Hematopoietic stem cells promote the expansion and function of adoptively transferred antitumor CD8 T cells

Abstract

Depleting host immune elements with nonmyeloablative regimens prior to the adoptive transfer of tumor-specific CD8(+) T cells significantly enhances tumor treatment. In the current study, superior antitumor efficacy was achieved by further increasing the intensity of lymphodepletion to a level that required HSC transplantation. Surprisingly, the HSC transplant and not the increased lymphodepletion caused a robust expansion of adoptively transferred tumor-specific CD8(+) T cells. The HSC-driven cell expansion of effector, but not of naive, CD8(+) T cells was independent of in vivo restimulation by MHC class I-expressing APCs. Simultaneously, HSCs also facilitated the reconstitution of the host lymphoid compartment, including inhibitory elements, not merely via the production of progeny cells but by enhancing the expansion of cells that had survived lymphodepletion. Profound lymphodepletion, by myeloablation or by genetic means, focused the nonspecific HSC boost preferentially toward the transferred tumor-specific T cells, leading to successful tumor treatment. These findings indicate that CD8(+) T cell-mediated tumor responses can be efficiently driven by HSCs in the myeloablative setting and have substantial implications for the design of new antitumor immunotherapies.

Figures

Figure 1. Myeloablative TBI (9 Gy) with HSC transplant significantly enhances ACT and drives T cell expansion.

(A) Increasing a nonmyeloablative regimen to a myeloablative one augmented ACT-mediated tumor treatment. C57BL/6 tumor-bearing mice were irradiated with 5 Gy or 9 Gy and received an HSC transplant (HSC); were left untreated as a control (NT); or received transfer of 1 × 106 effector pmel-1 CD8+ T cells and rhIL-2 (PI). Tumor treatment efficacy was strongly improved in myeloablated mice (P = 0.0036; 5 Gy PI versus 9 Gy PI HSC). Results for tumor area are the mean of measurements from 6 mice per group (±SEM). Data are representative of 3 independent experiments. (B) HSC transplant drives the proliferation of transferred pmel-1 CD8+ T cells in a myeloablated host. Cultured gene-marked (Thy1.1+) pmel-1 CD8+ T cells (1 × 106) were transferred with rhIL-2 into a nonmyeloablated or myeloablated host with or without an HSC transplant. At indicated days, the absolute numbers of adoptively transferred CD8+ T cells in the spleen of tumor-bearing mice were analyzed. Data shown represent 3 mice pooled per group. The experiment was performed 3 times, with similar results. (C) Increased serum levels of IL-7 and IL-15 were measured on day 5 after the HSC transplant in C57BL/6 mice by LINCOplex analysis. Data represent 3 mice pooled per group per time point. The experiment was performed twice, with similar results.

Figure 2. HSCs drive the proliferation and antitumor activity of naive and effector pmel-1 CD8+ T cells in mice treated with a myeloablative preparative regimen and an HSC transplant.

(A) The expansion of naive and effector pmel-1 CD8+ T cells is comparable in myeloablated hosts with HSC transplant. Gene-marked (Thy1.1+) naive or effector pmel-1 CD8+ T cells (1 × 106) were adoptively transferred with rhIL-2 into a nonmyeloablated host (left panel) or a myeloablated host receiving an HSC transplant (right panel). The absolute numbers of pmel-1 CD8+ T cells in the spleens of treated animals were enumerated on the days indicated. Spleens of 3 mice per group were pooled at each time point. This experiment was performed 3 times, with similar results. (B) Naive and effector pmel-1 CD8+ T cells elicit a similar tumor treatment in myeloablated hosts with HSC transplant. Naive or effector pmel-1 CD8+ T cells (1 × 106) were transferred with rhIL-2 in nonmyeloablated (left panel) or myeloablated hosts with HSC transplant (right panel). Both naive and effector pmel-1 CD8+ T cells mediated significant tumor treatment (9 Gy/HSC: P = 0.0003, NT versus naive PI; 9 Gy/HSC: P = 0.0002, NT versus activated PI; 9 Gy/HSC: P = 0.76, naive versus effector PI), but no tumor treatment was seen in nonmyeloablated animals. Results for tumor area are the mean of measurements from 5 mice per group (±SEM).

