Human T cells genetically modified to express chimeric antigen receptors (CAR) specific to the B cell tumor antigen CD19 can successfully eradicate systemic human CD19+ tumors in immunocompromised SCID (severe combined immunodeficient)‐Beige mice. However, in the clinical setting, CD4+ CD25hi T regulatory cells (Treg) present within the tumor microenvironment may be potent suppressors of tumor-targeted effector T cells. In order to assess the impact of Tregs on CAR-modified T cells in the SCID-Beige xenotransplant model, we isolated, genetically targeted and expanded natural T regulatory cells (nTreg). In vitro nTregs modified to express CD19-targeted CARs efficiently inhibited the proliferation of activated human T cells, as well as the capacity of CD19-targeted 19-28z+ effector T cells to lyse CD19+ Raji tumor cells. Intravenous infusion of CD19-targeted nTregs into SCID-Beige mice with systemic Raji tumors traffic to sites of tumor and recapitulate a clinically relevant hostile tumor microenvironment. Antitumor efficacy of subsequently infused 19-28z+ effector T cells was fully abrogated as assessed by long-term survival of treated mice. Optimal suppression by genetically targeted nTregs was dependent on nTreg to effector T-cell ratios and in vivo nTreg activation. Prior infusion of cyclophosphamide in the setting of this nTreg-mediated hostile microenvironment was able to restore the antitumor activity of subsequently infused 19-28z+ effector T cells through the eradication of tumor-targeted nTregs. These findings have significant implications for the design of future clinical trials utilizing CAR-based adoptive T-cell therapies of cancer. Cancer Res; 71(8); 2871–81. ©2011 AACR.

T cells may be genetically targeted to tumor antigens through the expression of chimeric antigen receptors (CAR) transduced using retroviral vectors (1). We have previously demonstrated that human T cells genetically modified to express a CD19-targeted CAR successfully eradicate established systemic human CD19+ B cell tumor cell lines in immune suppressed SCID (severe combined immunodeficient)–Beige mice (2). However, despite promising preclinical in vivo studies (2–5), results from initial clinical trials utilizing CAR-modified T cells have to date been disappointing (6–8).

A potential etiology of treatment failure in the clinical setting may be the suppression of targeted T cells by a hostile tumor microenvironment infiltrated with CD4+ CD25hi regulatory T cells (Treg) and myeloid derived suppressor cells, as well as tumor expression of inhibitory ligands (PD-L1) and cytokines (TGF-β and IL-10; refs. 9–11). This hostile tumor microenvironment is largely unaddressed in pre-clinical models utilizing immune compromised mice. To address this limitation, we sought to investigate the impact of Tregs, a potent endogenous suppressive element of the immune system, on the antitumor activity of adoptively transferred CAR-modified T cells in a previously established SCID-Beige mouse tumor model.

Natural Tregs (nTreg) are CD4+ T cells derived from the thymus and defined by a CD4+ CD25+ CD127 Foxp3+ phenotype. nTregs have been found to facilitate suppression of autoimmune T-cell responses and maintenance of peripheral tolerance (12–14), represent approximately 5% to 10% of peripheral CD4+ T cells in both mice and humans (13, 15), and express high levels of cytotoxic T lymphocyte associated antigen 4 (CTLA-4), glucocorticoid-induced TNFR-related protein, CD39, and CD73 (16–18). Patients with cancer, including B cell malignancies, have elevated numbers of Tregs in the peripheral blood and within the tumor microenvironment (19–21). Furthermore, in a variety of cancers, increased numbers of Tregs portend a poor prognosis (19, 22). Although the mechanism of suppression by Tregs appears to be multifactorial (23), it is clear that the presence of Tregs within the tumor microenvironment could markedly hinder the antitumor efficacy of adoptively transferred tumor-targeted effector T cells (24).

Many studies have been published implicating Tregs as the cause of failed antitumor immune responses using clinical correlates, Treg depleting strategies (22, 25), and systemic lymphodepletion (26, 27). Recently, investigators have developed protocols to readily isolate (28), stimulate, and expand enriched Treg populations for pre-clinical experimental purposes (29, 30).

In this report, we investigate the in vivo impact of nTregs on CD19-targeted CAR+ T-cell therapy in a previously established xenotransplant SCID-Beige tumor model of Burkitt lymphoma (2, 3) by recapitulating a clinically relevant tumor microenvironment hostile to effector T-cell function through the infusion of CD19-targeted nTregs. Systemic injection of targeted nTregs into SCID-Beige mice bearing established systemic Raji tumors prior to infusion of CD19-targeted CAR+ effector T cells wholly abolished effector T-cell antitumor benefit while prior treatment with cyclophosphamide effectively reversed in vivo nTreg-mediated suppression of CD19-targeted CAR+ effector T cells. Taken together, our data support the hypothesis that tumor specific nTregs may significantly compromise the antitumor efficacy of CAR-modified tumor-targeted effector T cells in the clinical setting and may, in part, explain the modest clinical outcomes reported in previously published clinical trials utilizing adoptively transferred CAR-modified T cells (6–8).

Cell lines and T cells

The Raji tumor cell line was cultured in RPMI 1640 (Life Technologies) supplemented with 10% heat-inactivated FCS (fetal calf serum), nonessential amino acids, HEPES buffer, pyruvate, and BME (Beta-mercaptoethanol; Life Techonologies). T cells were cultured in RPMI 1640 (Life Technologies) supplemented with 10% heat-inactivated FCS supplemented with 20IU IL-2/mL (R&D Systems). PG-13 and gpg29 retroviral producer cell lines were cultured in DMEM (Life Technologies) supplemented with 10% FCS, and NIH-3T3 artificial antigen-presenting cells (AAPC), were cultured in DMEM supplemented with 10% heat-inactivated donor calf serum. All media were supplemented with 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Life Technologies).

