A T cell repertoire timestamp is at the core of responsiveness to CTLA-4 blockade

Abstract

Biology of the response to anti-CTLA-4 involves the dynamics of specific T cell clones. Reasons for clinical success and failure of this treatment are still largely unknown. Here, we quantified the dynamics of the T cell receptor (TCR) repertoire, throughout 4 weeks involving treatment with anti-CTLA-4, in a syngeneic mouse model for colorectal cancer. These dynamics show an initial increase in clonality in tandem with a decrease in diversity, effects which gradually subside. Furthermore, response to treatment is tightly connected to the shared and public parts of the T cell repertoire. We were able to recognize time-dependent behaviors of specific TCR sequences and cell types and to show the response is dominated by specific motifs. We see that a single, specific time point might be useful to inform a physician of the true response to treatmentThe research further highlights the importance of temporal analyses of the immune response.

Keywords: Cell Biology: Bioinformatics; Immunology.

Graphical abstract

Figure 1

Experimental workflow C57BL/6 mice were implanted with freshly cultured MC38 colon tumor cells. One group (red arrow, horizontal, 15 mice) was treated with the anti-CTLA-4 clone 9H10 on days 1, 3, and 6, whereas the control group (5 mice) remained untreated. We have drawn blood samples once a week, starting day 0, during a period of 3 weeks (purple vertical lines). From these blood samples, we obtained PBMCs and processed their RNA to produce TCR repertoire sequencing. In addition, tumor (when available) and spleen samples were collected with animal sacrifice at the termination of the experiment. Tumor volumes were measured on days 0, 2, 5, 7, 9, 12, 14, 16, 19, and 21 (black vertical lines in the figure).

Figure 2

Changes in clonality and diversity subside over time and are distinctive to different response groups (A–C) Changes over time in tumor size (See also Table S1) (A), diversity index for beta chain (B), and clonality index for beta chain (C) in the control group and in the treated group. (D) Changes over time in the average tumor size. The treated mice in this panel are divided into two groups, according to their clonality index—low clonality (blue lines) and high clonality (orange lines). (E) The abundance levels (copy number) of the 10 most expanded clones in the control and treated groups in an amino acid view, in beta chain. (F and G) (F) Fold changes of the 10 most abundant clones in the different response groups (early, mid, late, see text) and (G) combined. See also Figure S1. Data of all panels are represented as mean ± SEM.

Figure 3

Repertoire-dependent response to treatment (A) Frequency of Public Exclusive clones in each of the response groups over time. Data are presented as means ± SEM (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; Student's t test). For the sake of legibility, the panel highlights a small set of some of the significant differences between the different levels. There are many additional significant contrasts. (B) Overlaps between samples. We used the 100 most abundant beta chain clones in each sample for the calculation. The metric we used is Morisita's index (see text). The panel highlights inner similarities within samples from the same animal. (C) The average overlap within and between groups. Each rectangle represents the average number of overlaps within all samples in a specific combination. To calculate an average overlap, we used the average over all sample combinations between two groups. (D) Changes, over time, in the averaged CR levels in the control (Rubelt et al.) and treated (blue) groups. Data are presented as means ± SEM. The panel indicates the significant increase in CR in the treated group at D14, in contrast with the significant decrease in CR in the control group on the same day. Interestingly, the curves meet at D21. See also figure S2.

Figure 4

Network analysis of clones Networks of a set of sequences assembled by collecting the 100 most abundant clones in each sample. Three different groups are visualized (mouse D = control; mouse L = early-responders; mouse H = late-responders). For each mouse, we see progress in time from left to right. The leftmost network is D0, whereas the rightmost network is D21. The size of the node represents the relative abundance of a clone within a mouse. We connected and clustered AA sequences according to their Levenshtein distance. An edge between two nodes appears when the distance is smaller or equal to 2.

Figure 5

Response and behavior are related to the presence of specific TCRs and cell types (A) The frequency of iNKT cells in alpha chain for all four different groups. (B) The frequency of MAIT cells in alpha chain. (C) The frequency iNKT cells in spleen samples in control and treated mice. (D) The frequency of CASSLELGGREQYF (the most shared clone observed, see Table S2) in the different groups. Data of all panels are represented as mean ± SEM. See also Figure S3.

Figure 6

Cluster of clone abundance Both TCR alpha (left) and TCR beta (right) show differences in clone sizes. See also Figure S4.