Patient characteristics at baseline are summarized in Table 1. Eighteen out of 29 patients (62.1%) had recurrence after gastrectomy. All patients received two or more lines of previous systemic chemotherapy before the nivolumab treatment and had no previous treatment with other ICIs.
The median duration of the nivolumab treatment was 1.9 months (range; 0.4 - 43.9 months). Median PFS and OS were 1.8 months (range; 0.4 - 45.5 months) and 6.7 months (range; 0.7 - 45.5 months), respectively. BOR was PR in 3 patients (10.3%), SD in 2 (6.9%), PD in 21 (72.4%), and NE in 3 (10.3%) (Table 2). All three cases of NE were clinically diagnosed as PD. Patients were classified into the responder group of CR, PR, and SD and the non-responder group of PD and NE for further analyses.
The expression frequency of each marker among PD-1+CD8+T cells in peripheral blood after the nivolumab treatment
Peripheral blood samples were collected from 29 patients before treatment, 25 patients after 2 weeks of treatment, and 19 patients after 6 weeks of treatment. PD-1+ T cells before and after the nivolumab treatment were detected with the anti-PD-1 antibody and anti-IgG4 antibody, respectively (Supplementary Fig. 1b). We initially evaluated the expression frequency of the surface or intracellular markers among PD-1+CD8+ T cells or PD-1-CD8+ T cells in peripheral blood before and after the nivolumab treatment, including activation marker CD103, naive marker CD45RA, immune checkpoint molecules Tim-3, ICOS and CD27, and proliferative marker Ki-67 (Fig. 1b, Supplementary Fig. 1c, 2). The frequency of each marker, except for CD45RA, was higher among PD-1+CD8+ T cells than among PD-1-CD8+ T cells at all time points. Furthermore, the frequencies of CD103, Tim-3, ICOS, and Ki-67 among PD-1+CD8+ T cells rapidly increased after the start of the nivolumab treatment and were sustained for 6 weeks (Fig. 1b, Supplementary Fig. 1c). On the other hand, changes in the frequency of each marker among PD-1-CD8+ T cells were smaller than those observed among PD-1+CD8+ T cells. Since a larger change was observed in the frequency of each marker, particularly among PD-1+CD8+ T cells, the ratio of the frequency of each marker among PD-1+CD8+ T cells 2 or 6 weeks after the start of the nivolumab treatment to that before treatment (% of each marker 2w or 6w/pre) was compared between the responder and non-responder groups (Fig. 2a, 2b). The ratio of the frequency of CD103 among PD-1+CD8+ T cells in peripheral blood 2 weeks after the start of the nivolumab treatment to that before treatment was significantly higher in the responder group than in the non-responder group (P=0.038). No significant differences were observed in the ratio of the frequencies of other markers among PD-1+CD8+ T cells in peripheral blood 2 or 6 weeks after the start of the nivolumab treatment to that before treatment between the responder and non-responder groups (Fig. 2a, 2b). When changes in the expression of markers on PD-1+CD8+ T cells before and after the nivolumab treatment were detected with the t-SNE analysis in one representative responder and one non-responder, CD103 expression on PD-1+CD8+ T cells 2 weeks after the start of the nivolumab treatment was more prominent in the responder than in the non-responder, and this cell population highly co-expressed ICOS, Ki-67, and CD27 with the lower expression of Tim-3 and CD45RA (Supplementary Fig. 3).
Analysis of functional characteristics of CD103+PD-1+CD8+ T cells
To characterize the phenotype and functions of the CD103+PD-1+CD8+ T cell subset, we analyzed proliferative activity and cytotoxicity in the following subsets: CD103+PD-1+CD8+ T cells, CD103-PD-1+CD8+ T cells, and PD-1-CD8+ T cells. Among the three groups, the highest frequency of Ki-67 was observed in CD103+PD-1+CD8+ T cells before the nivolumab treatment. Moreover, the frequency of Ki-67 increased 2 weeks and decreased 6 weeks after the start of the nivolumab treatment among all three subsets, with the largest increase in the frequency of Ki-67 occurring among CD103+PD-1+CD8+ T cells. (Fig. 3a). The majority of CD103+PD-1+CD8+ T cells expressed Ki-67 in the tSNE-analysis and a strong correlation was observed between the expression of CD103 and Ki-67 on PD-1+CD8+ T cells (Supplementary Fig. 3, 4a, 4b). Regarding cytotoxicity and the activation status, CD103+PD-1+CD8+ T cells had lower frequencies of perforin and granzyme than the other subsets at all time points, but higher frequencies of INF-γ, TNF-α, and IL-2 2 weeks after the start of the nivolumab treatment (Fig. 3b, 3c). These results suggest that CD103+PD-1+CD8+ T cell subsets are highly proliferative and less cytotoxic, but strongly activated.
We examined the differentiation status of T cells characterized by the naïve T cell marker, CD45RA, and the differentiation T cell marker, CD27 among the naive (CD45RA+CD27+), central memory (CD45RA-CD27+), effector memory (CD45RA-CD27-), and terminally differentiated (CD45RA+CD27-) subsets (Supplementary Fig. 5a) (8, 11-16). In this classification, the central memory subset was dominant in PD-1+CD8+ T cells, particularly in CD103+PD-1+CD8+ T cells, and the percentage of this subset significantly increased after the nivolumab treatment (Fig. 3d, Supplementary Fig. 5b).
The relationship between an increase in the subset of CD103+PD-1+CD8+ T cells and patient prognosis
When patients were divided into the high and low groups based on the median value of the frequency of CD103 among PD-1+CD8+ T cells 2 weeks after treatment, the high group had significantly better PFS than the low group (P=0.032, Fig. 4a). In the analysis for the ratio of the frequency of CD103 among PD-1+CD8+ T cells in peripheral blood 2 weeks after the start of the nivolumab treatment to that before treatment (%CD103 2w/pre), the high group also had significantly better PFS than the low group (P=0.007; Fig. 4b). Similar results were observed in the analysis of the actual cell number of CD103+PD-1+CD8+ T cells/µl 2 weeks after the start of treatment (Supplementary Fig. 6a, 6b). Furthermore, a multivariate analysis of clinicopathologic variables revealed that the ratio of the frequency of CD103 among PD-1+CD8+ T cells in peripheral blood 2 weeks after treatment to that before treatment (%CD103 2w/pre) was an independent prognostic factor for PFS (HR: 3.09, 95% CI: 1.19–8.79; P=0.020, Supplementary Table S1).
Evaluation of nivolumab-binding T cells in tumors and lymph nodes resected after the nivolumab treatment
Tumors, lymph nodes, and peripheral blood were collected from two individual patients (cases 1 and 2) who were treated with nivolumab, showed a durable response, and subsequently underwent gastrectomy. We detected the presence of nivolumab-binding CD8+ T cells in tumors, lymph nodes, and peripheral blood (Fig. 5a). The majority of nivolumab-binding CD8+ tumor-infiltrating lymphocytes (TILs) expressed CD103, while a relatively high percentage of nivolumab-binding T cells from lymph nodes expressed CD103 in contrast to a lower percentage in peripheral blood (Fig. 5b). Regarding the differentiation status, CD103+PD-1+CD8+ T cells in lymph nodes consisted of the highest percentage of central memory T cells, whereas effector memory T cells were more frequently observed among the CD103+PD-1+CD8+ T cells of TILs (Fig. 5c). The differentiation status of CD103+PD-1+CD8+ T cells in peripheral blood was between that of the lymph nodes and tumors. The frequency of Ki-67 among CD103+PD-1+CD8+ T cells was similar between the three samples (Fig. 5d).