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. 2007 Aug;117(8):2197-204.
doi: 10.1172/JCI32205.

Microbial translocation augments the function of adoptively transferred self/tumor-specific CD8+ T cells via TLR4 signaling

Chrystal M Paulos  1 , Claudia Wrzesinski, Andrew Kaiser, Christian S Hinrichs, Marcello Chieppa, Lydie Cassard, Douglas C Palmer, Andrea Boni, Pawel Muranski, Zhiya Yu, Luca Gattinoni, Paul A Antony, Steven A Rosenberg, Nicholas P Restifo
Affiliations

Microbial translocation augments the function of adoptively transferred self/tumor-specific CD8+ T cells via TLR4 signaling

Chrystal M Paulos et al. J Clin Invest. 2007 Aug.

Erratum in

  • J Clin Invest. 2007 Oct;117(10):3140

Abstract

Lymphodepletion with total body irradiation (TBI) increases the efficacy of adoptively transferred tumor-specific CD8(+) T cells by depleting inhibitory lymphocytes and increasing homeostatic cytokine levels. We found that TBI augmented the function of adoptively transferred CD8(+) T cells in mice genetically deficient in all lymphocytes, indicating the existence of another TBI mechanism of action. Additional investigation revealed commensal gut microflora in the mesenteric lymph nodes and elevated LPS levels in the sera of irradiated mice. These findings correlated with increased dendritic cell activation and heightened levels of systemic inflammatory cytokines. Reduction of host microflora using antibiotics, neutralization of serum LPS using polymyxin B, or removal of LPS signaling components using mice genetically deficient in CD14 and TLR4 reduced the beneficial effects of TBI on tumor regression. Conversely, administration of microbial ligand-containing serum or ultrapure LPS from irradiated animals to nonirradiated antibody-lymphodepleted mice enhanced CD8(+) T cell activation and improved tumor regression. Administration of ultrapure LPS to irradiated animals further enhanced the number and function of the adoptively transferred cells, leading to long-term cure of mice with large B16F10 tumors and enhanced autoimmune vitiligo. Thus, disruption of the homeostatic balance between the host and microbes can enhance cell-based tumor immunotherapy.

