Recipient-Type Leucocyte Infusion After Allohsct in Mice Slows Local Growth of Neuroblastoma and Elicits Activation of Intratumoral Cytotoxic NK Cells
Authors: Willems Leen , Rutgeerts Omer , Lenaerts Caroline , Van Gool Stefaan , Waer Mark , Billiau An 
Published: October 2019

Abstract
Abstract 4102
Donor leucocyte infusion (DLI) after alloHSCT can induce strong graft-versus-leukemia (GvL) effects, inspiring investigators to examine this approach for solid tumors resistant to conventional therapies (Grivas et al. Curr Clin Pharmacol. 2011). However, DLI produces graft-versus-host disease (GvHD). Recipient-type leucocyte infusion (RLI) is currently being explored clinically as a means to induce GvL without risk of GvHD (clinicaltrials.gov; Rubio et al. Blood 2003; De Somer et al. Haematologica. 2011). High-risk neuroblastoma carries a bleak prognosis despite aggressive treatment with chemo-, radiotherapy and autologous HSCT. Clinical observations in such patients suggest that alloHSCT may produce a graft-vs-neuroblastoma effect (Kanold et al. Bone Marrow Transplantation. 2008). In mice, alloHSCT delays local neuroblastoma growth, and adoptive transfer of tumor-pulsed donor dendritic cells and donor leucocytes enhances this effect (Ash et al. Cancer Immunol Immunother. 2009, Br J Cancer 2010). In this study, we show that not only DLI by itself, but also RLI enhances the local anti-neuroblastoma effect of alloHSCT in mice.
MHC-mismatched [C57BL/6 (H-2Kb) → A/J (H-2Kk)] bone marrow chimeras were given a subcutaneous inoculation with 1 × 106 Neuro2A cells (A/J neuroblastoma) on day 14 post HSCT. On day 21, we performed adoptive transfer of 10 × 106 donor splenocytes (DLI), 50 × 106 recipient splencoytes (RLI) and/or 1 × 106 recipient-type MACS-isolated DX5+ NK cells. We measured tumor volume twice weekly using a caliper (volume = width2 × length × 0,4). Validation of this model showed progressive tumor growth and mortality as a result of metastasis. Peripheral blood chimerism and tumor infiltrating lymphocytes were studied using flow cytometry.
AlloHSCT chimeras developed mixed donor T cell chimerism by day 21 and full donor chimerism by day 76 post HSCT. DLI induced a conversion to full donor T cell chimerism and RLI induced a complete loss of donor T cells chimerism, both within 1 week. AlloHSCT chimeras showed reduced local growth of subcutaneous neuroblastoma tumors relative to synHSCT chimeras. This delay in tumor growth was enhanced not only by DLI, but also by RLI; DLI provoked lethal GvHD whereas mice treated with RLI remained healthy. Within tumor-infiltrating lymphocytes, T and NK cell chimerism mirrored the systemic chimerism changes seen after RLI and DLI, associated with an increased intratumoral CD8/CD4 ratio, CD8+ T-cell IFN-γ-expression and NK-cell Granzyme B-expression. This indicates a close relation between lymphohematopoietic alloreactivity and the anti-tumor effect, and suggests that the anti-tumor mechanism of DLI and RLI involves not only CD8+ T cells but also cytotoxic NK cells. The baseline antitumor effect seen in alloHSCT chimeras was also accompanied by increased Granzyme B expression by intratumoral NK-cells, supporting a role for NK cells also in this baseline antitumor effect. In vivo Neuro2A-inoculation experiments in (poly(I:C)-treated) C57BL/6, TCR−/− C57BL/6 and A/J mice, and in vitro NK cytotoxicity experiments showed that 1° donor T cells critically resist Neuro2A cells, 2° donor NK cells exhibit spontaneous cytotoxic reactivity to Neuro2A that is enhanced by NK activation, but also that 3° syngeneic NK cells may acquire reactivity to Neuro2A cells provided they are activated. Interestingly, when RLI was given, the intratumoral NK-cell frequency declined markedly, and adoptive transfer of additional NK cells obtained from naïve A/J mice enhanced the local antitumor effect of RLI. This supports the hypothesis that, in addition to donor NK cells, also syngeneic NK cells can play a critical role in mediating a growth-limiting effect on local neuroblastoma. Along this line, we observed that also in synHSCT mice, which showed a reduced local neuroblastoma growth relative to naïve mice, the intratumoral NK cells showed increased FasL-expression.
These are the first experimental data showing that RLI after alloHSCT may induce immune-mediated anti-neuroblastoma effects, and that NK cells, in particular syngeneic NK cells, may play a critical role herein. Our data support the exploration of post alloHSCT adoptive cell therapy with recipient-type T and NK cells to produce enhanced immune antitumor effects for high-risk neuroblastoma, and potentially for other solid tumors that are unresponsive to traditional therapies, without the risk of GvHD.

Disclosures:
No relevant conflicts of interest to declare.