1. DenDritic cells expanD post cyclophosphamiDe anD meDiate robust anti-tumor responses
upon their stimulation with tlr3 agonist
Mohamed L. Salem, C. Marcela Díaz-Montero, Amir A. AL-Khami , Sabry EL-Naggar, and David J. Cole
Section of Surgical Oncology, Department of Surgery, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC, 29425, USA
abstract
Numerous preclinical and clinical studies would suggest that pre-conditioning a recipi-
Elimination of Figure 7. Depletion of DCs ab-
cytokine sink Aim
ent host via lymphodepletion can augment adoptive T cell therapy. Although several rogates the augmented T cell
Figure 1. An interactive model for responses post CTX therapy. WT
possibilities have been suggested, the precise mechanisms behind this augmentation
the mechanisms underlying the and DTR Tg mice (3 mice/group)
?
remain elusive. We show herein that treatment of naïve B6 mice with a standard Elimination
Role of
of regulatory impact of lymphodepletion on the were treated with PBS or CTX
lymphodepletion regimen (4 mg cyclophosphamide (CTX)) induces a marked expan-
sion of immature myeloid (CD11chighCD11bhighB220low) dendritic cells (DCs) in the
elements DCs enhanced Ag-specific responses and then adoptively transferred
of adoptively transferred T cells. 1 day later with pmel-1 cells. 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- Induction of survival mice were vaccinated with gp100/
cytokines
tion, indicating that they are biologically functional. In vivo, triggering toll-like receptors poly(I:C) on both days 2 and 12
(TLR) by their specific agonists at the peak of DC expansion induced an inflammatory post PBS or CTX treatment. On
milieu and activation of post CTX expanded DCs leading to their trafficking into lymph day 11, the DTR Tg mice were
nodes. Importantly, peptide vaccination combined with TLR3 agonist administration treated i.p. with 90 ng/mouse
at the peak of DC expansion strikingly increased the expansion and the anti-tumor diphtheria toxin to deplete DCs.
efficacy of adoptively transferred naïve pmel-1 CD8+ T cells. These effects were ab-
Models for adoptive T cell transfer The mice were sacrificed 2 days
solutely mediated by DCs. In conclusion, our data suggest that post CTX expansion post re-vaccination to analyze the
of DCs represents a novel mechanism contributing to the augmentation of adoptive numbers of DCs (A and B) and the
T cell therapy by lymphodepletion. Furthermore, these cells could potentially be ex- Finkelstein et al., J Leukoc Biol. 2004;76(2):333-337 expansion of pmel-1 cells (C).
ploited in vivo to foster more effective anti-tumor adoptive immunotherapy strategies.
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
introDuction protein mouse gp10025–33 (EGSRNQDWL) or human gp10025–33 (KVPRNQDWL).
APC, Antigen-presenting cell. Figure 8. Peptide boosting at
Preconditioning a host with lymphodepletion using either total body irradiation (TBI) or Figure 3. CTX preferentially induced expansion of circulating DC1 expressing imma- [A] [B]
the peak of post CTX DC expan-
450 100
chemotherapy (such as cyclophosphamide (CTX)) regimens can effectively augment ture phenotype. (A) Naïve B6 mice (n=4/group) were i.p. treated with PBS or CTX and 400
350
sion is essential to establish a
80
the anti-tumor efficacy of adoptively transferred T cells.1-6 bled to analyze the DC activation in PBL. (B) Shows the phenotypic characterization 300
therapeutic anti-tumor immunity of
60
250
Suggested mechanisms underlying this effect include: of DC1 (R3 and R5) and DC2 (R4, R6, and R7) gated from R2. (C) Shows the relative 200
self tumor Ag. (A) B6 mice (n=5/
40
Tumor area (mm2) Percent survival
150
1. Enhanced engraftment and survival of the transferred T cells by creation of an numbers of DC1 (CD11chighCD11bhighLy6GlowB220low) and DC2 (CD11chighCD1 100 20
50 group) were challenged with B16
*
immunological “niche” 7 with the induction of survival cytokines. 8, 9 1blowGr.1highB220high) in PBL. (D, left panel) Shows phagocytosis of DCs. (D, right 0 0
9 12 15 18 21 24
tumor and treated with CTX after
27 30 33 10 20 30 40
2. Elimination of the regulatory CD4+CD25+ T (Treg), NKT cells, and myeloid-de- panel) Shows the in vitro Ag presenting function of DCs sorted from PBL of PBS or Days post pmel-1 cell transfer
[C]
Days post pmel-1 cell transfer
10 days. Then, mice were vacci-
CTX treated mice.
