Increased intensity lymphodepletion enhances tumor treatment efficacy of adoptively transferred tumor-specific T cells

Abstract

Lymphodepletion before adoptive cell transfer (ACT)-based immunotherapies can enhance anti-tumor responses by augmenting innate immunity, by increasing access to homeostatic cytokines, and by depressing the numbers of regulatory T cells and myeloid-derived suppressor cells. Although it is clear that high-dose total body irradiation given together with hematopoietic stem cell (HSC) transplantation effectively enhances ACT, the relationship between the intensity of lymphodepletion and tumor treatment efficacy has not been systematically studied. Using the pmel-1 mouse model of self/tumor-reactive CD8 T cells, we observed a strong correlation between the intensity of the conditioning regimen and the efficacy of ACT-based treatments using linear regression analysis. This was the case for preparative total body irradiation administered either as a single dose (R=0.97, P<0.001) or in fractionated doses (R=0.94, P<0.001). Increased amounts of preparative total body irradiation were directly correlated with progressively more favorable ratios of transferred tumor-reactive CD8 T cells toward endogenous cells with the potential for inhibitory activity including: CD4 cells (potentially T regulatory cells); Gr1 cells (which are capable of functioning as myeloid-derived suppressor cells); and endogenous CD8 and natural killer 1.1 cells (that can act as "sinks" for homeostatic cytokines in the postablative setting). With increasing ablation, we also observed elevated lipopolysaccharide levels in the sera and heightened levels of systemic inflammatory cytokines. Thus, increased intensity lymphodepletion triggers enhanced tumor treatment efficacy and the benefits of high-dose total body irradiation must be titrated against its risks.

Figures

Figure 1. Tumor treatment curves of adoptively transferred T cells following increased intensity TBI

One million effector pmel-1 CD8+ T cells (P) were transferred with rhIL-2 (I) in tumor-bearing C57BL/6 mice which were either irradiated with a single dose of irradiation on the day of transfer or a fractionated dose of irradiation divided into six equal doses given twice daily at the amount indicated in the legend. All animals received an HSC transplant. Results for tumor area are the mean of measurements from 7 mice per group (+/−SEM). Statistical analysis is as follows. (A) For single-dose radiation for those treatment groups receiving PI: 0Gy vs. 5Gy (NS), 5Gy vs. 9Gy (P=0.009), 9Gy vs. 12Gy (NS), 5Gy vs. 12Gy (P=0.002). (B) For fractionated irradiation in groups receiving PI: 0Gy vs. 12Gy (P=0.006), 12Gy vs. 18Gy (NS), 18Gy vs. 24Gy (P=0.02), 12Gy vs. 24Gy (P=0.01). Data are representative of two independent experiments.

Figure 2. Linear regression analysis of tumor treatment efficacy vs. the amount of preparative TBI

Experiments were analyzed using linear regression analyses to study the relationship of radiation dose with tumor response. To perform the regression analysis, the tumor growth rates (mm2 d−1) were calculated then plotted against the amount of single-dose TBI administered or as the summed dose of fractionated TBI. Tumor response was elicited by using one million effector pmel-1 CD8+ T cells (P) transferred together with rhIL-2 (I) in tumor-bearing C57BL/6 mice which were either irradiated with (A) single-dose irradiation directly at the day of transfer or (B) fractionated doses divided equally over six doses given twice daily up to the amount indicated in the legend as described in Fig. 1. All animals received a syngeneic HSC transplant. Data are derived from two independently performed experiments. R2 values for linear regression of data sets in single-dose and fractionated radiation are shown.

Figure 3. Fraction of endogenous host immune cell subsets following TBI regimens

Thy1.2+ host mice received a preparative regimen with either single-dose irradiation at the day of T cell transfer or a fractionated (F) dose of irradiation over six times twice daily up to the amount indicated in the legend followed by the adoptive transfer of 1 x 106 Thy1.1+ effector pmel-1 CD8+ T cells (P) with rhIL-2 (I). Absolute numbers of Thy1.1+ pmel-1 vs.: (A) endogenous Thy1.2+ CD8+ T cells; (B) endogenous CD4+ T cells; (C) NK 1.1+ cells; and (D) Gr1+ cells were determined six days after the pmel-1 T cell transfer in the spleen. Error bars represent SEM. Experimental data are derived from three mice collected and individually evaluated. Experiment was repeated independently with similar results.

Figure 4. The ratio of adoptively transferred effector pmel-1 CD8+ T cells relative to returning host cells as a function of TBI dose

Flow cytometry data were analyzed using a linear least squares regression analysis. Three individual mice per group were used to calculate the ratios of adoptively transferred Thy1.1+ pmel-1 CD8+ T cells towards returning host cells six days after ACT. Thy1.2+ host mice received a preparative regimen with either (A) single-dose irradiation at the day of T cell transfer or (B) fractionated dose of irradiation divided into six equal doses given twice daily up to the amount indicated in the legend followed by the adoptive transfer of 1 x 106 Thy1.1+ effector pmel-1 CD8+ T cells with rhIL-2. Four quadrants are shown per panel. These include the ratios of the adoptively transferred Thy1.1+ cells to endogenous (Thy1.2+) CD8+ T cells, endogenous CD4+ T cells; as well as (endogenous) Gr1+ and NK 1.1+ cells as labeled. These ratios were then plotted against the dose of preparative TBI given and analyzed using linear regression with the least squares method. Experiment was repeated independently and similar results were obtained.

Figure 5. Measurements of systemic inflammatory cytokines and LPS levels in the sera of irradiated mice

One million effector pmel-1 T cells were adoptively transferred along with rhIL-2 in animals that received either a single dose of irradiation as indicated directly at the day of transfer (Panel A) or irradiated in six equal fractionated (F) doses of irradiation given twice daily up to the amount shown (Panel B). Six days after irradiation, sera of three mice were collected, pooled, and analyzed for inflammatory cytokines and chemokines. Shown are IL-6, IL-1α, and chemokine KC (Cxcl1). Microbial LPS was assessed using the limulus amebocyte lysate (LAL) assay.