Isolation of CD4+ CD25 effector T cells and CD4+ CD25+ nTregs

Peripheral blood from healthy donors, obtained under institutional review board–approved protocol 95-054, was fractionated in BD Vacutainer CPT tubes (BD Medical), to isolate peripheral blood mononuclear cells (PBMC). CD4+ CD25 responder T cells and CD4+ CD25+ nTregs were isolated from PBMCs using the CD4+ CD25+ Regulatory T-Cell Isolation Kit (Dynal brand; Invitrogen).

Retroviral genetic modification of T cells

Generation of retroviral producer PG-13 cell lines and gene transfer into effector T cells have been previously described (3, 31). For nTreg retroviral gene transfer, isolated nTregs were activated with Dynal CD3/CD28 Human Treg Expander magnetic beads (Invitrogen), cultured in RPMI media supplemented with IL-2 500IU and Rapamycin 100ng/mL (Sigma) for 48 hours (30, 32), and similarly transduced.

Expansion of CAR+ T cells

CAR+ effector T cells were expanded ex vivo on NIH-3T3 derived AAPCs as described previously (3). CAR+ nTregs were expanded with either Dynal CD3/CD28 Human Treg Expander beads (Invitrogen), or with AAPCs in RPMI medium supplemented with IL-2 and Rapamycin.

In vitro nTreg proliferation and suppression assay

5 × 105 T effector cells were labeled with 5 μmol/L carboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen) and cultured simultaneously with titrated numbers of purified autologous CAR+ CD4+ CD25+ Foxp3+ nTregs in 24-well tissue culture plates (Costar; ref.33). T-cell cocultures were stimulated with Dynabeads CD3/CD28 T-Cell Expander beads (Invitrogen) at a bead-to-responder T-cell ratio of 1:1 in the absence of exogenous IL-2 and proliferation was assessed by flow cytometry (FACS) at 72 hours.

Cytokine detection assays

Cytokine levels in tissue culture supernatant as well as serum were assessed using the multiplex Human Cytokine Detection System (Millipore Corp.) in conjunction with the Luminex IS100 system and IS 2.2 software (Luminex Corp.).

In vitro cytotoxicity assay

19-28z+ effector T cells were cocultured with Raji cells in RPMI media at 1:1 ratio with or without equal numbers of 19z1+ nTregs for 24 hours. Tumor lysis was subsequently assessed by FACS to detect residual CD19+ tumor cells. The GranToxiLux (OncoImmunin, Inc.) cytotoxicity assay was performed per manufacturer's instructions.

In vivo analyses of Treg function

We inoculated 8- to 12-week-old FOX CHASE C.B-17 (SCID-Beige) mice (Taconic) with Raji tumor cells by tail vein injection or subcutaneously as indicated. In the systemic tumor model, mice were injected by tail vein (i.v.) with 5 × 105 Raji tumor cells on day 1, and on day 5 were treated with a single i.v. infusion of 1 × 107 CAR+ nTregs, followed by a single i.v. infusion of 1 × 107 CAR+ effector T cells on day 6. For cyclophosphamide experiments, mice were injected by tail vein with 5 × 105 Raji tumor cells on day 1, on day 5 were treated with a single i.v. infusion of 1 × 107 CAR+ Tregs, followed by intraperitoneal (i.p.) injection of 100 mg/kg cyclophosphamide on day 6, and on day 7 injected with an i.v. dose of 1 × 107 CAR+ effector T cells. Mice were sacrificed when disease became clinically evident. All in vivo studies were done in the context of an Institutional Animal Care and Use Committee approved protocol (#00-05-065).

Bioluminescent imaging

For in vivo imaging of Raji tumor cells we utilized Raji tumor cells modified to express GFP-FFLuc (Clontech Laboratories). Imaging of nTregs was performed using nTregs modified with the previously described 19z1 IRES extGLuc bicistronic retroviral vector (34). Tumor and T cells were imaged using the Xenogen IVIS Imaging System (Xenogen; ref. 34).

Immunohistochemistry staining

Mouse bone marrow samples were fixed in 10% buffered formalin phosphate (Fisher Scientific). All tissues were processed by routine methods and embedded in paraffin wax. Five-micrometer sections were stained with H&E (Poly Scientific). Human T cells in the paraffin-embedded mouse tissues were detected using rabbit polyclonal sera specific to human CD3 (DakoCytomation).

Flow cytometry

We performed FACS with a FACScan cytometer with FlowJo software (Tree Star), using PE-labeled CAR-specific monoclonal antibody (12D11, MSKCC monoclonal antibody core facility), FITC-labeled human CD4 specific antibody (S3.5, Caltag), CD8 specific antibody (3B5, Caltag), CD62L specific antibody (DREG 56, BD Pharmingen), PE-labeled human CD25 specific antibody (CD25.3G10, Caltag), and APC-labeled human CD19 specific antibody (SJ25-C, Caltag). Foxp3 expression was assessed using the Human Regulatory T cell Staining Kit (eBioscience).

