(A) FACS analysis of nTregs isolated from peripheral blood utilizing the Dynal Regulatory T Cell Isolation Kit (Invitrogen). (B) FACS analyses of isolated nTregs as assessed by intracellular staining for Foxp3 expression. Blue line represents isotype control, green line represents CD4⁺ CD25^- non-Treg control and red line represents nTregs. (C) FACS analyses for Foxp3 and 19z1 expression of isolated nTregs at day 7 following isolation and 19z1 CAR retroviral gene transfer. Similar gene transfer was obtained for nTregs transduced with the 19-28z and Pz1 control CARs (data not shown). (D) nTreg expanded following activation with Dynal CD3/CD28 Human Treg Expander beads (day 0), transduction with the 19z1 CAR, and restimulation (arrow, day 14) with either Expander beads or 3T3(hCD19/CD80) AAPCs. Cell counts, normalized to total cell number, show similar expansion between the AAPC and CD3/CD28 bead activated groups. (E) Foxp3 expression is largely retained following restimulation with either Dynal CD3/CD28 Human Treg Expander beads or 3T3(hCD19/CD80) AAPCs, with the latter population demonstrating enhanced CAR expression. (F) Percentage of expanded T cells expressing the CAR is increased following stimulation on 3T3(hCD19/CD80) AAPCs, but stable following stimulation with Dynal CD3/CD28 Human Treg Expander beads (p < 0.01). (G) Absolute numbers of 19z1⁺ Tregs is increased following expansion on 3T3(hCD19/CD80) AAPCs when compared to expansion with Dynal CD3/CD28 Human Treg Expander beads.

(A) CSFE-labeled naïve T cells co-cultured with 19z1⁺ nTregs, Pz1⁺ nTregs, or 19z1⁺ Foxp3^- CD4⁺ CD25^- T cells at titrated effector to suppressor ratios were activated with CD3/CD28 Human T cell Expander beads. CFSE⁺ T cells were analyzed via flow cytometry on day 3 post activation. Percent proliferation was calculated using FlowJo software. 19z1⁺ and Pz1⁺ nTregs, in contrast to control non-Tregs, efficiently suppressed proliferation of naïve T cells. (B) 19z1⁺ nTregs inhibit secretion of IL-2 by activated naïve T cells in a dose dependent manner, as assessed by Luminex-based analyses of tissue culture supernatants at 24 hours post co-culture. (C) CFSE-labeled 19-28z⁺ effector T cells co-cultured with 19z1⁺ nTregs (solid bars) or Foxp3^- CD4⁺ CD25^- T cells at varying effector to suppressor ratios were activated on 3T3(hCD19/CD80) AAPCs for 3 days. 19z1⁺ nTregs, but not control non-Tregs, suppressed proliferation of 19-28z⁺ effector T cells. (D) 19z1⁺ nTregs inhibit 19-28z⁺ effector T cell cytotoxicity. CD19⁺ Raji tumor cells were co-cultured at a 1:1:1 ratio with 19-28z⁺ effector T cells and 19z1⁺ nTregs. At 24 hours, persistence of Raji tumor cells was assessed by FACS with corresponding FSC/SSC plots provided. Persistence of Raji tumor cells is evident when 19-28z⁺ effector T cells were co-cultured with 19z1⁺ nTregs, in contrast to coculture with 19-28z⁺ effector T cells in the absence of nTregs. (E) Standardized cytotoxicity assay of CD19⁺ Raji tumor cells co-cultured with 19-28z⁺ effector T cells alone or 19-28z⁺ effector T cells with 19z1⁺ nTregs (at a 1:1 ratio) at various effector to target (E:T) ratios. E:T ratios represent the ratio of 19-28z⁺ effector T cells to target Raji tumor cells.

(A) Dual bioluminescent imaging of Raji tumor and CAR⁺ nTregs show trafficking of 19z1⁺ nTregs, but not Pz1⁺ nTregs, to subcutaneous Raji tumors at 24 hours. SCID-Beige mice were injected subcutaneously with Raji(GFP-FFLuc) cells and 10 days later, with 19z1⁺ extGLuc⁺ or control Pz1⁺ extGLuc⁺ nTregs. Tumor cells were imaged using the FFLuc specific luciferin substrate, while T cells were imaged using the GLuc specific coelenterazine substrate. (B) Immunohistochemistry staining with an anti-human CD3 antibody confirms the presence of 19z1⁺ nTregs, but not Pz1⁺ nTregs, within Raji tumor microenvironment. (C) Bioluminescent imaging CAR⁺ nTregs show trafficking of 19z1⁺ nTregs, but not Pz1⁺ nTregs, to systemic Raji tumors at 24 hours, with predicted signal in the bone marrow of the femurs, tibia, and humeri (green arrows) of 19z1⁺ extGluc⁺ nTreg infused mice, as well as infiltration of these nTregs into the submandibular lymphnodes (orange arrow).

