T cell immunotherapies engage neutrophils to eliminate tumor antigen escape variants

Abstract

Cancer immunotherapies, including adoptive T cell transfer, can be ineffective because tumors evolve to display antigen-loss-variant clones. Therapies that activate multiple branches of the immune system may eliminate escape variants. Here, we show that melanoma-specific CD4⁺ T cell therapy in combination with OX40 co-stimulation or CTLA-4 blockade can eradicate melanomas containing antigen escape variants. As expected, early on-target recognition of melanoma antigens by tumor-specific CD4⁺ T cells was required. Surprisingly, complete tumor eradication was dependent on neutrophils and partly dependent on inducible nitric oxide synthase. In support of these findings, extensive neutrophil activation was observed in mouse tumors and in biopsies of melanoma patients treated with immune checkpoint blockade. Transcriptomic and flow cytometry analyses revealed a distinct anti-tumorigenic neutrophil subset present in treated mice. Our findings uncover an interplay between T cells mediating the initial anti-tumor immune response and neutrophils mediating the destruction of tumor antigen loss variants.

Keywords: CTLA-4; OX40; adoptive T cell therapies; anti-tumor neutrophils; antigenic heterogeneity; immune checkpoint blockade; immunotherapy; neutrophil extracellular traps; neutrophils; tumor hetergeneity.

Figure 1. Potent immunotherapies can eliminate antigen escape variants.

A) tSNE plots of transcripts of human melanoma antigens from 15 patients described in Tirosh et al. B) tSNE plots of melanoma cells of 3 individual patients showing TYRP1 levels. C) Melanoma biopsies (n=10) stained for TYRP1, S100, and DAPI. A Z-score was calculated using the fraction population of double positive TYRP1 and S100. Bar =100μm. D) Summary of a previously published experiment of a model of antigenically heterogeneous tumor. Green check marks are long term survival of the applied therapy while red X marks are cohorts that succumbed to tumor burden. E) Mice were implanted with B16:B78H1 mixture in the same flank and treated as described in the methods. Growth curves of individual mice over time Left and Kaplan-Meier Survival plots Right are shown. Inset numbers indicate the number of mice that succumbed to tumor burden. ***P < 0.001, ****P < 0.0001. Mouse experiments were repeated at least twice with equivalent results.

Figure 2:. Elimination of antigen escape variants does not require the adaptive immune system.

A) Mice were implanted with B16 cells. After 3 weeks, mice were injected with CTX. The next day mice were injected with Trp1-Luciferase (Trp1-Luc) cells and anti-OX40. Photon flux was calculated (p/s) on a selected region at the tumor site. Black line represents tumor size over time (mm²). Red line represents Trp1 infiltration over time. Symbols represent mean ± s.e.m. Representative images are shown. B) WT or MHC Class II KO mice were implanted with B16:B78H1 mixture. After 3 weeks, mice with injected with CTX, the next day, the mice were treated with Trp1 cells and anti-OX40. Cohorts of mice were injected 3X weekly with anti-MHC class II antibody. Growth curves of individual mice are depicted. C) Mice were treated as described. Three or 13 days after treatment, cohorts of mice received anti-CD4 antibody 3X weekly. Growth curves of tumors of individual mice are depicted. D) WT or Rag^−/− mice were treated as described. Growth curves of individual mice are shown. Inset numbers indicate the number of mice that succumbed to tumor burden. Experiments were repeated at least twice with equivalent results.

Figure 3:. Combination therapy increased activated neutrophils in the tumor.

Mice (5/group) were implanted with B16 cells and treated as described. After 10 days, tumor extracts were prepared and subjected to cytokine analysis. A) Volcano plot showing log fold change (FC) in cytokines concentration relative to the control vs. p-values (P). Dotted line represents P =0.05. B) Mice (5/group) were implanted with B16 cells. After 3 weeks, mice were treated as described. After 10 days, tumors were dissected and analyzed by Immunohistochemistry. Representative slides showing H&E, CD11b, and Ly6G staining. Bar= 80μm. Box-and-whisker plots are shown. 4–5 representative high-power fields from 5 tumors per condition were analyzed. Plots represent %CD11b area and Ly6G+ cells/mm². C) Mice (3–4/group) were treated as described. Single cell suspension was prepared from tumors at day 10 and analyzed by flow cytometry. Representative flow plots are shown for events gated on live CD45⁺CD11b⁺Ly6G^highLy6C^mid. Percentage of neutrophils/CD45+ cells ± s.e.m are quantified per treatment. Each symbol represents a mouse. D) Mice were treated as described. Left, representative micrographs of IgG and anti-OX40 treated mice groups where NETs are indicated by yellow arrows and MPO+ neutrophils are indicated with white arrows. Bar= 50μm Right, NETs were quantified by integrated intensity of Cit-H3 and MPO and the ratio of CitH3 to MPO was calculated. Box-and-whisker plots are shown. Two-five representative high-power fields from 5 different tumors per condition were analyzed. **P < 0.01, ***P < 0.001, ****P < 0.0001. Experiments were repeated at least twice with equivalent results.

Figure 4:. Neutrophils kill antigen escape variants.

