Adjuvant Effect of IL-12: Conversion of Peptide Antigen Administration from Tolerizing to Immunizing for CD8+ T Cells In Vivo

Clint S. Schmidt and Matthew F. Mescher
J Immunol September 1, 1999, 163 (5) 2561-2567;

Abstract

CD8+ T cells from TCR transgenic 2C mice, specific for SIYRYYGL peptide bound to H-2Kb, were adoptively transferred into C57BL/6 recipients to allow monitoring of their location, numbers, and phenotype upon peptide challenge. Recipients were primed by s.c. injection of SIYRYYGL alone or with CFA or IL-12, and the transferred cells then tracked by flow cytometry using the 1B2 mAb specific for the 2C TCR. Peptide alone induced a transient and weak expansion of 1B2+ cells in the draining lymph nodes (DLN) by day 3, but these cells were tolerant to secondary peptide challenge. In contrast, priming with CFA/peptide resulted in a large clonal expansion of 1B2+ cells in DLN by day 3, and the cells exhibited a CD25highCD44high phenotype, blast transformation, and lytic effector function. By day 5, 1B2+ cell numbers decreased in the DLN and increased in the spleen and blood. 1B2+ cells with a memory phenotype persisted through day 60 in the DLN, spleen, and blood and responded to secondary peptide challenge. Immunization with peptide, along with IL-12, mimicked the adjuvant effects of CFA with respect to phenotype, clonal expansion, effector function, and establishment of memory. IL-12 was not unique in providing this adjuvant effect however, since CFA/peptide immunization of IL-12-deficient recipient mice also resulted in 1B2+ T cell activation and clonal expansion. Thus, CFA or IL-12 can enhance Ag-specific CD8+ T cell responses to peptide, demonstrating that an inflammatory cytokine(s) can support activation and prevent tolerance induction.

In vivo presentation of soluble peptide Ag to naive T cells can result in either full activation and development of effector function, or, conversely, may induce tolerance in an Ag-specific manner. Many factors influence the two outcomes, such as the physical state of the Ag along with dose and route of immunization (1, 2, 3). It has been argued that the in vivo difference between a tolerogenic and an immunogenic T cell response depends on whether the cell senses “danger” in the environment (4, 5). Indeed, full effector function in vivo is only achieved when soluble peptide Ag is administered with adjuvant or as part of an intact organism (6, 7, 8). Thus the adjuvant, whether in the form of CFA, LPS, or other bacterial cell wall components, or aggregate protein, must incite an environment that is interpreted as “dangerous” by the host immune system. This awareness could be attributed to many causes, but a prevalent view is that inflammatory cytokines, produced by cells of the innate immune system and/or APC, provide the signals necessary for the induction of an effector response (5).

Previous CD4+ T cell studies have confirmed the importance of adjuvant to prevent tolerance in vivo, and have even begun to define molecular characteristics involved in full T cell activation (9, 10). In addition to costimulation and the resulting production of IL-2, IL-1 has been identified as a cytokine capable of inducing a strong in vivo peptide response for CD4+ T cells (11). In vitro studies have also identified some inflammatory mediators produced by professional APCs, including IL-1, IL-6, IL-12, and IFN-γ, that can positively influence a naive CD8+ T cell’s proliferation and/or differentiation to effector CTL (12, 13, 14, 15). Recent in vitro studies using latex microspheres coated with various combinations of class I MHC/peptide complexes and costimulatory molecules have shown that IL-12, but not IL-1, acts directly on the naive CD8+ T cell to promote clonal expansion and differentiation (16). The in vitro results demonstrating a direct effect of IL-12 on CD8+ T cells suggested that this cytokine might prevent Ag-specific tolerance upon peptide administration, and support an effective in vivo CD8+ T cell response.

