IL-12–induced polarization of adoptively transferred CD8+ T cells into type I effectors is not necessary for antitumor responses.
Based on known mechanisms, we hypothesized that IL-12 secreted in culture polarized pmel-1 CD8+ T cells into type I effectors and that these changes were responsible for the improvements in antitumor immunity. To test this hypothesis, we expressed similar levels of IL-12 in WT pmel-1 CD8+ T cells and Il12rb2–/– pmel-1 CD8+ T cells (Figure A). IL-12–engineered pmel-1 CD8+ T cells (IL-12 cells) possessing functional IL-12 receptors expressed a distinct CD62Lhi, IL-7Rαhi, IL-2Rαhi, and Sca1hi phenotype not present on Il12rb2–/– cells (Figure B). Furthermore, WT IL-12 cells were more activated and capable of secreting higher amounts of IFN-γ and TNF-α compared with Il12rb2–/– IL-12 cells (Figure C). We next adoptively transferred either 1 × 105 Il12rb2–/– or WT IL-12 cells into sublethally irradiated mice (5 Gy) bearing well-established subcutaneous B16 melanomas and, surprisingly, observed similar levels of antitumor immunity among the cell types (Figure D). We then transferred 1 × 105 WT IL-12 cells into WT or Il12rb2–/– host mice and observed significant impairment in tumor treatment in mice lacking the ability to respond to IL-12 (Figure E). Thus, polarization of T cells into type 1 effectors did not play a major role in enhancing antitumor immunity, but sensitization of endogenous components of the host’s immune system by IL-12 was necessary for successful treatment responses.
Antitumor immunity of IL-12–engineered pmel-1 CD8+ T cells (IL-12 cells) is not dependent on type I self polarization but does require an endogenous response to secreted IL-12.
IL-12–dependent sensitization of bone marrow–derived cells is required for antitumor immunity.
We next created bone marrow chimeras using C57BL/6 WT and Il12rb2–/– mice in order to better decipher the particular cell populations that may be sensitized by IL-12. We lethally irradiated WT mice and reconstituted them with Il12rb2–/– (Il12rb2–/–→WT) or WT (WT→WT) bone marrow and 6 weeks later implanted B16 subcutaneous melanomas. We then transferred IL-12 cells into sublethally irradiated mice and found that antitumor responses were impaired in Il12rb2–/–→WT mice but not in WT→WT mice (Figure A). We also created chimeras using host Il12rb2–/– mice reconstituted with bone marrow derived from Il12rb2–/– (Il12rb2–/–→Il12rb2–/–) or WT mice (WT→Il12rb2–/–) and implanted them with B16 subcutaneous melanomas 6 weeks later. We transferred IL-12 cells into sublethally irradiated mice and found that antitumor responses were rescued in Il12rb2–/– mice reconstituted with WT bone marrow (WT→Il12rb2–/–; Figure B). These experiments together demonstrated that IL-12 acted directly on bone marrow–derived cells in order to improve the antitumor immunity of transferred T cells.
IL-12–dependent sensitization of bone marrow–derived cells is critical for tumor regression.
IFN-γ secretion and sensitization by endogenous cells is required for antitumor immunity.
Given the requirement for sensitization of bone marrow–derived cells, we next tried to decipher the role of IFN-γ in tumor regression. We gene transduced open-repertoire WT or Ifng–/– CD8+ T cells with the pmel-1 TCR (P-TCR) and IL-12 (P-TCR/IL-12) and adoptively transferred equal numbers of double-positive cells (2.5 × 105) into sublethally irradiated WT or Ifng–/– mice bearing B16 melanomas (Figure A). Surprisingly, Ifng–/– P-TCR/IL-12 cells induced tumor regression to the same degree as WT P-TCR/IL-12 cells in WT hosts, indicating that IFN-γ secretion by transferred CD8+ T cells was not required for antitumor responses (Figure A). Within the same experiment, we also transferred WT P-TCR/IL-12 cells into WT or Ifng–/– mice and found that antitumor immunity was significantly impaired in mice unable to produce IFN-γ (Figure A). These results indicate that the production of IFN-γ by endogenous cells was necessary for tumor regression.
IL-12–induced IFN-γ production and sensitization by host cells are partially required for antitumor immunity.
We next assessed the importance of IFN-γ in imparting a direct effect on endogenous cells by transferring IL-12 cells into sublethally irradiated tumor-bearing Ifngr–/– mice and witnessed only a partial impairment in antitumor immunity when host cells were unable to respond to IFN-γ (Figure B). Taken together, these data illustrate the need for host cells to produce IFN-γ, but show that the requirement for IFN-γ receptors on endogenous cells is only partial. These findings suggest that the direct effects of IFN-γ on tumor cells may be mechanistically important for antitumor immunity in this model.
