Harnessing T cell exhaustion and trogocytosis to isolate patient-derived tumor-specific TCR

Abstract

To study and then harness the tumor-specific T cell dynamics after allogeneic hematopoietic stem cell transplant, we typed the frequency, phenotype, and function of lymphocytes directed against tumor-associated antigens (TAAs) in 39 consecutive transplanted patients, for 1 year after transplant. We showed that TAA-specific T cells circulated in 90% of patients but display a limited effector function associated to an exhaustion phenotype, particularly in the subgroup of patients deemed to relapse, where exhausted stem cell memory T cells accumulated. Accordingly, cancer-specific cytolytic functions were relevant only when the TAA-specific T cell receptors (TCRs) were transferred into healthy, genome-edited T cells. We then exploited trogocytosis and ligandome-on-chip technology to unveil the specificities of tumor-specific TCRs retrieved from the exhausted T cell pool. Overall, we showed that harnessing circulating TAA-specific and exhausted T cells allow to isolate TCRs against TAAs and previously not described acute myeloid leukemia antigens, potentially relevant for T cell-based cancer immunotherapy.

Fig. 1.. TAA-specific T cells accumulate exhausted T_SCM cells and contract before relapse.

Total circulating CD8⁺, TAA-specific and CMV-specific T cells were evaluated in N = 39 transplanted patients at sequential time points. (A to C) Number of circulating CMV- and TAA-specific T cells, in all time points (A), parsed according to antigen specificity (B) and to the time point after allo-HSCT (C); median and interquartile ranges. Mann-Whitney test (A). (D) Memory phenotype on total CD8⁺, CMV-specific, and TAA-specific T cells after allo-HSCT. Unpaired multiple Student’s t tests. Median and interquartile ranges. (E) Frequency of IRs expression on total CD8⁺, CMV-specific, and TAA-specific T cells. Unpaired multiple Student’s t tests. Median and interquartile ranges. (F and G) TAA- and CMV-specific events were analyzed with cytoChain. (F) Barnes-Hut Stochastic Neighbor Embedding (BH-SNE) biaxial plot of all events (left), then divided into CMV- and TAA-specific events (mid). Areas significantly enriched with either CMV- or TAA-specific events were highlighted (right). (G) Fold change with respect to dataset baseline, in each cluster significantly enriched for either CMV- or TAA-specific events. (H) Frequency of IRs coexpression on total CD8⁺, CMV-specific, and TAA-specific T cells. (I) IRs coexpression in TAA-specific T cells, in patients achieving long-term complete remission (CR) or experiencing relapse (REL). (J) Circulating exhausted T_SCM, parsed according to long-term disease status. Median and interquartile ranges. Mann-Whitney test. (K) Circulating TAA-specific T cells in patients with CR and REL. Two-way analysis of variance (ANOVA). Mean and SEM. T_Na, naïve T cells (CD45RA⁺CD62L⁺CD95⁻); T_SCM, stem cell memory T cells (CD45RA⁺CD62L⁺CD95⁺); T_CM, central memory T cells (CD45RA⁻CD62L⁺); T_EM, effector memory T cells (CD45RA⁻CD62L⁻); T_EMRA, terminal effector T cells (CD45RA⁺CD62L⁻). Dex CMV, CMV-specific T cells measured with dextramers. Dex tumor, TAA-specific T cells measured with dextramers. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 2.. Functional TAA-specific TCRs can be isolated from patients’ peripheral blood.

TAA-specific T cells were isolated, expanded, and then tested in vitro. (A) Dextramer⁺ events ex vivo (gray bars) and after in vitro sorting and expansion (black); N = 16 cell cultures, N = 10 patients. (B) CDR3-α (left) and CDR3-β (right) cell culture sequences. The ex vivo clonality is reported at the bottom. (C to G) The dominant TCRs from (B) were engineered into donor T cells and then tested in vitro. (C) Edited T cells from N = 4 healthy donors were cocultured with peptide-pulsed T cell lines (HLA-A*0201-restricted WT1_37–45, MAGE-A2_212–220 and survivin_96–104, HLA-A*0301-restricted hTERT_663–672). The normalized Green+Red area representing the amount of target cells undergoing apoptosis (caspase 3/7 activation) overtime upon coculture with T lymphocytes is reported. (D) Edited T cells were cocultured with T cell lines pulsed either with the appropriate peptide or with an unrelated peptide (N = 6 experiments). The elimination index after 3 days coculture is reported [1 − (alive target cells pulsed with the target peptide and cultured with edited T cells/alive target cells pulsed with an unrelated peptide and cultured with edited T cells)]. An elimination index equal to 1 indicates total killing of target cells. (E to G) WT1-, survivin-, and hTERT-redirected engineered T cells were challenged with WT1⁺, hTERT⁺, or survivin⁺ primary AML blasts expressing (target blasts, n = 3 per specificity) or not expressing (control blasts, n = 3) the appropriate HLA-restriction element. The normalized Green+Red area after coculture (E), the elimination index after 1 day of coculture (F) and the percentage of INF-γ and tumor necrosis factor–α–producing CD8⁺ T cells are reported (G). Two-way ANOVA was used to compare curves. Multiple unpaired Student’s t tests were used to compare groups in (F) and (G). *P < 0.05; **P < 0.01; ****P < 0.0001. The mean and the SEM are shown in (C), (E), and (G).

