Type 1 interferon licenses naïve CD8 T cells to mediate anti-viral cytotoxicity

Abstract

Naïve T cells, unlike memory T cells, exhibit very limited effector function in response to cognate antigen, but exposure to type 1 interferon (IFN) prior to cognate antigen allows for rapid manifestation of effector functions. A full assessment of the functions of these IFN-sensitized otherwise naïve T cells has not been made, nor has their capacity to be effector cells in vivo. We describe here that IFN-sensitized naïve T cells in the absence of cognate antigen adopt a partial activated phenotype distinguished by the upregulation of the surface activation marker CD69, effector-associated transcription factors Eomes and IRF4, and cytotoxicity effector molecule granzyme B. IFN-sensitized naive T cells lysed target cells in vivo and responded to low concentrations and affinities of cognate ligands. We suggest that this rapid and sensitive effector function of IFN-conditioned naïve CD8 T cells may play a role in pathogen control and help ward off superinfections.

Graphical Abstract

Introduction

High levels of type 1 interferons (IFN) are induced during infections with viruses and other pathogens, and they control infections by directly inhibiting viral replication and by modulating immune system functions (1-3). CD8 T cells are efficient regulators of viral infection, and type 1 IFN can contribute to the activation, differentiation, and proliferation of these cells by being a signal 3 cytokine (4-11). This contribution by IFN normally occurs after a naïve T cell encounters its MHC-expressed cognate peptide ligand (pMHC; signal 1) and co-stimulation by molecules such as B7.1 and 2 (signal 2). These T cells then bathe in the signal 3 inflammatory environment, which over 2-3 days drives naïve T cell proliferation and differentiation into effector cells. This requirement for cell division prior to effector function has now been challenged by some recent studies (12, 13), and we have shown that prior, or out-of-sequence, exposure to type 1 IFN before exposure of naïve T cells to signals 1 and 2 can drive T cells down an alternative differentiation pathway (14). Although the degree of clonal expansion is impaired in out-of-sequence CD8 T cells, they quickly become effector cells in regards to the synthesis of the transcription factor (TF) eomesodermin (Eomes) and the ability to produce IFNγ after exposure to their cognate ligand, much like the rapid activation of effector function seen with memory T cells (14-16). This may mean that only the T cells that get pMHC-engaged in the first one or two days of a viral infection would be expected to undergo the canonical differentiation pathway, whereas others, whether they be latecomer virus-specific T cells or T cells responding to a super-infecting pathogen, would be sensitized by type 1 IFN to become instant effector cells. Presumably, T cells derived from persistently infected or autoimmune hosts chronically producing type 1 IFN would be similarly affected and be activated through this non-canonical pathway. A full assessment of the functions of these IFN-sensitized otherwise naïve T cells has not been made, nor has their capacity to be effector cells in vivo. We define here the activation parameters of this type 1 IFN-induced sensitization and show that these sensitized naïve T cells elicit effector functions and lyse target cells in vivo. Further, these sensitized T cells can respond to low concentrations and affinities of cognate ligands, suggesting that sufficient numbers of non-proliferating naïve T cells may become functional in vivo to contribute to viral control.

Materials and Methods

Mice

C57BL/6J (WT B6) male mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Congenic (Ly5.1 or Thy1.1) P14 and OT-1 TCR-transgenic, B7.1/B7.2 double KO (B7 KO), and TAP1/TAP2 double KO (TAP KO), were bred in the Department of Animal Medicine at the University of Massachusetts Medical School (UMMS). All mice were maintained in accordance with the guidelines of the Institutional Animal Care and Use Committee of the UMMS.

Peptides and poly(I:C)

Poly(I:C), purchased from InvivoGen (San Diego, CA), was diluted in HBSS for a concentration of 1 μg/μl. Mice were inoculated with 200 μl HBSS or 200 μg poly(I:C) i.p. and spleens were harvested 1 day (18-22 hours) after treatment. Peptides used for intracellular cytokine staining and labeling splenocytes for in vivo cytotoxicity assay include GP33 (KAVYNFATC), K3L (YSLPNAGDVI), OVA (SIINFEKL) and altered peptide ligands Y3, T4, V4, and G4 (SIYNFEKL, SIITFEKL, SIIVFEKL, and SIIGFEKL) (26). Cells were incubated at a concentration of 1 μM unless otherwise noted.

