Elective Nodal Irradiation Attenuates the Combinatorial Efficacy of Stereotactic Radiation Therapy and Immunotherapy

Abstract

Purpose: In the proper context, radiotherapy can promote antitumor immunity. It is unknown if elective nodal irradiation (ENI), a strategy that irradiates tumor-associated draining lymph nodes (DLN), affects adaptive immune responses and combinatorial efficacy of radiotherapy with immune checkpoint blockade (ICB).Experimental Design: We developed a preclinical model to compare stereotactic radiotherapy (Tumor RT) with or without ENI to examine immunologic differences between radiotherapy techniques that spare or irradiate the DLN.Results: Tumor RT was associated with upregulation of an intratumoral T-cell chemoattractant chemokine signature (CXCR3, CCR5-related) that resulted in robust infiltration of antigen-specific CD8⁺ effector T cells as well as FoxP3⁺ regulatory T cells (Tregs). The addition of ENI attenuated chemokine expression, restrained immune infiltration, and adversely affected survival when combined with ICB, especially with anti-CLTA4 therapy. The combination of stereotactic radiotherapy and ICB led to long-term survival in a subset of mice and was associated with favorable CD8 effector-to-Treg ratios and increased intratumoral density of antigen-specific CD8⁺ T cells. Although radiotherapy technique (Tumor RT vs. ENI) affected initial tumor control and survival, the ability to reject tumor upon rechallenge was partially dependent upon the mechanism of action of ICB; as radiotherapy/anti-CTLA4 was superior to radiotherapy/anti-PD-1.Conclusions: Our results highlight that irradiation of the DLN restrains adaptive immune responses through altered chemokine expression and CD8⁺ T-cell trafficking. These data have implications for combining radiotherapy and ICB, long-term survival, and induction of immunologic memory. Clinically, the immunomodulatory effect of the radiotherapy strategy should be considered when combining stereotactic radiotherapy with immunotherapy. Clin Cancer Res; 24(20); 5058-71. ©2018 AACR.

Figure 1. A preclinical model of image-guided elective nodal irradiation (ENI) accurately targets the tumor-associated DLN

(A) Lymphatic tracking of fluorescent dye (IRDye® 800CW PEG contrast agent) following intratumoral administration on day 11 in tumor-bearing C57BL/6J mice inoculated by s.c. or i.d. flank injection with 5×10⁵ B16F10 or 1.5×10⁶ MC38 cells (n=2-3 mice per experiment, repeated twice). (B) Schematic of intratumoral dye injection, transit and accumulation in ipsilateral tumor-associated inguinal lymph node (DLN). (C) Representative images of fluorescent dye-guided surgery isolating ipsilateral inguinal DLN and coronal view cone-beam CT image with crosshairs overlying inguinal fat pad. (D) Immunofluorescence detection (63X magnification) of phospho-H2AX^ser139 (green) and DAPI (blue) nuclear staining in non-irradiated (control) and irradiated mice (Tumor RT or T+LN RT). Tumor and DLN tissue harvested 1 hour after 12Gy x1. (E) Representative H&E stain (4-20X magnification) of inguinal DLN harvested from untreated B16F10 tumor-bearing mouse (n=3) on day 14 post-inoculation depicting absence of tumor cells.

Figure 2. Elective nodal irradiation decreases tumor-infiltrating immune cell density relative to tumor-only stereotactic RT

Flank-tumor bearing mice untreated or irradiated (12Gy x1) to tumor-only (Tumor RT) or tumor and DLN (T+LN RT) on day 11 following s.c. injection of 1.5×10⁶ MC38 tumor cells (n=4-9 mice per group, repeated 2-3 times). Tumors were harvested for analysis on day 16, 120 hours after treatment. (A-C) Representative quantitative scatterplot and flow cytometry demonstrating absolute number tumor-infiltrating immune cells and percentage (CD8+ effector, CD44+ CD62L- CD8+; Treg, CD4+ FoxP3+; myeloid derived suppressor cells, CD11b+ Gr-1^hi) per gram tumor in MC38 tumor model. (D) Fold change of CD8 effector and Treg subsets by treatment group, normalized to control group. Quantitative bar graph of CD8 effector-to-Treg ratio by treatment group. Error bars represent SEM, ***: p < 0.001, **: p < 0.01, *: p < 0.05, treatment group comparisons by one-way ANOVA and post-hoc Tukey’s multiple comparison test.

