Unopposed production of granulocyte-macrophage colony-stimulating factor by tumors inhibits CD8+ T cell responses by dysregulating antigen-presenting cell maturation

Abstract

Tumor cells gene-modified to produce GM-CSF potently stimulate antitumor immune responses, in part, by causing the growth and differentiation of dendritic cells (DC). However, GM-CSF-modified tumor cells must be gamma-irradiated or they will grow progressively, killing the host. We observed that 23 of 75 (31%) human tumor lines and two commonly used mouse tumor lines spontaneously produced GM-CSF. In mice, chronic GM-CSF production by tumors suppressed Ag-specific CD8+ T cell responses. Interestingly, an inhibitory population of adherent CD11b(Mac-1)/Gr-1 double-positive cells caused the observed impairment of CD8+ T cell function upon direct cell-to-cell contact. The inhibitory cells were positive for some markers associated with Ag presenting cells, like F4/80, but were negative for markers associated with fully mature DC like DEC205, B7. 2, and MHC class II. We have previously reported that a similar or identical population of inhibitory "immature" APC was elicited after immunization with powerful recombinant immunogens. We show here that these inhibitory cells can be elicited by the administration of recombinant GM-CSF alone, and, furthermore, that they can be differentiated ex vivo into "mature" APC by the addition of IL-4 and GM-CSF. Thus, tumors may be able to escape from immune detection by producing "unopposed" GM-CSF, thereby disrupting the balance of cytokines needed for the maturation of fully functional DC. Further, CD11b/Gr-1 double-positive cells may function as "inhibitory" APC under the influence of GM-CSF alone.

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.

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.

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.

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.

FIGURE 5

Inhibitory cells can be differentiated into functionally mature, activating APC under the influence of combined GM-CSF and IL-4. To assess the influences of GM-CSF and IL-4 alone or in combination on the differentiation of inhibitory cells, we isolated Gr-1⁺ splenocytes from mice that had received TS/A tumor inoculation. Inhibitory cells were then exposed to either no cytokines, GM-CSF alone, IL-4 alone, or a combination of GM-CSF and IL-4. After 6 days of culture, these cells were tested for their function (A) and phenotype (B). A, Cytokine-treated inhibitory cells were added at a final concentration of 0.18 × 10⁶ cells (3%) to a MLC consisting of 3 × 10⁶ BALB/c splenocytes (H-2^d) together with an equal number of γ-irradiated C57BL/6n splenocytes (H-2^b). The MLC was assessed in a standard ⁵¹Cr release assay for activity after an additional 6 days of culture using an H-2^b target, MBL-2, and a control H-2^d target, CT26, at E:T ratios starting at 100:1, followed by 3-fold dilutions (100:1, 33:1, 11:1, 4:1, 1:1, 0.4:1). B, Phenotypic characterization of cells after the 6-day cytokine regimen. Isotype matched controls are shaded.