DenDritic cells expanD post cyclophosphamiDe anD meDiate robust anti-tumor responsesupon their stimulation with tlr3 agonist

Mohamed L. Salem, C. Marcela Díaz-Montero, Amir A. AL-Khami , Sabry EL-Naggar, and David J. ColeSection of Surgical Oncology, Department of Surgery, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC, 29425, USA

abstractNumerous preclinical and clinical studies would suggest that pre-conditioning a recipi-ent host via lymphodepletion can augment adoptive T cell therapy. Although several possibilities have been suggested, the precise mechanisms behind this augmentation remain elusive. We show herein that treatment of naïve B6 mice with a standard lymphodepletion regimen (4 mg cyclophosphamide (CTX)) induces a marked expan-sion of immature myeloid (CD11chighCD11bhighB220low) dendritic cells (DCs) in the peripheral blood from days 8 to 16 post treatment, (peaking on day 12). In vitro, these DCs demonstrated phagocytic and effective Ag presentation capability upon stimula-tion, indicating that they are biologically functional. In vivo, triggering toll-like receptors (TLR) by their specific agonists at the peak of DC expansion induced an inflammatory milieu and activation of post CTX expanded DCs leading to their trafficking into lymph nodes. Importantly, peptide vaccination combined with TLR3 agonist administration at the peak of DC expansion strikingly increased the expansion and the anti-tumor efficacy of adoptively transferred naïve pmel-1 CD8+ T cells. These effects were ab-solutely mediated by DCs. In conclusion, our data suggest that post CTX expansion of DCs represents a novel mechanism contributing to the augmentation of adoptive T cell therapy by lymphodepletion. Furthermore, these cells could potentially be ex-ploited in vivo to foster more effective anti-tumor adoptive immunotherapy strategies.

introDuctionPreconditioning a host with lymphodepletion using either total body irradiation (TBI) or chemotherapy (such as cyclophosphamide (CTX)) regimens can effectively augment the anti-tumor efficacy of adoptively transferred T cells.1-6

Suggested mechanisms underlying this effect include:1. Enhanced engraftment and survival of the transferred T cells by creation of an

immunological “niche” 7 with the induction of survival cytokines. 8, 9

2. Elimination of the regulatory CD4+CD25+ T (Treg), NKT cells, and myeloid-de-rived suppressor cells. 3, 10-12

3. Depletion of endogenous cells that compete with the transferred T cells for cytok-ines (“cytokine sink”) 7, 8, 13

We have recently reported that the adoptive transfer of T cells early after CTX treat-ment strikingly enhances the post vaccination T cell responses, and that analysis of the mechanisms underlying the beneficial effects of CTX showed that.14

1. Associated with the induction of the inflammatory cytokines IFN-a, MCP-1, and IL-6.2. Type I IFNs are critical.3. Creation of a space niche was not a critical factor.4. Associated with decreases in the numbers of Treg cells.5. Associated with rapid activation of DCs. (Similarly, activation of DCs was also

reported in the mice rendered lymphopenic by TBI due to the systemic release of lipopolysaccharide (LPS) after lymphopdepletion3).

6. Myeloid (CD11b+) cells were critical.

Recent studies, in addition to ours, showed that lymphodepletion mechanisms are not restricted to the homeostatic proliferation of the transferred T cells, the elimination of the endogenous suppressor cells, or the availability of endogenous cytokines 4, 14-17, indicating that additional unknown mechanisms might exist.

hypothesisGiven the observations of DC activation and the critical role of myeloid cells, we hy-pothesize that DCs might contribute to the beneficial effects of CTX preconditioning regimen.

aims1. Define the phenotypic and functional alteration in DCs post CTX therapy.2. Determine the role of DCs in mediation the Ag-specific responses of CD8+ T cells

post CTX therapy.

Elimination ofcytokine sink

Eliminationof regulatory

elements

Induction of survivalcytokines

Role ofDCs ?

Aim

Figure 1. An interactive model for the mechanisms underlying the impact of lymphodepletion on the enhanced Ag-specific responses of adoptively transferred T cells.

Models for adoptive T cell transfer

Figure 1. CTX preconditioning, adoptive T cell transfer, and vaccination. Recipient WT naïve Ly5.2 mice were treated with PBS or 4mg/mouse CTX and adoptively trans-ferred 1 day later by i.v. tail vein injection with 1 million of naive OT-1 or pmel-1 Ly5.1 cells. The mice were then vaccinated on day 2 and/or 12 with OVAp (in the case of OT-1 model) or gp100 melanoma peptide (in the case of pmel-1 model) along with or without poly(I:C). The mice were bled and sacrificed at the indicated time points to analyze the expansion and contraction of pmel-1 cells.

Figure 2. CTX treatment induced an increase in the relative and absolute numbers of circulating DCs during the restoration phase. Naïve B6 mice (n=4/group) were treated i.p. with PBS or CTX (4 mg/mouse) and sacrificed at the indicated time points. (A) A representative flow data showing % DCs (CD11c+CD11b+) in PBL at multiple time points post CTX treatment. (B) Shows the absolute numbers of DCs ± SD.

