Functional systemic CD4 immunity is required for clinical responses to PD-L1/PD-1 blockade therapy (2019)
Figure 7

Figure 7. Objective responders are found within G1 patients with PD-L1+ tumors. (A) Kaplan-Meier plot for PFS in patients undergoing immune checkpoint inhibitor therapies stratified by G1 / PD-L1+ tumors (blue) and remaining patients for whom their PD-L1 tumor status is known (red). (B) Same as (A) but including all patients in the study cohort. Remaining patients (red) also included G1 patients with PD-L1 low/negative tumors, G1 patients with unknown PD-L1 tumor status and G2 patients with either PD-L1+ or PD-L1 negative tumors.

EXPANDED FIGURE LEGENDS

Expanded figure 1. Validation dataset. (A) Distribution of circulating CD4 THD cells within CD4+ CD14negative cells in healthy donors (N=14) and in NSCLC patients constituting the validation set (N=32). G1 and G2 groups are indicated and separated by the mean (horizontal line). The means ± standard deviations of CD4 THD cells in G1 and G2 groups are shown on the right, as well as the association between G1 profiles and objective responses by the Fisher´s test. Differences between healthy donors and NSCLC patients were tested with the U of Mann-Whitney test. (B) ROC analysis of CD4 THD quantification in the validation dataset and objective responses. The cut-off value for identification of responses is shown in the graph. *, **, indicate significant (P<0.05) and highly significant (P<0.001) differences.

Expanded Figure 2. CD4 T cell profiling does not have significant prognostic value. Kaplan-Meier plot of relative time elapsed from diagnosis to the start of immunotherapy for G1 (blue) and G2 patient cohorts (red), as indicated. No significant differences were found.

Expanded figure 3. Ex vivo human lung adenocarcinoma-T cell recognition system. (A) Top, lentivector co-expressing an anti-CD3 single chain antibody gene (SC3) and blasticidine resistance for selection. SFFVp, spleen focus-forming virus promoter; UBIp, human ubiquitin promoter; LTR, long terminal repeat; SIN, U3-deleted LTR leading to a self-inactivating lentivector. Bottom, molecular structure of the SC3 molecule, which is anchored to the cell membrane by a transmembrane domain as indicated. OKT3 VL, variable region of the light chain from the anti-CD3 antibody OKT3; VH, variable region of the heavy chain from the anti-CD3 antibody OKT3. (B) Scheme of the cell-to-cell interactions mediated by the lentivector-modified A549 cell and T cells including SC3/CD3, PD-L/PD-1 and MHCII/LAG-3 interactions as indicated. (C) Representative flow cytometry density plots with the up-regulation of PD-1 expression in CD4 from NSCLC patients following co-incubation with A549-SC3 cell as indicated (right graph), or with unmodified A549 control (left graph). Percentages of PD-1+ T cells are shown within the graphs. (D) As in (C) but with CD8 T cells. Percentages of PD-1+ T cells are shown within the graphs.

Expanded Figure 4. CD4 THD cells in NSCLC patients are mainly non-senescent memory subsets. (A) Scatter plot graphs of the percentage of memory phenotypes in baseline CD4 THD cells according to CD62L-CD45RA expression (% CD45RAnegative CD62Lpositive central memory + % CD45RAnegative CD62Lnegative effector memory cells) in a sample of healthy donors, G1 and G2 patients, and in healthy donors as indicated. (B) Expression of the genotoxic damage maker H2AX by flow cytometry in CD4 T cell subsets from an aged-matched healthy donor, and NSCLC G1 and G2 patients as indicated. Percentage of positivity and mean fluorescent intensities are indicated for each population. Top, histogram analysis within CD27+ CD28+ CD4 T cells, and bottom, CD27negative CD28low/negative counterparts as indicated. (C) As in (B) but for CD57 expression. US, unstained control; NS, no significant differences (P>0.05); ***, highly significant differences (P<0.001).

Expanded Figure 5. Systemic CD8 responses in NSCLC patients. (A) Percentage of circulating highly differentiated CD8 cells in age-matched healthy donors and NSCLC patients before undergoing immunotherapies. Relevant statistical comparisons are shown. In green, objective responders (OR). In red, no OR. (B) ROC analysis of baseline CD8 THD quantification as a function of objective clinical responses. (C) Dot-plot of lung cancer antigen-specific CD8 T cells obtained before the start of immunotherapies and stimulated with A549-loaded autologous DCs in G1 and G2 patients, as indicated. (D) Left dot plot, percentage of CD28-negative CD8 T cells within TAA-specific CD8 subsets in G1 and G2 patients as indicated. Right dot plot, same as left but with CD28-positive subsets. Green, objective responders (OR) and red, no ORs. N, number of biological replicates (independent patients.

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