Adoptive transfers of splenocytes

Ly5.1 OT-1 or P14 splenocytes were isolated, removed of red blood cells by lysis with 0.84% ammonium chloride, and washed with HBSS. A total of 1-3×10⁷ transgenic splenocytes were resuspended in HBSS and transferred i.v. into Ly5.2 congenic mouse recipients.

In vivo cytotoxicity assay

Congenic P14 CD8 T cells were adoptively transferred into B6 mice as described in materials and methods. Mice were either inoculated with HBSS or poly(I:C) for ~1 day, followed by target cell transfer. Leukocytes from B6 mice were pulsed with 1 μM peptide at 37°C, 5% CO₂ for 1 hour. After peptide labeling, cells were dual labeled with 1 μM CellTrace Far Red DDAO (Molecular Probes), and various concentrations of CellTrace Violet (Molecular probes) to differentiate cells labeled with different peptides. Peptide pulsed target cells were adoptively transferred into recipient mice, and splenocytes were harvested ~20 hours post transfer. Specific lysis was calculated by the following formula:

$$\%\text{specific lysis}=100-((\text{Experimental}∕\text{Control})∕\text{Control}\times 100)$$

Surface, transcription factor, and intracellular cytokine staining

Splenocytes were stained with a combination of fluorescently labeled monoclonal antibodies (MAb) specific for CD8α (53-6.7), CD8β (YTS156.7.7), Vα2 TCR (B20.1), Ly5.1 (A20), Thy1.1 (HIS51), CD44 (IM7), CD127 (A7R34), CD62L (MEL-14), CD69 (H1.2F3), IFNAR1 (MAR1-5A3), and CD86 (GL1) for 20 min at 4°C. Cells were fixed with BD Cytofix for 5 min at RT and then resuspended in FACS buffer for collection or permeablized for intracellular transcription factor staining. Cells were permeablized for at least 1 hour at 4°C using the Foxp3 staining buffer kit (eBioscience) followed by intracellular staining for IRF4 (3E4), Eomes (Dan11mag), Tbet (eBio4B10), and granzyme B (GB11).

Intracellular cytokine staining was performed as described previously (14). Cells were stained with a combination of fluorescently labeled MAbs specific for TNF (MP6-XT22), IFNγ (XMG1.2), and granzyme B (GB11, Invitrogen). Stimulating in presence of CD107a (1D4B) and CD107b (ABL-93) identified cells undergoing antigen-induced degranulation. All MAbs were purchased from eBioscience (San Diego, CA), BioLegend (San Diego, CA), or BD Bioscience (San Diego, CA) unless otherwise noted.

All samples, freshly stained or previously fixed, were acquired using a BD Bioscience LSR II flow cytometer with FACS Diva software. Data were analyzed with FlowJo software (Tree Star Inc., Ashland, OR).

Statistical analysis

Where appropriate, Student's t test and linear regression were calculated using GraphPad InSt software. Significance was set at a P value of 0.05; * indicates a P of <0.05, ** a P of < 0.01, *** a P of <0.001, and **** a P of < 0.0001. Results are expressed as means +/− standard deviations.

Results and Discussion

Naïve T cells acquire an early activated phenotype associated with immediate effector function after poly(I:C) inoculation

