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. 2023 Apr 18;11(7):962–977. doi: 10.1158/2326-6066.CIR-22-0705

Figure 4.

Figure 4. Systemic treatment with mWTX-330 expands novel TCR clones and increases overall clonality of the TCR repertoire. EMT-6 tumor-bearing mice (n = 30 per group) were dosed twice a week for 2 weeks with mWTX-330 or vehicle. Tumors were collected at various time points for analysis (n = 6 per condition and time point). A, The frequency of polyfunctional CD8+ T cells was measured by examining coexpression of IFNγ, TNF, and Granzyme B after PMA/ionomycin restimulation. Tumors from vehicle mice at day 21 and 25 exceeded tumor burden and were not included in analysis. B, FFPE EMT-6 tumors from Day 11 were analyzed using the nanostring DSP system, and immunofluorescence staining of DAPI (blue), PanCK (pink), and CD8 (green) was performed. C, Volcano plot of transcripts differentially expressed by tumor-infiltrating CD8+ T cells between mWTX-330 and vehicle-treated mice. Pathway analysis was performed using the Nanostring DSP software and heat maps of genes associated with IL-12 signaling (D), IFNγ signaling (E), or TCR signaling (F) are reported (Supplementary Table S4). G–J, Live T cells were isolated from EMT-6 tumors on Day 11 and TCR sequencing was performed. G, The frequency of individual VDJ recombinations on the TCRβ chain. H, The frequency of the top 10 clones for each animal as a fraction of the total TCR repertoire. Each column represents the TCR repertoire of a single animal. I, The overall clonality index was calculated for these samples by treatment group. J, The top 100 TCR clones based on frequency were identified on the basis of CDR3 sequencing for each individual animal, and any clones that were present in the tumors of at least two of the vehicle-treated or two of the mWTX-330–treated animals were compared.

Systemic treatment with mWTX-330 expands novel TCR clones and increases overall clonality of the TCR repertoire. EMT-6 tumor-bearing mice (n = 30 per group) were dosed twice a week for 2 weeks with mWTX-330 or vehicle. Tumors were collected at various time points for analysis (n = 6 per condition and time point). A, The frequency of polyfunctional CD8+ T cells was measured by examining coexpression of IFNγ, TNF, and Granzyme B after PMA/ionomycin restimulation. Tumors from vehicle mice at day 21 and 25 exceeded tumor burden and were not included in analysis. B, FFPE EMT-6 tumors from Day 11 were analyzed using the nanostring DSP system, and immunofluorescence staining of DAPI (blue), PanCK (pink), and CD8 (green) was performed. C, Volcano plot of transcripts differentially expressed by tumor-infiltrating CD8+ T cells between mWTX-330 and vehicle-treated mice. Pathway analysis was performed using the Nanostring DSP software and heat maps of genes associated with IL-12 signaling (D), IFNγ signaling (E), or TCR signaling (F) are reported (Supplementary Table S4). G–J, Live T cells were isolated from EMT-6 tumors on Day 11 and TCR sequencing was performed. G, The frequency of individual VDJ recombinations on the TCRβ chain. H, The frequency of the top 10 clones for each animal as a fraction of the total TCR repertoire. Each column represents the TCR repertoire of a single animal. I, The overall clonality index was calculated for these samples by treatment group. J, The top 100 TCR clones based on frequency were identified on the basis of CDR3 sequencing for each individual animal, and any clones that were present in the tumors of at least two of the vehicle-treated or two of the mWTX-330–treated animals were compared.