IL-12–mediated improvements in antitumor immunity are independent of host T cells, B cells, and NK cells.
Based on our previous experiments and published mechanisms of IL-12, we hypothesized that endogenous CD8+
and NK cells were likely responsible for tumor destruction (17
). We therefore transferred IL-12 cells into sublethally irradiated tumor-bearing WT or Rag1–/–
mice that are devoid of endogenous T and B cells and found that antitumor responses remained robust in Rag1–/–
mice, indicating that endogenous T or B cells were not necessary to induce tumor regression (Figure A).
IL-12–induced sensitization of endogenous immunity is independent of host T, B, and NK cells, but increases tumor infiltration of adoptively transferred T cells.
We next transferred IL-12 cells into tumor-bearing sublethally irradiated WT or Rag1–/– mice depleted of NK cells (90%–95% NK cell elimination 3 weeks after adoptive transfer) and found that tumor destruction remained robust despite the lack of host T and B cells and a significant depletion in NK cells (Figure B). Based on our previous work indicating that IL-12 enhanced the infiltration of the adoptively transferred T cells within tumors and the clear benefits gained by IL-12 sensitization of bone marrow cells, we hypothesized that perhaps IL-12 might be altering bone marrow–derived APCs, leading to indirect effects on transferred T cells. We harvested tumor samples 3 and 7 days following the transfer of 1 × 105 mock-transduced cells (mock) or IL-12 cells into sublethally irradiated tumor-bearing Rag1–/– mice depleted of NK cells and found higher numbers of IL-12 cells infiltrating tumors compared with mock cells (Figure C).
To visualize infiltrating T cells within tumors, we captured immunofluorescent confocal images (transferred T cells, thy1.1+ green; blood vessels, CD31+ red) from tumor sections 7 days following cell transfer into sublethally irradiated mice and observed thy1.1+ transferred T cells infiltrating the extravascular space of tumors treated with IL-12 cells, but not mock cells (Figure D). We also subjectively witnessed disruptions in the vascular integrity of tumors in mice treated with IL-12 compared with mock cells, although this observation was more an association than a mechanistic finding. Thus, IL-12 within the tumor microenvironment indirectly led to the increased local proliferation of adoptively transferred tumor antigen–specific T cells. This was unexpected, but completely consistent with our data showing that IL-12 did not act directly on transferred T cells and instead sensitized bone marrow–derived cells independently of host T, B, and NK cells to indirectly enhance the ability of transferred T cells to infiltrate tumors.
An inflammatory gene signature and increased antigen processing and presentation within tumors treated with IL-12–engineered CD8+ T cells.
To understand how IL-12 triggered tumor regression, we performed whole transcriptome analysis of tumor samples from mice 3 and 7 days following treatment with IL-12 cells or mock cells (24
). We examined select genes encoding for cytokines, chemokines, chemokine receptors, inflammatory mediators, and costimulatory molecules and observed an over 10-fold increase in the expression for IL-1β, S100a8, and S100a9 from tumors of mice 3 days following treatment with IL-12 compared with mock cells (Table ). Interestingly, we also observed an increase in gene expression for Nlrp3
within IL-12–treated tumors, indicating a possible role for inflammasome formation in antitumor immunity.
Select genes significantly differentially expressed within tumors of mice treated with IL-12–engineered CD8+ T cells
Overall, we found 407 transcripts differentially expressed on days 3 and 7, with 360 genes upregulated and 47 genes downregulated (Figure A; fold change [FC] > 2, P value with false discovery rate [FDR] < 0.05, n = 4). We performed an unbiased gene-ontology enrichment analysis, and the highest enrichment score was assigned to immune system processes in tumors treated with IL-12 compared with mock cells (Supplemental Figure 2). Under the immune system category, pathway analysis identified antigen processing and presentation as the most differentially increased pathway (Figure B), and mRNA levels for several genes such as tap1, tap2, tpn, clip, lmp2, lmp7, H2-Ab1, H2-Q6, H2-Aa, H2-DMa, H2-K1, H2-D1, and H2-Ab1 were at least 2-fold higher in IL-12 compared with mock-treated tumors (Figure C). Additionally, the most differentially expressed transcript was Ifng (>62-fold) along with mRNA levels for genes commonly found in effector T cells (Gzmb, Il2ra, Gzma; Figure D).
Tumors from mice receiving IL-12 compared with mock cells display an increase in the intrinsic capabilities for antigen processing and presentation.
