Intrinsic TNF/TNFR2 interactions fine-tune the CD8 T cell response to respiratory influenza virus infection in mice.

TNF is an important inflammatory mediator and a target for intervention. TNF is produced by many cell types and is involved in innate inflammation as well as adaptive immune responses. CD8 T cells produce TNF and can also respond to TNF. Deficiency of TNF or TNFR2 has been shown to affect anti-viral immunity. However, as the complete knockout of TNF or its receptors has effects on multiple cell types as well as on lymphoid architecture, it has been difficult to assess the role of TNF directly on T cells during viral infection. Here we have addressed this issue by analyzing the effect of CD8 T cell intrinsic TNF/TNFR2 interactions during respiratory influenza infection in mice, using an adoptive transfer model in which only the T cells lack TNF or TNFR2. During a mild influenza infection, the capacity of the responding CD8 T cells to produce TNF increases from day 6 through day 12, beyond the time of viral clearance. Although T cell intrinsic TNF is dispensable for initial expansion of CD8 T cells up to day 9 post infection, intrinsic TNF/TNFR2 interactions potentiate contraction of the CD8 T cell response in the lung between day 9 and 12 post infection. On the other hand, TNF or TNFR2-deficient CD8 T cells in the lung express lower levels of IFN-γ and CD107a per cell than their wild type counterparts. Comparison of TNF levels on the TNFR2 positive and negative T cells is consistent with TNF/TNFR2 interactions inducing feedback downregulation of TNF production by T cells, with greater effects in the lung compared to spleen. Thus CD8 T cell intrinsic TNF/TNFR2 interactions fine-tune the response to influenza virus in the lung by modestly enhancing effector functions, but at the same time potentiating the contraction of the CD8 T cell response post-viral clearance

CD30 Is Dispensable for T-Cell Responses to Influenza Virus and Lymphocytic Choriomeningitis Virus Clone 13 but Contributes to Age-Associated T-Cell Expansion in Mice

CD30 is a tumor necrosis factor receptor (TNFR) family member whose expression is associated with Hodgkin’s disease, anaplastic large cell lymphomas, and other T and B lymphoproliferative disorders in humans. A limited number of studies have assessed the physiological role of CD30/CD30 ligand interactions in control of infection in mice. Here, we assess the role of CD30 in T-cell immunity to acute influenza and chronic lymphocytic choriomeningitis virus (LCMV) clone 13 infection, two viral infections in which other members of the TNFR superfamily are important for T-cell responses. We show that CD30 is expressed on activated but not resting CD4 and CD8 T cells in vitro, as well as on regulatory T cells and marginally on T helper 1 cells in vivo during influenza infection. Despite this, CD4 and CD8 T-cell expansion in response to influenza virus was comparable in CD30+/+ and CD30−/− littermates, with no discernable role for the pathway in the outcome of influenza infection. Similarly, during persistent infection with LCMV clone 13, CD30 plays no obvious role in CD4 or CD8 T-cell responses, the level of T-cell exhaustion or viral control. In contrast, in the steady state, we observed increased numbers of total CD4 and CD8 T cells as well as increased numbers of regulatory T cells in unimmunized older (~8 months) CD30+/+ but not in CD30−/− age-matched littermates. Naive T-cell numbers were unchanged in the aged CD30+/+ mice compared to their CD30−/− littermate controls, rather the T-cell expansions were explained by an increase in CD4+ and CD8+ CD44mid-hiCD62L− effector memory cells, with a similar trend in the central memory T-cell compartment. In contrast, CD30 did not impact the numbers of T cells in young mice. These data suggest a role for CD30 in the homeostatic regulation of T cells during aging, contributing to memory T-cell expansions, which may have relevance for CD30 expression in human T-cell lymphoproliferative diseases

TNFR2 expression is associated with reduced TNF production.

<p>OT-I and TNFR2^-/- OT-I cells recovered from lung and spleen 10 days post-infection as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068911#pone-0068911-g002" target="_blank">Figure 2</a> were restimulated at 37⁰C with OVA_257-64 peptide and Golgi Stop for five to six hours. OT-I cells were then stained for surface TNFR2, followed by staining of intracellular TNF. (<b>A</b>) Representative FACS plot of TNFR2 expression on OT-I cells, using TNFR2^-/- OT-I cells as a control, and TNF production by WT TNFR2⁺, WT TNFR2^- and TNFR2^-/- OT-I subsets. (<b>B</b>) Summary of TNF production in lung and spleen. Each symbol represents one mouse.</p

CD8 T cell intrinsic TNF enhances both IFN-γ production and degranulation.

