IL-12 Enhances CTL Synapse Formation and Induces Self-Reactivity

Mary A. Markiewicz, Erica L. Wise, Zachary S. Buchwald, Elizabeth E. Cheney, Ted H. Hansen, Anish Suri, Saso Cemerski, Paul M. Allen and Andrey S. Shaw
J Immunol February 1, 2009, 182 (3) 1351-1361; DOI: https://doi.org/10.4049/jimmunol.182.3.1351

Abstract

Immunological synapse formation between T cells and target cells can affect the functional outcome of TCR ligation by a given MHC-peptide complex. Although synapse formation is usually induced by TCR signaling, it is not clear whether other factors can affect the efficiency of synapse formation. Here, we tested whether cytokines could influence synapse formation between murine CTLs and target cells. We found that IL-12 enhanced synapse formation, whereas TGFβ decreased synapse formation. The enhanced synapse formation induced by IL-12 appeared to be functional, given that IL-12-treated cells could respond to weak peptides, including self-peptides, to which the T cells were normally unresponsive. These responses correlated with expression of functionally higher avidity LFA-1 on IL-12-treated CTLs. These findings have implications for the function of IL-12 in T cell-mediated autoimmunity.

The CTLs are critical for eliminating viral infections and intracellular pathogens, and they potentially play an important role in tumor immunity. In addition to their protective role, CTLs are also involved in pathogenic autoimmune responses. The primary signal used to determine which cells CTLs attack is engagement of the TCR with MHC class I-peptide complexes on potential target cells. The peptide bound to the MHC is critical to the CTL response. Although in general the TCR of a given T cell is thought to be specific for one optimal ligand, it is now clear that a single TCR can be stimulated by a broad range of different peptides (1, 2, 3, 4). These various peptide-MHC complexes bind to the TCR with varying affinities and elicit a variety of different outcomes (5, 6). Based on the functional responses that they evoke, peptides are classified as superagonists, agonists, weak agonists, antagonists, or null ligands for an individual TCR. Agonists induce the full functional repertoire of the T cell; weak agonists induce some, but not all T cell functions; antagonists do not induce T cell function, but inhibit agonist-induced responses; null peptides have no influence on T cell function.

Although it was originally thought that mature T cells recognize foreign Ags only in an infected host, it is now becoming clear that T cells can react to some proportion of self-peptides in the periphery. For example, T cells require low-affinity interactions with self-peptide-MHC complexes to undergo positive selection in the thymus (reviewed in Ref. 7). Similarly, both naive and activated (but not memory) T cells require a similar interaction to survive in the periphery (reviewed in Ref. 8). Further, it has recently been proposed that interactions with self-peptides are required to amplify signals when only small numbers of antigenic MHC-peptide complexes are present on the surface of APCs (9, 10). The recognition of self-peptides also underlies the pathogenesis of autoimmune disease and is the goal of many tumor immunotherapies.

Recent work in our laboratory suggests that formation of a mature immunological synapse can affect the sensitivity and specificity of a T cell response to a given peptide (11). A synapse is characterized by the concentration of TCRs and peptide-MHCs at the contact surface between a T cell and APC or target cell. In some cases, the synapse forms a characteristic bulls-eye pattern with the TCR and peptide-MHC concentrated at the center of the contact (referred to as the central supramolecular activation cluster or cSMAC)3 surrounded by a ring of LFA-1 and ICAM-1 molecules (the peripheral supramolecular activation cluster SMAC or pSMAC; Refs. 12, 13, 14). The results from our previous work (11, 15) suggest that by concentrating peptide-MHC in the center of the contact, TCR occupancy is enhanced allowing for stronger signaling. We hypothesize that the concentration of weak ligands in the synapse could amplify signaling by a weak agonist and might convert an antagonist into a weak agonist.

Until recently, TCR stimulation was thought to be required for synapse formation (11, 12, 13, 14). We demonstrated, however, that engagement of a surface receptor on CTL, namely NKG2D, can drive synapse formation (16). We postulated that NKG2D engagement might lower the threshold for synapse formation in an inflammatory situation, enhancing the sensitivity and specificity of an early immune response.

In this study, we set out to determine whether cytokines could influence synapse formation. We found that the proinflammatory cytokine IL-12 could strongly enhance synapse and cSMAC formation between CTLs and target cells, whereas the immunosuppressive cytokine TGFβ strongly inhibited synapse formation. Consistent with our hypothesis that concentration of weak agonists in the synapse could enhance signaling, we found that IL-12 treatment enhanced OT-1 CTL reactivity to a weak agonist and converted an antagonist peptide to a weak agonist. Surprisingly, we found that IL-12 could also stimulate killing of syngeneic target cells by both OT-1 and DUC18 CTL, suggesting that IL-12 induces CTL reactivity against self-peptide(s). Supporting this, we identified self-peptides that induced killing of target cells by IL-12-treated OT-1 or DUC18 CTL. These data suggest that the production of IL-12 during inflammation may function to enhance the sensitivity and broaden the specificity of T cells during the early phase of the immune response allowing for reactivity with self-Ags.

Materials and Methods

Mice

All mice were housed under specific pathogen-free conditions in the Washington University animal facilities in accordance with institutional guidelines. OT-1 TCR-transgenic C57BL/6 mice (17) were provided by Dr. H. Virgin (Washington University, St. Louis, MO). DUC18 TCR-transgenic BALB/c mice (18) were bred in house. C57BL/6J mice were purchased from The Jackson Laboratory.

