Using several tumor models, we demonstrate that mice deficient in Bcl11b in T cells, although having reduced numbers of T cells in the peripheral lymphoid organs, developed significantly less tumors compared with wild-type mice. Bcl11b−/− CD4+ T cells, with elevated TNF-α levels, but not the Bcl11b−/− CD8+ T cells, were required for the reduced tumor burden, as were NK1.1+ cells, found in increased numbers in Bcl11bF/F/CD4-Cre mice. Among NK1.1+ cells, the NK cell population was predominant in number and was the only population displaying elevated granzyme B levels and increased degranulation, although not increased proliferation. Although the number of myeloid-derived suppressor cells was increased in the lungs with metastatic tumors of Bcl11bF/F/CD4-Cre mice, their arginase-1 levels were severely reduced. The increase in NK cell and myeloid-derived suppressor cell numbers was associated with increased bone marrow and splenic hematopoiesis. Finally, the reduced tumor burden, increased numbers of NK cells in the lung, and increased hematopoiesis in Bcl11bF/F/CD4-Cre mice were all dependent on TNF-α. Moreover, TNF-α treatment of wild-type mice also reduced the tumor burden and increased hematopoiesis and the numbers and activity of NK cells in the lung. In vitro treatment with TNF-α of lineage-negative hematopoietic progenitors increased NK and myeloid differentiation, further supporting a role of TNF-α in promoting hematopoiesis. These studies reveal a novel role for TNF-α in the antitumor immune response, specifically in stimulating hematopoiesis and increasing the numbers and activity of NK cells.

Several immune populations are known to play key roles in antitumor immune responses, including CD8+ T or CTLs and NK cells. CTLs act through recognition of tumor-associated Ags, presented in the context of MHC class I, based on which vaccines have been developed [(14) and reviewed in Ref. 5]. However, melanoma cells and other tumor cells downregulate MHC class I, making the CTL-mediated immune response inefficient (6). NK cells with enhanced effector activity were shown to prevent metastasis (7). Following activation through engagement of activating NK receptors, NK cells lyse targeted tumor cells predominantly using perforin and granzymes (8, 9). Several activating NK receptors critical in the antitumor immune response have been described, including NKG2D belonging to NK group 2, important in tumor surveillance (10, 11), and NKp46, part of the natural cytotoxicity receptors (12), critical in preventing tumor progression (13). Conversely, other tumor-infiltrating populations, such as tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs), restrain the antitumor immune response (1417).

TNF-α is currently used for the treatment of advanced soft tissue sarcomas and metastatic melanomas (reviewed in Refs. 18, 19). TNF-α is a potent inhibitor of tumor-associated vasculature (20, 21) (reviewed in Refs. 18, 19); however, its impact on the antitumor immune response is less characterized.

Bcl11b is a C2H2 zinc finger transcriptional regulator (22, 23) important for positive selection and survival of double-negative 3 and double-positive thymocytes (24, 25). It has been demonstrated that deletion of Bcl11b at the double-negative 3 stage of thymic development or from double-positive (DP) thymocytes with the use of an inducible ERCre system resulted in generation of induced T-to-NK (ITNK) cells that possess antitumor activity (26), which opens an exciting avenue to develop potent antitumor immune cells. Following these findings, in this study we directly tested the antitumor immune response of in vivo generated Bcl11b−/− T cells using Bcl11bF/F/CD4-Cre mice, in which the gene is removed at the DP stage of T cell development (25). We demonstrate that Bcl11bF/F/CD4-Cre mice, despite the reduced numbers of T cells in the periphery (25), developed significantly fewer metastatic lung nodules compared with wild-type mice and showed lower tumor burdens in flank melanoma and flank Tramp tumor models. The reduction in the tumor burden was dependent on NK1.1+ cells and CD4+ T cells, but not on CD8+ T cells. The NK cells predominated and were the only NK1.1+ population upregulating granzyme B and exhibiting elevated degranulation. The increase in the NK population was dependent on TNF-α produced by Bcl11b−/− CD4+ T cells. Bcl11bF/F/CD4-Cre mice showed increased bone marrow and splenic hematopoiesis, which was also dependent on TNF-α. TNF-α treatment of wild-type mice with metastatic tumors reduced the tumor burden and caused increased NK cell numbers and increased splenic hematopoiesis, supporting a novel role for TNF-α in antitumor immune response.

Bcl11bF/F/CD4-Cre mice have been previously described (25, 27). Mice were housed under specific pathogen-free conditions. All the experiments were performed in accordance with animal protocols approved by the Institutional Animal Care and Use Committee of Albany Medical Center.

A total number of 0.5 × 106 B16-F10 (B16) melanoma cells was transferred i.v. into 8- to 10-wk-old Bcl11bF/F/CD4-Cre and wild-type mice. On day 10 posttransfer, mice were sacrificed. The lungs were flushed with PBS and collected into Fekete’s solution for counting melanoma nodules. Flank melanoma and flank Tramp tumors were induced by injecting of 5 × 106 B16 melanoma cells or Tramp C-2 tumor cells, s.c. The tumor size was measured from day 9 to day 25 for B16 melanoma and for 10 wk for Tramp C-2 tumors.

