The authors have declared that no competing interests exist.
Conceived and designed the experiments: TB FM SSA SC MC MvH. Performed the experiments: TB FM YC MH. Analyzed the data: TB FM. Contributed reagents/materials/analysis tools: SSA SC MC. Wrote the paper: TB FM MC SC MvH.
Current address: University Hospital Freiburg, Department of Gastroenterology and Hepatology, Freiburg, Germany.
Current address: University College London, Division of Infection and Immunity, London, Great Britain.
During acute viral infections, clearance of the pathogen is followed by the contraction of the anti-viral T cell compartment. In contrast, T cell responses need to be maintained over a longer period of time during chronic viral infections in order to control viral replication and to avoid viral spreading. Much is known about inhibitory signals such as through PD-1 that limit T cell activity during chronic viral infection, but little is known about the stimulatory signals that allow maintenance of anti-viral T cells. Here, we show that the co-stimulatory molecule OX40 (CD134) is critically required in the context of persistent LCMV clone 13 infection. Anti-viral T cells express high levels of OX40 in the presence of their cognate antigen and T cells lacking the OX40 receptor fail to accumulate sufficiently. Moreover, the emergence of T cell dependent germinal center responses and LCMV-specific antibodies are severely impaired. Consequently, OX40-deficient mice fail to control LCMV clone 13 infection over time, highlighting the importance of this signaling pathway during persistent viral infection.
A robust T cell response is the hallmark of an effective immune response to a variety of invading viruses. In many acute infections, the clearance of the viral pathogen is associated with a short and vigorous T cell response followed by development of pathogen-specific immune memory. However, some viruses can establish persistent infection in their respective host, during which an ongoing T cell response is required in order to prevent overwhelming viral replication. Little is known about the factors that sustain the T cell response in the persistent phase of a viral infection. In this report, we demonstrate that ligation of the OX40 molecule, which is expressed on T cells targeting the virus, is critically required in order to sustain the anti-viral immune response. We show that virus-specific, OX40-deficient T cells fail to accumulate sufficiently and consequently, mice lacking the OX40 receptor are incapable of controlling viral replication. Collectively our data establish OX40 as a crucial signaling molecule during a persistent viral infection.
Although persistent viral infections are typically associated with a dysfunctional and exhausted T cell signature
Wild type (WT) and OX40-deficient mice (OX40−/−) on a C57BL/6 background were challenged intravenously with 2×106 PFU of LCMV cl13
WT (black circles) and OX40−/− (white circles) mice were intravenously infected with 2×106 PFU of LCMV cl13. (A) Loss of body weight was measured twice weekly as a marker for disease severity. (B, C) Virus-specific splenic T cell populations in WT and OX40−/− mice were analyzed 20 days post infection using GP33-pentamers (B, CD8) and GP66-tetramers (C, CD4). (D–H) Splenocytes of infected mice were harvested 10, 20 and 50 days post infection. Intracellular cytokine stainings (ICCS) were performed following in vitro stimulation with immunodominant CD8 (D–F) and CD4 (G, H) epitopes as illustrated. (I) Representative ICCS staining following GP61 stimulation, gated on CD4 T cells. Data are derived from a total of 7 independent experiments, 1–2 experiments per time point.
Next, we wanted to assess whether the disrupted T cell response in OX40−/− mice would also impact T cell help to B cells. CD4 T cell interactions with B cells are required for the development of germinal centers (GCs) and the emergence of antibody producing plasma cells
(A–H) Follicular T helper cell (Tfh) and germinal center responses were analyzed 8 respectively 20 days post LCMV cl13 infection in WT (black bars and lines) and OX40−/− mice (white bars and dashed lines) and compared to naïve WT mice (grey bars). (A) Number of Bcl6+CXCR5+ Tfh cells per spleen, (B) representative FACS-plots gated on CD4 T cells. (C) Number of LCMV-specific follicular helper CD4 T cells per spleen, (D) representative FACS-plots gated on CD4 T cells. (E) Number of germinal center B cells per spleen, (F) representative FACS-plots gated on B cells showing germinal center B cell populations. (G) Number of plasma cells per spleen, (H) representative FACS-plots gated on B cells showing plasma cell populations. Representative results from 1 of 2–3 independent experiments are illustrated. (I) LCMV-specific IgG antibody titers were analyzed by Elisa on days 10, 20, 50, 80 and 120 post infection (n = 4–10 per group and time point). Data are derived from a total of 7 independent experiments, 1–2 experiments per time point.
