Jennifer A. Westwood

Cutting Edge: Tumor Rejection Mediated by NKG2D Receptor-Ligand Interaction Is Dependent upon Perforin

We have investigated the primary immunity generated in vivo by MHC class I-deficient and -competent tumor cell lines that expressed the NKG2D ligand retinoic acid early inducible-1 (Rae-1) β. Rae-1β expression on class I-deficient RMA-S lymphoma cells enhanced primary NK cell-mediated tumor rejection in vivo, whereas RMA-Rae-1β tumor cells were rejected by a combination of NK cells and CD8+ T cells. Rae-1β expression stimulated NK cell cytotoxicity and IFN-γ secretion in vitro, but not proliferation. Surprisingly, only NK cell perforin-mediated cytotoxicity, but not production of IFN-γ, was critical for the rejection of Rae-1β-expressing tumor cells in vivo. This distinct requirement for perforin activity contrasts with the NK cell-mediated rejection of MHC class I-deficient RMA-S tumor cells expressing other activating ligands such as CD70 and CD80. Thus, these results indicated that NKG2D acted as a natural cytotoxicity receptor to stimulate perforin-mediated elimination of ligand-expressing tumor cells.

Persistence and Efficacy of Second Generation CAR T Cell Against the LeY Antigen in Acute Myeloid Leukemia

In a phase I study of autologous chimeric antigen receptor (CAR) anti-LeY T-cell therapy of acute myeloid leukemia (AML), we examined the safety and postinfusion persistence of adoptively transferred T cells. Following fludarabine-containing preconditioning, four patients received up to 1.3 × 109 total T cells, of which 14-38% expressed the CAR. Grade 3 or 4 toxicity was not observed. One patient achieved a cytogenetic remission whereas another with active leukemia had a reduction in peripheral blood (PB) blasts and a third showed a protracted remission. Using an aliquot of In111-labeled CAR T cells, we demonstrated trafficking to the bone marrow (BM) in those patients with the greatest clinical benefit. Furthermore, in a patient with leukemia cutis, CAR T cells infiltrated proven sites of disease. Serial PCR of PB and BM for the LeY transgene demonstrated that infused CAR T cells persisted for up to 10 months. Our study supports the feasibility and safety of CAR-T-cell therapy in high-risk AML, and demonstrates durable in vivo persistence.

Cutting edge : TRAIL deficiency accelerates hematological malignancies

TNF apoptosis-inducing ligand is attracting considerable interest as a potential extrinsic tumor suppressor mechanism, although previous reports have conveyed somewhat contrasting views regarding the likely importance of this pathway. In this study, we provide the first evaluation of spontaneous tumor formation over the life span of TRAIL-deficient mice. Interestingly, >25% of these mice do develop lymphoid malignancies after 500 days of life. TRAIL suppressed the initiation and development of both tumors of lymphoid and stromal origin in the context of the loss of at least one p53 allele. Specific examination of the role of TRAIL in Her2/neu oncogene-driven mammary epithelial cancer revealed no critical role for TRAIL despite the inherent TRAIL sensitivity of such mammary carcinomas. Overall, the data indicate an important function of TRAIL in controlling carcinogenesis, but suggest that further examination of this pathway in epithelial malignancies is warranted.

Autoimmunity associated with immunotherapy of cancer

In this age of promise of new therapies for cancer, immunotherapy is emerging as an exciting treatment option for patients. Vaccines and cytokines are being tested extensively in clinical trials, and strategies using monoclonal antibodies and cell transfer are mediating dramatic regression of tumors in patients with certain malignancies. However, although initially advocated as being more specific for cancer and having fewer side effects than conventional therapies, it is becoming increasingly clear that many immunotherapies can lead to immune reactions against normal tissues. Immunotoxicities resulting from treatment can range from relatively minor conditions, such as skin depigmentation, to severe toxicities against crucial organ systems, such as liver, bowel, and lung. Treatment-related toxicity has correlated with better responses in some cases, and it is probable that serious adverse events from immune-mediated reactions will increase in frequency and severity as immunotherapeutic approaches become more effective. This review introduces immunotherapeutic approaches to cancer treatment, provides details of toxicities arising from therapy, and discusses future potential ways to avoid or circumvent these side effects.

Immune modulation of the tumor microenvironment for enhancing cancer immunotherapy

There is much promise in the use of immunotherapy for the treatment of cancer. Approaches such as those using antibodies or adoptive cell transfer can mediate complete tumor regression in a proportion of patients. However, the tumor microenvironment can inhibit immune responses leading to ineffective or suboptimal responses of tumors to immunotherapy in the majority of cases. As our knowledge of the tumor microenvironment increases, many strategies are emerging for changing the immunosuppressive nature of the tumor toward a microenvironment able to support immunity. These strategies aim to enhance the ability of immunotherapies to initiate effective immune responses able to destroy tumors. In this article, we review approaches that use immunomodulators specifically to modify the tumor microenvironment, and their use in combination with other immune-based strategies for cancer therapy.