To study the response of naïve CD8 T cells pre-exposed to signal 3 cytokine activation signals, antigen-specific congenic OT-1 and P14 transgenic CD8 T cells were adoptively transferred into B6 mice, which were then inoculated with HBSS or poly(I:C), as an inducer of type 1 IFN. These transgenic T cells remained small, as judged by flow cytometry, and our previous studies using the dye marker CFSE, which reduces in intensity when cells divide, have shown that P14 cells remain small and non-diving after poly(I:C) treatment or even after 12 days of infection by a non-crossreactive virus (15, 16). Representative histograms gated on host CD44lo (naïve) WT B6 and IFN alpha receptor 1 (AR1) KO, or on donor CD44lo OT-1 and P14 CD8 T cells from HBSS (shaded histograms)- or poly(I:C) (red open histograms)- treated mice are shown (Fig 1A-1B). Following poly(I:C) treatment, CD44lo WT B6 and donor transgenic CD8 T cells upregulated the early activation markers CD69 and CD86 and downregulated IFNAR1 and CD127, with a small population downregulating CD62L (Fig 1A). This poly(I:C)-induced activation phenotype in the absence of cognate antigen was seen for naïve transgenic T cells and also in naïve polyclonal CD8 T cell populations in WT B6 mice, but not in IFNAR KO polyclonal CD44lo CD8 T cells, indicating a role for type 1 IFN in poly(I:C)-induced early activation. The upregulation of CD69 was seen at the earliest time point tested, 12 hours after poly(I:C) inoculation, but by day 3 post treatment, CD44lo CD8 T cells were phenotypically similar to control-treated counterparts (data not shown), indicating that poly(I:C) transiently induces naïve CD8 T cells to acquire a partial activation phenotype. Poly(I:C) also induced changes in TFs in naïve T cells. The TF IRF4, which is normally upregulated through TCR activation (17), and the TF Eomes, normally associated with CD8 T cell memory (18), were upregulated in CD44lo CD8 T cells after poly(I:C) treatment in the absence of cognate antigen (Fig 1B). However, the CD8 T cell effector-associated T-box TF Tbet remained largely unchanged (19, 20). The upregulation of TFs also required type 1 IFN signals, as IFNAR KO CD44lo T cells did not induce IRF4 or Eomes expression. Thus, in the absence of stimulation with high-affinity cognate ligand, IFN alters naïve phenotype CD44lo CD8 T cells by upregulating CD69, CD86, IRF4, Eomes, as well as granzyme B (Fig 1) and by downregulating CD127 and CD62L. Not surprisingly, the type 1 IFN receptor is also down-regulated (Fig 1A), while the TF Tbet remains unchanged. Type 1 IFN-induced upregulation of CD69 in lymphocytes has been shown previously (21), and more recent studies have shown that type 1 IFN can directly induce Eomes expression in CD8 T cells (15, 22). However, to the best of our knowledge, antigen-independent IFN-mediated alterations of other phenotypic markers (CD62L) and TFs (IRF4 and Tbet) have yet to be described.

Figure 1. Naïve T cells acquire an early activated phenotype associated with immediate effector function after poly(I:C) inoculation.

Transgenic OT-1 or P14 CD8 T cells were adoptively transferred into WT B6 or IFNAR1 KO recipients. Mice were inoculated with HBSS (control) or poly(I:C). One day later, spleens were harvested and stained directly ex vivo (A-B) or after 5 hour ICS stimulation with or without GP33 peptide (C-F). A-B) Representative histograms gated on CD44lo donor OT-1 or P14 CD8 T cells or CD44lo host WT B6 or IFNAR CD8 T cells from HBSS-treated (shaded) or poly(I:C)-treated (open) mice expressing CD69, CD127, IFNAR1, CD62L, CD86 (A), IRF4, Eomes, and Tbet (B) are shown. C-F) The frequencies of CD44lo CD107a/b (C), granzyme B (D), TNF (E), and IFNγ (F) expressing P14 CD8 T cells from HBSS- (grey bars) or poly(I:C)- (red bars) inoculated mice are graphed. Data are representative of at least 2 independent experiments with 3-5 mice per group.

We next questioned how this transient activation of naïve CD8 T cells would affect their effector function when exposed to their high affinity cognate ligand ex vivo for 4-5 hrs. Poly(I:C)-treated CD44lo P14 CD8 T cells degranulated and secreted granzyme B upon ex vivo stimulation with their cognate ligand, GP33 (Fig 1C-D, respectively). Poly(I:C)-treated naïve P14 cells without ex vivo stimulation (No stim) had enhanced granzyme B expression, and upon stimulation with high-affinity GP33 ligand experienced a significant loss in granzyme B staining. These cells concomitantly expressed increased staining with the degranulation markers CD107a/b (Fig 1C), suggesting that granzyme B was being released or secreted.

