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Tetanus toxoid and CCL3 improve dendritic cell vaccines in mice and glioblastoma patients

Abstract

After stimulation, dendritic cells (DCs) mature and migrate to draining lymph nodes to induce immune responses1. As such, autologous DCs generated ex vivo have been pulsed with tumour antigens and injected back into patients as immunotherapy. While DC vaccines have shown limited promise in the treatment of patients with advanced cancers2,3,4 including glioblastoma5,6,7, the factors dictating DC vaccine efficacy remain poorly understood. Here we show that pre-conditioning the vaccine site with a potent recall antigen such as tetanus/diphtheria (Td) toxoid can significantly improve the lymph node homing and efficacy of tumour-antigen-specific DCs. To assess the effect of vaccine site pre-conditioning in humans, we randomized patients with glioblastoma to pre-conditioning with either mature DCs8 or Td unilaterally before bilateral vaccination with DCs pulsed with Cytomegalovirus phosphoprotein 65 (pp65) RNA. We and other laboratories have shown that pp65 is expressed in more than 90% of glioblastoma specimens but not in surrounding normal brain9,10,11,12, providing an unparalleled opportunity to subvert this viral protein as a tumour-specific target. Patients given Td had enhanced DC migration bilaterally and significantly improved survival. In mice, Td pre-conditioning also enhanced bilateral DC migration and suppressed tumour growth in a manner dependent on the chemokine CCL3. Our clinical studies and corroborating investigations in mice suggest that pre-conditioning with a potent recall antigen may represent a viable strategy to improve anti-tumour immunotherapy.

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Figure 1: Td pre-conditioning increases DC migration to VDLNs and is associated with improved clinical outcomes.
Figure 2: Td recall response activates CD4+ T cells to increase DC migration to VDLNs.
Figure 3: Td recall responses and induced CCL3 cooperate to facilitate DC migration to VDLNs.
Figure 4: Td pre-conditioning improves responses in tumour-bearing mice.

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Acknowledgements

The authors thank the staff who supported this study, including R. Schmittling, P. Norberg, W. Xie, P. Healy, D. Lally-Goss, S. McGehee-Norman, B. Perry, S. Snipes and R. Edward Coleman. This work was supported by grants from the National Institutes of Health (NIH) National Institute of Neurological Disorders and Stroke Specialized Program of Research Excellence in brain cancer (P50-CA108786, D.D.B. and J.H.S.) and SRC on Primary and Metastatic Tumors of the CNS (P50-NS20023, D.D.B. and J.H.S.) as well as NIH RO1 (R01-CA177476-01, J.H.S.; R01-NS067037, R01-CA134844, D.A.M.) and P01 (P01-CA154291-01A1, D.D.B. and J.H.S.) funding sources. Additional support is from the National Brain Tumor Society (D.A.M. and J.H.S.), the American Brain Tumor Association (D.A.M. and J.H.S.), Accelerate Brain Cancer Cure Foundation Young Investigator’s Award (D.A.M.), The Kinetics Foundation, (J.H.S.) Ben and Catherine Ivy Foundation (J.H.S.), and in part by Duke University’s Clinical & Translational Science Awards grant 1UL2 RR024128-01 from the National Institutes of Health National Center for Research Resources.

Author information

Authors and Affiliations

Authors

Contributions

D.A.M., G.E.A. and J.H.S. jointly conceived and implemented the clinical study; D.A.M. and K.A.B. jointly designed early DC migration studies in mice. K.A.B. conceived and designed the remainder of the mouse research; A.D., A.H.F., H.S.F., R.E.M., D.A.R., J.J.V., D.D.B. and J.H.S. were responsible for provision of clinical study resources, materials and patient access. S.K.N., E.A.R. and G.E.A. prepared human samples and conducted human in vitro experiments; K.A.B. performed all preclinical experiments and patient analyses; M.-N.H. provided additional support for preclinical experiments. D.A.M., K.A.B., M.D.G., K.L.C., E.A.R., G.E.A. and J.H.S. performed data analysis and interpretation; J.E.H. and A.C. provided statistical support for design and analysis of human and mouse studies. D.A.M., D.D.B. and J.H.S. contributed laboratory reagents and tools; and D.A.M., K.A.B., M.D.G., K.L.C. and J.H.S. wrote the paper. All authors gave their final approval to the manuscript.

Corresponding authors

Correspondence to Duane A. Mitchell or John H. Sampson.

