Research ArticleCELL BIOLOGY

Massive clonal expansion of medulloblastoma-specific T cells during adoptive cellular therapy

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Science Advances  27 Nov 2019:
Vol. 5, no. 11, eaav9879
DOI: 10.1126/sciadv.aav9879
  • Fig. 1 Clonal T cell expansion in murine splenocytes after ACT against medulloblastoma.

    (A) Experimental layout of ACT in tumor-bearing mice. Intracranial tumor–bearing hosts received 9 gray (Gy) total body irradiation for host conditioning followed by adoptive transfer of tumor-reactive T cells and weekly DC vaccines. Hosts also receive hematopoietic stem cell transfer after total body irradiation to protect from bone marrow failure. (B) Splenocytes of treated animals were harvested at 30, 50, and 120 days for long-term survivors, were analyzed for TCR sequencing, and revealed selective expansion of four TCR Vβ families. (C) Intrafamily analysis revealed that TCR Vβ 13-02 family T cells experienced clonal expansion of one clone. Other families did experience clonal expansion. (D) Fifteen TCR Vβ families from bulk tumor-reactive T cells were isolated using FACS, and each family was cocultured against target NSC tumor cells. IFN-γ secretion was measured as an indication of the recognition of cognate tumor antigen. (E) NSC express a luciferase reporter, allowing for bioluminescent imaging of tumor growth that was conducted in mice treated with ACT. (F) Relative expansion of adoptively transferred T cells in the TCR Vβ 13 family in murine peripheral blood mononuclear cells (PBMCs) was measured in concurrence with tumor growth using flow cytometry. (G) Relative expansion of adoptively transferred T cells in the TCR Vβ 4 family in murine PBMCs was also measured as a control as this Vβ family did not demonstrate prior antitumor reactivity. (H) Cerebellar NSC medulloblastoma-bearing mice received ACT with either bulk ex vivo expanded tumor-reactive T cells or tumor-reactive T cells that only express TCR Vβ 13 or Vβ 4. Experiments (D) to (G) were conducted at least three times with 7 to 10 mice per experiment. (H) was conducted twice with n = 7 mice per group.

  • Fig. 2 Selective expansion of the tumor-reactive TCR Vβ family in mice responsive to ACT.

    To generate antitumor T cells, total RNA is extracted from tumor cells and electroporated into syngeneic bone marrow–derived DCs. These cells are then cocultured with splenocytes from a previously immunized mouse with interleukin-2 for 5 to 7 days generating a polyclonal population of CD8+ T cells. After this ex vivo activation, 107 T cells are adoptively transferred into tumor-bearing mice followed by vaccination with 2.5 × 105 RNA-pulsed DCs. In the preclinical model of ACT, C57BL/6 mice receive orthotopic tumor followed by host conditioning with total body irradiation and hematopoietic stem cell transfer to protect from bone marrow failure. (A) Mice implanted with cerebellar NSC medulloblastoma were treated with ACT using DsRed+ tumor–reactive T cells. Spleens were harvested from all mice, and relative abundance of each TCR Vβ family was measured in both responders and nonresponders. Here, 25 mice are implanted with tumor and treated with ACT. The first five nonresponders that succumb to tumor are taken at humane end point and spleens were analyzed. The five responders are treated mice that demonstrate no evidence of tumor after 120 days. This experiment was repeated twice with the same results as shown. n = 5 to 7 mice per group. (B) Spleens of five asymptomatic long-term survivors were harvested at 90 days after ACT. DsRed+ T cells were isolated and separated by the TCR Vβ family. Each TCR Vβ family was cocultured in vitro against tumor cells, and IFN-γ secretion was measured. (C) Splenic T lymphocytes were harvested from nonresponders to therapy upon detection of tumor via bioluminescent imaging. DsRed+ T cells were FACS-isolated and sorted into TCR Vβ families and then used as effectors against the primary NSC cell line. IFN-γ was measured to determine antitumor reactivity.

  • Fig. 3 Identification of tumor-specific T lymphocytes in a preclinical glioma model.

    (A) Tumor-reactive T cells were generated in vitro and separated into 15 TCR Vβ families using sterile FACS isolation. T cells (4 × 105) per Vβ family were cocultured against 4 × 104 KR158B tumor target cells overnight, and supernatant IFN-γ was measured as an indication of the recognition of cognate tumor antigen. All conditions were conducted in triplicate, and the experiment was repeated an additional three times with the same results. (B) Fifteen mice received ACT using DsRed+ tumor–reactive T cells. Relative frequencies of TCR Vβ families within the adoptively transferred DsRed+ T cell population were compared between the first five nonresponders to therapy and five long-term survivors with no signs of tumor. (C) Spleens of the asymptomatic long-term survivors were also harvested for DsRed+ T cells, which were further separated by the TCR Vβ family using FACS. Each TCR Vβ family was cocultured in vitro against tumor cells as above, and IFN-γ secretion was measured.