Naïve quiescent CD8 T cells are reported to produce TNF after cognate ligand exposure in the absence of any IFN stimulation, and this is shown in Fig 1E (grey) (23). However, poly(I:C)-sensitized naïve T cells showed increased proportions of TNF- producing cells (Fig 1E, red) and produced more TNF on a per-cell basis, as shown by increased MFIs compared to controls, after cognate ligand exposure (15456±4865 vs. 5122±1245, n=4, p=0.0062). In contrast to their production of TNF, naïve T cells have traditionally been thought not to produce IFNγ prior to an extensive period of activation and cell division (24-26). However, we have shown that sensitization with type 1 IFN can enable IFNγ production from naïve phenotype T cells within 3-5 hours of cognate ligand stimulus, and this is confirmed here in Fig 1F (15). Thus, by several parameters, the poly(I:C)-sensitized naïve phenotype T cells rapidly exerted effector functions on exposure to cognate ligand. This sensitization process to cognate-ligand-induced effector function occurred as early as 12 hours after poly(I:C) exposure, under which conditions the naïve T cells did not express the proliferation marker Ki67, indicating that they were not dividing (data not shown). Furthermore, a lack of naïve transgenic P14 T cell division during IFN-induced inflammatory environments was shown in previous studies from our laboratory by lack of CFSE dilution up to 12 days post Pichinde virus infection (15).

Class I and costimulation mediate poly(I:C)-induced naïve T cell priming

Previous studies showed that P14 T cells could not be sensitized to immediate effector function in terms of the rapid production of IFNγ in beta 2 microglobulin KO or H2-Db KO hosts, but they could be sensitized in an H2-Kb KO host, which expressed the H2-Db recognized by P14 cells (15). This indicated that the cognate MHC Class I Db was needed for the process of sensitization. Because type 1 IFN is known to upregulate Class I antigen presentation machinery and costimulatory molecules (27), we hypothesized that IFN may enhance the stimulation of T cells by MHC-presented self-peptides, and that upregulation of all these factors may contribute to the sensitization of naïve T cells. Indeed, TAP KO mice, which have a deficiency in the generation of Class I MHC peptides that stabilize and are presented by MHC molecules, could not sensitize P14 T cells, in terms of the spontaneous up-regulation of CD69, Eomes, and IRF4 and in the production of IFNγ after stimulation with cognate GP33 ligand, as well as that in WT mice (Fig 2A-B).

Figure 2. Class I MHC and costimulation mediate poly(I:C)-induced naïve T cell priming.

Congenic P14 cells were adoptively transferred into WT B6, TAP KO, or B7 KO mice prior to HBSS or poly(I:C) inoculation. Splenocytes were harvested one day later and stained directly ex vivo (A) or after 5 hour ICS stimulation with GP33 (B-C). Representative flow plots gated on CD44lo P14 CD8 T cells from HBSS- (shaded histograms) or poly(I:C)-treated (open histograms) mice expressing CD69, Eomes, and IRF4 (A) or TNF and IFNγ (B) are depicted. Frequencies of CD44lo IFNγ+ P14 T cells from WT B6 or B7 KO priming hosts stimulated in the presence of WT HBSS- or poly(I:C)-primed splenocytes (C) are graphed. Data are representative of at least 2-4 independent experiments with 2-4 mice per group.

To determine if costimulation was required for the sensitization, P14 cells were transferred into B7.1/B7.2 KO mice (B7 KO) or WT B6 mice and tested for sensitization after HBSS or poly(I:C) treatment for one day. CD44lo P14 cells primed in the B7 KO environment did not produce IFNγ at the levels of cells primed in the WT environment after stimulation with cognate ligand ex vivo (Fig 2C). To determine if lack of costimulation during the ex vivo exposure to cognate ligand rather than during the IFN-induced sensitization process in vivo accounted for the failure in IFNγ production, B7-expressing cells were added to the ex vivo stimulated cultures, but the T cells still failed to produce IFNγ. This indicates that B7 costimulatory factors were needed in the IFN-induced sensitization process.