Ethics declarations

Competing interests

D.A.M., K.A.B. and J.H.S. have filed provisional patents related to the use of Td pre-conditioning as a method to improve immunization efficacy. D.A.M. has served as a paid member of the Schering Plough North American Investigators Advisory Board. S.K.N. is a co-inventor on a patent that describes the use of DCs transfected with tumour antigen encoding RNA that has been licensed by Argos Therapeutics through Duke University. S.K.N. has no financial interests in Argos Therapeutics and is not compensated by Argos Therapeutics. D.A.R. has served as paid speaker for Schering/Merck and Genentech/Roche. The remaining authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Schema of clinical trial.

SPECT/CT, single photon emission computed tomography/computed tomography; TMZ, temozolomide; XRT, external beam radiotherapy.

Extended Data Figure 2 Recall responses induced by other CD4+ T-cell-dependent protein antigens increase DC migration to VDLNs.

Primary immunization and vaccine site pre-conditioning with CD4+ T-cell-dependent protein antigens increase DC migration to VDLNs. Mice were immunized with either Haemophilus b conjugate (Hib) or pneumococcal 13-valent conjugate (PCV) intramuscularly, and 2 weeks later received vaccine site pre-conditioning with the recall antigen (recall) or saline (control). A separate cohort of mice received saline only throughout the immunization schedule (saline). Scatter plot shows biological replicates of individually processed right and left iLN per mouse (4 mice per group). Percentage migration of RFP+ DCs to VDLNs; one-way ANOVA, P < 0.0001; post-hoc Tukey t-test, PCV control versus recall, P < 0.05, Hib control versus recall, P < 0.05. Representative of n = 3 experiments; mean ± s.e.m.

Source data

Extended Data Figure 3 Bilateral migration of OVA-DCs after Td pre-conditioning or Td-activated CD4+ T-cell transfer.

Uptake of injected DCs to right and left iLNs 48 h after DC vaccination in Td-immune mice receiving Td pre-conditioning or naive mice administered Td-activated CD4+ T cells. Scatter plot shows biological replicates of individually processed right and left iLN per mouse (5 mice per group). CD4act ipsilateral versus contralateral, paired t-test, P = 0.41. Representative of n = 4 experiments; mean ± s.e.m.

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Extended Data Figure 4 Unilateral pre-conditioning with unpulsed DCs or TNF-α results in increased DC homing to ipsilateral draining inguinal lymph nodes.

Td-immune mice pre-conditioned with Td or saline before administration of OVA RNA-pulsed DC vaccine. Separate cohorts of naive mice received either 1 × 106 unpulsed DCs or 30 ng TNF-α on one side of the groin 24 h before the bilateral RFP+ DC vaccine. DC migration was quantified 24 h after vaccination. a, DC migration to ipsilateral lymph nodes (one-way ANOVA, P = 0.0018; post-hoc Tukey t-test, saline i.d. versus Td i.d., P = 0.007; saline i.d. versus TNF-α, P < 0.05; saline i.d. versus DCs, P < 0.05; Td i.d. versus TNF-α, P = 0.042; DCs versus Td i.d. and DCs versus TNF-α, P > 0.05. b, DC migration to contralateral lymph nodes; one-way ANOVA, P = 0.003; post-hoc Tukey t-test, saline i.d. versus DCs or TNF-α, P > 0.05; Td i.d. versus TNF-α, DCs or saline i.d., P < 0.05. n values are biological replicates of individually processed right and left iLNs per mouse (4 mice per group). Representative of n = 3 experiments; mean ± s.e.m.

Source data

Extended Data Figure 5 Serum cytokine and chemokine profile after Td pre-conditioning in patients and mice.

a, Serum cytokine panel of patients following vaccine site pre-conditioning with Td or unpulsed DCs; Wilcoxon rank sum, IFN-γ and IL-4, P < 0.05 (n = 6 patients). b, Similar to a in mice; Wilcoxon rank sum, all comparisons, P > 0.05 (Td recall n = 5, non-Td n = 6). c, Patient serum chemokines after vaccine site pre-conditioning. Patient CCL2 and CCL3 in Td recall (Td, n = 6) versus non-Td (unpulsed DC, n = 5); one-way ANOVA and Wilcoxon rank sum, P < 0.05. d, Mouse CCL22, CCL7 and CCL3 in Td recall (Td, n = 8 mice) and non-Td (saline, n = 8 mice); one-way ANOVA and Wilcoxon rank sum, P < 0.05. For ad, individual values represent biological replicates; mean ± s.e.m.

