Activation-Induced Nonresponsiveness: A Th-Dependent Regulatory Checkpoint in the CTL Response

Mice and cell lines

OT-I TCR-transgenic mice (25), originally a gift from Dr. F. Carbone (Monash Medical School, Victoria, Australia), were bred to Thy 1.1⁺ C57BL/6/PL mice. Lymph node cells from these mice were used for in vitro experiments and as the source of transgenic T cells for adoptive transfer. C57BL/6 were purchased from The Jackson Laboratory (Bar Harbor, ME). All animals were maintained under specific pathogen-free conditions. All experiments were performed in compliance with the relevant laws and institutional guidelines under a protocol approved by the Institutional Animal Care and Use Committee of the University of Minnesota (Minneapolis, MN). E.G7 tumor, EL-4 thymoma derived from the C57BL/6 mouse (H-2^b) and transfected with the gene for OVA (26), was maintained in vitro in complete RPMI medium containing 400 μg/ml G418. Cell lines were periodically passaged in vivo.

Proliferation assays

Cell-size latex microspheres bearing H-2K^b-OVA_257–264, H-2K^b- OVA_257–264/B7-1, and H-2K^b- OVA_257–264/B7-2 were prepared as described (27). Alternatively, microspheres were prepared by incubating 10⁷ latex microspheres with 2.0 μg of DimerX I H-2K^b fusion protein (BD PharMingen, San Diego, CA) in 1 ml of PBS for 20 m at 4°C. Microspheres were then blocked with BSA, washed, and pulsed with OVA_257–264 peptide at 2.0 μM in 1 ml of PBS for 2 h at 37°C. Lymph node OT-I cells were adherence depleted and the CD8 T cells were enriched by negative selection using CD8 Cellect columns (Cedarlane Laboratories, Hornby, Ontario, Canada). Purity of CD8 T cells was assessed by flow cytometry and was typically 90–95% with <1% CD4 T cells. A total of 5 × 10⁴ CD8 T cells were cultured with 1 × 10⁵ microspheres in 0.2 ml of complete RPMI medium. Cells were pulsed with 1 μCi of [³H]thymidine/well for the last 8 h of the indicated time and incorporation of radioactivity was determined. Results are expressed as the mean ± SD of triplicate samples. Anti-B7-1 (16-10A1) and anti-B7-2 (GL1) blocking Abs were used at 5 μg/ml. Anti-IL-2 receptor (PC61) blocking Ab was used at 10 μg/ml.

In vitro induction and reversal of AINR

Three to 4 days after initial stimulation of CD8 T cells, the AINR cells were harvested and dead cells were removed by Lympholyte M (Cedarlane Laboratories) gradient centrifugation. For all experiments, some of the cells were immediately restimulated to confirm that they were AINR, i.e., no longer responsive to stimulation with K^b-OVA/B7-1 microspheres, but still responsive to IL-2. Reversal of AINR was conducted by stimulating continued proliferation of the AINR CD8 T cells using 2.5 U/ml human rIL-2 for 1–2 days. In each experiment, a proliferation assay was again done at the end of the IL-2 culture period to confirm that AINR had been reversed. In all cases, proliferation assays were done using 5 × 10⁴ CD8 T cells (naive, AINR, or reversed) with 1 × 10⁵ microspheres in 0.2 ml of complete RPMI medium.

IL-2 mRNA measurements

CD8 T cells (naive, AINR, or reversed) and K^b-OVA/B7-1 microspheres were pelleted in 96-well V-bottom plates (1.5 × 10⁵ cells and 3 × 10⁵ microspheres/well) by centrifugation at 1250 × g for 10 min. The pellets were subsequently incubated at 37°C for 6–7 h. Trypan blue exclusion showed no significant change in cell viability at the end of the incubation period. Total RNA was isolated from the cells using the RNAqueous kit (Ambion, Austin, TX). IL-2 and actin mRNAs were determined by RT-PCR analysis using the same IL-2 and actin promoters and conditions as previously described, using a number of cycles confirmed to be within the linear amplification range for the assay (27). Experiments examining the effects of mitogen-activated protein kinase inhibitors on IL-2 mRNA expression were done by pretreating CD8 T cells with either DMSO (Fluka, Buchs, Switzerland), PD98059, or SB202190 (Calbiochem, San Diego, CA) at 37°C for 45 min before stimulation of the cells with microspheres.