Mice were i.p. injected with 200 μg anti-CD8a (53-6.72; BioXcell), anti-CD4 (GK1.5; BioXcell), anti-NK1.1 (PK-136; BioXcell), anti–TNF-α (XT3.11; BioXcell), anti–IFN-γ (XMG1.2; BioXcell), and anti–IL-17a (17F3; BioXcell) Abs or IgG 1 d before tumor cell injection, and further, the treatment was continued on days 2, 5, and 8 with 150 μg Abs. One microgram murine rTNF-α (PeproTech) or vehicle was i.p. injected, as above.

Lineage-negative (lin) bone marrow (BM) cells were enriched twice with the mouse lineage cell depletion kit (Miltenyi Biotec). Cells were cultured first in complete RPMI 1640 medium with 50 ng/ml stem cell factor, 5 ng/ml Flt3-L, 20 ng/ml IL-6, 0.5 ng/ml IL-7, ±50 ng/ml TNF-α for 6 d, following which cells were transferred in media with 20 ng/ml IL-15 (28, 29), ±TNF-α. For myeloid cell differentiation, lin BM cells were cultured on OP9 cells in α-MEM medium with 10 ng/ml IL-3, 10 ng/ml IL-7, 100 ng/ml stem cell factor, 100 ng/ml M-CSF, and 5 ng/ml Flt3L ± 20 ng/ml TNF-α for 10 d.

Cellular suspensions were stained, as previously described (30), using the following fluorophore-conjugated Abs: CD3ε (145-2c11), CD4 (GK1.5), CD8a (53-6.7), CD27 (LG.7F9), CD107a (ebio1D4B), CD127 (A7R34), NK1.1 (PK-136), NKp46 (29A1.1), c-Kit (2B8), Sca-1 (D7), Flt3 (A2F10), IFN-γ (XMG1.2), IL-17A (17B7), and TNF-α (MP6-XT22) from eBioscience. Anti-granzyme B (GB11) was from BioLegend. Intracellular cytokine staining was conducted, as previously described (31). FACS was performed on an upgraded (Cytek) eight-color FACSCalibur using FlowJo acquisition software, and the data were analyzed by FlowJo analysis software.

Lineage-positive populations were stained with the following mix of biotinylated anti-mouse Abs for: CD3ε, CD4, CD8α, CD11b/Mac1 (M1/70), CD11c (N418), CD19 (eBio1D3), B220 (RA3-6B2), GR-1 (RB6-8C5), NK1.1, Ter119 (TER-119), TCRβ (H57-597), γδTCR (eBioGL3), and FcεR1 (MAR-1), followed by streptavidin-conjugated allophycocyanin-eFluor 780. Cells were further stained with fluorophore-specific Abs, and the following populations were defined as: Lin/c-kit+/Sca-1 (LK), Lin/c-kit+Sca-1+ (LSK), granulocyte macrophage progenitors (GMP), based on CD16 and CD34 within the LK population, multipotent progenitors (MPP), and hematopoietic stem/progenitor cells (HSPC) based on Flt3 in the LSK population, PreProNKa and PreProNKb cells within the c-kitlowSca-1+ and c-kitSca-1+ cells, respectively, based on Flt3 and CD127 (3234).

The statistical significance between experimental groups was determined by unpaired two-tailed Student t test. The p values ≤0.05 were considered statistically significant. Results are presented as mean ± SD.

We employed several tumor models, specifically metastatic B16-F10 (B16) melanoma and flank grafts of B16 melanoma and Tramp prostate cancer cells, to test in vivo the contribution of Bcl11b−/− T cell populations in antitumor immune response, using Bcl11bF/F/CD4-Cre mice (25). Bcl11bF/F/CD4-Cre mice were previously shown to have reduced numbers of T cells in the periphery (25), yet the number of the tumor nodules in the lung was approximately eight times reduced in Bcl11bF/F/CD4-Cre mice i.v. transferred with B16 melanoma cells, compared with wild-type mice (Fig. 1A, Supplemental Fig. 1). Similarly, the tumor volumes were significantly reduced in the grafts initiated with melanoma or Tramp prostate cancer cells (Fig. 1B, 1C). Ex vivo removal of Bcl11b in DP thymocytes has been shown previously to generate CD8+ T cells reprogrammed to ITNK cells highly efficient in antitumor immune response when transferred into Rag2−/−gc−/− mice (26). To test the contribution of Bcl11b−/− CD8+ T cells to the antitumor immune response, we Ab-depleted CD8+ T cells (Supplemental Fig. 1B), which did not lead to a statistically distinguishable increase in lung metastases (Fig. 2A). Despite the fact that a large percentage of Bcl11b−/− CD8+ T cells in the lungs with metastatic tumors of Bcl11bF/F/CD4-Cre mice upregulated NK1.1 and NKp46 (Fig. 2B), these cells expressed only modest granzyme B levels and exhibited reduced degranulation based on surface expression of CD107a (Fig. 2B). For comparison, see levels of granzyme B and CD107a in NK cells (Fig. 3B). The reduced levels of granzyme B and surface CD107a could explain the modest contribution of Bcl11b−/− CD8+ T cells in the antitumor immune response, compared with the previously described reprogrammed Bcl11b−/− ITNK CD8+ T cells that were shown to express elevated levels of effector molecules (26). Thus, in the Bcll1bF/F/CD4-Cre system we observed enhanced antitumor control that did not depend on CD8+ T cells.

FIGURE 1.