Source data

Extended Data Figure 6 Td vaccine site pre-conditioning results in CCL3 upregulation in Td-immune hosts.

a, CCL3 production in skin site after Td pre-conditioning (Td ipsilateral versus contralateral). Representative of four independent experiments. b, CCL3 production in skin after Td recall response. c, CCL3 induction at skin site is abrogated with previous host depletion of CD4+ T cells. Bars in ac represent CCL3 protein detected in skin sites from n = 2 mice with n = 2 technical replicates performed per mouse. d, CCL3 remains increased at the Td pre-conditioning site in the skin after DC vaccination (24, 48 and 72 h, one-way ANOVA, P = 0.0001, Td plus Td i.d. and OVA-DC versus Td plus saline i.d. and OVA-DC, and saline plus saline i.d. and OVA-DC, P < 0.05, post-hoc Tukey t-test). Individual values represent biological replicates from n = 4 mice; mean ± s.e.m.

Source data

Extended Data Figure 7 Migratory DC subsets in wild-type and Ccl3−/− mice after induction of Td recall responses.

Both wild-type and Ccl3−/− mice were first immunized with Td and then challenged with Td pre-conditioning. Migration of endogenous DC subsets to inguinal lymph nodes contralateral to the site of Td pre-conditioning was assessed at days 4 and 8 after Td administration. a, Gating strategy used to quantify DC subsets in inguinal lymph nodes after skin pre-conditioning with Td. LC, Langerhans cells; MoDC, monocyte-derived DCs. b, Day-8 migration of LC population to non-draining inguinal lymph nodes in Ccl3−/− hosts is reduced in absence of CCL3; two-sample t-test, P = 0.046. Representative of three experiments. Individual values represent biological replicates from n = 4 mice; mean ± s.e.m.

Source data

Extended Data Figure 8 Anti-tetanus toxoid memory responses are induced and maintained in wild-type and Ccl3−/− mice throughout Td priming and boosting.

Wild-type and Ccl3−/− mice primed and boosted with Td. Serum from immunized mice was collected 2 weeks after each immunization before the next booster vaccine (for each boosting phase, wild-type versus Ccl3−/−, two-sample t-test, P > 0.05). i.m., intramuscular. Scatter plot showing averaged values from n = 4 mice with n = 2 technical replicates performed per mouse. Representative of three experiments; mean ± s.e.m.

Source data

Extended Data Figure 9 CCL21 levels in Td pre-conditioning skin sites and draining lymph nodes of wild-type and Ccl3−/− mice.

a, CCL21 levels in skin site of Ccl3−/− hosts after induction of Td recall response and CCL3 administration. Mixed model accounting for within-mouse correlation of measurements, F-test, P < 0.001; pairwise comparisons, WT Td plus Td i.d. (n = 4) versus Ccl3−/− Td plus Td i.d. (n = 3), P = 0.049; Ccl3−/− Td plus Td i.d. and CCL3 i.v. (n = 4) versus Ccl3−/− Td plus Td i.d. and versus Ccl3−/− plus CCL3 i.v. (n = 4), P = 0.044 and P = 0.0045, respectively. Scatter plot shows averaged values with n = 2 technical replicates performed per mouse. b, Bilateral iLN CCL21 levels in wild-type mice after Td recall with skin site pre-conditioning. Bars represent CCL21 protein within iLN ipsilateral and contralateral to the side of Td pre-conditioning from n = 2 mice with n = 2 technical replicates performed per iLN. Representative of three experiments. c, Increased lymph node CCL21 in Ccl3−/− hosts after CCL3 reconstitution and induction of Td recall response (all two group comparisons). CCL21 levels in bilateral iLNs of Ccl3−/− hosts after induction of Td recall response and CCL3 administration. Mixed model accounting for within-mouse correlation of measurements, F-test, P < 0.001; pairwise comparisons, WT Td plus Td i.d. (n = 4 iLNs) versus Ccl3−/− Td plus Td i.d. (n = 3 iLNs), P = 0.045; Ccl3−/− Td plus Td i.d. and CCL3 i.v. (n = 4 iLNs) versus Ccl3−/− Td plus Td i.d. and versus Ccl3−/− plus CCL3 i.v. (n = 4 iLNs), P = 0.0066 and P = 0.026, respectively. Scatter plot shows averaged values with n = 2 technical replicates performed per lymph node sampled. In a and c, mean ± s.e.m.

Source data

Extended Data Table 1 Clinical trial patient characteristics

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Mitchell, D., Batich, K., Gunn, M. et al. Tetanus toxoid and CCL3 improve dendritic cell vaccines in mice and glioblastoma patients. Nature 519, 366–369 (2015). https://doi.org/10.1038/nature14320

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