We confirmed the transcript level by real-time PCR assays for 2 known inflammatory mediators and found significantly higher expression of IL-10 and iNOS from tumors of mice treated with IL-12 compared with mock cells (Supplemental Figure 3). We also noted temporal changes within tumors, as 2,776 genes were differentially expressed between days 3 and 7 in tumors treated with IL-12 cells (Supplemental Figure 4). Although these results highlighted the dynamic nature of the tumor microenvironment prior to the regression of established lesions, analyzing gene transcripts differentially expressed consistently on both day 3 and day 7 provided valuable mechanistic insights. Given that tumor rejection involved IL-12–mediated sensitization of bone marrow–derived cells and that host T cells, B cells, and NK cells did not appear to be involved, data generated by the whole transcriptome analysis pointed toward antigen processing and presentation as an important mechanistic pathway differentially expressed in mice treated with IL-12 compared with mock cells.
Antitumor immunity of IL-12 cells is dependent on the functional reprogramming of myeloid-derived cells in situ to cross-present natural tumor antigens.
Based on our previous experiments, we hypothesized that IL-12 may have an impact on the antigen-presenting capabilities of bone marrow–derived stromal cells residing within tumors. We therefore looked for IL-12 receptor β2 (Il12rb2) expression in single-cell suspensions of whole tumors and found that the majority of cells capable of functionally responding to IL-12 were indeed CD11b+ myeloid–derived stromal cells (Figure A).
Enhanced antitumor immunity triggered by IL-12 is dependent on in vivo cross presentation of tumor antigens.
We next analyzed single-cell tumor suspensions from mice 1 week following treatment with mock or IL-12 cells and analyzed PI–, CD3–, B220–, NK1.1–, and CD11b+ myeloid–derived cells. Tumor-infiltrating myeloid cells from mice treated with IL-12 cells compared with mock cells appeared markedly different based on forward and side scatter on flow cytometric analysis (Figure B). To assess whether we had truly reprogrammed tumor-infiltrating myeloid-derived cells in situ, we performed a flow cytometry–based cell sort of PI–, CD3–, B220–, NK1.1–, and CD11b+ cells from established tumors of treated mice and carried out PCR-based arrays for transcripts common to APCs. We observed an over 8-fold increase in transcript levels for Ifng, Il2, Il8ra, Ccl11, and Ccl20 in 2 independent experiments from myeloid-derived cells isolated from multiple pooled tumors treated with IL-12 over mock cells (Supplemental Figure 5). Interestingly, we also measured over 4-fold increases in mRNA for B2m (Supplemental Figure 5) and recorded higher expression of H-2Db on CD11b+ myeloid–derived cells from tumors of mice treated with IL-12 over mock cells (Figure C).
To understand the functional importance of increased levels of MHC class I, we transferred IL-12 cells into sublethally irradiated tumor-bearing WT, B2m–/– (MHC class I deficient), or IAb–/– (MHC class II deficient) mice. We observed a significant impairment in antitumor immunity in mice lacking MHC class I but not MHC class II (Figure D). Importantly, untreated tumors grew at an identical pace in mice of all strains. These results clearly implicated the need for in vivo cross presentation for successful antitumor responses, an interesting finding given that we transferred cells that were highly activated in vitro and the tumors grown on mice were unmanipulated and fully capable of being directly recognized.
To further assess the importance of in vivo cross presentation, we transferred IL-12–engineered pmel-1 Ly 5.1+ (IL-12P-Ly5.1) gene–marked CD8+ T cells and open-repertoire IL-12 engineered thy1.1+ (IL-12OR-Thy1.1) gene–marked CD8+ T cells into the same WT or B2m–/– mouse (Figure E). We observed an increase in the proliferation of activated antigen-specific IL-12P-Ly 5.1 in WT mice compared with B2m–/– mice. Notably, we saw no IL-12P-Ly 5.1 within tumors of B2m–/– mice, but observed a clear population of cells within tumors of WT mice. Furthermore, we observed no defects in the longevity of open-repertoire IL-12OR-Thy1.1 CD8+ T cells in B2m–/– mice compared with WT mice (Figure E). To visualize infiltrating T cells within tumors in WT compared with B2m–/– mice, we captured immunofluorescent confocal images (transferred T cells, thy1.1+ green; blood vessels, CD31+ red) from tumor sections 7 days following the transfer of 1 × 105 IL-12 cells into sublethally irradiated WT or B2m–/– mice and observed thy1.1+ transferred T cells infiltrating the extravascular space of tumors in WT but not B2m–/– mice (Figure F). Thus, IL-12 secreted by tumor-specific CD8+ T cells triggered the functional reprogramming of in situ myeloid-derived cells, promoting the cross presentation of natural tumor antigens that enabled transferred T cells to infiltrate tumors and induce the regression of well-established lesions.
