A Head-to-Head Comparison between Cyclophosphamide and Ontak in Reducing the Influence of Tregs on Self (Tumor) Peptide-Specific CTL Responses

Introduction: Regulatory (suppressor) T cells limit immune responses including those directed against tumor-associated, self-antigens. Several approaches have been taken to reduce the numbers and/or function of these Tregs during vaccination against tumor-associated antigens. However, the immunomodulatory drugs used in these protocols have never been rigorously compared head-to-head for their ability to down-regulate Treg subsets and to up-regulate self peptide-specific responses. To this end FoxP3 promoter-GFP knock-in mice were used to track GFP⁺ Tregs following exposure to cyclophosphamide (CTX) or Ontak (Denileukin Diftitox/DAB289IL-2), two FDA-approved therapeutics currently being tested for their ability to boost anti-cancer vaccine responses. Transgenic mice expressing both this reporter construct and human HLA-A2 antigen were used to generate HLA-A2-restricted, CTL responses against a self-peptide derived from murine Blimp-1, a plasma cell-associated transcription factor representing a putative vaccine for plasma cell dyscrasias. The effects of CTX and Ontak on these responses also were determined.

Method: To track Tregs and to determine an optimal point for self-peptide immunization, FoxP3 promoter-GFP knock-in mice were pre-bled and injected i.p. with PBS, CTX (2mg once) or Ontak (5ug on days 1, 3, 15 and 18). Mice were bled periodically for up to 41 days and assessed for expression of FoxP3(GFP)⁺/CD25⁺ and FoxP3(GFP)⁺/CD25⁻ Tregs and other T cell subsets by flow cytometry. To determine the functional consequence of these treatments, “quadruple” transgenic/knock-out mice (mouse MHC-I^−/−, human HLA-A2 and β2 microgloblin transgenic, FoxP3 promoter-GFP knock-in) were treated with CTX or Ontak, mock immunized or immunized with murine Blimp-1 peptide at day 12 (CTX treatment) or day 23 (Ontak treatment), and boosted twice at 14 day intervals. Mice were sacrificed 14 days thereafter, spleen, lymph node and bone marrow assayed for Treg and other T cell subsets, and splenic cells re-challenged in vitro with Blimp-1 peptide. Cultures were evaluated 7 days later for CTL activity in a standard ⁵¹Cr-release killing assays using Blimp-1 or control peptide-pulsed HLA-A2⁺ human B lymphoma cells as targets.

Results: CTX dramatically decreased the absolute number of blood FoxP3⁺/CD25⁺ and FoxP3⁺/CD25⁻ Treg cells by day 3 (32 ± 2% and 18 ±3% of starting numbers on day 0 respectively) but had less of an effect on “responder” CD4⁺/FoxP3⁻ T cells (64 ± 4% at lowest on day3, p<0.001) or CD8⁺ T cells (68 ± 6% at lowest on day 3, p<0.001). FoxP3⁺ cells did not fully recover over a 40-day period while CD8⁺ T cells recovered by day 14. Ontak preferentially decreased FoxP3⁺/CD25⁺ Treg numbers (30 ± 4% of starting population) and had no effect on FoxP3⁺/CD25⁻ Tregs (96 ± 15% of starting numbers at day 5). FoxP3⁺/CD25⁺ Tregs did not completely recover at any point evaluated. Ontak modestly reduced the number of “responder” CD4⁺/FoxP3⁻ cells (64 ± 5% at day 5) but had little effect on CD8⁺ T cells (85 ± 7% of starting numbers at day 5). CD4⁺ and CD8⁺ T cells recovered by days 23 and 15 respectively after Ontak treatment. In some experiments, Ontak induced an increase in CD8⁺ cells above baseline levels 23–28 days after treatment. Despite specific killing of FoxP3⁺/CD25⁺ Tregs and a minimal effect on CD8⁺ T cells following Ontak treatment, Blimp-1 peptide-specific CTL responses were augmented only after CTX treatment.

Conclusion: CTX and Ontak influence different sub-populations of Tregs potentially impacting self (tumor) peptide-induced CTL responses differently. CTX appears more effective in augmenting Blimp-1-specific CTL responses in this study, possibly because of its ability to eliminate both CD25⁺ and CD25⁻ Tregs, results suggest that CD25⁻ Tregs play an important role in limiting self antigen-specific responses. In addition the data suggest that a combination of CTX and Ontak may be optimal in generating immune responses to peptides for immunotherapy of B cell malignancies and, perhaps, for other cancers.