Bcl11bF/F/CD4-Cre mice have significantly reduced tumors in metastatic melanoma and flank melanoma and Tramp syngeneic graft models. (A) Numbers of tumor nodules in lungs of Bcl11bF/F/CD4-Cre (knockout [KO]) and wild-type (WT) mice on day 10 posttumor injection. n = 9. (B) Size of the flank melanomas in Bcl11bF/F/CD4-Cre (KO) and wild-type (WT) mice measured on the indicated days. n = 5. (C) Size of the flank Tramp tumors in Bcl11bF/F/CD4-Cre (KO) and wild-type (WT) mice measured for 10 wk. n = 8. (A–C) The p values are indicated. Mean ± SD.

FIGURE 1.

Bcl11bF/F/CD4-Cre mice have significantly reduced tumors in metastatic melanoma and flank melanoma and Tramp syngeneic graft models. (A) Numbers of tumor nodules in lungs of Bcl11bF/F/CD4-Cre (knockout [KO]) and wild-type (WT) mice on day 10 posttumor injection. n = 9. (B) Size of the flank melanomas in Bcl11bF/F/CD4-Cre (KO) and wild-type (WT) mice measured on the indicated days. n = 5. (C) Size of the flank Tramp tumors in Bcl11bF/F/CD4-Cre (KO) and wild-type (WT) mice measured for 10 wk. n = 8. (A–C) The p values are indicated. Mean ± SD.

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FIGURE 2.

Bcl11b−/− CD4+ T cells, but not CD8+ T cells, have critical role in reduced metastatic lung melanoma tumor burden. (A) Numbers of tumor colonies in lungs of Bcl11bF/F/CD4-Cre mice treated with anti-CD4, anti-CD8 Abs, or IgG. The p values are indicated. Mean ± SD. (B and C) Frequencies of NK1.1, NKp46, granzyme B, and surface CD107a expressing CD8+ T (B) and CD4+ T (C) cells from lungs of Bcl11bF/F/CD4-Cre (knockout [KO]) and wild-type (WT) mice on day 10 posttumor injection, evaluated by FACS. Data are representative of six pairs of mice. (D) Frequencies of IFN-γ–, IL-17–, and TNF-α–producing CD4+ T cells in the lungs of Bcl11bF/F/CD4-Cre (KO) and wild-type (WT) mice, evaluated by FACS. Data are representative of six pairs of mice.

FIGURE 2.

Bcl11b−/− CD4+ T cells, but not CD8+ T cells, have critical role in reduced metastatic lung melanoma tumor burden. (A) Numbers of tumor colonies in lungs of Bcl11bF/F/CD4-Cre mice treated with anti-CD4, anti-CD8 Abs, or IgG. The p values are indicated. Mean ± SD. (B and C) Frequencies of NK1.1, NKp46, granzyme B, and surface CD107a expressing CD8+ T (B) and CD4+ T (C) cells from lungs of Bcl11bF/F/CD4-Cre (knockout [KO]) and wild-type (WT) mice on day 10 posttumor injection, evaluated by FACS. Data are representative of six pairs of mice. (D) Frequencies of IFN-γ–, IL-17–, and TNF-α–producing CD4+ T cells in the lungs of Bcl11bF/F/CD4-Cre (KO) and wild-type (WT) mice, evaluated by FACS. Data are representative of six pairs of mice.

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FIGURE 3.

NK cells express elevated levels of granzyme B and degranulation and are critical for reduced tumor burden in Bcl11bF/F/CD4-Cre mice. (A) Representative FACS data of NK1.1+CD3i−CD4CD8 NK cells and NK1.1+CD3i+CD4CD8 ITNK cells; (B) absolute numbers of NK1.1+CD3i−CD4CD8 NK cells and NK1.1+CD3i+CD4CD8 ITNK cells in lungs with metastatic melanoma from Bcl11bF/F/CD4-Cre (knockout [KO]) and wild-type (WT) mice. Data are representative of five independent experiments. (C) BrdU incorporation in lung NK1.1+CD3i−CD4CD8 NK cells and NK1.1+CD3i+CD4CD8 ITNK cells of mice with metastatic lung tumors. Mice were i.p. injected with 1 mg BrdU 6 h before being sacrificed. Data are representative of three independent experiments. (D and E) Levels of granzyme B and surface CD107a in NK1.1+CD3i−CD4CD8 NK cells (D) and NK1.1+CD3i+CD4CD8 ITNK cells (E) from lungs with metastatic melanoma of Bcl11bF/F/CD4-Cre (KO) and wild-type (WT) mice. (F) Numbers of tumor colonies in the lungs of Bcl11bF/F/CD4-Cre mice treated with anti-NK1.1 Abs or IgG. The p values are indicated. Mean ± SD. CD3i+ and CD3i− cells are positive and negative, respectively, for intracellular CD3.

FIGURE 3.