In vitro kinase assays and immunoblotting

CD8 T cells (naive, AINR, or reversed) were pelleted with ligand-bearing microspheres by centrifugation at 1250 × g in a swinging bucket rotor for 10 min at 4°C. The pellets were then incubated in a 37°C water bath for 2.5 min (extracellular signal-regulated kinase (ERK) assay), 10 min (c-Jun N-terminal kinase (JNK) assay), or 7.5 min (phospho-p38 Western blot) and the cells were then lysed by the addition of ice-cold Triton-X buffer. ERK and JNK activities were assayed as previously described (27) following precipitation with GST-Elk-1 (aa 307–428) and GST-c-jun (aa 1–69), respectively. For immunoblotting, whole cell lysates were resolved on a 12% SDS-PAGE gel and transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene, NH). Blots were blocked with 5% skim milk powder and incubated with an anti-phospho-p38 Ab (Cell Signaling Technology, Beverly, MA) overnight at 4°C. Detection of a HRP-conjugated secondary Ab was done using the SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL).

Adoptive transfer, tumor challenge, and IL-2 administration

Adoptive transfer of K^b/OVA-specific TCR-transgenic CD8 T cells from OT-I mice and analysis of responding cells was done as previously described in detail (15, 24). Briefly, lymph node cells from OT-I mice on the Thy 1.1⁺ C57BL/6/PL background were adoptively transferred into C57BL/6 mice by i.v. injection. Mice were challenged 1 day later by i.p. injection of 3 × 10⁶ E.G7 cells in 0.5 ml of PBS (day 0). At varying times, mice were sacrificed and cells were isolated from the spleen, draining lymph nodes (periaortic, mesenteric, axillary, and brachial) and the peritoneal cavity. Adoptively transferred OT-I cells in the resulting cell populations were identified and quantitated by flow cytometry by staining with anti-Thy 1.1-PE and anti-CD8α-CyChrome mAbs and CD25 levels were determined using a third anti-CD25-FITC mAb. Flow cytometry was done using a FACSCaliber and CellQuest software (BD Biosciences, San Jose, CA). When used, murine rIL-2 (R&D Systems, Minneapolis, MN) was administered by i.v. (tail vein) injection of 2000 IU/day on days 8 and 9 after inoculation with tumor.

Reversal of AINR by exogenous IL-2

In vitro induction of AINR was examined using CD8 T cells from OT-I mice that have a transgenic TCR specific for OVA_257–264 bound to H-2K^b. Stimulation of these cells with K^b/OVA_257–264 complexes immobilized on microspheres (K^b-OVA) was not sufficient to cause proliferation unless IL-2 was also added to the cultures. However, the need for exogenous IL-2 was overcome if B7-1 ligand was immobilized along with Ag on the microspheres (K^b-OVA/B7-1, Fig. 1⇓A); the B7-1 provides costimulation to allow autocrine production and use of IL-2 by the cells. Proliferation measured by [³H]thymidine incorporation peaks on day 3 and then declines and maximal numbers of OT-I cells are reached on day 4.

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FIGURE 1.

Naive CD8 T cells rapidly become AINR following initial stimulation. A, Freshly isolated OT-I CD8 T cells were stimulated with microspheres having immobilized BSA, K^b-OVA, or K^b-OVA/B7-1 and human rIL-2 was added as indicated. Proliferation was assessed by measuring [³H]thymidine incorporation during the last 8 h of the indicated day. B, Cells stimulated with K^b-OVA/B7-1 microspheres for 3.75 days were harvested, washed, and restimulated using the same stimuli as described in A and proliferation was measured as in A. Results are expressed as the mean ± SD of triplicate samples.

To examine responsiveness following the primary response, cells stimulated with K^b-OVA/B7-1 were recovered from the cultures after 3.5 days, washed, and placed back into culture at the same starting cell density as in the primary stimulation (5 × 10⁴/well). Unlike naive cells, these previously stimulated cells responded well when just IL-2 was added to the cultures; Ag alone stimulated no response nor did it increase the response to exogenous IL-2. Also, in marked contrast to the naive cells, the previously stimulated cells could not respond to Ag even when B7-1 was present to provide costimulation. Thus, the cells become AINR following an initial response, even when effective costimulation is present during the primary response. Although AINR cells cannot be stimulated to make IL-2, some TCR-dependent signaling remains intact because these cells can lyse targets and make IFN-γ in response to Ag (20).