<p>WT, TNF^-/- and TNFR2^-/- OT-I cells recovered from infected lungs and spleens as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068911#pone-0068911-g002" target="_blank">Figure 2</a>, were restimulated at 37⁰C with OVA_257-64 peptide and Golgi Stop for five to six hours. Cells were then analyzed for production of IFN-γ and expression of CD107a. (<b>A</b>) Representative FACS plots of IFN-γ and CD107a staining from day 9 post-infection in the lung. Cells that were not restimulated (No Antigen) as well as FMO staining were used as controls. (<b>B</b>) Percentage of OT-I cells that are positive for IFN-γ and CD107a on day 9 in the lung. (<b>C</b>) MFI of IFN-γ and CD107a for IFN-γ- and CD107a-positive OT-I cells, respectively. (<b>D</b>) MFI of IFN-γ and CD107a from spleen samples on day 9. Data are representative of five independent experiments for day 9-12 for TNF^-/- OT-I cells, and three for TNFR2^-/- OT-I cells, each using four mice per group.</p

Figure 1

<div><p><b>Characterization of adoptive transfer model with respect to weight loss, viral clearance</b> and <b>TNF/TNFR expression.</b></p> <p>(<b>A</b>) Ten thousand purified CD8 T cells from CD45.1⁺ OT-I or TNF^-/- CD45.1⁺ OT-I mice were injected intravenously into CD45.2⁺ wild type mice. A day later, the mice were infected with X31-OVA. (<b>B</b>) Infected recipients of either CD45.1⁺ OT-I or TNF^-/- CD45.1⁺ OT-I cells were monitored over time for weight loss, indicated as a percentage of original body weight. Data are representative of three independent experiments. (<b>C</b>) Lungs were excised from the animals at the indicated time points and analyzed for viral load as described in the methods. Data are representative of two independent experiments for day 3 and one for each day 6, 9 and 10, each using three to five mice per group. Day 3 and 6 or day 3 and 9 were done in the same experiment and results combined to show kinetics. (<b>D</b>, <b>E</b>) Cells from lung or spleen were restimulated with OVA_257-64 peptide and Golgi Stop as described in the methods, surface stained for CD8 and CD45.1, fixed and then intracellularly stained for TNF, with representative FACS plots of TNF-positive cells, previously gated on CD8⁺ CD45.1⁺ cells (<b>D</b>) and the percentage of TNF⁺ OT-I cells summarized (<b>E</b>). For detailed gating strategy see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068911#pone.0068911.s001" target="_blank">Figure S1</a>. Data are representative of two to four independent experiments for each time point, each using three to four mice per group. Data are pooled from experiments with day 6 and 9 or day 6 and 12 done in the same experiment. Error bars indicate SEM. (<b>F</b>) Representative histograms of TNFR2 and TNFR1 expression on lung and spleen OT-I cells day 7 post-infection. The dark line shows TNFR2 and TNFR1 on WT cells, and the shaded grey line represents TNFR2^-/- OT-I cells, or TNFR1 on the FMO sample. Data are representative of three independent experiments, each using three mice per group.</p></div

T cell intrinsic NOD2 is dispensable for CD8 T cell immunity.

NOD2 is an intracellular pattern recognition receptor that provides innate sensing of bacterial muramyl dipeptide by host cells, such as dendritic cells, macrophages and epithelial cells. While NOD2's role as an innate pathogen sensor is well established, NOD2 is also expressed at low levels in T cells and there are conflicting data as to whether NOD2 plays an intrinsic role in T cell function. Here we show that following adoptive transfer into WT hosts, NOD2(-/-) OT-I T cells show a small decrease in the number of OVA-specific CD8 T cells recovered at the peak of the response to respiratory influenza virus infection. On the other hand, no such defect was observed upon intranasal immunization with a replication defective adenovirus carrying the OVA epitope recognized by OT-I, or when OVA was delivered with LPS subcutaneously, or when influenza-OVA was delivered intraperitoneally. Thus we observed a selective defect in NOD2-deficient T cell responses only during a live viral infection. Moreover, there was no apparent defect when NOD2(-/-) OT-I T cells were stimulated in vitro. Finally, this selective defect in recovery of NOD2-deficient CD8 T cells was not observed in a non-transgenic respiratory infection model in which mixed bone marrow chimeras were used such that the NOD2(-/-) T cells were allowed to develop and respond in a NOD2-sufficient host. Taken together our data indicate that T cell intrinsic NOD2 is not required for CD8 T cell responses to antigen delivered under a variety of conditions in vitro and in vivo. However, CD8 T cells that have developed in the absence of NOD2 show a selective and modest impairment in their response to live respiratory influenza infection

Intrinsic TNF/TNFR2 signals contribute to contraction of the CD8 T cell response during influenza infection.

