Peptides, Abs, and cytokines

The following synthetic peptides were synthesized by Peptide Technologies (Washington, DC) to a purity of >99% as determined by HPLC and amino acid analyses: TPHPARIGL (amino acids 876−884 of β-gal, H-2L^d-restricted (27)), DAPIYTNV (amino acids 96−103 of β-gal, H-2K^b-restricted (28)). Rat mAb recognizing NLDC-145 (DEC-205) and F4/80 were purchased from BMA Biomedicals (Augst, Switzerland). FITC- or PE-labeled mAb recognizing mouse CD3, CD8, CD4, CD11b (Mac-1), CD45R/B220, CD11c, CD80 (B7−1), CD86 (B7−2), Ly-6G (Gr-1), anti-H-2 I-E^d/I-A^d, K^d, and the isotype-matched controls were purchased from PharMingen (San Diego, CA). The mAb 24G.2 (CD16/CD23; PharMingen), which recognizes the extracellular domain of the mouse Fcγ-RIII and RII, was used to block the nonspecific mAb binding. Recombinant mouse GM-CSF and IL-4 were purchased from PreproTech (Rocky Hill, NJ) and resuspended in HBSS containing 1% mouse serum (Sigma, St. Louis, MO).

Isolation and depletion of Gr-1⁺ splenocytes

A panning technique employing flasks coated with mouse anti-rat Abs (T-25 AIS MicroCell; Applied Immune Sciences, Santa Clara, CA) was used to enrich or eliminate Gr-1⁺ splenocytes from either tumor-bearing mice or mice treated with recombinant GM-CSF. Briefly, spleens were depleted of RBC through ACK lysis buffer (Biofluids) and resuspended in HBSS containing 1 mM EDTA and 10% mouse serum (HBSS-EDTA-MS). Cells were transferred on ice, incubated for 30 min with anti-Gr-1 Ab at a concentration of 10 μg/10⁷ cells, and washed with cold HBSS-EDTA. Cells were resuspended in ice-cold HBSS-EDTA-MS, transferred to flasks coated with the secondary Ab (anti-rat), and incubated for 1 h at 4°C. The nonadherent cells were then dislodged, and fluorescence labeling confirmed a >95% depletion. Cells that remained attached to the flasks were provided with CM and incubated for further studies. Adherent cells were usually recovered by gentle scraping in Versene solution (1:5000; Biofluids).

Maturation of CD11b⁺/Gr-1⁺ cells in vitro

Gr-1⁺ splenocytes were isolated as described above. Isolated cells were grown for 6 days in culture with 100 ng/ml GM-CSF, 100 ng/ml IL-4, a combination of both GM-CSF and IL-4, or in media alone. After 6 days of culture, the adherent cells were analyzed for phenotypic markers or added to a 6-day mixed lymphocyte stimulation. In the mixed lymphocyte reaction, BALB/c splenocytes (H-2^d) at 3 × 10⁶ were incubated with 3 × 10⁶ irradiated C57BL/6n splenocytes (H-2^b) in the presence or absence of 0.18 × 10⁶ Gr-1⁺ splenocytes (H-2^d). After 6 days of stimulation, the BALB/c lymphocytes were tested in a standard chromium release assay against syngeneic (CT26 or LSTRA) or allogeneic (MBL-2) targets.

Enrichment of spleen-derived DC

Spleens were collected aseptically, minced, and incubated for 30 min at room temperature in HBSS containing collagenase (1 mg/ml; Sigma). Spleens were depleted of RBC through ACK lysis buffer and washed. The single cell suspension was resuspended in CM and plated in a 150 × 25-mm tissue culture dish (Falcon, Cockeysville, MD). Splenocytes were incubated for 2 h at 37°C, and the nonadherent cells were removed by gentle washing. Adherent cells were further incubated for 18 h in CM containing 3 ng/ml mouse GM-CSF. At the end of the incubation, the supernatant was collected, and DC were finally enriched by centrifugation (1900 × g) over a 45% Percoll cushion. At this point, >80% of the cells were B7−2⁺, B220⁻, class II MHC ⁺, CD11c⁺.

GM-CSF-producing tumors induce CD11b⁺/Gr-1⁺ splenocytes that inhibit CD8⁺ T cells

To test the immune consequences of spontaneous GM-CSF production by tumors on the maturation of APC, two experimental tumor lines were grown in vivo. The colon adenocarcinoma, CT26.WT, and the mammary adenocarcinoma, TS/A, spontaneously produced significant levels of GM-CSF (average 50 and 250 pg/ml/10⁶ cells/day, respectively). Mice bearing unmanipulated s.c. TS/A tumors ~1 cm in diameter were splenectomized. A representative cytofluorometric analysis of spleen cells (shown in Fig. 1A) demonstrates an increase in the numbers of CD11b⁺/Gr-1⁺ cells. These cells comprised a mean of 22.3 ± 1.83% of all splenocytes in five independent experiments, compared with an average of 2.4 ± 0.43% in mice with a small but palpable tumor. Evaluation of splenocytes from tumor-bearing mice did not reveal substantial changes in the percentages of lymphocytes with T or B cell markers (31).