The heterodimeric cytokine IL-12 was originally identified as NK cell stimulating factor, and is known to stimulate CD4+ T cell differentiation to Th1 effectors (14, 17). IL-12 has also been shown to have anti-tumor effects in vivo, effects that at least in some cases can be attributed to production of IFN-γ (18, 19, 20). Clinical trials have also shown IL-12 to induce a higher CTL precursor frequency in humans, although this clinical effect was accompanied by significant side effects (21, 22). Aside from, but not excluding, the past studies, little is known about the effects of IL-12 on the in vivo etiology, kinetics, anatomy, and relative stability of a primary CD8+ T cell response. To examine this, we have used a system in which a small number of naive Ag-specific CD8+ T cells are adoptively transferred into naive, syngeneic recipients (23) to monitor their response to peptide immunization in the presence or absence of adjuvant and inflammatory cytokines. IL-12 was found to mimic the effects of CFA in preventing tolerance, and to be effective in supporting clonal activation, migration, development of effector function, and establishment of immunologic memory. While IL-12 administration is sufficient to support a strong response, it is not an absolute requirement, since a strong response can still be obtained using peptide and CFA in IL-12 (p40)-deficient mice.

Materials and Methods

Mice

2C TCR transgenic mice (24) were obtained as a kind gift from Dr. Dennis Loh (Washington University, St. Louis, MO) and were bred to wild-type C57BL/6 mice to generate mice heterozygous for the 2C TCR transgene. CD8+ T cells from these mice were used as donors in all adoptive transfer experiments, and C57BL/6 mice (Charles River Laboratories, Wilmington, MA) or IL-12 knockout (KO)3 mice on a C57BL/6 background (25) (The Jackson Laboratory, Bar Harbor, ME) were used as recipients. All mice were housed in a specific pathogen-free environment at all times.

Antibodies

The 1B2 mAb, specific for the transgenic 2C TCR, was from the 1B2 hybridoma (26) (a gift from Dr. Herman Eisen, Massachusetts Institute of Technology, Boston, MA) grown in vitro in complete RPMI media (RPMI 1640; Cellgro, Herndon, VA) with 10% FCS (Tissue Culture Biologicals, Tulare, CA), 0.2% l-glutamine, 0.1% penicillin/streptomycin, 0.1% HEPES (BioWhittaker, Walkersville, MD), 0.1% nonessential amino acids, 0.1% sodium pyruvate (Cellgro), and 0.05% 2-ME. The 1B2 mAb was purified from supernatants using a protein A-Sepharose column with elution by citric acid at pH 3.5. Fractions containing Ab were pooled, dialyzed into PBS, and biotinylated for 4 h at room temperature with 0.15 mg/ml biotin in DMSO and 0.15 mg/ml NaHCO3 per 1 mg/ml mAb. Other Abs used were anti-CD44-FITC (Pgp1), anti-CD25-FITC, anti-CD8α- CyChrome, streptavidin-APC, and rat IgG2a κ-FITC isotype control (all from PharMingen, San Diego, CA).

Cell lines

The thymoma EL4 (H-2b) was grown in vitro in complete RPMI medium (see above). Cultures were always 90–100% viable, as measured by trypan blue exclusion, and cells were washed with PBS before use in in vitro chromium release assays (see below).

Adoptive transfer of 2C transgenic cells

Lymph node cells (axillary, brachial, cervical, inguinal, periaortic, and mesenteric) from heterozygous 2C transgenic mice were removed, homogenized, ammonium chloride-treated to remove RBC, and adherence-depleted for 90 min. The nonadherent cells were washed in PBS and enriched for CD8+ cells using the CD8+ Cellect column purification kit (Biotex Laboratories, Edmonton, Canada). Before transfer, the purified population was analyzed by flow cytometry to determine the percentage of 1B2+ CD8+ cells and their phenotype with respect to CD25, CD44, and forward scatter (FSC) to insure that the transferred population was phenotypically naive. A total of 3–5 × 106 1B2+ CD8+ cells in 500 μl PBS was transferred via tail vein injection into age- and sex-matched naive 6- to 8-wk-old recipients. Recipient mice were then rested for 24 h before immunizations.

Immunizations

The synthetic peptide SIYRYYGL (27) (Chiron Mimotopes, Clayton, Victoria, Australia) was prepared in PBS and injected alone (peptide only) or emulsified in CFA (CFA/peptide) (Sigma, St. Louis, MO). All peptide immunizations involved the s.c. injection of 50 μg peptide in 300 μl per mouse distributed between two sites on the back. Recombinant murine IL-12 (Genetics Institute, Cambridge, MA) was administered i.p. at 1 μg in 100 μl PBS with 0.1% sterile mouse serum on days 0, 1, and 2. As controls, transferred animals were also immunized with PBS alone (Transfer Only).

