Brian G. Barnett

Bone Marrow Is a Reservoir for CD4+CD25+ Regulatory T Cells that Traffic through CXCL12/CXCR4 Signals

CD4+CD25+ regulatory T cells (Tregs) mediate peripheral T-cell homeostasis and contribute to self-tolerance. Their homeostatic and pathologic trafficking is poorly understood. Under homeostatic conditions, we show a relatively high prevalence of functional Tregs in human bone marrow. Bone marrow strongly expresses functional stromal-derived factor (CXCL12), the ligand for CXCR4. Human Tregs traffic to and are retained in bone marrow through CXCR4/CXCL12 signals as shown in chimeric nonobese diabetic/severe combined immunodeficient mice. Granulocyte colony-stimulating factor (G-CSF) reduces human bone marrow CXCL12 expression in vivo, associated with mobilization of marrow Tregs to peripheral blood in human volunteers. These findings show a mechanism for homeostatic Treg trafficking and indicate that bone marrow is a significant reservoir for Tregs. These data also suggest a novel mechanism explaining reduced acute graft-versus-host disease and improvement in autoimmune diseases following G-CSF treatment.

Regulatory T cells in ovarian cancer: biology and therapeutic potential.

Tumors express tumor‐associated antigens (TAA) and thus should be the object of immune attack. Nonetheless, spontaneous clearance of established tumors is rare. 1 Much work has demonstrated that tumors have numerous strategies either to prevent presentation of TAA, or to prevent TAA presentation in the context of T‐cell co‐signaling molecules. 1,2 Thus, it was thought that lack of TAA‐specific immunity was largely a passive process: tumors simply did not present enough TAA, or antigen‐presenting cells did not have sufficient stimulatory capacity. On this basis, attempts were made to bolster TAA‐specific immunity by using optimal antigen‐presenting cells or by growing TAA‐specific effector T cells ex vivo followed by adoptive transfer. 3 These approaches met with some success in mouse models of human tumors, and showed some early clinical efficacy in human trials, although long‐term efficacy remains to be established, and logistical problems are considerable. 4 These studies established the concept that experimentally induced TAA‐specific immunity is a rational and potentially efficacious means to treat cancer, including ovarian cancer. Nonetheless, recent work demonstrates that lack of naturally induced TAA‐specific immunity is not simply a passive process. 5–12 We discuss recent data clearly demonstrating that ‘tumors actively prevent induction of TAA‐specific immunity through induction of TAA‐specific tolerance’. 13 This tolerance is mediated in part by regulatory T cells (Tregs). Means to revert these tolerizing conditions represent a novel anticancer therapeutic stratagem. We discuss Tregs in this regard in human ovarian cancer and present evidence that depleting Treg in human cancer, including ovarian cancer, using denileukin diftitox (Ontak), improves immunity and may be therapeutic.

Altering regulatory T cell function in cancer immunotherapy: a novel means to boost the efficacy of cancer vaccines.

Cancers express tumor associated antigens that should elicit immune attack, but spontaneous immune rejection of established cancer is rare. Recent data demonstrate that specific and active tumor-mediated mechanisms hinder host anti-tumor immunity. CD4+CD25+ T regulatory cells (Tregs) are important mediators of active immune evasion in cancer. Disrupting tumor-mediated mechanisms hindering host immunity is a novel approach to tumor immunotherapy. Treg depletion improves endogenous anti-tumor immunity and the efficacy of active immunotherapy in animal models for cancer, suggesting that inhibiting Treg function could also improve the limited successes of human cancer immunotherapy. We have identified five strategies to block Treg activity: depletion, interference with trafficking, inhibition of differentiation, blockade of function or raising the effector T cell threshold for suppression. Discovery of additional regulatory cell populations expands the potential targets for these approaches. The fusion toxin denileukin diftitox (Ontak) reduces Treg numbers and function in the blood of some patients with cancer. We discuss specific strategies to block Treg activity and present some of our preliminary data in this area. Combining Treg depletion with active vaccination and other approaches poses additional challenges that are discussed.