As discussed in more detail below, observations of in vivo responses of OT-I T cells raised the possibility that the AINR state might be reversible upon proliferation of the cells in response to exogenous IL-2. This was examined using OT-I cells harvested after 3.5 days of stimulation with K^b-OVA/B7-1 microspheres. Less than 1% of the OT-I cells remain undivided at this time, as demonstrated in experiments examining dye dilution in cells labeled with CFSE before stimulation (data not shown). Following the initial stimulation, cells were washed and placed back into culture and IL-2 was added. After varying times from 0 to 4 days in culture with IL-2, cells were again harvested, washed, and placed into culture with various stimuli. As expected, the cells were AINR at 3.5 days and responded only if IL-2 was added; they did not respond to Ag or Ag and costimulation alone (Fig. 2⇓A). However, even after 1 day in culture with IL-2, the AINR cells had regained significant responsiveness that was further increased after 2 days and persisted through at least 4 days. The responsiveness of the “reversed” cells was distinctly different from that of naive cells in that Ag alone was sufficient to stimulate a strong proliferative response and coimmobilizing B7-1 to provide costimulation did not cause any further increase in the response. Essentially identical results have been obtained in experiments examining OT-I cells or CD8 T cells from C57BL/6 mice stimulated with microspheres having anti-TCR mAb and B7-1 immobilized on the surface (data not shown).

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FIGURE 2.

Growth in exogenous IL-2 reverses AINR. A, OT-I CD8 T cells were stimulated with K^b-OVA/B7-1 in primary culture (primary). After 3.5 days, AINR cells were harvested and either restimulated immediately (AINR), or were cultured in the presence of human rIL-2 for 1, 2, or 4 days, harvested, and restimulated with microspheres having immobilized BSA, K^b-OVA, or K^b-OVA/B7-1 and IL-2 was added as indicated. For each cell population, proliferation of 5 × 10⁴ cells/well was assessed by measuring [³H]thymidine incorporation during the last 8 h of a 2-day culture period. B, Naive CD8 T cells (left panel) were stimulated with K^b-OVA/B7-1 and K^b-OVA/B7-2 microspheres in the absence or presence of anti-B7-1 and anti-B7-2 blocking Abs, respectively. Reversed CD8 T cells (right panel; 3.5 day initial stimulation, 2 days in IL-2 for reversal) were stimulated with K^b-OVA or K^b-OVA/B7-1 in the absence or presence of anti-B7-1, anti-B7-2, or both mAb, as indicated. Proliferation was assessed by measuring [³H]thymidine incorporation during the last 8 h of a 2-day culture period. Results are expressed as the mean ± SD of triplicate samples.

Proliferation following reversal is autocrine IL-2 dependent but costimulation independent

Stimulation of the reversed cells with Ag alone suggested that these cells had lost a requirement for costimulation to support proliferation. However, activated T cells express B7 ligands and it was important to determine whether transcostimulation was being provided by cell-cell interactions in the culture. Anti-B7-1 and anti-B7-2 mAb effectively block costimulation of naive cells when the respective ligands are coimmobilized on microspheres along with Ag (Fig. 2⇑B). These same mAbs fail to block the response of reversed cells to Ag alone on the microspheres (Fig. 2⇑B), thus demonstrating that costimulation provided by B7 ligands on the T cells is not contributing to this response.

Because production of IL-2 by naive cells requires costimulation, the independence from costimulation following reversal of AINR raised the possibility that this response did not depend on IL-2. However, proliferation of the reversed cells in response to Ag alone was completely dependent on IL-2, as demonstrated by the effective blocking of the response that occurred when anti-IL-2R mAb was added to the cultures (Fig. 3⇓). Consistent with this, Ag alone is sufficient to up-regulate IL-2 mRNA expression in reversed cells (see below).

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FIGURE 3.

Proliferation following reversal of AINR depends upon autocrine IL-2 production. OT-I CD8 T cells were stimulated initially with K^b-OVA/B7-1 for 3.5 days and the AINR cells were cultured for an additional 2 days in IL-2 to reverse nonresponsiveness. These reversed cells were then restimulated using the indicated stimuli in the presence or absence of anti-IL-2R blocking mAb. Proliferation was assessed by measuring [³H]thymidine incorporation during the last 8 h of a day 2 culture. Results are expressed as the mean ± SD of triplicate samples.