While OX40−/− mice benefited clinically from the impaired T cell responses, as they lost markedly less weight than WT mice (
(A) Serum, (B) kidney, (C) lung and (D) liver samples were harvested on days 10, 20, 50, 80 and 120 post infection and viral loads were determined by plaque assay. Statistical analysis for viral titers was performed using log rank test, undetectable viral titers were defined as endpoint. Data are derived from a total of 8 independent experiments, 1–2 experiments per time point.
To analyze the OX40 expression kinetics on virus-specific CD4 and CD8 T cells during acute and persistent LCMV infection, we transferred naïve, congenically marked TCR transgenic (TCRtg) CD4 (smarta, smtg) and CD8 (P14) T cells into WT recipients and analyzed OX40 expression on days 3, 5, 7, and 20 post LCMV Armstrong or LCMV cl13 infection. While OX40 was not expressed on naïve P14 and smtg cells, we found that it was strongly induced on both populations 3 days following acute and persistent LCMV infection (
(A, B) Naïve GP61-specific TCRtg CD4 T cells (smarta, smtg) and GP33-specific TCRtg CD8 T cells (P14) were transferred into WT recipients prior to acute and persistent LCMV infection. OX40 expression on both populations was analyzed before and 3, 5, 7 and 20 days post infection by flow cytometry. (C) PD-1 and OX40 expression was analyzed on virus-specific CD4 T cells (smarta, smtg) 5, 7, 10 and 20 days post LCMV Armstrong and cl13 infection. Data are derived from a total of 7 independent experiments, 1–2 experiments per time point. (D) Equal numbers of CD45.1/2 mismatched WT and OX40−/− P14 cells were co-transferred into WT recipients prior to LCMV cl13 infection. Expression of OX40 and KLRG1 was determined on both populations 8 days post LCMV cl13 infection. FACS-plot gated on P14 T cells. (E) Fold reduction of the OX40−/− P14 compared to WT P14 in the KLRG1-low and KLRG1-high gate was analyzed 8 days post LCMV cl13 infection. Data are derived from a total of 2 independent experiments. (F) Expression of OX40 Ligand was determined by flow cytometry 5 days post infection on T cells (Thy1.2), B cells (CD19) and antigen presenting cell populations (CD11c+Thy1.2-CD19−; CD11b+Thy1.2-CD19−; F4/80+Thy1.2-CD19−) following collagenase digestion. Data are derived from a total of 2 independent experiments. (G and H) Changes in the OX40L expression levels on the same antigen presenting cell populations were examined following LCMV cl13 infection. Isotype MFI was subtracted from OX40L MFI and plotted in relation to expression levels on naïve cells. Time course experiment was performed once.
Next, we analyzed which cells could be capable of engaging the OX40 receptor through expression of the OX40 ligand (OX40L). OX40L is the only ligand that is known to activate the OX40 receptor and is typically expressed on activated antigen presenting cells, but also has been visualized on lymphoid tissue inducer (LTi) cells and activated/inflamed endothelium
To assess the direct impact of OX40 expression on virus-specific CD4 and CD8 T cells, we crossed TCRtg CD4 (smtg) and CD8 (P14) T cells onto the OX40−/− background. We then co-transferred equal numbers of naïve WT (CD45.1+CD45.2−) and OX40−/− (CD45.1+CD45.2+) cells into C57BL/6 (CD45.1−CD45.2+) recipients prior to LCMV cl13 infection or LCMV Armstrong infection. Strikingly, WT CD4 smtg cells accumulated to much higher numbers compared to OX40−/− smtg cells (
(A, B) Equal numbers of WT (CD45.1+CD45.2−) and OX40−/− (CD45.1+CD45.2+) smtg were transferred into the same WT recipients (CD45.1−CD45.2+) prior to LCMV cl13 infection. Splenocytes were harvested on days 5, 8, 10 and 20 post infection (n = 4–8 for each time point) and (A) frequencies of OX40−/− smtg and WT smtg were determined within the CD4 gate by flow cytometry. (B) Frequencies shown in FACS-plots are percent of CD45.1+ (transferred cells); plots are gated on CD4 T cells. (C–E) OX40+/+ (WT, black bars), OX40+/− (Het, grey bars) and OX40−/− (KO, white bars) smtg were separately transferred into WT recipients prior to LCMV cl13 infection and (C) frequencies of WT, Het and KO smtg cells within the CD4 gate were determined 10 days post infection (n = 5 per group). (D) OX40 expression on WT, Het and KO smtg cells was determined 10 days post infection by flow cytometry (n = 5 per group). (E) Representative FACS-plots showing transferred cells and OX40 expression gated on CD4 T cells. (F) Equal numbers of CD45.1/2 mismatched WT and OX40−/− P14 cells were co-transferred into WT recipients prior to LCMV cl13 infection and frequencies of OX40−/− P14 and WT P14 were determined within the transferred P14 population by flow cytometry on day 3, 5, 8 and 20 post infection (n = 4–5 for each time point). (G, H) Similar co-transfer experiments with CD4 and CD8 T cell were performed in order to compare the role of OX40 on a single cell level in acute and chronic LCMV infection. Graph shows percentage of transferred OX40−/− CD4 respectively OX40−/− CD8 T cells of all transferred T-Cells 8 days post infection. Data are derived from 2–3 independent CD4 smtg cell transfer experiments per time point and 1–3 independent CD8 P14 cell transfer experiments per time point.