Characterization and antigen-presenting capabilities of myeloid-derived subpopulations within tumors.
To analyze myeloid-derived stromal populations within tumors in a more detailed manner, we performed multicolor flow cytometry on single-cell suspensions from well-established subcutaneous B16 tumors shortly following (8 hours) a 5-Gy total body irradiation (TBI) preconditioning regimen and found a panoply of myeloid-derived cell populations residing within tumors. Due to the redundancy of cell-surface markers between many of the myeloid-derived cell subpopulations, we used highly multicolored flow cytometry to analyze unique subpopulations of cells within tumors. We first created single-cell suspensions of established B16 tumors and created a live gate by excluding cells staining positive for the vivid fixable violet live/dead stain.
We analyzed all endogenous cells within tumors by gating on CD45+
cells and found that the majority of endogenous cells were of myeloid origin and stained positive for CD11b (Figure A). The CD45+
population consisted mostly of lymphocytes, with a small fraction of NK cells (data not shown). Within the CD45+
gate, we observed a distinct population of dendritic cells that stained for CD11c and I-Ab
. We next analyzed the CD45+
population and observed a clear population of macrophages that stained positive for F4/80 (CD45+
). We then evaluated the fraction of myeloid cells that were CD45+
and detected distinct populations of cells expressing Ly6C and Ly6G. These cells, commonly characterized as MDSCs (ref. 34
), represent a heterogeneous population of cells from monocytic (MDSC-M) and granulocytic (MDSC-G) origins (Figure A). Within B16 tumors, we observed a distinct population of CD45+
cells of monocytic origin that we categorized as MDSC-M. There also existed a clear population of CD45+
cells that we characterized as granulocytic in origin (MDSC-G), although this categorization is still a matter of debate (Figure A and ref. 35
). Thus, bone marrow–derived myeloid cells composing the tumor stroma are a heterogeneous population of phenotypically distinct immune cells.
MDSCs, macrophages, and dendritic cells residing within B16 tumors of mice treated with IL-12 cells potently stimulate the proliferation of pmel CD8+ T cells.
We next sought to assess the ability of tumor-residing myeloid populations to stimulate the division and activation of CD8+ T cells. Single-cell suspensions of well-established B16 melanomas from mice treated with IL-12 or mock cells were sorted by flow cytometry for macrophages, dendritic cells, and MDSCs (sorting strategy depicted in Supplemental Figure 6). These different populations were then cocultured with CFSE-labeled CD8+ T cells isolated from untouched splenocytes of P-TCR Tg mice in a 10:1 ratio of T cells to APCs. Myeloid-derived populations isolated from B16 tumors of mice treated with mock cells failed to induce the division of naive pmel-1 CD8+ T cells (Figure B). In striking contrast, macrophages, dendritic cells, and MDSCs from B16 tumors in mice treated with IL-12 cells stimulated the in vitro division and activation of CD8+ T cells (Figure B). These results indicate that IL-12 triggered a programmatic change in the major professional APC populations present within tumors, although IL-12 sensitization of MDSCs appeared to be the most potent in stimulating CD8+ T cells.
Recognition of cross-presented tumor antigen by IL-12–expressing CD8+ T cells is sufficient to induce tumor regression.
We sought to further examine the importance of IL-12 in triggering the recognition of cross-presented antigen by antitumor T cells. We developed a model to examine the effects of in vivo cross presentation by implanting Cloudman S91 melanomas (H-2d) on C57BL/6 × DBA F1 (H-2b/d) mice followed by the adoptive transfer of pmel-1 CD8+ T cells (H-2b). To confirm the MHC restriction of our tumor lines, we added IFN-γ in vitro for 48 hours and witnessed an expected increase in H-2b expression in B16 melanoma cultures and an increase in H-2d expression in Cloudman S91 melanoma culture lines (Figure A). These results confirmed the identities of the tumors used in MHC mismatch experiments.
IL-12 cells (H-2b) are capable of causing complete regression of established Cloudman S91 melanomas (H-2d) on C57BL/6 × DBA F1 mice (H-2b/d).
To test the capacity of IL-12 cells to trigger killing of MHC mismatched tumors incapable of direct antigen presentation, we implanted Cloudman melanomas (H-2d) in C57BL/6 × DBA F1 H-2b/d mice and transferred IL-12 or mock cells (H-2b) into sublethally irradiated mice. We observed the regression of large established tumors following the transfer of IL-12 compared with mock cells (Figure B). The presence of IL-12 had a significant impact on improving the antitumor immunity of tumor-specific CD8+ T cells, indicating that IL-12 triggered changes within tumors to enhance the cross presentation of antigens in vivo.
