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Abstract
Background Radiotherapy (RT) in combination with CTLA-4 inhibition (CTLA4i) can expand and activate T-cells to reject tumors in both mice and some patients with tumors unresponsive to CTLA4i alone.1 2 However, only a subset of patients achieves long-term control of metastatic disease. Similar responses to RT+CTLA4i are seen in the 4T1 mouse model of triple negative breast cancer (TNBC), making it an ideal model to interrogate the interaction between RT and CTLA4i, and identify barriers to its effectiveness.
Methods Mice were inoculated in one or both flanks with 4T1 cells. In some experiments one tumor was removed for analysis before start of treatment with RT (3 × 8 Gy) and/or anti-CTLA-4 antibody (9H10, 3 × 200 ?g i.p.). The intratumoral T cell response was assessed using bulk and single cell RNA/TCR sequencing. The METABRIC dataset3 was used to associate gene expression signatures with patient survival. In some experiments, RT+CTLA4i was combined with PD-1, LAG-3, or CD40 Abs.
Results RT, alone and with CTLA4i, increased the TCR repertoire clonality and activated T cells density in the tumors (figure 1A-G). In untreated tumors, Gzmb+Prf1+Lag3+Pd1+Cd8+ T cells (cluster 0) were most common. CTLA4i ‘unlocked’ Ifng+Cd40lg+ Cd4+ T cells (cluster 2) while RT favored expansion/persistence of Cd8+ T cell clusters. In tumors of mice treated with RT+CTLA4i activated Treg cells (cluster 1) were decreased and Ifng+Cd40lg+Cd4+ T cells (cluster 2) increased. Relatively among CD8+ T cells, Ifng+Tnf+Cd8+ (cluster 4) was expanded at the expense of cluster 0 (figure 2A-F). Gene signatures defining clusters 0, 2, and 4 were associated with improved survival in the METABRIC TNBC patient cohort using a multivariate model (figure 2G-H). In mice, AH1-tumor antigen-specific CD8+ T cells occupied different transcriptional states, with a shift to cluster 4 in mice treated with RT+CTLA4i (figure 2I), suggesting that multiple functional T cell states are required for tumor rejection. Based on the T cell phenotypes expanded by RT+CTLA4i, antibodies to PD-1, LAG-3, and CD40 were tested for the ability to enhance RT+CTLA4i therapy. Only CD40-agonist improved significantly tumor control (figure 3A-B).
RT, alone and with CTLA4i, increased the TCR repertoire clonality and density of activated T cells in the tumors. (A) Design of the experiment enabling collection of pre- and post-treatment (pre-tx and post-tx) 4T1 tumor tissue that was analyzed using RNA- and TCR-sequencing. (B) Tumor growth curves. Statistical significance in tumor volume growth between groups was determined using 2-way repeated measures ANOVA between day 15–21 and t-test at day 21. (C) Shannon clonality of paired pre- and post-tx TCR repertoires. Pairwise and paired t-tests were used to evaluate statistical significance of differences between and within groups, respectively. (D) RNA-seq based gene expression heatmap of selected canonical T cell markers in post-tx tumors. (E) Linear regression between Cd3e and Cd4 or Cd8 gene expression in post-tx tumors. R2 and p indicate R-square and p-value for the models, respectively (F) Ingenuity Pathway Analysis Canonical Pathway and (G) Upstream Regulation analysis. Z-scores indicate predicted activation (> 2) or inhibition (< -2) of pathways and upstream regulators. (all panels) *, **, and ***, and #, ## and ### indicate p-values of pairwise and paired statistical tests, respectively. Tukey’s and Holm’s method for adjusting p-values corrected for multiple comparison was used for the ANOVA and t-tests, respectively. (Abbreviations) tx, treatment; RT, radiation therapy; CTLA4, CTLA-4 Ab therapy; TCR, T cell receptor
RT+CTLA-4i increased tumor infiltration by Gzmb+Prf1+Lag3+Pd1+Cd8+, Ifng+Cd40lg+Cd4+, and Ifng+Tnf+Cd8+ T cells in 4T1 tumors. (A) Design of experiment enabling single cell analysis of T cells infiltrating 4T1 tumors. (B) Based on gene expression levels, the T cells were divided into 17 clusters (indicated by colors) and visualized in 2D using UMAP dimensionality reduction algorithm. (C) Gene expression levels of selected high-level T cell markers. (D) Table with main phenotype, key genes representative for each cluster, and the distribution of T cells from each condition falling into the different clusters. (E) Proportion of Cd4+ and Cd8+ T cells for the different treatment groups. (F) The expression of cluster-specific gene signatures in bulk 4T1 tumors for clusters 0, 2, and 4. (G) Survival curves and (H) multivariate analysis of the association between survival and enrichment of the gene signatures of clusters 0, 2, and 4. (I) The positioning of the all AH1-dextramer+ Cd8+T cell clones within the UMAP plot. Color annotate cluster. (Abbreviations) tx, treatment; RT, radiation therapy; Untr., untreated; CTLA4, CTLA-4 Ab; TCR CDR3, T cell receptor complementary determining region 3; UMAP, Uniform Manifold Approximation and Projection for dimension reduction; AH1, tumor antigen in 4T1 tumors; SPSYVYHQF peptide derived from gp70 and restricted to H2-Ld
Agonistic CD40 treatment improves RT+CTLA-4 therapy. Individual tumor growth curves for untreated, RT+CTLA-4, or RT+CTLA-4+CD40 treated mice. Color annotate group. *, **, ***, and **** indicate p-values < 0.05, 0.01, 0.001, and 0.0001, respectively, calculated using a linear mixed-effects model. (A) and (B) represent two individual experiments. (Abbreviations) RT, radiation therapy; CTLA4, CTLA-4 Ab; CD40, anti-CD40 Ab; mm3, cubic millimeter; d, days
Conclusions Altogether, these results revealed that RT and CTLA4i have complementary effects and besides driving T cells into tumors shape CD4 and CD8 T cell functional differentiation towards subsets that are associated with improved survival in patients. Unexpectedly, inhibition of checkpoint receptors expressed by a large CD8 T cells cluster did not further improve responses to RT+CTLA4i, whereas agonistic CD40 therapy did, suggesting new therapeutic strategies.
Acknowledgements Grant support: R01CA198533
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