Statistical analysis

Statistical analysis utilizing the GraphPad Prism software (GraphPad Software) was done using log-rank analyses for survival and the Student's t-test and Wilcoxon rank sum test for line and bar graph comparisons.

nTregs are efficiently modified to express CARs by retroviral transduction

To assess whether human nTregs can be genetically manipulated despite their anergic nature and difficulty to maintain in culture (30, 35), we isolated nTregs using immunomagnetic sorting for the CD4+ CD25hi population from PBMCs consistently achieving CD4+ T‐cell populations that were more than 95% CD25hi, 70% to 90% Foxp3+ (Fig. 1A and B), CD62Lhi and CD127 (data not shown). CD3/CD28 bead activated nTregs were subsequently transduced with CD19-targeted CARs using retroviral supernatants, routinely resulting in more than 60% gene transfer (Fig 1C).

Figure 1.

Efficient transduction and expansion of CAR+ nTregs. 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. In contrast to isotype and non-Treg controls, nTregs express Foxp3. 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.

Figure 1.

Efficient transduction and expansion of CAR+ nTregs. 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. In contrast to isotype and non-Treg controls, nTregs express Foxp3. 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.

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We next compared CAR+ nTreg expansion in the context of high dose IL-2 and rapamycin, either by coculture on NIH-3T3 AAPCs [3T3(CD19/CD80); ref. 2] or through the addition of Dynal CD3/CD28 Human Treg Expander beads. CAR+ nTregs proliferated equally well under either condition with largely retained Foxp3 expression (Fig. 1D and E) while only expansion of CAR+ nTregs by coculture on 3T3(CD19/CD80) AAPCs (2) enriched the CAR+ nTreg fraction (Fig. 1E and F) resulting in a significantly increased absolute number of CAR+ nTregs (Fig. 1G). However, due to contamination by persistent 3T3 fibroblasts in AAPC expanded nTreg populations, we utilized nTregs generated by CD3/CD28 Human Treg Expander beads for further studies.

Expanded genetically modified nTregs inhibit naïve T‐cell proliferation and CAR+ effector T‐cell cytotoxicity in vitro

We next cocultured activated CFSE-labeled naïve T cells with varying numbers of 19z1+ nTregs, and as controls, nTregs transduced with the irrelevant Pz1 CAR specific to the prostate specific membrane antigen (31), and control 19z1+ non‐Treg CD4+ T cells. 19z1+ and Pz1+ nTregs, but not 19z1+ non-Treg control T cells, induced a potent inhibition of nonspecific T‐cell expansion even at low nTreg to effector T‐cell ratios (Fig. 2A). Consistent with our CFSE studies, IL-2 levels in nTreg cocultures decreased in a dose dependent manner (Fig. 2B), a finding similar to results published elsewhere (35, 36). Coculture of CFSE-labeled 19-28z+ T cells with titrated numbers of 19z1+ nTregs or control 19z1+ non-Tregs demonstrated that only 19z1+ nTregs suppressed 19-28z+ effector T‐cell expansion (Fig. 2C).

Figure 2.

CAR+ nTregs inhibit expansion of activated naïve T cells and cytotoxicity of 19-28z+ effector T cells. A, CSFE-labeled naïve T cells cocultured 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 postactivation. 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 postcoculture. C, CFSE-labeled 19-28z+ effector T cells cocultured 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 cocultured 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 (forward scatter)/SSC (side scatter) plots provided. Persistence of Raji tumor cells is evident when 19-28z+ effector T cells were cocultured 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 cocultured 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.

Figure 2.

CAR+ nTregs inhibit expansion of activated naïve T cells and cytotoxicity of 19-28z+ effector T cells. A, CSFE-labeled naïve T cells cocultured 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 postactivation. 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 postcoculture. C, CFSE-labeled 19-28z+ effector T cells cocultured 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 cocultured 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 (forward scatter)/SSC (side scatter) plots provided. Persistence of Raji tumor cells is evident when 19-28z+ effector T cells were cocultured 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 cocultured 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.

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To assess the role of 19z1+ nTregs on effector T‐cell cytotoxicity, 19-28z+ effector T cells were cocultured with 19z1+ nTregs at a 1:1 ratio for 24 hours followed by the addition of target CD19+ Raji tumor cells at a 1:1 ratio with effector 19-28z+ T cells. We found that 19z1+ nTregs inhibited killing of Raji tumor cells by 19-28z+ effector T cells (Fig. 2D). As expected, 19z1+ nTregs cocultured with CD19+ Raji tumors alone failed to eradicate Raji tumor cells consistent with the notion that CD19-targeted nTregs lack cytotoxic potential (data not shown). Similar to the above findings, nTregs markedly abrogated the lysis of Raji tumor cells by 19-28z+ effector T cells in a standard cytotoxicity assay (Fig. 2E).

19z1+ nTregs traffic to CD19+ Raji tumors

In order to assess whether CAR-modified nTregs efficiently traffic to Raji tumors in SCID-Beige mice, we employed dual bioluminescent imaging (BLI) enabling simultaneous imaging of both tumor cells and T cells within the same animal (34). SCID-Beige mice previously injected subcutaneously with Raji (GFP-FFLuc) tumor cells underwent BLI to verify detectable tumor (Fig. 3A). Subsequently, mice were infused i.v. with either 19z1+ or Pz1+ nTregs further modified to express extGLuc bioluminescent enzyme (extGLuc+ nTregs). BLI at 24 hours following nTreg infusion demonstrated 19z1+ extGLuc+ nTreg but not Pz1+ extGLuc+ nTreg signal localized to the Raji tumor (Fig. 3A). Immunohistochemistry studies confirmed the presence of 19z1+ extGLuc+, but not Pz1+ extGLuc+ nTregs within the Raji tumors (Fig. 3B). Similar results were obtained in mice bearing systemic Raji tumors, which primarily infiltrate the bone marrow and lymphnodes, demonstrating specific localization of 19z1+ extGLuc+ nTregs but not Pz1+ extGLuc+ nTregs to these sites at 24 hours (Fig. 3C). These findings indicate that by 24 hours, 19z1+ nTregs successfully traffic to CD19+ tumors, recapitulating a hostile tumor microenvironment which may be seen in the clinical setting.