  • Fig. 4 TCR Vβ 6+ T cells are required for efficacy of ACT against glioma.

    (A) Seven C57BL/6 mice received orthotopic KR158B tumor by implanting 104 tumor cells into the right caudate nucleus of the cortex. (B) This tumor line has a luciferase reporter that allowed for in vivo bioluminescent imaging of tumor growth of mice over time. Relative frequency of (C) TCR Vβ 6+. (D) Intracranial tumor–bearing mice received either ACT using bulk tumor-reactive T cell population, ACT using only TCR Vβ 6+ T cells, ACT using only TCR Vβ 8.1/8.2+ T cells, or ACT using only TCR Vβ 6+ and Vβ 8.1/8.2+ T cells. No significant differences in survival was found between bulk ACT and the group that received TCR Vβ 6+ T cells, n = 7 mice per group. (E) Intracranial tumor–bearing mice received either ACT, ACT with T cells depleted of Vβ 6+ T cells using FACS, ACT with T cells depleted of TCR Vβ 8.1/8.2+ T cells, or ACT with TCR Vβ 2+ T cells. We found a significant decrease in survival in the group where TCR Vβ 6+ cells were depleted as compared to bulk ACT, *P = 0.0003, n = 7 mice per group.

  • Fig. 5 PD-1 immune checkpoint blockade increases expansion of tumor-specific T cells.

    (A) Twenty-eight C57BL/6 mice received orthotopic KR158B glioma by implanting 104 tumor cells into the right caudate nucleus and then randomized into four groups. Mice received either no treatment, αPD-1 only, ACT only, or ACT + αPD-1 (n = 7 mice per group). There was no statistically significant increase in survival between ACT and ACT + αPD-1 groups, P = 0.1755. (B) KR158B tumor–bearing mice received ACT using DsRed+ tumor–reactive T cells alone, or (C) ACT + αPD-1. Tumor growth was followed weekly with bioluminescent in vivo imaging. At the same time points, peripheral blood was drawn to follow relative frequencies of DsRed+ TCR Vβ 6+ T cells over time in both groups, n = 7 mice per group.

  • Fig. 6 Clonal T cell expansion in recurrent medulloblastoma patient PBMCs after ACT.

    A patient with recurrent medulloblastoma was treated with ACT using autologous ex vivo activated T cells and experienced long-term survival with >2-year nonprogressing tumor. (A) TCR sequencing was conducted on the patient’s tumor-reactive T cells and on patient PBMCs taken at 2, 4, 6, and 16 weeks after ACT. All analysis and bioinformatics were conducted by Adaptive Biotechnologies (Seattle, WA). Productive frequencies of 59 TCR Vβ families were analyzed. Clonal analysis revealed hyperexpansion of five T cell clones, each expanding to greater than 5% productive frequency in PBMCs 16 weeks after treatment. Combined, these five clones make up 28.72% productive frequency of patient PBMCs. (B) Top four TCR Vβ families with the highest expression at 16 weeks after adoptive T cell transfer were plotted relative to the other 54 families at all time points (TCR Vβ 3, Vβ 5-01, Vβ 9-01, and Vβ 27-01). Clonal analysis of the 1000 clones within each family was conducted, revealing that the expanded families are largely composed of the expansion of a single clone. Each color on the graphs represents a single sequence. (C) TCR Vβ 9-01+ T cells from PBMCs were FACS-isolated and cocultured against tumor RNA–pulsed autologous DCs or GFP RNA–pulsed DCs in triplicate. IFN-γ secretion was measured.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/11/eaav9879/DC1

    Fig. S1. TILs in untreated mice.

    Fig. S2. Adoptive transfer of TCR Vβ6, 7, 8.1/8.2, or 11 does not provide survival benefit against NSC.

    Fig. S3. Relative expansion of TCR Vβ families in peripheral blood after ACT.

    Fig. S4. Tumor-reactive T cells retain function within tumor.

    Fig. S5. ACT in recurrent medulloblastoma and PNET.

    Fig. S6. Lack of single clonal expansion over time in nonresponder to ACT.

    Table S1. Clonal expansion in patient PBMCs after ACT.

    Table S2. Productive frequency of TCR Vβ families in patient MNCs before ACT, ex vivo expanded ttRNA T cells, and at follow-up after T cell infusion.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. TILs in untreated mice.
    • Fig. S2. Adoptive transfer of TCR Vβ6, 7, 8.1/8.2, or 11 does not provide survival benefit against NSC.
    • Fig. S3. Relative expansion of TCR Vβ families in peripheral blood after ACT.
    • Fig. S4. Tumor-reactive T cells retain function within tumor.
    • Fig. S5. ACT in recurrent medulloblastoma and PNET.
    • Fig. S6. Lack of single clonal expansion over time in nonresponder to ACT.
    • Table S1. Clonal expansion in patient PBMCs after ACT.
    • Table S2. Productive frequency of TCR Vβ families in patient MNCs before ACT, ex vivo expanded ttRNA T cells, and at follow-up after T cell infusion.

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