Cytokines and peptides

Recombinant mouse IL-12 (rmIL-12) and recombinant human TGFβ2 were purchased from BioSource International, Invitrogen. The following peptides were synthesized and purified as previously described (18): SIINFEKL, EIINFEKL, RGYNYEKL, SIIRFEKL, RTYTYEKL, ISFKFDHL, QYIHSANVL, RGYVYQGL; H-2Kd-binding peptides (19): KYQEVTNNL, SYLEMGHDI, VYNASNNEL, KYLTVKDYL, KYKDIYTEL, KYIHSANVL, KYMEDVTQI, KYKASENAI, AKPTGNVDIGSL, ESRVSDTGSAGLML, and IFIKPGADLSTGHDEL; FVQMMTAK, LYKESLTKL, GYVQSKEMI, TWNKLLTTI, HYFEDKENI, AYSIVIRQI, TYGLTPHYI, KYLSVQGAL, KYLSVQGQLF, MRYVASYLL, FGLLGLGSDGQPPVQK, GFGDLKTPAGLQVLND, NYGPMKGGSFGG, and NYGPMKGGSFGGRSSGSP. The following peptides were purchased from GenScript: PTYRFERL, AGYSFEKL, HTYDFEKL, RAKRYEKL, VGYMYETL.

Generation of CD3ζ-GFP CTL for synapse studies

The Plat E packaging cell line (20) was transfected with CD3ζ-GFP (provided by Dr. Mark Davis, Stanford University, Stanford, CA) expressed in the pMX vector (21) using Lipofectamine 2000 (Invitrogen). Viral supernatant was harvested 48 h later and put through a 0.45-μm pore size filter. Total splenocytes from OT-1 TCR-transgenic mice were cultured (5 × 106/ml) with SIINFEKL peptide (0.3 μM) and anti-CD3ε Ab (clone 2C11; 1 μg/ml). Eighteen hours after stimulation, 2 ml of the retroviral supernatant, along with Lipofectamine 2000 (1 μl/ml), were spun onto the cells (2200 rpm for 20 min at room temperature). After 4 h at 37°C, this spinfection was repeated a second time. Live T cells were then harvested 3 days later using Ficoll-Paque PLUS (GE Healthcare) and cultured with 20 ng/ml IL-12, 10 ng/ml TGFβ2, or no cytokine for another 24 h. The live cells harvested, either with or without cytokine treatment, were >95% CD8+Vα2+ (OT-1).

Formation and imaging of OT-1 CTL and target cell conjugates

RMA-S or EL-4 cells were cultured overnight with 10−6 M SIINFEKL peptide, 10−6 M EIINFEKL peptide, or no peptide in IMDM supplemented with 2% FCS. CD3ζ-GFP-expressing OT-1 CTL were mixed with RMA-S cells, or untransduced OT-1 CTL were mixed with EL-4 cells, in 100 μl IMDM with 10% FCS in 1.5-ml tubes, spun briefly, and incubated at 37°C for 30 min. The cells were then lightly resuspended in PBS with 2 μM Mg2+, 1 μM Ca2+, and 1% FCS. In the case of the EL-4 conjugates, the cells were stained with a TCRVα2-specific Ab (BD Biosciences). The cells were then flowed onto a parallel plate flow cell (Bioptechs) at 37°C. Cells were imaged using a Zeiss LSM510 confocal system. All images were taken with the pinhole open. Using the Zeiss LSM510 software, conjugates were scored as synapse positive when there was at least a 2-fold enrichment of CD3ζ-GFP at the cell-cell contact site compared with the rest of the T cell membrane. Conjugates with synapse formation in which the enrichment of CD3ζ-GFP was in a central area not larger than one-third of the total contact area were scored as cSMAC positive.

In vitro generation of CTL

After RBC lysis, OT-1 or DUC18 splenocytes (5 × 106/ml) were cultured in six-well plates (5–6 ml/well) in IMDM supplemented with 10% FCS and 55 μM 2-ME with 1 μM SIINFEKL (OT-1) or QYIHSANVL (DUC18). After 4 days, the cells were harvested by purification with Ficoll-Paque PLUS and placed in fresh medium containing 0 or 20 ng/ml rmIL-12. After 24 h, the cells were harvested and used in experiments. The live cells harvested, either with or without cytokine treatment, were >95% CD8+Vα2+ (OT-1) or CD8+Vβ8.3+ (DUC18).

Cytotoxicity assays

When appropriate, target cells were cultured overnight in IMDM supplemented with 2% FCS with peptide. Con A blasts were generated by culturing C57BL/6J or BALB/c splenocytes (2 × 107/10 ml) in complete IMDM supplemented with 2.5 μg/ml Con A for 48 h. Purified anti-H-2Kb Ab (clone B8-24-3) was used at 20 μg/ml. CTL cytotoxicity was measured using a standard chromium release assay. 51Cr-Labeled target cells were plated in 96-well U-bottom plates (10,000 cells/well) with CTL at varying E:T cell ratios. The plates were spun briefly and cultured for 4 h at 37°C. The supernatants were then collected and read on a MicroBeta counter (PerkinElmer), and the percent specific lysis was determined as: [(sample cpm − spontaneous cpm)/(maximum cpm − spontaneous cpm)] × 100.