Flow cytometric analysis of in vivo populations

Transferred and immunized mice were sacrificed at varying times after priming or rechallenge, and the lymph nodes and spleen were removed, homogenized, and ammonium chloride-treated to lyse RBC. PBL was drawn from the heart using a heparin-loaded syringe, and the RBC were lysed by ammonium chloride treatment. Brachial, axillary, and inguinal lymph nodes (LN) were pooled as draining lymph nodes (DLN). Each cell population (DLN, spleen, and PBL) was counted for total cell population using trypan blue to exclude dead cells. Isolated cells (1–2 × 106) from each site were stained with 1B2-biotin mAb, anti-CD8α-CyChrome, and either anti-CD44-FITC (Pgp1) or anti-CD25-FITC. After 20–30 min of incubation, cells were washed and streptavidin-APC was added for detection of 1B2-biotin. Stained cells were fixed with 1% formaldehyde and analyzed by flow cytometry using the CellQuest software package (Becton Dickinson, San Jose, CA), as described (23). All cytometer settings were identical for all time points within a given experiment. A total of 35–40 × 103 lymphocyte-gated events were collected and analyzed, and the percentage of 1B2+ CD8+ cells was multiplied by the total number of cells recovered from the site to determine the total number of 1B2+ CD8+ cells. All determinations were done in duplicate mice for each condition and time point. Values shown are averages, and error bars represent the range of the duplicates. Phenotypes of the 2C cells from various sites were determined by gating on the 1B2+CD8+ cells and collecting 200 events examining the FITC fluorescence of the various phenotype marker mAb. Gates denoting high and low expression of each surface marker were set based on the phenotype of naive T cells. Replacing the specific mAbs with isotype control mAbs resulted in almost no events (<1%) falling into the high gate for each marker.

Chromium release assay

Following immunizations, DLN cells of duplicate mice were harvested, pooled, homogenized, and enriched for CD8+ cells using the CD8+ Cellect column purification kit (Biotex Laboratories). These cells (of which 0.12–1.12% are 1B2+ CD8+) were then assayed using a standard 4-h 51Cr release assay with SIYRYYGL-pulsed EL4 target cells.

Results

Primary in vivo CD8+ T cell response to soluble peptide Ag in the presence or absence of CFA

The primary response of naive CD8+ T cells to soluble peptide Ag was monitored in vivo through the use of an adoptive transfer system utilizing 2C TCR transgenic CD8+ T cells that recognize the synthetic peptide SIYRYYGL in the context of H-2Kb (23, 24, 27). Phenotypically naive CD8+ 2C T cells (3–5 × 106) were injected i.v. into naive age/sex-matched C57BL/6 recipients, and the resulting 2C populations were monitored throughout the course of an immune response by the use of the anticlonotypic mAb 1B2 (26) and flow cytometric analysis. The 1B2+ CD8+ population comprises 0.2–0.4% of total LN cells 1 day after transfer and before immunization (23). Transferred mice were rested for 1 day and then immunized with SIYRYYGL peptide (s.c. 50 μg/mouse, distributed between two sites on the back) in the presence or absence of CFA. The amount of SIYRYYGL peptide used was based on previous in vivo dose/response titrations that resulted in optimal 1B2+CD8+ responses (data not shown).

Immunization with SIYRYYGL emulsified in CFA resulted in massive 1B2+ CD8+ clonal expansion in the DLN (inguinal, brachial, axillary) by day 3 (Fig. 1⇓A). The cells had increased levels of CD44 (Fig. 1⇓C), CD25, and blast transformation (49.7% CD25high, 74.8% FSChigh; Table I⇓), indicating that they were proliferating. To confirm that the increased 1B2+ numbers reflected clonal expansion and not simply increased migration to the site, donor cells were labeled with PKH26 lipophilic dye before adoptive transfer, and the fluorescence intensity was found to be highly diluted by day 3 as cells expanded (data not shown, and Ref. 16). 1B2+ CD8+ cell numbers peak at day 3, and the cells then migrate out of the DLN, and, by day 5, large numbers could be detected in the spleen and PBL. The numbers at these sites gradually decline as the primary response subsides, and, by day 20, only a small population of 1B2+CD8+ cells (4–6 × 104 in DLN) with a memory phenotype (54.6% CD44high) is still present (data not shown, and see below). The 2C cells detected in nondraining lymph nodes, such as mesenteric lymph nodes (MLN), did not proliferate in response to peptide Ag and retained a CD25−CD44low phenotype through day 20.