Figure 3. HSCs drive effector, but not naive, pmel-1 CD8+ T cell expansion and tumor treatment in the absence of host β2m.

(A) HSC-driven effector pmel-1 CD8+ T cell expansion and tumor treatment is MHC class I independent. Effector pmel-1 CD8+ T cells (1 × 106) were transferred together with rhIL-2 into 9-Gy irradiated β2m–/– or WT mice that had received the matched HSC transplant and had 10-day established B16 tumors. T cell expansion (left panels) and tumor treatment (right panels) were evaluated at the indicated days. Control groups were left untreated. Effector pmel-1 CD8+ T cells showed similar proliferation and tumor treatment in β2m–/– and WT mice (9 Gy effector PI: P = 0.9, WT versus β2m–/–; 9 Gy in β2m–/–: P = 0.001, NT versus effector PI). (B) In β2m–/– mice, only effector, but not naive, pmel-1 CD8+ showed HSC-driven T cell expansion and tumor treatment. Effector or naive pmel-1 CD8+ T cells were transferred into myeloablated tumor-bearing β2m–/– mice receiving a β2m–/– HCS transplant. Naive pmel-1 CD8+ T cells showed neither proliferation nor significant tumor treatment (P = 0.96, NT versus naive PI). Proliferation curves represent the numbers of gene-marked pmel-1 CD8+ T cells found in the spleens of 3 mice pooled per group per time point. Results for tumor area are the mean of measurements from 5 mice per group (±SEM). The data shown are representative of 3 independently performed experiments.

Figure 4. HSCs drive pmel-1 CD8+ T cell expansion in nonmyeloablated and myeloablated mice, but tumor treatment is only achieved in myeloablated mice.

(A and B) HSC transplantation–driven transgenic T cell expansion is not dependent on the intensity of immunodepletion. Mice received a preparative regimen of either 5 Gy or 9 Gy TBI, which was followed by effector pmel-1 CD8+ T cells (1 × 106) and rhIL-2 with or without HSC transplantation. (A) Absolute numbers of adoptively transferred congenic marked pmel-1 CD8+ T cells in the spleen were enumerated on the days indicated. Results shown were derived from pooled splenocytes of 3 mice per group per time point (A) or from 3 individual mice (±SEM) assessed on day 6 (B) (P = 0.0009, 9 Gy versus 9 Gy/HSC; P = 0.02, 5 Gy versus 5 Gy/HSC; P = 0.24, 5 Gy/HSC versus 9 Gy/HSC). (C) HSC-driven expansion of pmel-1 CD8+ T cells is therapeutically effective in myeloablated, but not in nonmyeloablated, WT mice. Animals bearing tumors established for 10 days were either left as controls (NT) or received, prior to the transfer of 1 × 106 effector pmel-1 CD8+ T cells and rhIL-2, either 5 Gy TBI, 5 Gy TBI and an HSC transplant, or 9 Gy TBI and an HSC transplant. Results for tumor area are the mean of measurements from 5 mice per group (±SEM). Results for each of the panels are representative of data from 3 independent experiments.

Figure 5. HSCs drive expansion of host cells surviving TBI, hindering the effectiveness of antitumor T cells.

