Myeloablative TMZ enhances T-cell function yet IL-2 remains limiting.

<p>To compare the levels of IL-2 secretion by OT-I T-cells relative to IFNγ and TNFα levels in the context of NMA (A) and MA (B) TMZ regimens we performed CBA analysis. Splenocytes from mice receiving TMZ followed by OT-I transfer and vaccine, were harvested on day 7 after vaccination and cultured in the presence of 1 µM SIINFEKL chicken Ovalbumin epitope (cognate) or negative peptide control (non-cognate) for 72 hours in T-cell media alone. Supernatant was harvested and samples were subjected to to CBA analysis as specified by the manufacturer for murine IFNγ, TNFα and IL-2. Representative experiments are shown; experiments were performed in at least duplicate. Statistical analysis was performed using unpaired, two-tailed student’s t-tests. Statistical significance was determined at a *p value ≤0.05.</p

Myeloablative TMZ is required for efficacious immunotherapy against intracerebral tumor.

<p>Antitumor efficacy of a vaccination in the context of lymphopenia induced by the different TMZ regimens and the impact of these TMZ regimens as a single agent were investigated in wild-type C57BL/6 mice (n = 7) inoculated with an intracerebral injection of 5×10³ B16F10-OVA melanoma cells. These mice received vehicle, NMA or MA TMZ treatment followed by vaccine (A) or no vaccine (B). On day 3 after tumor implantation, dose-intensifying TMZ regimens were initiated and administered for 5 consecutive days intraperitoneally. Vaccinated mice received an intravenous adoptive transfer of 1×10⁷ splenocytes consisting of 1∶1 ratio of OT-I and C57BL/6 splenocytes on the day after termination of TMZ treatment. Mice underwent subsequent intradermal vaccination with 100 µg of SIINFEKL chicken ovalbumin epitope emulsified in CFA. (B) In order to assess the effects of TMZ alone, mice were also treated with TMZ in the absence of vaccine. Mice were monitored for morbidity end-points approved by the Duke University IACUC and sacrificed when end points were met. The experiment was performed three times. Survival analysis was performed using the Gehan-Breslow-Wilcoxon Test. Statistical significance was determined at a *p value ≤0.05.</p

Myeloablative TMZ leads to elevated IL-2 serum levels, which are required for optimal antigen-specific T-cell expansion.

<p>The presence and requirement of endogenous IL-2 serum levels for optimal antigen-specific T-cell expansion following myeloablative TMZ was evaluated in wild-type C57BL/6 mice (n = 5 per group). To investigate the levels of endogenous IL-2, mice were treated with vehicle, NMA or MA TMZ for 5 consecutive days. Myeloablated mice received an HSC rescue on the day after cessation of TMZ treatment. (A) Serum IL-2 levels were analyzed 5 days after cessation of TMZ treatment by LINCOplex analysis. Statistical analysis was performed using Mann-Whitney test. Statistical significance was determined at a *p value ≤0.05. (B) To assess the role of IL-2 in potentiating antigen-specific T-cell responses, mice were infused with anti-IL-2 antibodies (S4B6 and JES6-1,250 µg per mouse, intraperitoneal), followed by OT-I ALT transfer and peptide vaccine in CFA. Anti-IL-2 antibodies were administered every other day for 7 doses. Mice were bled on day 7 and blood was stained with anti-CD8 and OVA tetramer w\as described before on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059082#pone-0059082-g003" target="_blank">Figure 3</a>. Absolute numbers per µl of blood were calculated using Flowcount^® beads from Beckman Coulter^©. Statistical analysis was performed using unpaired t- test. Statistical significance was determined at a *p value ≤0.05.</p

Myeloablative TMZ results in protracted depletion of CD4⁺Foxp3⁺

<p><b>T cells.</b> To evaluate, the impact of different TMZ regimens on CD4⁺Foxp3⁺ T_Regs C57BL/6 mice (n = 5) were treated with vehicle, NMA or MA TMZ regimens as stated before in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059082#pone-0059082-g001" target="_blank">Figure 1</a>. Mice were bled retro-orbitally on days 2, 7, 14 and 28 after termination of TMZ administration and submitted to flow cytometry analysis. Immediate effects of TMZ on CD4⁺Foxp3⁺ T_Regs (A), on day 2 and the recovery kinetics over time are shown (B). Absolute numbers per microliter of blood were calculated using Flowcount^® beads from Beckman Coulter^©. Representative experiments are shown; all experiments were performed in at least duplicate. Statistical analysis was performed using Mann-Whitney test. Statistical significance was determined at a *p value ≤0.05.</p

<i>In vivo</i> effects of daclizumab on the effector T-cell to regulatory T-cell ratio.