FIGURE 1.
FIGURE 1.

1B2+CD8+ cell numbers, location, and CD44 phenotype during the primary response to SIYRYYGL. Transferred 2C cells (3–5 × 106) were detected during the course of the primary response by flow cytometry using the 1B2 mAb specific for the 2C TCR and flow cytometry. A, 1B2+CD8+ cell numbers in the DLN, MLN, spleen, and PBL from days 1–8 after challenge with SIYRYYGL emulsified in CFA (50 μg peptide in 300 μl delivered s.c. to two sites on the back of the mouse). B, As in A, except after challenge with SIYRYYGL (50 μg) in PBS. Inset graph shows the same data plotted using a different scale on the y-axis. C, CD44 levels of 1B2+CD8+ populations from recipient mice after indicated immunizations. Data is representative of 10 independent experiments. Each time point and condition was performed in duplicate mice. Values shown represent the average of the duplicate mice and error bars represent the range.

View this table:
Table I.

Phenotype of 1B2+CD8+ cellsa

In contrast to immunization with adjuvant, 2C cells in mice immunized with SIYRYYGL alone in PBS showed only a low level of clonal expansion at all sites by day 3 (Fig. 1⇑B, note change of scale). By day 1, significant numbers of these cells in the DLN had acquired high levels of CD25 (34.7%) and CD44 (39.8%) and had undergone blast transformation (52.7% FSChigh) (Fig. 1⇑C, bottom, and Table I⇑), indicating that the 2C cells had indeed recognized and responded to the peptide challenge. This low-level response, however, was very transient, since 2C cell numbers in all sampled sites dropped to a point where accurate phenotypic analysis could not be made by day 8, and, by day 20, these cells were only detectable close to background (0.01% of total cells). To determine whether this response was specific, adoptively transferred mice were also immunized with an equal amount of SIINFEKL, an OVA peptide also bound by H-Kb. The 1B2+CD8+ cells in these mice remained essentially the same in both absolute numbers and phenotype as those found in transferred mice immunized with PBS (data not shown).

The reduced and transient CD8+ T cell response to soluble antigenic peptide immunization (as compared with CFA and peptide) is consistent with the adjuvant effect noted in previous studies examining CD4+ T cells in an adoptive transfer system. Immunization with peptide alone caused some CD4+ T cell clonal expansion, but this response was short-lived and resulted in long-term Ag-specific nonresponsiveness. In contrast, peptide immunization with adjuvant caused much larger clonal expansion (up to 3-fold higher than with peptide alone) along with development of effector function (9). The adjuvant effect is also present for CD8+ T cells, but the level of proliferation obtained with adjuvant in comparison to peptide alone is much greater (up to 25-fold greater with adjuvant; Fig. 1⇑, A and B). The fact that transferred 2C cells appeared to proliferate simultaneously in all sampled sites with peptide alone, as opposed to proliferating first in the DLN and subsequent migration to the spleen and PBL, as seen with CFA and peptide, may suggest that the adjuvant and peptide emulsification serves as an Ag depot to prevent the peptide from being distributed systemically in the animal. Comparable results to those shown in Fig. 1⇑ and Table I⇑ were obtained in 10 independent experiments. Additional experiments demonstrated that IFA had similar effects to CFA in this system (data not shown).

