Research ArticleCancer

Pediatric patients with acute lymphoblastic leukemia generate abundant and functional neoantigen-specific CD8+ T cell responses

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Science Translational Medicine  26 Jun 2019:
Vol. 11, Issue 498, eaat8549
DOI: 10.1126/scitranslmed.aat8549
  • Fig. 1 Putative cancer neoantigens elicit robust T cell responses.

    (A) Enriched CD8+ TILs from three patients were independently cocultured with aAPCs expressing a single patient-specific HLA molecule and pulsed with anti-human CD28/CD49d (1 μg/ml) and either 1 μM indicated 15-mer peptide or SEB (100 μg/ml). Cytokine production by CD8+ TILs was subsequently measured by intracellular cytokine staining (ICS). Normalized frequency (neoantigen peptide compared to SEB) of responding single, live CD8+ lymphocytes that produced cytokines (IFNγ or TNFα) after stimulation is plotted for each patient with ALL. (B) Representative flow cytometry plots from one patient depicting single, live CD8+ lymphocytes that are CD107a+ (left), IFNγ+ (middle), or TNFα+ (right) after irrelevant peptide, neoantigen peptide, or polyclonal (SEB; positive control) stimulation. Flow plots for negative controls (unstimulated cells lacking peptide stimulation and isotype controls) are also shown and were used to set gates. SSC-A, side scatter area. (C) For six patients, 1 × 106 to 2 × 106 bone marrow mononuclear cells (BMMCs) were stimulated with anti-human CD28/CD49d (1 μg/ml) and either 1 μM indicated 15-mer peptide, SEB (100 μg/ml), or 1× phorbol 12-myristate 13-acetate (PMA)/ionomycin (iono) cell stimulation cocktail (as a positive control; see Materials and Methods) and then subjected to ICS. Normalized frequency (see Materials and Methods) of responding single, live CD8+ T cells that produced cytokines (IFNγ or TNFα; left) or degranulated (right) after stimulation is plotted for each patient with ALL. (D) T cell response statistics for all putative neoantigens per patient (bar chart) and at the cohort level (pie charts). (E) For three patients, 2 × 106 BMMCs were pulsed with serial dilutions (1 μM to 10 pM) of the indicated 15-mer peptide, SEB (100 μg/ml), or 1× PMA/ionomycin cell stimulation cocktail and subjected to ICS. Normalized frequency of responding CD8+ T cells that produced cytokines (IFNγ or TNFα) after stimulation is plotted.

  • Fig. 2 Endogenous neoantigens are processed, presented, and induce CD8+ T cell responses.

    (A) Schematic depicting the generation of TMG constructs (step 1) and in vitro–transcribed (IVT) RNA (step 2) used to screen for recognition of putative somatic mutations. BMMCs were enriched for CD19+ cancer cells and CD8+ T cells (step 3). Cancer cells were then transfected with IVT RNA (step 4) and cocultured with enriched CD8+ T cells (step 5). Cocultured CD8+ T cells were subsequently subjected to ICS, and the frequency of cytokine producing cells was determined (step 6). Flow cytometry plots depict the representative purity after selection of CD19+ (left) and CD8+ T cells (right). (B) Representative flow cytometry plots from one patient depicting single, live CD8+ lymphocytes that are IFNγ+ or TNFα+ after coculture with autologous CD19+ cancer cells transfected with mock-TMG RNA, wild-type (WT) TMG RNA, mutant TMG RNA, or enriched CD8+ T cells stimulated with PMA/ionomycin. (C) Normalized frequency of IFNγ- and TNFα-producing CD8+ T cells after coculture with autologous tumor cells transfected with wild-type TMG RNA (open circles) or mutant TMG RNA (filled circles) from three patients. (D) Schematic depicting the generation of TMG constructs (step 1) used to transfect aAPCs (step 2) to screen for recognition of putative somatic mutations. BMMCs were enriched for CD8+ T cells (step 3). aAPCs transfected with TMG plasmid DNA were cocultured with enriched CD8+ T cells (step 4). Cocultured CD8+ T cells were interrogated by ICS, and the frequency of cytokine producing cells was determined (step 5). (E) Normalized frequency of IFNγ- and TNFα-producing CD8+ T cells after coculture with aAPCs transfected with wild-type TMG plasmid DNA (open circles) or mutant TMG plasmid DNA (filled circles) from three patients. (F) Sorted CD8+ TILs (2 × 104 to 3 × 104) from two patients were independently cocultured with either 8 × 104 autologous CD19+ tumor cells (dark gray shaded) or 8 × 104 aAPCs expressing patient-specific HLAs (negative control; light gray shaded). CD8+ TIL expansion and tumor reactivity were determined by flow cytometry after 21 days of coculturing by comparing the frequency of mutant tetramer-positive CD8+ TILs [ERG009, PE- and APC-conjugated HLA-A*30:02 PLCD3(311–319)MUT; ETV078, PE-conjugated HLA-A*03:01 GPR139(293–301)MUT] from autologous tumor and aAPC cocultures. Tetramer-positive gates were set on the basis of the binding of CD8+ TILs to irrelevant [ERG009, PE- and APC-conjugated HLA-A*24:02 CD101(884–892)IRR] or parent tetramers [ETV078, PE-conjugated HLA-A*03:01 GPR139(293–301)PAR]. FSC-A, forward scatter area; FSC-H, forward scatter height.