Intracellular cytokine staining

OT-1 CTL (105/well) and RMA-S cells (105/well) were plated in a 96-well U-bottom plate and cultured at 37°C for 16 h. Brefeldin A (Sigma-Aldrich) was added during the last 6 h of culture. The cells were then stained with a biotinylated-TCRVα2 Ab (BD Biosciences) followed by streptavidin-FITC (BD Biosciences), fixed with 4% paraformaldehyde in PBS, permeabilized with 0.1% saponin in PBS, and stained with a PE-labeled IFN-γ-specific Ab (BD Biosciences). The cells were analyzed using a FACScan (BD Biosciences).

Granzyme B ELISPOT

The Mouse Granzyme B Elispot Development Module was purchased from R&D Systems, and the assay was performed following the manufacturer’s instructions. Briefly, 96-well filter plates (Millipore) were coated with capture Ab overnight, blocked with PBS containing 1% BSA and 5% sucrose for 1 h at room temperature, and washed three times with PBS and once with IMDM. Untreated and IL-12-treated OT-1 CTL (100/well) and RMA-S cells ± peptide (1000/well) were added to the plates and incubated overnight at 37°C. The plates were washed three times with 0.05% Tween in PBS, and the detection Ab was added and incubated overnight at 4°C. The plates were then developed using the ELISPOT Blue Color Module (R&D Systems). The plates were then read on an Immunospot (Cellular Technology) ELISPOT reader.

LFA-1 measurements

LFA-1 surface expression.

OT-1 CTLs were stained with a purified LFA-1-specific Ab (BD Biosciences) followed by a PE-labeled anti-rat IgG secondary (Jackson ImmunoResearch Laboratories) or secondary Ab alone. The cells were then analyzed on a FACScan.

Plate adhesion assay.

Immunolon HB 96-well plates (Thermo-Scientific) were coated with recombinant mouse ICAM-1-Fc (R&D Systems) or BSA (Sigma-Aldrich) as control, (2 μg/ml) in 100 μl of PBS without Mg2+ and Ca2+ and left overnight at 4°C. The plate was blocked with 10 mg/ml heat-denatured BSA in PBS and incubated at 37°C for 2 h. Following one wash with PBS, the CTLs were plated at 2 × 106 cells/ml, 100 μl/well in IMDM (no FCS). The plate was briefly centrifuged, incubated for 1 h at 37°C, and washed gently once with PBS; and the cells in each well were counted using a hemocytometer. When PMA was used, the cells were preincubated with 20 ng/ml PMA for 15 min at 37°C. The percentage of cells that adhered to BSA-coated wells was subtracted as background.

Shear stress assay.

Tissue culture plates (35 mm; Corning Life Sciences) were coated with recombinant mouse ICAM-1-Fc (2 μg/ml in 1 ml of PBS) overnight at 4°C. These plates were used with a parallel plate flow chamber (Glycotech) and PHD 2000 infusion pump (Harvard Apparatus). CTLs (2 × 106) were flowed at a force of 1 dyne/cm2 for 2 min. The cells were visualized using the ×10 lens of a light microscope and camera (AmScope). The number of cells adhering to the plate was immediately determined in five different fields.

Results

Cytokines differentially affect CTL immune synapse formation

We hypothesized that the cytokine milieu surrounding CTLs could affect the efficiency of synapse formation between CTLs and target cells. We therefore tested whether two cytokines important for CTL function, IL-12 and TGFβ, could influence the efficiency of immunological synapse formation. We hypothesized that part of the differing effects of these cytokines on CTL function may be related to how they influence synapse formation. To test this hypothesis, we quantified synapse formation by transgenic OT-1 CTLs treated with these two cytokines.

OT-1 CTL were transduced with CD3ζ-GFP, treated with cytokine for 24 h, and allowed to form conjugates with histocompatible target cells (RMA-S). Live conjugates were then scored for synapse formation. Conjugates in which there was at least a 2-fold enrichment of CD3ζ-GFP at the cell-cell contact site compared with the rest of the T cell membrane were scored as positive for synapse formation (Fig. 1⇓A). In addition, conjugates with synapse formation in which the enrichment of CD3ζ-GFP was in a central area not larger than one-third of the total contact area were scored as having synapses with a cSMAC.

FIGURE 1.
FIGURE 1.

IL-12 enhances and TGFβ decreases Ag-driven synapse formation by OT-1 CTL. A, Examples of synapse-positive and cSMAC-positive conjugates formed between OT-1 CTLs and RMA-S cells pulsed with SIINFEKL peptide. B, Percentage of synapse-positive conjugates (±SD) formed between untreated, IL-12-treated, or TGFβ-treated OT-1 CTLs and RMA-S/SIINFEKL (10−6 M). No synapses were seen under any condition when conjugates were formed with RMA-S cells in the absence of exogenous peptide. C, Percentage of synapse-positive conjugates that were cSMAC-positive formed between untreated, IL-12-treated, or TGFβ-treated OT-1 CTLs and RMA-S/SIINFEKL (10−6 M). These results are the average of five independent experiments. ∗, p < 0.02; ∗∗, p < 0.0003; ∗∗∗, p < 0.03; two-sided Student t test.