(A) HSC transplants increase the numbers of splenic CD4+ T cells and Gr-1+, NK, and B cells found in irradiated mice. Absolute numbers of splenic CD4+, Gr1+, NK1.1+, or B220+ cells were determined 5 days after mice received 5 Gy or 9 Gy TBI with or without HSC transplant. Mice received 1 × 106 pmel-1 CD8+ T cells and rhIL-2. (B) HSC-driven recovery is derived from host cells, not HSC progeny. Thy1.1+ HSCs were transplanted into Thy1.2+ hosts that had received either 5 Gy or 9 Gy TBI and rhIL-2. Data shown are from 3 pooled spleens per group at day 7. (C and D) Returning host cells can abrogate pmel-1 CD8+ T cell tumor treatment. (C) Tumor-bearing myeloablated WT, CD4–/–, CD8–/–, or RAG–/– mice with a syngeneic HSC transplant were left untreated as control or received suboptimal amounts (5 × 105) of effector pmel-1 CD8+ T cells with rhIL-2. P = 0.017, 9 Gy PI WT versus 9 Gy CD4–/–; P = 0.004, 9 Gy PI WT versus 9 Gy CD8–/–; P = 0.0017, 9 Gy PI WT versus 9 Gy RAG–/–. (D) Tumor-bearing nonmyeloablated WT or RAG–/– mice were left untreated as control or received 1 × 106 effector pmel-1 CD8+ T cells and rhIL-2. NK1.1 cell–depleting (α-NK) or isotope control (ISO) antibodies were given every 5 days, starting at day 0 and ending on day 20 (P = 0.01, 5 Gy PI RAG–/– ISO versus 5 Gy PI RAG–/– α-NK). Results for tumor area are the mean of measurements from 5 mice per group (±SEM). Data are representative of 3 experiments.

Figure 6. The antitumor effectiveness of HSC-driven CD8+ T cell proliferation is dependent on the reduction of host inhibitory elements.

(A and B) Ratios of adoptively transferred effector pmel-1 CD8+ T cells (CD8+Ly5.1+) relative to returning host Treg cells (Foxp3+CD4+), host CD8+Th1.1+ cells, and host NK1.1+ cells are shifted toward pmel-1 CD8+ T cells in myeloablated mice. Thy1.2+ host mice received a preparative regimen of 5 Gy or 9 Gy with an HSC transplantation from Thy1.1+ mice, which was followed by the adoptive transfer of 1 × 106 effector (Ly5.1+) pmel-1 CD8+ T cells and rhIL-2. Control mice received 5 Gy TBI in the absence of an HSC transplant. Splenocytes obtained 1 week after HSC transplant were simultaneously analyzed for adoptively transferred gene-marked pmel-1 CD8+ and reconstituting host cells (left panel). Flow cytometry data from pooled splenocytes from 3 mice were used to calculate the ratios of adoptively transferred pmel-1 CD8+ T cells, and cumulative results from 3 independent experiments (3 mice per experiment) are shown in the right panel. The ratios of transferred pmel-1 T cells to host immune cell subsets were significantly higher after 9 Gy TBI with an HSC transplant than after 5 Gy irradiation with or without an HSC transplant. The levels of significance are as follows: for of NK cells (P = 0.0006, 5 Gy versus 9 Gy/HSC, P = 0.0005, 5 Gy/HSC versus 9 Gy/HSC), Foxp3+CD4+ Tregs (P = 0.0006, 5 Gy versus 9 Gy/HSC, P = 0.0005, 5 Gy/HSC versus 9 Gy/HSC) and CD8+ T cells (P = 0.003, 5 Gy versus 9 Gy/HSC, P = 0.002, 5 Gy/HSC versus 9 Gy/HSC). (B) HSC-driven pmel-1 CD8+ T cell proliferation can augment tumor treatment in RAG–/– mice. RAG–/– mice bearing 10-day tumors were irradiated with 5 Gy or 9 Gy with or without an HSC transplant and were treated with 1 × 106 effector pmel-1 CD8+ T cells with rhIL-2. Control mice were left untreated. Data are representative of 3 independent experiments, each with 5 mice per group. (C) Tumor treatment efficacy is associated with the severity of vitiligo observed 28 days after pmel-1 CD8+ T cell transfer.