NK cells express elevated levels of granzyme B and degranulation and are critical for reduced tumor burden in Bcl11bF/F/CD4-Cre mice. (A) Representative FACS data of NK1.1+CD3i−CD4CD8 NK cells and NK1.1+CD3i+CD4CD8 ITNK cells; (B) absolute numbers of NK1.1+CD3i−CD4CD8 NK cells and NK1.1+CD3i+CD4CD8 ITNK cells in lungs with metastatic melanoma from Bcl11bF/F/CD4-Cre (knockout [KO]) and wild-type (WT) mice. Data are representative of five independent experiments. (C) BrdU incorporation in lung NK1.1+CD3i−CD4CD8 NK cells and NK1.1+CD3i+CD4CD8 ITNK cells of mice with metastatic lung tumors. Mice were i.p. injected with 1 mg BrdU 6 h before being sacrificed. Data are representative of three independent experiments. (D and E) Levels of granzyme B and surface CD107a in NK1.1+CD3i−CD4CD8 NK cells (D) and NK1.1+CD3i+CD4CD8 ITNK cells (E) from lungs with metastatic melanoma of Bcl11bF/F/CD4-Cre (KO) and wild-type (WT) mice. (F) Numbers of tumor colonies in the lungs of Bcl11bF/F/CD4-Cre mice treated with anti-NK1.1 Abs or IgG. The p values are indicated. Mean ± SD. CD3i+ and CD3i− cells are positive and negative, respectively, for intracellular CD3.

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It has been reported that tumor-specific CD4+ T cells are able to eradicate tumors by developing cytotoxic activity (35). To test the contribution of CD4+ T cells in control of tumor growth, we Ab-depleted CD4+ T cells (Supplemental Fig. 1B), which caused a significant increase in the numbers of tumor nodules compared with IgG-treated Bcl11bF/F/CD4-Cre mice (Fig. 2A). Bcl11b−/− CD4+ T cells only minimally upregulated NK1.1, but not NKp46, and they neither expressed granzyme B nor evidenced degranulation (Fig. 2C). We previously demonstrated that CD4+ T cells of Bcl11bF/F/CD4-Cre mice had an activated phenotype (25, 31) and produced elevated levels of proinflammatory cytokines, specifically TNF-α, IFN-γ, and IL-17 (31). Elevated levels of TNF-α, IFN-γ, and IL-17 were also produced by Bcl11b−/− CD4+ T cells in the lungs of mice with metastatic lung tumors (Fig. 2D). These results taken together demonstrate that Bcl11b−/− CD4+ T cells play an important role in antitumor immune response, although without upregulation of cytotoxic markers.

NK cells are known to play a critical role in the antitumor immune response to B16 melanoma (36). This is because B16 melanoma cells express reduced levels of MHC class I (37); however, they express ligands for NKp46 and NKG2D receptors, which are recognized by and engage NK cells (reviewed in Ref. 12). Our previous results showed that Bcl11bF/F/CD4-Cre mice have a 3-fold increase in splenic NK cells (25). To identify the NK population, we used a gating strategy that eliminates not only CD8+ T cells but also cells that still have intracellular CD3. In this way we defined two populations: NK1.1+CD4CD8CD3i− cells, considered real NK cells, and NK1.1+CD4CD8CD3i+, which are CD8+ T cells that downregulated the coreceptor CD8, express CD3 only intracellularly, and are most likely a subpopulation of ITNKs. Both populations were increased in lungs with metastatic tumors of Bcl11bF/F/CD4-Cre mice, compared with control wild-type mice (Fig. 3A, 3B); however, the NK1.1+CD4CD8CD3i+ cells only represented ∼25% of the NK1.1+CD4CD8CD3i− NK population (Fig. 3A, 3B). Both populations had increased levels of the activating receptors NKp46 and NKG2D and reduced levels of inhibitory receptor Ly49C (Supplemental Fig. 2A, 2B). In addition, both populations showed modest incorporation of BrdU, suggesting limited proliferation (Fig. 3C). However, only the NK1.1+CD4CD8CD3i− NK population expressed elevated levels of granzyme B and showed high degranulation (Fig. 3D, 3E). To further demonstrate the role of NK1.1+ cells in the antitumor immune response in Bcl11bF/F/CD4-Cre mice, we Ab-depleted NK1.1+ cells (Supplemental Fig. 1B), which caused a significant increase in tumor burden (Fig. 3F). Collectively, these results demonstrate that NK1.1+ cells play a critical role in the antitumor immune response in Bcl11bF/F/CD4-Cre mice, within which the NK1.1+CD4CD8CD3i− NK population is predominant in number and is the only population that exhibited elevated granzyme B levels and degranulation.

TAMs and MDSCs are known to suppress the antitumor immune response, and one mechanism responsible for this effect is production of elevated levels of arginase-1 (17, 38). We therefore evaluated the CD11b+F4/80+CD11c cells in the metastatic lungs of Bcl11bF/F/CD4-Cre mice and found that these cells were similar numerically between Bcl11bF/F/CD4-Cre and wild-type mice; however, the levels of arginase-1 were reduced in Bcl11bF/F/CD4-Cre mice compared with wild mice (Supplemental Fig. 3A). MDSCs were elevated in numbers in the lung with metastatic melanoma; however, their arginase-1 levels were severely reduced (Supplemental Fig. 3B). These results collectively demonstrate that, although MDSCs are increased in the metastatic lungs of Bcl11bF/F/CD4-Cre mice, their arginase-1 levels, as well as that of TAMs, were substantially reduced, suggesting an altered suppressive function.

