Proliferation of PD-1+ CD8 T cells in peripheral blood after PD-1-targeted therapy in lung cancer patients

Abstract

Exhausted T cells in chronic infections and cancer have sustained expression of the inhibitory receptor programmed cell death 1 (PD-1). Therapies that block the PD-1 pathway have shown promising clinical results in a significant number of advanced-stage cancer patients. Nonetheless, a better understanding of the immunological responses induced by PD-1 blockade in cancer patients is lacking. Identification of predictive biomarkers is a priority in the field, but whether peripheral blood analysis can provide biomarkers to monitor or predict patients' responses to treatment remains to be resolved. In this study, we analyzed longitudinal blood samples from advanced stage non-small cell lung cancer (NSCLC) patients (n = 29) receiving PD-1-targeted therapies. We detected an increase in Ki-67+ PD-1+ CD8 T cells following therapy in ∼70% of patients, and most responses were induced after the first or second treatment cycle. This T-cell activation was not indiscriminate because we observed only minimal effects on EBV-specific CD8 T cells, suggesting that responding cells may be tumor specific. These proliferating CD8 T cells had an effector-like phenotype (HLA-DR+, CD38+, Bcl-2lo), expressed costimulatory molecules (CD28, CD27, ICOS), and had high levels of PD-1 and coexpression of CTLA-4. We found that 70% of patients with disease progression had either a delayed or absent PD-1+ CD8 T-cell response, whereas 80% of patients with clinical benefit exhibited PD-1+ CD8 T-cell responses within 4 wk of treatment initiation. Our results suggest that peripheral blood analysis may provide valuable insights into NSCLC patients' responses to PD-1-targeted therapies.

Keywords: CD8 T cells; PD-1; T-cell exhaustion; cancer immunotherapy; checkpoint inhibition.

Conflict of interest statement

Conflict of interest statement: R.A. is an inventor on patents held by Emory University that cover the topic of PD-1–directed immunotherapy. S.S.R. served on the scientific advisory board and received an honorarium from BMS, Merck, Genentech, and Astra Zeneca.

Figures

Fig. 1.

Proliferation of CD8 T cells can be detected in the blood of NSCLC patients after PD-1–targeted therapies. (A) Study design. (B) Dot plots show summary of gating strategy. (C) Dot plots show frequency of Ki-67+ cells among CD8 T cells, Foxp3neg CD4 T cells, and Foxp3+ CD4 Treg cells at baseline (pre) and 3 wk after treatment initiation (post) for one representative patient. Gates were determined based on naïve T cells. (D) Graph shows best fold increase in Ki-67+ cells among different T-cell populations (post PD-1 therapy compared with baseline). A 1.5-fold threshold is shown as gray shaded area. Lines represent the median fold increase for each population; n = 29. Patients with ≥1.5-fold increase in Ki-67+ CD8 T cells after PD-1–targeted therapy are indicated by dashed red rectangle.

Fig. S1.

Minimal changes on Ki-67+ T cells in the peripheral blood of healthy volunteers. PBMCs were isolated consecutively from two healthy subjects recruited through Emory Vaccine Center. The frequency of Ki-67+ cells among CD8 T cells, Foxp3neg CD4 T cells, and CD4 Treg cells was assessed at baseline and after 2 wk. Graph shows the fold increase in Ki-67+ cells among the different T-cell populations. Lines represent the median fold increase for each population. A 1.5-fold threshold is shown as a gray shaded area.

Fig. S2.

Complete blood counts are not affected by PD-1–targeted therapy. (A) Graphs show white blood cell (WBC) counts, absolute neutrophil counts (ANCs), and absolute lymphocyte counts (ALCs) at baseline. Patients were grouped according to clinical outcome. Yellow shaded areas indicate range of reference values. (B) Graphs show changes in complete blood counts (WBC, ANC, and ALC) as fold change (posttreatment/pretreatment). Each line represents one patient, and colors indicate clinical outcomes (blue, partial response; gray, stable disease; red, disease progression). Shaded gray areas indicate the mean fold increase for all time points. P, patient.

Fig. S3.