Figure 3.

CD19-targeted CAR+ nTregs traffic to CD19+ Raji tumor cells in vivo. A, dual BLI 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, BLI 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).

Figure 3.

CD19-targeted CAR+ nTregs traffic to CD19+ Raji tumor cells in vivo. A, dual BLI 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, BLI 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).

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Tumor infiltrating 19z1+ nTregs inhibit eradication of systemic CD19+ Raji tumors by 19-28z+ effector T cells

We have previously demonstrated that CD19-targeted effector T cells successfully eradicate systemic Raji tumors in SCID-Beige mice as assessed by long-term survival (2, 3). In SCID-Beige mice, Raji tumors have a primary tropism for the bone marrow and untreated mice reliably develop hind-limb paralysis at 3 to 5 weeks as a consequence of spinal cord compression by tumor expanding from vertebral bodies (2). To determine whether CD19-targeted nTregs within the tumor microenvironment could inhibit successful tumor eradication in this model, mice were injected systemically with Raji tumor cells on day 1, with 19z1+ nTregs on day 5, and with 19-28z+ effector T cells at a nTreg to T effector ratio of 1:1 on day 6. In all in vivo experiments, more than 80% of infused CAR+ effector T-cell populations retained a central memory phenotype (CD62Lhi CCR7+) and consisted of 50% to 65% CD8+ and 35% to 50% CD4+ T cells as assessed by FACS prior to infusion (data not shown). Prior infusion of 19z1+ nTregs, in contrast to Pz1+ nTregs, wholly abolished any antitumor effect by subsequently infused 19-28z+ effector T cells as assessed by overall survival (Fig 4A). To confirm the in vivo presence of Tregs, we further assessed the nTreg to effector T-cell ratio in the bone marrow of mice infused with 19z1+ nTregs followed by 19-28z+ effector T cells by FACS analysis at 24 hours following effector T‐cell infusion, demonstrating a 1:1 nTreg to effector T-cell ratio (Fig. 6B).

Figure 4.

CD19-targeted nTregs within the Raji tumor microenvironment suppress 19-28z+ effector T cell function in vivo. 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 represent 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.

Figure 4.

CD19-targeted nTregs within the Raji tumor microenvironment suppress 19-28z+ effector T cell function in vivo. 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 represent 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.

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Figure 5.

Optimal in vivo nTreg suppression is dependent on activation of nTregs within the tumor microenvironment. 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 coculture 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 cocultured 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 cocultures. 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 represent 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 (F) IL-2 (P < 0.005), and (G) IFNγ (P < 0.05), when compared to mice treated with 19-28z+ effector T cells alone. Figure 5F and G represents the combined data from 2 independent experiments with cohorts of 3 mice in each experiment.

Figure 5.

Optimal in vivo nTreg suppression is dependent on activation of nTregs within the tumor microenvironment. 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 coculture 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 cocultured 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 cocultures. 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 represent 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 (F) IL-2 (P < 0.005), and (G) IFNγ (P < 0.05), when compared to mice treated with 19-28z+ effector T cells alone. Figure 5F and G represents the combined data from 2 independent experiments with cohorts of 3 mice in each experiment.

Close modal
Figure 6.

Cyclophosphamide lymphodepletion following 19z1+ nTreg infusion, enhances 19-28z+ effector T‐cell tumor cell eradication, altering the nTreg to effector T‐cell ratio within the tumor microenvironment. 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 (100 mg/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 represent 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 vs. 0.9 without lymphodepletion, P < 0.005). Data represent the average from 2 independent experiments each with cohorts of 3 mice per treatment group.

Figure 6.

Cyclophosphamide lymphodepletion following 19z1+ nTreg infusion, enhances 19-28z+ effector T‐cell tumor cell eradication, altering the nTreg to effector T‐cell ratio within the tumor microenvironment. 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 (100 mg/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 represent 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 vs. 0.9 without lymphodepletion, P < 0.005). Data represent the average from 2 independent experiments each with cohorts of 3 mice per treatment group.

Close modal

To assess in vivo potency of nTreg effector suppression, we titrated the 19z1+ nTreg to effector 19-28z+ T‐cell ratio in Raji tumor bearing SCID-Beige mice. We observed that the 19z1+ nTregs were able to fully suppress the in vivo antitumor efficacy of effector 19-28z+ T cells, as assessed by survival, at a nTreg to effector T‐cell ratio as low as 1:8, but found recovery of effector T‐cell antitumor efficacy at a 1:16 nTreg to effector T‐cell ratio (Fig. 4B). Similar results were obtained when this experiment was conducted using 19-28z+ nTregs (data not shown).

Optimal suppression of 19-28z+ effector T cells requires nTreg activation within the tumor microenvironment

We next generated a CD19-targeted CAR lacking the ζ chain signaling domain termed 19(del) (Fig 5A). 19(del)+ nTregs failed to expand on 3T3(CD19/CD80) AAPCs verifying the lack of T‐cell activating signaling by the ζ chain-deleted 19(del) CAR (Fig 5B). However, despite loss of CAR signaling, 19(del)+ nTregs retained the capacity to traffic to CD19+ Raji tumors in vivo (Fig 5C) and the ability to potently suppress both naïve T-cell proliferation (Fig. 5D) and 19-28z effector T‐cell cytotoxicity in vitro (data not shown).