<p>CD45.1⁺ OT-I or TNF^-/- CD45.1⁺ OT-I cells were transferred into wild type mice, and intranasally infected with X31-OVA one day later. Recovery of OT-I cells from the mediastinal lymph node, spleen and lung was determined. The naïve mouse did not receive transferred cells. (<b>A</b>) Representative gating of OT-I cells from the lung on day 9 and 12 post-infection. (<b>B</b>) Summary of the percentage of OT-I cells of CD8 T cells recovered at the indicated time points. (<b>C</b>) The absolute number of OT-I cells shown here was calculated by total organ cell count multiplied by the proportion of live, CD8⁺ CD45.1⁺ cells. (<b>D</b>) Day 12 lung data from panel C are shown on an expanded scale to highlight the difference in the number of OT-I cells. Data are representative of four independent experiments for day 6, two for day 9 and three for day 12, each using three to five mice per group. Data were collected with day 6 and 9 or day 9 and 12 done in the same experiment. (<b>E</b>, <b>F</b>) CD45.1⁺ OT-I and TNFR2^-/- CD45.1⁺ OT-I cells were transferred into wild type mice, followed by intranasal infection with X31-OVA one day later. Recovery of OT-I cells in the lung and spleen was determined on day 12. (<b>E</b>) Representative FACS plots of OT-I cells from the lung and spleen. (<b>F</b>) Summary of the percentage and absolute number of OT-I and TNFR2^-/- OT-I cells of CD8 T cells in the spleen (left) and lung (right). Data in E, F are representative of three independent experiments, each using three to five mice per group.</p

LTβR signaling in dendritic cells induces a type I IFN response that is required for optimal clonal expansion of CD8+ T cells

During an immune response, antigen-bearing dendritic cells (DCs) migrate to the local draining lymph node and present antigen to CD4+ helper T cells. Antigen-activated CD4+ T cells then up-regulate TNF superfamily members including CD40 ligand and lymphotoxin (LT)αβ. Although it is well-accepted that CD40 stimulation on DCs is required for DC licensing and cross-priming of CD8+ T-cell responses, it is likely that other signals are integrated into a comprehensive DC activation program. Here we show that a cognate interaction between LTαβ on CD4+ helper T cells and LTβ receptor on DCs results in unique signals that are necessary for optimal CD8+ T-cell expansion via a type I IFN-dependent mechanism. In contrast, CD40 signaling appears to be more critical for CD8+ T-cell IFNγ production. Therefore, different TNF family members provide integrative signals that shape the licensing potential of antigen-presenting DCs

NOD2 deficient TCR Transgenic CD8 T cells have a normal percentage of IL-7α^Lo KLRG-1^Hi and IL-7α^Hi KLRG-1^Lo cells at the peak of respiratory influenza infection.

<p>10⁴ purified CD8 T cells from CD45.1⁺ WT or CD45.1⁺ NOD2^−/− OT-I mice were injected i.v. into naïve WT mice, and one day later mice were intranasally infected with Influenza A/X31-OVA as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056014#pone-0056014-g001" target="_blank">Figure 1</a>. On day 9, mice were sacrificed and the percent of SLEC (IL-7Rα^Lo KLRG-1^Hi) and MPEC (IL-7Rα^Hi KLRG-1^Lo) were analyzed on CD45.1⁺ OT-I recovered from the lungs, spleens and mediastinal lymph nodes (A, B). The data in A and B show the combined results of two independent experiments, each with 5 mice per group.</p

NOD2 is dispensable for TCR transgenic CD8 T cell expansion following intranasal delivery of adenoviral vectors.

<p>10⁴ purified CD8 T cells from CD45.1⁺ WT and CD45.1⁺ NOD2^−/− OT-I T cells were injected i.v. into naïve WT mice, and one day later mice were treated with 10⁹ pfu Ad-OVA intranasally. (A) On day 10, mice were sacrificed and the percent of OT-I out of the CD8 T cell population as well as the total number of OT-I T cells in the lungs, spleens and mediastinal lymph nodes were analyzed. (B) The effector function of the transferred OT-I T cells was analyzed by CD107a, TNFα, and IFNγ staining following a 5-hour restimulation with 1 µM SIINFEKL peptide. The data in A and B are summary of two experiments, each with 4–5 mice per group.</p