Open in a separate window

FIGURE 1

Tumor growth results in the induction of CD11b⁺/Gr-1⁺ cells that inhibit CD8⁺ T cells. A, Spleens of tumor-bearing mice contain large numbers of the CD11b⁺/Gr-1⁺ cells. Freshly harvested splenocytes were stained with FITC anti-Gr-1 and PE anti-CD11b mAbs. Only mice bearing palpable tumors 2−3 mm in diameter (left) or tumors with diameters >1 cm (right) are shown, as results in non-tumor-bearing mice and mice bearing a small but palpable tumor were identical. B, Inhibition of the anamnestic response in tumor-bearing mice is abrogated by the depletion of Gr-1⁺ cells. Three weeks after immunization with β-gal-rVV, BALB/c mice were injected s.c. with TS/A tumor cells. Duration of the tumor-bearing state was 35 days for mice with tumors >1 cm in diameter, and 15 days for tumors that were ~2−3 mm in diameter (open triangles). Splenocytes were collected, pooled, then cultured with β-gal peptide. Splenocytes originating from mice bearing tumors >1 cm² were depleted with anti-Gr-1 (filled circles) or with a control, IgG_2b, Ab (open squares) before the culture was established. Cytotoxicity shown was measured in a single assay after 6 days of incubation with the target peptide. Target cells were CT26.WT (H-2^d), CT26.WT pulsed with the L^d-restricted β-gal peptide (CT26. WT+pep), the lacZ-transfected, β-gal-expressing clone CT26.CL25, and the negative control E22 (lacZ-transfected β-gal-expressing EL4 cells, H-2^b haplotype). E:T cell ratios were 100:1, then 1:3 dilutions. No difference in β-gal-specific cytolytic activity between immune mice bearing a small tumor (control) vs normal immune mice was observed (not shown). These findings were obtained reproducibly in five independently performed experiments.

We sought to explore whether or not the growth of a GM-CSF-producing tumor had any impact on CD8⁺ T cell responses. Therefore, we immunized mice with an rVV encoding lacZ under the control of a strong promoter (25). This immunization has been previously shown to induce strong CD8⁺ T cell responses to the model Ag, β-gal (β-gal rVV). Mice were then allowed to develop a “memory” immune response. After 3 wk, mice were randomized to receive TS/A tumor cells that were allowed to grow s.c. for either 35 days, which resulted in tumor nodules >1 cm², or for only 15 days, which resulted in small but palpable tumor nodules. Mice with small but palpable tumors mounted a vigorous Ag-specific immune response to targets expressing the L^d-restricted, β-gal epitope (Fig. 1B, control). However, mice bearing large tumors failed to mount an anamnestic, β-gal-specific CD8⁺ T cell response.

To determine whether or not the Gr-1⁺ population that was so prevalent in mice bearing large tumors was responsible for the observed inhibition of Ag-specific CD8⁺ T cell responses, a fraction of the splenocytes from mice bearing large tumors was depleted with a Gr-1-specific Ab, or an isotype-controlled (IgG_2b) Ab before restimulation. The simple depletion of Gr-1⁺ cells normalized CD8⁺ T cell function. Indeed, the lytic levels observed were nearly as great as those seen in β-gal-immune mice bearing small, but palpable, tumors (Fig. 1B, compare anti-Gr-1 and control). Isotype-matched mAb did not normalize T cell function (Fig. 1B, IgG_2b). Experiments with the CT26 tumor cell line, which also naturally produces GM-CSF, yielded similar results (data not shown). Our results with mice bearing CT26 are consistent with those previously published by G. Nabel and colleagues (32) in which mice bearing this tumor were found to have large numbers of CD11b⁺ cells in their spleens and inhibition of allospecific responses. Therefore, the newly induced population of CD11b⁺/Gr-1⁺ cells appeared to be responsible for the abrogation of CD8⁺ T cell function.

We have previously shown that purified CD8⁺ T lymphocytes obtained from splenic preparations of mice with high levels of CD11b (Mac-1)⁺/Gr-1⁺ suppressor cells are normally responsive (24). Thus, the inhibition observed is not a quality of the CD8⁺ T cells, but is solely a quality of the CD11b/Gr-1 double-positive population.

GM-CSF, not the tumor-bearing state, drives the production of CD11b⁺/Gr-1⁺ cells

To determine whether or not the CD11b⁺/Gr-1⁺ cells were induced by GM-CSF or some other stimulus from the tumor, the murine melanoma, B16, which does not normally produce GM-CSF, was retrovirally transduced with the cDNA for mouse GM-CSF. Immediately following transduction, transduced B16 were cloned. To minimize clonotypic variability, several clones were isolated that released comparable amounts of the cytokine (600−900 pg/ml; Table II).

