Immunotransplantation preferentially expands T-effector cells over T-regulatory cells and cures large lymphoma tumors

Abstract

Ex vivo-expanded tumor-infiltrating lymphocytes infused into lymphodepleted recipients has clear antitumor efficacy. More practical sources of such antitumor lymphocytes would broaden the application of this approach. Previously, we described an in situ vaccination combining chemotherapy with intratumoral injection of CpG-enriched oligonucleotides, which induced T-cell immunity against established lymphoma. An ongoing clinical trial of this maneuver has demonstrated clinical responses in lymphoma patients. Here, we use this vaccine maneuver to generate immune cells for transfer into irradiated, syngeneic recipients. Transferred tumor-specific T-effector (T(eff)) cells preferentially expanded, increasing the T(eff)/T-regulatory (T(reg)) ratio in these "immunotransplantation" recipients and curing large and metastatic tumors. Donor T cells were necessary for tumor protection, and CD8 T-cell immune responses were enhanced by posttransplantation booster vaccination. Hematopoietic stem cell transplantation is a standard therapy for lymphoma. Therefore, in situ tumor vaccination followed by immunotransplantation of harvested tumor-specific T cells could be directly tested in clinical trials to treat otherwise resistant malignancies.

Figures

Figure 1

Treg increase in CpG/CTX-vaccinated mice and preferential Teff proliferation during immunotransplantation. (A) Mice received CpG/CTX vaccination as described. (B) On day 15 after vaccination, donor mice splenocytes were assessed for T-cell subsets by flow cytometry. (C) Splenocytes were taken from wild-type donor mice and labeled with 5 μg/mL CFSE and injected along with unlabeled BM cells into recipients that received either no (0 cGy) or lethal (900 cGy) TBI. (D) On day 15 after transplantation, splenocytes were taken from 3 recipient mice and separately measured by flow cytometry for CFSE, surface CD4, and intracellular foxP3. Data shown are gated on live, CFSE+, CD4+ cells. Data shown are representative of the 3 individual recipients.

Figure 2

Immunotransplantation enhances tumor-specific T-cell responses and protects against high-dose tumor challenge. (A) Mice received either no vaccine, vaccination with CpG/CTX, or syngeneic BM and CFSE-labeled splenocytes from vaccinated donors after either no irradiation (“full” recipients) or 900 cGy of TBI (“empty” recipients). On day 3 after transplantation, mice (10 per cohort) were challenged with 107 A20 cells subcutaneously. (B,D) On day 15 after transplantation, mice were bled and PBMCs tested for tumor-specific IFN-γ production. (B) Graphs are gated for CD3+ lymphocytes, and statistics are IFN-γ+ cells as a percentage of all CD44hi cells. (C) Tumor growth curves are composites for each cohort. Error bars represent plus or minus 1 SD. (D) (Top right panel) Mean fluorescent intensity (MFI) of IFN-γ–producing cells related to CFSE dilution. The MFI of IFN-γ+ cells (5.1) is lower than that of IFN-γ−, 12.1. (Bottom panels) “Empty” mice contain more (22.5%) CD3+CD44hi cells (defined as in panel A) than their “full” mice counterparts (1.7%). (E) On day 45 after transplantation, 9 mice were rebled and PBMCs were separately assayed as in panels B and D, except that gated are CD8+ cells and indicated are the proportion of IFN-γ+ cells as a percentage of CD44hi cells.

Figure 3

Immunotransplantation protects against systemic tumor burden. Donor mice received (A) no treatment or (B) CpG/CTX vaccination as described. On day 0 (7 days after the completion of the CpG/CTX vaccine), recipient mice received 9 Gy of TBI followed by transplantation of 5 × 106 BM cells and splenocytes from donors. Mice were challenged on day 1 after transplantation with 106 A20-LUC cells intravenously and followed clinically and per their bioluminescence. The same representative mice are shown over time. (C) Cohorts of recipient mice (n = 10) receiving the same treatments and tumor challenge with 106 wild-type A20 cells intravenously were followed for clinical signs of illness and survival.