A) Mice (5/group) were implanted with B16:B78H1 mix and treated as described. Neutrophils were cocultured with B78H1 cells in the presence of tumor extracts for 18h. B78H1 killing was measured by clonogenic assay at 1:1 target:effector ratio Left and flow cytometry measuring active caspase 3/7 at 1:10 target:effector ratio Right. Average ± s.e.m. was calculated from 4–6 different wells per condition. B) Trp1 cells were activated in culture with peptide and irradiated APCs. The next day, anti-OX40 or IgG was added to the cultures. After 6 days, the Trp1 cells were cocultured with B16 cells in a transwell assay at 1:10 target:effector ratio. On the top chamber, naïve neutrophils were cocultured with B78H1 for 16h. B78H1 killing was analyzed by flow cytometry. A diagram of the experimental setup is shown on the Left. Graphs represent the average ± s.e.m. % of active caspase 3/7 of 4 different wells per condition Right. C) Mice were treated as described. Cohorts of mice were injected with anti-Ly6G 3X weekly starting at day 3. Growth curves of individual mice Left, and Kaplan-Meier survival curves pooled from 2 independent experiments are depicted Right. Inset indicate the number of mice that succumbed to tumor burden. **P < 0.01, ***P < 0.001, ****P < 0.0001. Experiments were repeated at least twice with equivalent results.

Figure 5:. Tumor neutrophils from the triple combination therapy show a distinct transcriptional phenotype.

A) Mice were implanted with B16:B78H1 mix and treated as described. After 10 days, single-cell suspensions were prepared from tumors. Live⁺CD45⁺Ly6G⁺ cells were FACS-sorted, bulk RNA was prepared, sequenced, and analyzed. Left, PCA analysis of neutrophils from each mouse per treatment. Right, volcano plots depicting up and down-regulated genes per treatment. Lines are FC (Fold change) =2 and P (P-value) =0.05). B-D) Mice were implanted with B16:B78H1 mix and treated as described. A separate cohort of mice was implanted with B16:B78H1 mix a week later as untreated controls. After 10 days, single-cell suspensions were prepared from tumors. Live CD11b⁺CD45⁺ were sorted and analyzed by scRNA-seq as described in the methods. B) UMAP of each group showing different clusters. C) Heat map showing top genes expressed by each cluster (Refer to Supplemental Table 1). D) Detection of selected proteins derived from CITE-seq analysis on different regions of the UMAP.

Figure 6:. Tumor neutrophils from treated mice acquired a more mature anti-tumorigenic phenotype.

A-F) Analysis of scRNA-seq comparing IgG and anti-OX40 groups. A) UMAP from IgG and anti-OX40 groups are depicted Left. Pie charts showing the proportion of each cluster per treatment and the number of cells analyzed Right. B) Heat map showing top genes expressed by each cluster (Refer to Supplemental Table 2). C) Dot plots of selected enriched terms from GO overrepresentation analysis. The circle represents the counts of enriched genes in the signatures. P value is shown in the legend (refer to Supplemental Table 3). D) Expression of Cxcr2 and Cxcr4 transcripts projected into the described UMAP. E) Expression of selected GO or published gene signatures projected into the described UMAP. F) Cell trajectory and pseudotime inference for the neutrophil populations. G) Heatmaps of pseudo-temporally expressed genes in each curve showing the top 50 most variable genes in each lineage. H-I) Mice were treated as described and single cell suspensions were analyzed by flow cytometry (refer to Supplemental Figure 5B). H) Log fold change of median fluorescence intensity relative to the average for each marker of gated neutrophils pooled from 3 independent experiments. Differences between markers were P<0.05 or lower. I) Flow plots and quantification of selected markers gated on neutrophils. Bars represent averages ± s.e.m. Symbols represent individual mice. J) WT or iNOS KO mice were implanted with B16:B78H1 mix and treated as described. Growth curves of individual mice Left, and Kaplan-Meier survival curves pooled from 2 independent experiments. Right. Inset numbers indicate the number of mice that succumbed to tumor burden. K) WT or iNOS KO mice (5 mice/group) were implanted with B16 tumors and treated as described. At day 10 after treatment, Ly6G+ neutrophils were FACS-sorted from pooled tumors. Neutrophils were co-cultured with B78H1 cell for 18h. Killing of B78H1 cells was measured by clonogenic assay. Average ±s.e.m. was calculated from 4–6 different wells per condition. *P < 0.05, P < 0.01, *** P < 0.001, **** P < 0.00001. Flow cytometry and mouse experiments were repeated at least twice with equivalent results.

Figure 7:. Anti-tumor neutrophils are important for response to immunotherapies.

A) Cohorts of mice were implanted with MC38. At day 7, the mice were treated as described. Cohorts of mice received anti-Ly6G. Left, Schema of the experiment. Middle, Tumor curves of individual mice over time. Inset numbers indicate the number of mice with regressed tumors. Right, Survival curves pooled from 2 independent experiments. B) Biopsies from melanoma patients that were treated or not with ICB were stained for NETosis. Left, Schema of the experiment. Middle, Representative images. Yellow arrows indicate NETs and white arrows indicates MPO+ neutrophils. Bars = 50μm. Right, Semiquantitative supervised analysis of NETs per biopsy. Each dot represents one biopsy. NET score (Y axis): - = no NETs, −/+ = 5–10 NETs in the entire biopsy, + = few, ++ = intermediate, +++ = abundant number of NETs. C) Cohorts of mice were implanted with MC38 tumors and treated as described. Cohorts of mice received antiLy6G. Left, Schema of the experiment. Middle, Tumor curves of individual mice over time. Inset numbers indicate the number of mice that completely regressed their tumors. Right, Survival curves pooled from 2 independent experiments. Mouse experiments were repeated twice. D) Bulk RNAseq data from melanoma tumor biopsies generated from patients treated with anti-PD-1 alone (CTLA-4 naive) or anti-PD-1 in combination with anti-CTLA-4 (CTLA-4 experienced). Using the 10 top expressed genes for each signature in Cluster 0 and Cluster 1 normalized z-scores of gene expressions were generated and unpaired two-sided T-tests was performed. Box plots and violin plots are shown for patients with disease progression or not. * P<0.05.