Prospective identification of neoantigen-specific lymphocytes in the peripheral blood of melanoma patients

Abstract

Detection of lymphocytes that target tumor-specific mutant neoantigens--derived from products encoded by mutated genes in the tumor--is mostly limited to tumor-resident lymphocytes, but whether these lymphocytes often occur in the circulation is unclear. We recently reported that intratumoral expression of the programmed cell death 1 (PD-1) receptor can guide the identification of the patient-specific repertoire of tumor-reactive CD8(+) lymphocytes that reside in the tumor. In view of these findings, we investigated whether PD-1 expression on peripheral blood lymphocytes could be used as a biomarker to detect T cells that target neoantigens. By using a high-throughput personalized screening approach, we identified neoantigen-specific lymphocytes in the peripheral blood of three of four melanoma patients. Despite their low frequency in the circulation, we found that CD8(+)PD-1(+), but not CD8(+)PD-1(-), cell populations had lymphocytes that targeted 3, 3 and 1 unique, patient-specific neoantigens, respectively. We show that neoantigen-specific T cells and gene-engineered lymphocytes expressing neoantigen-specific T cell receptors (TCRs) isolated from peripheral blood recognized autologous tumors. Notably, the tumor-antigen specificities and TCR repertoires of the circulating and tumor-infiltrating CD8(+)PD-1(+) cells appeared similar, implying that the circulating CD8(+)PD-1(+) lymphocytes could provide a window into the tumor-resident antitumor lymphocytes. Thus, expression of PD-1 identifies a diverse and patient-specific antitumor T cell response in peripheral blood, providing a novel noninvasive strategy to develop personalized therapies using neoantigen-reactive lymphocytes or TCRs to treat cancer.

Figure 1

Frequency of PD-1 expression on circulating and tumor-resident CD8⁺ lymphocytes and prospective identification of circulating neoantigen-reactive cells in melanoma patients. (a) Expression of PD-1 on CD8⁺ lymphocytes in matched PBMC and tumors (n = 18). The median is plotted. ***P < 0.001; by Mann-Whitney U test. (b) Representative flow cytometry analysis for coexpression of PD-1 and TIM-3 (left), LAG-3 (middle) or 4–1BB (right) on CD8⁺ lymphocytes in the tumor (top) and PBMC (bottom) from one patient (of n = 18). The percentage of cells expressing each combination of receptors is shown. (c) Image showing gates used for flow cytometry–based sorting of the circulating CD8⁺ lymphocytes. SSCA, side-scatter area. (d–i) IFN-γ ELISPOT assays (d,f,h) and flow cytometry analysis for 4–1BB expression (e,g,i) showing reactivity of in vitro–expanded subsets (CD8⁺, CD8⁺PD-1^hi, CD8⁺PD-1⁻ and CD8⁺PD-1⁺) sorted from pretreatment PBMC from patients NCI-3998 (d,e), NCI-3784 (f,g) and NCI-3903 (h,i) to autologous DCs transfected with RNAs encoding an irrelevant TMG (Irrel.) or the indicated TMGs. Representative flow cytometry plots show the percentage of 4–1BB⁺ lymphocytes after coculture of circulating CD8⁺PD-1⁺ cells with the TMGs specified. Plotted cells were gated on live CD3⁺ lymphocytes. ‘>’ denotes greater than 500 spots/2 × 10⁴ cells. Experiments were performed without duplicates. All data are representative of at least two experiments.

Figure 2

Characterization of neoantigen-specific lymphocytes isolated from the circulating CD8⁺PD1⁺ subset of subject NCI-3998. (a–c) Reactivity of TMG1- (left), TMG3- (middle) and TMG5-reactive (right) lymphocytes to the indicated TMGs or to the individual mutant 25-mers encoded by the indicated TMG (a), serial dilutions of the wild-type (WT) or mutant (Mut) MAGEA6_E>K, PDS5A_Y>F;H>Y and MED13_P>S minimal peptides (b), and COS7 cells cotransfected with the corresponding constructs expressing the indicated TMG and the individual HLA alleles that encode the HLA class I molecules indicated on the x axis (such as HLA-A*01:01; denoted A*01:01) (c). In c, the mean ± s.d. is plotted. (d–f) Reactivity of autologous PBMC that were transduced with retroviruses encoding neoantigen-specific TCRs. The T cell population of origin and the rank of the TRA and TRB sequences used to construct each TCR is denoted (as ‘TCR (A rank number)/(B rank number)’). The constructed TCRs expressed mouse constant regions, enabling the detection of the TCR with antibodies specific for the mouse TRB constant region (mTRB). Analyses showing reactivity of two distinct MAGEA6_E>K-specific TCRs to TMG1 and the WT and Mut MAGEA6_E>K minimal epitopes (CD3⁺CD8⁺ cells are plotted and the percentage of mTRB⁺4–1BB⁺ cells is shown) (d), mutant neo-epitope recognition and HLA restriction of two different MED13_P>S-specific TCRs (e), and reactivity of a PDS5A_Y>F;H>Y-specific TCR to TMG3 and to the WT and Mut PDS5A_Y>F;H>Y peptides (f). Unless otherwise specified, experiments were performed without duplicates. All data are representative of at least two experiments.