(A) SCID-Beige mice were injected i.v. with Raji tumor cells on day 0, followed by CAR⁺ nTregs on day 5 (filled arrow) and CAR⁺ effector T cells on day 6 (open arrow). 19z1⁺ nTregs fully abrogated eradication of systemic Raji tumors by 19-28z⁺ effector T cells as assessed by survival over time when compared to mice treated with 19-28z⁺ effector T cells alone (p < 0.001). Pz1⁺ nTregs did not demonstrate significant suppression (p = 0.09 when compared to the 19-28z⁺ effector T cells alone cohort; p < 0.001 compared to the 19z1⁺ nTreg plus 19-28z⁺ effector T cells cohort). Data represents combined results from 2 independent experiments. (B) 19z1⁺ nTregs inhibited effector 19-28z⁺ T cells in a dose dependent manner with infused nTreg to effector T cell ratios of 1:1, 1:4, and 1:8 resulting in no long term surviving mice (all with p < 0.001, compared to 19-28z Teff alone cohort) while a 1:16 nTreg to effector T cell ratio allowed for a 50% long-term survival of treated mice (p = 0.02, compared to Pz1⁺ Teff treated control cohort). Survival of the 1:16 nTreg to effector T cell treated cohort was statistically similar to the 19-28z Teff alone control cohort (p = 0.3). Similar results were obtained in tumor bearing mice following prior infusion with 19-28z⁺ nTregs (data not shown). d: days since Raji tumor cell injection.

(A) Schematic of the 19(del) CAR. Black box, CD8 leader sequence; green box, (Gly₃Ser)₄ linker; LTR, long terminal repeat; SD, splice donor; SA, splice acceptor; arrows, start of transcription. (B) 19(del)⁺ nTregs, like control Pz1⁺ nTregs, but in contrast to 19z1⁺ nTregs, fail to expand following co-culture on 3T3(hCD19/CD80) AAPCs consistent with a lack of T cell activation mediated through the 19(del) CAR. (C) 19(del)⁺ extGLuc ⁺ nTregs retain the ability to traffic to Raji tumor in vivo. SCID-Beige mice bearing palpable Raji(GFP-FFLuc) tumors were injected i.v. with 19(del)⁺ extGLuc⁺ T cells. Mice were imaged at 24 hours post nTreg infusion. (D) 19(del) ⁺ nTregs retain the ability to inhibit expansion of activated naïve T cells. CFSE-labeled naïve T cells co-cultured with 19(del) ⁺ Tregs, 19z1⁺ Tregs, or 19z1⁺ CD4⁺ CD25^- non-Treg control T cells at varying effector to suppressor ratios were activated with Dynabeads CD3/CD28 Human T cell Expander beads. Both 19(del)⁺ nTregs and 19z1⁺ nTregs suppressed proliferation of naïve T cells when compared to the non-Treg control co-cultures. (E) Infusion of Raji tumor bearing mice with 19(del)⁺ nTregs partially inhibited antitumor efficacy of 19-28z⁺ effector T cells as assessed by long-term survival. Survival of mice previously infused with 19(del)⁺ nTregs, when compared to mice treated with 19-28z⁺ effector T cells alone, was significantly lower (p < 0.001) but significantly improved when compared to mice previously treated with 19z1⁺ nTregs (p < 0.001). Data represents combined results from 2 independent experiments. Closed arrow: nTreg infusion; open arrow, effector 19-28z⁺ T cell infusion; d, days since Raji tumor cell injection. At 24 hours following 19-28z⁺ effector T cell infusion, prior infusion with either 19z1⁺ or 19(del)⁺ nTregs significantly reduced serum levels of IL-2 (F) (p < 0.005), and IFNγ (G) (p < 0.05), when compared to mice treated with 19-28z⁺ effector T cells alone. Figure 5F and 5G represent the combined data from 2 independent experiments with cohorts of 3 mice in each experiment.

(A) SCID-Beige were mice injected i.v. with Raji tumor cells on day 0, followed by 19z1⁺ nTregs on day 5 (left filled arrow), i.p. injection of cyclophosphamide (100mg/kg) on day 6 (open arrow), and 19-28z or Pz1⁺ effector T cells on day 7 (right black arrow). Long term survival comparable to no-nTreg controls was observed in mice injected with 19z1⁺ nTregs followed by cyclophosphamide therapy and 19-28z⁺ effector T cell infusion which was significantly superior to similarly treated mice without prior lymphodepletion (p < 0.0001). Data represents combined results from 2 independent experiments. (B) Foxp3⁺ nTreg to effector cell ratios were assessed in tumor-involved tissues (bone marrow) by FACS at 24 hours following 19-28z⁺ effector T cell infusion. Prior cyclophosphamide therapy significantly altered the nTreg to effector T cell ratios in favor of the 19-28z⁺ effector T cells (0.14 with versus 0.9 without lymphodepletion, p < 0.005). Data represent the average from 2 independent experiments each with cohorts of 3 mice per treatment group.