We next asked whether the sensitization was likely elicited by professional antigen-presenting cells or whether it might be a consequence of aberrant stimulation by other cell types. Bone marrow (BM) chimera studies suggested that H2-Db on hematopoietic cells mediated IFN-induced naïve T cell priming. P14 cells inoculated into poly(I:C)-treated mice that received H2-Db KO BM did not upregulate the early activation marker CD69 to the same extent as P14 cells from mice that received WT BM priming (% of P14 cells that expressed CD69 after poly(I:C) treatment: 73.77±1.33 vs. 35.87±14.40, n=3, p=0.0105). Additionally, P14 cells from mice receiving H2-Db KO BM also showed reduced upregulation of Eomes after poly(I:C) priming (% Eomes+ cells: 71.47±3.07% vs. 55.97±0.15%, n=3, p=0.0009). These data suggest that H2-Db expression on Class I MHC-expressing hematopoietic cells mediates poly(I:C)-induced sensitization of naïve CD8 T cells. These results are consistent with the hypothesis that IFN sensitizes naïve T cells to partial activation by acting on the antigen presentation and costimulatory pathways of professional antigen-presenting cells. Since there was not a complete abrogation of the sensitization phenotype in the TAP KO or B7 KO mice after poly(I:C) inoculation, it is plausible that there could be some roles for other co-stimulatory factors or perhaps even for direct IFN effects on the CD8 T cells to mediate a partial activation phenotype.

Poly(I:C)-sensitized naïve T cells have a reduced threshold required for effector function

To determine if poly(I:C)-primed naïve CD8 T cells only responded to high-affinity, high-density antigen, or if they could respond to reduced affinities or densities, transgenic T cell-containing splenocytes were harvested and stimulated ex vivo with different concentrations of GP33 peptide and assayed for effector function. Poly(I:C)-sensitized (red) naïve P14 cells produced IFNγ (Fig 3A) and enhanced TNF (Fig 3B) in response to high-affinity, high-density antigen (1000 nM) and also in response to lower concentrations of ligands (250 nM, 64 nM, and 16 nM) to a significantly higher level over HBSS-treated controls (grey). Granzyme B expression levels were also determined after ex vivo stimulation with different concentrations of GP33 (Fig 3C). The frequency of poly(I:C)-primed CD44lo P14 cells expressing granzyme B was greatest without any ex vivo stimulation (NO), and upon increasing concentrations of GP33 stimulation, the proportion of granzyme B-expressing P14 cells decreased. At low peptide stimulation concentrations, such as 64 nM, significant reductions in granzyme B expression were still seen in the poly(I:C)-sensitized groups, suggesting release of granzyme B in response to low densities of high-affinity ligands. At no point did naïve P14 cells unexposed to poly(I:C) express granzyme B.

Figure 3. Primed naïve T cells elicit immediate effector functions in response to lower concentrations and affinity ligands.

Congenic P14 or OT-1 cells were transferred into WT B6 mice that were inoculated with HBSS (grey bars) or poly(I:C) (red bars). One day after inoculation, splenocytes were isolated and stimulated ex vivo with different concentrations of GP33 peptide (1000 nM, 250 nM, 64 nM, 16 nM or no peptide) (A-C) or different altered peptide ligands (OVA, Y3, T4, V4, G4, or no peptide) (D-F). The frequencies of CD44lo P14 (A-C) or OT-1 (D-F) cells that produced IFNγ (A, D), TNF (B, E), or granzyme B (C, F) are graphed. Data are representative of 2 individual experiments with 3-5 mice per group.

