Peripheral CD8+ T cell characteristics associated with durable responses to immune checkpoint blockade in patients with metastatic melanoma

Abstract

Immune checkpoint blockade (ICB) of PD-1 and CTLA-4 to treat metastatic melanoma (MM) has variable therapeutic benefit. To explore this in peripheral samples, we characterized CD8+ T cell gene expression across a cohort of patients with MM receiving anti-PD-1 alone (sICB) or in combination with anti-CTLA-4 (cICB). Whereas CD8+ transcriptional responses to sICB and cICB involve a shared gene set, the magnitude of cICB response is over fourfold greater, with preferential induction of mitosis- and interferon-related genes. Early samples from patients with durable clinical benefit demonstrated overexpression of T cell receptor-encoding genes. By mapping T cell receptor clonality, we find that responding patients have more large clones (those occupying >0.5% of repertoire) post-treatment than non-responding patients or controls, and this correlates with effector memory T cell percentage. Single-cell RNA-sequencing of eight post-treatment samples demonstrates that large clones overexpress genes implicated in cytotoxicity and characteristic of effector memory T cells, including CCL4, GNLY and NKG7. The 6-month clinical response to ICB in patients with MM is associated with the large CD8+ T cell clone count 21 d after treatment and agnostic to clonal specificity, suggesting that post-ICB peripheral CD8+ clonality can provide information regarding long-term treatment response and, potentially, facilitate treatment stratification.

Figures

Extended Data Fig.1. Extended transcriptomic response to ICB

a, Comparison of differential induction of genes by cICB (log fold change y axis) and sICB (log fold change x-axis) at day 63 (n=46 paired samples, 35 sICB, 11 cICB). b, Pathways preferentially upregulated (NES score >0) or supressed by cICB versus sICB as identified by Gene Set Enrichment analysis depicted in Figure 1e,f

Extended Data Fig. 2. Module pathways

Pathway analysis was performed using Gene Ontology Biological Processes across treatment response associated modules. For 7/9 modules, transcripts within the modules were significantly associated with discrete processes, with limited overlap between modules. These modules (M1:M4, M6:M8) and all associated pathways for them (FDR

Extended Data Fig. 3. Module expression

Graphs demonstrate average gene expression per module for each sample with red boxplots and associated points representing cICB samples (n=20 baseline, n=16 D21, n=5 D63) and green boxplots and associated points representing sICB samples (n=56 baseline, n=41 D21, n=26 D63). Controls are untreated healthy volunteers (n=24). All statistically significant differences (Tukey adjusted P

Extended Data Fig. 4. TCR qPCR

Validation of MiXCR results using quantitative PCR. For n=13 individuals, 2 TC R α and 2 β chains were identified according to whether or not MiXCR reported significant expansio n on day 21 versus baseline. Primers were designed to the complementarity determining region 3 (CDR3) sequences for each chain and quantitative PCR performed on pretreatment day 0 PBMC cDNA and PBMC cDNA from day 21. Ct values were normalised to total expression of CD3E. a, Clones identified as expanding in the MiXCR CD8+ RNA were found to show significantly more expansion (median 3.48 fold, IQR 1.85-12.79) than non expanding (median1.32 fold, IQR 0.97-1.88, Wilcoxo n signed-rank Test (two-sided) P=3x10-8). Lower and upper hinge of box on boxplots represent 25-75th percentiles, central line the median and the whiskers extend to largest and smallest values no greater than 1.5x interquartile range. b, MiXCR fold change per clone from CD8 cells was highly correlated with that determined from bulk PBMCs using quantitative PCR (Pearson correlation, two-sided T-statistic).

Extended Data Fig. 5. Extended clone size analysis

a, Day 21 post ICB clonal diversity is similar in patients who have 6 month response versus those with disease progression by this timepoint (two-sided T-test, n=69). b, As per Figure 3a, but with unique clones defined by the beta chain, left panel two-sided Wilcoxon signed-rank Test (n=25 controls, 49 patients, right panel one-sided Wilcoxon signed-rank Test, n=43 controls, 20 patients). c, Threshold for clone size associating with outcome, x-axis indicates size of clone with test comparing number of clones above that size according to clinical outcome, y-axis: -log10(p-value) from test. The difference between responding patients and progressing patients being maximal at clone size of 0.5% (two-sided Wilcoxon test, n=69). d, Across all samples there is no association between number of clones growing on day 21 (P

Extended Data Fig. 6. Temporal stability of clones

a, Clones from 42 individuals with samples at 3 timepoints were identified at day 0 pretreatment and classified according to size. The corresponding correlation for the same clones between day 21 and day 63 was assessed (Pearson correlation, two-sided T-statistic all P0.01% for intermediate) at later timepoints, the values on top of bars represent percentage of day 0 recovery.