Figure 4

Immunotransplantation of CD8 T cells is both necessary and sufficient for tumor protection. (A) Donor mice received CpG/CTX vaccination, and splenocyte cell subsets were purified from CpG/CTX vaccinated donors using mAb-conjugated ferromagnetic beads to either positively select or deplete specific populations. (B) Resulting populations were gated on live lymphocytes, and purity was assessed by flow cytometry. (C) CpG/CTX donor splenocyte subsets were used in immunotransplantation as described. Recipient mice received high-dose tumor challenge on day 3 after transplantation and were followed for bidimensional tumor size. Proportions of tumor-free mice are indicated. One mouse in the “CD4+CD8” group died within a week after transplantation but still had palpable tumor.

Figure 5

Donor requirements to transfer antitumor immunity. (A) Donor mice received no treatment, A20 tumor challenge, A20 tumor challenge followed by CTX, or A20 tumor challenge followed by CTX and intratumoral CpG. On day 0 (7 days after the completion of the CpG/CTX vaccine), recipient mice received 9 Gy of TBI followed by 5 × 106 BM cells and splenocytes from donors. One group received a simultaneous boost of 106 A20 cells, which were cultured with CpG-1826 at 3 μg/mL for 72 hours, then irradiated (50 Gy). On day 3 after transplantation, all cohorts of mice received high-dose tumor challenge. (B) On day 15, posttransplantation/transfer mice were bled and assayed by flow cytometry for tumor-specific CD8 T-cell responses as described. Indicated are the percentage of IFN-γ–producing CD8+ live lymphocytes (n = 3 per cohort). (C) Cohorts of mice were followed for bidimensional tumor size, and proportions of tumor-free mice are indicated. One mouse in the “no treatment donor” cohort (first column) showed minimal subcutaneous growth but manifested systemic disease (with hind-limb paralysis) and was killed on day 30.

Figure 6

Immunotransplantation-induced tumor immunity increases with time and with serial transplantations. (A) Donor mice received CpG/CTX vaccination, and recipient mice received TBI. (B) Donor splenocytes and BM were transferred to recipients that received 900, 600, or 300 cGy as depicted. (C) Recipient mice received 900 cGy of TBI followed by 5 × 106 BM cells and splenocytes from donors at donor/recipient ratios of 1:1, 1:4, or 1:8, as depicted. (B,C) Recipient mice received high-dose tumor challenge on day 3 after transplantation and were followed for bidimensional tumor size. Proportions of tumor-free mice are indicated. (D) Serial immunotransplantation. Cured immunotransplantation recipients from experiments conducted as in panel A were subsequently used as donors in a serial immunotransplantation. Secondary recipients received either no irradiation or 900 cGy of TBI as depicted, followed by 5 × 106 BM cells and splenocytes from cured immunotransplantation mice. On day 3 after (secondary) transplantation, recipients were challenged with 107 A20 cells subcutaneously and (E) followed for tumor growth.

Figure 7

Immunotransplantation cures mice with large tumors. (A) Donor mice received no treatment or CpG/CTX vaccination. Recipient mice were challenged with 107 A20 cells subcutaneous 14 days earlier, then received either no irradiation or 900 cGy of TBI followed by 5 × 106 BM cells and splenocytes from donors. Nonirradiated mice also received an intravenous boost of A20 cells stimulated with CpG followed by irradiation (CpG-A20 cells) at the time of transplantation. (B) On day 15 after transplantation, mice were bled, and IFN-γ–producing tumor-specific CD8 T cells were assayed by flow cytometry as described (n = 3). (C) Cohorts of mice (n = 10) were followed for bidimensional tumor size, and proportions of tumor-free mice are indicated. Three mice from the “no vaccine donor” cohort died within 3 days after transplantation, still with palpable tumor. (D) Tumor (400-mm2)–bearing mice were used as immunotransplantation recipients as in panels A to C from donors receiving no treatment or CpG/CTX vaccination; shown are photographs of the same mice over time. (E) Tumor (100 mm2)–bearing mice were used as immunotransplantation recipients and were killed on day 8 after transplantation. Excised tumors were stained for CD3 and visualized per standard immunoperoxidase protocol (original magnification ×20, representative of 10 fields examined).