to the cells at the time of CHX addition, where indicated. Cells were harvested and purified at 2 h after drug treatment. Data are representative of two experiments.
Figure 7.
Figure 7.
Cytokine regulation of TRAF1 levels in T cells. (a) Purified CD8 T cells from healthy donors were stimulated with either anti-CD3 (1 µg/ml)/CD28 (10 µg/ml) as a positive control or 20 ng/ml of IL-2, IL-7, IL-15, or IL-21, or media alone for 6 d. At day 6, cells were analyzed by flow cytometry. The right graph shows summary data, gated on CD45RA− CD8+ T cells, reported as the difference in median fluorescence intensity (dMFI) of TRAF1 compared with FMO controls, with each symbol representing a different donor. Statistical analysis was performed using one-way ANOVA. (b) PBMCs from HIV-infected donors were CFSE-labeled and incubated with cytokines as described in a. Data are shown gated on CD45RA−CFSElow (divided) CD8 T cells except for unstimulated samples, which were gated on CD45RA− CD8 T cells, as they did not undergo division. Data are reported as dMFI relative to FMO controls averaged for four different donors per group. Statistical analysis was performed using one-way ANOVA within each group of HIV donors (early, viral controller, and chronic). (c) TRAF1 levels in HIV-specific CD8 T cells in response to IL-7. To detect HIV-specific CD8 T cells over time, purified CD8 T cells were expanded in response to autologous monocytes pulsed with respective HIV peptides and 4-1BBL-AdV for 8 d with or without IL-7, as described in Materials and methods. The dMFI of TRAF1 against FMO in HIV-specific CD8 T cells for four donors, two early and two chronic, are reported. (d) Mice were infected with LCMV clone 13. At day 21-after infection, mice were treated with 10 µg/mouse of IL-7 or PBS. Tetramer+ and PD-1− CD8 T cells were sorted on day 23 as described in Fig. 4 and lysed for Western blot. CD8 T cells from uninfected TRAF1−/− and WT mice were used as controls. The left plot shows representative data and the bottom right plot shows the summary of TRAF1/actin ratio on the sorted cells which each data point representing an individual mouse. Data in d are the summary of two independent mouse experiments.
Figure 8.
Figure 8.
Combined treatment with IL-7 and agonistic anti–4-1BB increases the number of functional CD8 T cells and decreases viral load in a TRAF1-dependent manner. Mice were infected with LCMV clone 13 for 21 d and treated with either IL-7 alone, agonistic anti–4-1BB (3H3) alone, or in combination as indicated, using the following treatment regimen: 10 µg/mouse of IL-7 on day 21, 23, and 25, and 100 µg/mouse of 3H3 on day 25. Mice were sacrificed on d30 (a and b) or day 37 (c). (a) The number of tetramer+ CD8 T cells specific for LCMV epitopes was examined for each treatment group, with each data point representing a single mouse. (b) Splenocytes from mice in each group were subjected to LCMV peptide restimulation with brefeldin A, monensin, and anti-CD107a or isotype control for 6 h. Cells were then harvested for surface and intracellular staining and FACS analysis. (c) Organs were harvested at day 37, and viral titers were measured. Data in a–c are representative of three similar experiments for tetramer analysis and viral clearance, the latter presented as the median of seven or eight individual mice. The dotted line indicates the limit of detection of the assay. (d) WT and TRAF1−/− mice were treated with IL-7/anti–4-1BB or left untreated as described in a. At day 37 (12 d after treatment), the number of gp33-specific CD8 T cells were enumerated in the spleen (left). Viral titers were evaluated in spleen and liver (right). Data in d are pooled from two independent experiments with 7 TRAF1−/− and 10 WT mice per group.
Figure 9.
Figure 9.
Transfer of TRAF1-expressing memory T cells enhances viral control. WT P14 or TRAF1−/− P14 splenocytes were stimulated with GP33 peptide, and then washed and incubated with IL-15 for 6 d to generate memory like T cells as described in the Materials and methods. (a) Western blot analysis of TRAF1 levels in P14.TRAF1−/− and P14.WT T cells before transfer, compared with TRAF1 levels in LCMV tetramer+ (GP33, GP276, and NP396) CD8 T cells sorted from LCMV clone 13–infected mice at day 21 after infection (labeled as d21.tet+ in blot). (b and c) Mice were infected with 2 × 106 PFU/mouse LCMV clone 13. On day 21 post-infection, one million in-vitro-generated memory P14.WT or P14.TRAF1−/− T cells were transferred via the intravenous route. Mice were sacrificed 14 d later for analysis of T cells and viral titers. (b) Representative FACS plots are shown for GP33-specific CD8 T cell function as measured by production of IFN-γ and TNF in response to GP33 peptide restimulation as outlined in Materials and methods. Similar results were observed in three mice. In a separate and independent experiment, a higher percentage of GP33-tetramer+ CD8 T cells were found in P14.WT-transferred mice as compared with the P14.TRAF1−/−-transferred mice or mice without transfer. (c) Viral titer in kidney at 2 wk after P14 T cell transfer. Data are pooled from two independent experiments with a total of six mice without transfer, five mice with P14.WT transfer, and five mice with P14.TRAF1−/− transfer.
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