Bcl11b is not expressed in myeloid cells; however, it is expressed in NK cells (26). Importantly, the CD4-Cre system does not remove floxed alleles in NK cells, thus excluding a direct effect of Bcl11b deficiency on increased NK cell numbers. Considering the increased numbers of NK1.1+CD4CD8CD3i− NK cells, however, in the absence of proliferation, and of MDSCs in Bcl11bF/F/CD4-Cre mice and the fact that previously we observed that these mice had splenomegaly (25) and exhibited extramedullary hematopoiesis (D. Albu and D. Avram, unpublished observations), we investigated hematopoiesis both in BM and spleen in the mice with lung metastatic tumors and found an increase in the lin population both in the spleen and BM (Fig. 4A–C, 4E, 4F). The lin population was defined as CD3CD4CD8αCD11b/Mac1CD11cCD19B220GR-1NK1.1Ter119−TCRβTCRγδFcεR1. Specifically, HSPCs, MPPs, common myeloid progenitors (CMPs), and GMPs were all increased in numbers (Fig. 4A–C, 4E, 4F), as well as the NK cell progenitor populations, predominantly the PreProNKa (Fig. 4A–C, 4E, 4F). PreProNKa and PreProNKb cells were defined within the c-kitlowSca-1+ and c-kitSca-1+ cells, respectively, as Flt3CD127high (3234). Mature NK cells, comprising NK1.1+CD27+/−CD11b+ cells (39, 40), as well as total NK cells, had higher frequencies as well, both in BM and spleen of Bcl11bF/F/CD4-Cre mice compared with control wild-type mice (Fig. 4D, 4G). Thus, these results suggest that the increased numbers of NK1.1+CD4CD8CD3i− NK cells and MDSCs in the lung of Bcl11bF/F/CD4-Cre mice with metastatic tumors are most likely a consequence of increased hematopoiesis in the BM and extramedullary in the spleen.

FIGURE 4.

Bcl11bF/F/CD4-Cre mice have increased bone marrow and splenic hematopoiesis. (A) Diagram illustrating the gating strategy. Progenitor populations were evaluated in the lineage-negative (lin) CD3CD4CD8αCD11b/Mac1CD11cCD19B220 GR-1 NK1.1Ter119− TCRβ, γδTCRFcεR1 cells. LK are Lin/c-kit+/Sca-1; LSK are Lin/c-kit+Sca-1+. NK precursors, Pre-Pro-NKa and Pre-Pro-NKb, are CD127+Flt3 within the gated c-kitlowsca-1+ and c-kitsca-1+, respectively, populations. MPP and HSPC are Flt3+ and Flt3, respectively, within the LSK population. GMP and CMP were defined based on CD16 and CD34 in the LK population. Frequencies (B, E) and absolute numbers (C, F) of lin, Pre-Pro-NKa, Pre-Pro-NKb, GMP, CMP, MPP, and HSPC populations in spleen (B, C) and bone marrow (E, F) of Bcl11bF/F/CD4-Cre (knockout [KO]) and wild-type (WT) mice with lung metastatic melanoma. (D and G) Frequencies of NK1.1+CD3i−CD4CD8 cells and mature NK cells (CD11b+CD27+/−) in the spleen (D) and bone marrow (G). Data are representative of five independent experiments. The p values are indicated. Mean ± SD.

FIGURE 4.

Bcl11bF/F/CD4-Cre mice have increased bone marrow and splenic hematopoiesis. (A) Diagram illustrating the gating strategy. Progenitor populations were evaluated in the lineage-negative (lin) CD3CD4CD8αCD11b/Mac1CD11cCD19B220 GR-1 NK1.1Ter119− TCRβ, γδTCRFcεR1 cells. LK are Lin/c-kit+/Sca-1; LSK are Lin/c-kit+Sca-1+. NK precursors, Pre-Pro-NKa and Pre-Pro-NKb, are CD127+Flt3 within the gated c-kitlowsca-1+ and c-kitsca-1+, respectively, populations. MPP and HSPC are Flt3+ and Flt3, respectively, within the LSK population. GMP and CMP were defined based on CD16 and CD34 in the LK population. Frequencies (B, E) and absolute numbers (C, F) of lin, Pre-Pro-NKa, Pre-Pro-NKb, GMP, CMP, MPP, and HSPC populations in spleen (B, C) and bone marrow (E, F) of Bcl11bF/F/CD4-Cre (knockout [KO]) and wild-type (WT) mice with lung metastatic melanoma. (D and G) Frequencies of NK1.1+CD3i−CD4CD8 cells and mature NK cells (CD11b+CD27+/−) in the spleen (D) and bone marrow (G). Data are representative of five independent experiments. The p values are indicated. Mean ± SD.

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Given the elevated levels of proinflammatory cytokines in Bcl11bF/F/CD4-Cre mice, we investigated their roles in the protection against tumors by treatment of Bcl11bF/F/CD4-Cre mice with neutralizing Abs against TNF-α, IFN-γ, and IL-17. Whereas the treatment with TNF-α–neutralizing Abs resulted in a major increase in the tumor burden, treatment with anti–IFN-γ or anti–IL-17 Abs had only a minimal, and statistically nonsignificant, effect (Fig. 5A), demonstrating that TNF-α has a critical role in reduced tumor burden in Bcl11bF/F/CD4-Cre mice.

FIGURE 5.