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Figures

Figure 1
Figure 1. TBI enhances the function of adoptively transferred self/tumor-reactive pmel-1 T cells in mice genetically deficient in cytokine sinks and Tregs.
(A) TBI augmented antitumor responses in mice genetically deficient in cytokine sinks and Tregs. Rag2–/–γc–/– mice (deficient in T, B, and NK cells) bearing s.c. B16F10 tumors established for 10 days received nonmyeloablative 5 Gy TBI or were not irradiated (0 Gy). One day later, mice received an ACT treatment regimen consisting of the adoptive transfer of 105 cultured self/tumor reactive pmel-1 T cells, rFPhgp100 vaccination, and rhIL-2 or were left untreated (NT). Data (mean ± SEM; n = 5 per group) are representative of 4 independent experiments. (B) TBI enhanced autoimmune vitiligo in Rag2–/–γc–/– mice. Twenty-eight days after treatment, nonirradiated and irradiated Rag2–/–γc–/– mice were evaluated in a blinded fashion for the development of vitiligo. Each mouse was scored for degree of hypopigmentation on a scale of 0–5. Data (n = 14 per group) are representative of 2 independent experiments. Horizontal bars indicate means. (C) TBI enhanced the function of adoptively transferred pmel-1 CD8+ T cells. Five days after treatment, pmel-1–Thy1.1+ cells were isolated from spleens of irradiated and nonirradiated Rag2–/–γc–/– mice and were cocultured with irradiated splenocytes pulsed with 1 μM hgp10025–33. Secretion of IFN-γ, GM-CSF, TNF-α, and IL-2 in pmel-1 cells was analyzed. Unpulsed splenocytes were used as controls. Data (mean ± SEM; n = 3 per group) are representative of 2 independent experiments. †P = 0.05, ††P < 0.05, ‡P < 0.01, ‡‡P < 0.001 versus nonirradiated treated mice. Tx, treatment.
Figure 2
Figure 2. TBI activates the innate immune system and promotes translocation of microorganisms from the radiation-injured gastrointestinal tract.
(A and B) TBI induced activation of DCs. Splenocytes and inguinal LNs were isolated from 5 Gy irradiated or nonirradiated mice 1 day after TBI. Absolute numbers of activated CD11c+CD86high DCs in the spleens (A) and inguinal LNs (B) of TBI and nonirradiated C57BL/6 mice were enumerated. Data (n = 3–6 per group) are representative of 3 independent experiments. (C) TBI induced production of inflammatory cytokine IL-12p70. Serum was collected from nonirradiated and 5 Gy TBI mice 1 day after TBI, and IL-12p70 was measured by ELISA. Data (n = 3 per group) are representative of 2 independent experiments. (D) TBI damaged the colon. Colons of mice were analyzed 3 days after TBI and scored by a pathologist blinded to treatment group. Data (n = 3–6 per group) are representative of 2 independent experiments. (E) LPS was elevated in irradiated mice. Serum from nonirradiated and 5 Gy irradiated mice were collected and analyzed for the presence of LPS using a limulus amebocyte lysate assay 6 days after TBI. Data (n = 3–6 per group) are representative of 3 independent experiments. Horizontal bars indicate means. (F) Inflammatory cytokines were elevated in irradiated mice. Serum was collected from nonirradiated and 5 Gy TBI mice 1 day after TBI, and IL-6, IL-1β, and TNF-α cytokines were measured using ELISA. Data are representative of 2 independent experiments. ††P < 0.05, **P < 0.001 versus nonirradiated mice.
Figure 3
Figure 3. Ciprofloxacin treatment impairs the effectiveness of ACT therapy and reduces activation of the innate immune system in irradiated mice.
(A) Ciprofloxacin reduced the detectable level of LPS in serum. Serum from nonirradiated and 5 Gy irradiated mice left untreated or treated with ciprofloxacin (Cipro) was collected and analyzed for the presence of microbial LPS using a limulus amebocyte lysate assay. Data (n = 3 per group) are representative of 2 independent experiments. (B) Ciprofloxacin treatment reduced the absolute number of host DCs. One day after TBI, splenocytes were isolated from nonirradiated and 5 Gy irradiated mice left untreated or treated with ciprofloxacin. Absolute numbers of CD11c+CD86high DCs were determined in the spleens of nonirradiated and irradiated mice. Data (n = 3 per group) are representative of 2 independent experiments. Horizontal bars indicate means. (C) Treatment of irradiated hosts with ciprofloxacin reduced effectiveness of ACT treatment. C57BL/6 mice bearing s.c. B16F10 tumors established for 10 days received 5 Gy TBI. One day later, mice received an ACT treatment consisting of adoptive transfer of 106 cultured pmel-1 T cells, rFPhgp100 vaccination, and rhIL-2 or were left untreated. Administration of ciprofloxacin as indicated began 2 days prior to ACT and continued for 2 weeks after treatment. Data (mean ± SEM; n = 4–5 per group) are representative of 2 independent experiments. †P = 0.05, ††P < 0.05 versus 5 Gy TBI without ciprofloxacin; ‡‡P < 0.001 versus 5 Gy TBI plus treatment without ciprofloxacin.