Mice were vaccinated with β-gal rVV to establish β-gal-specific memory T cells (33, 34). Three weeks later, they were inoculated s.c. with either parental B16 (B16 wt) or one of the GM-CSF-producing clones. Tumors were allowed to grow for 14 days. Interestingly, no significant difference in the growth rate was observed between any of the GM-CSF-producing clones and the parental tumor, indicating that the levels of GM-CSF secretion were not sufficient to cause tumor rejection (Table II). Two weeks after tumor inoculation, we were able to detect increased serum levels of GM-CSF in mice bearing gene-modified tumors but not in mice bearing parental tumor, indicating that cytokine production had not been silenced in vivo (Table II). As seen with CT26 and TS/A, cytofluorometric analysis of spleens from mice bearing GM-CSF-transduced tumors demonstrated an increase in the percentage of the CD11b⁺ cells (Table II).

The lytic activity against β-gal after in vitro stimulation of the spleens with the K^b-restricted β-gal peptide in control mice were similar to those found in mice bearing the B16 wild-type tumor (B16 wt). In sharp contrast, growth of all the subclones producing GM-CSF (GM15−47) resulted in the inhibition of the anamnestic CD8⁺ T cell responses, as indicated by the reduction of the lytic units in the peptide-stimulated cultures. These data suggest that GM-CSF production by tumors cells can inhibit CD8⁺ T cell responses.

Recombinant GM-CSF administered alone induces CD11b⁺/Gr-1⁺ cells and inhibits CD8⁺ T cell function

To further test the hypothesis that GM-CSF was directly responsible for the increase in CD11b⁺/Gr-1⁺ cells, a 3-day cycle of recombinant mouse GM-CSF was administered twice daily to β-gal immune mice and naive controls. The administration of GM-CSF caused an increase in CD11b⁺/Gr-1⁺ cells in the spleens of treated mice (Fig. 2A) and inhibited the immune responses observed after stimulation with β-gal peptide (Fig. 2, B and C). These data demonstrated that a exposure to GM-CSF was sufficient to induce both the appearance of a distinct population of CD11b⁺/Gr-1⁺ cells and to induce CD8⁺ T cell inhibition.

Open in a separate window

FIGURE 2

Induction of inhibitory cells in vivo using recombinant GM-CSF alone. Administration of GM-CSF causes an increase in CD11b/Gr-1 double-positive splenocytes. BALB/c mice were injected either with saline alone (“Naive”), or saline containing β-gal-rVV (“β-gal immune”). After 3 wk, the two groups of mice were randomly separated into smaller groups of three animals each that were inoculated i.p. with HBSS alone (HBSS), or HBSS containing 5 μg of mouse GM-CSF (GM-CSF), twice daily for a total of 3 days. At the end of this cycle of inoculations, spleens were removed and cultured with β-gal peptide for 6 days. A, Cytofluorometric staining with FITC anti-Gr-1 and PE anti-CD11b mAbs of splenocytes from immune mice inoculated with GM-CSF (right panel) or with vehicle alone (left panel). Similar percentages of CD11b⁺/Gr-1⁺ cells were found in spleens of nonimmunized mice (data not shown). B and C, Functional consequence of CD11b⁺/Gr-1⁺ induction. The cytolytic response against a panel of β-gal-positive and -negative targets is shown in naive (B) and β-gal-immune (C) mice. E:T cell ratio was 100:1, then 3-fold dilutions (100:1, 33:1, 11:1, 4:1). Repeat experiments gave nearly identical results.

Inhibitory cells express some immature APC markers such as CD11b and F4/80 and are CD11c^low but lack the expression of costimulatory molecules and MHC class II

To characterize the inhibitory population of CD11b⁺ cells in greater detail, splenocytes were positively selected from the spleens of TS/A-bearing mice with anti-Gr-1 mAb and were cultured in vitro in standard medium. After 7 days of culture, a homogenous population of weakly adherent cells was isolated that retained its ability to inhibit T lymphocyte function (see Fig. 4A, “no cytokine”). We sought to characterize the phenotypes of these cells in more detail. For comparison, we simultaneously prepared a population of “classical” DC from naive mice using an established protocol (compare Fig. 3A with granulocyte-monocyte progenitor cells, Fig. 3B).