In order to assess the in vivo inhibitory capacity of 19(del)+ nTregs, SCID-Beige mice were injected i.v. with Raji tumor cells on day 1, injected with either 19z1+ or 19(del)+ nTregs on day 5, followed by injection of 19-28z+ effector T cells, at a 1:1 nTreg to effector T‐cell ratio, on day 6. As expected, 19z1+ nTregs conferred full suppression of 19-28z+ effector T cells, while 19(del)+ Tregs conferred only partial suppression (Fig. 5E). These findings suggest that optimal suppression requires both nTreg localization to and activation within the tumor microenvironment.

To further define the mechanism whereby 19(del)+ nTregs-mediated partial inhibition of 19-28z+ T‐cell effector function, we next measured serum levels of human IL-2 and IFNγ as well as the inhibitory human cytokines TGF-β and IL-10 in treated mice. Human TGF-β and IL-10 levels were consistently low to undetectable in all treated cohorts (data not shown), while human IL-2 and IFNγ levels were significantly and equally decreased in both 19z1 and 19(del)+ nTreg infused cohorts (Fig. 5F and G). This finding is consistent with sequestration of IL-2 by nTregs, a previously described mechanism of nTreg-mediated T‐cell suppression (35, 36), and inhibition of effector T‐cell activation as assessed by IFNγ levels. These findings were independent of nTreg activation status, and may explain, in part, the observed partial inhibition mediated by 19(del)+ nTregs.

Cyclophosphamide lymphodepletion eradicates 19z1+ nTregs and restores antitumor efficacy of 19-28z+ effector T cells

Lymphodepleting preconditioning regimens can enhance the antitumor efficacy of adoptively transferred cytotoxic T cells which may be mediated in part through the eradication of Tregs in the host (26, 27). In particular, cyclophosphamide chemotherapy has been shown to effectively eliminate Tregs (37–39). To this end, we next investigated whether cyclophosphamide therapy (100 mg/kg) following infusion of 19z1+ nTregs can abrogate effector T‐cell suppression by nTregs. SCID-Beige mice bearing systemic Raji tumors were injected with 19z1+ nTregs on day 5 following tumor cell infusion. On day 6, 24 hours prior to treatment with 19-28z+ T cells, mice were injected i.p. with cyclophosphamide. In contrast to mice infused with 19z1+ nTregs followed by 19-28z+ effector T cells with no long-term survival, mice infused sequentially with 19z1+ nTregs, i.p. cyclophosphamide, and 19-28z+ effector T cells demonstrated a prolonged long-term survival (67%) which compares favorably to mice treated with 19-28z+ effector T cells alone (78%; Fig. 6A). Furthermore, these studies demonstrated Raji tumors to be largely refractory to cyclophosphamide treatment at this dose level as evidenced by the poor survival of the cyclophosphamide followed by Pz1+ effector T‐cell–treated control cohort (Fig. 6A).

FACS analyses to assess the 19z1+ nTreg to 19-28z+ effector T‐cell ratios in the bone marrow of cyclophosphamide treated mice at 24 hours post 19-28z+ effector T‐cell infusion demonstrated a 0.14 nTreg to effector T‐cell ratio, in contrast to a 0.9 nTreg to effector T‐cell ratio seen in nonlymphodepleted mice (Fig. 6B). These data verify cyclophosphamide depletion of endogenous nTregs.

The generation of tumor-targeted T cells for adoptive therapy may be insufficient to achieve significant antitumor responses in the clinical setting. Specifically, the tumor itself may foster an environment capable of impairing targeted effector T‐cell function. Specifically, Tregs, which are relevant in both the clinic and in immune-competent animal models of disease (40, 41), are absent in the xenograft tumor models previously used to study modified human T cells in vivo.

Herein we report that isolated human nTregs from healthy donors may be efficiently transduced to express CARs and subsequently expanded in vitro. While other groups have examined the effects of induced Tregs (iTreg) toward similar ends (42), we opted to isolate and expand nTregs due to concerns regarding the stability of FOXP3 expression in iTregs (43). The resulting tumor-targeted nTregs successfully traffic to tumor in SCID-Beige mice recapitulating a hostile tumor microenvironment seen in the clinical setting. nTreg infiltrated tumors were markedly resistant to eradication by CAR-modified effector T cells even at low (1:8) nTreg to effector T‐cell ratios, while tumor resistance to effector T-cell eradication was no longer apparent at a ratio of 1:16, consistent with a dose dependent nTreg-mediated suppression. This dose dependent nature of inhibition, evidence of persistence of tumor-targeted nTregs within the tumors, and the inability of nontumor-targeted control Pz1+ nTregs to inhibit 19-28z+ effector T cells even at a 1:1 ratio, all support the notion that the suppression of tumor-targeted effector T cells is specifically dependent upon the presence of these nTregs within the tumor microenvironment.

While our in vitro data suggest that 19z1+ Tregs are capable of inhibiting both CAR-mediated cytotoxicity and proliferation, which of these factors predominates in vivo in this tumor model is currently unclear. However, our prior studies suggest that proliferation or persistence does not play a dominant role in this tumor model, as multiple infusions of 19-28z+ T cells are needed to achieve optimal antitumor efficacy in a pre B cell ALL tumor model (3). These data are consistent with limited in vivo persistence and proliferation of the CAR-modified cells in SCID-Beige mice favoring inhibited effector T‐cell cytotoxicity by colocalized nTregs as the primary mechanism of suppression observed in our studies.