Distribution of T-cell subsets and PD-1 expression on NSCLC patients and healthy subjects. Analysis was performed in whole blood obtained from NSCLC patients before PD-1–targeted therapy initiation. Healthy volunteers were recruited through Emory Vaccine Center. (A) Graphs show frequency of CCR7+CD45RA+ naïve (N), CCR7+CD45RAneg central memory (TCM), CCR7negCD45RAneg effector memory (TEM), and CCR7negCD45RA+ effector memory RA (TEMRA) on CD8 T cells (Left) and CD4 T cells (Right). (B) Graphs show frequency of PD-1+ T cells among the different subsets described in A, on CD8 T cells (Left) and CD4 T cells (Right). Gates were established based on naïve T cells. Lines represent the median. Unpaired Student t test.

Fig. 2.

Blockade of the PD-1 pathway induces proliferation of effector-like PD-1+ CD8 T cells. Analysis was performed at the best CD8 T-cell response time point on NSCLC patients with ≥1.5-fold increase in Ki-67+ CD8 T cells after treatment (n = 20). Gates were determined based on naïve CD8 T cells. (A) Graph shows frequency of PD-1+ cells among Ki-67+ CD8 T cells. Patients whose responding Ki-67+ CD8 T cells were mostly PD-1neg are indicated by gray circles. P, patient. (B) Dot plots show proliferation and PD-1 expression on CD8 T cells, on two representative patients with PD-1+ CD8 T-cell responses. (C) As in B, but depicted are two representative patients with PD-1neg CD8 T-cell responses. (D) Graph shows frequency of Bcl-2lo cells. (E) Dot plots show proliferation and Bcl-2 expression on responding PD-1+ Ki-67+ T cells (red dots) over the contour plot for total CD8 T cells on two representative patients. (F) Graph shows frequency of HLA-DR+CD38+ cells. (G) Dot plots show HLA-DR and CD38 expression as in E. (H) Graph shows frequency of CCR7neg CD45RAneg cells. (I) Dot plots show CCR7 and CD45RA expression as in E. (J) Graph shows frequency of granzyme B+ cells. (K) Dot plots show Ki-67 and granzyme B expression as in E. P, patient; C, treatment cycle.

Fig. S4.

Detection of therapeutic anti–PD-1 antibodies by indirect staining. Dot plots show staining of PBMCs isolated from NSCLC P20 at baseline (pre) and 2 wk after the second infusion with nivolumab, a human IgG4 anti–PD-1 (post). Dot plots show CD8 and PD-1 (anti–PD-1, anti–human-IgG4, or anti–human-IgG4 + anti–PD-1) expression on CD3+ live cells. Combined anti–human-IgG4 + anti–PD-1 staining was used to analyze all posttreatment PBMC samples from NSCLC patients receiving therapeutic anti–PD-1. Data are representative of NSCLC patients receiving nivolumab or pembrolizumab.

Fig. S5.

Phenotype of effector CD8 T cells induced by vaccination with live attenuated virus. (A) Dot plot shows gating of yellow fever (YF)-specific CD8 T cells by tetramer staining 14 d after s.c. administration of 17D live-attenuated yellow fever vaccine strain. (B) Dot plots show phenotype of YF-specific CD8 T cells (blue dots). Underlying contour plots show phenotype of total CD8 T cells.

Fig. S6.

Characterization of peripheral blood CD8 T-cell following PD-1–targeted therapy of NSCLC patients. (A) Graph shows Bcl-2 expression levels on Ki-67+ CD8 T cells (comparison between PD-1neg and PD-1+). Paired Student t test. (B) Graph shows frequency of Ki-67+ CD8 T cells that coexpress HLA-DR and CD38 (comparison between PD-1neg and PD-1+). Paired Student t test. (C) Graph shows frequency of granzyme B+ cells among the indicated CD8 T-cell populations. Gates were determined based on naïve T cells. Repeated-measures ANOVA and Tukey’s multiple comparisons test. MFI, mean fluorescence intensity.

Fig. 3.

PD-1+ Ki-67+ CD8 T cells responding to PD-1–targeted therapies express costimulatory molecules. Analysis was performed at the best CD8 T-cell response time point, on NSCLC patients with ≥1.5-fold increase in Ki-67+ CD8 T cells after treatment; n = 18. Gates were determined based on naïve CD8 T cells. (A) Graph shows frequency of CD28+ cells. (B) Graph shows frequency of CD27+ cells. (C) Dot plots show CD28 and CD27 expression on responding PD-1+ Ki-67+ T cells (red dots) over the contour plot for total CD8 T cells on two representative patients. (D) Graph shows frequency of ICOS+ cells. (E) Graph shows ICOS mean fluorescence intensity (MFI) on the indicated CD8 T-cell populations. Repeated-measures ANOVA and Tukey’s multiple-comparisons test. (F) Dot plots show proliferation and ICOS expression as in C. P, patient; C, treatment cycle.