  • Fig. 3 Antitumor CD8+ T cell responses are neoepitope specific and form immunodominance hierarchies.

    (A) Representative gating strategy from one patient used to identify and quantify neoepitope-specific CD8+ T cells in the bone marrow of patients with ALL. HLA tetramer staining of HLA-restricted CD8+ TILs is depicted for ERG009. (B) Frequency of CD3+CD8+ TILs from six patients binding irrelevant and/or parent tetramers (black bars) and mutant tetramers (colored bars) within the bone marrow of patient samples. Dashed lines distinguish tetramers complexed with irrelevant and/or parent peptides from tetramers complexed with nonamer and decamer mutant peptides. (C) T cell response statistics for all putative neoepitopes per patient (bar chart) and at the cohort level (pie charts). (D) Scatterplot depicting the relationship between the total CD8+ T cell and neoepitope-specific CD8+ T cell response from six patients in our cohort. Correlation coefficient (r) and P value were calculated using the Spearman rank-order correlation test. (E) Representative tetramer gating from three patients used to identify and quantify neoepitope-specific CD8+ T cells in an additional cohort of patients with the ETV6-RUNX1 gene fusion. Representative flow cytometry plots depict the frequency of CD8+ TIL binding irrelevant, mutant tetramers (red outline; cancer neoantigen bound to patient-specific HLA), and/or wild-type (parent self-peptide bound to patient-specific HLA) tetramers. (F) Frequency of CD3+CD8+ TILs binding irrelevant (black bars) and ETV6-RUNX1 fusion tetramers (colored bars) within the bone marrow of five additional patients containing the ETV6-RUNX1 gene fusion. Dashed lines distinguish tetramers complexed with irrelevant peptides from tetramers complexed with fusion peptides derived from ETV6-RUNX1.

  • Fig. 4 Characterization and confirmation of the PLCD3-neoepitope TCRαβ repertoire in patient ERG009.

    Ex vivo–stained CD8+ TILs from ERG009 were single cell–sorted on the basis of PLCD3 tetramer binding [HLA-A*30:02 PLCD3(311–319) mutant tetramer] for TCR analysis. (A) Clonotypic analysis of paired TCR CDR3 regions (CDR3α and CDR3β) using single-cell multiplexed polymerase chain reaction (PCR) on sorted PLCD3+ CD8+ TILs from patient ERG009. Numbers adjacent to the pie chart slices represent the number of PLCD3 tetramer-binding cells within clonally expanded populations. (B) Flow cytometry analysis of PLCD3 tetramer-binding CD8+ T cells. Dot plot depicts populations of PLCD3 tetramer-negative (shaded black) and sorted PLCD3 tetramer-positive (gated, shaded gray) CD8+ TILs from ERG009. Events corresponding to the top four clonotypes (shaded blue, purple, red, and green) from the sorted cells were overlaid onto the tetramer-positive population. (C) Flow cytometry plots depicting the frequency of SUP-T1 nontransduced cells (light gray), CCRF-CEM cells expressing an irrelevant TCR (dark gray), and SUP-T1 TCR transduced cells containing CDR3α and CDR3β corresponding to clones marked in 4A binding either an irrelevant (left), parent PLCD3 (middle), or mutant PLCD3 tetramer (right). (D) Flow cytometry histograms showing the frequency of TCR-expressing cells [as in (C)]. (E) Ratio of PLCD3 tetramer-positive cells [from (C)] to TCR-expressing cells [from (D)].