IL-12 mimics the CFA adjuvant effect during an in vivo peptide response

Based on its proinflammatory properties and previous in vitro results (14, 16), IL-12 was examined to determine whether it could replace CFA in the context of clonal expansion and development of effector function. Cells from the DLN, spleen, and PBL were examined on day 3 after immunization to correspond to the time of maximum clonal expansion seen in the CFA/peptide experiments described above. IL-12 (1 μg/day, 2.7 × 103 ± 1.2 × 103 U/μg) was administered i.p. on days 0, 1, and 2 in 100 μl PBS with 0.1% sterile mouse serum in addition to s.c. peptide immunization. IL-12/peptide supported nearly the same extent of clonal expansion as CFA/peptide in the DLN on day 3 (Fig. 2⇓). IL-12 also caused substantial clonal expansion in the spleen, most likely a consequence of both peptide and IL-12 being systemic, as opposed to the Ag remaining localized in CFA (Fig. 2⇓). The phenotypes of 1B2+CD8+ cells responding to IL-12/peptide were also nearly identical to those primed with CFA/peptide, with high levels of CD25, CD44, and blast transformation by day 3, and also showed decreased PKH26 fluorescence as described above (data not shown, and Ref. 16). To address whether this IL-12 effect was CD4+ T cell-dependent, CD4−/− mice were also used as naive adoptive transfer recipients. Proliferation and phenotype changes of transferred cells in response to CFA/peptide and IL-12/peptide were essentially the same as those observed in normal C57BL/6 recipients (data not shown), demonstrating that CD4+ T cells do not contribute in a detectable way to these responses.

FIGURE 2.
FIGURE 2.

1B2+CD8+ cell numbers in the DLN, spleen, and PBL on day 3 after immunizing with SIYRYYGL in the presence or absence of adjuvant or IL-12. Immunizations on day 0 include PBS alone (transfer only), SIYRYYGL in PBS (peptide only), SIYRYYGL in CFA (CFA/peptide), and SIYRYYGL + IL-12 (IL-12/peptide). All immunizations included 50 μg peptide in 300 μl CFA or PBS distributed between two sites on the back. Recombinant murine IL-12 (1 μg/mouse in 100 μl PBS with 0.1% sterile mouse serum) was injected i.p. on days 0, 1, and 2. Data is representative of seven independent experiments. Each time point and condition was performed in duplicate mice. Values shown represent the average of the duplicate mice and error bars represent the range.

Along with causing increased clonal expansion, IL-12 was also effective in supporting effector function development. CTL activity of 1B2+CD8+ cells was directly measured ex vivo using SIYRYYGL-pulsed target cells in a standard 4-h 51Cr release assay. DLNs of transferred and immunized animals were harvested and pooled for two mice on day 3, and total CD8+ cells were column-purified by negative selection (see Materials and Methods). The purified CD8+ T cells were then assayed for lytic activity at varying E:T ratios (Fig. 3⇓) and analyzed through flow cytometry to determine the percentages of 1B2+CD8+ T cells in this population. To establish that target cell lysis was indeed due to the transferred 1B2+ population, naive C57BL/6 mice that were not adoptively transferred were also immunized with CFA/peptide and assayed. IL-12/peptide and CFA/peptide did induce lytic effector function of transferred cells (30.6% and 19.2% specific lysis, respectfully) as measured at a 100:1 E:T ratio using the total CD8+ population. When corrected for the number of 1B2+CD8+ cells in the purifed CD8+ populations, the actual E:T ratios at 100:1 are as follows: transfer only, 0.12:1; peptide only, 0.32:1; IL-12/peptide, 1.33:1; CFA/peptide, 0.52:1; no transfer, CFA/peptide < 0.01:1. The CFA/peptide-primed, nontransfer control samples had little detectable killing above background, nor did the transfer only and peptide only samples show significant activity (<1.6% specific lysis; Fig. 3⇓). These data suggest that both IL-12 and CFA support development of effector function during peptide immunization and prevent tolerance induction.

FIGURE 3.
FIGURE 3.

CFA and/or IL-12 support development of lytic effector function of transferred 1B2+CD8+ cells primed with SIYRYYGL. Adoptively transferred mice were immunized with the indicated combinations of Ag on day 0, and lytic effector function was measured on day 3 by direct ex vivo 51Cr-release assay against SIYRYYGL-pulsed EL4 target cells. CD8+ cells were first purified from transferred animals by negative selection columns. Transferred 1B2+CD8+ cells represent a small percentage of the purified CD8+ cells; thus, at the 100:1 E:T data points, the actual 1B2+CD8+:target ratios are: transfer only, 0.12:1; peptide only, 0.32:1; IL-12/peptide, 1.33:1; CFA/peptide, 0.52:1; no transfer, CFA/peptide < 0.01:1. The “no transfer:CFA/peptide” group represents mice that did not receive an adoptive transfer of 1B2+CD8+ cells, but were immunized with CFA/peptide. Transfer only, peptide only, and no transfer:CFA/peptide samples all had specific lysis of <1.6%. Data is representative of four independent experiments. Duplicate mice were used for each immunization, and DLN cells were pooled before CD8+ column purification to increase the number of 1B2+CD8+ T cells in the assay culture.