We questioned whether IFN-sensitized T cells could also produce immediate effector function in response to lower affinity antigens by utilizing congenic OT-1 transgenic T cells for which peptides with different affinities have been defined. Splenocytes were stimulated ex vivo with a variety of peptides for which OT-1 T cells have a varying degree of affinity (order of peptide affinity: OVA>Y3>T4>V4>G4) (28). Naïve poly(I:C)-sensitized OT-1 CD8 T cells produced multiple cytokines in response to ligand stimulation to a significantly higher level over control-treated cells stimulated ex vivo with the same peptides. A higher frequency of naïve OT-1 T cells produced IFNγ to all of the ligands tested, including the lowest affinity, albeit to a very low level (Fig 3D). Not only was there an increased proportion of poly(I:C)-pretreated naïve OT-1 T cells that produced TNF in response to ligands (Fig 3E), but of the cells that produced TNF, poly(I:C)-pretreated OT-1 T cells had higher MFIs than HBSS-treated OT-1 cells, indicating that they produced more TNF on a per cell basis (TNF MFI ± S.D. after T4 stimulation: 10035±1240 vs. 5836±425, poly(I:C) vs. HBSS, n=4, p=0.0007). The loss of granzyme B staining in the poly(I:C)-pretreated CD44lo OT-1 T cells after ligand stimulation, as compared to unstimulated counterparts, indicates that granzyme B is released in response to even the lowest affinity ligands tested here (Fig 3F). Together, these data demonstrate that priming with poly(I:C) enables naïve T cells to produce immediate effector functions even in response to lower affinity ligands.

Poly(I:C)-primed naïve P14 CD8 T cells specifically lyse target cells in vivo

Having shown that primed naïve T cells degranulated and released granzyme B in response to antigen stimulation, we determined whether naïve CD8 T cells primed with poly(I:C) could exert cytolytic effector function in vivo by performing an in vivo cytotoxicity assay. Congenic P14 cells were transferred into B6 hosts that were inoculated with HBSS or poly(I:C). One day later, these same mice received a mixture of splenocytes that were pulsed without peptide, with an irrelevant K3L peptide, or with the cognate GP33 peptide. After a ~20 hour in vivo incubation, spleens were harvested, stained, and analyzed for specific lysis as judged by depletion of the peptide-pulsed targets. Representative flow plots are depicted in Figure 4A. K3L-pulsed target cells were not preferentially depleted in either the HBSS- or poly(I:C)-treated hosts. However, GP33-pulsed splenocytes were specifically depleted in poly(I:C)-treated (red) mice to a significant extent over HBSS-treated mice (grey) (Fig 4B). The vast majority of the donor P14 cells remained CD44lo, indicating that they maintained a naïve phenotype during the 20 hour in vivo cytotoxicity assay (Fig 4C). The poly(I:C)-primed donor P14 cells retained the early activated phenotype, originally seen 12 hours after poly(I:C) inoculation, during the in vivo cytotoxicity assay and remained CD69hi and CD62Llo. A positive correlation between P14 CD69 expression and % GP33 specific lysis but not between P14 CD69 expression and % K3L specific lysis was found (Fig 4D, GP33 R²=0.5892, p=0.0095; K3L R² =0.009837, p=0.7852). Moreover, a negative correlation between P14 CD62L expression and GP33 % specific lysis was identified (Fig 4E, GP33 R²=0.6890, p=0.0030). However, there was no correlation between P14 CD62L expression and % K3L-specific lysis (Fig 4E, K3L R²=0.0005151, p=0.9504), indicating specificity of the P14 cells for GP33-pulsed targets. Considering that naïve CD8 T cells were once considered to lack effector functions, it was remarkable that IFN-primed P14 cells exhibited specific cytotoxic effects in vivo. However, due to the relatively low specific lysis at these optimal conditions, titration of peptide dose or affinity was not performed.

Figure 4. Naïve T cells exhibit cytolytic capability in vivo after poly(I:C) priming.

Congenic P14 splenocytes were adoptively transferred into WT B6 mice that were inoculated with HBSS or poly(I:C). Peptide pulsed splenocytes were transferred into HBSS or poly(I:C) treated hosts for a 20 hour in vivo cytotoxicity assay. A) Representative histograms gated on donor splenocytes with the lowest concentration of CellTrace Violet representing no peptide-pulsed (Red), middle concentration, K3L peptide (Green), or highest concentration, GP33 peptide (Blue). B) Percent specific lysis was calculated for K3L- and GP33- specific cells. C) Representative flow plots gated on donor P14 cells expressing CD69 or CD62L from HBSS- and poly(I:C)-treated mice are shown. Correlation between % specific lysis and frequency of P14 cells expressing CD69 (D) or CD62L (E) is graphed. Data are representative of 2 individual experiments with n of 3-5 mice per group.