In order to directly compare the role of OX40 during acute and persistent LCMV infection, we performed co-transfer experiments with WT and OX40−/− TCRtg CD4 and CD8 T cells prior to LCMV Armstrong and cl13 infection. In agreement with the observation of reduced OX40 receptor expression during acute infection (
Signals through OX40 can influence several aspects of T cell biology, such as survival, proliferative capacities and the ability to secrete cytokines
(A) WT and OX40−/− P14 cells were labeled with CFSE prior to injection in WT recipients. Graph is showing CFSE dilution of congenically marked TCR transgenic (WT red, OX40−/− black) and unlabeled endogenous CD8 T cells (grey area) 5 days post infection. (B, C) CD45.1/2 mismatched WT and OX40−/− TCRtg cells were co-transferred into WT recipients prior to LCMV cl13 infection and BrdU was injected intraperitoneally on day 6 post infection according to the manufacturer's instructions. Cells were harvested and BrdU staining was detected by flow cytometry to visualize cells that had proliferated. Data are representative for 1 out of 2 independent experiments. (D) Intracellular cytokine staining (ICCS) for IFN-γ, IL-21 and IL-2 was performed 10 days post infection following peptide stimulation (n = 5 per group). (E) Representative FACS-plots showing IFN-γ and IL-21 production of WT, Het and KO smtg cells. (F) Intracellular cytokine staining (ICCS) for IFN-γ following GP33 peptide stimulation on congenically marked TCR transgenic CD8 T cells (P14) transferred into WT recipients was performed 5 and 8 days post infection (n = 4–5 per group). Data are derived from 2 independent CD4 smtg cell transfer experiments and 2 independent CD8 P14 cell transfer experiments.
(A–D) Fas, Annexin V and active caspase 3 analyses were performed to detect apoptotic signals in co-transferred TCRtg cells (WT red, OX40−/− black) and endogenous CD4 and CD8 T cells (grey area) after LCMV cl13 infection. (A, B) Representative Annexin V stainings and bar graphs showing Annexin V MFI on CD4 smtg cells on day 8 p.i. (A) and on CD8 P14 cells on day 10 p.i. (B). (C) Representative Fas staining on day 8 p.i. and bar graph showing Fas expression 5, 8, and 20 days post infection on CD4 smtg cells. (D) Representative intracellular staining for active caspase 3 and bar graph showing Caspase 3 MFI on day 8 p.i. on CD4 smtg cells. Data are derived from 3 independent CD4 smtg cell transfer experiments and one CD8 P14 transfer experiment. (E) Analysis of expression of anti-apoptotic molecules Bcl-2 and Bcl-xL on virus-specific CD4 T cells of WT and OX40−/− mice (WT red, OX40−/− black) via intracellular protein staining 8 and 20 days post persistent LCMV infection. Data are derived from 1–2 independent experiments per time point.