Although immunization with peptide in the absence of adjuvant (peptide only) does stimulate some limited clonal expansion (Fig. 1⇑B), this does not appear sufficient to induce lytic function in these cells (Fig. 3⇑). No killing activity was detectable in cells from the mice that received just peptide (peptide only) when lysis was examined at a 1B2+CD8+:target ratio, where killing was readily apparent by cells from mice immunized with peptide along with CFA or IL-12.

IL-12 is not required for the CFA adjuvant effect

While IL-12 can be necessary and sufficient to support a response to peptide Ag, it may not be the only means of supporting this response. To examine this, IL-12 (p40)-deficient mice (25) were used as naive recipients for the 2C adoptive transfer to determine whether a response could still be obtained to CFA/peptide immunization. Column-purified 1B2+CD8+ T cells from 2C mice were adoptively transferred into either IL-12 KO mice or naive C57BL/6 mice, to evaluate the effects of both CFA and IL-12 during peptide immunization. Fig. 4⇓ shows the absolute numbers of 1B2+CD8+ cells in the DLN on days 3 and 5 after immunization. The CFA/peptide response was still intact in the IL-12 KO mice, indicating that IL-12 is not unique in providing the adjuvant effect. IL-12 was also still able to support a strong proliferative response in these mice.

FIGURE 4.
FIGURE 4.

CFA + SIYRYYGL and IL-12 + SIYRYYGL administration to 2C transferred, IL-12 KO recipient mice results in 1B2+CD8+ T cell clonal expansion. IL-12 KO mice (25 ) and naive C57BL/6 mice were first transferred with 5 × 106 column-purified 1B2+CD8+ cells on day −1 and then immunized on day 0 with the indicated combinations of Ag. The resulting 1B2+CD8+ clonal expansion was measured on day 3 and day 5. Results are the averages of duplicate mice for each condition, and error bars indicate the range of values obtained. Essentially, identical results were obtained in two independent experiments.

IL-12 acts as an adjuvant to prevent tolerance, while supporting memory establishment

To directly analyze the effects of IL-12 and CFA on tolerance vs establishment of a memory population, transferred and immunized animals were given a secondary immunization 60 days after the primary challenge. The DLN, spleen, and PBL of the animals were then analyzed on day 63 (day 3 of secondary immunization) for 1B2+CD8+ clonal expansion. Animals that were transferred but only given PBS for both immunizations (PBS/PBS: primary immunizations for mice groups are listed first, followed by the secondary challenge) had a low but detectable level of 1B2+CD8+ cells in the DLN (3.1 × 103 cells/mouse) and spleen (7.0 × 103 cells/mouse) on day 63 (Fig. 5⇓). These cells, being naive, were capable of responding to CFA/peptide challenge (PBS/CFA + peptide). Animals that were immunized first with peptide alone and then received just PBS (peptide only/PBS) also had small populations of 1B2+CD8+ cells in the DLN (3.4 × 103 cells/mouse) and spleen (7.0 × 103 cells/mouse) (Fig. 5⇓), and these had a CD44high phenotype (39%), indicating that they had made some response to the initial immunization (data not shown). These cells, however, were incapable of responding to a secondary challenge of peptide even in the presence of CFA (peptide only/CFA + peptide). This lack of proliferation to secondary challenge with peptide was prevented by initially using CFA or IL-12 during the primary peptide immunization. This resulted in a larger 1B2+CD8+ population in the DLN by day 63 (6.5 × 104 for CFA + peptide/PBS and 3.6 × 104 for IL-12 + peptide/PBS), which is 11- to 19-fold larger than the population observed with peptide alone (peptide only/PBS). In addition, these cells had a phenotype characteristic of memory cells (>75% CD44 high; data not shown). Secondary challenge of these cells with peptide and CFA or IL-12 on day 60 resulted in significant proliferation within 3 days. If both peptide immunizations were accompanied by CFA (CFA + peptide/CFA + peptide), a 3-fold increase in 1B2+CD8+ cell numbers (over CFA + peptide/PBS) was observed in the DLN. If IL-12 was administered with peptide on both days 0 and 60 (IL-12 + peptide/IL-12 + peptide), the subsequent memory response dwarfed that seen with CFA, and even approached the levels seen in a primary response (nearly 22-fold higher than IL-12 + peptide/PBS; Fig. 5⇓). To insure that this 1B2+CD8+ response to secondary challenge is indeed a memory response, donor cells were labeled with PKH26 lipophilic dye before adoptive transfer and primary immunization, as mentioned above. The fluorescence intensity of 1B2+CD8+ cells was completely lost by day 8 as the primary response occurred, demonstrating that all detectable cells had responded to the initial Ag encounter (data not shown, and Ref. 16).

