Escape from suppression: tumor-specific effector cells outcompete regulatory T cells following stem-cell transplantation

Abstract

Immune reconstitution of autologous hematopoietic stem-cell transplant recipients with the progeny of mature T cells in the graft leads to profound changes in the emerging functional T-cell repertoire. In the steady state, the host is frequently tolerant to tumor antigens, reflecting dominant suppression of naive and effector T cells by regulatory T cells (T(regs)). We examined the relative frequency and function of these 3 components within the tumor-specific T-cell compartment during immune reconstitution. Grafts from tumor-bearing donors exerted a significant antitumor effect in irradiated, syngeneic tumor-bearing recipients. This was associated with dramatic clonal expansion and interferon-gamma (IFNgamma) production by previously tolerant tumor-specific T cells. While donor-derived T(regs) expanded in recipients, they did not inhibit the antigen-driven expansion of effector T cells in the early posttransplantation period. Indeed, the repopulation of tumor-specific effector T cells significantly exceeded that of T(regs), the expansion of which was limited by IL-2 availability. Although the intrinsic suppressive capacity of T(regs) remained intact, their diminished frequency was insufficient to suppress effector cell function. These findings provide an explanation for the reversal of tolerance leading to tumor rejection in transplant recipients and likely contribute to the efficacy of adoptive T-cell therapies in lymphopenic hosts.

Figure 1

Antitumor immunity visualized by A20HA-Luci imaging. (A) Experimental outline. Donor mice received 2.5 × 10⁶ CD4⁺-enriched HA-specific T cells with or without a tumor challenge (10⁶ A20HA-Luci intravenously 9 days before T-cell transfer). At 18 days after T-cell transfer, donor mice were killed, and their spleens and LNs were harvested and T cell–enriched to be transferred into transplant recipients. Recipients were challenged with or without 10⁶ A20HA-Luci intravenously 10 days prior to transplantation. Recipients underwent transplantation as described in “Methods.” A total of 3 tumor-bearing and 3 non–tumor-bearing mice were randomly removed from the donor pool and received plus or minus 10⁷ pfu VaccHA (subcutaneously). They were killed 6 days later to assess HA-specific T-cell function. (B) Percentage of HA-specific T-cell expansion in vivo and (C) IFNγ production in response to HA peptide in vitro in these non–tumor-bearing (NT) and tumor-bearing (T) mice are shown. Data represent mean plus or minus SE. (D,E) Photon emission was used as an indication of tumor size and dissemination. Images of 4 ear-tagged mice per group at the indicated time points are shown.

Figure 2

Endogenous activation of tumor-specific T cells in HSCT setting. Recipient mice with (A,B,D) or without (C) established tumor underwent transplantation on day 0 (D0), then were inoculated with or without 10⁷ pfu of VaccHA (subcutaneously) 15 days (A,B) or 35 days (C,D) after HSCT/adoptive transfer, and killed 6 days later. Percentage of HA-specific T cells (A) and IFNγ production in response to HA peptide (B-D) in recipient mice was measured. Values are means plus or minus SE of triplicate cultures from 3 mice in each group.

Figure 3

The frequency of antigen-specific T_regs in transplant recipients decreases immediately after HSCT. Donor mice (Thy1.2^+/+) with a 10-day established tumor burden received 2.5 × 10⁶ CD4⁺-enriched HA-specific T cells (Thy1.1⁺/1.2⁺). At 19 days after T-cell transfer, donor mice were killed, and their spleens and LNs were harvested and T-cell–enriched to be transferred into transplant recipients. Recipients (Thy1.1^+/+) were challenged with 1 × 10⁶ A20HA intravenously 10 days prior to HSCT and underwent transplantation as described in “Methods.” Half of the transplant recipients received daily injections of 10 μg/mouse IL-2 intraperitoneally. The frequency of HA-specific CD4⁺ T cells (Thy1.1⁺1.2⁺) expressing Foxp3 was determined by flow cytometry in the graft and in recipients killed 1 and 2 weeks after transplantation. Data represent means plus or minus SE.

Figure 4

Validation of function of T_regs and T_effs isolated from donors. (A) Experimental outline. Donor mice received 10⁶ A20HA intravenously, followed 10 days later by 2.5 × 10⁶ CFSE-labeled, CD4⁺-enriched HA-specific T cells. At 14 days after T-cell transfer, mice were vaccinated with 10⁷ pfu VaccHA (intraperitoneally) and were killed 5 days later. (B) Spleens and LNs were harvested and analyzed by fluorescence-activated cell sorter (FACS). T_regs (CFSE^lowGITR⁺) or T_effs (CFSE^lowGITR⁻) were sorted. (C-E) A total of 10 000 sorted T_regs or T_effs were cultured in vitro either alone or with 10 000 naive CD4⁺ cells from a 6.5/Rag^−/− mouse (naive responder [N]), along with 200 000 splenocytes from a WT BALB/c mouse, and stimulated with 10 μg/mL HA peptide. Proliferation (C) and cytokine production (D,E) were measured as described in “Methods.” Data represent mean (± SE) of triplicate cultures.

Figure 5

Tumor-specific T_effs outcompete T_regs during early immune reconstitution. Mice with a 10D established tumor burden were lethally irradiated and received transplants with BM along with T_regs only, T_effs only, or T_regs plus T_effs. Mice were killed 2 weeks after HSCT, and spleens were harvested. CD4⁺ T cell frequency was measured by flow cytometry (A) and absolute CD4⁺ cell numbers were calculated (B). CD4⁺ T-cell frequency as measured by flow cytometry is shown in panel A. Data in panel B represent mean (± SE) of 2–4 mice per group. (P = .1). A total of 50 000 tumor-purged splenocytes were cultured with 200 000 WT splenocytes to serve as APCs. HA peptide was added at the indicated concentrations. Proliferation (C) and IFNγ production (D) were measured as described in “Methods.” P values were calculated using the Student t test. Data in panels C and D represent mean (± SE) of triplicate cultures.

Figure 6

T_regs isolated from transplant recipients maintain their suppressive activity in vitro. Donor mice were prepared as in Figure 4. Tumor-bearing transplant recipients were killed 2 weeks after HSCT, and their spleens were harvested. T_regs were then sorted out of recipients and cultured in vitro with or without CD4⁺-enriched T cells from 6.5/Rag2^−/− mice as responder cells, as described in Figure 4. Proliferation (A) and IL-2 production (B) were measured. Data represent mean (± SE) of triplicate cultures.