The mechanism of in vivo nTreg suppression in our model further appears to be dependent, in part, on the activation status of nTregs at the tumor site since nTregs modified to express the 19(del) CAR retained a statistically significant ability to suppress in vivo effector T‐cell function when compared to mice treated with 19-28z+ effector T cells alone, but demonstrated statistically inferior suppression when compared to mice pretreated with 19z1+ nTregs (Fig. 5E). While both sets of CAR-modified nTregs were previously activated using CD3/28 beads and IL-2, only the 19z1+ nTregs receive additional intratumoral activation via a functional CAR. This phenomenon is consistent with prior literature reporting the requirement for activation to induce Treg-mediated suppression (36) although activation and suppression can be separated temporally (44). The mechanism of this retained but attenuated suppression may be partially mediated by direct contact of the nTreg with effector T cells since the 19(del)+ nTregs successfully traffic to the tumor (35). Additionally, we found that both 19z1+ and 19(del)+ nTreg infused mice demonstrated equal levels of IL-2 reduction in serum suggesting that 19(del)+ nTregs may further inhibit effector T‐cell function through a retained ability to sequester of IL-2, a known mechanism of Treg-mediated suppression (23).

Our data are consistent with the notion that tumor infiltrating Tregs may represent a significant obstacle to the successful application of this adoptive cell therapy. One relatively direct method of overcoming this obstacle would be to eliminate Tregs prior to CAR-modified T‐cell infusion. While several therapeutic options exist for this purpose, including anti-CD25 antibodies and immunotoxins (45), we chose cyclophosphamide due to its established ability to eliminate Tregs in the context of an antitumor response (46, 47) as well as the applicability of this approach to ongoing Phase I clinical trials at our center using autologous 19-28z modified T cells to treat patients with relapsed or refractory B cell malignances. In our studies, prior lymphodepletion with cyclophosphamide before adoptive cell therapy of SCID-Beige mice bearing systemic established Raji tumors infiltrated with 19z1+ nTregs abrogated the suppression of subsequently infused 19-28z+ effector T cells by significantly lowering the nTreg to effector T‐cell ratios from 0.90 to 0.14 within the tumor microenvironment (Fig. 6B). These data are furthermore consistent with our observed in vivo dose dependent nTreg-mediated suppression of effector T cells (Fig. 4B).

In conclusion, these data validate concerns that a hostile tumor microenvironment may markedly compromise CAR-modified effector T‐cell antitumor efficacy in the clinical setting. Further our data support the incorporation of lymphodepleting chemotherapy prior to infusion of CAR-modified tumor-targeted T cells in the modification of ongoing clinical trials and the design of future clinical trials. Finally, our data support the potential of CAR-modified nTregs as a novel approach for the treatment of autoimmune diseases.

No potential conflicts of interest were disclosed.