Extended Data Fig. 7. Effect of CMV

For a subset of cutaneous melanoma patients with day 21 data we were able to measure unequivocal CMV serology. a, CMV seropositivity is associated with a depletion of small clones (ratio 2% repertoire) (n=68 left panel, 53 right panel). b, Patients seropositive for CMV demonstrated significantly reduced CD8+ TCR diversity at day 21 (measured on TRB CDR3) (n=53, two-sided T-Test). c, Despite significant differences in diversity from CMV there was no difference in diversity between day 21 samples from progressing and responding patients (n=53, two-sided T-test). d, There is no association between CMV serology and number of large clones at day 21 (n=53, two-sided T-Test) For all boxplots lower and upper hinges of box represent 25-75th percentiles, central line the median and the whiskers extend to largest and smallest values no greater than 1.5x interquartile range.

Extended Data Fig. 8. Comparison of clonotypes to public clones

a, The complete dataset of clones were screened for public clonotypes for melanoma antigens, demonstrating that the size of clones matching these clonotypes in untreated melanoma patients is significantly greater than those in controls (Wilcoxon Test, n=106 patients, 68 controls) b, Melanoma patients showed no difference in mean EBV reactive clone size from controls (P>0.05) although the distribution of clones was skewed in non-melanoma patients and median clone size greater in patients (two-way Wilcoxon-Test). c, Treatment led to an small increase in median EBV reactive clonotype clone size across all patients, but d, the significance of this effect was greater for MAA clonotypes e, for samples with data for clone sizes at day 63 as well as day 21 (n=41 individuals) there was no further change in clone size at the later timepoint (two-way Wilcoxon-Test). For all boxplots lower and upper hinges of box represent 25-75th percentiles, central line the median and the whiskers extend to largest and smallest values no greater than 1.5x interquartile range.

Extended Data Fig. 9. Investigating flow correlates of clonal indices

a, for 72 samples from 19 patients, blinded flow cytometry data to assess CD8 subsets was integrated with the Shannon diversity index calculated for each sample. This demonstrated a strong positive association between TCM and diversity, whereas TEMRA was significantly anticorrelated with diversity (Pearson correlation, two way T-statistic). b, as per Figure 3e, except here large clone count was correlated with percentage CD4 subsets from each of the samples. Unlike for CD8 cells, there is no association between large clone count and percentage CD4 subset in the samples analysed (Pearson correlation, two way T-statistic).

Extended Data Fig. 10. Comparing MiXCR and 10X TCR data

a-h, for 8 patients, indicated by number, CD8+ cell samples were subject to 10X chromium single cell 5’ RNA sequencing providing T cell receptor sequencing and standard bulk sequencing (see methods). Clones were identified by their β chain and for each productive β chain identified the relative clonal proportion (frequency) was calculated. Clones were matched via the CDR3 amino acid sequence with β chains from the same samples mapped from bulk CD8+ cell RNA using MiXCR. Where the clone fell below detection limit in MiXCR a value of 0 was attributed. MiXCR clones were identified for 92.1% clones >0.1% population in 10X (6990/7597) and 99.4% clones >0.2% size (5816/5852). x-axis= 10X proportion, y axis MiXCR proportion, r calculated using Pearson correlation coefficient, all P0.5% total clonal population) identified from 10X and MiXCR approaches are correlated.

Figure 1. Transcriptomic response to ICB

1a) Transcripts differentially regulated between pre-treatment and 21 days post-sICB (n=40 paired samples, negative binomial Wald test, Benjamini Hochberg corrected P values); 1b) cICB 21 day response (n=15 paired samples, statistics as per a); 1c) log2 fold change effect of sICB (x-axis) versus cICB for all transcripts significantly regulated in either treatment at day 21, size of point indicates – PsICB/PcICB; 1d) summary of transcript number modulated by each treatment and genes differentially expressed comparing the response of cICB to sICB; 1e,1f) Rank based Gene set enrichment analysis of genes significantly more modulated by cICB identifies pathways preferentially induced or suppressed by combination treatment.