Human Leukocyte Antigen G Expression in Breast Cancer: Role in Immunosuppression

Human leukocyte antigen G (HLA-G) is an immunotolerant nonclassical major histocompatibility complex Class Ib molecule. It is expressed by trophoblastic placental cells during pregnancy to protect the fetus from maternal alloreactivity. HLA-G is overexpressed in tumors and involved in cancer immune evasion. Reverse transcription-polymerase chain reaction and immunohistochemistry (IHC) were used to examine HLA-G expression in normal mammary and breast cancer cell lines and normal and human breast cancer tissues. Reverse transcription-polymerase chain reaction confirmed that normal epithelial MCF-12A cells had no HLA-G mRNA expression, whereas cancer cell lines MCF-7, T47D, and MDA-MB-231 and NCI/Adr-Res had various levels of HLA-G mRNA expression. Twelve (12) normal and 38 breast cancer tissues were examined by IHC. Fifty-eight (58) percent (22/38) of cancers had medium to strong staining to HLA-G, whereas only 8% (1/12) of normal breast tissues had medium to strong staining, and the difference was significant (p < 0.05). HLA-G staining was found in the membranes and cytoplasm of cancer cells. In conclusion, breast cancer cells overexpress HLA-G mRNA and protein, and this probably contributes to immune evasion.

Regulatory T Cells: A New Frontier in Cancer Immunotherapy

Tumor-specific immune-mediate cancer therapy was documented in a mouse model about one and half century ago (1). Nonetheless, the success of immune-based cancer treatments in humans has remained quite modest despite advances in our understanding and technology. The current paradigm driving most immune strategies is that tumors express tumor-associated antigens (TAA), thereby making them the objects of immune attack. These TAA should then be captured by professional antigen-presenting cells, particularly dendritic cells, which in turn prime naïve T cells to become TAA-specific effector cells through T cell cosignaling molecules and other mediators. This paradigm predicts that the solution to improving the efficacy of tumor immunotherapy is to augment TAA expression, boost cosignaling, or increase the number of effector T cells or professional antigen-presenting cells. Experience shows, however, that with a few limited exceptions, such strategies do not yield durable clinical successes. Recent work, including from our group, now demonstrates that tumors employ a wide variety of active mechanisms to thwart what could be an otherwise effective host antitumor immune response (2–4). These tumor-associated mechanisms include production of factors such as VEGF, TGF-β, or IL-10; induction of dysfunctional dendritic cells; or dysfunctional T cell cosignaling (5). Much recent work implicates CD4CD25 regulatory T cells (Tregs) as an agent of this tumor-mediated anti-host defense (2, 6). CD4CD25 regulatory T cells normally mediate peripheral tolerance (7, 8). However, if they are abnormally elevated in numbers or function, they have the potential to perturb homeostatic immune functions or defeat a required immune response (6). In 1999, it was demonstrated that depletion of CD4CD25 T cells in a mouse model for cancer using PC61 antibody improved immune-mediated tumor rejection (9). Soon thereafter, CD4CD25 T cell depletion was shown to boost endogenous TAA-specific immunity as well as the efficacy of active immunization or antiCTLA-4 blockade (10, 11). CD4CD25 regulatory T cells are elevated in the peripheral blood of patients with a variety of cancers (2, 12–18). Nonetheless, most of the work performed until this time centered on mouse models of cancer.

Current management of acquired factor VIII inhibitors.

Purpose of reviewAcquired hemophilia is a rare autoimmune disease that can result in life-threatening bleeding if not treated effectively. Appropriate management requires the urgent treatment of bleeding episodes and prompt institution of immunosuppressive therapy for long-term inhibitor eradication. Recent findingsAcute bleeding episodes are generally best controlled with ‘bypassing’ hemostatic factor concentrates. Corticosteroid-based immunosuppressive therapy is effective in eliminating most acquired inhibitors; additional therapies, such as rituximab, are useful for patients who do not respond to standard immune-suppressing regimens. Up to 20% of patients relapse after immunomodulation and require additional treatment. A lack of controlled clinical data hampers the optimal selection of immunosuppressive therapy. SummaryPatients with acquired hemophilia remain at risk for severe hemorrhage until their inhibitors are permanently eradicated. Concurrent with bleed management, immunomodulation should be initiated with corticosteroid-based therapy in order to eliminate the autoantibody and restore normal hemostasis.