Figure 4
Figure 4. TLR4 signaling triggered by TBI improves the effectiveness of ACT therapy.
(A) Administration of PMB decreased the tumor treatment effectiveness of TBI. Mice bearing s.c. B16F10 tumors established for 10 days received 5 Gy TBI. One day later, mice received an ACT treatment consisting of adoptive transfer of 106 cultured pmel-1 T cells, rFPhgp100 vaccination, and rhIL-2 or were left untreated. For the duration of the experiment, mice were treated or not with PMB in their water. Data (mean ± SEM; n = 5 per group) are representative of 2 independent experiments. (B and C) The effectiveness of treatment was decreased in irradiated mice genetically deficient in CD14 (B) and TLR4 (C). WT, CD14–/–, and TLR4–/– tumor-bearing mice were irradiated and then received the ACT treatment described above or were left untreated. Data (mean ± SEM; n = 5 per group) are representative of 2 independent experiments. (D) Serum from irradiated mice improved treatment when transferred to nonirradiated antibody-lymphodepleted mice. Tumor-bearing mice received 5 Gy TBI or were left unirradiated. Alternatively, mice were depleted of lymphocytes with CD4 and NK antibodies and received 5 × 105 cultured pmel-1 T cells, rFPhgp100 vaccination, and IL-2 1 day later. Mice received serum with translocated LPS or serum removed of LPS with Detoxi-gel beads 1 day after ACT. Data (mean ± SEM; n = 4–5 per group) are representative of 2 independent experiments. ††P < 0.05, ‡P < 0.01 versus irradiated treated WT mice (A–C) or recipients of serum with LPS (D).
Figure 5
Figure 5. Depletion of cytokine sinks, removal of Tregs, and activation of the innate immune system recapitulate the effectiveness of TBI.
(A) Activation of the innate immune system with serum LPS, depletion of Tregs with anti-CD4 antibody, and removal of cytokine sinks with anti-NK antibody are required to improve the efficacy of adoptively transferred in nonirradiated mice. Tumor-bearing C57BL/6 mice received 5 Gy TBI or were left nonirradiated. Alternatively, mice were depleted of lymphocytes with CD4 and NK antibodies; 1 day later mice received 5 × 105 cultured pmel-1 T cells, rFPhgp100 vaccination, and IL-2. Serum for irradiated mice containing LPS was harvested and transferred into nonirradiated recipients. (B) Ultrapure LPS enhanced treatment in lymphodepleted nonirradiated mice. C57BL/6 mice were irradiated as a control. Mice received serum with translocated LPS or ultrapure LPS alone, CD4 alone, or NK-depleting antibody alone 1 day after ACT. Data (mean ± SEM; n = 4–5 per group) are representative of 2 independent experiments. (C) Ultrapure LPS recapitulated the effectiveness of TBI in Rag2–/–γc–/– mice genetically deficient in all lymphocytes. Data (mean ± SEM; n = 5 per group) are representative of 2 independent experiments. †P = 0.05 versus irradiated treated mice. The difference between the nonirradiated, treated, LPS-administered group and the irradiated treated group was not significant (P < 0.2).
Figure 6
Figure 6. Administration of ultrapure LPS after TBI significantly enhances antitumor immunity and autoimmunity.
(A) Ultrapure LPS augmented antitumor responses in irradiated mice. Mice bearing s.c. B16F10 tumors established for 10 days received 5 Gy TBI. One day later, mice received an ACT treatment consisting of adoptive transfer of 106 cultured pmel-1 T cells, rFPhgp100 vaccination, and rhIL-2 or were left untreated. The next day, mice received ultrapure LPS or were left untreated. Data (mean ± SEM; n = 5–10 per group) are representative of 10 independent experiments. (B) LPS enhanced autoimmune vitiligo in irradiated mice. One month after ACT treatment, irradiated mice treated with or without ultra-pure LPS were evaluated for the development of vitiligo. Mice were scored for the degree of hypopigmentation on a scale of 0–5. Data (n = 4–5 per group) are from 3 independent experiments. Horizontal bars indicate means. (C) Ultrapure LPS increased the absolute number of transferred pmel-1 T cells in the irradiated host. Absolute numbers of transferred pmel-1 T cells (CD8+Thy1.1+) in irradiated host. Data (mean ± SEM; n = 3–5 per group) are representative of 2 independent experiments. (D) Ultrapure LPS enhanced the function of adoptively transferred cells in irradiated mice. Five days after ACT treatment, pmel-1–Thy1.1+ splenocytes were cocultured with irradiated splenocytes pulsed with the indicated doses of hpg10025–33. Unpulsed splenocytes were used as controls. Data (mean ± SEM; n = 3 per group) are representative of 2 independent experiments. ††P < 0.05, ‡‡P < 0.001 versus irradiated treated mice.
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