No CTX, no pmel
rived suppressor cells. 3, 10-12 CTX alone
nated with gp100/poly(I:C) on day
PBS/Naive pmel-1+gp100/poly(I:C) on day 2
3. Depletion of endogenous cells that compete with the transferred T cells for cytok- 2 or on both day 2 and day 12 post
PBS/Naïve pmel-1+gp100+poly(I:C) on days 2&12
CTX/Naïve pmel-1+gp100/poly(I:C) on day 2
ines (“cytokine sink”) 7, 8, 13 CTX treatment. Two extra groups
CTX/Naïve pmel-1+gp100+poly(I:C) on days 2&12
We have recently reported that the adoptive transfer of T cells early after CTX treat- were challenged with B16 cells
and treated with PBS or CTX. (B)
2 vaccinations 1 vaccination
ment strikingly enhances the post vaccination T cell responses, and that analysis of Figure 1. CTX preconditioning, adoptive T cell transfer, and vaccination. Recipient on days 2 & 12 on day 2
the mechanisms underlying the beneficial effects of CTX showed that.14 WT naïve Ly5.2 mice were treated with PBS or 4mg/mouse CTX and adoptively trans- Survival of the tumor-bearing mice
1. Associated with the induction of the inflammatory cytokines IFN-a, MCP-1, and IL-6. ferred 1 day later by i.v. tail vein injection with 1 million of naive OT-1 or pmel-1 Ly5.1 in (A). (C) Shows vitiligo developed in recipients in (A) vaccinated once or twice.
2. Type I IFNs are critical. cells. The mice were then vaccinated on day 2 and/or 12 with OVAp (in the case of
3. Creation of a space niche was not a critical factor. OT-1 model) or gp100 melanoma peptide (in the case of pmel-1 model) along with
4. Associated with decreases in the numbers of Treg cells. or without poly(I:C). The mice were bled and sacrificed at the indicated time points to conclusions
5. Associated with rapid activation of DCs. (Similarly, activation of DCs was also analyze the expansion and contraction of pmel-1 cells.
 CTX treatment results in a significant increase in both the relative and absolute
reported in the mice rendered lymphopenic by TBI due to the systemic release of
numbers of DCs during the restoration phase.
lipopolysaccharide (LPS) after lymphopdepletion3).
 Post CTX expanded DCs preferentially express myeloid DC1, immature phenotype.
6. Myeloid (CD11b+) cells were critical.
 Post CTX expanded DCs are biologically functional.
Recent studies, in addition to ours, showed that lymphodepletion mechanisms are not  Prime-boost with peptide/poly(I:C) at the lymphopenic and restoration (DC expan-
restricted to the homeostatic proliferation of the transferred T cells, the elimination of sion peak) phases post CTX therapy is essential and sufficient to generate and
the endogenous suppressor cells, or the availability of endogenous cytokines 4, 14-17, establish the anti-tumor efficacy of adoptive cell transfer therapy.
indicating that additional unknown mechanisms might exist.  Our data indicate that DC expansion represents a novel mechanism behind the
beneficial effects of CTX to adoptive cell transfer therapy.
hypothesis References
Figure 4. Triggering TLR3 signaling at the peak of post CTX DC expansion creates an Figure 6. Stimulation of post CTX expanded DCs induces their migration to LNs aug-
Given the observations of DC activation and the critical role of myeloid cells, we hy- inflammatory milieu, augmenting the Ag-specific CD8+ T cell responses. CTX treated menting CD8+ T cell responses to self tumor Ag. Naïve B6 mice were inoculated with
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