<p>(A–B) Effector T-cells (CD4⁺ or CD8⁺) to T_Reg ratios were derived by dividing the absolute number of effector T-cells by the absolute number of T_Regs at the indicated time-points; the absolute number of cells was determined by a combination of CBC and FACS analysis. The ratios of effector T-cells to T_Regs as compared to baseline were generated by dividing the individual patient CD4⁺∶T_Reg or CD8⁺∶T_Reg ratio at every time point by the ratio at vaccine 1 (V-1 = baseline). A two sample t-test averaged over the V-2, V-3 and LP time-points was utilized to examine the difference between the daclizumab and saline groups in the CD4⁺∶T_Reg (p = 0.0757) and CD8⁺∶T_Reg (p = 0.0153) ratios.</p

<i>In vitro</i> effects of IL2Rα inhibition on CD4⁺, CD8⁺ and regulatory T-cells.

<p>Normal donor peripheral blood mononuclear cells (PBMCs) were cultured for 48 hours with increasing concentrations of daclizumab followed by an additional 14 days stimulation/expansion with CMV pp65 RNA-pulsed DCs along with IL-2 or IL-15. PBMC were then isolated and stimulated for 6 hours with SEB or pp65 peptide mix in the presence of CD28/CD49d costimulation and Brefeldin A. The IFN-γ secretion of (A) CD3⁺CD4⁺CD69⁺ or (B) CD3⁺CD8⁺CD69⁺ T-cells was determined by flow cytometry.</p

The frequency of T_Regs and anti-PEPvIII humoral responses are inversely correlated.

<p>Patient sera from peripheral blood (vaccine 4) and leukapheresis samples were analyzed for levels of anti-PEPvIII antibodies and humoral responses were plotted against the frequency of T_Regs (Foxp3⁺ of CD4⁺). Assuming the assessments within individuals are independent, the Spearman correlation coefficient for both saline and daclizumab treated patients overall is (R = −0.93, p<0.0001).</p

ZAP IT Patient Characteristics.

<p>> As of the lock date of the data, the indicated patient had 12 cycles of TMZ but continued with additional cycles of TMZ treatment.</p

Daclizumab remains bound to T_Regs a month after administration.

<p>(A) CD4⁺Foxp3⁺ T_Regs from day 35±2 leukapheresis samples (saline n = 3, daclizumab n = 3) were determined by flow cytometry and were additionally stained with anti-CD25 antibodies that bind the same CD25 eptiope as daclizumab (competing clone 2A3) or bind a separate epitope (non-competing clone MA251). The ratio of the frequency of CD4⁺CD25⁺Foxp3⁺ T_Regs as determined by MA251 or 2A3 binding was used as an indirect indicator of surface daclizumab expression. The percent change in the ratio was calculated from ratios determined from baseline (vaccine 1) samples, unpaired t-test *p = 0.0353. (B) CD4⁺CD25⁺Foxp3⁺ T_Regs were determined by FACS analysis of PBMC and examined for human antibody expression as a direct indicator of daclizumab binding to the surface of T_Regs. PBMCs from a saline and a daclizumab treated patient from vaccine 1 (Pre-Daclizumab) and leukapheresis at day 35±2 (Post-Daclizumab) time-points were assessed.</p

Regulatory T-cells are significantly depleted by a single infusion of daclizumab.

<p>The frequency of CD4⁺Foxp3⁺ T_Regs was determined by FACS analysis of peripheral blood samples drawn prior to vaccination (V) or leukapheresis (LP). Percent change was calculated in comparison to baseline (vaccine 1). For each follow-up assessment, percent change from baseline (vaccine 1) was computed. For statistical comparisons of the daclizumab and saline groups, the average percent change at vaccine 2 (V-2), vaccine 3 (V-3), and leukapheresis (LP) was computed for each patient. Daclizumab showed a significantly greater reduction in CD4⁺Foxp3⁺ regulatory T-cells (p = 0.0464).</p