  • Fig. 5 The transcriptional profiles of neoepitope-specific CD8+ TILs exhibit inter- and intrapatient heterogeneity.

    (A) Workflow of the experimental strategy for single-cell transcriptional studies. Bone marrow cells from three patients (ERG009, ETV001, and ETV078) were stained using an antibody cocktail and neoepitope-specific tetramers. CCR7CD45RO+ tetramer-binding CD8+ TILs were single-cell index–sorted for transcriptome studies. (B) Representative gating strategy from one patient depicting single cell–sorted CD8+ TIL subsets: tetramer-positive CCR7CD45RO+ (red outline) and tetramer-negative CCR7CD45RO+ (blue outline); 94 single cells from these two gates were sorted into 96-well plates. (C) Heatmap visualizing unscaled expression of genes (transcript expression threshold values; Et) and scaled surface protein data (median fluorescence intensity) for single cell–sorted neoepitope-specific CD8+ TILs (white indicates missing data) from three patients. Genes are ordered according to Ward’s method, and two clusters of relatively invariant genes were removed for ease of visualization. Top margin color bars represent, from top to bottom, groupings based on tetramer type (tetramer positive), hierarchical cluster number (clusters 1 to 3), and patient IDs (ERG009, ETV001, and ETV078). Bolded gene name colors represent transcription factors (black), inhibitory receptors (red), functional molecules (green), chemokine/chemokine receptors (blue), and transcriptional regulators (gray). (D) Representative bisulfite sequencing DNA methylation analysis of TCF7, TBX21, and IFNγ loci among bulk CCR7CD45RO+ neoepitope-specific CD8+ TILs from two patients.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/498/eaat8549/DC1

    Materials and Methods

    Fig. S1. Experimental pipeline used to identify cancer neoepitopes.

    Fig. S2. Phenotypic characterization of CD8+ TILs from human bone marrow samples.

    Fig. S3. Generation of aAPCs expressing single patient-specific HLA class I molecules.

    Fig. S4. Healthy donors exhibit negligible responses against endogenous neoantigens.

    Fig. S5. Neoepitope tetramers are patient specific and exhibit negligible nonspecific binding.

    Fig. S6. Phenotypic characterization of neoepitope-specific CD8+ TILs.

    Fig. S7. Patient-specific transcriptional profiles.

    Fig. S8. Phenotypic characterization of patient-specific CD19+ tumor cells.

    Table S1. Patient ALL subtypes.

    Table S2. FPKM values for HLA genes.

    Table S3. Patient-specific neoepitopes.

    Table S4. Tetramers used for specificity assays.

    Table S5. Fluidigm primer list.

    Table S6. Peptides for functional assays.

    Table S7. Mutant allele frequencies for sequenced mutations.

    Table S8. Single-cell indexed fluorescence-activated cell sorting median fluorescence intensity.

    Data file S1. Primary data.

    References (7181)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Experimental pipeline used to identify cancer neoepitopes.
    • Fig. S2. Phenotypic characterization of CD8+ TILs from human bone marrow samples.
    • Fig. S3. Generation of aAPCs expressing single patient-specific HLA class I molecules.
    • Fig. S4. Healthy donors exhibit negligible responses against endogenous neoantigens.
    • Fig. S5. Neoepitope tetramers are patient specific and exhibit negligible nonspecific binding.
    • Fig. S6. Phenotypic characterization of neoepitope-specific CD8+ TILs.
    • Fig. S7. Patient-specific transcriptional profiles.
    • Fig. S8. Phenotypic characterization of patient-specific CD19+ tumor cells.
    • Table S1. Patient ALL subtypes.
    • Table S2. FPKM values for HLA genes.
    • Table S3. Patient-specific neoepitopes.
    • Table S4. Tetramers used for specificity assays.
    • Table S5. Fluidigm primer list.
    • Table S6. Peptides for functional assays.
    • Table S7. Mutant allele frequencies for sequenced mutations.
    • References (7181)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S8 (Microsoft Excel format). Single-cell indexed fluorescence-activated cell sorting median fluorescence intensity.
    • Data file S1 (Microsoft Excel format). Primary data.

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