Immune tolerance induction for the eradication of inhibitors in patients with hemophilia A

Background: Both genetic and environmental factors contribute to the formation of alloantibodies that bind to functional domains on the Factor VIII (FVIII) molecule and inhibit its function. Patients with hemophilia A who develop high-titer inhibitors are at increased risk for serious hemorrhage and disability, particularly arthropathy, because bleeding events do not respond to standard therapy. Immune tolerance induction (ITI) is usually attempted to eradicate newly diagnosed inhibitors, restore replacement FVIII pharmacokinetics, and improve bleed management and quality of life. Objective: This paper summarizes regimens used for ITI, predictors of success and failure, and adjunctive therapies for patients failing ITI therapy. Methods: This is a systematic review of published reports on ITI regimens, data from registries capturing response rates and predictors of success, and reports of adjunctive treatments used to enhance ITI therapy. Results/conclusion: Many issues remain unresolved, chief among them optimal dose and dosing regimen, choice of FVIII product, and the role of adjunctive therapy. Resolution of these issues, as well as new approaches to inhibitor management, may come from ongoing basic science research and clinical trials.

Manipulating T regulatory cells in cancer immunotherapy

Malignant tumors express associated antigens that should make them objects of immune attack. Nonetheless, spontaneous immune clearance of established cancer is rare. Recent evidence demonstrates that cancers use active mechanisms to block host antitumor immunity. Significant evidence implicates CD4+CD25+ regulatory T cells (Tregs) as an important mechanism of active immune evasion in cancer. Animal models for cancer demonstrate that Treg depletion improves antitumor immunity and the success of immunotherapy. Thus, blocking Treg function could improve human cancer immunotherapy, where successes have been modest to date. The authors propose four means to block Treg activity: direct elimination; or interference with Treg trafficking, differentiation or function. The fusion toxin denileukin diftitox (Ontak®) eliminates Tregs in some human cancers. The authors discuss their preliminary experience with this agent in human cancer, and discuss progress attempting other strategies to block Treg activity. Reversing...

290 DEPLETING REGULATORY T CELLS IS ASSOCIATED WITH IMPROVED IMMUNITY AND TUMOR CLEARANCE IN HUMAN CANCER

Despite a compelling logic, tumor immunotherapy has generally failed. Work by us and others suggests that this failure owes in part to tumor-mediated immune dysfunction. CD4+CD25+ regulatory T cells (Tregs) are thought to contribute to this dysfunction. In support, we show that in ovarian cancer patients, Tregs block tumor-specific immunity, promote tumor growth and predict poor survival. We hypothesize that depleting Tregs will improve immunity and improve therapeutic outcomes in cancer patients. Ontak is a recombinant protein fusing the CD25-binding domain of IL-2 to the active domain of diphtheria toxin. It was designed to kill CD4+CD25+ leukemia cells. We tested the hypothesis that Ontak kills CD4+CD25+ Tregs and thus improves immunity in cancer patients. Patients 1-3 (advanced-stage cancer of the ovary, breast or lung) received a single intravenous infusion of 9 μg/kg Ontak. Patient 4 had relapsed stage IIIC ovarian cancer and received a single dose of Ontak at 12 μg/kg. Blood immune cells from before and after treatment were enumerated and cytokines were studied by flow cytometry. T cell suppression was also tested. Prior to infusion, the mean blood CD4+CD25+ cells/mm3 was 126, which was 26.8% of CD4+ T cells. This decreased to a mean of 78 cells/mm3 blood and mean of 19.0% of CD4+ T cells 3-5 days after Ontak. Also, interferon-γ-expressing T cells increased approximately 50% and FOXP3 (a functional marker for Tregs) was reduced after Ontak. In patient 4, CD4+CD25+ cells inhibited T cell proliferation in vitro prior to but not after treatment. Further, a single Ontak-mediated Treg depletion reduced blood CA-125 from 121 to 38 U/mL four weeks later in patient 4, suggesting clinical efficacy. Patient 4 then received 6 additional doses of Ontak at 12 μg/kg with a further decrease in blood CA-125 level to 16 U/mL four weeks after the final dose. Additionally, a PET/CT fusion demonstrated dramatic reduction of metastatic disease. The CA-125 level remains normal two months after the final Ontak dose. Our data demonstrate for the first time that Ontak efficiently depletes Tregs in patients with cancer, and Treg depletion is associated with improved T cell immunity, normalized CA-125 and reduced tumor metastasis. The data may help explain failures of current tumor immunotherapy protocols. It further suggests that Treg depletion or a combination with current immunotherapeutic regimens will be a novel strategy for treating patients with cancer.