TNF-α is critical for reduced tumor burden, increased numbers of NK cells, and increased hematopoiesis in Bcl11bF/F/CD4-Cre mice. (A) Fold increase of tumor colonies in lungs of Bcl11bF/F/CD4-Cre mice treated with anti–TNF-α, anti–IL-17a, anti–IFN-γ Abs, or IgG. (B) Absolute numbers of NK1.1+CD3i−CD4CD8 in the lung of Bcl11bF/F/CD4-Cre mice treated with anti–TNF-α Abs or IgG. (C) Absolute numbers of lin, Pre-Pro-NKa, Pre-Pro-NKb, GMP, CMP, MPP, and HSPC populations in the spleen of Bcl11bF/F/CD4-Cre mice with metastatic lung tumors treated with anti–TNF-α Abs or IgG. The gating strategies for progenitor cells are as described in Fig. 4. Data are representative of five independent experiments. The p values are indicated. Mean ± SD.

FIGURE 5.

TNF-α is critical for reduced tumor burden, increased numbers of NK cells, and increased hematopoiesis in Bcl11bF/F/CD4-Cre mice. (A) Fold increase of tumor colonies in lungs of Bcl11bF/F/CD4-Cre mice treated with anti–TNF-α, anti–IL-17a, anti–IFN-γ Abs, or IgG. (B) Absolute numbers of NK1.1+CD3i−CD4CD8 in the lung of Bcl11bF/F/CD4-Cre mice treated with anti–TNF-α Abs or IgG. (C) Absolute numbers of lin, Pre-Pro-NKa, Pre-Pro-NKb, GMP, CMP, MPP, and HSPC populations in the spleen of Bcl11bF/F/CD4-Cre mice with metastatic lung tumors treated with anti–TNF-α Abs or IgG. The gating strategies for progenitor cells are as described in Fig. 4. Data are representative of five independent experiments. The p values are indicated. Mean ± SD.

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The increased numbers of both NK cells and TNF-α were required for reduced tumor burden in Bcl11bF/F/CD4-Cre mice. Considering that increased NK cells is likely to occur through a bystander effect, we next investigated whether TNF-α plays a role in the increase in NK cell numbers and elevated hematopoiesis. Treatment with TNF-α–neutralizing Abs caused a marked decrease in the NK1.1+CD4CD8CD3i− NK cells in the lung of Bcl11bF/F/CD4-Cre mice with metastatic tumors (Fig. 5B), suggesting that TNF-α is essential for the elevated numbers of NK cells. Further evaluation of the effect of anti–TNF-α treatment on hematopoiesis revealed a reduction in the lin population, including HSPCs, MMPs, CMPs, and GMPs, as well as of the PreProNKa/b populations in the spleen (Fig. 5C). However, the treatment caused a less pronounced effect in the BM (data not shown). The more pronounced effect of TNF-α on extramedullary hematopoiesis may be due to the known role of TNF-α in mobilization (41). These results taken together demonstrate that TNF-α is critical for reduced tumor burden, elevated numbers of NK cells in the lung, and increased splenic hematopoiesis in Bcl11bF/F/CD4-Cre mice.

To further demonstrate the implication of TNF-α in the antitumor immune response, we treated wild-type mice with lung metastatic melanoma with rTNF-α. TNF-α treatment caused a significant reduction in the tumor burden and increased the numbers of NK cells (Fig. 6A, 6B) and MDSCs in the lung; however, the percentages of arginase-1+ TAMs and MDSCs were reduced (Supplemental Fig. 3C, 3D). Importantly, lung NK cells of TNF-α–treated mice had elevated granzyme B and degranulation (Fig. 6C). Additionally, lin cells, HSPCs, MPPs, CMPs, GMPs, as well as NK progenitors were increased in the spleen, although not significantly in the BM (Fig. 6D and data not shown). We further investigated whether TNF-α treatment caused increased proliferation of HSPCs and progenitors, and our results demonstrated that HSPCs, MPPs, GMPs, as well as PreProNKa/b populations of TNF-α–treated wild mice with lung metastatic melanoma incorporated more BrdU than untreated controls (Fig. 6E). Similarly, HSPCs and progenitors of Bcl11bF/F/CD4-Cre mice also had increased BrdU incorporation (Fig. 6E). Collectively, these results suggest that TNF-α promotes increased hematopoiesis through increased formation of progenitor cells, as well as increased proliferation of HSPCs and progenitor populations.

FIGURE 6.

TNF-α treatment of wild-type mice with lung metastatic melanoma inhibits tumor development and increases numbers and activity of NK cells in the lung and splenic hematopoiesis. (A) Numbers of tumor colonies in lungs of wild-type mice treated with 1 μg TNF-α or vehicle. (B) Absolute numbers of NK1.1+CD3i−CD4CD8 in the lung of wild-type mice treated with TNF-α or vehicle. (C) Levels of granzyme B and surface CD107a in NK1.1+CD3i−CD4CD8 NK cells in TNF-α–treated mice with metastatic tumors versus controls. (D) Absolute numbers of lin, Pre-Pro-NKa, Pre-Pro-NKb, GMP, CMP, MPP, and HSPC populations in the spleen of wild-type mice with lung metastatic melanoma treated with TNF-α or vehicle. Data are representative of five independent experiments. The p values are indicated. Mean ± SD. (E) Frequencies of cells that incorporated BrdU within HSPC, Pre-Pro-NKa and Pre-Pro-NKb, MPP, GMP, and CMP populations in the spleen of wild-type mice treated with TNF-α or vehicle and in Bcl11bF/F/CD4-Cre mice. Mice were i.p. injected with 1 mg BrdU 6 h before being sacrificed. The gating strategy for progenitor cells is described in Fig. 4. Data are representative of three independent experiments.