Fig. 4.

Increased expression of PD-1 and CTLA-4 on PD-1+ Ki-67+ CD8 T cells responding to PD-1–targeted therapies. Analysis was performed at the best CD8 T-cell response time point, on NSCLC patients with ≥1.5-fold increase in Ki-67+ CD8 T cells after treatment; n = 18. Gates were determined based on naïve CD8 T cells. (A) Graph shows PD-1 MFI on PD-1+ CD8 T cells (Ki-67neg and Ki-67+). Paired Student t test. (B) Graph shows CTLA-4 MFI on the indicated CD8 T-cell populations. Repeated-measures ANOVA and Tukey’s multiple-comparisons test. (C) Graph shows frequency of CTLA-4+. (D) Dot plots show proliferation and CTLA-4 expression on responding PD-1+ Ki-67+ T cells (red dots) over the contour plot for total CD8 T cells on two representative patients. P, patient; C, treatment cycle.

Fig. S7.

Minimal effects of PD-1–targeted therapies on EBV-specific PD-1+ CD8 T cells. (A, Left) Dot plots show PD-1 and EBV-TET staining on CD8 T cells. (A, Right) Dot plots show proliferation of PD-1+ EBV-TET+ CD8 T cells and PD-1+ EBV-TETneg CD8 T cells at baseline (pre) and at the best CD8 T-cell response posttreatment (post). Data from NSCLC patient 2. (B) Dot plots show EBV-TET staining on CD8 T cells at baseline (pre) and at the best CD8 T-cell response posttreatment (post). (C) As in B, but dot plots show CD38 and HLA-DR expression on EBV-specific CD8 T cells (blue dots) over the gray contour plot for total CD8 T cells. Numbers inside dot plots indicate the percentage of EBV-specific CD8 T cells that coexpress HLA-DR and CD38. (D) Dot plots show PD-1 expression on EBV-specific and total CD8 T cells at baseline. Data in B–D is from whole blood of three patients as indicated.

Fig. 5.

Early proliferation of PD-1+ CD8 T cells is correlative to clinical outcomes. (A) Spiderplot shows changes from baseline in the tumor burden as assessed by RECIST 1.1 (n = 27). (B) Graphs show fold increase in the proliferation of CD8 T cells at all time points analyzed. Patients whose responding Ki-67+ CD8 T cells were mostly PD-1neg are indicated by a gray circle. (C) Graph shows frequency of PD-1+ cells among Ki-67+ CD8 T cells induced within 4 wk of PD-1–targeted therapy initiation (patients in B Upper). (D) Graphs show frequency of different clinical outcomes on patients that displayed early PD-1+ CD8 T-cell responses and patients that had absent, delayed, or PD-1neg CD8 T-cell responses.

Fig. S8.

Clinical activity of PD-1–targeted therapies in NSCLC patients. (A) Waterfall plot shows maximum reduction from baseline (best response) in tumor burden assessed by RECIST 1.1. Highlighted by a dotted pattern are two patients (P30 and P44) who received previous treatment that may have contributed to tumor response: radiation treatment within 4 wk of PD-1–targeted therapy initiation. The +20% and −30% tumor burden changes are indicated by dashed lines (n = 29). (B) Survival curve of NSCLC patients according to RECIST 1.1 classification: disease progression (n = 7, no 12-mo follow-up data on P8, P24, and P32); stable disease (n = 7); partial response (n = 12).

Fig. S9.

Baseline proliferation of peripheral blood T cells on NSCLC patients. (A) Graph shows frequency of Ki-67+ cells among CD8 T cells, Foxp3neg CD4 T cells, and CD4 Treg cells at baseline. Gates were determined based on naïve T cells. Error bars show mean and SD. Outlier samples are indicated with patient number. (B) Graph shows frequency of Ki-67+ cells among CD8 T cells from patients grouped according to interval and type of previous treatments. Kruskal–Wallis test followed by Dunn’s multiple comparisons test.