Figure 3. CTX preferentially induced expansion of circulating DC1 expressing imma-ture phenotype. (A) Naïve B6 mice (n=4/group) were i.p. treated with PBS or CTX and bled to analyze the DC activation in PBL. (B) Shows the phenotypic characterization of DC1 (R3 and R5) and DC2 (R4, R6, and R7) gated from R2. (C) Shows the relative numbers of DC1 (CD11chighCD11bhighLy6GlowB220low) and DC2 (CD11chighCD11blowGr.1highB220high) in PBL. (D, left panel) Shows phagocytosis of DCs. (D, right panel) Shows the in vitro Ag presenting function of DCs sorted from PBL of PBS or CTX treated mice.

Figure 4. Triggering TLR3 signaling at the peak of post CTX DC expansion creates an inflammatory milieu, augmenting the Ag-specific CD8+ T cell responses. CTX treated B6 mice (n=4/group) were treated with PBS or poly(I:C) on day 12 post treatment. Mice were sacrificed 24 hours later and % DCs (A) and their expression of CD80 (B, upper panel) and CCR7 (B, lower panel) were determined. (C) Mice were treated as in A and bled 1 hour after treatments, and sera were collected to measure the levels of TNF-a , IL-6 and IL-10, and MCP-1. (D, left panel) Naive recipient mice (n=4/group) were treated with PBS or CTX, and 12 days later, mice were adoptively transferred with naïve OT-1 cells followed by vaccination with OVAp ± poly(I:C). (D, right panel) Shows the fold increases in the OT-1 expansion in the PBL.

Finkelstein et al., J Leukoc Biol. 2004;76(2):333-337

Figure 5. Interaction of pmel-1 CD8+ transgenic T cells with B16 melanoma. T cells express a TCR recognizing a H-2Db-restricted CD8+ epitope from the gp100/pmel-17 protein mouse gp10025–33 (EGSRNQDWL) or human gp10025–33 (KVPRNQDWL). APC, Antigen-presenting cell.

Figure 6. Stimulation of post CTX expanded DCs induces their migration to LNs aug-menting CD8+ T cell responses to self tumor Ag. Naïve B6 mice were inoculated with B16 tumor and treated 10 days later PBS or CTX and adoptively transferred with na-ïve pmel-1 cells. Mice were left without further manipulation, or vaccinated with gp100 ± poly(I:C) 12 days post PBS or CTX treatment. (B) shows the % and absolute num-bers of pmel-1 cells in PBL 5 days post vaccination. As described in (A), mice were vaccinated with gp100 ± poly(I:C) both on days 2 and 12 post PBS or CTX treatment and then sacrificed 3 days later to assess the % and absolute numbers of pmel-1 cells in PBL (C, left panel) and LNs (C, right panel) and % and absolute numbers of DCs in PBL (D, left panel) and LNs (D, right panel).

Figure 7. Depletion of DCs ab-rogates the augmented T cell responses post CTX therapy. WT and DTR Tg mice (3 mice/group) were treated with PBS or CTX and then adoptively transferred 1 day later with pmel-1 cells. The mice were vaccinated with gp100/poly(I:C) on both days 2 and 12 post PBS or CTX treatment. On day 11, the DTR Tg mice were treated i.p. with 90 ng/mouse diphtheria toxin to deplete DCs. The mice were sacrificed 2 days post re-vaccination to analyze the numbers of DCs (A and B) and the expansion of pmel-1 cells (C).

Figure 8. Peptide boosting at the peak of post CTX DC expan-sion is essential to establish a therapeutic anti-tumor immunity of self tumor Ag. (A) B6 mice (n=5/group) were challenged with B16 tumor and treated with CTX after 10 days. Then, mice were vacci-nated with gp100/poly(I:C) on day 2 or on both day 2 and day 12 post CTX treatment. Two extra groups were challenged with B16 cells and treated with PBS or CTX. (B) Survival of the tumor-bearing mice

in (A). (C) Shows vitiligo developed in recipients in (A) vaccinated once or twice.

conclusions CTX treatment results in a significant increase in both the relative and absolute

numbers of DCs during the restoration phase. Post CTX expanded DCs preferentially express myeloid DC1, immature phenotype. Post CTX expanded DCs are biologically functional. Prime-boost with peptide/poly(I:C) at the lymphopenic and restoration (DC expan-

sion peak) phases post CTX therapy is essential and sufficient to generate and establish the anti-tumor efficacy of adoptive cell transfer therapy.

Our data indicate that DC expansion represents a novel mechanism behind the beneficial effects of CTX to adoptive cell transfer therapy.

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Days post pmel-1 cell transfer

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9 12 15 18 21 24 27 30 33

Tumor area (mm2)

No CTX, no pmel

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PBS/Naive pmel-1+gp100/poly(I:C) on day 2

PBS/Naïve pmel-1+gp100+poly(I:C) on days 2&12

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CTX/Naïve pmel-1+gp100+poly(I:C) on days 2&12

1 vaccinationon day 2

10 20 30 400

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Days post pmel-1 cell transfer

Percent survival

[A]

[C]

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2 vaccinationson days 2 & 12

*