Altered requirements for activation of mitogen-activated protein kinases (MAPKs) following reversal of AINR

MAPKs are involved in regulating the transcription factors required for IL-2 gene expression in T cells and up-regulation of ERK, JNK, and p38 is defective in cells that have become AINR (27). Therefore, we examined cells that had recovered responsiveness following AINR to determine whether activation of the MAPKs was also restored. ERK and JNK activities were measured in lysates of stimulated cells using GST-Elk-1 and GST-c-jun fusion proteins as the substrates. Activation of p38 was assessed by Western blotting with a phospho-specific anti-p38 Ab to determine the amount of the active phosphorylated form of the enzyme.

ERK is activated in naive cells in response to Ag and its activity does not increase further upon costimulation with B7-1 and/or ICAM-I (Fig. 4⇓A). Activation was greatly reduced in AINR cells, but restored in reversed cells to the same levels as in naive cells. In contrast to ERK, JNK activation in naive cells requires costimulation (Fig. 4⇓B). Activation of JNK was also greatly reduced in AINR cells and restored in reversed cells. However, in the reversed cells, Ag alone was sufficient to fully activate JNK, and the presence of costimulatory ligands caused no further increase in activity. Active phospho-p38 increases in naive cells in response to just Ag (Fig. 4⇓C). AINR cells were profoundly defective in phospho-p38 formation, but activation was restored in reversed cells. Thus, activation of all three MAPKs is defective in AINR cells and restored in reversed cells, and some rewiring occurs during the reversal such that activation of JNK no longer depends upon costimulation as it does in naive cells.

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FIGURE 4.

Activation of MAPKs is restored upon reversal of AINR and costimulation requirements are altered. Naive, AINR, and reversed cells were either left unstimulated or stimulated with K^b-OVA or K^b-OVA/B7-1 microspheres for 2.5 min (ERK assay), 10 min (JNK assay), or 7.5 min (phospho-p38 Western blot) before being lysed by a Triton X-100 buffer. Times used for stimulation were those found to yield optimum activation of each enzyme. A, ERK kinase activity determined using GST-Elk-1 fusion protein as a substrate. B, JNK kinase activity determined using GST-c-Jun fusion protein as a substrate. C, Activation of p38 was assessed by Western blotting with a mAb specific for phospho-p38.

PD98059 is a specific inhibitor of mitogen-activated protein/extracellular signal-related kinase kinase and, therefore, ERK, and SB202190 blocks p38 activation and, at somewhat higher concentrations, JNK activation. Both block proliferation of naive CD8 T cells (27). The restoration of activation of the MAPKs, and the parallel loss of a costimulation requirement to activate JNK and induce proliferation, suggested that the MAPK pathway was also involved in stimulation of cells following reversal from AINR. This was confirmed in experiments demonstrating that both PD98059 (Fig. 5⇓, A and C) and SB202190 (Fig. 5⇓, B and D) blocked IL-2 mRNA up-regulation and proliferation by reversed cells upon stimulation with either Ag alone or Ag and B7-1. These results support the conclusion that recovery from AINR involves, at least in part, recovery of the ability to activate the MAPKs to signal for IL-2 production.

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FIGURE 5.

MAPK inhibitors block IL-2 mRNA up-regulation and proliferation by reversed cells in response to Ag or Ag and costimulation. A, Reversed cells were pretreated with PD98059 at 37°C for 45 min before being restimulated with K^b-OVA or K^b-OVA/B7-1 microspheres for 7 h. RNA was then isolated from the cells and RT-PCR was performed using IL-2 and β-actin-specific primers. B, The same as in A, but using SB202190. C, Reversed cells were restimulated with K^b-OVA or K^b-OVA/B7-1 microspheres in the presence of the indicated concentrations of PD98059 and proliferation was assessed by measuring [³H]thymidine incorporation during the last 8 h of a day 2 culture period. Results are expressed as the mean ± SD of triplicate samples. D, The same as in C, but using SB202190.

In vivo reversal of AINR by IL-2 administration

Adoptive transfer of T cells from TCR-transgenic mice into normal recipients provides a means of directly visualizing and quantitating in vivo responses; the transgenic cells can be identified for flow cytometric analysis but are present in small numbers so that the immune response is not completely skewed as it is in the intact transgenic animal (28). We have used this approach to examine the response of transferred OT-I cells to i.p. E.G7 tumor that expresses OVA (15, 24). Within 3–4 days of i.p. injection of the tumor, OT-I cells migrate to the peritoneal cavity, undergo activation and clonal expansion, and control tumor growth. Clonal expansion peaks at about day 5 and the OT-I cells then migrate out of the peritoneal cavity and are found in the spleen and lymph nodes, even though the Ag-expressing tumor is still growing in the peritoneal cavity. The OT-I cells that have responded and migrated to the spleen have developed AINR; they exhibit direct ex vivo killing activity and can proliferate in response to exogenous IL-2 but not in response to Ag and costimulation.