Co-stimulation of T cells is a central component of adaptive immunity. Numerous co-stimulatory pathways have been described and it has become evident that the biological relevance of each of those pathways is greatly dependent on the immunologic context
Although the role of OX40 has never been studied in a persistent viral infection with ongoing viral replication, much is known about the mechanisms by which OX40 can influence T cell responses. It has been demonstrated that OX40 promotes T cell survival, division and function in various immune models, including cancer models
Although OX40 has primarily been associated with CD4 T cell function, it became evident in recent years that it can also strongly influence CD8 T cells
Another observation of our study that is noteworthy is how OX40 deficiency impacts the antiviral immune response already at early stages post infection, however, loss of viral control in those mice occurs in a delayed fashion. The notion that changes in the immune response can precede differences in viral titers in the LCMV cl13 system has been described before
While OX40 has previously been shown to positively regulate antigen-specific immune responses, the degree to which OX40 influences adaptive immunity and, importantly, facilitates virus control in the context of persistent infection is remarkable. Indeed, the strong impact of OX40 on the cellular and humoral immunity in the persistent LCMV-system is in contrast to previous findings in the acute LCMV system, where the absence of OX40 primarily affected the magnitude of the CD4 T cell response but did not have an impact on the antiviral CD8 T cell response, antibody titers and virus control
While CD8 T cell responses have long been known to be critical for control of persistent LCMV-infection, the relevance of antibody responses in this context may have been underappreciated. Importantly, Pinschewer and colleagues showed that mice that were unable to produce LCMV-specific antibodies failed to control infection
Importantly, these findings could open new paths in the understanding of T cell failure during persistent viral infections in humans, i.e. chronic HCV infection. Particularly HCV-specific CD4 responses are very weak in persistently infected individuals
Collectively, our findings establish OX40 as a key factor in sustaining the cellular and humoral immunity during viral persistence and have important implications for the study of T cell dysfunction in persistent viral infections in humans.
C57BL/6 and B6.SJL mice were purchased from The Jackson Laboratory and housed at the La Jolla Institute (LIAI) as well as OX40−/− mice on a C57BL/6 background
Surface staining was performed on splenic single cell suspension with fluorescently-labeled or biotinylated antibodies against CD4, CD8, CD19, B220, CD150 (SLAM), CXCR5, PD-1, KLRG1, FAS, IgD, CD138, CD45.1, CD45.2, OX40L and fluorescently-labeled PNA as well as GP33-pentamers and GP66-tetramers. Streptavidin- APC and -PE were used to stain biotinylated antibodies. Bcl6, Bcl-2, Bcl-xL and active caspase 3 were stained intracellularly after permeabilization with Cytofix/Cytoperm (BD). For most experiments, LIVE/DEAD (Invitrogen) viability dye was used to exclude dead cells.
For ICCS, cells were stimulated with CD8- (GP33, GP276) and CD4-restricted (GP61) LCMV-epitopes (10 µg/ml) for five hours at 37°C in supplemented RPMI-medium (Invitrogen). Cells were permeabilized using Cytofix/Cytoperm (BD) and stained with fluorescently-labeled antibodies against IL-2, IFN-γ, and TNF. IL-21 staining was performed as described previously
Spleens were harvested from TCRtg mice (smtg or P14) on a WT and OX40−/− background. CD4 (smtg) and CD8 (P14) isolation was performed using purified rat antibodies against B220, CD11c, CD11b, CD16/32, I-A/I-E and CD4 or CD8 and sheep anti-rat Dynabeads (Invitrogen). For co-transfer experiments into the same or separate hosts, equal numbers of OX40+/+(WT), OX40+/−(Het) and/or OX40−/−(KO) cells (2,000 P14, 5,000 smtg; 1,000,000 for d3 analysis) were transferred into CD45.1/2 mismatched recipients. Cells were labeled with CFSE (BioChemika) prior to transfer in selected experiments. For analysis of proliferation at later time points we used the BrdU-Proliferation kit from BD.
Monolayers of Vero cells (ATCC #CRL-1587) were exposed to tissue homogenate or serum of individual mice in 1/10 dilutions. Plaque counts were performed 5 (organs) or 6 (serum) days post infection. Vero cells were cultured and maintained in supplemented DMEM (Invitrogen) at 37°C.
Elisa plates (Nunc) were coated overnight with LCMV infected cell lysate. LCMV-specific IgG was detected in serum samples using HRP goat anti-mouse IgG (Invitrogen). SureBlue Reserve TMB Kit (KPL) functioned as substrate. Absorbance was detected by a Spectra Max M2
Statistical analyses were performed and line art figures were designed using GraphPad Prism 5 software (GraphPad). If not specified otherwise, the unpaired, two tailed Student's t-test was used. For viral loads, the log rank test was applied with a defined endpoint of viral titers below the limit of detection. All error bars are SD. Values of p<0.05 were considered significant. *p<0.05, **p<0.01 and ***p<0.001.