In the absence of exogenous cytokine, OT-1 CTL efficiently generated synapses with target cells pulsed with the antigenic peptide, SIINFEKL (Fig. 1⇑B). Approximately 85% of conjugates scored positive for synapse formation; 23% of the synapses had a clearly defined cSMAC (Fig. 1⇑C). After treatment with IL-12, the CTL demonstrated a slightly increased efficiency of synapse formation (95%) with a significantly higher percentage of synapses that formed cSMACs (41%). In contrast, treatment with TGFβ significantly inhibited synapse formation (60%). These differences were not related to differences in the efficiency of conjugate formation, as pretreatment with IL-12 or TGF-β had no effects on conjugate formation (Supplemental Fig. 1A).4 Nor were they due to a change in TCR expression (Supplemental Fig. 1C). Lastly, synapse formation was Ag-specific, as no synapses were detected in the absence of antigenic peptide (data not shown).

These results demonstrate that cytokines can affect both the quantity and the quality of Ag-driven synapse formation. IL-12, a proinflammatory cytokine, significantly enhanced synapse and cSMAC formation. TGFβ, an immunosuppressive cytokine, significantly decreased synapse formation. These results lead to the hypothesis that the proinflammatory or immunosuppressive effects of these cytokines may be at least partially mediated via effects on synapse formation. To begin to address this question, we investigated how the enhanced synapse formation mediated by IL-12 affected CTL effector mechanisms.

IL-12-treated CTLs lyse histocompatible target cells

We measured the cytotoxic capabilities of untreated and IL-12-treated OT-1 CTL using the histocompatible cell line EL-4. To our surprise, we found that IL-12-treated CTL lysed these target cells in the absence of antigenic peptide (Fig. 2⇓A). The lysis appeared to be MHC restricted as the IL-12-treated CTLs did not kill the histoincompatible cell line P815 (Fig. 2⇓B). Unlike RMA-S cells, EL-4 cells induced a significant level of synapse formation by the OT-1 CTL in the absence of exogenous peptide (Fig. 2⇓C). A significant percentage of untreated CTL formed synapses with EL-4 (47%), with 15% of these forming a cSMAC. IL-12 increased the percentage of CTL that formed synapses (80%), as well as the percentage that formed cSMACs (30%). However, similar percentages of the two CTLs formed conjugates with EL-4 cells (Supplemental Fig. 1B).

FIGURE 2.
FIGURE 2.

IL-12-treated OT-1 CTLs lyse histocompatible target cells in a peptide-dependent manner. A and B, Untreated and IL-12-treated OT-1 CTLs were tested for cytotoxicity (±SD) against the histocompatible cell line EL-4 (H-2b; ±10−6 M SIINFEKL) and the histoincompatible cell line P815 (H-2Dd). These results are representative of multiple independent experiments. C, Percentage of synapse-positive and cSMAC-positive conjugates formed between untreated or IL-12-treated CTLs and unpulsed or SIINFEKL (10−6 M)-pulsed EL-4 cells. These results are representative of two independent experiments. D and E, Untreated and IL-12-treated DUC18 CTLs were tested for cytotoxicity (±SD) against the histocompatible cell line P815 (H-2Dd; ±10−6 M QYIHSANV) and the histoincompatible cell line EL-4 (H-2b). F, Surface expression of H-2Kb on unpulsed EL-4, RMA-S, and RMA-S pulsed with 10−6 M peptides. G, Lysis by IL-12-treated OT-1 CTL (±SD) of the surface H-2b high cell line EL-4, the surface H-2b low cell line RMA-S, or RMA-S pulsed with 10−6 M SIINFEKL or RGYVYQGL (control peptide). Data are representative of multiple independent experiments. Max, Maximum.

To determine whether this effect was unique to the OT-1 cells, we tested another CD8+ TCR-transgenic line from the DUC18 mice (BALB/c) that is specific for a mutated ERK2 peptide, QYIHSANVL, in the context of H-2Kd (18). Incubation of DUC18 CTLs with IL-12 allowed the CTLs to lyse the histocompatible P815 cell line in the absence of antigenic peptide, but not the histoincompatible cell line EL-4 (Fig. 2⇑, D and E). Together, these data suggest that IL-12 treatment can lead to MHC-restricted, Ag-independent killing by CTLs, most likely via self-peptide reactivity.

MHC class I expression is not sufficient for lysis of histocompatible target cells by IL-12-treated CTLs

To begin to test the hypothesis that the lysis of targets by IL-12-treated CTL was dependent on specific self-peptide(s) presented in class I MHC, we tested the ability of IL-12-treated OT-1 CTL to lyse the RMA-S cell line (23). Due to a deficiency in the peptide transporting TAP complex, these cells are unable to load peptide into class I MHC, resulting in very low MHC class I expression on the surface of these cells. IL-12-treated OT-1 CTL were unable to kill RMA-S cells in the absence of exogenous peptide (Fig. 2⇑, F and G), suggesting that killing required MHC class I surface expression.

To determine whether plasma membrane expression of syngeneic MHC class I molecules was sufficient to induce killing by IL-12-treated CTLs, regardless of the peptide presented, RMA-S cells were incubated with a peptide encoded by vesicular stomatitis virus, RGYVYQGL (24). This peptide binds efficiently to H-2Kb and can up-regulate MHC class I expression on RMA-S cells (Fig. 2⇑F). Although incubation with the vesicular stomatitis virus peptide up-regulated MHC class I, it was not sufficient to induce killing of RMA-S cells by IL-12-treated OT-1 CTLs, although these CTLs were able to kill EL-4 cells and RMA-S cells pulsed with SIINFEKL (Fig. 2⇑G). Together, these results suggest that specific peptide-MHC class I complex(s) on EL-4 cells was recognized by the IL-12-treated OT-1 CTLs.