This work was supported by CA138738, CA95152, CA059350, CA08748, CA86438, CA096945, CA094060, The Alliance for Cancer Gene Therapy, Damon Runyon Clinical Investigator Award (R.J. Brentjens), The Annual Terry Fox Run for Cancer Research (New York, NY) organized by the Canada Club of New York, Kate's Team, Mr. William H. Goodwin and Mrs. Alice Goodwin and the Commonwealth Cancer Foundation for Research and the Experimental Therapeutics Center of MSKCC, and the Geoffrey Beene Cancer Foundation. E. Hayman is a Howard Hughes Medical Institute award recipient.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Sadelain
M
,
Riviere
I
,
Brentjens
R
. 
Targeting tumours with genetically enhanced T lymphocytes
.
Nat Rev Cancer
2003
;
3
:
35
45
.
2.
Brentjens
RJ
,
Latouche
JB
,
Santos
E
,
Marti
F
,
Gong
MC
,
Lyddane
C
, et al
Eradication of systemic B-cell tumors by genetically targeted human T lymphocytes co-stimulated by CD80 and interleukin-15
.
Nat Med
2003
;
9
:
279
86
.
3.
Brentjens
RJ
,
Santos
E
,
Nikhamin
Y
,
Yeh
R
,
Matsushita
M
,
La Perle
K
, et al
Genetically targeted T cells eradicate systemic acute lymphoblastic leukemia xenografts
.
Clin Cancer Res
2007
;
13
:
5426
35
.
4.
Cheadle
EJ
,
Gilham
DE
,
Hawkins
RE
. 
The combination of cyclophosphamide and human T cells genetically engineered to target CD19 can eradicate established B-cell lymphoma
.
Br J Haematol
2008
;
142
:
65
8
.
5.
James
SE
,
Orgun
NN
,
Tedder
TF
,
Shlomchik
MJ
,
Jensen
MC
,
Lin
Y
, et al
Antibody-mediated B-cell depletion before adoptive immunotherapy with T cells expressing CD20-specific chimeric T-cell receptors facilitates eradication of leukemia in immunocompetent mice
.
Blood
2009
;
114
:
5454
63
.
6.
Morgan
RA
,
Dudley
ME
,
Wunderlich
JR
,
Hughes
MS
,
Yang
JC
,
Sherry
RM
, et al
Cancer regression in patients after transfer of genetically engineered lymphocytes
.
Science
2006
;
314
:
126
9
.
7.
Kershaw
MH
,
Westwood
JA
,
Parker
LL
,
Wang
G
,
Eshhar
Z
,
Mavroukakis
SA
, et al
A phase I study on adoptive immunotherapy using gene-modified T cells for ovarian cancer
.
Clin Cancer Res
2006
;
12
:
6106
15
.
8.
Till
BG
,
Jensen
MC
,
Wang
J
,
Chen
EY
,
Wood
BL
,
Greisman
HA
, et al
Adoptive immunotherapy for indolent non-Hodgkin lymphoma and mantle cell lymphoma using genetically modified autologous CD20-specific T cells
.
Blood
2008
;
112
:
2261
71
.
9.
Movahedi
K
,
Guilliams
M
,
Van den Bossche
J
,
Van den Bergh
R
,
Gysemans
C
,
Beschin
A
, et al
Identification of discrete tumor-induced myeloid-derived suppressor cell subpopulations with distinct T cell-suppressive activity
.
Blood
2008
;
111
:
4233
44
.
10.
Gajewski
TF
,
Meng
Y
,
Blank
C
,
Brown
I
,
Kacha
A
,
Kline
J
, et al
Immune resistance orchestrated by the tumor microenvironment
.
Immunol Rev
2006
;
213
:
131
45
.
11.
Hoechst
B
,
Ormandy
LA
,
Ballmaier
M
,
Lehner
F
,
Krüger
C
,
Manns
MP
, et al
A new population of myeloid-derived suppressor cells in hepatocellular carcinoma patients induces CD4(+)CD25(+)Foxp3(+) T cells
.
Gastroenterology
2008
;
135
:
234
43
.
12.
Liu
W
,
Putnam
AL
,
Xu-Yu
Z
,
Szot
GL
,
Lee
MR
,
Zhu
S
, et al
CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells
.
J Exp Med
2006
;
203
:
1701
11
.
13.
Sakaguchi
S
. 
Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and non-self
.
Nat Immunol
2005
;
6
:
345
52
.
14.
Suri-Payer
E
,
Amar
AZ
,
Thornton
AM
,
Shevach
EM
. 
CD4+CD25+ T cells inhibit both the induction and effector function of autoreactive T cells and represent a unique lineage of immunoregulatory cells
.
J Immunol
1998
;
160
:
1212
8
.
15.
Gavin
M
,
Rudensky
A
. 
Control of immune homeostasis by naturally arising regulatory CD4+ T cells
.
Curr Opin Immunol
2003
;
15
:
690
6
.
16.
Deaglio
S
,
Dwyer
KM
,
Gao
W
,
Friedman
D
,
Usheva
A
,
Erat
A
, et al
Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression
.
J Exp Med
2007
;
204
:
1257
65
.
17.
McHugh
RS
,
Whitters
MJ
,
Piccirillo
CA
,
Young
DA
,
Shevach
EM
,
Collins
M
, et al
CD4(+)CD25(+) immunoregulatory T cells: gene expression analysis reveals a functional role for the glucocorticoid-induced TNF receptor
.
Immunity
2002
;
16
:
311
23
.
18.
Read
S
,
Malmstrom
V
,
Powrie
F
. 
Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25(+)CD4(+) regulatory cells that control intestinal inflammation
.
J Exp Med
2000
;
192
:
295
302
.
19.
Beyer
M
,
Schultze
JL
. 
Regulatory T cells in cancer
.
Blood
2006
;
108
:
804
11
.
20.
Wolf
AM
,
Wolf
D
,
Steurer
M
,
Gastl
G
,
Gunsilius
E
,
Grubeck-Loebenstein
B
. 
Increase of regulatory T cells in the peripheral blood of cancer patients
.
Clin Cancer Res
2003
;
9
:
606
12
.
21.
Yang
ZZ
,
Novak
AJ
,
Ziesmer
SC
,
Witzig
TE
,
Ansell
SM
. 
Attenuation of CD8(+) T-cell function by CD4(+)CD25(+) regulatory T cells in B-cell non-Hodgkin's lymphoma
.
Cancer Res
2006
;
66
:
10145
52
.
22.
Curtin
JF
,
Candolfi
M
,
Fakhouri
TM
,
Liu
C
,
Alden
A
,
Edwards
M
, et al
Treg depletion inhibits efficacy of cancer immunotherapy: implications for clinical trials
.
PLoS ONE
2008
;
3
:
e1983
.
23.
Miyara
M
,
Sakaguchi
S
. 