FIGURE 5.
FIGURE 5.

CFA + SIYRYYGL and IL-12 + SIYRYYGL induce a memory population of 1B2+ cells capable of responding to secondary SIYRYYGL challenge. Naive C57BL/6 mice were adoptively transferred with 1B2+CD8+ cells on day −1, and immunized with the Ag combinations listed first on day 0. Sixty days later, the mice were rechallenged with the Ag combinations listed second, and 1B2+CD8+ numbers were measured on day 63 by flow cytometry using the 1B2 mAb. Results shown are averages of duplicate mice analyzed for each condition, and error bars indicate the range of values obtained. Essentially, identical results were obtained in two independent experiments.

Discussion

Through the use of a CD8+ TCR transgenic adoptive transfer model, we have attempted to dissect the in vivo anatomy of Ag-specific CD8+ T cell response to soluble peptide in the presence or absence of adjuvant or inflammatory mediators. Administration of just peptide has been previously shown to induce in vivo tolerance in CD4+ (9, 11) T cell populations. In vivo CD8+ models have also supported this phenomenon, although the peptide Ag is usually administered repeatedly to evoke the nonresponsive state (3, 28). Our data indicates that a single peptide immunization resulted in a weak proliferative response of 1B2+CD8+ cells (up to 25-fold less than with adjuvant) in all sampled sites by day 3 (Fig. 1⇑B). This disseminated response could possibly be due to the peptide being systemic after s.c. injection, as opposed to localized when administered with CFA. The initial response, however, is very transient, and few 1B2+CD8+ cells can be detected by day 20. Ex vivo effector CTL activity was not detected in these mice, but this could be a consequence of the small number of 1B2+CD8+ cells present in the entire CD8+ population. These cells were present in the DLN and spleen up to day 63, but appeared tolerized since they were incapable of responding to secondary peptide challenge even in the presence of adjuvant (<1.2-fold expansion; Fig. 5⇑).

In contrast, when peptide is administered with adjuvant (CFA), a large 1B2+CD8+ proliferative response is detected first in the DLN (Fig. 1⇑A), followed by migration to the spleen and peripheral blood. The initial proliferation is very localized to the DLN (axillary, brachial, and inguinal), while 1B2+CD8+ cells detected in nondraining (mesenteric) LN remain at a constant low level throughout the response. As the initial proliferative response subsides, the cell numbers decline to leave a small, stable population of cells with a memory phenotype (54.6% CD44high). In addition to proliferating, these 1B2+CD8+ cells also develop effector CTL activity by day 3, as measured by a direct ex vivo 51Cr-release assay (Fig. 3⇑). CFA/peptide immunization results in immunologic memory by day 60, as these same cells were capable of responding to secondary challenge with peptide and adjuvant by expanding up to 3-fold (Fig. 5⇑).