It is now established that type 1 IFN, mainly by its action on antigen-presenting cells in vivo, induces a low level activation of naïve CD8 T cells that enables them to produce IFNγ soon after exposure to their cognate ligand in vitro (15). Here we further characterize the activation state of these T cells in terms of TF and cell surface antigens and demonstrate that this sensitization is likely mediated by B7- expressing and self-peptide-displaying professional antigen-presenting cells. Importantly, in vivo cytotoxicity assays indicated that these IFN-sensitized naïve phenotype CD8 T cells can specifically lyse target cells in vivo. Of significance is that these sensitized naïve T cells have a low threshold for mediating effector function, as they can be triggered to degranulate and produce IFNγ in response to low concentrations or low affinities of cognate ligands. This leaves open the possibility that part of the short-term efficacy of type 1 IFN prophylaxis could be due to its effect on the naïve T cell population. The ability to be triggered by low concentrations of cognate ligands indicates that these IFN-sensitized naïve T cells could detect the early stages of a target cell infection, when virus-encoded peptides are just beginning to be expressed on the cell surface. Our published estimates of the total number of naïve CD8 T cell precursors to LCMV and vaccinia virus in non-immune mice are ~6,700 and ~13,900, respectively (29). This number of T cells is thought insufficient to control these virus infections until their clonal expansion, but it is now of interest to determine how well they could control infection if they were previously sensitized by IFN. The ability of IFN-sensitized naïve transgenic T cells to be triggered by ligands of lower affinity than those that could efficiently trigger non-sensitized naïve transgenic T cells suggests that prior exposure to IFN may also enable a larger number than expected of a polyclonal naïve T cell population to participate in a rapid effector response to infection.

The immune system seems mainly designed to initiate its response to infection while starting in a resting state, where signals 1 (antigen presentation), 2 (costimulation) and 3 (cytokine augmentation) occur in a prescribed order. There are, however, certain conditions where the IFN-mediated sensitization of naïve T cells would be expected to be a factor in viral pathogenesis. Those naïve T cells that do not engage antigen during the first 2 days of infection, but do so later on, i.e., latecomer T cells, would likely have been sensitized by IFN prior to exposure to their cognate ligand and would likely quickly thereafter participate in the effector response to infection. Further, if the acutely-infected host is exposed to another previously un-encountered pathogen, IFN-sensitized naïve T cells could likely exert a very rapid effector cell response against that superinfecting pathogen. Finally, T cells from individuals suffering from chronic infections or certain auto-immune diseases may also be expected to harbor IFN-sensitized naïve T cells, whose capacity to provide resistance to infection is thus far unexplored. It is difficult to parse out the relative contributions of the various anti-viral mechanisms that IFN can exert, but the clear demonstration in Figure 4 of the viral antigen-specific cytotoxic capacity of IFN-sensitized T cells in vivo argues that naïve T cells can contribute to viral control. The caveat is that in all of these situations it should be kept in mind that we are discussing an early and comparatively short lived stage of naïve T cell effector function, as it has been shown that the IFN-induced downregulation of the IFNAR inhibits T cells from receiving the signal 3 effects of IFN that allow for the most vigorous proliferative expansion (14). Nevertheless, the ability of these naïve T cells to exert immediate effector function in response to low density and low affinity antigens may be important for controlling antigen load early after infection and prior to naïve T cell clonal expansion.

Highlights.

1. Poly(I:C)-induced IFN licenses activation and rapid effector function of naïve T cells

2. IFN-sensitized naïve T cells have a reduced threshold required for effector function

3. Class I MHC and costimulation mediate poly(I:C)-induced naïve T cell priming

4. Poly(I:C)-primed naïve P14 CD8 T cells specifically lyse target cells in vivo