Oncolytic Virus and Anti–4-1BB Combination Therapy Elicits Strong Antitumor Immunity against Established Cancer

Oncolytic virotherapy using vaccinia virus (Vv) has shown some encouraging antitumor responses in mouse models and patients, but the breadth of efficacy in clinical trials has been somewhat limited. Given that antitumor effects have correlated with increased host immune responses, we hypothesized that improved therapeutic outcomes may be achieved by using oncolytic virus (OV) in combination with a potent immune agonist reagent. In this study, we carried out a preclinical evaluation of a genetically engineered strain of oncolytic vaccinia virus (Vvdd) for its capacity to induce antitumor responses when combined with an agonist antibody (Ab) specific for the costimulatory molecule 4-1BB (CD137). In immune-competent syngeneic mouse models of cancer, this combination therapy significantly reduced the growth of established subcutaneous tumors relative to either treatment alone. Importantly, the development of pulmonary metastatic lesions was also reduced. Tumor growth inhibition was associated with increased numbers of CD11b(+) and CD11c(+) myeloid cells in the tumor draining lymph nodes, greater infiltration of CD8(+) effector T and natural killer (NK) cells, and a more sustained presence of neutrophils at the tumor site. Depletion of T or NK cells or neutrophils reduced efficacy, confirming their contribution to an effective therapeutic response. We further extended this conclusion through results from IFNγ-deficient mice. In summary, our findings offered a proof-of-concept for a combinatorial approach to enhance the antitumor efficacy of an OV, suggesting a strategy to improve their use as an immunotherapeutic treatment for cancer.

Clinical application of genetically modified T cells in cancer therapy

Immunotherapies are emerging as highly promising approaches for the treatment of cancer. In these approaches, a variety of materials are used to boost immunity against malignant cells. A key component of many of these approaches is functional tumor‐specific T cells, but the existence and activity of sufficient T cells in the immune repertoire is not always the case. Recent methods of generating tumor‐specific T cells include the genetic modification of patient lymphocytes with receptors to endow them with tumor specificity. These T cells are then expanded in vitro followed by infusion of the patient in adoptive cell transfer protocols. Genes used to modify T cells include those encoding T‐cell receptors and chimeric antigen receptors. In this review, we provide an introduction to the field of genetic engineering of T cells followed by details of their use against cancer in the clinic.

Adoptive immunotherapy combined with intratumoral TLR agonist delivery eradicates established melanoma in mice

Toll-like receptor (TLR) agonists can trigger broad inflammatory responses that elicit rapid innate immunity and promote the activities of lymphocytes, which can potentially enhance adoptive immunotherapy in the tumor-bearing setting. In the present study, we found that Polyinosinic:Polycytidylic Acid [Poly(I:C)] and CpG oligodeoxynucleotide 1826 [CpG], agonists for TLR 3 and 9, respectively, potently activated adoptively transferred T cells against a murine model of established melanoma. Intratumoral injection of Poly(I:C) and CpG, combined with systemic transfer of activated pmel-1 T cells, specific for gp10025–33, led to enhanced survival and eradication of 9-day established subcutaneous B16F10 melanomas in a proportion of mice. A series of survival studies in knockout mice supported a key mechanistic pathway, whereby TLR agonists acted via host cells to enhance IFN-γ production by adoptively transferred T cells. IFN-γ, in turn, enhanced the immunogenicity of the B16F10 melanoma line, leading to increased killing by adoptively transferred T cells. Thus, this combination approach counteracted tumor escape from immunotherapy via downregulation of immunogenicity. In conclusion, TLR agonists may represent advanced adjuvants within the setting of adoptive T-cell immunotherapy of cancer and hold promise as a safe means of enhancing this approach within the clinic.

Tissues in different anatomical sites can sculpt and vary the tumor microenvironment to affect responses to therapy.

The tumor microenvironment can promote tumor growth and reduce treatment efficacy. Tumors can occur in many sites in the body, but how surrounding normal tissues at different anatomical sites affect tumor microenvironments and their subsequent response to therapy is not known.We demonstrated that tumors from renal, colon, or prostate cell lines in orthotopic locations responded to immunotherapy consisting of three agonist antibodies, termed Tri-mAb, to a much lesser extent than the same tumor type located subcutaneously. A tissue-specific response to Tri-mAb was confirmed by ex vivo separation of subcutaneous (SC) or orthotopic tumor cells from stromal cells, followed by reinjection of tumor cells into the opposite site. Compared with SC tumors, orthotopic tumors had a microenvironment associated with a type 2 immune response, related to immunosuppression, and an involvement of alternatively activated macrophages in the kidney model. Orthotopic kidney tumors were more highly vascularized than SC tumors. Neutralizing the macrophage- and Th2-associated molecules chemokine (C-C motif) ligand 2 or interleukin-13 led to a significantly improved therapeutic effect. This study highlights the importance of the tissue of implantation in sculpting the tumor microenvironment. These are important fundamental issues in tumor biology and crucial factors to consider in the design of experimental models and treatment strategies.

Cutting Edge: Novel Priming of Tumor-Specific Immunity by NKG2D-Triggered NK Cell-Mediated Tumor Rejection and Th1-Independent CD4+ T Cell Pathway

NKG2D is an activation receptor on NK cells and has been demonstrated as a primary cytotoxicity receptor for mouse NK cells. Primary rejection of class I-deficient RMA-S lymphoma cells expressing the NKG2D ligand, retinoic acid early inducible-1β, was critically dependent upon NK cell perforin and occurred independently of T cells. NKG2D-triggered NK cell rejection of RMA-S-retinoic acid early inducible-1β tumor primed a secondary tumor-specific T cell response mediated by both CD4+ and CD8+ T cells in the effector phase. Surprisingly, during the priming phase, CD4+ T cells, but not CD8+ T cells, were also required to generate this secondary T cell immunity; however, T cell priming was independent of Th1 cytokines, such as IFN-γ and IL-12. These data imply a novel pathway for priming T cell immunity, that is, stimulated upon NK cell-mediated cytotoxicity of NKG2D ligand-expressing tumor cells, dependent upon CD4+ T cells in the primary phase, and independent of conventional Th1-type immunity.