Open in a separate window

FIGURE 3

Phenotypic characterization of inhibitory cells and their comparison with “classical” DC. Comparison between mature DC (A) and the CD11b⁺/Gr-1⁺ cells from a tumor-bearing animal (B). Gr-1⁺ cells from the spleens of mice s.c. inoculated 35 days earlier with TS/A cells were enriched through panning and cultured for 7 days in complete medium. Before staining with the different mAb, nonadherent cells were removed, and adherent cells were collected in PBS, 1 mM EDTA by gentle scraping, then blocked for nonspecific staining with unlabelled anti-Fc Ab. The phenotypic pattern shown in this figure was also observed in repeat experiments, despite some variability in the percentage and intensity of staining with anti-CD11c. Isotype-matched control Abs are shaded.

Open in a separate window

FIGURE 4

A cell-cell contact is required for suppression of CD8⁺ T lymphocyte response. Gr-1⁺ cells were enriched through panning with a specific mAb from the spleens of mice that had received TS/A tumor inoculation. Following culture in medium without cytokines for 7 days, adherent cells were harvested and added at a final concentration of 0.18 × 10⁶ cells (3%) to a MLC consisting of BALB/c splenocytes and γ-irradiated C57BL/6n splenocytes. The Gr-1⁺-derived cells were either admixed in the same well (cell contact) or separated by a semipermeable membrane (no cell contact). Control cultures did not receive the third part population of suppressor cells (shown in the leftmost panel). After 5 days, cytolytic activity in the cultures was assayed against either a syngeneic (H-2^d) target, LSTRA, or an allogeneic (H-2^b) target, MBL-2. This experiment was repeated twice with similar results.

Immature markers for APC in the granulocyte-monocyte lineage stained positive including F4/80 and CD11b (also known as CD11b, integrin α_M chain and Ly-40) (Fig. 3B). The expression of CD11c (integrin α_χ chain) in the purified inhibitory cell was low, and cytofluorometric quantification of these cells varied somewhat from experiment to experiment with CD11c^dim cells comprising between 25 and 60% (compare with Fig. 4B, “no cytokine”). Interestingly, expression of Gr-1 (Ly-6G), another granulocyte-monocyte lineage marker, was present early but was consistently lost both when the cells were prepared from tumor-bearing mice or from mice that received a short course of GM-CSF.

Clearly, these inhibitory cells differed from “classical” mature DC and activated monocytes and macrophages most significantly by their lack of uniform expression of the costimulatory molecule B7−2 (CD86/B70/Ly-58). They also lacked the expression of DEC 205 (NLDC-145), a protein involved in carbohydrate endocytosis and Ag presentation (Fig. 3A vs 3B) (35). “Classical” DC, and activated macrophages for that matter, express high levels of MHC class II, whereas the inhibitory cells were consistently negative. While the cell surface marker CD16 (FcγRIII) is found in low quantities on DC and is a developmental marker on monocytes and macrophages, the inhibitory cells were negative. Normal levels of MHC class I were present, and there was no expression of T and B lymphocyte markers (data not shown), excluding the possibility that these CD11b⁺/Gr-1⁺ were CD8a⁺ as recently described for a subpopulation of DC with a suppressive function (36, 37). Cells with similar granulocyte-monocyte progenitor markers were found in C57BL/6n and BALB/c after induction, in vivo, with twice daily regimen of GM-CSF alone given for 3 days (data not shown).

Inhibitory cells prevent CD8⁺ T cell generation through a contact-dependent mechanism

We next investigated the mechanism used by CD11b⁺/Gr-1⁺ cells to inhibit CD8⁺ cell function. To determine whether soluble factors were responsible for the suppression, we did experiments in which inhibitory APC were cultured either together with, or in close proximity to, an allogeneic MLC. The anti-H-2^b MLC was generated by admixing BALB/c (H-2^d) splenocytes with irradiated C57BL/6n (H-2^b) splenocytes. Inhibitory cells, which were enriched from mice bearing TS/A tumors, were allowed to differentiate in vitro in medium devoid of exogenously added cytokines. These inhibitory cells were then added to MLC, either in the same well, or in a transwell. The effectors were then tested in a micro-cytotoxicity assay against syngeneic (LSTRA) or allogeneic (MBL-2) targets.

The secretion of soluble factors, such as inhibitory cytokines, by the CD11b⁺/Gr-1⁺ population may be expected to inhibit the allo-MLC reaction when the inhibitory cells were placed in diffusion chambers. However, as shown in Fig. 4, no such inhibition was observed, and the MLC functioned normally when the inhibitory cells were in transwells. Indeed, it appeared from this experiment that cell-cell contact was required for the immunosuppressive activity of the inhibitory cells, since suppression of allospecific cytolysis was observed only when the Gr-1⁺-derived population was not separated by a membrane from the population responsive to stimulation with allogeneic cells.

We thank P. Segato for editing the manuscript, D. R. Surman for help and advice with many aspects of the work presented here, P. Spiess and D. Jones for help with the animal experiments, M. Blalock for expert graphics, E. P. Shulman and K. R. Irvine for tissue culture preparations, and A. Mixon for the flow cytometry experiments.