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An innate-like Vδ1+ γδ T cell compartment in the human breast is associated with remission in triple-negative breast cancer

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Science Translational Medicine  09 Oct 2019:
Vol. 11, Issue 513, eaax9364
DOI: 10.1126/scitranslmed.aax9364
  • Fig. 1 Healthy human breast tissue harbors tissue-resident Vδ1+, Tc1-skewed γδ T cells.

    (A) Representative flow cytometry plots showing the gating strategy to identify lymphocytes including γδ T cell subsets isolated from human breast tissue and following grid culture. Lymphocytes were gated on size and scatter (1), followed by live-dead exclusion (2), a singlet gate (3), and CD45 (4) before subsetting (5 to 8). (B) Summary dot plots showing TCRγδ+ cells isolated from healthy human breast tissue, expressed as a percentage of recovered CD3+ cells (n = 29) (median indicated). In a subset of these, Vδ chain usage was quantified and expressed as percentage of pan-TCRγδ+ (n = 18) (medians indicated). (C) Expression of cell surface markers NKG2D, CD28, PD-1, CD103, and CD69 on Vδ1+ T cells (n = 9 to 11) (medians indicated). (D) Functional phenotype of tissue-resident Vδ1+ T cells. Dot plots showing intracellular cytokine staining for IFN-γ (n = 12), IL-13 (n = 8), IL-17A (n = 9), TNF (n = 10), and cell surface CD107a (n = 5) expression, after in vitro stimulation of bulk CD3+ cultures with PMA and ionomycin (4 hours) (medians indicated). (E) Summary data showing the percentages of breast-resident Vδ1+ or CD4 αβ T cells stained intracellularly for IL-17A. Cells were isolated by explant culture and then grown in two forms of IL-17–skewing media, followed by in vitro activation with PMA and ionomycin (4 hours) (n = 3, except for Vδ1+ cells grown in TGF-β–containing medium, where n = 2) (mean with SEM indicated).

  • Fig. 2 Breast tissue–resident Vδ1+ T cells are innate-like.

    (A) Summary data showing intracellular staining for IFN-γ (n = 19 for Vδ1+ and n = 15 for CD8+), TNF (n = 17 for Vδ1+ and n = 13 for CD8+), and cell surface CD107a (n = 10 for Vδ1+ and n = 5 for CD8+) expression, after in vitro activation of breast-resident Vδ1+ cells or of Vδ1+ and CD8+ αβ T cells from within the same cultures, exposed to plate-bound recombinant MICA (10 μg/ml) in the presence of brefeldin A (BFA). Plotted as percentage of parent Vδ1+ or CD8+ gate. (B) Summary data showing intracellular staining for IFN-γ (n = 6 for Vδ1+ and n = 6 for CD8+) and TNF (n = 5 for Vδ1+ and n = 5 for CD8+) after in vitro activation of breast tissue–resident Vδ1+ and CD8+ αβ T cells with low-dose plate-bound anti-CD3 antibody (50 ng/ml) with or without plate-bound recombinant MICA (10 μg/ml) in the presence of BFA. Where indicated, MICA-stimulated cells were pretreated with anti-human NKG2D antibody (plotted as percentage of parent Vδ1+ or CD8+ gate). (C) Summary data for breast-resident Vδ1+ T cells, showing intracellular IFN-γ production after in vitro activation with IL-12 (n = 3) or IL-18 (n = 3) or IL-12 + IL-18 (n = 9) and with medium alone (n = 9). For all panels, mean with SEM is indicated. **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001, Kruskal-Wallis with post hoc Dunn’s test corrected for multiple testing.

  • Fig. 3 Innate-like Vδ1+ Tc1 cells are a major subset of breast TILs.

    (A) γδ T cells were consistently isolated from human breast tumor samples obtained from mastectomies after grid culture. Dot plots showing numbers of TCRγδ+ cells isolated from paired nonmalignant breast tissue (•) or tumor (ο) expressed as a percentage of CD3+ T cells. γδ T cells were further phenotyped for Vδ1+, Vδ2+, and Vδ1Vδ2 TCR usage expressed as a percentage of TCRγδ+ T cells (n = 25) (medians indicated). (B) Breast tumor–infiltrating Vδ1+ T cells are functionally skewed. Dot plots showing intracellular cytokine staining for IFN-γ (n = 12), IL-13 (n = 11), IL-17A (n = 4), TNF (n = 9), and cell surface CD107a expression (n = 4), after in vitro activation with PMA (10 ng/ml) and ionomycin (1 μg/ml) in the presence of BFA (medians indicated). (C) Summary data showing intracellular staining for IFN-γ (n = 9 to 12, depending on activating condition), TNF (n = 7), and cell surface CD107a (n = 4 to 7, depending on activating condition) expression after in vitro activation of Vδ1+ and CD8+ αβ T cells with plate-bound recombinant MICA (10 μg/ml) in the presence of BFA or in vitro activation with IL-12 (100 ng/ml) and IL-18 (100 ng/ml). Where indicated, MICA-stimulated cells were pretreated with blocking anti-human NKG2D antibody (mean with SEM indicated). (D) Tumor cell lines, MCF7 and HCC1954, were incubated with negatively sorted γδ T cells (γδ) derived from nonmalignant breast tissue or breast tumor at E:T of 5:1, in the presence (γδ + αNKG2D) or absence (γδ) of a blocking anti-NKG2D antibody for 48 hours. Cell lines without effector γδ T cells were used as negative controls (Control). Dot plots show concentrations of caspase-cleaved cytokeratin 18 (cCK18). Each data point represents the mean of two technical replicates; the median values for those data points are indicated by a horizontal line (note that there were only three donors for the killing assay of HCC1954 cells by tumor-derived γδ T cells in the presence of anti-NKG2D). △ and ○ are two donors for which there were paired nonmalignant breast tissue and breast tumor. *P < 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001, Kruskal-Wallis with post hoc Dunn’s test corrected for multiple testing.