IL-12 treatment enhances peptide sensitivity of CTLs

Our data led us to hypothesize that IL-12 enhanced responses of CTLs to self-peptides for which they had weak affinity in the absence of exogenous cytokine. To begin to address this, we first determined whether IL-12 could increase CTL responses against normally weak peptides. To do this, we made use of a group of well-characterized altered peptide ligands for the OT-1 TCR (Refs. 25, 26, 27, 28 and Table I⇓). We first tested the effects of IL-12 on the sensitivity of OT-1 CTLs to an intermediate agonist peptide, SIIGFEKL. IL-12-treated OT-1 CTLs were indeed much more efficient at lysing RMA-S cells pulsed with this peptide than with untreated OT-1 CTLs (Fig. 3⇓A). These results demonstrate that IL-12 can increase the peptide sensitivity of CTLs to weak agonists.

FIGURE 3.
FIGURE 3.

IL-12 enhances peptide sensitivity and decreases peptide specificity of OT-1 CTLs. A, Untreated and IL-12-treated OT-1 CTLs were tested for cytotoxicity against RMA-S cells pulsed with the intermediate agonist SIIGFEKL (10−6 M) (±SD). Lysis of SIINFEKL at an E:T of 50:1 was 67% (no cytokine) and 78% (+IL-12). Lysis with control peptide at an E:T ratio of 50:1 was 6% for both types of CTL. B, Untreated and IL-12-treated CTL were tested for cytotoxicity against RMA-S cells pulsed with the weak agonist/antagonist EIINFEKL (10−6 M; ±SD). Lysis of SIINFEKL at E:T 75:1 was 95% (No cytokine) and 100% (+IL-12). Lysis with control peptide at E:T 75:1 was 5% for both types of CTL. C, Untreated and IL-12-treated OT-1 CTLs were tested for cytotoxicity against RMA-S cells pulsed with various concentrations of EIINFEKL at E:T 30:1 (±SD). Lysis of SIINFEKL at 10−6 M was 65% (no cytokine) and 70% (+IL-12). Lysis with control peptide at 10−6 M was 5% (no cytokine) and 15% (+IL-12). D, The percentage (±SD of three independent experiments) of untreated and IL-12-treated OT-1 CTLs that formed synapses with RMA-S cells pulsed with EIINFEKL (10−6 M). No cSMACs were formed. E, Percentage (±SD) of untreated and IL-12-treated OT-1 CTLs that expressed IFN-γ as measured by intracellular staining. F, Percentage (±SD) of untreated and IL-12-treated OT-1 CTLs that produced granzyme B as measured by an ELISPOT assay.

View this table:
Table I.

List of peptides tested for OT-1 CTL reactivity

We next tested the effect of IL-12 treatment on OT-1 CTL to the weak agonist/antagonist peptide EIINFEKL. Although the EIINFEKL peptide was unable to stimulate cytolysis by untreated CTL, treatment with IL-12 allowed OT-1 CTL to efficiently lyse EIINFEKL-pulsed RMA-S cells (Fig. 3⇑, B and C). To ensure that this killing was not due to antigenic peptide carried over from the initial T cell stimulation, we confirmed these findings using CTLs generated by stimulation with plate-bound anti-CD3 Ab (data not shown). In addition, by titrating the IL-12 concentration, we found that 1 ng/ml rmIL-12 was enough to induce a response from the CTL against this weak peptide (Supplemental Fig. 2). Corresponding with the lytic response, while IL-12 did not affect conjugate formation (data not shown), it greatly increased the percentage of OT-1 CTL that formed immune synapses with RMA-S cells pulsed with EIINFEKL (55% vs 5%; Fig. 3⇑D). However, even with IL-12, the EIINFEKL peptide did not induce cSMAC formation or IFN-γ production (Fig. 3⇑E and data not shown).

To determine whether IL-12 treatment induced more CTLs to kill or induced each CTL to lyse more EIINFEKL-pulsed target cells, we performed a granzyme B ELISPOT assay. IL-12 treatment resulted in a greater number of CTLs secreting lytic granules in response to RMA-S cells pulsed with EIINFEKL (Fig. 3⇑F). This suggested that IL-12 treatment created more CTLs capable of responding to EIINFEKL. These results suggest that IL-12 can enhance the response of CTL to some weak peptides, with the capability of converting an antagonist peptide into a weak agonist peptide.

We next tested two strong antagonist peptides, RGYNYEKL and SIIRFEKL. Because these peptides are able to positively select OT-1 T cells in the thymus, the OT-1 TCR must have at least some functional affinity for these peptides (25, 26). However, treatment with IL-12 did not allow these antagonist peptides to induce activation of OT-1 CTL (data not shown). Taken together, these results suggest that IL-12 can enhance the response of CTL to some weak peptides, altering the specificity of the CTL response within a small range of affinity.

IL-12-treated CTLs respond to normal self-peptides

The ability of IL-12 to induce reactivity to EL-4 cells suggested that IL-12 might allow CTL to respond to a self-peptide. To directly test this, we tested whether IL-12-treated OT-1 and DUC18 CTL could lyse nontransformed, syngeneic splenocytes. Splenocytes were activated using Con A, so that they could be labeled with 51Cr, and tested for their susceptibility to be killed by IL-12-treated OT-1 and DUC18 CTLs. Whereas killing by untreated OT-1 and DUC18 CTL required antigenic peptide, IL-12-treated OT-1 and DUC18 CTL were able to lyse syngeneic splenocytes in the absence of exogenous peptide (Fig. 4⇓). We confirmed that the killing by the IL-12-treated OT-1 CTLs was MHC restricted using a blocking Ab against H-2Kb. These results suggest that treatment with IL-12 allows CTLs to respond to self-peptide(s) present on syngeneic splenocytes.