Figure 2. Identification of transcriptomic correlates of long term response

2a) Transcripts differentially regulated between responders and progressors with direction showing relative expression in responders (n=144 samples from 69 patients, 67 pre-treatment and 77 post-treatment, negative binomial Wald test, Benjamini Hochberg corrected P values); 2b) GOBP pathway analysis of genes preferentially up-regulated (blue) and down-regulated (red) in responders (hypergeometric test); 2c) Boxplots of the most differentially regulated TCR genes between responders and progressors (144 samples, P values are uncorrected negative binomial Wald test returned from Deseq2); 2d) Results from Fisher’s exact test of enrichment of up-regulated TCR encoding versus all transcripts demonstrating no enrichment of TCR encoding genes in those regulated by cICB, whereas both TRAV and TRBV encoding genes are highly enriched amongst those up-regulated in responders (dotted line: OR=1, error bars represent 95% confidence interval); 2e) representative example of day 0 vs. day 21 clones from one patient showing both chains with filled points representing clones showing significant change in frequency; 2f) number of clones increasing in size (P<0.05) was significantly greater in cICB patients (n= 15 cICB, 30 sICB, two-sided Wilcoxon signed-rank Test); 2g) Reactome pathway analysis of genes positively associated with number of clones growing at day 21 (n=54, d21 samples) demonstrated increase in clone size to be strongly linked to expression of genes involved in mitosis (one-sided hypergeometric of genes correlated with clone growth, Supplementary Table 6); 2h) number of clones growing at day 21 (P<0.05) and outcome at six months (n=49 cutaneous melanoma patients, two-sided Wilcoxon signed-rank Test). Lower and upper hinge of box on boxplots represent 25-75th percentiles, central line the median and the whiskers extend to largest and smallest values no greater than 1.5x interquartile range).

Figure 3. Number of large clones is of prognostic importance

3a) Number of clones >0.5% repertoire at day 21 is significantly greater in responding patients than in control samples (clones identified by TRA chain, n=25 controls, 49 patients, two-sided Wilcoxon signed-rank Test, lower and upper hinge of box on boxplots represent 25-75th percentiles, central line the median and the whiskers extend to largest and smallest values no greater than 1.5x interquartile range); 3b) replication cohort (n=43 controls, 20 patients, one-sided Wilcoxon signed-rank Test, boxplot as per 3a); 3c) Day 21 large clone count associates with 6 month outcomes after sICB (left panel, n=42), cICB (centre panel, n=27), when considering all ICB (right panel, n=69, P values all one-sided Wilcoxon-signed rank Test, boxplot as per 3a); 3d) Shorter progression-free survival in patients with day 21 large clone count below median versus those with count above (P=0.003, two-sided Log-rank test, n=69); 3e) Kaplan-Meier survival curve demonstrates reduced overall survival in patients with day 21 large clone count below median versus those with count above (P=0.01, two-sided Log-rank test, n=69); 3f) correlation between large clone count and cell subset percentages from flow cytometry (72 samples, 19 patients, r reflects Pearson correlation, P value obtained from two-sided T-statistic).

Figure 4. Single cell sequencing demonstrates large clones have a distinct cytotoxic expression profile

4a) t-SNE plot of post-treatment CD8+ cell expression profiles from n=8 individuals (4 sICB, 4cICB patients, 12,699 cells total) with colours indicating cell clusters identified by Canonical Correlation Analysis; 4b) Heatmap of genes differentially expressed according to cluster identity as per 4a); 4c) barplot demonstrates total number of clones per sample with those coloured in orange representing large clones (>0.5% repertoire); 4d) number of cells that belong to small (green) and large clones per sample; 4e) t-SNE as per a) but cells are now coloured as to whether they belong to large or small clones; 4f) heatmap and boxplots (4g) demonstrating most significantly differentially expressed genes between large and small clones (lower and upper hinge of box on boxplots represent 25-75th percentiles, central line the median and the whiskers extend to largest and smallest values no greater than 1.5x interquartile range, violin component refers to kernel probability density and encompasses all cells)