Fig. 3.. The exhaustion signature can be exploited to isolate tumor-specific TCRs.

Bone-marrow infiltrating T cells from Pt#13-14-15 patients at the moment of relapse were sorted into a fraction not expressing (IR⁻) and a fraction expressing (IR⁺) multiple IRs and then expanded in vitro by means of leukemic-antigen presenting cells (L-APCs). (A) Schematics of the in vitro experimental protocol. (B) After sequential L-APC stimulations, the IR⁺ (red) and IR⁻ (blue) fractions from Pt#14 and Pt#15 were challenged with autologous leukemic blasts. The induction of HLA-DR on CD8⁺ edited T cells (left), expression of activated caspase 3 on target blasts (mid), and the elimination index (right) are reported. The elimination index was calculated according to the formula [1 − (number of alive blasts cultured with IR⁺ T cells/number of blasts cultured with IR⁻ T cells)]. (C) CDR3 α-(left) and β-chain (right) clonotype frequency in the IR⁺ and IR⁻ fractions, before and after sequential L-APCs stimulation, for all the three patients tested. The expanding dominant IR⁺ clone is highlighted in red. (D to G) In each patient, the dominant IR⁺ TCR identified at the end of the stimulation cultures was reconstructed and engineered into T cells derived from n = 1 (Pt#13) and n = 3 (Pt#14-15) different healthy donors. Edited T cells were then challenged with either autologous leukemic blasts or autologous healthy hematopoietic cells. The elimination index (D), the activation (E), degranulation (F), and cytokine production (G) abilities of engineered T cells are reported. The elimination index was calculated using the formula [1 − (number of alive target cells cocultured with engineered T cells/number of alive target cells cocultured alone)]. Two-way ANOVA was used to compare curves in (D), paired multiple Student’s t tests were used to compare groups in (E) to (G). The mean and the SEM are shown in (D) to (G). *P < 0.05; ***P < 0.001.

Fig. 4.. PeptiChip and trogocytosis allow to deorphanize tumor-specific T-Cell Receptors.

The trogocytosis effect was quantified in T cells engineered with the orphan TCRs of Fig. 3 and harnessed to perform ligandome analysis (PeptiChip). (A and B) Representative gates (A) and quantification (B) of the trogocytosis effect, defined as the percentage of blasts acquiring the CD3 T cell marker, in blast cultured with T cells engineered with Pt#13 to Pt#15 TCRs, at different effector:target ratios. (C) Experimental layout for the application of trogocytosis and PeptiChip for antigen discovery. (D) Number of 8- to 11-mers retrieved after ligandome analysis of total, CD3⁺, and CD3⁻ blast fractions cocultured with T cells engineered with either Pt#14 or Pt#15 TCRs, before (left) and after (right) quality control. The relevant epitopes further studied in the following panels are highlighted in red. (E and F) T cells derived from n = 5 different healthy donors and engineered with Pt#13 and Pt#14 TCRs were challenged in vitro with EBV-immortalized B cell lines sharing one or more HLA with the patients and pulsed with the isolated peptides. The elimination index (E) and the number of INF-γ⁺TFN-α⁺ CD8⁺ T cells (F) is reported. The elimination index was calculated using the formula [1 − (number of alive peptide-pulsed target cells cocultured with engineered T cells/number of alive non peptide-pulsed target cells cocultured with engineered T cells)]. (G and H) The expression of fatty acid–binding protein 5 (FAPB5) and histone 2B (H2B) isoforms (HIST1H2BA-B-C-G-H-K-L) were quantified in Pt#14 and Pt#15, respectively (G), and in a cohort of N = 32 matched diagnosis/relapse blast samples pairs (H). The mean and the SEM are shown in (B), (E), and (F). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.