Figure 3

Identification of neoantigens targeted by circulating CD8⁺PD-1⁺ cells isolated from subjects NCI-3784, NCI-3903 and NCI-3713. (a,b) Reactivity of NCI-3784 TMG3- (top), TMG5- (middle) and TMG8-reactive (bottom) CD3⁺CD8⁺ lymphocytes to autologous DCs pulsed with the individual mutant 25-mers encoded by the indicated TMG (a) or to autologous DCs pulsed with FLNA_R>C, KIF16B_L>P or SON_R>C 25-mers, or their WT counterparts (b). (c) Recognition of B cells pulsed with serial dilutions of the Mut KIF1PB_P>S minimal peptide or its WT counterpart by NCI-3903 TMG9-reactive CD3⁺CD8⁺ lymphocytes. (d) Frequency of the top five TRB clonotypes, as determined by deep-sequencing analysis of TRB from NCI-3903 TMG9-reactive lymphocytes. (e,f) IFN-γ ELISPOT assays (e) and frequency of 4–1BB expression (f) of NCI-3713 pretreatment PBMC that were sorted into CD8⁺, CD8⁺PD-1⁻, CD8⁺PD-1⁺ and CD8⁺PD-1^hi cells, expanded in vitro and cocultured with B cells pulsed with either DMSO as a control or the WT and Mut epitopes from the indicated proteins. Experiments were performed without duplicates. All data are representative of at least two experiments.

Figure 4

Recognition of tumors and self-antigens by TCRs or CD8⁺ T cells isolated from peripheral blood, and comparison of the specificity and TCR repertoire between circulating and tumor-infiltrating CD8⁺ T cell subsets. (a–c) Reactivity (as determined by 4–1BB upregulation on CD3⁺CD8⁺ cells) of retrovirally transduced lymphocytes from subject NCI-3998 expressing MAGEA6_E>K-, PDS5A_Y>F;H>Y- or MED13_P>S-specific TCRs (a), circulating FLNA_R>C-, KIF16B_L>P- or SON_R>C-specific lymphocytes from subject NCI-3784 (b) and KIF1BP_P>S-specific lymphocytes from subject NCI-3903 (c) that were cocultured with their corresponding autologous tumor cell lines (3998mel, 3784mel and 3903mel, respectively) pretreated with or without IFN-γ. (d) Reactivity of the circulating CD8⁺PD-1⁻ and CD8⁺PD-1⁺ lymphocytes from subjects NCI-3998, NCI-3784, NCI-3903, NCI-3926 and NCI-3713 to their corresponding autologous tumor cell line. Each dot represents the frequency of 4–1BB upregulation for one patient sample (n = 5). Mean ± s.e.m. is shown. (e–g) IFN-γ ELISPOT assays (e) and analysis of 4–1BB upregulation by flow cytometry (representative plots shown, gated on CD3⁺ cells) (f,g) of pretreatment PBMC CD8⁺ subsets from subjects (indicated below each graph) that were screened for recognition of the shared tumor antigens indicated. Experiments were performed without duplicates. All data are representative of at least two experiments. (h) Antigens recognized by circulating and tumor-infiltrating CD8⁺PD-1⁺ lymphocytes. Each rectangle represents a target antigen screened. (i) Deep-sequencing analysis of TRB from intratumoral CD8⁺ PD-1⁺ cells and matched pretreatment PBMC CD8⁺, CD8⁺PD-1⁻ and CD8⁺PD-1⁺ cells (n = 7) was used to determine TRB sequence overlap between the tumor-resident CD8⁺PD-1⁺ cells and the blood-derived CD8⁺, CD8⁺PD-1⁻ and CD8⁺PD-1⁺ cells (see Online Methods for calculation methodology). A TRB sequence overlap of 1 indicates 100% similarity between two populations. **P < 0.01 using Dunn’s test for multiple comparisons; n.s., not significant.