FIGURE 6.

TNF-α treatment of wild-type mice with lung metastatic melanoma inhibits tumor development and increases numbers and activity of NK cells in the lung and splenic hematopoiesis. (A) Numbers of tumor colonies in lungs of wild-type mice treated with 1 μg TNF-α or vehicle. (B) Absolute numbers of NK1.1+CD3i−CD4CD8 in the lung of wild-type mice treated with TNF-α or vehicle. (C) Levels of granzyme B and surface CD107a in NK1.1+CD3i−CD4CD8 NK cells in TNF-α–treated mice with metastatic tumors versus controls. (D) Absolute numbers of lin, Pre-Pro-NKa, Pre-Pro-NKb, GMP, CMP, MPP, and HSPC populations in the spleen of wild-type mice with lung metastatic melanoma treated with TNF-α or vehicle. Data are representative of five independent experiments. The p values are indicated. Mean ± SD. (E) Frequencies of cells that incorporated BrdU within HSPC, Pre-Pro-NKa and Pre-Pro-NKb, MPP, GMP, and CMP populations in the spleen of wild-type mice treated with TNF-α or vehicle and in Bcl11bF/F/CD4-Cre mice. Mice were i.p. injected with 1 mg BrdU 6 h before being sacrificed. The gating strategy for progenitor cells is described in Fig. 4. Data are representative of three independent experiments.

Close modal

We further investigated the impact of TNF-α treatment on differentiation of lin HSPCs and progenitors and their potential to form NK and myeloid cells in vitro. In vitro TNF-α treatment of the lin cells derived from wild-type and Bcl11bF/F/CD4-Cre mice with lung metastatic tumors enhanced the NK cell and myeloid cell formation, under the NK-promoting or myeloid-promoting conditions, respectively, compared with no treatment (Supplemental Fig. 4), further demonstrating the direct role of TNF-α in promoting the differentiation of HSPCs and progenitors.

In this study, we demonstrate that Bcl11bF/F/CD4-Cre mice have reduced tumor burden in three tumor models. We further demonstrate that the reduced tumor burden was dependent on the NK1.1+ cells and CD4+ T cells, but not on CD8+ T cells. This was surprising, as it was previously demonstrated that ex vivo removal of Bcl11b in DP thymocytes resulted in generation of CD8+ T cells reprogrammed to ITNK cells efficient in antitumor immune response when transferred into Rag2−/−gc−/− mice (26). In our study, Bcl11b−/− CD8+ T cells neither expressed elevated granzyme B levels nor exhibited enhanced degranulation. There are several possibilities that can explain why Bcl11b−/− CD8+ T cells of Bcl11bF/F/CD4-Cre mice failed in antitumor immune response compared with Bcl11b−/− CD8+ T cells generated ex vivo from DP thymocytes of Bcl11bF/F/ER-Cre mice, followed by transfer in Rag2−/−gc−/− mice. Perhaps the ex vivo generation of ITNK cells is more efficient, as it does not have to pass the rigorous control of thymic selection, which is likely to cause deletion of numerous reactive cells. In addition, the transfer in Rag2−/−gc−/− mice offers the opportunity of expansion related to lymphopenia in these mice, which is more strictly controlled in Bcl11bF/F/CD4-Cre mice. In addition, in Bcl11bF/F/CD4-Cre mice the CD8+ T cells are also exposed to other T cell populations and a different environment, which further can shape them differently than in Rag2−/−gc−/− mice. Interestingly, when we removed Bcl11b from mature CD8+ T cells using the dLck-iCre system, Bcl11b−/− CD8+ T cells not only had reduced expansion in response to Listeria monocytogenes and influenza challenge but they also exhibited reduced granzyme B levels and reduced cytotoxicity (42). However, when effector Bcl11b−/− CD8+ T cells were generated in vitro in the presence of IL-2, starting with naive CD8+ T cells of Bcl11bF/F/ER-Cre mice, in conditions in which the activation preceded the tamoxifen treatment, Bcl11b−/− CD8+ T cells did not downregulate perforin and granzyme B mRNA levels (42). Taken together, these data suggest that a specific in vivo environment, versus the more controlled in vitro environment, is likely to change the phenotype of the cells.