FIGURE 4.
FIGURE 4.

IL-12-treated CTL lyse syngeneic splenocytes. A and B, Cytotoxicity (±SD) of untreated and IL-12-treated OT-1 CTLs against Con A-blasted C57BL/6 splenocytes. C, Cytotoxicity (±SD) of untreated and IL-12-treated DUC18 CTL against Con A-blasted BALB/c splenocytes ± QYIHSANVL (10−6 M).

We next set out to identify self-peptides to which IL-12-treated OT-1 or DUC18 CTL were responsive. With the OT-1 CTLs, we tested self-peptides that can induce the positive selection of these T cells in the thymus (Refs. 25, 26, 27, 28 and Table I⇑). IL-12-treated OT-1 CTLs exhibited a low but significant level of lysis against RMA-S cells pulsed with three of the six peptides tested (Fig. 5⇓, A and B, and data not shown). In contrast, the untreated CTLs did not lyse RMA-S cells pulsed with any of these peptides.

FIGURE 5.
FIGURE 5.

IL-12-treated CTL react to self-peptides. A and B, Cytotoxicity (±SD) of untreated and IL-12-treated OT-1 CTL against RMA-S cells pulsed with the self-peptides AGYSFEKL, RYTYTEKL, or ISFKFDHL, or the antigenic peptide, SIINFEKL. C and D, Cytotoxicity (±SD) of untreated and IL-12-treated DUC18 CTL against T2-Kd cells pulsed with the wild-type ERK2 peptide, KYIHSANVL, or the antigenic peptide, QYIHSANVL. Results are representative of two independent experiments.

We also tested IL-12-treated DUC18 CTL responses against a panel of H-2Kd-binding self-peptides using the T2-Kd cell line (TAP1/2 negative) (29) pulsed with a panel of endogenous peptides known to be presented on H-2Kd (19). Due to low MHC class I expression on these cells, there was little lysis by either CTL against T2-Kd cells in the absence of exogenous peptide. Additionally, no peptides in this panel of self-peptides were able to induce killing by IL-12-treated DUC18 CTL (data not shown). Because the DUC18 T cells are specific for a mutated ERK2 peptide with a single amino acid substitution that does not affect binding to H-2Kd (30), we next tested whether IL-12 treatment would allow these cells to respond to the corresponding wild-type ERK2 peptide, KYIHSANVL. Indeed, IL-12-treated DUC18 CTLs efficiently lysed target cells pulsed with the wild-type ERK2 peptide, whereas untreated DUC18 CTLs had only a low reactivity to these targets (Fig. 5⇑, C and D). These data suggest that the self-peptide recognized by the IL-12-treated DUC18 CTLs is from the wild-type ERK2 peptide.

Together, these data suggest that IL-12 treatment allows CTLs to recognize a very restricted subset of self-peptides. These are peptides for which the CTL has a low affinity in the absence of IL-12, possibly the same peptides involved in the positive selection of the cells in the thymus.

LFA-1 on IL-12-treated CTLs is in a higher activation state

Interaction between the β2 integrin LFA-1 on T cells and ICAM-1 on APCs or target cells is required for T cell synapse formation (31). High levels of ICAM-1 alone are known to induce CTL to form ICAM-1 ring structures similar to pSMACs (32). Enhanced LFA-1-ICAM-1 interactions are also capable of enhancing T cell responses to class I MHC-peptide (33). Therefore, we wondered whether differences in LFA-1 could explain the enhanced synapse formation and altered peptide specificity of IL-12-treated CTLs.

We first determined that the level of LFA-1 on the surface of untreated and IL-12-treated CTL was similar by staining with an LFA-1-specific Ab followed by flow cytometry (Fig. 6⇓A). The function of LFA-1 is not only determined by expression level, but also by its activation state (reviewed in Ref. 34). Therefore, we performed two assays to compare the activation state of LFA-1 on untreated and IL-12-treated CTLs. First, we tested the ability of the CTL to bind to plate-bound ICAM-1 in a plate adhesion assay (Fig. 6⇓B). A greater percentage of IL-12-treated OT-1 CTLs adhered to ICAM-1 in this assay than did untreated OT-1 CTL. This difference was most apparent in the presence of the integrin activator PMA. We next tested the ability of the CTL to adhere to ICAM-1 under continuous flow conditions (1 dyne/cm2) using a parallel plate flow chamber. Under these more rigorous conditions, significantly more IL-12-treated CTLs were able to bind to ICAM-1 (Fig. 6⇓, C and D). These data demonstrated that IL-12 treatment enhanced the formation of a higher affinity LFA-1 on the CTLs. This increased LFA-1 affinity is likely responsible for the increased synapse formation and peptide sensitivity of the IL-12-treated CTLs.

FIGURE 6.
FIGURE 6.