The ability to reverse the AINR state in vitro by driving proliferation for 1–2 days with IL-2 predicted that it might also be possible to reverse the nonresponsive state in vivo with limited administration of IL-2 and achieve long-term responsiveness of the CD8 T cells once AINR was reversed. This was tested by challenging OT-I adoptive transfer recipients by i.p. injection of E.G7 tumor on day 0, allowing the initial response to occur unperturbed, and then administering low dose (2000 U/mouse) IL-2 systemically on days 8 and 9, the time at which the OT-I cells have become AINR (24). Mice were then sacrificed 10 days later (day 18 after tumor challenge) and the numbers of OT-I cells in the peritoneal cavity, spleen, and lymph nodes were determined, as well as the number of E.G7 tumor cells in the peritoneal cavity.

In adoptive transfer recipients that had not been challenged with tumor, OT-I cells were present in the spleen and lymph nodes in low numbers (Fig. 6⇓A, OT-I) and had a naive phenotype (data not shown). In mice challenged with tumor and left untreated (Fig. 6⇓A, OT-I/E.G7), some OT-I cells were present in the peritoneal cavity but greater numbers were found in the spleen and lymph nodes (Fig. 6⇓A) and tumor load was high (Fig. 6⇓B). In contrast, mice treated with IL-2 on days 8 and 9 had a high number of OT-I remaining in the peritoneal cavity by day 18 (Fig. 6⇓A, OT-I/E.G7 + IL-2, days 8–9), as well as elevated numbers in the spleen and lymph nodes (Fig. 6⇓A), and tumor load was substantially reduced in comparison to that in untreated mice (Fig. 6⇓B). Essentially the same results have been obtained in similar experiments done using recipient mice depleted of CD4 T cells by in vivo mAb administration or CD4 knockout recipients, thus ruling out a role for host CD4 T cells in reversal of the AINR state by IL-2 administration (data not shown).

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FIGURE 6.

In vivo reversal of CD8 T cell nonresponsiveness by IL-2. A, Lymph node cells from OT-I mice were adoptively transferred into C57BL/6/PL recipients. Mice were either left unchallenged (OT-I), or challenged 1 day later by i.p. injection of E.G7 tumor cells. One group received 2000 U of IL-2 on days 8–9 by i.v. injection as indicated. Mice were sacrificed on day 18 and the number of OT-I cells in the peritoneal cavity, spleen, and draining lymph nodes was determined. B, The number of E.G7 tumor cells in the peritoneal cavity on day 18 was determined for the groups of mice described in A. The groups had two mice each and error bars show the range of values obtained. Similar results were obtained in more than six independent experiments.

In addition to causing higher numbers of OT-I cells to be present at late times, brief IL-2 treatment resulted in prolonged responsiveness of the cells. Cells from both the lymph nodes and peritoneal cavities of mice treated on days 8 and 9 with IL-2 were continuing to respond at day 18, long after the exogenous IL-2 was gone, as evidenced by the fact that a large fraction of the cells had high forward scatter, indicating that they were blasting, and more than half expressed high CD25 (IL-2Rα) expression (Fig. 7⇓). In contrast, few of the day 18 OT-I cells from mice that had not received IL-2 exhibited high forward scatter or high CD25 expression (Fig. 7⇓). Thus, brief exposure to IL-2 is sufficient to reverse AINR and allow prolonged in vivo response to the tumor.

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FIGURE 7.

OT-I CD8 T cells undergo prolonged activation following in vivo reversal of AINR. Cells recovered from the draining lymph nodes and peritoneal cavities of mice described in Fig. 6⇑ were stained with anti-Thy 1.2 and anti-CD8 mAb to identify OT-I T cells (left panels). The OT-I populations identified by the boxes shown in the left panels were then analyzed for forward side scatter (middle panel) and CD25 expression (right panel). The numbers are the percentage of cells falling within the gates indicated by the horizontal bars. Similar results were obtained in more than four independent experiments.