  • Fig. 4 Vδ1+ T cells in TNBC are predictive of disease-free survival and OS.

    (A) Overall landscape of T cell subsets in nonmalignant breast tissue (“Tissue”) and matched tumor tissue (“Tumor”), determined by quantitative sequencing of rearranged TCR genes from patients undergoing mastectomy for TNBC. Absolute TCR copies (a surrogate for T cell numbers) are plotted per microgram of input DNA. Individual patients plotted: red, patients with relapsed disease; blue, patients in remission (median bar plotted). *P < 0.05 and **P ≤ 0.01, Wilcoxon matched pairs signed rank test. (B) Intratumoral presence of αβ T cells and γδ T cells in patients with subsequent relapsed disease versus those who remained in remission. *P < 0.05, Kolmogorov-Smirnov test. (C) PFS (months from surgery) split on median T cell subsets found in 11 TNBC tumors. *P < 0.05 and **P ≤ 0.01, Gehan-Breslow-Wilcoxon test. (D) OS (months from surgery) split on median T cell subsets found in 11 TNBC tumors. *P < 0.05 and **P ≤ 0.01, Gehan-Breslow-Wilcoxon test.

  • Fig. 5 Lack of tumor Vδ1+ TCR focusing relative to focusing of TCRα sequences.

    (A) Examples of circular tree plots of the Vδ1+ repertoire in paired nonmalignant tissue (“tissue”) and tumor tissue (“tumor”), where each circle represents a unique TCR clonotype and the size of the circle is proportional to the representation of the specified clone. Plots were generated from total Vδ1+ TCRs. (B) Examples of circular tree plots of the TCRα repertoire in paired nonmalignant tissue (“tissue”) and tumor tissue (“tumor”). Plots were generated from total TCRα+ TCRs. (C) Vδ1+ and TCRα+ sequences in nonmalignant and tumor tissue were down-sampled (within each patient) to equivalent numbers to calculate diversity metrics. The degree of repertoire focusing was assessed by the delta of the Gini coefficient and the delta D50 of sequences of Vδ1 chains and the top 10% in abundance of TCRα sequences in tumor versus paired nonmalignant repertoires. To test whether the degree of repertoire focusing was different between Vδ1+ and TCRα+ compartments in individual patients, the Wilcoxon matched pairs signed-rank test was used to compare ΔGini and ΔD50. All sequences were analyzed on the basis of amino acid sequence. n = 11.

  • Fig. 6 No detectable public intratumoral Vδ1+ clonotypes.

    (A) Intersections of Vδ1+ clonotypes between 11 patient tumor samples. Vertical bars represent the number of unique TCRs and the dot matrix represents sharing of TCRs across patients. A shared or public clonotype would be represented by at least two red dots (sharing between two patients) joined by a vertical red line. Private sequences are presented by an unconnected single red dot. (B) Intersections of Vδ2+ clonotypes between 11 patient tumor samples. All sequences were analyzed on the basis of amino acid sequence.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/513/eaax9364/DC1

    Fig. S1. Explant culture permitted the isolation of substantial numbers of human tissue-resident lymphocytes.

    Fig. S2. Vδ1+ T cells display innate-like responsiveness.

    Fig. S3. αβ and γδ T cells could be isolated from breast tumors and phenotypically resemble those from healthy tissue.

    Fig. S4. Both αβ and γδ T cells are enriched in tumors compared with paired nonmalignant tissue.

    Fig. S5. Vδ1+ T cells show no evidence of tumoral clonal focusing in contrast to αβ T cells.

    Fig. S6. There is limited Vδ1 repertoire overlap between tumor and paired nonmalignant tissue within patients.

    Table S1. Lymphocyte subtypes observed ex vivo after enzymatic digestion and in grid explant cultures.

    Table S2. Clinical features of KCL TNBC cohort.

    Table S3. Clonality metrics of down-sampled TCRs.

    Table S4. Clonality metrics of raw TCRs.

    Table S5. Public intratumoral phospho-antigen reactive Vδ2 CDR3 sequences and samples in which they were shared.

    Table S6. Antibodies and key reagents table.

    Data file S1. Primary data.

  • The PDF file includes:

    • Fig. S1. Explant culture permitted the isolation of substantial numbers of human tissue-resident lymphocytes.
    • Fig. S2. Vδ1+ T cells display innate-like responsiveness.
    • Fig. S3. αβ and γδ T cells could be isolated from breast tumors and phenotypically resemble those from healthy tissue.
    • Fig. S4. Both αβ and γδ T cells are enriched in tumors compared with paired nonmalignant tissue.
    • Fig. S5. Vδ1+ T cells show no evidence of tumoral clonal focusing in contrast to αβ T cells.
    • Fig. S6. There is limited Vδ1 repertoire overlap between tumor and paired nonmalignant tissue within patients.
    • Table S1. Lymphocyte subtypes observed ex vivo after enzymatic digestion and in grid explant cultures.
    • Table S2. Clinical features of KCL TNBC cohort.
    • Table S3. Clonality metrics of down-sampled TCRs.
    • Table S4. Clonality metrics of raw TCRs.
    • Table S5. Public intratumoral phospho-antigen reactive Vδ2 CDR3 sequences and samples in which they were shared.
    • Table S6. Antibodies and key reagents table.

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    Other Supplementary Material for this manuscript includes the following:

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