LFA-1 on IL-12-treated CTL is in a higher activation state. A, Untreated and IL-12-treated OT-1 CTL were stained with an LFA-1-specific (black lines) or control (gray lines) Ab. B, The percentage of untreated and IL-12-treated OT-1 CTLs capable of binding to ICAM-1 in a plate adhesion assay. Values are the average of six wells at each condition ±SD. Data are representative of multiple separate experiments. ∗, p < 0.01; ∗∗, p < 0.003; two-sided Student t test. C and D, The number of untreated and IL-12-treated OT-1 CTL that adhered to ICAM-1 under shear stress of 1 dyne/cm2 was determined. A representative field is pictured. Values are the average number of cells adhered in each of five different fields ±SD. Data are representative of two separate experiments.

Discussion

Here we tested whether cytokines could affect the quantity and quality of immunological synapse formation by CTL in response to antigenic peptide. We found that IL-12 enhanced, whereas TGFβ decreased, synapse and cSMAC formation by OT-1 CTL. Because both of these cytokines are present in inflammatory reactions in vivo, it seems likely that the effects that we saw are biologically relevant. IL-12 is predominantly produced by activated monocytes, macrophages, and dendritic cells and acts primarily on activated CD4+ and CD8+ T cells and NK cells to induce cell-mediated immunity (35). Alternatively, TGFβ can be produced by regulatory T cells (Treg), suppressing effector T cell function (36). Our data suggest that the differing effects of these two cytokines might be mediated via their effect on synapse formation. In further support of this, Esquerre et al. (37) recently reported that human Tregs can suppress Th function via TGFβ-mediated inhibition of synapse formation between Th cells and dendritic cells.

The enhancement of synapse formation by IL-12 correlated with enhanced LFA-1 avidity toward ICAM-1. LFA-1 interaction with ICAM-1 has a well-characterized role in both synapse formation and costimulation of both T and B cell responses (32, 38, 39). Dustin and coworkers previously reported that high densities of ICAM-1 could stimulate pSMAC formation by CTL in the absence of MHC (32), and recent studies demonstrate a role for LFA-1/ICAM-1 interaction in peptide sensitivity (40, 41). Thus, we were interested to test whether increased LFA-1 avidity might explain our phenotype. Our finding that IL-12 treatment resulted in enhanced LFA-1 avidity can explain both the enhanced synapse formation as well as the enhanced sensitivity to weak ligands.

Although we were surprised that the higher activation state of LFA-1 on IL-12-treated CTLs enhanced synapse formation without altering the efficiency of conjugate formation, this suggests that the resting avidity of LFA-1 on CTLs is high enough to induce maximal conjugate formation but that a higher avidity state is required to facilitate synapse formation. Such a disparity between conjugate and synapse formation requirements has been previously reported (37, 42). The cytokines might alter either the kinetics of synapse formation or the ability of the cells to form synapses entirely. Our current data do not allow us to discriminate between these two possibilities.

Studies suggest that generation of an immunological synapse can affect the sensitivity and/or specificity of a T cell response to a given peptide (40, 43). In particular, experiments with NKG2D, which we have shown also facilitates enhanced synapse and cSMAC formation (16), demonstrated a similar effect with enhanced recognition of weak peptides (43). Computational studies suggest that concentrating TCRs and peptide-MHC complexes in the synapse facilitates signaling, given that occupancy of the TCR is enhanced (11). This would be especially important when the number of antigenic peptide-MHC complexes is low.

The ability of IL-12 to facilitate synapse formation suggested that it would enhance signaling by weaker ligands for the TCR. We found that IL-12 treatment strongly amplified CTL responses of OT-1 cells to a weak agonist (SIIGFEKL) and converted an antagonist (EIINFEKL) into a weak agonist. This is consistent with our hypothesis that increased synapse formation leads to enhanced TCR sensitivity to peptides. It is interesting to speculate on the potentially contrasting roles for an antagonist peptide in the presence or absence of IL-12. In the absence of IL-12, the antagonist peptide might function to inhibit T cell responses; in the presence of IL-12, it could strongly enhance the response.

Although we have not demonstrated that the increased synapse formation seen with IL-12 directly leads to increased CTL killing, the enhanced synapse formation is indicative of stronger signaling. We propose that the increased LFA-1 avidity induced by IL-12 tightens the interaction between CTL and target cells, resulting in enhanced signaling in response to weak peptides. This would then lead to greater synapse formation resulting in even greater signaling.

Using two different CTLs, OT-1 and DUC18, we found that IL-12 treatment led to self-reactivity, suggesting that changes in the efficiency of synapse formation induced by IL-12 allowed for recognition of a self-peptide. This was shown by the facts that IL-12-treated OT-1 and DUC18 CTL could lyse syngeneic splenocytes, that OT-1 CTL could kill unpulsed EL-4 cells, and that DUC18 could kill unpulsed P815 cells. Ab blocking experiments confirmed that the killing was MHC dependent.

We identified putative self-peptides recognized by IL-12-treated OT-1 and DUC18 CTLs using a trial and error approach, testing endogenous peptides known to bind H-2Kb (for OT-1) and H-2Kd (for DUC18). We reasoned that good candidates were those peptides that induce positive selection in the thymus. Positively selecting peptides are thought to have intermediate affinities, too low to trigger T cell activation but higher than the vast majority of nonstimulatory endogenous peptides. Of the six endogenous peptides that we tested, three were able to stimulate IL-12-treated OT-1 CTLs.