Administration of IL-12 during peptide immunization resulted in massive clonal expansion similar to that seen with CFA/peptide (Fig. 2⇑). This proliferation was not limited to the DLN, as large numbers of 1B2+CD8+ cells could also be detected in the spleen. IL-12/peptide immunization also induced lytic effector development of 1B2+CD8+ cells, since cells isolated directly ex vivo were capable of killing peptide-pulsed EL4 target cells (Fig. 3⇑). In addition to preventing Ag-specific tolerance during a primary response (as measured by proliferation and effector function), IL-12 was also capable of generating a stable memory population that responded to secondary challenge with IL-12 and peptide (up to 22-fold expansion; Fig. 5⇑)

Although these results suggest that IL-12 is necessary and sufficient to prevent Ag-specific tolerance, the exact mechanisms of how this occurs in vivo are not known. Previous in vitro models have demonstrated that IL-12 acts directly on the CD8+ T cell, along with TCR ligation and costimulation, to induce proliferation and development of lytic effector function (16). Although the in vivo proliferation and CTL activity shown above are consistent with these previous studies, it is not known whether administered IL-12 acts directly on the 1B2+CD8+ T cell in the recipient animal. Given the broad immunological effects of IL-12, including Th1 differentiation and NK cell stimulation, this proinflammatory cytokine may serve to systematically increase levels of IFN-γ and/or effect the expression of costimulatory molecules present on the surface of APC. Recent evidence is suggesting that CD4+ T cells actually provide help to CD8+ T cells by conditioning APC, via CD40 ligation, to more effectively present Ag to CD8+ T cells and stimulate their response (29, 30, 31). It is possible that this involves induction of IL-12 production by the APC, known to be stimulated by CD40 ligation (32, 33), and that the IL-12 then supports CD8+ T cell proliferation and differentiation in response to Ag and costimulation on the APC.

Even though the effects of IL-12 during a peptide response appear to mimic those observed when CFA is used, IL-12 may only provide part of the overall adjuvant effect. Our data suggest that IL-12 does not fully account for the adjuvant effect of CFA, as the response is still intact in IL-12 KO mice (Fig. 4⇑). The exact mechanisms of how CFA prevents tolerance are not known, although increased costimulation or combinations of other cytokines may be responsible for the observed effects. IL-12 also had a strong effect (well above peptide alone) in these same IL-12 KO animals, further confirming that IL-12 prevents Ag-specific tolerance. The in vivo CD8+ T cell effects of IL-12 directly contrast with previous in vivo studies of CD4+ T cells, which indicate that IL-1, and not IL-12, is sufficient to promote clonal expansion and differentiation (11). Conversely, IL-1 was previously found to not promote these same events in CD8+ T cells (16). Thus, it appears that these two distinct populations of T cells are influenced differently with respect to inflammatory mediators.

The antitumor effects of IL-12 have been well-characterized; murine tumor models have focused heavily on delivery of this cytokine, ranging from locally with transduction and Ab-fusion protein experiments to systemic i.p. injections, all with promising results (34, 35, 36, 37). Other in vivo murine tumor models indicate that IL-12 may even contribute to CTL migration to the site of tumor load (38). Clinical trials have implicated IL-12 in promoting a higher CTL precursor frequency, although initial IL-12 protocols resulted in severe human toxicity (21, 22). In addition to the potential therapeutic value of IL-12 for established pathological conditions, the results reported here suggest that it may provide a very effective adjuvant for immunizing for protective CD8+ T cell responses. T cell activation, proliferation, lytic effector function, and memory establishment are all comparable to those induced by traditional adjuvants, and toxic effects may be minimal or absent with the limited amounts of IL-12 required for the adjuvant effect.

Acknowledgments

We thank Debra Lins for excellent technical assistance, and Drs. Marc Jenkins and Daniel Mueller for critical reading of the manuscript.

Footnotes

  • ↵1 This work was supported by National Institutes of Health Grants AI34824 and AI35296. C.S. was supported in part by National Institutes of Health Grant T32-AI07313.

  • ↵2 Address correspondence and reprint requests to Dr. Matthew F. Mescher, Center for Immunology, University of Minnesota, Box 334 Mayo, 420 Delaware Street S.E., Minneapolis, MN 55455. E-mail address: mesch001{at}maroon.tc.umn.edu

  • ↵3 Abbreviations used in this paper: KO, knockout; FSC, forward scatter; LN, lymph node; DLN, draining LN; MLN, mesenteric LN.

  • Received April 9, 1999.
  • Accepted June 21, 1999.

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The Journal of Immunology: 163 (5)
The Journal of Immunology
Vol. 163, Issue 5
1 Sep 1999
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