We sought to assess the differential impact of transferred IL-12–expressing cells on subpopulations of myeloid-derived cells capable of cross-presenting tumor-associated antigens for recognition by CD8+ T cells. We identified populations of myeloid-derived cells (macrophages, dendritic cells, MDSC-M, and MDSC-G) residing within Cloudman S91 melanomas (Figure C). Unlike the tumor cells in which they reside, the myelomonocytic cells from C57BL/6 × DBA F1 mice expressed both H-2b and H-2d and could therefore be recognized by IL-12 cells specific for gp10025–33 in the context of H-2b. We examined the presence of these cells within tumors 10 days following the adoptive transfer of either IL-12 or mock cells. Just prior to a major drop in tumor size, 10 days following the transfer of IL-12 cells, we observed a sudden decrease in CD11b+F4/80hi macrophages, CD11b+CD11chi dendritic cells, and CD11b+LyChiLy6Glo MDSC-M in tumors from mice treated with IL-12 cells compared with mock cells (Figure C). These changes were not observed at earlier time points such as day 3 and day 7 (data not shown).
The majority of myeloid-derived cells remaining within tumors of mice treated with IL-12 cells were a uniform population of CD11b+, Ly6CMid-hi, and Ly6Ghi cells (Figure C). Although the fractional percentage of these “double-positive” granulocytic cells appeared to be higher (Figure C), upon quantification, the absolute number of cells within tumors appeared to be unchanged (Figure D). We observed significant decreases in the number of CD11b+F4/80hi macrophages, CD11b+CD11chi dendritic cells, and CD11b+LyChiLy6Glo MDSC-M cells in tumors from mice treated with IL-12 cells compared with mock cells (Figure D). We additionally found that changes in the CD11b+ myeloid–derived populations occurred only in tumors and not in the spleens of C57BL/6 mice treated with IL-12 cells (Supplemental Figure 7). Unresolved by these experiments were whether decreased numbers of cells possessing the flow cytometric parameters identifying them as myelomonocytic cell subsets represented death of these cross-presenting cells or whether it indicated altered cell differentiation or migration. However, the changes that we observed in myeloid-derived subpopulations were confined to areas where tumor antigen was present and were not a part of a generalized phenomenon.
The role for CD11b+
, and Ly6Ghi
granulocytic cells within IL-12–treated tumors remains to be thoroughly explored. In light of recent studies highlighting the capability of polarizing tumor-infiltrating neutrophils to an N1 phenotype (36
), we hypothesized that perhaps the functional profile of Ly6Ghi
cells within IL-12–treated tumors may be altered. Given our earlier findings highlighting the need for endogenous cells to secrete IFN-γ (Figure ), we performed an intracellular stain for IFN-γ in addition to staining for the different myeloid surface markers. We found that CD11b+
cells, but not CD11b+
cells, from tumors of mice treated with IL-12 compared with mock cells, showed an increase in the ability to secrete IFN-γ (Supplemental Figure 8).
All experiments described so far were preceded by a 5-Gy TBI preconditioning regimen. To determine whether this pretreatment preparation in some manner had an impact on the observed in vivo effects of IL-12, we transferred IL-12 cells into nonirradiated Cd4–/– mice bearing B16 tumors and compared antitumor efficacy to that of WT mice that received the 5-Gy TBI pretreatment. We observed identical antitumor responses between irradiated WT mice and nonirradiated Cd4–/– mice, indicating that the effects of IL-12 on myeloid cells were independent of pretreatment regimens that were necessary to remove CD4+ T cell populations acting as immune suppressors or sinks (Supplemental Figure 9). We sought to further measure whether irradiation contributed to the effects of IL-12 on tumor-infiltrating myeloid-derived populations in vivo. We transferred cells into nonirradiated Rag1–/– mice and once again observed a decrease in the number of CD11b+F4/80hi macrophages, CD11b+CD11chi dendritic cells, and CD11b+Ly6ChiLy6Glo MDSC-M in tumors from mice 14 days following treatment with IL-12 cells compared with mock cells (Supplemental Figure 10). Overall, these experiments show the importance of IL-12 for enhancing the in vivo cross presentation of tumor antigens, a mechanism independent of NKp46+ innate lymphoid cells (Supplemental Figure 11), and suggest that the functional reprogramming of stromal cells such as macrophages, dendritic cells, and MDSCs plays an important role in the regression of established melanomas (Figure ).
Schematic diagram summarizing the proposed mechanism for tumor destruction induced by IL-12.