Figure 3. Tumor RT and T+LN RT have distinct radiation-induced intratumoral chemokine expression and CD8+ T-cell trafficking patterns

(A) Tumor-associated DLN of MC38 tumor-bearing mice harvested at 1hr, 48hrs and 120hrs after treatment with Tumor RT or T+LN RT on day 11. Quantitative bar graphs representing fold change of absolute number of CD4+, CD8+ and CD11b+ immune cell subsets in tumor-associated DLN over time, normalized to control (n=12 mice per group with 4 mice per time point, repeated twice). (B) Tumor lysate collected at same post-RT timepoints as Fig. 3A and analyzed by Luminex® multiplex immunoassay. Colorimetric heat map conditional formatting with blue to red indicating low to high chemokine/cytokine concentration [pg/mL], respectively. Error bars represent SEM, ***: p < 0.001, **: p < 0.01, *: p < 0.05, CD45+ immune cell and chemokine expression time course experiments analyzed by two-way ANOVA and post-hoc Tukey’s multiple comparison test.

Figure 4. Radiation-mediated tumor infiltration with functional antigen-specific CD8+ T-cells is restrained by elective nodal irradiation

2×10⁶ CFSE-labeled, OVA-specific CD8+ T-cells from donor Rag^−/−/OT-1 TCR transgenic CD45.2 mice were adoptively transferred (AT) into MC38-OVA tumor-bearing congenically-mismatched CD45.1 C57BL/6J mice on day 13. CD45.1 mice were treated on day 11 (48 hours before AT) with control, Tumor RT and T+LN RT with DLN and tumor harvested on day 16, 120 hours after RT (AT, n=5-9 mice per group, repeated 3 times). (A) Quantitative scatter plots of effector cytokine production by tumor-infiltrating OT-1, endogenous CD8+ T-cells via intracellular cytokine staining and ELISA (upper rows, L to R respectively). (B) Absolute number of tumor-infiltrating CD8+ CD44+ T-cells per gram tumor by H-2kb OVA (SIINFEKL) tetramer staining and representative flow cytometry plots (ELISA and tetramer staining, n=5 per group, repeated twice). (C) Bar graphs and representative flow plots of effector cytokine production (IFNɣ, TNFα) by AT CD45.2+ CD8+ T-cells in tumor-associated DLN. Error bars represent SEM, ***: p < 0.001, **: p < 0.01, *: p < 0.05, treatment group comparisons by one-way ANOVA and post-hoc Tukey’s multiple comparison test.

Figure 5. Elective nodal irradiation attenuates combinatorial efficacy between radiation and immunotherapy

(A) Treatment schema; Tumor RT or T+LN RT (12Gy x1) administered on day 11 and three doses (i.p. 200μg) of therapeutic antibody (isotype, αPD-1, αCTLA4) on days 10,12,14 in MC38-OVA tumor-bearing mice (n=6-7 per group, repeated twice). (B) Spider plots of tumor outgrowth (mm³) by treatment group, annotated with number of complete responses (CR) over total mice treated. (C) Percent survival by treatment group at day 90 after s.c. flank injection of MC38-OVA tumor cells. (D) Summary table of % CR and median survival by treatment corresponding to (B-C). Kaplan-Meier analysis with log-rank (Mantel-Cox) test for survival differences between treatment groups. ***: p < 0.001, **: p < 0.01, *: p < 0.05.

Figure 6. Favorable modulation of intratumoral CD8 effector-to-Treg ratio is associated with long-term survival

MC38-OVA tumor-bearing mice treated per schema in Fig. 5A, tumors harvested day 21 after s.c. flank injection (n=5 per group). (A-B) Representative flow cytometry (left, CD8 effectors; right, Tregs) and (C) quantitative scatterplots (upper and middle panels), demonstrating percentage and absolute number tumor-infiltrating immune cells per gram tumor by treatment group, respectively. (C) Quantitative bar graph (bottom panel) of CD8 effector-to-Treg ratio by treatment group. Error bars represent SEM, ***: p < 0.001, **: p < 0.01, *: p < 0.05, treatment group comparisons by one-way ANOVA and post-hoc Tukey’s multiple comparison test.

Figure 7. Improved resistance to re-challenge in animals treated with RT + αCTLA4

MC38-OVA tumor-bearing mice treated per schema in Fig. 5A. Tumors harvested day 21 after s.c. flank injection (n=5 per group). (A) Absolute number of antigen-specific tumor-infiltrating CD8+ CD44+ T-cells per gram tumor by H-2kb OVA (SIINFEKL) tetramer staining. (B) Measurement of cytokine concentration [pg/mL per gram tumor] by ELISA in TIL-derived supernatant. (C) Long-term survivors with complete responses previously treated with combined RT and ICB were re-challenged on the contralateral flank with 1.5×10⁶ MC38-OVA tumor cells 180 days after initial tumor s.c. implant; spider plots, % survival and % tumor clearance stratified by prior RT strategy or prior ICB received (treatment-naïve, n=5; Tumor RT, n=11 vs. T+LN RT, n=9; αPD-1, n=8 vs. αCTLA4, n=15). Error bars represent SEM, ***: p < 0.001, **: p < 0.01, *: p < 0.05, treatment group comparisons by one-way ANOVA and post-hoc Tukey’s multiple comparison test.