Bcl11bF/F/CD4-Cre mice had two populations of NK1.1+CD4CD8 cells, as follows: the real NK cells, negative for intracellular CD3 (NK1.1+CD3i−), and a population that expressed CD3 intracellularly (NK1.1+CD3i+). Importantly, of these two NK1.1+ populations, only the NK1.1+CD3i− exhibited increased granzyme B and elevated degranulation and also represented the numerically predominant population. Both populations expressed NKG2D- and NKp46-activating receptors with roles in tumor surveillance (10, 11) and tumor progression, respectively (13). These findings suggest that NK1.1+CD3i− NK cells constitute the predominant NK1.1+ population responsible for the reduced tumor burden in Bcl11bF/F/CD4-Cre mice. Interestingly, NK cell numbers were found increased in the spleen (25) and in the lungs with metastatic tumor of Bcl11bF/F/CD4-Cre mice, as demonstrated in this study. Bcl11b is expressed in NK cells; however, the CD4Cre system does not remove the gene in NK cells, suggesting that the increase in the NK population was caused by a bystander effect. The fact that Bcl11b−/− CD4+ T cells were shown to produce elevated levels of proinflammatory cytokines (31) prompted our investigation of their contribution to antitumor immune response. Whereas IFN-γ and IL-17 were dispensable, TNF-α was found critical for the reduced tumor burden in Bcl11bF/F/CD4-Cre mice and for the increased numbers of NK cells. Our results further demonstrated that TNF-α was responsible for the elevated hematopoiesis, including extramedullary hematopoiesis in the spleen, which explains the increased numbers of NK and MDSC cells in the lungs with metastatic tumors of Bcl11bF/F/CD4-Cre mice. In support of this notion, TNF-α treatment of wild-type mice with lung metastatic tumors not only reduced their tumor burden but increased the NK cells in the lung and caused elevated splenic hematopoiesis. Thus, TNF-α contributes to generation of immune populations, by increasing hematopoiesis, particularly extramedullary, therefore increasing generation of NK cells. Although the numbers of MDSCs were increased too, their arginase-1 levels were reduced, suggesting decreased suppressive activity. Additionally, TNF-α increased formation of NK and myeloid cells in vitro starting from lin HSPCs and progenitors. Whether TNF-α exerts this effect by simply promoting increased proliferation of HSPCs and progenitors or accelerates formation of progenitors from HSPCs requires further investigation. It will also be important to determine whether the reduced arginase-1 levels in MDSCs in Bcl11bF/F/CD4-Cre mice and in TNF-α–treated wild-type mice with tumors are a consequence of the tumor environment or altered generation of MDSCs.

We reason that a direct effect of TNF-α on killing tumors is less likely to occur, as it has been previously demonstrated that B16 cells are resistant to direct TNF-α–mediated killing (43). Moreover, knockdown of TNFαR1 in B16 cells had no impact on tumor burden in Bcl11bF/F/CD4-Cre mice or wild-type mice treated with TNF-α (data not shown). Thus, the major role of TNF-α in this system, in preventing tumor growth, appears to be mediated through enhanced hematopoiesis.

We previously demonstrated that the T regulatory (Treg) cells of Bcl11bF/F/CD4-Cre mice are less suppressive (31). Although we cannot exclude that their phenotype participated in reduced tumor burden in Bcl11bF/F/CD4-Cre mice, it was ultimately TNF-α that was responsible for the reduced tumor burden, increased NK cell numbers, and their generation through increased splenic hematopoiesis. If Treg cells rather than TNF-α were responsible for the reduced tumor burden in Bcl11bF/F/CD4-Cre mice, TNF-α neutralization alone would not have caused such a dramatic change in tumor burden, as in these conditions the Treg cells were still present in Bcl11bF/F/CD4-Cre mice. Additionally, we would not have expected to observe an impact of TNF-α treatment on wild-type mice, in which Treg cells were unaltered. Based on all these findings, we propose that TNF-α is effective in protection against tumors not only in blocking angiogenesis (20, 21) (reviewed in Refs. 18, 19) but also in shaping the antitumor immune response, by increasing splenic hematopoiesis and generation of NK cells. Previously, TNF-α was shown to increase mobilization of progenitor populations from BM, particularly of B cell progenitors, most likely through suppression of CXCL12 (41, 44), and to enhance generation of dendritic cells in vitro (45). Reports in the literature suggest a complex role of TNF-α on hematopoietic stem cells (HSCs). Whereas in vitro studies mostly demonstrated a suppressive effect on HSC renewal and maintenance (46, 47), in vivo studies suggested a stimulatory role of TNF-α on HSC maintenance (48) but a suppressive impact on actively cycling HSCs (49). Other studies further showed a beneficial effect on hematopoietic progenitors (44) and a favorable effect on engraftment (50). Our findings support the idea that in mice with lung metastatic tumors TNF-α production or its administration increases HSPC and other progenitor cell numbers, as well as their proliferation, resulting in increased NK numbers and efficient antitumor immune response. Importantly, the dose of TNF-α used in our treatments was much less than the doses observed to have toxicity (51, 52), and none of the treated mice died (data not shown). Our observation that TNF-α increases formation of NK cells from Lin HSPC and progenitor cells under NK differentiation conditions opens an avenue for therapeutic generation of NK cells for transplantation to promote antitumor immune responses.

We acknowledge Drs. Hung Le and Chandra S. Bapanpally for the flank melanoma and the flank TRAMP prostate graft experiments. We acknowledge Dr. Diana Albu for the preliminary observations of splenic extramedullary hematopoiesis. We gratefully acknowledge Drs. Neal G. Copeland, Nancy A. Jenkins (Houston Methodist Research Institute), and Pentao Liu (Sanger Institute) for mice. We acknowledge Adrian Avram for graphical presentation and Dr. Douglas Cohn, Victoria Boppert, and Hattie Wang for care of the mice.

This work was supported by Grants AI067846 and AI078273 (to D.A.) from the National Institutes of Health.

The online version of this article contains supplemental material.

Abbreviations used in this article:

BM

bone marrow

CMP

common myeloid progenitor

DP

double-positive

GMP

granulocyte macrophage progenitor

HSC

hematopoietic stem cell

HSPC

hematopoietic stem and progenitor cell

ITNK

induced T-to-NK

MDSC

myeloid-derived suppressor cell

MPP

multipotent progenitor

TAM

tumor-associated macrophage

Treg

T regulatory.

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The authors have no financial conflicts of interest.

Supplementary data