Natural regulatory T cells: mechanisms of suppression
.
Trends Mol Med
2007
;
13
:
108
16
.
24.
June
CH
. 
Adoptive T cell therapy for cancer in the clinic
.
J Clin Invest
2007
;
117
:
1466
76
.
25.
Rasku
MA
,
Clem
AL
,
Telang
S
,
Taft
B
,
Gettings
K
,
Gragg
H
, et al
Transient T cell depletion causes regression of melanoma metastases
.
J Transl Med
2008
;
6
:
12
.
26.
Dudley
ME
,
Wunderlich
JR
,
Yang
JC
,
Sherry
RM
,
Topalian
SL
,
Restifo
NP
, et al
Adoptive cell transfer therapy following non-myeloablative but lymphodepleting chemotherapy for the treatment of patients with refractory metastatic melanoma
.
J Clin Oncol
2005
;
23
:
2346
57
.
27.
Muranski
P
,
Boni
A
,
Wrzesinski
C
,
Citrin
DE
,
Rosenberg
SA
,
Childs
R
, et al
Increased intensity lymphodepletion and adoptive immunotherapy–how far can we go?
Nat Clin Pract Oncol
2006
;
3
:
668
81
.
28.
Wichlan
DG
,
Roddam
PL
,
Eldridge
P
,
Handgretinger
R
,
Riberdy
JM
. 
Efficient and reproducible large-scale isolation of human CD4+ CD25+ regulatory T cells with potent suppressor activity
.
J Immunol Methods
2006
;
315
:
27
36
.
29.
Battaglia
M
,
Stabilini
A
,
Migliavacca
B
,
Horejs-Hoeck
J
,
Kaupper
T
,
Roncarolo
MG
. 
Rapamycin promotes expansion of functional CD4+CD25+FOXP3+ regulatory T cells of both healthy subjects and type 1 diabetic patients
.
J Immunol
2006
;
177
:
8338
47
.
30.
Strauss
L
,
Whiteside
TL
,
Knights
A
,
Bergmann
C
,
Knuth
A
,
Zippelius
A
. 
Selective survival of naturally occurring human CD4+CD25+Foxp3+ regulatory T cells cultured with rapamycin
.
J Immunol
2007
;
178
:
320
9
.
31.
Gong
MC
,
Latouche
JB
,
Krause
A
,
Heston
WDW
,
Bander
NH
,
Sadelain
M
. 
Cancer patient T cells genetically targeted to prostate-specific membrane antigen specifically lyse prostate cancer cells and release cytokines in response to prostate-specific membrane antigen
.
Neoplasia
1999
;
1
:
123
7
.
32.
Battaglia
M
,
Stabilini
A
,
Roncarolo
MG
. 
Rapamycin selectively expands CD4+CD25+FoxP3 +regulatory T cells
.
Blood
2005
;
105
:
4743
8
.
33.
Oberg
HH
,
Wesch
D
,
Lenke
J
,
Kabelitz
D
. 
An optimized method for the functional analysis of human regulatory T cells
.
Scand J Immunol
2006
;
64
:
353
60
.
34.
Santos
EB
,
Yeh
R
,
Lee
J
,
Nikhamin
Y
,
Punzalan
B
,
Punzalan
B
, et al
Sensitive in vivo imaging of T cells using a membrane-bound Gaussia princeps luciferase
.
Nat Med
2009
;
15
:
338
44
.
35.
Takahashi
T
,
Kuniyasu
Y
,
Toda
M
,
Sakaguchi
N
,
Itoh
M
,
Iwata
M
, et al
Immunologic self-tolerance maintained by CD25+CD4+ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state
.
Int Immunol
1998
;
10
:
1969
80
.
36.
Thornton
AM
,
Shevach
EM
. 
CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production
.
J Exp Med
1998
;
188
:
287
96
.
37.
Matsushita
N
,
Pilon-Thomas
SA
,
Martin
LM
,
Riker
AI
. 
Comparative methodologies of regulatory T cell depletion in a murine melanoma model
.
J Immunol Methods
2008
;
333
:
167
79
.
38.
North
RJ
. 
Cyclophosphamide-facilitated adoptive immunotherapy of an established tumor depends on elimination of tumor-induced suppressor T cells
.
J Exp Med
1982
;
155
:
1063
74
.
39.
Berd
D
,
Mastrangelo
MJ
. 
Effect of low dose cyclophosphamide on the immune system of cancer patients: depletion of CD4+, 2H4+ suppressor-inducer T-cells
.
Cancer Res
1988
;
48
:
1671
5
.
40.
Beyer
M
,
Kochanek
M
,
Darabi
K
,
Popov
A
,
Jensen
M
,
Endl
E
, et al
Reduced frequencies and suppressive function of CD4+ CD25hi regulatory T cells in patients with chronic lymphocytic leukemia after therapy with fludarabine
.
Blood
2005
;
106
:
2018
25
.
41.
Serafini
P
,
Mgebroff
S
,
Noonan
K
,
Borrello
I
. 
Myeloid-derived suppressor cells promote cross-tolerance in B-cell lymphoma by expanding regulatory T cells
.
Cancer Res
2008
;
68
:
5439
49
.
42.
Loskog
A
,
Giandomenico
V
,
Rossig
C
,
Pule
M
,
Dotti
G
,
Brenner
MK
. 
Addition of the CD28 signaling domain to chimeric T-cell receptors enhances chimeric T-cell resistance to T regulatory cells
.
Leukemia
2006
;
20
:
1819
28
.
43.
Koenecke
C
,
Czeloth
N
,
Bubke
A
,
Schmitz
S
,
Kissenpfennig
A
,
Malissen
B
, et al
Alloantigen-specific de novo-induced Foxp3+ Treg revert in vivo and do not protect from experimental GVHD
.
Eur J Immunol
2009
;
39
:
3091
6
.
44.
Thornton
AM
,
Piccirillo
CA
,
Shevach
EM
. 
Activation requirements for the induction of CD4+CD25+ T cell suppressor function
.
Eur J Immunol
2004
;
34
:
366
76
.
45.
Koenecke
C
,
Ukena
SN
,
Ganser
A
,
Franzke
A
. 
Regulatory T cells as therapeutic target in Hodgkin's lymphoma
.
Expert Opin Ther Targets
2008
;
12
:
769
82
.
46.
Ghiringhelli
F
,
Larmonier
N
,
Schmitt
E
,
Parcellier
A
,
Cathelin
D
,
Garrido
C
, et al
CD4+CD25+ regulatory T cells suppress tumor immunity but are sensitive to cyclophosphamide which allows immunotherapy of established tumors to be curative
.
Eur J Immunol
2004
;
34
:
336
44
.
47.
Roux
S
,
Apetoh
L
,
Chalmin
F
,
Ladoire
S
,
Mignot
G
,
Puig
PE
, et al
CD4+CD25 +Tregs control the TRAIL-dependent cytotoxicity of tumor-infiltrating DCs in rodent models of colon cancer
.
J Clin Invest
2008
;
118
:
3751
61
.