In the case of the DUC18 CTL, we reasoned that the self-peptide might be the peptide that corresponds to the sequence from the wild-type ERK2 peptide. The DUC18 TCR-transgenic mouse contains a TCR cloned from the C18 cell line, a CTL line that specifically recognizes a mutated ERK2 peptide in the methylcholanthrene-induced CMS5 tumor cell line. This mutated peptide, QYIHSANVL, contains a glutamine instead of a lysine in the first position of the peptide. Previous studies showed that the DUC18 CTL (18) as well as the original C18 cell line (30) recognize the mutated ERK2 peptide but not the wild type. Because ERK2 is an abundant cellular protein that is ubiquitously expressed, we expect that the wild-type ERK2 peptide should also be ubiquitously present as a self-peptide. In this study, we found that IL-12 treatment induced significant lysis of target cells pulsed with the wild-type ERK2 peptide by DUC18 CTL. Thus, it seems likely that the wild-type ERK2 peptide is at least one of the self-peptides recognized by the IL-12-treated DUC18 CTL.

Together, these data suggest that IL-12 increases the sensitivity of CTLs against a restricted set of self-peptides for which they normally have a low but detectable affinity. These are likely self-peptides involved in the positive selection of the cells during thymic development and maintenance of the cells in the periphery.

While attempting to determine the mechanism by which IL-12 exerted its effects on CTL peptide reactivity, we explored whether there were other differences between untreated and IL-12-treated CTL in addition to LFA-1 avidity. We found no gross difference in the amount of tyrosine-phosphorylated proteins expressed by the two CTLs before stimulation. We tested the possibility that changes in the expression of inhibitory receptors could explain changes in sensitivity. Although we found that expression of the inhibitory receptor NKG2A was consistently lower on IL-12-treated OT-1 CTLs, Ab blocking experiments did not increase the sensitivity of OT-1 CTL to low-affinity peptides. Blocking another inhibitory receptor, PD-1, also had no effect in our system. Because our target cell lines do not express any CD28 or NKG2D ligands, neither of these costimulatory molecules is involved. Lastly, there was no difference between untreated and IL-12-treated CTL in the expression of the micro-RNA miR-181a, the expression of which has been shown to correlate with TCR peptide sensitivity by Li and coworkers (44). A microarray comparison of IL-12-treated and untreated CTL showed only 23 genes or expressed sequence tags that were differentially expressed (Supplemental Table I; National Center for Biotechnology Information Gene Expression Omnibus database accession number GSE13173). No obvious candidate genes identified could readily explain the altered specificity of IL-12-treated CTLs.

IL-12 is a key cytokine in Th1 differentiation, inducing the production of IFN-γ by T and NK cells. IL-12 enhances the generation of CTL by promoting transcription of cytolytic factors, including perforin and granzymes (45). In addition, IL-12 has been described as a third signal required for activated CD8+ T cell survival and for full CTL and memory cell differentiation (reviewed in Ref. 46). Because of all these effector functions, IL-12 is critical to the resistance of many intracellular pathogens (45), enhances antitumor immunity (47), and is involved in multiple autoimmune diseases (48). The importance of IL-12 in all of these diseases is generally believed to be due to its role in driving the generation of a Th1/Tc1 response. However, our data suggest that IL-12 could also be enhancing the affinity of CTL for peptides. Early in an immune reaction, this broadened specificity could be important in allowing a broader range of CTLs to participate, buying time while more Ag-specific T cells are expanding. These responses would also be beneficial to antitumor immunity, which is usually aimed at normal self-Ags. Alternatively, if these self-reactive responses are not controlled, autoimmunity could develop.

In murine studies, IL-12 treatment can result in CD8+ T cell-mediated tumor rejection, and IL-12 has also been shown to increase the number of CD8+ T cells infiltrating tumor sites in both murine and human studies (49). Further, IL-12 can act as a vaccine adjuvant in both mice and humans, resulting in effective T cell responses against self-peptides (50, 51, 52, 53). Our data suggest that one mechanism by which IL-12 may be acting as an antitumor agent is by allowing CTLs to respond to self-peptides expressed on tumor cells for which the CTLs have a low affinity in the absence of IL-12.

Our data lead to the hypothesis that the large amounts of IL-12 found in autoimmune sites could be leading to CTL responses against self-peptides. This is also a possible mechanism by which autoimmunity could be induced by a microbial infection, as has been suggested by several studies (54). Inflammatory sites induced by infection often contain large amounts of IL-12, and this could induce an autoimmune CTL response via normally low-affinity self-peptides. Such a CTL response is probably usually kept in check by Tregs that suppress autoreactive T cell responses (reviewed in Ref. 36). However, if Tregs are absent or deficient, as is the case in many autoimmune diseases, IL-12 could increase CTL-mediated damage.

Acknowledgments

We thank Drs. Kristin Hogquist and Matthew Mescher for helpful discussions.

Disclosures

The authors have no financial conflict of interest.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This work was partially supported by the American Cancer Society (to M.A.M.).

  • ↵2 Address correspondence and reprint requests to Dr. Andrey S. Shaw, Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110. E-mail address: shaw{at}pathology.wustl.edu

  • ↵3 Abbreviations used in this paper: cSMAC, central supramolecular activation cluster; pSMAC, peripheral supramolecular activation cluster; rmIL-12, recombinant mouse IL-12; Treg, regulatory T cell.

  • ↵4 The online version of this article contains supplemental material.

  • Received August 25, 2008.
  • Accepted November 17, 2008.

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